NNT mediates redox-dependent pigmentation via a UVB- and MITF-independent mechanism.

Ultraviolet (UV) light and incompletely understood genetic and epigenetic variations determine skin color. Here we describe an UV- and microphthalmia-associated transcription factor (MITF)-independent mechanism of skin pigmentation. Targeting the mitochondrial redox-regulating enzyme nicotinamide nucleotide transhydrogenase (NNT) resulted in cellular redox changes that affect tyrosinase degradation. These changes regulate melanosome maturation and, consequently, eumelanin levels and pigmentation. Topical application of small-molecule inhibitors yielded skin darkening in human skin, and mice with decreased NNT function displayed increased pigmentation. Additionally, genetic modification of NNT in zebrafish alters melanocytic pigmentation. Analysis of four diverse human cohorts revealed significant associations of skin color, tanning, and sun protection use with various single-nucleotide polymorphisms within NNT. NNT levels were independent of UVB irradiation and redox modulation. Individuals with postinflammatory hyperpigmentation or lentigines displayed decreased skin NNT levels, suggesting an NNT-driven, redox-dependent pigmentation mechanism that can be targeted with NNT-modifying topical drugs for medical and cosmetic purposes.

A UVB-responsive common variant at chromosome band 7p21.1 confers tanning response and melanoma risk via regulation of the aryl hydrocarbon receptor, AHR.

Genome-wide association studies (GWASs) have identified a melanoma-associated locus on chromosome band 7p21.1 with rs117132860 as the lead SNP and a secondary independent signal marked by rs73069846. rs117132860 is also associated with tanning ability and cutaneous squamous cell carcinoma (cSCC). Because ultraviolet radiation (UVR) is a key environmental exposure for all three traits, we investigated the mechanisms by which this locus contributes to melanoma risk, focusing on cellular response to UVR. Fine-mapping of melanoma GWASs identified four independent sets of candidate causal variants. A GWAS region-focused Capture-C study of primary melanocytes identified physical interactions between two causal sets and the promoter of the aryl hydrocarbon receptor (AHR). Subsequent chromatin state annotation, eQTL, and luciferase assays identified rs117132860 as a functional variant and reinforced AHR as a likely causal gene. Because AHR plays critical roles in cellular response to dioxin and UVR, we explored links between this SNP and AHR expression after both 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and ultraviolet B (UVB) exposure. Allele-specific AHR binding to rs117132860-G was enhanced following both, consistent with predicted weakened AHR binding to the risk/poor-tanning rs117132860-A allele, and allele-preferential AHR expression driven from the protective rs117132860-G allele was observed following UVB exposure. Small deletions surrounding rs117132860 introduced via CRISPR abrogates AHR binding, reduces melanocyte cell growth, and prolongs growth arrest following UVB exposure. These data suggest AHR is a melanoma susceptibility gene at the 7p21.1 risk locus and rs117132860 is a functional variant within a UVB-responsive element, leading to allelic AHR expression and altering melanocyte growth phenotypes upon exposure.

Capsaicin combined with stem cells improved mitochondrial dysfunction in PIG3V cells, an immortalized human vitiligo melanocyte cell line, by inhibiting the HSP70/TLR4/mTOR/FAK signaling axis.

