**Meet the editor**

Dr Philippe E. Spiess is an assistant professor with dual appointment in the department of Genitourinary Oncology at the Moffitt Cancer Center and the department of Urology at the University of South Florida in Tampa, Florida. Dr Spiess was born and raised in Montreal, Canada where he completed his urological training at McGill University. He subsequently completed his urologic

oncologic training at the M. D. Anderson Cancer Center in Houston, Texas. Dr Spiess has published over 80 peer reviewed scientific papers in urologic oncology in addition to being a nationally and internationally recognized expert in the management of genitourinary malignancies. He has dedicated much of his research efforts to optimizing the surgical management of locally advanced and locally recurrent prostate cancer. He currently sits on the NCCN Panel for bladder and penile cancer in addition to being the video section editor of the International Brazilian Journal of Urology.

Contents

**Preface IX** 

**Part 1 Cancer Biology 1** 

Daniel Djakiew

Chapter 1 **Epidemiology of Prostate Cancer:** 

**The Case of Ethnic German Migrants from the Former Soviet Union 3**  Volker Winkler and Heiko Becher

**A Suppressor of Growth and Cell Migration** 

**and Correlation TRAIL Expression in Prostate Cancer 45** 

Chapter 2 **NSAID Induction of p75NTR in the Prostate:** 

**Via the p38 MAPK Pathway 23** 

Yuanyuan Mi, Lijie Zhu and Ninghan Feng

**for the Treatment of Prostate Cancer 59**  Sanjay Kumar Saxena and Ashutosh Dash

**Compared to Hypoxia and Reoxygenation 77**  L. Terraneo, E. Finati, E. Virgili, G. Demartini, L. De Angelis,

R. Dall'Aglio, F. Fraschini, M. Samaja and R. Paroni

Chapter 3 **Polymorphism Analysis of TRAIL Gene** 

Chapter 4 **Development of Miniature 125I - Seeds** 

Chapter 5 **LNCaP Prostate Cancer Growth** *In Vivo***: Oncostatic Effects of Melatonin as** 

Chapter 6 **Cancer Detection from Transrectal Ultrasound Guided Biopsy in a Single Center 93**  Selvalingam S., Leong A.C., Natarajan C.,

Yunus R. and Sundram M.

Chapter 7 **Elderly and Early Prostate Cancer 101** 

**Part 2 Diagnostic Markers 91** 

K. Stamatiou

### Contents


#### **Part 2 Diagnostic Markers 91**


#### **Part 3 Therapeutic Novelties 151**


### Preface

In this book entitled "Prostate Cancer – Original Scientific Reports and Case Studies", we underscore active areas of scientific research within the field of prostate cancer. This textbook encompasses 3 sections pertaining to the topics of: 1) cancer biology, 2) diagnostic markers, and 3) therapeutic novelties. This book is an essential resource for healthcare professionals and scientist dedicated to the field of prostate cancer research. This book is a celebration of the significant advances made within this field over the past decade, with the hopes that this is the stepping stone for the eradication of this potentially debilitating and/or fatal malignancy.

As the editor-in-chief of this book, I would like to acknowledge the significant efforts made by the entire editorial team of InTech Open Access Publisher in the preparation of this book, particularly Mrs Radja, the publishing manager. The aim of the entire editorial team and contributing authors has been to generate the highest quality publication such that it can provide the armamentarium for our healthcare team and researchers of today and tomorrow with the necessary tools to optimize the care of our patients and potentially make major scientific discoveries.

I would like to dedicate this book to the loving memory of my uncle Jacques Amiel who took an active role in my upraising. His life is a testament that success is defined not only by our achievements in our respective specialties but as well by ensuring we surround ourselves with loving family and close friends. I want to as well dedicate this book to my patients who have not only entrusted me with their health and well being for many years but as well have taught me that heroism is alive and well in our society in the way they deal with their malignancy with unwavering courage and dignity every single day.

> **Philippe E. Spiess,** Editor-In-Chief Assistant Professor, Dept of Genitourinary Oncology H. Lee Moffitt Cancer Center Tampa, Florida USA

**Scott Eggener, M.D.**  Assistant Professor, Department of Urology University of Chicago, Chicago USA

#### **Vladimir Mourariev, M.D., Ph.D.**  Department of Urology, University of Cincinnati, Ohio USA

#### **Matthew Biagioli, M.D.**  Assistant Professor, Department of Radiation Oncology Moffitt Cancer Center, Tampa USA

#### **Kevin Zorn, M.D.;**

Assistant Professor, Department of Urology Universite de Montreal, Montreal Canada

#### **Shahrokh Shariat, M.D., Ph.D.;**

Associate Professor, Department of Urology Weill Cornell Medical Center, New York USA

#### **Dr Alejandro Rodriguez**

Assistant Professor, Department of Urology University of South Florida, Tampa USA

X Preface

**Scott Eggener, M.D.** 

**Matthew Biagioli, M.D.** 

**Kevin Zorn, M.D.;**

Moffitt Cancer Center, Tampa

Universite de Montreal, Montreal

**Shahrokh Shariat, M.D., Ph.D.;** 

**Dr Alejandro Rodriguez**

USA

USA

USA

Canada

USA

USA

University of Chicago, Chicago

**Vladimir Mourariev, M.D., Ph.D.** 

Assistant Professor, Department of Urology

Assistant Professor, Department of Urology

Associate Professor, Department of Urology Weill Cornell Medical Center, New York

Assistant Professor, Department of Urology

University of South Florida, Tampa

Department of Urology, University of Cincinnati, Ohio

Assistant Professor, Department of Radiation Oncology

**Part 1** 

**Cancer Biology** 

**Part 1** 

**Cancer Biology** 

**1** 

**Epidemiology of Prostate Cancer:** 

**from the Former Soviet Union** 

Volker Winkler and Heiko Becher

*Institute of Public Health University of Heidelberg* 

*Germany* 

 **The Case of Ethnic German Migrants** 

In Germany, prostate cancer is the leading cause of cancer (26%) and the third leading cause of death from cancer in males (10%). The mean age of disease and of death due to prostate cancer is 70.1 years and 77.5 years, respectively (Ziegler et al., 2009). Few people are diagnosed before the age of 50 years. A 70-year old man has a 6% risk of developing prostate cancer within the next ten years, whereas, the risk for a 40-year-old man is 0.1% (RKI, 2010). In 2006, approximately 238,500 men were diagnosed with prostate cancer

Currently, there is a statutory screening programme for prostate cancer in Germany. All men aged 45 years and over are asked once a year by their physician if they have any symptoms. This screening also includes an examination of the sexual organs, the lymph nodes, as well as a palpation examination of the prostate via the rectum. Presently, the

During the 1970s incidence was stable around 50 per 100,000 persons (Ziegler et al., 2009). Since 1980, the incidence has increased (see Figure 1). The yearly number of new prostate cancer cases in Germany has risen by 200% (from 1980-2006), which may partly be due to the demographic change. During the same period the age-standardized incidence rate (standardized to the European standard population) also increased by 110%. In 2006, the age-standardized incidence rate was 110.1 per 100,000 men (RKI, 2010). This increase is mainly due the use of new diagnostic methods, e.g. testing for PSA. Earlier diagnosis, in terms of both the cancer's stage of development and the patient's age, has led to much higher incidence rates in the age group 50- to 69-years and lower rates among over-75 year-olds. Additionally, the mean age at onset fell from 73 years in 1980 to 70 years in

On the other hand, age-standardized mortality rates have been more or less stable during the last decades and began to fall slightly since 1995. In 2006, the age-standardized mortality

prostate-specific antigen (PSA) blood test is not part of the statutory screening.

**1. Introduction** 

2006 (RKI, 2010).

**1.1 Epidemiology of prostate cancer** 

during the previous five years in Germany.

**1.1.2 Longitudinal trends in Germany** 

**1.1.1 Situation in Germany** 

## **Epidemiology of Prostate Cancer: The Case of Ethnic German Migrants from the Former Soviet Union**

Volker Winkler and Heiko Becher *Institute of Public Health University of Heidelberg Germany* 

#### **1. Introduction**

### **1.1 Epidemiology of prostate cancer**

#### **1.1.1 Situation in Germany**

In Germany, prostate cancer is the leading cause of cancer (26%) and the third leading cause of death from cancer in males (10%). The mean age of disease and of death due to prostate cancer is 70.1 years and 77.5 years, respectively (Ziegler et al., 2009). Few people are diagnosed before the age of 50 years. A 70-year old man has a 6% risk of developing prostate cancer within the next ten years, whereas, the risk for a 40-year-old man is 0.1% (RKI, 2010). In 2006, approximately 238,500 men were diagnosed with prostate cancer during the previous five years in Germany.

Currently, there is a statutory screening programme for prostate cancer in Germany. All men aged 45 years and over are asked once a year by their physician if they have any symptoms. This screening also includes an examination of the sexual organs, the lymph nodes, as well as a palpation examination of the prostate via the rectum. Presently, the prostate-specific antigen (PSA) blood test is not part of the statutory screening.

#### **1.1.2 Longitudinal trends in Germany**

During the 1970s incidence was stable around 50 per 100,000 persons (Ziegler et al., 2009). Since 1980, the incidence has increased (see Figure 1). The yearly number of new prostate cancer cases in Germany has risen by 200% (from 1980-2006), which may partly be due to the demographic change. During the same period the age-standardized incidence rate (standardized to the European standard population) also increased by 110%. In 2006, the age-standardized incidence rate was 110.1 per 100,000 men (RKI, 2010). This increase is mainly due the use of new diagnostic methods, e.g. testing for PSA. Earlier diagnosis, in terms of both the cancer's stage of development and the patient's age, has led to much higher incidence rates in the age group 50- to 69-years and lower rates among over-75 year-olds. Additionally, the mean age at onset fell from 73 years in 1980 to 70 years in 2006 (RKI, 2010).

On the other hand, age-standardized mortality rates have been more or less stable during the last decades and began to fall slightly since 1995. In 2006, the age-standardized mortality

Epidemiology of Prostate Cancer:

France Sweden USA Finland Austria Germany Denmark England Poland Hong Kong

to the development of malignancies.

**1.2 Migration and health 1.2.1 General aspects** 

cancer risk (Grönberg, 2003; Patel & Klein, 2009).

(RKI, 2010)

**1.1.4 Risk factors** 

The Case of Ethnic German Migrants from the Former Soviet Union 5

0 20 40 60 80 100 120 140 160 180

incidence mortality

Fig. 2. Age-standardized prostate cancer incidence and mortality rates per 100,000

(European Standard) in Germany in 2006 compared internationally (except France in 2005)

Risk factors and factors affecting disease progression are basically unknown. Clearly, male sex hormones play a role, without them prostate cancer would not develop. In addition, the aging process contributes to the development of prostate cancer as it does for all cancer sites. Cellular repair mechanisms become more and more error prone with age, which contributes

A genetic predisposition has been discussed, because of a higher incidence in several ethnic groups and disease at a younger age. A clustering of the disease among close relatives has also been shown, although there is no consensus on which inheritable genetic defects are involved. In spite of extensive research, reliable findings on risk factors relating to lifestyle, diet or the environment remain elusive. Possible lifestyle risk factors are high intakes of α-linolenic acid (a polyunsaturated fatty acid in vegetables and dairy products) and calcium. Common risk factors for various cancer sites such as tobacco smoking, alcohol consumption and low physical activity do not seem to affect prostate

Worldwide, there are many epidemiological studies of migrant populations that lead to new findings on the etiology of diseases (McCredie, 1998). Additionally, these studies help to develop targeted cancer prevention and early detection strategies for migrant groups. In general, research on migrants focuses on topics that are related to selection. There are push and pull factors influencing the migration process. Push factors make people more

Fig. 1. Age-standardized prostate cancer incidence and mortality rates per 100,000 (European Standard) in Germany (RKI, 2011)

Between 1984 and 1998, the 5-year survival rate in Germany was 82% (RKI, 2008). Currently, relative 5-year survival rates are about 90% (Ziegler et al., 2009). However, whether this slight improvement in survival is a result of earlier diagnosis due to screening in the last years is not clear. With regards to prognosis, a distinction must be made between slowly progressing forms and aggressive metastasizing forms, which occur in greater proportions among younger men (under 60).

#### **1.1.3 International comparison**

An international comparison of German prostate cancer mortality and incidence to selected international countries is displayed in Figure 2. The cancer mortality rate in Germany is among the lowest in Europe, whereas the incidence is around the European average. Internationally, some of the lowest prostate cancer rates with regard to mortality and incidence are seen in Hong Kong. Scandinavian countries are among those with the highest prostate cancer mortality worldwide. Prostate cancer mortality rates are also estimated to be very high in some African and South American countries (Ferlay, 2010). A country-specific comparison shows that high prostate cancer mortality rates do not necessarily mean high incidence rates and vice versa (RKI, 2010).

Fig. 2. Age-standardized prostate cancer incidence and mortality rates per 100,000 (European Standard) in Germany in 2006 compared internationally (except France in 2005) (RKI, 2010)

#### **1.1.4 Risk factors**

4 Prostate Cancer – Original Scientific Reports and Case Studies

rate was 21.2 per 100,000 men. The 30% increase in the number of deaths since 1980 is a

1980 1985 1990 1995 2000 2005

Fig. 1. Age-standardized prostate cancer incidence and mortality rates per 100,000

(European Standard) in Germany (RKI, 2011)

among younger men (under 60).

**1.1.3 International comparison** 

incidence rates and vice versa (RKI, 2010).

incidence mortality

Between 1984 and 1998, the 5-year survival rate in Germany was 82% (RKI, 2008). Currently, relative 5-year survival rates are about 90% (Ziegler et al., 2009). However, whether this slight improvement in survival is a result of earlier diagnosis due to screening in the last years is not clear. With regards to prognosis, a distinction must be made between slowly progressing forms and aggressive metastasizing forms, which occur in greater proportions

An international comparison of German prostate cancer mortality and incidence to selected international countries is displayed in Figure 2. The cancer mortality rate in Germany is among the lowest in Europe, whereas the incidence is around the European average. Internationally, some of the lowest prostate cancer rates with regard to mortality and incidence are seen in Hong Kong. Scandinavian countries are among those with the highest prostate cancer mortality worldwide. Prostate cancer mortality rates are also estimated to be very high in some African and South American countries (Ferlay, 2010). A country-specific comparison shows that high prostate cancer mortality rates do not necessarily mean high

result of demographic change.

0

20

40

60

80

100

Risk factors and factors affecting disease progression are basically unknown. Clearly, male sex hormones play a role, without them prostate cancer would not develop. In addition, the aging process contributes to the development of prostate cancer as it does for all cancer sites. Cellular repair mechanisms become more and more error prone with age, which contributes to the development of malignancies.

A genetic predisposition has been discussed, because of a higher incidence in several ethnic groups and disease at a younger age. A clustering of the disease among close relatives has also been shown, although there is no consensus on which inheritable genetic defects are involved. In spite of extensive research, reliable findings on risk factors relating to lifestyle, diet or the environment remain elusive. Possible lifestyle risk factors are high intakes of α-linolenic acid (a polyunsaturated fatty acid in vegetables and dairy products) and calcium. Common risk factors for various cancer sites such as tobacco smoking, alcohol consumption and low physical activity do not seem to affect prostate cancer risk (Grönberg, 2003; Patel & Klein, 2009).

#### **1.2 Migration and health**

#### **1.2.1 General aspects**

Worldwide, there are many epidemiological studies of migrant populations that lead to new findings on the etiology of diseases (McCredie, 1998). Additionally, these studies help to develop targeted cancer prevention and early detection strategies for migrant groups.

In general, research on migrants focuses on topics that are related to selection. There are push and pull factors influencing the migration process. Push factors make people more

Epidemiology of Prostate Cancer:

& Karagas, 1987).

pattern environment.

they were promised.

the last decades of the USSR.

(International Organization for Migration, 2005).

The Case of Ethnic German Migrants from the Former Soviet Union 7

country of origin, showing cancer site specific patterns for succeeding generations (Thomas

Results of an American study support the theory of a rather strong genetic influence on risk of prostate cancer. The study compared patterns of prostate cancer among black and white men (Chu et al., 2003). Black Americans had substantially higher prostate cancer rates than white Americans, but the longitudinal trends such as decreasing mortality, increasing incidence and survival were similar. Although this was not a typical migrant study, it compared different ethnic and thus genetic and lifestyle factors in a known risk

**1.3 Ethnic German migrants in Germany - background on the study population** 

patterns, focusing on cancer incidence and mortality, in the latter group.

ease of presentation we will use the term Aussiedler throughout the text.

the USSR worsened. Seen as an internal enemy, Stalin restricted their rights.

In the year 2005, only 2.9% of the global population were migrants, but migration is unequally distributed throughout the world. In past years, migration flows have shifted and in some cases, international migration is actually decreasing. Only two areas in the world have seen an increase in migration – North America and the Former Soviet Union (FSU)

Germany has long been a country of immigration. At present, there are two big groups of migrants, the Turks and ethnic Germans from countries of the FSU. We study disease

The 'Aussiedler' are ethnic German migrants and represent a unique group of diaspora migrants. Since 1993, the officially correct term for Aussiedler is Spätaussiedler, however for

The first Aussiedler came to Russia when Peter I (1689–1725) changed his politics towards Europe. They were the beginning of the urban German population in Russia. Tsarina Katharina II (1762–1796) promised the Aussiedler tax exemptions for 30 years, exoneration from military service, freedom of religion, autonomy and subsidy for resettlement. Many Germans living in regions still suffering from war migrated to Russia under these terms. During the first half of the 19th century approximately 55,000 German colonists settled in the Black Sea region. With time the Aussiedler lost several of the rights

For centuries these ethnic Germans lived abroad and were a relatively closed group of people. After the start of World War I the laws of liquidation were implemented. On the basis of these laws more than 200,000 German colonists were driven away. In 1922, after the October-revolution and civil war the Union of Soviet Socialist Republics (USSR) was founded. When the Nazi Party came into power in Germany the situation of the Germans in

Soon after the German aggression against the USSR in 1941 the deportation of the German population started. Following Stalin's decree about 1,200,000 ethnic Germans were deported into the eastern parts of the Soviet Union, predominantly to Siberia, Kazakhstan and in the Urals. Their civil rights were disregarded; they were detained and forbidden to speak German. Most had to work in labour camps in inhumane conditions. An estimated 700,000 Germans died due to bad working and living conditions and inadequate medical treatment. In particular, the Stalinism destroyed the independent German culture in Russia. In 1955, the discrimination was subsided, and the ethnic Germans were allowed to change their residence, but not to their former colony areas. The Aussiedler became partly assimilated in

willing to leave their country of origin, for example a poor economy, or political or religious persecution. Pull factors on the other hand attract migrants to a country like a good employment situation, labour demand, higher wages, higher living standards, political and religious freedom.

It has been suggested that migrants are not representative samples of their population of origin. Migrants are likely to be positively selected when they respond to pull factors in the country of destination and negatively selected in respond to push factors in the country of origin (Lee, 1966). With regards to health, this leads to the so called "healthy migrant effect". In general, people that are younger and healthier are more willing and able to migrate (Jasso et al., 2004). The elderly and people that are ill tend to stay in their country of origin. So, this selection results in migrants that tend to be healthier than their population of origin. It has been shown, that the healthy migrant effect diminishes dramatically with time (Fennelly, 2007).

In general, many different factors affect the health of migrant populations (Marmot et al., 1984): First, the migration itself can have an impact on health. This refers to positive or negative selective factors and to mental stress. Second, disease risk profiles in the country of origin may differ from the host country due to environmental factors for example, which may lead to disease. Third, destination effects which include physical and social environments, for example the integration politics in the destination country may influence migrant health strongly by making health care services easily accessible for migrant populations.

#### **1.2.2 Migration and cancer incidence & mortality**

Cancer is one of the leading causes of deaths in the industrialized world (World Health Organization [WHO], 2004) and the second leading cause of death in Germany (Federal Statistical Office of Germany [DeStatis], 2007). It has been demonstrated that migrant cancer incidence and mortality differs in general from cancer patterns in the respective host population.

Cancer is known to have a long latency period between exposure and disease onset. Important exposure factors can be traced back to childhood and young adulthood. This means short and medium term cancer mortality among first generation migrants is mainly influenced by country-of-origin factors (Parkin & Khlat, 1996).

The longer migrants live and adapt to their destination country, the more their cancer rates converge towards those in that country. This has been shown for stomach, colon and prostate cancer (McKay, 2003). Migrants from non-western countries to Europe were found to be more prone to cancers that are related to infections experienced in early life, such as liver, cervical and stomach cancer. In contrast, migrants of non-western origin were less likely to suffer from cancers related to a western lifestyle, e.g. colorectal and breast cancer (Arnold et al., 2010).

Evidence was found for a transition of cancer incidence and mortality patterns towards the host population among Turkish migrants in Germany (Zeeb et al., 2002). Convergence may occur due to diet acculturation, adaptation of new lifestyles or utilization of often superior health services. Higher mortality from cancers where incidence can be reduced by effective screening programs and those where survival depends on availability of treatment options, may decrease in a relatively shorter time. Another study analyzed differences in cancer rates between first and second generation migrants relative to the host country, stratified by

willing to leave their country of origin, for example a poor economy, or political or religious persecution. Pull factors on the other hand attract migrants to a country like a good employment situation, labour demand, higher wages, higher living standards, political and

It has been suggested that migrants are not representative samples of their population of origin. Migrants are likely to be positively selected when they respond to pull factors in the country of destination and negatively selected in respond to push factors in the country of origin (Lee, 1966). With regards to health, this leads to the so called "healthy migrant effect". In general, people that are younger and healthier are more willing and able to migrate (Jasso et al., 2004). The elderly and people that are ill tend to stay in their country of origin. So, this selection results in migrants that tend to be healthier than their population of origin. It has been shown, that the healthy migrant effect diminishes

In general, many different factors affect the health of migrant populations (Marmot et al., 1984): First, the migration itself can have an impact on health. This refers to positive or negative selective factors and to mental stress. Second, disease risk profiles in the country of origin may differ from the host country due to environmental factors for example, which may lead to disease. Third, destination effects which include physical and social environments, for example the integration politics in the destination country may influence migrant health strongly by making health care services easily accessible for

Cancer is one of the leading causes of deaths in the industrialized world (World Health Organization [WHO], 2004) and the second leading cause of death in Germany (Federal Statistical Office of Germany [DeStatis], 2007). It has been demonstrated that migrant cancer incidence and mortality differs in general from cancer patterns in the respective host

Cancer is known to have a long latency period between exposure and disease onset. Important exposure factors can be traced back to childhood and young adulthood. This means short and medium term cancer mortality among first generation migrants is mainly

The longer migrants live and adapt to their destination country, the more their cancer rates converge towards those in that country. This has been shown for stomach, colon and prostate cancer (McKay, 2003). Migrants from non-western countries to Europe were found to be more prone to cancers that are related to infections experienced in early life, such as liver, cervical and stomach cancer. In contrast, migrants of non-western origin were less likely to suffer from cancers related to a western lifestyle, e.g. colorectal and breast cancer

Evidence was found for a transition of cancer incidence and mortality patterns towards the host population among Turkish migrants in Germany (Zeeb et al., 2002). Convergence may occur due to diet acculturation, adaptation of new lifestyles or utilization of often superior health services. Higher mortality from cancers where incidence can be reduced by effective screening programs and those where survival depends on availability of treatment options, may decrease in a relatively shorter time. Another study analyzed differences in cancer rates between first and second generation migrants relative to the host country, stratified by

religious freedom.

migrant populations.

population.

(Arnold et al., 2010).

dramatically with time (Fennelly, 2007).

**1.2.2 Migration and cancer incidence & mortality** 

influenced by country-of-origin factors (Parkin & Khlat, 1996).

country of origin, showing cancer site specific patterns for succeeding generations (Thomas & Karagas, 1987).

Results of an American study support the theory of a rather strong genetic influence on risk of prostate cancer. The study compared patterns of prostate cancer among black and white men (Chu et al., 2003). Black Americans had substantially higher prostate cancer rates than white Americans, but the longitudinal trends such as decreasing mortality, increasing incidence and survival were similar. Although this was not a typical migrant study, it compared different ethnic and thus genetic and lifestyle factors in a known risk pattern environment.

#### **1.3 Ethnic German migrants in Germany - background on the study population**

In the year 2005, only 2.9% of the global population were migrants, but migration is unequally distributed throughout the world. In past years, migration flows have shifted and in some cases, international migration is actually decreasing. Only two areas in the world have seen an increase in migration – North America and the Former Soviet Union (FSU) (International Organization for Migration, 2005).

Germany has long been a country of immigration. At present, there are two big groups of migrants, the Turks and ethnic Germans from countries of the FSU. We study disease patterns, focusing on cancer incidence and mortality, in the latter group.

The 'Aussiedler' are ethnic German migrants and represent a unique group of diaspora migrants. Since 1993, the officially correct term for Aussiedler is Spätaussiedler, however for ease of presentation we will use the term Aussiedler throughout the text.

The first Aussiedler came to Russia when Peter I (1689–1725) changed his politics towards Europe. They were the beginning of the urban German population in Russia. Tsarina Katharina II (1762–1796) promised the Aussiedler tax exemptions for 30 years, exoneration from military service, freedom of religion, autonomy and subsidy for resettlement. Many Germans living in regions still suffering from war migrated to Russia under these terms. During the first half of the 19th century approximately 55,000 German colonists settled in the Black Sea region. With time the Aussiedler lost several of the rights they were promised.

For centuries these ethnic Germans lived abroad and were a relatively closed group of people. After the start of World War I the laws of liquidation were implemented. On the basis of these laws more than 200,000 German colonists were driven away. In 1922, after the October-revolution and civil war the Union of Soviet Socialist Republics (USSR) was founded. When the Nazi Party came into power in Germany the situation of the Germans in the USSR worsened. Seen as an internal enemy, Stalin restricted their rights.

Soon after the German aggression against the USSR in 1941 the deportation of the German population started. Following Stalin's decree about 1,200,000 ethnic Germans were deported into the eastern parts of the Soviet Union, predominantly to Siberia, Kazakhstan and in the Urals. Their civil rights were disregarded; they were detained and forbidden to speak German. Most had to work in labour camps in inhumane conditions. An estimated 700,000 Germans died due to bad working and living conditions and inadequate medical treatment. In particular, the Stalinism destroyed the independent German culture in Russia. In 1955, the discrimination was subsided, and the ethnic Germans were allowed to change their residence, but not to their former colony areas. The Aussiedler became partly assimilated in the last decades of the USSR.

Epidemiology of Prostate Cancer:

The Case of Ethnic German Migrants from the Former Soviet Union 9

very similar in Kazakhstan and in Ukraine. After a dip, the excess mortality increased sharply following the economic crisis of 1998. Mortality is largely due to vascular and external causes of death in adults (Men et al., 2003). In 2006, mortality was still high with about 1300 per 100,000 people. During the same period in Germany, all cause mortality

In 2008, the age-standardised mortality rate per 100,000 males for all cancers was 181.3 in Kazakhstan and 180.7 in the Russian Federation (Ferlay, 2010). In Germany, mortality for all cancer sites combined in the respective year was much lower with 133.2 per 100,000 males. An important reason for the lower cancer mortality in Germany compared to countries of the FSU is better survival. However, longitudinal trends in mortality for all cancer sites

A comparison of cancer incidence rates between Germany and the Aussiedler's countries of origin show much lower rates in the Former Soviet Union. However, it is likely that incidence rates are underestimated in the FSU as evidenced by mortality patterns and

Mortality from prostate cancer in FSU countries is lower compared to Germany, however, during recent years this difference has diminished (see Figure 4). In 2006, the agestandardised mortality rate per 100,000 males was 12.3 in Germany, 5.7 in Kazakhstan, 10.1

Incidence from prostate cancer is also much lower compared to Germany. In 2008, the age-standardised incidence rate was estimated to be 82.7 in Germany, 10.9 in Kazakhstan, 26.1 in the Russian Federation, and 20.3 in Ukraine (Ferlay, 2010). Low incidence in countries of the FSU is likely due to less prostate specific antigen (PSA) testing and may also represent a general underestimation of cancer incidence. This results in an incidence : mortality ratio of 7 in Germany and only 2 in Kazakhstan, 2.5 in the Russian Federation,

1990 1992 1994 1996 1998 2000 2002 2004 2006 Germany Kazakhstan Russian Federation Ukraine

Fig. 4. Age-standardized prostate cancer mortality rates per 100,000 (Segi Standard) (WHO,

declined continuously from around 850 to 650 per 100,000 people (WHO, 2011a).

developed in parallel between Germany and the FSU.

in the Russian Federation, and 9.3 in Ukraine (WHO, 2011b).

differences in diagnosis and treatment.

and 2 in Ukraine.

2011b)

Fig. 3. Immigration of the Aussiedler over time by country of origin (Federal Ministry of the Interior, 2009; Federal Office of Administration, 2011)

When the iron curtain fell around 1990, a wave of migration to Germany started (see Figure 3). Since then more than two million Aussiedler migrated to Germany from countries of the FSU, with most coming from Kazakhstan and the Russian Federation. There are few examples of a large migration of one ethnic group from one country to another in a similarly short period of time.

In 1993, the German government began to restrict the immigration of Aussiedler by implementing annual quotas, which were further reduced in 1996. In parallel the government eliminated several benefits previously offered to Aussiedler, e.g. special credits and unemployment benefits.

The number of Aussiedler immigrating to Germany has fallen rapidly in recent years. In 2010, only 2,350 Aussiedler migrated to Germany (Federal Office of Administration, 2011). Today, the Aussiedler comprise about 2.5% of the German population, representing a relatively large group within German society (Destatis, 2008a; Destatis, 2008b).

More information on the history of the Aussiedler can be found elsewhere (Federal Central Office for Political Education, 2000; Bade & Olmert, 1999; Eisfeld, 1999; Pohl, 2001).

#### **1.4 Comparing German incidence and mortality to the Former Soviet Union**

The health situation in the FSU has changed dramatically during the last thirty years. Since the late 1980s the FSU has been experiencing a mortality crisis, in temporal association with massive social changes.

In Russia between 1987 and 1994, increases were observed for all major causes of death, except for cancer (Leon et al., 1997). Age-standardized mortality for all causes of death rose from 1140 in 1987 to 1600 per 100,000 persons in 1994 (adjusted to Segi). Development was

1950 1960 1970 1980 1990 2000 2010

Fig. 3. Immigration of the Aussiedler over time by country of origin (Federal Ministry of the

When the iron curtain fell around 1990, a wave of migration to Germany started (see Figure 3). Since then more than two million Aussiedler migrated to Germany from countries of the FSU, with most coming from Kazakhstan and the Russian Federation. There are few examples of a large migration of one ethnic group from one country to another in a similarly

In 1993, the German government began to restrict the immigration of Aussiedler by implementing annual quotas, which were further reduced in 1996. In parallel the government eliminated several benefits previously offered to Aussiedler, e.g. special credits

The number of Aussiedler immigrating to Germany has fallen rapidly in recent years. In 2010, only 2,350 Aussiedler migrated to Germany (Federal Office of Administration, 2011). Today, the Aussiedler comprise about 2.5% of the German population, representing a

More information on the history of the Aussiedler can be found elsewhere (Federal Central

The health situation in the FSU has changed dramatically during the last thirty years. Since the late 1980s the FSU has been experiencing a mortality crisis, in temporal association with

In Russia between 1987 and 1994, increases were observed for all major causes of death, except for cancer (Leon et al., 1997). Age-standardized mortality for all causes of death rose from 1140 in 1987 to 1600 per 100,000 persons in 1994 (adjusted to Segi). Development was

relatively large group within German society (Destatis, 2008a; Destatis, 2008b).

Office for Political Education, 2000; Bade & Olmert, 1999; Eisfeld, 1999; Pohl, 2001).

**1.4 Comparing German incidence and mortality to the Former Soviet Union** 

Former FSU Poland Romania Other Total

0

Interior, 2009; Federal Office of Administration, 2011)

50 000

short period of time.

and unemployment benefits.

massive social changes.

100 000

150 000

200 000

250 000

300 000

350 000

very similar in Kazakhstan and in Ukraine. After a dip, the excess mortality increased sharply following the economic crisis of 1998. Mortality is largely due to vascular and external causes of death in adults (Men et al., 2003). In 2006, mortality was still high with about 1300 per 100,000 people. During the same period in Germany, all cause mortality declined continuously from around 850 to 650 per 100,000 people (WHO, 2011a).

In 2008, the age-standardised mortality rate per 100,000 males for all cancers was 181.3 in Kazakhstan and 180.7 in the Russian Federation (Ferlay, 2010). In Germany, mortality for all cancer sites combined in the respective year was much lower with 133.2 per 100,000 males. An important reason for the lower cancer mortality in Germany compared to countries of the FSU is better survival. However, longitudinal trends in mortality for all cancer sites developed in parallel between Germany and the FSU.

A comparison of cancer incidence rates between Germany and the Aussiedler's countries of origin show much lower rates in the Former Soviet Union. However, it is likely that incidence rates are underestimated in the FSU as evidenced by mortality patterns and differences in diagnosis and treatment.

Mortality from prostate cancer in FSU countries is lower compared to Germany, however, during recent years this difference has diminished (see Figure 4). In 2006, the agestandardised mortality rate per 100,000 males was 12.3 in Germany, 5.7 in Kazakhstan, 10.1 in the Russian Federation, and 9.3 in Ukraine (WHO, 2011b).

Incidence from prostate cancer is also much lower compared to Germany. In 2008, the age-standardised incidence rate was estimated to be 82.7 in Germany, 10.9 in Kazakhstan, 26.1 in the Russian Federation, and 20.3 in Ukraine (Ferlay, 2010). Low incidence in countries of the FSU is likely due to less prostate specific antigen (PSA) testing and may also represent a general underestimation of cancer incidence. This results in an incidence : mortality ratio of 7 in Germany and only 2 in Kazakhstan, 2.5 in the Russian Federation, and 2 in Ukraine.

Fig. 4. Age-standardized prostate cancer mortality rates per 100,000 (Segi Standard) (WHO, 2011b)

Epidemiology of Prostate Cancer:

Germany.

registry office.

Diseases (ICD).

**2.1.2 Saarland cohort** 

The Case of Ethnic German Migrants from the Former Soviet Union 11

The dataset contains information on name, date of birth, date of arrival in Germany, sex, country of origin, first city of residence and a unique code that identified members of the same family. After sample size calculation the cohort was restricted to a representative sample of 34,393 Aussiedler who were at least 15 years old when they migrated to

To ascertain vital status of each cohort member until the 31st December 2005 a follow up procedure was performed: Letters were sent to local registry offices in the cities of residence. In case of someone moving to another city, the registry provided the new city of residence and date of moving. The registry of the new city was then contacted until the individual was located. Changes of residence were recorded in a database with the exact date of moving. In the case of death, date and city of death were provided by the local

Cause of death was either ascertained through a record linkage system of the NRW regional statistical office or through the local health offices. The record linkage system has been described in detail by Klug and colleagues (2003). Local health offices provided an anonymous copy of the relevant death certificate. All copies of death certificates were then professionally coded at the Saarland Cancer Registry by International Classification of

The Aussiedler reception centre of the Saarland could not provide a dataset with the standard information on the Aussiedler as in NRW. As an alternative, all local refugee offices of the Saarland were contacted to ask for access to their available data on the Aussiedler. In order to be eligible for the Saarland cohort, migrants must have arrived in

All together information on 26,384 Aussiedler (more than 90% of all Aussiedler who settled in the Saarland during the respective period) was available. The dataset contains name, date of birth, date of German passport as an approximation for date of migration, sex, country of birth for about 70% of the cohort, and first city of residence. The final cohort consisted of a

Follow up and cause of death ascertainment used the same method as for the NRW cohort. Follow up for cancer incidence was done directly by the Saarland cancer registry. Most individuals were identified by name, sex and date of birth. However, many Aussiedler change names during the first years of stay in Germany complicating simple identification by name. To minimize this problem the name matching procedure was done phonetically. For some individuals, city of residence was used as an additional variable to ensure correct identification. 43 cases were excluded from the analysis because they were already

All analyses were restricted to the first cancer diagnosis; multiple tumours per individual

For evaluation of the Aussiedler's cancer incidence and mortality in comparison to the autochthonous German population, rates for comparison are needed. To analyse mortality, rates of the German population were used. Although these rates include the

Germany between 1990 and 2005 from countries of the FSU.

sample of 18,619 individuals without missing data.

diagnosed in their country of origin.

were not considered.

**2.1.3 Data for comparison** 

#### **1.5 Aims of the study and expected findings**

Our studies focus on the health profile of ethnic German migrants from the Former Soviet Union in Germany. The presented work focuses on mortality and incidence of prostate cancer.

We compare two cohorts of Aussiedler to the autochthonous German population to investigate the Aussiedler's overall health status with regard to all cause mortality, and overall cancer and prostate cancer incidence and mortality. For prostate cancer we also consider the influence of age and length of stay in Germany in order to differentiate between the effects of genetic versus life-style dependent factors.

The two study cohorts are located in different Federal States of Germany. The Saarland cohort provides information on cancer incidence and mortality of the Aussiedler, whereas the North Rhine Westphalian cohort provides information on mortality only.

Aussiedler are exposed to different kinds of risk factors in different times of their lives. Before migration they are exposed to risk factors in their countries of origin, which have different disease patterns than Germany. Later, the Aussiedler are exposed to the migration process itself which can cause mental stress and, finally, they are exposed to the German pattern of risk factors.

Since most Aussiedler migrated to Germany at the beginning of the 1990s the mortality crisis in countries of the FSU could have influenced their health status. High mortality rates in their countries of origin and physical as well as psychological stress caused by migration was thought to negatively affect the general health of the Aussiedler. On the other hand, the better health care system in Germany may have improved their health status, if they have access to it. Additionally, social integration may also influence their health status.

A previous study confirmed the hypothesis that Aussiedler experienced higher mortality only for specific causes of death. In contrast, overall mortality of the Aussiedler was lower compared to the German population (Becher et al., 2007).

In general, few migrant studies assess cancer incidence and even fewer investigate both cancer incidence and mortality. Most investigations that do are occupational cancer studies, which describe health risks associated with workplace exposures only.

Aussiedler are likely to have higher mortality rates for all cancers due to country of origin effects. With regards to prostate cancer, a slighty lower mortality compared to Germany is expected, and incidence rates should confirm the observed mortality pattern. A previous study showed no differences in incidence and mortality for all cancers and confirmed expectations for prostate cancer, although it had incomplete follow up (Winkler et al., 2009). It is likely that incidence and mortality rates of the Aussiedler and the German population converge with time. This has already been shown for stomach cancer (Ronellenfitsch et al., 2009).

#### **2. Materials and methods**

#### **2.1 Study population**

#### **2.1.1 North Rhine Westphalian cohort**

In 2001, the North Rhine Westphalian (NRW) cohort was established (Ronellenfitsch et al., 2004). In brief, routine information from the Aussiedler reception centre of NRW was collected to setup a cohort. The original dataset included all Aussiedler from countries of the FSU who settled in NRW between 1990 and 2001.

The dataset contains information on name, date of birth, date of arrival in Germany, sex, country of origin, first city of residence and a unique code that identified members of the same family. After sample size calculation the cohort was restricted to a representative sample of 34,393 Aussiedler who were at least 15 years old when they migrated to Germany.

To ascertain vital status of each cohort member until the 31st December 2005 a follow up procedure was performed: Letters were sent to local registry offices in the cities of residence. In case of someone moving to another city, the registry provided the new city of residence and date of moving. The registry of the new city was then contacted until the individual was located. Changes of residence were recorded in a database with the exact date of moving. In the case of death, date and city of death were provided by the local registry office.

Cause of death was either ascertained through a record linkage system of the NRW regional statistical office or through the local health offices. The record linkage system has been described in detail by Klug and colleagues (2003). Local health offices provided an anonymous copy of the relevant death certificate. All copies of death certificates were then professionally coded at the Saarland Cancer Registry by International Classification of Diseases (ICD).

#### **2.1.2 Saarland cohort**

10 Prostate Cancer – Original Scientific Reports and Case Studies

Our studies focus on the health profile of ethnic German migrants from the Former Soviet Union in Germany. The presented work focuses on mortality and incidence of prostate

We compare two cohorts of Aussiedler to the autochthonous German population to investigate the Aussiedler's overall health status with regard to all cause mortality, and overall cancer and prostate cancer incidence and mortality. For prostate cancer we also consider the influence of age and length of stay in Germany in order to differentiate

The two study cohorts are located in different Federal States of Germany. The Saarland cohort provides information on cancer incidence and mortality of the Aussiedler, whereas

Aussiedler are exposed to different kinds of risk factors in different times of their lives. Before migration they are exposed to risk factors in their countries of origin, which have different disease patterns than Germany. Later, the Aussiedler are exposed to the migration process itself which can cause mental stress and, finally, they are exposed to the German

Since most Aussiedler migrated to Germany at the beginning of the 1990s the mortality crisis in countries of the FSU could have influenced their health status. High mortality rates in their countries of origin and physical as well as psychological stress caused by migration was thought to negatively affect the general health of the Aussiedler. On the other hand, the better health care system in Germany may have improved their health status, if they have access to it. Additionally, social integration may also influence their

A previous study confirmed the hypothesis that Aussiedler experienced higher mortality only for specific causes of death. In contrast, overall mortality of the Aussiedler was lower

In general, few migrant studies assess cancer incidence and even fewer investigate both cancer incidence and mortality. Most investigations that do are occupational cancer studies,

Aussiedler are likely to have higher mortality rates for all cancers due to country of origin effects. With regards to prostate cancer, a slighty lower mortality compared to Germany is expected, and incidence rates should confirm the observed mortality pattern. A previous study showed no differences in incidence and mortality for all cancers and confirmed expectations for prostate cancer, although it had incomplete follow up (Winkler et al., 2009). It is likely that incidence and mortality rates of the Aussiedler and the German population converge with time. This has already been shown for stomach cancer

In 2001, the North Rhine Westphalian (NRW) cohort was established (Ronellenfitsch et al., 2004). In brief, routine information from the Aussiedler reception centre of NRW was collected to setup a cohort. The original dataset included all Aussiedler from countries of the

**1.5 Aims of the study and expected findings** 

between the effects of genetic versus life-style dependent factors.

compared to the German population (Becher et al., 2007).

which describe health risks associated with workplace exposures only.

the North Rhine Westphalian cohort provides information on mortality only.

cancer.

pattern of risk factors.

