**Part 3**

**Therapeutic Novelties** 

150 Prostate Cancer – Original Scientific Reports and Case Studies

Tabesh, A.; Kumar, V.; Pang, H.; Verbel, D. Kotsianti, A.; Teverovskiy M. & Saidi, O. (2005).

Tahir, M.A. & Bouridane, A. (2006). Novel Round-Robin Tabu Search Algorithm for Prostate

Yongguang, B. Ishii, N. & Du, X. (*2004*). Combining multiple k-nearest neighbour classifiers

*Proc. SPIE Med. Imag*., vol. 5747, pp.58–70.

*on information technology in biomedicine*, Vol. 10, No. 4.

*5th Int. Conf. Intell. Data Eng. Autom. Learn.*, U.K.

Automated prostate cancer diagnosis and Gleason grading of tissue microarrays,

Cancer Classification and Diagnosis Using Multispectral Imagery. *IEEE transactions* 

using different distance functions. *Lectures Notes in Computer Science (LNCS 3177),* 

**10** 

*Spain*

**New Selenoderivatives as Antitumoral Agents** 

Prostate cancer (PC) is the most common male malignancy in Western countries and the second most common urological malignancy (Knudsen & Vasioukhin, 2010). In 2008 the estimated new cases for PC in the European Union were 382.000 (Ferlay et al. 2010). The possibility of early detection is attractive to clinicians and potential patients in spite of the fact that until recently concrete evidence that screening would influence PC mortality was lacking (Schröder, 2010). There are many risk factors for PC occurrence. The family history, genetic and environmental factors and their interaction can contribute to develop PC (Colloca & Venturino, 2011). Other risk factors are age, ethnic-racial-geographic factors, named constitutional factors, though it is not possible to know what percentage of these neoplasms are a result of these risk factors (Ferris-i-Tortajada et al. 2011). The polymorphisms in genes associated with PC probably represent the most part of familial PC burden. The recent advances in genomic research have made it possible to identify several new genomic based biomarkers for PC. These markers are easy to measure and stable over time but only one biomarker, prostate specific antigen (PSA), is used in the clinical today (Aly et al. 2011). The PSA screening allows to detect PC years before the emergence of clinically evident disease, which usually represents locally advanced or metastatic cancer (Gjertson & Albertsen, 2011). Treatment options for advanced PC – including hormone ablation therapy, radiation and surgery – do not offer cure but delay the inevitable recurrence of the lethal hormone-refractory disease. Chemotherapy using available anticancer drugs, with the exception of the taxane drug docetaxel, for late stage PC does not offer any survival benefit. All of these treatments are costly and have significant side effects including impotence and incontinence, which negatively affect the quality of life of the patients. Prevention is an important strategy for limiting PC morbidity and mortality. Pharmacological and dietary interventions have potentials functions in reduction of incident cases and in inhibition of disease progression and recurrence (Silberstein & Parsons, 2010). 5-alpha reductase inhibitors remain the predominant therapy to reduce the future risk of a PC diagnosis. Dutasteride and finasteride are currently the only proven agents for PC risk reduction (Strope & Andriole, 2010). Among the potential dietary intervention efforts to use of the micro-nutrient selenium (Se) in PC clinical trials is emerging as an important highlight and the outcomes indicate that Se is a promising treatment. Furthermore, Se inhibits PC through multiple mechanisms, and it is beneficial in controlling the development of this disease (Abdulah

**1. Introduction** 

*Department of Organic and Pharmaceutical Chemistry, University of Navarra* 

Carmen Sanmartín, Juan Antonio Palop,

Beatriz Romano and Daniel Plano

### **New Selenoderivatives as Antitumoral Agents**

Carmen Sanmartín, Juan Antonio Palop,

Beatriz Romano and Daniel Plano

*Department of Organic and Pharmaceutical Chemistry, University of Navarra Spain*

#### **1. Introduction**

Prostate cancer (PC) is the most common male malignancy in Western countries and the second most common urological malignancy (Knudsen & Vasioukhin, 2010). In 2008 the estimated new cases for PC in the European Union were 382.000 (Ferlay et al. 2010). The possibility of early detection is attractive to clinicians and potential patients in spite of the fact that until recently concrete evidence that screening would influence PC mortality was lacking (Schröder, 2010). There are many risk factors for PC occurrence. The family history, genetic and environmental factors and their interaction can contribute to develop PC (Colloca & Venturino, 2011). Other risk factors are age, ethnic-racial-geographic factors, named constitutional factors, though it is not possible to know what percentage of these neoplasms are a result of these risk factors (Ferris-i-Tortajada et al. 2011). The polymorphisms in genes associated with PC probably represent the most part of familial PC burden. The recent advances in genomic research have made it possible to identify several new genomic based biomarkers for PC. These markers are easy to measure and stable over time but only one biomarker, prostate specific antigen (PSA), is used in the clinical today (Aly et al. 2011). The PSA screening allows to detect PC years before the emergence of clinically evident disease, which usually represents locally advanced or metastatic cancer (Gjertson & Albertsen, 2011). Treatment options for advanced PC – including hormone ablation therapy, radiation and surgery – do not offer cure but delay the inevitable recurrence of the lethal hormone-refractory disease. Chemotherapy using available anticancer drugs, with the exception of the taxane drug docetaxel, for late stage PC does not offer any survival benefit. All of these treatments are costly and have significant side effects including impotence and incontinence, which negatively affect the quality of life of the patients. Prevention is an important strategy for limiting PC morbidity and mortality. Pharmacological and dietary interventions have potentials functions in reduction of incident cases and in inhibition of disease progression and recurrence (Silberstein & Parsons, 2010). 5-alpha reductase inhibitors remain the predominant therapy to reduce the future risk of a PC diagnosis. Dutasteride and finasteride are currently the only proven agents for PC risk reduction (Strope & Andriole, 2010). Among the potential dietary intervention efforts to use of the micro-nutrient selenium (Se) in PC clinical trials is emerging as an important highlight and the outcomes indicate that Se is a promising treatment. Furthermore, Se inhibits PC through multiple mechanisms, and it is beneficial in controlling the development of this disease (Abdulah

New Selenoderivatives as Antitumoral Agents 155

processes. The direction of future studies lies in clarifying the effects of these products and exploring the biological mechanisms responsible for the prevention of prostate cancer (Fairweather-Tait et al. 2011). This chapter includes information on twenty eight general chemical structures containing Se that have shown either anticancer, chemopreventive or apoptotic activities. Thus, Se derivatives emerge as promising downstream candidates for

Some studies have shown that the selenium-based compound methylseleninic acid (MSeA, Figure 2) can disrupt AR signaling in PC cells by reaction with reduced glutathione within the PC cell (Husbeck et al., 2006). On the other hand, it was observed that a combination of MSeA with bicalutamide produced a robust downregulation of PSA through the identification of *hTERT*/telomerase as an important AR target. Telomerase activation has been reported in >90% of prostate cancer samples, but not in normal or benign prostatic hyperplasia tissues. Telomerase activation play an essential role in cell survival and oncogenesis, and inhibition of telomerase has been shown to suppress growth and tumorigenic potential of PC (S.A. Liu et al., 2010). Other mechanisms have demonstrated that the growth inhibitory effect could be attributed to cell cycle modulation and apoptosis induction provoked by MSeA by activation the forkhead box O1 (FOXO1) (H.T. Zhang et al., 2010). This compound has shown efficacy in transgenic adenocarcinoma of mouse prostate model (Wang et al., 2009). Too, it has been investigated in mice model treated with this Se form and were observed changes in its proteome (Zhang et al., 2011). In addition, MSeA exerted a dose-dependent inhibition of DU145 xenograft growth without genotoxic properties (Li et al., 2008). Moreover, in advanced and hormone refractory prostate cancer the efficacy of MSeA is based on down regulating hypoxia inducible factor 1α (HIF-1α) accompanied of a reduction of vascular endothelial growth factor (VEGF) and glucose transporter 1 (GLUT1) (I. Sinha et al., 2011). On the other hand, MSeA inactivated protein kinase C (PKC), particularly the promitogenic and prosurvival epsilon isoenzyme, acting through a redox modification of vicinal cysteine sulfhydryls in the catalytic domain of PKC (Gundimeda et al., 2008). Some metabolites of MSeA such as methylselenol may contribute to their anticancer activities. For example, an upregulation of cyclin dependent kinase inhibitor (CDKI) proteins p21Cip 1 and/or p27Kip 1 was observed in DU145 prostate cancer cells (Wang et al., 2010). Too, a novel mechanism of Se action has been proposed for methylselenol due to its ability to inhibit histone deacetylase (HDAC) (Kassam et al., 2011). Recently, speciation analysis showed that MSeA was completely transformed during the incubations while metabolic conversion of the other Se

Sodium selenite (Figure 2) is another compound that has been studied in relation to PC and it may modulate the androgen receptor through the repression of interleukin-6 (IL-6) (Gazi et al., 2007). Too, it was observed that selenite decreased HDAC activity and increased levels of acetylated lysine 9 on histone H3, but decreased levels of methylated H3-Lys 9 (Xiang et al., 2008). Other mechanisms of action have been postulated such as an increase of the activity of the tumor suppressor protein (PTEN) and of the thioredoxin reductase (TR) (Berggren et al., 2009). Too, selenite is able to induce cell death and apoptosis by production of superoxide in mitochondria in LNCaP cells (Xiang et al., 2009). In 2010 was reported that

cancer therapy.

**2. Selenoderivatives against prostate cancer** 

compounds was limited (Lunoe et al., 2011).

**2.1 Methylseleninic acid, sodium selenite and sodium selenate**

et al. 2011). Se is an essential trace element for humans, animals and some bacteria and it is important for many cellular processes, cardiovascular disease, central nervous system pathologies and may prevent cancer (Dennert et al. 2011). The evidence that Se is a cancer preventive agent includes that from geographic, animal, prospective and intervention studies (Tabassum et al. 2010, Schmid et al. 2011).

Furthermore, literature reports have consistently shown that the different effects of different chemical forms and dose of Se (Algotar et al. 2011) on signaling and expression of transcripts in PC cells might have important implications in the outcome of ongoing PC prevention clinical trials. These include the forms of Se present in the diet and in the body, their functions and mechanisms of action, and methods employed in assessing an individual's Se nutritional status – both in general and in epidemiological studies into the risk of cancer in relation to diet, as well as in connection with long-term trials for investigating the disease-preventive potential of selenium supplementation. Several mechanisms have been suggested to mediate the anticancer effects of Se. The major ones are reduction of DNA damage; oxidative stress; inflammation; induction of phase II conjugating enzymes that detoxify carcinogens; enhancement of immune response; incorporation into selenoproteins; alteration in DNA methylation status of tumor suppressor genes; inhibition of cell cycle and angiogenesis and induction of apoptosis. The specific mechanisms for PC are the inhibition of androgen receptor (AR) signaling, reduction in the mRNA, and protein levels of the AR, recruitment of corepressors to the AR elements in the promoters of androgen responsive genes, inhibition of signaling pathways like NF-*k*B, IL-6, Stat3, and induction of apoptosis (Nadiminty & Gao, 2008) (Figure 1).

Fig. 1. Schematic representation of cellular processes targeted by Se and some specific molecular targets in each pathway. Figure from Nadiminty & Gao, 2008 with permission from John Wiley and Sons.

The rapid advance in the knowledge of different selenoproteins and their biological functions has opened up new possibilities to increase our understanding of the biological effects of Se supplementation (Rebsch et al. 2006). Selenoprotein deficiency leads to the accelerated development of lesions associated with PC progression, implicating selenoproteins in cancer risk and development and raising the possibility that Se prevents cancer by modulating the levels of these selenoproteins. Recently, it has been reported that the new discovered selenoprotein, SEP15, which is highly expressed in the prostate, may play a role either independently or by modifying the effects of Se in PC survival (Penney et al. 2010). Moreover, further research and additional trials of this type are needed to define the benefits and risks of different types and doses of Se supplements that in the future may be implemented for public health reasons. Another necessary focus for future research is a better understanding of the mechanisms by which Se interferes with the carcinogenic

et al. 2011). Se is an essential trace element for humans, animals and some bacteria and it is important for many cellular processes, cardiovascular disease, central nervous system pathologies and may prevent cancer (Dennert et al. 2011). The evidence that Se is a cancer preventive agent includes that from geographic, animal, prospective and intervention

Furthermore, literature reports have consistently shown that the different effects of different chemical forms and dose of Se (Algotar et al. 2011) on signaling and expression of transcripts in PC cells might have important implications in the outcome of ongoing PC prevention clinical trials. These include the forms of Se present in the diet and in the body, their functions and mechanisms of action, and methods employed in assessing an individual's Se nutritional status – both in general and in epidemiological studies into the risk of cancer in relation to diet, as well as in connection with long-term trials for investigating the disease-preventive potential of selenium supplementation. Several mechanisms have been suggested to mediate the anticancer effects of Se. The major ones are reduction of DNA damage; oxidative stress; inflammation; induction of phase II conjugating enzymes that detoxify carcinogens; enhancement of immune response; incorporation into selenoproteins; alteration in DNA methylation status of tumor suppressor genes; inhibition of cell cycle and angiogenesis and induction of apoptosis. The specific mechanisms for PC are the inhibition of androgen receptor (AR) signaling, reduction in the mRNA, and protein levels of the AR, recruitment of corepressors to the AR elements in the promoters of androgen responsive genes, inhibition of signaling pathways like NF-*k*B, IL-6, Stat3, and

Fig. 1. Schematic representation of cellular processes targeted by Se and some specific molecular targets in each pathway. Figure from Nadiminty & Gao, 2008 with permission

The rapid advance in the knowledge of different selenoproteins and their biological functions has opened up new possibilities to increase our understanding of the biological effects of Se supplementation (Rebsch et al. 2006). Selenoprotein deficiency leads to the accelerated development of lesions associated with PC progression, implicating selenoproteins in cancer risk and development and raising the possibility that Se prevents cancer by modulating the levels of these selenoproteins. Recently, it has been reported that the new discovered selenoprotein, SEP15, which is highly expressed in the prostate, may play a role either independently or by modifying the effects of Se in PC survival (Penney et al. 2010). Moreover, further research and additional trials of this type are needed to define the benefits and risks of different types and doses of Se supplements that in the future may be implemented for public health reasons. Another necessary focus for future research is a better understanding of the mechanisms by which Se interferes with the carcinogenic

studies (Tabassum et al. 2010, Schmid et al. 2011).

induction of apoptosis (Nadiminty & Gao, 2008) (Figure 1).

from John Wiley and Sons.

processes. The direction of future studies lies in clarifying the effects of these products and exploring the biological mechanisms responsible for the prevention of prostate cancer (Fairweather-Tait et al. 2011). This chapter includes information on twenty eight general chemical structures containing Se that have shown either anticancer, chemopreventive or apoptotic activities. Thus, Se derivatives emerge as promising downstream candidates for cancer therapy.

#### **2. Selenoderivatives against prostate cancer**

#### **2.1 Methylseleninic acid, sodium selenite and sodium selenate**

Some studies have shown that the selenium-based compound methylseleninic acid (MSeA, Figure 2) can disrupt AR signaling in PC cells by reaction with reduced glutathione within the PC cell (Husbeck et al., 2006). On the other hand, it was observed that a combination of MSeA with bicalutamide produced a robust downregulation of PSA through the identification of *hTERT*/telomerase as an important AR target. Telomerase activation has been reported in >90% of prostate cancer samples, but not in normal or benign prostatic hyperplasia tissues. Telomerase activation play an essential role in cell survival and oncogenesis, and inhibition of telomerase has been shown to suppress growth and tumorigenic potential of PC (S.A. Liu et al., 2010). Other mechanisms have demonstrated that the growth inhibitory effect could be attributed to cell cycle modulation and apoptosis induction provoked by MSeA by activation the forkhead box O1 (FOXO1) (H.T. Zhang et al., 2010). This compound has shown efficacy in transgenic adenocarcinoma of mouse prostate model (Wang et al., 2009). Too, it has been investigated in mice model treated with this Se form and were observed changes in its proteome (Zhang et al., 2011). In addition, MSeA exerted a dose-dependent inhibition of DU145 xenograft growth without genotoxic properties (Li et al., 2008). Moreover, in advanced and hormone refractory prostate cancer the efficacy of MSeA is based on down regulating hypoxia inducible factor 1α (HIF-1α) accompanied of a reduction of vascular endothelial growth factor (VEGF) and glucose transporter 1 (GLUT1) (I. Sinha et al., 2011). On the other hand, MSeA inactivated protein kinase C (PKC), particularly the promitogenic and prosurvival epsilon isoenzyme, acting through a redox modification of vicinal cysteine sulfhydryls in the catalytic domain of PKC (Gundimeda et al., 2008). Some metabolites of MSeA such as methylselenol may contribute to their anticancer activities. For example, an upregulation of cyclin dependent kinase inhibitor (CDKI) proteins p21Cip 1 and/or p27Kip 1 was observed in DU145 prostate cancer cells (Wang et al., 2010). Too, a novel mechanism of Se action has been proposed for methylselenol due to its ability to inhibit histone deacetylase (HDAC) (Kassam et al., 2011). Recently, speciation analysis showed that MSeA was completely transformed during the incubations while metabolic conversion of the other Se compounds was limited (Lunoe et al., 2011).

Sodium selenite (Figure 2) is another compound that has been studied in relation to PC and it may modulate the androgen receptor through the repression of interleukin-6 (IL-6) (Gazi et al., 2007). Too, it was observed that selenite decreased HDAC activity and increased levels of acetylated lysine 9 on histone H3, but decreased levels of methylated H3-Lys 9 (Xiang et al., 2008). Other mechanisms of action have been postulated such as an increase of the activity of the tumor suppressor protein (PTEN) and of the thioredoxin reductase (TR) (Berggren et al., 2009). Too, selenite is able to induce cell death and apoptosis by production of superoxide in mitochondria in LNCaP cells (Xiang et al., 2009). In 2010 was reported that

New Selenoderivatives as Antitumoral Agents 157

and alpha-tocopherol do not inhibit prostate carcinogenesis in the testosterone plus estradiol treated NBL rat model (Ozten et al., 2010). However, the selected combination of silymarin and SeMet significantly reduced two markers of lipid metabolism known associated with PC progression (Vidlar et al., 2010). In order to improve the activity and safety inorganic and organic hybrid nanoparticles are potentially useful in biomedicine, mainly for tumor treatments (Choi et al., 2010). Se nanoparticles are safer compared with SeMet isolated and was observed an inhibition of the growth of prostate LNCaP cancer cells partially through caspases mediated apoptosis, Akt kinase modulation and by

The first selenocyanate described was 1,4-phenylenebis(methylene)selenocyanate (*p*-XSC) (Figure 4). The most recent studies postulate that this compound is capable of altering cofilin-2, single-stranded mitochondrial DNA binding protein, chaperonin 10, nucleoside diphosphate kinase 6 and chain A Horf 6 human peroxidase enzyme in LNCaP cells and in its androgen independent clone (AI) (R. Sinha et al., 2008). Too, this compound can induce apoptosis, inhibits AR expression and decreases Akt phosphorylation (Facompre et al., 2010). Other organic selenocyanates have emerged during the last years. So phenylalkyl isoselenocyanates (Figure 4), isosteric selenium analogues of naturally occurring phenylalkyl isothiocyanates, have shown a reduction in tumor size associated to apoptosis. The structure activity relationship studies concluded that an increase in the alkyl chain length is critical for the activity being n = 4, named ISC-4, the optimal (Sharma et al., 2008). In 2011, these same authors have reported that ISC-4 activates prostate apoptosis response protein 4 (Par-4) (Sharma et al., 2011). As a continuation of the synthesis of novel alkyl selenocyanates in 2010 was described the synthesis of substituted naphthalimide based organoselenocyanates (Figure 4) with the alkyl chain length n = 5 and investigated their systematic toxicity profile in mice by consideration changes in body weight, hepatotoxicity and nephrotoxicity resulting less toxic than other selenium forms but retaining the efficacy (Roy et al., 2010). Numerous studies have been conducted to elucidate the mechanism underlying the antitumor effects associated with cyclooxygenase 2 (COX-2) inhibitors. However, this mechanism has not yet been clearly defined. Nonsteroidal antiinflammatory drugs (NSAIDs) have been shown to retard the progression of PC in men and NSAIDs have been used in clinical trials for prostate cancer. Celecoxib (Celebrex), a specific COX-2 inhibitor, reduces prostate tumors in experimental models mainly through cell cycle regulation and angiogenesis. However, the growth inhibitory properties of Celecoxib may be COX-2 independent. Considering this possible effect a novel strategy

disrupting AR (Kong et al., 2011).

**2.3 Selenocyanate derivatives**

Fig. 3. Methylselenocysteine and selenomethionine structures.

sodium selenite inhibited the lipopolysaccharide (LPS)-induced TLR4-NF-kB signaling in PC-3 (Pei et al., 2010). Sodium selenite can act alone or in combination with other treatments for PC. So, this compound significantly enhances the effect of radiation on well established hormone-independent prostate tumors and does not sensitize the intestinal epithelial cells to radiation. These results suggest that may increase the therapeutic index of radiation therapy (Tian et al., 2010). In addition, the effectiveness on PC treatment of the association between sodium selenite and docetaxel has resulted as a new strategy in PC therapeutic approach (Freitas et al., 2011). Too, combination of genistein and selenite has shown synergistic effects on apoptosis, cell cycle arrest associated signaling pathways in p53 expression (Zhao et al., 2009). Actually, other inorganic forms of Se as sodium selenate (Figure 2), where Se is in oxidation state + 6, are in Phase I studies and have shown antiangiogenic properties (Corcoran et al., 2010).

Fig. 2. Methylseleninic acid, sodium selenite and sodium selenate structures.

#### **2.2 Methylselenocysteine and selenomethionine**

Se may exert its beneficial effects through incorporation into selenoproteins including, glutathione peroxidases, selenoprotein P, iodothyronine deiodinases and thioredoxin reductases. There are more than 30 selenoproteins that have been identified in humans and they are involved in a range of cellular functions including immune function and protection against lipid and DNA damage. The cancer preventive mechanisms of action of methylselenocysteine (MeSeCys) (Figure 3) in human prostate cells are variable. A mechanism of action proposed for MeSeCys is that can alter the expression of several types of collagen gene and protein expression and thus may impact on the extracellular matrix and alter prostate cell progression and invasion (Hurst et al., 2008). Other authors affirm that the effect is due to methylselenol, a metabolite active in a study carried out in the transgenic adenocarcinoma mouse prostate model by oral administration of MeSeCys (J. Zhang et al., 2010). This hypothesis has been reinforced and completed in 2011 with the inclusion of new metabolites, the α-keto acids analogues of MeSeCys (Pinto et al., 2011). Related to selenomethionine (SeMet) (Figure 3) one of the mechanism that is gaining interest is the HDAC inhibition by metabolites of SeMet accompanied of redox signaling proteins modulation (J.I. Lee et al., 2009). Too, in combination with genistein induced growth arrest with modulation of expression of matrix metalloproteinase-2 (MMP-2) (Kumi-Diaka et al., 2010). On the other hand, this compound has been employed in order to reduce the toxic effects of di(2-ethylhexyl)phthalate (DEHP), an abundant plasticizer environmental contaminant that causes alterations in endocrine and spermatogenic functions mediated by induction of reactive oxygen species (ROS) and activation of nuclear p53 and p21 proteins in LNCaP cells. The SeMet supplementation reduced ROS production with modulation of intracellular redox status that is related to response against testicular toxicity (Erkekoglu et al., 2011). If we consider the possibility of combination between SeMet and other compound for modulating PC development the results are drug dependent. So, SeMet

sodium selenite inhibited the lipopolysaccharide (LPS)-induced TLR4-NF-kB signaling in PC-3 (Pei et al., 2010). Sodium selenite can act alone or in combination with other treatments for PC. So, this compound significantly enhances the effect of radiation on well established hormone-independent prostate tumors and does not sensitize the intestinal epithelial cells to radiation. These results suggest that may increase the therapeutic index of radiation therapy (Tian et al., 2010). In addition, the effectiveness on PC treatment of the association between sodium selenite and docetaxel has resulted as a new strategy in PC therapeutic approach (Freitas et al., 2011). Too, combination of genistein and selenite has shown synergistic effects on apoptosis, cell cycle arrest associated signaling pathways in p53 expression (Zhao et al., 2009). Actually, other inorganic forms of Se as sodium selenate (Figure 2), where Se is in oxidation state + 6, are in Phase I studies and have shown antiangiogenic properties

Fig. 2. Methylseleninic acid, sodium selenite and sodium selenate structures.

Se may exert its beneficial effects through incorporation into selenoproteins including, glutathione peroxidases, selenoprotein P, iodothyronine deiodinases and thioredoxin reductases. There are more than 30 selenoproteins that have been identified in humans and they are involved in a range of cellular functions including immune function and protection against lipid and DNA damage. The cancer preventive mechanisms of action of methylselenocysteine (MeSeCys) (Figure 3) in human prostate cells are variable. A mechanism of action proposed for MeSeCys is that can alter the expression of several types of collagen gene and protein expression and thus may impact on the extracellular matrix and alter prostate cell progression and invasion (Hurst et al., 2008). Other authors affirm that the effect is due to methylselenol, a metabolite active in a study carried out in the transgenic adenocarcinoma mouse prostate model by oral administration of MeSeCys (J. Zhang et al., 2010). This hypothesis has been reinforced and completed in 2011 with the inclusion of new metabolites, the α-keto acids analogues of MeSeCys (Pinto et al., 2011). Related to selenomethionine (SeMet) (Figure 3) one of the mechanism that is gaining interest is the HDAC inhibition by metabolites of SeMet accompanied of redox signaling proteins modulation (J.I. Lee et al., 2009). Too, in combination with genistein induced growth arrest with modulation of expression of matrix metalloproteinase-2 (MMP-2) (Kumi-Diaka et al., 2010). On the other hand, this compound has been employed in order to reduce the toxic effects of di(2-ethylhexyl)phthalate (DEHP), an abundant plasticizer environmental contaminant that causes alterations in endocrine and spermatogenic functions mediated by induction of reactive oxygen species (ROS) and activation of nuclear p53 and p21 proteins in LNCaP cells. The SeMet supplementation reduced ROS production with modulation of intracellular redox status that is related to response against testicular toxicity (Erkekoglu et al., 2011). If we consider the possibility of combination between SeMet and other compound for modulating PC development the results are drug dependent. So, SeMet

**2.2 Methylselenocysteine and selenomethionine**

(Corcoran et al., 2010).

and alpha-tocopherol do not inhibit prostate carcinogenesis in the testosterone plus estradiol treated NBL rat model (Ozten et al., 2010). However, the selected combination of silymarin and SeMet significantly reduced two markers of lipid metabolism known associated with PC progression (Vidlar et al., 2010). In order to improve the activity and safety inorganic and organic hybrid nanoparticles are potentially useful in biomedicine, mainly for tumor treatments (Choi et al., 2010). Se nanoparticles are safer compared with SeMet isolated and was observed an inhibition of the growth of prostate LNCaP cancer cells partially through caspases mediated apoptosis, Akt kinase modulation and by disrupting AR (Kong et al., 2011).

Fig. 3. Methylselenocysteine and selenomethionine structures.

#### **2.3 Selenocyanate derivatives**

The first selenocyanate described was 1,4-phenylenebis(methylene)selenocyanate (*p*-XSC) (Figure 4). The most recent studies postulate that this compound is capable of altering cofilin-2, single-stranded mitochondrial DNA binding protein, chaperonin 10, nucleoside diphosphate kinase 6 and chain A Horf 6 human peroxidase enzyme in LNCaP cells and in its androgen independent clone (AI) (R. Sinha et al., 2008). Too, this compound can induce apoptosis, inhibits AR expression and decreases Akt phosphorylation (Facompre et al., 2010). Other organic selenocyanates have emerged during the last years. So phenylalkyl isoselenocyanates (Figure 4), isosteric selenium analogues of naturally occurring phenylalkyl isothiocyanates, have shown a reduction in tumor size associated to apoptosis. The structure activity relationship studies concluded that an increase in the alkyl chain length is critical for the activity being n = 4, named ISC-4, the optimal (Sharma et al., 2008). In 2011, these same authors have reported that ISC-4 activates prostate apoptosis response protein 4 (Par-4) (Sharma et al., 2011). As a continuation of the synthesis of novel alkyl selenocyanates in 2010 was described the synthesis of substituted naphthalimide based organoselenocyanates (Figure 4) with the alkyl chain length n = 5 and investigated their systematic toxicity profile in mice by consideration changes in body weight, hepatotoxicity and nephrotoxicity resulting less toxic than other selenium forms but retaining the efficacy (Roy et al., 2010). Numerous studies have been conducted to elucidate the mechanism underlying the antitumor effects associated with cyclooxygenase 2 (COX-2) inhibitors. However, this mechanism has not yet been clearly defined. Nonsteroidal antiinflammatory drugs (NSAIDs) have been shown to retard the progression of PC in men and NSAIDs have been used in clinical trials for prostate cancer. Celecoxib (Celebrex), a specific COX-2 inhibitor, reduces prostate tumors in experimental models mainly through cell cycle regulation and angiogenesis. However, the growth inhibitory properties of Celecoxib may be COX-2 independent. Considering this possible effect a novel strategy

New Selenoderivatives as Antitumoral Agents 159

tissue can be modified in their expression by coincubation with ebselen (Wartenberg et al., 2010). Too, it has been used for protecting PC-3 cells against apoptosis induction provoked by curcumin, a potent anticancer agent (Hilchie et al., 2010). On the other hand, ebselen has been reported as a covalent inactivator of α-methylacyl coenzyme A racemase (AMACR), a metabolic enzyme whose overexpression has been shown to be a diagnostic indicator of prostatic adenocarcinoma and other solid tumors and has been employed as reference drug for screening of approximately 5000 unique compounds as AMACR inhibitors (Wilson et al., 2011). In structural relation with ebselen is the organoselenium compound 1,2-bis-[1,2 benzisoselenazolone-3(2*H*)-ketone]ethane (BBSKE) (Figure 5), which has shown an inhibitory effect on the growth of a variety of human cancer cells, provokes S phase arrest accompanied by increases in the protein levels of cyclin A, E and p21 and decreases in levels of cyclin B1, D1 and Cdk4 (Shi et al., 2003a, 2003b). A recent study carried out in rats affirms that the metabolites of BBSKE can act as antitumoral agents (Zhou et al., 2010). Too, in association with cisplatin increases the sensitivity of the colon cancer cell line LoVo towards cisplatin via regulation of G1 phase and reversal of G2/M phase arrest (Fu et al., 2011). The formulation as copolymer micelles allows the accumulation into tumor efficiently due to an

D-501036, 2,5-bis(5-hydroxymethyl-2-selenienyl)-3-hydroxymethyl-*N*-methylpyrrole (Figure 5), has been identified as a novel antineoplastic agent with a broad spectrum of antitumoural activity against several human cancer cells and has an IC50 value in the nanomolar range. This compound induces cell death associated with the DNA damagemediated induction of ataxia telangiectasia-mutated activation without interfering with topoisomerase-I and topoisomerase-II function (Juang et al., 2007). Another mechanism that has been proposed for the activity of D-501036 is angiogenesis inhibition. Although antiangiogenesis strategies have generated a great deal of enthusiasm for therapeutic applications, it is still unknown whether these systems would be feasible for prevention. The possibility of interfering very early in tumour progression by modulating the cancer angiogenic switch is appealing, though there is increasing evidence for close correlation between inflammation, the micro-environment and tumour-associated neo-angiogenesis

In 2010, a new series of heterocyclic organoselenium compounds were synthesized and evaluated as possible chemopreventive agents in human prostate cancer LNCaP cells. Two of this 3-selena-1-dethiacephem derivatives (Figure 5) strongly activated nuclear factor E2 related factor 2 (Nrf2)/ antioxidant response element (ARE) signaling that regulate expression of phase II antioxidant and detoxifying enzymes such as glutathione peroxidase (GPX), γ-glutamylcysteine synthetase (γ-GCS), heme oxygenase-1 (HO-1), NADPH quinone oxidoreductase (NQO-1), and glutathione S-transferase (GST) expression. These two compounds also possessed a potent antioxidant activity. Furthermore, both compounds were capable of inhibiting cell growth via cell cycle arrest. Related to structure activity relationship the presence of the exo-olefin (carbon–carbon double bond) as well as the aliphatic substitution at the imine part is critical for these compounds to activate Nrf2/ARE signaling (Terazawa et al., 2010). Other interesting derivatives are 2-substituted selenazolidine-4(*R*)-carboxylic acids (Figure 5). There are numerous studies that concern the induction of a protective hepatic enzyme related to gluthathione-S-tranferase and gluthathione peroxidase and it seemed of interest to evaluate these compounds in PC cells

increase in water solubility (M. Liu et al., 2010).

