Human Epidermal Melanocytes Research Articles

Dysregulation of autophagy in melanocytes contributes to hypopigmented macules in tuberous sclerosis complex

BackgroundTuberous sclerosis complex (TSC) gene mutations lead to constitutive activation of the mammalian target of rapamycin (mTOR) pathway, resulting in a broad range of symptoms. Hypopigmented macules are the earliest sign. Although we have already confirmed that topical rapamycin treatment (an mTOR inhibitor) protects patients with TSC against macular hypopigmentation, the pathogenesis of such lesions remains poorly understood. ObjectiveRecently emerging evidence supports a role for autophagy in skin pigmentation. Herein, we investigated the impact of autophagic dysregulation on TSC-associated hypopigmentation. MethodsSkin samples from 10 patients with TSC, each bearing characteristic hypopigmented macules, and 6 healthy donors were subjected to immunohistochemical and electron microscopic analyses. In addition, TSC2-knockdown (KD) was investigated in human epidermal melanocytes by melanin content examination, real-time PCR, western blotting analyses, and intracellular immunofluorescence staining. ResultsActivation of the mTOR signaling pathway decreased melanocytic pigmentation in hypopigmented macules of patients with TSC and in TSC2-KD melanocytes. In addition, LC3 expression (a marker of autophagy) and autophagosome counts increased, whereas, intracellular accumulation of autophagic degradative substrates (p62 and ubiquitinated proteins) was evident in TSC2-KD melanocytes. Furthermore, depigmentation in TSC2-KD melanocytes was accelerated by inhibiting autophagy (ATG7-KD or bafilomycin A1-pretreatment) and was completely reversed by induction of autophagy via mTOR-dependent (rapamycin) or mTOR-independent (SMER28) exposure. Finally, dysregulation of autophagy, marked by increased LC3 expression and accumulation of ubiquitinated proteins, was also observed in melanocytes of TSC-related hypopigmented macules. ConclusionOur data demonstrate that melanocytes of patients with TSC display autophagic dysregulation, which thereby reduced pigmentation, serving as the basis for the hypomelanotic macules characteristic of TSC.

Different Levels of Skin Whitening Activity among 3,6-Anhydro-l-galactose, Agarooligosaccharides, and Neoagarooligosaccharides.

3,6-Anhydro-l-galactose (AHG), a major monomeric constituent of red macroalgae (Rhodophyta), was recently reported to possess skin whitening activity. Moreover, AHG-containing oligosaccharides, such as agarooligosaccharides (AOSs) and neoagarooligosaccharides (NAOSs), have various physiological activities, including anti-inflammatory, antioxidant, and skin moisturizing effects. In this study, AHG and NAOSs were produced from agarose by enzymatic reactions catalyzed by an endo-type β-agarase, an exo-type β-agarase, and a neoagarobiose hydrolase. In a cell proliferation assay, AHG, AOSs, and NAOSs at 12.5, 25, and 50 μg/mL concentrations did not exhibit cytotoxicity toward murine B16 melanoma cells or human epidermal melanocytes. In an in vitro skin whitening activity assay of AHG, AOSs, and NAOSs at 50 μg/mL, AHG showed the highest skin whitening activity in both murine B16 melanoma cells and human epidermal melanocytes; this activity was mediated by the inhibition of melanogenesis. Neoagarotetraose and neoagarohexaose also exhibited in vitro skin whitening activity, whereas neoagarobiose and AOSs with degrees of polymerization of 3 (agarotriose), 5 (agaropentaose), and 7 (agaroheptaose) did not. Therefore, AHG is responsible for the skin whitening activity of agar-derived sugars, and the structural differences among the AHG-containing oligosaccharides may be responsible for their different skin whitening activities.

The expression pattern of keratin 24 in tissue-engineered dermo-epidermal human skin substitutes in an in vivo model.