(Ronellenfitsch et al., 2009).

**2. Materials and methods** 

**2.1.1 North Rhine Westphalian cohort** 

FSU who settled in NRW between 1990 and 2001.

**2.1 Study population** 

health status.

The Aussiedler reception centre of the Saarland could not provide a dataset with the standard information on the Aussiedler as in NRW. As an alternative, all local refugee offices of the Saarland were contacted to ask for access to their available data on the Aussiedler. In order to be eligible for the Saarland cohort, migrants must have arrived in Germany between 1990 and 2005 from countries of the FSU.

All together information on 26,384 Aussiedler (more than 90% of all Aussiedler who settled in the Saarland during the respective period) was available. The dataset contains name, date of birth, date of German passport as an approximation for date of migration, sex, country of birth for about 70% of the cohort, and first city of residence. The final cohort consisted of a sample of 18,619 individuals without missing data.

Follow up and cause of death ascertainment used the same method as for the NRW cohort. Follow up for cancer incidence was done directly by the Saarland cancer registry. Most individuals were identified by name, sex and date of birth. However, many Aussiedler change names during the first years of stay in Germany complicating simple identification by name. To minimize this problem the name matching procedure was done phonetically. For some individuals, city of residence was used as an additional variable to ensure correct identification. 43 cases were excluded from the analysis because they were already diagnosed in their country of origin.

All analyses were restricted to the first cancer diagnosis; multiple tumours per individual were not considered.

#### **2.1.3 Data for comparison**

For evaluation of the Aussiedler's cancer incidence and mortality in comparison to the autochthonous German population, rates for comparison are needed. To analyse mortality, rates of the German population were used. Although these rates include the

Epidemiology of Prostate Cancer:

covariable i.

**3. Results** 

less than 5%.

**3.1 Descriptive results** 

an unknown destination.

malignant neoplasm (ICD-9: 140-208; except 173).

**2.2.3 Multivariate analysis: Poisson regression** 

estimated by maximum likelihood estimation.

performed with SAS® version 9.2.

The Case of Ethnic German Migrants from the Former Soviet Union 13

It is possible to measure effects of different covariables e.g. age, length of stay in Germany, etc. on the SMR and SIR by categorization, but this method is limited because of small sample sizes in subcategories. Another approach classically used in cohort studies is a Poisson regression model, which assesses the effects of different covariables simultaneously. It is based on the Poisson distribution, which is an approximation of the binomial

After transformation, the Poisson model estimating the SMR and the SIR can be written as given in equation 2. α is the intercept, βi is the regression coefficient, and xi is the vector of

log( ) log( ) *OE x i i ii*

The Poisson model is a generalized linear model characterized by the dependence of the outcome on a linear predictor through a non-linear link function. The predictors βi can be

More detailed information on the statistical methods can be found elsewhere (Breslow and Day, 1987). Data management was done by using Microsoft Access® and analysis was

Descriptive characteristics of both cohorts and results of the follow up procedure are presented in Table 1. The Saarland cohort was approximately half the size of the cohort in NRW. Females were slightly overrepresented in both cohorts. The arrival period for entering the cohort was four years longer for the Saarland cohort. The NRW study population was restricted by age at migration of 15 years or older, whereas the Saarland cohort had no age restriction. Thus, the Saarland cohort was on average younger. Country of origin distribution was similar for both cohorts: around 55% of the Aussiedler came from Kazakhstan, 37% from the Russian Federation. Other countries of the FSU contributed each

Overall, the NRW cohort accumulated 344,486.1 PY and the Saarland cohort 147,165.2 PY. Follow up of the NRW cohort was complete for 96.7% of the cohort members with a mean follow up time of 10.1 years. Overall 2,580 (7.5%) cohort members died. Causes of death were known for 94.8% of deceased persons. 1,138 (3.3%) persons were lost to follow-up within the observation period, which means their last date of contact was censored. Individuals were lost follow-up due to different reasons, if they moved abroad or moved to

Vital status was known for 77.4% of individuals in the Saarland cohort. Mean follow up time was 8 years. 87% of individuals lost to follow-up were censored on the day of leaving the study area because they moved to another Federal State. Since the Saarland is a relatively small state people are much more likely to move into another state than in the NRW. During the observation period 780 (4.2%) persons died. Cause of death is known for all types of cancer. Between 1990 and 2005, 448 members of the Saarland cohort were diagnosed with a

  (2)

distribution applied in large samples where the probability of the outcome is small.

Aussiedler as a part of the German population, this should not bias the results of the comparison. For cardiovascular disease mortality it has been shown that the Aussiedler's influence on German mortality is limited to approximately 1% (Deckert et al., 2010). German mortality rates were calculated using the WHO mortality database (WHO, 2011b). Before 1998 causes of death are coded with 9th revision of ICD, thereafter the 10th is used in Germany.

A comparison to German incidence is not possible for the period between 1990 and 2005, since nation-wide information on cancer incidence is not available. For those years German incidence is estimated on basis of the Saarland Cancer Registry. Therefore, we directly compare cancer incidence of the Saarland Aussiedler cohort to the Saarland population. The Saarland Cancer Registry provided data on Saarland population figures and number of cancer cases (Saarland Cancer Registry, 2008). Cancer incidence data is coded in ICD9 only.

#### **2.2 Statistical methods**

#### **2.2.1 Calculation of person-time**

In most cohort studies it is necessary to calculate the actual time-at-risk for each individual as person-time. The person-time is used to either calculate mortality or incidence rates of the cohort or to perform indirect standardization or multivariate analysis.

Person-time was calculated in person-years (PY) by a SAS® macro. The macro uses the three time variables of age, length of stay in Germany and calendar-year. The macro calculates and distributes the person-years exactly to the day. Age and length of stay are categorized in one year intervals. Afterwards age is categorized into five year age groups up to 85 and older.

#### **2.2.2 Indirect standardization**

For comparing Aussiedler incidence and mortality with the German/Saarland population, indirect standardization was used. Compared to the method of direct standardization, the indirect method is advantageous when the stratum-specific rates of one of the populations to be compared are based on small numbers. In this case one can use the more stable rates of the larger population for the indirect standardization, thus gaining robustness with regard to sampling variation (Breslow and Day, 1987).

The standardized mortality ratio (SMR), and the standardized incidence ratio (SIR) are given by the observed number of events O (incident cases or number of deaths) divided by the number of events which one would expect E if the cohort had the mortality rate of the population used for standardization. Equation 1 shows the SMR as an example.

$$\text{SMRR} = \frac{O}{E} = \frac{\sum\_{i=1}^{i} O\_i}{\sum\_{i=1}^{i} py\_i \lambda\_i} \tag{1}$$

Oi gives the number of deaths in stratum i of the cohort. pyi gives the person-years in stratum i and λi the rate stratum j of the population used for standardization. All 95% confidence intervals (95% CI) were calculated using the exact method (Breslow and Day, 1987).

#### **2.2.3 Multivariate analysis: Poisson regression**

It is possible to measure effects of different covariables e.g. age, length of stay in Germany, etc. on the SMR and SIR by categorization, but this method is limited because of small sample sizes in subcategories. Another approach classically used in cohort studies is a Poisson regression model, which assesses the effects of different covariables simultaneously. It is based on the Poisson distribution, which is an approximation of the binomial distribution applied in large samples where the probability of the outcome is small.

After transformation, the Poisson model estimating the SMR and the SIR can be written as given in equation 2. α is the intercept, βi is the regression coefficient, and xi is the vector of covariable i.

$$\log(O\_i) = \log(E\_i) + \alpha + \beta\_i \ge\_i \tag{2}$$

The Poisson model is a generalized linear model characterized by the dependence of the outcome on a linear predictor through a non-linear link function. The predictors βi can be estimated by maximum likelihood estimation.

More detailed information on the statistical methods can be found elsewhere (Breslow and Day, 1987). Data management was done by using Microsoft Access® and analysis was performed with SAS® version 9.2.

#### **3. Results**

12 Prostate Cancer – Original Scientific Reports and Case Studies

Aussiedler as a part of the German population, this should not bias the results of the comparison. For cardiovascular disease mortality it has been shown that the Aussiedler's influence on German mortality is limited to approximately 1% (Deckert et al., 2010). German mortality rates were calculated using the WHO mortality database (WHO, 2011b). Before 1998 causes of death are coded with 9th revision of ICD, thereafter the 10th

A comparison to German incidence is not possible for the period between 1990 and 2005, since nation-wide information on cancer incidence is not available. For those years German incidence is estimated on basis of the Saarland Cancer Registry. Therefore, we directly compare cancer incidence of the Saarland Aussiedler cohort to the Saarland population. The Saarland Cancer Registry provided data on Saarland population figures and number of cancer cases (Saarland Cancer Registry, 2008). Cancer incidence data is

In most cohort studies it is necessary to calculate the actual time-at-risk for each individual as person-time. The person-time is used to either calculate mortality or incidence rates of the

Person-time was calculated in person-years (PY) by a SAS® macro. The macro uses the three time variables of age, length of stay in Germany and calendar-year. The macro calculates and distributes the person-years exactly to the day. Age and length of stay are categorized in one year intervals. Afterwards age is categorized into five year age groups

For comparing Aussiedler incidence and mortality with the German/Saarland population, indirect standardization was used. Compared to the method of direct standardization, the indirect method is advantageous when the stratum-specific rates of one of the populations to be compared are based on small numbers. In this case one can use the more stable rates of the larger population for the indirect standardization, thus gaining robustness with regard

The standardized mortality ratio (SMR), and the standardized incidence ratio (SIR) are given by the observed number of events O (incident cases or number of deaths) divided by the number of events which one would expect E if the cohort had the mortality rate of the

1

*py* 

*O*

*i i*

(1)

*i i i i*

1

*i*

Oi gives the number of deaths in stratum i of the cohort. pyi gives the person-years in stratum i and λi the rate stratum j of the population used for standardization. All 95% confidence intervals (95% CI) were calculated using the exact method (Breslow and Day,

population used for standardization. Equation 1 shows the SMR as an example.

*<sup>O</sup> SMR E*

cohort or to perform indirect standardization or multivariate analysis.

is used in Germany.

coded in ICD9 only.

up to 85 and older.

1987).

**2.2.2 Indirect standardization** 

to sampling variation (Breslow and Day, 1987).

**2.2 Statistical methods** 

**2.2.1 Calculation of person-time** 

#### **3.1 Descriptive results**

Descriptive characteristics of both cohorts and results of the follow up procedure are presented in Table 1. The Saarland cohort was approximately half the size of the cohort in NRW. Females were slightly overrepresented in both cohorts. The arrival period for entering the cohort was four years longer for the Saarland cohort. The NRW study population was restricted by age at migration of 15 years or older, whereas the Saarland cohort had no age restriction. Thus, the Saarland cohort was on average younger. Country of origin distribution was similar for both cohorts: around 55% of the Aussiedler came from Kazakhstan, 37% from the Russian Federation. Other countries of the FSU contributed each less than 5%.

Overall, the NRW cohort accumulated 344,486.1 PY and the Saarland cohort 147,165.2 PY. Follow up of the NRW cohort was complete for 96.7% of the cohort members with a mean follow up time of 10.1 years. Overall 2,580 (7.5%) cohort members died. Causes of death were known for 94.8% of deceased persons. 1,138 (3.3%) persons were lost to follow-up within the observation period, which means their last date of contact was censored. Individuals were lost follow-up due to different reasons, if they moved abroad or moved to an unknown destination.

Vital status was known for 77.4% of individuals in the Saarland cohort. Mean follow up time was 8 years. 87% of individuals lost to follow-up were censored on the day of leaving the study area because they moved to another Federal State. Since the Saarland is a relatively small state people are much more likely to move into another state than in the NRW. During the observation period 780 (4.2%) persons died. Cause of death is known for all types of cancer. Between 1990 and 2005, 448 members of the Saarland cohort were diagnosed with a malignant neoplasm (ICD-9: 140-208; except 173).

Epidemiology of Prostate Cancer:

78.1

67.8

Saarland cohort

whole observation period.

Saarland population.

The Case of Ethnic German Migrants from the Former Soviet Union 15

observation. Three of the deceased men died from prostate cancer, one case (no. 30) was not diagnosed before death, and the cause of death is known from the death certificate only (DCO).

47.9

44.1

47.4

62.8

67.8

60.1

1990 1991 1992 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005

migration to Germany date of diagnosis prostate cancer death non prostate cancer death moved out of study area

This analysis of the Aussiedler in Germany focuses on prostate cancer. However, to place this in context of the Aussiedler's general health situation all cause mortality and mortality and incidence for all malignant neoplasms is presented. SMR is calculated for both cohorts together in comparison to the German population. SIR is based on the Saarland cohort in comparison to the Saarland population. Figure 6 shows SMR and SIR calculated for the

All cause mortality is significantly reduced for both sexes of the Aussiedler. In contrast, the mortality from all neoplasms is also reduced among females, but equal to the German population for males. Incidence of all cancer sites is somewhat lower for males compared to females, but not significantly reduced for either sex in comparison to the

Fig. 5. Overview of all 35 incident prostate cancer cases with age at diagnosis from the

**3.2 Comparing mortality and incidence of the Aussiedler to Germany** 

57.7

63.4

68.7

71.8

70.3

70.1

72.5

58.1

83.6

65.9

74.6

69.1

61.8

63.7

53.1

56.2

57.6

63.0

66.5

66.5

68.8

73.2

83.0

78.9

55.0

76.3

81.2


#### Descriptive results of the follow-up procedure


Table 1. Descriptive results of the two Aussiedler cohorts, from North-Rhine Westphalia and Saarland

During the observation period, 28 men died due to prostate cancer in both cohorts. Their mean age of deaths was 76.9 years (Range: 60.8 - 92.1). In the Saarland cohort 35 men were diagnosed with prostate cancer. Mean age of diagnosis was 67.6 years (Range: 45.3 - 85.8;). Figure 5 displays all 35 incident prostate cancer cases, starting with their migration to Germany, their age at diagnosis and their final status. Most cases were alive at the end of the observation period. Two cases moved out of the study area and nine died during the

Number of cohort members 34,393 18,619

Immigration period 1990-2001 1990-2005

 1990-1993 14,728 6,933 1994-1997 11,441 6,536 1998-2001/5 8,224 5,150

Age restriction 15+ -

(standard deviation; range) 40.0 (17.0; 15-97) 32.4 (19.8; 0-103)

Males 38.4 (16.0; 15-93) 30.9 (19.0; 0-95)

Females 41.5 (17.7; 15-97) 33.8 (20.4; 0-103)

Descriptive results of the follow-up procedure

Table 1. Descriptive results of the two Aussiedler cohorts, from North-Rhine Westphalia and

During the observation period, 28 men died due to prostate cancer in both cohorts. Their mean age of deaths was 76.9 years (Range: 60.8 - 92.1). In the Saarland cohort 35 men were diagnosed with prostate cancer. Mean age of diagnosis was 67.6 years (Range: 45.3 - 85.8;). Figure 5 displays all 35 incident prostate cancer cases, starting with their migration to Germany, their age at diagnosis and their final status. Most cases were alive at the end of the observation period. Two cases moved out of the study area and nine died during the

End of follow-up date 31-12-2005 31-12-2005

Mean time of follow-up 10.1 years 8.0 years

Person-years 346,671.5 148,313.1 Males 167,882.0 71703.4 Females 178,789.4 76,609.6 Alive 89.2% 73.2% Dead 7.5% 4.2% Lost to follow-up 3.3% 22.6%

Mean age at migration

Saarland

Migrant cohort in the Federal State of NRW

Males (%) 16,734 (48.7%) 8,977 (48.2%) Females (%) 17,659 (51.3%) 9,642 (51.8%)

Migrant cohort in the Federal State of Saarland observation. Three of the deceased men died from prostate cancer, one case (no. 30) was not diagnosed before death, and the cause of death is known from the death certificate only (DCO).

migration to Germany date of diagnosis prostate cancer death non prostate cancer death moved out of study area

Fig. 5. Overview of all 35 incident prostate cancer cases with age at diagnosis from the Saarland cohort

#### **3.2 Comparing mortality and incidence of the Aussiedler to Germany**

This analysis of the Aussiedler in Germany focuses on prostate cancer. However, to place this in context of the Aussiedler's general health situation all cause mortality and mortality and incidence for all malignant neoplasms is presented. SMR is calculated for both cohorts together in comparison to the German population. SIR is based on the Saarland cohort in comparison to the Saarland population. Figure 6 shows SMR and SIR calculated for the whole observation period.

All cause mortality is significantly reduced for both sexes of the Aussiedler. In contrast, the mortality from all neoplasms is also reduced among females, but equal to the German population for males. Incidence of all cancer sites is somewhat lower for males compared to females, but not significantly reduced for either sex in comparison to the Saarland population.

Epidemiology of Prostate Cancer:

prostate cancer

**4. Discussion** 

age

including longitudinal and age effects.

important risk factors such as lifestyle.

populations of countries from the FSU.

applicable to this group of migrants.

The Case of Ethnic German Migrants from the Former Soviet Union 17

intercept -0.47 0.0165

younger than 60 years 0.83 0.0401 older than 60 years\* 0 -

Table 2. Parameter estimates of the multivariable Poisson Model for the SIR function of

The aim of the study was to analyse prostate cancer mortality and incidence among ethnic German migrants who came to Germany from the FSU after 1990. Additionally, we highlighted some general aspects of the Aussiedler's health profile to place prostate cancer incidence and mortality into a broader context. We analyzed two cohorts of migrants in terms of all cause mortality, overall cancer and prostate cancer mortality and incidence

Methodological aspects of the cohorts and the statistical analysis have been discussed in detail elsewhere, they have been shown to be representative samples of the Aussiedler. representative (Klug et al., 2003; Ronellenfitsch et al., 2004; Becher et al., 2007; Winkler et al., 2009). In brief, both cohort studies have the pros and cons of historical cohort studies. It is possible to give valid estimates of Aussiedler mortality and incidence in terms of SMR and SIR. Indirect standardization is more appropriate for rare outcomes than direct standardization, resulting in the calculation of rates (McMichael and Giles, 1988). A limitation of this study is that we did not have access to information on potentially

Results for all causes of deaths were significantly lower compared to the autochthon German population. Overall SMR is reduced for the Aussiedler, therefore, they seem to be healthier or more resistant than the Germans and therefore they are much healthier than

Whether this is due to the healthy migrant effect is not immediately clear. For groups like the Aussiedler who have a legal right to migrate to Germany without fulfilling any prerequisites (at least in times when the majority immigrated), it may be assumed that the impact of self-selection on mortality trends is attenuated due to the small number of people staying in the country of origin. The assumption that almost all ethnic Germans migrated to Germany is supported by the continuously declining numbers of newly arriving Aussiedler. On the other hand, declining numbers of migrants may also be due to changes in German law. In addition, there are no official statistics about the number of ethnic Germans for the USSR nor for the FSU and estimations are highly controversial. There are estimates that approximately one million ethnic Germans live in the FSU (Ohliger, 1998). However, an analysis of family size in the NRW cohort shows that the Aussiedler tend to migrate with their whole complete family. Therefore, we think the healthy migrant theory is not

**Estimate** *p***-value** 

\*reference category

Fig. 6. Standardized mortality (SMR) and incidence (SIR) ratios for all causes, all cancer sites (except non melanoma skin cancer), and prostate cancer of the Aussiedler

Mortality from prostate cancer is strongly reduced among male Aussiedler with an SMR of 0.58 (95% CI: 0.40-0.83) for both cohorts combined. Cohort specific analysis shows a somewhat higher mortality among the NRW cohort compared to the Saarland cohort. In both cohorts mortality due to prostate cancer is significantly reduced.

Prostate cancer incidence is also reduced among the Aussiedler, however, the result is not significant with 0.75 (95% CI: 0.54-1.04).

#### **3.3 Longitudinal effects on prostate cancer mortality and incidence of the Aussiedler**

Results from the univariate analysis of prostate cancer mortality and incidence do not take into account the effect of different covariables, which might influence the SMR and SIR. Various covariables where considered to model longitudinal effects: age, calendar year, year of immigration, length of stay in Germany; cohort was considered for the analysis of mortality.

Multivariate Poisson regression did not show any significant effect of the considered covariables on mortality (data not shown). This is in contrast to the analysis of prostate cancer incidence. While the covariables length of stay and year of immigration did not reveal significant effects on the SIR, calendar year was nearly significant (estimate: -0.1213; p-value: 0.0650). Age, however, influenced the SIR. Table 2 shows the result of a model considering age as a dichotomised variable (age below 60 and over 60 years). The modelled SIR of Aussiedler being older than 60 years is 0.63 (exp(-0.47)). In contrast, the SIR of males aged below 60 years is 1.43 (exp(-0.46+0.83)).


Table 2. Parameter estimates of the multivariable Poisson Model for the SIR function of prostate cancer

#### **4. Discussion**

16 Prostate Cancer – Original Scientific Reports and Case Studies

Fig. 6. Standardized mortality (SMR) and incidence (SIR) ratios for all causes, all cancer sites

Mortality from prostate cancer is strongly reduced among male Aussiedler with an SMR of 0.58 (95% CI: 0.40-0.83) for both cohorts combined. Cohort specific analysis shows a somewhat higher mortality among the NRW cohort compared to the Saarland cohort. In

Prostate cancer incidence is also reduced among the Aussiedler, however, the result is not

**3.3 Longitudinal effects on prostate cancer mortality and incidence of the Aussiedler**  Results from the univariate analysis of prostate cancer mortality and incidence do not take into account the effect of different covariables, which might influence the SMR and SIR. Various covariables where considered to model longitudinal effects: age, calendar year, year of immigration, length of stay in Germany; cohort was considered for the analysis of

Multivariate Poisson regression did not show any significant effect of the considered covariables on mortality (data not shown). This is in contrast to the analysis of prostate cancer incidence. While the covariables length of stay and year of immigration did not reveal significant effects on the SIR, calendar year was nearly significant (estimate: -0.1213; p-value: 0.0650). Age, however, influenced the SIR. Table 2 shows the result of a model considering age as a dichotomised variable (age below 60 and over 60 years). The modelled SIR of Aussiedler being older than 60 years is 0.63 (exp(-0.47)). In contrast, the SIR of males

(except non melanoma skin cancer), and prostate cancer of the Aussiedler

both cohorts mortality due to prostate cancer is significantly reduced.

**all causes all cancer sites\* prostate cancer** 

**\* except non melanoma skin cancer**

0.40

mortality.

SMR male SIR male SMR female SIR female

significant with 0.75 (95% CI: 0.54-1.04).

aged below 60 years is 1.43 (exp(-0.46+0.83)).

0.60

0.80

1.00

1.20

The aim of the study was to analyse prostate cancer mortality and incidence among ethnic German migrants who came to Germany from the FSU after 1990. Additionally, we highlighted some general aspects of the Aussiedler's health profile to place prostate cancer incidence and mortality into a broader context. We analyzed two cohorts of migrants in terms of all cause mortality, overall cancer and prostate cancer mortality and incidence including longitudinal and age effects.

Methodological aspects of the cohorts and the statistical analysis have been discussed in detail elsewhere, they have been shown to be representative samples of the Aussiedler. representative (Klug et al., 2003; Ronellenfitsch et al., 2004; Becher et al., 2007; Winkler et al., 2009). In brief, both cohort studies have the pros and cons of historical cohort studies. It is possible to give valid estimates of Aussiedler mortality and incidence in terms of SMR and SIR. Indirect standardization is more appropriate for rare outcomes than direct standardization, resulting in the calculation of rates (McMichael and Giles, 1988). A limitation of this study is that we did not have access to information on potentially important risk factors such as lifestyle.

Results for all causes of deaths were significantly lower compared to the autochthon German population. Overall SMR is reduced for the Aussiedler, therefore, they seem to be healthier or more resistant than the Germans and therefore they are much healthier than populations of countries from the FSU.

Whether this is due to the healthy migrant effect is not immediately clear. For groups like the Aussiedler who have a legal right to migrate to Germany without fulfilling any prerequisites (at least in times when the majority immigrated), it may be assumed that the impact of self-selection on mortality trends is attenuated due to the small number of people staying in the country of origin. The assumption that almost all ethnic Germans migrated to Germany is supported by the continuously declining numbers of newly arriving Aussiedler. On the other hand, declining numbers of migrants may also be due to changes in German law. In addition, there are no official statistics about the number of ethnic Germans for the USSR nor for the FSU and estimations are highly controversial. There are estimates that approximately one million ethnic Germans live in the FSU (Ohliger, 1998). However, an analysis of family size in the NRW cohort shows that the Aussiedler tend to migrate with their whole complete family. Therefore, we think the healthy migrant theory is not applicable to this group of migrants.

Epidemiology of Prostate Cancer:

the development of prostate cancer.

2010), pp. 2647-59, ISSN 1879-0852

ISBN 978-3-89971-120-2, Osnabrück

ISBN 978-9-28320-182-3, Lyon

enreport2008Bevoelkerung,property=file.pdf

ung/Bevoelkentwicklung042010,property=file.pdf

16, ISSN 0008-543X

2458

from

Available from:

978-3784423821, München

zur politischen Bildung, Bonn

**6. Acknowledgment** 

**7. References** 

The Case of Ethnic German Migrants from the Former Soviet Union 19

Prostate cancer mortality and incidence is lower among the Aussiedler and somehow reflects the situation in the FSU. Analysis did not reveal any short-term convergence of the Aussiedler's prostate cancer to German rates as would be expected in lifestyle driven cancer sites. Therefore, our results support the hypothesis of a relatively strong genetic influence on

Many thanks to Robin Nesbitt for language editing and critical thoughts on the manuscript.

Arnold, M.; Razum, O. & Coebergh, JW. (2010). Cancer risk diversity in non-western

Bade, KJ. & Oltmer, J. (1999). *Aussiedler: deutsche Einwanderer aus Osteuropa* (Volume 8), IMIS,

Becher, H.; Razum, O.; Kyobutungi, C.; Laki, J.; Ott, JJ.; Ronellenfitsch, U. & Winkler, V.

Chu, KC.; Tarone, RE. & Freeman, HP. (2003). Trends in prostate cancer mortality among

Deckert, A.; Winkler, V.; Paltiel, A.; Razum, O. & Becher, H. (2010). Time trends in

Destatis. (2008). Bevölkerung, In: *Auszug aus dem Datenreport 2008*, 19.05.2011, Available

http://www.destatis.de/jetspeed/portal/cms/Sites/destatis/Internet/DE/Conte

Destatis. (2008). Bevölkerungsentwicklung 2008, In: *Wirtschaft und Statistik*, 03.04.2011,

http://www.destatis.de/jetspeed/portal/cms/Sites/destatis/Internet/DE/Conte

Eisfeld A. (1999). *Die Russlanddeutschen* (Volume 2.2), Stiftung Ostdeutscher Kulturrat, ISBN

Federal Central Office for Political Education. (2000). *Aussiedler* (Volume 267), Informationen

migrants to Europe: An overview of the literature. *Eur J Cancer,* 46, 14, (September

(2007). Mortalität von Aussiedlern aus der ehemaligen Sovietunion: Ergebnisse einer Kohortenstudie. *Deutsches Ärzteblatt,* 104, 23, (June 2007), pp. A-1655-62 Breslow, NE. & Day, NE. (1987). *Statistical Methods in Cancer Research. The Design and* 

*Analysis of Cohort Studies* (Volume II), International Agency for Research on Cancer,

black men and white men in the United States. *Cancer,* 97, 6, (March 2003), pp. 1507-

cardiovascular disease mortality in Russia and Germany from 1980 to 2007 - are there migration effects? *BMC Public Health,* 10, 488, (August 2010), ISSN 1471-

nt/Publikationen/Querschnittsveroeffentlichungen/Datenreport/Downloads/Dat

nt/Publikationen/Querschnittsveroeffentlichungen/WirtschaftStatistik/Bevoelker

The lower overall mortality is largely due to lower cardiovascular disease mortality, which is the predominant cause of death. Reasons for the reduced mortality remain unclear, but may be the result of genetic selection. For centuries the Aussiedler lived as a relatively closed group of people, which was only partly assimilated in the last decades of the USSR.

Among males, incidence and mortality due to all malignant neoplasms is neither different from the autochthon population nor different from each other. However, male cancer incidence is lower compared to mortality and to female cancer incidence. A possible explanation may be that in general males do not utilize health services as well as females. This may also lead to lower mortality among females.

Additionally, smaller differences between SIR and SMR may be explained by differences in the underlying populations for comparison. SMR was calculated on the basis of German rates and SIR on the basis of rates from the Saarland population.

Overall cancer incidence and mortality of the Aussiedler is comparable to the German population. Although there are larger differences for specific cancer sites (data not shown). Mortality for all cancer sites among females is lower, which is largely due to low mortality due to breast and lung cancer

The analysis of prostate cancer mortality and incidence revealed several interesting points. First, there was no difference between the mean age of death from prostate cancer in the Aussiedler and the Saarland population, at 76.9 and 77.5 years of age, respectively. However, mean age of diagnosis was 2.5 years earlier among Aussiedler, but this difference was not significant, which might be due to the limited number of observations.

Overall evaluation of prostate cancer shows lower mortality in Aussiedler than in the German population. Multivariate analysis did not reveal any longitudinal trends or differences in age patterns of dying from prostate cancer. However, this could be due to the relatively small number of observed deaths.

Prostate cancer diagnosis is lower among Aussiedler, but clearly higher than mortality. Poisson regression also revealed that Aussiedler below 60 have significantly more prostate cancer diagnoses than the Saarland population of this age. Longitudinal covariables had no significant effect on the SIR.

#### **5. Conclusion**

The Aussiedler are a unique group of diaspora migrants. There are few examples in the world of the migration of a large ethnic group from one country to another in a similarly short time period. Kazakhstan and Russia, the main countries of origin, have very different disease patterns than Germany, which may influence the risk profile of the Aussiedler. Studying the health risks of the Aussiedler not only helps to improve the health care they receive in Germany but also has wider implications for understanding the etiology of disease. The strength of this study is its cohort design. However, the retrospective cohort design relies on data from public registries and does not include information on individual risk factors such as lifestyle, which may also be important.

Results were in contrast to expectations based on country of origin data. Aussiedler have a lower mortality due to all causes of death, which cannot yet be explained completely. Cancer mortality and cancer incidence also differ from FSU countries, but are relatively equal to German rates, however, there are big differences in cancer site specific rates.

Prostate cancer mortality and incidence is lower among the Aussiedler and somehow reflects the situation in the FSU. Analysis did not reveal any short-term convergence of the Aussiedler's prostate cancer to German rates as would be expected in lifestyle driven cancer sites. Therefore, our results support the hypothesis of a relatively strong genetic influence on the development of prostate cancer.

#### **6. Acknowledgment**

Many thanks to Robin Nesbitt for language editing and critical thoughts on the manuscript.

#### **7. References**

18 Prostate Cancer – Original Scientific Reports and Case Studies

The lower overall mortality is largely due to lower cardiovascular disease mortality, which is the predominant cause of death. Reasons for the reduced mortality remain unclear, but may be the result of genetic selection. For centuries the Aussiedler lived as a relatively closed group of people, which was only partly assimilated in the last decades of

Among males, incidence and mortality due to all malignant neoplasms is neither different from the autochthon population nor different from each other. However, male cancer incidence is lower compared to mortality and to female cancer incidence. A possible explanation may be that in general males do not utilize health services as well as females.

Additionally, smaller differences between SIR and SMR may be explained by differences in the underlying populations for comparison. SMR was calculated on the basis of German

Overall cancer incidence and mortality of the Aussiedler is comparable to the German population. Although there are larger differences for specific cancer sites (data not shown). Mortality for all cancer sites among females is lower, which is largely due to low mortality

The analysis of prostate cancer mortality and incidence revealed several interesting points. First, there was no difference between the mean age of death from prostate cancer in the Aussiedler and the Saarland population, at 76.9 and 77.5 years of age, respectively. However, mean age of diagnosis was 2.5 years earlier among Aussiedler, but this difference

Overall evaluation of prostate cancer shows lower mortality in Aussiedler than in the German population. Multivariate analysis did not reveal any longitudinal trends or differences in age patterns of dying from prostate cancer. However, this could be due to the

Prostate cancer diagnosis is lower among Aussiedler, but clearly higher than mortality. Poisson regression also revealed that Aussiedler below 60 have significantly more prostate cancer diagnoses than the Saarland population of this age. Longitudinal covariables had no

The Aussiedler are a unique group of diaspora migrants. There are few examples in the world of the migration of a large ethnic group from one country to another in a similarly short time period. Kazakhstan and Russia, the main countries of origin, have very different disease patterns than Germany, which may influence the risk profile of the Aussiedler. Studying the health risks of the Aussiedler not only helps to improve the health care they receive in Germany but also has wider implications for understanding the etiology of disease. The strength of this study is its cohort design. However, the retrospective cohort design relies on data from public registries and does not include information on individual

Results were in contrast to expectations based on country of origin data. Aussiedler have a lower mortality due to all causes of death, which cannot yet be explained completely. Cancer mortality and cancer incidence also differ from FSU countries, but are relatively

equal to German rates, however, there are big differences in cancer site specific rates.

was not significant, which might be due to the limited number of observations.

This may also lead to lower mortality among females.

due to breast and lung cancer

significant effect on the SIR.

**5. Conclusion** 

relatively small number of observed deaths.

risk factors such as lifestyle, which may also be important.

rates and SIR on the basis of rates from the Saarland population.

the USSR.


 http://www.destatis.de/jetspeed/portal/cms/Sites/destatis/Internet/DE/Conte nt/Publikationen/Querschnittsveroeffentlichungen/Datenreport/Downloads/Dat enreport2008Bevoelkerung,property=file.pdf

Destatis. (2008). Bevölkerungsentwicklung 2008, In: *Wirtschaft und Statistik*, 03.04.2011, Available from: http://www.destatis.de/jetspeed/portal/cms/Sites/destatis/Internet/DE/Conte

nt/Publikationen/Querschnittsveroeffentlichungen/WirtschaftStatistik/Bevoelker ung/Bevoelkentwicklung042010,property=file.pdf


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**2** 

Daniel Djakiew

*USA* 

**NSAID Induction of p75NTR in the Prostate:** 

**Via the p38 MAPK Pathway** 

*Georgetown University Medical School* 

 **A Suppressor of Growth and Cell Migration** 

Chronic consumption of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) has been associated with a reduced incidence of prostate cancer (Nelson & Harris, 2000). Pathologic inflammation can induce oxidative stress generating free radicals that can subsequently react with infectious agents and surrounding cells during induction of innate immunity. Different forms of reactive oxygen species can form DNA adducts by halogenation, nitration and oxygenation of bases (Kang & Sowers, 2008). DNA repair of reactive oxygen species damaged bases can seal in mutations that may eventually lead to transformation of cells and carcinogenesis (Burrows, 2009). NSAIDs inhibit inflammation (Masferrer et al., 1995, Tegeder et al., 2001) and therefore may reduce the incidence of carcinogenesis by preventing free radical transformation of cells. NSAID inhibition of the cyclo-oxygenases (COXs) reduces inflammation, however, the mechanism of action of NSAIDs associated with reduced risk of prostate cancer appears independent of COX inhibition (Quann et al., 2007a; Quann et al., 2007b). NSAIDs represent a diverse category of pharmacological compounds with the common biological activity to reduce inflammation, temperature and pain, but with diverse chemical structures that undoubtedly interact with multiple target molecules with multiple mechanisms of action. In the prostate, selected aryl propionic acid NSAIDS such as ibuprofen and r-flurbiprofen (profens) inhibit epithelial cell growth in a COX independent manner (Quann et al., 2007a; Quann et al., 2007b). A mechanism by which these profens inhibit prostate growth appears to be via re-induction of the p75 neurotrophin receptor (p75NTR) which has been shown to exhibit both tumor suppressor and metastasis suppressor activity (Krygier & Djakiew, 2001a; Krygier & Djakiew, 2002). In pathologic human prostate cancer tissues, the p75NTR protein exhibits focal loss of expression which is further lost with malignant progression of tissues (Perez et al., 1997). Significantly, treatment of prostate tumor cells with profen NSAIDs promotes re-expression of the p75NTR protein that inhibits growth (Quann et al., 2007a; Quann et al., 2007b) consistent with p75NTR dependent tumor and metastasis suppressor activity (Krygier & Djakiew, 2001;2002). The COX independent pathway by which profen NSAIDs induce p75NTR re-expression involves rapid phosphorylation of p38 MAPK and down stream effectors leading to re-expression of p75NTR protein and suppressor activity (Quann et al., 2007b). This review discusses the body of evidence for the inhibitory role of the p75NTR in prostate growth; the pathologic loss of

**1. Introduction** 

Ziegler, H.; Stabenow, R.; Holleczek B. & Stegmaier C. (21.08.2009). *Krebs im Saarland, Ministerium für Justiz*, 03.05.2010, Availaible from: http://www.krebsregister.saarland.de/krebsatlas/EKRS\_Krebsatlas\_21082009.pdf

### **NSAID Induction of p75NTR in the Prostate: A Suppressor of Growth and Cell Migration Via the p38 MAPK Pathway**

Daniel Djakiew *Georgetown University Medical School USA* 

#### **1. Introduction**

22 Prostate Cancer – Original Scientific Reports and Case Studies

Ziegler, H.; Stabenow, R.; Holleczek B. & Stegmaier C. (21.08.2009). *Krebs im Saarland,* 

http://www.krebsregister.saarland.de/krebsatlas/EKRS\_Krebsatlas\_21082009.pdf

*Ministerium für Justiz*, 03.05.2010, Availaible from:

Chronic consumption of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) has been associated with a reduced incidence of prostate cancer (Nelson & Harris, 2000). Pathologic inflammation can induce oxidative stress generating free radicals that can subsequently react with infectious agents and surrounding cells during induction of innate immunity. Different forms of reactive oxygen species can form DNA adducts by halogenation, nitration and oxygenation of bases (Kang & Sowers, 2008). DNA repair of reactive oxygen species damaged bases can seal in mutations that may eventually lead to transformation of cells and carcinogenesis (Burrows, 2009). NSAIDs inhibit inflammation (Masferrer et al., 1995, Tegeder et al., 2001) and therefore may reduce the incidence of carcinogenesis by preventing free radical transformation of cells. NSAID inhibition of the cyclo-oxygenases (COXs) reduces inflammation, however, the mechanism of action of NSAIDs associated with reduced risk of prostate cancer appears independent of COX inhibition (Quann et al., 2007a; Quann et al., 2007b). NSAIDs represent a diverse category of pharmacological compounds with the common biological activity to reduce inflammation, temperature and pain, but with diverse chemical structures that undoubtedly interact with multiple target molecules with multiple mechanisms of action. In the prostate, selected aryl propionic acid NSAIDS such as ibuprofen and r-flurbiprofen (profens) inhibit epithelial cell growth in a COX independent manner (Quann et al., 2007a; Quann et al., 2007b). A mechanism by which these profens inhibit prostate growth appears to be via re-induction of the p75 neurotrophin receptor (p75NTR) which has been shown to exhibit both tumor suppressor and metastasis suppressor activity (Krygier & Djakiew, 2001a; Krygier & Djakiew, 2002). In pathologic human prostate cancer tissues, the p75NTR protein exhibits focal loss of expression which is further lost with malignant progression of tissues (Perez et al., 1997). Significantly, treatment of prostate tumor cells with profen NSAIDs promotes re-expression of the p75NTR protein that inhibits growth (Quann et al., 2007a; Quann et al., 2007b) consistent with p75NTR dependent tumor and metastasis suppressor activity (Krygier & Djakiew, 2001;2002). The COX independent pathway by which profen NSAIDs induce p75NTR re-expression involves rapid phosphorylation of p38 MAPK and down stream effectors leading to re-expression of p75NTR protein and suppressor activity (Quann et al., 2007b). This review discusses the body of evidence for the inhibitory role of the p75NTR in prostate growth; the pathologic loss of

NSAID Induction of p75NTR in the Prostate:

Djakiew et al., 1996).

prostate epithelial cell growth (Dalal & Djakiew, (1997).

