Microphthalmia-associated Transcription Factor Protein Research Articles

The MITF/mir-579-3p regulatory axis dictates BRAF-mutated melanoma cell fate in response to MAPK inhibitors

Therapy of melanoma has improved dramatically over the last years thanks to the development of targeted therapies (MAPKi) and immunotherapies. However, drug resistance continues to limit the efficacy of these therapies. Our research group has provided robust evidence as to the involvement of a set of microRNAs in the development of resistance to target therapy in BRAF-mutated melanomas. Among them, a pivotal role is played by the oncosuppressor miR-579-3p. Here we show that miR-579-3p and the microphthalmia-associated transcription factor (MITF) influence reciprocally their expression through positive feedback regulatory loops. In particular we show that miR-579-3p is specifically deregulated in BRAF-mutant melanomas and that its expression levels mirror those of MITF. Luciferase and ChIP studies show that MITF is a positive regulator of miR-579-3p, which is located in the intron 11 of the human gene ZFR (Zink-finger recombinase) and is co-transcribed with its host gene. Moreover, miR-579-3p, by targeting BRAF, is able to stabilize MITF protein thus inducing its own transcription. From biological points of view, early exposure to MAPKi or, alternatively miR-579-3p transfection, induce block of proliferation and trigger senescence programs in BRAF-mutant melanoma cells. Finally, the long-term development of resistance to MAPKi is able to select cells characterized by the loss of both miR-579-3p and MITF and the same down-regulation is also present in patients relapsing after treatments. Altogether these findings suggest that miR-579-3p/MITF interplay potentially governs the balance between proliferation, senescence and resistance to therapies in BRAF-mutant melanomas.

Prognostic significance of melanogenesis pathway and its association with the ultrastructural characterisation of melanosomes in uveal melanoma

BackgroundPigmentation could be a relevant prognostic factor in uveal melanoma (UM) development. Microphthalmia-associated transcription factor (MITF) regulates melanin synthesis by activating tyrosinase-related protein 2 (TYRP2) and silver protein (SILV) that...

Role of microphthalmia-associated transcription factor in uveal melanoma and its association with clinicopathological parameters.

153 Background: High melanin content could lead to high metastatic potential in Uveal Melanoma (UM). In the Asian population, Microphthalmia-Associated Transcription Factor (MITF) regulates the melanogenesis pathway and activates Silver Protein (SILV) for eumelanin synthesis. MITF also stimulates the hypoxia–inducible factor 1-alpha (HIF-1α) gene that regulates hypoxia around the tumor microenvironment. Although their oncogenic potential has been observed in a variety of malignancies, MITF and SILV have not been investigated in UM in the Asian population. Therefore, our study aimed to detect the prognostic significance of MITF, SILV, and HIF1-α gene/protein expression levels in UM patients. Methods: Immunohistochemistry of MITF, SILV & HIF-1 α was performed on 82 UM tissue samples. Western blot of all three markers was carried out on representative cases. The mRNA expression level was done by qRT-PCR in 70 fresh UM cases. Cox proportional hazard model and log-rank test were used to determine the prognostic outcome of these markers. Results: MITF and SILV proteins were expressed in 56% and 70% of cases, respectively. High pigmentation, BAP1 loss, and HIF-1α expression were statistically correlated with MITF and SILV protein expression. Upregulation of both genes was found in more than 50% of cases at the mRNA level. This was also statistically significant with high pigmentation and HIF-1α mRNA expression. High expression of MITF protein showed reduced disease-free survival. Conclusions: Our study highlighted the fact that MITF and SILV could be potential biomarkers in UM development. Hence, it might play a key role in future therapeutic target.

Paeoniflorin found in Paeonia lactiflora root extract inhibits melanogenesis by regulating melanin-related signal transduction in B16F10 cells.