(El-Sayed et al., 2007, Poerschke & Moos 2011).

causing the adverse outcomes of prostate cancer (Araldi et al., 2008).

has been proposed based on to combine selenium and COX-2 inhibitor. Considering that sulfonamide moiety and pyrazole ring are important for the proapoptotic activity of Celecoxib against PC, the Selenocoxib-1 (Figure 4) was synthesized. The structural modifications introduced were the replacement of the trifluoromethyl group by methyleneselenocyanate fragment and the elimination of the methyl group. The study carried out against PAIII cells derived from a metastatic prostate tumor that arose spontaneously in a Lobund-Wistar (LW) rat. In addition, human metastatic prostate cancer cells, PC-3M, were tested for antitumor effect of Selenocoxib-1 *in vitro*. Selenocoxib-1 induced apoptosis in a dose-dependent manner in the PAIII cells and resulted more effective against PC than Celecoxib (Desai et al., 2010a). Other modulations have been introduced maintaining the methyl group in order to obtain Selenocoxib-2 (Figure 4) but it has not been studied as antitumoral yet (Desai et al., 2010b).

Fig. 4. Chemical structures for selenocyanate derivatives.

#### **2.4 Heterocycles containing selenium**

Ebselen (Figure 5) is one of the most relevant heterocyclic compounds derived from selenium. Ebselen is a glutathione peroxidase mimetic seleno-organic compound that attenuates the H2O2 level. In this process a hydroxylamine spin trap reacts with oxygencentered radicals, including superoxide. It was seen that this compound blocked the expression of the disintegrin and metalloprotease ADAM9 in LNCaP or C4-2 PC cells through inhibition of ROS production (Sung et al., 2006). In other studies Ebselen was used as an external agent for reverting biochemical processes such as glycolysis. For example, ABC transporters like P-glycoprotein (P-gp/ABCB1), that are membrane proteins responsible for the transport of toxic compounds out of non-malignant cells and tumor

has been proposed based on to combine selenium and COX-2 inhibitor. Considering that sulfonamide moiety and pyrazole ring are important for the proapoptotic activity of Celecoxib against PC, the Selenocoxib-1 (Figure 4) was synthesized. The structural modifications introduced were the replacement of the trifluoromethyl group by methyleneselenocyanate fragment and the elimination of the methyl group. The study carried out against PAIII cells derived from a metastatic prostate tumor that arose spontaneously in a Lobund-Wistar (LW) rat. In addition, human metastatic prostate cancer cells, PC-3M, were tested for antitumor effect of Selenocoxib-1 *in vitro*. Selenocoxib-1 induced apoptosis in a dose-dependent manner in the PAIII cells and resulted more effective against PC than Celecoxib (Desai et al., 2010a). Other modulations have been introduced maintaining the methyl group in order to obtain Selenocoxib-2 (Figure 4) but it

has not been studied as antitumoral yet (Desai et al., 2010b).

Fig. 4. Chemical structures for selenocyanate derivatives.

Ebselen (Figure 5) is one of the most relevant heterocyclic compounds derived from selenium. Ebselen is a glutathione peroxidase mimetic seleno-organic compound that attenuates the H2O2 level. In this process a hydroxylamine spin trap reacts with oxygencentered radicals, including superoxide. It was seen that this compound blocked the expression of the disintegrin and metalloprotease ADAM9 in LNCaP or C4-2 PC cells through inhibition of ROS production (Sung et al., 2006). In other studies Ebselen was used as an external agent for reverting biochemical processes such as glycolysis. For example, ABC transporters like P-glycoprotein (P-gp/ABCB1), that are membrane proteins responsible for the transport of toxic compounds out of non-malignant cells and tumor

**2.4 Heterocycles containing selenium** 

tissue can be modified in their expression by coincubation with ebselen (Wartenberg et al., 2010). Too, it has been used for protecting PC-3 cells against apoptosis induction provoked by curcumin, a potent anticancer agent (Hilchie et al., 2010). On the other hand, ebselen has been reported as a covalent inactivator of α-methylacyl coenzyme A racemase (AMACR), a metabolic enzyme whose overexpression has been shown to be a diagnostic indicator of prostatic adenocarcinoma and other solid tumors and has been employed as reference drug for screening of approximately 5000 unique compounds as AMACR inhibitors (Wilson et al., 2011). In structural relation with ebselen is the organoselenium compound 1,2-bis-[1,2 benzisoselenazolone-3(2*H*)-ketone]ethane (BBSKE) (Figure 5), which has shown an inhibitory effect on the growth of a variety of human cancer cells, provokes S phase arrest accompanied by increases in the protein levels of cyclin A, E and p21 and decreases in levels of cyclin B1, D1 and Cdk4 (Shi et al., 2003a, 2003b). A recent study carried out in rats affirms that the metabolites of BBSKE can act as antitumoral agents (Zhou et al., 2010). Too, in association with cisplatin increases the sensitivity of the colon cancer cell line LoVo towards cisplatin via regulation of G1 phase and reversal of G2/M phase arrest (Fu et al., 2011). The formulation as copolymer micelles allows the accumulation into tumor efficiently due to an

increase in water solubility (M. Liu et al., 2010). D-501036, 2,5-bis(5-hydroxymethyl-2-selenienyl)-3-hydroxymethyl-*N*-methylpyrrole (Figure 5), has been identified as a novel antineoplastic agent with a broad spectrum of antitumoural activity against several human cancer cells and has an IC50 value in the nanomolar range. This compound induces cell death associated with the DNA damagemediated induction of ataxia telangiectasia-mutated activation without interfering with topoisomerase-I and topoisomerase-II function (Juang et al., 2007). Another mechanism that has been proposed for the activity of D-501036 is angiogenesis inhibition. Although antiangiogenesis strategies have generated a great deal of enthusiasm for therapeutic applications, it is still unknown whether these systems would be feasible for prevention. The possibility of interfering very early in tumour progression by modulating the cancer angiogenic switch is appealing, though there is increasing evidence for close correlation between inflammation, the micro-environment and tumour-associated neo-angiogenesis causing the adverse outcomes of prostate cancer (Araldi et al., 2008).

In 2010, a new series of heterocyclic organoselenium compounds were synthesized and evaluated as possible chemopreventive agents in human prostate cancer LNCaP cells. Two of this 3-selena-1-dethiacephem derivatives (Figure 5) strongly activated nuclear factor E2 related factor 2 (Nrf2)/ antioxidant response element (ARE) signaling that regulate expression of phase II antioxidant and detoxifying enzymes such as glutathione peroxidase (GPX), γ-glutamylcysteine synthetase (γ-GCS), heme oxygenase-1 (HO-1), NADPH quinone oxidoreductase (NQO-1), and glutathione S-transferase (GST) expression. These two compounds also possessed a potent antioxidant activity. Furthermore, both compounds were capable of inhibiting cell growth via cell cycle arrest. Related to structure activity relationship the presence of the exo-olefin (carbon–carbon double bond) as well as the aliphatic substitution at the imine part is critical for these compounds to activate Nrf2/ARE signaling (Terazawa et al., 2010). Other interesting derivatives are 2-substituted selenazolidine-4(*R*)-carboxylic acids (Figure 5). There are numerous studies that concern the induction of a protective hepatic enzyme related to gluthathione-S-tranferase and gluthathione peroxidase and it seemed of interest to evaluate these compounds in PC cells (El-Sayed et al., 2007, Poerschke & Moos 2011).

New Selenoderivatives as Antitumoral Agents 161

feature the presence of an alpha-methylidene-gamma-butyrolactone moiety in their structures (Arantes et al., 2009). This hypothesis has been corroborated in other tumoral cell lines. In addition, the bioactive α-santonin derivatives are selective against cancer cells

It is well established that various human diseases, including PC, are associated with a disturbed intracellular redox balance and oxidative stress (OS). Se based agents (Figure 6) turn the oxidizing redox environment present in certain cancer cells into a lethal cocktail of reactive species that push these cells over a critical redox threshold and ultimately kill them through apoptosis. The main advantage is that this kind of toxicity is highly selective: normal, healthy cells remain largely unaffected, since changes to their naturally low levels of oxidizing species produce little effect though the biochemical pathways triggered by these agents need to be studied in more detail such as redox modulator like cysteine-containing Bcl proteins, which control apoptosis at an early stage, certain caspases, which execute apoptotic mechanisms further downstream, are also redox sensitive (Jamier et al., 2010).

Fig. 6. Chemical structures for selenide and diselenide derivatives.

Selenium- and sulfur-containing compounds have been widely studied as potential antioxidants for the prevention or reduction of oxidative DNA damage and the organoselenium compounds are of particular interest because they appear to be more bioavailable relative to inorganic Se compounds. The Se and sulfur antioxidant activity has been explained using copper-mediated DNA damage studies and UV-vis spectroscopy that have allowed identifying a copper coordination through a novel metal bond. For this reason has been described the synthesis of relevant copper selone complexes with tris(pyrazolyl)methane or tris(pyrazolyl)borate ligands (Figure 7) (Kimani et al., 2010). The determination of redox potential for Cu-Selone complexes indicated that Se coordination to copper in biological systems may prevent the reduction of Cu2+ by NADH required for the

**2.6 Selenium and metal complexes** 

catalytic formation of damaging hydroxyl radical.

(Arantes et al., 2010).

Fig. 5. Chemical structures for heterocycles containing selenium.

#### **2.5 Selenide and diselenide derivatives**

The selenide function is present in a lot of organoselenium compounds. Many of the above described derivatives possess this type of bond (i.e. MeSeCys, SeMet,). One compound does not described in the above sections is *p*-xylylbis(methylselenide) (*p*-XMS) (Figure 6), a organoselenium compound that modifies the growth, secretion of PSA and the intracellular redox status and genomic profiles (Pinto et al., 2007). Too, the selenide function is present linked to nucleosides. The natural nucleosides may be used as Se-carriers. The synthesis and antitumor activity of novel nucleosides derivatized from uridine and thymidine (Figure 6), with a selenomethyl group at various positions has been described. In general, the activity against PC cells is position-dependent. Compounds with the selenomethyl group in position 5' are more active than the corresponding in 2' or 3'. The probable explanation is that it is easier to metabolize the Se-nucleoside containing the primary selenomethyl than the secondary selenomethyl, thereby generating more methylselenol (Lin et al., 2009). The natural products continue to be a rich source of new promising substances for cancer therapy. Sesquiterpene lactones (SQLs) are a class of naturally occurring plant terpenoids of Asteraceae family, known for their various biological activities such as cytotoxicity against different tumor cell lines. Many authors have linked this activity mainly to the α-methyleneγ-lactone functionality, which is prone to react with suitable nucleophiles, e.g., sulfhydryl groups of cysteine, in a Michael addition mechanism. These reactions are nonspecific, leading to the inhibition of a large number of enzymes or factors involved in key biological in spite of it is well known, however, that the α-methylene-γ-lactone moiety is not an absolute requirement for cytotoxicity. In this context emerge other interesting compounds such as the alpha-santonin derivatives (Figure 6) a sesquiterpene lactone isolated from *Artemisia santonica*. The compounds with higher activity showed as common structural

The selenide function is present in a lot of organoselenium compounds. Many of the above described derivatives possess this type of bond (i.e. MeSeCys, SeMet,). One compound does not described in the above sections is *p*-xylylbis(methylselenide) (*p*-XMS) (Figure 6), a organoselenium compound that modifies the growth, secretion of PSA and the intracellular redox status and genomic profiles (Pinto et al., 2007). Too, the selenide function is present linked to nucleosides. The natural nucleosides may be used as Se-carriers. The synthesis and antitumor activity of novel nucleosides derivatized from uridine and thymidine (Figure 6), with a selenomethyl group at various positions has been described. In general, the activity against PC cells is position-dependent. Compounds with the selenomethyl group in position 5' are more active than the corresponding in 2' or 3'. The probable explanation is that it is easier to metabolize the Se-nucleoside containing the primary selenomethyl than the secondary selenomethyl, thereby generating more methylselenol (Lin et al., 2009). The natural products continue to be a rich source of new promising substances for cancer therapy. Sesquiterpene lactones (SQLs) are a class of naturally occurring plant terpenoids of Asteraceae family, known for their various biological activities such as cytotoxicity against different tumor cell lines. Many authors have linked this activity mainly to the α-methyleneγ-lactone functionality, which is prone to react with suitable nucleophiles, e.g., sulfhydryl groups of cysteine, in a Michael addition mechanism. These reactions are nonspecific, leading to the inhibition of a large number of enzymes or factors involved in key biological in spite of it is well known, however, that the α-methylene-γ-lactone moiety is not an absolute requirement for cytotoxicity. In this context emerge other interesting compounds such as the alpha-santonin derivatives (Figure 6) a sesquiterpene lactone isolated from *Artemisia santonica*. The compounds with higher activity showed as common structural

Fig. 5. Chemical structures for heterocycles containing selenium.

**2.5 Selenide and diselenide derivatives**

feature the presence of an alpha-methylidene-gamma-butyrolactone moiety in their structures (Arantes et al., 2009). This hypothesis has been corroborated in other tumoral cell lines. In addition, the bioactive α-santonin derivatives are selective against cancer cells (Arantes et al., 2010).

It is well established that various human diseases, including PC, are associated with a disturbed intracellular redox balance and oxidative stress (OS). Se based agents (Figure 6) turn the oxidizing redox environment present in certain cancer cells into a lethal cocktail of reactive species that push these cells over a critical redox threshold and ultimately kill them through apoptosis. The main advantage is that this kind of toxicity is highly selective: normal, healthy cells remain largely unaffected, since changes to their naturally low levels of oxidizing species produce little effect though the biochemical pathways triggered by these agents need to be studied in more detail such as redox modulator like cysteine-containing Bcl proteins, which control apoptosis at an early stage, certain caspases, which execute apoptotic mechanisms further downstream, are also redox sensitive (Jamier et al., 2010).

Fig. 6. Chemical structures for selenide and diselenide derivatives.

#### **2.6 Selenium and metal complexes**

Selenium- and sulfur-containing compounds have been widely studied as potential antioxidants for the prevention or reduction of oxidative DNA damage and the organoselenium compounds are of particular interest because they appear to be more bioavailable relative to inorganic Se compounds. The Se and sulfur antioxidant activity has been explained using copper-mediated DNA damage studies and UV-vis spectroscopy that have allowed identifying a copper coordination through a novel metal bond. For this reason has been described the synthesis of relevant copper selone complexes with tris(pyrazolyl)methane or tris(pyrazolyl)borate ligands (Figure 7) (Kimani et al., 2010). The determination of redox potential for Cu-Selone complexes indicated that Se coordination to copper in biological systems may prevent the reduction of Cu2+ by NADH required for the catalytic formation of damaging hydroxyl radical.

New Selenoderivatives as Antitumoral Agents 163

this reason, and in order to improve the potency of our compounds, we decided to introduce some structural modifications. Among these modifications was the preparation of new compounds with related structures based on aroyl and heteroaroyl selenylacetic acid derivatives (Figure 8). The most promising derivatives against PC-3 cancer cells were the corresponding phenyl, 3,5-dimethoxyphenyl and benzyl with TGI values of 6.8, 4.0 and 2.9 μM (Sanmartín et al., 2009). Too, there is current interest in heterocyclic compounds that contain a Se atom in the ring. Bearing this fact in mind, and as a continuation of our previous work, we proposed the synthesis 1,2,5-selenadiazolo[3,4 *d*]pyridines and 1,2,5-benzoselenadiazolo derivatives (Figure 8). The most promising molecule was a pyridine derivative (Plano et al., 2010b). Other explored structures were compounds with selenocyanate and diselenide moieties. Moreover, we evaluated their antioxidant-prooxidant properties so as their cytotoxic activities against PC-3 resulting eighteen of the fifty-nine compounds evaluated more potent than etoposide (Plano et al., 2010a). Taking into account that oxidation state for Se is related to antitumoral effect we have synthesized and evaluated an original series consisting of a small group of compounds which possess Se in the +4 states oxidation instead of Se +2 (Figure 8) (Plano

Fig. 8. Selenoderivatives obtained and evaluated by our research group.

modification in ErB4, GS3Ka and PKCA was detected (unpublished results).

Considering that Se has been associated with an anticancer effect via the modulation of some kinase such as Akt (J.H. Lee et al., 2008) some of these compounds have been studied as kinase modulators. Some of them modulate CK1A and GS3Ka expression and a weak

The preliminary results from the biological screening of these novel compounds are very encouraging and these systems could offer an excellent framework in this field and may

et al., 2010a).

Fig. 7. Chemical structure for Cu-Selone complexes. Reprinted with permission from Kimani et al., 2010. Copyright 2010 American Chemical Society.

#### **2.7 A case study: Novel selenoderivatives as cytotoxic agents and apoptosis inducers in prostate cancer cells**

In the last four years, several articles have been published by our research group related to the design, synthesis and biological evaluation of novel compounds containing Se as cytotoxic agents and apoptosis inducers. In addition, mounting evidence suggests that selenium (Se) works by inhibiting important early steps in carcinogenesis in a variety of experimental models and the anticancer activity is dependent on the chemical form of selenium. Se occurs in both organic and inorganic forms. Based on these findings we envisaged a new investigation that involves the synthesis of new compounds that incorporate the Se-containing moiety.

#### **2.7.1 Structures and biological results**

Initially, the rationale behind the design of these compounds was to maintain molecular symmetry, a structural property that is frequently present in cytotoxic and pro-apoptotic drugs (Sanmartín et al., 2006). The structures synthesised correspond to molecules with a central nucleus made up of an alkyl imidothiocarbamate (alkyl isothiourea) or alkyl imidoselenocarbamate (alkyl isoselenourea) connected by a carbonyl group on each side to two identical lateral aromatic or heteroaromatic rings mono, bi or polycyclics (Figure 8). The sulfur and selenium substituents were varied (methyl, ethyl, benzyl and isopropyl) to determine the effect of the alkyl chain length and the ramifications that this has on the activity (Plano et al., 2007, Ibáñez et al., 2011). The best results in PC-3 were obtained for the compound with X = Se, Y = C, R = CH3 and R' = 4-CH3. This compound was the most potent (IC50 = 1.85 μM) and was 4.5 times more active than standard methylseleninic acid (IC50 = 8.38 μM) and 7.3 times more active than etoposide (IC50 = 13.6 μM), an agent used in the treatment of PC. In addition, the novel compound was less toxic than the reference and apoptotic inducer in MCF-7 and CCRF-CEM. For the heteroaromatic rings thienyl and quinolinyl were the most interesting. During the course of our work a great number of different structural classes of selenocompounds were reported (Sanmartín et al., 2008). For

Fig. 7. Chemical structure for Cu-Selone complexes. Reprinted with permission from Kimani

**2.7 A case study: Novel selenoderivatives as cytotoxic agents and apoptosis inducers** 

In the last four years, several articles have been published by our research group related to the design, synthesis and biological evaluation of novel compounds containing Se as cytotoxic agents and apoptosis inducers. In addition, mounting evidence suggests that selenium (Se) works by inhibiting important early steps in carcinogenesis in a variety of experimental models and the anticancer activity is dependent on the chemical form of selenium. Se occurs in both organic and inorganic forms. Based on these findings we envisaged a new investigation that involves the synthesis of new compounds that

Initially, the rationale behind the design of these compounds was to maintain molecular symmetry, a structural property that is frequently present in cytotoxic and pro-apoptotic drugs (Sanmartín et al., 2006). The structures synthesised correspond to molecules with a central nucleus made up of an alkyl imidothiocarbamate (alkyl isothiourea) or alkyl imidoselenocarbamate (alkyl isoselenourea) connected by a carbonyl group on each side to two identical lateral aromatic or heteroaromatic rings mono, bi or polycyclics (Figure 8). The sulfur and selenium substituents were varied (methyl, ethyl, benzyl and isopropyl) to determine the effect of the alkyl chain length and the ramifications that this has on the activity (Plano et al., 2007, Ibáñez et al., 2011). The best results in PC-3 were obtained for the compound with X = Se, Y = C, R = CH3 and R' = 4-CH3. This compound was the most potent (IC50 = 1.85 μM) and was 4.5 times more active than standard methylseleninic acid (IC50 = 8.38 μM) and 7.3 times more active than etoposide (IC50 = 13.6 μM), an agent used in the treatment of PC. In addition, the novel compound was less toxic than the reference and apoptotic inducer in MCF-7 and CCRF-CEM. For the heteroaromatic rings thienyl and quinolinyl were the most interesting. During the course of our work a great number of different structural classes of selenocompounds were reported (Sanmartín et al., 2008). For

et al., 2010. Copyright 2010 American Chemical Society.

**in prostate cancer cells**

incorporate the Se-containing moiety.

**2.7.1 Structures and biological results** 

this reason, and in order to improve the potency of our compounds, we decided to introduce some structural modifications. Among these modifications was the preparation of new compounds with related structures based on aroyl and heteroaroyl selenylacetic acid derivatives (Figure 8). The most promising derivatives against PC-3 cancer cells were the corresponding phenyl, 3,5-dimethoxyphenyl and benzyl with TGI values of 6.8, 4.0 and 2.9 μM (Sanmartín et al., 2009). Too, there is current interest in heterocyclic compounds that contain a Se atom in the ring. Bearing this fact in mind, and as a continuation of our previous work, we proposed the synthesis 1,2,5-selenadiazolo[3,4 *d*]pyridines and 1,2,5-benzoselenadiazolo derivatives (Figure 8). The most promising molecule was a pyridine derivative (Plano et al., 2010b). Other explored structures were compounds with selenocyanate and diselenide moieties. Moreover, we evaluated their antioxidant-prooxidant properties so as their cytotoxic activities against PC-3 resulting eighteen of the fifty-nine compounds evaluated more potent than etoposide (Plano et al., 2010a). Taking into account that oxidation state for Se is related to antitumoral effect we have synthesized and evaluated an original series consisting of a small group of compounds which possess Se in the +4 states oxidation instead of Se +2 (Figure 8) (Plano et al., 2010a).

Fig. 8. Selenoderivatives obtained and evaluated by our research group.

Considering that Se has been associated with an anticancer effect via the modulation of some kinase such as Akt (J.H. Lee et al., 2008) some of these compounds have been studied as kinase modulators. Some of them modulate CK1A and GS3Ka expression and a weak modification in ErB4, GS3Ka and PKCA was detected (unpublished results).

The preliminary results from the biological screening of these novel compounds are very encouraging and these systems could offer an excellent framework in this field and may

New Selenoderivatives as Antitumoral Agents 165

The authors wish to express their gratitude to the Ministerio de Educación y Ciencia, Spain

Abdulah R, *et al.* (2011) Molecular targets of selenium in prostate cancer prevention

Algotar AM*, et al.* (2011) Dose-dependent effects of selenized yeast on total selenium levels

Aly M, Wiklund F, & Grönberg H (2011) Early detection of prostate cancer with emphasis

Araldi EMV*, et al.* (2008) Natural and synthetic agents targeting inflammation and

Arantes FFP*, et al.* (2010) Synthesis of novel alpha-santonin derivatives as potential cytotoxic

Arantes FFP*, et al.* (2009) Synthesis and cytotoxic activity of alpha-santonin derivatives.

Berggren M*, et al.* (2009) Sodium selenite increases the activity of the tumor suppressor

Choi HS*, et al.* (2010) Design considerations for tumour-targeted nanoparticles. *Nature Nanotechnology,* Vol.5, No.1, (January 2010), pp. 42-47, ISSN 1748-3387. Colloca G & Venturino A (2011) The evolving role of familial history for prostate cancer. *Acta Oncologica,* Vol.50, No.1, (January 2011), pp. 14-24, ISSN 0284-186X. Corcoran NM, *et al.* (2010) Open-label, phase I dose-escalation study of sodium selenate, a

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**4. Acknowledgment** 

**5. References** 

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ultimately lead to discovery of potent antitumour agent. Thus, the search for new drugs with Se continues to be a great challenge in medical science.

#### **3. Conclusion**

It is clear from the studies discussed above that Se compounds do have effects on growth, cell cycle and apoptosis and that such compounds offer great promise as anticancer and apoptotic agents in many tumoral processes, mainly for PC. In this chapter we have summarized information on more than twenty eight structures that contain Se – most of which were published in the last three years – and possess cytotoxic activity against PC. This list of compounds and references is by no means exhaustive and merely hints at the hundreds of other citations due to the ever increasing amount of work carried out in this field. As a result of these studies, Se derivatives are rapidly emerging as valid chemotherapeutic agents. However, various organic and inorganic selenium compounds used in some studies have produced variable results when they are tested in animal models and human subjects and more investigations are urgently needed in order to ascertain the safety in their use. We have included some structures that have not yet been evaluated in prostate cancer cells because we believe that the study of these compounds would be of interest. Although several possible mechanisms have been proposed to explain the anticancer and apoptotic properties of selenium compounds, the results described here suggest the following preliminary considerations:


This class of compound offers a great deal of promise to broaden significantly the horizons of modern apoptosis and anticancer drug discovery for the potential treatment of prostate cancer. Animal data, epidemiological data, and intervention trials have shown a clear role for selenium derivatives in both the prevention of specific cancers and antitumourigenic effects in postinitiation phases of cancer through apoptosis induction. Accordingly, in recent years there has been substantial interest directed toward the synthesis of seleniumcontaining derivatives that could be used as cytotoxic, cancer chemopreventive and apoptotic agents. However, a great deal of further research is needed to unravel the precise manner in which selenium compounds act.

#### **4. Acknowledgment**

The authors wish to express their gratitude to the Ministerio de Educación y Ciencia, Spain (SAF 2009-07744) for financial support.

#### **5. References**

164 Prostate Cancer – Original Scientific Reports and Case Studies

ultimately lead to discovery of potent antitumour agent. Thus, the search for new drugs

It is clear from the studies discussed above that Se compounds do have effects on growth, cell cycle and apoptosis and that such compounds offer great promise as anticancer and apoptotic agents in many tumoral processes, mainly for PC. In this chapter we have summarized information on more than twenty eight structures that contain Se – most of which were published in the last three years – and possess cytotoxic activity against PC. This list of compounds and references is by no means exhaustive and merely hints at the hundreds of other citations due to the ever increasing amount of work carried out in this field. As a result of these studies, Se derivatives are rapidly emerging as valid chemotherapeutic agents. However, various organic and inorganic selenium compounds used in some studies have produced variable results when they are tested in animal models and human subjects and more investigations are urgently needed in order to ascertain the safety in their use. We have included some structures that have not yet been evaluated in prostate cancer cells because we believe that the study of these compounds would be of interest. Although several possible mechanisms have been proposed to explain the anticancer and apoptotic properties of selenium compounds, the results described here

1. The chemical form is a determinant factor for the activity and the metabolism is

2. The effect of some selenium compounds mainly depends on the dose and the oxidation state of selenium. For inorganic selenium compounds the +4 oxidation state gives the highest anticarcinogenic properties and for organic selenium compounds the activity is

3. The existence of diverse responses for the same chemical structure suggests several mechanisms of action. For example, sodium selenite induced apoptosis by redox processes, decreased HDAC activity, increased of PTEN activity. The expectation of a broad therapeutic benefit from agents that target only one member of either pathway may

be overly simplistic due to the complex interrelated network governing apoptosis. 4. Experimental evidence shows that molecular symmetry, as a broad concept, could be a positive factor for cancer prevention and apoptosis (sodium selenite, methylseleninic acid, *p*-XSC, *p*-XMS, BBSKE). The importance of molecular symmetry in cytotoxic and pro-apoptotic activities was reported by us in 2006. Recently, we described a new series of symmetrical organoselenium compounds that are potent as cytotoxic agents in

This class of compound offers a great deal of promise to broaden significantly the horizons of modern apoptosis and anticancer drug discovery for the potential treatment of prostate cancer. Animal data, epidemiological data, and intervention trials have shown a clear role for selenium derivatives in both the prevention of specific cancers and antitumourigenic effects in postinitiation phases of cancer through apoptosis induction. Accordingly, in recent years there has been substantial interest directed toward the synthesis of seleniumcontaining derivatives that could be used as cytotoxic, cancer chemopreventive and apoptotic agents. However, a great deal of further research is needed to unravel the precise

with Se continues to be a great challenge in medical science.

suggest the following preliminary considerations:

mainly observed for the +2 oxidation state.

required for anticarcinogenic activity.

prostate cancer cells.

manner in which selenium compounds act.

**3. Conclusion** 


New Selenoderivatives as Antitumoral Agents 167

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**11** 

*USA*

**Stem Like Cells and Androgen** 

**Deprivation in Prostate Cancer** 

Yao Tang, Mohammad A. Khan, Bin Zhang and Arif Hussain *University of Maryland School of Medicine and Baltimore VA Medical Center* 

Cancer stem cells (CSC) have been hypothesized to contribute to tumor initiation and recurrence, but the very existence of CSC is currently under debate. Increased expression of stem cell markers in cancer tissues after various treatments has been observed in both experimental animal models and patients for a number of cancer types. Cancer cells that express stem cell markers are generally called stem-like cells (SLC) since the exact origin of

Using human LNCaP prostate cancer cell-based mouse xenografts as well as a transgenic model of prostate cancer (TRAMP), we studied the possible origin of SLC and their potential role in cancer recurrence after androgen deprivation therapy (ADT). We found that the proportion of SLC within a tumor can change over time, particularly after anti-cancer therapy (Tang et al., 2010). A significant increase in the SLC population occurred in tumors soon after ADT (surgical castration), but then returned to basal levels when the tumors resumed growth after the initial response to ADT. Several stem cell markers were found to be elevated during this period. This phenomenon was observed in both LNCaP xenografts and in TRAMP mice. These observations suggest that ADT may induce a 'stemness' stage in tumors which, although transient, could allow tumor cells to adapt to the anti-tumor effects of ADT and enhance their survival. A similar phenomenon was observed in LNCaP xenografts after docetaxel treatment. We believe stemness may have a biological function in self protection; it may be one pathway by which tumor cells can survive and recur after anti-

The concept of cancer stem cells (CSC) is built upon the hypothesis that tumor tissues harbor a very small population of cells that is responsible for tumor initiation and recurrence due to its capacity for self-renewal and multilineage differentiation, as well as relative drug resistance. However, the frequency of CSC can be highly variable among different tumor types, and even among tumors of the same type (Visvader & Lindeman, 2008). For instance, in clinical samples, the CSC population in melanomas (ABCB5+) ranges between 1.6 - 20% (Schatton et al., 2008), and in colorectal carcinomas (CD133+) between 1.8 - 24.5% (O'Brien et al., 2007). The factors governing the different frequencies of CSC within

**1. Introduction** 

cancer treatment.

**2. Cancer stem cell or cancer cell stemness** 

these cells is often not clear.

clinical trial. *Biomedical Papers-Olomouc,* Vol.154, No.3, (September 2010), pp. 239- 244, ISSN 1213-8118.


