**Flavonoids: Promising Natural Products for Treatment of Skin Cancer (Melanoma)**

Raimundo Gonçalves de Oliveira Júnior, Christiane Adrielly Alves Ferraz, Mariana Gama e Silva, Érica Martins de Lavor, Larissa Araújo Rolim, Julianeli Tolentino de Lima, Audrey Fleury, Laurent Picot, Jullyana de Souza Siqueira Quintans, Lucindo José Quintans Júnior and Jackson Roberto Guedes da Silva Almeida

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67573

#### Abstract

Melanoma, which is the most malignant skin cancer type, has got one of the fastest increasing incidence rates of all cancer types in the world. When belatedly diagnosed, melanoma is extremely invasive and metastatic. Although there are effective drugs used to treat melanoma, some cell lines have proven resistant to chemotherapy. In this context, several research groups on natural products have investigated the anticancer effect of new natural molecules in the treatment of melanoma. Flavonoids have shown to play an important role in chemoprevention and inhibition of the proliferation, migration, and invasion of melanoma cells. In this chapter, we present a systematic review performed through a literature search over a period of 20 years, using specialized databases. Analysis of all selected manuscripts demonstrated that at least 97 flavonoids have already been investigated for the treatment of melanoma using in vitro or in vivo models. Most of the bioactive flavonoids belong to the classes of flavones (38.0%), flavonols (17.5%), or isoflavonoids (17.5%). Apigenin, diosmin, fisetin, luteolin, and quercetin were considered as the most studied flavonoids for melanoma treatment. In general, flavonoids have shown to be a promising source of molecules with great potential for the treatment of melanoma.

© 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.

Keywords: cancer, melanoma treatment, herbal medicines, medicinal plants, flavonoids

### 1. Introduction

Natural products have contributed significantly to new drugs discovery. Historically, natural products derived from plants, microorganisms, and animals have been a promising source of medicinal preparations and molecules with therapeutic potential, for various diseases, including cancer treatment. The study of natural products also contributed to the provision of unique chemical structures, which were chemically modified, resulting in the development of new drugs [1–3].

An analysis of the new medicines approved by the US Food and Drug Administration (FDA) between 1981 and 2010 revealed that 34% of those drugs were based on small molecules from natural compounds or derivatives of natural compounds (semisynthetic products). This includes drugs such as statins, tubulin-binding anticancer, and immunosuppressant drugs. In this context, it is evident the contribution of natural products for drug discovery [3–5].

In the search for new anticancer drugs, natural products have provided many structural models with different mechanisms of action, for the treatment of melanoma regional or distant metastatic melanoma. Vinblastine from Vinca rosea and paclitaxel, which originates from a Chinese plant, is an example of anticancer agent obtained from natural sources. The therapy also includes drugs with different mechanisms of action, such as immunomodulatory agents, BRAF, and MEK inhibitors, and most recently, use of vaccines [6, 7]. However, even with recent advances in anticancer therapy, there is still a demand to develop new effective anticancer drugs for the melanoma treatment [8].

Despite the diversity of treatments for melanoma, the high resistance of tumor cells to conventional therapies drives the search for new anticancer agents that have less toxic effects, and greater effectiveness, incentive to develop new therapies that can be used individually or in combination with other drugs bringing therapeutic benefits for the patient. The polyphenolic compounds like flavonoids possess a large spectrum of pharmacological activity, including anticancer activity. These secondary metabolites have molecular mechanisms of action in tumor cells already understood, acting in enzymes and receptors associated and signal transduction pathways relating to cellular proliferation, differentiation, apoptosis, inflammation, angiogenesis, and metastasis [9–11].

### 2. Pathological aspects of skin cancer (melanoma)

### 2.1. Definition

Melanomas are malignant skin tumors deriving from melanocytes, the melanin-producing cells, that typically occur in the skin but may rarely occur in mucous membranes (vulva, vagina, and rectum), or uvea, the pigmented layer of the eye, lying beneath the sclera and cornea, and comprising the iris, choroid, and ciliary body. Melanomas account for less than 2% of skin cancers but are responsible for 80% of the mortality of patients with skin cancer [12]. They are classified in several subtypes, according to their tissue origin, tumor form, spreading and infiltrating behavior, metastatic potential, etc. These includes (a) superficial spreading melanoma, that tend to start growing outwards rather than downwards into the skin, (b) nodular melanoma, that tends to grow downwards, deeper into the skin, (c) lentigo maligna melanoma, that develops from very slow growing pigmented areas of skin called lentigo maligna or Hutchinson's melanotic freckle, (d) acral lentiginous melanoma, most commonly found on the palms of the hands and soles of the feet or around the big toenail, and (e) amelanotic melanoma, that usually have no, or very little color, occasionally are pink or red, or have light brown or gray around the edges [13].

#### 2.2. Etiology

Keywords: cancer, melanoma treatment, herbal medicines, medicinal plants,

Natural products have contributed significantly to new drugs discovery. Historically, natural products derived from plants, microorganisms, and animals have been a promising source of medicinal preparations and molecules with therapeutic potential, for various diseases, including cancer treatment. The study of natural products also contributed to the provision of unique chemical structures, which were chemically modified, resulting in the development of new

An analysis of the new medicines approved by the US Food and Drug Administration (FDA) between 1981 and 2010 revealed that 34% of those drugs were based on small molecules from natural compounds or derivatives of natural compounds (semisynthetic products). This includes drugs such as statins, tubulin-binding anticancer, and immunosuppressant drugs. In

In the search for new anticancer drugs, natural products have provided many structural models with different mechanisms of action, for the treatment of melanoma regional or distant metastatic melanoma. Vinblastine from Vinca rosea and paclitaxel, which originates from a Chinese plant, is an example of anticancer agent obtained from natural sources. The therapy also includes drugs with different mechanisms of action, such as immunomodulatory agents, BRAF, and MEK inhibitors, and most recently, use of vaccines [6, 7]. However, even with recent advances in anticancer therapy, there is still a demand to develop new effective antican-

Despite the diversity of treatments for melanoma, the high resistance of tumor cells to conventional therapies drives the search for new anticancer agents that have less toxic effects, and greater effectiveness, incentive to develop new therapies that can be used individually or in combination with other drugs bringing therapeutic benefits for the patient. The polyphenolic compounds like flavonoids possess a large spectrum of pharmacological activity, including anticancer activity. These secondary metabolites have molecular mechanisms of action in tumor cells already understood, acting in enzymes and receptors associated and signal transduction pathways relating to cellular proliferation, differentiation, apoptosis, inflammation,

Melanomas are malignant skin tumors deriving from melanocytes, the melanin-producing cells, that typically occur in the skin but may rarely occur in mucous membranes (vulva, vagina, and rectum), or uvea, the pigmented layer of the eye, lying beneath the sclera and

this context, it is evident the contribution of natural products for drug discovery [3–5].

flavonoids

162 Natural Products and Cancer Drug Discovery

1. Introduction

drugs [1–3].

cer drugs for the melanoma treatment [8].

angiogenesis, and metastasis [9–11].

2.1. Definition

2. Pathological aspects of skin cancer (melanoma)

The precise etiology of melanoma depends on several individual factors and is probably multifactorial in most cases [12]. Sun exposure (particularly UVB radiation) and genetic susceptibility (including faulty DNA repair) have been reported as major environmental and genetic factors associated with the risk of melanoma initiation and promotion [14, 15]. Precursor lesions, particularly dysplastic nevi/atypical moles, probably play a critical role in melanoma initiation [16]. Moreover, the distribution of melanoma among various work forces suggests that occupational risk factors could play an important role in the etiology of this cancer. For example, melanoma incidence is significantly higher in populations working in printing and press, petrochemical, and telecommunications industries [17].

#### 2.3. Progression

The development of a melanoma tumor is considered a multistage process that involves various genetic and epigenetic alterations. From a histopathological point of view, the following steps can be considered: (a) common acquired nevi and dysplastic nevi, (b) radial growth phase melanoma, in which melanocytes undergo changes that enable them to survive and proliferate (c) vertical growth phase melanoma, in which tumor cells deeply invade into the dermis/hypodermis, and (d) malignant metastatic melanoma, in which the cells may eventually invade the endothelium and migrate to distant tissues [18–20]. When diagnosed in the early stages, melanoma can be easily treated by surgical excision of the primary tumor [21]. However, when the disease is at an advanced stage the treatment is very difficult because the cancer cells have a high capacity to cause metastases (including brain metastases) and acquire resistance to conventional therapy [22, 23]. The progression of cutaneous melanoma in the skin can be classified using the Clark method [18, 19] (Figure 1).

This classification is not very used, contrary to the TNM one, which is regularly revised by the American Joint Committee on Cancer (AJCC) [24, 25]. The TNM classification is based on the thickness of the primary tumor (T, also known as Breslow's index), presence of metastatic cells in the neighboring lymph nodes (N), and localization of metastasis in the body (M) (Table 1). The T criterion is subdivided into four categories from T1 to T4 discriminating melanomas from 1 –4 mm thick. Each category is subdivided into two subgroups that report the presence (a) or not (b) of ulceration of the primary tumor. The survival rate decreases with an increase in


Figure 1. Progression stages of the cutaneous melanoma, according to the Clark skin infiltration classification [18, 19].


Table 1. The TNM classification of melanoma progression (adapted from Ref. [26]).

the thickness of the tumor, which may, however, be smaller in the presence of ulceration. The classification N evaluates the number of neighboring lymph nodes containing metastatic melanoma cells. This criterion is subdivided into four categories from N0 to N3, as well as three subgroups according to the presence of small metastases detected after biopsy (a), large metastasis detected after clinical examination (b) and metastases in transit (c) (melanoma cells located between the primary tumor and the lymph node region in lymph channels). The classification M, with four categories from M0 to M1c, evaluates the localization of metastases in the organism, as well as the increase in serum lactate dehydrogenase concentration [24].

According to the TNM parameters, four melanoma progression stages can be defined (Table 2).

The only efficient treatment is the early surgical resection of the primary melanoma, when tumor cells have not already spread to nearby lymph nodes (stages I and II). Advanced and metastatic melanoma (stages III and IV) has very poor prognosis as most chemotherapeutical agents used to treat cancers are ineffective in killing melanoma cells, which are constitutively or adaptively resistant to proapoptotic drugs [22]. Melanoma is also resistant to radiotherapy [27, 28]. The overall positive responses to melanoma monotherapy using conventional anticancer drugs are weak and range from 4 to 26% [22]. Additionally, melanoma tumor displays pronounced neoangiogenesis [29] and a high ability to escape immune cell that explain why the 5-year survival rate for metastatic melanoma ranges from 5 to 10%, with a median survival of less than 8 months [30, 31]. Brain metastasis is present in 75% of stage IV melanoma patients, and constitutes a major cause of mortality because of the low permeability of the blood-brain barrier to chemotherapeutic drugs [30].

#### 2.4. Epidemiology

Figure 1. Progression stages of the cutaneous melanoma, according to the Clark skin infiltration classification [18, 19].

T1 ≤1.0 mm a. Without ulceration and mitosis < 1/mm2

b. With ulceration or mitosis ≥ 1/mm2

b. With ulceration

b. With ulceration

b. With ulceration

b. Macrometastasis

b. Macrometastasis

Normal

c. In transit without metastatic nodule

T classification Tumor thickness Ulceration-mitosis

T2 1.01–2.0 mm a. Without ulceration

T3 2.01–4.0 mm a. Without ulceration

T4 >4.0 mm a. Without ulceration

N1 1 a. Micrometastasis

N2 2–3 a. Micrometastasis

N0 0 –

N3 ≥4 –

M0 0 – M1a Subcutaneous tissue and/or metastatic lymph nodes Normal

M1c Distant metastasis High

Table 1. The TNM classification of melanoma progression (adapted from Ref. [26]).

