Human Epidermal Melanocyte Cells Research Articles

Targeting phosphorylation circuits on CREB and CRTCs as the strategy to prevent acquired skin hyperpigmentation.

Background: The cAMP response element-binding protein (CREB) and CREB-regulated transcription coactivators (CRTCs) cooperate in the transcriptional activation of microphthalmia-associated transcription factor subtype M (MITF-M) that is a master regulator in the biogenesis, pigmentation and transfer of melanosomes at epidermal melanocytes. Here, we propose the targeting of phosphorylation circuits on CREB and CRTCs in the expression of MITF-M as the rationale to prevent skin hyperpigmentation by elucidating the inhibitory activity and mechanism of yakuchinone A (Yaku A) on facultative melanogenesis. Methods: We employed human epidermal melanocyte cell, mouse skin, and mouse melanoma cell, and applied Western blotting, reverse transcription-polymerase chain reaction, immunoprecipitation and confocal microscopy to conduct this study. Results: This study suggested that α-melanocyte stimulating hormone (α-MSH)-induced melanogenic programs could switch on the axis of protein kinase A-salt inducible kinases (PKA-SIKs) rather than that of PKA-AMP activated protein kinase (PKA-AMPK) during the dephosphorylation of CRTCs in the expression of MITF-M. SIK inhibitors rather than AMPK inhibitors stimulated melanin production in melanocyte cultures in the absence of extracellular melanogenic stimuli, wherein SIK inhibitors increased the dephosphorylation of CRTCs but bypassed the phosphorylation of CREB for the expression of MITF-M. Treatment with Yaku A prevented ultraviolet B (UV-B)-irradiated skin hyperpigmentation in mice and inhibited melanin production in α-MSH- or SIK inhibitor-activated melanocyte cultures. Mechanistically, Yaku A suppressed the expression of MITF-M via dually targeting the i) cAMP-dependent dissociation of PKA holoenzyme at the upstream from PKA-catalyzed phosphorylation of CREB coupled with PKA-SIKs axis-mediated dephosphorylation of CRTCs in α-MSH-induced melanogenic programs, and ii) nuclear import of CRTCs after SIK inhibitor-induced dephosphorylation of CRTCs. Conclusions: Taken together, the targeting phosphorylation circuits on CREB and CRTCs in the expression of MITF-M could be a suitable strategy to prevent pigmentary disorders in the skin.

Heat-Killed Lactobacillus rhamnosus ATCC 7469 Improved UVB-Induced Photoaging Via Antiwrinkle and Antimelanogenesis Impacts.

Exposure of ultraviolet B (UVB) radiation is the main factor from the environment to cause skin photoaging. Lactobacillus rhamnosus ATCC 7469, is a probiotic strain with a good track record for enhancing human health. The present study conducted the impacts of heat-killed L.rhamnosus ATCC 7469 (RL) on photoaging invitro using mouse skin fibroblast (MSF) cells and human epidermal melanocytes (HEM) exposed to UVB. The results showed that (1) RL-protected UVB-induced cytotoxicity relating to absorb UVB and reduce DNA damage. (2) RL exerted the antiwrinkle impact involved in two aspects. Firstly, RL downregulated MMP-1, 2, 3 expressions associating with MAPK signaling, resulting in the increased the protein expression of COL1A1, further booting type I collagen abundant thereby promoting the antiwrinkle impact in MSF cells. Secondly, RL reduced ROS content, further decreasing oxidative damage relating to Nrf2/Sirt3/SOD2 signaling, thereby promoting the antiwrinkle impact in MSF cells. (3) RL suppressed tyrosinase and TYRP-2 activity and/or levels associating with PKA/CREB/MITF signaling, thereby promoting antimelanogenesis impact in HEM cells. In conclusion, our findings suggest that RL could reduce photoaging caused by UVB via antiwrinkle and antimelanogenesis properties and may be a potential antiphotoaging beneficial component, which is applied in the cosmetic industry.

