**An Update On Natural Compounds and Their Modern Formulations for the Management of Malignant Melanoma**

Danciu Corina, Soica Codruta, Antal Diana, Alexandra Popescu, Roxana Ghiulai, Ioana Zinuca Pavel, Stefana Avram, Minda Daliana and Cristina Dehelean

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67647

#### Abstract

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The chapter includes a brief presentation of the types of skin cancer. The most aggressive type of skin cancer, melanoma, is discussed from the point of view of incidence, molecular, and immunohistochemical mechanism along with the most important biomarkers for identification. Recent studies containing active phytocompounds with chemopreventive activity pointing toward phytochemicals used for melanoma prevention and therapy are reviewed. Modern physicochemical formulations for the enhancement of bioavailability of some active phytochemicals with chemopreventive activity for malignant melanoma are discussed.

Keywords: malignant melanoma, mechanism, biomarkers, active phytocompounds, modern formulations

### 1. Introduction

Skin cancer, with its many forms, is one of the most prevalent diseases today. In the last decades, there was a rising incidence in the reported cases of skin cancer and is estimated that one out of three Caucasians may develop a type of skin cancer in their lifetime [1, 2]. There are three common types of skin cancer, namely basal cell carcinoma (BCC), squamous cell carcinoma (SCC), and melanoma. Basal and squamous cell carcinomas are also known as nonmelanoma skin cancer (NMSC) [1, 3].

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Melanoma and non-melanoma skin cancers represent the most common malignancies worldwide [2, 3]. Wehner et al. indicated that approximately 30% of white people are likely to develop NMSC in their lifetime as a result of increased exposure to ultraviolet radiation [4]. It is considered that exposure to high doses of ultraviolet B radiation can cause basal cell carcinoma, while squamous cell carcinoma can be triggered after a chronic exposure to UV [2]. Melanoma in the first place and squamous cell carcinoma in the second place are tumor types with a high risk of metastasis, while basal cell carcinoma rarely does. Usually, these types of cancer affect people with white skin, blue eyes, and red hair [5]. Regarding gender, men are more likely to develop a type of NMSC in their lifetime than women [1].

As already mentioned above, one of the most important risk factors for developing skin cancer is considered exposure to ultraviolet radiation (UV) [6]. There are three types of UV radiation depending on the wavelength: UVA, UVB, and UVC. UV radiation especially ultraviolet B radiation is known to cause oxidative stress, inflammation, DNA damage, and apoptotic cell death in the skin [6, 7]. UV exposure also determines immunosuppression and photoaging of the skin [8]. People who use indoor tanning especially at an early age (before 25 years) are exposed to a higher risk of developing a form of skin cancer in their lifetime [4]. Among UV exposure, ozone depletion, genetics, and immune suppression are also incriminated in the rising incidence of skin cancer [9]. Another factor considered responsible for skin cancer growth is long-term exposure to arsenic, a class I human carcinogen found in industrial, agricultural, and medicinal substances [10, 11]. PUVA therapy is a treatment used for severe skin diseases which consists of psoralens (P) and exposure to ultraviolet A radiation (UVA). PUVA may induce skin cancer by photomutagenicity and photoinduced immunosuppression. It is important to evaluate the risk-benefit ratio before starting with the PUVA therapy [10, 12, 13]. Exposure to ionizing radiation, inorganic metals, and polycyclic hydrocarbons are also risk factors for skin cancer development [10].

#### 1.1. Basal cell carcinoma

Basal cell carcinoma (BCC) is considered the most common type of skin cancer affecting people worldwide [5, 3]. It develops in the basal layer of the skin, from keratinocyte stem cells. Usually BCC appears de novo on hair-bearing skin, more often in adults. The most frequent BCC is considered the nodular/nodulo-ulcerative type which is commonly found on the neck and head. It looks like a red or pink papule with raised edges, telangiectasias, pearly appearance, and central small ulcers. Nodular BCC develops slowly but in time it can invade the tissue around it producing significant damage [14].

Exposure to high doses and intermittent UV radiation can trigger BCC [5]. Most cases of BCC were reported in Australia and are mainly attributed to UV radiation [3]. UVB is considered the most harmful environmental factor that can induce DNA mutations after exposure. BCC can develop through alteration of p53 and PTCH1 tumor-suppressor genes.

In general, BCC is found on the arms, head, neck, or the back and is more prevalent in people with light hair, blue or green eyes, and freckles. Other factors reported to increase the risk of BCC are genetic conditions, ionized radiation, genodermatoses, exposure to chemical carcinogens, organic and inorganic solvents, and organophosphate compounds [14, 15]. In some cases of superficial BCC, human papillomavirus (HPV) is considered to be a risk factor in cancer growth. Also, immunosuppression caused by other diseases is considered to have a role in NMSC development [14].

There are various types of treatment options for BCC. The therapeutic approach depends on the location and the size of the tumor. Also, it is important to know the morphology and histologic subtype of the tumor and whether or not it has spread to nearby tissues. The most common therapeutic option is surgical excision of the skin [14, 15]. In most of the cases, the method is applicable if the lesion is less than 2 cm in diameter and is found in the trunk or extremities. An advantage of surgical excision is that after surgery, the lesion can be histologically analyzed and the tumor subtype can be determined. Also, there are disadvantages of using this method, including the possibility of infection and the presence of scars [14]. When surgery is not possible due to tumor location or because it is locally advanced, radiation therapy is an option [15]. The method can be used when the tumor is located on the ears, nose, eyelids, and lips. Other therapeutical options include photodynamic therapy, cryosurgery [14, 16], and topical therapies with imiquimod, an immune response modifier or 5-fluorouracil, a pyrimidine analog [15]. It is important to properly treat BCC because it has a high tendency to reappear if it remains untreated. Even if treated, in 18% of the cases, BCC will reappear in a few years after treatment [14].

### 1.2. Squamous cell carcinoma

Melanoma and non-melanoma skin cancers represent the most common malignancies worldwide [2, 3]. Wehner et al. indicated that approximately 30% of white people are likely to develop NMSC in their lifetime as a result of increased exposure to ultraviolet radiation [4]. It is considered that exposure to high doses of ultraviolet B radiation can cause basal cell carcinoma, while squamous cell carcinoma can be triggered after a chronic exposure to UV [2]. Melanoma in the first place and squamous cell carcinoma in the second place are tumor types with a high risk of metastasis, while basal cell carcinoma rarely does. Usually, these types of cancer affect people with white skin, blue eyes, and red hair [5]. Regarding gender,

As already mentioned above, one of the most important risk factors for developing skin cancer is considered exposure to ultraviolet radiation (UV) [6]. There are three types of UV radiation depending on the wavelength: UVA, UVB, and UVC. UV radiation especially ultraviolet B radiation is known to cause oxidative stress, inflammation, DNA damage, and apoptotic cell death in the skin [6, 7]. UV exposure also determines immunosuppression and photoaging of the skin [8]. People who use indoor tanning especially at an early age (before 25 years) are exposed to a higher risk of developing a form of skin cancer in their lifetime [4]. Among UV exposure, ozone depletion, genetics, and immune suppression are also incriminated in the rising incidence of skin cancer [9]. Another factor considered responsible for skin cancer growth is long-term exposure to arsenic, a class I human carcinogen found in industrial, agricultural, and medicinal substances [10, 11]. PUVA therapy is a treatment used for severe skin diseases which consists of psoralens (P) and exposure to ultraviolet A radiation (UVA). PUVA may induce skin cancer by photomutagenicity and photoinduced immunosuppression. It is important to evaluate the risk-benefit ratio before starting with the PUVA therapy [10, 12, 13]. Exposure to ionizing radiation, inorganic metals, and polycyclic hydrocarbons are

Basal cell carcinoma (BCC) is considered the most common type of skin cancer affecting people worldwide [5, 3]. It develops in the basal layer of the skin, from keratinocyte stem cells. Usually BCC appears de novo on hair-bearing skin, more often in adults. The most frequent BCC is considered the nodular/nodulo-ulcerative type which is commonly found on the neck and head. It looks like a red or pink papule with raised edges, telangiectasias, pearly appearance, and central small ulcers. Nodular BCC develops slowly but in time it can invade the

Exposure to high doses and intermittent UV radiation can trigger BCC [5]. Most cases of BCC were reported in Australia and are mainly attributed to UV radiation [3]. UVB is considered the most harmful environmental factor that can induce DNA mutations after exposure. BCC

In general, BCC is found on the arms, head, neck, or the back and is more prevalent in people with light hair, blue or green eyes, and freckles. Other factors reported to increase the risk of BCC are genetic conditions, ionized radiation, genodermatoses, exposure to chemical carcinogens, organic and inorganic solvents, and organophosphate compounds [14, 15]. In some cases

can develop through alteration of p53 and PTCH1 tumor-suppressor genes.

men are more likely to develop a type of NMSC in their lifetime than women [1].

also risk factors for skin cancer development [10].

tissue around it producing significant damage [14].