The use of autologous tissue-engineered skin substitutes is a promisingapproach to cover large skin defects in patients. Preclinical investigation is pivotal to test and improve the quality of these bio-engineered substitutes. In the skin, the epidermis, formed mainly by keratinocytes, provides the first physical barrier protecting from the environment. Proper keratinocyte differentiation and, thus, formation of astratified epidermis is essential for this function. Keratins, the main structural support of keratinocytes, play a vital role regarding differentiation of keratinocytes. Here, we examined the expression pattern of a recently described keratinocyte differentiation marker, namely Keratin 24, in our skin substitutes. Human epidermal keratinocytes, melanocytes, dermal fibroblasts, palmar fibroblasts or sweat gland cells were used to prepare skin substitutes. Fibroblast-containing collagen hydrogels were prepared, and keratinocytes or sweat gland cells and melanocytes were seeded onto the hydrogels. The generated tissue-engineered dermo-epidermal skin analogs were transplanted onto full-thickness skin wounds created on the back of immuno-incompetent rats. The skin substitutes were excised at different time points and histologically examined with regard to Keratin 24 expression. We observed the expression of Keratin 24 in keratinocytes of the upper stratum spinosum of the epidermis. In particular, we observed an intensified expression of Keratin 24 13weeks after transplantation compared to 4weeks after transplantation. Importantly, we noticed a markedly higher presence of Keratin 24 in more spinous layers if we used palmar fibroblasts or sweat gland cells in our skin substitutes compared non-palmar fibroblasts or epidermal keratinocytes. Our observations prove that the keratinocyte differentiation marker Keratin 24 is expressed in our dermo-epidermal skin substitutes in a normal pattern. This highlights that our bio-engineered skinanalogs mature and reach homeostasis in an in vivo assay. These findings harbor favorable implications regarding future clinical application.

Light-emitting diode 585 nm photomodulation inhibiting melanin synthesis and inducing autophagy in human melanocytes

BackgroundMelasma is a common hyperpigmentation skin disease on face. Light-emitting diode (LED) photomodulation (585nm) is reported to be effective for the treatment of melasma. However, whether and how LED photomodulation would influence melanogenesis of human epidermal melanocytes (HEMs) is unknown. ObjectiveTo evaluate the effects of LED photomodulation (585nm) on melanogenesis in HEMs. MethodsHEMs were irradiated with fluences of 0, 5, 10 and 20J/cm2 585nm LED light. After 5-day treatment, cell viability was analyzed by CCK-8 assay, and apoptosis was assessed by Annexin V APC assay. Melanin content and tyrosinase activity were measured by spectrophotometer. Melanosome stage and autophagosomes were determined under transmission electron microscope (TEM). The formation of autophagic punctate structures was observed under confocal microscope. RT-PCR and western blotting were used to assess the expression of relative mRNA and protein levels. ResultsYellow light LED 585nm had no effects on HEMs cell viability and apoptosis. Treatment with LED 585nm from 5J/cm2 to 20J/cm2 inhibited melanosome maturation, decreased melanin content and tyrosinase activity. Inhibition was accompanied by the decreased expression of tyrosinase (TYR), tyrosinase-related protein-1 (TRP-1) and microphthalmia-associated transcription factor (MITF) on both mRNA and protein levels. Autophagosomes were observed under TEM. Autophagic punctate structures of microtubule-associated protein light chain 3 (LC3) proteins were induced by LED 585nm light. The configuration change of LC3 from LC3-I to LC3-II, and the degradation of p62 protein were observed after LED 585nm. Furthermore, we also revealed that the anti-melanogenic effect of LED 585nm photomodulation was reversed by 3-Methyladenine (3-MA), which inhibits autophagy by blocking autophagosome formation via the inhibition of type III Phosphatidylinositol 3-kinases (PI-3K). ConclusionsOur finding demonstrated that LED photomodulation with 585nm wavelength suppressed melanin content in HEMs, and the effect was caused by its dose-dependent inhibition on melanogenesis and the induction of HEMs autophagy. This may provide new insights into the efficacy of LED photomodulation in the treatment of hyperpigmentation disorders.

The Autophagy Receptor Adaptor p62 is Up-regulated by UVA Radiation in Melanocytes and in Melanoma Cells.