BACKGROUND: Vitiligo is a common autoimmune skin disease. Capsaicin has been found to exert a positive effect on vitiligo treatment, and mesenchymal stem cells (MSCs) are also confirmed to be an ideal cell type. This study aimed to explore the influence of capsaicin combined with stem cells on the treatment of vitiligo and to confirm the molecular mechanism of capsaicin combined with stem cells in treating vitiligo. METHODS AND RESULTS: PIG3V cell proliferation and apoptosis were detected using CCK-8 and TUNEL assays, MitoSOX Red fluorescence staining was used to measure the mitochondrial ROS level, and JC-1 staining was used to detect the mitochondrial membrane potential. The expression of related genes and proteins was detected using RT‒qPCR and Western blotting. Coimmunoprecipitation was used to analyze the protein interactions between HSP70 and TLR4 or between TLR4 and mTOR. The results showed higher expression of HSP70 in PIG3V cells than in PIG1 cells. The overexpression of HSP70 reduced the proliferation of PIG3V cells, promoted apoptosis, and aggravated mitochondrial dysfunction and autophagy abnormalities. The expression of HSP70 could be inhibited by capsaicin combined with MSCs, which increased the levels of Tyr, Tyrp1 and DCT, promoted the proliferation of PIG3V cells, inhibited apoptosis, activated autophagy, and improved mitochondrial dysfunction. In addition, capsaicin combined with MSCs regulated the expression of TLR4 through HSP70 and subsequently affected the mTOR/FAK signaling pathway CONCLUSIONS: Capsaicin combined with MSCs inhibits TLR4 through HSP70, and the mTOR/FAK signaling pathway is inhibited to alleviate mitochondrial dysfunction and autophagy abnormalities in PIG3V cells.

Combination of 308-nm excimer laser and piperine promotes melanocyte proliferation, migration, and melanin content production via the miR-328/SFRP1 axis.

OBJECTIVE: Both piperine and a 308-nm excimer laser have significant curative effects on vitiligo. This study mainly explored the molecular mechanism of a 308-nm excimer combined with piperine in regulating melanocyte proliferation. METHODS: Epidermal melanocytes were cultured in piperine solution, and the cells were irradiated by an XTRAC excimer laser treatment system at 308-nm output monochromatic light. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were for detecting the expression levels of genes or proteins. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and Transwell method was for assessing cell viability and migration capacity. The content of melanin was also detected. RESULTS: The combination of the 308-nm excimer laser and piperine enhanced the cell proliferation, migration, and melanin production of melanocytes and upregulated the level of miR-328, and restraint of miR-328 reversed the influence of the 308-nm excimer laser and piperine. Secreted frizzled-related protein 1 (SFRP1) is a direct target gene of miR-328, and miR-328 can inhibit the expression of SFRP1 and elevate the protein level of the Wnt/ß-catenin signaling pathway. CONCLUSION: The 308-nm excimer laser combined with piperine may be more efficient than piperine alone in the remedy of vitiligo, and the miR-328/SFRP1 and Wnt/ß-catenin pathways are participated in the proliferation, migration, and melanin synthesis of melanocytes.

Exploring the Influence of Cold Plasma on Epidermal Melanogenesis In Situ and In Vitro.

Epidermal melanin synthesis determines an individual's skin color. In humans, melanin is formed by melanocytes within the epidermis. The process of melanin synthesis strongly depends on a range of cellular factors, including the fine-tuned interplay with reactive oxygen species (ROS). In this context, a role of cold atmospheric plasma (CAP) on melanin synthesis was proposed due to its tunable ROS generation. Herein, the argon-driven plasma jet kINPen® MED was employed, and its impact on melanin synthesis was evaluated by comparison with known stimulants such as the phosphodiesterase inhibitor IBMX and UV radiation. Different available model systems were employed, and the melanin content of both cultured human melanocytes (in vitro) and full-thickness human skin biopsies (in situ) were analyzed. A histochemical method detected melanin in skin tissue. Cellular melanin was measured by NIR autofluorescence using flow cytometry, and a highly sensitive HPLC-MS method was applied, which enabled the differentiation of eu- and pheomelanin by their degradation products. The melanin content in full-thickness human skin biopsies increased after repeated CAP exposure, while there were only minor effects in cultured melanocytes compared to UV radiation and IBMX treatment. Based on these findings, CAP does not appear to be a useful option for treating skin pigmentation disorders. On the other hand, the risk of hyperpigmentation as an adverse effect of CAP application for wound healing or other dermatological diseases seems to be neglectable.

α-Chitosan and ß-Oligochitosan Mixtures-Based Formula for In Vitro Assessment of Melanocyte Cells Response.