M1b Lung metastasis

164 Natural Products and Cancer Drug Discovery

viscera metastasis

M classification Site Seric LDH

N classification Metastatic lymph nodes Size of metastatic lymph nodes

Since 2005, World Health Organization (WHO) has identified the cutaneous melanoma as a priority public health concern as 132,000 new cases are registered every year in the world [32]. Since the early 1970s, the incidence of malignant melanoma has increased significantly, for example an average 4% every year in the United States [12, 14, 33]. The melanoma death rate in


Table 2. Melanoma progression stages based on the TNM classification (adapted from Ref. [26]).

2012 was the highest in Australia and New Zealand (3.5/100,000) and Europe (2.3 per 100,000 people). In 2014, 76,100 new cases were diagnosed in the United States and 9710 patients with cutaneous melanoma died, according to the American Cancer Society. The wide disparity in melanoma incidence throughout the world depends upon the variation of early sun-exposure behaviors, recreational and vacation histories, nevus phenotypes and skin phototypes, distribution of melanoma risk genotypes, and discrepancies in epidemiological registrations between countries [34]. According to the WHO, a large number of atypical nevi (moles) are the strongest risk factor for malignant melanoma in fair-skinned populations. Malignant melanoma is more common among people with a pale complexion, blue eyes, and red or fair hair. It is over 20 times more frequent in White people compared to African-Americans and the risk increases with the age, although it also affects young adults, especially women. High, intermittent exposure to solar UV appears to be a significant risk factor for the development of malignant melanoma [14, 35], particularly for White people living in tropical regions [36, 37]. The incidence of malignant melanoma in White populations generally increases with decreasing latitude, with the highest recorded incidence occurring in Australia, where the annual rates are 10 and over 20 times the rates in Europe for women and men, respectively. Several epidemiological studies support a positive association with history of sunburn, particularly sunburn at an early age [34]. The role of cumulative sun exposure in the development of malignant melanoma is equivocal. However, malignant melanoma risk is higher in people with a history of nonmelanoma skin cancers and solar keratoses, both of which are indicators of cumulative UV exposure [38].

#### 2.5. Immunity and immunotherapy of melanomas

Activation of genes in transformed melanocytes leads to the expression or overexpression of tumour-associated antigens. Several melanoma-associated antigens (MAA) have been identified and classified according to their tissue expression and structure [39, 40]. These include proteic antigens that can be recognized as MHC-I—peptide complexes by cytolytic T lymphocytes, membrane gangliosides, and conformational antigens inducing strong humoral responses by B-lymphocytes. MAGE-1, 2, 3 and 4 antigens are expressed by metastatic melanoma while their expression is absent in melanocytes and weak in primary melanoma tumors, indicating that the corresponding genes are activated during malignant transformation and progression [41]. Other MAA such as Melan-A/melanoma antigen recognized by T-cells (MART-1), tyrosinase, Pme117/gp100, gp75/tyrosine-related protein (TRP)-1 and AIM-2 are expressed in normally differentiated melanocytes and melanoma cells but absent in other tumor cells, suggesting the possibility to target them for a specific destruction of melanoma tumors [42, 43]. The expression of various gangliosides present in the membranes of melanocytes and melanoma cells (GM3, GD3, GM2, GD2 and O-acetyl GD3) is also significantly increased during malignant transformation [44]. Given that malignant melanoma is one of the most immunogenic tumor and that melanomas are highly resistant to chemotherapy and radiotherapy, immunotherapy appears as one of the most promising and relevant strategies to destroy melanoma tumors and metastatic cells.

Promising results have been reported using ex-vivo stimulation of tumor-infiltrating lymphocytes by cytokines and MAA, potentiation of T-cell cytotoxic activity by blocking CTL-A4 co-inhibitory receptor (using monoclonal antibodies), CAR-T strategies, and combination of immunotherapy with chemotherapeutics (e.g., dacarbazine/CTL-A4 blockade) [22, 31, 45–49]. Interferon-α and interleukin-2 monotherapeutic treatments give an overall positive response in 13–25% patients, and constitute a first-line therapy for nonmetastatic patients. Ipilimumab, an anti-CTLA-4 monoclonal antibody, targeting a T-cell receptor decreasing T-cell activation and cytotoxicity, allows a long-term survival benefit in one-third of metastatic melanoma patients, and a complete remission in patients [50]. As a consequence, a high research effort is dedicated to the development of new antibodies activating antitumoral immunity and to the discovery of new natural drugs with cytostatic, antimetastatic, and/or antiangiogenic activity that could stimulate the immune system and be used in chemoimmunotherapy protocols to synergize with chemotherapeutic drugs and immune effectors.

In this view, only a few natural molecules have proved their efficacy to limit tumor growth and inhibit the invasiveness of highly aggressive melanoma cells in in vitro and in vivo models. The efficacy of such molecules is related to their antiangiogenic activity (e.g., resveratrol [51], curcumin [52]), to their capacity to induce melanoma cell death regardless of their apoptosissensitivity (e.g., narciclasine [53], carotenoids [54–59]), to their ability to target components of apoptotic pathways to overcome melanoma cells resistance to anticancer drugs (e.g., epigallocatechin gallate [60–62]), or to their strong stimulatory effect on antitumoral immunity (e.g. Lentinula edodes polysaccharides [63]). Considering the clinical efficacy of melanoma immunotherapy, combined to the high potential of natural compounds to limit melanoma growth and restore melanoma sensitivity to apoptosis inducers without impairing antitumoral immunity, an important research effort should be undertaken to assess the efficacy of original natural cytostatic compounds, highlight the molecular and cellular mechanisms involved in their pharmacological action, and study if these molecules favor in vivo melanoma rejection via their immune regulatory properties. Considering the fast growth of melanoma and failure of current treatments, the identification and clinical development of such efficient molecules will obviously have a significant impact on patient survival rate and duration.

### 3. Molecular and cellular pathways involved in melanoma biogenesis and progression

### 3.1. Implication of the MAPK pathway

2012 was the highest in Australia and New Zealand (3.5/100,000) and Europe (2.3 per 100,000 people). In 2014, 76,100 new cases were diagnosed in the United States and 9710 patients with cutaneous melanoma died, according to the American Cancer Society. The wide disparity in melanoma incidence throughout the world depends upon the variation of early sun-exposure behaviors, recreational and vacation histories, nevus phenotypes and skin phototypes, distribution of melanoma risk genotypes, and discrepancies in epidemiological registrations between countries [34]. According to the WHO, a large number of atypical nevi (moles) are the strongest risk factor for malignant melanoma in fair-skinned populations. Malignant melanoma is more common among people with a pale complexion, blue eyes, and red or fair hair. It is over 20 times more frequent in White people compared to African-Americans and the risk increases with the age, although it also affects young adults, especially women. High, intermittent exposure to solar UV appears to be a significant risk factor for the development of malignant melanoma [14, 35], particularly for White people living in tropical regions [36, 37]. The incidence of malignant melanoma in White populations generally increases with decreasing latitude, with the highest recorded incidence occurring in Australia, where the annual rates are 10 and over 20 times the rates in Europe for women and men, respectively. Several epidemiological studies support a positive association with history of sunburn, particularly sunburn at an early age [34]. The role of cumulative sun exposure in the development of malignant melanoma is equivocal. However, malignant melanoma risk is higher in people with a history of nonmelanoma skin cancers and solar keratoses, both of which are indicators

Activation of genes in transformed melanocytes leads to the expression or overexpression of tumour-associated antigens. Several melanoma-associated antigens (MAA) have been identified and classified according to their tissue expression and structure [39, 40]. These include proteic antigens that can be recognized as MHC-I—peptide complexes by cytolytic T lymphocytes, membrane gangliosides, and conformational antigens inducing strong humoral responses by B-lymphocytes. MAGE-1, 2, 3 and 4 antigens are expressed by metastatic melanoma while their expression is absent in melanocytes and weak in primary melanoma tumors, indicating that the corresponding genes are activated during malignant transformation and progression [41]. Other MAA such as Melan-A/melanoma antigen recognized by T-cells (MART-1), tyrosinase, Pme117/gp100, gp75/tyrosine-related protein (TRP)-1 and AIM-2 are expressed in normally differentiated melanocytes and melanoma cells but absent in other tumor cells, suggesting the possibility to target them for a specific destruction of melanoma tumors [42, 43]. The expression of various gangliosides present in the membranes of melanocytes and melanoma cells (GM3, GD3, GM2, GD2 and O-acetyl GD3) is also significantly increased during malignant transformation [44]. Given that malignant melanoma is one of the most immunogenic tumor and that melanomas are highly resistant to chemotherapy and radiotherapy, immunotherapy appears as one of the most promising and relevant strategies

Promising results have been reported using ex-vivo stimulation of tumor-infiltrating lymphocytes by cytokines and MAA, potentiation of T-cell cytotoxic activity by blocking CTL-A4

of cumulative UV exposure [38].

166 Natural Products and Cancer Drug Discovery

2.5. Immunity and immunotherapy of melanomas

to destroy melanoma tumors and metastatic cells.

The receptor tyrosine kinase MAPK pathway triggers a signaling cascade that regulates cell growth, proliferation, differentiation, and survival in response to a wide variety of extracellular stimuli including hormones, cytokines, and growth factors through the activation of tyrosine kinase receptors. As mutations of components of the MAPK pathway are associated with increased activity of ERK1/2 proteins [64], deregulation of this pathway contribute to both development and progression of melanoma. In particular, mutations in B-RAF, a member of the RAF kinase family, have been identified in up to 70% of malignant melanoma [65].

Binding of a ligand to the membrane bound tyrosine kinases receptors (RTKs) or integrins adhesion to extracellular matrix triggers the activation of the RAS GTPases which further lead to activation/transduction of the MAPK signaling pathway. Ras GTPases are small proteins bound to the cytoplasmic membrane. The RAS gene encodes three isoforms with tissuespecific pattern: HRAS, KRAS, and NRAS [64]. Downstream targets of RAS proteins are the PI3K/Akt pathway and the serine threonine kinase RAF proteins [66, 67]. Activated B-RAF then leads to the activation of the MEK/ERKs kinases, which targets a variety of signaling pathways such as cell growth, proliferation, protein synthesis, and apoptosis.

The RAF kinase family consists of three cytoplasmic proteins (A-RAF, B-RAF, and C-RAF) which participate in the MAPK transduction pathway. Unlike, c-RAF and A-RAF, mutations in B-RAF have been identified in up to 70% of malignant melanoma [68, 65]. Most frequent activating somatic mutations in B-RAF occur at the V599E where a valine replaces a glutamic acid [68]. Identification of such activating mutations in B-RAF proteins leads to the development of new drugs, such as B-RAF inhibitors, as anticancer strategies [69].

These oncogenic B-RAF proteins are able to transform fibroblastic cell line and lead to hyperactivation of the ERK proteins [68]. Constitutive ERK leads to increased proliferation apoptosis resistance in melanoma cells [69]. Interestingly, suppression of the tumor suppressor PTEN and activating mutations in B-RAF are both necessary in melanoma development highlighting the importance of the PI3K/Akt pathway upregulation in melanoma growth and apoptosis resistance [70].