### **Stem Like Cells and Androgen Deprivation in Prostate Cancer**

Yao Tang, Mohammad A. Khan, Bin Zhang and Arif Hussain *University of Maryland School of Medicine and Baltimore VA Medical Center USA*

#### **1. Introduction**

170 Prostate Cancer – Original Scientific Reports and Case Studies

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3(2H)-ketone) -ethane, a novel anti-cancer agent in rat. *Yaoxue Xuebao,* Vol.45, No.5,

Cancer stem cells (CSC) have been hypothesized to contribute to tumor initiation and recurrence, but the very existence of CSC is currently under debate. Increased expression of stem cell markers in cancer tissues after various treatments has been observed in both experimental animal models and patients for a number of cancer types. Cancer cells that express stem cell markers are generally called stem-like cells (SLC) since the exact origin of these cells is often not clear.

Using human LNCaP prostate cancer cell-based mouse xenografts as well as a transgenic model of prostate cancer (TRAMP), we studied the possible origin of SLC and their potential role in cancer recurrence after androgen deprivation therapy (ADT). We found that the proportion of SLC within a tumor can change over time, particularly after anti-cancer therapy (Tang et al., 2010). A significant increase in the SLC population occurred in tumors soon after ADT (surgical castration), but then returned to basal levels when the tumors resumed growth after the initial response to ADT. Several stem cell markers were found to be elevated during this period. This phenomenon was observed in both LNCaP xenografts and in TRAMP mice. These observations suggest that ADT may induce a 'stemness' stage in tumors which, although transient, could allow tumor cells to adapt to the anti-tumor effects of ADT and enhance their survival. A similar phenomenon was observed in LNCaP xenografts after docetaxel treatment. We believe stemness may have a biological function in self protection; it may be one pathway by which tumor cells can survive and recur after anticancer treatment.

#### **2. Cancer stem cell or cancer cell stemness**

The concept of cancer stem cells (CSC) is built upon the hypothesis that tumor tissues harbor a very small population of cells that is responsible for tumor initiation and recurrence due to its capacity for self-renewal and multilineage differentiation, as well as relative drug resistance. However, the frequency of CSC can be highly variable among different tumor types, and even among tumors of the same type (Visvader & Lindeman, 2008). For instance, in clinical samples, the CSC population in melanomas (ABCB5+) ranges between 1.6 - 20% (Schatton et al., 2008), and in colorectal carcinomas (CD133+) between 1.8 - 24.5% (O'Brien et al., 2007). The factors governing the different frequencies of CSC within

Stem Like Cells and Androgen Deprivation in Prostate Cancer 173

deprivation and chemotherapy on prostate cancer (Y. Tang et al., 2006, 2008, 2009, 2010). Tumor tissues were collected at different time points before and after various treatments, and the expression patterns of several stem cell markers were evaluated in an effort to better

LNCaP is a hormone-sensitive human prostate cancer cell line. Withdrawal of androgens *in vitro* interrupts its growth and induces apoptosis. *In vivo*, LNCaP tumors in male SCID mice cease growth for up to 2-3 weeks after surgical castration (bilateral orchiectomy), which is invariably followed by accelerated tumor growth (Figure 1A). Immunohistochemical (IHC) analysis of proliferation-related (Ki67) and angiogenesisrelated (CD105) markers in castrated and non-castrated mice is shown in Figures 1B and 1C. It is apparent that a significant decrease in overall proliferation occurs only in the castrated mice between one and two weeks after castration, followed then by recovery of proliferative potential. This recovery of cell proliferation appears to parallel increased angiogenesis, which is observed at the IHC level between two and three weeks post castration. In non-castrated mice, on the other hand, tumors continue to grow unabated over time, with no observable changes in cell proliferation or overall angiogenesis noted

To determine whether SLC play any role in the above model, we also studied several stem cell markers. Recent studies suggest that SLC are a heterogeneous population with diverse biological properties, and that multiple subpopulations with stem cell-like characteristics can coexist in the same tumor (Hermann et al., 2007; Ma et al., 2008; Hope et al., 2004). Since there is no single specific 'standard' marker for identifying cancer stem cells, we selected several antigens that have been implicated in one way or the other in stem cell biology; specifically, CD44, CD133, and c-Kit (CD117) for IHC studies, and human ALDH (aldehyde dehydrogenase), Shh (sonic hedgehog), p63, BCRP (breast cancer resistant protein), Notch1 and bcl-2 for western blot analyses. Tumor samples were collected at days 5, 10, 15, 20, 25, and 30 post castration, and tumors from non-castrated mice collected at similar time points

By IHC, expressions of c-Kit (Figure 1D) and CD44 (Figure 1E), but not CD133, were significantly increased in LNCaP xenograft tumors at the day 15 time point after castration but not at other time points in either castrated or control mice. Representative images of c-Kit and CD44 at day-15 (Cas-15) and day 30 (Cas-30) are shown in Figure 1E. Interestingly, we noted that the distribution of CD44+ and c-Kit+ cells in tumor tissues was different. Most of the CD44+ cells were found in the periphery of tumor islands, whereas c-Kit+ cells were present within the tumor mass (Figure 1E). This suggests that these two proteins can be expressed in different cells within the LNCaP tumors, and may potentially signify the presence of different subtypes of SLC in these tumors. In untreated control mice, on the other hand, SLC markers do not change significantly over time despite continued tumor

Data for several proteins evaluated by western blots are summarized in Table 1. It is apparent from these initial studies that there is a trend for Shh, Notch, ALDH, BCRP and p63 to be over expressed at least 2-fold at day 15 compared to the other time points post castration in most or all the tumor samples tested, while Bcl2 is over expressed in 2 of the 4

understand treatment response and potential mechanisms of tumor recurrence.

**3.1 LNCaP xenograft based studies** 

served as controls.

growth, as shown in Figure 1D.

tumors evaluated at this time point.

over this period of continued growth (Figures 1A-C).

tumors are not clearly delineated, although communication with surrounding cells and stroma, alterations in pH, chemokines/cytokines in the microenvironment, locoregional angiogenesis, and host response to local tissue damage could all potentially affect the CSC population to varying degrees.

A number of cell surface markers have been used to identify CSC in human cell cultures and *in vivo* experiments, including CD44, CD133, and c-Kit, among others. The relevance of some of the common stem cell markers such as CD44 with respect to stem-cell like properties and growth characteristics, however, is not altogether clear, particularly in established long term cell culture lines. For instance, in human prostate cancer cell lines, CD44 has been used to identify CSC; isolated CD44+ cells from prostate cancer cell lines and xenograft tumors show stem-like functions in terms of self-renewal, clonogenicity, tumorigenicity, as well as tumor metastasis (Patrawala et al., 2006; H. Li et al., 2008). However, significant differences in CD44 expression can exist between various prostate cancer cell lines in culture. For instance, by flowcytometry, CD44+ cells can represent 80- 90% of the population in PC3 and DU145 cell lines (Patrawala et al., 2006; H. Li et al., 2008), but in LNCaP they are undetectable. The side population (SP) assay, which is based on exclusion of vital dyes, has also been used to identify a small subpopulation of cells enriched in self-renewal function particularly that derived from the bone marrow (Goodell et al., 1996). No difference in SP fractions is observed among PC3, DU145 and LNCaP cell lines, and their relevance to the overall biology of these cancer cells is not clear.

A recent comprehensive analysis of stem cell makers in the NCI60 Tumor Cell Line Panel demonstrates the presence of these various markers, but they are expressed in rather complex combinatorial patterns in cancer cell lines of different lineages (Stuelten et al., 2010). This and other studies suggest that established immortal cancer cell lines harbor SLC subpopulations, but also underscore the complexity of stem cell biology. Although mounting evidence supports the existence of CSC in various types of tumors (Baker, 2008; Dalerba et al., 2007; Lobo et al., 2007; C. Tang et al., 2007; Huntly & Gilliland, 2005), how cells with stem cell like-properties affect the growth characteristics and/or metastatic potential of tumors still remains an open question (Dalerba et al., 2007; Fabian et al., 2009; Jordan, 2009; Marotta & Polyak 2009; Clevers, 2011).

Another term, stemness, is frequently used in stem cell studies, but its exact definition has not been universally accepted (Leychkis et al., 2009; Hoffmann & Tsonis, 2011). Epithelialmesenchymal and mesenchymal-epithelial transition states are also relevant not only to embryogenesis but to tumorigenesis; how these may relate to stemness is an area of active investigation ((Yang & Weinberg, 2008). In general, stemness represents a *state* in which cells are characterized by self-renewal and plasticity. In cancer tissue, the stemness state may be a transiently acquired property by a subpopulation of tumor cells that likely also involves input from surrounding cells. Biological or pharmacological stress, or changes in the tumor microenvironment, could serve as potential triggers for inducing this state, which may then allow for adaptation, survival and eventual disease progression.

#### **3. Anti-tumor therapy and stem like cells in prostate cancer**

Over the last several years we have utilized an LNCaP-based xenograft model and a genetically engineered transgenic mouse model, TRAMP, to study the effects of androgen deprivation and chemotherapy on prostate cancer (Y. Tang et al., 2006, 2008, 2009, 2010). Tumor tissues were collected at different time points before and after various treatments, and the expression patterns of several stem cell markers were evaluated in an effort to better understand treatment response and potential mechanisms of tumor recurrence.

#### **3.1 LNCaP xenograft based studies**

172 Prostate Cancer – Original Scientific Reports and Case Studies

tumors are not clearly delineated, although communication with surrounding cells and stroma, alterations in pH, chemokines/cytokines in the microenvironment, locoregional angiogenesis, and host response to local tissue damage could all potentially affect the CSC

A number of cell surface markers have been used to identify CSC in human cell cultures and *in vivo* experiments, including CD44, CD133, and c-Kit, among others. The relevance of some of the common stem cell markers such as CD44 with respect to stem-cell like properties and growth characteristics, however, is not altogether clear, particularly in established long term cell culture lines. For instance, in human prostate cancer cell lines, CD44 has been used to identify CSC; isolated CD44+ cells from prostate cancer cell lines and xenograft tumors show stem-like functions in terms of self-renewal, clonogenicity, tumorigenicity, as well as tumor metastasis (Patrawala et al., 2006; H. Li et al., 2008). However, significant differences in CD44 expression can exist between various prostate cancer cell lines in culture. For instance, by flowcytometry, CD44+ cells can represent 80- 90% of the population in PC3 and DU145 cell lines (Patrawala et al., 2006; H. Li et al., 2008), but in LNCaP they are undetectable. The side population (SP) assay, which is based on exclusion of vital dyes, has also been used to identify a small subpopulation of cells enriched in self-renewal function particularly that derived from the bone marrow (Goodell et al., 1996). No difference in SP fractions is observed among PC3, DU145 and LNCaP cell lines, and their relevance to the overall biology of these cancer cells is not

A recent comprehensive analysis of stem cell makers in the NCI60 Tumor Cell Line Panel demonstrates the presence of these various markers, but they are expressed in rather complex combinatorial patterns in cancer cell lines of different lineages (Stuelten et al., 2010). This and other studies suggest that established immortal cancer cell lines harbor SLC subpopulations, but also underscore the complexity of stem cell biology. Although mounting evidence supports the existence of CSC in various types of tumors (Baker, 2008; Dalerba et al., 2007; Lobo et al., 2007; C. Tang et al., 2007; Huntly & Gilliland, 2005), how cells with stem cell like-properties affect the growth characteristics and/or metastatic potential of tumors still remains an open question (Dalerba et al., 2007; Fabian et al., 2009;

Another term, stemness, is frequently used in stem cell studies, but its exact definition has not been universally accepted (Leychkis et al., 2009; Hoffmann & Tsonis, 2011). Epithelialmesenchymal and mesenchymal-epithelial transition states are also relevant not only to embryogenesis but to tumorigenesis; how these may relate to stemness is an area of active investigation ((Yang & Weinberg, 2008). In general, stemness represents a *state* in which cells are characterized by self-renewal and plasticity. In cancer tissue, the stemness state may be a transiently acquired property by a subpopulation of tumor cells that likely also involves input from surrounding cells. Biological or pharmacological stress, or changes in the tumor microenvironment, could serve as potential triggers for inducing this state, which may then

Over the last several years we have utilized an LNCaP-based xenograft model and a genetically engineered transgenic mouse model, TRAMP, to study the effects of androgen

Jordan, 2009; Marotta & Polyak 2009; Clevers, 2011).

allow for adaptation, survival and eventual disease progression.

**3. Anti-tumor therapy and stem like cells in prostate cancer** 

population to varying degrees.

clear.

LNCaP is a hormone-sensitive human prostate cancer cell line. Withdrawal of androgens *in vitro* interrupts its growth and induces apoptosis. *In vivo*, LNCaP tumors in male SCID mice cease growth for up to 2-3 weeks after surgical castration (bilateral orchiectomy), which is invariably followed by accelerated tumor growth (Figure 1A). Immunohistochemical (IHC) analysis of proliferation-related (Ki67) and angiogenesisrelated (CD105) markers in castrated and non-castrated mice is shown in Figures 1B and 1C. It is apparent that a significant decrease in overall proliferation occurs only in the castrated mice between one and two weeks after castration, followed then by recovery of proliferative potential. This recovery of cell proliferation appears to parallel increased angiogenesis, which is observed at the IHC level between two and three weeks post castration. In non-castrated mice, on the other hand, tumors continue to grow unabated over time, with no observable changes in cell proliferation or overall angiogenesis noted over this period of continued growth (Figures 1A-C).

To determine whether SLC play any role in the above model, we also studied several stem cell markers. Recent studies suggest that SLC are a heterogeneous population with diverse biological properties, and that multiple subpopulations with stem cell-like characteristics can coexist in the same tumor (Hermann et al., 2007; Ma et al., 2008; Hope et al., 2004). Since there is no single specific 'standard' marker for identifying cancer stem cells, we selected several antigens that have been implicated in one way or the other in stem cell biology; specifically, CD44, CD133, and c-Kit (CD117) for IHC studies, and human ALDH (aldehyde dehydrogenase), Shh (sonic hedgehog), p63, BCRP (breast cancer resistant protein), Notch1 and bcl-2 for western blot analyses. Tumor samples were collected at days 5, 10, 15, 20, 25, and 30 post castration, and tumors from non-castrated mice collected at similar time points served as controls.

By IHC, expressions of c-Kit (Figure 1D) and CD44 (Figure 1E), but not CD133, were significantly increased in LNCaP xenograft tumors at the day 15 time point after castration but not at other time points in either castrated or control mice. Representative images of c-Kit and CD44 at day-15 (Cas-15) and day 30 (Cas-30) are shown in Figure 1E. Interestingly, we noted that the distribution of CD44+ and c-Kit+ cells in tumor tissues was different. Most of the CD44+ cells were found in the periphery of tumor islands, whereas c-Kit+ cells were present within the tumor mass (Figure 1E). This suggests that these two proteins can be expressed in different cells within the LNCaP tumors, and may potentially signify the presence of different subtypes of SLC in these tumors. In untreated control mice, on the other hand, SLC markers do not change significantly over time despite continued tumor growth, as shown in Figure 1D.

Data for several proteins evaluated by western blots are summarized in Table 1. It is apparent from these initial studies that there is a trend for Shh, Notch, ALDH, BCRP and p63 to be over expressed at least 2-fold at day 15 compared to the other time points post castration in most or all the tumor samples tested, while Bcl2 is over expressed in 2 of the 4 tumors evaluated at this time point.

Stem Like Cells and Androgen Deprivation in Prostate Cancer 175

We also carried out pilot studies with docetaxel chemotherapy in the LNCaP xenografts (Figure 2). Specifically, LNCaP-bearing SCID mice were treated with docetaxel when tumour volumes reached approximately 300 cc (docetaxel was given IP at 8 mg/kg every 3 to 4 days X 4 doses over a two week period). Tumors were harvested from the mice at

**A***. Tumor volumes at different time points. Arrows indicate start and end of docetaxel treatments.* **B***.* 

*Equal amounts of protein from 3-5 tumors at each time point were analyzed with western blots. After* 

Table 1. The expression of stem cell markers in LNCaP xenografts post castration.

*actin, at least double the mean of the band intensities with respect to the non-castrated control* 

Fig. 2. Evaluation of LNCaP tumors in docetaxel-treated mice.

*samples in each reaction was considered to be overexpressed. ND: not done.* 

*normalization to* 

*Representative western blot. Expressions of ALDH and Shh at different time points from start of treatment were determined in tumor samples (n=5 per time point).* **C***. IHC. c-Kit expression in various tumor tissues. The selected images are representative of 3-5 tumors per time point. Five to 10 images per slide were taken and digitized using MCID 7.0 software. Data were analyzed with SigmaPlot and plotted using Microsoft Excel.* 

**A**. *Relative tumor volumes at different time points. Male SCID mice (6-8 wks of age), were inoculated sq with 5 x 106 LNCaP cells per flank. The tumor volumes are shown as the average tumor volume at each time point (5-10 mice) divided by the average tumor volume at day 0.* **B-D**. *Protein expression patterns are summarized as histographs based on IHC data. Cryosections or FFPE (formalin-fixed paraffin-embedded) tissue sections of tumors from each time point (n=4-5) were analyzed for the expression of Ki67* (**B**), *CD105* (**C**)*, and c-Kit* (**D**)*. Five to ten images per section were taken randomly and digitized using the autoscan function of MCID 7.0 software which was set with respect to grain counts (CD105, Ki67) or positively staining cells (c-Kit). Data were analyzed using SigmaPlot. E. Representative images demonstrating expressions of CD44 and c-Kit in tumors at days 15 (Cas-15) and 30 (Cas-30) post castration. (Amplification: 200x).* 

Fig. 1. Evaluation of LNCaP tumors in castrated and non-castrated mice.

Grain counts

0 0.5 1 1.5 2

Cell counts

**Ki67**

**c-Kit**

Days after castration

Non-castrated Castrated

0 5 10 15 20 25 30

Non-castrated Castrated

Days after castration

0 5 10 15 20 25 30

**A**. *Relative tumor volumes at different time points. Male SCID mice (6-8 wks of age), were inoculated sq with 5 x 106 LNCaP cells per flank. The tumor volumes are shown as the average tumor volume at each time point (5-10 mice) divided by the average tumor volume at day 0.* **B-D**. *Protein expression patterns are summarized as histographs based on IHC data. Cryosections or FFPE (formalin-fixed paraffin-embedded) tissue sections of tumors from each time point (n=4-5) were analyzed for the expression of Ki67* (**B**), *CD105* (**C**)*, and c-Kit* (**D**)*. Five to ten images per section were taken randomly and digitized using the autoscan function of MCID 7.0 software which was set with respect to grain counts (CD105, Ki67) or positively staining cells (c-Kit). Data were analyzed using SigmaPlot. E. Representative images demonstrating expressions of CD44 and c-Kit in tumors at* 

*days 15 (Cas-15) and 30 (Cas-30) post castration. (Amplification: 200x).* 

Days after castration

0 5 10 15 20 25 30

0 5 10 15 20 25 30 Days after castration

**C D** 

Non-castrated Castrated

Non-castrated Castrated

**A B**

**CD105**

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

Relative volume change

Grain counts

0.0 2.0 4.0 6.0 8.0 10.0

Fig. 1. Evaluation of LNCaP tumors in castrated and non-castrated mice.

We also carried out pilot studies with docetaxel chemotherapy in the LNCaP xenografts (Figure 2). Specifically, LNCaP-bearing SCID mice were treated with docetaxel when tumour volumes reached approximately 300 cc (docetaxel was given IP at 8 mg/kg every 3 to 4 days X 4 doses over a two week period). Tumors were harvested from the mice at

**A***. Tumor volumes at different time points. Arrows indicate start and end of docetaxel treatments.* **B***. Representative western blot. Expressions of ALDH and Shh at different time points from start of treatment were determined in tumor samples (n=5 per time point).* **C***. IHC. c-Kit expression in various tumor tissues. The selected images are representative of 3-5 tumors per time point. Five to 10 images per slide were taken and digitized using MCID 7.0 software. Data were analyzed with SigmaPlot and plotted using Microsoft Excel.* 

Fig. 2. Evaluation of LNCaP tumors in docetaxel-treated mice.


*Equal amounts of protein from 3-5 tumors at each time point were analyzed with western blots. After normalization to actin, at least double the mean of the band intensities with respect to the non-castrated control samples in each reaction was considered to be overexpressed. ND: not done.* 

Table 1. The expression of stem cell markers in LNCaP xenografts post castration.

Stem Like Cells and Androgen Deprivation in Prostate Cancer 177

We previously reported that castration of these mice at 12 weeks of age resulted in two different outcomes with respect to anti-tumor response (Y. Tang et al., 2008). In one group of mice, significant locoregional disease progression occurred subsequent to the castration without any evidence of tumor shrinkage such that the mice ended up with large prostatic tumors (designated Castration-Large or Cas-L); in these mice, the average genitourinary (GU) organ weight (which included prostate gland and seminal vesicles) to total body weight (G/B) ratios were 25.43 ± 5.25 (Figure 3A) (Y. Tang et al., 2008). Other mice (designated Castration-Small or Cas-S) had a positive response to castration in that prominent shrinkage of the prostate gland and other GU organs occurred so that the average G/B ratios were around 1.41 ± 1.31 (Figure 3A); despite a positive response to castration the prostate glands still harbored cancer cells. Analysis of two SLC markers, Sca1 (which is mouse-specific) and CD133, in mice responding positively to castration (i.e. small G/B ratios) revealed significant increase of both markers at weeks 5 (Cas-S 5wk) and 10 (Cas-S 10wk) (Figure 3B). By contrast, in Cas-L tumors and in tumors from non-castrated

The above studies indicate that enhanced expression of SLC-related proteins can occur in prostate tumors in response to anti-proliferative/cytotoxic treatments such as androgen deprivation or chemotherapy. Further, this SLC response appears to be transient in nature in that it is generally limited to the time period of maximal anti-tumor response to treatment. The increased expression of these SLC-related proteins observed in our tumor samples could be due to more proteins being expressed per cell but within a limited number of the cells, or alternatively more cells that express these proteins (but not necessarily at significantly higher levels than baseline) may be recruited, or it could be a combination of these two processes. Regardless of the specifics of the underlying processes associated with each individual protein in terms of patterns and mechanisms of expression, our results indicate that the *overall*  expressions of several SLC markers in tumors change as a function of time post treatment. These transient elevations of a relatively broad range of stem cell markers indicate a complex tissue response in which not only tumor cells but other surrounding cells could be involved. We hypothesize that this transient period represents a stemness stage during which cancer

Elevations of stem cell related proteins have also been observed by others. For instance, CD44 over expression in breast cancer patients after chemotherapy (X. Li et al., 2008), CD133 over expression in human glioblastoma after radiation (Bao et al., 2006), and ALDH1 over expression and increased enzyme activity in human colorectal xenograft tumors after chemotherapy (Dylla et al., 2008) have been reported. Other forms of stress such as hypoxia and products of metabolism, including lactate and ketones, have also recently been shown to induce stemness in tumor tissues (Kim et al., 2009; Martinez-Outschoorn et al., 2011). Thus, emerging data suggest a correlation between SLC and anti-cancer therapy or other forms of cellular stress, although the exact role of the SLC population in tumor recurrence

Xin et al have shown that prostate glands in C57BL6 wild type mice undergoing castration are enriched in Sca1+ cells, and which are found primarily in the relatively treatmentresistant G0 phase of the cell cycle (Xin et al., 2005). These cells are capable of regenerating tubular structures containing basal cells and luminal cells in a dissociated prostate

mice, Sca1 and CD133 levels remained low (Figure 3B).

cells and surrounding cells can adapt to a changed microenvironment.

**4. Discussion** 

remains unclear.

different time points post docetaxel treatment as shown in Figure 2A, and evaluated for ALDH and Shh expression by western blots and for c-Kit expression by IHC (Figures 2B and 2C). Although our analysis is limited, interestingly, as is the case with castrated mice, increased levels of ALDH, Shh and c-Kit were observed in the docetaxel treated mice at points of maximal anti-tumor response (Figures 2A-C). These initial studies suggest that docetaxel treatment can also induce enhanced expression of some of the proteins associated with SLC, although the kinetics of this response are somewhat different than observed in tumors from castrated mice. Thus, there is a trend towards enhanced expression of SLCrelated proteins in both castrated mice and docetaxel treated mice at points of maximal antitumor response (i.e. when tumors are at their smallest sizes).

#### **3.2 TRAMP based studies**

A similar phenomena of enhanced SLC expression post castration was also observed in the TRAMP model in which probasin promoter-driven T antigen expression in mouse prostatic epithelia induces prostate cancers in male mice as they mature sexually (by 8 to 12 weeks of age)( Greenberg et al., 1995; Y. Tang et al. 2008).

**A***. Effects of castration on genitourinary (GU) organs. Based on G/B ratios (GU organs versus mouse body weight), TRAMP mice were divided into two groups after castration – those with G/B ratios ≤ 3.0 (Cas-S) and others with G/B ratios > 20 (Cas-L).* **B***. IHC. Expressions of CD133 and Sca-1 in TRAMP mouse prostates. These proteins are highly expressed in Cas-S tumors around 5 (Cas-S 5wk; n=3) and 10 wks (Cas-S 10wk; n=3) post castration, but not in Cas-L tumors (n=3). Prostate tumors from non-castrated mice (intact; n=3) and normal prostate tissues from wild-type siblings (n=3) served as controls.* 

Fig. 3. Evaluation of tumors in TRAMP mice.

We previously reported that castration of these mice at 12 weeks of age resulted in two different outcomes with respect to anti-tumor response (Y. Tang et al., 2008). In one group of mice, significant locoregional disease progression occurred subsequent to the castration without any evidence of tumor shrinkage such that the mice ended up with large prostatic tumors (designated Castration-Large or Cas-L); in these mice, the average genitourinary (GU) organ weight (which included prostate gland and seminal vesicles) to total body weight (G/B) ratios were 25.43 ± 5.25 (Figure 3A) (Y. Tang et al., 2008). Other mice (designated Castration-Small or Cas-S) had a positive response to castration in that prominent shrinkage of the prostate gland and other GU organs occurred so that the average G/B ratios were around 1.41 ± 1.31 (Figure 3A); despite a positive response to castration the prostate glands still harbored cancer cells. Analysis of two SLC markers, Sca1 (which is mouse-specific) and CD133, in mice responding positively to castration (i.e. small G/B ratios) revealed significant increase of both markers at weeks 5 (Cas-S 5wk) and 10 (Cas-S 10wk) (Figure 3B). By contrast, in Cas-L tumors and in tumors from non-castrated mice, Sca1 and CD133 levels remained low (Figure 3B).

#### **4. Discussion**

176 Prostate Cancer – Original Scientific Reports and Case Studies

different time points post docetaxel treatment as shown in Figure 2A, and evaluated for ALDH and Shh expression by western blots and for c-Kit expression by IHC (Figures 2B and 2C). Although our analysis is limited, interestingly, as is the case with castrated mice, increased levels of ALDH, Shh and c-Kit were observed in the docetaxel treated mice at points of maximal anti-tumor response (Figures 2A-C). These initial studies suggest that docetaxel treatment can also induce enhanced expression of some of the proteins associated with SLC, although the kinetics of this response are somewhat different than observed in tumors from castrated mice. Thus, there is a trend towards enhanced expression of SLCrelated proteins in both castrated mice and docetaxel treated mice at points of maximal anti-

A similar phenomena of enhanced SLC expression post castration was also observed in the TRAMP model in which probasin promoter-driven T antigen expression in mouse prostatic epithelia induces prostate cancers in male mice as they mature sexually (by 8 to 12 weeks of

**A***. Effects of castration on genitourinary (GU) organs. Based on G/B ratios (GU organs versus mouse body weight), TRAMP mice were divided into two groups after castration – those with G/B ratios ≤ 3.0 (Cas-S) and others with G/B ratios > 20 (Cas-L).* **B***. IHC. Expressions of CD133 and Sca-1 in TRAMP mouse prostates. These proteins are highly expressed in Cas-S tumors around 5 (Cas-S 5wk; n=3) and 10 wks (Cas-S 10wk; n=3) post castration, but not in Cas-L tumors (n=3). Prostate tumors from non-castrated mice (intact; n=3) and normal* 

tumor response (i.e. when tumors are at their smallest sizes).

age)( Greenberg et al., 1995; Y. Tang et al. 2008).

*prostate tissues from wild-type siblings (n=3) served as controls.* 

Fig. 3. Evaluation of tumors in TRAMP mice.

**3.2 TRAMP based studies** 

The above studies indicate that enhanced expression of SLC-related proteins can occur in prostate tumors in response to anti-proliferative/cytotoxic treatments such as androgen deprivation or chemotherapy. Further, this SLC response appears to be transient in nature in that it is generally limited to the time period of maximal anti-tumor response to treatment.

The increased expression of these SLC-related proteins observed in our tumor samples could be due to more proteins being expressed per cell but within a limited number of the cells, or alternatively more cells that express these proteins (but not necessarily at significantly higher levels than baseline) may be recruited, or it could be a combination of these two processes. Regardless of the specifics of the underlying processes associated with each individual protein in terms of patterns and mechanisms of expression, our results indicate that the *overall*  expressions of several SLC markers in tumors change as a function of time post treatment. These transient elevations of a relatively broad range of stem cell markers indicate a complex tissue response in which not only tumor cells but other surrounding cells could be involved. We hypothesize that this transient period represents a stemness stage during which cancer cells and surrounding cells can adapt to a changed microenvironment.

Elevations of stem cell related proteins have also been observed by others. For instance, CD44 over expression in breast cancer patients after chemotherapy (X. Li et al., 2008), CD133 over expression in human glioblastoma after radiation (Bao et al., 2006), and ALDH1 over expression and increased enzyme activity in human colorectal xenograft tumors after chemotherapy (Dylla et al., 2008) have been reported. Other forms of stress such as hypoxia and products of metabolism, including lactate and ketones, have also recently been shown to induce stemness in tumor tissues (Kim et al., 2009; Martinez-Outschoorn et al., 2011). Thus, emerging data suggest a correlation between SLC and anti-cancer therapy or other forms of cellular stress, although the exact role of the SLC population in tumor recurrence remains unclear.

Xin et al have shown that prostate glands in C57BL6 wild type mice undergoing castration are enriched in Sca1+ cells, and which are found primarily in the relatively treatmentresistant G0 phase of the cell cycle (Xin et al., 2005). These cells are capable of regenerating tubular structures containing basal cells and luminal cells in a dissociated prostate

Stem Like Cells and Androgen Deprivation in Prostate Cancer 179

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regeneration system, demonstrating their plasticity and role in prostate regeneration. Moreover, enriched SLC populations have been reported in several tumor-free tissues following local damage (Beltrami et al., 2003; Amcheslavsky et al., 2009). Thus, stemness may represent a protective response by tissue cells, including cancerous cells, to damage or environmental change. This stemness state may in turn allow cells to survive, adapt and grow. Stemness is not an unusual characteristic; many cells possess this ability. As indicated by Zipori et al, all cells possess the molecular machinery that enables them to return to a relatively undifferentiated stem cell-like state when appropriately challenged (Zipori, 2009). Several recent studies have also documented de-differentiation in mature cells (Brawley & Matunis, 2004; Monje et al., 2010; Red-Horse et al., 2010; Shoshani & Zipori, 2011) During tumor recurrence/progression, the de-differentiated SLC can re-differentiate back into the original tumor, or under other conditions, they could trans-differentiate to a different phenotype. Our TRAMP mice castrated at 12 weeks of age all eventually developed prostate cancer, including distant metastasis in over 70% of the animals (Y. Tang et al., 2008, 2009). In several of these mice, neuroendocrine carcinoma (NEC) partially or completely replaced the original adenocarcinomas in the prostate gland and/or distant metastatic sites (Y. Tang et al., 2009); this was rarely observed in the non-castrated mice. Thus, in TRAMP mice regrowth after castration can lead to 'differentiation' along the same original adenocarcinoma pathway or trans-differentiation along the NEC pathway.

