Melanocytes as instigators and victims of oxidative stress.

Epidermal melanocytes are particularly vulnerable to oxidative stress owing to the pro-oxidant state generated during melanin synthesis, and to the intrinsic antioxidant defenses that are compromised in pathologic conditions. Melanoma is thought to be oxidative stress driven, and melanocyte death in vitiligo is thought to be instigated by a highly pro-oxidant state in the epidermis. We review the current knowledge about melanin and the redox state of melanocytes, how paracrine factors help counteract oxidative stress, the role of oxidative stress in melanoma initiation and progression and in melanocyte death in vitiligo, and how this knowledge can be harnessed for melanoma and vitiligo treatment.

Introduction

Oxidative stress results from overproduction of pro-oxidant species in cells, and/or reduction of cellular antioxidant capacity, and can damage nucleic acids, lipids, and proteins, leading to mutagenesis or cell death (Sander ). Reactive oxygen species (ROS) are produced by mitochondria and peroxisomes during normal cellular metabolic processes. The ROS production may be accentuated under pathologic conditions, such as inflammation and cancer, as well as, upon exposure to exogenous factors, such as ultraviolet rays (UV), or chemicals (Klaunig and Kamendulis, 2004; Klaunig ; Sander ; Zhang ). Skin is the largest organ that interfaces with the environment, and a major source of ROS that are induced by sun exposure. Epidermal melanocytes are particularly vulnerable to excessive ROS production due to their specialized function: melanin synthesis, which is stimulated by sun exposure, during the process of tanning, and by inflammation that results in postinflammatory hyperpigmentation (Figure 1). Oxidative stress can disrupt the homeostasis of melanocytes, compromising their survival or leading to their malignant transformation (Casp ; Fried and Arbiser, 2008; Gavalas ; Govindarajan ; Guan ; Picardo ; Schallreuter ).

Figure 1

Induction of ROS by endogenous and exogenous sources and antioxidant defences that restore normal redox state in melanocytes.

Melanin and the redox state of melanocytes

Melanin synthesis involves oxidation reactions and superoxide anion (O2−) and hydrogen peroxide (H2O2) generation, which subject melanocytes to oxidative stress (Koga ; Simon ). Confinement of melanin synthesis to melanosomes protects other cellular components from oxidative damage. Tyrosinase, the rate-limiting enzyme for melanin synthesis, oxidizes tyrosine to dopa, and dopa to dopaquinone, a specific orthoquinone that can react with nucleophilic compounds such as thiols or amino groups. The catalytic activity of tyrosinase results in the generation of O2− (Koga ; Tomita ). Dopaquinone is converted into dopachrome through a redox exchange. After spontaneous decarboxylation, dopachrome generates either dihydroxyindole (5,6-DHI), which is oxidized into indole quinone, or produces dihydroxyindole carboxylic acid (5,6-DHICA) after tautomerisation by tyrosinase-related protein 2 (TRP2), and 5,6-DHICA is then converted into the corresponding quinone. Moreover, TRP2 protects against oxidative stress by increasing glutathione levels, and reducing the toxicity of quinones and DNA damage induced by free radicals (Michard ). The redox cycling from indoles to quinones generates ROS (Nappi and Vass, 1996). Polymerization of these reactive quinones finally leads to the formation of the brown/black eumelanin. The red-yellow pheomelanin differs from eumelanin in that it has a higher ratio of sulfur to quinones, and its synthesis involves the generation of cysteinyl-dopa (instead of dopa), which is converted into benzothiazine derivatives. These differences account for the higher sunlight-induced pro-oxidant property of pheomelanin compared to eumelanin.