Indeed, the tumor suppressor PTEN is downregulated in melanoma and this is associated with PI3K/Akt hyperactivation [71]. Apoptosis resistance could be mediated through activation of the NF-KB pathway, target of hyperactive ERK proteins [72]. Oncogenic B-RAF also leads to inhibition of the LKB1-AMPK pathway, a central signaling pathway at a crossroad between metabolism and proliferation regulation through, in particular, inhibition of the mTOR pathway. This study highlights a new pathway in tumor growth regulation [73]. Finally, expression of MCL-1, a member of the Bcl-2 pathway, whose alternative splicing leads to proteins with either pro- and antiapoptotic activities, is increased in melanoma metastasis associated with oncogenic B-RAF [74]. Oncogenic B-RAF may also trigger the antiapoptosis pathway through inhibition of the proapoptotic Bim proteins [75].

Besides, downregulation of a downstream target of the MAPK pathway, MITF (microphthalmiaassociated transcription factor), the master regulator of melanocyte development, survival, and function, is associated with poor diagnosis and melanoma progression [76]. Finally, cKIT, a tyrosine kinase receptor, might trigger proliferation signals in melanoma through activation of the MAPK pathway [77].

Others signaling pathways contribute to melanoma progression and metastasis such as the noncanonical Wnt signaling [78] and deregulation of the cyclin-dependent kinase inhibitor 2A (CDKN2A) pathway involved proliferation and apoptosis control [23].

### 3.2. Implication of extracellular vesicles (EV) in melanoma biogenesis and progression

Extracellular vesicles (EVs) are small vesicles released by most cell types in the extracellular environment, and as a consequence can be retrieved from various body fluids, especially plasma. EV might split into apoptotic bodies (>1 μm), microparticles (100 nm to 1 μm) released after membrane blebbing and exosomes (<100 nm), vesicles with an endosomal origin release after multivesicular bodies fuse with the plasma membrane [79]. EVs are biological vectors that convey lipids, different classes of proteins (cytoskeleton, adhesion, raft associated proteins, histones, chaperones [80], glycoproteins, and chemokines [81] or even morphogens such as Hedgehog proteins [82]. EVs also harbor nucleic acids [83] able to modulate the differentiation of the target cells [84]. EVs communicate with their target cells via receptor-ligand interaction, through transfer of membrane proteins [85, 86], can fuse with the plasma membrane [87], or transfer their components into target cells via phagocytosis [88] or endocytosis [89]. The ability of EVs released from antigen presenting cells to convey MHC class II proteins [90] highlights their immunomodulatory properties and their potential as therapeutic agents in anticancer strategies [91]. Moreover, as they carry tumor antigens via MCH class I proteins, EVs can initiate antitumor response in vitro [92] and in vivo [93]. Nevertheless, the composition of EVs and the message they convey depend both on the cells they originate from and the conditions triggering their release. Indeed, EVs from dendritic cells can also suppress immune response in inflammatory diseases models such as DTH (delayed-type hypersensitivity) mice [94]. This ability to attenuate immune response might be associated with the capacity of EV to induce expression of molecules able to inactivate T-cells or suppress immune response [95].

In addition to their immunomodulatory properties, the role of EVs in inflammation, angiogenesis, and proliferation has been widely demonstrated [96–98]. This suggests an implication for EVs in tumor survival and progression. In this study, exosomal markers, such as CD63, could be found also on a wide range of subpopulations of EVs, and as long as there is no determination of the cellular origin of vesicles, we chose to use the term EV to refer to both exosomes and microparticles.

### 3.3. Role of circulating EV in melanoma biogenesis and progression

Circulating EV can trigger inflammatory pathways in target cells [96], stimulate angiogenesis [82, 99, 100], protect against apoptosis [101], or stimulate proliferation [102].

Plasma levels of EV harboring CD63 in melanoma-engrafted SCID mice correlate to tumor size, suggesting a role of the tumor in EV secretion [103]. However, other suggests that circulating rates of EV do not differ between melanoma and healthy patients [104–106] but instead, EV protein composition might differ. In particular, plasmatic EVs from melanoma patients are enriched in platelet-derived EV involved in neovascularization (CD42a harboring EV) and antitumour immune responses (CD8 harboring EV) [105]. Furthermore, circulating endothelial and platelet derived-EV (EEV) and procoagulant EV are significantly higher in melanoma patients [107]. Such procoagulant EVs stimulate proinflammatory cytokines secretion by macrophages and drive melanoma metastasis in vivo [108] reinforcing the implication of EV in melanoma progression.

### 3.4. A role of EV in melanoma metastasis

to activation/transduction of the MAPK signaling pathway. Ras GTPases are small proteins bound to the cytoplasmic membrane. The RAS gene encodes three isoforms with tissuespecific pattern: HRAS, KRAS, and NRAS [64]. Downstream targets of RAS proteins are the PI3K/Akt pathway and the serine threonine kinase RAF proteins [66, 67]. Activated B-RAF then leads to the activation of the MEK/ERKs kinases, which targets a variety of signaling

The RAF kinase family consists of three cytoplasmic proteins (A-RAF, B-RAF, and C-RAF) which participate in the MAPK transduction pathway. Unlike, c-RAF and A-RAF, mutations in B-RAF have been identified in up to 70% of malignant melanoma [68, 65]. Most frequent activating somatic mutations in B-RAF occur at the V599E where a valine replaces a glutamic acid [68]. Identification of such activating mutations in B-RAF proteins leads to the develop-

These oncogenic B-RAF proteins are able to transform fibroblastic cell line and lead to hyperactivation of the ERK proteins [68]. Constitutive ERK leads to increased proliferation apoptosis resistance in melanoma cells [69]. Interestingly, suppression of the tumor suppressor PTEN and activating mutations in B-RAF are both necessary in melanoma development highlighting the importance of the PI3K/Akt pathway upregulation in melanoma growth and

Indeed, the tumor suppressor PTEN is downregulated in melanoma and this is associated with PI3K/Akt hyperactivation [71]. Apoptosis resistance could be mediated through activation of the NF-KB pathway, target of hyperactive ERK proteins [72]. Oncogenic B-RAF also leads to inhibition of the LKB1-AMPK pathway, a central signaling pathway at a crossroad between metabolism and proliferation regulation through, in particular, inhibition of the mTOR pathway. This study highlights a new pathway in tumor growth regulation [73]. Finally, expression of MCL-1, a member of the Bcl-2 pathway, whose alternative splicing leads to proteins with either pro- and antiapoptotic activities, is increased in melanoma metastasis associated with oncogenic B-RAF [74]. Oncogenic B-RAF may also trigger the antiapoptosis pathway through

Besides, downregulation of a downstream target of the MAPK pathway, MITF (microphthalmiaassociated transcription factor), the master regulator of melanocyte development, survival, and function, is associated with poor diagnosis and melanoma progression [76]. Finally, cKIT, a tyrosine kinase receptor, might trigger proliferation signals in melanoma through activation of

Others signaling pathways contribute to melanoma progression and metastasis such as the noncanonical Wnt signaling [78] and deregulation of the cyclin-dependent kinase inhibitor 2A

3.2. Implication of extracellular vesicles (EV) in melanoma biogenesis and progression

Extracellular vesicles (EVs) are small vesicles released by most cell types in the extracellular environment, and as a consequence can be retrieved from various body fluids, especially plasma. EV might split into apoptotic bodies (>1 μm), microparticles (100 nm to 1 μm) released after membrane blebbing and exosomes (<100 nm), vesicles with an endosomal origin release

(CDKN2A) pathway involved proliferation and apoptosis control [23].

pathways such as cell growth, proliferation, protein synthesis, and apoptosis.

ment of new drugs, such as B-RAF inhibitors, as anticancer strategies [69].

apoptosis resistance [70].

168 Natural Products and Cancer Drug Discovery

the MAPK pathway [77].

inhibition of the proapoptotic Bim proteins [75].

EV release is exacerbated in human malignant [109] and murine [110] melanoma cell lines. Furthermore, in comparison with murine melanocyte cell line, metastastic melanoma cell lines secrete highly procoagulant EV harboring phosphatidylserine and enriched in tissue factor proteins suggesting that melanocyte transformation into cancer cells is associated with the secretion of such EVs [110]. Besides, Wnt5a, a noncanonical Wnt signaling ligand in involved melanoma progression [78] induces the release of melanoma exosomes enriched in proangiogenic proteins and pro-inflammatory cytokines [111].

Proteomic analysis of human malignant melanoma cell lines A375 reveals an enrichment in proteins involved in angiogenesis and matrix remodeling such as annexin A1 and hyaluronan and proteoglycan link protein 1 (HAPLN1) [109]. Analysis of EV microRNA content reveals enrichment in miRNA involved in cell growth, proliferation, and apoptosis. Uptake of such EV promotes the invasion ability of normal melanocytes [109]. Furthermore, tumor-derived EV harbor FAS ligand involved in antitumor response through lymphocytes apoptosis [112]. Finally, human (SK-Mel28/-202/-265/-35) and mouse (B16-F10) cell line–derived exosomes are enriched in TYRP2 (tyrosinase-related protein-2), VLA-4 and Hsp90 proteins. Indeed, B16F10-derived exosomes are enriched in prooncogenic proteins such as the oncogene MET which has been described a role in cell transformation, proliferation, survival, invasion, and metastasis [113– 115]. BM cell treatment with such exosomes led to an increase in tumor size compared to nontreated mice. Compared to EV derived from B16F1, a poor metastatic cell line, injection of B16F10 EV led to increased metastatic lesions and a wider tissue distribution (brain, bone) [106]. This is in agreement with previous studies suggesting that highly metastasis cells are enriched in oncogene Met72 and are more deleterious than B16F1-derived EV [116]. These data strongly suggest that EV from melanoma cells is able to suppress antitumor response and stimulate tumor progression but also their ability to trigger melanoma invasion and metastasis. However, different populations of EV have distinct procoagulant properties [117]. Thus, it is therefore necessary to identify the cell origin of EV in order to determine their role in cancer progression.

### 3.5. Role of microRNAs in melanoma progression

MicroRNAs are noncoding small RNAs able to bind target mRNAs, through their 3<sup>0</sup> UTRs leading to their degradation. Binding of microRNAs to their targets allows regulating a wide variety of cellular mechanisms such as proliferation, angiogenesis, inflammation, and survival.

A role for microRNAs in melanoma progression was first demonstrated through different miRNA expression signatures associated with the developmental lineage and differentiation state of solid tumors [118]. Furthermore, a microarray analysis demonstrates a specific targeting between A375 cell line and the A375 cells-derived EV of 28 miRNAs involved in cellular growth, development, and proliferation [109]. Relevance of microRNAs implication in melanoma development was illustrated by the fact that miRNAS loci are retrieved in genomic regions altered in melanoma [119]. MITF (microphthalmia-associated transcription factor) the master regulator of melanocyte development, survival, and function, which is often dysregulated in melanoma is a target of miR-137 [120] and miR-182 [121]. Finally, a number of microRNAs such as miR-214 [122] and miR-223 [123], but also miR-137, miR-182, miR-221/ 222, and miR-34a, have been involved in melanoma progression (for a review see [124]). In particular, miR-221 and miR-222 are involved in tumor proliferation and an increased in invasion and migration abilities through targeting of p27Kip1/CDKN1B (cyclin-dependent kinase inhibitor 1B) and the tyrosine kinase receptor c-KIT receptor [124, 125].

However, five members of the Let-7 family are downregulated in primary melanoma suggesting that these microRNAs might trigger anticancer responses. In particular, Let7b which targets cyclins exerts antitumoral responses through inhibition of cancer cycle progression [126].

