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Cancer Research Frontiers. 2018; 4(1): 101-130. doi: 10.17980/2018.101

Role of Reactive Oxygen Species and Targeted Therapy in Metastatic Melanoma

Rosalin Mishra*, Hima Patel*, Long Yuan, Joan T. Garrett#

Address: James L. Winkle College of Pharmacy, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, Ohio 45267-0514.

*These authors contributed equally.

 

#Corresponding author: Joan T. Garrett, James L. Winkle College of Pharmacy, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, Ohio 45267-0514. Phone: 513-558-6662; Fax: 513-558-4372; Email: joan.garrett@uc.edu

Citation: Rosalin Mishra, et al. Role of Reactive Oxygen Species and Targeted Therapy in Metastatic Melanoma. Cancer Research Frontiers. 2018; 4(1): 101-130. doi: 10.17980/2018.101

Copyright: @ 2018 Rosalin Mishra, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Competing Interests: The authors declare no competing financial interests.

Received June 29, 2018; Revised Sept 12, 2018; Accepted Sept 14, 2018. Published Oct 11, 2018.

 

Abstract

Reactive oxygen species (ROS) play a significant role in various stages of melanoma development including melanocyte transformation, melanin production, melanoma cell metabolism, metastasis and immune response against melanoma progression. Several molecular and enzymatic signaling cascades control ROS levels and have differential regulation depending on the cell type. The equilibrium between ROS production and scavenging is crucial for maintaining cellular homeostasis and this balance is often altered in tumor cells. ROS is generated in cancer cells due to mitochondrial dysfunction, enhanced metabolic rates, increased cellular signaling, enhanced peroxisome activities and genetic alterations. In this review, we discuss the source and mechanisms of ROS generation. We also highlight the role of ROS in the process of melanomagenesis. This review provides an overview of ROS-dependent anticancer therapies including ROS scavenging antioxidants and ROS boosting therapies which have presented promising outcomes both in in vitro and in vivo melanoma models. We summarize how the understanding of ROS-targeted signaling plays a crucial role in melanoma prognosis and drug resistance. Hence, the knowledge of ROS in melanoma etiology and progression can be exploited in clinical practice for development of better therapies for melanoma treatment.

Keywords: Melanoma, ROS, BRAF, antioxidant, targeted therapy

 

Introduction

Melanoma is a highly malignant form of skin cancer derived from melanin producing melanocytes which protect the skin from UV-induced radiation damage. Although melanoma cases represent only 4% of all skin cancers, it accounts for 80% of all skin cancer related deaths. Melanoma five year survival rates are at only 15% for patients with advanced disease, which is incurable, underscoring the urgent need for more optimal therapies. According to the American Cancer Society, about 91,270 new cases of melanoma will be diagnosed in the US in the year 2018, and 9,320 people are expected to die from melanoma in 2018 (1). The etiology of melanoma is poorly understood, however various molecular and cellular mechanisms are known to contribute towards melanoma development. Melanoma metastasis is characterized by several intermediate processes including angiogenesis, extravasation, evasion of immune surveillance, presence of prothrombotic embolism, adhesion and organ specific colonization (2). Melanoma metastases can develop in regional lymph nodes as in-transit lesions or a satellite or in distant organs. Lymph flow and chemotaxis regulate the homing of melanoma cells to other distant sites. Molecular markers such as p53, BRAF, N-RAS, cyclin-dependent kinase 4 (CDK4), cyclin-dependent kinase inhibitor 2A (CDKN2A), c-KIT, melanocortin 1 receptor (MC1R), mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1) and cadherins contribute to melanoma metastasis (2, 3). Several signaling pathways including mitogen activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK) and phosphoinositide 3-kinase (PI3K) cascades induce the development of melanoma through various genomic alterations. Approximately 50% of melanomas contain a BRAFT1799A transversion, encoding the constitutively active BRAFV600E oncoprotein. This led to the clinical development of selective adenosine triphosphate (ATP)-competitive RAF kinase inhibitors, including vemurafenib and dabrafenib, targeting the mutant BRAF protein (4, 5). Combination therapy including BRAF and MEK inhibitors has become the standard of care for BRAF-mutant melanoma with response rates of ~70% (6, 7). However, most of the patients acquire resistance and eventually exhibit relapse and disease progression. There is evidence suggesting that increased ROS in tumor cells leads to oxidative stress and initiate signaling pathways leading to persistent tumor cell survival, vascularization and metastasis which ultimately results in the resistance to clinically relevant drugs (8).

 

ROS is generated due to enhanced metabolism of transformed cancer cells, immune reaction against developing tumors and DNA damage induced due to ultraviolet radiations. There are several incongruities regarding the role of ROS in regulating cell growth and the mechanism leading to its generation. ROS acts as a double edge sword which exhibits beneficial or deleterious effects depending on cell type, levels and types of ROS involved or genetic background of the living systems. Low/moderate ROS levels are essential for normal cell survival and proliferation; however increased ROS production can lead to oxidative stress which in turn damages cellular proteins, DNA and lipid components. In cancerous cells, enhanced ROS production can lead to activation of signaling cascades and metabolic activities that promote ROS adaptation, ultimately leading to upregulation of antioxidants to maintain the redox homeostasis (9, 10). The balance between benign and deleterious effects of ROS is controlled by redox regulation and is a decisive factor for survival of both normal and cancer cells. Elevated production of ROS and an altered redox status has long been observed in cancer cells including melanoma. High ROS triggers the metastatic potential of melanoma via inducing DNA changes (mutations and epigenetic alterations), activating cell proliferation, stimulating the adhesion of circulating tumor cells (CTCs) to the blood vessels to promote extravasation, escaping immune surveillance, damaging the microenvironment that secrete chemokines to destroy the melanoma cells. It also induces the activation of pro-metastatic molecules such as urokinase plasminogen activator receptor (uPAR), C-X-C chemokine receptor type 4 (CXCR4), interleukin-8 (IL-8), epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and very late antigen 4 (VLA-4) expression which promote melanoma metastasis (11). Migratory bone marrow-derived cells (BMDCs) and tumor-associated macrophages fuse with melanoma cells and contribute to melanoma metastasis via signaling molecules which regulate epithelial-mesenchymal transition (EMT) pathways (12). Recent observations demonstrate that BRAF inhibitors induce ROS generation in melanoma cells through peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α)-induced mitochondria biogenesis (13). Furthermore irrespective of PGC1α status, ROS production is induced by BRAF inhibition (14). Hence, antioxidants or therapies that utilize elevated ROS levels can be used as monotherapy or as combination therapy with standard of care treatments may serve as a promising alternative for patients with malignant melanoma.

 

Transformation of melanocytes to malignant melanoma

Malignant melanoma is a neoplasm which arises from malignant transformation of melanocytes present at the basal epidermis of skin but also found at other sites including mucosal membrane (maxillary gingiva, hard palate, lip, esophagus, throat, vulva, perianal region) and eye (uvea and retina). Melanoma is highly heterogeneous in outcome. All the histological and clinical patterns of melanoma are primarily caused by UV irradiation, with the incidence being markedly augmented in patients with chronic sun exposure and repeated episodes of sunburn (15). Several signaling cascades are differently involved between types and subtypes of melanoma classified according to anatomic site or sun exposure (16). Consequently, a mutation in BRAF and N-RAS commonly prevails in melanoma that is found at sites intermittently exposed to UV, while KIT mutations are found at chronically sun-exposed or sun-protected sites like mucous membranes (17, 18). Ultraviolet (UV) radiations can induce DNA damage through direct and indirect mechanisms. These radiations are subdivided into longer wavelength UVA, shorter UVB and UVC. In addition, UV radiation locally induces several cytokines, proopiomelanocortin peptides, enkephalins, urocortins, corticotropin producing hormones and release them into local circulation to exert systemic effects including activation of the central hypothalamic-pituitary-adrenal axis, opioidogenic effects and immunosuppression (19). UVC radiations possess minimal harmful effects as these are readily absorbed by ozone layer in the atmosphere. UVB radiations are absorbed by the outer epidermis, cause DNA breaks, carcinogenesis in the keratinocytes via formation of mutagenic cyclobutane pyrimidine dimers and photoproducts. This could lead to development of non-melanoma skin cancer (20). Also, UVB radiations cause indirect DNA damage via oxidative stress generation (21). UVA radiations are highly penetrating which are readily absorbed by keratinocytes, melanocytes and dermal fibroblasts (22). UVA induced melanocytes transformation is through two different mechanisms, depending on the different precursor lesions which include different gene mutations and stages of transformation as discussed (23). Melanomas associated with chronic sun exposure due to UVA radiation don’t arise from preexisting nevi, but from melanoma in situ or dysplastic lesions containing several mutations in proto-oncogene and tumor suppressor gene including tumor protein p53 (TP53), neurofibromatosis type I (NF1), and phosphatase and tensin homolog (PTEN). However, benign nevi harboring 80% of BRAFV600E mutation remain indolent for decades due to immune surveillance. These nevi require activation of additional mutations in telomerase reverse transcriptase (TERT) and CDKN2A for their malignant transformation (23-25). UVA and UVB radiations both contribute to light induced carcinogenesis and immune suppression (26). A recent study demonstrates that malignant transformation of melanocytes in melanoma cell lines and patient tumors follows a two-dimensional differentiation trajectory which is attributed to the embryonic history of melanocytes derived from four subtype clusters. The first cluster is the undifferentiated subtype due to enrichment of invasive phenotype genes including those involved in cell migration and adhesion. The second is defined as neural crest-like subtype because of unique enrichments for neural crest-related gene sets. The third subtype is transitory subtype due to mixed enrichment of pigmentation-associated genes and neural crest-associated gene types. The fourth is the most differentiated melanocytic subtype due to loss of neural crest signature and enrichment of pigment-associated gene sets. This differentiation model can be linked with subtype-specific sensitivity to iron-dependent oxidative stress and non-apoptotic cell death termed as ferroptosis. In addition, this differentiation pattern plays a key role in recurrent innate/acquired resistance mechanisms to kinase targeted therapies and immunotherapies in the clinic (27). Figure 1 illustrates the transformation of normal melanocytes to malignant melanoma and the associated signaling mechanism.

 

 

Figure 1. Transformation of normal melanocytes to malignant melanoma and associated signaling mechanism. Factors contributing to malignant transformation of normal melanocytes. It depicts normal and hyperactivated MAPK pathway and consequences of altered ROS levels in normal melanocytes versus malignant melanoma cells.

 

Melanin synthesis and its hormonal regulation

Melanin represents a group of natural pigments classified as eumelanin, pheomelanin, neuromelanin which are synthesized inside melanocytes as the end products during multistep transformation of L-tyrosine. Melanin biosynthesis is initiated either directly from L-tyrosine or the hydroxylation of L-phenylalanine to L-tyrosine that is further hydroxylated to L-dihydroxyphenylalanine (L-DOPA). L-DOPA is oxidized to dopaquinone, a common intermediate to eu- and pheomelanogenic pathways (28). Dopaquinone is transformed into leukodopachrome during the process of eumelanogenesis followed by several oxido-reduction reactions with production of the intermediates dihydroxyindole (DHI) and DHI carboxylic acid (DHICA) along with the final synthesis of eumelanin. Dopaquinone conjugates with cysteine or glutathione (GSH) to yield cysteinyldopa and glutathionyldopa along with the final production of pheomelanin during the process of pheomelanogenesis. The types of intermediates and rate of reaction are tightly regulated by physicochemical milieu including pH, metal ions and tyrosinase related protein type 1 (TRP1) and tyrosinase related protein type 2 (TRP2) (28, 29). L-tyrosine and L-DOPA not only serve as substrate and intermediate in the process of melanogenesis but also act as positive regulators and inducers in the melanogenic pathway through receptor or non-receptor-mediated processes (30). Based on L-DOPA and L-tyrosine concentrations, membrane-associated binding proteins lead to formation of melanosomes and delivery of elements necessary for melanogenesis through endocytic signaling pathways (31). There are several hormones including melanocortins (MSH) and adrenocorticotropic hormone (ACTH) and their receptors which regulate the process of melanogenesis. Several other growth factors and neurotransmitters also modulate various steps involved in melanogenesis (32). The systemic administration of ACTH, α-MSH and ß-MSH induce pigmentation in sun-exposed areas of human skin (33, 34). In melanoma cells, α-MSH and ß-MSH trigger melanogenesis by activating tyrosinase and post-dopa oxidase steps (35). In cultured melanocytes derived from human skin, minimal concentration of α-MSH, ß-MSH and ACTH stimulate melanocyte proliferation, cyclic adenosine monophosphate (cAMP) synthesis and tyrosinase activity via interaction with MC1R (36). ß-endorphin (a secretory hormone) and its µ-opiate receptor are widely expressed in human epidermal melanocytes and closely associated with melanocyte proliferation, dendricity and pigmentation. ß-endorphin/µ-opiate receptor complex signals via protein kinase C (PKC) ß-isoforms or at gene transcriptional level to upregulate tyrosinase activity with stimulation of melanogenesis (37). Endothelin, a vasoconstrictive peptide synthesized by endothelial cells plays a key role in melanocyte proliferation and differentiation (38). Cultured human melanocytes stimulated by endothelin results in increased tyrosinase activity via activation of PKC, cAMP and PKA signaling pathways (39). Histamine, a neurotransmitter known to be involved in inflammatory response regulates the ratio of eumelanin-to-pheomelanin in cultured human melanocytes (40). Different histamine receptor agonists differentially regulate tyrosinase activity and the process of melanogenesis (41). The catecholamine agonists including those against epinephrine, norepinephrine modulate tyrosinase activity and melanin synthesis (42). Stem cell factor (SCF) also known as KIT ligand or mast cell growth factor is encoded by c-kit proto-oncogene which regulates melanocyte differentiation and melanin pigmentation (43, 44). Several steroid hormones and their receptors modulate melanogenesis by regulating tyrosinase activity. Estrogen effect on tyrosinase activity is influenced by multiple factors including cultured conditions of human melanocytes (45, 46). Vitamin D and its derivatives differentially regulate melanocyte proliferation, tyrosinase activity or melanogenesis. A study found that vitamin D3 induces the tyrosinase activity (47) and another report suggests no significant effect on its activity (48). Another study indicates that human melanocytes stimulated with vitamin D3 do not affect melanin synthesis, melanocytes proliferation or tyrosinase activity whereas vitamin D3 derivatives including 25(OH)D3 and 1,25(OH)2D3 suppress tyrosinase activity via stimulation of endothelin B receptor expression (49). Melanogenesis is negatively regulated by several G- protein coupled ligands (serotonin, melatonin, dopamine and acetycholine) and their receptors, melanocortin antagonists (agouti proteins, melanin concentrating hormones), cytokines including interleukin-1 (IL-1), interleukin-6 (IL-6), interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα) and their receptors. Other potent inhibitors of melanogenesis are ceramide-2, triiodothyronine (T3) and vitamin E (32).