A Suppressor of Growth and Cell Migration Via the p38 MAPK Pathway 25

BDNF also is also expressed by human prostate stromal cells (Dalal & Djakiew, 1997). Hence, the two neurotrophins, NGF and BDNF appear to function as paracrine factors for

The p75NTR is expressed by normal human prostate epithelial cells (MacGrogan et al., 1992; Graham et al., 1992). Immunoblot (Pflug et al., 1992), immunofluorescence (Graham et al., 1992), and immunohistochemical (Pflug et al., 1992) studies have shown that p75NTR protein expression progressively declines in human prostate cancer (Perez et al., 1997; Pflug et al., 1995; Djakiew et al., 1996). The p75NTR is expressed in PIN tissue (Perez et al., 1997), and shows a gradual decline in the percentage of cells that retain expression with increasing Gleason score of pathologic prostate tissues (Perez et al., 1997). Hence, loss of p75NTR expression appears to be correlated with cancer grade in organ-confined disease (Perez et al., 1997). The p75NTR is also absent in three human cancer cell lines derived from metastases (Pflug et al., 1992). Loss of p75NTR expression in prostate cancer may be related to its role in the induction of programmed cell death (Pflug & Djakiew, 1998; Djakiew et al., 1996). In this context, the p75 neurotrophin receptor (p75NTR) is a 75 kD cell surface receptor glycoprotein that shares both structural and sequence homology with the tumor necrosis factor receptor super-family of proteins (Chao, 1994; Chapman, 1995). Some of these proteins (e.g. p75NTR, p55TNFR, Fas, DRs3-5) have similar sequence motifs of defined elongated structure (Chao, 1994) designated "death domains" based upon their apoptosis inducing function (Chao, 1994). Hence, re-expression of p75NTR by stable and transient transfection showed that the p75NTR inhibits growth of prostate epithelium *in vitro*, at least in part, by induction of programmed cell death (Pflug & Djakiew, 1998). Hence, loss of p75NTR expression appears to eliminate a potential programmed cell death pathway in prostate cancer cells (Figure 2), thereby facilitating the growth of these cancer cells during carcinogenesis (Perez et al., 1997;

Since expression of the p75NTR is lost during malignant progression and transformation of the prostate (Figure 2), NGF mediated growth of cancer cells has been shown to occur via the family of high affinity Trk receptors (Pflug & Djakiew, 1998; Dalal & Djakiew, 1997; Pflug et al., 1995). Differential expression of TrkA, TrkB and TrkC occurs in normal, organ confined and metastatic prostate cancer tissue (Chapman, 1995) and cell lines (Dalal & Djakiew, 1997) suggesting the presence of a neurotophin mediated proliferative stimulus via the Trk receptors (Figure 2). Expression of the TrkA receptor has been observed in normal prostate epithelial cells, organ confined prostate adenocarcinoma tissues (Djakiew et al., 1996; Pflug et al., 1995; Djakiew et al., 1996; Dionne et al., 1998), in prostate tumors that have metastasized to the bone (Dionne et al., 1998), as well as in several human prostate tumor cell lines derived from metastases (Djakiew et al., 1996). Interestingly, although normal prostate epithelial cells do the not express either TrkB or TrkC (Dionne et al., 1998), these receptors are expressed in metastatic prostate cancer of the bone (Dionne et al., 1998). Hence, it appears that normal prostate epithelial cells express TrkA, and that cancer cells continue to express TrkA within primary tumors and at metastatic foci, while TrkB and TrkC expression is subsequently up-regulated within metastatic prostate tumors (Dionne et al., 1998). Pharmacological inhibition of the Trk family of receptors has provided a basis for their role in mediating a proliferative stimulus is tumor cells. In this context, the indolocarbazole kinase inhibitors selectively antagonize Trk receptors at nanomolar concentrations (Berg et al., 1992) and inhibit NGF-stimulated Trk phosphorylation in cancer cell lines (Delsite & Djakiew, 1996). Concurrently, Trk selective indolocarbazoles inhibit growth of cancer cell lines *in vitro* (Delsite & Djakiew, 1996), and *in vivo* (Dionne et al., 1998).

p75NTR expression during progression to prostate cancer, and the ability of profen NSAIDs to re-induce p75NTR protein expression through the p38 MAPK pathway with concomitant tumor and metastasis suppressor activity which provides a basis for NSAID associated reduced risk of prostate cancer.

Fig. 1. Affinities of the neurotrophin ligands, nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) for the family of tropomyosin receptor kinases (Trks) and the p75 neurotrophin receptor (p75NTR). Solid arrows show primary affinities, while dotted arrows show secondary affinities.

#### **2. Expression of neurotrophins and their receptors (p75NTR and Trks) in the prostate**

The progression of prostate cancer is accompanied by modifications in the expression of growth factors and their receptors (Bostwick et al, 2004). Amongst these are nerve growth factor (NGF) and its receptors, p75NTR and the Trk family (Djakiew, 2000). The neurotrophin family of ligands (Leibrock et al., 1989, Maisonpierre et al., 1990, Hallbook et al., 1991) consisting of nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) which is identical to NT-5, all of which can bind with similar affinity to the p75NTR (Bothwell, 1995) and with differential affinities to the Trk family (TrkA, TrkB, TrkC) of high affinity receptors (Figure 1). TrkA preferentially binds with NGF, but also binds BDNF. TrkB preferentially binds BDNF (Soppert et al., 1991), but also binds NT-3 and NT-4 (Berkemeier et al., 1991) and TrkC preferentially binds NT-3 (Lamballe et al., 1991), but also binds NT-4 (Figure 1). NGF immunoreactive protein has been localized to normal epithelium (MacGrogan, 1992; Paul et., 1992) and the stroma of normal, BPH (Djakiew et al., 1991) and cancer tissues of the human prostate (MacGrogan et al., 1992; Djakiew, 1991; Graham et al., 1992). Exogenous NGF has been shown to stimulate proliferation *in vitro* (Delsite & Djakiew, 1999; Angelsen et al., 1998; Pflug & Djakiew, 1998), and anchorage independent growth of several prostate tumor cell lines (Chung et al., 1992).

p75NTR expression during progression to prostate cancer, and the ability of profen NSAIDs to re-induce p75NTR protein expression through the p38 MAPK pathway with concomitant tumor and metastasis suppressor activity which provides a basis for NSAID associated

Fig. 1. Affinities of the neurotrophin ligands, nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) for the family of tropomyosin receptor kinases (Trks) and the p75 neurotrophin receptor (p75NTR). Solid arrows show primary affinities, while dotted arrows show secondary affinities.

**2. Expression of neurotrophins and their receptors (p75NTR and Trks) in the** 

The progression of prostate cancer is accompanied by modifications in the expression of growth factors and their receptors (Bostwick et al, 2004). Amongst these are nerve growth factor (NGF) and its receptors, p75NTR and the Trk family (Djakiew, 2000). The neurotrophin family of ligands (Leibrock et al., 1989, Maisonpierre et al., 1990, Hallbook et al., 1991) consisting of nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) which is identical to NT-5, all of which can bind with similar affinity to the p75NTR (Bothwell, 1995) and with differential affinities to the Trk family (TrkA, TrkB, TrkC) of high affinity receptors (Figure 1). TrkA preferentially binds with NGF, but also binds BDNF. TrkB preferentially binds BDNF (Soppert et al., 1991), but also binds NT-3 and NT-4 (Berkemeier et al., 1991) and TrkC preferentially binds NT-3 (Lamballe et al., 1991), but also binds NT-4 (Figure 1). NGF immunoreactive protein has been localized to normal epithelium (MacGrogan, 1992; Paul et., 1992) and the stroma of normal, BPH (Djakiew et al., 1991) and cancer tissues of the human prostate (MacGrogan et al., 1992; Djakiew, 1991; Graham et al., 1992). Exogenous NGF has been shown to stimulate proliferation *in vitro* (Delsite & Djakiew, 1999; Angelsen et al., 1998; Pflug & Djakiew, 1998), and anchorage independent growth of several prostate tumor cell lines (Chung et al., 1992).

reduced risk of prostate cancer.

**prostate** 

BDNF also is also expressed by human prostate stromal cells (Dalal & Djakiew, 1997). Hence, the two neurotrophins, NGF and BDNF appear to function as paracrine factors for prostate epithelial cell growth (Dalal & Djakiew, (1997).

The p75NTR is expressed by normal human prostate epithelial cells (MacGrogan et al., 1992; Graham et al., 1992). Immunoblot (Pflug et al., 1992), immunofluorescence (Graham et al., 1992), and immunohistochemical (Pflug et al., 1992) studies have shown that p75NTR protein expression progressively declines in human prostate cancer (Perez et al., 1997; Pflug et al., 1995; Djakiew et al., 1996). The p75NTR is expressed in PIN tissue (Perez et al., 1997), and shows a gradual decline in the percentage of cells that retain expression with increasing Gleason score of pathologic prostate tissues (Perez et al., 1997). Hence, loss of p75NTR expression appears to be correlated with cancer grade in organ-confined disease (Perez et al., 1997). The p75NTR is also absent in three human cancer cell lines derived from metastases (Pflug et al., 1992). Loss of p75NTR expression in prostate cancer may be related to its role in the induction of programmed cell death (Pflug & Djakiew, 1998; Djakiew et al., 1996). In this context, the p75 neurotrophin receptor (p75NTR) is a 75 kD cell surface receptor glycoprotein that shares both structural and sequence homology with the tumor necrosis factor receptor super-family of proteins (Chao, 1994; Chapman, 1995). Some of these proteins (e.g. p75NTR, p55TNFR, Fas, DRs3-5) have similar sequence motifs of defined elongated structure (Chao, 1994) designated "death domains" based upon their apoptosis inducing function (Chao, 1994). Hence, re-expression of p75NTR by stable and transient transfection showed that the p75NTR inhibits growth of prostate epithelium *in vitro*, at least in part, by induction of programmed cell death (Pflug & Djakiew, 1998). Hence, loss of p75NTR expression appears to eliminate a potential programmed cell death pathway in prostate cancer cells (Figure 2), thereby facilitating the growth of these cancer cells during carcinogenesis (Perez et al., 1997; Djakiew et al., 1996).

Since expression of the p75NTR is lost during malignant progression and transformation of the prostate (Figure 2), NGF mediated growth of cancer cells has been shown to occur via the family of high affinity Trk receptors (Pflug & Djakiew, 1998; Dalal & Djakiew, 1997; Pflug et al., 1995). Differential expression of TrkA, TrkB and TrkC occurs in normal, organ confined and metastatic prostate cancer tissue (Chapman, 1995) and cell lines (Dalal & Djakiew, 1997) suggesting the presence of a neurotophin mediated proliferative stimulus via the Trk receptors (Figure 2). Expression of the TrkA receptor has been observed in normal prostate epithelial cells, organ confined prostate adenocarcinoma tissues (Djakiew et al., 1996; Pflug et al., 1995; Djakiew et al., 1996; Dionne et al., 1998), in prostate tumors that have metastasized to the bone (Dionne et al., 1998), as well as in several human prostate tumor cell lines derived from metastases (Djakiew et al., 1996). Interestingly, although normal prostate epithelial cells do the not express either TrkB or TrkC (Dionne et al., 1998), these receptors are expressed in metastatic prostate cancer of the bone (Dionne et al., 1998). Hence, it appears that normal prostate epithelial cells express TrkA, and that cancer cells continue to express TrkA within primary tumors and at metastatic foci, while TrkB and TrkC expression is subsequently up-regulated within metastatic prostate tumors (Dionne et al., 1998). Pharmacological inhibition of the Trk family of receptors has provided a basis for their role in mediating a proliferative stimulus is tumor cells. In this context, the indolocarbazole kinase inhibitors selectively antagonize Trk receptors at nanomolar concentrations (Berg et al., 1992) and inhibit NGF-stimulated Trk phosphorylation in cancer cell lines (Delsite & Djakiew, 1996). Concurrently, Trk selective indolocarbazoles inhibit growth of cancer cell lines *in vitro* (Delsite & Djakiew, 1996), and *in vivo* (Dionne et al., 1998).

NSAID Induction of p75NTR in the Prostate:

A Suppressor of Growth and Cell Migration Via the p38 MAPK Pathway 27

ectopically expressed the p75NTR protein exhibited a retardation of cell cycle progression characterized by accumulation of cells in G1 phase with a corresponding reduction of cells in the S phase of the cell cycle (Krygier & Djakiew, 2001a). In rank order, prostate cancer cells that expressed increased levels of p75NTR protein exhibited increased rates of apoptosis and reduced rates of proliferation (Krygier & Djakiew, 2001a). When the same series of tumor cells were injected into the flanks of SCID mice the growth of prostate tumors was suppressed in proportion to increased p75NTR expression levels (Krygier & Djakiew, 2001a) thereby functionally demonstrating that the p75NTR exhibits tumor suppressor activity (Krygier & Djakiew, 2001a). Further evidence for the tumor suppressor function of p75NTR was show utilizing a gene therapy strategy based upon intra-tumoral injection into xenografts of PC-3 prostate tumor cells of a lipoplex containing the p75NTR gene. Administration of the p75NTR gene into subcutaneous PC-3 xenografts suppressed in a dosedependent manner the growth of tumors (Allen et al., 2004). Within the gene therapy treated tumors re-expression of the p75NTR gene product was associated with increased apoptosis and reduced proliferation of tumor cells (Allen et al., 2004), the net effect of which was to reduce overall growth and tumor volume. Utilizing the same prostate tumor cells that exhibited a rank order (dose-dependent) increase in p75NTR expression for growth of subcutaneous tumors in SCID mice (Krygier & Djakiew, 2001a), treatment of these tumors with NGF stimulated both proliferation as indicated by PCNA expression, and apoptosis as indicated by TUNEL assay, the net result of which was no change in the overall growth of the tumors (Krygier & Djakiew, 2002). However, NGF was found to increase the formation of smaller secondary satellite tumors, both contiguous and non-contiguous with respect to the primary tumor mass, indicating dose-dependent induction of metastasis (Krygier & Djakiew , 2002). Significantly, the formation of satellite tumors was suppressed by the expression of p75NTR thus showing that p75NTR is a tumor suppressor of growth and a metastasis suppressor of NGF stimulated migration (Djakiew et al, 1993) of human prostate tumor cells (Krygier & Djakiew, 2002). To better understand the molecular mechanism of p75NTR on tumor and metastasis suppression a cDNA microarray composed of approximately 6,000 human cancer-related genes was used to determine the gene expression pattern altered by re-introduction of p75NTR into PC-3 prostate tumor cells (Nalbandian et al., 2005). Comparison of the transcripts in the *neo* control and p75NTR-transfected cells revealed 52 differentially expressed genes, of which 21 were up-regulated and 31 were down-regulated in the presence of p75NTR. The known biological functions of these p75NTR regulated genes suggested a role in the regulation of differentiation as well as cell adhesion, signal transduction, apoptosis, tumor cell invasion and metastasis (Nalbandian et al., 2005). Quantitative real-time polymerase chain reaction and immunoblot analysis confirmed increased CRABPI and IGFBP5 protein levels and decreased level of PLAUR protein with increasing p75NTR protein expression. Indeed, CRABPI was elevated far more than any other genes (Nalbandian et al., 2005). In this context, the retinoids, ATRA and 9-*cis* RA, that bind CRABPI, promoted functional cell differentiation in p75NTR PC-3 cells, but not in *neo* control PC-3 cells. Subsequent examination of the retinoic acid receptors expression levels demonstrated an absence of RAR-β in the *neo* control cells and re-expression in the p75NTR expressing cells, consistent with previous findings where RAR-β is believed to play a critical role as a tumor suppressor gene which is lost during de-differentiation of prostate epithelial cells. Whereas the RAR-α and -γ protein levels remained unchanged, RXR-α and -β also exhibited increasing protein levels with re-expression of the p75NTR protein (Nalbandian et al., 2005). Moreover, the ability of p75NTR siRNA to knockdown levels of RAR-β, RXR-α, and

Interestingly, Trk family mutations within the human prostate have not been identified (George et al., 1998). However, the absence of mutations in otherwise genetically unstable prostate tumor DNA suggests that intact Trk family signaling pathways may be important in prostate cancer development (George et al., 1998) consistent with a role as a protooncogene.

Fig. 2. Schematic diagram showing expression of NGF and BDNF in prostatic stroma for the paracrine regulation of epithelial cell growth. Pre-malignant epithelial cells express both TrkA and the p75NTR, whereas metastatic tumor cells have lost expression of the p75NTR and gained expression of both TrkB and TrkC.

#### **3. Ectopic re-expression of p75NTR induces tumor suppressor, metastasis suppressor and differentiation phenotypes in prostate tumor cells**

Whereas the gene encoding p75NTR is intact in prostate cancer cells (Krygier & Djakiew, 2001b), expression of the p75NTR protein is suppressed (Perez et al., 1997; Pflug et al., 1992; Pflug et al., 1995). Moreover, transient transfection of two constructs of p75NTR into prostate cell lines that do not express the protein, one construct containing the full 2-kb 3' untranslated region and another that contains just a few hundred bases of the 3' untranslated region, showed that only the truncated construct allowed expression of the p75NTR protein (Krygier & Djakiew, 2001b). This lead to the conclusion that elements of the 3' untranslated region of p75NTR contribute to mRNA stability and p75NTR protein expression (Krygier & Djakiew, 2001b). Utilizing the truncated p75NTR expression vector that allows protein expression, a series of stable prostate cell lines were developed that express different levels of p75NTR protein (Pflug et al., 1992, Pflug et al., 1995). These cancer cells that

Interestingly, Trk family mutations within the human prostate have not been identified (George et al., 1998). However, the absence of mutations in otherwise genetically unstable prostate tumor DNA suggests that intact Trk family signaling pathways may be important in prostate cancer development (George et al., 1998) consistent with a role as a proto-

Fig. 2. Schematic diagram showing expression of NGF and BDNF in prostatic stroma for the paracrine regulation of epithelial cell growth. Pre-malignant epithelial cells express both TrkA and the p75NTR, whereas metastatic tumor cells have lost expression of the p75NTR

**3. Ectopic re-expression of p75NTR induces tumor suppressor, metastasis** 

Whereas the gene encoding p75NTR is intact in prostate cancer cells (Krygier & Djakiew, 2001b), expression of the p75NTR protein is suppressed (Perez et al., 1997; Pflug et al., 1992; Pflug et al., 1995). Moreover, transient transfection of two constructs of p75NTR into prostate cell lines that do not express the protein, one construct containing the full 2-kb 3' untranslated region and another that contains just a few hundred bases of the 3' untranslated region, showed that only the truncated construct allowed expression of the p75NTR protein (Krygier & Djakiew, 2001b). This lead to the conclusion that elements of the 3' untranslated region of p75NTR contribute to mRNA stability and p75NTR protein expression (Krygier & Djakiew, 2001b). Utilizing the truncated p75NTR expression vector that allows protein expression, a series of stable prostate cell lines were developed that express different levels of p75NTR protein (Pflug et al., 1992, Pflug et al., 1995). These cancer cells that

**suppressor and differentiation phenotypes in prostate tumor cells** 

and gained expression of both TrkB and TrkC.

oncogene.

ectopically expressed the p75NTR protein exhibited a retardation of cell cycle progression characterized by accumulation of cells in G1 phase with a corresponding reduction of cells in the S phase of the cell cycle (Krygier & Djakiew, 2001a). In rank order, prostate cancer cells that expressed increased levels of p75NTR protein exhibited increased rates of apoptosis and reduced rates of proliferation (Krygier & Djakiew, 2001a). When the same series of tumor cells were injected into the flanks of SCID mice the growth of prostate tumors was suppressed in proportion to increased p75NTR expression levels (Krygier & Djakiew, 2001a) thereby functionally demonstrating that the p75NTR exhibits tumor suppressor activity (Krygier & Djakiew, 2001a). Further evidence for the tumor suppressor function of p75NTR was show utilizing a gene therapy strategy based upon intra-tumoral injection into xenografts of PC-3 prostate tumor cells of a lipoplex containing the p75NTR gene. Administration of the p75NTR gene into subcutaneous PC-3 xenografts suppressed in a dosedependent manner the growth of tumors (Allen et al., 2004). Within the gene therapy treated tumors re-expression of the p75NTR gene product was associated with increased apoptosis and reduced proliferation of tumor cells (Allen et al., 2004), the net effect of which was to reduce overall growth and tumor volume. Utilizing the same prostate tumor cells that exhibited a rank order (dose-dependent) increase in p75NTR expression for growth of subcutaneous tumors in SCID mice (Krygier & Djakiew, 2001a), treatment of these tumors with NGF stimulated both proliferation as indicated by PCNA expression, and apoptosis as indicated by TUNEL assay, the net result of which was no change in the overall growth of the tumors (Krygier & Djakiew, 2002). However, NGF was found to increase the formation of smaller secondary satellite tumors, both contiguous and non-contiguous with respect to the primary tumor mass, indicating dose-dependent induction of metastasis (Krygier & Djakiew , 2002). Significantly, the formation of satellite tumors was suppressed by the expression of p75NTR thus showing that p75NTR is a tumor suppressor of growth and a metastasis suppressor of NGF stimulated migration (Djakiew et al, 1993) of human prostate tumor cells (Krygier & Djakiew, 2002). To better understand the molecular mechanism of p75NTR on tumor and metastasis suppression a cDNA microarray composed of approximately 6,000 human cancer-related genes was used to determine the gene expression pattern altered by re-introduction of p75NTR into PC-3 prostate tumor cells (Nalbandian et al., 2005). Comparison of the transcripts in the *neo* control and p75NTR-transfected cells revealed 52 differentially expressed genes, of which 21 were up-regulated and 31 were down-regulated in the presence of p75NTR. The known biological functions of these p75NTR regulated genes suggested a role in the regulation of differentiation as well as cell adhesion, signal transduction, apoptosis, tumor cell invasion and metastasis (Nalbandian et al., 2005). Quantitative real-time polymerase chain reaction and immunoblot analysis confirmed increased CRABPI and IGFBP5 protein levels and decreased level of PLAUR protein with increasing p75NTR protein expression. Indeed, CRABPI was elevated far more than any other genes (Nalbandian et al., 2005). In this context, the retinoids, ATRA and 9-*cis* RA, that bind CRABPI, promoted functional cell differentiation in p75NTR PC-3 cells, but not in *neo* control PC-3 cells. Subsequent examination of the retinoic acid receptors expression levels demonstrated an absence of RAR-β in the *neo* control cells and re-expression in the p75NTR expressing cells, consistent with previous findings where RAR-β is believed to play a critical role as a tumor suppressor gene which is lost during de-differentiation of prostate epithelial cells. Whereas the RAR-α and -γ protein levels remained unchanged, RXR-α and -β also exhibited increasing protein levels with re-expression of the p75NTR protein (Nalbandian et al., 2005). Moreover, the ability of p75NTR siRNA to knockdown levels of RAR-β, RXR-α, and

NSAID Induction of p75NTR in the Prostate:

independently to promote suppressor activity.

A Suppressor of Growth and Cell Migration Via the p38 MAPK Pathway 29

deletion constructs that lack much of the intracellular death domain were shown to rescue p75NTR down-regulation of MKK4 and phosphorylated JNK (Allen et al., 2005) thereby further establishing a link between the JNK pathway downstream of p75NTR (Allen et al., 2005). At the functional level, ectopic re-expression of p75NTR reduces cell survival (Allen et al., 2005), and dominant negative antagonism of IKK or MKK4 partially rescues survival (Allen et al., 2005). Hence, it appears that both the NFB and JNK pathways promote cell survival and that p75NTR down regulation of these pathways inhibits survival in prostate cancer cells, thereby providing a signal transduction pathway for the observed suppressor activities of the p75NTR protein (Krygier and Djakiew, 2001a; Krygier and Djakiew, 2002). The observation that both signaling pathways bifurcate from the p75NTR suggests a redundancy whereby robust down regulation of either pathway may function

Ectopic re-expression of p75NTR alters the cell cycle kinetics of prostate tumor cells (Krygier & Djakiew, 2001a). Cell cycle initiation and progression is cooperatively regulated by several classes of cyclin-dependent kinases (cdks). The expression of cyclin D1-cdk6 (Boonstra, 2003; Sherr, & Roberts, 1995) complexed with PCNA has been shown to promote hyperphosphorylation of Rb during progression through early to mid-G1 (Satyanarayana & Rudolph, 2004). Conversely, p16INK4a binds and induces an allosteric conformational change in cdk6 that inhibits the binding of ATP thereby disrupting the formation of the cdk6-cyclin D1 complex (Satyanarayana & Rudolph, 2004; Golsteyn, 2005). Prostate cancer cells that express rank-order (dose-dependent) increased levels of p75NTR show suppression of cdk6, PCNA and hypophosphorylated Rb, and up-regulation of p16INK4a levels indicates that p75NTR selectively regulates specific components of the holoenzyme complex (Khwaja et al., 2006) associated with retarded progression through early to mid-G1 of the cell cycle (Meyer et al., 2002). The rescue of cdk6, PCNA and phosphorylated Rb levels, and conversely suppression of p16INK4a levels, by both a death domain deleted dominant–negative antagonist of p75NTR and by NGF ligand, show a p75NTR-dependent regulation of early to mid-G1 in prostate tumor cells (Khwaja, et al., 2006). Beyond mid-G1, near the G1/S restriction point, the expression of cyclin E complexed with cdk2 has been shown to

promote hyper-phosphorylation of Rb (Boonstra, 2003; Shintani et al., 2002).

A rank-order increase in p75NTR suppression of cyclin E and cdk2 in prostate tumor cells (Khwaja et al., 2006) indicate that progression through the G1/S restriction point is regulated by p75NTR protein expression. (Figure 3). Since the Rb protein is a major effector of cell proliferation through its ability to regulate entry into the S phase (Sellers & Kaelin, 1996; , Huang et al., 2002), and the cyclin E/cdk2 holoenzyme complex hyper-phosphorylates Rb during the G1/S transition, the observation that p75NTR expression can stabilize hypophosphorylated Rb and diminish the phosphorylation of Rb is consistent with a mechanism by which p75NTR retards progression through the G1/S restriction point of the cell cycle (Khwaja et al., 2006). Furthermore, hypophosphorylated Rb has been shown to bind the E2F1 transcription factor (Harbour & Dean, 2000), so that the Rb/E2F1 complex can no longer promote transcription of PCNA preventing progression into the S phase of the cell cycle. Hence, the observation that p75NTR associated hypo-phosphorylation of Rb in conjunction with suppression of E2F1 and PCNA expression (Westwood et al., 2002) further supports a role of p75NTR in G1/S restriction point cytostasis (Khwaja et al., 2006). Beyond the G1/S restriction point, expression of the cyclin A/cdk2 holoenzyme complex has been shown to maintain hyper-phosphorylation of Rb during the S phase of the cell cycle (Sherr, 1996). Hence,

RXR-β support the specificity of the functional involvement of p75NTR in differentiation. Hence, re-expression of the p75NTR appears to partially reverse de-differentiation of prostate cancer cells by up-regulating expression of CRABPI for localized sequestration of retinoids that are available to newly up-regulated RAR-β, RXR-α, and RXR-β proteins (Nalbandian et al., 2005). Hence, the p75NTR has been shown to exhibit tumor suppressor (Krygier & Djakiew, 2001a), metastasis suppressor (Krygier & Djakiew, 2002) and differentiation (Nalbandian et al., 2005) functions all of which contribute to an anti-cancer phenotype in the prostate.

#### **4. Effectors of signal transduction, cell cycle and apoptosis following constitutive p75NTR expression in the prostate**

Ectopic re-expression of p75NTR down regulates the NFB and JNK pathways leading to reduced survival of tumor cells (Allen et al., 2005). As a member of the TNF receptor superfamily the p75NTR has been shown to mediate signal transduction through it's intracellular death domain. Expression of two adaptor proteins, TRAF2 and RIP are down-regulated following restoration of p75NTR protein by stable transfection of prostate tumor cells (Allen et al., 2005). Significantly, TRAF2 has previously been implicated as an upstream signaling molecule of both the NFB and JNK pathways (MacEwan, 2002), both of which function as potent effectors of transcription. Similarly, RIP has also been shown to interact with death receptors that signal through both the NFB and JNK pathways (Harper et al, 2003). Moreover, deletion constructs that lack much of the intracellular death domain were shown to rescue p75NTR down-regulation of RIP (Allen et al., 2005). This p75NTR-dependent reduction in RIP protein appeared to be a consequence of caspase-8 cleavage of RIP, since a caspase-8 inhibitor rescued the p75NTR-dependent reduction in the levels of RIP. This observation is consistent with previous reports where caspase-8 cleavage of RIP was shown to prevent activation of the NFB pathway (Lin et al., 1999; Martinon et al., 2000) and suggests that caspase-8 cleavage of RIP is capable of preventing the previously reported RIP activation of the JNK pathway (Kelliher et al., 1998). Hence, both the NFB and JNK pathways appear to be regulated by p75NTR expression (Allen et al., 2005). With regard to the NFB signaling bifurcation, ectopic re-expression of p75NTR reduces levels of IKKs, with consequent reduced phosphorylation levels of IB, stabilized levels of unphosphorylated IB, and reduced levels of RelA (Allen et al., 2005). Moreover, the activity of the IKKs is regulated through the serine/threonine kinase activity of upstream full length RIP (Hur et al., 2003). Hence, the p75NTR-dependent reduction in the levels of RIP, via caspase-8 cleavage of RIP, leads to an overall reduction in signal transduction components required for activation of NFB (Allen et al., 2005). Several of these p75NTR dependent changes in signaling components are rescued with dominant negative death domain deletion constructs, thereby further establishing a link between the NFB pathway downstream of p75NTR (Allen et al., 2005). With regard to the JNK signaling bifurcation, ectopic restoration of p75NTR levels is associated with a reduction in the expression of the MKK4 kinase and a reduction in the phosphorylated (active) form of JNK. Since MKK4 phosphorylation of JNK has been shown to promote nuclear translocation (Cobb, 1999; Gonzalez et al., 2000) where activated JNK may function as an effector of transcription for growth (Gee et al., 2000; Herr & Debatin, 2001), p75NTR associated suppression of JNK phosphorylation and suppression of translocation is consistent with the suppression of the JNK signaling pathway. Moreover,

RXR-β support the specificity of the functional involvement of p75NTR in differentiation. Hence, re-expression of the p75NTR appears to partially reverse de-differentiation of prostate cancer cells by up-regulating expression of CRABPI for localized sequestration of retinoids that are available to newly up-regulated RAR-β, RXR-α, and RXR-β proteins (Nalbandian et al., 2005). Hence, the p75NTR has been shown to exhibit tumor suppressor (Krygier & Djakiew, 2001a), metastasis suppressor (Krygier & Djakiew, 2002) and differentiation (Nalbandian et al., 2005) functions all of which contribute to an anti-cancer

**4. Effectors of signal transduction, cell cycle and apoptosis following** 

Ectopic re-expression of p75NTR down regulates the NFB and JNK pathways leading to reduced survival of tumor cells (Allen et al., 2005). As a member of the TNF receptor superfamily the p75NTR has been shown to mediate signal transduction through it's intracellular death domain. Expression of two adaptor proteins, TRAF2 and RIP are down-regulated following restoration of p75NTR protein by stable transfection of prostate tumor cells (Allen et al., 2005). Significantly, TRAF2 has previously been implicated as an upstream signaling molecule of both the NFB and JNK pathways (MacEwan, 2002), both of which function as potent effectors of transcription. Similarly, RIP has also been shown to interact with death receptors that signal through both the NFB and JNK pathways (Harper et al, 2003). Moreover, deletion constructs that lack much of the intracellular death domain were shown to rescue p75NTR down-regulation of RIP (Allen et al., 2005). This p75NTR-dependent reduction in RIP protein appeared to be a consequence of caspase-8 cleavage of RIP, since a caspase-8 inhibitor rescued the p75NTR-dependent reduction in the levels of RIP. This observation is consistent with previous reports where caspase-8 cleavage of RIP was shown to prevent activation of the NFB pathway (Lin et al., 1999; Martinon et al., 2000) and suggests that caspase-8 cleavage of RIP is capable of preventing the previously reported RIP activation of the JNK pathway (Kelliher et al., 1998). Hence, both the NFB and JNK pathways appear to be regulated by p75NTR expression (Allen et al., 2005). With regard to the NFB signaling bifurcation, ectopic re-expression of p75NTR reduces levels of IKKs, with consequent reduced phosphorylation levels of IB, stabilized levels of unphosphorylated IB, and reduced levels of RelA (Allen et al., 2005). Moreover, the activity of the IKKs is regulated through the serine/threonine kinase activity of upstream full length RIP (Hur et al., 2003). Hence, the p75NTR-dependent reduction in the levels of RIP, via caspase-8 cleavage of RIP, leads to an overall reduction in signal transduction components required for activation of NFB (Allen et al., 2005). Several of these p75NTR dependent changes in signaling components are rescued with dominant negative death domain deletion constructs, thereby further establishing a link between the NFB pathway downstream of p75NTR (Allen et al., 2005). With regard to the JNK signaling bifurcation, ectopic restoration of p75NTR levels is associated with a reduction in the expression of the MKK4 kinase and a reduction in the phosphorylated (active) form of JNK. Since MKK4 phosphorylation of JNK has been shown to promote nuclear translocation (Cobb, 1999; Gonzalez et al., 2000) where activated JNK may function as an effector of transcription for growth (Gee et al., 2000; Herr & Debatin, 2001), p75NTR associated suppression of JNK phosphorylation and suppression of translocation is consistent with the suppression of the JNK signaling pathway. Moreover,

**constitutive p75NTR expression in the prostate** 

phenotype in the prostate.

deletion constructs that lack much of the intracellular death domain were shown to rescue p75NTR down-regulation of MKK4 and phosphorylated JNK (Allen et al., 2005) thereby further establishing a link between the JNK pathway downstream of p75NTR (Allen et al., 2005). At the functional level, ectopic re-expression of p75NTR reduces cell survival (Allen et al., 2005), and dominant negative antagonism of IKK or MKK4 partially rescues survival (Allen et al., 2005). Hence, it appears that both the NFB and JNK pathways promote cell survival and that p75NTR down regulation of these pathways inhibits survival in prostate cancer cells, thereby providing a signal transduction pathway for the observed suppressor activities of the p75NTR protein (Krygier and Djakiew, 2001a; Krygier and Djakiew, 2002). The observation that both signaling pathways bifurcate from the p75NTR suggests a redundancy whereby robust down regulation of either pathway may function independently to promote suppressor activity.

Ectopic re-expression of p75NTR alters the cell cycle kinetics of prostate tumor cells (Krygier & Djakiew, 2001a). Cell cycle initiation and progression is cooperatively regulated by several classes of cyclin-dependent kinases (cdks). The expression of cyclin D1-cdk6 (Boonstra, 2003; Sherr, & Roberts, 1995) complexed with PCNA has been shown to promote hyperphosphorylation of Rb during progression through early to mid-G1 (Satyanarayana & Rudolph, 2004). Conversely, p16INK4a binds and induces an allosteric conformational change in cdk6 that inhibits the binding of ATP thereby disrupting the formation of the cdk6-cyclin D1 complex (Satyanarayana & Rudolph, 2004; Golsteyn, 2005). Prostate cancer cells that express rank-order (dose-dependent) increased levels of p75NTR show suppression of cdk6, PCNA and hypophosphorylated Rb, and up-regulation of p16INK4a levels indicates that p75NTR selectively regulates specific components of the holoenzyme complex (Khwaja et al., 2006) associated with retarded progression through early to mid-G1 of the cell cycle (Meyer et al., 2002). The rescue of cdk6, PCNA and phosphorylated Rb levels, and conversely suppression of p16INK4a levels, by both a death domain deleted dominant–negative antagonist of p75NTR and by NGF ligand, show a p75NTR-dependent regulation of early to mid-G1 in prostate tumor cells (Khwaja, et al., 2006). Beyond mid-G1, near the G1/S restriction point, the expression of cyclin E complexed with cdk2 has been shown to promote hyper-phosphorylation of Rb (Boonstra, 2003; Shintani et al., 2002).

A rank-order increase in p75NTR suppression of cyclin E and cdk2 in prostate tumor cells (Khwaja et al., 2006) indicate that progression through the G1/S restriction point is regulated by p75NTR protein expression. (Figure 3). Since the Rb protein is a major effector of cell proliferation through its ability to regulate entry into the S phase (Sellers & Kaelin, 1996; , Huang et al., 2002), and the cyclin E/cdk2 holoenzyme complex hyper-phosphorylates Rb during the G1/S transition, the observation that p75NTR expression can stabilize hypophosphorylated Rb and diminish the phosphorylation of Rb is consistent with a mechanism by which p75NTR retards progression through the G1/S restriction point of the cell cycle (Khwaja et al., 2006). Furthermore, hypophosphorylated Rb has been shown to bind the E2F1 transcription factor (Harbour & Dean, 2000), so that the Rb/E2F1 complex can no longer promote transcription of PCNA preventing progression into the S phase of the cell cycle. Hence, the observation that p75NTR associated hypo-phosphorylation of Rb in conjunction with suppression of E2F1 and PCNA expression (Westwood et al., 2002) further supports a role of p75NTR in G1/S restriction point cytostasis (Khwaja et al., 2006). Beyond the G1/S restriction point, expression of the cyclin A/cdk2 holoenzyme complex has been shown to maintain hyper-phosphorylation of Rb during the S phase of the cell cycle (Sherr, 1996). Hence,

NSAID Induction of p75NTR in the Prostate:

A Suppressor of Growth and Cell Migration Via the p38 MAPK Pathway 31

include both pro- and anti-apoptotic members (Puthalakath & Strasser, 2002). In this context, a rank-order increase in p75NTR levels (Khwaja et al., 2006) was associated with a decrease in the mitochondrial pro-survival effector, Bcl-xL, and concomitantly, an increase in proapoptotic effectors (Smac, Bax, Bak, Bad). In the absence of NGF ligand the ability of a death domain deleted dominant–negative antagonist of p75NTR to rescue p75NTR-dependent changes in Smac and BH family proteins, and the addition of NGF having a comparable effect, suggest a ligand independent p75NTR-potentiated apoptosis in prostate cells that occurs via a mitochondrial stress pathway (Khwaja et al., 2006). Release of Smac from the intermembranous space of the mitochondria into the cytosol (Shiozaki & Shi, 2004) also initiates apoptosis where it competes with caspases for binding to XIAP. Indeed, to protect against inadvertent activation of apoptosis, the IAP family of proteins (Shiozaki & Shi, 2004) have been shown to bind and prevent activation of caspases. Hence, p75NTR-dependent down-regulation of XIAP in prostate cells allows for activation of the caspase cascade. Significantly, a rank-order increase in p75NTR levels was shown to activate both caspase-9 and caspase-7 (Khwaja et al., 2006). In the intrinsic mitochondrial dependent pathway, the assembly of the apoptosome requires the recruitment of the initiator caspase-9 (Shiozaki & Shi, 2004) which subsequently activates the effector caspase-7. Interestingly, a specific peptide inhibitor of procaspase-9 cleavage was shown to prevent cleavage/activation of procaspase-7. Since the initiator procaspase-9 is a proximate component of the caspase cascade, it is clear that procaspase-7 is downstream from procaspase-9 during mitochondrial mediated p75NTR potentiation of apoptosis (Shiozaki & Shi, 2004). Moreover, in p75NTR expressing prostate cells the activation of caspase-7 appears to facilitate subsequent cleavage of PARP. PARP cleavage has been shown to be a key event in the execution phase of apoptosis leading to cellular demise (Lazebnik et al., 1994). Clearly, the ability of a death domain deleted dominant–negative antagonist of p75NTR and NGF ligand to rescue p75NTR potentiated changes in XIAP, caspase-9, caspase-7, PARP and apoptotic nuclear fragmentation show that mitochondrial mediated apoptosis is dependent, in part, on p75NTR in the prostate. Moreover, the effect of p75NTR-dependent down regulation of NFB and JNK survival pathways, regulation of cell cycle regulatory molecules of the cyclin/cdk holoenzyme complex that produce stasis in G1 and/or inhibition of progression to S phase of the cell cycle and regulation of mitochondrial effector proteins that activate the caspase cascade leading to apoptosis, in combination, all inhibit growth of prostate cells as a function of it's tumor suppressor activity (Krygier & Djakiew, 2001a; Khwaja et al., 2006).

**5. Constitutive p75NTR expression regulates protease activity in the prostate** 

Ectopic re-expression of p75NTR down regulates protease activities in prostate cancer cells (Nalbandian & Djakiew, 2006). The urokinase plasminogen activator (uPA) and its receptor (uPAR) are associated with tumor malignancy through an extracellular cascade of proteolysis (Andreasen et al., 2000; Reuning et al., 1998) including activation of the type IV collagen matrix metalloproteinases (MMP-2 and MMP-9) during invasion and tumor progression (Mazzier et al., 1997). Ectopic re-expression of p75NTR reduces enzymatic protein levels and activity of uPA, MMP-2 and MMP-9 in prostate tumor cells (Nalbandian & Djakiew, 2006). Conversely, expression of an MMP-9 antagonist, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) exhibits an increase in protein levels with an increase in p75NTR levels. Whereas, levels of TIMP-2 were not detectable (Nalbandian & Djakiew, 2006) transient transfection with an inducible death domain deleted dominant–negative antagonist of p75NTR rescued uPA, MMP-

observations that p75NTR-potentiated suppression of cdk2 and cyclin A support a selective effect of p75NTR expression on maintaining hypo-phosphorylation of Rb (Khwaja et al., 2006), thereby retarding progression through the S phase of the cell cycle (Figure 3). Significantly, the rescue of cyclin E, cdk2, PCNA, phosphorylated Rb, E2F1 and PCNA by both a death domain deleted dominant–negative antagonist of p75NTR and also by NGF ligand (Khwaja et al., 2006) show a p75NTR-dependence of the regulation of progression through both the G1/S restriction point and the S phase in prostate tumor cells. Hence, it seems clear that p75NTR expression selectively alters specific cell cycle regulatory molecules that retard progression through early to mid-G1, the G1/S restriction point and the S phase of the cell cycle (Figure 3). Moreover, in the absence of ligand, the ability of p75NTR to retard cell cycle progression is dependent on the intracellular death domain, and that addition of NGF ligand attenuates inhibition of cell cycle progression at the level of the cyclin/cdk holoenzyme complex and related effects on Rb expression and tumor cell proliferation. Hence, p75NTR-dependent regulation of specific cyclindependent kinases and associated cell cycle effectors to retard progression of prostate tumors through the G1 phase and entry into S phase of the cell cycle (Figure 3) provides a biochemical basis upon which p75NTR inhibits prostate cell proliferation (Khwaja et al., 2006).

Fig. 3. Effects of p75NTR on regulation of the cell cycle. Re-expression of p75NTR induces midG1 suppression of cdk6 and up-regulation of p16Ink4a. During the G1/S transition reexpression of p75NTR induces suppression of cdk2 and cyclin E. Similarly, during S phase re-expression of p75NTR induces suppression of cdk2 and cyclin A. The net effect of these changes is to diminish phosphorylation of Rb thereby preventing progression of the cell cycle in favor of cessation.