MicroRNAs can also be transported via EV and regulate the pathway in distant target cells. In particular, circulating EV from metastatic melanoma patients harbors a specific miRNA signature. Indeed, those EVs are enriched in oncogenics miRNAs mir17 and miR19a suggesting a role for miRNAs-associated EV in tumor progression and metastasis [127]. On the other hand, circulating EV in advanced melanoma patients shows a decrease in miR-125b which downregulation has been described in melanoma progression [128].

Finally, deep-RNA sequencing allows identifying an enrichment of 23 specific microRNAs in small EV including miR-199a-3p, miR-150-5p, miR-142-3p, and miR-486-5p known to be involved in melanoma progression or identified in melanoma metastasis or patient blood samples [129]. In particular, miR-214 has been associated with melanoma metastasis [122]. Interestingly, in silico analysis reveals that some of these miRNAs could target the BRAF pathway which is often deregulated in melanoma [129]. Metastatic cell lines secrete EV enriched in the oncogenic miR-222. Furthermore, miR-222 associated with EV can be transferred into target cells and promote tumorigenesis through activation the Akt/PI3K pathway [130].

### 3.6. Identification of new markers for melanoma diagnosis and prognosis

progression [78] induces the release of melanoma exosomes enriched in proangiogenic proteins

Proteomic analysis of human malignant melanoma cell lines A375 reveals an enrichment in proteins involved in angiogenesis and matrix remodeling such as annexin A1 and hyaluronan and proteoglycan link protein 1 (HAPLN1) [109]. Analysis of EV microRNA content reveals enrichment in miRNA involved in cell growth, proliferation, and apoptosis. Uptake of such EV promotes the invasion ability of normal melanocytes [109]. Furthermore, tumor-derived EV harbor FAS ligand involved in antitumor response through lymphocytes apoptosis [112]. Finally, human (SK-Mel28/-202/-265/-35) and mouse (B16-F10) cell line–derived exosomes are enriched in TYRP2 (tyrosinase-related protein-2), VLA-4 and Hsp90 proteins. Indeed, B16F10-derived exosomes are enriched in prooncogenic proteins such as the oncogene MET which has been described a role in cell transformation, proliferation, survival, invasion, and metastasis [113– 115]. BM cell treatment with such exosomes led to an increase in tumor size compared to nontreated mice. Compared to EV derived from B16F1, a poor metastatic cell line, injection of B16F10 EV led to increased metastatic lesions and a wider tissue distribution (brain, bone) [106]. This is in agreement with previous studies suggesting that highly metastasis cells are enriched in oncogene Met72 and are more deleterious than B16F1-derived EV [116]. These data strongly suggest that EV from melanoma cells is able to suppress antitumor response and stimulate tumor progression but also their ability to trigger melanoma invasion and metastasis. However, different populations of EV have distinct procoagulant properties [117]. Thus, it is therefore necessary to identify the cell origin of EV in order to determine their role in cancer progression.

MicroRNAs are noncoding small RNAs able to bind target mRNAs, through their 3<sup>0</sup>

kinase inhibitor 1B) and the tyrosine kinase receptor c-KIT receptor [124, 125].

However, five members of the Let-7 family are downregulated in primary melanoma suggesting that these microRNAs might trigger anticancer responses. In particular, Let7b which targets cyclins exerts antitumoral responses through inhibition of cancer cycle progression [126].

leading to their degradation. Binding of microRNAs to their targets allows regulating a wide variety of cellular mechanisms such as proliferation, angiogenesis, inflammation, and survival. A role for microRNAs in melanoma progression was first demonstrated through different miRNA expression signatures associated with the developmental lineage and differentiation state of solid tumors [118]. Furthermore, a microarray analysis demonstrates a specific targeting between A375 cell line and the A375 cells-derived EV of 28 miRNAs involved in cellular growth, development, and proliferation [109]. Relevance of microRNAs implication in melanoma development was illustrated by the fact that miRNAS loci are retrieved in genomic regions altered in melanoma [119]. MITF (microphthalmia-associated transcription factor) the master regulator of melanocyte development, survival, and function, which is often dysregulated in melanoma is a target of miR-137 [120] and miR-182 [121]. Finally, a number of microRNAs such as miR-214 [122] and miR-223 [123], but also miR-137, miR-182, miR-221/ 222, and miR-34a, have been involved in melanoma progression (for a review see [124]). In particular, miR-221 and miR-222 are involved in tumor proliferation and an increased in invasion and migration abilities through targeting of p27Kip1/CDKN1B (cyclin-dependent

UTRs

and pro-inflammatory cytokines [111].

170 Natural Products and Cancer Drug Discovery

3.5. Role of microRNAs in melanoma progression

Circulating concentrations of lactate dehydrogenase [79], S100 and MIA (Melanoma Inhibitory Activity), two small proteins expressed by melanoma cells, are significantly higher in melanoma patients [131] and thus are widely used a proteins markers in order to monitor melanoma progression. LDH concentrations might be a better prognosis factor to classify advanced melanoma [132, 133].

Other circulating factors such as circulating nucleic acids or EV could be used in melanoma detection as a prognosis factor in advanced stages of diseases. Indeed, circulating EVs from stage III to stage IV are enriched TYRP2 (tyrosinase-related protein-2), a specific melanoma protein, VLA-4 (very late antigen 4) and HSP90. Furthermore, these enriched EVs correlated with poor survival prognosis [106]. These authors identified a specific exosomes protein signature that could be used as a prognosis marker in stages III and IV melanoma patients [106].

Besides, circulating EV carries melanoma markers such as S100B and MIA proteins. Concentrations of EV-S100B and EV-MIA are higher in stage IV melanoma patients and such EV was associated to poor prognosis in patients [104]. Detection of such EV could be used as an additional diagnosis and prognosis marker of melanoma patients. In contrast, these authors did not find an increase in TYRP2 containing exosomes in plasma of melanoma patients. This discrepancy could be due to difference in EV isolation, or EV concentrations/number analysis (NTA analysis vs. EVprotein concentration determination). Finally, circulating EV enriched in oncogenes miRNAs mir17 and miR19a could be used as predictive markers in melanoma patients [127].

In addition, some microRNAs detected in patient metastasis such as miR-150, miR-342-3p, miR-455-3p, miR-145, miR-155, and miR-497 could be used as a specific signature to predict postsurvival recurrence with a high expression of miR-145, miR-155 in metastatic tissue associated with longer survival [134]. Finally, identification of a specific signature of 16 differentially expressed microRNAs in patient blood samples represents a new noninvasive tool in diagnosis applications [135]. Finally, other authors suggest that microRNAs from blood patients could be used to monitor melanoma recurrence [136, 137].

### 4. Current melanoma treatment

The treatment options for regional or distant metastatic melanoma have expanded in recent years and are directly influenced by disease stage at diagnosis and the extent of metastases. The therapy used includes several drugs with different mechanisms of action, including chemotherapies, immunomodulatory agents, the serine/threonine protein kinase BRAF, mitogen-activated protein kinase (MEK) inhibitors, and most recently, use of vaccines [6, 7]. The primary treatment of this cancer type is surgical excision, sentinel lymph node dissection, radical lymph node dissection, and isolated limb perfusion [138, 139].

Chemotherapy may now be considered a second or third line in patients with resistance to immunotherapy and targeted therapy [140]. Tumor cells may evade the immune attack by some mechanisms, such as impaired antigen presentation, expression of factors with immunosuppressive properties, such as transforming growth factor-beta (TGF-β), vascular endothelial growth factor (VEGF), interleukin-2 (IL-2), and induction of resistance to apoptosis. In addition, melanoma cells further express receptors on the cell surface which function as checkpoints to the immune system response, as the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1). The ipilimumab is an anti-CTLA-4 monoclonal antibody and the nivolumab and pembrolizumab are also monoclonal antibodies directed against the PD-1 receptor, that blocking the inhibitory ligand's suppression of immune response. Thus, the main objectives of immunotherapy are to activate an immune response through the immunostimulation of IL-2, the upregulation of tumor-inhibitory T cells, and the inhibition of the immune control points [141, 142].

In addition to the immunological approach, targeted therapies have also been employed in the treatment of melanoma, such as BRAF and MEK inhibitors. The BRAF gene is responsible for encoding the B-raf protein that participates in the regulation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway, which regulates cell proliferation, differentiation, and cell cycle progression [142]. The discovery that BRAF was mutated in about 50% of melanomas led to the development of BRAF kinase inhibitors as vemurafenib and dabrafenib. However, most patients acquire resistance mechanisms to BRAF kinase inhibition [141, 143]. In view of the development of resistance to single BRAF blockade, several combination schemes have been developed, as the combination therapy with MEK inhibitors trametinib and cobimetinib [144–146].

Despite these advances, about 80% of patients develop resistance to the current standard of treatment with the combination of a selective BRAF and MEK inhibitors, which stimulates research for new treatment alternatives. The use of triple combining therapy has also been the subject of investigations and demonstrated prolonged responses [147]. Preclinical assays performed with the triple combination of BRAF and MEK inhibitors and anti-PD-1 demonstrated high antitumor activity and phase I/II clinical studies have shown promise in BRAFV600-mutated melanoma [148]. Moreover, vaccines have also been investigated and in 2015 the Food and Drug Administration (FDA) approved the Talimogene laherparepvec (T-VEC), an oncolytic virus derived from herpes simplex type 1, which can selectively replicate within tumors and produce granulocyte macrophage colony stimulating factor (GM-CSF) which promotes increased antitumor immune response [149].

### 5. Chemical and biological aspects of flavonoids

4. Current melanoma treatment

172 Natural Products and Cancer Drug Discovery

The treatment options for regional or distant metastatic melanoma have expanded in recent years and are directly influenced by disease stage at diagnosis and the extent of metastases. The therapy used includes several drugs with different mechanisms of action, including chemotherapies, immunomodulatory agents, the serine/threonine protein kinase BRAF, mitogen-activated protein kinase (MEK) inhibitors, and most recently, use of vaccines [6, 7]. The primary treatment of this cancer type is surgical excision, sentinel lymph node dissection,

Chemotherapy may now be considered a second or third line in patients with resistance to immunotherapy and targeted therapy [140]. Tumor cells may evade the immune attack by some mechanisms, such as impaired antigen presentation, expression of factors with immunosuppressive properties, such as transforming growth factor-beta (TGF-β), vascular endothelial growth factor (VEGF), interleukin-2 (IL-2), and induction of resistance to apoptosis. In addition, melanoma cells further express receptors on the cell surface which function as checkpoints to the immune system response, as the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1). The ipilimumab is an anti-CTLA-4 monoclonal antibody and the nivolumab and pembrolizumab are also monoclonal antibodies directed against the PD-1 receptor, that blocking the inhibitory ligand's suppression of immune response. Thus, the main objectives of immunotherapy are to activate an immune response through the immunostimulation of IL-2, the upregulation of tumor-inhibitory T cells,

In addition to the immunological approach, targeted therapies have also been employed in the treatment of melanoma, such as BRAF and MEK inhibitors. The BRAF gene is responsible for encoding the B-raf protein that participates in the regulation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway, which regulates cell proliferation, differentiation, and cell cycle progression [142]. The discovery that BRAF was mutated in about 50% of melanomas led to the development of BRAF kinase inhibitors as vemurafenib and dabrafenib. However, most patients acquire resistance mechanisms to BRAF kinase inhibition [141, 143]. In view of the development of resistance to single BRAF blockade, several combination schemes have been developed, as the combination therapy with MEK

Despite these advances, about 80% of patients develop resistance to the current standard of treatment with the combination of a selective BRAF and MEK inhibitors, which stimulates research for new treatment alternatives. The use of triple combining therapy has also been the subject of investigations and demonstrated prolonged responses [147]. Preclinical assays performed with the triple combination of BRAF and MEK inhibitors and anti-PD-1 demonstrated high antitumor activity and phase I/II clinical studies have shown promise in BRAFV600-mutated melanoma [148]. Moreover, vaccines have also been investigated and in 2015 the Food and Drug Administration (FDA) approved the Talimogene laherparepvec (T-VEC), an oncolytic virus derived from herpes simplex type 1, which can selectively replicate within tumors and produce granulocyte macrophage colony stimulating factor (GM-CSF)

radical lymph node dissection, and isolated limb perfusion [138, 139].

and the inhibition of the immune control points [141, 142].

inhibitors trametinib and cobimetinib [144–146].

which promotes increased antitumor immune response [149].