 

Melanogenesis in regulating melanoma behavior and therapy

Melanogenesis is a tightly regulated process leading to melanin synthesis which protects the epidermis and normal melanocytes against UV-induced damage via acting as radioprotector, light filter and scavenger of free radicals, metal cations and toxic chemicals. These properties although exhibit beneficial effects on normal skin however make the melanotic melanomas resistant to chemo-, radio- and phototherapy (32). It also affects epidermal homeostasis and melanoma behavior via modulating various signaling pathways (50). Induction of melanin pigmentation in cultured melanoma cells results in robust upregulation of HIF-1-dependent target gene expression involved in the process of angiogenesis (vascular endothelial growth factor A; VEGFA), cellular metabolism including glucose metabolism (glucose transporter 1; GLUT1) and also stimulates key enzymes in the glycolytic pathway including pyruvate dehydrogenase kinase 1 (PDK1), hexokinase 2 (HK2), aldolase A (ALDOA), lactose dehydrogenase A (LDHA). In addition, melanogenesis stimulates several stress, steroidogenic, immune and growth-related genes having putative hypoxia response elements (HREs) binding sites indicating the role of melanogenesis in HIF-1-independent pathways. High concentration of melanogenic precursors in culture media of hamster AbC1 and SK-MEL-188 melanoma cells leads to overall increase in HIF-1α and but not HIF-2α expression. Nuclear HIF-1α expression is significantly higher in excisions of advanced melanotic compared to amelanotic melanomas (51). Melanin synthesis is linked to higher disease advancement in patients with metastatic melanoma and reduces the outcome of radiotherapy (52). It also shortens overall and disease-free survival of patients with metastatic melanoma (53). The process of melanogenesis acts as a double edge sword where it generates melanin to scavenge ROS and other toxic agents on one hand; while on the other induces ROS, quinone, semiquinone intermediates production which display genotoxic, cytotoxic or mutagenic activities and act as potent immunosuppressant. The intermediates produced in the melanogenesis process exhibit immunosuppressive effects by shutting off T- and B-cell immune activities or the selective lymphotoxic effects of levodopa or products of its autoxidation (30, 32, 54-56).

 

Reactive Oxygen Species and its source

ROS are a group of chemically reactive molecules comprising of a family of radical and non-radical species derived from partial reduction of molecular oxygen. A radical species is a free electron-containing species that includes superoxide anion (O2•ˉ), its conjugated hydroperoxyl radical (HO2•ˉ), hydroxyl (•OH), carbonate (CO3•ˉ), peroxyl (RO2•) and the alkoxyl radical (RO•). Non-radical species like hydrogen chloride (HCl), hydrogen peroxide (H2O2), reactive aldehydes, fatty acid hydroperoxides (FaOOH), reactive aldehydes and singlet oxygen which can be readily reduced into free electron-containing species (57). The way ROS reacts with different compounds varies, based on the processing into higher reactive species and their diffusion capabilities. Certain ROS including H2O2 and O2•ˉ readily diffuse away from the site of formation into extracellular space and are more stable as these molecules don’t interact with other biomolecules. Hydroxide free radicals on the other hand are highly reactive with a very short half-life. •OH radicals produced from the reaction of iron and hydrogen peroxide are very reactive as they readily react with species in the immediate environment in vivo (58). The ROS generation traditionally is attributed as a byproduct of cellular metabolism during mitochondrial electron transport chain (ETC) (59). Other cellular compartments including peroxisomes, melanosomes, and enzymes including NADPH oxidases (NOX) family members are also known to contribute to ROS generation (60). Isolated mitochondria are known to produce O2•ˉ through autoxidation of the flavin component of complex I nicotinamide adenine dinucleotide phosphate hydrogenase (NADPH) and/or autooxidation of the semiquinone at complex III. Approximately 1-5% of total oxygen consumed in aerobic metabolism leads to formation of O2• ˉ which is produced by complex I and is released into mitochondrial matrix via electron leaks. Complex III releases O2•ˉ both into inner mitochondrial space and matrix (61, 62). Mitochondrial DNA mutation is not the major cause for ROS synthesis and cancer development in melanoma. However, the mitochondria plays a key role in defective metabolic regulation and mitochondrial-derived ROS directly participate in the process (63). In addition, some reports suggest the role of mitochondrial-derived ROS in cancer metastasis (64, 65).

 

Another source of ROS is NADPH oxidases, a NOX family member which contain membrane-bound enzymes catalyzing the controlled production of O2•ˉ via coupling NADPH-derived electrons to oxygen. NADPH oxidase complex contains cytochrome B559 with two subunits of gp91phox, p22phox and four cytoplasmic proteins, p47phox, p67phox, p40phox and the small guanosine triphosphate (GTP) binding Ras-related C3 botulinum toxin substrate 1 (RAC1) and RAC2 (66, 67). The NOX family of enzymes includes 7 family members (Nox1-5, Duox1 and Duox2) and acts as a major contributor of cytosolic ROS. Several pieces of evidence suggest the key role of cytosolic ROS in melanoma progression. Nox1 overexpression in Wm3211 primary melanoma cells induces ROS production, increases cell invasion via upregulation of matrix metallopeptidase 2 (MMP-2) protein expressions and regulation of the EMT pathway (68). Hyaluronic acid (HA) stimulates Rac1 activity, induces Nox1 activity which enhances ROS generation and B16F10 cell motility (69). H2O2 induces ROS production via Rac1/Nox1-dependent mechanism which in turn induces the pro-metastatic property of mouse non-invasive B16F0 melanoma cells (70). Nox4 expression is associated with melanoma development and Nox4-generated ROS contributes to melanoma growth via regulating the G2-M cell cycle arrest (71). Superoxide anions are also generated through electron transfer reaction catalyzed by enzymes including xanthine oxidases, cyclooxygenases, monoamine oxidases, lipoxygenases and components of the cytochrome P450 system. Peroxisomes under physiological conditions generate H2O2, but not O2•ˉ (72). Several studies indicate that melanocytes and melanoma cells demonstrate a unique redox regulation that led to discovery of ROS generating roles of melanin and melanosomes in melanoma progression (73-75).

 

Role of ROS in initiation and melanoma progression

ROS is involved in several stages of melanomagenesis which includes ROS-induced malignant transformation of hypoxic melanocytes, melanoma cell metabolism, immune response towards melanoma, melanin synthesis and melanoma metastasis. Hypoxia inducible factor-1 alpha (HIF-1α) activation is necessary for AKT-dependent transformation of melanocytes residing in the mildly hypoxic environment of skin epidermis (76). AKT induces the expression of the ROS generating enzyme Nox4 in melanoma cells and also stabilizes cells that generate ROS due to severe mitochondrial damage (77). MAPK signaling is utilized by melanoma cells to regulate ROS synthesis which in turn maintains melanoma cell survival by modulating apoptotic signaling pathways (78). ROS constitutively activates the (nuclear factor kappa-light-chain-enhancer of activated B cells) NF-ĸB pathway to promote melanoma progression (79). ROS activates another transcription factor, activator protein-1 (AP-1) which plays a crucial role in Ras-induced oncogenic transformation (80, 81). It is reported that ROS-induced cell death of melanoma cells contributes to vasculogenic mimicry, the formation of microvascular channels by tumor cells (82). Metabolically active melanoma cells can impair ROS homeostasis by modulating the activity of growth promoting signaling pathways. ROS generated by NADPH oxidase increases melanoma cell proliferation via NF-ĸB pathway activation (83). Inflammatory immune cells including mast cells, monocytes/macrophages infiltrate the microenvironment of melanoma during early and late stage of tumorigenesis. These cells are capable of producing ROS as a cytotoxic mediator to kill cancer cells. ROS generated by these inflammatory cells can induce permanent genomic alterations such as mutations, rearrangements and deletions leading to development of genetically different tumor cells which exhibit resistance to oxidative stress pressure (84). UV radiation-induced melanin synthesis is a complex phenomenon which results in formation of basic monomers for red/light brown phaeomelanin. Cysteine is a necessary amino acid involved in phaeomelanin production that plays an important part in defense mechanism against ROS signaling. More production of phaeomelanin leads to depletion of glutathione and causes oxidative stress. As a result phaeomelanin and 5-S-cysteinyldopa become pro-oxidants in melanoma cells compared to normal melanocytes (85). Binding of iron to melanin oxidizes melanin which reacts with O2 to form H2O2, O2•ˉ and other free radicals which perturb the ROS homeostasis (86). UVA induces thioredoxin interacting protein (Txnip) expression which consequently suppresses Trx (thioredoxin) activity. This process results in enhanced ROS and transendothelial cell migration of melanoma cells which promotes early stage melanoma metastasis. High ROS inhibits Txnip expression, thereby pushing cells to inhibit ROS levels (87). The in vivo metastasis is also dependent on interaction of ROS with the tumor microenvironment and activation of HIF-1α regulated genes (88, 89).

 

ROS and redox signaling in melanoma

Tumor cells exposed to high ROS levels activate several redox sensors in order to adapt to the oxidative stress for maintenance of homeostasis. Apurinic/apyrimidinic endonuclease 1 (APE-1)/Ref-1 activates several transcription factors such as AP-1, HIF-1α, NF-ĸB and p53 which modulate cell survival, proliferation, and apoptotic signaling pathways (90). Increased APE-1/Ref-1 is associated with decreased ROS and reduced DNA damage lesions induced due to oxidative stress. However, a prolonged oxidative stress leads to continuous activation of APE-1/Ref-1 that switches cellular signaling to sustained proliferation. Yang et al. shows that enhanced ROS and APE-1/Ref-1 contribute significantly to malignant transformation by increased soft agar colony formation and anchorage-independent growth (91). The same group also demonstrates that abnormal high levels of APE-1/Ref-1 in melanoma cells versus normal melanocytes can lead to melanoma cell proliferation and drug resistance (92). Ras-ERK pathway is activated by superoxide anion level which is associated with global DNA hypermethylation responsible for malignant transformation of melanocytes (93). Another MAPK signaling pathway, p38 is activated in melanocytes exposed to oxidative stress and UV-induced irradiation. Activated p38 in turn induces the tumor suppressor p16INK4A which in turn decreases ROS levels (94). The p38 MAPK pathway is reported to play a significant role in ROS-induced apoptosis (95, 96).

 

Antioxidant therapy in melanoma

Redox homeostasis in normal or tumor cells including melanoma is determined by the balance between the ROS production and detoxification rates by various antioxidant defense systems. The body has a number of ways by which it can induce formation of antioxidants in response to increased oxidative stress. The maintenance of intracellular redox state is crucial for regulation of signal transduction pathways in both normal and cancer cells. Based on this, an option for addressing increased ROS levels would be to augment the levels of antioxidants in melanoma patients. Literature both supports and opposes the role of antioxidants in melanoma development. Antioxidant defense systems are broadly categorized into enzymatic and non-enzymatic sub-types. Enzymatic antioxidants defense system consists of superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR) and catalase (CAT). SODs convert the O2•ˉ into the lesser reactive species H2O2 (97). There are three SODs involved in the superoxide neutralization process depending on the site of production and diffusion. MnSOD2 is present in the mitochondria, Cu,Zn-SOD1 is present in the cytoplasm, Cu,ZnSOD3 is located in the extracellular space (98). SOD inhibits the vemurafenib-induced upregulation of intracellular O2•ˉ and nitric oxide (NO) production, thereby salvaging cell proliferation in human melanoma A375 cells harboring BRAFV600E mutation (99). Increased MnSOD2 expression alters the malignant phenotypes of melanoma cells in culture, reduces colony formation and tumor growth in nude mice model (100). Catalase decomposes H2O2 to H2O and O2 in peroxisomes. Polyethylene glycol (PEG)-conjugated catalase shows a beneficial effect in in vitro and in vivo melanoma models in which it lowers the expression of epidermal growth factor (EGF) and EGFR in the lungs of rodents harboring metastatic melanoma tumor cells. PEG-catalase inhibits the initial stages in metastasis by reducing the number of surviving tumor cells and is also significantly effective in later stages of melanoma progression (101). Taken together, PEG-catalase inhibits the survival, adhesion, invasion and proliferation both in in vitro and in vivo melanoma models by inducing tumor dormancy and hence prolonging tumor survival period (102). Glutathione catalyzes the reduction of H2O2 into H2O via oxidation of glutathione. Glutathione is present in both reduced (GSH) and in oxidized (GSSG) forms. The ratio of GSH/GSSG tightly regulates redox homeostasis in the mammalian cells (103). High GSH levels protect metastatic murine melanoma B16 cells from in vivo and in vitro sinusoidal cell-mediated oxidative stress, thereby contributing to metastatic cell survival within the hepatic microvasculature (104). Peroxiredoxins (Prxs) are a family of peroxidases which reduce H2O2 into H2O. Prxs isoforms are maintained in the reduced state by the thioredoxin reduction system in conjunction with GSH (105). The thioredoxin enzyme system consists of thioredoxin (Trx), thioredoxin reductase (TrxR) and NADPH. Ectopic expression of Prx2 inhibits melanoma cell migration and proliferation via negatively regulating ERK/Src pathway which increases the E-cadherin/β-catenin complexes in the adherens junction. Also, Prx2 expression inversely correlates with the metastatic capacity of melanoma cells (106).