Chitosan is a natural polymer with numerous biomedical applications. The cellular activity of chitosan has been studied in various types of cancer, including melanoma, and indicates that these molecules can open new perspectives on antiproliferative action and anticancer therapy. This study analyzes how different chitosan conformations, such as α-chitosan (CH) or ß-oligochitosan (CO), with various degrees of deacetylation (DDA) and molar mass (MM), both in different concentrations and in CH-CO mixtures, influence the cellular processes of SK-MEL-28 melanocytes, to estimate the reactivity of these cells to the applied treatments. The in vitro evaluation was carried out, aiming at the cellular metabolism (MTT assay), cellular morphology, and chitinase-like glycoprotein YKL-40 expression. The in vitro effect of the CH-CO mixture application on melanocytes is obvious at low concentrations of α-chitosan/ß-oligochitosan (1:2 ratio), with the cell's response supporting the hypothesis that ß-oligo-chitosan amplifies the effect. This oligochitosan mixture, favored by the ß conformation and its small size, penetrates faster into the cells, being more reactive when interacting with some cellular components. Morphological effects expressed by the loss of cell adhesion and the depletion of YKL-40 synthesis are significant responses of melanocytes. ß-oligochitosan (1.5 kDa) induces an extension of cytophysiological effects and limits the cell viability compared to α-chitosan (400-900 kDa). Statistical analysis using multivariate techniques showed differences between the CH samples and CH-CO mixtures.

Laccase-Treated Polystyrene Surfaces with Caffeic Acid, Dopamine, and L-3,4-Dihydroxyphenylalanine Substrates Facilitate the Proliferation of Melanocytes and Embryonal Carcinoma Cells NTERA-2.

This study presents the effects of treating polystyrene (PS) cell culture plastic with oxidoreductase enzyme laccase and the catechol substrates caffeic acid (CA), L-DOPA, and dopamine on the culturing of normal human epidermal melanocytes (NHEMs) and human embryonal carcinoma cells (NTERA-2). The laccase-substrate treatment improved PS hydrophilicity and roughness, increasing NHEM and NTERA-2 adherence, proliferation, and NHEM melanogenesis to a level comparable with conventional plasma treatment. Cell adherence dynamics and proliferation were evaluated. The NHEM endpoint function was quantified by measuring melanin content. PS surfaces treated with laccase and its substrates demonstrated the forming of polymer-like structures. The surface texture roughness gradient and the peak curvature were higher on PS treated with a combination of laccase and substrates than laccase alone. The number of adherent NHEM and NTERA-2 was significantly higher than on the untreated surface. The proliferation of NHEM and NTERA-2 correspondingly increased on treated surfaces. NHEM melanin content was enhanced 6-10-fold on treated surfaces. In summary, laccase- and laccase-substrate-modified PS possess improved PS surface chemistry/hydrophilicity and altered roughness compared to untreated and plasma-treated surfaces, facilitating cellular adherence, subsequent proliferation, and exertion of the melanotic phenotype. The presented technology is easy to apply and creates a promising custom-made, substrate-based, cell-type-specific platform for both 2D and 3D cell culture.

Petanin Potentiated JNK Phosphorylation to Negatively Regulate the ERK/CREB/MITF Signaling Pathway for Anti-Melanogenesis in Zebrafish.

Petanin, an acylated anthocyanin from the Solanaceae family, shows potential in tyrosinase inhibitory activity and anti-melanogenic effects; however, its mechanism remains unclear. Therefore, to investigate the underlying mechanism of petanin's anti-melanogenic effects, the enzyme activity, protein expression and mRNA transcription of melanogenic and related signaling pathways in zebrafish using network pharmacology, molecular docking and molecular dynamics simulation were combined for analysis. The results showed that petanin could inhibit tyrosinase activity and melanogenesis, change the distribution and arrangement of melanocytes and the structure of melanosomes, reduce the activities of catalase (CAT) and peroxidase (POD) and enhance the activity of glutathione reductase (GR). It also up-regulated JNK phosphorylation, inhibited ERK/RSK phosphorylation and down-regulated CREB/MITF-related protein expression and mRNA transcription. These results were consistent with the predictions provided through network pharmacology and molecular docking. Thus, petanin could inhibit the activity of tyrosinase and the expression of tyrosinase by inhibiting and negatively regulating the tyrosinase-related signaling pathway ERK/CREB/MITF through p-JNK. In conclusion, petanin is a good tyrosinase inhibitor and anti-melanin natural compound with significant market prospects in melanogenesis-related diseases and skin whitening cosmetics.