Flavonoids are phenolic constituents commonly found in a variety of fruits, vegetables, and medicinal plants. They add color, flavor, and aroma to plants, and play an important role in protection mechanisms against pathogens, ultraviolet radiation, and herbivores. Flavonoids comprise an important class of secondary metabolites, with numerous possibilities of chemical structures [150, 151].

Flavonoids have a basic phenylbenzopyrone skeleton (C6-C3-C6), admitting several substitution possibilities. In accordance with the substitution pattern of A, B, and C rings of the basic structure, flavonoids can be classified as chalcones, aurones, flavones, flavonols, flavanols, flavanones, isoflavones, flavanonols, among others [10]. The most common classes of flavonoids are shown in Figure 2.

Flavonoids may include the polyhydroxylated or polymethoxylated form. There are more than 8000 flavonoids identified, some of the most abundant are quercetin, catechin, and kaempferol, which are often combined with glycosidic units (commonly glucose, galactose, and rhamnose) through C-C or C-O-C bonds [152, 153]. The structural diversity of flavonoids directly influences their chemical, physical and pharmacological properties.

Figure 2. Basic skeleton (C6-C3C6) and main classes of flavonoids. This figure was adapted from Ref. [10].

Several studies have demonstrated the therapeutic properties of flavonoids obtained from plants or through synthesis. In fact, the structural diversity of flavonoids contributes to the diverse pharmacological activities reported for these compounds. In vitro and in vivo assays have shown the antioxidant, anti-inflammatory, antinociceptive, cardioprotective, photoprotective, antidepressant, antimicrobial, and cytotoxic effects of flavonoids [154, 155].

Recently, flavonoids have been shown to be potent antitumor agents. These compounds showed promising effect against different tumor cell lines, including human melanoma cells [156, 157]. In addition, flavonoids typically exhibit low toxicity in biological systems, which make them an alternative therapy compared with traditional anticancer drugs [158–161].

### 6. Bioactivity of flavonoids on melanoma

In this section, the authors present a systematic review performed through a literature search over a period of 20 years (January 1996–December 2016). This literature search was performed through specialized databases (PUBMED, LILACS, SCIELO, Science Direct, and Web of Science) using different combinations of the following keywords: flavonoid, flavonoid derivative, melanoma, skin cancer, treatment, and anticancer therapy. We did not contact investigators and we did not attempt to identify unpublished data.

Manuscripts were selected based on the inclusion criteria: articles published in English, Portuguese, Spanish, or French and articles with keywords in the title, abstract, or keywords, as well as studies involving anticancer activity of natural flavonoids necessarily against melanoma in in vitro or in vivo models. Other review articles, meta-analysis, abstracts, conferences, editorial/ letters, case reports, conference proceedings, or articles that did not meet the inclusion criteria were excluded from this systematic review.

For the selection of the manuscripts, two independent investigators (RGOJ and CAAF) first selected the articles according to the title, then to the abstract, and finally through an analysis of the full-text publication. A consensus between the investigators was reached as a clarification for in order to clarify all disagreements. The selected articles were manually reviewed with the purpose of identifying and excluding the works that did not fit the criteria described above.

The primary search identified 164 articles. However, among these, 39 manuscripts were indexed in two or more databases and were considered only once, resulting in 125 articles. After an initial screening of titles, abstracts, full text, and time of publication, 43 articles were selected, while the remainder did not meet the inclusion criteria (n = 82). Although many articles presented promising anticancer activity for plant extracts rich in flavonoids, we considered only articles that showed anticancer activity of the isolated flavonoids on melanoma cell lines. Investigations involving synthetic flavonoids were also excluded from this review. A flowchart illustrating the progressive study selection and numbers at each stage is shown in Figure 3.

Analysis of all selected manuscripts demonstrated that at least 97 flavonoids have already been investigated for the treatment of melanoma using in vitro or in vivo models. Most of the bioactive flavonoids belong to the classes of flavones (38%), flavonols (17.5%), or isoflavonoids

Figure 3. Flowchart of included studies for the systematic review.

Several studies have demonstrated the therapeutic properties of flavonoids obtained from plants or through synthesis. In fact, the structural diversity of flavonoids contributes to the diverse pharmacological activities reported for these compounds. In vitro and in vivo assays have shown the antioxidant, anti-inflammatory, antinociceptive, cardioprotective, photoprotective, antide-

Recently, flavonoids have been shown to be potent antitumor agents. These compounds showed promising effect against different tumor cell lines, including human melanoma cells [156, 157]. In addition, flavonoids typically exhibit low toxicity in biological systems, which make them an alternative therapy compared with traditional anticancer drugs [158–161].

In this section, the authors present a systematic review performed through a literature search over a period of 20 years (January 1996–December 2016). This literature search was performed through specialized databases (PUBMED, LILACS, SCIELO, Science Direct, and Web of Science) using different combinations of the following keywords: flavonoid, flavonoid derivative, melanoma, skin cancer, treatment, and anticancer therapy. We did not contact investigators

Manuscripts were selected based on the inclusion criteria: articles published in English, Portuguese, Spanish, or French and articles with keywords in the title, abstract, or keywords, as well as studies involving anticancer activity of natural flavonoids necessarily against melanoma in in vitro or in vivo models. Other review articles, meta-analysis, abstracts, conferences, editorial/ letters, case reports, conference proceedings, or articles that did not meet the inclusion criteria

For the selection of the manuscripts, two independent investigators (RGOJ and CAAF) first selected the articles according to the title, then to the abstract, and finally through an analysis of the full-text publication. A consensus between the investigators was reached as a clarification for in order to clarify all disagreements. The selected articles were manually reviewed with the purpose of identifying and excluding the works that did not fit the criteria described above.

The primary search identified 164 articles. However, among these, 39 manuscripts were indexed in two or more databases and were considered only once, resulting in 125 articles. After an initial screening of titles, abstracts, full text, and time of publication, 43 articles were selected, while the remainder did not meet the inclusion criteria (n = 82). Although many articles presented promising anticancer activity for plant extracts rich in flavonoids, we considered only articles that showed anticancer activity of the isolated flavonoids on melanoma cell lines. Investigations involving synthetic flavonoids were also excluded from this review. A flowchart illustrating the

Analysis of all selected manuscripts demonstrated that at least 97 flavonoids have already been investigated for the treatment of melanoma using in vitro or in vivo models. Most of the bioactive flavonoids belong to the classes of flavones (38%), flavonols (17.5%), or isoflavonoids

progressive study selection and numbers at each stage is shown in Figure 3.

pressant, antimicrobial, and cytotoxic effects of flavonoids [154, 155].

6. Bioactivity of flavonoids on melanoma

174 Natural Products and Cancer Drug Discovery

and we did not attempt to identify unpublished data.

were excluded from this systematic review.

(17.5%), which has aroused the interest of several research groups in natural and synthetic products in the world. All information about these flavonoids for the treatment of melanoma was reported in Box 1. Next, we highlight the anticancer properties of the main tested flavonoids on melanoma cells (apigenin, diosmin, fisetin, luteolin, and quercetin).

#### 6.1. Bioactivity of apigenin on melanoma

Apigenin (4<sup>0</sup> ,5,7,-trihydroxyflavone) is a nonmutagenic and low-toxicity dietary flavonoid commonly present in many fruits, vegetables, and medicinal plants. This flavone has a broad spectrum of antiproliferative activities against many types of cancer cells, including melanoma. Recent studies have demonstrated that apigenin inhibits cell growth through cell cycle arrest and apoptosis in malignant human melanoma cell lines. Hasnat et al. [169] showed that treatment with 50 μM apigenin significantly reduced viable cell percentages in A375 and A2058 human melanoma cells. Treatment with apigenin for 24 h also decreased human melanoma cell numbers in a dose-dependent manner. A similar result was observed by Spoerlein et al. [170], who evaluated the cytotoxic potential and the effect of apigenin on the cell cycle of 518A2 human melanoma cells. Apigenin also caused a dose-dependent decrease in the percentage of transwell-migrated cells, and ∼90 and ∼70% inhibitions of cell migration were recorded upon treatment with 20 μM of apigenin, respectively, for A2058 and A375 cells [169].

The cytotoxic effects of apigenin were related to its ability to reduce integrin protein levels and inhibit the phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK1/2). Furthermore, apigenin treatment increased apoptotic factors such as caspase-3 and cleaved poly(ADP-ribose) polymerase in a dose-dependent manner. Cao et al. [172] have also demonstrated that apigenin suppressed STAT3 phosphorylation, decreased STAT3 nuclear localization, and inhibited STAT3 transcriptional activity. Apigenin also downregulated STAT3 target genes MMP-2, MMP-9, VEGF, and Twist1, which are involved in cell migration and invasion. In this same investigation, it was determined the in vivo antimetastatic effect of



Aurones

2,6-Dihydroxy-2-[(4-

In vitro

B16-BL6

ND

Preliminary

IC50 > 100 μM.

 test determined

NE

[162]

176 Natural Products and Cancer Drug Discovery

(MM)

hydroxyphenyl)methyl]-3-

benzofuranone

Anthocyanins

Cyanidin-3-

glucopyranoside

 (C-3-G)

O-β-

In vitro M14 (HM)

 5 or 10 μM

A treatment with a single dose of

C-3-G decreased cell without affecting cell viability and without inducing apoptosis

or necrosis. C-3-G treatment also induced increase of cAMP levels

and expression and activity resulting in an enhanced melanin synthesis

and

Biflavonoids

Pteridium III

Chalcones

20,40-Dihydroxychalcone

4,40-Dihydroxy-20

methoxychalcone

Isoliquiritigenin

 (ISL)

In vitro

B16-BL6

ND

(MM)

In vitro

B16F0 (MM) 5–25 μg/ml

and

in vivo (mice)


In vitro

B16-BL6

ND

(MM)

In vitro

B16-BL6

ND

Preliminary

equal to 44.3 μM.

Preliminary

equal to 56.3 μM.

Preliminary

equal to 80.5 μM.

A significant time-dependent

proliferation

cell inhibition rate ranged from 18

to 79% and 35 to 91% after 24 and 48 h of ISL treatment (5, 10, 15, 20,

and 25 μg/mL),

respectively.