Skin pigmentation is modulated by various processes, with melanogenesis playing a key role. Melanin is synthesized by the catalysis of melanogenesis-related enzymes, such as tyrosinase and tyrosine-related proteins TRP-1 and TRP-2. Paeoniflorin is the main bioactive component of Paeonia suffruticosa Andr., Paeonia lactiflora., or Paeonia veitchii Lynch and has been used for centuries for its anti-inflammatory, anti-oxidant, and anti-carcinogenic properties. In this study, melanin biosynthesis in mouse melanoma (B16F10) cells was induced using α-melanocyte-stimulating hormone (α-MSH), and then cells were co-treated with paeoniflorin to evaluate its potential anti-melanogenic effect. α-MSH stimulation increased melanin content, tyrosinase activity, and melanogenesis-related markers in a dose-dependent manner. However, treatment with paeoniflorin reversed α-MSH-induced upregulation of melanin content and tyrosinase activity. Furthermore, paeoniflorin inhibited cAMP response element-binding protein activation and TRP-1, TRP-2, and microphthalmia-associated transcription factor protein expression in α-MSH-stimulated B16F10 cells. Overall, these findings show the potential of paeoniflorin as a depigmenting agent for cosmetic products.

Anti-Melanogenic Effects of Fractioned Cynanchum atratum by Regulation of cAMP/MITF Pathway in a UVB-Stimulated Mice Model.

Based on traditional pharmacological applications and partial in vitro data, Cynanchum atratum (CA) is proposed to act on skin whitening. However, its functional evaluation and underlying mechanisms have yet to be identified. This study aimed to examine the anti-melanogenesis activity of CA fraction B (CAFB) on UVB-induced skin hyperpigmentation. Forty C57BL/6j mice were exposed to UVB (100 mJ/cm2, five times/week) for eight weeks. After irradiation, CAFB was applied to the left ear once a day for 8 weeks (the right ear served as an internal control). The results showed that CAFB significantly reduced melanin production in the ear skin, as indicated by the gray value and Mexameter melanin index. In addition, CAFB treatment notably decreased melanin production in α-MSH-stimulated B16F10 melanocytes, along with a significant reduction in tyrosinase activity. Cellular cAMP (cyclic adenosine monophosphate), MITF (microphthalmia-associated transcription factor), and tyrosinase-related protein 1 (TRP1) were also noticeably downregulated by CAFB. In conclusion, CAFB is a promising ingredient for treating skin disorders caused by the overproduction of melanin and its underlying mechanisms involving the modulation of tyrosinase, mainly mediated by the regulation of the cAMP cascade and MITF pathway.

Phenolic acids in Panax ginseng inhibit melanin production through bidirectional regulation of melanin synthase transcription via different signaling pathways

BackgroundOur previous investigation indicated that the preparation of Panax ginseng Meyer (P. ginseng) inhibited melanogenesis. It comprised salicylic acid (SA), protocatechuic acid (PA), p-coumaric acid (p-CA), vanillic acid (VA), and caffeic acid (CA). In this investigation, the regulatory effects of P. ginseng phenolic acid monomers on melanin production were assessed. MethodsIn vitro and in vivo impact of phenolic acid monomers were assessed. ResultsSA, PA, p-CA and VA inhibited tyrosinase (TYR) to reduce melanin production, whereas CA had the opposite effects. SA, PA, p-CA and VA significantly downregulated the melanocortin 1 receptor (MC1R), cycle AMP (cAMP), protein kinase A (PKA), cycle AMP-response element-binding protein (CREB), microphthalmia-associated transcription factor (MITF) pathway, reducing mRNA and protein levels of TYR, tyrosinase-related protein 1 (TYRP1), and TYRP2. Moreover, CA treatment enhanced the cAMP, PKA, and CREB pathways to promote MITF mRNA level and phosphorylation. It also alleviated MITF protein level in α-MSH-stimulated B16F10 cells, comparable to untreated B16F10, increasing the expression of phosphorylation glycogen synthase kinase 3β (p-GSK3β), β-catenin, p-ERK/ERK, and p-p38/p38. Furthermore, the GSK3β inhibitor promoted p-GSK3β and p-MITF expression, as observed in CA-treated cells. Moreover, p38 and ERK inhibitors inhibited CA-stimulated p-p38/p38, p-ERK/ERK, and p-MITF increase, which had negative binding energies with MC1R, as depicted by molecular docking. ConclusionP. ginseng roots' phenolic acid monomers can safely inhibit melanin production by bidirectionally regulating melanin synthase transcription. Furthermore, they reduced MITF expression via MC1R/cAMP/PKA signaling pathway and enhanced MITF post-translational modification via Wnt/mitogen-activated protein kinase signaling pathway.