UVA (315-400 nm) is the most abundant form of UV radiation in sunlight and indoor tanning beds. However, much remains to be understood about the regulation of the UVA damage response in melanocytes and melanoma. Here, we show that UVA, but not the shorter waveband UVB (280-315 nm), up-regulates adaptor protein p62 in an Nrf2- and reactive oxygen species (ROS)-dependent manner, suggesting a UVA-specific effect on p62 regulation. UVA-induced p62 up-regulation was inhibited by a mitochondria-targeted antioxidant or Nrf2 knockdown. In addition, p62 knockdown inhibited UVA-induced ROS production and Nrf2 up-regulation. We also report here a novel regulatory feedback loop between p62 and PTEN in melanoma cells. PTEN overexpression reduced p62 protein levels, and p62 knockdown increased PTEN protein levels. As compared with normal human skin, p62 was up-regulated in human nevus, malignant melanoma and metastatic melanoma. Furthermore, p62 was up-regulated in melanoma cells relative to normal human epidermal melanocytes, independent of their BRAF or NRAS mutation status. Our results demonstrated that UVA up-regulates p62 and induces a p62-Nrf2 positive feedback loop to counteract oxidative stress. Additionally, p62 forms a feedback loop with PTEN in melanoma cells, suggesting p62 functions as an oncogene in UVA-associated melanoma development and progression.

An inhibitory mechanism of action of a novel syringic-acid derivative on α-melanocyte–stimulating hormone (α-MSH)-induced melanogenesis

AimsTo report the effects of a novel syringic-acid derivative, (R)-ethyl-2-acetamido-3-(4-hydroxy-3,5-dimethoxybenzoylthio)propanoate (EABTO), on melanin synthesis and to identify its mechanism of action in B16F1 melanoma cells. MethodsThe effects of EABTO on melanin synthesis in B16F1 cells and human epidermal melanocytes and the influence on cell-free tyrosinase activity were evaluated. EABTO-induced cellular signaling cascades were studied by western blotting. Key findingsEABTO effectively decreased melanin synthesis in a dose-dependent manner but had no effect on cell-free tyrosinase activity. EABTO significantly decreased the expression of melanogenic enzymes such as tyrosinase, tyrosinase-related protein 1 (TRP-1), and TRP-2. EABTO decreased the amounts of phosphorylated cAMP response element–binding protein (CREB) and cyclic adenosine monophosphate (cAMP), thereby inhibiting expression of microphthalmia-associated transcription factor (MITF). Moreover, EABTO upregulated phosphorylated ERK. A specific ERK pathway inhibitor, PD98059, reduced EABTO-induced ERK phosphorylation and restored the expression of MITF and melanin content. SignificanceEABTO inhibits melanogenesis in B16F1 melanoma cells via suppression of the cAMP–CREB pathway and activation of ERK, thus decreasing expression of MITF and of melanogenic enzymes.

Antitumor activity and expression profiles of genes induced by sulforaphane in human melanoma cells

PurposeHuman melanoma is a highly aggressive incurable cancer due to intrinsic cellular resistance to apoptosis, reprogramming, proliferation and survival during tumour progression. Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, plays a role in carcinogenesis in many cancer types. However, the cytotoxic molecular mechanisms and gene expression profiles promoted by SFN in human melanoma remain unknown.MethodsThree different cell lines were used: two human melanoma A375 and 501MEL and human epidermal melanocytes (HEMa). Cell viability and proliferation, cell cycle analysis, cell migration and invasion and protein expression and phosphorylation status of Akt and p53 upon SFN treatment were determined. RNA-seq of A375 was performed at different time points after SFN treatment.ResultsWe demonstrated that SFN strongly decreased cell viability and proliferation, induced G2/M cell cycle arrest, promoted apoptosis through the activation of caspases 3, 8, 9 and hampered migration and invasion abilities in the melanoma cell lines. Remarkably, HEMa cells were not affected by SFN treatment. Transcriptomic analysis revealed regulation of genes involved in response to stress, apoptosis/cell death and metabolic processes. SFN upregulated the expression of pro-apoptotic genes, such as p53, BAX, PUMA, FAS and MDM2; promoted cell cycle inhibition and growth arrest by upregulating EGR1, GADD45B, ATF3 and CDKN1A; and simultaneously acted as a potent inhibitor of genotoxicity by launching the stress-inducible protein network (HMOX1, HSPA1A, HSPA6, SOD1).ConclusionOverall, the data show that SFN cytotoxicity in melanoma derives from complex and concurrent mechanisms during carcinogenesis, which makes it a promising cancer prevention agent.

Expression of monoacylglycerol lipase as a marker of tumour invasion and progression in malignant melanoma.