New Butyroside D from Argan Press Cake Possess Anti-Melanogenesis Effect via MITF Downregulation in B16F10 and HEM Cells.

Hyperpigmentation is a skin condition where patches of skin become darker in color due to excess melanin production upon UV exposure leading to melasma, which are lentigines or post inflammatory hyperpigmentation that psychologically affecting a great number of people. The present study investigates the anti-melanogenic effect of Butyroside D and the underling mechanism. After the confirmation of the non-cytotoxic effect of Butyroside D on B16F10 cells, we proceeded with analyzing the impact of the treatment at low and high concentration (i.e., 0.2 μM and 2 μM) using gene profiling analysis and examined the differentiation in gene expression. Our results identify cyclic adenosine monophosphate (cAMP), Wnt/β-catenin and Mitogen-Activated Protein Kinase (MAPK) signaling pathways to be downregulated upon treatment with Butyroside D. These pathways were targeted to further validate the effect of Butyroside D on membrane receptors melanocortin 1 receptor (MC1R) and receptor tyrosine kinase (c-Kit), related microphthalmia-associated transcription factor (MITF) and consequently tyrosinase (TYR), and tyrosine-related protein-1 (TYRP-1) that were all shown to be downregulated and, therefore, leading to the repression of melanin biosynthesis. Finally, the anti-melanogenic effect of Butyroside D was confirmed on human epidermal melanocytes (HEM) cells by inhibiting the activation of cAMP pathway generally mediated through α-melanocyte-stimulating hormone (α-MSH) and MC1R. Overall, this study suggests the potential applicability of this purified compound for the prevention of hyperpigmentation conditions.

The molecular mechanisms of vulpinic acid induced programmed cell death in melanoma.

Malignant melanoma is an aggressive skin tumor with a rapidly increasing incidence and there is not yet a successful treatment strategy. Vulpinic acid (VA) is derived from secondary metabolites from lichen species. In the current study, we, for the first time, investigated the anti-cancer effects of VA and the underlying mechanism VA induced programmed cell death in melanoma. The anti-cancer effects of VA on melanoma cells were evaluated by the xCELLigence system, flow cytometry, caspase-3 activity and RT-PCR analysis. Our results showed that VA had a strong anti-proliferative effect on A-375 melanoma cells without damaging human epidermal melanocyte cells. Additionally, VA promoted apoptotic cell death through G2/M arrest and the activation of both intrinsic and extrinsic apoptosis pathways according to the analysis of 88 genes associated with apoptosis by qRT-PCR. Our findings suggest that VA could become an alternative topical and transdermal treatment strategy in the treatment of maligned melanoma cancer. However, further investigations are needed to assess the underlying molecular mechanism of VA mediated apoptotic cell death in the treatment of melanoma.

Dehydroglyasperin D Suppresses Melanin Synthesis through MITF Degradation in Melanocytes.

Licorice (Glycyrrhiza) has been used as preventive and therapeutic material for hyperpigmentation disorders. Previously, we isolated noble compounds including dehydroglyasperin C (DGC), dehydroglyasperin D (DGD) and isoangustone A (IAA) from licorice hexane/ethanol extracts. However, their anti-melanogenic effects and underlying molecular mechanisms are unknown. The present study compared effects of DGC, DGD and IAA on pigmentation in melan-a melanocytes and human epidermal melanocytes (HEMn). DGD exerted the most excellent anti-melanogenic effect, followed by DGC and IAA at non-cytotoxic concentrations. In addition, DGD significantly inhibited tyrosinase activity in vitro cell-free system and cell system. Western blot result showed that DGD decreased expression of microphthalmia-associated transcription factor (MITF), tyrosinase and tyrosinase-related protein-1 (TRP-1) in melan-a cells and HEMn cells. DGD induced phosphorylation of MITF, ERK and Akt signal pathway promoting MITF degradation system. However, DGD did not influence p38 and cAMP-dependent protein kinase (PKA)/CREB signal pathway in melan-a cells. These result indicated that DGD inhibited melanogenesis not only direct regulation of tyrosinase but also modulating intracellular signaling related with MITF level. Collectively, these results suggested a protective role for DGD against melanogenesis.