 ISL

 was observed. The

 reduction in cell

concentration-

 and

ISL increased reactive oxygen species (ROS) formation during

B16F0 cell specific target was evaluated.

differentiation,

 but no

 test determined

 IC50

NE

 test determined

 IC50

NE

 test determined

 IC50

NE

[162]

[162]

[162]

[165]

(MM)

In vitro A375 (HM)

 ND

Preliminary

equal to 106.7 μM.

 test determined

 IC50

NE

[164]

melanosome

 maturation.

upregulation

 of tyrosinase

proliferation

Upregulation

differentiation

MART-1 in treated cells respect to

the untreated control was

recorded.

 antigen Melan-A/

 of the melanoma

[163]

Model Cell line

Concentration

 or dose

Effectiveness

Possible

mechanisms

 of action

 Reference Flavonoids: Promising Natural Products for Treatment of Skin Cancer (Melanoma) http://dx.doi.org/10.5772/67573 177



6-Prenylapigenin

Albanin Apigenin

In vitro

B16F10

ND

(MM)

In vitro A375 and

0–50 μM

> A2058 (HM)

In vitro B16 (MM)

 ND

In vitro B16 (MM)

Model Cell line

 ND

Concentration

 or dose

Effectiveness

Preliminary

equal to 32.5 μM.

Preliminary

equal to 84.7 μM.

Preliminary

equal to 26 μM. Treatment with 50 μM apigenin

Apigenin reduced integrin

[169]

protein levels and inhibited the

phosphorylation

kinase (FAK) and

signal regulated kinase (ERK1/2).

Furthermore,

increased apoptotic factors such

as caspase-3 and cleaved poly

(ADP-ribose)

dose dependent manner.

Apigenin induced an arrest of the

[170]

cell cycle of 518A2 melanoma

cells at the G2/M transition and

also attenuated and secretion of the metastasis

relevant matrix metalloproteinases

MMP-9.

NE

[171]

In vitro In vitro

B16F10

0–40 μM (in vitro tests) and

and

(MM), A375

150 mg/kg (in vivo tests)

> in vivo

and G361

(mice)

(HM)

MDA-MB-435 (HM)

1–50 μM

Preliminary

IC50 > 50 μM. Apigenin (5 and 10 μM) also

Apigenin suppressed

phosphorylation,

STAT3 nuclear localization

inhibited STAT3 activity. Apigenin also

downregulated

genes MMP-2, MMP-9, VEGF and

Twist1, which are involved in cell

migration and invasion.

 STAT3 target

 decreased

 and transcriptional

 STAT3

[172]

dose-dependently

B16F10, A375 G361 cell migration

and invasion. mice had significant fewer

metastatic nodules.

Apigenin-treated

 inhibited

 test determined

 MMP-2 and

 the expression

 polymerase

 in a

 apigenin treatment

 of focal adhesion

extracellular

significantly

percentages

melanoma cells. Apigenin

exhibited inhibition of melanoma cell migration, unlike untreated

controls.

In vitro

518A2 (HM) ND

Preliminary

IC50 > 50 μM. The flavonoid also

reduced cell migration.

 test determined

dose-dependent

 in both types of

 reduced viable cell

 test determined

 IC50

NE

 test determined

 IC50

NE

 test determined

 IC50

NE

Possible

mechanisms

 of action

 Reference

[168]

[168]

[167]

178 Natural Products and Cancer Drug Discovery

#### Flavonoids: Promising Natural Products for Treatment of Skin Cancer (Melanoma) http://dx.doi.org/10.5772/67573 179



Desmethoxylcentaureidin

Diosmin

In vivo

B16F10

0.2% solution (ad libitum as

(mice)

In vivo

B16F10

551 mg/kg/day

 Diosmin alone

(mice)

(MM)

or combined with different

doses of IFN-α

(MM)

drink)

In vitro

B16F10

ND

(MM)

Model Cell line

Concentration

 or dose

Effectiveness

Preliminary

equal to 64 μM. Animals treated with diosmin

NE

> presented a reduction in the

number of subpleural mestases in

comparison

control group.

IFN-α antiinvasive

activity in our study, while

diosmin showed an antiinvasive

activity similar to the lower dose

of IFN-α used. diosmin and IFN-α have shown

synergistic effect.

Group treated with diosmin

NE

[178]

showed the greatest reduction

(52%) in the number of metastatic

nodules.

Diosmin decreased the number of

NE

[179]

metastatic nodules (52%),

implantation

and invasion (45%) index.

Preliminary

equal to 16 μM.

Preliminary

equal to 58 μM.

Preliminary

from 33 to 85 μM.

Preliminary

equal to 44 μM.

Preliminary

equal to 11.76 μM.

 test determined

 IC50

NE

 test determined

 IC50

NE

 test determined

 IC50

NE

 test determined

 IC50

NE

 test determined

 IC50

NE

[167]

[167]

[180]

[167]

[181]

 (79%), growth (67%)

In vivo

B16F10

0.2% solution (ad libitum as

(mice)

In vivo

B16F10

20 mg/day

(mice)

Eupafolin

Eupatilin

In vitro

B16F10

ND

(MM)

In vitro

B16F10

104

–108 M

(MM)

Eupatorin Isolinariin A

In vitro C32 (HM)

 ND

In vitro

B16F10

ND

(MM)

In vitro

B16F10

ND

(MM)

(MM)

(MM)

drink)

Combination

 of

 and

showed a

dose-dependent

NE

[177]

antiproliferative

 to the negative

 test determined

 IC50

NE

Possible

mechanisms

 of action

 Reference

[167]

[176]

180 Natural Products and Cancer Drug Discovery



Model Cell line

In vitro

B16F10

 5–50 μM (in vitro tests) and

Luteolin suppressed induced changes in the cells in a

dose-dependent

experimental

mice, treatment with luteolin

reduced metastatic in the lungs by 50%.

Preliminary

equal to 170 μM.

Preliminary

equal to 7.8 μM.

Preliminary

equal to 64 μM.

Preliminary

equal to 7.17 μM. Tangeretin decreased the number

NE

> of metastatic nodules,

implantation,

invasion index.

 growth and

 test determined

 IC50

NE

 test determined

 IC50

NE

 test determined

 IC50

NE

 test determined

 IC50

NE

[168]

[168]

[167]

[181]

[179]

colonization

 metastasis model

 manner. In

 the hypoxia-

> 10–20 mg (in vivo tests)

and

in vivo (mice)

Morin Norartocarpin Pectolinarigenin

Pectolinarin

Tangeretin Flavanones

3,7-Dihydroxy-6-

In vitro

B16-BL16

ND

Preliminary

IC50 > 100 μM.

Preliminary

IC50 > 100 μM.

Preliminary

IC50 equal to 6.7 μM.

Preliminary

IC50 equal to 99.9 μM.

Preliminary

IC50 > 100 μM.

 test determined

NE

 test determined

NE

 test determined

NE

 test determined

NE

 test determined

NE

[162]

[162]

[162]

[162]

[162]

(MM)

methoxyflavanone

3,7-Dihydroxyflavanone

7-Hydroxy-6-

In vitro

B16-BL16

ND

(MM)

methoxyflavanone

7-Hydroxyflavanone

Alnustinol

In vitro

B16-BL16

ND

(MM)

In vitro

B16-BL16

ND

(MM)

In vitro

B16-BL16

ND

(MM)

In vivo

B16F10

20 mg/day

(mice)

(MM)

In vitro C32 (HM)

 ND

In vitro

B16F10

ND

(MM)

In vitro B16 (MM)

 ND

In vitro B16 (MM)

 ND

Concentration

 or dose

Effectiveness

Possible increased the level of intracellular

ROS, leading to ROS mediated

apoptosis and ER stress.

Luteolin inhibited the hypoxia-

induced transition in malignant melanoma

cells both in vitro and in vivo via

the regulation of β3 integrin.

epithelial-mesenchymal

[184]

182 Natural Products and Cancer Drug Discovery

mechanisms

 of action

 Reference

#### Flavonoids: Promising Natural Products for Treatment of Skin Cancer (Melanoma) http://dx.doi.org/10.5772/67573 183



Model Cell line

In vitro

A375 (HM)

 10 and 20 μM, in

with sorafenib (2 and 5 μM)

combination

Combination

sorafenib) more effectively reduced the migration and

invasion of melanoma cells both in vitro and

in raft cultures compared to

individual agents. In addition,

fisetin potentiated

antiinvasive

effects of sorafenib in vivo.

 and

 the antimetastatic

ZEB1 protein expression

compared to

expression of MMP-2 and MMP-9

in xenograft tumors was further

reduced in compared to individual agents.

Fisetin decreased cell viability

[188]

with G1-phase arrest and

disruption of

signaling.

Wnt/β-catenin

In vitro

451Lu (HM) 20–100 μM (in vitro tests) and

IC50 was estimated to be 80, 37.2,

and 17.5 μM at 24, 48, and 72

hours of treatment, respectively. A smaller average tumor volume

was consistently treated with fisetin. This was

more marked in animals receiving

1 mg fisetin than in animals

receiving the 2 mg dose,

indicating a nonlinear dose

response.

The efficacy of fisetin in the

Fisetin treatment induced

[189]

endoplasmic

in highly aggressive A375 and

451Lu human melanoma cells, as

revealed by

stress markers including IRE1a,

XBP1s, ATF4 and GRP78. Both

extrinsic and intrinsic apoptosis

upregulation

 of ER

 reticulum (ER) stress

induction of apoptosis varied

with cell type as A375 cells were

more susceptible

treatment compared to 451Lu

cells. Results confirm apoptosis as

the primary mechanism

which fisetin inhibits melanoma

cell growth.

 through

 to fisetin

In vitro

451Lu and

20–80 μM

> A375 (HM)

 observed in mice

and

1 and 2 mg/kg (in vivo tests)

> in vivo

(mice)

combination

 treatment

monotherapy.

 The

BRAF-mutated

 treatment (fisetin

þ

Combination

sorafenib) promoted a decrease in

N cadherin, vimentin and

fibronectin

cadherin both in vitro and in

xenograft tumors.

combination

inhibited Snail1, Twist1, Slug and

 therapy effectively

Furthermore,

 and an increase in E-

 treatment (fisetin

þ

[187]

184 Natural Products and Cancer Drug Discovery

and

in vivo (mice)

Concentration

 or dose

Effectiveness

Possible effect of fisetin on AKT was mediated indirectly, through

targeting interrelated

 pathways.

mechanisms

 of action

 Reference



Isorhamnetin Isorhamnetin-3-

glucoside Kaempferol

In vitro In vitro

SK-MEL-2

ND

(HM)

SK-MEL-2

ND

(HM)

Kaempferol-3-

Myricetin Quercetin

In vitro

B16F10

3.156–50 μM

(MM)

In vitro

B16F10

25–50 μM (in vitro tests) and

7.5–15 mg/kg (in vivo tests), in

combination

doses of

 with different

sulforaphane.

and

(MM)

in vivo (mice)

In vitro

B16F10

3.156–50 μM

(MM)

O-rhamnoside In vitro

MDA-MB-435 (HM)

1–50 μM

Preliminary

equal to 1.5 μM.

Preliminary

equal to 6.9 μM.

Preliminary

equal to 33.9 μM.

Preliminary

equal to μM.

Preliminary

IC50 > 50 μM. Quercetin and

combination

proliferation

melanoma cells more effectively

than either compound

These compounds

significantly

melanoma growth as compared

to their individual use in a mouse

model.

Exposure to quercetin resulted in

inhibition of melanoma cells, induction of cell

proliferation

 of

of STAT3 signaling by interfering

with STAT3

phosphorylation,

 and

Quercetin inhibited the activation

[198]

In vitro

A375,

0–60 μM (in vitro tests) and

and

A2058 (HM)

100 mg/kg (in vivo tests)

 suppressed

 used alone.

> in

combination

 and migration of

 inhibit the

sulforaphane

 in

This combined effect was

predominantly

in MMP

mouse tumors.