Comparative Analysis of Olive-Derived Phenolic Compounds' Pro-Melanogenesis Effects on B16F10 Cells and Epidermal Human Melanocytes.

Olive leaf contains plenty of phenolic compounds, among which oleuropein (OP) is the main component and belongs to the group of secoiridoids. Additionally, phenolic compounds such as oleocanthal (OL) and oleacein (OC), which share a structural similarity with OP and two aldehyde groups, are also present in olive leaves. These compounds have been studied for several health benefits, such as anti-cancer and antioxidant effects. However, their impact on the skin remains unknown. Therefore, this study aims to compare the effects of these three compounds on melanogenesis using B16F10 cells and human epidermal cells. Thousands of gene expressions were measured by global gene expression profiling with B16F10 cells. We found that glutaraldehyde compounds derived from olive leaves have a potential effect on the activation of the melanogenesis pathway and inducing differentiation in B16F10 cells. Accordingly, the pro-melanogenesis effect was investigated by means of melanin quantification, mRNA, and protein expression using human epidermal melanocytes (HEM). This study suggests that secoiridoid and its derivates have an impact on skin protection by promoting melanin production in both human and mouse cell lines.

Study on the Skincare Effects of Red Rice Fermented by Aspergillus oryzae In Vitro.

Red rice, a variety of pigmented grain, serves dual purposes as both a food and medicinal resource. In recent years, we have witnessed an increasing interest in the dermatological benefits of fermented rice extracts, particularly their whitening and hydrating effects. However, data on the skincare advantages derived from fermenting red rice with Aspergillus oryzae remain sparse. This study utilized red rice as a substrate for fermentation by Aspergillus oryzae, producing a substance known as red rice Aspergillus oryzae fermentation (RRFA). We conducted a preliminary analysis of RRFA's composition followed by an evaluation of its skincare potential through various in vitro tests. Our objective was to develop a safe and highly effective skincare component for potential cosmetic applications. RRFA's constituents were assessed using high-performance liquid chromatography (HPLC), Kjeldahl nitrogen determination, the phenol-sulfuric acid method, and enzyme-linked immunosorbent assay (ELISA). We employed human dermal fibroblasts (FB) to assess RRFA's anti-aging and antioxidative properties, immortalized keratinocytes (HaCaT cells) and 3D epidermal models to examine its moisturizing and reparative capabilities, and human primary melanocytes (MCs) to study its effects on skin lightening. Our findings revealed that RRFA encompasses several bioactive compounds beneficial for skin health. RRFA can significantly promote the proliferation of FB cells. And it markedly enhances the mRNA expression of ECM-related anti-aging genes and reduces reactive oxygen species production. Furthermore, RRFA significantly boosts the expression of Aquaporin 3 (AQP3), Filaggrin (FLG), and Hyaluronan Synthase 1 (HAS1) mRNA, alongside elevating moisture levels in a 3D epidermal model. Increases were also observed in the mRNA expression of Claudin 1 (CLDN1), Involucrin (IVL), and Zonula Occludens-1 (ZO-1) in keratinocytes. Additionally, RRFA demonstrated an inhibitory effect on melanin synthesis. Collectively, RRFA contains diverse ingredients which are beneficial for skin health and showcases multifaceted skincare effects in terms of anti-aging, antioxidant, moisturizing, repairing, and whitening capabilities in vitro, highlighting its potential for future cosmetic applications.

Adipose tissue-derived Muse cells promote autophagy and oxidative stress tolerance in human epidermal melanocytes.