1.1. Basal cell carcinoma

212 Natural Products and Cancer Drug Discovery

Squamous cell carcinoma (SCC) also arises from keratinocytes and is the second most common type of skin cancer [17, 18]. SCC can be localized in the epidermis, known as in situ SCC, or it can extend to the dermis or deeper in the skin, known as invasive SCC. UV exposure is the main risk factor incriminated for SCC development. Cumulative high doses of UV radiation are responsible for SCC evolution [17], also non-healing wounds or chronic lesions that were linked with previous cases of chronic immuno-inflammation are associated with SCC [5]. SCC is found at a higher prevalence at people who smoke, compared to non-smokers, probably as a result of the immunosuppressive effects caused by smoking. Viruses, such as human papillomavirus (HPV), human immunodeficiency virus (HIV), or human herpes virus (HHV), also increase the risk for SCC. Exposure to chemical agents, including arsenic and polycyclic aromatic hydrocarbons, can lead in time to SCC. People with genetic disorders, such as xeroderma pigmentosum and oculocutaneous albinism, are more likely to develop SCC [17].

This type of cancer is usually found in people older than 50 years and is located on the head and neck, being more frequent in males, than in females [17]. Usually, it does not metastasize, but when it does, the survival rate is poor, between 25 and 40% [18].

SCC treatment depends on the location of the tumor, the size, and whether or not it has metastasized. For high-risk tumors, Mohs micrographic surgery (microscopically oriented histographic surgery) is considered the best method, while for low-risk SCC, surgical excision is the most common therapeutic option. In order to have a low recurrence rate, safety margins must be also excised. They vary between 4 and 10 mm according to tumor size. Cryosurgery is another surgical method used for small tumors [17, 19]. Nonsurgical treatments are also effective therapies for SCC, namely radiation therapy, topical application of 5-fluorouracil and imiquimod, and photodynamic therapy. There are studies, which indicate that non-steroidal anti-inflammatory drugs are useful in the prevention and treatment of SCC [17]. Other drugs used rather in the prevention than in the treatment of SCC are retinoids. The results of Tran et al. showed that the topical administration of retinoids neutralizes vitamin A depletion induced by exposure to UVB radiation and thus has a beneficial role on recovery [20]. Recurrence rate is around 5 years after treatment, so it is important that the patients are closely monitorized [17].

### 1.3. Melanoma

Melanoma is a melanocytic neoplasia, and it is thought to be the rarest form of skin cancer and the most aggressive one [1]. It is considered that melanocytic nevi are precursors for melanoma. They are benign nevi, usually found in body areas exposed to the sun and can undergo transformation due to several risk factors and then progress into dysplastic nevi, which can further turn into melanoma [21].

In order to establish whether or not a melanocytic nevus has a malignant transformation, five characteristics can be followed, also known as the ABCDE of melanoma. The first is Asymmetry —usually a benign melanocytic nevus is symmetric; Border—the margins of the nevus are irregular; Color—the pigmentation is not equable on the entire surface of the nevus; Diameter —the melanocytic nevus is bigger than 6 mm, Evolution [1].

Exposure to UV radiation is considered one of the most important factors in melanoma development. Among it, genetic conditions, multiple nevi, and exposure to some chemical agents are incriminated as risk factors [22, 23]. People who have first-degree relatives with melanoma are exposed to a higher risk in developing this type of skin cancer. Immunosuppression is also thought to have a role in melanoma growth. Other risk factors include the exposure to some pesticides, such as parathion, carbaryl, maneb/mancozeb, and benomyl [23].

Commonly, melanoma appears on males older than 54 years and females older than 45 years and is mostly found on the trunk, face, and ears. In the last years, the number of melanomas found in women aged 15–24 has increased [23]. Indoor tanning is considered to have an important role in this higher incidence rate [24].

There are different forms of melanoma, the most common being melanoma of intermittently sun-exposed skin, acral (and mucosal) melanoma, lentigo maligna melanoma, and nodular melanoma. Melanoma of intermittently sun-exposed skin is generally found in Caucasian people and is related with BRAF mutations and melanocytic nevi. It affects more women than men, and is located in the lower extremities and the trunk [22]. Acral (and mucosal) melanomas can appear in areas of the body that are not frequently exposed to UV radiation such as the soles of the feet and the palms of the hands. This type is found in black people and is associated with chromosomal aberrations [5, 22]. Lentigo maligna (solar) melanoma develops after chronic exposure to UV radiation. It is found in people older than 60 years and is correlated with genomic aberrations and the lack of BRAF mutations. Nodular melanoma is a rapid growing tumor that can have pagetoid spread, commonly found in people older than 30 years [22].


Melanoma has a high metastatic potential through lymphatic and vascular channels. Early diagnosis is very important because in the early stages it can be removed surgically. In order to reduce the risk of tumor recurrence, along with the tumoral tissue, part of the surrounding normal structure is also removed. The American Academy of Dermatology recommends the following excisional margins (cm) according to tumor thickness (mm) [1]:

When there is evidence of metastasis, systemic chemotherapy is used. Frequently used drugs are cisplatin, mitomycin, doxorubicin, and temozolomide. Immunotherapies are also an option for metastatic melanomas. This includes administration of interferon-alpha or interleukin 2 [1].

Patients with high-risk melanomas must be physically monitored every 3 months for the first 3 years, considered the period when metastases are more likely to appear and thereafter biannually or annually [22].

### 2. Melanoma: incidence, molecular, and immunohistochemical mechanism

### 2.1. Incidence and mortality of melanoma

and imiquimod, and photodynamic therapy. There are studies, which indicate that non-steroidal anti-inflammatory drugs are useful in the prevention and treatment of SCC [17]. Other drugs used rather in the prevention than in the treatment of SCC are retinoids. The results of Tran et al. showed that the topical administration of retinoids neutralizes vitamin A depletion induced by exposure to UVB radiation and thus has a beneficial role on recovery [20]. Recurrence rate is around 5 years after treatment, so it is important that the patients are closely

Melanoma is a melanocytic neoplasia, and it is thought to be the rarest form of skin cancer and the most aggressive one [1]. It is considered that melanocytic nevi are precursors for melanoma. They are benign nevi, usually found in body areas exposed to the sun and can undergo transformation due to several risk factors and then progress into dysplastic nevi, which can

In order to establish whether or not a melanocytic nevus has a malignant transformation, five characteristics can be followed, also known as the ABCDE of melanoma. The first is Asymmetry —usually a benign melanocytic nevus is symmetric; Border—the margins of the nevus are irregular; Color—the pigmentation is not equable on the entire surface of the nevus; Diameter

Exposure to UV radiation is considered one of the most important factors in melanoma development. Among it, genetic conditions, multiple nevi, and exposure to some chemical agents are incriminated as risk factors [22, 23]. People who have first-degree relatives with melanoma are exposed to a higher risk in developing this type of skin cancer. Immunosuppression is also thought to have a role in melanoma growth. Other risk factors include the exposure to some pesticides, such as parathion, carbaryl, maneb/mancozeb, and benomyl [23]. Commonly, melanoma appears on males older than 54 years and females older than 45 years and is mostly found on the trunk, face, and ears. In the last years, the number of melanomas found in women aged 15–24 has increased [23]. Indoor tanning is considered to have an

There are different forms of melanoma, the most common being melanoma of intermittently sun-exposed skin, acral (and mucosal) melanoma, lentigo maligna melanoma, and nodular melanoma. Melanoma of intermittently sun-exposed skin is generally found in Caucasian people and is related with BRAF mutations and melanocytic nevi. It affects more women than men, and is located in the lower extremities and the trunk [22]. Acral (and mucosal) melanomas can appear in areas of the body that are not frequently exposed to UV radiation such as the soles of the feet and the palms of the hands. This type is found in black people and is associated with chromosomal aberrations [5, 22]. Lentigo maligna (solar) melanoma develops after chronic exposure to UV radiation. It is found in people older than 60 years and is correlated with genomic aberrations and the lack of BRAF mutations. Nodular melanoma is a rapid growing tumor that can have pagetoid spread, commonly found in people older than 30

monitorized [17].

1.3. Melanoma

years [22].

further turn into melanoma [21].

214 Natural Products and Cancer Drug Discovery

—the melanocytic nevus is bigger than 6 mm, Evolution [1].

important role in this higher incidence rate [24].

From the total number of all skin cancer cases, melanoma represents less than 2%, nevertheless being the major cause of death from skin cancer. Despite this steadily increasing incidence of melanoma, the mortality caused by this pathology stabilized in the last 20 years, on the strength of advances in medical care. At present, the survival rate of 5 years exceeds 90%, being associated with the moment of diagnosis [25].

It was predicted that 73,870 patients would be diagnosed with this condition in 2015. The most affected segment was the white people of non-Hispanic origin. According to American Cancer Society, the incidence rates vary from 1 per 100,000 annual cases in blacks to 4 in Hispanics, and 25 in non-Hispanic whites. Although it can appear independently of age, usually the elders are more affected by melanoma, being diagnosed at an average age of 62 years [25]. By the age of 50, women are more affected than men; however, by age 65 the incidence is two times higher in men than in women, tripling by the age of 80. These disparities are pursuant to ultraviolet radiation, either solar or artificial. During 1930, the incidence of melanoma was 1 in 1500 American people, reaching 1 in 68 in 2002. Through the years 2002–2012, the incidence extended with 1.8% per year. This enhancement of new cases of melanoma is partially linked to the development of new, modern, diagnostic methods, and also to the rising of the average life expectancy [25, 26].