In Silico Study of Naringenin as Melanogenesis Inducer in Vitiligo

Introduction: Vitiligo is a pigmentation disorder characterized by loss of skin color (depigmentation) due to melanocyte dysfunction and loss. Melanocytes produce melanin pigment through a melanogenesis process. Melanocyte survival and melanogenesis process are influenced by Microphthalmia Associated- Transcription Factor (MITF) and several proteins, including WNT, β-catenin, tyrosinase, Tyrosinase- Related Protein-1 (TRP1), and Tyrosinase-Related Protein-2 (TRP2). The current therapy for vitiligo is still unsatisfactory. Naringenin is one of Rhizophora mucronata compound, one type of mangrove plant often found in the eastern coastal area of Surabaya City. Objective: To investigate the naringenin’s potency in melanogenesis and to predict the pharmacokinetics or toxicity of naringenin by in silico study. Methods: This is a computational study using a molecular docking method to observe the interaction of naringenin with WNT, β-catenin, MITF, tyrosinase, TRP-1, and TRP-2 proteins. Pharmacokinetic or toxicity prediction of naringenin using the pkCSM method. Psoralen was used as a control. Results: Naringenin binds to all these proteins in the same region as psoralen, indicating that naringenin can stimulate melanogenesis. Naringenin has lower binding energy than psoralen on all proteins (except β-catenin), indicating that naringenin's interaction with these proteins is stronger than psoralen. Pharmacokinetic and toxicity predictions show that naringenin has good absorption or permeation, is not mutagenic, is not hepatotoxic, and does not cause skin sensitization. Conclusion: This computational study concludes that naringenin has melanogenesis inducer potency and good pharmacokinetics.

Phenolic from apple blossom "Hongro" inhibits the expression of proteins related to melanogenesis in B16F10 melanoma cells.

This study aimed to assess apple blossom extracts as potential natural whitening agents due to their ability to inhibit melanogenesis. Ethanol extracts of apple blossom (ABE) were assessed for biological activity in the B16F10 mouse melanoma cell line. ABE toxicity was assessed by thiazolyl blue tetrazolium bromide (MTT) assay. Levels of melanogenic enzyme expression in response to ABE supplementation were assessed by western blotting. Also assessed purified kaempferol, one of the phenolic compounds extracted from apple blossom, was evaluated using western blot analysis. The expression levels of cellular tyrosinase, microphthalmia-associated transcription factor (MITF), tyrosinase-related protein (TRP)-1, and TRP-2 proteins related to melanogenesis decreased in a dose-dependent manner with ABE treatment of cells. Using nuclear magnetic resonance, we identified kaempferol in the ABE. Treatment of cells with purified kaempferol decreased the expression levels of tyrosinase and the MITF protein to a similar degree as that observed with ABE treatment. This suggests that the efficacy of melanogenesis-related inhibition demonstrated by ABE was due to kaempferol. ABE has an inhibitive effect on melanogenic enzymes and potentially can be applied to functional foods and cosmetics having a whitening effect as a natural material.

Echinochrome A Inhibits Melanogenesis in B16F10 Cells by Downregulating CREB Signaling.

Excessive increase in melanin pigment in the skin can be caused by a variety of environmental factors, including UV radiation, and can result in spots, freckles, and skin cancer. Therefore, it is important to develop functional whitening cosmetic reagents that regulate melanogenesis. In this study, we investigated the effects of echinochrome A (Ech A) on melanogenesis in the B16F10 murine melanoma cell line. We triggered B16F10 cells using α-MSH under Ech A treatment to observe melanin synthesis and analyze expression changes in melanogenesis-related enzymes (tyrosinase, tyrosinase-related protein 1 (TYRP1), and tyrosinase-related protein 2 (TYRP2)) at the mRNA and protein levels. Furthermore, we measured expression changes in the microphthalmia-associated transcription factor (MITF), CREB, and pCREB proteins. Melanin synthesis in the cells stimulated by α-MSH was significantly reduced by Ech A. The expression of the tyrosinase, TYRP1, and TYRP2 mRNA and proteins was significantly decreased by Ech A, as was that of the MITF, CREB, and pCREB proteins. These results show that Ech A suppresses melanin synthesis by regulating melanogenesis-related enzymes through the CREB signaling pathway and suggest the potential of Ech A as a functional agent to prevent pigmentation and promote skin whitening.