Accumulating evidence suggests that the lipid lytic enzyme monoacylglycerol lipase (MAGL) promotes tumour invasion and metastasis through up-regulation of pro-tumorigenic signalling lipids in several tumour cell lines. However, the expression status of MAGL in clinical melanoma tissues and its clinicopathological significance remain unclear. To correlate the tumour expression status of MAGL with the clinicopathological information of patients with malignant melanoma. Polymerase chain reaction (PCR) array screening was performed, and the results were validated using immunocytochemical analysis of tumour and non-tumour melanocytic cell lines. Immunohistochemical staining for MAGL was performed for 74 melanoma samples, including 48 primary and 26 metastatic tumours, in which the expression of MAGL was determined by evaluating the percentage of MAGL-positive tumour cells and the MAGL staining intensity. Finally, we analysed the association of MAGL expression status with tumour progression, tumour thickness and vascular invasion of the primary lesion. Immunocytochemical analysis revealed that MAGL was expressed in all 12 melanoma cell lines, but not in normal human epidermal melanocytes. In the immunohistochemical analysis, positive staining for MAGL was noted in 32 of 48 (64.5%) primary lesions, 14 of 17 (82.4%) lymph node metastatic lesions and 7 of 9 (77.8%) skin metastatic lesions. Metastatic tumours had a significantly higher staining intensity (P = 0.033 for lymph node, P = 0.010 for skin). In the analysis of primary lesions, higher MAGL expression correlated with greater tumour thickness (P = 0.015) and the presence of vascular invasion (P = 0.017). On further evaluation of MAGL-positive primary lesions, staining intensity of MAGL tended to be higher in deeper areas of the tumour mass. The expression of MAGL in tumour cells reflects the aggressiveness of melanoma cells and may serve as a marker of tumour progression.

Abstract 5442: Down-regulation of miRNA-214 expression by bioactive molecules mitigates invasive potential of melanoma cells through ALCAM/TFAP2 signaling

Abstract Melanoma is responsible for 80% of skin cancer related deaths in the United States and represents fifth most common neoplasia in humans. Over the past decades, the incidence of melanoma has increased by 3% to 8% per year in western countries. Therefore, it is essential to understand the molecular regulatory pathways that contribute to melanoma aggressiveness and metastatic dissemination. MicroRNAs (miRNAs) are small noncoding regulatory RNAs that contribute to tumor formation and progression, for their ability to regulate gene expression post-transcriptionally by either inhibiting protein translation or degrading target mRNAs. miR-214 is overexpressed in various types of cancers and is associated with the regulation of numerous carcinogenic processes including cell differentiation, senescence, angiogenesis and cell migration/invasion, etc. In the present study, we demonstrated that miR-214 shows distinct over expression in melanoma cell lines (A375, Hs294t, SK-Mel119, and SK-Mel28) as compared to its expression in normal human epidermal melanocytes (NHEM), and this overexpression of miR-214 in melanoma cells was associated with the invasive potential than NHEM. Treatment of melanoma cells with an inhibitor of miR-214 (50 nM) resulted in inhibition of cell invasion ability. We also determined the effects of bioactive phytochemical grape seed proanthocyanidins (GSPs) on the expression levels of miRNA-214 in melanoma cell lines (A375 and Hs294t) and its underlying molecular targets. Treatment of A375 and Hs294t cells with GSPs resulted in suppression of the levels of miRNA-214, which leads to decrease in migration potential of melanoma cell lines. By using 3D melanoma progression model, we also verified this suppressive effect of GPSs on melanoma invasion. The transition from the non-invasive to the invasive and metastatic stage is accompanied by the loss of AP-2 transcription factors (TFAP-2) and expression of activated leukocyte cell adhesion molecule (ALCAM) and those are associated with miRNA-214 overexpression. Here, we observed that the treatment of GSPs decreases ALCAM expression and restore TFAP-2 levels in melanoma cell lines. Further, dietary administration of GSPs significantly reduce the metastasis potential of A375 cells in different vital organs such as lung, liver, kidney, and spleen, and this suppressive effect of GSPs on invasive potential of A375 cells was associated with the reduction in the levels of miRNA-214 and ALCAM. Our results indicate that modulation of miR-214 influences in vitro cell movement and extravasation from blood vessels in vivo. Together, these studies suggest that an identification of metastasis related miRNAs may provide potential diagnostic biomarkers and better understanding of the complex functions of miRNAs, as well as therapeutic targets for clinical application. Note: This abstract was not presented at the meeting. Citation Format: Ram Prasad, Santosh K. Katiyar. Down-regulation of miRNA-214 expression by bioactive molecules mitigates invasive potential of melanoma cells through ALCAM/TFAP2 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5442. doi:10.1158/1538-7445.AM2017-5442