A recent study showed that certain differentiated cells in breast tissue *spontaneously* converted to a stem cell-like state (Chaffer et al., 2011). This challenges the scientific dogma that differentiation is a one way path, i.e. once cells specialize they cannot return to a SLC state on their own. Thus, a considerable degree of plasticity exists among cells in their ability to reprogram themselves to a more permissive state not only when appropriately challenged, but under certain conditions this seems to occur spontaneously. The state of 'stemness' that occurs in response to androgen deprivation in our xenograft and transgenic prostate cancer models appears to be transient. Interestingly, recent work has shown that cancer cells in culture exposed to various drugs can undergo histone deacetylation mediated chromatin changes that result in transient reversible drug tolerant states (Sharma et al., 2010).

#### **5. Conclusion**

The transient nature of various adaptive responses noted above suggests that to maximize anti-cancer treatments, not only more effective agents need to be developed, but also the relative timing of these treatments with respect to the transient cellular states need to be taken into account. Thus, given that various adaptive cellular processes are dynamic, real time detection and targeting of cancer cells undergoing de-differentiation may improve the efficacy of anti-cancer therapies.

#### **6. Acknowledgment**

This work was supported by a Merit Review Award, Department of Veterans Affairs (A. H.).

#### **7. References**

Amcheslavsky, A., Jiang, J. and Ip, Y.T., 2009. Tissue damage-induced intestinal stem cell division in Drosophila. Cell Stem Cell, Vol.4, No.1, (January 9, 2009), pp. 49-61, ISSN 1875-9777

regeneration system, demonstrating their plasticity and role in prostate regeneration. Moreover, enriched SLC populations have been reported in several tumor-free tissues following local damage (Beltrami et al., 2003; Amcheslavsky et al., 2009). Thus, stemness may represent a protective response by tissue cells, including cancerous cells, to damage or environmental change. This stemness state may in turn allow cells to survive, adapt and grow. Stemness is not an unusual characteristic; many cells possess this ability. As indicated by Zipori et al, all cells possess the molecular machinery that enables them to return to a relatively undifferentiated stem cell-like state when appropriately challenged (Zipori, 2009). Several recent studies have also documented de-differentiation in mature cells (Brawley & Matunis, 2004; Monje et al., 2010; Red-Horse et al., 2010; Shoshani & Zipori, 2011) During tumor recurrence/progression, the de-differentiated SLC can re-differentiate back into the original tumor, or under other conditions, they could trans-differentiate to a different phenotype. Our TRAMP mice castrated at 12 weeks of age all eventually developed prostate cancer, including distant metastasis in over 70% of the animals (Y. Tang et al., 2008, 2009). In several of these mice, neuroendocrine carcinoma (NEC) partially or completely replaced the original adenocarcinomas in the prostate gland and/or distant metastatic sites (Y. Tang et al., 2009); this was rarely observed in the non-castrated mice. Thus, in TRAMP mice regrowth after castration can lead to 'differentiation' along the same original

adenocarcinoma pathway or trans-differentiation along the NEC pathway.

result in transient reversible drug tolerant states (Sharma et al., 2010).

**5. Conclusion** 

efficacy of anti-cancer therapies.

ISSN 1875-9777

**6. Acknowledgment** 

**7. References** 

A recent study showed that certain differentiated cells in breast tissue *spontaneously* converted to a stem cell-like state (Chaffer et al., 2011). This challenges the scientific dogma that differentiation is a one way path, i.e. once cells specialize they cannot return to a SLC state on their own. Thus, a considerable degree of plasticity exists among cells in their ability to reprogram themselves to a more permissive state not only when appropriately challenged, but under certain conditions this seems to occur spontaneously. The state of 'stemness' that occurs in response to androgen deprivation in our xenograft and transgenic prostate cancer models appears to be transient. Interestingly, recent work has shown that cancer cells in culture exposed to various drugs can undergo histone deacetylation mediated chromatin changes that

The transient nature of various adaptive responses noted above suggests that to maximize anti-cancer treatments, not only more effective agents need to be developed, but also the relative timing of these treatments with respect to the transient cellular states need to be taken into account. Thus, given that various adaptive cellular processes are dynamic, real time detection and targeting of cancer cells undergoing de-differentiation may improve the

This work was supported by a Merit Review Award, Department of Veterans Affairs (A. H.).

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**12** 

*Japan* 

**Injection Site Granulomas Resulting from** 

*Toho University, Medical Center, Ohashi Hospital, Department of Dermatology* 

Leuprorelin acetate is a luteinizing hormone releasing hormone (LHRH) agonist and was launched in 1997. It has been used for sex hormone-dependent diseases such as prostate cancer, endometriosis, premenopausal breast cancer, and central precocious puberty. In Japan, a preparation for administration at monthly intervals (Leuprin®) has been available since 1999, and a continuous sustained release preparation for administration every 3 months (Leuprin SR®) since 2002. In recent years, there has been a considerable increase in the frequency of foreign body granulomas, particularly in patients in whom a 1-monthly preparation was changed to a 3-monthly continuous sustained release preparation. We

Case 1: A 77-year-old Japanese male had been treated for prostate cancer with monthly subcutaneous `injections of depot leuprorelin acetate since August 2005. In December 2005, his treatment was changed to the 3-monthly leuprorelin acetate preparation. One month later, he presented with a nodule at the injection site. The nodule was 15 mm in diameter, firm and tethered to the overlying skin **(Photograph 1)**. Skin biopsy revealed a granuloma with epithelioid cells, and multinucleated giant cells were observed in the subcutaneous tissue by hematoxylin–eosin staining. The granuloma contained vacuoles of various sizes, and portions of it were phagocytosed **(Photograph 3)**. In addition, inflammatory cell infiltration was observed, mainly of lymphocytes and neutrophils, along with eosinophils

His treatment was then changed to goserelin acetate (Zoladex®) for subsequent

Case 2: A 78-year-old Japanese male had been treated for prostate cancer with monthly subcutaneous injections of depot leuprorelin acetate since October 2002. In February 2003, his treatment was changed to the 3-monthly preparation. After the sixth administration, he

Laboratory tests showed no abnormalities in blood count, blood biochemistry, and urine. Serum PSA was high at 5.14 ng/ mL, while γ-SM was within normal limits (0.4 ng/ mL). The nodule was 30 × 35 mm in diameter, painless, firm, and tethered to the overlying skin

report two such cases that we encountered and present a literature review.

**1. Introduction** 

**2. Case** 

**(Photograph 4)**.

**(Photograph 2)**.

injections,no problems occurred thereafter.

presented with a nodule at the injection site.

**Administration of Leuprorelin Acetate** 

Taku Suzuki and Hideki Mukai

Clinical and Experimental Pathology, Vol.3, No.2, (November 2009), ISSN 1936- 2625


### **Injection Site Granulomas Resulting from Administration of Leuprorelin Acetate**

Taku Suzuki and Hideki Mukai

*Toho University, Medical Center, Ohashi Hospital, Department of Dermatology Japan* 

#### **1. Introduction**

182 Prostate Cancer – Original Scientific Reports and Case Studies

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2625

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Cells and the Molecular Basis of the Stem State, K. Turksen, (Ed.), pp. 200-206.

Leuprorelin acetate is a luteinizing hormone releasing hormone (LHRH) agonist and was launched in 1997. It has been used for sex hormone-dependent diseases such as prostate cancer, endometriosis, premenopausal breast cancer, and central precocious puberty. In Japan, a preparation for administration at monthly intervals (Leuprin®) has been available since 1999, and a continuous sustained release preparation for administration every 3 months (Leuprin SR®) since 2002. In recent years, there has been a considerable increase in the frequency of foreign body granulomas, particularly in patients in whom a 1-monthly preparation was changed to a 3-monthly continuous sustained release preparation. We report two such cases that we encountered and present a literature review.

#### **2. Case**

Case 1: A 77-year-old Japanese male had been treated for prostate cancer with monthly subcutaneous `injections of depot leuprorelin acetate since August 2005. In December 2005, his treatment was changed to the 3-monthly leuprorelin acetate preparation. One month later, he presented with a nodule at the injection site. The nodule was 15 mm in diameter, firm and tethered to the overlying skin **(Photograph 1)**. Skin biopsy revealed a granuloma with epithelioid cells, and multinucleated giant cells were observed in the subcutaneous tissue by hematoxylin–eosin staining. The granuloma contained vacuoles of various sizes, and portions of it were phagocytosed **(Photograph 3)**. In addition, inflammatory cell infiltration was observed, mainly of lymphocytes and neutrophils, along with eosinophils **(Photograph 4)**.

His treatment was then changed to goserelin acetate (Zoladex®) for subsequent injections,no problems occurred thereafter.

Case 2: A 78-year-old Japanese male had been treated for prostate cancer with monthly subcutaneous injections of depot leuprorelin acetate since October 2002. In February 2003, his treatment was changed to the 3-monthly preparation. After the sixth administration, he presented with a nodule at the injection site.

Laboratory tests showed no abnormalities in blood count, blood biochemistry, and urine. Serum PSA was high at 5.14 ng/ mL, while γ-SM was within normal limits (0.4 ng/ mL).

The nodule was 30 × 35 mm in diameter, painless, firm, and tethered to the overlying skin **(Photograph 2)**.

Injection Site Granulomas Resulting from Administration of Leuprorelin Acetate 185

Fig. 2. Case 2 presented with a subcutaneous nodule with an injection scar on the right upper arm. The solitary nodule was 30 35 mm in size, slightly firm, and mobile.

Fig. 3. It showed several eosinophils with multinucleated giant cells in HE staining (original

magnification ×400).

Skin biopsy showed a granuloma with epithelioid cells and multinucleated giant cells, and lymphocytes were observed from the subdermal layer to the subcutaneous tissue **(Photograph 5)**. His treatment was changed to goserelin acetate (Zoladex®) for subsequent injections, and no problems occurred thereafter.

Intradermal and patch tests were not performed in either patient because we were unable to obtain consent from them.

Fig. 1. Case 1 presented with a subcutaneous nodule (dotted area) in the right navel area. The solitary nodule was 15 mm in diameter, slightly firm, and only slightly mobile.

Skin biopsy showed a granuloma with epithelioid cells and multinucleated giant cells, and lymphocytes were observed from the subdermal layer to the subcutaneous tissue **(Photograph 5)**. His treatment was changed to goserelin acetate (Zoladex®) for subsequent

Intradermal and patch tests were not performed in either patient because we were unable to

Fig. 1. Case 1 presented with a subcutaneous nodule (dotted area) in the right navel area. The solitary nodule was 15 mm in diameter, slightly firm, and only slightly mobile.

injections, and no problems occurred thereafter.

obtain consent from them.

Fig. 2. Case 2 presented with a subcutaneous nodule with an injection scar on the right upper arm. The solitary nodule was 30 35 mm in size, slightly firm, and mobile.

Fig. 3. It showed several eosinophils with multinucleated giant cells in HE staining (original magnification ×400).

Injection Site Granulomas Resulting from Administration of Leuprorelin Acetate 187

distant metastasis such as to the bone. Among the endocrine therapies, the first choice for treatment in the initial stage is an LHRH agonist or a combination of an LHRH agonist and

Fig. 5. In the subdermal layer beneath the subcutaneous fat tissue, there was a granuloma, part of which contained multinucleated giant cells (hematoxylin–eosin staining; original

Leuprorelin acetate is a derivative of LHRH with a substituted amino acid sequence; it is a superagonist with an activity approximately 100-fold higher than the natural form. Continuous administration of leuprorelin acetate causes downregulation of the LHRH receptor, has an antagonist-like action, and depresses gonadal function. Therefore, it is used for treating sex hormone-dependent diseases. At present, a preparation of leuprorelin acetate designed for administration at intervals of 3 months is indicated for prostate cancer

If these symptoms are severe, they may require consultation with a physician. Other more

The side effect profile of systemic LHRH agonist injections is below

fainting and fast and irregular breathing and shortness of breath

an anti-androgen.

magnification ×100).

and premenopausal breast cancer.

 fast and irregular heart beat bone, muscles, and joint pain

numbness and tingling in the hands and feet

common symptoms that are less severe include:

 pains in the chest or tightness of chest and wheezing pain in the groin or the legs, especially the calves

 swelling of the eyes and the eyelids skin rash or hives and itching

Fig. 4. It showed an epithelial granuloma with foreign body multinucleated giant cells containing microspheres and scattered eosinophils. The granuloma with multinucleated giant cells, eosinophils, and numerous vacuoles was located in the subcutaneous tissue (hematoxylin–eosin staining; original magnification ×200).

#### **3. Discussion**

Treatments for prostate cancer include hormone therapy, surgical treatment, radiation therapy, and chemotherapy. Although treatment plans differ according to the stage of disease, endocrine therapy is used for clinically localized cancer, locally invasive cancer, and

Fig. 4. It showed an epithelial granuloma with foreign body multinucleated giant cells containing microspheres and scattered eosinophils. The granuloma with multinucleated giant cells, eosinophils, and numerous vacuoles was located in the subcutaneous tissue

Treatments for prostate cancer include hormone therapy, surgical treatment, radiation therapy, and chemotherapy. Although treatment plans differ according to the stage of disease, endocrine therapy is used for clinically localized cancer, locally invasive cancer, and

(hematoxylin–eosin staining; original magnification ×200).

**3. Discussion** 

distant metastasis such as to the bone. Among the endocrine therapies, the first choice for treatment in the initial stage is an LHRH agonist or a combination of an LHRH agonist and an anti-androgen.

Fig. 5. In the subdermal layer beneath the subcutaneous fat tissue, there was a granuloma, part of which contained multinucleated giant cells (hematoxylin–eosin staining; original magnification ×100).

Leuprorelin acetate is a derivative of LHRH with a substituted amino acid sequence; it is a superagonist with an activity approximately 100-fold higher than the natural form. Continuous administration of leuprorelin acetate causes downregulation of the LHRH receptor, has an antagonist-like action, and depresses gonadal function. Therefore, it is used for treating sex hormone-dependent diseases. At present, a preparation of leuprorelin acetate designed for administration at intervals of 3 months is indicated for prostate cancer and premenopausal breast cancer.

The side effect profile of systemic LHRH agonist injections is below


If these symptoms are severe, they may require consultation with a physician. Other more common symptoms that are less severe include:

Injection Site Granulomas Resulting from Administration of Leuprorelin Acetate 189

of insertion of the injection needle. All caese were single .The underlying disease was prostate cancer in all cases except four with central precocious puberty. Regarding the cases of prostate cancer, we found that the range of patient ages was 60–89 years, with a mean of 76.3 years, which is consistent with the mean age of prostate cancer onset. The 1-monthly preparation was used in 12 patients, the 3-monthly preparation in 36 patients, and the preparation type was unknown in 18 patients. Among the patients who received the 3 monthly preparation, 24 had a treatment history with LHRH agonists including leuprorelin acetate. Most of these patients (19 of them) had previously received the 1-monthly preparation. The injection site was the upper arm in 30 patients, the abdomen in 15 patients, the buttock in 1 patient, and unknown in 20 patients. Among those whose injection sites were known, either the injection sites were changed or the dermatological symptoms were observed at another site in 16 patients. Histologically, all cases had foreign-body

Various opinions on the cause of local reactions to depot leuprorelin acetate have been suggested, including the base used and the nature of the preparation itself. Manasco et al. stated that an identical reaction was observed when base alone was injected, and that natural LHRH did not cause a similar reaction. However, Neely et al. administered an alternative drug without the base and observed erythema. In an animal experiment, they also observed a strong reaction when a high concentration of leuprorelin acetate was combined with the base. Therefore, the local reaction was thought to be associated not only

The leuprorelin acetate preparation contained controlled release solid dispersion microspheres comprising lactic acid–glycolic acid copolymers, which slowly disseminated throughout the body. Subsequently, there was a continuous inflammatory response to the leuprorelin acetate, and multinucleated giant cells appeared as a result of the recognition of foreign bodies because of the long-term presence of the microspheres themselves. Therefore, histologically, a foreign-body granuloma was formed. The injection dose and needle insertion depth could have also contributed to this finding. There was a 3-fold difference in the leuprorelin acetate content between the 1-monthly and 3-monthly preparations. Therefore, the 3-monthly preparation was more likely to cause an inflammatory response. Regarding the needle insertion depth, injections are administered intramuscularly in Europe and the USA, and subcutaneously in Japan. The difference in the number of reported cases between Europe and the USA on one hand and Japan on the other hand may be related to

The number of prostate cancer cases is increasing in Japan with a concomitant increase in the frequency of use of leuprorelin acetate. The number of reported cases with granuloma may increase in the future. If a granuloma develops as a result of the use of this preparation, we believe that dermatologists should be obliged to provide appropriate advice to other physicians, such as a change of drug. And physician should pay attention to perform the injection of LA in enough subcutaneous depth and exchange injection sites everytimes.

Adachi H, Hashimoto J,Hota H.Two cases of granuloma due to leuprorelin acetate

Arai Y, Ebihara K,Okubo Y.et al A case of granuloma leuprorelin acetate subcutaneous

subcutaneous injection. Jpn J Urology ; 106: 456, 2006(in Japanese)

injection. Jpn J Dermatol; 116: 963, 2006(in Japanese)

granulomatous tissue reaction.

with the base, but also with the preparation itself.

this difference in needle insertion depth.

**4. References** 


The side effect profile of local LHRH agonist injections is below2


Previous reports of foreign body granuloma formation resulting from the use of leuprorelin acetate are summarized in Table 1. Sixty-six cases have been reported globally, and most of these reports (58 cases, 87.9%) are from Japan. As mentioned below, the difference in the number of reports between Japan and other countries is believed to be related to the depth


Table 1. Summary of 66 cases

of insertion of the injection needle. All caese were single .The underlying disease was prostate cancer in all cases except four with central precocious puberty. Regarding the cases of prostate cancer, we found that the range of patient ages was 60–89 years, with a mean of 76.3 years, which is consistent with the mean age of prostate cancer onset. The 1-monthly preparation was used in 12 patients, the 3-monthly preparation in 36 patients, and the preparation type was unknown in 18 patients. Among the patients who received the 3 monthly preparation, 24 had a treatment history with LHRH agonists including leuprorelin acetate. Most of these patients (19 of them) had previously received the 1-monthly preparation. The injection site was the upper arm in 30 patients, the abdomen in 15 patients, the buttock in 1 patient, and unknown in 20 patients. Among those whose injection sites were known, either the injection sites were changed or the dermatological symptoms were observed at another site in 16 patients. Histologically, all cases had foreign-body granulomatous tissue reaction.

Various opinions on the cause of local reactions to depot leuprorelin acetate have been suggested, including the base used and the nature of the preparation itself. Manasco et al. stated that an identical reaction was observed when base alone was injected, and that natural LHRH did not cause a similar reaction. However, Neely et al. administered an alternative drug without the base and observed erythema. In an animal experiment, they also observed a strong reaction when a high concentration of leuprorelin acetate was combined with the base. Therefore, the local reaction was thought to be associated not only with the base, but also with the preparation itself.

The leuprorelin acetate preparation contained controlled release solid dispersion microspheres comprising lactic acid–glycolic acid copolymers, which slowly disseminated throughout the body. Subsequently, there was a continuous inflammatory response to the leuprorelin acetate, and multinucleated giant cells appeared as a result of the recognition of foreign bodies because of the long-term presence of the microspheres themselves. Therefore, histologically, a foreign-body granuloma was formed. The injection dose and needle insertion depth could have also contributed to this finding. There was a 3-fold difference in the leuprorelin acetate content between the 1-monthly and 3-monthly preparations. Therefore, the 3-monthly preparation was more likely to cause an inflammatory response. Regarding the needle insertion depth, injections are administered intramuscularly in Europe and the USA, and subcutaneously in Japan. The difference in the number of reported cases between Europe and the USA on one hand and Japan on the other hand may be related to this difference in needle insertion depth.

The number of prostate cancer cases is increasing in Japan with a concomitant increase in the frequency of use of leuprorelin acetate. The number of reported cases with granuloma may increase in the future. If a granuloma develops as a result of the use of this preparation, we believe that dermatologists should be obliged to provide appropriate advice to other physicians, such as a change of drug. And physician should pay attention to perform the injection of LA in enough subcutaneous depth and exchange injection sites everytimes.

#### **4. References**

188 Prostate Cancer – Original Scientific Reports and Case Studies

Previous reports of foreign body granuloma formation resulting from the use of leuprorelin acetate are summarized in Table 1. Sixty-six cases have been reported globally, and most of these reports (58 cases, 87.9%) are from Japan. As mentioned below, the difference in the number of reports between Japan and other countries is believed to be related to the depth

Others: 8 cases

Upper arm: 30cases Abdomen: 15cases Buttock: 1case Unknown: 20cases

central precocious puberty: 4 cases

1-month depot preparation:12cases 1~about60times(average:27.8times) 3-months depot preparation :36cases

<past history of LH-RH agonist:24cases> month depot preparation: 19cases (once~five years; average12.8times) bicalutamide:3cases(unspecified) goserelin acetate:2cases(unspecified)

2~7times(average:2.6times)

burning, itching, redness, and swelling at the injection site

gynecomastia (swelling or tenderness and pain of the breasts)

inability to maintain an erection satisfactory for intercourse

The side effect profile of local LHRH agonist injections is below2

country Japan: 58 cases

disease prostate cancer: 62 cases

age 1~89(average: 76.3)

hot flashes

blurred vision

trouble sleeping

 erythema nodule induration dermatitis

dizziness and headaches

decreased size of testicles

decrease in interest in sex

lesion

Kind and the number of times of

(except for 18 cases of unknown)

Table 1. Summary of 66 cases

drugs

swelling or feet and lower legs

nausea and vomiting and constipation


Injection Site Granulomas Resulting from Administration of Leuprorelin Acetate 191

Ota K,Terao A,et al.Subcutaneous granuloma with formation caused by a slow-release

Ouchi T, Naho MIYATA,Makoto S.et al Granuloma caused by subcutaneous injection of

Quella S, Loprinzi CL, Dose M. A qualitative approach to defining "hot flashes" in men.

Role of Estrogen in Normal Male Function: Clinical Implications for Patients with Prostate

Sadahira C,Yoneda K.Kubota Y. et al. A cases of granuloma due to leuprorelin acetate. Japanese Journal of Dermatoallergology; 13:138-143, 2005(in Japanese) Saimoto H,Horikawa S,Nagai N.et al A case of subcutaneous allergy granuloma due to leuprorelin acetate. Acta Urologica Japonica;50:834,2004(in Japanese) Sakamoto R,Kanekura T,Kanzaki T.et al Granulomas induced by subcutaneous injection of

Shimizu H,Shimoura S,Sarayama Y.et al Granuloma The granuloma by the acetic acid Lew

Taguchi S,Ishi Y.Granuloma due to leuprorelin acetate. Jpn J Dermatol;114:1440,2004(in

Takahashi G,Hashimoto Y.Iizuka H.et al A case of granuloma due to leuprorelin acetate

Takakura Y. DDS type injecton.Journal of Practical Pharmacy;56:2437-2440,2005(in Japanese) Tanaka E, Tanaka A.Hori K. Granuloma due to leuprorelin acetate subcutaneous injection.

Tanaka S. Tamura M.Three cases of granuloma due to leuprorelin acetate. rinsho

Taneda T,Kanno T,Kanamaru H.et al Surgical manegement of inflammatory granuloma

Tonini G,Forleo V,Rustico M.et al Local reactions to lutenizing hormone releasing hormone

Watanabe T, Yamada N.Yamamoto O.Histopathological Examination of the histopathology

Yasukawa K,Sugawara H,Kato N.et al Leuprorelin acetate granulomas:case reports and

Yamashita F.,Hirai S,Ikeda S.et al A case of foreign granuloma due to leuprorelin acetate 3 month preparations. Hifubyo Shinryo; 27: 1277-1280,2005 (in Japanese)

hypodermic injection Jpn J Dermatol;116:803,2006(in Japanese)

review of the literture. Br J Dermatol; 152: 1045-1047,2005

which developed following subcutaneous injection of leuprorelin acetate. Acta

of six granuloma that resulted from acetic acid Lew professional re phosphorous

injection. Rinsho hihuka ; 47: 782-783, 2005(in Japanese)

Rinsho hihuka ; 48: 411-415, 2006(in Japanese)

Urologica Japonica;51:487-489,2005(in Japanese)

analog therapy. J Pediatr ; 126: 159-160, 1995

Japanese)

Dermatol;33:719-721,2006

Urol Nurs. 1994; 14: 155–158

leuprorelin acetate, J Dermatol;33:43-45,2006

Kiyo;50:199-202,2004(in Japanese)

derma;47:788-792,2005(in Japanese)

Japanese)

leuprorelin acetate preparation. Nishinihon Journal of Urology;68: 267-269,2006(in

leuprorelin acetate product:Case report and histopathological findings; J

Cancer on Androgen Deprivation Therapy. The Journal of Urology 185, 17-23, 2011

professional re phosphorous pharmaceutical administration is formed by a base remaining in subcutis for a long term. Jpn J Dermatol ; 116: 781,2006(in Japanese) Sugano Y, Fujii K,Ogou N.A case of lipogranukoma induced by subcutaneous injection of the drug for prostate carcinoma. Jpn J Dermato;107:899,1997(in Japanese) Tachibana M,Yamano Z,Chimogaki H,Hamami G.et al Cutaneous epitheloid granulomas

caused by subcutaneous infusion of leuprorelin acetate:a case report.Hinyokika


Egi M, Maeda M.Furuya K.Skin disorder by leuprorelin acetate.Nishinihon Journal of

Ferran M,Baena V,Pujol RM.et al Depot Leuprorelin Acetate-induced Granulomatous

Fujita R, Sakuma S,Komiya H. A case of subcutaneous tumor due to leuprorelin acetate onemonth preparation.Nishinihon Journal of Urology ;68:159-161,2006(in Japanese) Goto N, Mori R.Kudo H.Three cases due to leuprorelin acetate. Skin Research; 4: 507, 2005(in

Hatcho Y,Ide Y,Masuda M.et al Two cases of granuloma due to leuprorelin acetate subcutaneous injection. Rinsho hihuka;48:1055-1057,2006(in Japanese) Hirashima N, Shinogi T,Narisawa H.et al A case of cutaneous injury induced by

Ishigami T, Urano,Hujii Y.Four cases of skin disorder due to leuprorelin acetate. Nishinihon

Kato A,Noro S,Kawana S.et al A case of granuloma due to leuprorelin acetate. rinsho derma

Kawase A,Mizoguchi M,Iwamoto T,et al.A case of foreign body granuloma induced by injecton of leuprorelin acetate. Jpn J Dermatol;114:574,2004(in Japanese) Koura S, Watanabe T.A suspected case of granuloma due to leuprorelin acetate. Nishinihon

Liu XS, Folia C, Gomella LG. Pharmacology for common urologic diseases: 2011 review for

Manasco PK,Pescovitz OH, Blizzard RM.Local reactions to depot leuprolide therapy for

Marumo K, Baba S, Murai M. Erectile function and nocturnal penile tumescence in patients

Mizoguchi K, Hamasaki Y,Igawa H.et al A case of drug induced granulomatous reaction by

Muya M, Takijiri C,Shirahara S.Two cases of granuloma caused by subcutaneous injection of

Nagata K,Shinoda S,Yonehara S.et al Two cases of granuloma due to leuprorelin acetate.

Navon L, Morag A. Advanced prostate cancer patients' ways of coping with the hormonal

Neely EK, Hintz RL, Parker B et al.Two-year results of treatment with depot leuprolide

Nomoto H, Ishida K,Kitagawa T.et al Granuloma due to leuprorelin acetate subcutaneous

Ohara N, Mihara S,Usui T.et al A case of granuloma due to leuprorelin acetate . rinsho

therapy's effect on body, sexuality, and spousal ties. Qual Health Res. 2003;13:1378

with prostate cancer undergoing luteinizing hormone-releasing hormone agonist

leuprorelin acetate for prostate cancer. Jpn J Dermatol; 114: 163-167, 2004(in

Manifested as Persistent Suppurative Nodules. Acta Derm Venerol ; 86: 453-455,

subcutaneous injection of leuprolide acetate. Nishinihon Journal of Dermatology

Dermatology ;66:206,2004(in Japanese)

;63:384-386,2001(in Japanese)

;60:1013-1016,2006(in Japanese)

therapy. Int J Urol. 1999;6:19

Japanese)

Journal of Dermatology;68:223,2006(in Japanese)

Journal of Dermatology ; 68: 220, 2006(in Japanese)

the primary care physician. Can J Urol. 2011 ;18 Suppl:24-38

central precocious puberty.et al. J Pediatr ; 123: 334-335, 1993

leuprorelin acetate. Rinsho Hifuka;53:801-803,1999(in Japanese)

acetate for central precocious puberty. J Pediatr;121:634-640,1992

injection. Jpn J Dermatol; 116: 1089, 2006(in Japanese)

derma ;48:781-783,2006(in Japanese)

Rinsho Hihuka ; 47: 784-787, 2005(in Japanese)

2006

Japanese)


**13** 

*Japan* 

**New Botanical Materials with** 

*Tokyo University of Marine Science and Technology* 

Enlargement of prostate, which affects 50% of men aged 60 and 90% of men by age 80, is commonly referred to as benign prostate hyperplasia (BPH) (Russell & Wilson, 1994). BPH is a slow, progressive enlargement of the fibromuscular and epithelial structures of the prostate gland (Cristoni et al., 2000). Substantial evidence indicates that the androgens testosterone (T) and dihydrotestosterone (DHT) contribute to the production of BPH (Lowe et al., 2003). The principal serum androgen T is converted by 5alpha-reductase (5aR) to DHT. DHT binds to androgen receptor (AR) in the prostate, where it initiates DNA synthesis (Marks, 2004). This action, in turn induces protein synthesis and abnormal growth of prostate. Current clinical evidence indicates that either the inhibition of 5aR or the inhibition of the binding of DHT to AR reverses the symptoms of BPH in human males. The effective drugs, finasteride as a 5aR inhibitor and flutamide as a binding inhibitor to AR are utiliezed clinically for treatment of BPH. Alternatively functional foods are eclectic selection as dietary approaches to prevent BPH in middle-aged men. Natural products are frequently used to care BPH in preference to therapeutic agents that can cause severe sideeffects. Plant extracts such as the lipid extracts of saw palmetto berry extract (SPE) have also been found to reduce the conversion of testosterone (T) to dihydrotestosterone (DHT) by inhibiting 5alpha-reductase (5aR) both in vitro and in vivo (Elliot, 2001). However, few products of natural origin besides SPE are believed to be effective against enlargement of the prostate. The scientifically-proven food materials are expected to be of benefit for

In our preliminary study, the suppressive effects of natural food materials have been found in BHT model mice. We introduce here the suppressive effects against BHT of two materials, i.e., "banana peel" and "leaf of *Houttuynia cordata*". The effects of these materials in the androgen-responsive LNCaP human prostate cancer cell line and in effects of BHT model mice were examined. These data presented here demonstrate that these materials inhibit the

The chemicals used and their sources were as follows: testosterone propionate and dihydrotestosterone from Wako Pure Chemical Industries (Osaka, Japan); flutamide from

growth of the prostate and HCE has anti-androgenic activity.

**1. Introduction** 

prevention of BPH.