To investigate the effect of human adipose tissue-derived multilineage-differentiating stress-enduring (Muse) cells on the oxidative stress injury of human epidermal melanocytes (HEMs) in vitro. HEMs were treated with H2O2 to establish an oxidative stress injury model and then were co-cultured with adipose tissue-derived Muse cells. Immunohistochemistry, flow cytometry and Western blotting were used to assess changes in autophagy flux, apoptosis, expression of melanin synthesis related proteins and proliferation of melanocytes. Our findings demonstrate that co-culture with Muse cells significantly increased the tolerance of HEMs to oxidative stress, enhanced autophagy flux and reduced apoptosis. The expression of proteins related to the formation of melanin increased as did cell proliferation. Treatment with the autophagy inhibitor, 3-methyladenine (3MA), partially counteracted the improvement of oxidative stress tolerance in melanocytes elicited by co-culture with Muse cells. Muse cells promote autophagy and oxidative stress tolerance of melanocytes.

Neurog1-Derived Peptides RMNE1 and DualPep-Shine Penetrate the Skin and Inhibit Melanin Synthesis by Regulating MITF Transcription.

Anti-pigmentation peptides have been developed as alternative skin-lightening agents to replace conventional chemicals that have adverse effects on the skin. However, the maximum size of these peptides is often limited by their low skin and cell penetration. To address this issue, we used our intra-dermal delivery technology (IDDT) platform to identify peptides with hypo-pigmenting and high cell-penetrating activity. Using our cell-penetrating peptides (CPPs) from the IDDT platform, we identified RMNE1 and its derivative RMNE3, "DualPep-Shine", which showed levels of α-Melanocyte stimulating hormone (α-MSH)-induced melanin inhibition comparable to the conventional tyrosinase inhibitor, Kojic acid. In addition, DualPep-Shine was delivered into the nucleus and regulated the gene expression levels of melanogenic enzymes by inhibiting the promoter activity of microphthalmia-associated transcription factor-M (MITF-M). Using a 3D human skin model, we found that DualPep-Shine penetrated the lower region of the epidermis and reduced the melanin content in a dose-dependent manner. Furthermore, DualPep-Shine showed high safety with little immunogenicity, indicating its potential as a novel cosmeceutical ingredient and anti-pigmentation therapeutic agent.

Toll-like receptor 2 and 6 agonist fibroblast-stimulating lipopeptide increases expression and secretion of CXCL1 and CXCL2 by uveal melanocytes.

Fibroblast-stimulating lipopeptide (FSL-1) can activate Toll-like receptor 2 and 6 (TLR2/6), which recognize relevant molecules from gram-positive pathogens, fungus, and mycoplasma, and elevates the expression of CXCL1 and CXCL2, neutrophil chemoattractants, in certain types of cells. This effect has not previously been reported in the uveal melanocytes (UM). This study was designed to test the hypothesis that FSL-1 can induce the expression and secretion of CXCL1 and CXCL2 via activation of TLR2/6 in cultured human UM and producing an acute non-infectious uveitis reaction in the mouse. Flow cytometry and fluorescent immunostaining were used to measure the effect of FSL-1 on the expression of TLR2/6 in UM. Real time PCR and ELISA analysis were used to assess the ability of FSL-1 to elevate CXCL1/CXCL2 levels in cell lysates and conditioned media of UM, respectively. Flow cytometry measured phosphorylated MAPK and activated NF-κB signals in UM, with and without FSL-1 treatment. ELISA analysis tested the impact of various signal inhibitors (NF-κB, p38 MAPK, JNK1/2 and ERK1/2) and TLR2/6 antagonists on FSL-1-induced CXCL1/CXCL2 levels in cultured UM. The effects of neutralizing antibodies to TLR2 on FSL-1-induced mouse uveitis were tested in an experimental animal model. FSL-1 induced the expression of TLR2/6 proteins in cultured UM. FSL-1 significantly elevated the CXCL1 and CXCL2 proteins and mRNA levels in cultured UM time- and dose-dependently. FSL-1 mainly activated NF-κB, JNK, and expression of TLR2. FSL-1-induced expression of CXCL1 and CXCL2 was blocked by NF-κB, JNK, ERK inhibitors and TLR2 antagonists. Intravitreal injection of FSL-1 induced acute non-infectious mouse uveitis, which was significantly reduced in severity by a TLR2 antagonist. These results suggest that UM may play a role in the immune reaction, which targets invading pathogens, especially gram-positive bacteria. On the other hand, an excessive reaction to molecules from gram-positive bacteria may promote an inflammatory state of non-infectious uveitis.