 

Trxs are small 12 kDa redox proteins with a disulfide reducible site. It reduces into Trx-(SH)2 by thioredoxin reductase in the presence of the electron donor, NADPH. Trx isoforms are located in the cytoplasm, nucleus and mitochondria of mammalian cells (107). Trx1 sensitizes melanoma to inhibit glycolytic metabolism which results in inhibition of in vivo melanoma metastasis (108). The intracellular expression of Trx and TrxR along with endogenous TNFα expression correlates with the resistance to TNFα-induced cytotoxicity in melanoma cells (109). Melanoma cells secreted Trx induces regulatory T cells (Tregs) infiltration and triggers the survival of Tregs in tumor microenvironment of murine metastatic melanoma model by suppressing antitumor immune response. This mechanism suggests that Trx antibody therapy could be promising for melanoma treatment in clinic (110). Non-enzymatic antioxidants consist of carotenoids, ascorbic acid (vitamin C), vitamin D derivatives, flavonoids, N-acetyl cysteine (NAC), α-tocopherol (vitamin E), thioredoxin and others antioxidants. Antioxidant supplements administration such as NAC and vitamin E increases melanoma cell invasion and lymph node metastasis in malignant melanoma murine model (111). Mounting evidences from clinical trial indicate that supplementing diet with antioxidants can increase cancer risks (112). Studies exploring the benefits of antioxidants as dietary supplements failed to correlate that higher intake of antioxidants would lower risk of melanoma development (113, 114). Women receiving vitamin C, vitamin E and ß-carotenoid as dietary supplements demonstrate higher incidence of skin cancer compared to men in a cohort of French population (115). Another study indicates that retinol (a component of vitamin A) supplements in diet might have a significant preventative effect in melanoma among women, however there was no correlation established between melanoma risks with intake of vitamin A (116). Vitamin D3 (1,25(OH)2D3) and novel CYP11A1-derived hydroxyderivatives of D3 show anti-melanoma and protective properties against UVB- induced DNA damage while a defect in the signaling supports melanomagenesis. 1,25(OH)2D3 suppresses in vitro cell proliferation, colony formation of cultured human and rodent melanoma cell lines. Low levels of 25(OH)2D3 is associated with poor patient outcome and advanced melanoma progression. Another vitamin D derivative, 20(OH)D3 attenuates in vivo tumor growth of human melanoma cells in immunodeficient mice. Thus inadequate amount of vitamin D, decreased expression of the vitamin D receptors and defects in the enzymes modulating vitamin D activity affect melanomagenesis and tumor progression (117). CYP11A1-derived secosteroids demonstrate protective activity against oxidative stress and UVB-induced DNA damage by attenuating ROS, NO and H2O2 generation in keratinocytes, melanocytes, melanoma cells. These derivatives inhibit the generation of cyclobutane pyrimidine dimers in response to UVB radiation, increase phospho-p53 expression at ser-15 position, regulate the GSH level and upregulate the genes encoding enzymes responsible for defense against oxidative stress (118). A phase II trial is initiated in patient with cutaneous melanoma to evaluate the feasibility, safety and toxicity of oral administration of vitamin D (119). Lumisterol, a stereoisomer of ergosterol is produced as a photochemical by-product during the synthesis of vitamin D1. Lumisterol and its hydroxyl derivatives suppress cell proliferation of human skin cells in a cell-type dependent manner along with inhibition of melanoma cell proliferation in both monolayer and soft agar. 20-hydroxylumisterol stimulates the expression of genes associated with keratinocytes differentiation and anti-oxidative process (120). Resveratrol is a naturally occurring polyphenolic antioxidant abundantly present in grapes, red wine and plant extracts. A study found resveratrol exerts its anti-cancer effects by diminishing malignancy of highly invasive B16F10 and BL6 murine melanoma cells. Oral administration of resveratrol suppresses primary tumor volume, AKT activation and lung metastasis in syngeneic melanoma mouse model (121). It induces G1/S cell cycle arrest, inhibits the proliferation of A375 and SK-MEL-31 cells via caspase-3/9 activation, downregulation of Bcl-2 and upregulation of Bax protein expression (122). Resveratrol triggers the autophagy in B16 cells through ceramide accumulation and AKT/mTOR pathway inhibition (123). In combination with ursolic acid (UA) and chloroquine, resveratrol reduces the viability of B16F10 and A375 cells (124). Co-treatment of resveratrol and 5-fluorouracil (5-FU) suppresses cell growth and angiogenesis in B16 tumors (125). Resveratrol is readily absorbed in human and animal models but undergoes rapid metabolism to form sulphate and glucuronide metabolite which lowers its bioavailability to exhibit effective preventive efficacy for melanoma treatment (126-128). Further investigation is required to demonstrate if repeated dosing of the drug can overcome this problem. Melatonin (N-acetyl-5-methoxy tryptamine) is a hormone synthesized by the pineal gland known to regulate circadian rhythmicity and lower vertebrate skin pigmentation (129). It exhibits a broad spectrum of functional properties which include regulation of apoptosis, direct and indirect antioxidative effects, DNA damage repair, immunomodulatory and antitumor activities. Topical application of melatonin and its derivative N1 -acetyl-N2 -formyl-5-methoxykynurenine (AFMK) protect the human and porcine epidermal cells against UVB-induced 8-hydroxy-2′-deoxyguanosine (8-OHdG) formation, DNA damage and cell death with enhancement in p53ser15 expression. Melatonin de-monstrates photoprotective action in both pre- and post-UVB treated human and porcine epidermal cells. Melatonin and its derivatives increase the expression of antioxidative enzymes including glutamylcysteine synthetase (GCS), CAT, glutathione-s-transferase P 1 (GSTP1), GPx and Cu/MnSOD post-UVB irradiation of HaCaT keratinocytes cells. The exogenous application of melatonin or its derivatives prior to UV exposure results in the improved genomic, cellular and tissue integrity against UVB-induced carcinogenesis (130). Melatonin and its metabolites have a modulatory effect on mitochondrion redox and bioenergetic homeostasis (131). Melatonin reduces cell proliferation and induces melanogenesis in SK-MEL-1 cells via p38 MAPK-dependent signaling pathway (132). Melatonin increases the antitumor activity of fisetin (a bio-flavonoid widely present in plants) in MeWo and SK-MEL-28 cells. Melatonin in combination with fisetin induces PARP cleavage, triggers the release of cytochrome-c, enhances the suppression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression, represses the nuclear localization of p300 and NF-κB and abrogates the binding of NF-κB on COX-2 promoter. It also triggers the inhibition of cell proliferation, cell viability and colony formation in combination with fisetin (133). Melatonin differentially suppresses the proliferation of melanoma cell lines (SBCE2, WM-98, WM-164 and SKMEL-188) having specific pattern of melatonin cellular receptor and cytosolic binding protein expression (134).

 

Other potential ROS targeting therapies in melanoma

Natural therapies from plant source

Dihydromyricetin (DHM) is a flavonoid derived from Ampelopsisgross dentata with a broad range of biological and pharmacological properties which exhibits anti-inflammatory, antioxidant and anticancer effects. Treatment with DHM upregulates the expression of autophagic markers (LC3, Beclin-1 and p62) and induces apoptosis in vitro via regulating NF-ĸB signaling mechanism. DHM triggers ROS generation in a time and dose-dependent manner which plays a significant role in DHM-induced NF-ĸB signaling in human melanoma SK-MEL-28 cells (135). It attenuates melanogenesis by suppressing melanin production and also downregulates MAPK, PKA and PKC signaling cascades (136).

 

Jacaranone is a benzoquinone isolated from the leaves of Pentacalia desiderabilis which induces apoptosis by generating ROS. It has redox cycling capability in which reduced semiquinones formed initiate a signaling cascade that ultimately generates H2O2 and hydroxyl free radicals. It demonstrates protective and anti-tumor effects at low therapeutic doses. Jacaranone increases oxidative stress via inducing ROS formation which leads to mitochondrial damage both in in vitro and in vivo melanoma models. It brings about mitochondrial dysfunction by alteration of the mitochondrial permeability which results in mitochondrial depolarization. Jacaranone treatment significantly increases the levels of pro-apoptotic caspase 2, caspase 3, caspase 8, caspase 9 and Bax in human melanoma A2058 and SK-MEL-28 cell lines. Jacaranone-induced ROS downregulates AKT, activates p38, stimulates apoptosis in vitro and delays in vivo tumor growth in a dose dependent manner in B16F10 cells (137).

 

Isoegomaketone (IK) is an essential oil component of Perilla frutescens which inhibits in vivo tumor growth and induces ROS-mediated apoptosis through mitochondria-dependent/independent pathways in B16 cells. IK-induced apoptosis in these cells is a consequence of upregulation in the Bax/Bcl-2 ratio. It activates the intrinsic caspase 3/9- mediated apoptotic pathway. ROS generation leads to nuclear translocation of apoptosis inducing factor (AIF) which regulates downstream signaling (138). IK-induced ROS generation modulates cell growth inhibition, PI3K/AKT signaling pathway and triggers apoptosis through caspase‑dependent and -independent pathways in SK-MEL-2 cells (139). Another anticancer drug, celastrol suppresses growth and induces apoptosis in B16 melanoma cells via the activation of ROS-mediated caspase-dependent and -independent signaling and the inhibition of of PI3K/AKT pathway (140).

 

Cryptotanshinone (CT), a diterpene is an active component isolated form Salvia miltiorrhiza bunge. It induces apoptosis through the induction of ROS-dependent mitochondrial apoptotic pathway. In addition, it impairs cell proliferation, migration and invasion of A375 cells via downregulation of matrix metallopeptidase-9 (MMP-9) expression. CT triggers ROS-induced apoptosis via increased expression of cleaved caspase 3 and pro-apoptotic protein, Bax along with inhibition of anti-apoptotic Bcl-2 expression (141). Generation of ROS including H2O2 and O2 by CT boosts the apoptotic effects of TNF-related apoptosis-inducing ligand (TRAIL) in vitro and restores the sensitivity of the cells to TRAIL. CT induces the expression of death receptor 5 (DR5) in TRAIL-resistant melanoma cells as a response to ROS-mediated CCAAT/enhancer-binding protein homologous protein (CHOP) activation in A375 cells (142).

 

Cudraflavone C, a naturally occurring flavonol extracted from the roots of Artocarpus species demonstrates anticancer properties in melanoma cells (143). It induces cellular and mitochondrial ROS generation which results in cellular cytotoxicity and cell cycle arrest in B16 cells. Cudraflavone C stimulates A375.S2 cellular apoptosis via enhancing mitochondrial ROS generation, activating MAPKs (p38, ERK, JNK) and upregulating levels of pro-apoptotic protein (Puma, Bax, Bad, Bid, Apaf-1, cytochrome C, caspase 9 and caspase 3/7) expression (144).

 

Icariside II (IS), a flavonol glycoside and a metabolite of icariin, derived from Herba epimedii exhibits anti-proliferative effect. IS inhibits A375 cell proliferation, induces ROS generation and causes G0/G1 and G2/M cell cycle arrest via activation of p38/p53 pathways along with inhibition of cyclin E, CDK2, cyclin B1 and p-CDK1 expression. NAC abrogates ROS-induced inhibition of A375 cell proliferation and cell cycle arrest (145). IS downregulates cFLIP expression, an anti-apoptotic protein and enhances TRAIL-induced apoptosis via inhibition of ROS-dependent STAT3 and NF-ĸB signaling. An increase in ROS level activates AKT and decreases phosphorylated STAT3 levels in A375 cells (146).