In addition to cell cycle effects, ectopic re-expression of p75NTR has been shown to modify mitochondrial effector proteins that activate the caspase cascade leading to apoptosis (Khwaja, et al., 2006). Mitochondrial mediated apoptosis is facilitated by members of the Bcl-2 homology (Puthalakath & Strasser, 2002) family of proteins (O'Neill et al., 2004) that

observations that p75NTR-potentiated suppression of cdk2 and cyclin A support a selective effect of p75NTR expression on maintaining hypo-phosphorylation of Rb (Khwaja et al., 2006), thereby retarding progression through the S phase of the cell cycle (Figure 3). Significantly, the rescue of cyclin E, cdk2, PCNA, phosphorylated Rb, E2F1 and PCNA by both a death domain deleted dominant–negative antagonist of p75NTR and also by NGF ligand (Khwaja et al., 2006) show a p75NTR-dependence of the regulation of progression through both the G1/S restriction point and the S phase in prostate tumor cells. Hence, it seems clear that p75NTR expression selectively alters specific cell cycle regulatory molecules that retard progression through early to mid-G1, the G1/S restriction point and the S phase of the cell cycle (Figure 3). Moreover, in the absence of ligand, the ability of p75NTR to retard cell cycle progression is dependent on the intracellular death domain, and that addition of NGF ligand attenuates inhibition of cell cycle progression at the level of the cyclin/cdk holoenzyme complex and related effects on Rb expression and tumor cell proliferation. Hence, p75NTR-dependent regulation of specific cyclindependent kinases and associated cell cycle effectors to retard progression of prostate tumors through the G1 phase and entry into S phase of the cell cycle (Figure 3) provides a biochemical

basis upon which p75NTR inhibits prostate cell proliferation (Khwaja et al., 2006).

Fig. 3. Effects of p75NTR on regulation of the cell cycle. Re-expression of p75NTR induces midG1 suppression of cdk6 and up-regulation of p16Ink4a. During the G1/S transition reexpression of p75NTR induces suppression of cdk2 and cyclin E. Similarly, during S phase re-expression of p75NTR induces suppression of cdk2 and cyclin A. The net effect of these changes is to diminish phosphorylation of Rb thereby preventing progression of the cell

In addition to cell cycle effects, ectopic re-expression of p75NTR has been shown to modify mitochondrial effector proteins that activate the caspase cascade leading to apoptosis (Khwaja, et al., 2006). Mitochondrial mediated apoptosis is facilitated by members of the Bcl-2 homology (Puthalakath & Strasser, 2002) family of proteins (O'Neill et al., 2004) that

cycle in favor of cessation.

include both pro- and anti-apoptotic members (Puthalakath & Strasser, 2002). In this context, a rank-order increase in p75NTR levels (Khwaja et al., 2006) was associated with a decrease in the mitochondrial pro-survival effector, Bcl-xL, and concomitantly, an increase in proapoptotic effectors (Smac, Bax, Bak, Bad). In the absence of NGF ligand the ability of a death domain deleted dominant–negative antagonist of p75NTR to rescue p75NTR-dependent changes in Smac and BH family proteins, and the addition of NGF having a comparable effect, suggest a ligand independent p75NTR-potentiated apoptosis in prostate cells that occurs via a mitochondrial stress pathway (Khwaja et al., 2006). Release of Smac from the intermembranous space of the mitochondria into the cytosol (Shiozaki & Shi, 2004) also initiates apoptosis where it competes with caspases for binding to XIAP. Indeed, to protect against inadvertent activation of apoptosis, the IAP family of proteins (Shiozaki & Shi, 2004) have been shown to bind and prevent activation of caspases. Hence, p75NTR-dependent down-regulation of XIAP in prostate cells allows for activation of the caspase cascade. Significantly, a rank-order increase in p75NTR levels was shown to activate both caspase-9 and caspase-7 (Khwaja et al., 2006). In the intrinsic mitochondrial dependent pathway, the assembly of the apoptosome requires the recruitment of the initiator caspase-9 (Shiozaki & Shi, 2004) which subsequently activates the effector caspase-7. Interestingly, a specific peptide inhibitor of procaspase-9 cleavage was shown to prevent cleavage/activation of procaspase-7. Since the initiator procaspase-9 is a proximate component of the caspase cascade, it is clear that procaspase-7 is downstream from procaspase-9 during mitochondrial mediated p75NTR potentiation of apoptosis (Shiozaki & Shi, 2004). Moreover, in p75NTR expressing prostate cells the activation of caspase-7 appears to facilitate subsequent cleavage of PARP. PARP cleavage has been shown to be a key event in the execution phase of apoptosis leading to cellular demise (Lazebnik et al., 1994). Clearly, the ability of a death domain deleted dominant–negative antagonist of p75NTR and NGF ligand to rescue p75NTR potentiated changes in XIAP, caspase-9, caspase-7, PARP and apoptotic nuclear fragmentation show that mitochondrial mediated apoptosis is dependent, in part, on p75NTR in the prostate. Moreover, the effect of p75NTR-dependent down regulation of NFB and JNK survival pathways, regulation of cell cycle regulatory molecules of the cyclin/cdk holoenzyme complex that produce stasis in G1 and/or inhibition of progression to S phase of the cell cycle and regulation of mitochondrial effector proteins that activate the caspase cascade leading to apoptosis, in combination, all inhibit growth of prostate cells as a function of it's tumor suppressor activity (Krygier & Djakiew, 2001a; Khwaja et al., 2006).

#### **5. Constitutive p75NTR expression regulates protease activity in the prostate**

Ectopic re-expression of p75NTR down regulates protease activities in prostate cancer cells (Nalbandian & Djakiew, 2006). The urokinase plasminogen activator (uPA) and its receptor (uPAR) are associated with tumor malignancy through an extracellular cascade of proteolysis (Andreasen et al., 2000; Reuning et al., 1998) including activation of the type IV collagen matrix metalloproteinases (MMP-2 and MMP-9) during invasion and tumor progression (Mazzier et al., 1997). Ectopic re-expression of p75NTR reduces enzymatic protein levels and activity of uPA, MMP-2 and MMP-9 in prostate tumor cells (Nalbandian & Djakiew, 2006). Conversely, expression of an MMP-9 antagonist, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) exhibits an increase in protein levels with an increase in p75NTR levels. Whereas, levels of TIMP-2 were not detectable (Nalbandian & Djakiew, 2006) transient transfection with an inducible death domain deleted dominant–negative antagonist of p75NTR rescued uPA, MMP-

NSAID Induction of p75NTR in the Prostate:

A Suppressor of Growth and Cell Migration Via the p38 MAPK Pathway 33

inhibitors consistently induce apoptosis in prostate cancer cells regardless of COX-2 expression (Zha et al., 2001; Jacobs et al., 2007; Roberts et al., 2002; Hsu et al., 2000; Johnson et al., 2001). Indeed, NSAIDS that do not inhibit the cyclo-oxygenases (Rflurbiprofen) or NSAIDs that inhibit COX null tumor cells demonstrates an alternate COX independent mechanism of action for selected NSAID inhibition of some tumor cells. In this context, NSAIDs exhibit selective activity to induce p75NTR-dependent cell death (Khwaja et al., 2004). The propionic acids, ibuprofen and r-flurbiprofen, as well as the indolacetic acid, indomethacin, exhibit greater pharmacological activity to induce p75NTRdependent cell death than the salicyclate, aspirin, or acetaminophen (Khwaja et al., 2004). Within the aryl propionic acid NSAIDs, r-flurbiprofen exhibits greater activity to induce p75NTR in descending rank-order than ibuprofen, oxaprozin, fenoprofen, naproxen and least of all ketoprofen (Quann et al., 2007a). Significantly, this activity appears relatively selective for induction of p75NTR (Quann et al., 2007a), since r-flurbiprofen and ibuprofen do not induce expression of Fas, p55TNFR, DR3, DR4, DR5 or DR6 in prostate cancer cells (Quann et al., 2007a). Some other NSAIDs have also been reported to induce expression of

Fig. 4. Classification of the Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) into three categories of carboxylic acids, enolic acids and COX-2 inhibitors. Sub-classifications of

For instance, sulindac sulfide induces DR5 associated cell death in prostate cancer cells (Huang et al., 2001) and celocoxibs induce DR5 in both prostate and colon cancer cells (He et al., 2008). Moreover, aryl propionic acid induction of p75NTR appears somewhat selective for urogenital cancer cell lines such as prostate, bladder, kidney, ovary, and colon (Khwaja et al., 2004), whereas cell lines derived from the lung, or breast do not exhibit induction of the p75NTR protein (Khwaja et al., 2004). Hence, induction of p75NTR appears to represent a COX independent mechanism for NSAID associated reduced risk

certain TNF receptor super family members, particularly DR5.

NSAIDs are further delineated with specific examples.

of prostate cancer.

2, and MMP-9 protein levels and protease activities, and conversely suppressed TIMP-1 levels (Nalbandian & Djakiew, 2006). Since ectopic p75NTR signal transduction has been shown to suppress the NFB and JNK pathways (Allen et al., 2005), antagonism of signaling intermediaries in these pathways, using dominant negative IKKβ or dominant negative MKK-4, respectively, was shown to further decrease expression of uPA, MMP-2, and MMP-9 protein and enzymatic activity levels, and conversely up-regulate levels of TIMP-1. Hence, expression of uPA, MMP-2, MMP-9, and TIMP-1 are directly regulated by expression of p75NTR and its downstream signal transduction cascade. This suggests that the metastasis suppressor activity of p75NTR (Krygier & Djakiew, 2002) is mediated, in part, by down-regulation of specific proteases (uPA, type IV collagenases) implicated in cell migration and metastasis (Nalbandian & Djakiew, 2006).

#### **6. NSAIDS selectively induce pharmacological re-expression of the p75NTR**

NSAIDs represent a diverse and often structurally unrelated group of compounds that exhibit a range of anti-inflammatory, anti-pyretic and analgesic activities. NSAIDs can be classified into three broad categories as carboxylic acids, enolic acids, and COX-2 inhibitors, or coxibs (Figure 4). The carboxylic acid group is further divided into salicylic acids and esters, acetic acids, and propionic acids. The enolic acids are divided into pyrazolones and oxicams. Many NSAIDs used to treat inflammation inhibit cyclooxygenase (COX) activity. Two well known isoforms of COX exist, COX-1 and COX-2. COX-1 is considered to be a house keeping gene, and is expressed constitutively and ubiquitously at low levels. COX-2 is highly inducible in response to cytokines, hormones, and growth factors. The COX enzymes catalyze the conversion of arachidonic acid to various prostaglandins, which play a role in biological processes including immune response, blood pressure regulation, angiogenesis, ovulation, pain and inflammatory responses. Nonselective NSAIDs inhibit both COX-1 and COX-2, and are frequently associated with gastrointestinal side effects. Whereas, NSAIDs that selectively inhibit COX-2 have significantly decreased gastrointestinal side effects, they often exhibit enhanced cardiovascular toxicity (Dubois et al., 2004). Overexpression of COX-2 is observed in several cancer types including colon, breast, pancreas, and lung (Sarkar et al., 2007; Mascaux et al., 2006). Increased COX-2 expression is believed to contribute to tumorigenesis through several mechanisms including stimulation of growth, promotion of angiogenesis, increased inflammation, increased invasion and migration, immune suppression, and inhibition of apoptosis (Liao et al., 2007). Hence, NSAID inhibition of COX-2 activity may suppress these mechanisms that contribute to tumorigenesis. Consistently, several reports have linked long term NSAID use to decreased cancer risk for colon (Thun et al., 1991), bladder (Castelao et al., 2000), and prostate (Nelson & Harris, 2000) and possibly other organ specific sites. Although it has been the subject of multiple reports, there is not a consensus concerning the expression and role of COX-2 in prostate cancer. Several studies show that COX-2 is overexpressed, while others found that it is low or absent in most prostate cancers (Madaan et al., 2000; Kirschenbaum et al., 2000; Yoshimura et al., 2000). In addition, expression of COX-2 is absent in the metastatic human prostate cancer cell lines LNCaP, DU145, and PC-3 (Yoshimura et al., 2000). Although reports of COX-2 expression in prostate cancer vary, long term NSAID use is associated with decreased prostate cancer risk (Nelson & Harris, 2000), and several COX

2, and MMP-9 protein levels and protease activities, and conversely suppressed TIMP-1 levels (Nalbandian & Djakiew, 2006). Since ectopic p75NTR signal transduction has been shown to suppress the NFB and JNK pathways (Allen et al., 2005), antagonism of signaling intermediaries in these pathways, using dominant negative IKKβ or dominant negative MKK-4, respectively, was shown to further decrease expression of uPA, MMP-2, and MMP-9 protein and enzymatic activity levels, and conversely up-regulate levels of TIMP-1. Hence, expression of uPA, MMP-2, MMP-9, and TIMP-1 are directly regulated by expression of p75NTR and its downstream signal transduction cascade. This suggests that the metastasis suppressor activity of p75NTR (Krygier & Djakiew, 2002) is mediated, in part, by down-regulation of specific proteases (uPA, type IV collagenases) implicated in cell migration and metastasis (Nalbandian

**6. NSAIDS selectively induce pharmacological re-expression of the p75NTR** 

NSAIDs represent a diverse and often structurally unrelated group of compounds that exhibit a range of anti-inflammatory, anti-pyretic and analgesic activities. NSAIDs can be classified into three broad categories as carboxylic acids, enolic acids, and COX-2 inhibitors, or coxibs (Figure 4). The carboxylic acid group is further divided into salicylic acids and esters, acetic acids, and propionic acids. The enolic acids are divided into pyrazolones and oxicams. Many NSAIDs used to treat inflammation inhibit cyclooxygenase (COX) activity. Two well known isoforms of COX exist, COX-1 and COX-2. COX-1 is considered to be a house keeping gene, and is expressed constitutively and ubiquitously at low levels. COX-2 is highly inducible in response to cytokines, hormones, and growth factors. The COX enzymes catalyze the conversion of arachidonic acid to various prostaglandins, which play a role in biological processes including immune response, blood pressure regulation, angiogenesis, ovulation, pain and inflammatory responses. Nonselective NSAIDs inhibit both COX-1 and COX-2, and are frequently associated with gastrointestinal side effects. Whereas, NSAIDs that selectively inhibit COX-2 have significantly decreased gastrointestinal side effects, they often exhibit enhanced cardiovascular toxicity (Dubois et al., 2004). Overexpression of COX-2 is observed in several cancer types including colon, breast, pancreas, and lung (Sarkar et al., 2007; Mascaux et al., 2006). Increased COX-2 expression is believed to contribute to tumorigenesis through several mechanisms including stimulation of growth, promotion of angiogenesis, increased inflammation, increased invasion and migration, immune suppression, and inhibition of apoptosis (Liao et al., 2007). Hence, NSAID inhibition of COX-2 activity may suppress these mechanisms that contribute to tumorigenesis. Consistently, several reports have linked long term NSAID use to decreased cancer risk for colon (Thun et al., 1991), bladder (Castelao et al., 2000), and prostate (Nelson & Harris, 2000) and possibly other organ specific sites. Although it has been the subject of multiple reports, there is not a consensus concerning the expression and role of COX-2 in prostate cancer. Several studies show that COX-2 is overexpressed, while others found that it is low or absent in most prostate cancers (Madaan et al., 2000; Kirschenbaum et al., 2000; Yoshimura et al., 2000). In addition, expression of COX-2 is absent in the metastatic human prostate cancer cell lines LNCaP, DU145, and PC-3 (Yoshimura et al., 2000). Although reports of COX-2 expression in prostate cancer vary, long term NSAID use is associated with decreased prostate cancer risk (Nelson & Harris, 2000), and several COX

& Djakiew, 2006).

inhibitors consistently induce apoptosis in prostate cancer cells regardless of COX-2 expression (Zha et al., 2001; Jacobs et al., 2007; Roberts et al., 2002; Hsu et al., 2000; Johnson et al., 2001). Indeed, NSAIDS that do not inhibit the cyclo-oxygenases (Rflurbiprofen) or NSAIDs that inhibit COX null tumor cells demonstrates an alternate COX independent mechanism of action for selected NSAID inhibition of some tumor cells. In this context, NSAIDs exhibit selective activity to induce p75NTR-dependent cell death (Khwaja et al., 2004). The propionic acids, ibuprofen and r-flurbiprofen, as well as the indolacetic acid, indomethacin, exhibit greater pharmacological activity to induce p75NTRdependent cell death than the salicyclate, aspirin, or acetaminophen (Khwaja et al., 2004). Within the aryl propionic acid NSAIDs, r-flurbiprofen exhibits greater activity to induce p75NTR in descending rank-order than ibuprofen, oxaprozin, fenoprofen, naproxen and least of all ketoprofen (Quann et al., 2007a). Significantly, this activity appears relatively selective for induction of p75NTR (Quann et al., 2007a), since r-flurbiprofen and ibuprofen do not induce expression of Fas, p55TNFR, DR3, DR4, DR5 or DR6 in prostate cancer cells (Quann et al., 2007a). Some other NSAIDs have also been reported to induce expression of certain TNF receptor super family members, particularly DR5.

Fig. 4. Classification of the Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) into three categories of carboxylic acids, enolic acids and COX-2 inhibitors. Sub-classifications of NSAIDs are further delineated with specific examples.

For instance, sulindac sulfide induces DR5 associated cell death in prostate cancer cells (Huang et al., 2001) and celocoxibs induce DR5 in both prostate and colon cancer cells (He et al., 2008). Moreover, aryl propionic acid induction of p75NTR appears somewhat selective for urogenital cancer cell lines such as prostate, bladder, kidney, ovary, and colon (Khwaja et al., 2004), whereas cell lines derived from the lung, or breast do not exhibit induction of the p75NTR protein (Khwaja et al., 2004). Hence, induction of p75NTR appears to represent a COX independent mechanism for NSAID associated reduced risk of prostate cancer.

NSAID Induction of p75NTR in the Prostate:

**MAPK pathway** 

A Suppressor of Growth and Cell Migration Via the p38 MAPK Pathway 35

The mRNA stabilizing effects of the p38 MAPK pathway (Ronkina et al., 2007) also involves the RNA binding protein HuR (Tran et al., 2003; Lin et al., 2006; Jin et al., 2007; Song et al., 2005). Its ability to stabilize target mRNAs is linked to its subcellular localization, and activation of p38 MAPK and MK2 have been shown to cause translocation of HuR from the nucleus to the cytoplasm, resulting in increased mRNA stability of a number of p38 MAPK regulated genes (Tran et al., 2003; Lin et al., 2006; Jin et al., 2007; Song et al., 2005). HuR has repeatedly been shown to stabilize transcripts containing the AUUUA sequence (Tran et al., 2003; Song et al., 2005). The human p75NTR transcript contains AUUUA sites located in the 3'UTR at positions 2946 and 3124, suggesting they may be involved in regulating p75NTR expression. Significantly, treatment with R-flurbiprofen or ibuprofen results in an increase in the cytoplasmic level of HuR and binding to the p75NTR transcript (Quann et al., 2007b) although this is not the sole mechanism responsible for increased p75NTR expression. Indeed, eIF4E is also phosphorylated by kinases downstream of p38 MAPK (Scheper & Proud, 2002). eIF4E has also been shown to control the nuclear export as well as translation of a subset of transcripts (Culjkovic et al., 2007). eIF4E has been linked to the control of mRNA stability by removal of the 5'-cap during mRNA degradation (von der Haar et al., 2004). R-flurbiprofen or ibuprofen treatment increases the level of phosphorylated eIF4E (Quann et al., 2007b) involved in translation initiation and this appears to occur at least partially through the p38 MAPK pathway since the increase in phosphorylation is substantially inhibited in the presence of p38 MAPK siRNA. Therefore, modulation of eIF4E activity is another mechanism by which the p38 MAPK pathway may control post-transcriptional events in response to R-flurbiprofen or ibuprofen treatment (Figure 6). R-flurbiprofen or ibuprofen stabilization of the p75NTR transcript allows levels of the p75NTR protein to accumulate (Quann et al., 1997a, Quann et al., 197b) that then exhibit biological activity to inhibit prostate cancer cell survival and induce apoptosis (Quann et al., 1997a) consistent with its tumor suppressor activity (Krygier & Djakiew, 2001a). Significantly, NSAID induction of p38 MAPK dependent p75NTR expression and tumor suppressor activity may represent a new pathway to medicinal drug design. Rather that inhibit kinase dependent proliferation pathways, pharmacological induction of the p75NTR tumor suppressor, as shown with NSAIDs (Figure 6), may represent an alternate approach to the targeting of cancer cell survival pathways. Presumably, target molecules that activate this pathway appear to occur immediately proximal to MKK6 and the p38 MAPK (Figure 5).

**8. NSAIDs induce expression of NAG-1 down stream of p75NTR via the p38** 

R-flurbiprofen and ibuprofen treatment of prostate cancer cells induces expression of the NSAID activated gene-1 (Nag-1) protein, a divergent member of the transforming growth factor beta (TGF-β) family (Wynne & Djakiew, 2010). Moreover, a selective pharmacological inhibitor of p38 MAPK and p38 MAPK specific siRNA, both reduce Nag-1 induction following NSAID treatment. Hence, NSAID induced Nag-1 expression is regulated by the p38 MAPK pathway (Figure 6). Interestingly, p75NTR specific siRNA pretreatment abrogates Nag-1 induction by NSAIDs (Wynne & Djakiew, 2010) thereby demonstrating that Nag-1 is downstream of p75NTR induction (Figure 6). Functionally, decreased survival of NSAID treated cells is rescued by p75NTR specific siRNA (Quann et al., 2007a; Wynne & Djakiew, 2010) but not by Nag-1 siRNA. Transwell chamber and *in vitro* wound healing assays demonstrate decreased cell migration upon NSAID treatment (Wynne & Djakiew, 2010). Pre-treatment of prostate cancer cells with p75NTR and Nag-1 specific siRNA shows that

#### **7. NSAID induced phosphorylation of p38 MAPK is necessary for induction of p75NTR expression**

NSAIDs exhibit a range of efficacy to inhibit survival of prostate cancer cells (Andrews et al., 2002). Induction of p75NTR dependent cell death by selected NSAIDs (R-flurbiprofen, ibuprofen) occurs through hyper-phosphorylation of p38 MAPK (Quann et al., 2007b). Although p75NTR is transcribed at a high level (Quann et al., 2007b; Krygier & Djakiew, 2001b), prostate cancer cells have very little p75NTR mRNA or protein due to increased mRNA instability mediated through the 3'UTR (Krygier & Djakiew, 2001b). Since, increased 3'UTR length provides increased potential for post-transcriptional regulation through the 3'UTR (Mazumder et al., 2003) R-flurbiprofen and ibuprofen appear to modulate mRNA stability as a determinant in regulating the expression level of p75NTR protein (Quann et al., 2007b). Significantly, activity of the p38 MAPK pathway is an important regulator of mRNA stability (Zarubin & Han, 2005; Kennedy et al., 2007; Gaestel, 2006). Pretreatment of prostate cancer cells with a p38 MAPK selective pharmacological inhibitor or siRNA knockdown of p38 MAPK prior to R-flurbiprofen and ibuprofen treatment prevents induction of p75NTR protein. In addition, the phytoestrogens biochanin A and to a lesser extent genestein, inhibit ibuprofen induced phosphorylation of p38 MAPK which in turn suppresses p75NTR expression and increases cell survival (El Touny et al., 2010). R-flurbiprofen and ibuprofen cause increased p38 MAPK phosphorylation within 5 minutes of treatment (Quann et al., 2007b). Whereas, p38 MAPK can be phosphorylated by MKK6 and MKK3, R-flurbiprofen and ibuprofen induce hyperphosphorylation of only MKK6 but not MKK3 within 30 seconds of treatment (Figure 5) indicating that the target molecule of these NSAIDs is immediately proximal to MKK6. Significantly, several oxidative stress pathways converge upstream of MKK6, thereby providing a basis by which NSAID inhibition of inflammation (Masferrer et al., 1995; Tegeder et al., 2001) may regulate p38 MAPK dependent expression of the p75NTR tumor suppressor to reduce the incidence of prostate cancer. Downstream of p38 MAPK R-flurbiprofen and ibuprofen induced activation of the kinase MK2 (Quann et al., 2007b). MK2 and the closely related MK3 are known to be responsible for mediating the mRNA stabilizing effects of the p38 MAPK pathway (Ronkina et al., 2007). Moreover, siRNA knockdown of both MK2 and MK3 together prevents induction of p75NTR by R-flurbiprofen or ibuprofen to a greater extent than knockdown of either MK2 or MK3 separately, indicating that p38 MAPK is able to induce p75NTR by acting through both MK2 and MK3 (Quann et al., 2007b).

Fig. 5. Western blot showing the time course in seconds (s), minutes (m) and hours (h) of the phosphorylation forms of MKK6, but not MKK3, in PC-3 prostate cancer cells treated with rflurbiprofen (Flu), or ibuprofen (Ibu). The positive controls for the phosphorylated forms of both MKK6 and MKK3 are shown to the right.

**7. NSAID induced phosphorylation of p38 MAPK is necessary for induction of** 

NSAIDs exhibit a range of efficacy to inhibit survival of prostate cancer cells (Andrews et al., 2002). Induction of p75NTR dependent cell death by selected NSAIDs (R-flurbiprofen, ibuprofen) occurs through hyper-phosphorylation of p38 MAPK (Quann et al., 2007b). Although p75NTR is transcribed at a high level (Quann et al., 2007b; Krygier & Djakiew, 2001b), prostate cancer cells have very little p75NTR mRNA or protein due to increased mRNA instability mediated through the 3'UTR (Krygier & Djakiew, 2001b). Since, increased 3'UTR length provides increased potential for post-transcriptional regulation through the 3'UTR (Mazumder et al., 2003) R-flurbiprofen and ibuprofen appear to modulate mRNA stability as a determinant in regulating the expression level of p75NTR protein (Quann et al., 2007b). Significantly, activity of the p38 MAPK pathway is an important regulator of mRNA stability (Zarubin & Han, 2005; Kennedy et al., 2007; Gaestel, 2006). Pretreatment of prostate cancer cells with a p38 MAPK selective pharmacological inhibitor or siRNA knockdown of p38 MAPK prior to R-flurbiprofen and ibuprofen treatment prevents induction of p75NTR protein. In addition, the phytoestrogens biochanin A and to a lesser extent genestein, inhibit ibuprofen induced phosphorylation of p38 MAPK which in turn suppresses p75NTR expression and increases cell survival (El Touny et al., 2010). R-flurbiprofen and ibuprofen cause increased p38 MAPK phosphorylation within 5 minutes of treatment (Quann et al., 2007b). Whereas, p38 MAPK can be phosphorylated by MKK6 and MKK3, R-flurbiprofen and ibuprofen induce hyperphosphorylation of only MKK6 but not MKK3 within 30 seconds of treatment (Figure 5) indicating that the target molecule of these NSAIDs is immediately proximal to MKK6. Significantly, several oxidative stress pathways converge upstream of MKK6, thereby providing a basis by which NSAID inhibition of inflammation (Masferrer et al., 1995; Tegeder et al., 2001) may regulate p38 MAPK dependent expression of the p75NTR tumor suppressor to reduce the incidence of prostate cancer. Downstream of p38 MAPK R-flurbiprofen and ibuprofen induced activation of the kinase MK2 (Quann et al., 2007b). MK2 and the closely related MK3 are known to be responsible for mediating the mRNA stabilizing effects of the p38 MAPK pathway (Ronkina et al., 2007). Moreover, siRNA knockdown of both MK2 and MK3 together prevents induction of p75NTR by R-flurbiprofen or ibuprofen to a greater extent than knockdown of either MK2 or MK3 separately, indicating that p38 MAPK is able to induce

p75NTR by acting through both MK2 and MK3 (Quann et al., 2007b).

both MKK6 and MKK3 are shown to the right.

Fig. 5. Western blot showing the time course in seconds (s), minutes (m) and hours (h) of the phosphorylation forms of MKK6, but not MKK3, in PC-3 prostate cancer cells treated with rflurbiprofen (Flu), or ibuprofen (Ibu). The positive controls for the phosphorylated forms of

**p75NTR expression** 

The mRNA stabilizing effects of the p38 MAPK pathway (Ronkina et al., 2007) also involves the RNA binding protein HuR (Tran et al., 2003; Lin et al., 2006; Jin et al., 2007; Song et al., 2005). Its ability to stabilize target mRNAs is linked to its subcellular localization, and activation of p38 MAPK and MK2 have been shown to cause translocation of HuR from the nucleus to the cytoplasm, resulting in increased mRNA stability of a number of p38 MAPK regulated genes (Tran et al., 2003; Lin et al., 2006; Jin et al., 2007; Song et al., 2005). HuR has repeatedly been shown to stabilize transcripts containing the AUUUA sequence (Tran et al., 2003; Song et al., 2005). The human p75NTR transcript contains AUUUA sites located in the 3'UTR at positions 2946 and 3124, suggesting they may be involved in regulating p75NTR expression. Significantly, treatment with R-flurbiprofen or ibuprofen results in an increase in the cytoplasmic level of HuR and binding to the p75NTR transcript (Quann et al., 2007b) although this is not the sole mechanism responsible for increased p75NTR expression. Indeed, eIF4E is also phosphorylated by kinases downstream of p38 MAPK (Scheper & Proud, 2002). eIF4E has also been shown to control the nuclear export as well as translation of a subset of transcripts (Culjkovic et al., 2007). eIF4E has been linked to the control of mRNA stability by removal of the 5'-cap during mRNA degradation (von der Haar et al., 2004). R-flurbiprofen or ibuprofen treatment increases the level of phosphorylated eIF4E (Quann et al., 2007b) involved in translation initiation and this appears to occur at least partially through the p38 MAPK pathway since the increase in phosphorylation is substantially inhibited in the presence of p38 MAPK siRNA. Therefore, modulation of eIF4E activity is another mechanism by which the p38 MAPK pathway may control post-transcriptional events in response to R-flurbiprofen or ibuprofen treatment (Figure 6). R-flurbiprofen or ibuprofen stabilization of the p75NTR transcript allows levels of the p75NTR protein to accumulate (Quann et al., 1997a, Quann et al., 197b) that then exhibit biological activity to inhibit prostate cancer cell survival and induce apoptosis (Quann et al., 1997a) consistent with its tumor suppressor activity (Krygier & Djakiew, 2001a). Significantly, NSAID induction of p38 MAPK dependent p75NTR expression and tumor suppressor activity may represent a new pathway to medicinal drug design. Rather that inhibit kinase dependent proliferation pathways, pharmacological induction of the p75NTR tumor suppressor, as shown with NSAIDs (Figure 6), may represent an alternate approach to the targeting of cancer cell survival pathways. Presumably, target molecules that activate this pathway appear to occur immediately proximal to MKK6 and the p38 MAPK (Figure 5).

#### **8. NSAIDs induce expression of NAG-1 down stream of p75NTR via the p38 MAPK pathway**

R-flurbiprofen and ibuprofen treatment of prostate cancer cells induces expression of the NSAID activated gene-1 (Nag-1) protein, a divergent member of the transforming growth factor beta (TGF-β) family (Wynne & Djakiew, 2010). Moreover, a selective pharmacological inhibitor of p38 MAPK and p38 MAPK specific siRNA, both reduce Nag-1 induction following NSAID treatment. Hence, NSAID induced Nag-1 expression is regulated by the p38 MAPK pathway (Figure 6). Interestingly, p75NTR specific siRNA pretreatment abrogates Nag-1 induction by NSAIDs (Wynne & Djakiew, 2010) thereby demonstrating that Nag-1 is downstream of p75NTR induction (Figure 6). Functionally, decreased survival of NSAID treated cells is rescued by p75NTR specific siRNA (Quann et al., 2007a; Wynne & Djakiew, 2010) but not by Nag-1 siRNA. Transwell chamber and *in vitro* wound healing assays demonstrate decreased cell migration upon NSAID treatment (Wynne & Djakiew, 2010). Pre-treatment of prostate cancer cells with p75NTR and Nag-1 specific siRNA shows that

NSAID Induction of p75NTR in the Prostate:

**10. Acknowledgements** 

**11. References** 

A Suppressor of Growth and Cell Migration Via the p38 MAPK Pathway 37

that activate the caspase cascade leading to apoptosis. The metastasis suppressor activity of p75NTR is manifest as down-regulation of urokinase plasminogen activator and type IV collagenases (MMP-2, MMP-9) as well as increased expression of the tissue inhibitor of matrix metalloproteinase-1 (Nalbandian & Djakiew, 2006). The differentiation activity of p75NTR is manifest as up-regulated expression of CRABPI for localized sequestration of retinoids that are available to newly up-regulated RAR-β, RXR-α, and RXR-β proteins (Nalbandian et al., 2005). Moreover, certain aryl propionic acid type NSAIDs (profens) restore p75NTR protein levels that inhibit growth via a COX independent mechanism of action. Indeed, these profens induce hyperphosphorylation of p38 MAPK that initiates a signal transduction cascade leading to mRNA stability of the p75NTR transcript and increased protein levels (Quann et al., 2007a; Quann et al., 2007b). Profen NSAIDs also induce Nag-1 expression downstream of p75NTR (Wynne & Djakiew, 2010). Functionally, profen dependent induction of p75NTR inhibits growth and downstream Nag-1 inhibition of cell migration consistent with the tumor suppressor (Krygier & Djakiew, 2001a) and metastasis suppressor (Krygier & Djakiew, 2002) activities of p75NTR expression, respectively. Hence, these profen NSAIDs induce multiple activities of the p75NTR and downstream Nag-1 consistent with observations that chronic consumption of NSAIDs is associated with a reduced incidence of prostate cancer (Nelson & Harris, 2000).

The author thanks his former graduate students (Drs. Robert Delsite, Beth Pflug, Scott Krygier, Jeff Allen, Angele Nalbandian, Emily Quann, Shehla Wynne) and research associates (Drs. Rhaki Dalal, Ashia Tabassum, Fatima Khwaja, Lara El Touny) for their dedication and tireless efforts that contributed to much of this work. A significant amount of this research was supported by the National Institutes of Health (DK52626). Research support was also provided

Allen, J., Khwaja, F., Byers, S. & Djakiew, D. (2005). The p75NTR Mediates a Bifurcated Signal

Andreasen, P., Egelund, R. & Petersen, H. (2000). The plasminogen activation system in

Andrews, J., Djakiew, D., Krygier, S. & Andrews, P. (2002). Superior effectiveness of

Angelsen, A., Sandvik, A., Syversen, U., Stridsberg, M. & Waldum, H. (1998). NGF-beta, NE-

*Nephrology,* Vol.32, No.1 (February 1998), pp 7-13, PMID 9561567

Transduction Cascade through the NFB and JNK Pathways to Inhibit Cell Survival. *Experimental Cell Research,* Vol.304, No.1, (March 2005), pp69-80, PMID 15707575 Allen, J., Khwaja, F. & Djakiew, D. (2004). Gene therapy of prostate xenograft tumors with a

p75NTR lipoplex. *Anticancer Research,* Vol.24, No.5A, (September-October 2004),

tumor growth, invasion, and metastasis. *Cellular and Molecular Life Sciences,* Vol.57,

ibuprofen compared with other NSAIDs for reducing the survival of human prostate cancer cells. Cancer Chemotherapy and Pharmacology, Vol. 50, No.4,

cells and prostatic cancer cell lines. A study of neuroendocrine expression in the human prostatic cancer cell lines DU-145, PC-3, LNCaP, and TSU-pr1 following stimulation of the nerve growth factor-beta. *Scandinavian Journal of Urology and* 

by the Department of Defense (PC060409) to graduate students (EQ, SW).

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NSAID inhibition of cell migration is mediated by Nag-1 and p75NTR (Wynne & Djakiew, 2010). Additionally, prostate cancer cells stably expressing Nag-1 exhibit decreased migration relative to the parental cell line (Wynne & Djakiew, 2010), thereby independently confirming a role for this protein in reduced prostate cancer cell migration. Hence, it appears that NSAID induction of Nag-1 functions in the inhibition of cell migration, but not survival (Figure 6). Interestingly, NSAIDs have been linked to metastasis suppression in a variety of cancers including prostate cancer (Lloyd et al., 2003; Jin et al., 2010; Kamei et al., 2009). Hence, NSAID induction of Nag-1 via the p38 MAPK pathway may contribute to the metastasis suppressor activity of p75NTR (Krygier & Djakiew, 2002).

Fig. 6. Schematic diagram showing the biochemical pathway by which profen NSAIDs positively stimulate and the phytoestrogen, biochanin A, negatively inhibits phosphorylation of the p38 MAPK pathway leading to p75NTR expression with consequent tumor suppressor and metastasis suppressor activity.

#### **9. Summary and conclusions**

Paracrine regulated growth of the prostate is mediated, in part, by neurotrophin dependent interactions with the Trk family of receptors and the p75NTR (Djakiew, 2000). In the normal prostate neurotrophins stimulate proliferation via the Trk family of receptors and abrogate p75NTR apoptotic activity. However, p75NTR protein expression progressively declines in human prostate cancer (Perez et al., 1997; Pflug et al., 1995; Djakiew et al., 1996). Hence, in the absence of p75NTR inhibition of growth, prostate tumor cells respond to neurotrophins with a stiochiometry that favors Trk family dependent proliferation. Even though the p75NTR protein is no longer expressed in many prostate cancer cells the gene has remained intact (Krygier & Djakiew, 2001b). Indeed, reduced p75NTR protein in cancer cells occurs via loss of mRNA stability (Krygier & Djakiew, 2001b). Ectopic re-expression of p75NTR in cancer cells induces tumor suppressor (Krygier & Djakiew, 2001a), metastasis suppressor (Krygier & Djakiew, 2002) and differentiation phenotypes (Nalbandian et al., 2005). The tumor suppressor activity of p75NTR is manifest as a suppression of the NFB and JNK pathways, modification of cyclindependent holoenzyme complexes resulting in accumulations of cells in G1 and restriction of entry into the S1 phase of the cell cycle, and modification of mitochondrial effector proteins that activate the caspase cascade leading to apoptosis. The metastasis suppressor activity of p75NTR is manifest as down-regulation of urokinase plasminogen activator and type IV collagenases (MMP-2, MMP-9) as well as increased expression of the tissue inhibitor of matrix metalloproteinase-1 (Nalbandian & Djakiew, 2006). The differentiation activity of p75NTR is manifest as up-regulated expression of CRABPI for localized sequestration of retinoids that are available to newly up-regulated RAR-β, RXR-α, and RXR-β proteins (Nalbandian et al., 2005). Moreover, certain aryl propionic acid type NSAIDs (profens) restore p75NTR protein levels that inhibit growth via a COX independent mechanism of action. Indeed, these profens induce hyperphosphorylation of p38 MAPK that initiates a signal transduction cascade leading to mRNA stability of the p75NTR transcript and increased protein levels (Quann et al., 2007a; Quann et al., 2007b). Profen NSAIDs also induce Nag-1 expression downstream of p75NTR (Wynne & Djakiew, 2010). Functionally, profen dependent induction of p75NTR inhibits growth and downstream Nag-1 inhibition of cell migration consistent with the tumor suppressor (Krygier & Djakiew, 2001a) and metastasis suppressor (Krygier & Djakiew, 2002) activities of p75NTR expression, respectively. Hence, these profen NSAIDs induce multiple activities of the p75NTR and downstream Nag-1 consistent with observations that chronic consumption of NSAIDs is associated with a reduced incidence of prostate cancer (Nelson & Harris, 2000).

#### **10. Acknowledgements**

36 Prostate Cancer – Original Scientific Reports and Case Studies

NSAID inhibition of cell migration is mediated by Nag-1 and p75NTR (Wynne & Djakiew, 2010). Additionally, prostate cancer cells stably expressing Nag-1 exhibit decreased migration relative to the parental cell line (Wynne & Djakiew, 2010), thereby independently confirming a role for this protein in reduced prostate cancer cell migration. Hence, it appears that NSAID induction of Nag-1 functions in the inhibition of cell migration, but not survival (Figure 6). Interestingly, NSAIDs have been linked to metastasis suppression in a variety of cancers including prostate cancer (Lloyd et al., 2003; Jin et al., 2010; Kamei et al., 2009). Hence, NSAID induction of Nag-1 via the p38 MAPK pathway may contribute to the

Fig. 6. Schematic diagram showing the biochemical pathway by which profen NSAIDs

phosphorylation of the p38 MAPK pathway leading to p75NTR expression with consequent

Paracrine regulated growth of the prostate is mediated, in part, by neurotrophin dependent interactions with the Trk family of receptors and the p75NTR (Djakiew, 2000). In the normal prostate neurotrophins stimulate proliferation via the Trk family of receptors and abrogate p75NTR apoptotic activity. However, p75NTR protein expression progressively declines in human prostate cancer (Perez et al., 1997; Pflug et al., 1995; Djakiew et al., 1996). Hence, in the absence of p75NTR inhibition of growth, prostate tumor cells respond to neurotrophins with a stiochiometry that favors Trk family dependent proliferation. Even though the p75NTR protein is no longer expressed in many prostate cancer cells the gene has remained intact (Krygier & Djakiew, 2001b). Indeed, reduced p75NTR protein in cancer cells occurs via loss of mRNA stability (Krygier & Djakiew, 2001b). Ectopic re-expression of p75NTR in cancer cells induces tumor suppressor (Krygier & Djakiew, 2001a), metastasis suppressor (Krygier & Djakiew, 2002) and differentiation phenotypes (Nalbandian et al., 2005). The tumor suppressor activity of p75NTR is manifest as a suppression of the NFB and JNK pathways, modification of cyclindependent holoenzyme complexes resulting in accumulations of cells in G1 and restriction of entry into the S1 phase of the cell cycle, and modification of mitochondrial effector proteins

positively stimulate and the phytoestrogen, biochanin A, negatively inhibits

tumor suppressor and metastasis suppressor activity.

**9. Summary and conclusions** 

metastasis suppressor activity of p75NTR (Krygier & Djakiew, 2002).

The author thanks his former graduate students (Drs. Robert Delsite, Beth Pflug, Scott Krygier, Jeff Allen, Angele Nalbandian, Emily Quann, Shehla Wynne) and research associates (Drs. Rhaki Dalal, Ashia Tabassum, Fatima Khwaja, Lara El Touny) for their dedication and tireless efforts that contributed to much of this work. A significant amount of this research was supported by the National Institutes of Health (DK52626). Research support was also provided by the Department of Defense (PC060409) to graduate students (EQ, SW).