Abstract 820: Potential pro-proliferative role of SIRT6 in melanoma

Abstract Melanoma is one of the deadliest form of skin cancer that can rapidly metastasize to become lethal, if not diagnosed early or left untreated. In 2016, approximately 76,380 new melanoma cases and 10,130 melanoma-related deaths are predicted in the United States. Current preventive and therapeutic strategies have not been sufficiently effective in the management of melanoma. Therefore, novel molecular targets and treatments are required for an effective management of this neoplasm. In our laboratory, we are assessing the role and functional and therapeutic significance of sirtuin family of proteins in melanoma. Sirtuins (SIRTs) have been conserved through evolution from prokaryotic to eukaryotic cells. SIRTs are nicotinamide adenine dinucleotide (NAD+) dependent protein deacetylases and belong to class III of the histone deacetylase (HDAC) family. Seven members of the mammalian SIRT family are known to date, and despite structural similarities, each SIRT has their own biological niche, performing unique functions via regulating critical mechanisms in the cell. The role of SIRTs in cancer is somewhat controversial, as they have exhibited conflicting functions (tumor promoter vs. tumor suppressor) depending on cell and tissue contexts. The sirtuin SIRT6, a predominantly nuclear protein, has been shown to conduct ADP-ribosyl transferase and histone deacetylase activities. SIRT6 plays key roles in DNA repair, inflammation and metabolic diseases such as cancer. Currently, the role of SIRT6 in melanoma is not known. The objective of this study was to determine the role of SIRT6 in melanoma. Using a panel of human melanoma cell lines (A375, Hs 294T, G361, SK-MEL-2, SK-MEL-28, SK-MEL-31, WM115 and WM35) differing in genetic complexity and disease progression stage, and normal human epidermal melanocytes (NHEMs), we determined the endogenous expression levels of SIRT6. We found that compared to NHEMs, SIRT6 is significantly upregulated in melanoma cell lines, at mRNA as well as protein levels, as shown by quantitative Real-Time PCR and western blot analyses. Further, employing a human tissue microarray (TMA) coupled with quantitative Vectra™ analysis, we determined the expression profile of SIRT6 protein in human melanoma and melanocytic nevus tissues. Our data demonstrated that SIRT6 is significantly overexpressed in human melanoma tissues when compared to nevi. Furthermore, lentiviral short hairpin RNA-mediated knockdown of SIRT6 in human melanoma cells was found to result in a marked anti-proliferative response in melanoma cells. Taken together, our data suggest that SIRT6 overexpression could potentially be a contributing factor in melanoma progression. Further detailed studies are underway to understand the functional significance of SIRT6 in melanoma development and progression. Citation Format: Liz Garcia-Peterson, Mary A. Ndiaye, Chandra K. Singh, Wei Huang, Nihal Ahmad. Potential pro-proliferative role of SIRT6 in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 820. doi:10.1158/1538-7445.AM2017-820

Cannabidiol upregulates melanogenesis through CB1 dependent pathway by activating p38 MAPK and p42/44 MAPK

Melanogenesis plays a critical role in the protection of skin against external stresses such as ultraviolet irradiation and oxidative stressors. This study was aimed to investigate the effects of cannabidiol on melanogenesis and its mechanisms of action in human epidermal melanocytes. We found that cannabidiol increased both melanin content and tyrosinase activity. The mRNA levels of microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein (TRP) 1, and TRP2 were increased following cannabidiol treatment. Likewise, cannabidiol increased the protein levels of MITF, TRP 1, TRP 2, and tyrosinase. Mechanistically, we found that cannabidiol regulated melanogenesis by upregulating MITF through phosphorylation of p38 mitogen-activated protein kinase (MAPK) and p42/44 MAPK, independent of cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling. In addition, the melanogenic effect of cannabidiol was found to be mediated by cannabinoid CB1 receptor, not by CB2 receptor. Taken together, these findings indicate that cannabidiol-induced melanogenesis is cannabinoid CB1 receptor-dependent, and cannabidiol induces melanogenesis through increasing MITF gene expression which is mediated by activation of p38 MAPK and p42/44 MAPK. Our results suggest that cannabidiol might be useful as a protective agent against external stresses.