 

Curcumin is a primary bioactive component obtained from rhizome of Curcuma longa is known to be significantly effective against melanoma growth and progression. Curcumin induces ROS production to activate mammalian STE-20-like kinase 1 (MST1) and induces apoptosis via JNK pathway activation. Additionally, MST1 mediates curcumin-induced Foxo 3a activation and nuclear translocation, where it upregulates Bim-1 and causes cellular apoptosis in B16 and WM-115 cells. ROS scavenger NAC attenuates curcumin-induced JNK and MST1 activation (147). Curcumin alone or in combination with 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) activates JNK signaling and inhibits PI3K/AKT pathway through the production of ROS which in turn mediates DNA damage and apoptosis (148, 149). It prevents proliferation and induces cell death in melanoma cells via regulating cellular redox levels. It induces oxidative stress through ROS production, reducing GSH levels, wrecking mitochondria membrane potential leading to cytochrome C release causing activation of intrinsic cellular apoptosis in A375 cells. All these effects are reversed in presence of NAC as NAC is an anti-oxidant and scavenges the high ROS generated by curcumin treatment. ROS generated in response to curcumin treatment also targets HIF-1α, induces apoptosis via regulating p53 and caspase dependent signaling mechanism (150). Natural borneol synergizes with curcumin to induce apoptosis in A375 cells via inhibition of MAPK/AKT signaling and activation of JNK/caspase-dependent mechanism. It potentiates curcumin to trigger intracellular ROS overburst leading to DNA damage with upregulation of activated p53, ataxia-telangiectasia-mutated (ATM) and pBRCA1 levels (151).

 

Piperine, an alkaloid obtained from Piper nigrum and Piper longum demonstrates anti-proliferative effect in repressing the in vivo tumor growth using murine melanoma B6 and B16F10 cells (152). Piperine treatment generates ROS which causes cell cycle arrest in SK-MEL-28 and B16F10 cells. Piperine treatment causes DNA damage marked by increase in phosphorylation of ataxia telangiectasia and Rad3-related (ATR) and checkpoint kinase 1 (Chk1) at serine 428 and 296 respectively. Blocking this ROS by tiron protects these cells from piperine-mediated cell cycle arrest in G1 phase and apoptosis (153). It induces apoptosis in A375 and SK-MEL-28 cells via ROS-mediated mitochondrial membrane potential disruption and activation of JNK pathway. This disrupted mitochondrial membrane potential leads to upregulation of cleaved caspase 3, p21, p27 and Bax accompanied with downregulation of Bcl-2 protein expression to mediate apoptosis by intrinsic pathway (154).

 

Zerumbone, a monocyclic sesquiterpene derived from Zingiber zerumbet shows anti-proliferative and anti-migratory effects in CHL-1 melanoma cell line. Treatment with zerumbone increases ROS levels, decreases mitochondrial matrix potential and reduces mitochondrial transcription factor A mRNA levels. This alters the mitochondrial bioenergetics of these melanoma cells by reduction in the ATP and mitochondrial DNA levels (155).

 

Jolkinolide B is a bioactive diterpenoid extracted from the roots of Euphorbia fischeriana steud which induces ROS production, regulates glycolytic pathway, induces mitochondrial-dependent apoptosis, decreases lactic acid, ATP production in murine melanoma B16F10 cells and suppress in vivo tumor growth. Downregulation in the mRNA expression of HK2, lactate dehydrogenase A (LDHA) and GLUT1 promote apoptosis and suppress cell proliferation in response to ROS production. ROS generated in response to Jolkinolide B treatment decreases the mitochondrial membrane potential, upregulates mRNA expression of pro-apoptotic protein such as Bax, downregulates of mRNA levels of anti-apoptotic molecules such as Bcl-2, caspase 3 and caspase 9 (156).

 

Volatile oil from ginger, obtained from Zingiber officinale has an inhibitory effect on cell proliferation and melanogenesis of murine B16 cells. The volatile oil contains various alkene-containing substances (zingiberene and iso-horn teaene) which readily oxidizes and have antioxidant activities to scavenge ROS and inhibit lipid peroxidation. This oil suppresses melanin synthesis and upregulates the levels of antioxidants such as SOD, GSH and CAT in B16 cells. The inhibitory effect of this oil is attributed to its ability to suppress TRP-1, TRP-2, p38 and microphthalmia-associated transcription factor (MITF) activity (157).

 

Natural therapy from fungal source

Trichodimerol is a secondary metabolite isolated from Trichotechium species, a marine fungus that induces anti-proliferative effect and apoptosis in A375 S2 cells. It causes sub-G1 cell cycle arrest and induces apoptosis via increasing activated caspase 3 and caspase 7 levels. It induces ROS production which mediates the anti-proliferative and pro-apoptotic effects on A375 S2 cells along with activation of p38 and inhibition of ERK (158).

 

Natural therapy from animal source

 Mastoparan is an α-helical, 14-amino acid amphipathic and cationic cell penetrating peptide obtained from venom of wasp Vespula lewisii. It demonstrates anti-proliferative effect in a murine model of B16F10-Nex2 cells. It induces oxidative stress by excessive ROS generation in B16F10-Nex2 cells which disrupts the mitochondrial membrane potential and membrane integrity, releases pro-apoptotic molecules into cytosol which leads to activation of caspase-dependent apoptosis. It decreases the expression of Bcl-XL and phospho-Bad (at serine 112) with an increase in the levels of pro-apoptotic proteins such as Bim, Bak, and cytochrome c which triggers apoptosis via intrinsic pathway. Mastoparan also attenuates the growth of subcutaneous melanoma in syngeneic mice and increases their overall survival (159).

 

ROS targeting therapies from synthetic agents

Disulfiram, a member of the dithiocarbamate family and a copper chelator is currently used for treatment of alcoholism (160). Disulfiram inhibits cell proliferation of M-14, WM-278, WM-1552c melanoma cells, targets spreading of melanoma through superficial and nodular routes via increased ROS production and activates the extrinsic apoptotic pathway (161). It induces apoptosis in A375, C81-46a, c81-61 melanoma cell lines by a redox-associated mechanism, in which it chelates the copper along with depleting and/or oxidizing cellular glutathione to generate oxidative stress which induces apoptosis. Disulfiram augments cell death by reducing the mitochondrial membrane polarization and by decreasing the GSH/GSSG ratio (162, 163). It is tested in phase I/II clinical trial in patients with stage IV melanoma (164). A phase II trial investigating the benefits of co-administration of disulfiram with chelated zinc for patients with refractory disseminated malignant melanoma who have failed to show a response to first line therapy has been completed (165).

 

Choline tetrathiomolybdate (ATN-224) is a second generation ammonium tetrathiomolybdate analogue with a high copper binding affinity that inhibits SOD1. Inhibition of SOD1 in tumor cells attenuates angiogenesis, cancer cell proliferation in in vitro and in vivo melanoma models by various pathways such as inhibition of VEGF, induction of p-ERK and signaling molecules as PKB/AKT and NF-ĸB expression (166, 167). ATN-224 in combination with a DNA alkylating agent, temozolomide demonstrates additive cytotoxicity in five melanoma cell lines including M14, WM3211, YUZAZ6, SK-Mel-5 and A375. ATN-224 synergizes with buthionine sulfoximine to show its effect in vitro, where as it demonstrates moderate antagonistic effect with arsenic trioxide or disulfiram, both of which are known to interfere with glutathione recycling (168). It reduces serum copper levels in phase I clinical trial in patient with metastatic melanoma with a recommended phase II dose of 300 mg/day (169). A phase II trial evaluating the safety and efficacy of ATN-224 in combination with temozolomide for patients with advance melanoma has been conducted (170).

Elesclomol is identified from a phenotypic screening of small molecules displaying potent pro-apoptotic activities. Concomitant treatment of elesclomol increases intracellular ROS content and HSP70 RNA levels in Hs294T melanoma cells. Also, elesclomol induces apoptosis via oxidative stress induced cytochrome c release and caspase 3-dependent pathways. This effect is nullified by NAC in Hs294T cells (171). Generation of ROS by elesclomol is in part dependent on chelation and redox recycling of copper in the mitochondria via electron transport system. Elesclomol chelates copper outside of cells leads to elesclomol-Cu II complex formation. This complex selectively transports the copper to mitochondria where Cu II is reduced to Cu I, followed by subsequent ROS production (172, 173). A phase III trial investigating the combination of elesclomol with paclitaxel in patients with advanced melanoma has been terminated as the combination did not improve the progression free survival (PFS) significantly compared to paclitaxel single agent (174, 175). A phase III trial exploring the advantage of combination of elesclomol with paclitaxel vs paclitaxel alone in stage IV metastatic melanoma patients showed minimal improvement in the PFS without any statistical significance (176).

 

SC-514 is an IKKβ inhibitor which induces ROS generation. Increased ROS levels result in enhanced DNA crosslinking efficiency triggered by fotemustine, an alkylating agent and nitrosourea family member. SC-514 enhances ATM phosphorylation and sensitizes fotemustine-induced cell death in A375, G361, A2058, SK-MEL2, SK-MEL-5, SK-MEL-28, Hs294T, IGR-1, MeWo, Colo829, Malma-3M melanoma cell lines. SC-514 synergizes with fotemustine to reduce tumor size and malignancy in vivo (177).

 

TRAM-34 is clotrimazole analog and selective calcium (Ca2+)-dependent potassium (K+) channel inhibitor (KCa3.1). This sensitizes A375 cells to vemurafenib-induced cell death via caspase 3-dependent signaling activation. This synergistic combination alters the mitochondrial membrane potential, increases intracellular ROS levels and is effective in vemurafenib-resistant cells. The antioxidant vitamin E overcomes the effect of activated caspase signaling and ROS production (178). TRAM-34 also increases TRAIL-induced apoptosis via controlled release of second mitochondria-derived activator of caspases (SMAC) and anti-apoptotic cellular inhibitors of apoptosis protein (cIAP) (179).

 

Zinc induces apoptosis by increasing ROS levels and via modulation of p53 and FAS/FAS ligand (FASL) protein expression in WM 266-4 cells. p53 regulates the intracellular redox state and induces ROS-dependent apoptosis (180). Zinc oxide nanoparticles known to possess semiconductor properties induce oxidative stress, enhance ROS production and deplete GSH in A375 cells. These particles induce genotoxic and apoptotic response via caspase 3-dependent mechanism (181). ZnO nanoparticles also induce oxidative stress in Cloudman S91 melanoma cancer cells. Exposure to these nanoparticles leads to a spontaneous increase in the ROS levels and membrane lipid peroxidation along with a decrease in the level of GSH, SOD and catalase (182).

 

AC-1001 H3 CDR peptide is a murine monoclonal antibody-derived peptide in which the heavy chain complementary determining region (VH CDR3) on immunoglobulin domain inhibits lung metastasis in a syngeneic mice model generated using B16F10‐Nex2 cells. It alters the mitochondrial membrane potential and induces ROS production. This ROS induces cytotoxic effect in vitro in human A2058 and murine B16F10 melanoma cells via activation of intrinsic apoptotic pathway. It increases expression of LC3/LC3II along with Beclin1 which are early signs of autophagy (183).

 

Di-methyl-ampal-thio-ester (DIMATE) is an isoform specific competitive irreversible inhibitor of aldehyde dehydrogenase (ALDH) isoforms 1 and 3. Epigenetic mechanism upregulates ALDH1A3 significantly in melanoma cells versus normal melanocytes. Elevated levels of ALDH1 activity correlates with increased ROS levels in several melanoma and patient-derived cell lines. Elevated levels of ROS in turn cause generation and accumulation of apoptogenic aldehydes such as 4-hydroxynoneal (4-HNE) and malondialdehyde (MDA) ultimately leading to cellular apoptosis marked by increased Bax and reduced Bcl-2 levels. DIMATE reduces in vivo tumor growth and targets slow cycling patient-derived cell population consisting of the tumorigenic and chemo-resistant melanoma tumor cells (184).

 

PD-0332991 (Palbociclib), a CDK4/6 inhibitor enhances TRAIL sensitivity via inducing ROS generation. TRAIL sensitivity correlates positively with the induced cell cycle arrest in TRAIL-sensitive/resistant or partially acquired resistant melanoma cell lines. In addition to cell cycle arrest, apoptosis is induced in these cells due to loss of mitochondrial membrane potential, ROS production, upregulation of Bcl-2 and PUMA levels in response to combined treatment of PD-0332991 and TRAIL (185).

 

Cerium oxide (CeO2) nanoparticles enter the general circulation and reach the distant tissues/organs readily. These particles induce ROS production which leads to DNA damage, apoptosis via ROS-triggered mitochondrial pathway marked by an increase in caspase 3 levels, chromosomal condensation and fragmentation in vitro. This nanoparticle possesses genotoxic property that stimulates single and double strand DNA breaks (186, 187). Co-administration of nanoparticles with doxorubicin enhances anti-tumor property of doxorubicin by increasing the cytotoxicity and ROS formation leading to oxidative damage in A375 cells (188). A375 cells treated CeO2 alters the intracellular redox status of the cells in response to a surge in the levels of ROS. This leads to activation of the apoptotic pathway by releasing cytochrome c, activating caspase 3 and inducing PARP cleavage. It also leads to decrease in tumor growth in in vivo mice model (189).

 

Lomefloxacin, a fluoroquinolone and a topoisomerase II inhibitor is a synthetic antibiotic used to treat infections. Treatment of COLO829 melanoma cells with this drug leads to overproduction of ROS and depletes the level of intracellular glutathione. Overproduction of ROS disturbs the intracellular redox balance thus inducing oxidative stress leading to oligonucleosomal DNA fragmentation. Loss of mitochondrial membrane potential causes S, G2/M cell cycle arrest and apoptosis (190).