Effects of tea polyphenols on UVA-induced melanogenesis via inhibition of α-MSH-MC1R signalling pathway.

IntroductionUltraviolet (UV) irradiation is a major environmental factor affecting photoaging, which is characterized by skin wrinkle formation and hyperpigmentation. Although many factors are involved in the melanogenesis progress, UV is thought to play a major role in tanning. The pathway of α-melanocyte-stimulating hormone (α-MSH)-melanocortin receptor 1 (MC1R) is associated with UV-induced melanogenesis. Thus, α-MSH antagonists may have applications in the prevention of melanogenesis.AimTo investigate the effects of tea polyphenols (TPS) on pigmentation, and further explore the underlying mechanism.Material and methodsHuman keratinocyte cell line (HaCaT) cells and Human epidermal melanocytes (HEM) were exposed to UVA and treated with different concentrations of TPS or Nonapeptide-1 acetate salt (N-1A). Then, cell viability, melanin content, and tyrosinase activity of both kinds of cells were detected. Quantification of α-MSH in HaCaT cells and HEM cells determined by ELISA assays. Immunohistochemistry of HEM cells was employed to further investigate the expression of melanogenesis-related proteins.ResultsThe different concentrations of TPS were found to decrease the melanin content, tyrosinase activity and melanogenesis-related proteins such as microphthalmia-associated transcription factor (MITF), tyrosinase-related protein (TRP)1, and TRP2. Besides, TPS inhibited α-MSH-MC1R signalling through directly suppressed α-MSH expression rather than the down-regulated expression level of MC1R.ConclusionsOur findings indicate that TPS may be a potential whitening agent for use in cosmetics and the medical treatment of hyperpigmentation disorders.

TCF21 regulates miR-10a-5p/LIN28B signaling to block the proliferation and invasion of melanoma cells.

Background and aimSome research has suggested that miRNA-10a (miR-10a-5p) had an inhibitory function in proliferation and invasion of cancers. Whereas the role of miR-10a-5p in melanoma has not been fully explored. This study aims to confirm LIN28B as the targeted gene of miR-10a-5p which was explored in melanoma cells. In addition, upstream regulatory molecule of miR-10a-5p was also investigated in melanoma cells.MethodsReal-time Quantitative polymerase chain reaction (RT-qPCR) was adopted to analyze miR-10a-5p expression level in melanoma and the normal human epidermal melanocyte cells. Several biological assays were performed to evaluate miR-10a-5p influences on cell proliferation, migration and invasion ability in A375 and B16-F10 cells. Gene prediction of miRNA targeting and a dual luciferase assay were applied to assess miR-10a-5p-targeted LIN28B. Western blot assessed the impacts of miR-10a-5p on the protein expression of LIN28B. Western blot analyzed the TCF21 effects on the expression of LIN28B and RT-qPCR assessed the influence of TCF21 on the expression level of miRNA-10a. In addition, Chromatin Immunoprecipitation (ChIP) Assay and JASPAR databases were employed to explore the regulatory relationship between TCF21 and miR-10a-5p.ResultsWe discovered that miR-10a-5p expression was lower in melanoma cells and high expression of miR-10a-5p suppressed the proliferation, migration and invasion abilities of melanoma cells. We also discovered that miR-10a-5p targeted the LIN28B mRNA 3′UTR area and diminished LIN28B protein expression. We found that LIN28B expression was strongly decreased by TCF21 upregulation in the two melanoma cells. The qRT-PCR assay showed that miR-10a-5p expression level was obviously boosted by increased TCF21 expression. The results also demonstrated that TCF21 directly regulated miR-10a-5p at transcript levels.ConclusionTCF21 induced miRNA-10a targeting LIN28B could affect the progression and growth of melanoma.

DUXAP8 knockdown inhibits the development of melanoma by regulating the miR-3182/NUPR1 pathway.