9 expression in the

 due to a decrease

 test determined

NE

 test determined

 IC50

NE

 test determined

 IC50

NE

 test determined

 IC50

NE

 test determined

 IC50

NE

[171]

[196]

[196]

[174]

[174]

[197]

O-β-D-

In vitro

SK-MEL-2

ND

Preliminary

IC50 > 40 μg/ml for both cell lines.

 test determined

NE

[189]

(HM) and

B16F1 (MM)

In vitro

SK-MEL-2

ND

Preliminary

IC50 > 40 μg/ml for both cell lines.

 test determined

> (HM) and

B16F1 (MM)

Model Cell line

Concentration

 or dose

Effectiveness

Possible the levels of STAT3 -targeted

genes. Icaritin also inhibited IGF-

1-induced STAT3 activation

through IGF-1R level.

NE

[189]

186 Natural Products and Cancer Drug Discovery

downregulation

 of total

mechanisms

 of action

 Reference 187



pretreatment.

Preliminary

IC50 >100 μM.

Preliminary

IC50 >100 μM.

Preliminary

equal to 24.1 μM.

Preliminary

IC50 >100 μM.

Preliminary

IC50 >100 μM.

 test determined

NE

 test determined

NE

 test determined

 IC50

NE

 test determined

NE

 test determined

NE

[162]

[162]

[162]

[162]

[162]



Flavanols

Epigallocatechin

Flavanonols

Aromadendrin

Pinobanksin Pinobanksin

Silymarin

In vitro

A375-S2

1 · 10�5 to 1 · 10�4 M

(HM)

5-methylether

In vitro

B16BL6

ND

(MM)

In vitro

B16BL6

ND

(MM)

In vitro M14 (HM)

 1–40 μM

The compound

inhibit cell

μM Preliminary

IC50 > 200 μM.

Preliminary

IC50 equal to 187 μM.

It was assessed the effect of silymarin on anti-Fas agonistic antibody CH11 treated human

malignant melanoma,

cells. significantly

apoptosis in

A375-S2 cells.

Isoflavonoids

20-Hydroxybiochanin

40-Methoxy-20,3,7-

In vitro

B16-BL6

ND

(MM)

trihydroxyisoflavanone

7-

Biochanin A

Calycosin

In vitro

B16-BL6

ND

(MM)

In vitro

B16-BL6

ND

(MM)

O-Methylvestitol

In vitro

B16-BL6

ND

(MM)

 A

In vitro

B16-BL6

ND

Preliminary

IC50 >100 μM.

Preliminary

IC50 >100 μM.

Preliminary

equal to 24.1 μM.

Preliminary

IC50 >100 μM.

Preliminary

IC50 >100 μM.

 test determined

NE

 test determined

NE

 test determined

 IC50

NE

 test determined

NE

 test determined

NE

[162]

[162]

[162]

[162]

[162]

(MM)

 induced cell CH11-treated

Pretreatment

 with silymarin

 A375-S2

 test determined

NE Caspase-8,

caspase inhibitors partially reversed silymarin induced

apoptosis of

The expression of

proteins with death domain

(FADD),

was increased by silymarin

pretreatment.

procaspase-8

 and

�3

CH11-treated

 cells. Fas-associated

�9,

�3 and pan

[201]

 test determined

NE

[175]

[175]

concentrations.

 was not able to

NE

[166]

proliferation

 at 1–40

In vitro

CHL-1 and

0.1–200 μM

Preliminary

equal to 10.3 and 51.2 μM for

CHL-1 and WM266-4 cells.

 test determined

 IC50

Expression

apoptosis markers.

 of ER stress and

[200]

188 Natural Products and Cancer Drug Discovery

WM266-4

(HM)

Model Cell line

Concentration

 or dose

Effectiveness

Possible

mechanisms

 of action

 Reference Flavonoids: Promising Natural Products for Treatment of Skin Cancer (Melanoma) http://dx.doi.org/10.5772/67573 189


Box 1.Anticancer activity of flavonoids on melanoma cell lines.

apigenin in an experimental lung metastasis model. Apigenin-treated mice had significant fewer metastatic nodules when compared to the vehicle control group, suggesting apigenin inhibits the metastasis potential of B16F10 melanoma cells in vivo mouse model (Figure 4).

#### 6.2. Bioactivity of diosmin on melanoma

Flavonoid Pratensein

Vestitol Vestitone Violanone Xenognosin

ND: not described; NE: not evaluated; HM: human melanoma cell line; MM: murine melanoma cell line.

Box 1.

Anticancer

 activity of flavonoids

 on melanoma

 cell lines.

 B

In vitro

B16-BL6

ND

(MM)

In vitro

B16-BL6

ND

(MM)

In vitro

B16-BL6

ND

(MM)

In vitro

B16-BL6

ND

(MM)

Model Cell line

In vitro

B16-BL6

ND

(MM)

Concentration

 or dose

Effectiveness

Preliminary

IC50 >100 μM.

Preliminary

equal to 57.4 μM.

Preliminary

IC50 >100 μM.

Preliminary

IC50 >100 μM.

Preliminary

IC50 equal to 34.1 μM.

 test determined

NE

 test determined

NE

 test determined

NE

 test determined

 IC50

NE

 test determined

Possible

NE

mechanisms

 of action

 Reference

[162]

[162]

[162]

190 Natural Products and Cancer Drug Discovery

[162]

[162]

Diosmin is a glycosylated flavonoid commonly used as an active constituent of several pharmaceutical products, mainly for cardiovascular diseases treatment. Diosmin is used in the treatment of venous insufficiency, because of its vasoprotector and venotonic properties. In addition, it acts as an antioxidant, anti-inflammatory, and antimutagenic molecule, regulating the activity of several enzymes, including cyclooxygenases and cytochrome P450 proteins [177, 179]. Interestingly, the anticancer effects of diosmin have also been studied [176, 178], suggesting that this flavonoid presents a broad spectrum of pharmacological activities.

Conesa et al. [179] performed a comparative study with three different flavonoids (tangeretin, rutin, and diosmin) using an experimental model of B16F10 melanoma cell-induced pulmonary metastasis. The greatest reduction in the number of metastatic nodules (52%) was obtained with diosmin treatment. Similarly, diosmin presented a relevant decreasing in implantation, growth, and invasion index (79.40, 67.44, and 45.23%, respectively). These results were confirmed by another study developed by Martínez et al. [178], suggesting diosmin is an effective agent against metastatic stages of melanoma.

The antimetastatic effect of diosmin has also been evaluated in combination with IFN-α [176, 177], an important cytokine that has shown the significant effect in the treatment of metastatic melanoma in high doses. In both investigations, it was verified that synergistic antiproliferative and antimetastatic effects shown by the combination of the flavonoid and the lowest dose of IFN-α, which was similar to that produced by the highest dose of the cytokine alone. These results suggest that diosmin may be used in combination with IFN-α in an attempt to reduce its therapeutic dose, thereby reducing the side effects promoted by continued cytokine use.

Figure 4. Apigenin inhibited murine melanoma B16F10 cell lung metastasis. B16F10 melanoma cells were injected into the tail vein of the C57BL/6 mice. These mice then received intragastric administration of vehicle or apigenin (150 mg/kg/ day) for 24 consecutive days. Lung metastasis of B16F10 melanoma cells in the mouse model (upper) and the metastasis nodules number in the lungs (bottom) were shown. Data were mean SD, n = 8, \*p < 0.05. This figure was taken from Ref. [172].

### 6.3. Bioactivity of fisetin on melanoma

Fisetin (3,7,3<sup>0</sup> ,40 -tetrahydroxyflavone) is a flavonol also found in many fruits and vegetables, such as strawberries, apples, persimmons, kiwi, onions, and cucumbers. This flavonoid has shown a relevant neuroprotective effect, aiding in memory and cognition processes, as well as reducing behavioral deficits. Recently, the effect of fisetin on anticancer therapy has also been studied [186].

Investigation conducted by Syed et al. [188] determined an IC50 value of 38.1 and 20.3 μM against A375 human melanoma cell line, at 24 and 48 h after treatment. In a subsequent study, Syed et al. [191] have demonstrated that fisetin induces apoptosis in melanoma cells. The efficacy of fisetin in the induction of apoptosis varied with cell type and preliminary results confirmed apoptosis as the primary mechanism through which fisetin inhibits melanoma cell growth. The possible mechanisms involved include upregulation of ER stress markers such as IRE1a, XBP1s, ATF4, and GRP78. In addition, both extrinsic and intrinsic apoptosis pathways are involved in fisetin cytotoxic effects.

The effect of fisetin was also evaluated on the growth of metastatic 451Lu human melanoma cells, which exhibit constitutiveWnt signaling in addition to harboring a mutation in the B-Raf gene. The IC50 value was estimated to be 17.5 μM at 72 h of treatment in the MTTassay. In an in vivo model, a smaller average tumor volume was consistently observed in mice treated with fisetin. This was more marked in animals receiving 1 mg fisetin than in animals receiving the 2 mg dose, indicating a nonlinear dose response. The authors attributed this effect to a decreasing of cell viability with G1-phase arrest and disruption of Wnt/β-catenin signaling mediated by fisetin [186].

A recent report evaluated the effect of fisetin in combination with sorafenib, a multi-kinase inhibitor of mutant and wild-type BRAF and CRAF kinases, on melanoma cell invasion and metastasis. In this study, fisetin potentiated the anti-invasive and antimetastatic effects of sorafenib in vivo, suggesting that this flavonoid can be used as an alternative agent in melanoma therapy reducing doses of anticancer drugs used for this purpose [187].

#### 6.4. Bioactivity of luteolin on melanoma

Luteolin is a common flavone that exists in many types of plants including fruits, vegetables, and medicinal herbs. This flavonoid presents potential for cancer prevention and therapy [174]. Concerning to melanoma treatment, George et al. [182] showed that luteolin possesses relevant cytotoxicity against A375 human melanoma cell line, with an IC50 value of 115.1 μM in a preliminary test. Luteolin also inhibited colony formation and induced apoptosis in a dose and time-dependent manner by disturbing cellular integrity. Accumulation of cells in the G0/ G1 (60.4–72.6%) phase for A375 cells after 24 h treatment indicated cell cycle arresting potential of this flavonoid, suggesting that luteolin inhibits cell proliferation and promotes cell cycle arrest and apoptosis in human melanoma cells. A similar result was demonstrated by Casagrande and Darbon [204], who highlighted the involvement of the regulation of cyclindependent kinases CDK2 and CDK1 in the antiproliferative effect of luteolin on OCM-1 human melanoma cells.

In a recent investigation, the inhibitory effect of luteolin on melanoma cell proliferation was related to ER stress induced. In this context, luteolin increased the expression of the ER stress-related proteins, such as protein kinase RNA-like ER kinase, phosphorylation eukaryotic translation initiation factor 2α, activating transcription factor (ATF) 6, CCAAT/enhancer-binding proteinhomologous protein (CHOP), and cleaved caspase 12. In addition, luteolin increased the level of intracellular ROS, leading to ROS-mediated apoptosis and ER stress, suggesting that luteolin induces apoptosis by ER stress via increasing ROS levels [183].

Anticancer potential of luteolin has also evaluated in vivo. In experimental metastasis model, mice treatment with luteolin (10 or 20 mg/kg) reduced metastatic colonization in the lungs by 50%. This treatment increased E-cadherin expression while reduced the expression of vimentin and β3 integrin in the tumor tissues [184]. These results encourage the use of luteolin as an anticancer chemopreventive and chemotherapeutic agent.

#### 6.5. Bioactivity of quercetin on melanoma

6.3. Bioactivity of fisetin on melanoma

are involved in fisetin cytotoxic effects.