**2. Experimental** 

**2.1 Drugs and chemicals** 

**Anti-Androgenic Activity** 

Tomoyuki Koyama


## **New Botanical Materials with Anti-Androgenic Activity**

Tomoyuki Koyama *Tokyo University of Marine Science and Technology Japan* 

#### **1. Introduction**

192 Prostate Cancer – Original Scientific Reports and Case Studies

Yamashita F.,Hirai S,Ikeda S.et al A case of foreign granuloma due to leuprorelin acetate 3 month preparations. Hifubyo Shinryo;27:1277-1280,2005(in Japanese) Yasukawa K,Sugawara H,Kato N.et al Leuprorelin acetate granulomas:case reports and

Zippe CD, Raina R, Thukral M, et al. Management of erectile dysfunction following radical

review of the literture. Br J Dermatol; 152: 1045-1047, 2005

prostatectomy. Curr Urol Rep. 2001;2:495–503

Enlargement of prostate, which affects 50% of men aged 60 and 90% of men by age 80, is commonly referred to as benign prostate hyperplasia (BPH) (Russell & Wilson, 1994). BPH is a slow, progressive enlargement of the fibromuscular and epithelial structures of the prostate gland (Cristoni et al., 2000). Substantial evidence indicates that the androgens testosterone (T) and dihydrotestosterone (DHT) contribute to the production of BPH (Lowe et al., 2003). The principal serum androgen T is converted by 5alpha-reductase (5aR) to DHT. DHT binds to androgen receptor (AR) in the prostate, where it initiates DNA synthesis (Marks, 2004). This action, in turn induces protein synthesis and abnormal growth of prostate. Current clinical evidence indicates that either the inhibition of 5aR or the inhibition of the binding of DHT to AR reverses the symptoms of BPH in human males.

The effective drugs, finasteride as a 5aR inhibitor and flutamide as a binding inhibitor to AR are utiliezed clinically for treatment of BPH. Alternatively functional foods are eclectic selection as dietary approaches to prevent BPH in middle-aged men. Natural products are frequently used to care BPH in preference to therapeutic agents that can cause severe sideeffects. Plant extracts such as the lipid extracts of saw palmetto berry extract (SPE) have also been found to reduce the conversion of testosterone (T) to dihydrotestosterone (DHT) by inhibiting 5alpha-reductase (5aR) both in vitro and in vivo (Elliot, 2001). However, few products of natural origin besides SPE are believed to be effective against enlargement of the prostate. The scientifically-proven food materials are expected to be of benefit for prevention of BPH.

In our preliminary study, the suppressive effects of natural food materials have been found in BHT model mice. We introduce here the suppressive effects against BHT of two materials, i.e., "banana peel" and "leaf of *Houttuynia cordata*". The effects of these materials in the androgen-responsive LNCaP human prostate cancer cell line and in effects of BHT model mice were examined. These data presented here demonstrate that these materials inhibit the growth of the prostate and HCE has anti-androgenic activity.

#### **2. Experimental**

#### **2.1 Drugs and chemicals**

The chemicals used and their sources were as follows: testosterone propionate and dihydrotestosterone from Wako Pure Chemical Industries (Osaka, Japan); flutamide from

New Botanical Materials with Anti-Androgenic Activity 195

and long axes of the prostates were then measured with vernier calipers, and the seminal vesicles were removed and weighed. In particular, the weights of the seminal vesicles were sensitive to androgenic effects in our mice model. Finasteride (FI, 2 mg/kg) and flutamide (FL, 10 mg /kg) were used as positive control for the assay system. As shown in Fig. 1B, orally administration of flutamide suppressed weight of the seminal vesicles in BPH model

The LNCaP human prostate cancer cell line is a well-established androgen-dependent cell line. (Goldman et al., 2004) AR-positive human prostate cancer LNCaP cells were obtained from the Riken BRC Cell Bank (Tsukuba, Japan). The cells were plated onto a 96-well plate at a density of 2 × 105 /well and supplemented with 10% charcoal stripped fetal bovine serum (CSFBS) obtained from Invitrogen Japan (Tokyo). Twenty-four h later, the cells were treated with either vehicle control or androgens (T or DHT) in the presence and the absence of each concentration of assessed samples for another 3 d. Sample was dissolved in ethanol and added to the cells after further dilution so that the final volume of ethanol was 1% or less. After culture, cell proliferation was determined to measure cell viability by 3-[4,5 dimethyl thiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. (Hamid et al., 2004) The cells were treated with 1 mg/ml of MTT for 2 h, and precipitated dye was dissolved

Data were analyzed statistically by Student's *t*-test to determine significant differences in the data among the groups. The *p* values less than 0.05 were considered significant. The values

Fig. 2. Banana peel extract (BPEx) prepared from fresh peel part from organically-cultivated

bananas showed suppressive effects for enlargement of prostate in BPH model mice

mice with dose dependent manner (1-25 mg/kg/day) for 10 d.

**2.4 Cell culture and growth studies of human prostate cancer cells** 

into dimethylsulfoxide. The absorbance of each well was measured at 570 nm.

**2.5 Statistical analysis** 

**3. Banana peel** 

were expressed as mean ± S.E.

Sigma–Aldrich Japan (Tokyo); finasteride from LKT Labs. (St. Paul, MN); and pentobarbital from Dainippon Sumitomo Pharma (Osaka, Japan). Other chemicals were of analytical grade.

#### **2.2 Animal experiments**

Male ddY mice (6-8 weeks of age) were purchased from SLC Inc., Shizuoka, Japan. The room was maintained at 24±1°C and 50±10% humidity under a 12 h light/dark cycle (lights on from 8:00 AM to 8:00 PM), and the animals had free access to water and food. Animal studies were conducted according to the 2006 guidelines entitled Notification No. 88 of the Ministry of the Environment in Japan and Guidelines for Animal Experimentation of Tokyo University of Marine Science and Technology with the approval of the Animal Care and Use Committee of Tokyo University of Marine Science and Technology.

#### **2.3 Growth suppression of mouse prostates and seminal vesicles in testosteroneinduced BPH model mice**

Assay of growth suppression in castrated mouse prostates and seminal vesicles was performed based on the OECD protocol. (Yamasaki et al., 2003) The testes of ddY mice were removed at 7 weeks of age under anesthesia by intraperitoneal injection of pentobarbital (50 mg/ml/kg). After 1 d, testosterone propionate (TP) was injected intraperitoneally into the mice once daily for 10 d. Enlargement of the reproductive organs in the mice was demonstrated dose-dependently due to TP in our preliminary studies as shown in Fig. 1A. The growth of these organs is androgen-dependent (Franck-Lissbrant et al., 1998).

Fig. 1. TP-induced regrowth of seminal vesicles (A) and its suppressive effects by flutamide (B) in castrated mice

Testosterone propionate (TP; 2, 10, and 50 mg/kg) was injected i.p. into castrated mice (7 weeks-old) once daily for 10 days. Flutamide (1, 5, and 25 mg/kg) suspended in 1% ethanol and orally administered once daily. The seminal vesicles were removed and weighed and compared with castrated mice as a control. Cast: castrated control, Cont: control (TPtreated), and FL: flutamide. Each value represents the mean ± S.E., *n*=5. \*\*: *p*<0.01,\*\*\*: *p*<0.005 *vs* control.

To evaluate the effects on reproductive organs, each of various doses of sample extract was suspended in 1% ethanol and orally administered to the BPH model mice once daily. After 10 d, the mice were weighed, and sacrificed by cervical dislocation. The lengths of the short

Sigma–Aldrich Japan (Tokyo); finasteride from LKT Labs. (St. Paul, MN); and pentobarbital from Dainippon Sumitomo Pharma (Osaka, Japan). Other chemicals were of analytical grade.

Male ddY mice (6-8 weeks of age) were purchased from SLC Inc., Shizuoka, Japan. The room was maintained at 24±1°C and 50±10% humidity under a 12 h light/dark cycle (lights on from 8:00 AM to 8:00 PM), and the animals had free access to water and food. Animal studies were conducted according to the 2006 guidelines entitled Notification No. 88 of the Ministry of the Environment in Japan and Guidelines for Animal Experimentation of Tokyo University of Marine Science and Technology with the approval of the Animal Care and Use

**2.3 Growth suppression of mouse prostates and seminal vesicles in testosterone-**

The growth of these organs is androgen-dependent (Franck-Lissbrant et al., 1998).

Assay of growth suppression in castrated mouse prostates and seminal vesicles was performed based on the OECD protocol. (Yamasaki et al., 2003) The testes of ddY mice were removed at 7 weeks of age under anesthesia by intraperitoneal injection of pentobarbital (50 mg/ml/kg). After 1 d, testosterone propionate (TP) was injected intraperitoneally into the mice once daily for 10 d. Enlargement of the reproductive organs in the mice was demonstrated dose-dependently due to TP in our preliminary studies as shown in Fig. 1A.

 Fig. 1. TP-induced regrowth of seminal vesicles (A) and its suppressive effects by flutamide

Testosterone propionate (TP; 2, 10, and 50 mg/kg) was injected i.p. into castrated mice (7 weeks-old) once daily for 10 days. Flutamide (1, 5, and 25 mg/kg) suspended in 1% ethanol and orally administered once daily. The seminal vesicles were removed and weighed and compared with castrated mice as a control. Cast: castrated control, Cont: control (TPtreated), and FL: flutamide. Each value represents the mean ± S.E., *n*=5. \*\*: *p*<0.01,\*\*\*:

To evaluate the effects on reproductive organs, each of various doses of sample extract was suspended in 1% ethanol and orally administered to the BPH model mice once daily. After 10 d, the mice were weighed, and sacrificed by cervical dislocation. The lengths of the short

Committee of Tokyo University of Marine Science and Technology.

**2.2 Animal experiments** 

**induced BPH model mice** 

(B) in castrated mice

*p*<0.005 *vs* control.

and long axes of the prostates were then measured with vernier calipers, and the seminal vesicles were removed and weighed. In particular, the weights of the seminal vesicles were sensitive to androgenic effects in our mice model. Finasteride (FI, 2 mg/kg) and flutamide (FL, 10 mg /kg) were used as positive control for the assay system. As shown in Fig. 1B, orally administration of flutamide suppressed weight of the seminal vesicles in BPH model mice with dose dependent manner (1-25 mg/kg/day) for 10 d.

#### **2.4 Cell culture and growth studies of human prostate cancer cells**

The LNCaP human prostate cancer cell line is a well-established androgen-dependent cell line. (Goldman et al., 2004) AR-positive human prostate cancer LNCaP cells were obtained from the Riken BRC Cell Bank (Tsukuba, Japan). The cells were plated onto a 96-well plate at a density of 2 × 105 /well and supplemented with 10% charcoal stripped fetal bovine serum (CSFBS) obtained from Invitrogen Japan (Tokyo). Twenty-four h later, the cells were treated with either vehicle control or androgens (T or DHT) in the presence and the absence of each concentration of assessed samples for another 3 d. Sample was dissolved in ethanol and added to the cells after further dilution so that the final volume of ethanol was 1% or less. After culture, cell proliferation was determined to measure cell viability by 3-[4,5 dimethyl thiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. (Hamid et al., 2004) The cells were treated with 1 mg/ml of MTT for 2 h, and precipitated dye was dissolved into dimethylsulfoxide. The absorbance of each well was measured at 570 nm.

#### **2.5 Statistical analysis**

Data were analyzed statistically by Student's *t*-test to determine significant differences in the data among the groups. The *p* values less than 0.05 were considered significant. The values were expressed as mean ± S.E.

#### **3. Banana peel**

Fig. 2. Banana peel extract (BPEx) prepared from fresh peel part from organically-cultivated bananas showed suppressive effects for enlargement of prostate in BPH model mice

New Botanical Materials with Anti-Androgenic Activity 197

seminal vesicles, and showed almost maximal effect at 200 mg/kg. These results indicate that some components in banana peel will be required to suppress prostate enlargement in

 Fig. 3. Effects of extracts of peel (Pe) and edible part (Ed) of banana fruit on TP-induced

The effects of BPEx on the proliferation of prostate cancer cells (LNCaP cells) were investigated. LNCaP cells show most of the characteristics of human prostatic carcinoma, such as dependence on androgens, the presence of ARs, and the production of acid phosphatase and prostate-specific antigen. The LNCaP cell line is used as an attractive model for in vitro studies of the biology of human prostate cancer. LNCaP cells were incubated with different concentrations of BPEx (3.13–100 μg/ml) with and without T or DHT for 3 d. In the absence of BPEx, T alone stimulated LNCaP cell number about 20.5 ± 0.5 x 105 /well, and DHT alone stimulated LNCaP cell numbers to about 30.4 ± 2.1 x 105 /well.

Fig. 4. Inhibitory effects of BPEx on the proliferation of LNCaP cells induced by testosterone

this model.

(A) (B)

regrowth of prostates and seminal vesicles in castrated mice

**3.4 Inhibitory effects of BPEx on prostate cancer cells** 

(T) or dihydrotestosterone (DHT)

#### **3.1 Source material**

The common banana (Musa spp.) is a tropical fruit that grows in the western hemisphere. Primarily viewed as a food source, the banana has a fleshy inside portion surrounded by an outer typically yellow peel (Fig. 2). The fleshy inside portion, or pulp, is edible when raw, and the peel is usually discarded. When ripe, bananas have a deep yellow rind with brown spots, and a creamy pulp, which is easily digested. Bananas are rich in carbohydrates and contain relatively large amounts of vitamins A, B and C and the minerals potassium and phosphorous (Proteggente et al., 2002; Blades et al., 2003). However, banana peel has not been studied nutritionally and pharmaceutically as a source of bioactive compounds. In this report, we examined the effects of banana peel extract (BPEx) on androgen-induced enlargement of accessory reproductive organs in castrated mice. To elucidate the mechanisms of action, the effects of BPEx on the androgen-responsive LNCaP human prostate cancer cell line were investigated. The data presented here indicate that BPEx inhibits growth of the prostate and that BPEx has anti-androgenic activity through inhibition of 5aR.

Fresh banana peel (2.4 kg) of organically-cultivated bananas were cut and extracted with methanol at room temperature for 2 days. The extracts were filtered, concentrated under a vacuum, and freeze-dried. BPEx (56 g) was stored in a refrigerator before assay.

#### **3.2 Suppressive effect of benign prostate hyperplasia in mouse**

Our research group has been shown that BPEx treatment (10 d of administration at 0, 50, 100, and 200 mg/kg per orally) dose-dependently reduced the prostate size and the weights of seminal vesicles in the BPH model mice. Then, the effects of BPEx on the growth of mouse prostates and seminal vesicles were studied as compared with those of flutamide and finasteride in the BPH model mice for 10 d. The short and long axes of the ventral prostates and the weights of the seminal vesicles were severely reduced, and when TP (2 mg/kg i.p.) was injected, significant growth of prostates and seminal vesicles was induced. BPEx (200 mg/kg) produced a reduction in prostate weight. Finasteride (2 mg/kg) and flutamide (10 mg/kg), well-known anti-androgens, showed larger reductions in prostate weight. Similar results were observed with regard to seminal vesicle weights. When DHT (6 mg/kg, i.p.) was injected in place of T, significant growth of the prostate and seminal vesicles was induced. BPEx did not inhibit these effects of DHT (Akamine, 2009). These results indicate that orally treatment of BPEx suppress the action of testosterone against enlargement of the reproductive organs in vivo.

#### **3.3 Effects of parts of banana fruit on mouse prostate and seminal vesicles**

Testosterone propionate (TP, 2 mg/kg, i.p.) and sample solution (p.o.) were treated to castrated mice once daily for 10 d (Fig. 3). The lengths of the long axes (A) of the prostates of mice were measured vernier calipers, and seminal vesicles (B) were removed and weighed. Cast, castration only; Cont, control; Pe, banana peel (200 mg/kg); Ed, edible part (200 mg/kg). Each value represents the mean ± S.E., *n*=5. \*: *p*<0.05; \*\*: *p*<0.01 *vs*. control (TPtreated without samples).

Fresh banana fruits separated into two parts, peel and edible part to evaluate suppressive effects of the extracts ("Pe" and "Ed", respectively) on BPH model mice. The inhibitory effects of BPEx on the growth of mouse prostates and seminal vesicles were estimated after 10 d of administration. BPEx dose-dependently reduced the prostate size and the weights of

The common banana (Musa spp.) is a tropical fruit that grows in the western hemisphere. Primarily viewed as a food source, the banana has a fleshy inside portion surrounded by an outer typically yellow peel (Fig. 2). The fleshy inside portion, or pulp, is edible when raw, and the peel is usually discarded. When ripe, bananas have a deep yellow rind with brown spots, and a creamy pulp, which is easily digested. Bananas are rich in carbohydrates and contain relatively large amounts of vitamins A, B and C and the minerals potassium and phosphorous (Proteggente et al., 2002; Blades et al., 2003). However, banana peel has not been studied nutritionally and pharmaceutically as a source of bioactive compounds. In this report, we examined the effects of banana peel extract (BPEx) on androgen-induced enlargement of accessory reproductive organs in castrated mice. To elucidate the mechanisms of action, the effects of BPEx on the androgen-responsive LNCaP human prostate cancer cell line were investigated. The data presented here indicate that BPEx inhibits growth of the prostate and that BPEx has anti-androgenic activity through

Fresh banana peel (2.4 kg) of organically-cultivated bananas were cut and extracted with methanol at room temperature for 2 days. The extracts were filtered, concentrated under a

Our research group has been shown that BPEx treatment (10 d of administration at 0, 50, 100, and 200 mg/kg per orally) dose-dependently reduced the prostate size and the weights of seminal vesicles in the BPH model mice. Then, the effects of BPEx on the growth of mouse prostates and seminal vesicles were studied as compared with those of flutamide and finasteride in the BPH model mice for 10 d. The short and long axes of the ventral prostates and the weights of the seminal vesicles were severely reduced, and when TP (2 mg/kg i.p.) was injected, significant growth of prostates and seminal vesicles was induced. BPEx (200 mg/kg) produced a reduction in prostate weight. Finasteride (2 mg/kg) and flutamide (10 mg/kg), well-known anti-androgens, showed larger reductions in prostate weight. Similar results were observed with regard to seminal vesicle weights. When DHT (6 mg/kg, i.p.) was injected in place of T, significant growth of the prostate and seminal vesicles was induced. BPEx did not inhibit these effects of DHT (Akamine, 2009). These results indicate that orally treatment of BPEx suppress the action of testosterone against enlargement of the

vacuum, and freeze-dried. BPEx (56 g) was stored in a refrigerator before assay.

**3.3 Effects of parts of banana fruit on mouse prostate and seminal vesicles** 

Testosterone propionate (TP, 2 mg/kg, i.p.) and sample solution (p.o.) were treated to castrated mice once daily for 10 d (Fig. 3). The lengths of the long axes (A) of the prostates of mice were measured vernier calipers, and seminal vesicles (B) were removed and weighed. Cast, castration only; Cont, control; Pe, banana peel (200 mg/kg); Ed, edible part (200 mg/kg). Each value represents the mean ± S.E., *n*=5. \*: *p*<0.05; \*\*: *p*<0.01 *vs*. control (TP-

Fresh banana fruits separated into two parts, peel and edible part to evaluate suppressive effects of the extracts ("Pe" and "Ed", respectively) on BPH model mice. The inhibitory effects of BPEx on the growth of mouse prostates and seminal vesicles were estimated after 10 d of administration. BPEx dose-dependently reduced the prostate size and the weights of

**3.2 Suppressive effect of benign prostate hyperplasia in mouse** 

**3.1 Source material** 

inhibition of 5aR.

reproductive organs in vivo.

treated without samples).

seminal vesicles, and showed almost maximal effect at 200 mg/kg. These results indicate that some components in banana peel will be required to suppress prostate enlargement in this model.

Fig. 3. Effects of extracts of peel (Pe) and edible part (Ed) of banana fruit on TP-induced regrowth of prostates and seminal vesicles in castrated mice

#### **3.4 Inhibitory effects of BPEx on prostate cancer cells**

The effects of BPEx on the proliferation of prostate cancer cells (LNCaP cells) were investigated. LNCaP cells show most of the characteristics of human prostatic carcinoma, such as dependence on androgens, the presence of ARs, and the production of acid phosphatase and prostate-specific antigen. The LNCaP cell line is used as an attractive model for in vitro studies of the biology of human prostate cancer. LNCaP cells were incubated with different concentrations of BPEx (3.13–100 μg/ml) with and without T or DHT for 3 d. In the absence of BPEx, T alone stimulated LNCaP cell number about 20.5 ± 0.5 x 105 /well, and DHT alone stimulated LNCaP cell numbers to about 30.4 ± 2.1 x 105 /well.

Fig. 4. Inhibitory effects of BPEx on the proliferation of LNCaP cells induced by testosterone (T) or dihydrotestosterone (DHT)

New Botanical Materials with Anti-Androgenic Activity 199

We examined the effects of *H. cordata* extract (HCE) in the androgen-responsive effects in BPH model mice and LNCaP human prostate cancer cell line. The data presented here demonstrate that HCE inhibits the growth of the prostate with anti-androgenic activity.

Testosterone propionate (TP, 2 mg/kg, i.p.) and sample solution (p.o.) were treated to castrated mice once daily for 10 d. The seminal vesicles were removed and weighed. Cast, castration only; Cont, TP treated control; HCE: *Houttuynia cordata* extract (20, 100, and 500 mg/kg). Each data presents as mean±S.E., *n*=6, \*: *p*<0.05, \*\*: *p*<0.01, \*\*\*: *p*<0.005 vs control

The suppressive effects of HCE on reproductive organs were investigated in testosteroneinduced BPH model mice. The assay to evaluate the effects of HCE on castrated mice prostates and seminal vesicles was performed based on the Hershberger assay as a mean of rapidly developing in vivo. The testes of ddY mice were removed at 7 weeks of age under anesthesia with intraperitoneal injection of pentobarbital (50 mg/ml/kg). After one day of orchiectmy, testosterone propionate (TP) 2 mg/kg was injected intraperitoneally (i.p.) into the mice once daily for 10 days. HCE was administered once daily. After 10 days, mice were weighted and sacrificed by cervical dislocation. The lengths of the long axes of prostate were

As shown in Fig. 6, HCE suppress prostates and seminal in a dose-dependent manner at 20,

**Cast Cont 20 100 500**

Fig. 6. Effects of extracts of HCE on TP-induced regrowth of prostates and seminal vesicles

In the further experiments, the suppressive effects of HCE were compared with flutamide (FL, 10 mg/kg/day) and finasteride (FI, 2 mg/kg/day) in BPH model mice. As shown in Fig. 7, the size of long-axis length of prostates (A) and the weight of seminal vesicles (B) were severely reduced by HCE ingestion, and when T was injected, a significant growth of prostates and seminal vesicles was induced. Flutamide and finasteride are well known anti-androgen drugs, showed a larger reduction in prostate and seminal vesicle. HCE showed suppressive effect on reproductive organs as same as these medicine at these

\* \*\*

\*\*

measured by vernier calipers, and seminal vesicles were removed and weighed.

**4.2 Suppressive effect of benign prostate hyperplasia in mouse** 

(Cont) as shown in Fig. 6.

100, and 500 mg/kg.

in castrated mice

experimental conditions.

0.0

1.0

\*\*\*

**Weight** (**g/kg**)

2.0

3.0

Treatment of LNCaP cells with BPEx in the presence of T resulted in dose-dependent inhibition of cell growth. In the presence of T, both finasteride and flutamide inhibited cell proliferation. However, in the presence of DHT, while flutamide inhibited cell proliferation, finasteride did not. These results indicate that BPEx will suppress on T did not affect on DHT in vivo and in vitro experiments. Fig. 4 showed the inhibitory effects of anti-androgenic samples at 25 µg/ml on LNCaP cells. Scince BPEx showed similar profile with that of FI and not FL, BPEx was estimate to inhibit BPH by its inhibitory activity against 5aR.

Proliferation of prostate cancer cells (LNCaP) were evaluated cell viability (% of control) by MTT assay after 3 days incubation with or without samples at 25 µg/ml in the presence of testosterone (T: 10 mg/ml) or dihydrotestosterone (DHT: 10 mg/ml). BPEx: banana peel extract; FI: finasteride; FI: flutamide. Each data presents as mean±S.E. (*n*=4).

#### **4. Leaf of** *Houttuynia cordata*

Fig. 5. *Houttuynia cordata* extract (HCE) prepared from fresh leaf part showed suppressive effects for enlargement of prostate in BPH model mice

#### **4.1 Source material**

*Houttuynia cordata* Thunb., which is called dokudami in Japanese, is widely distributed in eastern Asia, and it has a thin leafstalk and heart-shaped leaf (Fig. 5). It is used in folk medicine for diuresis and detoxification. Thus far, it has been reported that *H. cordata* contains many flavonoids (quercitrin, isoquercitrin, rutin, etc.), alkaloids (aristolactam B, norcepharadione B, splendidine, etc.), and volatile components of essential oils (methyl-nnonyl ketone, lauraldehyde, β-myrcene, etc.) (Meng et al., 2005; Kim et al., 2001; Xu et al., 2005). The extracts and components exhibit diuretic, anti-obesity (Miyata et al., 2009), antioxidative (Kusirisin et al., 2009), antibacterial (Kim et al., 2008), anti-inflammatory (Lu et al., 2006), and apoptotic effects (Tang et al., 2009). The effects for benign prostate hyperplasia (BPH) of this material have been not investigated.

The fresh *Houttuynia cordata* were cut and extracted with 99.5% methanol at room temperature for 2 days. The extract were filtered, and concentrated under a vacuum. The extract was stored in the refrigerator before assay.

Treatment of LNCaP cells with BPEx in the presence of T resulted in dose-dependent inhibition of cell growth. In the presence of T, both finasteride and flutamide inhibited cell proliferation. However, in the presence of DHT, while flutamide inhibited cell proliferation, finasteride did not. These results indicate that BPEx will suppress on T did not affect on DHT in vivo and in vitro experiments. Fig. 4 showed the inhibitory effects of anti-androgenic samples at 25 µg/ml on LNCaP cells. Scince BPEx showed similar profile with that of FI and not FL, BPEx was estimate to inhibit BPH by its inhibitory activity

Proliferation of prostate cancer cells (LNCaP) were evaluated cell viability (% of control) by MTT assay after 3 days incubation with or without samples at 25 µg/ml in the presence of testosterone (T: 10 mg/ml) or dihydrotestosterone (DHT: 10 mg/ml). BPEx: banana peel

Fig. 5. *Houttuynia cordata* extract (HCE) prepared from fresh leaf part showed suppressive

*Houttuynia cordata* Thunb., which is called dokudami in Japanese, is widely distributed in eastern Asia, and it has a thin leafstalk and heart-shaped leaf (Fig. 5). It is used in folk medicine for diuresis and detoxification. Thus far, it has been reported that *H. cordata* contains many flavonoids (quercitrin, isoquercitrin, rutin, etc.), alkaloids (aristolactam B, norcepharadione B, splendidine, etc.), and volatile components of essential oils (methyl-nnonyl ketone, lauraldehyde, β-myrcene, etc.) (Meng et al., 2005; Kim et al., 2001; Xu et al., 2005). The extracts and components exhibit diuretic, anti-obesity (Miyata et al., 2009), antioxidative (Kusirisin et al., 2009), antibacterial (Kim et al., 2008), anti-inflammatory (Lu et al., 2006), and apoptotic effects (Tang et al., 2009). The effects for benign prostate

The fresh *Houttuynia cordata* were cut and extracted with 99.5% methanol at room temperature for 2 days. The extract were filtered, and concentrated under a vacuum. The

effects for enlargement of prostate in BPH model mice

hyperplasia (BPH) of this material have been not investigated.

extract was stored in the refrigerator before assay.

extract; FI: finasteride; FI: flutamide. Each data presents as mean±S.E. (*n*=4).

against 5aR.

**4. Leaf of** *Houttuynia cordata*

**4.1 Source material** 

We examined the effects of *H. cordata* extract (HCE) in the androgen-responsive effects in BPH model mice and LNCaP human prostate cancer cell line. The data presented here demonstrate that HCE inhibits the growth of the prostate with anti-androgenic activity.

#### **4.2 Suppressive effect of benign prostate hyperplasia in mouse**

Testosterone propionate (TP, 2 mg/kg, i.p.) and sample solution (p.o.) were treated to castrated mice once daily for 10 d. The seminal vesicles were removed and weighed. Cast, castration only; Cont, TP treated control; HCE: *Houttuynia cordata* extract (20, 100, and 500 mg/kg). Each data presents as mean±S.E., *n*=6, \*: *p*<0.05, \*\*: *p*<0.01, \*\*\*: *p*<0.005 vs control (Cont) as shown in Fig. 6.

The suppressive effects of HCE on reproductive organs were investigated in testosteroneinduced BPH model mice. The assay to evaluate the effects of HCE on castrated mice prostates and seminal vesicles was performed based on the Hershberger assay as a mean of rapidly developing in vivo. The testes of ddY mice were removed at 7 weeks of age under anesthesia with intraperitoneal injection of pentobarbital (50 mg/ml/kg). After one day of orchiectmy, testosterone propionate (TP) 2 mg/kg was injected intraperitoneally (i.p.) into the mice once daily for 10 days. HCE was administered once daily. After 10 days, mice were weighted and sacrificed by cervical dislocation. The lengths of the long axes of prostate were measured by vernier calipers, and seminal vesicles were removed and weighed.

As shown in Fig. 6, HCE suppress prostates and seminal in a dose-dependent manner at 20, 100, and 500 mg/kg.

Fig. 6. Effects of extracts of HCE on TP-induced regrowth of prostates and seminal vesicles in castrated mice

In the further experiments, the suppressive effects of HCE were compared with flutamide (FL, 10 mg/kg/day) and finasteride (FI, 2 mg/kg/day) in BPH model mice. As shown in Fig. 7, the size of long-axis length of prostates (A) and the weight of seminal vesicles (B) were severely reduced by HCE ingestion, and when T was injected, a significant growth of prostates and seminal vesicles was induced. Flutamide and finasteride are well known anti-androgen drugs, showed a larger reduction in prostate and seminal vesicle. HCE showed suppressive effect on reproductive organs as same as these medicine at these experimental conditions.

New Botanical Materials with Anti-Androgenic Activity 201

T+HCE

T+FL

T+FI

DHT+HCE

DHT+FL

DHT+FI

1 10 100

Fig. 8. Inhibitory effects of HCE, Flutamide (FL), and Finasteride (FI) on LNCaP cell proliferation in the presence of testosterone (T) or dihydrotestosterone (DHT). Each point

We studied the in vivo potency of BPEx and HCE, as new botanical materials in immature castrated mice. We evaluated these biological effects in the androgen-responsive LNCaP

BPEx inhibited TP-induced growth of prostates and seminal vesicles in castrated mice, although it was less potent than finasteride at the same doses. While BPEx inhibited the action of TP, it did not inhibit DHT-induced organ growth. In addition, BPEx inhibited cell proliferation in the presence of T, but not in the presence of DHT. These results suggest that BPEx suppressed the growth of prostates and seminal vesicles by inhibiting the conversion of T to DHT, rather than by blocking the binding of androgen and its receptor. BPEx may have reduced prostate size and seminal vesicle weight by inhibiting 5aR. In further study, we found that treatment of LNCaP cells with BPEx inhibited Tinduced cell proliferation (Akamine et al., 2009). The inhibition of this effect of T might have be due, at least in part, to inhibition of 5aR or to antagonism of androgen binding to the AR (Lazier et al., 2004). Since 25 μg/ml of BPEx inhibited 5aR activity by about 30%, we expected to find that this dose would inhibit T action. Similar results have been found with finasteride, a well-known inhibitor of 5aR (McConnell et al., 1992; Sudduth & Koronkowski, 1993), at doses above 25 μg/ml. Similar results were also found with flutamide, an antagonist of androgen binding to the AR (Bergman, & Eriksson, 1996; Sundblad et al., 2005). On the other hand, treatment of LNCaP cells with BPEx in the

Concentration (µg/ml)

0

//

shows the mean of the separated duplicate experiments.

human prostate cancer cell line and in effects of BHT model mice.

20

40

60

Growth rate (%)

**5. Discussion** 

80

100

120

Fig. 7. Evaluation of HCE on T-induced regrowth in castrated mouse prostates and seminal vesicles. (A) long-axis length of prostates, (B) weight of seminal vesicles.