Double homeobox A pseudogene 8 (DUXAP8) has been reported to regulate the growth of several types of cancers, such as breast cancer and ovarian cancer. However, its role in melanoma remains unclear. In the present study, the mechanism through which DUXAP8 regulates melanoma progression was explored. The expression levels of DUXAP8 were determined in 43 samples from patients with melanoma in different stages, as well as human epidermal melanocytes cells and malignant melanoma cell lines using reverse transcription-quantitative PCR (RT-qPCR). The prognosis of patients was analyzed using the Kaplan-Meier method. The relationship between lncRNA DUXAP8 expression and microRNA (miR)-3182 or nuclear protein 1 transcriptional regulator (NUPR1) levels was analyzed using Pearson's correlation. Luciferase reporter and RNA pull-down were used to examine the interactions between these molecules. Proliferation was assessed using Cell Counting-Kit-8. Transwell assays were used to examine cell migration and invasion. lncRNA DUXAP8 was upregulated in melanoma tissue and cells compared with normal tissues and cells. The levels of DUXAP8 inversely correlated with survival time of patients with melanoma. Knockdown of lncRNA DUXAP8 inhibited proliferation, migration and invasion of melanoma cells. lncRNA DUXAP8 targeted miR-3182, while miR-3182 targeted NUPR1. The overexpression of NUPR1 reversed the effects of DUXAP8 knockdown or miR-3182 mimic on melanoma progression. In conclusion, lncRNA DUXAP8 downregulation inhibits the development of melanoma by regulating the miR-3182/NUPR1 axis.

Oxyresveratrol-induced Activation of Nrf2/HO-1 Signaling Pathway Enhances Ability of Resveratrol to Inhibit UVB-induced Melanin

Abstract Objective: This study was performed to investigate the relationship between the human melanogenesis and antioxidant systems and to further confirm the synergistic effect of oxyresveratrol (OXYR) and resveratrol (RES) in human epidermal melanocyte cell line. Methods: The human epidermal melanocyte line PIG1 cells were divided into the UV groups and control group, treated with different doses of UVB and without UVB, respectively. MTT assay and flow cytometry were used to detect cell viability and apoptosis. The expression of Nrf2/HO-1 and melanogenesis-associated proteins/genes was measured by Western blotting and real-time qPCR (RT-qPCR). pCMV6-XL5-Nrf2 was used to upregulate the expression of Nrf2. Subsequently, the proteins/genes levels of Nrf2/HO-1 and tyrosinase (TYR), melanin/eumelanin content, and reactive oxygen species (ROS) were analyzed. Isobologram analysis and cell experiment were used to analyze whether OXYR and RES inhibit TYR synergistically. Western blotting, RT-qPCR, and NaOH splitting method were used to determine the Nrf2/HO-1 and melanogenesis-associated proteins/genes expression and melanin content to evaluate the efficacy of OXYR and RES. Results: The activated Nrf2 and HO-1 eliminated ROS produced by UVB irradiation. The melanogenesis-associated proteins/genes of melanocyte-inducing transcription factor (MITF, P < 0.01 on protein expression), TYR (both P < 0.01), TYR-related protein (TRP)-1 (both P < 0.05), and TRP2 (P < 0.05 on mRNA expression) were activated in PIG1 cells by UVB irradiation. Simultaneously, the upregulation of Nrf2 significantly reduced melanogenesis formation (P < 0.001) and TYR level (P < 0.01 on protein expression). Moreover, OXYR and RES synergistically inhibited TYR activity (P < 0.001) and reduced melanin content (P < 0.001). Conclusions: A microbalance exists between Nrf2/HO-1 signaling and melanogenesis production in the UVB-induced responses of melanocytes. Simultaneously, OXYR enhances the ability of RES to inhibit melanin production.

MiR-143 acts as an inhibitor of migration and proliferation as well as an inducer of apoptosis in melanoma cancer cells in vitro.