#### **11. References**


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**3** 

*China* 

**Polymorphism Analysis of TRAIL Gene and** 

Prostate cancer (PCa) is the most common male non-dermatological cancer in Europe and the United States of America (USA), and the sixth leading cause of cancer related-deaths, accounting for 14% (903,500) of total new diagnosed cancer cases and 6% (258,400) of whole cancer deaths in males in 2008 [1]. Because the increased use of screening techniques testing serum concentrations of prostate-specific antigen (PSA) has meant that PCa is more commonly diagnosed and can be detected at an earlier stage, the incidence rates recorded primarily in the developed countries, such as Oceania, Europe and North America, were hight. In contrast, males of African individuals in the Caribbean region have the highest PCa mortality rates in

Death rates for PCa have been decreasing in many developed countries, including Australia, Canada, USA, the United Kingdom, Italy and Norway in part due to the improved treatment with curative intent [4-6]. Recently, one European-based trial on the efficacy of PSA testing could reduce the rate of death from PCa by 20% [7]. In contrast to the trends of western countries, incidence and mortality rates are rising in several Asian and central/eastern-European countries, such as Japan, China and Poland, suggesting an increasingly westernized lifestyle in these regions [4, 5]. The underlying etiology of PCa remains poorly understood, with both genetic predisposition and environmental factors (diet, lifestyle, older age, race, family history and hormone) likely to play an important role [8-10]. Despite this strong evidence for a genetic component in PCa, little progress has been

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) is a novel member of the TNF super-family and was first identified by Wiley in 1995 [12]. TRAIL is mapped to the long arm of chromosome 3q26 in humans and is composed of five exons. It encodes 1.77 kb mRNA. Similar to FasL, TRAIL is also a type II membrane protein which induces apoptosis in a wide variety of cancer cells and spares normal cells [12]. TRAIL-induced apoptosis is a multi-step process: it binds to death receptor 4 (DR4) and DR5 cell surface receptors leading

the world, which is thought to reflect partly difference in genetic susceptibility [2, 3].

**1. Introduction** 

made to identify a major gene or genes [11].

\* Corresponding author: Ninghan Feng, MD, PhD.

**Correlation TRAIL Expression** 

Yuanyuan Mi1,2, Lijie Zhu2 and Ninghan Feng1,\* *1Department of Urology, The First Affiliated Hospital* 

**in Prostate Cancer** 

*of Nantong University, Wuxi* 

*of Nanjing Medical University, Nanjing* 

*2Department of Urology, Third Affiliated Hospital* 


### **Polymorphism Analysis of TRAIL Gene and Correlation TRAIL Expression in Prostate Cancer**

Yuanyuan Mi1,2, Lijie Zhu2 and Ninghan Feng1,\*

*1Department of Urology, The First Affiliated Hospital of Nanjing Medical University, Nanjing 2Department of Urology, Third Affiliated Hospital of Nantong University, Wuxi China* 

#### **1. Introduction**

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B., Kaplan, D., Hunter, T., Nikolics, K. & Parada, L. (1991). The neurotrophic factors brain-derived neurotrophic factor and neurotrophin-3 are ligands for the trkB tyrosine kinase receptor. *Cell,* Vol.65, No.5, (May 1991), pp 895-903, PMID 1645620 Tegeder, I., Pfeilschifter, J., & Geisslinger, G. (2001). Cyclooxygenase-independent actions of

cyclooxygenase inhibitors. *Federation of American Society of Experimental Biology* 

cancer. *The New England Journal of Medicine,* Vol.325, No.23, (December 1991), pp

receptor mRNAs by HuR is linked to its cytoplasmic accumulation induced by activated mitogen-activated protein kinase-activated protein kinase 2. *Molecular and Cellular Biology,* Vol.23, No.20, (October 2003), pp 7177-7188, PMID 14517288 von der Haar, T., Gross, J., Wagner, G. & McCarthy, J. (2004). The mRNA cap-binding

protein eIF4E in post-transcriptional gene expression. *Nature Structure and* 

growth factor (bFGF)-induced cell death is mediated through a caspase-dependent and p53-independent cell death receptor pathway. *Oncogene,* Vol.21, No.5, (January

mediated by Nag-1 induction via the p38 MAPK-p75NTR pathway. *Molecular Cancer* 

N., Hla, T. & Wada, S. (2000). Expression of cyclooxygenase-2 in prostate

Marzo, A. & Isaacs, W. (2001). Cyclooxygenase-2 is up-regulated in proliferative inflammatory atrophy of the prostate, but not in prostate carcinoma. *Cancer* 

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2002), pp 809-824, PMID 11850809

PMID 15337122

Prostate cancer (PCa) is the most common male non-dermatological cancer in Europe and the United States of America (USA), and the sixth leading cause of cancer related-deaths, accounting for 14% (903,500) of total new diagnosed cancer cases and 6% (258,400) of whole cancer deaths in males in 2008 [1]. Because the increased use of screening techniques testing serum concentrations of prostate-specific antigen (PSA) has meant that PCa is more commonly diagnosed and can be detected at an earlier stage, the incidence rates recorded primarily in the developed countries, such as Oceania, Europe and North America, were hight. In contrast, males of African individuals in the Caribbean region have the highest PCa mortality rates in the world, which is thought to reflect partly difference in genetic susceptibility [2, 3].

Death rates for PCa have been decreasing in many developed countries, including Australia, Canada, USA, the United Kingdom, Italy and Norway in part due to the improved treatment with curative intent [4-6]. Recently, one European-based trial on the efficacy of PSA testing could reduce the rate of death from PCa by 20% [7]. In contrast to the trends of western countries, incidence and mortality rates are rising in several Asian and central/eastern-European countries, such as Japan, China and Poland, suggesting an increasingly westernized lifestyle in these regions [4, 5]. The underlying etiology of PCa remains poorly understood, with both genetic predisposition and environmental factors (diet, lifestyle, older age, race, family history and hormone) likely to play an important role [8-10]. Despite this strong evidence for a genetic component in PCa, little progress has been made to identify a major gene or genes [11].

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) is a novel member of the TNF super-family and was first identified by Wiley in 1995 [12]. TRAIL is mapped to the long arm of chromosome 3q26 in humans and is composed of five exons. It encodes 1.77 kb mRNA. Similar to FasL, TRAIL is also a type II membrane protein which induces apoptosis in a wide variety of cancer cells and spares normal cells [12]. TRAIL-induced apoptosis is a multi-step process: it binds to death receptor 4 (DR4) and DR5 cell surface receptors leading

<sup>\*</sup> Corresponding author: Ninghan Feng, MD, PhD.

Polymorphism Analysis of TRAIL Gene and Correlation TRAIL Expression in Prostate Cancer 47

signaling involves the mitochondrial pathway via caspase-8-mediated cleavage of Bid to t-Bid. Proapoptotic signaling through the intrinsic pathway is further regulated by pro apoptotic and anti apoptotic members of the Bcl-2 family. Receptor tyrosine kinase (RTK) signaling and chemotherapy or radiotherapy can further modulate the intrinsic proapoptotic pathway through targeting Bcl-2 family members. Under certain circumstances, DR4 or DR5 signaling can promote alternative signaling pathways such as JNK, MAPK or NFkB, which may require recruitment of RIP1 and TRAF2 or TRAFs5 to form secondary signaling complexes. Depicted in blue are inhibitors that may enhance proapoptotic signaling by PARAs by targeting mechanisms of resistance in tumor cells. (This picture was cited from Yang et al. [50] Current

Several single nucleotide polymorphisms (SNPs) present along the TRAIL gene located in the 3q26 region have been found in both healthy and disease individuals, including four SNPs in the 5' regulatory region [16], two SNPs within exons, and five SNPs in the 3' untranslated regions[17-18]. TRAIL gene polymorphisms were also identified in patients

Recently, a SNP of -716A>G polymorphism (rs12488654) in the promoter region of TRAIL gene has been found to be associated with breast cancer with functional implications both in vitro and vivo studies [22]. To date, there have been no data about the association between this polymorphism and PCa, so we first explored the role of the TRAIL A>G polymorphism in PCa patients in southern Chinese Han descent. Moreover, we detected the serum levels of TRAIL expression with different genotypes in cases to characterize the functional

One hundred and eighty-seven PCa patients were newly diagnosed between November 2009 and May 2010 in the First Affiliated Hospital of Nanjing Medical University (Jiangsu Province Hospital) in Nanjing, China. All PCa cases were between 51 and 94 years of age and were diagnosed with the disease within the last one year; all controls were between 47 and 96 years of age. All cases were diagnosed with PCa through needle biopsy (ultrasoundguided transrectal needle biopsy of prostate, 13-fold biopsy) or operation (radical prostatectomy and transurethral resection of the prostate). All the patients were southern Chinese Han descent. The control group (n = 237) was age-matched and the subjects were healthy checkup examinees without cancer history and were collected in the same period. Controls were excluded if they ever had abnormal appearance of pathology, abnormal prostate-specific antigen test (i.e., ≥ 4 ng/ml), abnormal digital rectal examination, other previous cancer diagnosis, symptom of any prostate disease or abnormal appearance of other auxiliary examination including computed tomography urography (CTU), magnetic resonance urography (MRU), positron emission tomographic (PET), transrectal

After informed consent was obtained, 2 ml peripheral blood sample was collected and each subject was asked to finish a questionnaire including age, weight, height, race, tobacco use, alcohol use, family history of cancer and so on. In our present research, smoking more than five cigarettes per day for more than 5 years was defined as smoking; drinking habit was defined as drinking at least three times per week and lasting more than 10 years; family history of cancer was defined as cancer in first-degree relatives (parents, siblings, or

Opinion in Cell Biology. 2010)

**2. Materials and methods** 

ultrasonography and so on.

**2.1 Study population** 

with multiple sclerosis [19, 20] and fatty liver disease [21].

consequences of TRAIL -716 A>G polymorphism.

to the formation of the death inducing signaling complex (DISC) that recruits caspase-8 via the adaptor protein Fas-associated with death domain protein (FADD). The formation of DISC and recruitment of caspase-8 leads to proteolytic activation of caspase-3 and caspase-7 leading to DNA fragmentation and apoptosis [13-15] (Fig. 1).

Fig. 1. TRAIL pathway for cancer therapy. DR4 and DR5 activation by PARAs (either trimeric rhApo2L/TRAIL or agonistic DR4 or DR5-specific antibodies) or Apo2L/TRAIL expressed by innate immune cells. FADD is recruited to DR4 or DR5 located within lipid raft containing regions of the membrane, which promotes receptor clustering and autocatalytic processing of the apoptosis initiating proteases caspase-8 or caspase-10 to form the active DISC. Caspase-8 can be polyubiquitylated at the DISC by a cullin-3/Rbx1-based E3 ubiquitin ligase, which facilitates caspase-8 activation. This process is negatively regulated by the de-ubiquitinating enzyme, A20. The signaling adaptor p62 can bind to ubiquitilated caspase-8 and translocate it to ubiquitin-rich foci, which may also enhance its activity. In many cancer cells, proapoptotic

signaling involves the mitochondrial pathway via caspase-8-mediated cleavage of Bid to t-Bid. Proapoptotic signaling through the intrinsic pathway is further regulated by pro apoptotic and anti apoptotic members of the Bcl-2 family. Receptor tyrosine kinase (RTK) signaling and chemotherapy or radiotherapy can further modulate the intrinsic proapoptotic pathway through targeting Bcl-2 family members. Under certain circumstances, DR4 or DR5 signaling can promote alternative signaling pathways such as JNK, MAPK or NFkB, which may require recruitment of RIP1 and TRAF2 or TRAFs5 to form secondary signaling complexes. Depicted in blue are inhibitors that may enhance proapoptotic signaling by PARAs by targeting mechanisms of resistance in tumor cells. (This picture was cited from Yang et al. [50] Current Opinion in Cell Biology. 2010)

Several single nucleotide polymorphisms (SNPs) present along the TRAIL gene located in the 3q26 region have been found in both healthy and disease individuals, including four SNPs in the 5' regulatory region [16], two SNPs within exons, and five SNPs in the 3' untranslated regions[17-18]. TRAIL gene polymorphisms were also identified in patients with multiple sclerosis [19, 20] and fatty liver disease [21].

Recently, a SNP of -716A>G polymorphism (rs12488654) in the promoter region of TRAIL gene has been found to be associated with breast cancer with functional implications both in vitro and vivo studies [22]. To date, there have been no data about the association between this polymorphism and PCa, so we first explored the role of the TRAIL A>G polymorphism in PCa patients in southern Chinese Han descent. Moreover, we detected the serum levels of TRAIL expression with different genotypes in cases to characterize the functional consequences of TRAIL -716 A>G polymorphism.

#### **2. Materials and methods**

#### **2.1 Study population**

46 Prostate Cancer – Original Scientific Reports and Case Studies

to the formation of the death inducing signaling complex (DISC) that recruits caspase-8 via the adaptor protein Fas-associated with death domain protein (FADD). The formation of DISC and recruitment of caspase-8 leads to proteolytic activation of caspase-3 and caspase-7

Fig. 1. TRAIL pathway for cancer therapy. DR4 and DR5 activation by PARAs (either trimeric rhApo2L/TRAIL or agonistic DR4 or DR5-specific antibodies) or Apo2L/TRAIL expressed by innate immune cells. FADD is recruited to DR4 or DR5 located within lipid raft containing regions of the membrane, which promotes receptor clustering and autocatalytic processing of the apoptosis initiating proteases caspase-8 or caspase-10 to form the active DISC. Caspase-8 can be polyubiquitylated at the DISC by a cullin-3/Rbx1-based E3 ubiquitin ligase, which facilitates caspase-8 activation. This process is negatively regulated by the de-ubiquitinating enzyme, A20. The signaling adaptor p62 can bind to ubiquitilated caspase-8 and translocate it to ubiquitin-rich foci, which may also enhance its activity. In many cancer cells, proapoptotic

leading to DNA fragmentation and apoptosis [13-15] (Fig. 1).

One hundred and eighty-seven PCa patients were newly diagnosed between November 2009 and May 2010 in the First Affiliated Hospital of Nanjing Medical University (Jiangsu Province Hospital) in Nanjing, China. All PCa cases were between 51 and 94 years of age and were diagnosed with the disease within the last one year; all controls were between 47 and 96 years of age. All cases were diagnosed with PCa through needle biopsy (ultrasoundguided transrectal needle biopsy of prostate, 13-fold biopsy) or operation (radical prostatectomy and transurethral resection of the prostate). All the patients were southern Chinese Han descent. The control group (n = 237) was age-matched and the subjects were healthy checkup examinees without cancer history and were collected in the same period. Controls were excluded if they ever had abnormal appearance of pathology, abnormal prostate-specific antigen test (i.e., ≥ 4 ng/ml), abnormal digital rectal examination, other previous cancer diagnosis, symptom of any prostate disease or abnormal appearance of other auxiliary examination including computed tomography urography (CTU), magnetic resonance urography (MRU), positron emission tomographic (PET), transrectal ultrasonography and so on.

After informed consent was obtained, 2 ml peripheral blood sample was collected and each subject was asked to finish a questionnaire including age, weight, height, race, tobacco use, alcohol use, family history of cancer and so on. In our present research, smoking more than five cigarettes per day for more than 5 years was defined as smoking; drinking habit was defined as drinking at least three times per week and lasting more than 10 years; family history of cancer was defined as cancer in first-degree relatives (parents, siblings, or

Polymorphism Analysis of TRAIL Gene and Correlation TRAIL Expression in Prostate Cancer 49

One hundred and eighty-seven patients and 237 cancer-free controls were enrolled in our study. The distribution of relevant demographic and clinical characteristics is presented in Table 1. Baseline characteristics were similar between cases and controls, except that the frequency of relatives with cancer from the case group was higher, compared to nonrelatives (27.27% vs. 15.61%, *P* = 0.003); there were more subjects who had larger body mass index (>23 kg/m2) among the cases than among the controls (60.43% vs. 50.21%, *P* = 0.036), the frequency of ever alcohol drinking in cases was higher than in controls (34.22% vs. 20.68%, *P* = 0.002) and the mean ± SD PSA levels of PCa patients and control subjects were

Characteristics Csaes (n=187) Controls (n=237) *<sup>P</sup>*-Value n % n %

Mean ± SD 80.45 ±262.25 2.14 ±1.42 <0.001

Age (year) 0.687 ≤70 55 29.41 74 31.22 >70 132 70.59 163 68.78 BMI (kg/m2) 0.036 ≤23 74 39.57 118 49.79 >23 113 60.43 119 50.21 Cigarette smoking 0.839 Never 81 43.32 105 44.30 Ever 106 56.68 132 55.70 Alcohol drinking 0.002 Never 123 65.78 188 79.32 Ever 64 34.22 49 20.68 Family history of cancers 0.003 No 136 72.73 200 84.39 Yes 51 27.27 37 15.61

**3. Results** 

PSA(ng/ml)

Clinical stage

Gleason score

BMI: body mass index

**3.1 Characteristics of the study population** 

80.45 ± 262.25 and 2.14 ± 1.42 ng/ml, respectively (*P* < 0.001).

Localized 86 46.00 Advanced 101 54.00

<7 50 26.74 = 7 70 37.43 >7 67 35.82

Table 1. Demographic characteristic of PCa cases and controls

**3.2 Genotype distributions of TRAIL -716 A>G polymorphism and risk of PCa** 

The distribution of TRAIL -716 A>G in the control group was 21.10% for AA homozygote, 51.48% for AG heterozygote, 27.42% for GG homozygote, and was in Hardy–Weinberg equilibrium (χ2 = 0.268, *P* = 0.604). As shown in Table 2, the TRAIL -716 A>G polymorphism

children); disease stage was determined by pathologic findings, pelvic computed tomography, magnetic resonance image and radio-nucleotide bone scans, the tumor stage was determined using tumor-node-metastasis (TNM) classification and graded according to WHO guidelines; pathologic grade was recorded as the Gleason score.

#### **2.2 Genotyping**

Polymorphisms were analyzed by polymorphism chain reaction and ligase detection reaction (PCR-LDR). Each PCR reaction was done in a total volume of 15 ul, which contains 1 ul genomic DNA, 2.5 pmol of each promer, 10× buffer 1.5 ul, MgCl2 1.5 ul, 0.3 ul of dNTP (MBI, Inc.), 0.25 ul of Taq DNA Polymerase (MBI, Inc.) and ddH2O 9.95 ul. PCR was subjected to 35 thermal cycles at 94°C 15 sec, 56°C for 15 sec, and 72°C for 60 sec conducted on the ABI 9600 (ABI, Inc.). Primers were 5'-TGACGACTTCTTCCTCTTTGC-3' (sense) 5'- GATAGTGACAGCGAGACATTG-3' (antisense). The probes for LDR were: 5'-P-GTAGGAAGTAGTTGACACACTCAGATTT-FAM-3' with common phosphorylated 5'-end and 6-carboxy X-uorescein (FAM) labeled 3'-end, the A-specific probe 5' TTTTCATGCCTGTGTGTTAGGCTGCACAA-3', the G-specific probe 5' tttttTTCATGCCTGTGTGTTAGGCTGCACAG-3'. For each PCR product, the ligation reaction was performed in a final volume of 10 ul, which contains 3 ul PCR product, 10×Taq DNA ligase buffer 1 ul, 5 U of Taq DNA ligase (NEB, Inc.,), 0.1 pmol of each probe, and ddH2O 5.575 ul. The LDR parameters were as follows: 25 thermal cycles at 94 °C for 30 sec and 60°C for 30 min. The LDR reaction products were analyzed on ABI 3730 DNA Sequencer (ABI, Inc.,). To confirm the accuracy of PCR-LDR genotyping method, direct DNA sequencing of randomly selected PCR products was performed. The proportion of the sequencing samples were about 5%, the results of the PCR-LDR genotyping showed 100% concordance to direct DNA sequencing of the randomly selected PCR products.

#### **2.3 Enzyme-linked immunosorbent assay (ELISA)**

Blood was collected in standard cubes without anticoagulant and was immediately centrifuged for 20 min, at 3,000 rpm. Serums were stored at -808°C until serum TRAIL levels were measured by ELISA kit (R&D Systems, Inc.). The optical density was determined by measuring the absorbance at 450 nm. The absorbance was correlated against a standard curve.

#### **2.4 Statistical analysis**

Hardy–Weinberg equilibrium (HWE) was tested among controls using the Pearson chisquare test. Differences in the distributions of demographic characteristics, selected variables and frequencies of genotypes of TRAIL -716 A>G polymorphism between the cases and controls were evaluated by using the student's t-test (for continuous variables) or chi-square (χ2) test (for categorical variables). The odds ratios (OR) and 95% confidence internals (CI) were calculated by logistic regression analysis to quantify the association between TRAIL -716 A>G polymorphism and risk of PCa with the adjustment for potential covariates (age, BMI, cigarette smoking, alcohol drinking and family history of cancers). The correlation between the serum TRAIL levels and genotypes of TRAIL -716 A>G polymorphism were evaluated by oneway ANOVA. A *P*-value < 0.05 was considered statistically significant and all statistical tests were two sided. All statistical analyses were performed with Statistics Analysis System software (Version 9.1.3; SAS Institute, Inc., Cary, NC).

### **3. Results**

48 Prostate Cancer – Original Scientific Reports and Case Studies

children); disease stage was determined by pathologic findings, pelvic computed tomography, magnetic resonance image and radio-nucleotide bone scans, the tumor stage was determined using tumor-node-metastasis (TNM) classification and graded according to

Polymorphisms were analyzed by polymorphism chain reaction and ligase detection reaction (PCR-LDR). Each PCR reaction was done in a total volume of 15 ul, which contains 1 ul genomic DNA, 2.5 pmol of each promer, 10× buffer 1.5 ul, MgCl2 1.5 ul, 0.3 ul of dNTP (MBI, Inc.), 0.25 ul of Taq DNA Polymerase (MBI, Inc.) and ddH2O 9.95 ul. PCR was subjected to 35 thermal cycles at 94°C 15 sec, 56°C for 15 sec, and 72°C for 60 sec conducted on the ABI 9600 (ABI, Inc.). Primers were 5'-TGACGACTTCTTCCTCTTTGC-3' (sense) 5'- GATAGTGACAGCGAGACATTG-3' (antisense). The probes for LDR were: 5'-P-GTAGGAAGTAGTTGACACACTCAGATTT-FAM-3' with common phosphorylated 5'-end and 6-carboxy X-uorescein (FAM) labeled 3'-end, the A-specific probe 5' TTTTCATGCCTGTGTGTTAGGCTGCACAA-3', the G-specific probe 5' tttttTTCATGCCTGTGTGTTAGGCTGCACAG-3'. For each PCR product, the ligation reaction was performed in a final volume of 10 ul, which contains 3 ul PCR product, 10×Taq DNA ligase buffer 1 ul, 5 U of Taq DNA ligase (NEB, Inc.,), 0.1 pmol of each probe, and ddH2O 5.575 ul. The LDR parameters were as follows: 25 thermal cycles at 94 °C for 30 sec and 60°C for 30 min. The LDR reaction products were analyzed on ABI 3730 DNA Sequencer (ABI, Inc.,). To confirm the accuracy of PCR-LDR genotyping method, direct DNA sequencing of randomly selected PCR products was performed. The proportion of the sequencing samples were about 5%, the results of the PCR-LDR genotyping showed 100%

concordance to direct DNA sequencing of the randomly selected PCR products.

Blood was collected in standard cubes without anticoagulant and was immediately centrifuged for 20 min, at 3,000 rpm. Serums were stored at -808°C until serum TRAIL levels were measured by ELISA kit (R&D Systems, Inc.). The optical density was determined by measuring the absorbance at 450 nm. The absorbance was correlated against a standard

Hardy–Weinberg equilibrium (HWE) was tested among controls using the Pearson chisquare test. Differences in the distributions of demographic characteristics, selected variables and frequencies of genotypes of TRAIL -716 A>G polymorphism between the cases and controls were evaluated by using the student's t-test (for continuous variables) or chi-square (χ2) test (for categorical variables). The odds ratios (OR) and 95% confidence internals (CI) were calculated by logistic regression analysis to quantify the association between TRAIL -716 A>G polymorphism and risk of PCa with the adjustment for potential covariates (age, BMI, cigarette smoking, alcohol drinking and family history of cancers). The correlation between the serum TRAIL levels and genotypes of TRAIL -716 A>G polymorphism were evaluated by oneway ANOVA. A *P*-value < 0.05 was considered statistically significant and all statistical tests were two sided. All statistical analyses were performed with Statistics Analysis System

**2.3 Enzyme-linked immunosorbent assay (ELISA)** 

software (Version 9.1.3; SAS Institute, Inc., Cary, NC).

WHO guidelines; pathologic grade was recorded as the Gleason score.

**2.2 Genotyping** 

curve.

**2.4 Statistical analysis** 

#### **3.1 Characteristics of the study population**

One hundred and eighty-seven patients and 237 cancer-free controls were enrolled in our study. The distribution of relevant demographic and clinical characteristics is presented in Table 1. Baseline characteristics were similar between cases and controls, except that the frequency of relatives with cancer from the case group was higher, compared to nonrelatives (27.27% vs. 15.61%, *P* = 0.003); there were more subjects who had larger body mass index (>23 kg/m2) among the cases than among the controls (60.43% vs. 50.21%, *P* = 0.036), the frequency of ever alcohol drinking in cases was higher than in controls (34.22% vs. 20.68%, *P* = 0.002) and the mean ± SD PSA levels of PCa patients and control subjects were 80.45 ± 262.25 and 2.14 ± 1.42 ng/ml, respectively (*P* < 0.001).


BMI: body mass index

Table 1. Demographic characteristic of PCa cases and controls

#### **3.2 Genotype distributions of TRAIL -716 A>G polymorphism and risk of PCa**

The distribution of TRAIL -716 A>G in the control group was 21.10% for AA homozygote, 51.48% for AG heterozygote, 27.42% for GG homozygote, and was in Hardy–Weinberg equilibrium (χ2 = 0.268, *P* = 0.604). As shown in Table 2, the TRAIL -716 A>G polymorphism

Polymorphism Analysis of TRAIL Gene and Correlation TRAIL Expression in Prostate Cancer 51

In addition, as show in Table 4, the association between TRAIL -716 A>G polymorphism and PCa did not vary by cigarette smoking and alcohol drinking. However, the association appeared stronger in subgroups of BMI >23kg/m2 (OR = 0.58, 95%CI = 0.31-0.89), age ≤70 years (OR = 0.32, 95%CI = 0.12-0.87) and no family history of cancers (OR = 0.86, 95%CI =

Genotypes(case/control)

AA genotype AG/GG genotype n % n %

> 143 /187

12.16 42/65 76.36/

22.88 62/91 83.78/

19.33 81/96 71.68/

24.76 61/79 75.31/

 82/ 108

 94/ 149

22.45 49/38 76.56/

 102/ 156

16.22 41/31 80.39/

 101/ 122

76.47/

76.52/

77.36/

76.42/

75.00/

21.10

25.15

18.18

20.74

22.00

Table 4. Association and stratification between TRAIL-716A/G and PCa risk.

aTwo-sided χ2 test for the distributions of genotypes frequencies between the cases and controls. bOdd ratios (ORs) were obtained from a logistic regression model with adjusting for age, BMI, cigarette smoking, alcohol drinking, family history of cancers; 95%CI, 95% confidence interval. BMI: body mass

**3.4 Association of TRAIL -716 A>G polymorphism with expression levels of TRAIL**  We collected 83 tumor serum samples obtained from in present study with different genotypes of the TRAIL -716 A>G polymorphism, and the distribution of the AA, AG, and

*P*-valuea

78.90 0.577 0.87

87.84 0.026 0.32

74.85 0.575 1.17

77.12 0.165 1.78

80.67 0.042 0.58

75.24 0.967 0.99

81.82 0.535 0.81

79.26 0.513 0.83

77.55 0.944 0.97

78.00 0.034 0.86

83.78 0.972 1.02

Adjusted OR(95%CI) b

(0.54-1.41)

(0.12-0.87)

(0.67-2.04)

(0.79-3.99)

(0.31-0.89)

(0.49-1.97)

(0.41-1.58)

(0.48-1.45)

(0.38-2.45)

(0.51-0.96)

(0.31-3.32)

0.51-0.96).

Variables <sup>N</sup>

Age(years)

BMI (kg/m2)

Cigarette smoking

Alcohol drinking

Family history of cancers

index.

(case/control)

Total 187/237 44/50 23.53/

≤ 70 55/74 13/9 23.64/

>70 132/163 31/41 23.48/

≤ 23 74/118 12/27 16.22/

>23 113/119 32/23 28.32/

Never 81/105 20/26 24.69/

Ever 106/132 24/24 22.64/

Never 123/188 29/39 23.58/

Ever 64/49 15/11 23.44/

No 136/200 34/44 25.00/

Yes 51/37 10/6 19.61/

was not associated with total PCa. After adjusting for potential covariates (age, BMI, cigarette smoking, alcohol drinking, family history of cancers), compared with AA homozygote, subjects carrying GG homozygote did not have any association between cases and controls (OR = 0.94, 95%CI = 0.69-1.27, *P* = 0.397). In addition, no association was also found between subjects carrying AG/GG genotypes and AA homozygote (OR = 0.87, 95%CI = 0.54-1.41, *P* = 0.577).


aThe genotype frequencies among the control subjects were in agreement with the Hardy-Weinberg

equilibrium (χ2 = 0.268, P = 0.604). bTwo-sided χ2 test for the distributions of genotypes frequencies between the cases and controls. cOdd ratios (ORs) were obtained from a logistic regression model with adjusting for age, BMI, cigarette smoking, alcohol drinking, family history of cancers; 95%CI, 95% confidence interval.

Table 2. Genotypes in patients with PCa and controls

#### **3.3 Stratified analysis**

The association between genotypes and PCa risk stratified by disease stage (Localized: T1- 2N0M0; Advanced: T3-4NXMX or TXN1MX or TXNXM1), pathologic grade (Gleason score < 7, = 7 and >7) and serum PSA level (≤ 20 and >20) is shown in Table 3. These associations were in the same direction for advanced, higher grade disease and PSA level but were not statistically significant.


aTwo-sided χ2 test for the distributions of genotypes frequencies between the cases and controls. bOdd ratios (ORs) were obtained from a logistic regression model with adjusting for age, BMI, cigarette smoking, alcohol drinking, family history of cancers; 95%CI, 95% confidence interval. cLocalized: T1-2N0M0; Advanced: T3-4NXMX or TXN1MX or TXNXM1 [according to the international

tumor-node-metastasis (TNM) staging system for PCa].

Table 3. TRAIL-716A/G and clinico-pathological characteristics in patients with PCa

was not associated with total PCa. After adjusting for potential covariates (age, BMI, cigarette smoking, alcohol drinking, family history of cancers), compared with AA homozygote, subjects carrying GG homozygote did not have any association between cases and controls (OR = 0.94, 95%CI = 0.69-1.27, *P* = 0.397). In addition, no association was also found between subjects carrying AG/GG genotypes and AA homozygote (OR = 0.87, 95%CI

Genotype PCa, No. (%) Controlsa, No. (%) *P*-valueb Adjusted OR(95%CI)c

AA 44(23.53) 50(21.10) 1.00(reference) AG 98(52.41) 122(51.48) 0.712 0.89(0.54-1.47) GG 45(24.06) 65(27.42) 0.397 0.94(0.69-1.27) AA 44(23.53) 50(21.10) 1.00(reference) AG+GG 143(76.47) 187(78.90) 0.577 0.87(0.54-1.41) aThe genotype frequencies among the control subjects were in agreement with the Hardy-Weinberg

equilibrium (χ2 = 0.268, P = 0.604). bTwo-sided χ2 test for the distributions of genotypes frequencies between the cases and controls. cOdd ratios (ORs) were obtained from a logistic regression model with adjusting for age, BMI, cigarette

The association between genotypes and PCa risk stratified by disease stage (Localized: T1- 2N0M0; Advanced: T3-4NXMX or TXN1MX or TXNXM1), pathologic grade (Gleason score < 7, = 7 and >7) and serum PSA level (≤ 20 and >20) is shown in Table 3. These associations were in the same direction for advanced, higher grade disease and PSA level but were not

Variables TRAIL-716A/G *P*-valuea Adjusted OR (95% CI)b

Control 50(21.10) 187(78.90) 1.00(reference)

Localized 15(17.65) 70(82.35) 0.644 1.17(0.60-2.27) Advanced 29(28.43) 73(71.57) 0.158 0.67(0.38-1.17)

<7 6(12.00) 44(88.00) 0.117 2.14(0.83-5.53) = 7 19(27.14) 51(72.86) 0.226 0.68(0.36-1.27) >7 19(28.36) 48(71.64) 0.117 0.59(0.31-1.14)

≤ 20 16(17.58) 75(82.42) 0.626 1.17(0.62-2.23) >20 28(28.87) 69(71.13) 0.121 0.64(0.36-1.13)

aTwo-sided χ2 test for the distributions of genotypes frequencies between the cases and controls. bOdd ratios (ORs) were obtained from a logistic regression model with adjusting for age, BMI, cigarette

smoking, alcohol drinking, family history of cancers; 95%CI, 95% confidence interval. cLocalized: T1-2N0M0; Advanced: T3-4NXMX or TXN1MX or TXNXM1 [according to the international

Table 3. TRAIL-716A/G and clinico-pathological characteristics in patients with PCa

smoking, alcohol drinking, family history of cancers; 95%CI, 95% confidence interval.

AA,No.(%) AG/GG,No.(%)

= 0.54-1.41, *P* = 0.577).

**3.3 Stratified analysis** 

statistically significant.

Clinical stagec

Gleason score

PSA

Total 187 237

Table 2. Genotypes in patients with PCa and controls

tumor-node-metastasis (TNM) staging system for PCa].

In addition, as show in Table 4, the association between TRAIL -716 A>G polymorphism and PCa did not vary by cigarette smoking and alcohol drinking. However, the association appeared stronger in subgroups of BMI >23kg/m2 (OR = 0.58, 95%CI = 0.31-0.89), age ≤70 years (OR = 0.32, 95%CI = 0.12-0.87) and no family history of cancers (OR = 0.86, 95%CI = 0.51-0.96).


aTwo-sided χ2 test for the distributions of genotypes frequencies between the cases and controls. bOdd ratios (ORs) were obtained from a logistic regression model with adjusting for age, BMI, cigarette smoking, alcohol drinking, family history of cancers; 95%CI, 95% confidence interval. BMI: body mass index.

Table 4. Association and stratification between TRAIL-716A/G and PCa risk.

#### **3.4 Association of TRAIL -716 A>G polymorphism with expression levels of TRAIL**

We collected 83 tumor serum samples obtained from in present study with different genotypes of the TRAIL -716 A>G polymorphism, and the distribution of the AA, AG, and

Polymorphism Analysis of TRAIL Gene and Correlation TRAIL Expression in Prostate Cancer 53

SNP in a regulatory region may have an influence on gene transcription, a SNP located in a RNA splice site may affect RNA splicing, a SNP in the 3'-untranslated region of a gene may have an effect on mRNA stability, and a SNP in the coding region may result in an amino acid substitution in the encoded protein. It is thought that SNPs contribute to interindividual variability in susceptibility to common diseases such as cancer [35, 36]. So far, some published meta-analyses have confirmed that a number of SNPs are associated with increased or decreased PCa risk in different races, such as A49T in steroid-5-alpha-reductase, alpha polypeptide 2 (SRD5A2) gene [37], Gly388Arg in fibroblast growth factor receptor 4 (FGFR4) gene [38], -160C/A in E-cadherin (CDH1) gene [39], Val16Ala in manganese superoxide dismutase (MnSOD) gene [40], C677T in

Several studies have investigated the possible role of anti-tumor gene polymorphisms and the prevalence of PCa. This impairment of host factors might result in susceptibility or resistance to tumor progression. The transcription factor Sp3 (stimulatory protein 3) exhibits a similar DNA binding affinity for Sp1 consensus sequence [42-44] and represses the Sp1 mediated trans-activation of promoters with two or more Sp1 sites [45-47]. TRAIL has two Sp1 consensus sequences in the basal promoter [48, 49]. AA genotype at -716 in TRAIL promoter with additional Sp1 consensus sequence can decrease TRAIL expression due to the repression caused by binding of Sp3, whereas, GG genotype background at the same position can increase TRAIL expression because of the lower probability of Sp3 driven repression. To date, only one study [22] showed the association between TRAIL -716 A>G polymorphism and cancer risk: individuals with -716 GG genotype were at a greater risk of developing breast cancer, in addition, G allele resulted in a higher expression than the A allele to regulate the expression of TRAIL in four different cancer cell lines (HeLa, MCF-

To the best of our knowledge this is the first study investigating the genetic association of polymorphism of the -716 site in TRAIL gene with PCa and the expression of TRAIL with different genotypes in serum of cases in southern Chinese Han descent. No statistically significant association was observed between TRAIL -716 A>G polymorphism and PCa. Moreover, when stratifying the case group by clinical characteristics, the present study also did not find any association among PSA, Gleason and clinical stage. There must be some factors that would contribute to this discrepancy. First, TRAIL -716 A>G polymorphism might play a different role in different cancers. Second, multiple genes and environmental factors may lead to cancer formation. Third, race may be related to cancer. Either through common risk factors or other genes in linkage disequilibrium with TRAIL suggests that a possible role of ethnic differences is in genetic backgrounds and the

Furthermore, we found that the decreased risk associated with the AG/GG genotypes was more pronounced in: subjects with age ≤70 years (OR = 0.32, 95%CI = 0.12-0.87) and no family history of cancers (OR = 0.86, 95%CI = 0.51-0.96). It confirmed the concept that younger age and no family history of cancers might be protective factors for PCa. In addition, we found that the OR for AG/GG genotypes was 0.58(95%CI = 0.31-0.89) among subjects with BMI >23kg/m2. This finding may reflect that PCa formation may be subject to a variety of environmental and genetic factors. In these subgroups, other high level of

Except for above associated results, we detected the expression of TRAIL in the serums of the cases. We found that the protective genotypes AG/GG were associated with higher

genetic susceptibilities or other unknown risk factors may influence our results.

5,10-methylenetetrahydrofolate reductase (MTHFR) gene [41].

7, HepG2, HT1080).

environment they lived in.

GG genotypes was 27 (32.53%), 44 (53.01%) and 12 (14.46%), respectively. Moreover, serum TRAIL levels in PCa patients with AG/GG genotypes were significantly higher than those with AA genotypes (901.18 ± 189.58 µg/L vs. 819.13 ± 111.00 µg/L, *P* = 0.041; Fig. 2)

Fig. 2. Analysis of serum TRAIL levels in three groups of PCa cases with mean values (horizontal lines, mean values). \* *P* = 0.041 compared with the AG/GG and AA genotypes.

#### **4. Discussion**

Recently, Kuribayashi et al. [23] indicated a direct regulation of TRAIL gene by p53 protein. Moreover, early growth response protein (EGR) [24], interferon regulatory factor 1 (IRF1) [25], NF-kB [26], SP1 [27] and PU1 [28] have been implicated in the regulation of TRAIL. TRAIL is present in various tissues, particular in the prostate, spleen and lung.

TRAIL binds to two different types of receptors: death receptors and decoy receptors. TRAIL can also bind to osteoprotegerin (OPG) (a soluble inhibitor of receptor activator of NF-kB ligand) at low affinity. To date, four human receptors specific for TRAIL have been recognized: the death receptors TRAIL-R1 (also know as DR4), TRAIL-R2 (also known as DR5), the putative decoy receptors TRAIL-R3 (DcR1) and TRAIL-R4 (DcR2). TRAIL-R1 (DR4) is expressed at very low levels in most human tissues including the spleen, thymus, liver, peripheral blood leukocytes, activated T cells, small intestine and some tumor cell lines. TRAIL-R2 (DR5) is ubiquitiously distributed both in normal and tumor cell lines but is more abundant in spleen, peripheral blood leukocytes, activated lymphocytes and hepatocyes [29-31].

TRAIL has attained the centre stage in anti-tumor drug discovery because of its efficacy in killing tumor cells without lethal toxicity in pre-clinical models apart from the inherent property to activate both the extrinsic and intrinsic apoptotic pathways [32-34].

Single nucleotide polymorphisms (SNPs) are the most abundant form of genetic variation in the human genome. By convention, a point mutation is referred to as a SNP when the frequency of the minor (rarer) allele exceeds 1% in at least one population. For example, a

GG genotypes was 27 (32.53%), 44 (53.01%) and 12 (14.46%), respectively. Moreover, serum TRAIL levels in PCa patients with AG/GG genotypes were significantly higher than those

with AA genotypes (901.18 ± 189.58 µg/L vs. 819.13 ± 111.00 µg/L, *P* = 0.041; Fig. 2)

Fig. 2. Analysis of serum TRAIL levels in three groups of PCa cases with mean values (horizontal lines, mean values). \* *P* = 0.041 compared with the AG/GG and AA genotypes.

TRAIL is present in various tissues, particular in the prostate, spleen and lung.

property to activate both the extrinsic and intrinsic apoptotic pathways [32-34].

Recently, Kuribayashi et al. [23] indicated a direct regulation of TRAIL gene by p53 protein. Moreover, early growth response protein (EGR) [24], interferon regulatory factor 1 (IRF1) [25], NF-kB [26], SP1 [27] and PU1 [28] have been implicated in the regulation of TRAIL.

TRAIL binds to two different types of receptors: death receptors and decoy receptors. TRAIL can also bind to osteoprotegerin (OPG) (a soluble inhibitor of receptor activator of NF-kB ligand) at low affinity. To date, four human receptors specific for TRAIL have been recognized: the death receptors TRAIL-R1 (also know as DR4), TRAIL-R2 (also known as DR5), the putative decoy receptors TRAIL-R3 (DcR1) and TRAIL-R4 (DcR2). TRAIL-R1 (DR4) is expressed at very low levels in most human tissues including the spleen, thymus, liver, peripheral blood leukocytes, activated T cells, small intestine and some tumor cell lines. TRAIL-R2 (DR5) is ubiquitiously distributed both in normal and tumor cell lines but is more abundant in spleen, peripheral blood leukocytes, activated lymphocytes and

TRAIL has attained the centre stage in anti-tumor drug discovery because of its efficacy in killing tumor cells without lethal toxicity in pre-clinical models apart from the inherent

Single nucleotide polymorphisms (SNPs) are the most abundant form of genetic variation in the human genome. By convention, a point mutation is referred to as a SNP when the frequency of the minor (rarer) allele exceeds 1% in at least one population. For example, a

**4. Discussion** 

hepatocyes [29-31].

