Microphthalmia-associated Transcription Factor Expression Research Articles

Advantages of nanofibrous membranes for culturing of primary RPE cells compared to commercial scaffolds.

Dysfunction of the retinal pigment epithelium (RPE) causes numerous forms of retinal degeneration. RPE replacement is a modern option to save vision. We aimed to test the results of transplanting cultured RPEs on biocompatible membranes. We cultivated porcine primary RPE cells isolated from cadaver eyes from the slaughterhouse on two types of membranes: commercial polyester scaffolds Transwell (Corning Inc., Kenneburg, ME, USA) with 0.4 µm pore size and prepared Poly (L-lactide-co-DL-lactide) (PDLLA) nanofibrous membranes with an average pore size of 0.4 µm. Five types of assays were used for the analysis: immunocytochemistry (ICC), phagocytosis assay, Western blotting, real-time qPCR (RT-qPCR) and electron microscopy. RT-qPCR demonstrated that RPEs cultured on nanofibrous membranes have higher expressions of BEST1 (bestrophin 1), RLBP1 (retinaldehyde-binding protein 1), RPE65 (retinal pigment epithelium-specific 65 kDa protein), PAX6 (transcription factor PAX6), SOX9 (transcription factor SOX9), DCT (dopachrome tautomerase) and MITF (microphthalmia-associated transcription factor). ICC of the RPEs cultured on nanofibrous membranes showed more intensive staining of markers such as BEST1, MCT1 (monocarboxylate transporter 1), Na+ /K+ ATPase, RPE65 and acetylated tubulin in comparison with commercial ones. Additionally, the absence of α-SMA proved the stability of the RPE polarization state and the absence of epithelial-to-mesenchymal transition. RPE possessed high phagocytic activity. Electron microscopy of both membranes confirmed a confluent layer of RPE cells and their genuine morphological structure, which was comparable to native RPEs. Retinal pigment epitheliums cultured on polylactide nanofibrous membranes improved the final quality of the cell product by having better maturation and long-term survival of the RPE monolayer compared to those cultured on commercial polyester scaffolds. PDLLA-cultured RPEs are a plausible source for the replacement of non-functioning RPEs during cell therapy.

Wound Healing-Promoting and Melanogenesis-Inhibiting Activities of Angelica polymorpha Maxim. Flower Absolute In Vitro and Its Chemical Composition.

Angelica polymorpha Maxim. (APM) is used in traditional medicine to treat chronic gastritis, rheumatic pain, and duodenal bulbar ulcers. However, it is not known whether APM has epidermis-associated biological activities. Here, we investigated the effects of APM flower absolute (APMFAb) on responses associated with skin wound healing and whitening using epidermal cells. APMFAb was obtained by solvent extraction and its composition was analyzed by GC/MS. Water-soluble tetrazolium salt, 5-bromo-2′-deoxyuridine incorporation, Boyden chamber, sprouting, and enzyme-linked immunosorbent assays and immunoblotting were used to examine the effects of APMFAb on HaCaT keratinocytes and B16BL6 melanoma cells. APMFAb contained five compounds and induced keratinocyte migration, proliferation, and type IV collagen synthesis. APMFAb also induced the phosphorylations of ERK1/2, JNK, p38 mitogen-activated protein kinase, and AKT in keratinocytes. In addition, APMFAb decreased serum-induced B16BL6 cell proliferation and inhibited tyrosinase expression, melanin contents, and microphthalmia-associated transcription factor expression in α-melanocyte-stimulating hormone-stimulated B16BL6 cells. These findings demonstrate that APMFAb has beneficial effects on skin wound healing by promoting the proliferation, migration, and collagen synthesis of keratinocytes and on skin whitening by inhibiting melanin synthesis in melanoma cells. Therefore, we suggest that APMFAb has potential use as a wound healing and skin whitening agent.

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.