In the US, during the year 2015, there were estimated 73,870 new cases of melanoma from which 42,670 cases were in male and 31,200 in female. The total melanoma deaths in US, in 2015, reached 9940, whence 6640 cases in men and 3300 in women. In the State of California, the total number of new melanoma cases reaches the upper limit (8560 cases), being followed by Florida (5480 cases), while the minimum was observed in the District of Columbia (80 cases), followed by Alaska (100) [27].

In Europe, 100,000 new cases of melanoma were diagnosed during 2012, mostly in the central and eastern part. The incidence of melanoma deaths was high in these regions of Europe (36%); from a total of 22,000 deaths, 8000 cases were found in the Central and Eastern Europe. However, the most cases of melanoma in Europe were in the Nordic regions and the Netherlands [28].

A recent study has compared the incidence and mortality rate in 11 countries from Europe: Bosnia and Herzegovina, Bulgaria, Croatia, Cyprus, Czech Republic, Malta, Romania, Serbia, Slovenia, and Turkey. Regarding the distribution of incidence upon age and sex, it was observed that younger females and males had the same incidence rates, although it increased in middleaged and older men in comparison with females from the same category. The incidence rate of melanoma was increased in most of the countries, the higher trend concerning the young people (25–49 years) being observed in Slovenia, though in men aged 50–69, the incidence was important in Slovenia, Slovakia, Bulgaria, and Serbia. After the age of 70, the rate was raised in men, independently on the level of the incidence in the country. In women, the rate of melanoma was increased in most regions of Europe, especially in Slovenia, whatever the age. For 50–69 aged females, the incidence was higher in Bulgaria and Romania, while for women older than 70 years, a greater incidence in Serbia and Slovakia was remarked. For this category of women, the incidence was also significant in Bulgaria, Croatia, and the Czech Republic. Regarding the melanoma mortality rates in the 11 countries of Europe previously mentioned, men were more affected than women in most countries. In younger groups, the mortality rate was non-significantly decreased, in both men and women. Men of middle and older age had a relevant mortality in Serbia. Also, the elders were affected in Turkey and Czech Republic. On the other hand, women aged 50–69 had elevated rates of mortality in most countries, particularly in Slovenia. The mortality increased with age, mainly in Serbia and Slovenia [29].

#### 2.2. Molecular and immunohistochemical mechanism in melanoma

From the embryonic neural crest cells, throughout development, melanoblasts are first taken into dermis, and then these precursors migrate toward the epidermis to settle in its basal epithelial layer, on the basal lamina. Melanoblasts arise into dermis by 11 weeks. Melanin pigment is produced at around 12 weeks [30].

Differentiated melanocytes are hold tightly in the basement membrane, when they arrive in contact with keratinocytes. At higher epidermal layers, they are unable to survive, unless turned into nevi or melanomas. Keratinocytes play an important role in the maintenance of melanocytes homeostasis. The impairment of this homeostasis affects the epidermal melanin unit, giving rise to an unceasing proliferation of melanocytes. It is presumed that melanoma cells get rid of the keratinocytes control by several mechanisms: (a) down-regulation of some

molecules (like E-cadherin) that play a role in their communication and adhesion to keratinocytes; (b) up-regulation of certain molecules that participate in cell signaling significant for melanoma-melanoma and melanoma-fibroblast interplays, like N-cadherin, zonula occludens protein-1, and MCAM (melanoma cell adhesion molecule); and (c) impaired expression of several proteins that bind the extracellular matrix with the repercussions for the retention into the basement membrane [31, 32].

In the US, during the year 2015, there were estimated 73,870 new cases of melanoma from which 42,670 cases were in male and 31,200 in female. The total melanoma deaths in US, in 2015, reached 9940, whence 6640 cases in men and 3300 in women. In the State of California, the total number of new melanoma cases reaches the upper limit (8560 cases), being followed by Florida (5480 cases), while the minimum was observed in the District of Columbia (80

In Europe, 100,000 new cases of melanoma were diagnosed during 2012, mostly in the central and eastern part. The incidence of melanoma deaths was high in these regions of Europe (36%); from a total of 22,000 deaths, 8000 cases were found in the Central and Eastern Europe. However, the most cases of melanoma in Europe were in the Nordic regions and the Nether-

A recent study has compared the incidence and mortality rate in 11 countries from Europe: Bosnia and Herzegovina, Bulgaria, Croatia, Cyprus, Czech Republic, Malta, Romania, Serbia, Slovenia, and Turkey. Regarding the distribution of incidence upon age and sex, it was observed that younger females and males had the same incidence rates, although it increased in middleaged and older men in comparison with females from the same category. The incidence rate of melanoma was increased in most of the countries, the higher trend concerning the young people (25–49 years) being observed in Slovenia, though in men aged 50–69, the incidence was important in Slovenia, Slovakia, Bulgaria, and Serbia. After the age of 70, the rate was raised in men, independently on the level of the incidence in the country. In women, the rate of melanoma was increased in most regions of Europe, especially in Slovenia, whatever the age. For 50–69 aged females, the incidence was higher in Bulgaria and Romania, while for women older than 70 years, a greater incidence in Serbia and Slovakia was remarked. For this category of women, the incidence was also significant in Bulgaria, Croatia, and the Czech Republic. Regarding the melanoma mortality rates in the 11 countries of Europe previously mentioned, men were more affected than women in most countries. In younger groups, the mortality rate was non-significantly decreased, in both men and women. Men of middle and older age had a relevant mortality in Serbia. Also, the elders were affected in Turkey and Czech Republic. On the other hand, women aged 50–69 had elevated rates of mortality in most countries, particularly in

Slovenia. The mortality increased with age, mainly in Serbia and Slovenia [29].

From the embryonic neural crest cells, throughout development, melanoblasts are first taken into dermis, and then these precursors migrate toward the epidermis to settle in its basal epithelial layer, on the basal lamina. Melanoblasts arise into dermis by 11 weeks. Melanin

Differentiated melanocytes are hold tightly in the basement membrane, when they arrive in contact with keratinocytes. At higher epidermal layers, they are unable to survive, unless turned into nevi or melanomas. Keratinocytes play an important role in the maintenance of melanocytes homeostasis. The impairment of this homeostasis affects the epidermal melanin unit, giving rise to an unceasing proliferation of melanocytes. It is presumed that melanoma cells get rid of the keratinocytes control by several mechanisms: (a) down-regulation of some

2.2. Molecular and immunohistochemical mechanism in melanoma

pigment is produced at around 12 weeks [30].

cases), followed by Alaska (100) [27].

216 Natural Products and Cancer Drug Discovery

lands [28].

Recent studies revealed the up-regulation of CCN3 protein in melanocyte-keratinocyte coculture, influencing some important characteristics in the physiology of melanocytes. This matricellular protein is overexpressed in some malignant diseases being related to the evolution of prostate cancer, carcinoma of renal cells, and Ewing's sarcoma; moreover, its expression is linked to the differentiation of tumors in rhabdomyosarcoma and cartilage malignancies [33–35]. Depending on the type of cancer, CCN3 influence divergently the genesis of tumor. Therefore, in gliomas it manifests antiproliferative effects, as well as in chronic myeloid leukemia cells. By contrast, it stimulates the invasion of Ewing's sarcoma cells [36, 37]. Regarding the effects of CCN3 on melanocytes, it reduces their proliferation and is essential for the melanocyte network organization [38]. In addition to this, it enhances the adherence of melanocytes to the type-IV collagen from basement membrane. Studies in vivo show that CCN3 overexpression is associated with an extremely attenuated invasion of melanoma cells, through the inhibition of MMP (matrix metalloproteinase) expression [31, 39].

The progression of melanoma comprises five distinct phases. First of all, hyperplasia of melanocytes gives rise to nevi; these can be acquired or congenital. The second phase is characterized by dysplastic nevi along with cytological and architectural abnormality. The third one corresponds to radial growth phase (RGP) melanoma, distinguished by the presence of tumor cells within the epidermis, or their invasion through the superficial dermis, having a reduced ability to spread from the primary site. The last stage is the vertical growth phase (VGP), which is featured by the profound invasion of tumor cells into the dermis and hypodermic tissue, augmenting the systemic dissemination risk. Lastly, metastasis occurs, being the most advanced phase of this condition, as a result of the altered homeostasis in the skin. The impaired mechanisms of homeostatic control determine the development of melanoma, since these mechanisms control the proliferation of cells, as well as their differentiation and apoptosis [33, 41].