In the skin, the balance between the pro- and antioxidant properties of melanin are mainly determined by the relative eumelanin and pheomelanin contents, the levels of melanin intermediates, the concentrations of reactive metals within the melanosome microenvironment (Di Donato ; Liu ). There are conflicting reports about the role of melanin or melanin intermediates as pro- or antioxidants. Constitutive pigmentation is reported to correlate directly with catalase activity in cultured human melanocytes, and with the levels of thioredoxin reductase in human skin (Maresca ). Generation of H2O2 in response to UV correlates inversely with constitutive pigmentation, suggesting an anti-oxidant effect of melanin(Song ). In comparison to keratinocytes, the induction of 8-hydroxydeoxyguanosine (8-OHdG), a major form of oxidative DNA damage, and expression of several base-excision repair (BER) genes are higher in melanocytes (Mouret ). Paradoxically, cultured human melanocytes with high melanin content are reported to be more vulnerable to UVA-induced, but less susceptible to hydrogen peroxide-induced oxidative DNA damage than their counterparts with low melanin content (Hoogduijn ; Wang ). Stimulation of melanogenesis in human melanocytes or mouse melanoma cells is reported to increase UVA-induced DNA damage (Kvam and Tyrrell, 1999; Marrot ; Wenczl ). In contrast, stimulation of melanogenesis in cultured human melanocytes by α-melanocortin (α-MSH) increases the activity and protein levels of catalase, and markedly reduces UV-induced H2O2 generation (Maresca ; Song ). In human melanoma cells, increased pigmentation protects against UV- or hydrogen peroxide- induced mitochondrial DNA damage (Swalwell ). The controversy about the pro-oxidant versus the antioxidant effects of melanin and its intermediates is fuelled by reports using purified melanin or melanin intermediates exogenously added to cultured cells or naked DNA (Kipp and Young, 1999; Kovacs ; Tomita ). Although these data support the oxidative nature of melanin, the experimental conditions used are unlikely to be physiologically relevant, since melanin is normally confined in melanosomes.

Activation of antioxidant defenses in melanocytes by paracrine factors

The homeostasis of epidermal human melanocytes is maintained primarily by a complex paracrine network consisting of growth factors and cytokines synthesized by epidermal keratinocytes and dermal fibroblasts, and modulated by UV. The keratinocyte-derived endothelin-1 is a potent mitogen and melanogenic factor that reduces H2O2 generation and apoptosis in UV-irradiated human melanocytes (Imokawa ; Kadekaro ; Tada ). The melanocortins α-MSH and adrenocorticotropic hormone (ACTH) are synthesized by keratinocytes and melanocytes, and stimulate eumelanin synthesis as well as melanocyte survival and proliferation by binding and activating the melanocortin 1 receptor (MC1R). The MC1R is a Gs protein-coupled receptor expressed on the cell surface of melanocytes. Treatment of cultured human melanocytes with α-MSH results in rapid reduction in the generation of H2O2 in response to UV exposure, consistent with earlier findings by Haycock et al. (Haycock ; Kadekaro ; Kadekaro ; Song ). Additionally, α-MSH increases the protein and activity levels of catalase, and counteracts the inhibitory effect of UV on this enzyme (Song ). Subsequently, treatment with α-MSH reduces the induction of 8-oxodG and enhances its repair in UV-irradiated melanocytes, and also reduces oxidative DNA damage induced by H2O2 (Kadekaro ; Song ). The antioxidant effects of α-MSH require binding and activation of MC1R, are absent in melanocytes expressing loss-of-function MC1R, and are inhibited by agouti signaling protein, the physiological MC1R antagonist (Song ). These results establish the significance of the activated MC1R in protection of melanocytes from oxidative stress.

Activation of p53 is an important mechanism by which the activated MC1R reduces oxidative stress in melanocytes. It is noteworthy that p53 regulates pigmentation by increasing the expression of tyrosinase in human melanocytes, and pro-opiomelanocortin, the precursor for melanocortins, in mouse keratinocytes (Cui ). Activation of the MC1R by α-MSH binding augments the UV-induced accumulation of p53 in human melanocytes by increasing phosphorylation of p53 on Ser15. Treatment with α-MSH also increases the levels of the BER enzymes OGG1 and APE-1 by a p53-dependent mechanism (Kadekaro ).