Kaempferol, the melanogenic component of Sanguisorba officinalis, enhances dendricity and melanosome maturation/transport in melanocytes

Kaempferol, a representative flavonoid constituent of Sanguisorba officinalis, promotes melanogenesis, but the underlying mechanisms remain unknown. Here, we evaluated the effects of kaempferol on melanocytes morphology and behavior and determined the mechanisms regulating kaempferol-induced pigmentation. We observed that kaempferol increased melanin contents and dendritic length and stimulated melanocyte migration both in vitro and vivo. It significantly enhanced the expression of microphthalmia-associated transcription factor (MITF) and downstream enzymes of melanin biosynthesis—tyrosinase (TYR), tyrosinase-related protein (TRP-1), and dopachrome tautomerase (DCT). It also induced melanosome maturation (increased stage III and IV melanosomes) and melanin transfer to dendritic tips; this was evidenced as follows: kaempferol-treated melanocytes exhibited the perimembranous accumulation of HMB45-positive melanosomes and increased the expression of Rab27A, RhoA, and Cdc42, which improved melanosome transport to perimembranous actin filaments. These results jointly indicated that kaempferol promotes melanogenesis and melanocyte growth. Additionally, kaempferol stimulated the phosphorylation of P38/ERK MAPK and downregulated p-PI3K, p-AKT, and p-P70s6K expression. Pre-incubation with P38 (SB203580) and ERK (PD98059) signaling inhibitors reversed the melanogenic and dendritic effects and MITF expression. PI3K/AKT inhibitor augmented kaempferol-induced melanin content and dendrite length. In summary, kaempferol regulated melanocytes’ dendritic growth and melanosome quantity, maturation, and transport via P38/ERK MAPK and PI3K/AKT signaling pathways.

Quantitative proteomic analysis uncovers inhibition of melanin synthesis by silk fibroin via MITF/tyrosinase axis in B16 melanoma cells

AimsSilk fibroin (SF), a natural product from silkworms, has been used to promote anti-inflammation, induce wound healing, and reduce melanin production. However, the underlying regulatory mechanism of SF on melanin production remains unknown. The aim of this study was to investigate the distinct regulatory mechanism of SF in B16 melanoma cells by applying quantitative proteomic approach. Materials and methodsB16 melanoma cells were treated with PBS, KA or SF for 48 h, respectively. Cell viability, melanin content, and tyrosinase activity were examined. A label-free quantitative proteomic approach was utilized to investigate the regulatory mechanism of SF. The differentially expressed proteins and their related biological processes were subsequently identified by bioinformatics methods. Furthermore, the identified differentially expressed proteins were validated by western blot. Key findingsBoth SF and KA were able to suppress the melanin synthesis of B16 melanoma cells without appreciable toxicity; yet, SF had a distinct effect on mushroom tyrosinase activity in vitro. Moreover, quantitative proteomic approach identified 141 proteins differentially expressed only in SF/Con group. Bioinformatic analysis of these proteins revealed that oxidation-reduction process, RNA processing, fatty acid degradation, as well as melanin biosynthetic process were enriched with SF treatment. The proteins associated with melanin biosynthetic process, including microphthalmia-associated transcription factor (MITF) and tyrosinase, were down-regulated in SF group, which was confirmed by western blot. SignificanceSF inhibited melanin synthesis in B16 melanoma cells via down-regulation of MITF and tyrosinase expression, which provides a rationale for future utilization of SF.

The Ethanol Extract of Musa sapientum Linn. Peel Inhibits Melanogenesis through AKT Signaling Pathway