 

Vorinostat, a histone deacetylate inhibitor is effective against BRAF-inhibitors or BRAF plus MEK inhibitors resistance in in vitro, in vivo and clinical studies against melanoma. Treatment with vorinostat suppresses SLC7A11 gene (gene encoding for a precursor of ROS scavenger glutathione) which adds to elevated ROS levels and induces DNA damage and cell death (191).

 

Other potential ROS-targeting agents in melanoma

Calpain 3 (p94), a gene product of CAPN3 belongs to superfamily of calcium-regulated intracellular cysteine proteases is predominantly expressed in skeletal muscles. Melanoma tissues express higher CAPN3 compared to other tumor types (192). Calpain 3 induces ROS production in vitro and p53 plays a vital role in its regulation. It increases the expression of oxidative stress-related gene, RANTES (codes for CCL5 chemokine) which impairs cell proliferation and induces cell death in HT-144 and A375 cells. Active calpain-3 (variant hmp84) leads to accumulation of p53, modulates oxidative stress and induces DNA damage (193).

Recent study indicate that aged fibroblasts secret a Wnt antagonist, sFRP2 which regulate multiple signaling mechanism in melanoma cells that decreases β-catenin and MITF levels and leads to the loss of a key redox effector, APE1. Loss of APE1 expression inhibits the response of melanoma cells to ROS-induced DNA damage and increases the resistance of melanoma cells to BRAF-targeted therapies. Hence, sFRP2 could be a promising target in ROS-mediated signaling for treatment of metastatic melanoma (194).

 

 

Figure 2. ROS production and targeted therapies. Prdx: Reduced peroxiredoxin; Prx: Oxidized peroxiredoxin; GSH: Reduced glutathione; GSSG: Oxidized glutathione; Trx: Thioredoxin; ECSOD: Extracellular superoxide dismutase; SOD: Superoxide dismutase; NADPH: Nicotinamide adenine dinucleotide phosphate. ROS targeting agents includes: ROS scavenger (Volatile oil from ginger); Non-enzymatic antioxidants (Vitamin C, Vitamin E, ß-carotene, Retinol, Vitamin D3 and its derivatives, Lumisterol and its derivatives, Resveratrol alone or in combination with Ursolic acid and Chloroquinone/5- Flurouracil, Melatonin alone or in combination with Fisetin) and enzymatic anti-oxidants (Superoxide dismutase; Catalases and peroxidases). ROS inducers obtained from natural source includes Flavonoid: Dihydromyricetin; Benzoquinone: Jacaranone; Essential oil: Isoegomaketone; Celastrol; Diterpene: Cryptotanshinone; Flavonol: Cudraflavone C; Flavonol glycoside: Icariside II; Rhizome: Curcumin alone or in combination with 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) and Borneol; Alkaloid: Piperine; Monosyclic sesquiterpene: Zerumbone; Diterpenoid: Jolkinolide B; Fungal source: Trichodimerol; Animal source: Mastoparan. Synthetic agents that induce ROS include: Copper chelator: Disulfiram; Elesclomol; IKKβ (Inhibitor of nuclear factor kappa-B kinase subunit beta) inhibitor: SC-514; TRAM-34 alone or in combination with vemurafenib; Zinc and Zinc oxide nanoparticles; Murine momoclonal antibody-derived peptide: AC-1001 H3 CDR; Aldehyde dehydrogenase inhibitor: DIMATE; Cyclin dependent kinase 4/6 inhibitor: PD-0332991; Cerium oxide nanoparticles alone or in combination with Doxorubicin; Fluoroquinolone antibiotic: Lomefloxacin; Histone deacetylase inhibitor: Vorinostat; Wnt antagonist: sFRP2. SOD1 inhibitor (ATN-224 alone or in combination with Buthionine sulfoximine (BSO)/arsenic trioxide/temozolamide/disulfiram.

 

To summarize, five drugs including vitamin D and its derivatives, disulfiram, ATN-224, ATN-224 in combination with temozolomide and elesclomol have undergone various stages of clinical trial. In addition, preliminary clinical studies have been performed using retinol and vitamin C in combination with vitamin E and ß-carotene in patient with metastatic melanoma. Table 1 summarizes ROS targeted therapies and the associated signaling pathways. Figure 2 summarizes the ROS production and the targeted therapies.

 

Conclusion and future prospectus

Oxidative stress and ROS are crucial etiological factors for melanoma progression. The pleiotrophic effects of ROS induce diverse cellular responses that contribute to melanoma progression. Two main therapeutic approaches have been exploited to regulate ROS levels to control oxidative stress and cancer cell metastasis. One strategy was to interrupt ROS-mediated signaling cascade and cancer metastasis via antioxidant based ROS scavenging mechanism. However, single antioxidant therapy failed in clinical testing owing to increase tumor development. Also, endogenous antioxidants synthesized in cancer cells including melanoma as a result of adaptive response to increased ROS ultimately mask the effect of exogenous antioxidants. Another approach used was to induce ROS production in the cancer cells and to inhibit the cellular antioxidant levels. In preclinical studies, this approach resulted in cytotoxicity of the cancer cells with increased endogenous ROS production. Most cancer cells adapt to altered redox mechanism induced by ROS generating agents and eventually develop resistance. A combinatorial approach of epigenetic therapy with drugs that are capable of preventing generation and chronic accumulation of ROS along with standard chemotherapeutic regimens or radiotherapy might be crucial in overriding intrinsic melanoma resistance. Another recent approach is the use of drugs which induce ROS accumulation in melanoma cells to trigger ferroptosis, an iron-dependent form of regulated non-apoptotic cell death. Several studies are ongoing to explore combinatorial approach of ferroptosis-inducing drugs with conventional immunotherapy or kinase inhibitors to overcome resistance in de-differentiating melanoma cells. Exploring the benefits of these prospective therapies for treatment of patients with metastatic melanoma requires further investigation.

 

Acknowledgements

This work is funded by university of Cincinnati Pilot Translational Research and Innovative Core Grant Program.

 