Melanoma is a serious form of skin cancers begins in the melanocyte. Micro-RNAs are small noncoding RNA with 19 to 25 nucleotides in length involves in the regulation of a wide range of biological processes. MicroRNAs are affected by an aberrant epigenetic alteration in the tumors that may lead to their dysregulation and formation of cancer. Recently, dysregulation of numerous microRNAs has been reported in different types of cancer. The present study focused on the role of miR-143 in carcinogenesis of melanoma cancer. Here, we evaluated the expression level of miR-143 in three melanoma cell lines in comparison with the normal human epidermal melanocyte cell line. Then, miR-143 gene plasmid transfected into the WM115 cell line, for having the lowest expression of miR-143. In addition, the effect of miR-143 transfection on mRNA and protein levels of metastasis-related genes was performed along with MTT assay, wound healing assay, and flow cytometry. The results showed that mRNA and protein expression levels of metastasis-related genes including MMP-9, E-cadherin, Vimentin, and CXCR4 have been reduced following transfection of miR-143. Moreover, the results of the scratch test showed that miR-143 re-expression inhibited cell migration. Also, the role of miR-143 in the induction of apoptosis and inhibition of proliferation by flow cytometry and MTT was confirmed. As a result, the present study showed that miR-143 was involved in metastatic and apoptotic pathways, suggesting that miR-143 acts as a tumor-suppressor microRNA in melanoma cancer.

MUC4 isoforms expression profiling and prognosis value in Chinese melanoma patients.

Mucin 4 (MUC4), a type I membrane-bound mucin, blocks apoptosis, promotes invasion, proliferation and migration and causes chemo-resistance in epithelial cancers. However, the expression profiling and clinical implications of MUC4 alternative splicing during cancer pathogenesis, including melanoma, remain obscure. We examined the mRNA expression profiling of MUC4 isoforms in gastrointestinal cancer cell lines, melanoma cell lines, human epidermal melanocyte cells, as well as 138 cases of human melanoma tissues by RT-qPCR. Then we analyzed the relationship of mRNA expression of MUC4 isoforms to clinicopathological characteristics and survival of patients. The dynamic mRNA expression profiling of MUC4 isoforms was found in melanoma. We identified MUC4 isoform f was highly expressed in melanoma cell lines but negative in gastrointestinal cancer cell lines. Clinical analysis based on 138 cases of human melanomas showed that MUC4 isoform d was related with melanoma subtypes (p = 0.028) and TNM stage (p = 0.036). MUC4 isoform e was related with tumor thickness (p = 0.004) and T stage (p = 0.036). The Kaplan-Meier assay showed that the median overall survival (OS) for patients with MUC4 isoform f high expression was significantly shorter than that of patients with low expression (p = 0.024). And the median PFS of the patients with high expression of MUC4 isoform d or e was significantly shorter than that of with low expression (p = 0.012 and 0.035, respectively). Multivariate analysis indicated that high level of MUC4 isoform f was an independent prognostic factor for OS, and MUC4 isoform d was an independent prognostic factor for PFS of patients treated with chemotherapy. In conclusion, our results indicate that the dynamic MUC4 isoforms expressed in melanoma, and MUC4 isoform d and f might be served as a novel prognostic indicator of melanoma patients.

Melanogenesis effects of rice protein hydrolysate and its characteristic peptides Leu-Leu-Lys, Leu-Pro-Lys, and pyroGlu-Lys on UVB-induced human epidermal melanocyte cells.

This study assessed the melanogenesis effects of rice protein hydrolysate (RPH) and explored the underlying molecular mechanism of its characteristic peptides. In this investigation, human epidermal melanocyte (PIG1) cells were used to establish a UVB-induced model to evaluate the effect of RPH on melanin content, tyrosinase activity, and reactive oxygen species (ROS) levels. High performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was employed to identify the peptide composition (2-4 amino acids) in RPH. Enzymatic hydrolysis was employed to screen the characteristic peptides Leu-Leu-Lys (LLK), Leu-Pro-Lys (LPK), and pyroGlu-Lys (pEK), while their effect on the molecular mechanism involved in the melanin synthesis process was further explored using quantitative real-time polymerase chain reaction (PCR) and western blotting. The results indicated that RPH reduced the melanin content, tyrosinase activity, and ROS production in PIG1 cells. The selected peptides LLK, LPK, and pEK from RPH reduced the expression of tyrosinase-related protein 1 (TRP-1) and tyrosinase-related protein 2 (TRP-2) and affected melanin synthesis by regulating the JNK/β-Trcp/NFκB-p65/MITF signaling pathway at the mRNA and protein levels. This study shows that RPH plays a vital role in the melanogenesis process, therefore, providing a theoretical basis for the use of RPH as a novel additive product.