Hyperpigmentation caused by melanin overproduction can be induced by UV radiation. The quest for effective depigmenting agents continues because many anti-melanin agents have restricted use and/or produce side-effects. The present study was aimed to investigate the inhibitory activity of Musa sapientum Linn. (AA group) peel ethanol extracts (MPE) on α-melanocyte stimulating hormone (α-MSH)-induced melanin production. In addition, the molecular mechanism related to this process was examined in B16F10 mouse melanoma cells. The results indicated that MPE remarkably inhibited melanogenesis in α-MSH-stimulated B16F10 cells. Microphthalmia-associated transcription factor (MITF) and tyrosinase expressions were suppressed by MPE in a concentration-dependent manner. In addition, MPE significantly decreased the expression of melanosome transfer protein markers (Rab27a and Pmel17) in a dose-dependent manner. This study found that the elevated phosphorylation of AKT in the B16F10 cells was diminished by MPE treatment. Furthermore, microtubule-associated protein 1 light chain 3 (LC3)-II and p62 (autophagy markers) were affected after the B16F10 cells were treated with MPE. This study demonstrated that MPE might be an effective agent for anti-melanogenesis through the AKT pathway, subsequently diminishing MITF expression and tyrosinase enzyme family production. The findings indicated that MPE could potentially serve as a depigmenting agent in cosmeceuticals.

Promising anti-melanogenic impacts of Portulaca oleracea on B16F1 murine melanoma cell line: An in-vitro vision

Portulaca oleracea (P. oleracea) has been used in folk medicine for treatment of diverse disorders for centuries. In the present study, we aimed to evaluate the anti-melanogenic properties of P. oleracea ethanolic extract on B16F1 murine melanoma cell line. The effect of different concentrations of P. oleracea and arbutin as positive control were investigated on the levels of cell viability, cellular tyrosinase, extracellular and intracellular melanin content as well as tyrosinase, tyrosinase related protein (Trp)-1, Trp-2, and microphthalmia-associated transcription factor (MITF) expression levels using real time-PCR, in B16F1 murine melanoma cell line. All different concentrations of P. oleracea (1–100 μg/ml) and arbutin (10 μM) did not significantly change the viability of B16F1 cells. Our results showed that α−melanocyte-stimulating hormones (α-MSH) (100 nM) significantly increased the levels of tyrosinase activity, extracellular, and intracellular melanin content as well as tyrosinase, Trp-1, Trp-2, and MITF expression levels in B16F1 cells. In contrast, arbutin (10 μM) significantly suppressed these changes following the α-MSH stimulation in B16F1 cells. Moreover, P. oleracea (1–100 μg/ml) remarkably mitigated the levels of tyrosinase activity, extracellular and intracellular melanin content as well as tyrosinase, Trp-1, Trp-2, and MITF expression levels following the α-MSH stimulation in B16F1 cells in a concentration-dependent manner. The plant possessed anti-melanogenic effects through alleviating tyrosinase, TRP-1, TRP-2, and MITF expression levels as well as melanin content and it may be a promising candidate for pigmentation disorders. However, further clinical investigations are required to confirm our results.

Codonopsis pilosula Extract Protects Melanocytes against H2O2-Induced Oxidative Stress by Activating Autophagy

Recently, as the anti-aging role of melanin in the skin and the inhibition of melanin production has been identified, the development of materials capable of maintaining skin homeostasis has been attracting attention. In this study, we further investigated the anti-melanogenic effect of Codonopsis pilosula extract (CPE) and, under oxidative stress, the cytoprotective effect in Melan-a melanocytes exposed to H2O2. First, CPE treatment significantly reduced melanin production by inhibiting melanogenesis-associated proteins, including microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related protein 2 (TRP 2), as a result of the phosphorylation of MAPK/JNK in Melan-a cells. Next, to investigate the protective effects of the CPE on oxidative-stress-induced skin injury and its molecular mechanism, we determined the effect of CPE after inducing oxidative stress by exposing melanocytes to H2O2. CPE protected cells from H2O2-induced cytotoxicity by reducing the expression of the gene encoding the Bax pro-apoptotic protein, whereas it induced the genes encoding the B-cell lymphoma 3 (Bcl2) family and MITF, which is a transcriptional regulator that promotes melanocyte differentiation. Furthermore, our results show that CPE enhanced the production of autophagy-related proteins such as Beclin-1 and light chain 3 (LC3) II; this was substantially reversed by 3-methyladenin (MA, an autophagy inhibitor) pretreatment. Collectively, our findings demonstrate that CPE treatment exhibits not only an anti-melanogenic effect in normal melanocytes, but also a cytoprotective effect in melanocytes subjected to oxidative stress by inducing autophagy and MITF expression. Therefore, we believe that CPE is a potent candidate for cell maintenance in melanocytes.