Throughout the development stages of melanoma, CCN3 protein expression is down-regulated. Immunohistochemical techniques performed on melanoma cells evidentiated the inversely proportional interdependence between CCN3 expression and tumor dimension. The transduction of CCN3 in progressive melanomas inhibited the activity of matrix metalloproteinases MMP 2 and 9, resulting in an intense reduction of tumor invasion. Hence, the absence of CCN3 expression in melanomas is related to an invasive phenotype [31].

Melanomas can appear from melanocytes that have the role in the production of melanin pigment, and also in its storage and distribution to keratinocytes. There are other melanomas that arise from nevi, particularly dysplastic or giant congenital nevi. Melanomas are divided into four clinic-histological types: superficial-spreading melanoma, nodular form, lentigo maligna, and acral lentiginous type. Different factors are involved in the development of melanoma, such as genetic triggers, methylation of DNA, acetylation, or milieu factors [40, 41]. Impairment in tumor-suppressor genes such as mutation, deletion, translocation, or activation of proto-oncogenes has a significant role in mutagenesis. Mutation or deletion of CDKN2A antioncogene and CDK4 proto-oncogene is related to the development of familial melanoma, which constitute 8–12% of total melanomas [42]. This type of melanoma is characterized by an abnormal regulation of cell cycle by cyclin-dependent enzymes. In this case, the 9p21 chromosome has a germline mutation in the locus of CDKN 2A gene, affecting two proteins p16 and p14 or Arf that have a suppressive role. The p16 protein has an inhibitory effect on cyclin D/CDK4 and CDK6, influencing therefore pRB (retinoblastoma protein) which, through liberation of E2F, change the G1 phase of cell cycle into S phase [43, 44]. It was discovered lately that E2F1 protein, as well, manifests a suppressor function through p53 or p73. Between 10 and 30% of familial melanoma cases are due to CDKN2A mutation. NRAS and p16 mutations are also incriminated in some isolated cases of melanoma. Mutation of CDKN2A can lead to multiple melanomas. This alteration seldom appears in actinic keratosis, skin carcinomas, and other types of cancers [45, 46].

The p14 protein, encoded by CDKN2A reading frame, affects the p53 protein through the influence on other protein, Mdm2. The p14 deprivation lowers the activity of p53 [47]. Studies taken on mice models have shown that p14 can determine apoptosis of oncogene-activated melanocytes [48, 49].

It is known that pigmentation of the skin that depends on the amount of melanin is in direct relation with the development of melanoma. It is 10 times more frequently in whites than in blacks; however, people affected by albinism are exceptionally affected. The highly polymorphic melanocortin 1-receptor gene (MC1R) situated on 16q24 chromosome controls sun sensitivity [50, 51]. It codifies the transmembrane receptor coupled with G protein. The pituitary polypeptide POMC (pro-opiomelanocortin) is cleaved to give rise to different peptide hormones, including MSH (melanocyte-stimulating hormone), that acts through this receptor, enhancing cAMP and therefore stimulating tyrosinase enzyme activity. As a fact, keratinocytes can also produce these molecules. The synthesis of dark eumelanin and yellow cysteine-abundant pheomelanin is augmented by MSH. It was observed that people with MC1R variations evolve to earlier melanoma [52]. New studies have revealed that MITF (microphthalmia-associated transcription factor) gene found on 3p14 chromosome has a role in the promotion of melanocyte cell cycle progression [53].

### 3. The most important biomarkers for the identification of malignant melanoma

In order to diagnose malignant melanoma, there are several methods and techniques that when combined can give a certain degree of surety to the diagnosis.

As previously discussed, the first step in diagnosing malignant melanoma is the visual inspection of the lesion and the assessment of the well-known ABCDE signs (Asymmetry, irregular Borders, uneven Color, large Diameter, Evolution) [54]. These findings are combined with dermatoscopical inspection of the lesion in order to confirm or to infirm the naked eye observations. If these findings lead to a suspicion of malignancy, then most likely a wide-excision surgery is performed and upon the assessment, a sentinel lymph node biopsy is also performed

[55]. In order to establish a certain diagnosis, there are various sets of markers specific for this type of cancer, which enable the obtaining of a definite diagnosis and/or the prognosis and staging of the disease, which can be identified by several methods [56].

There are different types of biomarkers to investigate malignant melanoma such as the microscopical markers identified upon the examination of the tumor and the immunohistochemical biomarkers identified upon isolation from the tumor/tissue section. Another type of malignant melanoma markers is the serological biomarkers that are identified from the peripheral blood samples of the patients by various methods.

#### 3.1. Microscopical biomarkers in malignant melanoma

Impairment in tumor-suppressor genes such as mutation, deletion, translocation, or activation of proto-oncogenes has a significant role in mutagenesis. Mutation or deletion of CDKN2A antioncogene and CDK4 proto-oncogene is related to the development of familial melanoma, which constitute 8–12% of total melanomas [42]. This type of melanoma is characterized by an abnormal regulation of cell cycle by cyclin-dependent enzymes. In this case, the 9p21 chromosome has a germline mutation in the locus of CDKN 2A gene, affecting two proteins p16 and p14 or Arf that have a suppressive role. The p16 protein has an inhibitory effect on cyclin D/CDK4 and CDK6, influencing therefore pRB (retinoblastoma protein) which, through liberation of E2F, change the G1 phase of cell cycle into S phase [43, 44]. It was discovered lately that E2F1 protein, as well, manifests a suppressor function through p53 or p73. Between 10 and 30% of familial melanoma cases are due to CDKN2A mutation. NRAS and p16 mutations are also incriminated in some isolated cases of melanoma. Mutation of CDKN2A can lead to multiple melanomas. This alteration seldom appears in actinic keratosis, skin carcinomas, and other types of cancers [45, 46].

The p14 protein, encoded by CDKN2A reading frame, affects the p53 protein through the influence on other protein, Mdm2. The p14 deprivation lowers the activity of p53 [47]. Studies taken on mice models have shown that p14 can determine apoptosis of oncogene-activated

It is known that pigmentation of the skin that depends on the amount of melanin is in direct relation with the development of melanoma. It is 10 times more frequently in whites than in blacks; however, people affected by albinism are exceptionally affected. The highly polymorphic melanocortin 1-receptor gene (MC1R) situated on 16q24 chromosome controls sun sensitivity [50, 51]. It codifies the transmembrane receptor coupled with G protein. The pituitary polypeptide POMC (pro-opiomelanocortin) is cleaved to give rise to different peptide hormones, including MSH (melanocyte-stimulating hormone), that acts through this receptor, enhancing cAMP and therefore stimulating tyrosinase enzyme activity. As a fact, keratinocytes can also produce these molecules. The synthesis of dark eumelanin and yellow cysteine-abundant pheomelanin is augmented by MSH. It was observed that people with MC1R variations evolve to earlier melanoma [52]. New studies have revealed that MITF (microphthalmia-associated transcription factor) gene found on 3p14 chromosome has a role in the promotion of melanocyte cell cycle progression [53].

3. The most important biomarkers for the identification of malignant

when combined can give a certain degree of surety to the diagnosis.

In order to diagnose malignant melanoma, there are several methods and techniques that

As previously discussed, the first step in diagnosing malignant melanoma is the visual inspection of the lesion and the assessment of the well-known ABCDE signs (Asymmetry, irregular Borders, uneven Color, large Diameter, Evolution) [54]. These findings are combined with dermatoscopical inspection of the lesion in order to confirm or to infirm the naked eye observations. If these findings lead to a suspicion of malignancy, then most likely a wide-excision surgery is performed and upon the assessment, a sentinel lymph node biopsy is also performed

melanocytes [48, 49].

218 Natural Products and Cancer Drug Discovery

melanoma

Breslow thickness refers to the thickness of the tumor and was demonstrated to be the most important prognostic histopathological factor. This parameter is of great importance and is used to stage malignant melanoma [57].

Tumor ulceration is the loss of epidermal integrity of the melanoma and is used for staging malignant melanoma and was linked to rapid tumor growth and metastasis [58].

The mitotic rate refers to the number of mitoses per square millimeter. A high mitotic rate is correlated with high metabolic cell activity and predicts rapid tumor growth and metastasis. Also, increased mitotic rate was linked with decreased survival [59].


#### 3.2. Immunohistochemical and serological biomarkers in malignant melanoma

#### Immunohistochemical biomarkers


### 4. Recent studies concerning active phytocompounds targeting malignant melanoma

Although synthesized to serve the plant in modulating its interaction with the environment, many natural compounds act as drugs when used by humans by binding to various enzymes and receptors. In the area of anticancer medications, it has been shown that about 75% of the medications are naturally derived or inspired [86]. Phytocompounds hold an important potential in the chemoprevention and treatment of melanoma, including metastasis [87, 88]. The main advantage of natural products relies in their multi-target activity, with one compound being able to interact with multiple proteins and receptors. Antimelanoma activities have been pointed out for both phytocompounds from nutraceuticals (polyphenols, polysaccharides, and carotenoids), as well as for highly active secondary metabolite classes like the alkaloids or the terpenes. Additionally, there are a number of crude extracts or enriched fractions with anti-melanoma actions, for which the active compound(s) could not be singled out yet. A promising field in the actual usage of plantderived compounds is their synergistic activity when administered in combination with synthetic anticancer medications [89].