SNP in a regulatory region may have an influence on gene transcription, a SNP located in a RNA splice site may affect RNA splicing, a SNP in the 3'-untranslated region of a gene may have an effect on mRNA stability, and a SNP in the coding region may result in an amino acid substitution in the encoded protein. It is thought that SNPs contribute to interindividual variability in susceptibility to common diseases such as cancer [35, 36].

So far, some published meta-analyses have confirmed that a number of SNPs are associated with increased or decreased PCa risk in different races, such as A49T in steroid-5-alpha-reductase, alpha polypeptide 2 (SRD5A2) gene [37], Gly388Arg in fibroblast growth factor receptor 4 (FGFR4) gene [38], -160C/A in E-cadherin (CDH1) gene [39], Val16Ala in manganese superoxide dismutase (MnSOD) gene [40], C677T in 5,10-methylenetetrahydrofolate reductase (MTHFR) gene [41].

Several studies have investigated the possible role of anti-tumor gene polymorphisms and the prevalence of PCa. This impairment of host factors might result in susceptibility or resistance to tumor progression. The transcription factor Sp3 (stimulatory protein 3) exhibits a similar DNA binding affinity for Sp1 consensus sequence [42-44] and represses the Sp1 mediated trans-activation of promoters with two or more Sp1 sites [45-47]. TRAIL has two Sp1 consensus sequences in the basal promoter [48, 49]. AA genotype at -716 in TRAIL promoter with additional Sp1 consensus sequence can decrease TRAIL expression due to the repression caused by binding of Sp3, whereas, GG genotype background at the same position can increase TRAIL expression because of the lower probability of Sp3 driven repression. To date, only one study [22] showed the association between TRAIL -716 A>G polymorphism and cancer risk: individuals with -716 GG genotype were at a greater risk of developing breast cancer, in addition, G allele resulted in a higher expression than the A allele to regulate the expression of TRAIL in four different cancer cell lines (HeLa, MCF-7, HepG2, HT1080).

To the best of our knowledge this is the first study investigating the genetic association of polymorphism of the -716 site in TRAIL gene with PCa and the expression of TRAIL with different genotypes in serum of cases in southern Chinese Han descent. No statistically significant association was observed between TRAIL -716 A>G polymorphism and PCa. Moreover, when stratifying the case group by clinical characteristics, the present study also did not find any association among PSA, Gleason and clinical stage. There must be some factors that would contribute to this discrepancy. First, TRAIL -716 A>G polymorphism might play a different role in different cancers. Second, multiple genes and environmental factors may lead to cancer formation. Third, race may be related to cancer. Either through common risk factors or other genes in linkage disequilibrium with TRAIL suggests that a possible role of ethnic differences is in genetic backgrounds and the environment they lived in.

Furthermore, we found that the decreased risk associated with the AG/GG genotypes was more pronounced in: subjects with age ≤70 years (OR = 0.32, 95%CI = 0.12-0.87) and no family history of cancers (OR = 0.86, 95%CI = 0.51-0.96). It confirmed the concept that younger age and no family history of cancers might be protective factors for PCa. In addition, we found that the OR for AG/GG genotypes was 0.58(95%CI = 0.31-0.89) among subjects with BMI >23kg/m2. This finding may reflect that PCa formation may be subject to a variety of environmental and genetic factors. In these subgroups, other high level of genetic susceptibilities or other unknown risk factors may influence our results.

Except for above associated results, we detected the expression of TRAIL in the serums of the cases. We found that the protective genotypes AG/GG were associated with higher

Polymorphism Analysis of TRAIL Gene and Correlation TRAIL Expression in Prostate Cancer 55

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serum TRAIL expression when compared with the AA genotype. Previous one study [22] have reported that the G allele resulted in a higher expression than the A allele. Our work confirmed the findings of this study. Furthermore, since TRAIL can be measured in the blood and the serum level has been found significantly different in different genotypes in PCa cases, this may be a novel tumor marker and provide a future screening target. We need further investigations on the molecular mechanisms of how genetic variants might affect the TRAIL expression.

This study has several potential limitations. First of all, it is well known that sporadic and familial PCa have frequently quite different epidemiological and molecular peculiarities, clinical evolution and prognosis, so it is better to analysis these two kinds of PCa, respectively, however, we got together as a whole case group. Second, the numbers of cases/controls in our studies were not sufficiently large for a comprehensive analysis. Third, the control group in our study contained not only the healthy old matched man but also the benign prostatic hyperplasia (BPH), which was not the strict 'control'.

#### **5. Conclusions**

Our study suggested that a functional polymorphism -716 A>G in the TRAIL gene may play a role in the development of PCa in southern Chinese Han descent, and the protective genotypes AG/GG of -716 A>G were associated with increased TRAIL expression in serum, which makes it a potential role in early detection for PCa. Moreover, further investigations with larger sample size are needed to confirm this relationship and to elucidate the mechanism responsible for this association.

#### **6. Acknowledgments**

This study was supported by Natural Science Foundation of Jiangsu Province (No. BK2010577) and the foundation of medical key department of Jiangsu Province— Department of General Surgery of Jiangsu Province Hospital. We also thank professor Avi Ashkenazi (Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA) with kindly help.

#### **7. Conflict of interest**

The authors declare that they have no conflict of interest related to the publication of this manuscript.

#### **8. References**


serum TRAIL expression when compared with the AA genotype. Previous one study [22] have reported that the G allele resulted in a higher expression than the A allele. Our work confirmed the findings of this study. Furthermore, since TRAIL can be measured in the blood and the serum level has been found significantly different in different genotypes in PCa cases, this may be a novel tumor marker and provide a future screening target. We need further investigations on the molecular mechanisms of how genetic variants might

This study has several potential limitations. First of all, it is well known that sporadic and familial PCa have frequently quite different epidemiological and molecular peculiarities, clinical evolution and prognosis, so it is better to analysis these two kinds of PCa, respectively, however, we got together as a whole case group. Second, the numbers of cases/controls in our studies were not sufficiently large for a comprehensive analysis. Third, the control group in our study contained not only the healthy old matched man but also the

Our study suggested that a functional polymorphism -716 A>G in the TRAIL gene may play a role in the development of PCa in southern Chinese Han descent, and the protective genotypes AG/GG of -716 A>G were associated with increased TRAIL expression in serum, which makes it a potential role in early detection for PCa. Moreover, further investigations with larger sample size are needed to confirm this relationship and to elucidate the

This study was supported by Natural Science Foundation of Jiangsu Province (No. BK2010577) and the foundation of medical key department of Jiangsu Province— Department of General Surgery of Jiangsu Province Hospital. We also thank professor Avi Ashkenazi (Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA) with

The authors declare that they have no conflict of interest related to the publication of this

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**4** 

*India* 

**Development of Miniature 125I - Seeds** 

**for the Treatment of Prostate Cancer** 

*Radiopharmaceuticals Division, Bhabha Atomic Research Centre, Trombay, Mumbai* 

Cancer of prostate is one of the very common diseases of ageing men with incidence rises up 40% over the age of 75-80 years. It has also been noticed recently, that incidences of prostate cancers are on increase specially in many developing countries mainly because of long survival time due to better health care facilities available now-a-days and also due to changes in lifestyles of population of these countries. As compared with white men, black men have a 40% higher risk of the disease and twice the rate of death. The mortality due to prostate cancer has steadily declined for a decade, and it decreased by 4% per year between 1999 and 2003. This decrease may be attributable to several factors, including earlier detection of cancer and improved local and possibly systemic treatment [1]. Thanks to the recent advancements in diagnostic tools, more and more patients are being diagnosed with potentially curable localized prostate cancer at a time where radical local treatment is deemed to be appropriate. For disease that is likely confined to the prostate and the immediate surrounding area, surgery, external beam radiation (EBRT) and seed implantation are the primary treatment options. In recent years, seed implantation has become more popular as a treatment option as it is simpler, less traumatic and the duration of relief is comparable. It has been estimated that up to 50% of patients with early stage

prostate cancer are now receiving ultra sound guided seed implantation [2].

Seed implantation for prostate cancer began in 1911. Louis Pasteur suggested that surgical insertion of radium seed into the prostate may eradicate this malignancy [3]. A number of techniques were subsequently used with limited success. In the 1960s, Drs. Scardino and Carlton at Baylor College of Medicine, Houston, reintroduced permanent prostate brachytherapy using 198Au interstitial implantation in combination with external beam

At about the same time, Dr. Whitmore and colleagues at Memorial Sloan Kettering Cancer Center (MSKCC) also began to insert 125I seeds through an open incision as a sole treatment [5]. Unfortunately, these early techniques did not allow for clear visualization of the seeds as they were being inserted into the prostate and, as a result, there was often poor dose coverage of the prostate gland. However, some important information was obtained from these early seed implantation approaches. Local control was better in patients who received

**1. Introduction** 

**2. Historical background** 

radiation therapy (EBRT)[4].

Sanjay Kumar Saxena and Ashutosh Dash


### **Development of Miniature 125I - Seeds for the Treatment of Prostate Cancer**

Sanjay Kumar Saxena and Ashutosh Dash *Radiopharmaceuticals Division, Bhabha Atomic Research Centre, Trombay, Mumbai India* 

#### **1. Introduction**

58 Prostate Cancer – Original Scientific Reports and Case Studies

[49] Wang Q, Ji Y, Wang X, Evers BM. Isolation and molecular characterization of the 50-

[50] Yang A, Wilson NS, Ashkenazi A. Proapoptotic DR4 and DR5 signaling in cancer cells:

toward clinical translation. Curr Opin Cell Biol. 2010;22:837-844.

2000;276:466–471.

upstream region of the human TRAIL gene. Biochem Biophys Res Commun.

Cancer of prostate is one of the very common diseases of ageing men with incidence rises up 40% over the age of 75-80 years. It has also been noticed recently, that incidences of prostate cancers are on increase specially in many developing countries mainly because of long survival time due to better health care facilities available now-a-days and also due to changes in lifestyles of population of these countries. As compared with white men, black men have a 40% higher risk of the disease and twice the rate of death. The mortality due to prostate cancer has steadily declined for a decade, and it decreased by 4% per year between 1999 and 2003. This decrease may be attributable to several factors, including earlier detection of cancer and improved local and possibly systemic treatment [1]. Thanks to the recent advancements in diagnostic tools, more and more patients are being diagnosed with potentially curable localized prostate cancer at a time where radical local treatment is deemed to be appropriate. For disease that is likely confined to the prostate and the immediate surrounding area, surgery, external beam radiation (EBRT) and seed implantation are the primary treatment options. In recent years, seed implantation has become more popular as a treatment option as it is simpler, less traumatic and the duration of relief is comparable. It has been estimated that up to 50% of patients with early stage prostate cancer are now receiving ultra sound guided seed implantation [2].

#### **2. Historical background**

Seed implantation for prostate cancer began in 1911. Louis Pasteur suggested that surgical insertion of radium seed into the prostate may eradicate this malignancy [3]. A number of techniques were subsequently used with limited success. In the 1960s, Drs. Scardino and Carlton at Baylor College of Medicine, Houston, reintroduced permanent prostate brachytherapy using 198Au interstitial implantation in combination with external beam radiation therapy (EBRT)[4].

At about the same time, Dr. Whitmore and colleagues at Memorial Sloan Kettering Cancer Center (MSKCC) also began to insert 125I seeds through an open incision as a sole treatment [5]. Unfortunately, these early techniques did not allow for clear visualization of the seeds as they were being inserted into the prostate and, as a result, there was often poor dose coverage of the prostate gland. However, some important information was obtained from these early seed implantation approaches. Local control was better in patients who received

Development of Miniature 125I - Seeds for the Treatment of Prostate Cancer 61

production and processing procedures for 125I have been developed and regular production of

The Indian pursuit of developing technology for 125I brachytherapy sources was driven mainly by three considerations, namely, (a) well-established and ease of reliable production of 125I in several GBq quantities in the research reactors in BARC, (b) need to provide 125Ibrachytherapy sources at an affordable cost to meet the domestic needs, (c) help to ease reliance on import and to promote the beneficial use of 125I- brachytherapy sources in the

> **Average Energy (keV)**

(100%) 28.5 90% in

(100%) 21 90% in 58

(100%) 30.4 90% in 33

The encapsulated source's outer dimensions are ~ 4.75 mm length and 0.8mmdia. The active core is situated within the shell of the capsules, often of 50 micron thickness. Iodine belongs to the halogen group of elements which is highly reactive. Although several stable compounds of iodides are reported in the literature, most of them have a definite solubility in water/saline. The main challenge, therefore, is to develop a non-leachable source core containing 125I incorporated in a solid substrate at very high specific activity. Production of the source core in a highly reproducible manner within acceptable dimensional tolerances is yet another challenge. An innovative strategy has to be devised to develop the necessary technology for fabrication of 125I source core, capsules and encapsulation technique.

Table 1. Radiation Characteristics of radionuclides used in prostate seed implantation

Preparation of 125I-brachytherapy sources addresses the following main issues:

Two types of source core are used for the preparation of 125I seed; namely

Silver is chosen as the basic matrix for immobilization of iodine which functions both as the active support and as the x-ray marker. Iodine-125 can be used directly in its elemental form, or as iodide, iodate, hypoiodate, or other ionic forms, or in the form of compounds such as aliphatic or aromatic iodo labeled compounds. The choice of anion(s) depend on

a. Immobilization of 125I in a suitable solid matrix.

**5. Preparation of 125 I minature source cores** 

c. Fabrication of titanium capsules of suitable dimensions. d. Hermetic Sealing of the capsules by Laser welding. e. Quality assurance of the encapsulated sources.

b. Quality evaluation of the sources.

 Rod/wire type sources Spherical type sources

**Dose Delivery**

204 days 145 Gy

days 125 Gy

days 115 Gy

**Total** 

**Dose Production methods** 

124Xe(n,)125Xe125I Nuclear Reactor

> 103Rh(p,n)103Pd Cyclotron

130Ba(n,)131Ba131Cs Nuclear Reactor

this isotope has been commenced by Radiopharmaceuticals Division, BARC, Mumbai.

country.

**Isotope T1/2**

**Specific Activity (TBq/g)** 

125I 60d 650 EC

103Pd 17d 2763 EC

131Cs 9 d 3808 EC

**4. Source requirements** 

**Mode of Decay** 

high-quality implants and who had low-grade and early-stage cancer. The subsequent development of the transperineal, ultrasound guided approach provided a means to more accurately place seeds and thereby improve dose coverage. In the 1980s, several investigators were exploring new brachytherapy approaches to the treatment of prostate cancer. Drs. Syed and Puthawala pioneered a temporary seed technique of placing the needles while visualizing them through an open laparotomy [6]. In 1983, Dr. Holm introduced the use of transrectal ultrasound to visualize the permanent placement of 125I seeds via needles inserted through the perineum directly into the prostate[7]. Drs Blasko and Ragde [8] began the first transperineal ultrasound-guided approach in the United States. The transperineal ultrasound-guided, approach resulted in increased accuracy of seed placement and relatively even distribution of seeds throughout the prostate. This marked a major advance in prostate brachytherapy in that it allowed more precise planning of the implant prior to the procedure. These advances also significantly increased the accuracy of seed placement and insured that the prostate would receive the proper number, strength, and positioning of radioactive sources.

The first transrectal ultrasound-guided, template-guided 125I implant procedure was carried out at the Seattle Prostate Institute in late 1985 and is now being practiced around the world. The original Seattle approach has been modified and improved several times since the original implants. As this procedure has become more popular, many technical improvements have been added to improve the consistency and quality of the procedure. The availability of better imaging techniques such as transrectal ultrasound, fluoroscopy, high quality CT scan, etc. have now made permanent prostate implants much more refined. Today, the implant is planned prior to the procedure either on the day of or several weeks prior to the implant. Typically, the implant is completed in a 45-90 minute outpatient procedure under spinal anesthesia or light general anesthesia [8].

In addition to the availability of loose sealed radioactive sources, seeds incorporating radionuclides such as 125I ,103Pd and 131Cs are now available in continuous strand form, increasing the likelihood that the seeds will remain in place after implantation. About 60-140 radioactive seeds encompassing the entire prostate gland are used to eradicate the tumor. While slight differences in technique are expected to grow as more and more physicians perform this procedure and as more technical advances are made, the basic approach is quite similar and it remains to be determined whether any single technique will prove superior in controlling the cancer.

#### **3. Selection of radionuclide**

Beginning in 1967, 125I became the first radioisotope sealed within a titanium capsule popularly known as seed. While its use continues to this day, many patients and doctors in recent years have chosen shorter half-life isotopes other than 125I such as 103Pd and 131Cs.

Radionuclides used in the prostate radiotherapy are 125I, 103Pd or 131Cs. The radiation characteristics of these three isotopes are given in Table 1. The high energy (10-50 MeV) cyclotron produced 103Pd is not yet available in India. The reactor production of 131Cs is difficult due to low percentage abundance of 130Ba (~1%) in natural targets and the logistics and cost considerations of this isotope do not permit its use at present. On the other hand, 125I with its relatively longer half-life and suitable gamma energy coupled with ease of production is a cost effective isotope and can be easily produced by (n,γ) reaction of natural 124Xe gas in a special set up provided in the research reactors (DHRUVA) of BARC, Mumbai, India. The production and processing procedures for 125I have been developed and regular production of this isotope has been commenced by Radiopharmaceuticals Division, BARC, Mumbai.

The Indian pursuit of developing technology for 125I brachytherapy sources was driven mainly by three considerations, namely, (a) well-established and ease of reliable production of 125I in several GBq quantities in the research reactors in BARC, (b) need to provide 125Ibrachytherapy sources at an affordable cost to meet the domestic needs, (c) help to ease reliance on import and to promote the beneficial use of 125I- brachytherapy sources in the country.


Table 1. Radiation Characteristics of radionuclides used in prostate seed implantation

#### **4. Source requirements**

60 Prostate Cancer – Original Scientific Reports and Case Studies

high-quality implants and who had low-grade and early-stage cancer. The subsequent development of the transperineal, ultrasound guided approach provided a means to more accurately place seeds and thereby improve dose coverage. In the 1980s, several investigators were exploring new brachytherapy approaches to the treatment of prostate cancer. Drs. Syed and Puthawala pioneered a temporary seed technique of placing the needles while visualizing them through an open laparotomy [6]. In 1983, Dr. Holm introduced the use of transrectal ultrasound to visualize the permanent placement of 125I seeds via needles inserted through the perineum directly into the prostate[7]. Drs Blasko and Ragde [8] began the first transperineal ultrasound-guided approach in the United States. The transperineal ultrasound-guided, approach resulted in increased accuracy of seed placement and relatively even distribution of seeds throughout the prostate. This marked a major advance in prostate brachytherapy in that it allowed more precise planning of the implant prior to the procedure. These advances also significantly increased the accuracy of seed placement and insured that the prostate would receive the proper number,

The first transrectal ultrasound-guided, template-guided 125I implant procedure was carried out at the Seattle Prostate Institute in late 1985 and is now being practiced around the world. The original Seattle approach has been modified and improved several times since the original implants. As this procedure has become more popular, many technical improvements have been added to improve the consistency and quality of the procedure. The availability of better imaging techniques such as transrectal ultrasound, fluoroscopy, high quality CT scan, etc. have now made permanent prostate implants much more refined. Today, the implant is planned prior to the procedure either on the day of or several weeks prior to the implant. Typically, the implant is completed in a 45-90 minute outpatient

In addition to the availability of loose sealed radioactive sources, seeds incorporating radionuclides such as 125I ,103Pd and 131Cs are now available in continuous strand form, increasing the likelihood that the seeds will remain in place after implantation. About 60-140 radioactive seeds encompassing the entire prostate gland are used to eradicate the tumor. While slight differences in technique are expected to grow as more and more physicians perform this procedure and as more technical advances are made, the basic approach is quite similar and it remains to be determined whether any single technique will prove

Beginning in 1967, 125I became the first radioisotope sealed within a titanium capsule popularly known as seed. While its use continues to this day, many patients and doctors in recent years have chosen shorter half-life isotopes other than 125I such as 103Pd and 131Cs. Radionuclides used in the prostate radiotherapy are 125I, 103Pd or 131Cs. The radiation characteristics of these three isotopes are given in Table 1. The high energy (10-50 MeV) cyclotron produced 103Pd is not yet available in India. The reactor production of 131Cs is difficult due to low percentage abundance of 130Ba (~1%) in natural targets and the logistics and cost considerations of this isotope do not permit its use at present. On the other hand, 125I with its relatively longer half-life and suitable gamma energy coupled with ease of production is a cost effective isotope and can be easily produced by (n,γ) reaction of natural 124Xe gas in a special set up provided in the research reactors (DHRUVA) of BARC, Mumbai, India. The

strength, and positioning of radioactive sources.

superior in controlling the cancer.

**3. Selection of radionuclide** 

procedure under spinal anesthesia or light general anesthesia [8].

The encapsulated source's outer dimensions are ~ 4.75 mm length and 0.8mmdia. The active core is situated within the shell of the capsules, often of 50 micron thickness. Iodine belongs to the halogen group of elements which is highly reactive. Although several stable compounds of iodides are reported in the literature, most of them have a definite solubility in water/saline. The main challenge, therefore, is to develop a non-leachable source core containing 125I incorporated in a solid substrate at very high specific activity. Production of the source core in a highly reproducible manner within acceptable dimensional tolerances is yet another challenge. An innovative strategy has to be devised to develop the necessary technology for fabrication of 125I source core, capsules and encapsulation technique. Preparation of 125I-brachytherapy sources addresses the following main issues:


### **5. Preparation of 125 I minature source cores**

Two types of source core are used for the preparation of 125I seed; namely


Silver is chosen as the basic matrix for immobilization of iodine which functions both as the active support and as the x-ray marker. Iodine-125 can be used directly in its elemental form, or as iodide, iodate, hypoiodate, or other ionic forms, or in the form of compounds such as aliphatic or aromatic iodo labeled compounds. The choice of anion(s) depend on

Development of Miniature 125I - Seeds for the Treatment of Prostate Cancer 63

The potential of the cell was kept at 233 mV. The electrodeposition was carried out under

Zhang et al. [11] have described a method of depositing 125I using chlorinated silver rod. In brief, the procedure involved coating a layer of AgCl on the silver rod of 3 mm long and 0.5 mm in diameter. The activated silver rods were put into 4 mol/l nitric acid and heated for 5 minutes. They were then put into a mixed solution of sodium hypochlorite and hydrochloric acid. The chlorinated silver rods were put in Na125I solution at pH > 6.5 to deposit required

Han et al. [12] have described a method of depositing 125I using a ceramic rods. The ceramic rods of 3 mm long and 0.5 mm sizes were immersed in conc. HF solution for 4 hours, scratched with a specially made pin along the length at regular intervals to made miniature horizontal cavities of ~ 0.1 mm depth. they were then immersed aqueous AgNO3 solution to absorb the solution. Impregnation of 125I on to the treated rod was carried out by the controlled addition of Na125I solution at pH > 9 to deposit required amount of 125I. Extending this theme, Park et a1.[13] have investigated the possibility of adsorption of 125I on a Ag + Al2O3 rod as a carrier body. The adsorption capacity was more than 95% after 4

Our group have used a novel method to adsorb radioiodine(125I) on silver wires, by precoating the wires with palladium[14-15]. The experimental conditions such as amount of radioactivity, carrier concentration, reaction time, reaction temperature, reaction volume, pH of the reaction mixture, etc. were systematically optimized to achieve best results. More than 80% of the initial radioactivity could be firmly deposited on the source core. The sources with extremely good reproducibility and consistency with respect to activity content

The microstructures of plain silver wire and palladium coated silver wires are shown in Fig. 2 (a) and (b) respectively, indicating the presence of huge sites for sorption of iodine on

(a) (b)

Fig. 2. (a) SEM of Plain Silver wire Fig. 2 (b) SEM of Palladium Coated Silver wire

ultra violet radiation.

amount of 125I.

**5.1.2 Iodination of the silver rod** 

**5.1.3 Absorption of 125I on a ceramic matrix** 

**5.1.4 Physico-chemical adsorption** 

palladium cated silver wires.

hours at a volume of 50 μl containing about 5 mCi of 125I.

and other quality parameters could be produced.

the methodology intended to use. Owing to volatile nature of elemental iodine, iodide ions are used for source preparation. Iodine-125 as iodide may be physically trapped in or on the substrate, by adsorption, or may be chemically attached to it in some way. The radioactive source core should be of an overall size and dimensions to fit inside a conventional seed container suitable for encapsulation. The range of desired activity is about 0.3 to 4.0 mCi per seed.

#### **5.1 Preparation of rod/wire type source cores**

Silver wires of guranted purity of 3 mm length & 0.5mm dia are used to incorporate Iodine-125. The following methodologies were explored for preparing 125I-silver rod source core.


#### **5.1.1 Electro deposition**

Anodic electro deposition of radio iodine (125I) was carried out in a quartz bath size [1.2 cm (dia.), 2.5 cm (ht)] with platinum cathode (1mm). The wires were arranged in the cell as shown in Fig.1. Various experimental parameters such as the current used, radioiodine concentration in the cell and time for deposition were optimized to obtain maximum activity on the silver wire. By this method, more than 85% of the initial radioactivity could be firmly deposited on the source at 20 µA current for 25-30 min duration on the silver wires. These sources with extremely good reproducibility and consistency with respect to activity content, could by this method [9].

Fig. 1. Schematic diagram of the electro-deposition set-up.

Cieszykowska et al.[10] have also developed an electrochemical method of depositing 125I form an electrolyte containing 0.01 M NaOH. In brief, 10 ml of solution containing appropriate amount of I-125 was taken in a platinum crucible which served as cathode. The cathode consists of a single silver bar of 3 mm long and 0.5 mm in diameter. The volume of the electrolyte used was 10 mL and the distance between the electrodes was about 20 mm. The potential of the cell was kept at 233 mV. The electrodeposition was carried out under ultra violet radiation.

#### **5.1.2 Iodination of the silver rod**

62 Prostate Cancer – Original Scientific Reports and Case Studies

the methodology intended to use. Owing to volatile nature of elemental iodine, iodide ions are used for source preparation. Iodine-125 as iodide may be physically trapped in or on the substrate, by adsorption, or may be chemically attached to it in some way. The radioactive source core should be of an overall size and dimensions to fit inside a conventional seed container suitable for encapsulation. The range of desired activity is about 0.3 to 4.0 mCi

Silver wires of guranted purity of 3 mm length & 0.5mm dia are used to incorporate Iodine-125. The following methodologies were explored for preparing 125I-silver rod

Anodic electro deposition of radio iodine (125I) was carried out in a quartz bath size [1.2 cm (dia.), 2.5 cm (ht)] with platinum cathode (1mm). The wires were arranged in the cell as shown in Fig.1. Various experimental parameters such as the current used, radioiodine concentration in the cell and time for deposition were optimized to obtain maximum activity on the silver wire. By this method, more than 85% of the initial radioactivity could be firmly deposited on the source at 20 µA current for 25-30 min duration on the silver wires. These sources with extremely good reproducibility and consistency with respect to activity

Cieszykowska et al.[10] have also developed an electrochemical method of depositing 125I form an electrolyte containing 0.01 M NaOH. In brief, 10 ml of solution containing appropriate amount of I-125 was taken in a platinum crucible which served as cathode. The cathode consists of a single silver bar of 3 mm long and 0.5 mm in diameter. The volume of the electrolyte used was 10 mL and the distance between the electrodes was about 20 mm.

per seed.

source core.

1. Electrodeposition

**5.1.1 Electro deposition** 

content, could by this method [9].

Fig. 1. Schematic diagram of the electro-deposition set-up.

**5.1 Preparation of rod/wire type source cores** 

2. Physical & physico-chemical adsorption

Zhang et al. [11] have described a method of depositing 125I using chlorinated silver rod. In brief, the procedure involved coating a layer of AgCl on the silver rod of 3 mm long and 0.5 mm in diameter. The activated silver rods were put into 4 mol/l nitric acid and heated for 5 minutes. They were then put into a mixed solution of sodium hypochlorite and hydrochloric acid. The chlorinated silver rods were put in Na125I solution at pH > 6.5 to deposit required amount of 125I.

#### **5.1.3 Absorption of 125I on a ceramic matrix**

Han et al. [12] have described a method of depositing 125I using a ceramic rods. The ceramic rods of 3 mm long and 0.5 mm sizes were immersed in conc. HF solution for 4 hours, scratched with a specially made pin along the length at regular intervals to made miniature horizontal cavities of ~ 0.1 mm depth. they were then immersed aqueous AgNO3 solution to absorb the solution. Impregnation of 125I on to the treated rod was carried out by the controlled addition of Na125I solution at pH > 9 to deposit required amount of 125I. Extending this theme, Park et a1.[13] have investigated the possibility of adsorption of 125I on a Ag + Al2O3 rod as a carrier body. The adsorption capacity was more than 95% after 4 hours at a volume of 50 μl containing about 5 mCi of 125I.

#### **5.1.4 Physico-chemical adsorption**

Our group have used a novel method to adsorb radioiodine(125I) on silver wires, by precoating the wires with palladium[14-15]. The experimental conditions such as amount of radioactivity, carrier concentration, reaction time, reaction temperature, reaction volume, pH of the reaction mixture, etc. were systematically optimized to achieve best results. More than 80% of the initial radioactivity could be firmly deposited on the source core. The sources with extremely good reproducibility and consistency with respect to activity content and other quality parameters could be produced.

The microstructures of plain silver wire and palladium coated silver wires are shown in Fig. 2 (a) and (b) respectively, indicating the presence of huge sites for sorption of iodine on palladium cated silver wires.

Fig. 2. (a) SEM of Plain Silver wire Fig. 2 (b) SEM of Palladium Coated Silver wire

Development of Miniature 125I - Seeds for the Treatment of Prostate Cancer 65

A mixture of pre-cooled (5°C) solution of hexamethylenetetraamine and urea (3 M) with aluminum nitrate solution was dispersed as droplets into hot oil to bring about the formation of aluminium hydroxide in to solid gel sphere form. The spheres were dried in an air oven at 100°C and heat treated at 700°C for 5 h in a furnace to obtain alumina spheres. Spheres of uniform sizes were selected by passing through a 600 m mesh. The experimental conditions such as amount of radioactivity, carrier concentration, reaction time, reaction temperature, reaction volume etc. were systematically optimised. By this method, more than 95% of the initial radioactivity could be firmly deposited on the source core and 0.6-0.8 mCi of radioiodine could be adsorbed on the alumina microsphere [9].

Silver beads of dimension of 0.5 mm() were pre-coated with palladium for the incorporation of radioiodine (125I). The experimental conditions such as amount of radioactivity, carrier concentration, reaction time, reaction temperature, reaction volume, pH of the reaction mixture, etc. were systematically optimized [16]. By this method, more than 83 % of the initial radioactivity could be irreversibly adsorbed on the palladium coated on the source core and radioactive sources in the range of 20-251 MBq ( 0.5-0.6 mCi) can be prepared. The sources with extremely good reproducibility and consistency w.r.t. activity content and other quality parameters could be produced. All the sources were measured with calibrated ionization chambers and seed strength was quoted with an overall

The physicochemical adsorption of radioiodine on alumina microspheres is easy and also less expensive. Quantitative adsorption of activity on the spheres is possible by using radioactive iodine in iodate (IO−3) form. However, it was found that the percentage leachability of radioactivity from the spheres was more than desirable. Further developments are needed to assess the potential of this approach on a reliable and

The physicochemical adsorption of Pd-silver microspheres is easy and 125I-beads could be prepared in a nonleachable form. The radioactive sources upto ~111MBq (3 mCi) activity

The capsule matrix should have chemical compatibility with the source core which will be encapsulated inside. The capsule material need to be of low atomic weight so that it would attenuate radiation to a minimum extent. Titanium's unique combination of attributes such as light weight, high strength to weight ratio, corrosion resistance, amenability for easy welding, biocompatibility, and durability in extreme environments make it an excellent material for capsule fabrication. Titanium is used extensively for medical and dental implants because it is biocompatible with the human body. It is completely inert and immune to corrosion by all uids and tissues of the body, making it ideal for encapsulation. The wall thickness of the capsule should be adequate to provide requisite mechanical strength to retain the source core, in order to reduce the risk of radioactive contamination in

can be prepared by arranging six individual beads in a well-arranged geometry.

**5.2.1 Alumina microspheres** 

**5.2.2 Metallic microspheres** 

uncertainty of 10%.

continuous basis.

**5.2.3 Comparison of techniques** 

**6. Fabrication of titanium capsules** 

#### **5.1.5 Comparison of techniques**

The electrodeposition method of preparing 125I source core is a straight forward procedure and used by many commercial manufacturers of these sources. Quantitative firm and uniform deposition could be obtained by the optimized parameters, with low leaching of activity. However this method is labour intensive, require high skill and would be exposing personnel to long time to radiation. The electro deposition method is attractive only if automated system where radioactive iodine is electrodeposited on a long piece of silver wire of the required diameter and subsequently precision cut in to the required dimensions by remotely operated electro mechanical cutting devices, is available.

The inherent drawbacks of absorption of 125I on a ceramic matrix and iodination of the silver rod, include a tedious time-consuming matrix preparation procedure, need for strict adherence to the operational protocol, and the requirement for well-trained, skilled operator.

Chemisorption of 125I activity on the PdCl2 treated silver wire as more suitable for adsorption of 125I than plain silver wires in terms of quantitative adsorption and nonleachability of 125I activity. The procedure is straightforward and easily performed. The workload to use this protocol is small. The incidence of serious error may be low. The mild experimental conditions of adsorption at neutral to alkaline pH facilitates the safe handling of high amounts of radioactivity for the preparation of therapeutic sources without the release of air activity. The stability of 125I on Pd coated silver wire is due to the formation of insoluble palladous iodide on the surface of the wire and accounts for low leachability. This procedure is routinely used by Radiopharmaceuticals Division, BARC, Mumbai. Fig. 3 depicts the laboratory used for the regular production of 125I source core.

Fig. 3. Laboratory for the production of 125I source core

#### **5.2 Preparation of spherical type source cores**

A linear assembly of six alumina microspheres or six palladium coated silver spheres of 0.5 mm() were used for making spherical seed sources.

#### **5.2.1 Alumina microspheres**

64 Prostate Cancer – Original Scientific Reports and Case Studies

The electrodeposition method of preparing 125I source core is a straight forward procedure and used by many commercial manufacturers of these sources. Quantitative firm and uniform deposition could be obtained by the optimized parameters, with low leaching of activity. However this method is labour intensive, require high skill and would be exposing personnel to long time to radiation. The electro deposition method is attractive only if automated system where radioactive iodine is electrodeposited on a long piece of silver wire of the required diameter and subsequently precision cut in to the required dimensions by

The inherent drawbacks of absorption of 125I on a ceramic matrix and iodination of the silver rod, include a tedious time-consuming matrix preparation procedure, need for strict adherence to the operational protocol, and the requirement for well-trained, skilled

Chemisorption of 125I activity on the PdCl2 treated silver wire as more suitable for adsorption of 125I than plain silver wires in terms of quantitative adsorption and nonleachability of 125I activity. The procedure is straightforward and easily performed. The workload to use this protocol is small. The incidence of serious error may be low. The mild experimental conditions of adsorption at neutral to alkaline pH facilitates the safe handling of high amounts of radioactivity for the preparation of therapeutic sources without the release of air activity. The stability of 125I on Pd coated silver wire is due to the formation of insoluble palladous iodide on the surface of the wire and accounts for low leachability. This procedure is routinely used by Radiopharmaceuticals Division, BARC, Mumbai. Fig. 3

A linear assembly of six alumina microspheres or six palladium coated silver spheres of 0.5

remotely operated electro mechanical cutting devices, is available.

depicts the laboratory used for the regular production of 125I source core.

Fig. 3. Laboratory for the production of 125I source core

**5.2 Preparation of spherical type source cores** 

mm() were used for making spherical seed sources.

**5.1.5 Comparison of techniques** 

operator.

A mixture of pre-cooled (5°C) solution of hexamethylenetetraamine and urea (3 M) with aluminum nitrate solution was dispersed as droplets into hot oil to bring about the formation of aluminium hydroxide in to solid gel sphere form. The spheres were dried in an air oven at 100°C and heat treated at 700°C for 5 h in a furnace to obtain alumina spheres. Spheres of uniform sizes were selected by passing through a 600 m mesh. The experimental conditions such as amount of radioactivity, carrier concentration, reaction time, reaction temperature, reaction volume etc. were systematically optimised. By this method, more than 95% of the initial radioactivity could be firmly deposited on the source core and 0.6-0.8 mCi of radioiodine could be adsorbed on the alumina microsphere [9].

#### **5.2.2 Metallic microspheres**

Silver beads of dimension of 0.5 mm() were pre-coated with palladium for the incorporation of radioiodine (125I). The experimental conditions such as amount of radioactivity, carrier concentration, reaction time, reaction temperature, reaction volume, pH of the reaction mixture, etc. were systematically optimized [16]. By this method, more than 83 % of the initial radioactivity could be irreversibly adsorbed on the palladium coated on the source core and radioactive sources in the range of 20-251 MBq ( 0.5-0.6 mCi) can be prepared. The sources with extremely good reproducibility and consistency w.r.t. activity content and other quality parameters could be produced. All the sources were measured with calibrated ionization chambers and seed strength was quoted with an overall uncertainty of 10%.

#### **5.2.3 Comparison of techniques**

The physicochemical adsorption of radioiodine on alumina microspheres is easy and also less expensive. Quantitative adsorption of activity on the spheres is possible by using radioactive iodine in iodate (IO−3) form. However, it was found that the percentage leachability of radioactivity from the spheres was more than desirable. Further developments are needed to assess the potential of this approach on a reliable and continuous basis.

The physicochemical adsorption of Pd-silver microspheres is easy and 125I-beads could be prepared in a nonleachable form. The radioactive sources upto ~111MBq (3 mCi) activity can be prepared by arranging six individual beads in a well-arranged geometry.

#### **6. Fabrication of titanium capsules**

The capsule matrix should have chemical compatibility with the source core which will be encapsulated inside. The capsule material need to be of low atomic weight so that it would attenuate radiation to a minimum extent. Titanium's unique combination of attributes such as light weight, high strength to weight ratio, corrosion resistance, amenability for easy welding, biocompatibility, and durability in extreme environments make it an excellent material for capsule fabrication. Titanium is used extensively for medical and dental implants because it is biocompatible with the human body. It is completely inert and immune to corrosion by all uids and tissues of the body, making it ideal for encapsulation. The wall thickness of the capsule should be adequate to provide requisite mechanical strength to retain the source core, in order to reduce the risk of radioactive contamination in

Development of Miniature 125I - Seeds for the Treatment of Prostate Cancer 67

Although the tungsten inert gas (TIG) process is the most common method for welding SS capsules, this is not suited to our type of application.TIG welding cause molten titanium to flow down and the weld was observed to be porous. The most likely cause of porosity is the trapping of gas bubbles between dendrites during solidification and presence of hydrogen from moisture in the arc environment. This resulted in the leakage of radioactivity from the welded capsules significantly higher than the prescribed limit. Hence an alternative method

In order to circumvent these problems, a pulsed laser welding system was used for the encapsulation of capsules. This system consists of a laser head, power supply unit, chiller unit and welding system. As the welding is carried out in pulses, the heat input to the 125I source is reduced. The Nd:YAG laser installed inside the fume-hood in our laboratory is shown in the Fig.6. The output of the laser is taken through the ports and it is connected to

Fig. 5. Loading of 125I source core inside the capsules

of welding is to be adopted.

the welding head using optical fiber.

Fig. 6. Laser Welding System

the event of source rupture during handling. At the same time it should not be too thick to attenuate the radiation emanating from the source. The wall thickness of the capsule is configured to provide adequate mechanical strength as well as required radiation output. About 0.05 mm thick titanium tubes is sufficient to allow gamma rays and low energy Xrays to pass through for providing therapeutic effect. It is preferred that the capsule have an open end and a closed end. The capsule is preferably sealed with a suitable end cap using techniques such as laser/electron beam welding.

The titanium Capsules of the required dimensions 4.75mm(l) x 0.8 mm() x 0.05mm(t) along with suitable caps(lids) of 0.8mm() are generally used. Fig.4 shows the cross-sectional view of titanium encapsulated wire source and microspheres.

Fig. 4. Cross sectional view of encapsulated 125I- sources.

#### **7. Laser welding of titanium capsules**

The welding of capsules containing radioactive sources requires that the following conditions are met:


The activities of the 125I rods/spheres were measured and the sources with activity within 5 % of the targeted activity (generally 111 MBq/source) were segregated and used for encapsulation. The rods were inserted individually into the titanium capsule followed by placement of a cap over it with the aid of magnifying glass. The source loading procedure adapted in radiopharmaceuticals Division of Bhabha Atomic Research Center is depicted in Fig.5.

the event of source rupture during handling. At the same time it should not be too thick to attenuate the radiation emanating from the source. The wall thickness of the capsule is configured to provide adequate mechanical strength as well as required radiation output. About 0.05 mm thick titanium tubes is sufficient to allow gamma rays and low energy Xrays to pass through for providing therapeutic effect. It is preferred that the capsule have an open end and a closed end. The capsule is preferably sealed with a suitable end cap using

The titanium Capsules of the required dimensions 4.75mm(l) x 0.8 mm() x 0.05mm(t) along with suitable caps(lids) of 0.8mm() are generally used. Fig.4 shows the cross-sectional view

**Microspheres Rod type** 

 The welding has to be carried out in a well ventilated shielded enclosure The process of welding should be feasible in a remotely operated system.

The welding should not change the geometry of the capsules.

The welding of capsules containing radioactive sources requires that the following

Loss of activity during the welding should be minimum(minimum heat input to the

Efficiency of welding should be such that there should not be leakage of activity after

The activities of the 125I rods/spheres were measured and the sources with activity within 5 % of the targeted activity (generally 111 MBq/source) were segregated and used for encapsulation. The rods were inserted individually into the titanium capsule followed by placement of a cap over it with the aid of magnifying glass. The source loading procedure adapted in radiopharmaceuticals Division of Bhabha Atomic Research Center is depicted

techniques such as laser/electron beam welding.

of titanium encapsulated wire source and microspheres.