The combination of sucrose dilaurate and sucrose laurate suppresses HMGB1: an enhancer of melanocyte dendricity and melanosome transfer to keratinocytes.

BACKGROUND: Hyperpigmented spots are common issues in all ethnicities, involving multiple intrinsic and extrinsic factors such as UVB exposure, hormone balance, inflammatory status and ageing. OBJECTIVES: To determine (i) melanocyte dendricity in multiple facial spot types, (ii) impact of High Mobility Group Box 1 (HMGB1), and the combination of sucrose dilaurate and sucrose laurate (SDL) on melanogenesis and melanocyte dendricity, and (iii) SDL effect on facial spots in a human use test. METHODS: Facial spot and adjacent non-spot skin biopsies were collected from Chinese women (age 20-70). Histological assessment of melanocyte dendricity was performed for 3 spot types (solar lentigo, melasma and postinflammatory hyperpigmentation) by immunofluorescent staining for c-kit/MITF. Keratinocyte, melanocyte and melanocyte-keratinocyte co-culture models were used to assess HMGB1 release by UVB radiation, the effects of HMGB1 and SDL on melanin production, melanocyte dendricity and melanosome transfer. The effect of an SDL-containing moisturizer on appearance of facial hyperpigmented spots was assessed against a vehicle control in an 8-week human use test. RESULTS: Melanocytes in spot areas are more dendritic than melanocytes in adjacent non-spot skin across three investigated spot types. In cell culture models, a moderate UVB-radiation exposure caused release of HMGB1 from keratinocytes. HMGB1 did not alter melanin production in melanocytes, but enhanced melanocyte dendricity and melanosome transfer. SDL reduced HMGB1 release from keratinocytes, inhibited melanin production, reversibly suppressed melanocyte dendricity and reduced melanosome transfer. In the human use test, SDL-containing moisturizer reduced appearance of spots versus vehicle. CONCLUSION: Increased melanocyte dendricity was observed in multiple types of facial spots. Addition of HMGB1 protein increased melanocyte dendricity and melanosome transfer in cell cultures, implicating potential involvement in spot formation. SDL suppressed melanin production, melanocyte dendricity and melanosome transfer in vitro and reduced appearance of spots in the use test, suggesting SDL is an effective solution to address hyperpigmented spot concerns.

Deoxyvasicinone with Anti-Melanogenic Activity from Marine-Derived Streptomyces sp. CNQ-617.

The tricyclic quinazoline alkaloid deoxyvasicinone (DOV, 1) was isolated from a marine-derived Streptomyces sp. CNQ-617, and its anti-melanogenic effects were investigated. Deoxyvasicinone was shown to decrease the melanin content of B16F10 and MNT-1 cells that have been stimulated by α-melanocyte-stimulating hormone (α-MSH). In addition, microscopic images of the cells showed that deoxyvasicinone attenuated melanocyte activation. Although, deoxyvasicinone did not directly inhibit tyrosinase (TYR) enzymatic activity, real-time PCR showed that it inhibited the mRNA expression of TYR, tyrosinase-related protein 1 (TRP-1), and tyrosinase-related protein 2 (TRP-2). In the artificial 3D pigmented skin model MelanodermTM, deoxyvasicinone brightened the skin significantly, as confirmed by histological examination. In conclusion, this study demonstrated that the marine microbial natural product deoxyvascinone has an anti-melanogenic effect through downregulation of melanogenic enzymes.