Additionally, activation of MC1R by α-MSH regulates intracellular redox status by up regulating the expression of antioxidant genes, including heme oxygenase-1 (HO-1), ferritin, and peroxiredoxin-1 (Kadekaro ; Song ). α-MSH activates a number of transcription factors known to regulate the redox state of melanocytes. In normal human melanocytes and melanoma cells, the redox sensor APE-1 is a target of Mitf, the master regulator of melanocyte survival and function (Liu ). Treatment of human melanocytes with α-MSH up regulates Mitf as well as APE-1 (Kadekaro ; Kadekaro ). Melanocytes also express Nrf-2, an important transcription factor that up regulates the expression of genes for phase II detoxification enzymes, and its main target HO-1 (Jain ; Jian ; Kaspar ; Marrot ; Taguchi ). Additionally, α-MSH increases the expression of Nrf-2 gene and its target genes HO-1, γ-glutamylcysteine-synthetase, and glutathione-S-transferase Pi in cultured human melanocytes, and abrogates the inhibitory effects of UV on Nrf-2 and its targets (Kokot ). Another transcription factor that is activated by α-MSH is NFκB, known to be activated by TNF-α and ROS (Haycock ; Ichiyama ; Manna and Aggarwal, 1998). Treatment of melanocytes with α-MSH inhibits UV-induced apoptosis by increasing the protein levels of Bcl2, a known target of NFκB and Mitf (Bohm ; Kadekaro ).

Significance of oxidative stress in melanoma

Sunlight is a major inducer of ROS formation in the skin, and a major contributor to skin cancer (Sander ). Irradiation of the skin by UVA and/or UVB impairs natural antioxidant defenses, and induces high levels of ROS. Acute exposure to UV is a main etiological factor for melanomagenesis. Irradiation of cultured human melanocytes with UV (75% UVB, 25%UVA) results in rapid dose-dependent generation of H2O2 (van der Kemp )(van der Kemp ), and subsequent decrease in catalase activity and protein levels, and reduced HO-1 expression (Kadekaro ; Kadekaro ; Kadekaro ; Kokot ; Song ). Exposure of human OGG1 protein, an important BER enzyme, to UVB results in its inactivation (van der Kemp ).

There is increasing evidence for the significance of oxidative stress in initiation and progression of melanoma. The role of oxidative stress in melanoma is supported by the findings that mutations in several melanoma-associated genes result from, or exacerbate, oxidative stress. The activating V600EBRAF mutation, a somatic mutation commonly expressed in nevi and melanoma, may be oxidative stress-induced (Landi ). In melanocytes, p16 is an important regulator of oxidative stress, and its depletion in cultured human melanocytes significantly increases ROS levels (Jenkins ). Melanocytes are more sensitive to p16 depletion than either keratinocytes or fibroblasts, which may impart the association of p16 mutations with melanoma. Loss of PTEN is associated with melanoma progression, presumably due to increased superoxide anion resulting from sustained activation of Akt (Govindarajan ). Loss-of-function alleles of the MC1R that are associated with increased melanoma risk cause sustained oxidative stress in human melanocytes due to inability to respond to α-MSH (Kadekaro ). In addition, oxidative stress can impair nucleotide excision repair, the main repair pathway for UV-induced DNA photoproducts, via lipid peroxidation products that inactivate DNA repair enzymes (Feng ; Feng ). Null polymorphisms of GSTM1 and GSTT1 that belong to the glutathione S-transferase family of antioxidant genes, have been associated with high risk of melanoma, especially in subjects with history of sunburns in childhood (Fortes ). One SNP in the glutathione S-transferase gene GSTP1, which reduces the activity of the enzyme, has been associated with melanoma susceptibility, and with further increase in melanoma risk when co-expressed with MC1R variant alleles (Ibarrola-Villava ). These results strongly suggest that oxidative stress is a driver of melanomagenesis (Cassidy ).

There is increasing evidence for aberrant redox state in melanoma. Melanocytes derived from melanoma patients display increased sensitivity to oxidizing agents due to endogenous antioxidant imbalance (Grammatico ; Meyskens ; Picardo ; Picardo ). Melanoma tumor cells have higher intracellular levels of O2− compared to normal melanocytes, and aberrantly activate the transcription factors NF-κB and AP-1 (Meyskens ). Moreover, melanoma tumor cells express higher levels of neuronal nitric oxide synthase, thus generate higher levels of nitric oxide than normal melanocytes, and this increase correlates with the disease stage in melanoma (Yang ). The significance of oxidative stress in melanoma is further supported by the finding that the antioxidant N-acetylcysteine inhibits tumor formation in the HGF-survivin melanoma mouse model (Cotter ), and selective inhibitors of neuronal nitric oxide synthase inhibit melanoma cell growth and metastatic potential (Yang ). Accordingly, antioxidants are being considered for prevention, as well as treatment of melanoma.