829 Folic acid protects melanocytes from oxidative damage by Keap1-Nrf2 pathway

This study aims to investigate whether folic acid could protect human melanocyte against oxidative damage and to elucidate the underlying pharmacological mechanism. And we demonstrate that folic acid could protect melanocytes form oxidative damage. Folic acid is of great therapeutic potential in the treatment of vitiligo. Vitiligo is a common skin disease characterized by the loss of functional melanocytes. Previous studies have indicated that oxidative stress plays a pivotal role in the onset of vitiligo. Thus, antioxidant therapy is a promising therapeutic strategy to prevent or even reverse the progression of depigmentation. Folic acid is a conventional antioxidant, but whether folic acid can be used to treat vitiligo is unclear. PIG1 cells that are an immortalized human epidermal melanocyte cell line were cultured in Medium 254. H2O2 at 800 μM was chosen to induce oxidative stress. CCK8 assay was used to estimate the cell survival rate. Flow cytometry was used to measure the intracellular ROS level and apoptosis rate. The nuclear translocation of Nrf2 was tested by immunofluorescence. Western blot and qRT-PCR were used to analyze the mRNA and protein expression levels of Keap1, Nrf2, HO-1, SOD2 and NQO1. We initially found that folic acid was able to ameliorate H2O2-induced oxidative damage in human melanocytes. In parallel, folic acid lessened the accumulation of intracellular ROS by potentiating the activity of antioxidant enzymes. Furthermore, folic acid protected melanocyte against oxidative stress by activating Nrf2 and its downstream genes HO-1, SOD2 and NQO1, and interfering with Nrf2 diminished the protection of folic acid against H2O2-induced apoptosis. At last, the down-regulation of Keap1 contributed to the activation of Nrf2 induced by folic acid.

The Inhibition of Melanogenesis Via the PKA and ERK Signaling Pathways by Chlamydomonas reinhardtii Extract in B16F10 Melanoma Cells and Artificial Human Skin Equivalents.

Abnormal melanin synthesis results in several hyperpigmentary disorders such as freckles, melanoderma, age spots, and other related conditions. In this study, we investigated the antimelanogenic effects of an extract from the microalgae Chlamydomonas reinhardtii (CE) and potential mechanisms responsible for its inhibitory effect in B16F10, normal human epidermal melanocyte cells, and human skin-equivalent models. The CE extract showed significant dose-dependent inhibitory effects on α-melanocyte-stimulating, hormone-induced melanin synthesis in cells. Additionally, the CE extract exhibited suppressive effects on the mRNA and protein expression of microphthalmia-associated transcription factor, tyrosinase, tyrosinase-related protein-1, and tyrosinase-related protein-2. The CE extract also inhibited the phosphorylation of protein kinase A and extracellular signal-related kinase, which function as upstream regulators of melanogenesis. Using a three-dimensional, reconstructed pigmented epidermis model, the CE-mediated, anti-pigmentation effects were confirmed by Fontana-Masson staining and melanin content assays. Taken together, CE extract can be used as an anti-pigmentation agent.