Thioxothiazolidin derivative, 4-OST, inhibits melanogenesis by enhancing the specific recruitment of tyrosinase-containing vesicles to lysosome.

Tyrosinase catalyzes the rate-limiting step in melanin synthesis. Melanin is synthesized from l-tyrosin in the melanosomes, where tyrosinase and other melanogenic factors are recruited via the vesicle transport system. Genetic and biochemical approaches have revealed a correlation between impairments in the vesicle transport system and albinism. However, the specificity of the individual transport systems for the corresponding melanogenic factors has not been well elucidated yet. Here, we report that the thioxothiazolidin derivative, 4-OST (4-[(5E)-5-[(4-fluorophenyl)methylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]-4-azatricyclo [5.2.1.02 ,6]dec-8-ene-3,5-dione: CAS RN. 477766-87-3) strongly inhibited melanogenesis in mouse melanoma B16F10 cells. 4-OST reduces tyrosinase protein levels without affecting its messenger RNA levels or enzymatic activity. Although a reduction in tyrosinase protein level was observed in the presence of a protein synthesis inhibitor, the reduction may be coupled with protein synthesis. Similarly, GIF-2202 (a derivative of 4-OST) lowers tyrosinase protein levels without affecting the levels of another melanogenic enzyme, tyrosinase-related protein 1 (TYRP1) level. The reduction in tyrosinase protein level is associated with an increase in the levels of the lysosomal proteinase cathepsin S. Chloroquine, a lysosome inhibitor, restored the tyrosinase protein level downregulated by GIF-2202, although no effects of other inhibitors (against proteasome, autophagy, or exocytosis) were observed. In addition, GIF-2202 segregated the immunofluorescence signals of tyrosinase from those of TYRP1. Chloroquine treatment resulted in co-localization of tyrosinase and cathepsin S signals near the perinuclear region, suggesting that 4-OST and GIF-2202 may alter the destination of the tyrosinase vesicle from the melanosome to the lysosome. 4-OST and GIF-2202 can be new tools for studying the tyrosinase-specific vesicle transport system.

Loss of Melanopsin (OPN4) Leads to a Faster Cell Cycle Progression and Growth in Murine Melanocytes.

Skin melanocytes harbor a complex photosensitive system comprised of opsins, which were shown, in recent years, to display light- and thermo-independent functions. Based on this premise, we investigated whether melanopsin, OPN4, displays such a role in normal melanocytes. In this study, we found that murine Opn4KO melanocytes displayed a faster proliferation rate compared to Opn4WT melanocytes. Cell cycle population analysis demonstrated that OPN4KO melanocytes exhibited a faster cell cycle progression with reduced G0-G1, and highly increased S and slightly increased G2/M cell populations compared to the Opn4WT counterparts. Expression of specific cell cycle-related genes in Opn4KO melanocytes exhibited alterations that corroborate a faster cell cycle progression. We also found significant modification in gene and protein expression levels of important regulators of melanocyte physiology. PER1 protein level was higher while BMAL1 and REV-ERBα decreased in Opn4KO melanocytes compared to Opn4WT cells. Interestingly, the gene expression of microphthalmia-associated transcription factor (MITF) was upregulated in Opn4KO melanocytes, which is in line with a higher proliferative capability. Taken altogether, we demonstrated that OPN4 regulates cell proliferation, cell cycle, and affects the expression of several important factors of the melanocyte physiology; thus, arguing for a putative tumor suppression role in melanocytes.

Isolindleyin exerts anti-melanogenic effects in human epidermal melanocytes via direct binding to tyrosinase.