Abstract 2157: Potential correlations between PLK1, BRAF and MITF in melanoma

Abstract Melanoma is one of the most aggressive types of cancer and extremely difficult to treat after metastasis. BRAFV600E-activating mutations give rise to ∼80% of melanocytic nevi, yet only one-third of melanocytic nevi result in melanoma, suggesting the involvement of other factors. Polo-like kinase 1 (PLK1), an important regulator of cell cycle progression, is overexpressed in melanoma and its expression has been shown to correlate with patient prognosis. Microphthalmia-associated transcription factor (MITF) is a melanocytic lineage-specific transcription factor that regulates a variety of genes critical for melanin synthesis as well as melanoma progression. The goal of this study was to define potential interactions between PLK1, BRAFV600E, and MITF in human melanoma. First, we employed a commercially available human tissue microarray (TMA) coupled with high-throughput, multispectral Vectra scanning and inForm analysis to study a number of clinical tissue cores (nevus, malignant and metastatic melanoma). The TMA was simultaneously immunostained for PLK1, BRAFV600E, MITF, proliferation marker Ki67, melanoma biomarker S100 and DAPI, and was subjected to Vectra scanning and inForm analyses. Using Simple Linear Regression analyses, we found significant correlations among each pair of the selected four proteins (PLK1, BRAFV600E, MITF and Ki67) with correlation co-efficient ranging 0.24-0.84. To analyze if PLK1, and BRAFV600E are contributing to cell proliferation (Ki67 expression) or affecting MITF expression, we employed a Multiple Linear Regression analysis. Our data suggested that high expressions of both BRAFV600E and PLK1 are correlated positively with the expression of Ki67. However, when fitting both PLK1 and BRAFV600E versus MITF, only high PLK1 had significant positive correlation with MITF, while BRAFV600E did not show correlation with MITF. These results suggest that PLK1 and MITF could contribute to melanoma progression independent to BRAFV600E. To further validate our findings, we analyzed The Cancer Genome Atlas (TCGA) database containing a large melanoma cohort of 432 melanoma patients with information on overall survival (OS). To visualize the survival plots, the expression level of PLK1 and MITF was sorted and equally separated to two groups using the median value as a cutoff. Kaplan-Meier analyses showed that high mRNA expression of both PLK1 and MITF were individually associated with significant reductions in OS. Interestingly, when we sorted the data for both high PLK1 and high MITF in the same patient, the OS was shorter than that of patients with low PLK1 and low MITF. Overall, our study suggests an association between PLK1 and MITF pathways during melanoma progression, which may affect overall survival in melanoma patients. Thus, concomitant targeting of PLK1 and MITF could provide an advantage over monotherapy towards melanoma management. However, in-depth studies are required to validate our findings. Citation Format: Gagan Chhabra, Shengqin Su, Chandra K. Singh, Mary A. Ndiaye, Nihal Ahmad. Potential correlations between PLK1, BRAF and MITF in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2157.

MITF Promotes Cell Growth, Migration and Invasion in Clear Cell Renal Cell Carcinoma by Activating the RhoA/YAP Signal Pathway.