MTOR-mediated Na+/Ca2+ exchange affects cell proliferation and metastasis of melanoma cells

Melanoma is a common malignant tumor, which is associated with high mortality rate. The multiple-drug resistance of tumor cells often results in failure of chemotherapy. The aim of our study is to investigate the expression of Nav 1.6 in human melanoma cells and human epidermal melanocytes. Additionally, the effect of Na+channels on Ca+ current and mTOR activity in melanoma cells were also analyzed. The protein expression levels of Nav1.6 in human melanocyte PIG1, WM266 and WM115 cells were investigated by western blot. After treatment of Na+ channel inhibitor Tetroadotoxin (TTX) or mTOR inhibitor rapamycin (RAPA), the electrophysiological activity (Na+ current and Ca2+ current) in WM266 and WM115 cells was detected by patch clamp technique. The expression of mTORC1 phosphorylates S6 kinase (p-S6), cell invasion and migration, cell proliferation and cell apoptosis were also performed. Results shown that Nav 1.6 was overexpressed in WM266 and WM115 cells, and the inhibition of Na+ channel by TTX reduced Na+ current. Both TTX and RAPA suppressed Ca2+ current and the expression of p-S6, thus inducing Na+ channel which activates the mTOR-Ca2+ signaling pathway. Both TTX and RAPA suppressed cell invasion, migration and proliferation, and promoted cell apoptosis of WM266 cells. Thus, the Nav1.6 sodium channel promotes cell proliferation and invasion through mTOR-mediated Na+/Ca2+ exchange in melanoma. The observations will provide a new perspective for understanding the malignant biological behavior of melanoma cells, and potentially provide a new drug target.

Keap1 knockdown in melanocytes induces cell proliferation and survival via HO-1-associated β-catenin signaling

BackgroundNrf2-Keap1 signaling pathway protects cells against photo-oxidative stress. Yet in recent works, its role in melanogenesis together with cell protection functions against oxidative stress has been gaining interest. However, its effect on melanogenesis still has contradictory results from different studies. ObjectiveThe aims of our study were to investigate the effect of Keap1 silencing in melanocyte on melanogenesis and its associated mechanism. MethodsPrimary human epidermal melanocytes and melan-a cell line were used for this experiment. RNA sequencing was done to identify genes involved in melanocyte biology using Keap1 knockdown through siRNA techniques. And melanogenesis and the expression of melanogenesis-associated molecules were evaluated in Keap1 silenced melanocyte to examine the effects of Keap1 on melanogenesis, melanocyte growth, and related pathways. ResultsRNA-sequencing data revealed that Keap1 knockdown in primary human epidermal melanocytes (PHEMs) induced cell survival-related gene expression. Additionally, siRNA-mediated inhibition of Keap1 led to upregulation of MITF and melanogenesis-associated molecules along with Nrf2 activation in PHEMs. HO-1, a major gene that is upregulated in RNA-sequencing using Keap1-silenced PHEMs, protected melanocytes against H2O2-induced cell death and upregulated MITF and β-catenin expression. Further, increased expression of melanogenesis-associated molecules after Keap1 silencing was validated to occur through HO-1-associated β-catenin activation in a Keap1 and HO-1 double knockdown experiment. ConclusionThis work suggests that Keap1 silencing in melanocytes induced melanogenesis and the expression of melanogenesis-associated molecules through HO-1-associated β-catenin activation. Keap1 downregulation in melanocytes is important for cell proliferation and survival.