Analysis of the differentially expressed genes and characterization of PIMITF genes of the leopard coral grouper (Plectropomus leopardus)

Leopard coral grouper (Plectropomus leopardus) is an indispensable kind of fish in the coral reef ecosystems and marine aquaculture, and the body color of leopard coral grouper is exhibited in black, brown or red. In order to explore the molecular mechanism affecting the body color of the leopard coral grouper, the illumina sequencing technology was used here, the transcriptome of the skin and brain tissues of the red and black leopard coral grouper were sequenced and the differentially expressed genes between the different body colors of the two tissues were further analyzed in this study. Our results showed that the expressional level of multiple genes in the pathways related to pigment synthesis exhibited significant differences. In addition, encoding gene of microphthalmia-associated transcription factor (MITF) were identified related to the pigment synthesis, phylogenetic analysis and three-dimensional structure predictions on the MITF protein were performed as well. In conclusion, a relatively complete transcriptome data set of different body colors and different tissues in leopard coral grouper were established in this study. The analysis of genes related to the melanin synthesis pathway here will provide a further understanding of the internal mechanism of body color transition in vertebrate.

PD-L1 expression is regulated by microphthalmia-associated transcription factor (MITF) in nodular melanoma

Malignant melanoma (MM) is known to avoid the host’s immune response. Studies on in vitro melanoma cell lines link the microphthalmia-associated transcription factor (MITF) with the regulation of the PD-L1 expression. It seems that MITF affects the activation of the gene responsible for PD-L1 protein expression. Several proteins, including Bcl-2 and Cyclin D1, play major roles in malignant melanoma cell cycle regulation and survival. Our study aims to assess the relationship between MITF, Bcl-2, and cyclin D1 protein expression and the expression of the PD-L1 molecule. Additionally, we examined the association of BRAF mutation, MITF, and CCND1 gene amplification with PD-L1 protein expression. We performed immunohistochemical staining on fifty-two tumour samples from patients diagnosed with nodular melanoma (NM). BRAF V600 mutation, MITF, and CCND1 gene amplification analyses were analyzed by the Sanger sequencing and QRT-PCR methods, respectively. Statistical analyses confirmed the significant inverse correlation between cyclin D1 and PD-L1 expression (p = 0.001) and correlation between PD-L1 and MITF protein expression (p = 0.023). We found a statistically significant inverse correlation between the present MITF gene amplification and PD-L1 (p = 0.007) and MITF protein expression (p = 3.4 ×10–6), respectively. Our study, performed on clinical NM materials, supports the in vitro study findings providing a rationale for the potential MITF-dependent regulation of PD-L1 expression in malignant melanoma.

Hinokitiol-induced decreases of tyrosinase and microphthalmia-associated transcription factor are mediated by the endoplasmic reticulum-associated degradation pathway in human melanoma cells

Tyrosinase (TYR) is a key enzyme for melanin production. We previously showed that hinokitiol, a naturally occurring seven-membered ring terpenoid, potently inhibits human TYR activity. Interestingly, hinokitiol was recently reported to decrease expression of TYR and microphthalmia-associated transcription factor (MITF), which is a main transcription factor of the TYR gene, in murine melanoma cells. However, the mechanisms by which hinokitiol decreases the intracellular levels of TYR and MITF have not been fully elucidated. Here, we investigated the underlying mechanisms of the decreases using cultured human melanoma cells. As a result, hinokitiol treatment decreased TYR protein level in a time- and dose-dependent manner in G361 human melanoma cells, while MITF protein level was decreased only at higher concentrations after 3 days treatment. Notably, the mRNA levels of TYR and MITF were slightly increased by hinokitiol treatment. Therefore, we focused on the degradation of TYR and MITF in endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway. Importantly, co-treatment of ERAD inhibitor with hinokitiol restored the protein levels of TYR and MITF to approximately 30% and 20% of total those in untreated control cells, respectively. Hinokitiol affected the ER homeostasis as well as degradation of TYR and MITF in two human melanoma cell lines, G361 and HT-144, but the changes of ER-stress markers under the hinokitiol treatment were different in the two human melanoma cell lines. Taken together, these observations indicate that hinokitiol may induce ER stress and trigger the degradation of unfolded newly synthesizing TYR and MITF via the ERAD pathway.