Simple SummaryMicrophthalmia-associated transcription factor (MITF) has been reported to play a role in the progression of melanoma and other cancer types. However, the biological role of MITF in clear cell renal cell carcinoma (ccRCC) is largely unknown. In this study, we elucidate the role of MITF in the progression of ccRCC. MITF- and MITF-mediated signaling pathways were investigated in ccRCC cell through MITF knockdown as well as overexpression of MITF in vitro and in vivo. MITF contributed to cell proliferation, migration, invasion and tumor growth in ccRCC through activation of the RhoA/YAP signaling pathways. This study suggests that MITF has potential as a therapeutic target in ccRCC.Microphthalmia-associated transcription factor (MITF) is a basic helix-loop-helix leucine zipper transcription factor involved in the lineage-specific regulation of melanocytes, osteoclasts and mast cells. MITF is also involved in the progression of melanomas and other carcinomas, including the liver, pancreas and lung. However, the role of MITF in clear cell renal cell carcinoma (ccRCC) is largely unknown. This study investigates the functional role of MITF in cancer and the molecular mechanism underlying disease progression in ccRCC. MITF knockdown inhibited cell proliferation and shifted the cell cycle in ccRCC cells. In addition, MITF knockdown reduced wound healing, cell migration and invasion compared with the controls. Conversely, MITF overexpression in SN12C and SNU482 cells increased cell migration and invasion. Overexpression of MITF activated the RhoA/YAP signaling pathway, which regulates cell proliferation and invasion, and increased YAP signaling promoted cell cycle-related protein expression. Additionally, tumor formation was impaired by MITF knockdown and enhanced by MITF overexpression in vivo. In summary, MITF expression was associated with aggressive tumor behavior, and increased the migratory and invasive capabilities of ccRCC cells. These effects were reversed by MITF suppression. These results suggest that MITF is a potential therapeutic target for the treatment of ccRCC.

Effect of Phlorotannins Isolated From Eisenia bicyclis on Melanogenesis in Mouse B16 Melanoma Cells

Phlorotannins isolated from brown algae, such as Eisena bicyclis, have positive physiological effects, including anti-cancer, anti-inflammatory, and anti-Alzheimer’s disease. Although phlorotannins have been shown to inhibit tyrosinase, an enzyme essential for melanogenesis, their effect on melanogenesis remains unexplored. Thus, we isolated phlorotannins from E. bicyclis and examined their effects on α-melanocyte-stimulating hormone (α-MSH)-induced melanogenesis in murine B16 melanoma cells. Both fucofuroeckol-A (FF-A) and phlorofucofuroeckol-A (PFF-A) suppressed α-MSH-induced melanogenesis. Neither inhibited human tyrosinase (TYR) activity, but both inhibited tyrosinase-related protein-2 activity. FF-A downregulated the expression of microphthalmia-associated transcription factor and TYR, which subsequently suppressed melanin production. These results suggest that phlorotannins could be beneficial as melanin control drugs for hyperpigmentation disorders.

A prepared platelet-rich plasma extract, namely Self-Growth Colony, inhibits melanogenesis by down-regulating microphthalmia-associated transcription factor in skin melanocyte.

During melanogenesis, melanocytes produce melanin through enzymatic reactions. Microphthalmia-associated transcription factor (MITF) is a major regulator in controlling the expressions of melanogenic enzymes tyrosinase (TYR), tyrosine-related protein-1 (TRP1), and dopachrome tautomerase (DCT). Self-Growth Colony (SGC) is prepared from human platelet-rich plasma (PRP) having an enrichment of growth factors, and which has claimed skin regeneration function. In this study, we aim to identify and investigate the novel role of SGC in skin melanogenesis. MTT assay was performed to determine the cytotoxicity of applied SGC. Melanin assay was adopted to quantify the intracellular melanin after SGC treatment. Promoter-driven luciferase assay, real-time PCR, and Western blotting were performed to determine the expressions of melanogenic enzymes and MITF in SGC-treated cultured Melan-A cells, being treated with or without UV induction. Ex vivo mouse skin was treated with SGC, and then was subjected to Western blotting and histochemical staining. We identified that SGC inhibited melanogenesis in cultured melanocytes and ex vivo mouse skin. The phenomena were attributed to a reduction of MITF expression, which subsequently down-regulated the melanogenic enzymes, that is, TYR, TRP1, and DCT. Moreover, ERK signaling was activated in the SGC-inhibited melanogenesis. The findings suggest that SGC extracting from human blood can be a safe and potential agent in promoting skin whitening.

Molecular and Biochemical Basis of Minocycline-Induced Hyperpigmentation-The Study on Normal Human Melanocytes Exposed to UVA and UVB Radiation.