6.4. Bioactivity of luteolin on melanoma

human melanoma cells.


as strawberries, apples, persimmons, kiwi, onions, and cucumbers. This flavonoid has shown a relevant neuroprotective effect, aiding in memory and cognition processes, as well as reducing behavioral deficits. Recently, the effect of fisetin on anticancer therapy has also been studied [186]. Investigation conducted by Syed et al. [188] determined an IC50 value of 38.1 and 20.3 μM against A375 human melanoma cell line, at 24 and 48 h after treatment. In a subsequent study, Syed et al. [191] have demonstrated that fisetin induces apoptosis in melanoma cells. The efficacy of fisetin in the induction of apoptosis varied with cell type and preliminary results confirmed apoptosis as the primary mechanism through which fisetin inhibits melanoma cell growth. The possible mechanisms involved include upregulation of ER stress markers such as IRE1a, XBP1s, ATF4, and GRP78. In addition, both extrinsic and intrinsic apoptosis pathways

The effect of fisetin was also evaluated on the growth of metastatic 451Lu human melanoma cells, which exhibit constitutiveWnt signaling in addition to harboring a mutation in the B-Raf gene. The IC50 value was estimated to be 17.5 μM at 72 h of treatment in the MTTassay. In an in vivo model, a smaller average tumor volume was consistently observed in mice treated with fisetin. This was more marked in animals receiving 1 mg fisetin than in animals receiving the 2 mg dose, indicating a nonlinear dose response. The authors attributed this effect to a decreasing of cell viability with

A recent report evaluated the effect of fisetin in combination with sorafenib, a multi-kinase inhibitor of mutant and wild-type BRAF and CRAF kinases, on melanoma cell invasion and metastasis. In this study, fisetin potentiated the anti-invasive and antimetastatic effects of sorafenib in vivo, suggesting that this flavonoid can be used as an alternative agent in mela-

Luteolin is a common flavone that exists in many types of plants including fruits, vegetables, and medicinal herbs. This flavonoid presents potential for cancer prevention and therapy [174]. Concerning to melanoma treatment, George et al. [182] showed that luteolin possesses relevant cytotoxicity against A375 human melanoma cell line, with an IC50 value of 115.1 μM in a preliminary test. Luteolin also inhibited colony formation and induced apoptosis in a dose and time-dependent manner by disturbing cellular integrity. Accumulation of cells in the G0/ G1 (60.4–72.6%) phase for A375 cells after 24 h treatment indicated cell cycle arresting potential of this flavonoid, suggesting that luteolin inhibits cell proliferation and promotes cell cycle arrest and apoptosis in human melanoma cells. A similar result was demonstrated by Casagrande and Darbon [204], who highlighted the involvement of the regulation of cyclindependent kinases CDK2 and CDK1 in the antiproliferative effect of luteolin on OCM-1

In a recent investigation, the inhibitory effect of luteolin on melanoma cell proliferation was related to ER stress induced. In this context, luteolin increased the expression of the ER stress-related

G1-phase arrest and disruption of Wnt/β-catenin signaling mediated by fisetin [186].

noma therapy reducing doses of anticancer drugs used for this purpose [187].

,40

192 Natural Products and Cancer Drug Discovery

Fisetin (3,7,3<sup>0</sup>

Quercetin is a noncarcinogenic dietary flavonoid with low toxicity, has been shown to exert antioxidant, anti-inflammatory, neuroprotective, and antimelanoma activities [193]. A preliminary study showed that quercetin presents a weak cytotoxic effect on B16F10 murine melanoma cells, with an IC50 value > 50 μM [174]. However, Casagrande and Darbon [204] and Kim et al. [196] showed that quercetin presents a considerable antiproliferative effect on OCM-1 and SK-MEL-2 human melanoma cells, with an IC50 value between 4.7 and 19 μM. In these investigations, the authors demonstrated that the presence of hydroxyl group at the 3<sup>0</sup> -position of the ring B in quercetin favors the cytotoxic effect and a G1 cell cycle arrest. The involvement of the regulation of cyclin-dependent kinases CDK2 and CDK1 may also be present in its anticancer effect.

Cao et al. [198] evaluated the involvement of STAT3 signaling in the inhibitory effects of quercetin on melanoma cell growth, migration, and invasion. Quercetin treatment promoted inhibition in proliferation of melanoma cells, induction of cell apoptosis, and suppression of migratory and invasive properties. Furthermore, mechanistic study indicated that quercetin inhibits the activation of STAT3 signaling by interfering with STAT3 phosphorylation, and reducing STAT3 nuclear localization. In an animal model, quercetin inhibited murine B16F10 cells lung metastasis, indicating that quercetin possesses antitumor potential.

### 7. Brief structure-activity relationship (SAR) considerations

Nagao et al. [167] evaluated the cytotoxic activity of 21 flavones and the effect of the substitution patterns on their anticancer potential, although the authors highlight that the number of compounds examined might not be sufficient to determine the structure-activity relationships. Generally, the data show that the growth inhibitory activity of one flavone against the three different tumor cell lines (including a murine melanoma cell line) is not always the same, suggesting differences in the sensitivity of tumor cells to flavones.

The influences of ring A substituents against B16F10 cells were examined. Comparing the antiproliferative activity of four 3<sup>0</sup> ,40 -di-OH-flavones, the order of contribution was found to be 5-OH-6,7-di-OCH3 > 5,6,7-tri-OH > 5,7-di-OH-6-OCH3 > 5,7-di-OH. In contrast, in the 30 -OH-4<sup>0</sup> -OCH3-flavones (desmethoxycentaureidin, eupatorin, and 5,6,3<sup>0</sup> -trihydroxy-7,4<sup>0</sup> dimethoxyflavone), the order is 5,6-di-OH-7-OCH3 > 5-OH-6,7-di-OCH3 > 5,7-di-OH-6-OCH3. In the 3<sup>0</sup> ,40 -di-OCH3-flavones (eupatilin and 5,6-dihydroxy-7,3<sup>0</sup> ,40 -trimethoxyflavone), the order is 5,6-di-OH-7-OCH3 > 5,7-di-OH-6-OCH3, and in the 3<sup>0</sup> -OCH3-4<sup>0</sup> -OH-flavones (jaceosidin and cirsilineol), it is 5,7-di-OH-6-OCH3 > 5-OH-6,7-di-OCH3. In addition, for ring B substituents, 3<sup>0</sup> ,40 di-OH and 3<sup>0</sup> ,40 -di-OH-5<sup>0</sup> -OCH3 showed a greater effect than the others, but the influence of 3<sup>0</sup> ,50 di-OCH3-4<sup>0</sup> -OH appears to be not relevant [167].

Another investigation evaluated the effects of polyhydroxylated flavonoids on the growth of B16F10 melanoma cells. In general, the results suggest that the presence of a C2–C3 double bond and three adjacent hydroxyl groups in the A- or B-rings confers greater antiproliferative activity [174]. Casagrande and Darbon [204] investigated the effects of a series of flavonoids on cell proliferation and cell cycle distribution in human melanoma cells (OCM-1). Interestingly, the presence of a hydroxyl group at the 3<sup>0</sup> -position of the ring B in quercetin and luteolin was correlated to a G1 cell cycle arrest while its absence in kaempferol and apigenin was correlated to a G2 block.

The presence of isoprenoid units in the cytotoxic effect of flavonoids has also been evaluated for melanoma cells [168]. The results indicated that isoprenoid substitutions in flavonoids enhance their cytotoxic potential, and that the position of attachment and the number of isoprenoidsubstituent moieties per molecule influence flavonoid cytotoxicity. This is probably related to their lipophilicity and affinity properties, which favor penetration into the cell membrane.

### Acknowledgements

The authors thank FACEPE (Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco) for financial support.

### Author details

Raimundo Gonçalves de Oliveira Júnior<sup>1</sup> , Christiane Adrielly Alves Ferraz1 , Mariana Gama e Silva1 , Érica Martins de Lavor<sup>1</sup> , Larissa Araújo Rolim<sup>1</sup> , Julianeli Tolentino de Lima1 , Audrey Fleury<sup>2</sup> , Laurent Picot<sup>2</sup> , Jullyana de Souza Siqueira Quintans<sup>3</sup> , Lucindo José Quintans Júnior<sup>3</sup> and Jackson Roberto Guedes da Silva Almeida<sup>1</sup> \*

\*Address all correspondence to: jackson.guedes@univasf.edu.br

1 Center for Studies and Research of Medicinal Plants, Federal University of San Francisco Valley, Petrolina, Pernambuco, Brazil

2 Faculty of Science and Technology, University of La Rochelle, La Rochelle, France

3 Department of Physiology, Federal University of Sergipe (DFS/UFS), São Cristóvão, Sergipe, Brazil

### References

30 -OH-4<sup>0</sup>

In the 3<sup>0</sup>

di-OH and 3<sup>0</sup>

di-OCH3-4<sup>0</sup>

a G2 block.

,40

,40

194 Natural Products and Cancer Drug Discovery

Acknowledgements

Author details

Audrey Fleury<sup>2</sup>

Silva1

Brazil

Pernambuco) for financial support.

Raimundo Gonçalves de Oliveira Júnior<sup>1</sup>

, Laurent Picot<sup>2</sup>

Júnior<sup>3</sup> and Jackson Roberto Guedes da Silva Almeida<sup>1</sup>

\*Address all correspondence to: jackson.guedes@univasf.edu.br

, Érica Martins de Lavor<sup>1</sup>

Valley, Petrolina, Pernambuco, Brazil


presence of a hydroxyl group at the 3<sup>0</sup>



is 5,6-di-OH-7-OCH3 > 5,7-di-OH-6-OCH3, and in the 3<sup>0</sup>


dimethoxyflavone), the order is 5,6-di-OH-7-OCH3 > 5-OH-6,7-di-OCH3 > 5,7-di-OH-6-OCH3.

cirsilineol), it is 5,7-di-OH-6-OCH3 > 5-OH-6,7-di-OCH3. In addition, for ring B substituents, 3<sup>0</sup>

Another investigation evaluated the effects of polyhydroxylated flavonoids on the growth of B16F10 melanoma cells. In general, the results suggest that the presence of a C2–C3 double bond and three adjacent hydroxyl groups in the A- or B-rings confers greater antiproliferative activity [174]. Casagrande and Darbon [204] investigated the effects of a series of flavonoids on cell proliferation and cell cycle distribution in human melanoma cells (OCM-1). Interestingly, the

correlated to a G1 cell cycle arrest while its absence in kaempferol and apigenin was correlated to

The presence of isoprenoid units in the cytotoxic effect of flavonoids has also been evaluated for melanoma cells [168]. The results indicated that isoprenoid substitutions in flavonoids enhance their cytotoxic potential, and that the position of attachment and the number of isoprenoidsubstituent moieties per molecule influence flavonoid cytotoxicity. This is probably related to their lipophilicity and affinity properties, which favor penetration into the cell membrane.

The authors thank FACEPE (Fundação de Amparo à Ciência e Tecnologia do Estado de

, Jullyana de Souza Siqueira Quintans<sup>3</sup>

, Larissa Araújo Rolim<sup>1</sup>

1 Center for Studies and Research of Medicinal Plants, Federal University of San Francisco

3 Department of Physiology, Federal University of Sergipe (DFS/UFS), São Cristóvão, Sergipe,

2 Faculty of Science and Technology, University of La Rochelle, La Rochelle, France

, Christiane Adrielly Alves Ferraz1

\*

, Julianeli Tolentino de Lima1

,40





, Mariana Gama e

,

, Lucindo José Quintans




,40 -

,50 -


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