References

  1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018. CA: a cancer journal for clinicians. 2018;68(1):7-30.
  2. Zbytek B, Carlson JA, Granese J, Ross J, Mihm MC, Jr., Slominski A. Current concepts of metastasis in melanoma. Expert review of dermatology. 2008;3(5):569-85.
  3. Liu Y, Sheikh MS. Melanoma: Molecular Pathogenesis and Therapeutic Management. Molecular and cellular pharmacology. 2014;6(3):228.
  4. Chapman PB, Hauschild A, Robert C, Haanen JB, Ascierto P, Larkin J, et al. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. The New England journal of medicine. 2011;364(26):2507-16.
  5. Luke JJ, Flaherty KT, Ribas A, Long GV. Targeted agents and immunotherapies: optimizing outcomes in melanoma. Nature reviews Clinical oncology. 2017;14(8):463-82.
  6. Long GV, Flaherty KT, Stroyakovskiy D, Gogas H, Levchenko E, de Braud F, et al. Dabrafenib plus trametinib versus dabrafenib monotherapy in patients with metastatic BRAF V600E/K-mutant melanoma: long-term survival and safety analysis of a phase 3 study. Annals of oncology : official journal of the European Society for Medical Oncology. 2017;28(7):1631-9.
  7. Long GV, Eroglu Z, Infante J, Patel S, Daud A, Johnson DB, et al. Long-Term Outcomes in Patients With BRAF V600-Mutant Metastatic Melanoma Who Received Dabrafenib Combined With Trametinib. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2018;36(7):667-73.
  8. Raza MH, Siraj S, Arshad A, Waheed U, Aldakheel F, Alduraywish S, et al. ROS-modulated therapeutic approaches in cancer treatment. Journal of cancer research and clinical oncology. 2017;143(9):1789-809.
  9. de Sa Junior PL, Camara DAD, Porcacchia AS, Fonseca PMM, Jorge SD, Araldi RP, et al. The Roles of ROS in Cancer Heterogeneity and Therapy. Oxidative medicine and cellular longevity. 2017;2017:2467940.
  10. Liou GY, Storz P. Reactive oxygen species in cancer. Free radical research. 2010;44(5):479-96.
  11. Joosse A, De Vries E, van Eijck CH, Eggermont AM, Nijsten T, Coebergh JW. Reactive oxygen species and melanoma: an explanation for gender differences in survival? Pigment cell & melanoma research. 2010;23(3):352-64.
  12. Pawelek JM, Chakraborty AK. Fusion of tumour cells with bone marrow-derived cells: a unifying explanation for metastasis. Nature reviews Cancer. 2008;8(5):377-86.
  13. Haq R, Shoag J, Andreu-Perez P, Yokoyama S, Edelman H, Rowe GC, et al. Oncogenic BRAF regulates oxidative metabolism via PGC1alpha and MITF. Cancer cell. 2013;23(3):302-15.
  14. Corazao-Rozas P, Guerreschi P, Jendoubi M, Andre F, Jonneaux A, Scalbert C, et al. Mitochondrial oxidative stress is the Achille’s heel of melanoma cells resistant to Braf-mutant inhibitor. Oncotarget. 2013;4(11):1986-98.
  15. Bataille V. Sun exposure, sunbeds and sunscreens and melanoma. What are the controversies? Current oncology reports. 2013;15(6):526-32.
  16. Curtin JA, Fridlyand J, Kageshita T, Patel HN, Busam KJ, Kutzner H, et al. Distinct sets of genetic alterations in melanoma. The New England journal of medicine. 2005;353(20):2135-47.
  17. Heppt MV, Siepmann T, Engel J, Schubert-Fritschle G, Eckel R, Mirlach L, et al. Prognostic significance of BRAF and NRAS mutations in melanoma: a German study from routine care. BMC cancer. 2017;17(1):536.
  18. Curtin JA, Busam K, Pinkel D, Bastian BC. Somatic activation of KIT in distinct subtypes of melanoma. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2006;24(26):4340-6.
  19. Slominski AT, Zmijewski MA, Plonka PM, Szaflarski JP, Paus R. How UV Light Touches the Brain and Endocrine System Through Skin, and Why. Endocrinology. 2018;159(5):1992-2007.
  20. Kim Y, He YY. Ultraviolet radiation-induced non-melanoma skin cancer: Regulation of DNA damage repair and inflammation. Genes & diseases. 2014;1(2):188-98.
  21. Horikawa-Miura M, Matsuda N, Yoshida M, Okumura Y, Mori T, Watanabe M. The greater lethality of UVB radiation to cultured human cells is associated with the specific activation of a DNA damage-independent signaling pathway. Radiation research. 2007;167(6):655-62.
  22. Birch-Machin MA, Swalwell H. How mitochondria record the effects of UV exposure and oxidative stress using human skin as a model tissue. Mutagenesis. 2010;25(2):101-7.
  23. Shain AH, Bastian BC. From melanocytes to melanomas. Nature reviews Cancer. 2016;16(6):345-58.
  24. Gray-Schopfer V, Wellbrock C, Marais R. Melanoma biology and new targeted therapy. Nature. 2007;445(7130):851-7.
  25. Leonardi GC, Falzone L, Salemi R, Zanghi A, Spandidos DA, McCubrey JA, et al. Cutaneous melanoma: From pathogenesis to therapy (Review). International journal of oncology. 2018;52(4):1071-80.
  26. Reichrath J, Rass K. Ultraviolet damage, DNA repair and vitamin D in nonmelanoma skin cancer and in malignant melanoma: an update. Advances in experimental medicine and biology. 2014;810:208-33.
  27. Tsoi J, Robert L, Paraiso K, Galvan C, Sheu KM, Lay J, et al. Multi-stage Differentiation Defines Melanoma Subtypes with Differential Vulnerability to Drug-Induced Iron-Dependent Oxidative Stress. Cancer cell. 2018;33(5):890-904 e5.
  28. Simon JD, Peles D, Wakamatsu K, Ito S. Current challenges in understanding melanogenesis: bridging chemistry, biological control, morphology, and function. Pigment cell & melanoma research. 2009;22(5):563-79.
  29. Park HY, Kosmadaki M, Yaar M, Gilchrest BA. Cellular mechanisms regulating human melanogenesis. Cellular and molecular life sciences : CMLS. 2009;66(9):1493-506.
  30. Slominski A, Zmijewski MA, Pawelek J. L-tyrosine and L-dihydroxyphenylalanine as hormone-like regulators of melanocyte functions. Pigment cell & melanoma research. 2012;25(1):14-27.
  31. Slominski A, Paus R. Towards defining receptors for L-tyrosine and L-dopa. Molecular and cellular endocrinology. 1994;99(2):C7-11.
  32. Slominski A, Tobin DJ, Shibahara S, Wortsman J. Melanin pigmentation in mammalian skin and its hormonal regulation. Physiological reviews. 2004;84(4):1155-228.
  33. Lerner AB, McGuire JS. Effect of alpha- and betamelanocyte stimulating hormones on the skin colour of man. Nature. 1961;189:176-9.
  34. Levine N, Sheftel SN, Eytan T, Dorr RT, Hadley ME, Weinrach JC, et al. Induction of skin tanning by subcutaneous administration of a potent synthetic melanotropin. Jama. 1991;266(19):2730-6.
  35. Pawelek JM, Chakraborty AK, Osber MP, Orlow SJ, Min KK, Rosenzweig KE, et al. Molecular cascades in UV-induced melanogenesis: a central role for melanotropins? Pigment cell research. 1992;5(5 Pt 2):348-56.
  36. Suzuki I, Cone RD, Im S, Nordlund J, Abdel-Malek ZA. Binding of melanotropic hormones to the melanocortin receptor MC1R on human melanocytes stimulates proliferation and melanogenesis. Endocrinology. 1996;137(5):1627-33.
  37. Kauser S, Schallreuter KU, Thody AJ, Gummer C, Tobin DJ. Regulation of human epidermal melanocyte biology by beta-endorphin. The Journal of investigative dermatology. 2003;120(6):1073-80.
  38. Yada Y, Higuchi K, Imokawa G. Effects of endothelins on signal transduction and proliferation in human melanocytes. The Journal of biological chemistry. 1991;266(27):18352-7.
  39. Imokawa G, Miyagishi M, Yada Y. Endothelin-1 as a new melanogen: coordinated expression of its gene and the tyrosinase gene in UVB-exposed human epidermis. The Journal of investigative dermatology. 1995;105(1):32-7.
  40. Lassalle MW, Igarashi S, Sasaki M, Wakamatsu K, Ito S, Horikoshi T. Effects of melanogenesis-inducing nitric oxide and histamine on the production of eumelanin and pheomelanin in cultured human melanocytes. Pigment cell research. 2003;16(1):81-4.
  41. McEwan MT, Parsons PG. Regulation of tyrosinase expression and activity in human melanoma cells via histamine receptors. The Journal of investigative dermatology. 1991;97(5):868-73.
  42. Howe J, Costantino R, Slominski A. On the putative mechanism of induction and regulation of melanogenesis by L-tyrosine. Acta dermato-venereologica. 1991;71(2):150-2.
  43. Costa JJ, Demetri GD, Harrist TJ, Dvorak AM, Hayes DF, Merica EA, et al. Recombinant human stem cell factor (kit ligand) promotes human mast cell and melanocyte hyperplasia and functional activation in vivo. The Journal of experimental medicine. 1996;183(6):2681-6.
  44. Luo D, Chen H, Searles G, Jimbow K. Coordinated mRNA expression of c-Kit with tyrosinase and TRP-1 in melanin pigmentation of normal and malignant human melanocytes and transient activation of tyrosinase by Kit/SCF-R. Melanoma research. 1995;5(5):303-9.
  45. Ranson M, Posen S, Mason RS. Human melanocytes as a target tissue for hormones: in vitro studies with 1 alpha-25, dihydroxyvitamin D3, alpha-melanocyte stimulating hormone, and beta-estradiol. The Journal of investigative dermatology. 1988;91(6):593-8.
  46. Jee SH, Lee SY, Chiu HC, Chang CC, Chen TJ. Effects of estrogen and estrogen receptor in normal human melanocytes. Biochemical and biophysical research communications. 1994;199(3):1407-12.
  47. Tomita Y, Torinuki W, Tagami H. Stimulation of human melanocytes by vitamin D3 possibly mediates skin pigmentation after sun exposure. The Journal of investigative dermatology. 1988;90(6):882-4.
  48. Mansur CP, Gordon PR, Ray S, Holick MF, Gilchrest BA. Vitamin D, its precursors, and metabolites do not affect melanization of cultured human melanocytes. The Journal of investigative dermatology. 1988;91(1):16-21.
  49. Watabe H, Soma Y, Kawa Y, Ito M, Ooka S, Ohsumi K, et al. Differentiation of murine melanocyte precursors induced by 1,25-dihydroxyvitamin D3 is associated with the stimulation of endothelin B receptor expression. The Journal of investigative dermatology. 2002;119(3):583-9.
  50. Slominski RM, Zmijewski MA, Slominski AT. The role of melanin pigment in melanoma. Experimental dermatology. 2015;24(4):258-9.
  51. Slominski A, Kim TK, Brozyna AA, Janjetovic Z, Brooks DL, Schwab LP, et al. The role of melanogenesis in regulation of melanoma behavior: melanogenesis leads to stimulation of HIF-1alpha expression and HIF-dependent attendant pathways. Archives of biochemistry and biophysics. 2014;563:79-93.
  52. Brozyna AA, Jozwicki W, Roszkowski K, Filipiak J, Slominski AT. Melanin content in melanoma metastases affects the outcome of radiotherapy. Oncotarget. 2016;7(14):17844-53.
  53. Brozyna AA, Jozwicki W, Carlson JA, Slominski AT. Melanogenesis affects overall and disease-free survival in patients with stage III and IV melanoma. Human pathology. 2013;44(10):2071-4.
  54. Slominski A, Paus R, Mihm MC. Inhibition of melanogenesis as an adjuvant strategy in the treatment of melanotic melanomas: selective review and hypothesis. Anticancer research. 1998;18(5B):3709-15.
  55. Slominski A, Zbytek B, Slominski R. Inhibitors of melanogenesis increase toxicity of cyclophosphamide and lymphocytes against melanoma cells. International journal of cancer. 2009;124(6):1470-7.
  56. Slominski AT, Carlson JA. Melanoma resistance: a bright future for academicians and a challenge for patient advocates. Mayo Clinic proceedings. 2014;89(4):429-33.
  57. Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. The international journal of biochemistry & cell biology. 2007;39(1):44-84.
  58. Valko M, Rhodes CJ, Moncol J, Izakovic M, Mazur M. Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chemico-biological interactions. 2006;160(1):1-40.
  59. Wallace DC. Mitochondria and cancer. Nature reviews Cancer. 2012;12(10):685-98.
  60. Liu-Smith F, Dellinger R, Meyskens FL, Jr. Updates of reactive oxygen species in melanoma etiology and progression. Archives of biochemistry and biophysics. 2014;563:51-5.
  61. Collins Y, Chouchani ET, James AM, Menger KE, Cocheme HM, Murphy MP. Mitochondrial redox signalling at a glance. Journal of cell science. 2012;125(Pt 4):801-6.
  62. Muller FL, Liu Y, Van Remmen H. Complex III releases superoxide to both sides of the inner mitochondrial membrane. The Journal of biological chemistry. 2004;279(47):49064-73.
  63. Filipp FV, Ratnikov B, De Ingeniis J, Smith JW, Osterman AL, Scott DA. Glutamine-fueled mitochondrial metabolism is decoupled from glycolysis in melanoma. Pigment cell & melanoma research. 2012;25(6):732-9.
  64. Ishikawa K, Hayashi J. A novel function of mtDNA: its involvement in metastasis. Annals of the New York Academy of Sciences. 2010;1201:40-3.
  65. Koshikawa N, Hayashi J, Nakagawara A, Takenaga K. Reactive oxygen species-generating mitochondrial DNA mutation up-regulates hypoxia-inducible factor-1alpha gene transcription via phosphatidylinositol 3-kinase-Akt/protein kinase C/histone deacetylase pathway. The Journal of biological chemistry. 2009;284(48):33185-94.
  66. Altenhofer S, Kleikers PW, Radermacher KA, Scheurer P, Rob Hermans JJ, Schiffers P, et al. The NOX toolbox: validating the role of NADPH oxidases in physiology and disease. Cellular and molecular life sciences : CMLS. 2012;69(14):2327-43.
  67. Lassegue B, San Martin A, Griendling KK. Biochemistry, physiology, and pathophysiology of NADPH oxidases in the cardiovascular system. Circulation research. 2012;110(10):1364-90.
  68. Liu F, Gomez Garcia AM, Meyskens FL, Jr. NADPH oxidase 1 overexpression enhances invasion via matrix metalloproteinase-2 and epithelial-mesenchymal transition in melanoma cells. The Journal of investigative dermatology. 2012;132(8):2033-41.
  69. Kim Y, Lee YS, Choe J, Lee H, Kim YM, Jeoung D. CD44-epidermal growth factor receptor interaction mediates hyaluronic acid-promoted cell motility by activating protein kinase C signaling involving Akt, Rac1, Phox, reactive oxygen species, focal adhesion kinase, and MMP-2. The Journal of biological chemistry. 2008;283(33):22513-28.
  70. Park SJ, Kim YT, Jeon YJ. Antioxidant dieckol downregulates the Rac1/ROS signaling pathway and inhibits Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein 2 (WAVE2)-mediated invasive migration of B16 mouse melanoma cells. Molecules and cells. 2012;33(4):363-9.