### 4.1. Polyphenols

Immunohistochemical biomarkers

220 Natural Products and Cancer Drug Discovery

2.8 Chondroitin sulfate proteoglycan 4

2.10 P53 (cellular tumor antigen p53)

Serological biomarkers 3.1 LDH (lactate dehydrogenase)

3.2 CRP (C-reactive

3.3 MIA (melanomainhibiting activity)

3.4 VEGF (vascular endothelial growth

protein)

factor)

3.6 miRNA (MicroRNAs)

3.11 CTC (circulating tumor cells)

3.12 Melanomainitiating cells

2.9 EFGR (epidermal growth factor receptor)

2.7 Vimentin Ectodermal, neural, and pancreatic progenitor

anaerobic conditions [77]

cancer [65]

[77, 78]

angiogenesis [78]

3.5 BRAF Raf kinases family involved in RAF-MEK-ERK

3.7 GM3 Ganglioside (glycosphingolipid) involved in

3.8 OPN (osteopontin) Glycol-phosphoprotein implicated in reduction

3.10 YKL-40 Heparin secreted during the late stages of cell differentiation [84]

marrow [82]

cell marker, overexpression in cancer [72]

Tumor suppressor "guardian of the genome" (promotes cell cycle arrest, apoptosis, senescence, and DNA repair) [75, 76]

Enzyme that converts lactate into pyruvate in

Nonspecific marker of inflammation, infection,

Protein associated with in vitro increased invasiveness, extravasation, and metastasis [77]

Protein that stimulates vasculogenesis and

signal transduction pathway (cell growth, proliferation, and differentiation) [79]

RNA molecules involved in proliferation, differentiation, stress responses, apoptosis

cell proliferation, differentiation, apoptosis, embryogenesis, and oncogenesis [81]

of apoptosis, promotion of tumor growth, tumor-promoting stromal cell in the bone

3.9 IL-8 (interleukin-8) Chemokine produced by malignant cells [82] Associated with tumor burden, stage

Located in melanocytes, promotes cell adhesion, growth, migration, motility [65]

Biomarker type Significance/location Specificity/involvement in prognosis

BRAF transmembrane tyrosine kinase [73] Progression and metastasis [74]

Tumor growth, invasion, and poor prognosis

Inactivated or functionally impaired in

High levels are correlated with metastasis, decreased survival rate, negative outcome after

Predictor for disease progression in high levels

Elevated levels, marker for advanced stage (III

Elevated levels linked to poor progression-free

BRAF mutation, BRAF-V600E, associated with increased tumor thickness, ulceration, and

Disease progression and risk of recurrence

Low levels (early stage) linked to invasive proliferation; higher levels (late stage) linked to

Rapid melanoma growth, angiogenesis, and

of the disease, survival, and response to

for selecting the systemic therapy [77]

Overexpression associated with poor survival

cell migration and invasion [81]

aggressive metastasis [83]

and IV) and poorer prognosis [77]

Invasion and metastasis [65]

[72]

melanoma [75]

treatment [77]

survival [78]

[77, 78]

therapy [82]

[84]

Cells from melanoma metastatic tumors [77] Linked to inferior survival; predictive marker

Cancer stem cells [85] Can lead the specific targeted systemic therapy [85]

reduced survival [80]

[65]

The potential of polyphenolic compounds (Figure 1) as anti-melanoma agents was recognized as early as 1995, when Menon and coworkers found that orally administered curcumin, catechin, and rutin significantly prolonged the life span of animals with experimental melanoma [90]. The anticancer potential of these food constituents is supported by epidemiological studies [91]. Their general mechanisms of action may be summarized as follows: antioxidative, photoprotectant, induction of apoptosis, and inhibition of angiogenesis. Antioxidative capacities are seminal for the protective activity against UV-induced oxidative stress [92]. The ability to act as UV filters is related to the physiologic function of certain flavonoids present in epicuticular waxes protecting the plant epidermis against UV radiation [93]. Apoptosis is mainly promoted by polyphenols via regulation of Bcl-2 family proteins and caspase-3 activation, while the anti-migratory effects are mainly obtained by down-regulation of MMPs expression.

Intensely researched polyphenols in the context on melanoma include flavonoids (quercetin, apigenin, silymarin, genistein, catechins, anthocyanins, biflavonoids), curcumin, and resveratrol [94]. Quercetin (1), an abundant dietary flavonol, acts via all above-cited mechanisms. It decreases the viability and promotes the apoptosis of ultraviolet UVB-irradiated B16F10 melanoma cells by several mechanisms: it increases the levels of reactive oxygen species, depolarizes mitochondrial membrane potential, induces the imbalance of calcium homeostasis, attenuates MEK-ERK signaling, and changes the ratio of Bcl-2, Bax, and Bim expression in favor of cell death elicitation [95]. It offers photoprotection against melanogenesis through a regulatory effect on the Nrf2-ARE pathway [96]. Furthermore, quercetin inhibits melanoma cell migration in a setup of experimentally activated receptor tyrosine kinase c-Met, known to be involved in the acquisition of metastatic phenotypes [97].

Figure 1. Chemical structure of representative polyphenols targeting malignant melanoma. 1: quercetin, 2: (–)-epigallocatechin-3-gallate, 3: curcumin; 4: resveratrol.

Apigenin, a flavone from the point of view of chemical structure, offers an effective photoprotection against UVA- and UVB-induced skin carcinogenesis [98], fights metastasis by preventing the activation of MMP-2, MMP-9, VEGF, and Twist1 genes through down-regulation of STAT2 signaling [99], promotes apoptosis via caspase-3 and cleaved poly(ADP-ribose) polymerase [100]. In addition, apigenin blocks the COX-2 pathway and increases terminal differentiation in the epidermis [101].

Silymarin, a complex of flavonolignans from the achenes of milk thistle, is a natural product known especially for its hepatoprotective, anti-oxidative and anti-inflammatory effects [102]. When applied locally, silymarin was able to inhibit carcinogenesis induced by UV-radiation and to decrease tumor multiplicity and growth. It produces cell cycle arrest in melanoma cells via inhibition of kinase activities of (MEK)-1/2 and RSK-2 [103], induces apoptosis and counteracts metastasis via down-regulation of MMPs expression [104]. Silymarin is a prominent inhibitor of Wnt/β-catenin translocation in melanoma cells, beside few other phytocompounds like the flavonoid fisetin, the triterpene lupeol, and the alkaloid tryptanthrin; aberrant Wnt signaling occurs in one-third of melanomas [105].

Catechins are flavanols devoid of the 3-oxo group specific to other flavonoids; they may especially be found in woody plants like wine and the tea plant. Catechins as well as their esters with gallic acid, in particular epigallocatechin-3-gallate (EGCG, 2), are the subject of intense research in cancer prevention and treatment. In the field of anti-melanoma activities, the chemopreventive effect of ECGC is mainly achieved by a photoprotective effect exerted through interleukin 12 induction followed by DNA repair, inhibition of angiogenesis, and the stimulation of cytolytic T cells [106]. The proapoptotic effects are exerted by EGCG through down-regulation of inhibitory proteins such as Bcl-2, D1, and cdk2, as well as the up-regulation of the proapoptosis protein Bax. Important tumor-suppressor proteins (p16, p21, and p2) are induced by EGCG [107, 108]. More recently, the discussed polyphenol was shown to bind to the cell surface receptor 67LR, overexpressed in melanoma [109]. As an agonist of 67LR, EGCG is able to suppress melanoma tumor growth by activating a specific signaling pathway (cAMP/PKA/PP2A) via regulation of miRNA-let-7b expression in melanoma cells [110]. Moreover, EGCG displays an excellent antimetastatic profile, reducing the number of lung metastases in mice [111]. Molecular targets in this regard are COX-2, PGE2 receptors, the impairment of epithelial-to-mesenchymal transition [112], reduction of angiogenesis promoters and down-regulation of MMP2 activity and ERK1/2 pathway [113].

Curcumin (3), an orange-colored constituent of turmeric rhizomes (Curcuma sp.), is an extensively investigated compound with anti-inflammatory and anticancer activities. In human melanoma cell lines (A375 and C8161), it reduces cell viability, has an antiproliferative effect arresting mitosis at the transition of G2/M phase, suppresses cell invasion, and induces autophagy [114]. The anti-metastatic effect was demonstrated in vivo using C57BL/6 mice; the down-regulation of metalloproteinases and collagenases activities, modulation of integrin receptors [115] and interference with the STAT3 pathway support this observation [116].

Resveratrol (4), a stilbene found mainly in grapes and Polygonum cuspidatum, inhibits melanoma proliferation in vitro, arresting cell divisions at the G1/S transition and induces apoptosis targeting Bcl-2-associated X protein, B-cell lymphoma 2, and caspases -9 and -3 [117]. Through the suppression of β-catenin and STAT3-pathway, resveratrol reduces the levels of survivin, a protein which is essential for the survival of melanoma cells. Furthermore, the compound has anti-migratory activities mediated by the deactivation of the proto-oncogenic Akt [118]. The low bioavailability of resveratrol triggered the obtainment of several analogs, as well as the preference for the use of the natural pterostilbene having a longer half-life [87].