Fig. 4. Cross sectional view of encapsulated 125I- sources.

welding.( Welding should cause less porosity)

**7. Laser welding of titanium capsules** 

conditions are met:

active source)

in Fig.5.

Fig. 5. Loading of 125I source core inside the capsules

Although the tungsten inert gas (TIG) process is the most common method for welding SS capsules, this is not suited to our type of application.TIG welding cause molten titanium to flow down and the weld was observed to be porous. The most likely cause of porosity is the trapping of gas bubbles between dendrites during solidification and presence of hydrogen from moisture in the arc environment. This resulted in the leakage of radioactivity from the welded capsules significantly higher than the prescribed limit. Hence an alternative method of welding is to be adopted.

In order to circumvent these problems, a pulsed laser welding system was used for the encapsulation of capsules. This system consists of a laser head, power supply unit, chiller unit and welding system. As the welding is carried out in pulses, the heat input to the 125I source is reduced. The Nd:YAG laser installed inside the fume-hood in our laboratory is shown in the Fig.6. The output of the laser is taken through the ports and it is connected to the welding head using optical fiber.

Fig. 6. Laser Welding System

Development of Miniature 125I - Seeds for the Treatment of Prostate Cancer 69

Quality is "the degree to which a set of inherent characteristics fulfils requirements." Control is "the need or expectation that is stated, generally implied or obligatory." Iodine-125 sources produced are subjected to numerous checks obligatory by regulatory authorities. The emphasis given is on the physical and chemical aspects. The schematic

The estimation of source activity/ strength for the seed was carried out by using a calibrated well-type ionization chambers. The charge collected per unit time by keeping the source at the position of peak response in the well chamber was multiplied by the calibration factor to estimate the activity content of source. The factor recommended by AAPM for converting the strength of 125I-seeds from apparent activity (mCi) to source strength (i.e. air kerma strength) is 1.27 mGy/h-m2 per mCi (irrespective of internal construction of the source) and

diagrm of an assembly of laser welded 125I seed is shown in Fig. 10.

Fig. 9. SEM of welded capsule

**8. Quality control of sealed sources** 

Fig. 10. Assembly of laser welded 125I seed

**8.1 Source strength measurement** 

Major advantages of laser beam welding are low welding stresses, low risk of distortion, creation of minimal heat affected zone with minimal 125I contamination and capability of welding of varying mass that allows hermetic sealing of Ti capsules. Nd:YAG pulsed lasers have the ability to weld hard materials like Ti and produce an aesthetic weld with high depth/width ratio free from any weld buildup that eliminates many secondary operations such as grinding or honing. It has also high welding speed, good reproducibility, flexibility and the process can be easily be remotized and automated. Due to the extreme reactivity of titanium metal, it is essential to shield the molten pool and the hot metal from contact with air. Argon is used as inert gas protection.

Prior to welding, the welding parameters such as energy of laser pulse, frequency, pulse duration and rotational speed of sample are systematically optimized to obtain quality welds with negligible leakage. The laser-welding operations were carried out remotely using PC-based controlled system. Fig. 7 depicts a typical welded 125I- seed.

Fig. 7. Encapsulated 125I- brachytherapy sources

The metallography test of welded capsules was carried out by optical metallography (The metallograph of a welded source is shown in Fig. 8.

Fig. 8. Optical micrograph (50 times magnified) of a welded capsule.

The penetration depth in the samples was evaluated by Scanning Electron Microscopy(SEM). The SEM micrograph is depicted in Fig. 9. The penetration depth was found to be ~ 2-3 times the wall thickness of the capsules. The welded samples showed high integrity and superior metallurgical quality.

Fig. 9. SEM of welded capsule

68 Prostate Cancer – Original Scientific Reports and Case Studies

Major advantages of laser beam welding are low welding stresses, low risk of distortion, creation of minimal heat affected zone with minimal 125I contamination and capability of welding of varying mass that allows hermetic sealing of Ti capsules. Nd:YAG pulsed lasers have the ability to weld hard materials like Ti and produce an aesthetic weld with high depth/width ratio free from any weld buildup that eliminates many secondary operations such as grinding or honing. It has also high welding speed, good reproducibility, flexibility and the process can be easily be remotized and automated. Due to the extreme reactivity of titanium metal, it is essential to shield the molten pool and the hot metal from contact with

Prior to welding, the welding parameters such as energy of laser pulse, frequency, pulse duration and rotational speed of sample are systematically optimized to obtain quality welds with negligible leakage. The laser-welding operations were carried out remotely

The metallography test of welded capsules was carried out by optical metallography (The

The penetration depth in the samples was evaluated by Scanning Electron Microscopy(SEM). The SEM micrograph is depicted in Fig. 9. The penetration depth was found to be ~ 2-3 times the wall thickness of the capsules. The welded samples showed high

using PC-based controlled system. Fig. 7 depicts a typical welded 125I- seed.

air. Argon is used as inert gas protection.

Fig. 7. Encapsulated 125I- brachytherapy sources

integrity and superior metallurgical quality.

metallograph of a welded source is shown in Fig. 8.

Fig. 8. Optical micrograph (50 times magnified) of a welded capsule.

#### **8. Quality control of sealed sources**

Quality is "the degree to which a set of inherent characteristics fulfils requirements." Control is "the need or expectation that is stated, generally implied or obligatory." Iodine-125 sources produced are subjected to numerous checks obligatory by regulatory authorities. The emphasis given is on the physical and chemical aspects. The schematic diagrm of an assembly of laser welded 125I seed is shown in Fig. 10.

Fig. 10. Assembly of laser welded 125I seed

#### **8.1 Source strength measurement**

The estimation of source activity/ strength for the seed was carried out by using a calibrated well-type ionization chambers. The charge collected per unit time by keeping the source at the position of peak response in the well chamber was multiplied by the calibration factor to estimate the activity content of source. The factor recommended by AAPM for converting the strength of 125I-seeds from apparent activity (mCi) to source strength (i.e. air kerma strength) is 1.27 mGy/h-m2 per mCi (irrespective of internal construction of the source) and

Development of Miniature 125I - Seeds for the Treatment of Prostate Cancer 71

Fig. 12. (a) Specially designed autoradiography gadget; b) developed autoradiography film

It was mandatory to check all the sealed sources to ascertain leak tightness of the sealing.

The fabricated sealed sources are placed in nearly boiling water kept in a glass beaker for about 2 minutes. Appearance of bubbles from the sealed sources indicates improper sealing

The fabricated sealed sources are immersed in a closed glass container containing ethylene glycol and the pressure inside the chamber is reduced to 100 mm of Hg. Any leak from the

One sealed source is immersed in 20 mL of water taken in a glass beaker and heated to 50oC for 5 hours. The source is removed, the water concentrated to 1mL and the activity released is estimated in a well type NaI(Tl) counter. Activity measuring 185 Bq /source is considered to be the limit for acceptance of leak tightness. When the activities detected in the immersion test of the sealed source is less than 185 Bq, the sealed source capsule is considered to be leak

i. The surface of each source must be checked regularly for visible damage. Sources with

sources is shown by a string of bubbles, and such sources are rejected.

The following steps should be taken during Quality Control of 125I seeds.

damaged surface must not be touched for wipe test.

of the sources.

*Bubble test* 

*Pressure test* 

tight.

**8.6 Immersion test** 

**8.5 Leakage test** 

and such sources are discarded.

The following tests are performed on all the sources.

the same was used as a reference for the studies carried out at our end. A source calibration accuracy of ~ 3% relative to existing air kerma standards seems reasonable. The radiation equipment should not be used whenever the level is exceeded.

#### **8.2 Leachability**

Source cores ( Un-encapsulated) were immersed in 100 mL of still distilled water at ambient temperature for 48 hours. At the end of 48 hours, the total leached out radioactivity was assayed to determine the leachability of bare sources should be less than 0.01% of the total seed activity. The physico-chemical adsorption method [14] adapted by us was found to achieve this limit and thus complied with regulatory norms.

#### **8.3 Swipe test**

The sealed sources are tested for surface contamination or presence of any loose activity by swiping the sources using alcohol immersed cotton wool and checking the radioactive content in a NaI(Tl) scintillation counter. When the activities detected in the swipe is less than 185 Bq, the sealed source capsule is considered to be contamination free.

#### **8.4 Uniformity of activity**

Uniformity of deposition of 125I activity was examined by autoradiography using a specially designed gadget (Fig. 11.) A circular disc [ 4.4 cm (), 1.4cm thick] made of brass with a central hole of 3mm diameter and 8 mm depth was taken and eight equidistant tunnels (45o angle between each successive tunnels) of uniform aperture were drilled through the central hole. One source was placed at a time in the central hole and autoradiographed simultaneously by wrapping a strip of photographic film all along the side of brass disc.

#### Fig. 11. Gadget for Auto-radiography

The film gets exposed from eight equidistant directions through the holes. The Optical density distribution of the exposed film at different angles was measured by B/W transmission densitometer. The variation in OD values at different positions should be ±10 %. Sources prepared by the physico-chemical adsorption methodology [14] in our laboratory has variation within ± 5% (Fig.12).

Fig. 12. (a) Specially designed autoradiography gadget; b) developed autoradiography film of the sources.

#### **8.5 Leakage test**

It was mandatory to check all the sealed sources to ascertain leak tightness of the sealing. The following tests are performed on all the sources.

#### *Bubble test*

70 Prostate Cancer – Original Scientific Reports and Case Studies

the same was used as a reference for the studies carried out at our end. A source calibration accuracy of ~ 3% relative to existing air kerma standards seems reasonable. The radiation

Source cores ( Un-encapsulated) were immersed in 100 mL of still distilled water at ambient temperature for 48 hours. At the end of 48 hours, the total leached out radioactivity was assayed to determine the leachability of bare sources should be less than 0.01% of the total seed activity. The physico-chemical adsorption method [14] adapted by us was found to

The sealed sources are tested for surface contamination or presence of any loose activity by swiping the sources using alcohol immersed cotton wool and checking the radioactive content in a NaI(Tl) scintillation counter. When the activities detected in the swipe is less

Uniformity of deposition of 125I activity was examined by autoradiography using a specially designed gadget (Fig. 11.) A circular disc [ 4.4 cm (), 1.4cm thick] made of brass with a central hole of 3mm diameter and 8 mm depth was taken and eight equidistant tunnels (45o angle between each successive tunnels) of uniform aperture were drilled through the central hole. One source was placed at a time in the central hole and autoradiographed simultaneously by wrapping a strip of photographic film all along the side of brass disc.

The film gets exposed from eight equidistant directions through the holes. The Optical density distribution of the exposed film at different angles was measured by B/W transmission densitometer. The variation in OD values at different positions should be ±10 %. Sources prepared by the physico-chemical adsorption methodology [14] in our laboratory

than 185 Bq, the sealed source capsule is considered to be contamination free.

equipment should not be used whenever the level is exceeded.

achieve this limit and thus complied with regulatory norms.

**8.2 Leachability** 

**8.3 Swipe test** 

**8.4 Uniformity of activity** 

Fig. 11. Gadget for Auto-radiography

has variation within ± 5% (Fig.12).

The fabricated sealed sources are placed in nearly boiling water kept in a glass beaker for about 2 minutes. Appearance of bubbles from the sealed sources indicates improper sealing and such sources are discarded.

#### *Pressure test*

The fabricated sealed sources are immersed in a closed glass container containing ethylene glycol and the pressure inside the chamber is reduced to 100 mm of Hg. Any leak from the sources is shown by a string of bubbles, and such sources are rejected.

#### **8.6 Immersion test**

One sealed source is immersed in 20 mL of water taken in a glass beaker and heated to 50oC for 5 hours. The source is removed, the water concentrated to 1mL and the activity released is estimated in a well type NaI(Tl) counter. Activity measuring 185 Bq /source is considered to be the limit for acceptance of leak tightness. When the activities detected in the immersion test of the sealed source is less than 185 Bq, the sealed source capsule is considered to be leak tight.

The following steps should be taken during Quality Control of 125I seeds.

i. The surface of each source must be checked regularly for visible damage. Sources with damaged surface must not be touched for wipe test.

Development of Miniature 125I - Seeds for the Treatment of Prostate Cancer 73

In order to evaluate the ability of the sealed source to withstand high impacts, a steel billet of 50 grams weight is dropped over the sealed sources from a height of one meter. The integrity the sealed source is then examined by visual examination as well as conducting leakage tests and surface contamination tests. If a wipe test or leakage tests detects less than 185Bq (0.005 μCi) of removable radioactive material, the sealed sources are consider

In view of radioactive nature of 125I, safe and appropriate radioactive procedures should be adopted during the whole preparation process. Although gamma/X-ray emanating from 125I have little penetrating power, they are hazardous if 125I is ingested or inhaled. For this reason, it is recommended to wear protective clothing and safety glasses when working with 125I. Radiation monitoring of personnel should be accomplished with TLD dosimeters worn while working. The room or area used for the preparation of 125I sources must be posted with a sign having the words "Caution - Radioactive Materials". Ventilation of the source preparation laboratory should be sufficient to quickly reduce the presence of gaseous radioactive products. It is useful to locate a surveillance monitor in the source preparation laboratory which can give a warning signal if there is significant increase in the radiation level resulting from radioactive contamination on the work place. The sensitivity of the monitor should be easily adjustable. The ALARA principle should be observed with regard to the radiation exposure of the operating staff**.** Reduction of the dose during the preparation of 25I-brachytherapy sources can be achieved by a combination of the

i. Reduce the time of exposure during handling of activity, as the total dose is proportional with time. Materials and equipment used for source preparation must be set ready. Procedures must be practiced with non-active, dummy material to gain

ii. Keep the distance as large as possible. Radioactive materials should not be touched by hand. It is recommended to use long forceps or tweezers to handle the source cores. The

iii. Reduce the amount of radioactive materials handling as far as possible. Measures must be taken to reduce the exposure from the radioactive materials not used for source preparation. Each source must be subjected quality control separately, while any other

iv. Use the shielding material that is available. Examples are the shields at the preparation

Radioactive sources cannot be left in the laboratory hood. All fabricated sources must be registered. The register must contain information on the activity on a given date and eventually the batch number and the results of the QC checks. The sources have to be locked away safely in a storage container. The radiation level at 1 m from the container surface should be less than 1 mSv. h-1 and at the surface less than 2 mSv.h-1. Storage containers must be fire resistant and carefully locked when in use to prevent access by unauthorized persons. The radiation symbols should be clearly visible on each container. A logbook must

inverse square law is the most effective method of dose reduction.

source is stored in a shielded container and set aside at some distance.

show the date of dispatch and the destination of the sources such as user's address.

hood or movable shields besides the ion chamber.

**9.3 Impact test** 

withstanding high impacts.

**10. Radiological safety** 

following principles:

experience.


#### **9. Classification performance testing**

For use of radioactive seeds in interstitial applications, where sources of higher radioactive strength of the order of 30 GBq to 370 GBq and comprising of radiologically more hazardous and longer lived radionuclides such as 137Cs, 192 Ir etc. are used, the integrity of sources upto a temperature of 600ºC is necessarily evaluated. In case of 125I-seeds, the strength of a typical implant of 100 seeds is very low ( 1.85-3.7 GBq) as compared to the strength of 137Cs or 192 Ir sources. However, the volatile nature of iodine makes the source vulnerable to air borne release of radioactivity at higher temperature exposure conditions. In view of the low levels of radioactivity, low radiotoxicity and less risk of accidents or hazards associated with 125I seeds, the exemption for test at 600ºC seeds was sought from Atomic Energy Regulatory Board, India. 125I -seeds were tested upto 400°C, a temperature that was experimentally found to release the radioactivity within the permissible levels of 185 Bq. Release of radioactivity from sources after subjecting them to various classification performance evaluation tests was found to be well within the permissible level of 185 Bq and the source design was approved by Atomic Energy Regulatory Board, India under class C-43211. Classification performance testing of indigenous 125I-seeds was carried out for ensuring their safety in brachytheray applications [17] and following tests were performed.

#### **9.1 Temperature and thermal shock tests**

Sealed sources were tested for their integrity under extreme temperature as well as quick changes in temperature. Two sources are heated to of 400 ºC in a controlled manner and kept at this temperature for one hour. Two other sources are heated to 400 ºC for 15 minutes and then quenched to 20ºC. Two other sources are cooled to - 40 ºC and kept at this temperature for 20 minutes. All these sources are subjected to tests for leakage in hot water and surface contamination as described above. When the activities detected are less than 185 Bq, the sealed source pass this test.

#### **9.2 Pressure test**

In order to assess the strength of the sources to withstand extreme pressures, two sealed sources are subjected to a pressure of 2 MPa. In a separate set-up, two sources are subjected to a vacuum of 25 kPa. After repeating two cycles of both these tests for 5 minutes, tests for leakage in hot water and surface contamination are carried out. When the activities detected in the swipe as well as in water are less than 185 Bq, the sealed sources were consider withstanding extreme pressures.

#### **9.3 Impact test**

72 Prostate Cancer – Original Scientific Reports and Case Studies

iv. Ion chamber used for measuring activity should be used solely for that purpose only

v. Swipe test should be carried out using a dedicated surface contamination detector which can easily show even the smallest amount of radioactive material. The table used for keeping the surface contamination detector must be separated from the source preparation laboratory. The instruments may get contaminated and must be decontaminated regularly. The verification of the absence of radiation in the surface

For use of radioactive seeds in interstitial applications, where sources of higher radioactive strength of the order of 30 GBq to 370 GBq and comprising of radiologically more hazardous and longer lived radionuclides such as 137Cs, 192 Ir etc. are used, the integrity of sources upto a temperature of 600ºC is necessarily evaluated. In case of 125I-seeds, the strength of a typical implant of 100 seeds is very low ( 1.85-3.7 GBq) as compared to the strength of 137Cs or 192 Ir sources. However, the volatile nature of iodine makes the source vulnerable to air borne release of radioactivity at higher temperature exposure conditions. In view of the low levels of radioactivity, low radiotoxicity and less risk of accidents or hazards associated with 125I seeds, the exemption for test at 600ºC seeds was sought from Atomic Energy Regulatory Board, India. 125I -seeds were tested upto 400°C, a temperature that was experimentally found to release the radioactivity within the permissible levels of 185 Bq. Release of radioactivity from sources after subjecting them to various classification performance evaluation tests was found to be well within the permissible level of 185 Bq and the source design was approved by Atomic Energy Regulatory Board, India under class C-43211. Classification performance testing of indigenous 125I-seeds was carried out for ensuring their

safety in brachytheray applications [17] and following tests were performed.

Sealed sources were tested for their integrity under extreme temperature as well as quick changes in temperature. Two sources are heated to of 400 ºC in a controlled manner and kept at this temperature for one hour. Two other sources are heated to 400 ºC for 15 minutes and then quenched to 20ºC. Two other sources are cooled to - 40 ºC and kept at this temperature for 20 minutes. All these sources are subjected to tests for leakage in hot water and surface contamination as described above. When the activities detected are less than

In order to assess the strength of the sources to withstand extreme pressures, two sealed sources are subjected to a pressure of 2 MPa. In a separate set-up, two sources are subjected to a vacuum of 25 kPa. After repeating two cycles of both these tests for 5 minutes, tests for leakage in hot water and surface contamination are carried out. When the activities detected in the swipe as well as in water are less than 185 Bq, the sealed sources were consider

ii. All quality control results must be documented.

and should be kept separately.

**9. Classification performance testing** 

**9.1 Temperature and thermal shock tests** 

185 Bq, the sealed source pass this test.

withstanding extreme pressures.

**9.2 Pressure test** 

iii. For each type of test separate instrument should be used.

contamination detector should be a routine task.

In order to evaluate the ability of the sealed source to withstand high impacts, a steel billet of 50 grams weight is dropped over the sealed sources from a height of one meter. The integrity the sealed source is then examined by visual examination as well as conducting leakage tests and surface contamination tests. If a wipe test or leakage tests detects less than 185Bq (0.005 μCi) of removable radioactive material, the sealed sources are consider withstanding high impacts.

#### **10. Radiological safety**

In view of radioactive nature of 125I, safe and appropriate radioactive procedures should be adopted during the whole preparation process. Although gamma/X-ray emanating from 125I have little penetrating power, they are hazardous if 125I is ingested or inhaled. For this reason, it is recommended to wear protective clothing and safety glasses when working with 125I. Radiation monitoring of personnel should be accomplished with TLD dosimeters worn while working. The room or area used for the preparation of 125I sources must be posted with a sign having the words "Caution - Radioactive Materials". Ventilation of the source preparation laboratory should be sufficient to quickly reduce the presence of gaseous radioactive products. It is useful to locate a surveillance monitor in the source preparation laboratory which can give a warning signal if there is significant increase in the radiation level resulting from radioactive contamination on the work place. The sensitivity of the monitor should be easily adjustable. The ALARA principle should be observed with regard to the radiation exposure of the operating staff**.** Reduction of the dose during the preparation of 25I-brachytherapy sources can be achieved by a combination of the following principles:


Radioactive sources cannot be left in the laboratory hood. All fabricated sources must be registered. The register must contain information on the activity on a given date and eventually the batch number and the results of the QC checks. The sources have to be locked away safely in a storage container. The radiation level at 1 m from the container surface should be less than 1 mSv. h-1 and at the surface less than 2 mSv.h-1. Storage containers must be fire resistant and carefully locked when in use to prevent access by unauthorized persons. The radiation symbols should be clearly visible on each container. A logbook must show the date of dispatch and the destination of the sources such as user's address.

Development of Miniature 125I - Seeds for the Treatment of Prostate Cancer 75

[1] Localized Prostate Cancer-Patrick C. Walsh, Theodore L. DeWeese, and Mario A.

[2] Prostate Canvcer Brachytherapy: Clinical and Financial Imperatives for Permanent

[3] Porter AT, Blasko JC, Grimm PD, Reddy SM, Ragde H: Brachytherapy for prostate

[4] Scardino P, Carlton C: Combined interstitial and external irradiation for prostatic cancer,

[5] Whitmore WF, Jr., Hilaris B, Grabstald H: Retropubic implantation to iodine 125 in the

[6] Puthawala A, Syed A, Tansey L: Temporary iridium implant in the management of

[7] Holm HH, Juul N, Pedersen JF, Hansen H, Stroyer I: Transperineal I-125odine seed

[8] Blasko JC, Radge H, Schumacher D: Transperineal Percutaneous Iodine-125

[9] R. B. Manolkar, S. U. Sane, K. T Pillai, M. A. Majali, Comparison of methods for

[10] Izabela Cieszykowska, Andrzej Piasecki, Mieczysław Mielcarski, An approach to the preparation of iodine-125 seed-type sources, NUKLEONIKA 2005;50(1):17-22 [11] Chunfu Zhang, Yongxian Wang, Haibin Tian, Duan Zhiyin Preparation of iodine-125

[12] H. S. Han, U. J. Park, A. Dash, The absorption of iodine-131 on a ceramic matrix, Journal of Radioanalytical and Nuclear Chemistry, Vol. 262, No. 3 (2004) 703.705. [13] U. J. Park, J. S. Lee, K. J. Son, H. S. Han and S. S. Nam, The adsorption of 125I on a

Radioanalytical and Nuclear Chemistry, Vol. 277, No. 7 (2008) 429-432. [14] Saxena SK, Shanta A., Rajurkar Nilima S, Majali MA, Studies on the production and

[15] Mathew C., Majali MA, Balakrishnan SA., A novel approach for the adsorption of 125I

[16] Sanjay Kumar Saxena, S.D.Sharma, Ashutosh Dash, Meera Venkatesh, Development of

and prostate cancer Applied Radiation and Isotopes, 67 (2009) 1421–1425

ocular and prostate cancers. Appl. Radiat. Isot. 2006; 64: 441-447.

carcinoma of the prostate. Endocurie Hyper Oncol 1985; 1:25-33

Guidance. Endocurietherapy Hyperthermia Oncol 1987; 3:131-139

Implantation, in Oncology Roundtable Annual Meeting. Washington, D.C., The

in Principles and Management of Urologic Cancer. Edited by Javadpour N.

treatment of prostatic cancer. J Urol 1972; 108:918-20 19. Blasko JC, Grimm PD, Ragde H: Brachytherapy and organ preservation in the management of carcinoma

implantation in prostatic cancer guided by transrectal ultrasonography. J Urol 1983;

Implantation For Prostatic Carcinoma Using Transrectal Ultrasound and Template

preparation of 125I brachytherapy source cores for the treatment of eye cancer, Applied Radiation and Isotopes, Volume 59, Issues 2-3, August-September 2003,

seed,Part I: Iodination of the silver rod, Journal of Radioanalytical and Nuclear

Ag+Al2O3 rod as a carrier body for a brachytherapy source, Journal of

quality assurance of miniature 125 I radioactive sources suitable for treatment of

on silver wire as matrix for brachytherapy for the treatment of eye and prostate

a new design 125I-brachytherapy seed for its application in the treatment of eye

Eisenberger- N Engl J Med 2007;357:2696-705.

cancer . CA Cancer J Clin 1995; 45:165-78

Chemistry, Vol. 252, No. 1 (2002) 161–163

cancer. Appl. Radiat. Isot. 2002; 57:359-367.

Baltimore, Williams & Wilkins, 1983, pp 392-408.

of the prostate. Semin. Radiat. Oncol. 1993; 3:240-249

Advisory Board Co., 2000, p 15

**13. References** 

130:283-286

Pages 145-150

#### **11. Conclusion**

The technique of permanent seed implantation in management of prostate carcinoma is historically proven and is being widely used in many advanced countries. Modern transrectal ultrasound-guided, interstitial permanent brachytherapy is a single outpatient treatment for the majority of men with early-stage prostate cancer. It has documented fiveand ten-year biochemical, overall, and disease-specific relapse-free survival rates that equal the best that radical prostatectomy has thus far achieved. These favorable findings have established permanent prostate brachytherapy as a primary treatment option for early stage prostate cancer.

The potential utility of various methods for the preparation of 125I-brachytherapy sources for prostate cancer have been documented. Quality control of 125I-brachytherapy sources after preparation is an important part. Many publications give recommendations on frequencies of quality control procedures without describing the procedures. It is, however, of extreme importance for the general process of quality assurance that these procedures are well defined and understood by the source manufactures. The quality control procedure depicted in this manuscript can be applied universally irrespective of the source preparation method adapted. The procedures presented here are a set of minimum requirements and can serve as guidelines for developing a QC protocol. It should be noted, however, that if any national set of requirements exists, these should be followed. It is envisaged that any one of the source preparation strategy would serve for ensuring easy availability 125Ibrachytherapy sources particularly in Institution with Radiochemistry Laboratory facility where commercial sources are too expensive. Availability 125I-brachytherapy sources at local level would promote the beneficial use of permanent seed implantation technique.

#### **12. Acknowledgements**

The authors express their gratitude to all who have contributed in the preparation of this chapter. The authors are highly grateful to Prof. M. R. A. Pillai, Head, Radiopharmaceuticals Division for his keen interest, guidance, encouragement, valuable scientific discussions and support. The authors also wish to thank Dr. Meera Venkatesh, former Head, Radiopharmaceuticals Division for her encouragement and administrative support. The authors wish to thank Prof. V. Venugopal, Director, Radiochemistry and Isotope Group and Prof. K.L. Ramkumar, Associate Director, Radiochemistry and Isotope Group for their administrative support. The authors are indebted to their ex-colleagues Mr. S.A.Balakrishnan, Dr. M.A. Majali, Mr. C. Mathew who have started the work and brought to the this shape. The authors also thanks Mr. P. Sreeramakrishnan and Mr. A.S.Tapase of Isotope Applications Division, BARC for their contribution in the autoradiography analysis, Mr. K.C. Sahoo and Mr. E.Ramadasan of Post Irradiation Examination Division, BARC for the metallography analysis of welded capsules,Dr. K.T. Pillai, Fuel Chemistry Division for his contribution in making the alumina microspheres, Dr. C.G.S.Pillai, Chemistry Division, BARC for his contribution in the SEM analysis. The authors are grateful to Dr. (Mrs.) A. Shanta and Dr. S.D.Sharma of Radiological Physics & Advisory Division of our Institute for establishing clinical dosimetric parameters. The authors are grateful to the Engineers of Centre for Design and Manufacture for undertaking the fabrication of titanium capsules.

#### **13. References**

74 Prostate Cancer – Original Scientific Reports and Case Studies

The technique of permanent seed implantation in management of prostate carcinoma is historically proven and is being widely used in many advanced countries. Modern transrectal ultrasound-guided, interstitial permanent brachytherapy is a single outpatient treatment for the majority of men with early-stage prostate cancer. It has documented fiveand ten-year biochemical, overall, and disease-specific relapse-free survival rates that equal the best that radical prostatectomy has thus far achieved. These favorable findings have established permanent prostate brachytherapy as a primary treatment option for early stage

The potential utility of various methods for the preparation of 125I-brachytherapy sources for prostate cancer have been documented. Quality control of 125I-brachytherapy sources after preparation is an important part. Many publications give recommendations on frequencies of quality control procedures without describing the procedures. It is, however, of extreme importance for the general process of quality assurance that these procedures are well defined and understood by the source manufactures. The quality control procedure depicted in this manuscript can be applied universally irrespective of the source preparation method adapted. The procedures presented here are a set of minimum requirements and can serve as guidelines for developing a QC protocol. It should be noted, however, that if any national set of requirements exists, these should be followed. It is envisaged that any one of the source preparation strategy would serve for ensuring easy availability 125Ibrachytherapy sources particularly in Institution with Radiochemistry Laboratory facility where commercial sources are too expensive. Availability 125I-brachytherapy sources at local

level would promote the beneficial use of permanent seed implantation technique.

The authors express their gratitude to all who have contributed in the preparation of this chapter. The authors are highly grateful to Prof. M. R. A. Pillai, Head, Radiopharmaceuticals Division for his keen interest, guidance, encouragement, valuable scientific discussions and support. The authors also wish to thank Dr. Meera Venkatesh, former Head, Radiopharmaceuticals Division for her encouragement and administrative support. The authors wish to thank Prof. V. Venugopal, Director, Radiochemistry and Isotope Group and Prof. K.L. Ramkumar, Associate Director, Radiochemistry and Isotope Group for their administrative support. The authors are indebted to their ex-colleagues Mr. S.A.Balakrishnan, Dr. M.A. Majali, Mr. C. Mathew who have started the work and brought to the this shape. The authors also thanks Mr. P. Sreeramakrishnan and Mr. A.S.Tapase of Isotope Applications Division, BARC for their contribution in the autoradiography analysis, Mr. K.C. Sahoo and Mr. E.Ramadasan of Post Irradiation Examination Division, BARC for the metallography analysis of welded capsules,Dr. K.T. Pillai, Fuel Chemistry Division for his contribution in making the alumina microspheres, Dr. C.G.S.Pillai, Chemistry Division, BARC for his contribution in the SEM analysis. The authors are grateful to Dr. (Mrs.) A. Shanta and Dr. S.D.Sharma of Radiological Physics & Advisory Division of our Institute for establishing clinical dosimetric parameters. The authors are grateful to the Engineers of Centre for Design and Manufacture for

**11. Conclusion** 

prostate cancer.

**12. Acknowledgements** 

undertaking the fabrication of titanium capsules.


**5** 

*Italy* 

**LNCaP Prostate Cancer Growth** *In Vivo***:** 

**Compared to Hypoxia and Reoxygenation** 

Oxygen (O2) is often thought of as a double-edged sword: all forms of life actually need O2 for survival as the terminal acceptor of electrons in oxidative phosphorylation, but excess O2 might increase formation of reactive O2 species (ROS). Whereas on one hand ROS trigger uncontrolled burst of free radicals that lead to potentially lethal injury, on the other hand they act as messengers that elicit cell protection and improve survival through a variety of mechanisms. Although one can easily expect a link of O2 with tumor growth and metastatic

Lack of O2, or hypoxia, has been extensively studied in cancer because the growth of solid tumors requires a local vascular network that supplies O2 and nutrients to tumor cells. In the classical view, when cell proliferation exceeds angiogenesis, the vasculature might become unable to sustain the O2 needs of tumor cells, which therefore have to cope with an environment chronically deficient in O2 as a result of diffusion-limited O2 supply (Vaupel, 2004). This triggers various mechanisms, most of which are mediated by over-expression of the hypoxia-inducible factor-1 (HIF-1). HIF-1 stimulates a variety of mechanisms aimed at survival of hypoxic tissue, for example the angiogenic switch, which provides growth factors for the development of circulation to feed the growing tumor, as well as antiapoptotic and cell cycle factors. Therefore, tumor hypoxia emerges as a major contributor to the malignant phenotype (Hockel *et al.*, 2001) and a cause of resistance to radiation therapy

There are several instances whereby the O2 supply to tissues is altered. The term "hypoxia" is sometimes improperly attributed to any of them, but there are important differences with

**1. Introduction** 

**1.1 Hypoxia in cancer** 

(Brown *et al.*, 1998).

 \*

potential, there is no univocal role for O2 in cancer.

dramatically different phenotypes, as explained in Table 1.

These Authors contributed equally to the work

**Oncostatic Effects of Melatonin as** 

L. Terraneo\*, E. Finati\*, E. Virgili, G. Demartini,

L. De Angelis, R. Dall'Aglio, F. Fraschini,

M. Samaja and R. Paroni *University of Milan, Milan* 


### **LNCaP Prostate Cancer Growth** *In Vivo***: Oncostatic Effects of Melatonin as Compared to Hypoxia and Reoxygenation**

L. Terraneo\*, E. Finati\*, E. Virgili, G. Demartini, L. De Angelis, R. Dall'Aglio, F. Fraschini, M. Samaja and R. Paroni *University of Milan, Milan Italy* 

#### **1. Introduction**

76 Prostate Cancer – Original Scientific Reports and Case Studies

[17] Saxena S.K.,Yogendra Kumar, Manoj Kumar, Ashutosh Dash and Meera Venkatesh

[18] Regulations for the Safe transport of Radioactive material, 2005 Edition, International

[19] International Standard ISO 2919:1999, Radiation protection- Sealed radioactive sources-

[20] International Standard ISO 9978:1992, Radiation protection-Sealed radioactive sources-

(ICMP-2008) ,49-50

Leakage test methods

Atomic Energy Agency (IAEA) No. TS-R-1

General requirements and classification

(2008),Quality Assurance and Classification Performance Testing of 125Ibrachytherapy Seeds, Proceedings of International Conference on Medical Physics

> Oxygen (O2) is often thought of as a double-edged sword: all forms of life actually need O2 for survival as the terminal acceptor of electrons in oxidative phosphorylation, but excess O2 might increase formation of reactive O2 species (ROS). Whereas on one hand ROS trigger uncontrolled burst of free radicals that lead to potentially lethal injury, on the other hand they act as messengers that elicit cell protection and improve survival through a variety of mechanisms. Although one can easily expect a link of O2 with tumor growth and metastatic potential, there is no univocal role for O2 in cancer.

#### **1.1 Hypoxia in cancer**

Lack of O2, or hypoxia, has been extensively studied in cancer because the growth of solid tumors requires a local vascular network that supplies O2 and nutrients to tumor cells. In the classical view, when cell proliferation exceeds angiogenesis, the vasculature might become unable to sustain the O2 needs of tumor cells, which therefore have to cope with an environment chronically deficient in O2 as a result of diffusion-limited O2 supply (Vaupel, 2004). This triggers various mechanisms, most of which are mediated by over-expression of the hypoxia-inducible factor-1 (HIF-1). HIF-1 stimulates a variety of mechanisms aimed at survival of hypoxic tissue, for example the angiogenic switch, which provides growth factors for the development of circulation to feed the growing tumor, as well as antiapoptotic and cell cycle factors. Therefore, tumor hypoxia emerges as a major contributor to the malignant phenotype (Hockel *et al.*, 2001) and a cause of resistance to radiation therapy (Brown *et al.*, 1998).

There are several instances whereby the O2 supply to tissues is altered. The term "hypoxia" is sometimes improperly attributed to any of them, but there are important differences with dramatically different phenotypes, as explained in Table 1.

<sup>\*</sup> These Authors contributed equally to the work

LNCaP Prostate Cancer Growth *In Vivo*:

2003; Kotler *et al.*, 1998).

Fig. 1. Structure of melatonin, MW 232.2 Da.

dihydrotestosterone-induced calcium influx model (Xi *et al.*, 2000).

**1.4 Solid lipid nanoparticles and cryopass laser therapy** 

Oncostatic Effects of Melatonin as Compared to Hypoxia and Reoxygenation 79

vessels, and brain (Beyer *et al.*, 1998). This suggests some significant actions of melatonin on the cell biology of these target tissues outside the central nervous system as well (Cardinali *et al.*, 1997; Pang *et al.*, 1993). In addition, melatonin is known to exhibit antioxidant properties against the deleterious effects of reactive oxygen and nitrogen species (ROS and RNS, respectively) that are independent of its many receptor-mediated effects (Korkmaz *et al.*, 2009; Ochoa *et al.*, 2011). Melatonin has been reported to scavenge hydrogen peroxide (H2O2), hydroxyl radical (HO•), nitric oxide (NO•), peroxynitrite anion (ONOO-

hypochlorous acid (HOCl), singlet molecular oxygen [O2(1g)] and superoxide anion (O2

(Allegra *et al.*, 2003; Reiter, 1998; Reiter *et al.*, 2001; Tan *et al.*, 2000). Melatonin has also been shown to possess genomic actions, through regulation of the expression of several genes including glutathione peroxidase, superoxide dismutase, and catalase, both under physiological conditions and under conditions of elevated oxidative stress (Allegra *et al.*,

A multitude of literature reports have documented a direct modulatory effect of melatonin on benign and malignant cell proliferation and an anti-tumor effect was reported both in vitro and in vivo in human tumors of the reproductive tissues (Cos *et al.*, 1998; Shiu *et al.*, 1999). Among these, a significant role of melatonin, by itself and by interaction with sex steroids, has been found in the pathobiology of prostate cancer and benign prostatic hyperplasia (Laudon *et al.*, 1996; Lupowitz *et al.*, 1999). Of the various experimental prostate cancer models, hormone (androgen)-sensitive and hormone (androgen)-insensitive metastatic human prostate cancer cell lines are widely used for the experimental evaluation of pharmacological agents with therapeutic potential for the disease (Cho *et al.*, 2011). In particular, the growth of the androgen-independent but androgen-sensitive (responsive) human LNCaP prostate cancer cells has been demonstrated to be inhibited by the pineal gland indoleamine hormone both in vitro and in vivo in a nude mice xenograft model (Siu *et al.*, 2002; Xi *et al.*, 2000). The antiproliferative action of melatonin seems to be mediated in part by means of MT1 receptor activation and partly by means of attenuation of

Cytostatic and antitumoral drugs activity is often impaired by low plasma solubility, poor systemic absorbance, rapid metabolism, non-specific tissue distribution and toxic effects. The search for advanced methods to deliver these molecules to target tissues and to

overcome the failure shown even with very active drugs is therefore mandatory.

),

•-)


Table 1. Different types of hypoxia and examples of correlated pathologies.

#### **1.2 Prostate cancer**

Prostate cancer is the most common neoplasia and the second most frequent cause of male cancer death in the developed world and in many Western countries (Hsing *et al.*, 2000). A malignant tumor derived from the interaction of genetic and environmental factors, potentially curative treatment options are available for management of early localized disease, whereas palliative hormonal therapy in the form of medical or surgical castration is the mainstay of treatment for patients with advanced prostate cancer. Approximately 80% of castrated patients will suffer from a relapse of the disease within 2 years, with progression of the tumor from a hormone-dependent to a hormone-independent stage (Wilding, 1995), which is associated with unfavorable prognosis.

In addition, prostate cancer may also represent an useful workbench to investigate the relationship between hypoxia and cancer, because HIF-1 is over-expressed compared with normal prostate epithelium (Zhong *et al.*, 1999) and its up-regulation is recognized as an early event in carcinogenesis (Zhong *et al.*, 2004). Furthermore, androgens and androgen receptors modulate HIF-1 levels (Kimbro *et al.*, 2006), and hypoxia increases androgen receptor activity in LNCaP cells in *vitro* via HIF-1 (Park *et al.*, 2006).

#### **1.3 Melatonin and prostate cancer**

Melatonin (N-acetyl-5-methoxytryptamine, **Figure 1**), a neurohormone synthesized during night time in the pineal gland, mediates many physiological, endocrinological and behavioral processes, including the well-known biorhythmic regulation of organism physiology, through its action on the biological clock at the hypothalamic suprachiasmatic nucleus (Tamarkin *et al.*, 1985). Melatonin increases sleepiness, decreases core temperature, and increases peripheral temperature in humans (Brzezinski, 1997; Burgess *et al.*, 2001; Lewy *et al.*, 1996). The melatonin's regulatory roles are mediated through high affinity G protein– coupled receptors that reside primarily in the eye, kidney, gastrointestinal tract, blood

*Name Relative experimental situation Correlated pathologies* 

Chronic obstructive

Some cases of immature capillary network with pulsing perfusion changes

Obstructive sleep apnea, sickle cell anemia crises, asthma, immature capillary network with pulsing perfusion changes

altitude

pulmonary disease, congenital heart disease, cancer-derived anemia, blood O2 carrying failure, CO poisoning, high

Prolonged O2 supply/demand unbalance without interruption, typical of decreased tissue perfusion for altered geometry of O2 diffusion

from capillary to cell

Animals housed in hypoxic chambers that are opened for cleaning and animal feeding

Repetitive hypoxic events, typical of immature capillary network with pulsing perfusion changes

Table 1. Different types of hypoxia and examples of correlated pathologies.

receptor activity in LNCaP cells in *vitro* via HIF-1 (Park *et al.*, 2006).