To overcome dermatological concerns causing abnormally excessive melanin synthesis, highly effective and safe skin depigmentation compounds have been identified in the cosmetic and pharmaceutical industries. Among several methods used to achieve skin depigmentation, inhibition of tyrosinase is one of the most effective, since tyrosinase is a crucial enzyme in melanogenesis. Herein, isolindleyin, a novel inhibitor of human tyrosinase, was introduced and evaluated for its anti-melanogenic effects in human epidermal melanocytes. The results revealed that isolindleyin was directly bound to tyrosinase and it suppressed melanin synthesis. The binding mode between isolindleyin and the active sites of human tyrosinase was investigated using computational molecular docking at the atomic level. Isolindleyin binding was found to be stabilized by hydrophobic interactions between His 367 and Val 377 and by hydrogen bonds between Ser 380 and Asn 364. The results of this study revealed the anti-melanogenic effects of isolindleyin that could contribute toward overcoming dermatological concerns that cause abnormally excessive melanin synthesis.

Expression of DNA Methyltransferase 1 Is a Hallmark of Melanoma, Correlating with Proliferation and Response to B-Raf and Mitogen-Activated Protein Kinase Inhibition in Melanocytic Tumors.

Aberrant DNA methylation is an epigenetic hallmark of melanoma, but the expression of DNA methyltransferase (Dnmt)-1 in melanocytic tumors is unknown. Dnmt1 expression was analyzed in primary melanocytes, melanoma cell lines, and 83 melanocytic tumors, and its associations with proliferation, mutational status, and response to B-Raf and mitogen-activated protein kinase kinase (MEK) inhibition were explored. Dnmt1 expression was increased incrementally from nevi [mean fluorescence intensity (MFI), 48.1; interquartile range, 41.7 to 59.6] to primary melanomas (MFI, 68.8; interquartile range, 58.4 to 77.0) and metastatic melanomas (MFI, 87.5; interquartile range, 77.1 to 114.5) (P < 0.001). Dnmt1 expression was correlated with Ki-67 expression (Spearman correlation, 0.483; P < 0.001) and was independent of BRAF mutation status (P = 0.55). In BRAF-mutant melanoma, Dnmt1 was down-regulated during response to B-Raf and MEK inhibition and was again up-regulated on drug resistance in vitro and in vivo. Degradation of Dnmt1 by the histone deacetylase inhibitor suberoylanilide hydroxamic acid was associated with decreased cell viability in B-Raf inhibitor-sensitive and -resistant cell lines. This study demonstrates that Dnmt1 expression is correlated with proliferation in melanocytic tumors, is increased with melanoma progression, and is associated with response to B-Raf and MEK inhibition. Given its strong expression in metastatic melanoma, Dnmt1 may be a promising target for combined epigenetic and immunotherapy.

Antioxidant and anti-aging carbon quantum dots using tannic acid.

Overexpression of collagenase, elastase, and tyrosinase is caused by external factors such as ultraviolet (UV) radiation and stress, resulting in wrinkle formation and freckles through the loss of skin elasticity and skin pigmentation. In this study, we developed novel carbon quantum dots (CQDs) with antioxidant and anti-aging properties using tannic acid as a carbon source through a simple microwave-assisted pyrolysis method. The synthesized tannic acid-derived CQDs (T-CQDs) showed bright blue fluorescence (QY = 28.2 ± 4.0%), exhibiting maximum emission at 430 nm under 350 nm excitation. Even though small amount of the T-CQDs (3µg ml-1) was used, they exhibited excellent free radical scavenging ability (82.8 ± 4.3%). Also, the T-CQDs (10µg ml-1) revealed remarkable inhibitory activity against skin aging-related collagenase (77.6 ± 4.8%), elastase (52.6 ± 1.0%), and tyrosinase (44.2 ± 1.3%), demonstrating their antioxidant and anti-aging effects. Furthermore, their antioxidant and anti-aging properties were superior to those of tannic acid, L-ascorbic acid, and quercetin used as positive controls. Finally, the T-CQDs effectively suppressed UV-induced reactive oxygen species generation by 30% at the cellular levels and showed high cell viability (99.7 ± 0.8%) even at 500µg ml-1. These results demonstrate that the T-CQDs with superior antioxidant, anti-aging properties, and low cytotoxicity can be utilized as novel anti-aging materials in cosmetic and nanomedicine fields.