  71. Yamaura M, Mitsushita J, Furuta S, Kiniwa Y, Ashida A, Goto Y, et al. NADPH oxidase 4 contributes to transformation phenotype of melanoma cells by regulating G2-M cell cycle progression. Cancer research. 2009;69(6):2647-54.
  72. Valko M, Izakovic M, Mazur M, Rhodes CJ, Telser J. Role of oxygen radicals in DNA damage and cancer incidence. Molecular and cellular biochemistry. 2004;266(1-2):37-56.
  73. Meyskens FL, Jr., McNulty SE, Buckmeier JA, Tohidian NB, Spillane TJ, Kahlon RS, et al. Aberrant redox regulation in human metastatic melanoma cells compared to normal melanocytes. Free radical biology & medicine. 2001;31(6):799-808.
  74. Meyskens FL, Jr., Farmer P, Fruehauf JP. Redox regulation in human melanocytes and melanoma. Pigment cell research. 2001;14(3):148-54.
  75. Farmer PJ, Gidanian S, Shahandeh B, Di Bilio AJ, Tohidian N, Meyskens FL, Jr. Melanin as a target for melanoma chemotherapy: pro-oxidant effect of oxygen and metals on melanoma viability. Pigment cell research. 2003;16(3):273-9.
  76. Harris AL. Hypoxia–a key regulatory factor in tumour growth. Nature reviews Cancer. 2002;2(1):38-47.
  77. Govindarajan B, Sligh JE, Vincent BJ, Li M, Canter JA, Nickoloff BJ, et al. Overexpression of Akt converts radial growth melanoma to vertical growth melanoma. The Journal of clinical investigation. 2007;117(3):719-29.
  78. Verhaegen M, Bauer JA, Martin de la Vega C, Wang G, Wolter KG, Brenner JC, et al. A novel BH3 mimetic reveals a mitogen-activated protein kinase-dependent mechanism of melanoma cell death controlled by p53 and reactive oxygen species. Cancer research. 2006;66(23):11348-59.
  79. Ueda Y, Richmond A. NF-kappaB activation in melanoma. Pigment cell research. 2006;19(2):112-24.
  80. Xanthoudakis S, Miao G, Wang F, Pan YC, Curran T. Redox activation of Fos-Jun DNA binding activity is mediated by a DNA repair enzyme. The EMBO journal. 1992;11(9):3323-35.
  81. Smeal T, Binetruy B, Mercola DA, Birrer M, Karin M. Oncogenic and transcriptional cooperation with Ha-Ras requires phosphorylation of c-Jun on serines 63 and 73. Nature. 1991;354(6353):494-6.
  82. Vartanian A, Baryshnikov AY. Crosstalk between apoptosis and antioxidants in melanoma vasculogenic mimicry. Advances in experimental medicine and biology. 2007;601:145-53.
  83. Brar SS, Kennedy TP, Whorton AR, Sturrock AB, Huecksteadt TP, Ghio AJ, et al. Reactive oxygen species from NAD(P)H:quinone oxidoreductase constitutively activate NF-kappaB in malignant melanoma cells. American journal of physiology Cell physiology. 2001;280(3):C659-76.
  84. Coussens LM, Werb Z. Inflammation and cancer. Nature. 2002;420(6917):860-7.
  85. Pavel S, van Nieuwpoort F, van der Meulen H, Out C, Pizinger K, Cetkovska P, et al. Disturbed melanin synthesis and chronic oxidative stress in dysplastic naevi. European journal of cancer. 2004;40(9):1423-30.
  86. Meyskens FL, Jr., Farmer PJ, Anton-Culver H. Etiologic pathogenesis of melanoma: a unifying hypothesis for the missing attributable risk. Clinical cancer research : an official journal of the American Association for Cancer Research. 2004;10(8):2581-3.
  87. Cheng GC, Schulze PC, Lee RT, Sylvan J, Zetter BR, Huang H. Oxidative stress and thioredoxin-interacting protein promote intravasation of melanoma cells. Experimental cell research. 2004;300(2):297-307.
  88. Rofstad EK, Mathiesen B, Henriksen K, Kindem K, Galappathi K. The tumor bed effect: increased metastatic dissemination from hypoxia-induced up-regulation of metastasis-promoting gene products. Cancer research. 2005;65(6):2387-96.
  89. Rofstad EK, Mathiesen B, Galappathi K. Increased metastatic dissemination in human melanoma xenografts after subcurative radiation treatment: radiation-induced increase in fraction of hypoxic cells and hypoxia-induced up-regulation of urokinase-type plasminogen activator receptor. Cancer research. 2004;64(1):13-8.
  90. Liu H, Colavitti R, Rovira, II, Finkel T. Redox-dependent transcriptional regulation. Circulation research. 2005;97(10):967-74.
  91. Yang S, Misner BJ, Chiu RJ, Meyskens FL, Jr. Redox effector factor-1, combined with reactive oxygen species, plays an important role in the transformation of JB6 cells. Carcinogenesis. 2007;28(11):2382-90.
  92. Yang S, Meyskens FL. Apurinic/apyrimidinic endonuclease/redox effector factor-1(APE/Ref-1): a unique target for the prevention and treatment of human melanoma. Antioxidants & redox signaling. 2009;11(3):639-50.
  93. Molognoni F, de Melo FH, da Silva CT, Jasiulionis MG. Ras and Rac1, frequently mutated in melanomas, are activated by superoxide anion, modulate Dnmt1 level and are causally related to melanocyte malignant transformation. PloS one. 2013;8(12):e81937.
  94. Jenkins NC, Liu T, Cassidy P, Leachman SA, Boucher KM, Goodson AG, et al. The p16(INK4A) tumor suppressor regulates cellular oxidative stress. Oncogene. 2011;30(3):265-74.
  95. Lee S, Kim YK, Shin TY, Kim SH. Neurotoxic effects of bisphenol AF on calcium-induced ROS and MAPKs. Neurotoxicity research. 2013;23(3):249-59.
  96. Selimovic D, Hassan M, Haikel Y, Hengge UR. Taxol-induced mitochondrial stress in melanoma cells is mediated by activation of c-Jun N-terminal kinase (JNK) and p38 pathways via uncoupling protein 2. Cellular signalling. 2008;20(2):311-22.
  97. Borgstahl GE, Parge HE, Hickey MJ, Johnson MJ, Boissinot M, Hallewell RA, et al. Human mitochondrial manganese superoxide dismutase polymorphic variant Ile58Thr reduces activity by destabilizing the tetrameric interface. Biochemistry. 1996;35(14):4287-97.
  98. Zelko IN, Mariani TJ, Folz RJ. Superoxide dismutase multigene family: a comparison of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) gene structures, evolution, and expression. Free radical biology & medicine. 2002;33(3):337-49.
  99. Yu L, Gao LX, Ma XQ, Hu FX, Li CM, Lu Z. Involvement of superoxide and nitric oxide in BRAF(V600E) inhibitor PLX4032-induced growth inhibition of melanoma cells. Integrative biology : quantitative biosciences from nano to macro. 2014;6(12):1211-7.
  100. Church SL, Grant JW, Ridnour LA, Oberley LW, Swanson PE, Meltzer PS, et al. Increased manganese superoxide dismutase expression suppresses the malignant phenotype of human melanoma cells. Proceedings of the National Academy of Sciences of the United States of America. 1993;90(7):3113-7.
  101. Hyoudou K, Nishikawa M, Umeyama Y, Kobayashi Y, Yamashita F, Hashida M. Inhibition of metastatic tumor growth in mouse lung by repeated administration of polyethylene glycol-conjugated catalase: quantitative analysis with firefly luciferase-expressing melanoma cells. Clinical cancer research : an official journal of the American Association for Cancer Research. 2004;10(22):7685-91.
  102. Hyoudou K, Nishikawa M, Kobayashi Y, Umeyama Y, Yamashita F, Hashida M. PEGylated catalase prevents metastatic tumor growth aggravated by tumor removal. Free radical biology & medicine. 2006;41(9):1449-58.
  103. Forman HJ, Zhang H, Rinna A. Glutathione: overview of its protective roles, measurement, and biosynthesis. Molecular aspects of medicine. 2009;30(1-2):1-12.
  104. Anasagasti MJ, Martin JJ, Mendoza L, Obrador E, Estrela JM, McCuskey RS, et al. Glutathione protects metastatic melanoma cells against oxidative stress in the murine hepatic microvasculature. Hepatology. 1998;27(5):1249-56.
  105. Poole LB, Hall A, Nelson KJ. Overview of peroxiredoxins in oxidant defense and redox regulation. Current protocols in toxicology. 2011;Chapter 7:Unit7 9.
  106. Lee DJ, Kang DH, Choi M, Choi YJ, Lee JY, Park JH, et al. Peroxiredoxin-2 represses melanoma metastasis by increasing E-Cadherin/beta-Catenin complexes in adherens junctions. Cancer research. 2013;73(15):4744-57.
  107. Sengupta R, Holmgren A. Thioredoxin and glutaredoxin-mediated redox regulation of ribonucleotide reductase. World journal of biological chemistry. 2014;5(1):68-74.
  108. Cassidy PB, Honeggar M, Poerschke RL, White K, Florell SR, Andtbacka RH, et al. The role of thioredoxin reductase 1 in melanoma metabolism and metastasis. Pigment cell & melanoma research. 2015;28(6):685-95.
  109. Barral AM, Kallstrom R, Sander B, Rosen A. Thioredoxin, thioredoxin reductase and tumour necrosis factor-alpha expression in melanoma cells: correlation to resistance against cytotoxic attack. Melanoma research. 2000;10(4):331-43.
  110. Wang X, Dong H, Li Q, Li Y, Hong A. Thioredoxin induces Tregs to generate an immunotolerant tumor microenvironment in metastatic melanoma. Oncoimmunology. 2015;4(9):e1027471.
  111. Le Gal K, Ibrahim MX, Wiel C, Sayin VI, Akula MK, Karlsson C, et al. Antioxidants can increase melanoma metastasis in mice. Science translational medicine. 2015;7(308):308re8.
  112. Alpha-Tocopherol BCCPSG. The effect of vitamin E and beta carotene on the incidence of lung cancer and other cancers in male smokers. The New England journal of medicine. 1994;330(15):1029-35.
  113. Feskanich D, Willett WC, Hunter DJ, Colditz GA. Dietary intakes of vitamins A, C, and E and risk of melanoma in two cohorts of women. British journal of cancer. 2003;88(9):1381-7.
  114. Millen AE, Tucker MA, Hartge P, Halpern A, Elder DE, Guerry Dt, et al. Diet and melanoma in a case-control study. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology. 2004;13(6):1042-51.
  115. Hercberg S, Ezzedine K, Guinot C, Preziosi P, Galan P, Bertrais S, et al. Antioxidant supplementation increases the risk of skin cancers in women but not in men. The Journal of nutrition. 2007;137(9):2098-105.
  116. Asgari MM, Brasky TM, White E. Association of vitamin A and carotenoid intake with melanoma risk in a large prospective cohort. The Journal of investigative dermatology. 2012;132(6):1573-82.
  117. Slominski AT, Brozyna AA, Skobowiat C, Zmijewski MA, Kim TK, Janjetovic Z, et al. On the role of classical and novel forms of vitamin D in melanoma progression and management. The Journal of steroid biochemistry and molecular biology. 2018;177:159-70.
  118. Slominski AT, Janjetovic Z, Kim TK, Wasilewski P, Rosas S, Hanna S, et al. Novel non-calcemic secosteroids that are produced by human epidermal keratinocytes protect against solar radiation. The Journal of steroid biochemistry and molecular biology. 2015;148:52-63.
  119. Saw RP, Armstrong BK, Mason RS, Morton RL, Shannon KF, Spillane AJ, et al. Adjuvant therapy with high dose vitamin D following primary treatment of melanoma at high risk of recurrence: a placebo controlled randomised phase II trial (ANZMTG 02.09 Mel-D). BMC cancer. 2014;14:780.
  120. Slominski AT, Kim TK, Hobrath JV, Janjetovic Z, Oak ASW, Postlethwaite A, et al. Characterization of a new pathway that activates lumisterol in vivo to biologically active hydroxylumisterols. Scientific reports. 2017;7(1):11434.
  121. Bhattacharya S, Darjatmoko SR, Polans AS. Resveratrol modulates the malignant properties of cutaneous melanoma through changes in the activation and attenuation of the antiapoptotic protooncogenic protein Akt/PKB. Melanoma research. 2011;21(3):180-7.
  122. Wu Z, Liu B, E C, Liu J, Zhang Q, Liu J, et al. Resveratrol inhibits the proliferation of human melanoma cells by inducing G1/S cell cycle arrest and apoptosis. Molecular medicine reports. 2015;11(1):400-4.
  123. Wang M, Yu T, Zhu C, Sun H, Qiu Y, Zhu X, et al. Resveratrol triggers protective autophagy through the ceramide/Akt/mTOR pathway in melanoma B16 cells. Nutrition and cancer. 2014;66(3):435-40.
  124. Junco JJ, Mancha-Ramirez A, Malik G, Wei SJ, Kim DJ, Liang H, et al. Ursolic acid and resveratrol synergize with chloroquine to reduce melanoma cell viability. Melanoma research. 2015;25(2):103-12.
  125. Lee SH, Koo BS, Park SY, Kim YM. Anti-angiogenic effects of resveratrol in combination with 5-fluorouracil on B16 murine melanoma cells. Molecular medicine reports. 2015;12(2):2777-83.
  126. Boocock DJ, Faust GE, Patel KR, Schinas AM, Brown VA, Ducharme MP, et al. Phase I dose escalation pharmacokinetic study in healthy volunteers of resveratrol, a potential cancer chemopreventive agent. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology. 2007;16(6):1246-52.
  127. Marier JF, Vachon P, Gritsas A, Zhang J, Moreau JP, Ducharme MP. Metabolism and disposition of resveratrol in rats: extent of absorption, glucuronidation, and enterohepatic recirculation evidenced by a linked-rat model. The Journal of pharmacology and experimental therapeutics. 2002;302(1):369-73.
  128. Walle T, Hsieh F, DeLegge MH, Oatis JE, Jr., Walle UK. High absorption but very low bioavailability of oral resveratrol in humans. Drug metabolism and disposition: the biological fate of chemicals. 2004;32(12):1377-82.
  129. Slominski AT, Hardeland R, Zmijewski MA, Slominski RM, Reiter RJ, Paus R. Melatonin: A Cutaneous Perspective on its Production, Metabolism, and Functions. The Journal of investigative dermatology. 2018;138(3):490-9.
  130. Skobowiat C, Brozyna AA, Janjetovic Z, Jeayeng S, Oak ASW, Kim TK, et al. Melatonin and its derivatives counteract the ultraviolet B radiation-induced damage in human and porcine skin ex vivo. Journal of pineal research. 2018;65(2):e12501.
  131. Slominski AT, Zmijewski MA, Semak I, Kim TK, Janjetovic Z, Slominski RM, et al. Melatonin, mitochondria, and the skin. Cellular and molecular life sciences : CMLS. 2017;74(21):3913-25.
  132. Cabrera J, Negrin G, Estevez F, Loro J, Reiter RJ, Quintana J. Melatonin decreases cell proliferation and induces melanogenesis in human melanoma SK-MEL-1 cells. Journal of pineal research. 2010;49(1):45-54.
  133. Yi C, Zhang Y, Yu Z, Xiao Y, Wang J, Qiu H, et al. Melatonin enhances the anti-tumor effect of fisetin by inhibiting COX-2/iNOS and NF-kappaB/p300 signaling pathways. PloS one. 2014;9(7):e99943.