Prostate cancer is the most common neoplasia and the second most frequent cause of male cancer death in the developed world and in many Western countries (Hsing *et al.*, 2000). A malignant tumor derived from the interaction of genetic and environmental factors, potentially curative treatment options are available for management of early localized disease, whereas palliative hormonal therapy in the form of medical or surgical castration is the mainstay of treatment for patients with advanced prostate cancer. Approximately 80% of castrated patients will suffer from a relapse of the disease within 2 years, with progression of the tumor from a hormone-dependent to a hormone-independent stage (Wilding, 1995),

In addition, prostate cancer may also represent an useful workbench to investigate the relationship between hypoxia and cancer, because HIF-1 is over-expressed compared with normal prostate epithelium (Zhong *et al.*, 1999) and its up-regulation is recognized as an early event in carcinogenesis (Zhong *et al.*, 2004). Furthermore, androgens and androgen receptors modulate HIF-1 levels (Kimbro *et al.*, 2006), and hypoxia increases androgen

Melatonin (N-acetyl-5-methoxytryptamine, **Figure 1**), a neurohormone synthesized during night time in the pineal gland, mediates many physiological, endocrinological and behavioral processes, including the well-known biorhythmic regulation of organism physiology, through its action on the biological clock at the hypothalamic suprachiasmatic nucleus (Tamarkin *et al.*, 1985). Melatonin increases sleepiness, decreases core temperature, and increases peripheral temperature in humans (Brzezinski, 1997; Burgess *et al.*, 2001; Lewy *et al.*, 1996). The melatonin's regulatory roles are mediated through high affinity G protein– coupled receptors that reside primarily in the eye, kidney, gastrointestinal tract, blood

*Chronic hypoxia* 

*CH with repeated reoxygenation* 

*Intermittent hypoxia* 

**1.2 Prostate cancer** 

which is associated with unfavorable prognosis.

**1.3 Melatonin and prostate cancer** 

vessels, and brain (Beyer *et al.*, 1998). This suggests some significant actions of melatonin on the cell biology of these target tissues outside the central nervous system as well (Cardinali *et al.*, 1997; Pang *et al.*, 1993). In addition, melatonin is known to exhibit antioxidant properties against the deleterious effects of reactive oxygen and nitrogen species (ROS and RNS, respectively) that are independent of its many receptor-mediated effects (Korkmaz *et al.*, 2009; Ochoa *et al.*, 2011). Melatonin has been reported to scavenge hydrogen peroxide (H2O2), hydroxyl radical (HO•), nitric oxide (NO•), peroxynitrite anion (ONOO- ), hypochlorous acid (HOCl), singlet molecular oxygen [O2(1g)] and superoxide anion (O2 •-) (Allegra *et al.*, 2003; Reiter, 1998; Reiter *et al.*, 2001; Tan *et al.*, 2000). Melatonin has also been shown to possess genomic actions, through regulation of the expression of several genes including glutathione peroxidase, superoxide dismutase, and catalase, both under physiological conditions and under conditions of elevated oxidative stress (Allegra *et al.*, 2003; Kotler *et al.*, 1998).

Fig. 1. Structure of melatonin, MW 232.2 Da.

A multitude of literature reports have documented a direct modulatory effect of melatonin on benign and malignant cell proliferation and an anti-tumor effect was reported both in vitro and in vivo in human tumors of the reproductive tissues (Cos *et al.*, 1998; Shiu *et al.*, 1999). Among these, a significant role of melatonin, by itself and by interaction with sex steroids, has been found in the pathobiology of prostate cancer and benign prostatic hyperplasia (Laudon *et al.*, 1996; Lupowitz *et al.*, 1999). Of the various experimental prostate cancer models, hormone (androgen)-sensitive and hormone (androgen)-insensitive metastatic human prostate cancer cell lines are widely used for the experimental evaluation of pharmacological agents with therapeutic potential for the disease (Cho *et al.*, 2011). In particular, the growth of the androgen-independent but androgen-sensitive (responsive) human LNCaP prostate cancer cells has been demonstrated to be inhibited by the pineal gland indoleamine hormone both in vitro and in vivo in a nude mice xenograft model (Siu *et al.*, 2002; Xi *et al.*, 2000). The antiproliferative action of melatonin seems to be mediated in part by means of MT1 receptor activation and partly by means of attenuation of dihydrotestosterone-induced calcium influx model (Xi *et al.*, 2000).

#### **1.4 Solid lipid nanoparticles and cryopass laser therapy**

Cytostatic and antitumoral drugs activity is often impaired by low plasma solubility, poor systemic absorbance, rapid metabolism, non-specific tissue distribution and toxic effects. The search for advanced methods to deliver these molecules to target tissues and to overcome the failure shown even with very active drugs is therefore mandatory.

LNCaP Prostate Cancer Growth *In Vivo*:

melatonin administration.

**2. Methods** 

as described below.

12/12 h light/dark cycle was maintained.

where they were treated accordingly.

experimental situations (**Figure 2**).

pharmacological treatments able to modulate these pathways.

Oncostatic Effects of Melatonin as Compared to Hypoxia and Reoxygenation 81

cells. We demonstrated that systemic chronic *in vivo* hypoxia promotes prostate cancer growth regardless of HIF-1 expression level and neovascularization. We have also assessed that altering HIF-1 expression by use of non-pharmacological agents (intermittent hypoxia with reoxygenation) alter the phenotype of tumor growth. These observations suggest an important role for hypoxia dependent pathways that do not involve HIF-1, and for the

Pharmacologic concentrations of melatonin inhibit *in vitro* expression of HIF-1 protein under both normoxic and hypoxic conditions in DU145, PC-3, and LNCaP prostate cancer cells (Park *et al.*, 2009). This effect, perhaps a result of the antioxidant activity of melatonin against ROS induced by hypoxia, and the subsequent suppression of HIF-1 transcriptional activity decreases VEGF expression in HCT116 human colon cancer cell line (Park *et al.*, 2010). Therefore, the main aim of this preliminary study was to focus into the role of melatonin as a therapeutic/adjuvant agent that reduces tumor growth in vivo as a proof-of-concept for further studies assessing melatonin interference with the hypoxia signaling paths. To this purpose, we used the *in-vivo* model of nude mice xenograft with human LNCaP prostate cancer cells under a variety of conditions spanning from chronic hypoxia with/out reoxygenation, and compared different routes of

*Cells.* LNCaP cells (80-90% confluence) were maintained in RPMI-1640 medium containing 10% (v/v) heat-inactivated fetal bovine serum and L-glutamine, and cultured in 5% CO2. To ensure that LNCaP cells were not injured when passing through G26 needles during xenografts, we verified that their vitality did not decreased by more 2-3% per each passage. To obtain positive controls for HIF-1 immunostaining, LNCaP cells were incubated for 24 h in the presence of 100 M CoCl2, washed, fixed in formalin and stained

*Mice.* Seven-week old Foxn1nu/nu mice (Harlan, n=46), weighing 25-30 g at the entry into the study, were cared in accordance to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, revised 1996). Water and bedding were heat-sterilized, whereas food was sterilized by 60Co -irradiation. Mice had free access to water and conventional laboratory diet until 24 h before sacrifice. A

*Xenografts.* LNCaP cells were resuspended in ice-cold Matrigel (1:1) at a final concentration of 3106/0.1 ml. Mice were inoculated in each flank with 0.1 ml of cells using a 26G insulin syringe. The next day, mice were transferred into the gas chamber,

*In vivo measurements of tumor growth.* Body weight and tumor volume were measured three times a week during the various treatments. The tumor volume was calculated as lengthwidthheight0.5236, as measured by a caliper. Data are expressed as the ratio (tumor volume)/(body weight) to compensate different rates of growth in the various

*Sacrifice.* At the end of the observation period, mice were anesthetized by i.p. Na-thiopental (10 mg/100 g body weight) plus heparin (500 units), then they were thoracotomized to

Solid-lipid nanoparticles (SLN) is a technology able to produce sub-micrometric lipidic particles characterized by an average diameter <500 nm, with a narrow size distribution and a spherical shape. The several advantages of this technology are listed in Table 2 (Mehnert *et al.*, 2001). SLN formulation of antitumor drugs is predicted to be advantageous with respect to other formulations because it allows greater uptake into the malignant cell, with consequent intracellular accumulation and higher efficacy, still keeping the level of free circulating drug as low as possible, thereby preventing nonspecific unwanted side effects.

#### *Main advantages of SLN formulation for drugs delivery.*

*To control and direct the release of the drug against a specific target organ* 

*To modify favorably drugs pharmacokinetics* 

*To target directly to lymph bypassing the entero-hepatic circulation following oral administration* 

*To obtain a stealth formulation that avoids recognition by the reticuloendothelial system To allow oral administration of drugs poorly soluble and not easily adsorbed in the gastrointestinal tract* 

*To overcome the blood-brain barrier* 

*To permit administration routes as transdermal or eye topical* 

Table 2. Main advantages to administer drugs as solid-lipid nanoparticles (SLN).

Criopass therapy, is a procedure used to actively deliver drugs across the dermal barrier. It is based on topical application of a frozen drug emulsion in 1.5% hydroxymethylcellulose by means of a laser source that gives energy to penetrate the dermal barrier and deliver the active principle to the target area. The low energy photon flux generated by a laser beam hit the drug molecules frozen in the crystal lattice, exciting the electrons in the outer orbital. The drug molecules, when melt at the ice-skin interface, release the accumulated potential energy transforming into kinetic energy, which speeds up the passage of the drug across the skin membrane and allows to reach the target area. The last step consists in a laser scan on the area of drug application to optimize the adsorption through the skin and facilitate the drug to reach the desired site of activity. This treatment is particularly advantageous for treatment of bones cartilage, producing significant drug accumulation in a tissue difficult to be reached by traditional administration techniques. The main advantages of this noninvasive and painless treatment are speed of absorption (15-20 s), high capacity of penetration (6 cm depth), suitability for polar and non-polar molecules delivery, improved drug bioavailability and high specificity.

#### **1.5 Aims**

Solid tumors contain underperfused regions where hypoxia might induce adaptation and cell proliferation. Often, this response is mediated by HIF-1 over-expression in hypoxic cells. We demonstrated that systemic chronic *in vivo* hypoxia promotes prostate cancer growth regardless of HIF-1 expression level and neovascularization. We have also assessed that altering HIF-1 expression by use of non-pharmacological agents (intermittent hypoxia with reoxygenation) alter the phenotype of tumor growth. These observations suggest an important role for hypoxia dependent pathways that do not involve HIF-1, and for the pharmacological treatments able to modulate these pathways.

Pharmacologic concentrations of melatonin inhibit *in vitro* expression of HIF-1 protein under both normoxic and hypoxic conditions in DU145, PC-3, and LNCaP prostate cancer cells (Park *et al.*, 2009). This effect, perhaps a result of the antioxidant activity of melatonin against ROS induced by hypoxia, and the subsequent suppression of HIF-1 transcriptional activity decreases VEGF expression in HCT116 human colon cancer cell line (Park *et al.*, 2010). Therefore, the main aim of this preliminary study was to focus into the role of melatonin as a therapeutic/adjuvant agent that reduces tumor growth in vivo as a proof-of-concept for further studies assessing melatonin interference with the hypoxia signaling paths. To this purpose, we used the *in-vivo* model of nude mice xenograft with human LNCaP prostate cancer cells under a variety of conditions spanning from chronic hypoxia with/out reoxygenation, and compared different routes of melatonin administration.

#### **2. Methods**

80 Prostate Cancer – Original Scientific Reports and Case Studies

Solid-lipid nanoparticles (SLN) is a technology able to produce sub-micrometric lipidic particles characterized by an average diameter <500 nm, with a narrow size distribution and a spherical shape. The several advantages of this technology are listed in Table 2 (Mehnert *et al.*, 2001). SLN formulation of antitumor drugs is predicted to be advantageous with respect to other formulations because it allows greater uptake into the malignant cell, with consequent intracellular accumulation and higher efficacy, still keeping the level of free circulating drug as low as possible, thereby preventing non-

*Main advantages of SLN formulation for drugs delivery.* 

*To control and direct the release of the drug against a specific target organ* 

*To permit administration routes as transdermal or eye topical* 

*To target directly to lymph bypassing the entero-hepatic circulation following oral* 

Table 2. Main advantages to administer drugs as solid-lipid nanoparticles (SLN).

*To obtain a stealth formulation that avoids recognition by the reticuloendothelial system To allow oral administration of drugs poorly soluble and not easily adsorbed in the* 

Criopass therapy, is a procedure used to actively deliver drugs across the dermal barrier. It is based on topical application of a frozen drug emulsion in 1.5% hydroxymethylcellulose by means of a laser source that gives energy to penetrate the dermal barrier and deliver the active principle to the target area. The low energy photon flux generated by a laser beam hit the drug molecules frozen in the crystal lattice, exciting the electrons in the outer orbital. The drug molecules, when melt at the ice-skin interface, release the accumulated potential energy transforming into kinetic energy, which speeds up the passage of the drug across the skin membrane and allows to reach the target area. The last step consists in a laser scan on the area of drug application to optimize the adsorption through the skin and facilitate the drug to reach the desired site of activity. This treatment is particularly advantageous for treatment of bones cartilage, producing significant drug accumulation in a tissue difficult to be reached by traditional administration techniques. The main advantages of this noninvasive and painless treatment are speed of absorption (15-20 s), high capacity of penetration (6 cm depth), suitability for polar and non-polar molecules delivery, improved

Solid tumors contain underperfused regions where hypoxia might induce adaptation and cell proliferation. Often, this response is mediated by HIF-1 over-expression in hypoxic

specific unwanted side effects.

*administration* 

*gastrointestinal tract* 

*To overcome the blood-brain barrier* 

drug bioavailability and high specificity.

**1.5 Aims** 

*To modify favorably drugs pharmacokinetics* 

*Cells.* LNCaP cells (80-90% confluence) were maintained in RPMI-1640 medium containing 10% (v/v) heat-inactivated fetal bovine serum and L-glutamine, and cultured in 5% CO2. To ensure that LNCaP cells were not injured when passing through G26 needles during xenografts, we verified that their vitality did not decreased by more 2-3% per each passage. To obtain positive controls for HIF-1 immunostaining, LNCaP cells were incubated for 24 h in the presence of 100 M CoCl2, washed, fixed in formalin and stained as described below.

*Mice.* Seven-week old Foxn1nu/nu mice (Harlan, n=46), weighing 25-30 g at the entry into the study, were cared in accordance to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, revised 1996). Water and bedding were heat-sterilized, whereas food was sterilized by 60Co -irradiation. Mice had free access to water and conventional laboratory diet until 24 h before sacrifice. A 12/12 h light/dark cycle was maintained.

*Xenografts.* LNCaP cells were resuspended in ice-cold Matrigel (1:1) at a final concentration of 3106/0.1 ml. Mice were inoculated in each flank with 0.1 ml of cells using a 26G insulin syringe. The next day, mice were transferred into the gas chamber, where they were treated accordingly.

*In vivo measurements of tumor growth.* Body weight and tumor volume were measured three times a week during the various treatments. The tumor volume was calculated as lengthwidthheight0.5236, as measured by a caliper. Data are expressed as the ratio (tumor volume)/(body weight) to compensate different rates of growth in the various experimental situations (**Figure 2**).

*Sacrifice.* At the end of the observation period, mice were anesthetized by i.p. Na-thiopental (10 mg/100 g body weight) plus heparin (500 units), then they were thoracotomized to

LNCaP Prostate Cancer Growth *In Vivo*:

laser scan.

Oncostatic Effects of Melatonin as Compared to Hypoxia and Reoxygenation 83

Then, 15 mL of the suspension or 0.72 mg melatonin was transferred in suitable devices and

The final dose administered topically was about ~0.120 mg melatonin/mouse/treatment, i.e. 4 mg/Kg. For the administration, the stick containing frozen melatonin (or saline for the control group) was delivered topically for 2.4 min in the correspondence of the xenografts with the frozen stick connected to the laser beam. The duration of the treatment was kept within 2.4 min, because topical application of the frozen stick in small-size animals causes hypothermia (when necessary, mice were kept on a heating plate at 37°C during the treatment, not shown). The concentration of melatonin was selected in order to deliver the wanted amount in 2.4 min. After this first phase, mice were placed in a home-made device, immobilized and exposed for 15 min to the laser scan (the wide of the laser beam scan was set to the minimum value, so to cover only the small tumor area exposed on the mice back) (*Figure 4B*). We used an instrument LASERICE Med C.I.R.C.E. S.r.L., Magnago, Milano (230 V, 50 Hz, 150 mA) constituted by a device for freezing the drug emulsion and by a scanner connected to photodiode laser bean with = 635 nm, maximum power <5 mW, collimation lens <20 mV. For our experiments, we selected the software designed for veterinary use in small animals. The software automatically selects the most suitable laser power and

A B

Fig. 4. (A) Topical administration of melatonin by a frozen stick connected to a low energy laser beam. (B) Experimental setup showing exposure of immobilized mice to high-energy

*Effects of hypoxia with/out reoxygenation.* Data relative to this set of experiments have been published in (Terraneo *et al.*, 2010) and are here reported to enable comparing with the groups with melatonin. Briefly, mice xenografted with LNCaP cells as described above were exposed to either chronic hypoxia (10% O2) or hypoxia with reoxygenation (3 times/week for 1 h), with normoxia as control (n=17, 19 and 20, respectively). Hypoxia was induced by using the hypoxic chambers described elsewhere that prevent any unwanted contact of the

animal with room air during cleaning operations and sacrifice (Milano *et al.*, 2002).

frozen at -20°C overnight. Each frozen stick (*Figure 4A*) was used to treat 6 mice.

frequency to target the drug at the right depth into the tissues.

withdraw a blood sample into a heparinized syringe from the left ventricle, and tumors were quickly excised from surrounding skin.

Fig. 2. Picture of a mouse at the end of the observation window. The tumors are clearly visible on both flanks.

*Hemoglobin.* Hemoglobin (Hb) concentration was measured in blood, by diluting 10 l of well-stirred blood in 1 ml of Drabkin reagent, followed by incubation for 30 min at room temperature and absorbance reading at =540 nm. The concentration was calculated assuming λ=11.05 cm-1 mM-1.

*Effects of melatonin i.p..* For this set of experiments, mice xenografted with LNCaP cells as described above were treated with melatonin i.p. (30 µg, 1 mg/Kg), given either dissolved in in 100 µL isotonic saline (n=7) or encapsulated in 100 µL SLN (n=7). Mice treated with 100 µL saline acted as control. Whereas melatonin-saline was prepared fresh, melatonin-SLN was obtained from Nanovector S.r.L., Torino, Italy (Gasco *et al.*, 2007). Timing of treatments is given in *Figure 3*.

Fig. 3. Timing of melatonin treatments.

*Effects of melatonin-laser.* For this set of experiments, mice xenografted with LNCaP cells as described above were exposed to laser treatment with (n=11) or without (n=11) melatonin. Melatonin was prepared fresh every week by emulsifying 0.048 mg melatonin/mL of 1.5 % hydroxymethylcellulose for 7 min with a Ultraturrex at the maximum speed in ice and dark.

withdraw a blood sample into a heparinized syringe from the left ventricle, and tumors

Fig. 2. Picture of a mouse at the end of the observation window. The tumors are clearly

*Hemoglobin.* Hemoglobin (Hb) concentration was measured in blood, by diluting 10 l of well-stirred blood in 1 ml of Drabkin reagent, followed by incubation for 30 min at room temperature and absorbance reading at =540 nm. The concentration was calculated

*Effects of melatonin i.p..* For this set of experiments, mice xenografted with LNCaP cells as described above were treated with melatonin i.p. (30 µg, 1 mg/Kg), given either dissolved in in 100 µL isotonic saline (n=7) or encapsulated in 100 µL SLN (n=7). Mice treated with 100 µL saline acted as control. Whereas melatonin-saline was prepared fresh, melatonin-SLN was obtained from Nanovector S.r.L., Torino, Italy (Gasco *et al.*, 2007). Timing of treatments

*Effects of melatonin-laser.* For this set of experiments, mice xenografted with LNCaP cells as described above were exposed to laser treatment with (n=11) or without (n=11) melatonin. Melatonin was prepared fresh every week by emulsifying 0.048 mg melatonin/mL of 1.5 % hydroxymethylcellulose for 7 min with a Ultraturrex at the maximum speed in ice and dark.

were quickly excised from surrounding skin.

visible on both flanks.

is given in *Figure 3*.

assuming λ=11.05 cm-1 mM-1.

Fig. 3. Timing of melatonin treatments.

Then, 15 mL of the suspension or 0.72 mg melatonin was transferred in suitable devices and frozen at -20°C overnight. Each frozen stick (*Figure 4A*) was used to treat 6 mice.

The final dose administered topically was about ~0.120 mg melatonin/mouse/treatment, i.e. 4 mg/Kg. For the administration, the stick containing frozen melatonin (or saline for the control group) was delivered topically for 2.4 min in the correspondence of the xenografts with the frozen stick connected to the laser beam. The duration of the treatment was kept within 2.4 min, because topical application of the frozen stick in small-size animals causes hypothermia (when necessary, mice were kept on a heating plate at 37°C during the treatment, not shown). The concentration of melatonin was selected in order to deliver the wanted amount in 2.4 min. After this first phase, mice were placed in a home-made device, immobilized and exposed for 15 min to the laser scan (the wide of the laser beam scan was set to the minimum value, so to cover only the small tumor area exposed on the mice back) (*Figure 4B*). We used an instrument LASERICE Med C.I.R.C.E. S.r.L., Magnago, Milano (230 V, 50 Hz, 150 mA) constituted by a device for freezing the drug emulsion and by a scanner connected to photodiode laser bean with = 635 nm, maximum power <5 mW, collimation lens <20 mV. For our experiments, we selected the software designed for veterinary use in small animals. The software automatically selects the most suitable laser power and frequency to target the drug at the right depth into the tissues.

Fig. 4. (A) Topical administration of melatonin by a frozen stick connected to a low energy laser beam. (B) Experimental setup showing exposure of immobilized mice to high-energy laser scan.

*Effects of hypoxia with/out reoxygenation.* Data relative to this set of experiments have been published in (Terraneo *et al.*, 2010) and are here reported to enable comparing with the groups with melatonin. Briefly, mice xenografted with LNCaP cells as described above were exposed to either chronic hypoxia (10% O2) or hypoxia with reoxygenation (3 times/week for 1 h), with normoxia as control (n=17, 19 and 20, respectively). Hypoxia was induced by using the hypoxic chambers described elsewhere that prevent any unwanted contact of the animal with room air during cleaning operations and sacrifice (Milano *et al.*, 2002).

LNCaP Prostate Cancer Growth *In Vivo*:

and Bonferroni post-test).

laser+melatonin.

**3.2 Effect of melatonin laser** 

Oncostatic Effects of Melatonin as Compared to Hypoxia and Reoxygenation 85

Fig. 6. Time course (A) and time necessary for the xenografted tumors to be palpable (B) in mice exposed to either laser only or laser+melatonin (2.04 mg melatonin/mouse in 17 administrations over a 42-day period). Data are expressed as meanSEM from 12, 13 and 12 tumors, respectively. No significant difference between the two treatments was observed,

but both laser groups are different from Saline control (\*, P<0.05).

Fig. 5. Time course (A) and time necessary for the tumors to be palpable (B) in mice

melatonin/mouse in 17 administrations over a 42 day period), whereas saline-treated mice represent the control group. Data are expressed as mean SEM from 12, 10 and 8 tumors, respectively. \*, P<0.05 from untreated; #, P<0.05 from melatonin SLN (ANOVA

This set of experiments aims at assessing whether administering melatonin topically by treatment with laser results into an oncostatic situation as that described above for i.p. melatonin. The xenograft rate of success was in the range 75-95%. Whereas **Figure 6A** reports the time course of the successful xenografts, **Figure 6B** reports the time after the xenograft necessary for the xenografted tumors to be palpable. Whereas laser+melatonin results into a oncostatic situation (compare with the saline-treated as reported in Figure 4), it is difficult to discern an effect due to melatonin in comparison with that driven by

treated i.p. with either melatonin in saline or melatonin in SLN (0.51 mg

*Statistics.* Data are expressed as meanSEM. Significance level was P=0.05 (two-tailed). To detect differences among the groups, we performed one-way ANOVA. If this test resulted significant, the differences between selected pairs of data were tested using the Bonferroni procedure (Instat 3, GraphPad software).

### **3. Results**

**Table 3** shows the main characteristics of the mice considered in this study. No significant differences were detected among the various melatonin groups as far as the changes in body weight and Hb concentration are concerned. By contrast, hypoxia depressed the gain in body weight and increased the blood Hb concentration, as expected. The marked decrease in body weight in the mice exposed to hypoxia forced us to shorten the 42-day observation window to 28 days.


Table 3. Main characteristics of the mice considered in this study. Data from the hypoxia groups are published in (Terraneo *et al.*, 2010). \*, P<0.05 with respect to body weight at entry (unpaired two-tailed Student's t-test); #, P<0.05 with respect to the relative control (ANOVA and Bonferroni post-test).

#### **3.1 Effect of melatonin i.p.**

This set of experiments aims at assessing whether the traditional way to administer melatonin results into an oncostatic situation, and whether administration of the same amount of melatonin is oncostatic as well. The xenograft rate of success was in the range 55- 90%. Whereas **Figure 5A** reports the time course of the successful xenografts, **Figure 5B** reports the time after the xenograft necessary for the xenografted tumors to be palpable. Data confirm that i.p. treatment with melatonin in saline has important oncostatic potential. Of interest, melatonin does not delay appreciably the time of appearance of the tumors after the xenograft, but rather it decreases the growth rate. Melatonin in SLN has less oncostatic potential than melatonin in saline when given in the same amount, probably as the result of less delivery efficiency than in the melatonin-saline group.

Fig. 5. Time course (A) and time necessary for the tumors to be palpable (B) in mice treated i.p. with either melatonin in saline or melatonin in SLN (0.51 mg melatonin/mouse in 17 administrations over a 42 day period), whereas saline-treated mice represent the control group. Data are expressed as mean SEM from 12, 10 and 8 tumors, respectively. \*, P<0.05 from untreated; #, P<0.05 from melatonin SLN (ANOVA and Bonferroni post-test).

#### **3.2 Effect of melatonin laser**

84 Prostate Cancer – Original Scientific Reports and Case Studies

*Statistics.* Data are expressed as meanSEM. Significance level was P=0.05 (two-tailed). To detect differences among the groups, we performed one-way ANOVA. If this test resulted significant, the differences between selected pairs of data were tested using the Bonferroni

**Table 3** shows the main characteristics of the mice considered in this study. No significant differences were detected among the various melatonin groups as far as the changes in body weight and Hb concentration are concerned. By contrast, hypoxia depressed the gain in body weight and increased the blood Hb concentration, as expected. The marked decrease in body weight in the mice exposed to hypoxia forced us to shorten the 42-day observation

> *Body weight at entry, g*

**Effects of melatonin i.p.**  *Saline* 6/6 26.630.20 26.770.80 1203 *Melatonin-saline* 7/7 27.710.32 32.170.46\*# 1196 *Melatonin-SLN* 7/7 27.960.25 31.940.90\*# 1193 **Effects of melatonin laser**  *Laser* 11/11 27.240.31 29.700.91\* 12611 *Laser+melatonin* 11/11 27.400.30 30.580.74\* 1196 **Effects of hypoxia (28 days of treatment)**  *Normoxia* 19/20 26.830.39 31.170.61\* 1343 *Chronic hypoxia* 16/17 280.46 27.380.52# 1973#

*reoxygenation* 9/9 27.550.37 23.920.28\*# 2055# Table 3. Main characteristics of the mice considered in this study. Data from the hypoxia groups are published in (Terraneo *et al.*, 2010). \*, P<0.05 with respect to body weight at entry (unpaired two-tailed Student's t-test); #, P<0.05 with respect to the relative control (ANOVA

This set of experiments aims at assessing whether the traditional way to administer melatonin results into an oncostatic situation, and whether administration of the same amount of melatonin is oncostatic as well. The xenograft rate of success was in the range 55- 90%. Whereas **Figure 5A** reports the time course of the successful xenografts, **Figure 5B** reports the time after the xenograft necessary for the xenografted tumors to be palpable. Data confirm that i.p. treatment with melatonin in saline has important oncostatic potential. Of interest, melatonin does not delay appreciably the time of appearance of the tumors after the xenograft, but rather it decreases the growth rate. Melatonin in SLN has less oncostatic potential than melatonin in saline when given in the same amount, probably as the result of

*Final body weight, g (42 days of treatment)* 

*Blood [Hb], g/L* 

procedure (Instat 3, GraphPad software).

*n Surviving/total* 

less delivery efficiency than in the melatonin-saline group.

**3. Results** 

window to 28 days.

*Hypoxia with* 

and Bonferroni post-test).

**3.1 Effect of melatonin i.p.** 

This set of experiments aims at assessing whether administering melatonin topically by treatment with laser results into an oncostatic situation as that described above for i.p. melatonin. The xenograft rate of success was in the range 75-95%. Whereas **Figure 6A** reports the time course of the successful xenografts, **Figure 6B** reports the time after the xenograft necessary for the xenografted tumors to be palpable. Whereas laser+melatonin results into a oncostatic situation (compare with the saline-treated as reported in Figure 4), it is difficult to discern an effect due to melatonin in comparison with that driven by laser+melatonin.

Fig. 6. Time course (A) and time necessary for the xenografted tumors to be palpable (B) in mice exposed to either laser only or laser+melatonin (2.04 mg melatonin/mouse in 17 administrations over a 42-day period). Data are expressed as meanSEM from 12, 13 and 12 tumors, respectively. No significant difference between the two treatments was observed, but both laser groups are different from Saline control (\*, P<0.05).

LNCaP Prostate Cancer Growth *In Vivo*:

hypoxia agrees with the described findings.

Oncostatic Effects of Melatonin as Compared to Hypoxia and Reoxygenation 87

expression in three human cancer cell lines (PANC-1, HeLa and A549), at high pharmacological concentrations it markedly reduces expression of VEGF and HIF-1 induced by CoCl2 in cultured cancer cells (Dai *et al.*, 2008). A breakthrough in understanding the mechanisms underlying the oncostatic properties of melatonin came along with unraveling the relationship between melatonin and the hypoxia signaling path. Melatonin indeed inhibits HIF-1 protein expression in normoxic and hypoxic DU145, PC-3 and LNCaP prostate cancer cells without affecting HIF-1 mRNA levels (Park *et al.*, 2009). In HCT116 human colon cancer cells, melatonin destabilizes HIF-1, suppresses its transcriptional activity, thereby decreasing VEGF expression (Park *et al.*, 2010). In turn, these features block in vitro tube formation and invasion and migration of human umbilical vein endothelial cells induced by hypoxia media, indicating that melatonin plays a pivotal role in tumor suppression via inhibition of HIF-1-mediated angiogenesis. Our observation that melatonin inhibits LNCaP prostate cancer growth as opposite to enhanced growth in

*Chronic hypoxia.* Despite the acknowledged role of hypoxia in cancer biology, mainly acquired through *in vitro* and clinical studies, accurate analysis of existing literature revealed that the effects of *chronic* hypoxia *in vivo* had not been investigated experimentally as well. To mimic chronic hypoxia, we recently developed an experimental model whereby prostate tumorbearing mice are exposed to various forms of hypoxia *in vivo*, while continuously monitoring tumor growth and finally assessing the molecular and cellular phenotypes (Terraneo *et al.*, 2010). We found that, although hypoxia *in vivo* promotes prostate cancer growth, this could not be entirely ascribed to HIF-1, in the favor of other molecular pathways, such as those involving phosphatidyl inositol-3-phosphate/protein kinase B (Akt) pathway. This finding bears important implications especially when designing effective therapies: as a matter of facts, on clinical ground therapies targeting HIF-1 do not appear particularly successful (Fox *et al.*, 2011). Comparing the effects led by chronic

hypoxia with/out reoxygenation contributed to univocally assess the latter issue.

growth, the effect of HIF-1 on metastatic potential is still to be investigated.

*Chronic hypoxia with reoxygenation.* Reoxygenating the tumors during hypoxia increased HIF-1 cytosolic level >10-fold more than in chronic hypoxia, yet tumor growth was essentially similar to that of chronic hypoxia (Terraneo *et al.*, 2010). Higher HIF-1 in reoxygenated tumors with respect to chronic hypoxia could be a consequence of shorter normalization time since the last exposure to hypoxia (2-3 days), or of reoxygenation-induced HIF-1 stabilization or enhanced synthesis. As the reoxygenation of hypoxic tissues causes considerable oxidative stress (Milano *et al.*, 2004), the associated enhanced generation of mitochondrial ROS may stabilize HIF-1 (Chandel *et al.*, 2000). It has also been proposed that the persistent oxidative stress promoted by ROS during the reoxygenation further amplifies HIF-1 activation in a feed-forward mechanism through a mechanism involving mTOR (Semenza *et al.*, 2007). Therefore, although this finding was key to suppose non-centrality of HIF-1 in cancer

*Therapeutic potential and future perspectives*. The role of melatonin as anti-proliferative factor in the management of prostate cancer is currently under study by several research groups. However, its signaling mechanism and its crosstalk with other positive or negative growth regulator factors as HIF-1, sex steroids, epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) and others is still under debate and deserves to be thoroughly investigated. Moreover, to elucidate the biological effects of melatonin under

#### **3.3 Effect of hypoxia**

This set of experiments aims at assessing whether inducing local hypoxia increases the growth of prostate tumors as published elsewhere (Terraneo *et al.*, 2010). By decreasing arterial PO2 from 85 to 34 mmHg, the selected hypoxia severity (10% O2, equivalent to an altitude of about 5000 m) decreases the total O2 arterial content by 35%, despite the increased blood Hb concentration. The xenograft success rate was 56-83% without any significant effect of hypoxia nor its way of administration. **Figure 7A** reports the time course of the successful xenografts, and **Figure 7B** reports the time after the xenograft necessary for the tumors to be palpable. Chronic hypoxia resulted into significant increase of the tumor growth rate. Interestingly, an operation aimed at increasing the cytosolic abundance of HIF-1 by approximately 10 times, e.g., exposing mice to repeated reoxygenation events during hypoxia, did not affected appreciably the tumor growth rate. This suggests that mechanisms other than those mediated by HIF-1 might have determined the increased tumor growth rate in hypoxic mice.

Fig. 7. Time course (A) and time necessary for the xenografted tumors to be palpable (B) in mice exposed to either laser only or laser+melatonin (n g melatonin/mouse in n administrations over a 42 day period). Data are expressed as meanSEM from n, n and n tumors, respectively. \*, P<0.05 from normoxia (ANOVA and Bonferroni post-test).

#### **4. Discussion**

*Melatonin.* After the first observation that in MCF-7 cells *in vitro*, 1 nM melatonin reduces tumor cells invasiveness in Falcon invasion chambers (Cos *et al.*, 1998), the oncostatic properties of melatonin have been thoroughly investigated. Melatonin acts synergistically with castration in inhibiting growth of androgen-sensitive LNCaP tumor through opposite changes in cyclin D1 levels induced by activated MT1 and EGF receptors (Siu *et al.*, 2002). As MT1 receptors are clearly involved in androgen-sensitive LNCaP, but not in androgeninsensitive PC-3 cells (Xi *et al.*, 2001), it is likely that the antiproliferative action of melatonin in LNCaP tumor growth is associated with MT1 receptor protein expression. Furthermore, the clear relationship found in MCF-7 xenografts between melatonin and telomerase activity, responsible of telomere elongation that activated in most human cancers (Leon-Blanco *et al.*, 2003), suggests that melatonin also influences telomerase decreasing its activity in the tumors. Although melatonin at physiologic concentration has no impact on VEGF

This set of experiments aims at assessing whether inducing local hypoxia increases the growth of prostate tumors as published elsewhere (Terraneo *et al.*, 2010). By decreasing arterial PO2 from 85 to 34 mmHg, the selected hypoxia severity (10% O2, equivalent to an altitude of about 5000 m) decreases the total O2 arterial content by 35%, despite the increased blood Hb concentration. The xenograft success rate was 56-83% without any significant effect of hypoxia nor its way of administration. **Figure 7A** reports the time course of the successful xenografts, and **Figure 7B** reports the time after the xenograft necessary for the tumors to be palpable. Chronic hypoxia resulted into significant increase of the tumor growth rate. Interestingly, an operation aimed at increasing the cytosolic abundance of HIF-1 by approximately 10 times, e.g., exposing mice to repeated reoxygenation events during hypoxia, did not affected appreciably the tumor growth rate. This suggests that mechanisms other than those mediated by HIF-1 might have determined the increased tumor growth

Fig. 7. Time course (A) and time necessary for the xenografted tumors to be palpable (B) in

administrations over a 42 day period). Data are expressed as meanSEM from n, n and n tumors, respectively. \*, P<0.05 from normoxia (ANOVA and Bonferroni post-test).

*Melatonin.* After the first observation that in MCF-7 cells *in vitro*, 1 nM melatonin reduces tumor cells invasiveness in Falcon invasion chambers (Cos *et al.*, 1998), the oncostatic properties of melatonin have been thoroughly investigated. Melatonin acts synergistically with castration in inhibiting growth of androgen-sensitive LNCaP tumor through opposite changes in cyclin D1 levels induced by activated MT1 and EGF receptors (Siu *et al.*, 2002). As MT1 receptors are clearly involved in androgen-sensitive LNCaP, but not in androgeninsensitive PC-3 cells (Xi *et al.*, 2001), it is likely that the antiproliferative action of melatonin in LNCaP tumor growth is associated with MT1 receptor protein expression. Furthermore, the clear relationship found in MCF-7 xenografts between melatonin and telomerase activity, responsible of telomere elongation that activated in most human cancers (Leon-Blanco *et al.*, 2003), suggests that melatonin also influences telomerase decreasing its activity in the tumors. Although melatonin at physiologic concentration has no impact on VEGF

mice exposed to either laser only or laser+melatonin (n g melatonin/mouse in n

**3.3 Effect of hypoxia** 

rate in hypoxic mice.

**4. Discussion** 

expression in three human cancer cell lines (PANC-1, HeLa and A549), at high pharmacological concentrations it markedly reduces expression of VEGF and HIF-1 induced by CoCl2 in cultured cancer cells (Dai *et al.*, 2008). A breakthrough in understanding the mechanisms underlying the oncostatic properties of melatonin came along with unraveling the relationship between melatonin and the hypoxia signaling path. Melatonin indeed inhibits HIF-1 protein expression in normoxic and hypoxic DU145, PC-3 and LNCaP prostate cancer cells without affecting HIF-1 mRNA levels (Park *et al.*, 2009). In HCT116 human colon cancer cells, melatonin destabilizes HIF-1, suppresses its transcriptional activity, thereby decreasing VEGF expression (Park *et al.*, 2010). In turn, these features block in vitro tube formation and invasion and migration of human umbilical vein endothelial cells induced by hypoxia media, indicating that melatonin plays a pivotal role in tumor suppression via inhibition of HIF-1-mediated angiogenesis. Our observation that melatonin inhibits LNCaP prostate cancer growth as opposite to enhanced growth in hypoxia agrees with the described findings.

*Chronic hypoxia.* Despite the acknowledged role of hypoxia in cancer biology, mainly acquired through *in vitro* and clinical studies, accurate analysis of existing literature revealed that the effects of *chronic* hypoxia *in vivo* had not been investigated experimentally as well. To mimic chronic hypoxia, we recently developed an experimental model whereby prostate tumorbearing mice are exposed to various forms of hypoxia *in vivo*, while continuously monitoring tumor growth and finally assessing the molecular and cellular phenotypes (Terraneo *et al.*, 2010). We found that, although hypoxia *in vivo* promotes prostate cancer growth, this could not be entirely ascribed to HIF-1, in the favor of other molecular pathways, such as those involving phosphatidyl inositol-3-phosphate/protein kinase B (Akt) pathway. This finding bears important implications especially when designing effective therapies: as a matter of facts, on clinical ground therapies targeting HIF-1 do not appear particularly successful (Fox *et al.*, 2011). Comparing the effects led by chronic hypoxia with/out reoxygenation contributed to univocally assess the latter issue.

*Chronic hypoxia with reoxygenation.* Reoxygenating the tumors during hypoxia increased HIF-1 cytosolic level >10-fold more than in chronic hypoxia, yet tumor growth was essentially similar to that of chronic hypoxia (Terraneo *et al.*, 2010). Higher HIF-1 in reoxygenated tumors with respect to chronic hypoxia could be a consequence of shorter normalization time since the last exposure to hypoxia (2-3 days), or of reoxygenation-induced HIF-1 stabilization or enhanced synthesis. As the reoxygenation of hypoxic tissues causes considerable oxidative stress (Milano *et al.*, 2004), the associated enhanced generation of mitochondrial ROS may stabilize HIF-1 (Chandel *et al.*, 2000). It has also been proposed that the persistent oxidative stress promoted by ROS during the reoxygenation further amplifies HIF-1 activation in a feed-forward mechanism through a mechanism involving mTOR (Semenza *et al.*, 2007). Therefore, although this finding was key to suppose non-centrality of HIF-1 in cancer growth, the effect of HIF-1 on metastatic potential is still to be investigated.

*Therapeutic potential and future perspectives*. The role of melatonin as anti-proliferative factor in the management of prostate cancer is currently under study by several research groups. However, its signaling mechanism and its crosstalk with other positive or negative growth regulator factors as HIF-1, sex steroids, epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) and others is still under debate and deserves to be thoroughly investigated. Moreover, to elucidate the biological effects of melatonin under

LNCaP Prostate Cancer Growth *In Vivo*:

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#### **5. Conclusion**

*In vivo* systemic hypoxia promotes prostate cancer growth regardless of HIF-1 expression level and neovascularization, suggesting an important role for hypoxia-dependent pathways that do not involve HIF-1, as the phosphatidyl inositol-3-phosphate signaling cascade. Melatonin experiments may not only provide a useful probe to assess the effects of HIF-1 on tumor growth, but may also represent the basis for the future introduction of this natural molecule as adjuvant active component in novel therapeutic strategies for the treatment of malignant prostate cancer in humans, for the prevention of cancer relapses, or simply for the amelioration of the quality of life of the oncologic patient.

#### **6. Acknowledgments**

We thanks to Dr. Enrico Bonizzoni and Dr. Emilio Bonizzoni C.I.R.CE. S.r.L., Magnago, Milano, who kindly borrowed the Laser equipment.

#### **7. References**


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**5. Conclusion** 

**6. Acknowledgments** 

**7. References** 


**Part 2** 

**Diagnostic Markers** 