Identification of ACA‐28, a 1′‐acetoxychavicol acetate analogue compound, as a novel modulator of ERK MAPK signaling, which preferentially kills human melanoma cells

The extracellular signal-regulated kinase (ERK) signaling pathway is essential for cell proliferation and is frequently deregulated in human tumors such as melanoma. Melanoma remains incurable despite the use of conventional chemotherapy; consequently, development of new therapeutic agents for melanoma is highly desirable. Here, we carried out a chemical genetic screen using a fission yeast phenotypic assay and showed that ACA-28, a synthetic derivative of 1'-acetoxychavicol acetate (ACA), which is a natural ginger compound, effectively inhibited the growth of melanoma cancer cells wherein ERK MAPK signaling is hyperactivated due to mutations in the upstream activating regulators. ACA-28 more potently inhibited the growth of melanoma cells than did the parental compound ACA. Importantly, the growth of normal human epidermal melanocytes (NHEM) was less affected by ACA-28 at the same 50% inhibitory concentration. In addition, ACA-28 specifically induced apoptosis in NIH/3T3 cells which were oncogenically transformed with human epidermal growth factor receptor-2 (HER2/ErbB2), but not in the parental cells. Notably, the ACA-28-induced apoptosis in melanoma and HER2-transformed cells was abrogated when ERK activation was blocked with a specific MEK inhibitor U0126. Consistently, ACA-28 more strongly stimulated ERK phosphorylation in melanoma cells, as compared in NHEM. ACA-28 might serve as a promising seed compound for melanoma treatment.

730 Wavelength specific gene expression clustering identified by cap analysis of gene expression in normal human epidermal melanocytes

730 Wavelength specific gene expression clustering identified by cap analysis of gene expression in normal human epidermal melanocytes

Melanocyte-protective effect of afzelin is mediated by the Nrf2-ARE signalling pathway via GSK-3β inactivation.

Vitiligo is an acquired condition characterized by depigmented, cutaneous lesions that result from the death of pigment-producing cells, melanocytes. The occurrence of oxidative stress has been proposed as a pathogenetic mechanism for melanocyte degeneration in vitiligo. Therefore, in this study, we investigated the cytoprotective effects of afzelin against oxidative stress and its mechanism of action in human epidermal melanocytes. We found that afzelin significantly inhibited hydrogen peroxide-induced cell death, cellular reactive oxygen species production and lipid peroxidation in melanocytes. In an antioxidant response element (ARE)-luciferase reporter assay, afzelin increased ARE-luciferase reporter activity in a concentration-dependent manner. Consistently, the expression of antioxidant genes, including NF-E2-related factor 2 (Nrf2), haem oxygenase-1 (HO-1) and catalase, was enhanced by afzelin treatment. Nuclear translocation of Nrf2 also increased in response to afzelin treatment. In addition, the phosphorylation of glycogen synthase kinase-3β (GSK-3β) was induced by afzelin treatment. The enhancement of HO-1 gene expression by afzelin treatment was reduced by Nrf2-siRNA expression. Furthermore, we found that the expression of Nrf2-siRNA significantly attenuated the cytoprotective effect of afzelin against hydrogen peroxide. These data suggest that the cytoprotective effects of afzelin against hydrogen peroxide may be mediated by Nrf2-ARE signalling via GSK-3β inactivation. Our data suggest the novel use of afzelin for the prevention of oxidative stress-induced damage in melanocytes and its potential as a therapeutic agent for vitiligo.

UCHL1 Regulates Melanogenesis through Controlling MITF Stability in Human Melanocytes

Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is involved in many signaling pathways via the ubiquitin-proteasome system. UCHL1 is expressed in the human skin and serves as a neuronal marker; however, its functions in melanogenesis remain unknown. Here, we investigated the role of UCHL1 in melanogenesis and elucidated the underlying mechanism using human melanocytes. UCHL1 downregulation by small interfering RNA resulted in upregulation of microphthalmia-associated transcription factor (MITF), tyrosinase, dopachrome tautomerase, tyrosinase-related protein-1, and melanin. In contrast, overexpression of UCHL1 in melanocytes via adenovirus transfection led to downregulation of tyrosinase, dopachrome tautomerase, and tyrosinase-related protein-1 and decreased melanin contents. Furthermore, UCHL1 reduced the protein, but not mRNA, levels of MITF, the upstream regulator of tyrosinase, dopachrome tautomerase, and tyrosinase-related protein-1. Inhibition of de novo protein synthesis and treatment of normal human primary epidermal melanocytes with proteasome inhibitor MG132 revealed that UCHL1 negatively regulates the stability of MITF by binding to the ubiquitinated protein. Finally, overexpression of MITF via an adenovirus restored the level of melanogenesis reduced by UCHL1. Collectively, our findings indicate a role of UCHL1 in regulating skin pigmentation. Suppression of MITF activity by UCHL1 via protein degradation might aid in the development of new therapeutic approaches for melanoma or dyspigmentation disorders.