Minocycline is a drug which induces skin hyperpigmentation. Its frequency reaches up to 50% of treated patients. The adverse effect diminishes the great therapeutic potential of minocycline, including antibacterial, neuroprotective, anti-inflammatory and anti-cancer actions. It is supposed that an elevated melanin level and drug accumulation in melanin-containing cells are related to skin hyperpigmentation. This study aimed to evaluate molecular and biochemical mechanism of minocycline-induced hyperpigmentation in human normal melanocytes, as well as the contribution of UV radiation to this side effect. The experiments involved the evaluation of cyto- and phototoxic potential of the drug using cell imaging with light and confocal microscopes as well as biochemical and molecular analysis of melanogenesis. We showed that minocycline induced melanin synthesis in epidermal melanocytes. The action was intensified by UV irradiation, especially with the UVB spectrum. Minocycline stimulated the expression of microphthalmia-associated transcription factor (MITF) and tyrosinase (TYR) gene. Higher levels of melanin and increased activity of tyrosinase were also observed in treated cells. Moreover, minocycline triggered the supranuclear accumulation of tyrosinase, similar to UV radiation. The decreased level of premelanosome protein PMEL17 observed in all minocycline-treated cultures suggests disorder of the formation, maturation or distribution of melanosomes. The study revealed that minocycline itself was able to enhance melanin synthesis. The action was intensified by irradiation, especially with the UVB spectrum. Demonstrated results confirmed the potential role of melanin and UV radiation minocycline-induced skin hyperpigmentation.

Stimulation of melanin synthesis by UVB is mediated by NO/cGMP/PKG cascade targeting PAK4 in vitro.

The coat color of mammals is primarily determined by the type, quantity, and distribution of melanin in the skin and hair. As an endogenous gas molecule, nitric oxide (NO) regulates tyrosinase production by modulating the cGMP-dependent protein kinase (PKG) pathway, which enhances melanin synthesis. However, some interrelationships have not been fully elucidated. In the present study, mouse melanocytes co-cultured with mouse keratinocytes in vitro, or as monocultures, were used as research models. The results indicated that ultraviolet B irradiation increased nitric oxide synthase (NOS) activity and NO production, and increased PKG, p21-activated kinase 4 (PAK4), and microphthalmia-associated transcription factor (MITF) levels, as well as tyrosinase (TYR), tyrosinase-related protein 1 and 2 expression, and melanin synthesis. During PKG inhibition, the expression of NO-regulated PAK4 and MITF was decreased. Pigment production was also affected, but remained higher than that in the control and NO inhibitor groups. These findings suggest that ultraviolet light regulates melanin production by activating the NO/cGMP/PKG pathway, which mediates the expression of PAK4, affecting melanin synthesis. On this basis, further elucidation of this regulatory network may improve our understanding of patterns of animal hair color formation.

α-Melanocyte-Stimulating Hormone Triggers Melanogenesis Via Activation of the Aryl Hydrocarbon Receptor Pathway in B16F10 Mouse Melanoma Cells.

Melanin is a group of natural pigments that determines the human skin color and provides fundamental protection against the harmful impacts of physical and chemical stimuli. The aim of this study was to establish the regulatory role of aryl hydrocarbon receptor (AhR) in α-melanocyte-stimulating hormone (α-MSH) induced melanogenesis. In the present study, following knockdown of AhR, murine B16F10 cells were treated with α-MSH (200 nM) and tyrosinase activities, cellular melanin content, mRNA levels of several important genes involved in melanogenesis including AhR, CTNNB1, TYR2, and microphthalmia-associated transcription factor (MITF) were measured as endpoints. Exposure to α-MSH led to elevated expression of AhR, CTNNB1, MITF, and TYR in accordance with increased tyrosinase enzyme activity as well as a significant rise in the total melanin content. Our results suggest that AhR plays a regulatory role in α-MSH-stimulated melanogenesis.