Testosterone propionate (TP, 2 mg/kg, i.p.) and sample solution (p.o.) were treated to castrated mice once daily for 10 d. The lengths of the long axes (A) of the prostates of mice were measured vernier calipers, and seminal vesicles (B) were removed and weighed. Cast: Castration without T-treatment (50 mg/kg, i.p.), Cont: Control, HCE: *H. cordata* extract (100 mg/kg), FI : Finasteride 2 mg/kg, FL: Flutamide 10 mg/kg. Each value represents the means±S.E., *n*=5. \*:*p*<0.05, \*\*:*p*<0.01, \*\*\*:*p*<0.005 vs Control.

#### **4.3 Inhibitory effects of HCE on LNCaP cells**

The effects of HCE on the proliferation of prostate cancer cells (LNCaP cells) were investigated. After incubation, the cell numbers in T-treated and DHT-treated groups were defined as 100%. As shown in Fig. 8 finasteride, 5aR inhibitor, showed dose-dependent inhibition in T-treated and DHT-treated LNCaP cells. However, its inhibitory effects on DHT-treated group are relatively weak. In contrast, flutamide, androgen receptor antagonist, showed inhibition both in T alone and DHT alone groups at the similar concentrations. On the other hand, the treatment of HCE to LNCaP cells in the presence of T and DHT resulted in the concentration-dependent inhibition of cell proliferation at the same concentrations as same as case of flutamide treatment. These results indicate that HCE suppress enlargement of prostate with a different mechanism with finasteride, 5aR inhibitor. Proliferation of prostate cancer cells (LNCaP) were evaluated cell viability (% of control) by MTT assay after 3 days incubation with or without samples at 6.25, 25, 100 µg/ml in the presence of testosterone (T: 10 mg/ml) or dihydrotestosterone (DHT: 10 mg/ml). HCE: *Houttuynia cordata* extract; FI: finasteride; FI: flutamide. Each data presents as mean±S.E. (*n*=4).

Fig. 8. Inhibitory effects of HCE, Flutamide (FL), and Finasteride (FI) on LNCaP cell proliferation in the presence of testosterone (T) or dihydrotestosterone (DHT). Each point shows the mean of the separated duplicate experiments.

#### **5. Discussion**

200 Prostate Cancer – Original Scientific Reports and Case Studies

**0.0**

**0.0**

**1.0**

**2.0**

**3.0**

\*\*\*

vesicles. (A) long-axis length of prostates, (B) weight of seminal vesicles.

means±S.E., *n*=5. \*:*p*<0.05, \*\*:*p*<0.01, \*\*\*:*p*<0.005 vs Control.

**4.3 Inhibitory effects of HCE on LNCaP cells** 

mean±S.E. (*n*=4).

**1.0**

**Length (mm)**

(A)

(B)

**Weight (g/kg)**

**2.0**

**3.0**

\*\*\* \*\*\* \*\* \*\*

Cast Cont HCE FL FI

Cast Cont HCE FL FI

Fig. 7. Evaluation of HCE on T-induced regrowth in castrated mouse prostates and seminal

Testosterone propionate (TP, 2 mg/kg, i.p.) and sample solution (p.o.) were treated to castrated mice once daily for 10 d. The lengths of the long axes (A) of the prostates of mice were measured vernier calipers, and seminal vesicles (B) were removed and weighed. Cast: Castration without T-treatment (50 mg/kg, i.p.), Cont: Control, HCE: *H. cordata* extract (100 mg/kg), FI : Finasteride 2 mg/kg, FL: Flutamide 10 mg/kg. Each value represents the

The effects of HCE on the proliferation of prostate cancer cells (LNCaP cells) were investigated. After incubation, the cell numbers in T-treated and DHT-treated groups were defined as 100%. As shown in Fig. 8 finasteride, 5aR inhibitor, showed dose-dependent inhibition in T-treated and DHT-treated LNCaP cells. However, its inhibitory effects on DHT-treated group are relatively weak. In contrast, flutamide, androgen receptor antagonist, showed inhibition both in T alone and DHT alone groups at the similar concentrations. On the other hand, the treatment of HCE to LNCaP cells in the presence of T and DHT resulted in the concentration-dependent inhibition of cell proliferation at the same concentrations as same as case of flutamide treatment. These results indicate that HCE suppress enlargement of prostate with a different mechanism with finasteride, 5aR inhibitor. Proliferation of prostate cancer cells (LNCaP) were evaluated cell viability (% of control) by MTT assay after 3 days incubation with or without samples at 6.25, 25, 100 µg/ml in the presence of testosterone (T: 10 mg/ml) or dihydrotestosterone (DHT: 10 mg/ml). HCE: *Houttuynia cordata* extract; FI: finasteride; FI: flutamide. Each data presents as

\* \* \*

We studied the in vivo potency of BPEx and HCE, as new botanical materials in immature castrated mice. We evaluated these biological effects in the androgen-responsive LNCaP human prostate cancer cell line and in effects of BHT model mice.

BPEx inhibited TP-induced growth of prostates and seminal vesicles in castrated mice, although it was less potent than finasteride at the same doses. While BPEx inhibited the action of TP, it did not inhibit DHT-induced organ growth. In addition, BPEx inhibited cell proliferation in the presence of T, but not in the presence of DHT. These results suggest that BPEx suppressed the growth of prostates and seminal vesicles by inhibiting the conversion of T to DHT, rather than by blocking the binding of androgen and its receptor. BPEx may have reduced prostate size and seminal vesicle weight by inhibiting 5aR. In further study, we found that treatment of LNCaP cells with BPEx inhibited Tinduced cell proliferation (Akamine et al., 2009). The inhibition of this effect of T might have be due, at least in part, to inhibition of 5aR or to antagonism of androgen binding to the AR (Lazier et al., 2004). Since 25 μg/ml of BPEx inhibited 5aR activity by about 30%, we expected to find that this dose would inhibit T action. Similar results have been found with finasteride, a well-known inhibitor of 5aR (McConnell et al., 1992; Sudduth & Koronkowski, 1993), at doses above 25 μg/ml. Similar results were also found with flutamide, an antagonist of androgen binding to the AR (Bergman, & Eriksson, 1996; Sundblad et al., 2005). On the other hand, treatment of LNCaP cells with BPEx in the

New Botanical Materials with Anti-Androgenic Activity 203

due to an anti-androgen effect, such as inhibition of AR. These results suggest that HCE suppressed the growth of prostates and seminal vesicles by blocking the binding to AR, rather then by conversion of T to DHT. HCE may have reduced prostate size and seminal

There are generally two ways to suppress prostate regrowth in animal experiments: by inhibiting 5aR activity and by blocking the binding of androgen and its receptor. An androgen antagonist can suppress DHT-induced prostate and seminal vesicle regrowth. Therefore, blocking DHT from binding to androgen receptors in the prostate and seminal vesicle was considered to be possible mechanism of action (other than 5aR inhibition) of the extract of HCE. To examine this possibility, the effects of HCE on LNCaP cells growth induced by DHT were investigated. These results suggest that HCE inhibited prostate growth by inhibiting androgen receptor rather than by having a direct effect on the 5aR, although the detailed mechanism unclear. In this study, we found that HCE may have antiandrogenic activities through in vitro androgen binding to the AR and in vivo growth suppression of prostate and seminal vesicles from castrated mice. Active compounds showing anti-androgen activity in HCE are of scientific interest. Since, the clinical implications of this activity are currently unknown, further research is needed before is

For several years, SPEx has been used as a popular phytotherapeutic agent in the treatment of BPH, but its active component and mechanism of action have not been fully elucidated (Hill & Kyprianou, 2004). Banana peel is usually considered useless and is discarded, but the anti-androgenic activity of BPEx might be useful in the treatment of BPH patients. And leaf of H. cordata have been used as folk medicine, however, never used for treatment BPH with suppressive effect of androgenic functions. Since the clinical implications of this activity are currently unknown, further research is needed before

In this chapter, the androgen-responsive effects of the two natural extracts from banana peel extract (BPEx) and leaf of *H. cordata* extract (HCE) were shown in BPH model mice and LNCaP human prostate cancer cell line. The data presented here revealed that BPEx was estimated to inhibit the growth of the prostate in BPH model mice by its inhibitory activity against 5-alpha reductase (5aR). And HCE was estimated to inhibit BPH in mice by its antiandrogenic activity different from 5aR inhibition. Therefore, these new botanical materials can be promising most likely candidates as potential material for preventing benign prostate hyperplasia, and it is able to continue to be one of the best preventive medicinal foods to

Akamine, K.; Koyama, T. & Yazawa, K. (2009) Banana peel extract suppresses prostate gland

Bergman, L. & Eriksson, E. (1996) Marked symptom reduction in two women with bulimia

*Psychiatrica Scandinavica*, Vol. 94, pp. 137-139, ISSN: 0001-690X

enlargement in testosterone-treated mice. *Biosci. Biotech. Biochem*. Vol. 73, No. 9, pp.

nervosa treated with the testosterone receptor antagonist flutamide. *Acta* 

vesicle weight by blocking the binding of androgen and its receptor.

needed before HCE can for the treatment of BPH.

BPEx can be used in the treatment of BPH.

keep our good health in the future.

1911-1914, ISSN: 0916-8451

**6. Conclusion** 

**7. References** 

presence of DHT did not result in a dose-dependent inhibition of cell growth. These results suggest that inhibition of cell growth in the presence of BPEx was not the result of a cell cytotoxic effect, but rather was due to an anti-androgen effect, such as inhibition of 5aR. According to a previous report describing discrimination between cytotoxic and cytostatic effects of integrants on LNCaP cells (Romijn et al., 1988), cytotoxic effects are detectable as a decrease in MTT conversion to a level below that of the starting cells. In our experiment, growth of androgen-induced LNCaP cells was suppressed without decreasing from the starting level in the presence of BPEx during the experimental period. Therefore, a cytostatical effect was estimated for suppression of BPEx on LNCaP cell growth. However, since no index markers related to apoptotic signaling were measured in our experiments, the possibility of a non-cytostatical effect should be considered and confirmed in further detailed investigation. There are generally two ways to suppress prostate regrowth in animal experiments: by inhibiting 5aR activity, and through the use of an androgen receptor antagonist (Imperato-McGinley et al., 1992). An androgen antagonist can suppress DHT-induced prostate and seminal vesicle regrowth (Geller et al., 1981) Therefore, blocking DHT from binding to androgen receptors in the prostate and seminal vesicle is considered to be a possible mechanism of action (other than 5aR inhibition) (Nakayama et al., 1997). To examine this possibility, the effects of BPEx on prostate growth induced by DHT were investigated. If the suppression of prostate growth is caused only by inhibition of 5aR, DHT-induced prostatic regrowth should not be suppressed. Ten d after castration, the weights of mouse prostates were markedly reduced, and prostate size was restored by i.p. injections of TP or DHT. BPEx had no effect on the sizes of the prostate and seminal vesicle of castrated mice that received DHT, whereas flutamide, an androgen receptor antagonist, significantly reduced prostate weight (Fig. 3). In our study, blood levels of testosterone and samples were not measured, but in vivo and in vitro experiments gave mutually correlated results with regard with their mechanism of action. These results suggest that BPEx inhibited prostate growth by inhibiting 5aR activity rather than by exerting a direct effect on the androgen receptor, although the detailed mechanism including bioavailability remains unclear. Recently, there is research reports on components of the banana peel: dietary fiber and pectin (Happi Emaga et al., 2008), and phytosterol from unripe pulp and peel (Oliveira et al., 2008) have been reported. In particular, phytosterols are able to be the active principle in BPEx, due to their anti-androgen activity based on structural similarity. Investigation to elucidate the active components in banana peel is in progress. In this study, we found that BPEx can have anti-androgenic activities through in vitro 5aR inhibitory activity and in vivo growth suppression of prostates and seminal vesicles from castrated mice.

In this study, we have shown that HCE inhibited the T-induced growth of prostates and seminal vesicles in castrated mice and that HCE inhibited T- and DHT-induced cell proliferation in NLCaP cells at the same concentration. The inhibition of this effect of T may be due, at least in part, to the inhibition of 5aR or to antagonism of androgen binding to the AR. And treatment of LNCaP cells with HCE in the presence of DHT result in the dosedependent inhibition of cell growth. These results were seen with flutamide, which is an antagonist of androgen binding to the AR. These results suggested that the inhibition of cell proliferation in the presence HCE was not the result of a cell cytotoxic effect, but rather was

presence of DHT did not result in a dose-dependent inhibition of cell growth. These results suggest that inhibition of cell growth in the presence of BPEx was not the result of a cell cytotoxic effect, but rather was due to an anti-androgen effect, such as inhibition of 5aR. According to a previous report describing discrimination between cytotoxic and cytostatic effects of integrants on LNCaP cells (Romijn et al., 1988), cytotoxic effects are detectable as a decrease in MTT conversion to a level below that of the starting cells. In our experiment, growth of androgen-induced LNCaP cells was suppressed without decreasing from the starting level in the presence of BPEx during the experimental period. Therefore, a cytostatical effect was estimated for suppression of BPEx on LNCaP cell growth. However, since no index markers related to apoptotic signaling were measured in our experiments, the possibility of a non-cytostatical effect should be considered and confirmed in further detailed investigation. There are generally two ways to suppress prostate regrowth in animal experiments: by inhibiting 5aR activity, and through the use of an androgen receptor antagonist (Imperato-McGinley et al., 1992). An androgen antagonist can suppress DHT-induced prostate and seminal vesicle regrowth (Geller et al., 1981) Therefore, blocking DHT from binding to androgen receptors in the prostate and seminal vesicle is considered to be a possible mechanism of action (other than 5aR inhibition) (Nakayama et al., 1997). To examine this possibility, the effects of BPEx on prostate growth induced by DHT were investigated. If the suppression of prostate growth is caused only by inhibition of 5aR, DHT-induced prostatic regrowth should not be suppressed. Ten d after castration, the weights of mouse prostates were markedly reduced, and prostate size was restored by i.p. injections of TP or DHT. BPEx had no effect on the sizes of the prostate and seminal vesicle of castrated mice that received DHT, whereas flutamide, an androgen receptor antagonist, significantly reduced prostate weight (Fig. 3). In our study, blood levels of testosterone and samples were not measured, but in vivo and in vitro experiments gave mutually correlated results with regard with their mechanism of action. These results suggest that BPEx inhibited prostate growth by inhibiting 5aR activity rather than by exerting a direct effect on the androgen receptor, although the detailed mechanism including bioavailability remains unclear. Recently, there is research reports on components of the banana peel: dietary fiber and pectin (Happi Emaga et al., 2008), and phytosterol from unripe pulp and peel (Oliveira et al., 2008) have been reported. In particular, phytosterols are able to be the active principle in BPEx, due to their anti-androgen activity based on structural similarity. Investigation to elucidate the active components in banana peel is in progress. In this study, we found that BPEx can have anti-androgenic activities through in vitro 5aR inhibitory activity and in

vivo growth suppression of prostates and seminal vesicles from castrated mice.

In this study, we have shown that HCE inhibited the T-induced growth of prostates and seminal vesicles in castrated mice and that HCE inhibited T- and DHT-induced cell proliferation in NLCaP cells at the same concentration. The inhibition of this effect of T may be due, at least in part, to the inhibition of 5aR or to antagonism of androgen binding to the AR. And treatment of LNCaP cells with HCE in the presence of DHT result in the dosedependent inhibition of cell growth. These results were seen with flutamide, which is an antagonist of androgen binding to the AR. These results suggested that the inhibition of cell proliferation in the presence HCE was not the result of a cell cytotoxic effect, but rather was due to an anti-androgen effect, such as inhibition of AR. These results suggest that HCE suppressed the growth of prostates and seminal vesicles by blocking the binding to AR, rather then by conversion of T to DHT. HCE may have reduced prostate size and seminal vesicle weight by blocking the binding of androgen and its receptor.

There are generally two ways to suppress prostate regrowth in animal experiments: by inhibiting 5aR activity and by blocking the binding of androgen and its receptor. An androgen antagonist can suppress DHT-induced prostate and seminal vesicle regrowth. Therefore, blocking DHT from binding to androgen receptors in the prostate and seminal vesicle was considered to be possible mechanism of action (other than 5aR inhibition) of the extract of HCE. To examine this possibility, the effects of HCE on LNCaP cells growth induced by DHT were investigated. These results suggest that HCE inhibited prostate growth by inhibiting androgen receptor rather than by having a direct effect on the 5aR, although the detailed mechanism unclear. In this study, we found that HCE may have antiandrogenic activities through in vitro androgen binding to the AR and in vivo growth suppression of prostate and seminal vesicles from castrated mice. Active compounds showing anti-androgen activity in HCE are of scientific interest. Since, the clinical implications of this activity are currently unknown, further research is needed before is needed before HCE can for the treatment of BPH.

For several years, SPEx has been used as a popular phytotherapeutic agent in the treatment of BPH, but its active component and mechanism of action have not been fully elucidated (Hill & Kyprianou, 2004). Banana peel is usually considered useless and is discarded, but the anti-androgenic activity of BPEx might be useful in the treatment of BPH patients. And leaf of H. cordata have been used as folk medicine, however, never used for treatment BPH with suppressive effect of androgenic functions. Since the clinical implications of this activity are currently unknown, further research is needed before BPEx can be used in the treatment of BPH.

### **6. Conclusion**

In this chapter, the androgen-responsive effects of the two natural extracts from banana peel extract (BPEx) and leaf of *H. cordata* extract (HCE) were shown in BPH model mice and LNCaP human prostate cancer cell line. The data presented here revealed that BPEx was estimated to inhibit the growth of the prostate in BPH model mice by its inhibitory activity against 5-alpha reductase (5aR). And HCE was estimated to inhibit BPH in mice by its antiandrogenic activity different from 5aR inhibition. Therefore, these new botanical materials can be promising most likely candidates as potential material for preventing benign prostate hyperplasia, and it is able to continue to be one of the best preventive medicinal foods to keep our good health in the future.

#### **7. References**


New Botanical Materials with Anti-Androgenic Activity 205

Lowe, F. C.; McConnell, J. D.; Hudson, P. B.; Romas, N.A.; Boake, R.; Lieber, M.; Elhilali, M.;

McConnell, J. D.; Wilson, J. D.; George, F. W.; Geller, J.; Pappas, F.; Stoner, E. (1992)

Miyata, M.; Koyama, T. & Yazawa, K. (2010). Water extract of *Houttuynia cordata* Thunb.

Nakayama, O.; Hirosumi, J.; Chida, N.; Takahashi, S.; Sawada, K.; Kojo, H. & Notsu, Y.

Oliveira, L.; Freire, C.S.; Silvestre, A. J. & Cordeiro, N. (2008) Lipophilic extracts from

Proteggente, A. R.; Pannala, A. S.; Paganga, G.; Van Buren, L.; Wagner, E.; Wiseman, S.; Van

Romijn, J. C.; Verkoelen, C. F. & Schroeder, F. H. (1988) Application of the MTT assay to

Russell, D. W. & Wilson J. D. (1994). Steroid 5 alpha-reductase: two genes/two enzymes. *Annual Review of Clinical Biochemistry*, Vol 63, pp. 25-61, PMID: 7979239 Sudduth, S. L. & Koronkowski, M. J. (1993) Finasteride- The first 5-alpha-reductase inhibitor.

Sundblad, C.; Landen, M.; Eriksson, T.; Bergman, L. & Eriksson, E. (2005) Effects of the

Tang, Y. J.; Yang, J. S.; Lin, C. F.; Shyu, W. C.; Tsuzuki, M.; Lu, C. C.; Chen, Y. F. & Lai, K. C.

Xu, C. J.; Liang, Y. Z.; Chau, F. T. (2005). Identification of essential components of *Houttuynia* 

*Endocrinology and Metabolism*, Vol. 74, No. 3, 505-508, ISSN: 0021-972X Meng, J.; Leung, K. S.; Jiang, Z.; Dong, X.; Zhao, Z. & Xu, L. J. (2005). Establishment of

*Nutr Sci Vitaminol,* Vol. 56, No. 2, 150-156, ISSN: 0301-4800

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composition. *Free Radic Res*., Vol. 36, pp. 217-233, ISSN: 1071-5762

cytotoxic effects. *Prostate,* Vol. 12, pp. 99-110, ISSN: 0270-4137

*Pharmacotherapy*, Vol. 13, pp. 309-329, ISSN 0277-0008

*Psychopharmacology*, Vol. 25, pp. 85-88, ISSN: 0271-0749

approach. *Talanta,* Vol. 68, 108-115, ISSN: 0039-9140

S11-S21, ISSN: 1523-6161

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**14** 

*Taiwan* 

**Inhibition of Advanced Prostate Cancer by Androgens and Liver X Receptor Agonists** 

*2Translational Center for Glandular Malignancies, National Health Research Institutes* 

Prostate cancer is the most frequently diagnosed non-cutaneous tumor of men in western countries. National Cancer Institute estimated that more than 217,000 people were diagnosed and 32,000 people died of prostate cancer in the United States in 2010. Currently, primary therapies for prostate cancer include radical prostatectomy, radiation therapy, highintensity focused ultrasound, chemotherapy, cryosurgery, hormonal therapy, and combination of different treatments. Approximately 20-40% of patients treated with radical prostatectomy will have tumor recurrence and elevation of serum prostate-specific antigen (PSA) (Sadar 2011). More than 80% of patients who died from prostate cancer developed bone metastases, primary metastatic sites include bones and lymph nodes (Bubendorf et al

In 1941, Huggins and Hodges reported that androgen ablation therapy caused regression of primary and metastatic prostate cancer (Huggins C 1941). Since then, androgen ablation therapy, using luteinizing hormone-releasing hormone agonists (LH-RH) or bilateral orchiectomy, has become one of the primary treatment for prostate cancer (Seruga and Tannock 2008). More than 80% of men with these advanced prostate cancers respond to androgen ablation therapy, resulting in tumors shrinkage and reduction of serum PSA (Seruga and Tannock 2008). Anti-androgens are frequently used in conjunction with androgen ablation therapy as a combined androgen blockade to improve therapeutic outcome (Klotz et al 2004). However, 80-90% of the patients who receive androgen ablation therapy ultimately develop recurrent tumors in 12-33 months. The median overall survival of patients after tumor relapse is 1-2 years (Fowler et al 1998, Hellerstedt and Pienta 2002). In addition, androgen deprivation therapy is associated with several undesired side-effects, including sexual dysfunction, osteoporosis, hot flashes, fatigue, gynecomastia, anemia, depression, cognitive dysfunction, increased risk of diabetes, and cardiovascular diseases (Keating et al 2006, Keating et al 2010, Saigal et al 2007, Seruga and Tannock 2008). Androgen deprivation therapy using LH-RH agonists was reported to increase risk of incident diabetes, incident coronary heart disease, myocardial infarction, sudden cardiac death, and stroke (Keating et al 2006, Keating et al 2010, Saigal et al 2007). Combined

**1. Introduction** 

2000, Ibrahim et al 2010, Keller et al 2001).

Chih-Pin Chuu1,2, Hui-Ping Lin1,2, Ching-Yu Lin1,2,

*3Department of Life Sciences, National Central University* 

*1Affiliated University, Institute of Cellular and System Medicine* 

Chiech Huo1,2,3 and Liang-Cheng Su1,2

Yamasaki, K.; Sawaki, M.; Ohta, R.; Okuda, H.; Katayama, S.; Yamada, T.; Ohta, T.; Kosaka, T. & Owens, W. (2003) OECD validation of the Hershberger assay in Japan: Phase 2 dose response of methyltestosterone, vinclozolin, and *p,p*'-DDE. *Environ Health Perspect*, Vol. 111, pp. 1912-1919, ISSN: 0091-6765

### **Inhibition of Advanced Prostate Cancer by Androgens and Liver X Receptor Agonists**

Chih-Pin Chuu1,2, Hui-Ping Lin1,2, Ching-Yu Lin1,2, Chiech Huo1,2,3 and Liang-Cheng Su1,2 *1Affiliated University, Institute of Cellular and System Medicine 2Translational Center for Glandular Malignancies, National Health Research Institutes 3Department of Life Sciences, National Central University Taiwan* 

#### **1. Introduction**

206 Prostate Cancer – Original Scientific Reports and Case Studies

Yamasaki, K.; Sawaki, M.; Ohta, R.; Okuda, H.; Katayama, S.; Yamada, T.; Ohta, T.; Kosaka,

*Perspect*, Vol. 111, pp. 1912-1919, ISSN: 0091-6765

T. & Owens, W. (2003) OECD validation of the Hershberger assay in Japan: Phase 2 dose response of methyltestosterone, vinclozolin, and *p,p*'-DDE. *Environ Health* 

> Prostate cancer is the most frequently diagnosed non-cutaneous tumor of men in western countries. National Cancer Institute estimated that more than 217,000 people were diagnosed and 32,000 people died of prostate cancer in the United States in 2010. Currently, primary therapies for prostate cancer include radical prostatectomy, radiation therapy, highintensity focused ultrasound, chemotherapy, cryosurgery, hormonal therapy, and combination of different treatments. Approximately 20-40% of patients treated with radical prostatectomy will have tumor recurrence and elevation of serum prostate-specific antigen (PSA) (Sadar 2011). More than 80% of patients who died from prostate cancer developed bone metastases, primary metastatic sites include bones and lymph nodes (Bubendorf et al 2000, Ibrahim et al 2010, Keller et al 2001).

> In 1941, Huggins and Hodges reported that androgen ablation therapy caused regression of primary and metastatic prostate cancer (Huggins C 1941). Since then, androgen ablation therapy, using luteinizing hormone-releasing hormone agonists (LH-RH) or bilateral orchiectomy, has become one of the primary treatment for prostate cancer (Seruga and Tannock 2008). More than 80% of men with these advanced prostate cancers respond to androgen ablation therapy, resulting in tumors shrinkage and reduction of serum PSA (Seruga and Tannock 2008). Anti-androgens are frequently used in conjunction with androgen ablation therapy as a combined androgen blockade to improve therapeutic outcome (Klotz et al 2004). However, 80-90% of the patients who receive androgen ablation therapy ultimately develop recurrent tumors in 12-33 months. The median overall survival of patients after tumor relapse is 1-2 years (Fowler et al 1998, Hellerstedt and Pienta 2002). In addition, androgen deprivation therapy is associated with several undesired side-effects, including sexual dysfunction, osteoporosis, hot flashes, fatigue, gynecomastia, anemia, depression, cognitive dysfunction, increased risk of diabetes, and cardiovascular diseases (Keating et al 2006, Keating et al 2010, Saigal et al 2007, Seruga and Tannock 2008). Androgen deprivation therapy using LH-RH agonists was reported to increase risk of incident diabetes, incident coronary heart disease, myocardial infarction, sudden cardiac death, and stroke (Keating et al 2006, Keating et al 2010, Saigal et al 2007). Combined

Inhibition of Advanced Prostate Cancer by Androgens and Liver X Receptor Agonists 209

development of androgen-independency phenotype following androgen ablation therapy, and elevation of AR mRNA and protein are both necessary and sufficient progression of prostate cancer towards androgen-independency (Culig et al 1999, Joly-Pharaboz et al 1995). Elevated AR expression in androgen-independent prostate cancer cells or recurrent hormone-refractory tumors has been observed in our progression model (Chuu et al 2005, Chuu et al 2006, Kokontis et al 1994, Kokontis et al 1998, Kokontis et al 2005, Umekita et al 1996) and several other groups (Chen et al 2004a, de Vere White et al 1997, Edwards et al 2003, Ford et al 2003, Gregory et al 2001, Hara et al 2003, Holzbeierlein et al 2004, Kim et al 2002, Linja et al 2001, Shi et al 2004, Singh et al 2004, Visakorpi et al 1995, Wang et al 2001, Zhang et al 2003). Mechanisms contribute to the progression towards androgenindependency including AR gene amplification, AR mutation, bypass of androgenic activation of AR, or bypass AR signaling for cell survival and proliferation (Feldman and

LNCaP is one of the most commonly used cell line for prostate cancer research, which was derived from a human lymph node metastatic lesion of prostate adenocarcinoma (Chuu et al 2007, Horoszewicz et al 1980). LNCaP expressed AR and inducible PSA. Previously, we cultured androgen-sensitive LNCaP 104-S cells in androgen-depleted conditions *in vitro* to establish relapsed hormone-refractory prostate cancer cells mimic clinical situation in which prostate cancer recurs during androgen deprivation (Kokontis et al 1994a, Kokontis et al 1998b). After 3 months in medium depleted with androgens, most LNCaP 104-S cells underwent G1 cell cycle arrest and apoptosis. A few colonies of cells, named 104-I cells, evolved that proliferated very slowly in the absence of androgen (Kokontis et al 1994). After approximately 11 months, cells called 104-R1 cells emerged that grew much more rapidly in the absence of androgen. After 20 months, 104-R2 cells evolved which proliferated in the absence of androgen at a rate comparable to the proliferation rate of 104-S cells grown in

During the transition of 104-S cells to 104-R1 and 104-R2 cells, AR mRNA and protein level elevated several folds (Chuu et al 2005, Chuu et al 2006, Kokontis et al 1994, Kokontis et al 1998). Proliferation of 104-R1 and 104-R2 cells is androgen-independent but is unexpectedly suppressed by physiological concentrations of androgen both *in vitro* and *in vivo* (Chuu et al 2005, Chuu et al 2006, Kokontis et al 1994, Kokontis et al 1998b, Kokontis et al 2005, Umekita et al 1996). When 104-R1 cells were incubated for several weeks in a high concentration of synthetic androgen R1881 (20 nM), cells named R1Ad adapted after a period of growth arrest (Kokontis et al 1998). Growth of R1Ad cells is slow and not dependent on androgen

To further mimic the clinical situation of combined androgen deprivation and anti-androgen therapy, LNCaP 104-S cells were incubated with 5 M Casodex (biculatimide) in androgendeprived medium. After four weeks, Casodex-resistant colonies appeared at low frequency (1 in 1.4x105) as most of the cells appeared to undergo senescent cell death. The relapsed cells, called CDXR, had increased AR expression and were repressed by androgen (Kokontis et al 2005). Unlike 104-R1 cells, most CDXR cells grown in 10 nM R1881 underwent apoptosis 6 to 8 days after R1881 exposure. However, 1 in 1.9x103 cells relapsed as androgen-insensitive that were not repressed by R1881 or Casodex. These sublines,

**3. Androgenic suppression of advanced prostate cancer cells** 

Feldman 2001).

**3.1 Androgenic suppression** *in vitro*

androgen (Kokontis et al 1994, Kokontis et al 1998).

but is stimulated by 10 nM R1881.

androgen blockade was associated with increased risk of incident coronary heart disease (Keating et al 2010). Orchiectomy was associated with coronary heart disease and myocardial infarction (Keating et al 2010). Therefore, shortening the period of androgen ablation therapy may protect the patients.

Liver X receptors (LXRs) are ligand-activated transcriptional factors that belong to the nuclear receptor superfamily. LXRs are important regulators of cholesterol, fatty acid, and glucose homeostasis (Chuu et al 2007). There are two LXR isoforms. LXR expression is most abundant in liver, kidney, intestine, fat tissue, macrophages, lung, and spleen, while LXR is ubiquitously expressed (Chuu et al 2007, Edwards et al 2002, Willy et al 1995). A specific group of oxysterols are natural ligands for LXRs (Chuu et al 2007, Forman et al 1997, Janowski et al 1996). LXR agonists are effective for treatment of murine models of atherosclerosis, diabetes, and Alzheimer's disease (Alberti et al 2001, Blaschke et al 2004, Cao et al 2003, Chuu et al 2007, Edwards et al 2002, Efanov et al 2004, Joseph et al 2002, Joseph et al 2003, Koldamova et al 2005, Peet et al 1998, Song et al 2001, Song and Liao 2001). Our and other groups' previous studies suggested that androgen and LXR agonists may suppress tumor growth of hormone-refractory prostate cancer cells (Chuu et al 2006, Chuu et al 2007, Chuu and Lin 2010, Fukuchi et al 2004b). We thus discuss the possibility of manipulating androgen/androgen receptor (AR) signaling and LXR signaling as a treatment for advanced prostate cancers.