### 4.2. Polysaccharides

Apigenin, a flavone from the point of view of chemical structure, offers an effective photoprotection against UVA- and UVB-induced skin carcinogenesis [98], fights metastasis by preventing the activation of MMP-2, MMP-9, VEGF, and Twist1 genes through down-regulation of STAT2 signaling [99], promotes apoptosis via caspase-3 and cleaved poly(ADP-ribose) polymerase [100]. In addition, apigenin blocks the COX-2 pathway and increases terminal differentiation in

Figure 1. Chemical structure of representative polyphenols targeting malignant melanoma. 1: quercetin, 2: (–)-epigallo-

Silymarin, a complex of flavonolignans from the achenes of milk thistle, is a natural product known especially for its hepatoprotective, anti-oxidative and anti-inflammatory effects [102]. When applied locally, silymarin was able to inhibit carcinogenesis induced by UV-radiation and to decrease tumor multiplicity and growth. It produces cell cycle arrest in melanoma cells via inhibition of kinase activities of (MEK)-1/2 and RSK-2 [103], induces apoptosis and counteracts metastasis via down-regulation of MMPs expression [104]. Silymarin is a prominent inhibitor of Wnt/β-catenin translocation in melanoma cells, beside few other phytocompounds

the epidermis [101].

catechin-3-gallate, 3: curcumin; 4: resveratrol.

222 Natural Products and Cancer Drug Discovery

The potential of polysaccharide fractions obtained from terrestrial and marine organisms in cancer treatment receives an increasing attention. These compounds are potent immunomodulators, leading to the release of cytokines which may play important roles in the defense against cancer [119]. Their main mechanism of action includes activation of natural killer cells, induction of inflammatory caspases, and down-regulation of MMPs expression, but as a general feature their mechanism of action is much less known than in the case of plant polyphenols. An important advantage of polysaccharides is their very low toxicity.

The sulfated and partially acetylated fucoidans from brown algae Coccophora langsdorfii and Fucus evanescens, consisting of (1!3)- and (1!4)-linked α-l-fucopyranose residues, produced a significant inhibition of colony formation of SK-MEL-5 and SK-MEL-28 melanoma cells [120].

Beta-glucans are constituents of cell walls in certain fungi, algae, and vascular plants such as oat or barley. Oral administration of yeast-derived beta-glucan to mice inoculated with B16 murine melanoma cells significantly reduced tumor weight, pulmonary metastasis, and survival rate. These effects are mediated by the activation of NK cells. An important finding was that beta-glucan supplementation was devoid of hematopoietic toxicity, unlike chemotherapeutic drugs such as 5-fluorouracil [121]. Low-molecular-weight beta-glucan prepared from oat decreased the cell viability of cancer cell lines Me45 and A431, while normal keratinocytes were not affected. The cytotoxic effect was mediated by induction of caspase-12 expression. This natural product had the advantage of a good solubility in water and a low viscosity, in comparison with other glucans [122]. Polysaccharides from higher plants, like raspberry fruits, have as well been shown to inhibit melanoma growth, and to have a synergistic activity with docetaxel in vivo, reducing at the same time liver and kidney injuries inflicted by the latter [123]. Pectins from corn demonstrated their ability to inhibit cancer cell growth and metastasis by modulating specific markers such as galectin-3, VEGF, MMP-2 and MMP-9, and NF-κB [124].

### 4.3. Volatile oils

Volatile oils are complex mixtures of monoterpenes, sesquiterpes, aromatic compounds, and their derivatives, which are produced by plants in order to protect themselves against pathogens, herbivores, or, on the contrary, to act as signals for pollinators and disseminators [125]. In the search for innovative strategies in melanoma treatment, these natural products have as well been investigated. The main mechanisms of action known so far are represented by induction of apoptosis (via increasing the expression of p53 and caspase-3) and reduction of angiogenesis with down-regulation of MMPs.

The volatile oil of the tea tree plant (Melaleuca alternifolia) and its main component terpinen-4-ol (5) were evaluated in an experimental setting of human melanoma M14 WT cells and M14 adriamycin-resistant cells. Concentration as low as 0.005–0.03% proved to induce apoptosis, and conversely the resistant cell variant was more sensitive [126]. More recently, Greay and coworkers [127] applied a topical formulation containing 10% tea tree oil and found a significant delay in the growth of established subcutaneous B16-F10 melanoma, while systemic toxicity was absent. The volatile oil of Tridax procumbens, a common annual Asteraceae in most tropical regions, displayed anti-metastatic effects by the inhibition of lung nodule formation by B16F-10 cells in C57BL/6 mice. Furthermore, it induced apoptosis increasing the expression of p53 and caspase-3 and reduced angiogenesis [128]. Curcuma zedoaria volatile oil suppressed as well melanoma growth and lung metastasis, with the down-regulation of MMPs [129].

### 4.4. Alkaloids

against cancer [119]. Their main mechanism of action includes activation of natural killer cells, induction of inflammatory caspases, and down-regulation of MMPs expression, but as a general feature their mechanism of action is much less known than in the case of plant poly-

The sulfated and partially acetylated fucoidans from brown algae Coccophora langsdorfii and Fucus evanescens, consisting of (1!3)- and (1!4)-linked α-l-fucopyranose residues, produced a significant inhibition of colony formation of SK-MEL-5 and SK-MEL-28 melanoma

Beta-glucans are constituents of cell walls in certain fungi, algae, and vascular plants such as oat or barley. Oral administration of yeast-derived beta-glucan to mice inoculated with B16 murine melanoma cells significantly reduced tumor weight, pulmonary metastasis, and survival rate. These effects are mediated by the activation of NK cells. An important finding was that beta-glucan supplementation was devoid of hematopoietic toxicity, unlike chemotherapeutic drugs such as 5-fluorouracil [121]. Low-molecular-weight beta-glucan prepared from oat decreased the cell viability of cancer cell lines Me45 and A431, while normal keratinocytes were not affected. The cytotoxic effect was mediated by induction of caspase-12 expression. This natural product had the advantage of a good solubility in water and a low viscosity, in comparison with other glucans [122]. Polysaccharides from higher plants, like raspberry fruits, have as well been shown to inhibit melanoma growth, and to have a synergistic activity with docetaxel in vivo, reducing at the same time liver and kidney injuries inflicted by the latter [123]. Pectins from corn demonstrated their ability to inhibit cancer cell growth and metastasis by modulating specific markers such as galectin-3, VEGF, MMP-2

Volatile oils are complex mixtures of monoterpenes, sesquiterpes, aromatic compounds, and their derivatives, which are produced by plants in order to protect themselves against pathogens, herbivores, or, on the contrary, to act as signals for pollinators and disseminators [125]. In the search for innovative strategies in melanoma treatment, these natural products have as well been investigated. The main mechanisms of action known so far are represented by induction of apoptosis (via increasing the expression of p53 and caspase-3) and reduction of

The volatile oil of the tea tree plant (Melaleuca alternifolia) and its main component terpinen-4-ol (5) were evaluated in an experimental setting of human melanoma M14 WT cells and M14 adriamycin-resistant cells. Concentration as low as 0.005–0.03% proved to induce apoptosis, and conversely the resistant cell variant was more sensitive [126]. More recently, Greay and coworkers [127] applied a topical formulation containing 10% tea tree oil and found a significant delay in the growth of established subcutaneous B16-F10 melanoma, while systemic toxicity was absent. The volatile oil of Tridax procumbens, a common annual

phenols. An important advantage of polysaccharides is their very low toxicity.

cells [120].

224 Natural Products and Cancer Drug Discovery

and MMP-9, and NF-κB [124].

angiogenesis with down-regulation of MMPs.

4.3. Volatile oils

Alkaloids are nitrogen-containing plant metabolites with usually very intense pharmacologic effects at even low dosages. Consequently, their safety profile is very different from the one of compounds discussed above. The main compounds from this class, which are able to target melanoma, include berberine, harmine, paclitaxel, and glycoalkaloids from Solanaceae. While the number of reports on the anti-melanoma potential of alkaloids is rather scarce in comparison with that of polyphenols, the mechanisms of action are well studied. The anti-migratory properties of alkaloids in malignant melanoma have been the subject of a comprehensive review [87]. Alkaloids may induce apoptosis via the inhibition of the Ras-signaling cascade at various downstream points, blocking both PI3K/AKT and MAP/ERK pathways. Metastasis is mainly counteracted by blocking the COX pathway and angiogenesis. NF-κB inhibition is as well a target of anti-melanoma alkaloids.