The association of aberrant melanin synthesis with oxidative stress and melanoma has been investigated by several research teams. Dysplastic nevi that are precursors for melanoma have increased ROS, and high pheomelanin, sulfur, iron, and calcium levels, and DNA damage (Pavel ; Salopek ; Smit ). Noonan et al.(2012) reported that frequency of UVA-induced melanoma tumors in HGF mice increases with skin pigmentation via an oxidative process involving melanin photoreactivity (Noonan ). Conversely, tumor formation in HGF mice is inhibited by the antioxidant N-acetylcysteine (Cotter ). In human skin, UVA-induced pigmentation was found to lack photoprotective properties (Miyamura ), indicating that exposure to UVA (e.g. in tanning beds) is not a safe practice. Recently, Mitra et al.(2012) observed that recessive yellow mice, with loss of function mc1r, and co-expressing activating BRAFv600E mutation develop more invasive melanoma tumors than their albino counterparts, and that pheomelanin results in oxidative DNA damage (Mitra ). They concluded that oxidative DNA damage resulting from pheomelanin synthesis is causal for melanoma, independently of UV exposure. How these findings apply to human pigmentation and melanoma deserves to be investigated, since human melanocytes synthesize both, eumelanin and pheomelanin, unlike recessive yellow mouse melanocytes that only synthesize pheomelanin. The ratio of these pigments should determine the overall effects on the redox state of melanocytes particularly upon UV exposure. Also, eumelanin and pheomelanin and their intermediates might differ chemically in human vs. mouse melanocytes, which might impact their pro- or antioxidant properties. Given that eumelanin is a scavenger of ROS (Meredith and Sarna, 2006), it can be concluded that reduction of eumelanin, as in individuals with fair skin, or absence of eumelanin, as in recessive yellow mice, potentiates melanoma risk by increasing the vulnerability of melanocytes to oxidative stress.

Oxidative stress and loss of melanocytes in vitiligo

Vitiligo is a depigmentary disease that occurs in approximately 0.5% of the world population, and is characterized by loss of melanocytes in the epidermis by an autoimmune mechanism (Spritz, 2013; Taieb and Picardo, 2007). However, there is strong evidence for the role of oxidative stress as a key factor in the onset and progression of the disease. Increased sensitivity of melanocytes from vitiligo patients to UVB-induced cell death as compared to normal melanocytes was attributed to their compromised capacity to cope with increased oxidative stress (Jimbow ). Further evidence supports the exaggerated sensitivity of melanocytes from non-lesional vitiligo skin to chemical or physical oxidative stress (Boissy and Manga, 2004; Maresca ). Vitiligo patients are known to have very high levels of H2O2 (1 mM) and peroxynitrite in their epidermis, concomitant with reduced levels and activity of catalase, which affects the immune response (Maresca ; Schallreuter ; Schallreuter ; Schallreuter ). High levels of H2O2 inactivate and reduce the levels of methionine sulfoxide reductase (MSR) A and B, and thioredoxin/thioredoxin reductase, thus contributing to oxidative stress and melanocyte death in vitiligo (Schallreuter ; Zhou ). Also, high levels of H2O2 in the epidermis are found to oxidize proopiomelanocortin-derived bioactive peptides ACTH and α-MSH, both of which have antioxidant and survival effects on human melanocytes, and this effect can be mitigated by treatment with pseudocatalase (Kadekaro ; Kadekaro ; Spencer ). These findings suggest that the pro-oxidant state of vitiligo skin is causal for melanocyte death.

The transcription factor Nrf-2 is implicated in the pathogenesis of vitiligo. An allelic variant of the Nrf-2 gene, A−650, is thought to be a risk factor for vitiligo (Guan ). More recently, Natarajan et al.(2010) reported increased transcript levels of Nrf-2, as well as its targets NQO-1, γ-glutamyl cysteine ligase catalytic and modulatory subunits (GCLC and GCLM, respectively) in vitiligo lesional epidermis, as compared to non-lesional skin (Natarajan ). However, induction of Nrf-2, and its target genes HO-1, NQO-1, GCLC, and GCLM by the electrophilic compounds curcumin and santalol is evident in non-lesional, but not in lesional vitiligo skin, further confirming the disruption of redox homeostasis in vitiligo (Natarajan ). Treatment of vitiligo patients with PUVA increases the expression of the Nrf-2 target HO-1 in the skin (Elassiuty ). Comparison of cultured non-lesional vitiligo melanocytes to their normal counterpart shows that the former exhibit greater induction of HO-1 than the latter in response to exposure to UVA or the phenolic compound 4-Tertuary butylphenol, demonstrating increased sensitivity of vitiligo-derived melanocytes to oxidative stress.