Inhibition of melanogenesis by Gaillardia aristata flower extract

The purpose of the study was to determine the anti-melanogenic and anti-oxidant properties of Gaillardia aristata flower extract (GAE). Melanogenesis inhibition by GAE was investigated in cultivated cells and in a human skin model. In cultivated cells, the melanogenesis regulatory effect of GAE was evaluated using melanin content, intracellular tyrosinase activity and anti-oxidant characteristics. In addition, the expression of melanogenesis-related proteins was determined by Western blotting and real-time PCR. GAE reduced the amount of melanin in B16F 10 and normal human epidermal melanocyte cells and suppressed intracellular tyrosinase activity in a dose-dependent pattern. Also, GAE significantly decreased the expression of melanogenesis-related proteins (microphthalmia associated transcription factor, tyrosinase, tyrosinase- related protein-1, and dopachrome tautomerase). Real-time PCR results revealed a down-regulation of the mRNAs of these proteins. GAE possessed anti-oxidant characteristics as free radical-scavenging capacity and reducing power. In the three-dimensional human skin model, GAE applied to hyperpigmented skin significantly increased the degree of skin lightening within 2 weeks of treatment. The safety of GAE on human skin was confirmed. The results indicate the potential of GAE for use in suppressing skin pigmentation.

Inhibition of melanogenesis by Gaillardia aristata flower extract.

BackgroundThe purpose of the study was to determine the anti-melanogenic and anti-oxidant properties of Gaillardia aristata flower extract (GAE).MethodsMelanogenesis inhibition by GAE was investigated in cultivated cells and in a human skin model. In cultivated cells, the melanogenesis regulatory effect of GAE was evaluated using melanin content, intracellular tyrosinase activity and anti-oxidant characteristics. In addition, the expression of melanogenesis-related proteins was determined by western blot assay and real-time PCR.ResultsGAE reduced the amount of melanin in B16F10 and normal human epidermal melanocyte cells and suppressed intracellular tyrosinase activity in a dose-dependent pattern. Also, GAE significantly decreased the expression of melanogenesis-related proteins (microphthalmia associated transcription factor, tyrosinase, tyrosinase-related protein-1, and dopachrome tautomerase). Real-time PCR results revealed a down-regulation of the mRNAs of these proteins. GAE possessed anti-oxidant characteristics as free radical-scavenging capacity and reducing power. In the three-dimensional human skin model, GAE applied to hyperpigmented skin significantly increased the degree of skin lightening within 2 weeks of treatment. The safety of GAE on human skin was confirmed.ConclusionsThese results indicate the potential of GAE for use in suppressing skin pigmentation. We proposed GAE as a new candidate of anti-melanogenic and antioxidant agents that could be used for cosmetic skin care products.

Regulation of type I-interferon responses in the human epidermal melanocyte cell line SKMEL infected by the Ross River alphavirus

Melanocytes are melanin-producing cells and with emerging innate immune functions including the expression of antiviral interferon-type I cytokines. We herein ascertained the susceptibility of the human melanocytes to Ross River alphavirus (RRV) infection and analyzed the subsequent immune responses. We demonstrated for the first time that (1) SKMEL-28 melanocyte cell line was susceptible to RRV infection and displaying major cytopathic activities and (2) RRV interfered with the interferon-type I response by altering nuclear translocation of pSTAT1 and pSTAT2 in infected SKMEL-28. These results suggest that the human melanoma cell line SKMEL-28 is a valuable model to analyze the mechanisms involved in severe skin manifestations and melanocyte’s immunity at the portal of entry of major infection by arboviruses.

Effects of Camellia reticulata, Chloranthus japonicas, Dodonaea viscosa and Paris polyphylla on the proliferation of and tyrosinase activity in a melanocyte cell line