#### **2. Androgens and androgen receptor in prostate cancer**

Androgens include testosterone, dehydroepiandrosterone, androstenedione, androstenediol, androsterone, and dihydrotestosterone (DHT). Androgens are mainly produced by testes, while the rest amount of androgens are produced from the adrenal glands. Androgens are important for growth and survival of the prostate cells. Testosterone is the main circulating androgen in human body, while DHT is the more potent androgen (Anderson and Liao 1968, Kokontis and Liao 1999, Liang and Liao 1992). 90% of the free testosterone enters prostate cells is converted to dihydrotestosterone (DHT) by the enzyme 5-reductase (Liang and Liao 1992). The average serum testosterone level declines with age from approximately 620-670 ng/dl at age 25-44 to 470-520 ng/dl at age 65-84 (Vermeulen 1996). Low serum testosterone level was associated with an increased risk of prostate cancer (Morgentaler and Rhoden 2006), and prostate tumors arising in a low testosterone environment appeared to be more aggressive (Hoffman et al 2000, Lane et al 2008), suggesting a potential therapeutic role for androgen in advanced prostate cancer treatment.

Androgen receptor (AR) is an androgen-activated transcription factor and belongs to the steroid nuclear receptor family. AR is composed of an N-terminal domain, a central DNAbinding domain, and a C-terminal ligand-binding domain (Chang et al 1988a, Chang et al 1988b, Feldman and Feldman 2001). After binding ligand DHT, AR dissociates from heatshock proteins, phosphorylates, dimerizes, transocates into the nucleus, and binds to androgen-response elements (ARE) in the promoter regions of its target genes under the regulation of co-activators and co-repressors (Feldman and Feldman 2001). Target genes of AR regulate growth, survival, and the production of prostate-specific antigen (PSA) in prostate cells.

Gene microarray study of seven different human prostate cancer xenograft models demonstrated that increase of AR mRNA is the only change consistently associated with the development of androgen-independency phenotype following androgen ablation therapy, and elevation of AR mRNA and protein are both necessary and sufficient progression of prostate cancer towards androgen-independency (Culig et al 1999, Joly-Pharaboz et al 1995). Elevated AR expression in androgen-independent prostate cancer cells or recurrent hormone-refractory tumors has been observed in our progression model (Chuu et al 2005, Chuu et al 2006, Kokontis et al 1994, Kokontis et al 1998, Kokontis et al 2005, Umekita et al 1996) and several other groups (Chen et al 2004a, de Vere White et al 1997, Edwards et al 2003, Ford et al 2003, Gregory et al 2001, Hara et al 2003, Holzbeierlein et al 2004, Kim et al 2002, Linja et al 2001, Shi et al 2004, Singh et al 2004, Visakorpi et al 1995, Wang et al 2001, Zhang et al 2003). Mechanisms contribute to the progression towards androgenindependency including AR gene amplification, AR mutation, bypass of androgenic activation of AR, or bypass AR signaling for cell survival and proliferation (Feldman and Feldman 2001).

#### **3. Androgenic suppression of advanced prostate cancer cells**

#### **3.1 Androgenic suppression** *in vitro*

208 Prostate Cancer – Original Scientific Reports and Case Studies

androgen blockade was associated with increased risk of incident coronary heart disease (Keating et al 2010). Orchiectomy was associated with coronary heart disease and myocardial infarction (Keating et al 2010). Therefore, shortening the period of androgen

Liver X receptors (LXRs) are ligand-activated transcriptional factors that belong to the nuclear receptor superfamily. LXRs are important regulators of cholesterol, fatty acid, and glucose homeostasis (Chuu et al 2007). There are two LXR isoforms. LXR expression is most abundant in liver, kidney, intestine, fat tissue, macrophages, lung, and spleen, while LXR is ubiquitously expressed (Chuu et al 2007, Edwards et al 2002, Willy et al 1995). A specific group of oxysterols are natural ligands for LXRs (Chuu et al 2007, Forman et al 1997, Janowski et al 1996). LXR agonists are effective for treatment of murine models of atherosclerosis, diabetes, and Alzheimer's disease (Alberti et al 2001, Blaschke et al 2004, Cao et al 2003, Chuu et al 2007, Edwards et al 2002, Efanov et al 2004, Joseph et al 2002, Joseph et al 2003, Koldamova et al 2005, Peet et al 1998, Song et al 2001, Song and Liao 2001). Our and other groups' previous studies suggested that androgen and LXR agonists may suppress tumor growth of hormone-refractory prostate cancer cells (Chuu et al 2006, Chuu et al 2007, Chuu and Lin 2010, Fukuchi et al 2004b). We thus discuss the possibility of manipulating androgen/androgen receptor (AR) signaling and LXR signaling as a treatment for advanced

Androgens include testosterone, dehydroepiandrosterone, androstenedione, androstenediol, androsterone, and dihydrotestosterone (DHT). Androgens are mainly produced by testes, while the rest amount of androgens are produced from the adrenal glands. Androgens are important for growth and survival of the prostate cells. Testosterone is the main circulating androgen in human body, while DHT is the more potent androgen (Anderson and Liao 1968, Kokontis and Liao 1999, Liang and Liao 1992). 90% of the free testosterone enters prostate cells is converted to dihydrotestosterone (DHT) by the enzyme 5-reductase (Liang and Liao 1992). The average serum testosterone level declines with age from approximately 620-670 ng/dl at age 25-44 to 470-520 ng/dl at age 65-84 (Vermeulen 1996). Low serum testosterone level was associated with an increased risk of prostate cancer (Morgentaler and Rhoden 2006), and prostate tumors arising in a low testosterone environment appeared to be more aggressive (Hoffman et al 2000, Lane et al 2008), suggesting a potential therapeutic

Androgen receptor (AR) is an androgen-activated transcription factor and belongs to the steroid nuclear receptor family. AR is composed of an N-terminal domain, a central DNAbinding domain, and a C-terminal ligand-binding domain (Chang et al 1988a, Chang et al 1988b, Feldman and Feldman 2001). After binding ligand DHT, AR dissociates from heatshock proteins, phosphorylates, dimerizes, transocates into the nucleus, and binds to androgen-response elements (ARE) in the promoter regions of its target genes under the regulation of co-activators and co-repressors (Feldman and Feldman 2001). Target genes of AR regulate growth, survival, and the production of prostate-specific antigen (PSA) in

Gene microarray study of seven different human prostate cancer xenograft models demonstrated that increase of AR mRNA is the only change consistently associated with the

ablation therapy may protect the patients.

**2. Androgens and androgen receptor in prostate cancer** 

role for androgen in advanced prostate cancer treatment.

prostate cancers.

prostate cells.

LNCaP is one of the most commonly used cell line for prostate cancer research, which was derived from a human lymph node metastatic lesion of prostate adenocarcinoma (Chuu et al 2007, Horoszewicz et al 1980). LNCaP expressed AR and inducible PSA. Previously, we cultured androgen-sensitive LNCaP 104-S cells in androgen-depleted conditions *in vitro* to establish relapsed hormone-refractory prostate cancer cells mimic clinical situation in which prostate cancer recurs during androgen deprivation (Kokontis et al 1994a, Kokontis et al 1998b). After 3 months in medium depleted with androgens, most LNCaP 104-S cells underwent G1 cell cycle arrest and apoptosis. A few colonies of cells, named 104-I cells, evolved that proliferated very slowly in the absence of androgen (Kokontis et al 1994). After approximately 11 months, cells called 104-R1 cells emerged that grew much more rapidly in the absence of androgen. After 20 months, 104-R2 cells evolved which proliferated in the absence of androgen at a rate comparable to the proliferation rate of 104-S cells grown in androgen (Kokontis et al 1994, Kokontis et al 1998).

During the transition of 104-S cells to 104-R1 and 104-R2 cells, AR mRNA and protein level elevated several folds (Chuu et al 2005, Chuu et al 2006, Kokontis et al 1994, Kokontis et al 1998). Proliferation of 104-R1 and 104-R2 cells is androgen-independent but is unexpectedly suppressed by physiological concentrations of androgen both *in vitro* and *in vivo* (Chuu et al 2005, Chuu et al 2006, Kokontis et al 1994, Kokontis et al 1998b, Kokontis et al 2005, Umekita et al 1996). When 104-R1 cells were incubated for several weeks in a high concentration of synthetic androgen R1881 (20 nM), cells named R1Ad adapted after a period of growth arrest (Kokontis et al 1998). Growth of R1Ad cells is slow and not dependent on androgen but is stimulated by 10 nM R1881.

To further mimic the clinical situation of combined androgen deprivation and anti-androgen therapy, LNCaP 104-S cells were incubated with 5 M Casodex (biculatimide) in androgendeprived medium. After four weeks, Casodex-resistant colonies appeared at low frequency (1 in 1.4x105) as most of the cells appeared to undergo senescent cell death. The relapsed cells, called CDXR, had increased AR expression and were repressed by androgen (Kokontis et al 2005). Unlike 104-R1 cells, most CDXR cells grown in 10 nM R1881 underwent apoptosis 6 to 8 days after R1881 exposure. However, 1 in 1.9x103 cells relapsed as androgen-insensitive that were not repressed by R1881 or Casodex. These sublines,

Inhibition of Advanced Prostate Cancer by Androgens and Liver X Receptor Agonists 211

expression of AR (Chuu et al 2006). Low serum level of testosterone (130 60 ng/dl), stop tumor growth of 104-Rrel tumors but tumor growth resumed in 4 weeks. High serum level of testosterone (2970 495 ng/dl), which is approximately 5-fold higher than normal level, caused regression of 104-Rrel tumors growth. However, all 104-Rrel cells adapted to androgen and relapsed after 4 weeks as androgen-stimulated 104-Radp tumors (Chuu et al 2006) (Figure 2). Growth of the LNCaP 104-R1 tumors was also suppressed by androgen, but all tumors adapted to androgenic suppression and relapsed as androgen-stimulated R1Ad tumors in 5-6 weeks (Chuu et al 2005). Growth of R1Ad tumors was stimulated by testosterone and removal of testosterone totally stopped the tumor growth (Chuu et al 2005, Chuu et al 2006). Both 104-Radp and R1Ad tumors express very little AR and PSA mRNA and protein or serum PSA level (Figure 2), similar to R1Ad cells observed in cell culture

Fig. 2. Progression of androgen-dependent LNCaP 104-S tumors to androgen-independent 104-Rrel tumors, and androgenic growth suppression of 104-Rrel tumors. (A) Mice were injected subcutaneously with androgen-dependent 104-S cells. After allowing tumors to grow for 7 weeks, mice were separated into control (filled circles, 14 mice with 19 tumors) and castration groups (open circles, 24 mice with 36 tumors) and the time was designated as week 1 (Chuu et al 2006). (B) Mice in the castrated group in (A) at the 14th week were

separated into 3 groups including a control group (open circles, 6 mice with 9 tumors), a low dosage testosterone treatment group that received a subcutaneous implant of a 20 mg TP/cholesterol (1:9) pellet (filled squares, 9 mice with 12 tumors), and a high-dosage

testosterone treatment group that received a subcutaneous implant of a 20 mg pure TP pellet (filled circles, 10 mice with 12 tumors) (Chuu et al 2006). Tumor volumes are expressed as the mean + standard error. (C) PSA, AR, and actin protein levels in 104-S tumor (in intact mice), 104-Rrel-T tumors, 104-Radp-1+T tumors, and 104Radp-T were assayed by Western blot (Chuu et al 2005). (D) Serum PSA level of mice with 104-S tumors (in intact mice), 104- Rrel-T tumors, 104-Rrel+T tumors, Radp+T tumors, Radp-T tumors was determined by Elisa

Both early and late treatment of androgen caused regression of CDXR3 tumors. 70% of tumors regress completely and the rest of tumors relapse after 60-90 days of treatment (Kokontis et al 2005) (Figure 3). The relapsed tumors show diminished expression of AR and no longer require androgen for growth, essentially identical to the behavior of IS3 cells that emerged after androgen exposure *in vitro* (Kokontis et al 2005). It is worthwhile to notice that 100% of 104-R1 tumor being treated with testosterone relapsed in 4-5 weeks, while only 30% of CDXR tumors relapsed after 9-13 weeks after testosterone treatment (Chuu et al

(Chuu et al 2005, Chuu et al 2006, Kokontis et al 1998).

kit (Chuu et al 2005).

designated IS, showed greatly reduced AR expression (Kokontis et al 2005). Growth of IS cells was not stimulated by R1881 or suppressed by Casodex. 104-R2 cells, like CDXR cells, gave rise to androgen-insensitive cells after androgen treatment (unpublished data). Therefore, during progression from 104-R1 to 104-R2 stages, the cells appear to pass a point where cells can no longer recover responsiveness to androgen, but instead progress to androgen insensitivity (Liao et al 2005). Direct progression of 104-S cells to the CDXR stage by selection in androgen-depleted medium containing anti-androgen seems to bypass this intermediate 104-R1 stage. Androgen-suppressive phenotype and elevated AR of hormonerefractory LNCaP cells was observed by several other groups (Culig et al 1999, Joly-Pharaboz et al 1995, Joly-Pharaboz et al 2000, Shi et al 2004, Soto et al 1995). The progression model of LNCaP is shown in Figure 1.

Fig. 1. The LNCaP cell line progression model. (A) AR expression level increases during the progression from androgen-dependent LNCaP 104-S cells to androgen-independent 104-R1, 104-R2, and CDXR cells. Proliferation of LNCaP 104-R1, 104-R2, CDXR cells are suppressed by androgen, but these cells can adapt to androgenic suppression and evolve as R1Ad, R2Ad, and IS cells. R1Ad, R2Ad, and IS cells express very little AR. (B) Effect of 96 h treatment of synthetic androgen (0, 0.1, 1, 10 nM) R1881 on 104-S, 104-R1, 104-R2, CDXR, R1Ad, R2Ad, IS cells was assayed by 96-well proliferation assay.

LNCaP cells express a mutant AR (T877A) that displays relaxed ligand binding specificity (Kokontis et al 1991, Veldscholte et al 1990), however, androgenic suppression is not limited to LNCaP cells. ARCaP is an AR-positive, tumorigenic, and highly metastatic cell line derived from the ascites fluid of a patient with advanced metastatic disease. Proliferation of ARCaP cells is suppressed by androgen (Zhau et al 1996). MDA PCa 2b-hr was generated *in vitro* from bone metastasis-derived, androgen-dependent MDA PCa 2b human PC cells with higher AR proteins. Proliferation of MDA PCa 2b-hr was inhibited by testosterone concentration higher than 3.5 nM or Casodex (Hara et al 2003). PC-3 is a commonly used human prostate cancer cell lines established from bone-derived metastases with no AR expression (Chuu et al 2007). Physiological concentration of DHT caused growth inhibition, G1 cell cycle arrest, and apoptosis in PC-3 cells over-expressing full length wild-type AR (Heisler et al 1997, Litvinov et al 2004, Yuan et al 1993).

#### **3.2 Androgenic suppression** *in vivo*

Castration causes regression of 104-S xenografts but tumor relapsed after 8 weeks as androgen-independent relapsed tumors 104-Rrel with elevated mRNA and protein

designated IS, showed greatly reduced AR expression (Kokontis et al 2005). Growth of IS cells was not stimulated by R1881 or suppressed by Casodex. 104-R2 cells, like CDXR cells, gave rise to androgen-insensitive cells after androgen treatment (unpublished data). Therefore, during progression from 104-R1 to 104-R2 stages, the cells appear to pass a point where cells can no longer recover responsiveness to androgen, but instead progress to androgen insensitivity (Liao et al 2005). Direct progression of 104-S cells to the CDXR stage by selection in androgen-depleted medium containing anti-androgen seems to bypass this intermediate 104-R1 stage. Androgen-suppressive phenotype and elevated AR of hormonerefractory LNCaP cells was observed by several other groups (Culig et al 1999, Joly-Pharaboz et al 1995, Joly-Pharaboz et al 2000, Shi et al 2004, Soto et al 1995). The progression

Fig. 1. The LNCaP cell line progression model. (A) AR expression level increases during the progression from androgen-dependent LNCaP 104-S cells to androgen-independent 104-R1, 104-R2, and CDXR cells. Proliferation of LNCaP 104-R1, 104-R2, CDXR cells are suppressed by androgen, but these cells can adapt to androgenic suppression and evolve as R1Ad, R2Ad, and IS cells. R1Ad, R2Ad, and IS cells express very little AR. (B) Effect of 96 h treatment of synthetic androgen (0, 0.1, 1, 10 nM) R1881 on 104-S, 104-R1, 104-R2, CDXR,

LNCaP cells express a mutant AR (T877A) that displays relaxed ligand binding specificity (Kokontis et al 1991, Veldscholte et al 1990), however, androgenic suppression is not limited to LNCaP cells. ARCaP is an AR-positive, tumorigenic, and highly metastatic cell line derived from the ascites fluid of a patient with advanced metastatic disease. Proliferation of ARCaP cells is suppressed by androgen (Zhau et al 1996). MDA PCa 2b-hr was generated *in vitro* from bone metastasis-derived, androgen-dependent MDA PCa 2b human PC cells with higher AR proteins. Proliferation of MDA PCa 2b-hr was inhibited by testosterone concentration higher than 3.5 nM or Casodex (Hara et al 2003). PC-3 is a commonly used human prostate cancer cell lines established from bone-derived metastases with no AR expression (Chuu et al 2007). Physiological concentration of DHT caused growth inhibition, G1 cell cycle arrest, and apoptosis in PC-3 cells over-expressing full length wild-type AR

Castration causes regression of 104-S xenografts but tumor relapsed after 8 weeks as androgen-independent relapsed tumors 104-Rrel with elevated mRNA and protein

R1Ad, R2Ad, IS cells was assayed by 96-well proliferation assay.

(Heisler et al 1997, Litvinov et al 2004, Yuan et al 1993).

**3.2 Androgenic suppression** *in vivo*

model of LNCaP is shown in Figure 1.

expression of AR (Chuu et al 2006). Low serum level of testosterone (130 60 ng/dl), stop tumor growth of 104-Rrel tumors but tumor growth resumed in 4 weeks. High serum level of testosterone (2970 495 ng/dl), which is approximately 5-fold higher than normal level, caused regression of 104-Rrel tumors growth. However, all 104-Rrel cells adapted to androgen and relapsed after 4 weeks as androgen-stimulated 104-Radp tumors (Chuu et al 2006) (Figure 2). Growth of the LNCaP 104-R1 tumors was also suppressed by androgen, but all tumors adapted to androgenic suppression and relapsed as androgen-stimulated R1Ad tumors in 5-6 weeks (Chuu et al 2005). Growth of R1Ad tumors was stimulated by testosterone and removal of testosterone totally stopped the tumor growth (Chuu et al 2005, Chuu et al 2006). Both 104-Radp and R1Ad tumors express very little AR and PSA mRNA and protein or serum PSA level (Figure 2), similar to R1Ad cells observed in cell culture (Chuu et al 2005, Chuu et al 2006, Kokontis et al 1998).

Fig. 2. Progression of androgen-dependent LNCaP 104-S tumors to androgen-independent 104-Rrel tumors, and androgenic growth suppression of 104-Rrel tumors. (A) Mice were injected subcutaneously with androgen-dependent 104-S cells. After allowing tumors to grow for 7 weeks, mice were separated into control (filled circles, 14 mice with 19 tumors) and castration groups (open circles, 24 mice with 36 tumors) and the time was designated as week 1 (Chuu et al 2006). (B) Mice in the castrated group in (A) at the 14th week were separated into 3 groups including a control group (open circles, 6 mice with 9 tumors), a low dosage testosterone treatment group that received a subcutaneous implant of a 20 mg TP/cholesterol (1:9) pellet (filled squares, 9 mice with 12 tumors), and a high-dosage testosterone treatment group that received a subcutaneous implant of a 20 mg pure TP pellet (filled circles, 10 mice with 12 tumors) (Chuu et al 2006). Tumor volumes are expressed as the mean + standard error. (C) PSA, AR, and actin protein levels in 104-S tumor (in intact mice), 104-Rrel-T tumors, 104-Radp-1+T tumors, and 104Radp-T were assayed by Western blot (Chuu et al 2005). (D) Serum PSA level of mice with 104-S tumors (in intact mice), 104- Rrel-T tumors, 104-Rrel+T tumors, Radp+T tumors, Radp-T tumors was determined by Elisa kit (Chuu et al 2005).

Both early and late treatment of androgen caused regression of CDXR3 tumors. 70% of tumors regress completely and the rest of tumors relapse after 60-90 days of treatment (Kokontis et al 2005) (Figure 3). The relapsed tumors show diminished expression of AR and no longer require androgen for growth, essentially identical to the behavior of IS3 cells that emerged after androgen exposure *in vitro* (Kokontis et al 2005). It is worthwhile to notice that 100% of 104-R1 tumor being treated with testosterone relapsed in 4-5 weeks, while only 30% of CDXR tumors relapsed after 9-13 weeks after testosterone treatment (Chuu et al

Inhibition of Advanced Prostate Cancer by Androgens and Liver X Receptor Agonists 213

R1 growth (Kokontis et al 1994). Therefore, androgen regulate cell cycle and proliferation of

Fig. 4. Effect of androgen on cell proliferation, cell cycle, and cell cycle-related proteins in androgen-dependent 104-S and androgen-independent 104-R1 cells. (A) LNCaP 104-S and 104-R2 cells were treated with increasing concentration of synthetic androgen R1881 for 96 hours. Relative cell number was determined by 96-well proliferation assay and was

normalized to cell number of 104-S cells at 0.1 nM R1881. (B) Percentage of 104-S and 104-R1 cells in S phase determined by flow cytometry. LNCaP 104-S and 104-R2 cells were treated with increasing concentration of synthetic androgen R1881 for 96 hours. Values represent the mean +/- Standard Error derived from 5 independent experiments. (C) Protein expression of androgen receptor (AR), prostate specific antigen (PSA), p21cip, p27Kip, retinoblastoma protein (Rb), c-myc, S phase kinase-associated protein 2 (Skp2) were determined by Western bloting assay in 104-S and 104-R1 cells treated 96 hrs with different

Clinical and basic studies showed that in comparison with continuous androgen ablation (CAB) therapy, intermittent androgen suppression (IAS) therapy substantially prolongs the time to development of castration-resistant prostate cancer (Akakura et al 1993, Mathew 2008, Sato et al 1996, Szmulewitz et al 2009). Intermittent androgen ablation therapy is a strategy to periodically perform and terminate the androgen ablation therapy, allowing the endogenous testosterone level to elevate during the period between ablation therapies. IAS therapy delayed the androgen-independent progression of Shionogi mammary carcinoma (Akakura et al 1993) and LNCaP xenograft (Sato et al 1996). Pether et al. reported in a clinical trial of 102 patients that there is a trend toward extended times to progression and death compared to CAB treatment, and growth of advanced prostate tumors was delayed in ~50% patients treated with IAS (Pether et al 2003). Bruchovsky et al. showed that IAS

concentration of R1881. -actin was used as loading control.

**4. Androgen treatment of advanced prostate cancer in clinical** 

LNCaP cells via AR, Skp2, c-Myc, and p27Kip1.

2005, Kokontis et al 2005). This is probably due to the apoptosis induced in CDXR cells but not in 104-R1 cells by androgen (Kokontis et al 1998, Kokontis et al 2005). Regression and relapse after androgen treatment of LNCaP xenograft was also observed by other group (Joly-Pharaboz et al 2000) and ARCaP xenograft (Zhau et al 1996).

Fig. 3. Regression and relapse of LNCaP CDXR-3 tumor xenografts in nude mice treated with testosterone (A) LNCaP CDXR-3 tumor xenografts in castrated male nude mice were allowed to grow until they reached an average volume of 400 mm3 on the 38th day. All mice carrying tumors received a subcutaneous implant of a 20mg testosterone. The mice in the control group were implanted with a 20 mg testosterone pellet either at early stage (50 days after inoculation) or late stage (92 days after inoculation). Open triangle represent tumors relapsed, while open squares represent tumors disappeared after androgen treatment. Tumor volumes are expressed as the mean standard error.

#### **3.3 Molecular mechanism of androgenic suppression**

Antiandrogen Casodex (bicalutamide) does not affect proliferation of 104-R1 and 104-R2 cells but blocked androgenic repression of growth as well as androgenic induction of PSA (Kokontis et al 1998). Knockdown of AR expression in CDXR3 cells by shRNA relieved androgenic repression of growth (Kokontis et al 2005). Retroviral overexpression of AR in IS cells restored the androgen-repressed phenotype in these cells (Kokontis et al 2005). These observations confirmed that androgen cause growth inhibition via AR.

Synthetic androgen R1881 increases S phase population in androgen-dependent LNCaP 104- S cells but induces G1 arrest in androgen-independent LNCaP cells (such as 104-R1m 104- R2, CDXR, etc.) within 24 hours of treatment (Joly-Pharaboz et al 2000, Kokontis et al 1994, Kokontis et al 1998, Kokontis et al 2005, Soto et al 1995) (Figure 4). Cell cycle inhibitors p21waf1/cip1 and p27Kip1 were induced by androgen in 104-R1 and 104-R2 cells (Kokontis et al 1998a) (Figure 4). In contrast, expression of p21waf1/cip1 and p27Kip1 was repressed by androgen in 104-S cells. Androgen down-regulates F-box protein S phase kinase-associated protein 2 (Skp2), a protein mediating the ubiquitination and degradation of p27Kip1. Androgen also decreases c-Myc at the protein and mRNA level in hours in 104-R1 cells (Figure 5). Enforced retroviral overexpression of c-Myc blocks androgenic repression of 104-

2005, Kokontis et al 2005). This is probably due to the apoptosis induced in CDXR cells but not in 104-R1 cells by androgen (Kokontis et al 1998, Kokontis et al 2005). Regression and relapse after androgen treatment of LNCaP xenograft was also observed by other group

Fig. 3. Regression and relapse of LNCaP CDXR-3 tumor xenografts in nude mice treated with testosterone (A) LNCaP CDXR-3 tumor xenografts in castrated male nude mice were allowed to grow until they reached an average volume of 400 mm3 on the 38th day. All mice carrying tumors received a subcutaneous implant of a 20mg testosterone. The mice in the control group were implanted with a 20 mg testosterone pellet either at early stage (50 days after inoculation) or late stage (92 days after inoculation). Open triangle represent tumors relapsed, while open squares represent tumors disappeared after androgen treatment.

Antiandrogen Casodex (bicalutamide) does not affect proliferation of 104-R1 and 104-R2 cells but blocked androgenic repression of growth as well as androgenic induction of PSA (Kokontis et al 1998). Knockdown of AR expression in CDXR3 cells by shRNA relieved androgenic repression of growth (Kokontis et al 2005). Retroviral overexpression of AR in IS cells restored the androgen-repressed phenotype in these cells (Kokontis et al 2005). These

Synthetic androgen R1881 increases S phase population in androgen-dependent LNCaP 104- S cells but induces G1 arrest in androgen-independent LNCaP cells (such as 104-R1m 104- R2, CDXR, etc.) within 24 hours of treatment (Joly-Pharaboz et al 2000, Kokontis et al 1994, Kokontis et al 1998, Kokontis et al 2005, Soto et al 1995) (Figure 4). Cell cycle inhibitors p21waf1/cip1 and p27Kip1 were induced by androgen in 104-R1 and 104-R2 cells (Kokontis et al 1998a) (Figure 4). In contrast, expression of p21waf1/cip1 and p27Kip1 was repressed by androgen in 104-S cells. Androgen down-regulates F-box protein S phase kinase-associated protein 2 (Skp2), a protein mediating the ubiquitination and degradation of p27Kip1. Androgen also decreases c-Myc at the protein and mRNA level in hours in 104-R1 cells (Figure 5). Enforced retroviral overexpression of c-Myc blocks androgenic repression of 104-

(Joly-Pharaboz et al 2000) and ARCaP xenograft (Zhau et al 1996).

Tumor volumes are expressed as the mean standard error.

observations confirmed that androgen cause growth inhibition via AR.

**3.3 Molecular mechanism of androgenic suppression** 

R1 growth (Kokontis et al 1994). Therefore, androgen regulate cell cycle and proliferation of LNCaP cells via AR, Skp2, c-Myc, and p27Kip1.

Fig. 4. Effect of androgen on cell proliferation, cell cycle, and cell cycle-related proteins in androgen-dependent 104-S and androgen-independent 104-R1 cells. (A) LNCaP 104-S and 104-R2 cells were treated with increasing concentration of synthetic androgen R1881 for 96 hours. Relative cell number was determined by 96-well proliferation assay and was normalized to cell number of 104-S cells at 0.1 nM R1881. (B) Percentage of 104-S and 104-R1 cells in S phase determined by flow cytometry. LNCaP 104-S and 104-R2 cells were treated with increasing concentration of synthetic androgen R1881 for 96 hours. Values represent the mean +/- Standard Error derived from 5 independent experiments. (C) Protein expression of androgen receptor (AR), prostate specific antigen (PSA), p21cip, p27Kip, retinoblastoma protein (Rb), c-myc, S phase kinase-associated protein 2 (Skp2) were determined by Western bloting assay in 104-S and 104-R1 cells treated 96 hrs with different concentration of R1881. -actin was used as loading control.

#### **4. Androgen treatment of advanced prostate cancer in clinical**

Clinical and basic studies showed that in comparison with continuous androgen ablation (CAB) therapy, intermittent androgen suppression (IAS) therapy substantially prolongs the time to development of castration-resistant prostate cancer (Akakura et al 1993, Mathew 2008, Sato et al 1996, Szmulewitz et al 2009). Intermittent androgen ablation therapy is a strategy to periodically perform and terminate the androgen ablation therapy, allowing the endogenous testosterone level to elevate during the period between ablation therapies. IAS therapy delayed the androgen-independent progression of Shionogi mammary carcinoma (Akakura et al 1993) and LNCaP xenograft (Sato et al 1996). Pether et al. reported in a clinical trial of 102 patients that there is a trend toward extended times to progression and death compared to CAB treatment, and growth of advanced prostate tumors was delayed in ~50% patients treated with IAS (Pether et al 2003). Bruchovsky et al. showed that IAS

Inhibition of Advanced Prostate Cancer by Androgens and Liver X Receptor Agonists 215

et al 1996). A few synthetic LXR agonists have been developed, including non-steroidal LXR agonists T0901317 (Schultz et al 2000) and GW3965 (Collins et al 2002), and steroidal LXR

LXRs are important regulators of cholesterol, fatty acid, and glucose homeostasis (Chuu et al 2007). Oral administration of an LXR agonist has an overall hypolipidemic effect in hypercholesterolemic rats, mice, and hamsters (Song and Liao 2001). LXR-/- mice are healthy when fed with a low-cholesterol diet. However, LXR-/- mice develop enlarged fatty livers, hepatocellular degeneration, high hepatic cholesterol levels, and impaired liver function when fed a high-cholesterol diet (Alberti et al 2001, Edwards et al 2002, Peet et al 1998). LXRβ-/- mice are unaffected by a high-cholesterol diet, suggesting that LXR and LXR have separate roles. LXR and LXR regulate cholesterol transport. LXRs induces expression of the cholesterol transporters ATP-binding cassette transporter A1 and G1 (ABCA1 and ABCG1) (Edwards et al 2002, Nakamura et al 2004, Venkateswaran et al 2000) as well as cholesterol acceptor apolipoprotein E (ApoE) (Chawla et al 2001). Treatment with LXR agonists (hypocholamide, T0901317, or GW3965) lowers the cholesterol level in serum and liver and inhibits the development of atherosclerosis in murine disease models

LXRs regulate fatty acid synthesis by modulating the expression of sterol regulatory element-binding protein-1c (SREBP-1c) (Repa et al 2000, Yoshikawa et al 2001) and downstream lipogenic genes, including acetyl CoA carboxylase and FAS (Liang et al 2002). LXRs also regulate insulin signaling in liver (Chen et al 2004b, Tobin et al 2002). LXR-/- LXR-/- double knockout mice lack insulin-mediated induction of an entire class of enzymes involved in both fatty acid and cholesterol metabolism (Tobin et al 2002). Treatment with T0901317 stimulates insulin secretion in pancreatic beta cells, reduces plasma glucose, and improves glucose tolerance and insulin resistance in murine and rat

LXR signaling is important for brain function as well. LXRs regulate lipid homeostasis in the brain. LXR-/- LXR-/- mice develop neurodegenerative changes in brain tissue (Wang et al 2002). Knockout of LXR, but not LXR, results in adult-onset motor neuron degeneration in male mice (Andersson et al 2005), suggesting a different role of LXR from LXR. Treatment with T0901317 decreases amyloidal beta production in an Alzheimer's disease

Based on our recent observations using several prostate cancer cell lines, we discovered that LXR agonists suppress proliferation of human prostate cancer cell lines. Treatment of PC-3, DU-145, and LNCaP sublines (104-S, 104-R1, 104-R2, CDXR, R1Ad, IS) cells with LXR agonists (22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, or T0901317) suppresses the proliferation of these cells (Chuu and Lin 2010, Fukuchi et al 2004b, Vigushin et al 2004). LXR agonists treatment causes growth inhibition in prostate cancer cells via induction of G1 cell cycle arrest (Chuu and Lin 2010, Fukuchi et al 2004b). T0901317 decreases the percentage of cells in S-phase and increases the percentage of cells in G1-phase. T0901317 suppresses

agonists hypocholamide (Song and Liao 2001) and YT-32 (Kaneko et al 2003)].