Berberine, an isoquinoline alkaloid (6), occurs in several medicinal plants of the Berberidaceae, Ranunculaceae, and Papaveraceae families [130]. While best known for antimicrobial, antiinflammatory, anti-hyperglycemic, and anti-hyperlipidemic effects [131], berberine is one of the most active alkaloids in melanoma. It has been shown to inhibit kinases like PI3K, ERK, and GSK3β [132]. Moreover, in combination with doxorubicin, it intensely reduces proliferation and increases apoptosis, effect demonstrated both in vitro and in vivo [133]. Berberine inhibits the migration of melanoma cells via the reduction of COX-2/PGE2 receptors expression [134] and has anti-angiogenetic properties [135]. Harmine is an indole alkaloid contained by only a few plant species, including Peganum harmala and Banisteriopsis caapi. While its ability to bind monoamino-oxidase A and to elicit psychoactive effects is known since several decades, the potential anticancer properties of this compound have only been explored in recent years. In melanoma, it displays important anti-migratory effects through the inhibition of NF-κB and other transcription factors (CREB, ATF-2) [136]; it reduces lung metastasis in C57BL/6 mice and down-regulates the expression of pro-metastatic genes (MMP-9, ERK, and VEGF) [137].

Paclitaxel is a pseudoalkaloid, first isolated from the bark of the Pacific yew (Taxus brevifolia), approved for the clinical treatment of several solid tumors with poor prognosis including melanoma [138]. It stabilizes microtubules, disrupting the dynamic equilibrium between free and polymerized tubulin; as a consequence, cell division is arrested in the G2/M transition of mitosis [139]. Additionally, paclitaxel has antiangiogenic effects, which are potentiated by COX-2 inhibitors [140]. Disadvantages of this compound include its extreme hydrophobicity, which could be overcome in modern nanoformulations, as well as drug resistance to paclitaxel due to the induction of the efflux protein P-gp and unusual expression of class III isotype of βtubulin [139].

Glycoalkaloids from Solanaceae, including α-solanine and solamargine, displayed antimelanoma effects in recent reports. Interestingly, solamargine inhibited selectively the growth of WM239 and WM115 melanoma cells, in comparison to benign WM35 cells. Cellular necrosis occurred upon permeabilization of the lysosomal membrane, which triggered, in turn, the extrinsic pathway of mitochondrial destruction. The intrinsic apoptosis pathway was as well disrupted by this compound [141]. α-Solanine displays anti-proliferative effects, induces apoptosis, and suppresses melanoma cell invasion, mainly via the reduction of MMP-2 and MMP-9 activities. The potential of this compound in the treatment of metastatic melanoma is further substantiated by the inhibition of JNK and PI3K kinases as well as the reduction of NF-κB activity [142]. Two chemical structures of representative natural compounds targeting melanoma, other than polyphenols, are presented in Figure 2.

Figure 2. Chemical structures of representative natural compounds targeting melanoma, other than polyphenols. 5: Terpinen-4-ol, the major monoterpene in tea tree volatile oil, 6: Berberine, an intensely researched alkaloid.

### 5. Modern physicochemical formulations for the enhancement of bioavailability of selected active phytochemicals with chemopreventive activity for malignant melanoma

Formulation development can be regarded as a determining aspect in the delivery of antineoplastic natural compounds due to their physicochemical properties, in particular water solubility, which limit their bioavailability [143]. A variety of formulation strategies have been reported highlighting the main challenges that arise in the development of optimized drug delivery in the anticancer field [144], including skin cancer. The most studied natural anticancer compounds in terms of bioavailability improvement are different types of polyphenols (curcumin, silymarin, and resveratrol) and alkaloids (paclitaxel). In Tables 1–4, we aim to summarize the main reported attempts to increase the bioavailability of the above-mentioned compounds through various modulations of their physicochemical parameters as well as modern pharmaceutical formulations.


due to the induction of the efflux protein P-gp and unusual expression of class III isotype of β-

Glycoalkaloids from Solanaceae, including α-solanine and solamargine, displayed antimelanoma effects in recent reports. Interestingly, solamargine inhibited selectively the growth of WM239 and WM115 melanoma cells, in comparison to benign WM35 cells. Cellular necrosis occurred upon permeabilization of the lysosomal membrane, which triggered, in turn, the extrinsic pathway of mitochondrial destruction. The intrinsic apoptosis pathway was as well disrupted by this compound [141]. α-Solanine displays anti-proliferative effects, induces apoptosis, and suppresses melanoma cell invasion, mainly via the reduction of MMP-2 and MMP-9 activities. The potential of this compound in the treatment of metastatic melanoma is further substantiated by the inhibition of JNK and PI3K kinases as well as the reduction of NF-κB activity [142]. Two chemical structures of representative natural compounds targeting mela-

5. Modern physicochemical formulations for the enhancement of

5: Terpinen-4-ol, the major monoterpene in tea tree volatile oil, 6: Berberine, an intensely researched alkaloid.

bioavailability of selected active phytochemicals with chemopreventive

Figure 2. Chemical structures of representative natural compounds targeting melanoma, other than polyphenols.

Formulation development can be regarded as a determining aspect in the delivery of antineoplastic natural compounds due to their physicochemical properties, in particular water solubility, which limit their bioavailability [143]. A variety of formulation strategies have been reported highlighting the main challenges that arise in the development of optimized drug delivery in the anticancer field [144], including skin cancer. The most studied natural anticancer compounds in terms of bioavailability improvement are different types of polyphenols (curcumin, silymarin, and resveratrol) and alkaloids (paclitaxel). In Tables 1–4, we aim to summarize the main reported attempts to increase the bioavailability of the above-mentioned compounds through various modulations of their physicochemical parameters as well as

noma, other than polyphenols, are presented in Figure 2.

activity for malignant melanoma

modern pharmaceutical formulations.

tubulin [139].

226 Natural Products and Cancer Drug Discovery



Curcumin Sustained-release

MicroActive

formulation):



acids



Curcumin

þ

Meriva®,

Oral albino rats, 340 mg/kg

administration

 to male Wistar

Reduced curcumin

gastrointestinal

Significant increased

(compared

Indications: studies targeting sites other than the

gastrointestinal

 tract

Chemoprophylaxis

intervention

 to an

plasma/liver

unformulated

 curcumin);

 levels

 mucosa;

concentration

 in the

[150]

(patented complex of curcumin with soy

phosphatidylcholine):







 20%

curcuminoids

Randomized,

crossover human study in healthy

volunteers (hard gel capsules,

376 mg

curcuminoids)

double-blind,


absorption compared to

standard curcumin, 5.8-fold increase over a

phytosome

over

(curcumin

from turmeric rhizome)

formulation

Selectively delivered to

spleen and bone marrow;

Potential intravenous

for antioxidant

therapies

 and

anti-inflammatory

 delivery system

macrophages

 in

[152]

þ

volatile oils

formulation,

 34.9-fold increase

unformulated

[151]

Curcumin

þ PVP (polyvinylpyrrolidone)




 agents

(polyvinylpyrrolidone)

þ

cellulosic

derivatives

þantioxidant

Lipid based

phospholipid

nanospheres

vesicles/lipid-

(molar ratio

DMPC =

10/1/0.06/1.9):

1,2-Dimyristoyl-sn-

glycero-3-phosphocholine;

SA = l-glutamic acid, N-(3-

carboxy-1-oxopropyl)-,

dihexadecyl

 ester);

 1,5-

nanoparticles:

Phospholipid

 vesicles:

IV mg/kg)

administration

 to male Wistar rats (2


 agents

microcrystalline

 cellulose

demethoxycurcumin

bisdemethoxycurcumin

phosphatidylcholine

curcuminoid

 mixture

phosphatidylcholine

 cellulose

triglycerides

 esters of fatty

curcuminoids

®

Curcumin

(patented

Oral capsules healthy humans, 500 mg curcumin

(single dose)

administered

 to

9.7-fold increase in

period: 12 h)

bioavailability

 (test

[149]

228 Natural Products and Cancer Drug Discovery

formulation

 of curcumin

formulations

Components

Method of application

Indications/remarks


elimination.

degradation

 and

Ref.

Table 1. Curcumin formulations.




Silymarin Self-microemulsifying

system (SMEDDS) Semisolid dispersion binary system using

Silymarin:Gelucire

Oral Zealand albino rabbits (280 mg/kg)

administration

 to New

> 1:1 and 1:3 (w/w)

Gelucire 44/14 as carrier

Solid dispersion of silymarin with a


Influence of preparation

methodology

of silymarin

 of solid dispersions





co-precipitation

preparation)

Silymarin loaded chitosan

nanoparticles

 -40 mg chitosan

Oral Albino mice (CCl4

model), 50 mg/kg

administration

 to Swiss hepatotoxicity




tripolyphosphate


(for pH adjustment)


gelation method of

> Solid dispersion and silica

of silymarin with

material and release enhancer

nanoparticles


dispersion:


K30:soy bean lecithin:

acrylic resin IV:

1–3:0.3–0.8:0.2–0.5


 (w:w)

slow-release

 matrix

 solution (ionic preparation)

Oral

administration

 in Beagle dogs Continuous

 controlled release for 72 h;

[159]

Half-life 2.3 times longer

 method of

hydrophilic

 polymer (HPMC E15 LV)

 drug delivery

formulations

Components -10% ethyl linoleate


 EL

Method of application

Oral mongrel dogs (50 mg/kg, single

dose)

administration

 to male

3 months stability;

Faster release; 2.2-fold enhanced oral absorption,

bioavailability;

Indication: Oral Linear increase of silymarin solubility and

dissolution

 degree depending

concentration

 -

pharmacokinetic

Co-precipitation

soluble and stable amorphous

dispersion;

The best results: 1:1 for the kneading method

and 1:3 for other methods; Increased silymarin dissolution:

precipitation

fold)

Mean dissolution

precipitation,

17.4 min for kneading

Sustained release

Improved

pharmacological

 activity

[158]

 14.2 min for spray drying and

 time: 8.3 min for co-

>

kneading (1.5-fold);

 (2.5-fold)

> spray drying (1.9-

 co-

 method revealed the best

[157]

 solid

 profile

>

significant superior

 on the career

[156]

administration

 of silymarin

230 Natural Products and Cancer Drug Discovery

 increased

Indications/remarks

Ref.