Objective To estimate the whitening effect of extracts of Camellia reticulata, Chloranthus japonicas, Dodonaea viscosa and Paris polyphylla. Methods A human epidermal melanocyte cell line HEM-m was cultured in vitro, and classified into several groups to be treated with 7 concentrations（10, 25, 50, 100, 200, 400, 800 mg/L）of different plant extracts, including Camellia reticulata leaf extract, 70% ethanol-eluted fraction of Camellia reticulata leaf extract, extract of Camellia reticulata alabastrum, 70% ethanol-eluted fraction of extract of Camellia reticulata alabastrum, Chloranthus japonicas extract, Dodonaea viscosa extract, and Paris polyphylla extract. At the same time, the negative control group, blank control group and positive control group（treated with 100 mg/L arbutin）were set up. After 72 hours of treatment, MTT assay was performed to detect cell proliferation, and dopa-oxidation assay to estimate tyrosinase activity in melanocytes. Results As far as the inhibitory effect on the proliferation of HEM-m cells was concerned, 400 mg/L Camellia reticulata leaf extract, 70% ethanol-eluted fraction of Camellia reticulata leaf extract at 10-100 mg/L, extract of Camellia reticulata alabastrum at 10-25 mg/L, 70% ethanol-eluted fraction of extract of Camellia reticulata alabastrum at 10 mg/L and Dodonaea viscosa extract at 10-25 mg/L were equivalent to（all P > 0.05）, 800 mg/L Camellia reticulata leaf extract was significantly weaker than（P 0.05）, stronger than 70% ethanol-eluted fraction of extract of Camellia reticulata alabastrum at 10-25 mg/L（P < 0.05）, but weaker than the other concentrations of these plant extracts（P < 0.05 or 0.01）. Conclusion Special concentrations of Camellia reticulate and Dodonaea viscose can inhibit tyrosinase activity. Key words: Plant extracts; Melanocytes; Cell proliferation; Tyrosinase

Endocannabinoids Stimulate Human Melanogenesis via Type-1 Cannabinoid Receptor

We show that a fully functional endocannabinoid system is present in primary human melanocytes (normal human epidermal melanocyte cells), including anandamide (AEA), 2-arachidonoylglycerol, the respective target receptors (CB(1), CB(2), and TRPV1), and their metabolic enzymes. We also show that at higher concentrations AEA induces normal human epidermal melanocyte apoptosis (∼3-fold over controls at 5 μM) through a TRPV1-mediated pathway that increases DNA fragmentation and p53 expression. However, at lower concentrations, AEA and other CB(1)-binding endocannabinoids dose-dependently stimulate melanin synthesis and enhance tyrosinase gene expression and activity (∼3- and ∼2-fold over controls at 1 μM). This CB(1)-dependent activity was fully abolished by the selective CB(1) antagonist SR141716 or by RNA interference of the receptor. CB(1) signaling engaged p38 and p42/44 mitogen-activated protein kinases, which in turn activated the cyclic AMP response element-binding protein and the microphthalmia-associated transcription factor. Silencing of tyrosinase or microphthalmia-associated transcription factor further demonstrated the involvement of these proteins in AEA-induced melanogenesis. In addition, CB(1) activation did not engage the key regulator of skin pigmentation, cyclic AMP, showing a major difference compared with the regulation of melanogenesis by α-melanocyte-stimulating hormone through melanocortin 1 receptor.

Effect of Novel Marine Nutraceuticals on IL-1α-Mediated TNF-αRelease from UVB-Irradiated Human Melanocyte-Derived Cells

UV-induced inflammation and reactive oxygen species formation are involved in the development of melanoma. Natural products like 5β-scymnol and CO2-supercritical fluid extract (CO2-SFE) of mussel oil contain anti-inflammatory and antioxidant properties that may aid in reducing the deleterious effects of UV radiation. Therefore, their effect on the release of the proinflammatory cytokine, tumour necrosis factor-α (TNF-α), from UVB-irradiated human melanocytic cells was examined. Human epidermal melanocytes (HEM) and MM96L melanoma cells were exposed to UVB radiation and IL-1α. Cell viability and TNF-α levels were determined 24 hours after-irradiation while p38 mitogen-activated protein kinase (MAPK) activation was observed at 15 min after-irradiation. When α-tocopherol, CO2-SFE mussel oil, and 5β-scymnol were added to the UVB-irradiated HEM cells treated with IL-1α, TNF-α levels fell by 53%, 65%, and 76%, respectively, while no inhibition was evident in MM96L cells. This effect was not due to inhibition of the intracellular p38 MAPK signalling pathway. These compounds may be useful in preventing inflammation-induced damage to normal melanocytes.