In vitiligo, oxidative stress-induced death of melanocytes is exacerbated by abnormal levels and/or activities of other antioxidant and BER enzymes. Catalase allelic variants have been associated with vitiligo, and the levels of several antioxidant enzymes, such as catalase, glutathione peroxidase, and glutathione reductase have been found to be altered in vitiligo, which account for sustained high levels of hydrogen peroxide in the epidermis (Casp ; Gavalas ; Park ). Salem et al. (2009) showed that in both lesional and non-lesional vitiligo skin, the levels of the BER enzymes OGG1, APE1, and DNA polymerase β are increased (Salem ). In addition to high levels of hydrogen peroxide, high levels of inducible nitric oxide synthase (iNos) in lesional and non-lesional skin, and increased 8-oxoG in the skin and plasma of vitiligo patients, can be detected, further indicating generalized oxidative stress in vitiligo.

Targeting oxidative stress pathways for treatment of melanoma and vitiligo

A major benefit to understanding redox-related mechanisms occurring in healthy and diseased melanocytes is the capacity to harness these pathways for effective, targeted therapies and prevention measures. Repigmentation of depigmented skin of Vitiligo, characterized by high levels of epidermal hydrogen peroxide and peroxynitrite, is achieved by reducing hydrogen peroxide, such as with application of narrow band UVB-activated pseudocatalase (Schallreuter ). For treatment of melanoma, characterized by aberrant redox state, two different strategies were proposed (Fruehauf and Meyskens, 2007). The first strategy is to use agents that increase ROS scavenging to reduce melanoma tumor growth via inhibiting hydrogen peroxide signaling, which mediates the proliferative effects of growth factors and inhibits the activity of protein tyrosine phosphatases, such as PTEN. Over expression of superoxide dismutase, glutathione peroxidase, or catalase reduces tumor cell growth (Finch ; Liu ; Venkataraman ). There is increasing evidence for the efficacy of antioxidants as chemopreventative agents that inhibit melanoma onset and progression. Administration of the antioxidant NAC or selenium delays the onset of UV-induced melanoma tumors (Cassidy ; Cotter ). Honikiol, a potent scavenger of superoxide and peroxyl radicals, inhibits melanoma cell growth in vitro (Dikalov ; Mannal ). Selective inhibitors of nitric oxide reduce melanoma cell growth and metastasis (Yang ). Selenium, which increases glutathione peroxidase activity and the levels of glutathione, also decreases the size of human melanoma xenografts in vivo, and inhibits the growth of human melanoma cells in vitro (Cassidy ). Treatment of human melanoma cell lines with cAMP inducers, such as α-MSH, inhibits their proliferation, due in part to inhibition of oxidative stress (Lyons ). The second strategy is to treat melanoma tumors with agents that trigger apoptosis by compromising ROS scavenging. Such agents include butathionine sulfoximine, which depletes GSH, and disulfiram, which inhibits copper, zinc superoxide dismutase, both of which inhibit melanoma cell proliferation in vitro (Cen ; Fruehauf ). Additionally, quercetin, motexafin gadolinium, melphalan and cisplatin, which inhibit thioredoxin, are effective in killing cancer cells (Hashemy ; Lu ; Witte ). Resveratrol, known to inhibit APE-1/Ref-1 endonuclease activity sensitizes melanoma cells to DNA alkylating agents (Yang ). Combined therapy utilizing some of the above agents can synergistically inhibit melanoma tumor growth. Understanding the complexity of oxidative stress pathways in pigmentation production, melanocyte proliferation and malignant transformation has enormous potential to expand our armamentarium of clinically effective compounds and offer enormous promise for patients suffering from pigmentary disorders and melanoma.