Protective effects of tacalcitol against oxidative damage in human epidermal melanocytes.

Oxidative damage may lead to the dysfunction of melanocytes (MCs) and is one of the causative mechanisms involved in the pathogenesis of vitiligo. This study was designed to investigate the protective effects of the vitamin D3 analog tacalcitol on oxidative damage induced by hydrogen peroxide (H2 O2 ) in human epidermal MCs. Human epidermal MCs were cultured and identified by l-DOPA staining and HMB-45 immunohistochemical staining. The model of oxidative damage induced by H2 O2 was established, and the cells were treated with tacalcitol. The viability of MCs was determined using an MTS assay. Morphological changes in cell dendrites were observed by microscopy, and the rate of change of dendrites was calculated. The reactive oxygen species (ROS) level in MCs was determined using immunofluorescence microscopy. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels in MCs were determined using the WST-1 and TBA methods, respectively. In comparison with the control group, the viability of MCs and SOD activity were significantly decreased in the H2 O2 group (P<0.05) and significantly increased in the tacalcitol group (P<0.05). In comparison with the control group, the rate of change of cell dendrites and levels of ROS and MDA were significantly increased in the H2 O2 group (P<0.05) and significantly decreased in the tacalcitol group (P<0.05). Tacalcitol can reduce oxidative damage induced by H2 O2 in MCs by inhibiting intracellular ROS overproduction, increasing SOD activity, and decreasing the level of MDA, thereby reducing cell apoptosis.

Pheomelanin content of cultured human melanocytes from lightly and darkly pigmented skin: A pyrolysis-gas chromatography/tandem mass spectrometry study

The diverse susceptibility of human skin with various degree of constitutive pigmentation to UV-induced damage, including carcinogenesis, may result from the differences in both total melanin content and the pigment composition, in particular the proportion of potentially harmful pheomelanin. The aim of this study was to compare a pheomelanin content of human epidermal melanocytes derived from lightly and darkly pigmented neonatal skin, using the previously developed method based on the thermal degradation of the pigment. Melanin was isolated from the cultured cells and pyrolysed at 500°C with a microfurnace-type device. The obtained pyrolysates were analyzed for the presence of the marker degradation products using a gas chromatography/tandem mass spectrometry system operating in a multiple reaction monitoring mode. The pheomelanin content was calculated from a calibration curve, generated with the use of a series of synthetic melanin pigments with known percentages of incorporated pheomelanin. We have found that the pheomelanin content of human epidermal melanocytes from neonatal skin does not exceed 10pg per cell, and depends on the degree of constitutive pigmentation. Heavily pigmented melanocytes derived from dark skin produce approximately four times as much pheomelanin as the cells from light skinned donors.

Upregulation of Mitf by Phenolic Compounds-Rich Cymbopogon schoenanthus Treatment Promotes Melanogenesis in B16 Melanoma Cells and Human Epidermal Melanocytes.

Melanin provides inherent protection against skin cancer by absorbing broad-spectrum radiant energy of UV radiation. Cutaneous malignant melanoma incidence has recently been observed to increase and the frequency is closely associated with the skin color, highlighting the importance of skin pigmentation. Here, we showed how melanin biosynthesis is enhanced by treatment with phenolic compounds-rich Cymbopogon schoenanthus (CYM) in B16 murine melanoma cells and human epidermal melanocytes (HEM). CYM increased the melanin content of the cells by upregulating the expression of tyrosinase (TYR), tyrosinase-related protein 1 (TRP1), and dopachrome tautomerase (DCT) at the protein and mRNA levels, comparable to the effect of α-melanocyte-stimulating hormone (MSH), in both B16 cells and HEM. Moreover, global gene expression analysis showed that at least 44 pigmentation-associated genes were modulated, including the microphthalmia-associated transcription factor (Mitf) and its transcriptional regulators (Sox10, Pax3, and Lef1). Upregulation of copper transport-associated gene Atp7b indicates that CYM also promotes tyrosinase activity. CYM upregulated Mitf and possibly activates tyrosinase enzyme, providing evidence for its possible use to promote melanogenesis and as a therapeutic agent against hypopigmentation disorders.