Tanshinone IIA, a melanogenic ingredient basis of Salvia miltiorrhiza Bunge

Background: The roots of Salvia miltiorrhiza Bunge (Lamiaceae) have been often used to treat vitiligo in clinical for many years. However, the main ingredient basis of efficacy has not been known. Objectives: We investigated whether the two main constituents of S. miltiorrhiza, major hydrophobic compound tanshinone IIA (Tan IIA) and the major hydrophilic compound salvianolic acid B (Sal B), had the same melanogenic activity. Methods: To testify the potential roles of Tan IIA and Sal B in pigmentation, tyrosinase (Tyr) activity, melanin synthesis ability, and the molecular mechanisms stimulating melanin production were determined in B16F10 melanoma cells. Results: Tan IIA promoted melanogenesis and enhanced Tyr activity at its maximum concentration (10 μM), whereas that of Sal B had no effect. Furthermore, the color of cell pellets and morphological observation of B16F10 cells were visibly darkened. Regarding molecular mechanisms, Western blot results showed that Tan IIA (1, 3, and 10 μM) dose dependently increased the level of phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase MAPK, respectively. However, it decreased phosphorylation in extracellular signal-regulated protein kinase 1/2 MAPK signaling. Meanwhile, Tan IIA promoted the expression of microphthalmia-associated transcription factor and Tyr. Conclusion: Tan IIA might be a melanogenic ingredient basis of S. miltiorrhiza to increase the Tyr activity by activating the upstream MAPK signaling pathways, thereby contributing to pigmentary processing.

Acriflavine, a Potent Inhibitor of HIF-1α, Disturbs Glucose Metabolism and Suppresses ATF4-Protective Pathways in Melanoma under Non-Hypoxic Conditions

Simple SummaryHypoxia is a common feature in solid tumors such as melanoma, contributing locally and systemically to tumor progression. Although the hypoxia response in tumor cells is well understood, the role of constitutively activated hypoxia-inducible factor (HIF)-1α in normoxic conditions is less known. Here, we used acriflavine, a chemical inhibitor of HIF-1α, to investigate the role of this transcription factor on the progression of melanoma under normoxic conditions. The data indicated that acriflavine disturbs glucose metabolism and induces melanoma cell death under normoxia. As a result, we describe a possible clinical option that may target melanoma cells irrespective of the hypoxic microenvironment of the tumors. However, the translational importance of these findings should be confirmed in pre-clinical models.Hypoxia-inducible factor (HIF)-1α is constitutively expressed in melanoma cells under normoxic conditions and its elevated expression correlates with the aggressiveness of melanoma tumors. Here, we used acriflavine, a potent inhibitor of HIF-1α dimerization, as a tool to investigate whether HIF-1α-regulated pathways contribute to the growth of melanoma cells under normoxia. We observed that acriflavine differentially modulated HIF-1α-regulated targets in melanoma under normoxic conditions, although acriflavine treatment resulted in over-expression of vascular endothelial growth factor (VEGF), its action clearly downregulated the expression of pyruvate dehydrogenase kinase 1 (PDK1), a well-known target of HIF-1α. Consequently, downregulation of PDK1 by acrifavine resulted in reduced glucose availability and suppression of the Warburg effect in melanoma cells. In addition, by inhibiting the AKT and RSK2 phosphorylation, acriflavine also avoided protective pathways necessary for survival under conditions of oxidative stress. Interestingly, we show that acriflavine targets activating transcription factor 4 (ATF4) for proteasomal degradation while suppressing the expression of microphthalmia-associated transcription factor (MITF), a master regulator of melanocyte development and a melanoma oncogene. Since acriflavine treatment results in the consistent death of melanoma cells, our results suggest that inhibition of HIF-1α function in melanoma could open new avenues for the treatment of this deadly disease regardless of the hypoxic condition of the tumor.

Microphthalmia-Associated Transcription Factor-Dependent Melanoma Cell Adhesion Molecule Activation Promotes Peritoneal Metastasis of Ovarian Cancer.