(Blaschke et al 2004, Joseph et al 2002, Song et al 2001, Song and Liao 2001).

obesity models (Cao et al 2003, Efanov et al 2004, Joseph et al 2003).

**6.1 Anti-proliferative effect of LXR agonists in cancer cells** 

mouse model (Koldamova et al 2005).

**6. Anti-cancer effect of LXR agonists** 

**5.2 Role of LXR signaling in metabolism** 

therapy cause repeated differentiation of tumor with recovery of apoptotic potential, inhibition of tumor growth by rapid restoration of serum testosterone, and restraint of tumor growth by subnormal levels of serum testosterone (Bruchovsky et al 2000). They concluded that IAS is a viable treatment option for men with prostate cancer which affords an improved quality of life as well as reduced toxicity and costs (Bruchovsky et al 2000, Morris et al 2009, Pether et al 2003).

A few studies have shown that androgen is safe and potentially effective for treatment of advanced prostate cancer. Mathew reported that the testosterone level in a prostate cancer patient undergone radical prostatectomy and LH-RH therapy remained at castrated levels and serum PSA was undetectable for 15 years. PSA levels then began to rise and the patient was given testosterone replacement therapy to attain a normal range of serum testosterone. After an initial flare, PSA levels gradually declined over 18 months. After 27 months, PSA level started to increase. When testosterone replacement therapy was discontinued, PSA levels dropped (Mathew 2008). The observation was similar to the transition from 104-R1 to R1Ad phenotype under androgen treatment in our LNCaP progression model (Chuu et al 2005, Kokontis et al 1998). Szmulewitz et al. reported that 15 prostate cancer patients with progressive disease following androgen ablation, anti-androgen therapy, and withdrawal without minimal metastatic disease were randomized to treatment with three doses of transdermal testosterone of 2.5, 5.0, or 7.5 mg/day, resulting in increase of serum testosterone concentrations to 305 ng/dl, 308 ng/dl, and 297 ng/dl, respectively. The conclusion of this study is that testosterone is a feasible and reasonably well-tolerated therapy for men with early hormone-refractory prostate cancer (Szmulewitz et al 2009). Morris et el. performed a phase 1 clinical trial to determine the safety of high-dose exogenous testosterone in patients with castration-resistant metastatic prostate cancer. Cohorts of 3-6 patients with progressive castration-resistant prostate cancer who had been castrated for at least 1 yr received testosterone by skin patch or topical gel for 1 week, 1 month, or until disease progression. No adverse effect was reported. The serum testosterone ranged from 330-870 ng/dl (Morris et al 2009). This study suggested that patients with advanced prostate cancer can be safely treated with exogenous testosterone. Researchers suggested that maximizing testosterone serum levels in selected patients with androgen receptor over-expression may improve the treatment outcome.

#### **5. Liver X receptor (LXR) signaling**

#### **5.1 LXR and LXR**

Liver X receptors are ligand-activated transcriptional factors that belong to the nuclear receptor superfamily. There are two LXR isoforms, LXR and LXR (Chuu et al 2007). Although LXR and LXR share high similarity in their DNA- and ligand-binding domains, expression of these proteins in various tissues differs. LXR expression is restricted to liver, kidney, intestine, fat tissue, macrophages, lung, and spleen (Edwards et al 2002, Willy et al 1995). LXR is ubiquitously expressed (Song et al 1994). LXR and LXR form heterodimers with the obligate partner 9-cis retinoic acid receptor (RXR) (Chuu et al 2007, Song et al 1994, Willy et al 1995). The LXR/RXR heterodimer can be activated with either an LXR agonist (oxysterols) or a RXR agonist (cis-retinoic acid). Oxysterols are oxygenated derivatives of cholesterol. Oxysterols, such as 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and cholestenoic acid, are natural ligands for LXR (Chuu et al 2007, Forman et al 1997, Janowski et al 1996). A few synthetic LXR agonists have been developed, including non-steroidal LXR agonists T0901317 (Schultz et al 2000) and GW3965 (Collins et al 2002), and steroidal LXR agonists hypocholamide (Song and Liao 2001) and YT-32 (Kaneko et al 2003)].

#### **5.2 Role of LXR signaling in metabolism**

214 Prostate Cancer – Original Scientific Reports and Case Studies

therapy cause repeated differentiation of tumor with recovery of apoptotic potential, inhibition of tumor growth by rapid restoration of serum testosterone, and restraint of tumor growth by subnormal levels of serum testosterone (Bruchovsky et al 2000). They concluded that IAS is a viable treatment option for men with prostate cancer which affords an improved quality of life as well as reduced toxicity and costs (Bruchovsky et al 2000,

A few studies have shown that androgen is safe and potentially effective for treatment of advanced prostate cancer. Mathew reported that the testosterone level in a prostate cancer patient undergone radical prostatectomy and LH-RH therapy remained at castrated levels and serum PSA was undetectable for 15 years. PSA levels then began to rise and the patient was given testosterone replacement therapy to attain a normal range of serum testosterone. After an initial flare, PSA levels gradually declined over 18 months. After 27 months, PSA level started to increase. When testosterone replacement therapy was discontinued, PSA levels dropped (Mathew 2008). The observation was similar to the transition from 104-R1 to R1Ad phenotype under androgen treatment in our LNCaP progression model (Chuu et al 2005, Kokontis et al 1998). Szmulewitz et al. reported that 15 prostate cancer patients with progressive disease following androgen ablation, anti-androgen therapy, and withdrawal without minimal metastatic disease were randomized to treatment with three doses of transdermal testosterone of 2.5, 5.0, or 7.5 mg/day, resulting in increase of serum testosterone concentrations to 305 ng/dl, 308 ng/dl, and 297 ng/dl, respectively. The conclusion of this study is that testosterone is a feasible and reasonably well-tolerated therapy for men with early hormone-refractory prostate cancer (Szmulewitz et al 2009). Morris et el. performed a phase 1 clinical trial to determine the safety of high-dose exogenous testosterone in patients with castration-resistant metastatic prostate cancer. Cohorts of 3-6 patients with progressive castration-resistant prostate cancer who had been castrated for at least 1 yr received testosterone by skin patch or topical gel for 1 week, 1 month, or until disease progression. No adverse effect was reported. The serum testosterone ranged from 330-870 ng/dl (Morris et al 2009). This study suggested that patients with advanced prostate cancer can be safely treated with exogenous testosterone. Researchers suggested that maximizing testosterone serum levels in selected patients with androgen

Liver X receptors are ligand-activated transcriptional factors that belong to the nuclear receptor superfamily. There are two LXR isoforms, LXR and LXR (Chuu et al 2007). Although LXR and LXR share high similarity in their DNA- and ligand-binding domains, expression of these proteins in various tissues differs. LXR expression is restricted to liver, kidney, intestine, fat tissue, macrophages, lung, and spleen (Edwards et al 2002, Willy et al 1995). LXR is ubiquitously expressed (Song et al 1994). LXR and LXR form heterodimers with the obligate partner 9-cis retinoic acid receptor (RXR) (Chuu et al 2007, Song et al 1994, Willy et al 1995). The LXR/RXR heterodimer can be activated with either an LXR agonist (oxysterols) or a RXR agonist (cis-retinoic acid). Oxysterols are oxygenated derivatives of cholesterol. Oxysterols, such as 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and cholestenoic acid, are natural ligands for LXR (Chuu et al 2007, Forman et al 1997, Janowski

Morris et al 2009, Pether et al 2003).

receptor over-expression may improve the treatment outcome.

**5. Liver X receptor (LXR) signaling** 

**5.1 LXR and LXR**

LXRs are important regulators of cholesterol, fatty acid, and glucose homeostasis (Chuu et al 2007). Oral administration of an LXR agonist has an overall hypolipidemic effect in hypercholesterolemic rats, mice, and hamsters (Song and Liao 2001). LXR-/- mice are healthy when fed with a low-cholesterol diet. However, LXR-/- mice develop enlarged fatty livers, hepatocellular degeneration, high hepatic cholesterol levels, and impaired liver function when fed a high-cholesterol diet (Alberti et al 2001, Edwards et al 2002, Peet et al 1998). LXRβ-/- mice are unaffected by a high-cholesterol diet, suggesting that LXR and LXR have separate roles. LXR and LXR regulate cholesterol transport. LXRs induces expression of the cholesterol transporters ATP-binding cassette transporter A1 and G1 (ABCA1 and ABCG1) (Edwards et al 2002, Nakamura et al 2004, Venkateswaran et al 2000) as well as cholesterol acceptor apolipoprotein E (ApoE) (Chawla et al 2001). Treatment with LXR agonists (hypocholamide, T0901317, or GW3965) lowers the cholesterol level in serum and liver and inhibits the development of atherosclerosis in murine disease models (Blaschke et al 2004, Joseph et al 2002, Song et al 2001, Song and Liao 2001).

LXRs regulate fatty acid synthesis by modulating the expression of sterol regulatory element-binding protein-1c (SREBP-1c) (Repa et al 2000, Yoshikawa et al 2001) and downstream lipogenic genes, including acetyl CoA carboxylase and FAS (Liang et al 2002). LXRs also regulate insulin signaling in liver (Chen et al 2004b, Tobin et al 2002). LXR-/- LXR-/- double knockout mice lack insulin-mediated induction of an entire class of enzymes involved in both fatty acid and cholesterol metabolism (Tobin et al 2002). Treatment with T0901317 stimulates insulin secretion in pancreatic beta cells, reduces plasma glucose, and improves glucose tolerance and insulin resistance in murine and rat obesity models (Cao et al 2003, Efanov et al 2004, Joseph et al 2003).

LXR signaling is important for brain function as well. LXRs regulate lipid homeostasis in the brain. LXR-/- LXR-/- mice develop neurodegenerative changes in brain tissue (Wang et al 2002). Knockout of LXR, but not LXR, results in adult-onset motor neuron degeneration in male mice (Andersson et al 2005), suggesting a different role of LXR from LXR. Treatment with T0901317 decreases amyloidal beta production in an Alzheimer's disease mouse model (Koldamova et al 2005).

#### **6. Anti-cancer effect of LXR agonists**

#### **6.1 Anti-proliferative effect of LXR agonists in cancer cells**

Based on our recent observations using several prostate cancer cell lines, we discovered that LXR agonists suppress proliferation of human prostate cancer cell lines. Treatment of PC-3, DU-145, and LNCaP sublines (104-S, 104-R1, 104-R2, CDXR, R1Ad, IS) cells with LXR agonists (22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, or T0901317) suppresses the proliferation of these cells (Chuu and Lin 2010, Fukuchi et al 2004b, Vigushin et al 2004).

LXR agonists treatment causes growth inhibition in prostate cancer cells via induction of G1 cell cycle arrest (Chuu and Lin 2010, Fukuchi et al 2004b). T0901317 decreases the percentage of cells in S-phase and increases the percentage of cells in G1-phase. T0901317 suppresses

Inhibition of Advanced Prostate Cancer by Androgens and Liver X Receptor Agonists 217

correlates with the sensitivity of different cancer cells to 22(R)-hydroxycholesterol treatment. The effective concentrations for 22(R)-hydroxycholesterol to suppress cancer cell growth is within its known physiological range and is much lower than the concentrations to activate other nuclear receptors (Janowski et al 1996). LXR-ABCG1 signaling was reported to regulate sterol metabolism (Bensinger et al 2008). Activation of LXR inhibited the proliferation of T-cells but had no effect on cell viability (Bensinger et al 2008). Since T0901317 did not inhibit the proliferation of CAOV3 ovarian cancer cells treated with siRNA against LXR or LXR (Scoles et al 2010), it is possible that 22(R)-hydroxycholesterol inhibited cell proliferation mainly through activation of LXR, while inhibition of T0901317 may be caused by both LXR and LXR activation. We did not observe T0901317 to cause cancer cell growth inhibition at 300 nM (data not shown). It is unclear why the concentration needed for T0901317 to suppress the proliferation of human cancer cells is 15-fold higher than the effective concentration for T0901317 to activate LXR (20 nM) (Schultz et al 2000). The concentration of T0901317 observed to cause growth inhibition of ovarian cancer cell lines by Scoles et al. was 10-50 nM when the researchers used 0.1% FBS (Scoles et al 2010). We used 10% FBS in our study, it is possible that some proteins or growth factors in serum

In our progression model, expression of LXR and its target gene ABCA1 is higher in androgen-dependent LNCaP 104-S cells than in androgen-independent LNCaP 104-R1 and 104-R2 cells (Fukuchi et al 2004a). Expression of the LXR, ABCA1, and sterol 27 hydroxylase (CYP27) genes, all target genes of LXR, decreases during prostate cancer progression towards androgen-independency in athymic mice (Chuu et al 2006). The change in expression of genes involved in LXR signaling suggests a potential role of LXR signaling during prostate cancer progression. LXR agonists treatment on LNCaP sublines suggested that androgen-dependency and expression of AR level did not affect the growth inhibition caused by LXR agonists, thus LXR agonists may inhibit different progression stages of

We found that suppression of ABCA1 expression by androgen coincided with increased proliferation of androgen-dependent LNCaP 104-S cells (Fukuchi et al 2004a). Thus, under androgen-depleted conditions, ABCA1 levels are high and proliferation of 104-S cells is inhibited. During progression, the surviving androgen-independent relapsed tumor cells appear to escape ABCA1 suppression by down-regulating expression of LXR target genes. T0901317 induces expression of the ABCA1 gene in 104-S tumors in athymic mice (Fukuchi et al 2004b). Compared to the control group, T0901317 treatment delays the development of androgen-independent relapsed tumors for 4 weeks in athymic mice bearing 104-S tumors after castration (Chuu et al 2006) (Figure 5). This result indicates that treatment with an LXR

Our LNCaP progression model may provide the molecular explanation for IAS treatment. As most relapsed prostate tumors after androgen ablation therapy express AR and expression of mRNA and protein level of AR are frequently elevated (de Vere White et al 1997, Ford et al 2003, Linja et al 2001), restoration of endogenous testosterone level by IAS

agonist may retard development of androgen-independent prostate cancer.

may hinder the suppressive effect of T0901317.

prostate tumors in patients (Chuu and Lin 2010).

**7. Conclusion** 

**6.2 Inhibition of prostate cancer progression by LXR agonists** 

the expression of Skp2 and causes the accumulation of p27Kip1. Overexpression of Skp2 in PC-3 cells or knockdown of p27Kip1 in LNCaP cells increases the resistance of cells to T0901317 treatment (Chuu and Lin 2010, Fukuchi et al 2004b). Daily oral administration of T0901317 (10 mg/kg) suppresses growth of androgen-dependent LNCaP 104-S prostate tumors in athymic mice, resulting in a 2-fold difference in mean tumor volume between the control and the T0901317 treatment group (Fukuchi et al 2004b) (Figure 5).

Fig. 5. Inhibition of proliferation and progression of prostate cancer by the LXR agonists T0901317. (A) Mice carrying 104-S tumors were administered 10 mg/kg T0901317 (filled circle, 10 mice with 13 tumors) or vehicle alone (open circle, 10 mice with 15 tumors) by gavage once a day during the experiment period, resulting in a more than 2-fold difference in mean tumor volume between vehicle and T0901317-treated tumors after 4 weeks. Relative tumor volumes were expressed as mean SE. (Fukuchi et al 2004b). (B) After castration, mice carrying 104-S tumors were administered 10 mg/kg T0901317 (filled circle, 9 mice with 15 tumors) or vehicle alone (open circles, 9 mice with 13 tumors) by gavage five times a week during the experiment period, resulting in a 4-week delay in time required for development of androgenindependent relapsed tumors between vehicle and T0901317-treated group. Relative tumor volumes were expressed as mean SE. See reference 8 for details.

T0901317 and 22(R)-hydroxycholesterol also suppresses the proliferation of several commonly used human cancer cell lines, including breast cancer MCF-7 cells, hepatoma HepG2 cells, non-small lung cancer H1299 cells, cervical cancer HeLa cells, epidermoid carcinoma A431 cells, osteosarcoma saos-2 cells, melanoma MDA-MB-435 cells, squamous carcinoma SCC13 cells, CAOV3 and SKOV3 ovarian cancer cells, as well as T and B cells of chronic lymphoblastic leukemia (CLL) (Chuu and Lin 2010, Fukuchi et al 2004b, Geyeregger et al 2009, Scoles et al 2010, Vedin et al 2009). Expression of LXR mRNA in these cancer cells correlates with the cancer cells' sensitivity to 22(R)-hydroxycholesterol treatment (Chuu and Lin 2010), suggesting that G1 cell cycle arrest induced by LXR agonists in cancer cells is partially mediated through LXR gene regulation (Fukuchi et al 2004b).

The EC50 for 22(R)-hydroxycholesterol in suppressing the proliferation of cancer cells (Chuu and Lin 2010) is comparable to the concentration required for 22(R)-hydroxycholesterol to activate LXR (1.5 M) (Janowski et al 1996), this may explain why the level of LXR

correlates with the sensitivity of different cancer cells to 22(R)-hydroxycholesterol treatment. The effective concentrations for 22(R)-hydroxycholesterol to suppress cancer cell growth is within its known physiological range and is much lower than the concentrations to activate other nuclear receptors (Janowski et al 1996). LXR-ABCG1 signaling was reported to regulate sterol metabolism (Bensinger et al 2008). Activation of LXR inhibited the proliferation of T-cells but had no effect on cell viability (Bensinger et al 2008). Since T0901317 did not inhibit the proliferation of CAOV3 ovarian cancer cells treated with siRNA against LXR or LXR (Scoles et al 2010), it is possible that 22(R)-hydroxycholesterol inhibited cell proliferation mainly through activation of LXR, while inhibition of T0901317 may be caused by both LXR and LXR activation. We did not observe T0901317 to cause cancer cell growth inhibition at 300 nM (data not shown). It is unclear why the concentration needed for T0901317 to suppress the proliferation of human cancer cells is 15-fold higher than the effective concentration for T0901317 to activate LXR (20 nM) (Schultz et al 2000). The concentration of T0901317 observed to cause growth inhibition of ovarian cancer cell lines by Scoles et al. was 10-50 nM when the researchers used 0.1% FBS (Scoles et al 2010). We used 10% FBS in our study, it is possible that some proteins or growth factors in serum may hinder the suppressive effect of T0901317.

#### **6.2 Inhibition of prostate cancer progression by LXR agonists**

In our progression model, expression of LXR and its target gene ABCA1 is higher in androgen-dependent LNCaP 104-S cells than in androgen-independent LNCaP 104-R1 and 104-R2 cells (Fukuchi et al 2004a). Expression of the LXR, ABCA1, and sterol 27 hydroxylase (CYP27) genes, all target genes of LXR, decreases during prostate cancer progression towards androgen-independency in athymic mice (Chuu et al 2006). The change in expression of genes involved in LXR signaling suggests a potential role of LXR signaling during prostate cancer progression. LXR agonists treatment on LNCaP sublines suggested that androgen-dependency and expression of AR level did not affect the growth inhibition caused by LXR agonists, thus LXR agonists may inhibit different progression stages of prostate tumors in patients (Chuu and Lin 2010).

We found that suppression of ABCA1 expression by androgen coincided with increased proliferation of androgen-dependent LNCaP 104-S cells (Fukuchi et al 2004a). Thus, under androgen-depleted conditions, ABCA1 levels are high and proliferation of 104-S cells is inhibited. During progression, the surviving androgen-independent relapsed tumor cells appear to escape ABCA1 suppression by down-regulating expression of LXR target genes. T0901317 induces expression of the ABCA1 gene in 104-S tumors in athymic mice (Fukuchi et al 2004b). Compared to the control group, T0901317 treatment delays the development of androgen-independent relapsed tumors for 4 weeks in athymic mice bearing 104-S tumors after castration (Chuu et al 2006) (Figure 5). This result indicates that treatment with an LXR agonist may retard development of androgen-independent prostate cancer.

#### **7. Conclusion**

216 Prostate Cancer – Original Scientific Reports and Case Studies

the expression of Skp2 and causes the accumulation of p27Kip1. Overexpression of Skp2 in PC-3 cells or knockdown of p27Kip1 in LNCaP cells increases the resistance of cells to T0901317 treatment (Chuu and Lin 2010, Fukuchi et al 2004b). Daily oral administration of T0901317 (10 mg/kg) suppresses growth of androgen-dependent LNCaP 104-S prostate tumors in athymic mice, resulting in a 2-fold difference in mean tumor volume between the

control and the T0901317 treatment group (Fukuchi et al 2004b) (Figure 5).

Fig. 5. Inhibition of proliferation and progression of prostate cancer by the LXR agonists T0901317. (A) Mice carrying 104-S tumors were administered 10 mg/kg T0901317 (filled circle, 10 mice with 13 tumors) or vehicle alone (open circle, 10 mice with 15 tumors) by gavage once a day during the experiment period, resulting in a more than 2-fold difference in mean tumor volume between vehicle and T0901317-treated tumors after 4 weeks. Relative tumor volumes were expressed as mean SE. (Fukuchi et al 2004b). (B) After castration, mice carrying 104-S tumors were administered 10 mg/kg T0901317 (filled circle, 9 mice with 15 tumors) or vehicle alone (open circles, 9 mice with 13 tumors) by gavage five times a week during the experiment

period, resulting in a 4-week delay in time required for development of androgen-

cells is partially mediated through LXR gene regulation (Fukuchi et al 2004b).

volumes were expressed as mean SE. See reference 8 for details.

independent relapsed tumors between vehicle and T0901317-treated group. Relative tumor

T0901317 and 22(R)-hydroxycholesterol also suppresses the proliferation of several commonly used human cancer cell lines, including breast cancer MCF-7 cells, hepatoma HepG2 cells, non-small lung cancer H1299 cells, cervical cancer HeLa cells, epidermoid carcinoma A431 cells, osteosarcoma saos-2 cells, melanoma MDA-MB-435 cells, squamous carcinoma SCC13 cells, CAOV3 and SKOV3 ovarian cancer cells, as well as T and B cells of chronic lymphoblastic leukemia (CLL) (Chuu and Lin 2010, Fukuchi et al 2004b, Geyeregger et al 2009, Scoles et al 2010, Vedin et al 2009). Expression of LXR mRNA in these cancer cells correlates with the cancer cells' sensitivity to 22(R)-hydroxycholesterol treatment (Chuu and Lin 2010), suggesting that G1 cell cycle arrest induced by LXR agonists in cancer

The EC50 for 22(R)-hydroxycholesterol in suppressing the proliferation of cancer cells (Chuu and Lin 2010) is comparable to the concentration required for 22(R)-hydroxycholesterol to activate LXR (1.5 M) (Janowski et al 1996), this may explain why the level of LXR

Our LNCaP progression model may provide the molecular explanation for IAS treatment. As most relapsed prostate tumors after androgen ablation therapy express AR and expression of mRNA and protein level of AR are frequently elevated (de Vere White et al 1997, Ford et al 2003, Linja et al 2001), restoration of endogenous testosterone level by IAS

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treatment or treatment with exogenous testosterone will suppress the proliferation of the AR-rich relapsed prostate cancer cell according, similar to the observations in LNCaP 104- R1, 104-R2, CDXR, and in other relapsed prostate cancer cell models. Patients showed no response to IAS treatment might have tumors with very low or no AR expression. At the beginning of IAS or testosterone treatment, serum PSA level will increase dramatically (Mathew 2008), similar to the stimulated PSA expression in 104-R1, 104-R2, and CDXR cells. The AR-rich relapsed prostate cancer cells will then undergo G1 cell cycle arrest and/or apoptosis, causing the regression of tumor and decrease of serum PSA level. The regression of tumors can continue for weeks or months before the prostate cancer cells adapt to the androgenic suppression, possibly by down-regulating AR. The adapted cells are probably similar to R1Ad cells in patients receiving androgen ablation therapy (LH-RH agonists) or similar to IS cells in patients receiving combined treatment of LH-RH agonists and antiandrogens. The PSA secretion stimulated by androgen in R1Ad or IS cells is very low, so the serum PSA level will remain low until the adapted tumors start to grow, either stimulated by testosterone like R1Ad cells or by androgen-insensitive growth like IS cells. IAS will delay the growth of R1Ad-like but not IS-like tumors, therefore, only the subgroup of patients carrying R1Ad-like tumors will respond to the subsequent cycles of IAS treatment. As 104-R1 cells will progress to 104-R2 cells in androgen-depleted medium and 104-R2 cells, like CDXR cells, will generate IS-like cells following androgen treatment, patients receiving a few cycle of IAS treatment will ultimately develop IS-like tumors which don't respond to further IAS treatment. Alternative therapies, such as green tea catechin epigallocatechin 3 gallate (EGCG) or liver X receptor agonists might be able to suppress growth of these androgen-insensitive prostate tumors.

Patients develop relapsed androgen-independent prostate tumors after androgen ablation therapy should be biopsied for expression level of AR protein in tumors. IAS and/or administration of androgen at a concentration 5-fold higher than the physiologic concentration will benefit patients with AR-rich relapsed tumors by suppressing tumor growth, improving quality of life, and reducing risks for cardiovascular diseases and diabetes. Combined treatment of androgen ablation therapy with anti-androgen may cause a more rapid and irreversible selection of CDXR-like advanced prostate cancer cells, although androgen treatment may cause regression and disappearance of these tumors (Kokontis et al 2005). Androgen deprivation therapy alone, on the other hand, may promote a slow adaptation to androgen-independence. LXR agonists suppress the proliferation of multiple human prostate cancer cell lines via reduction of Skp2 and induction of p27Kip, thus cause G1 cell cycle arrest. LXR agonist T0901317 treatment also delays the progression of androgen-dependent LNCaP xenograft towards androgenindependency in castrated nude mice. It is therefore possible to modulate LXR signaling as an adjuvant therapy for treatment of all stages of prostate cancer. In conclusion, manipulating androgen/AR might be a potential therapy for AR-positive advanced prostate cancer, and LXR agonists might be an adjuvant therapy for treatment of advanced prostate cancer.

#### **8. Acknowledgements**

This work is supported by CS-100-PP-12 (NHRI), DOH100-TD-C-111-014 (DOH), and NSC 99-2320-B-400-015-MY3 (NSC) in Taiwan for C.-P.Chuu.

#### **9. References**

218 Prostate Cancer – Original Scientific Reports and Case Studies

treatment or treatment with exogenous testosterone will suppress the proliferation of the AR-rich relapsed prostate cancer cell according, similar to the observations in LNCaP 104- R1, 104-R2, CDXR, and in other relapsed prostate cancer cell models. Patients showed no response to IAS treatment might have tumors with very low or no AR expression. At the beginning of IAS or testosterone treatment, serum PSA level will increase dramatically (Mathew 2008), similar to the stimulated PSA expression in 104-R1, 104-R2, and CDXR cells. The AR-rich relapsed prostate cancer cells will then undergo G1 cell cycle arrest and/or apoptosis, causing the regression of tumor and decrease of serum PSA level. The regression of tumors can continue for weeks or months before the prostate cancer cells adapt to the androgenic suppression, possibly by down-regulating AR. The adapted cells are probably similar to R1Ad cells in patients receiving androgen ablation therapy (LH-RH agonists) or similar to IS cells in patients receiving combined treatment of LH-RH agonists and antiandrogens. The PSA secretion stimulated by androgen in R1Ad or IS cells is very low, so the serum PSA level will remain low until the adapted tumors start to grow, either stimulated by testosterone like R1Ad cells or by androgen-insensitive growth like IS cells. IAS will delay the growth of R1Ad-like but not IS-like tumors, therefore, only the subgroup of patients carrying R1Ad-like tumors will respond to the subsequent cycles of IAS treatment. As 104-R1 cells will progress to 104-R2 cells in androgen-depleted medium and 104-R2 cells, like CDXR cells, will generate IS-like cells following androgen treatment, patients receiving a few cycle of IAS treatment will ultimately develop IS-like tumors which don't respond to further IAS treatment. Alternative therapies, such as green tea catechin epigallocatechin 3 gallate (EGCG) or liver X receptor agonists might be able to suppress growth of these

Patients develop relapsed androgen-independent prostate tumors after androgen ablation therapy should be biopsied for expression level of AR protein in tumors. IAS and/or administration of androgen at a concentration 5-fold higher than the physiologic concentration will benefit patients with AR-rich relapsed tumors by suppressing tumor growth, improving quality of life, and reducing risks for cardiovascular diseases and diabetes. Combined treatment of androgen ablation therapy with anti-androgen may cause a more rapid and irreversible selection of CDXR-like advanced prostate cancer cells, although androgen treatment may cause regression and disappearance of these tumors (Kokontis et al 2005). Androgen deprivation therapy alone, on the other hand, may promote a slow adaptation to androgen-independence. LXR agonists suppress the proliferation of multiple human prostate cancer cell lines via reduction of Skp2 and induction of p27Kip, thus cause G1 cell cycle arrest. LXR agonist T0901317 treatment also delays the progression of androgen-dependent LNCaP xenograft towards androgenindependency in castrated nude mice. It is therefore possible to modulate LXR signaling as an adjuvant therapy for treatment of all stages of prostate cancer. In conclusion, manipulating androgen/AR might be a potential therapy for AR-positive advanced prostate cancer, and LXR agonists might be an adjuvant therapy for treatment of

This work is supported by CS-100-PP-12 (NHRI), DOH100-TD-C-111-014 (DOH), and NSC

androgen-insensitive prostate tumors.

advanced prostate cancer.

**8. Acknowledgements** 

99-2320-B-400-015-MY3 (NSC) in Taiwan for C.-P.Chuu.


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### *Edited by Philippe E. Spiess*

This book encompasses three sections pertaining to the topics of cancer biology, diagnostic markers, and therapeutic novelties. It represents 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.

Prostate Cancer - Original Scientific Reports and Case Studies

Prostate Cancer

Original Scientific Reports and Case Studies

*Edited by Philippe E. Spiess*

Photo by wacomka / iStock