[155]



Paclitaxel

Paclitaxel

(microemulsion/Taxol®)

silymarin

þ

formulations

Components




(insufficient

 data)

microemulsion

Method of application

Oral -Taxol®

þ10–20 mg/kg

silymarin;


microemulsion

þ20 mg/kg

silymarin

Paclitaxel

curcumin

Paclitaxel cyclosporin

 A

(Genetaxyl®)

þ

Genetaxyl®

(paclitaxel 6 mg/mL)

þ

cyclosporin

Genetaxyl®:


Genaxol®:


Paclitaxel

Crystalline

Self micro emulsifying

formulations

þcyclosporine

 A

 (SMEOFs)

 oily

Paclitaxel 1.5% (% w/v):



nanosuspension

 -paclitaxel (20 mg/mL): -0.1% (w/w) Cremophor

phosphate saline

 EL in

xenograft female

SCID-beige

(20 mg/kg)



administration

 to humans [170]

 knockout mice [169];

administration

 to wild-type and

Satisfying oral

exposure and oral

comparable

 to Taxol® (40%);

bioavailability

 -

bioavailability

 of paclitaxel was

> the systemic

[169],

[170]

 mice

IV

administration

 to tumor bearing

Weaker Cremophor

 standard

antiproliferative

 effects than the

[168]

formulations

þ

Cremophor

 EL

 Taxol®: Paclitaxel in Cremophor

dehydrated

 ethanol (1:1, v/v)

 EL –

cancer patients

Intraperitoneal

administration

 to

↓ Plasma

Prolonged peritoneal activity

concentration

 and

bioavailability;

[167]

 EL

 EL

 A;

(nanoemulsion)

þ



(insufficient

 data)

nanoemulsion

Oral bearing nu/nu mice

(50 mg/kg

curcumin

þ20 mg/kg

paclitaxel)

Oral (60/120/180

A, 10 mg/kg

IV: 175 mg/m2

3 h infusion

,

 mg/m2) þoral cyclosporin

administration

 to cancer patients

Less than

exposure with increasing doses;

Limited paclitaxel

entrapment;

Cremophor

EL ↓ -<sup>↓</sup> median time to peak

concentration;


absorption of

paclitaxel;


metabolism

 of

concentration:

bioavailability

 by micellar

proportional

 increase in systemic

[166]

administration

 to SKOV3 tumor-

5.2-fold increased oral relative

anti-tumor

 activity of paclitaxel (3.2-fold enhanced

accumulation

 in the tumor tissue)

bioavailability

 and

[165]

administration

 to rats:

Silymarin association

bioavailability

The highest absolute

silymarin

Faster absorption for paclitaxel

compared to Taxol®

þ

paclitaxel

microemulsion;

microemulsion

232 Natural Products and Cancer Drug Discovery

 of paclitaxel; bioavailability

 (19%) -

> for

 increased the oral

Indications/Remarks

Ref.

[164]



Paclitaxel

formulations

Components -Cosurfactant:


ether/ 300, 1:1( w/w)

Organic phase:

In vitro studies

Diffusion controlled release;

[173]

234 Natural Products and Cancer Drug Discovery

Low toxicologic

 profile;

Gastrointestinal

Suitable formulation

 stability

 for oral drug delivery




0.005



Water phase: -ultrapure water (mL) q.s. 10

(niosomes prepared through

film hydration method)


In vitro studies

Longer half life, slower release rate;

4.5-fold increase in cytotoxicity melanoma SK-MEL-3 cell line, in comparison

the free drug; Possible alternative to the

therapy

conventional

 on human

[174]

 with




(1:6.5:9:28, w/w) -deionized water and ethanol

99.4% (67.5:32.5, v/v) (pegylated ethosomes

prepared by reverse phase

evaporation

(mg/g):

Oral Dawley rats

(10 mg/kg)

administration

 to male Sprague-

3-fold higher oral

Taxol®-verapamil

(=P-glycoprotein

Potential formulation

bioavailability

association

 of the drug, avoiding verapamil

 inhibitor)

 to augment the oral

association);

bioavailability

 (same results for

[175]






> Solid lipid

modified with

cyclodextrin

 (HPCD)

nanoparticles

 surface-


Oral Dawley male rats (25 mg/kg)

administration

 to male Sprague-

Oral -fast release of paclitaxel

(nearly complete

release);


administration:

[176]




 188

hydroxypropyl-β-

Solutol®HS15

Paclitaxel-loaded

nanocapsules

 lipid

 technique)

Paclitaxel-loaded

ethosomes

 pegylated

 (Ml) – 10;

 (M) –

 (M) – 0.0475

Niosomal Niosomes with nonionic surfactants

formulation:

polyethylene

 glycol

 glycol monoethyl

Method of application

Indications/Remarks

Ref.


Table 3. Paclitaxel formulations.


Paclitaxel

formulations

Components


(Poly(lactic-co-glycolic

acid))


Aqueous phase: -PVA (polyvinyl alcohol)

1%, 3%, 5% (w/v) (emulsification-solvent

diffusion method)

 -lack of data

Oral/IV

administration

 in rats/mice

 Prolonged High oral absorption;

Optimized

administration;

Indication: delivery vehicles (carriers) for oral or

intravenous

 paclitaxel

administration

pharmacokinetic

 profile after IV

gastrointestinal

 release;

[180]

(solvent/evaporation

technique)

Stabilized Pluronic micelles

Table 3.

Paclitaxel

formulations.

Method of application

Indications/Remarks

Higher line when compared to blank

similar to paclitaxel solution

antiproliferative

 activity on Caco-2-cell-

nanoparticles

 but

236 Natural Products and Cancer Drug Discovery

Ref.


Table 4. Resveratrol formulations.

### Author details

Danciu Corina<sup>1</sup> \*, Soica Codruta<sup>2</sup> , Antal Diana<sup>3</sup> , Alexandra Popescu<sup>4</sup> , Roxana Ghiulai<sup>2</sup> , Ioana Zinuca Pavel<sup>1</sup> , Stefana Avram1 , Minda Daliana<sup>1</sup> and Cristina Dehelean<sup>5</sup>

\*Address all correspondence to: corina.danciu@umft.ro

1 Department of Pharmacognosy, Faculty of Pharmacy, University of Medicine and Pharmacy "Victor Babes", Timisoara, Romania

2 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Medicine and Pharmacy "Victor Babes", Timisoara, Romania

3 Department of Pharmaceutical Botany, Faculty of Pharmacy, University of Medicine and Pharmacy "Victor Babes", Timisoara, Romania

4 Department of Physical Chemistry, Faculty of Pharmacy, University of Medicine and Pharmacy "Victor Babes", Timisoara, Romania

5 Department of Toxicology, Faculty of Pharmacy, University of Medicine and Pharmacy "Victor Babes", Timisoara, Romania

### References

Resveratrol

formulations

60/40, 100/0 wt%



d. Stabilization:


(seeded Lack of data

Encapsulation

Zinc pectinate beads

Zinc acetate =



temperature



(ionotropic

Zinc-pectin-chitosan

microparticles

Resveratrol

þquercetin

þcurcumin

 -quercetin -resveratrol


(applied alone/in

combination,

Table 4.

Resveratrol

formulations.

 at 50 μM)

 composite

 gelation method)



 3:1

In vivo studies on rats

In vitro studies

Increased oral absorption without interfering with the absorption of quercetin

 of resveratrol

 and curcumin,

[187]

 Specific drug release in the colon

[186]

concentration

 5% (% w/v)

 ratio of 3:1

 time 0.5 h -drying at room

 solution pH 1.5

concentration

 5% (% w/v)

cross-linking

 agent;

In vitro studies

 within yeast cell

polymerization

 technique) In vitro studies

Increased Good stability, enhanced

solubility, sustained release)

Better delayed release for

calcium-pectinate

Encapsulation

pectinate beads

 of a great amount of resveratrol

 in zinc-

 beads;

zinc-pectinate

 beads than

[185]

DPPH-radical

 scavenging

 activity;

[184]

238 Natural Products and Cancer Drug Discovery

bioavailability

 (increased

Components

Method of application

Indications/remarks

Ref.


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