Ovarian cancer (OvCa) is one of the leading causes of death due to its high metastasis rate to the peritoneum. Recurrent peritoneal tumors also develop despite the use of conventional platinum-based chemotherapies. Therefore, it is still important to explore the factors associated with peritoneal metastasis, as these predict the prognosis of patients with OvCa. In this study, we investigated the function of microphthalmia-associated transcription factor (MITF), which contributes to the development of melanoma, in epithelial ovarian cancer (OvCa). High MITF expression was significantly associated with a poor prognosis in OvCa. Notably, MITF contributed to the motility and invasion of OvCa cells, and specifically with their peri-mesothelial migration. In addition, MITF-positive cells expressed the melanoma cell adhesion molecule (MCAM/CD146), which was initially identified as a marker of melanoma progression and metastasis, and MCAM expression was regulated by MITF. MCAM was also identified as a significant prognostic factor for poor progression-free survival in patients with OvCa. Collectively, our results suggest that MITF is a novel therapeutic target that potentially promotes peritoneal metastasis of OvCa.

Melanogenesis Effect of 7-acetoxy-4-methylcoumarin in B16F10 Melanoma Cells

The increased interest in anti-whitening dyes has enhanced the research interest to identify efficient melanogenic activators. Melanogenesis is the process of melanin production by melanocytes in the hair follicles and skin, which is mediated by several enzymes, such as microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), tyrosinase-related protein (TRP)-1, and TRP-2. This study investigated the melanogenesis-stimulating effect of 4-Methylumbelliferone (4MUMB) and its synthetic derivatives, 7-acetoxy-4-methylcoumarin (7A4MC) and 4-methylheriniarin (4MH) in B16F10 melanoma cells. The cytotoxicity of these compounds was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, followed by the assessment of the melanin content and the intracellular TYR activity. Finally, the expression levels of the key enzymes involved in melanogenesis were investigated. 7A4MC increased melanin production in B16F10 cells relative to that by 4MUMB and 4MH treated cells in a dose-dependent manner without significant cytotoxicity. Concomitantly, 7A4MC significantly increased TYR activity and enhanced the expression of MITF, which significantly induced the expression of TRP-1, TRP-2, and TYR. Furthermore, 7A4MC stimulated melanogenesis via increased phosphorylation of c-Jun N-terminal kinases (JNK) and reduced phosphorylation of protein kinase B (AKT). These results confirmed the melanogenesis-inducing effects of 7A4MC and indicated its potential use as an anti-hair bleaching agent in cosmetics industries.

Additive Effect of a Combination of Artocarpus lakoocha and Glycyrrhiza glabra Extracts on Tyrosinase Inhibition in Melanoma B16 Cells.

Artocarpus lakoocha (Al) and Glycyrrhiza glabra (Gg) extracts have been reported to show tyrosinase inhibitory activity and melanin pigment reduction. This is the first study to assess the combination of Al and Gg extracts in enhancing inhibition of tyrosinase and reduction of melanin pigments. Al and Gg extracted by maceration in 70% and 95% ethanol were analyzed for oxyresveratrol and glabridin using Ultra High Performance Liquid Chromatography. Extracts of Al and Gg singly and combinations of Al95 and Gg95 were tested for cytotoxicity, tyrosinase inhibitory activity, and reduction of melanin pigments in melanoma B16 cells. Al95 had higher antioxidant, tyrosinase inhibitory activity and reduced more melanin pigments in B16 cells compared to Al70, and exhibited higher levels of oxyresveratrol. Gg95 inhibited oxidative stress and mushroom tyrosinase better than Gg70, and exhibited higher levels of glabridin. Combinations of Al95 and Gg95 at various ratios (concentration of 0.1 mg/mL) were not cytotoxic to B16 cells. Interestingly, Al95 and Gg95 combined at a ratio 9:1 reduced melanin pigment up to 53% in B16 cells. This combination of Al95 and Gg95 extracts exhibited the additive effect of reducing melanin pigments by suppressing the expression of microphthalmia-associated transcription factor (MITF), tyrosinase (TYR) and tyrosinase-related protein-2 (TRP-2) in B16 cells. The combination of Al and Gg extracts could be developed as skin care products for hyperpigmentation treatment.