  134. Fischer TW, Zmijewski MA, Zbytek B, Sweatman TW, Slominski RM, Wortsman J, et al. Oncostatic effects of the indole melatonin and expression of its cytosolic and nuclear receptors in cultured human melanoma cell lines. International journal of oncology. 2006;29(3):665-72.
  135. Zhou DZ, Sun HY, Yue JQ, Peng Y, Chen YM, Zhong ZJ. Dihydromyricetin induces apoptosis and cytoprotective autophagy through ROS-NF-kappaB signalling in human melanoma cells. Free radical research. 2017;51(5):517-28.
  136. Huang HC, Liao CC, Peng CC, Lim JM, Siao JH, Wei CM, et al. Dihydromyricetin from Ampelopsis grossedentata inhibits melanogenesis through down-regulation of MAPK, PKA and PKC signaling pathways. Chemico-biological interactions. 2016;258:166-74.
  137. Massaoka MH, Matsuo AL, Figueiredo CR, Farias CF, Girola N, Arruda DC, et al. Jacaranone induces apoptosis in melanoma cells via ROS-mediated downregulation of Akt and p38 MAPK activation and displays antitumor activity in vivo. PloS one. 2012;7(6):e38698.
  138. Kwon SJ, Lee JH, Moon KD, Jeong IY, Ahn DU, Lee MK, et al. Induction of apoptosis by isoegomaketone from Perilla frutescens L. in B16 melanoma cells is mediated through ROS generation and mitochondrial-dependent, -independent pathway. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association. 2014;65:97-104.
  139. Kwon SJ, Lee JH, Moon KD, Jeong IY, Yee ST, Lee MK, et al. Isoegomaketone induces apoptosis in SK-MEL-2 human melanoma cells through mitochondrial apoptotic pathway via activating the PI3K/Akt pathway. International journal of oncology. 2014;45(5):1969-76.
  140. Lee JH, Won YS, Park KH, Lee MK, Tachibana H, Yamada K, et al. Celastrol inhibits growth and induces apoptotic cell death in melanoma cells via the activation ROS-dependent mitochondrial pathway and the suppression of PI3K/AKT signaling. Apoptosis : an international journal on programmed cell death. 2012;17(12):1275-86.
  141. Ye T, Zhu S, Zhu Y, Feng Q, He B, Xiong Y, et al. Cryptotanshinone induces melanoma cancer cells apoptosis via ROS-mitochondrial apoptotic pathway and impairs cell migration and invasion. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie. 2016;82:319-26.
  142. Tse AK, Chow KY, Cao HH, Cheng CY, Kwan HY, Yu H, et al. The herbal compound cryptotanshinone restores sensitivity in cancer cells that are resistant to the tumor necrosis factor-related apoptosis-inducing ligand. The Journal of biological chemistry. 2013;288(41):29923-33.
  143. Arung ET, Yoshikawa K, Shimizu K, Kondo R. Isoprenoid-substituted flavonoids from wood of Artocarpus heterophyllus on B16 melanoma cells: cytotoxicity and structural criteria. Fitoterapia. 2010;81(2):120-3.
  144. Lee CW, Yen FL, Ko HH, Li SY, Chiang YC, Lee MH, et al. Cudraflavone C Induces Apoptosis of A375.S2 Melanoma Cells through Mitochondrial ROS Production and MAPK Activation. International journal of molecular sciences. 2017;18(7).
  145. Wu J, Song T, Liu S, Li X, Li G, Xu J. Icariside II inhibits cell proliferation and induces cell cycle arrest through the ROS-p38-p53 signaling pathway in A375 human melanoma cells. Molecular medicine reports. 2015;11(1):410-6.
  146. Du J, Wu J, Fu X, Tse AK, Li T, Su T, et al. Icariside II overcomes TRAIL resistance of melanoma cells through ROS-mediated downregulation of STAT3/cFLIP signaling. Oncotarget. 2016;7(32):52218-29.
  147. Yu T, Ji J, Guo YL. MST1 activation by curcumin mediates JNK activation, Foxo3a nuclear translocation and apoptosis in melanoma cells. Biochemical and biophysical research communications. 2013;441(1):53-8.
  148. Yu T, Li J, Qiu Y, Sun H. 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) facilitates curcumin-induced melanoma cell apoptosis by enhancing ceramide accumulation, JNK activation, and inhibiting PI3K/AKT activation. Molecular and cellular biochemistry. 2012;361(1-2):47-54.
  149. Kocyigit A, Guler EM. Curcumin induce DNA damage and apoptosis through generation of reactive oxygen species and reducing mitochondrial membrane potential in melanoma cancer cells. Cellular and molecular biology. 2017;63(11):97-105.
  150. Liao W, Xiang W, Wang FF, Wang R, Ding Y. Curcumin inhibited growth of human melanoma A375 cells via inciting oxidative stress. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie. 2017;95:1177-86.
  151. Chen J, Li L, Su J, Li B, Chen T, Wong YS. Synergistic apoptosis-inducing effects on A375 human melanoma cells of natural borneol and curcumin. PloS one. 2014;9(6):e101277.
  152. Raj L, Ide T, Gurkar AU, Foley M, Schenone M, Li X, et al. Selective killing of cancer cells by a small molecule targeting the stress response to ROS. Nature. 2011;475(7355):231-4.
  153. Fofaria NM, Kim SH, Srivastava SK. Piperine causes G1 phase cell cycle arrest and apoptosis in melanoma cells through checkpoint kinase-1 activation. PloS one. 2014;9(5):e94298.
  154. Song X, Gao T, Lei Q, Zhang L, Yao Y, Xiong J. Piperlongumine Induces Apoptosis in Human Melanoma Cells Via Reactive Oxygen Species Mediated Mitochondria Disruption. Nutrition and cancer. 2018;70(3):502-11.
  155. Yan H, Ren MY, Wang ZX, Feng SJ, Li S, Cheng Y, et al. Zerumbone inhibits melanoma cell proliferation and migration by altering mitochondrial functions. Oncology letters. 2017;13(4):2397-402.
  156. Gao C, Yan X, Wang B, Yu L, Han J, Li D, et al. Jolkinolide B induces apoptosis and inhibits tumor growth in mouse melanoma B16F10 cells by altering glycolysis. Scientific reports. 2016;6:36114.
  157. Wang LX, Qian J, Zhao LN, Zhao SH. Effects of volatile oil from ginger on the murine B16 melanoma cells and its mechanism. Food & function. 2018;9(2):1058-69.
  158. Yao Y, Hong Z, Niu Y, Li W-Q, Li J, Yan Q-S, et al. P25 Trichodimerol induces apoptosis in A375-S2 human melanoma cells through modulation of ERK and p38 activities mediated by reactive oxygen species. Biochemical Pharmacology. 2017;139:133.
  159. de Azevedo RA, Figueiredo CR, Ferreira AK, Matsuo AL, Massaoka MH, Girola N, et al. Mastoparan induces apoptosis in B16F10-Nex2 melanoma cells via the intrinsic mitochondrial pathway and displays antitumor activity in vivo. Peptides. 2015;68:113-9.
  160. Conticello C, Martinetti D, Adamo L, Buccheri S, Giuffrida R, Parrinello N, et al. Disulfiram, an old drug with new potential therapeutic uses for human hematological malignancies. International journal of cancer. 2012;131(9):2197-203.
  161. Morrison BW, Doudican NA, Patel KR, Orlow SJ. Disulfiram induces copper-dependent stimulation of reactive oxygen species and activation of the extrinsic apoptotic pathway in melanoma. Melanoma research. 2010;20(1):11-20.
  162. Cen D, Brayton D, Shahandeh B, Meyskens FL, Jr., Farmer PJ. Disulfiram facilitates intracellular Cu uptake and induces apoptosis in human melanoma cells. Journal of medicinal chemistry. 2004;47(27):6914-20.
  163. Cen D, Gonzalez RI, Buckmeier JA, Kahlon RS, Tohidian NB, Meyskens FL, Jr. Disulfiram induces apoptosis in human melanoma cells: a redox-related process. Molecular cancer therapeutics. 2002;1(3):197-204.
  164. John P. Fruehauf, University of California, Irvine. Disulfiram in Patients With Metastatic Melanoma. ClinicalTrials.gov 2017.NCT00256230.
  165. University of Utah. Disulfiram and chelated zinc for the rx of disseminated mets mel that has failed first line therapy.ClinicalTrials.gov 2016.NCT02101008.
  166. Juarez JC, Betancourt O, Jr., Pirie-Shepherd SR, Guan X, Price ML, Shaw DE, et al. Copper binding by tetrathiomolybdate attenuates angiogenesis and tumor cell proliferation through the inhibition of superoxide dismutase 1. Clinical cancer research : an official journal of the American Association for Cancer Research. 2006;12(16):4974-82.
  167. Donate F, Juarez JC, Burnett ME, Manuia MM, Guan X, Shaw DE, et al. Identification of biomarkers for the antiangiogenic and antitumour activity of the superoxide dismutase 1 (SOD1) inhibitor tetrathiomolybdate (ATN-224). British journal of cancer. 2008;98(4):776-83.
  168. Trapp V, Lee K, Donate F, Mazar AP, Fruehauf JP. Redox-related antimelanoma activity of ATN-224. Melanoma research. 2009;19(6):350-60.
  169. Lowndes SA, Adams A, Timms A, Middleton M, Hayward C, Reich SD, et al. Phase I study of ATN-224 in patients (pts) with advanced solid tumours. Journal of Clinical Oncology. 2006;24(18_suppl):2065-.
  170. Attenuon. Randomized trial of ATN-224 and temozolomide in advanced melanoma. ClinicalTrials.gov 2007.NCT00383851.
  171. Kirshner JR, He S, Balasubramanyam V, Kepros J, Yang CY, Zhang M, et al. Elesclomol induces cancer cell apoptosis through oxidative stress. Molecular cancer therapeutics. 2008;7(8):2319-27.
  172. Nagai M, Vo NH, Shin Ogawa L, Chimmanamada D, Inoue T, Chu J, et al. The oncology drug elesclomol selectively transports copper to the mitochondria to induce oxidative stress in cancer cells. Free radical biology & medicine. 2012;52(10):2142-50.
  173. Blackman RK, Cheung-Ong K, Gebbia M, Proia DA, He S, Kepros J, et al. Mitochondrial electron transport is the cellular target of the oncology drug elesclomol. PloS one. 2012;7(1):e29798.
  174. O’Day SJ, Eggermont AMM, Chiarion-Sileni V, Kefford R, Grob JJ, Mortier L, et al. Final Results of Phase III SYMMETRY Study: Randomized, Double-Blind Trial of Elesclomol Plus Paclitaxel Versus Paclitaxel Alone As Treatment for Chemotherapy-Naive Patients With Advanced Melanoma. Journal of Clinical Oncology. 2013;31(9):1211-8.
  175. Synta Pharmaceuticals Corp. Elesclomol (STA-4783) with paclitaxel versus paclitaxel alone in melanoma. ClinicalTrilas.gov 2014.NCT00522834.
  176. Hauschild A, Eggermont AM, Jacobson E, O’Day SJ. Phase III, randomized, double-blind study of elesclomol and paclitaxel versus paclitaxel alone in stage IV metastatic melanoma (MM). Journal of Clinical Oncology. 2009;27(18S):LBA9012-LBA.
  177. Tse AK, Chen YJ, Fu XQ, Su T, Li T, Guo H, et al. Sensitization of melanoma cells to alkylating agent-induced DNA damage and cell death via orchestrating oxidative stress and IKKbeta inhibition. Redox biology. 2017;11:562-76.
  178. Bauer D, Werth F, Nguyen HA, Kiecker F, Eberle J. Critical role of reactive oxygen species (ROS) for synergistic enhancement of apoptosis by vemurafenib and the potassium channel inhibitor TRAM-34 in melanoma cells. Cell death & disease. 2017;8(2):e2594.
  179. Quast SA, Berger A, Buttstadt N, Friebel K, Schonherr R, Eberle J. General Sensitization of melanoma cells for TRAIL-induced apoptosis by the potassium channel inhibitor TRAM-34 depends on release of SMAC. PloS one. 2012;7(6):e39290.
  180. Provinciali M, Pierpaoli E, Bartozzi B, Bernardini G. Zinc Induces Apoptosis of Human Melanoma Cells, Increasing Reactive Oxygen Species, p53 and FAS Ligand. Anticancer research. 2015;35(10):5309-16.
  181. Alarifi S, Ali D, Alkahtani S, Verma A, Ahamed M, Ahmed M, et al. Induction of oxidative stress, DNA damage, and apoptosis in a malignant human skin melanoma cell line after exposure to zinc oxide nanoparticles. International journal of nanomedicine. 2013;8:983-93.
  182. Wahab R, Dwivedi S, Umar A, Singh S, Hwang IH, Shin HS, et al. ZnO nanoparticles induce oxidative stress in Cloudman S91 melanoma cancer cells. Journal of biomedical nanotechnology. 2013;9(3):441-9.
  183. Rabaca AN, Arruda DC, Figueiredo CR, Massaoka MH, Farias CF, Tada DB, et al. AC-1001 H3 CDR peptide induces apoptosis and signs of autophagy in vitro and exhibits antimetastatic activity in a syngeneic melanoma model. FEBS open bio. 2016;6(9):885-901.
  184. Perez-Alea M, McGrail K, Sanchez-Redondo S, Ferrer B, Fournet G, Cortes J, et al. ALDH1A3 is epigenetically regulated during melanocyte transformation and is a target for melanoma treatment. Oncogene. 2017;36(41):5695-708.
  185. Quast SA, Steinhorst K, Plotz M, Eberle J. Sensitization of Melanoma Cells for Death Ligand TRAIL Is Based on Cell Cycle Arrest, ROS Production, and Activation of Proapoptotic Bcl-2 Proteins. The Journal of investigative dermatology. 2015;135(11):2794-804.
  186. Ali D, Alarifi S, Alkahtani S, AlKahtane AA, Almalik A. Cerium Oxide Nanoparticles Induce Oxidative Stress and Genotoxicity in Human Skin Melanoma Cells. Cell biochemistry and biophysics. 2015;71(3):1643-51.
  187. Pesic M, Podolski-Renic A, Stojkovic S, Matovic B, Zmejkoski D, Kojic V, et al. Anti-cancer effects of cerium oxide nanoparticles and its intracellular redox activity. Chemico-biological interactions. 2015;232:85-93.
  188. Sack M, Alili L, Karaman E, Das S, Gupta A, Seal S, et al. Combination of conventional chemotherapeutics with redox-active cerium oxide nanoparticles–a novel aspect in cancer therapy. Molecular cancer therapeutics. 2014;13(7):1740-9.
  189. Alili L, Sack M, von Montfort C, Giri S, Das S, Carroll KS, et al. Downregulation of tumor growth and invasion by redox-active nanoparticles. Antioxidants & redox signaling. 2013;19(8):765-78.
  190. Beberok A, Wrzesniok D, Szlachta M, Rok J, Rzepka Z, Respondek M, et al. Lomefloxacin Induces Oxidative Stress and Apoptosis in COLO829 Melanoma Cells. International journal of molecular sciences. 2017;18(10).
  191. Wang L, Leite de Oliveira R, Huijberts S, Bosdriesz E, Pencheva N, Brunen D, et al. An Acquired Vulnerability of Drug-Resistant Melanoma with Therapeutic Potential. Cell. 2018;173(6):1413-25 e14.
  192. Weeraratna AT, Becker D, Carr KM, Duray PH, Rosenblatt KP, Yang S, et al. Generation and analysis of melanoma SAGE libraries: SAGE advice on the melanoma transcriptome. Oncogene. 2004;23(12):2264-74.
  193. Moretti D, Del Bello B, Allavena G, Corti A, Signorini C, Maellaro E. Calpain-3 impairs cell proliferation and stimulates oxidative stress-mediated cell death in melanoma cells. PloS one. 2015;10(2):e0117258.
  194. Kaur A, Webster MR, Marchbank K, Behera R, Ndoye A, Kugel CH, 3rd, et al. sFRP2 in the aged microenvironment drives melanoma metastasis and therapy resistance. Nature. 2016;532(7598):250-4.

 

 

 

 

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