User guide to MiT-TFE isoforms and post-translational modifications.

The microphthalmia-associated transcription factor (MITF) is at the core of melanocyte and melanoma fate specification. The related factors TFEB and TFE3 have been shown to be instrumental for transcriptional regulation of genes involved in lysosome biogenesis and autophagy, cellular processes important for mediating nutrition signals and recycling of cellular materials, in many cell types. The MITF, TFEB, TFE3, and TFEC proteins are highly related. They share many structural and functional features and are targeted by the same signaling pathways. However, the existence of several isoforms of each factor and the increasing number of residues shown to be post-translationally modified by various signaling pathways poses a difficulty in indexing amino acid residues in different isoforms across the different proteins. Here, we provide a resource manual to cross-reference amino acids and post-translational modifications in all isoforms of the MiT-TFE family in humans, mice, and zebrafish and summarize the protein accession numbers for each isoform of these factors in the different genomic databases. This will facilitate future studies on the signaling pathways that regulate different isoforms of the MiT-TFE transcription factor family.

Lactobacillus helveticus-Fermented Milk Whey Suppresses Melanin Production by Inhibiting Tyrosinase through Decreasing MITF Expression.

Whey obtained from milk fermented by the Lactobacillus helveticus CM4 strain (LHMW) has been shown to improve skin barrier function and increase skin-moisturizing factors. In this study, we investigated the effects of LHMW on melanin production to explore the additional impacts of LHMW on the skin. We treated mouse B16 melanoma cells with α-melanocyte-stimulating hormone (α-MSH) alone or simultaneously with LHMW and measured the amount of melanin. The amount of melanin in B16 cells treated with α-MSH significantly increased by 2-fold compared with that in control cells, and tyrosinase activity was also elevated. Moreover, treatment with LHMW significantly suppressed the increase in melanin content and elevation of tyrosinase activity due to α-MSH. LHMW also suppressed the α-MSH-induced increased expression of tyrosinase, tyrosinase-related protein 1 (TRP1), and dopachrome tautomerase (DCT) at the protein and mRNA levels. Furthermore, the mRNA and protein microphthalmia-associated transcription factor (MITF) expression levels were significantly increased with treatment with α-MSH alone, which were also suppressed by LHMW addition. LHMW suppression of melanin production is suggested to involve inhibition of the expression of the tyrosinase gene family by lowering the MITF expression level. LHMW may have promise as a material for cosmetics with expected clinical application in humans.

The role of MITF and Mcl-1 proteins in the antiproliferative and proapoptotic effect of ciprofloxacin in amelanotic melanoma cells: In silico and in vitro study

Mcl-1 is a potent antiapoptotic protein which is amplified in many human cancer, while microphthalmia associated transcription factor (MITF) promotes cell proliferation and has pro-survival role. The study was designed to examine whether the interaction between ciprofloxacin, one of the fluoroquinolones derivative, and MITF/Mcl-1 proteins affects C32 melanoma cells viability, proliferation and induces apoptosis. Preliminary molecular docking studies, Western blot analysis and fluorescence image cytometry were applied to demonstrate the signaling pathway underlying antiproliferative and proapoptotic effect of the drug. In silico analysis showed that ciprofloxacin possess the ability to form complexes with MITF and Mcl-1proteins. This phenomenon was confirmed by in vitro experimental model where the drug was found to decrease MITF and increase Mcl-1 expression at the protein level. Moreover, we found that ciprofloxacin decreases the cell viability and exerts anti-proliferative effect on amelanotic C32 melanoma cells. Image cytometric studies showed that the tested drug induced GSH depletion and apoptosis via intrinsic death pathway leading to DNA fragmentation. Analysis of the cell cycle distribution revealed that ciprofloxacin caused a block in the G2/M phase. This is the first study that characterized the role of MITF and Mcl-1 proteins in the antiproliferative and pro-apoptotic effect of ciprofloxacin towards amelanotic melanoma cells, opening the possibility to use of this drug as a potential agent for the treatment of melanoma.

Eribulin Suppresses Clear Cell Sarcoma Growth by Inhibiting Cell Proliferation and Inducing Melanocytic Differentiation Both Directly and Via Vascular Remodeling.

Clear cell sarcoma (CCS) is a rare but chemotherapy-resistant and often fatal high-grade soft-tissue sarcoma (STS) characterized by melanocytic differentiation under control of microphthalmia-associated transcription factor (MITF). Eribulin mesilate (eribulin) is a mechanistically unique microtubule inhibitor commonly used for STS treatment, particularly liposarcoma and leiomyosarcoma. In this study, we examined the antitumor efficacy of eribulin on four human CCS cell lines and two mouse xenograft models. Eribulin inhibited CCS cell proliferation by inducing cell-cycle arrest and apoptosis, shrunk CCS xenograft tumors, and increased tumor vessel density. Eribulin induced MITF protein upregulation and stimulated tumor cell melanocytic differentiation through ERK1/2 inactivation (a MITF negative regulator) in vitro and in vivo Moreover, tumor reoxygenation, probably caused by eribulin-induced vascular remodeling, attenuated cell growth and inhibited ERK1/2 activity, thereby upregulating MITF expression and promoting melanocytic differentiation. Finally, downregulation of MITF protein levels modestly debilitated the antiproliferative effect of eribulin on CCS cells. Taken together, eribulin suppresses CCS through inhibition of cell proliferation and promotion of tumor differentiation by acting both directly on tumor cells and indirectly through tumor reoxygenation.

MITF: an evolutionarily conserved transcription factor in the sea urchin Paracentrotus lividus.

Microphthalmia-associated transcription factor (MITF) is a member of MYC superfamily, associated with melanocyte cells, as it was discovered in depigmented mice. However, over the last years it was found to be involved in many cellular signaling pathways, among which oncogenesis, osteoclast differentiation, and stress response. In mammals, Mitf gene mutations can cause diverse syndromes affecting pigmentation of eyes or skin, bone defects and melanomas. As MITF protein homologs were also found in some invertebrates, we have isolated and characterized the MITF cDNAs from the sea urchin Paracentrotus lividus, referred to as Pl-Mitf. The in silico study of the secondary and tertiary structure of Pl-Mitf protein showed high conserved regions mostly lying in the DNA binding domain. To understand the degree of evolutionary conservation of MITF, a phylogenetic analysis was performed comparing the Pl-Mitf deduced protein with proteins from different animal species. Moreover, the analysis of temporal and spatial expression pattern of Pl-Mitf mRNA showed that it was expressed from the onset of gastrulation of the sea urchin embryo to the pluteus larva, specifically in primary mesenchymes cells (PMCs), the sea urchin skeletogenic cells, and in the forming archenteron, the larval gut precursor. In silico protein-protein interactions analysis was used to understand the association of MITF with other proteins. Our results put in evidence the conservation of the MITF protein among vertebrates and invertebrates and may provide new perspectives on the pathways underlying sea urchin development, even if further functional analyses are needed.

Tetrahydrocurcumin Inhibits α-MSH-induced Melanogenesis via GSK3β Activation in B16F10 Melanoma Cells

Tetrahydrocurcumin (THC) has been used as a component of skin whitening cream. However, very little is known about the mechanisms for how THC regulates melanogenesis. The aim of the present study was to investigate mechanisms involved in the regulation of melanogenesis by THC using B16F10 murine melanoma cells. B16F10 cells were treated with various concentrations of THC (0–30 µg/mL) in the presence of 0.1 nM ±-melanocyte stimulating hormone (±-MSH). Total melanin content; intracellular tyrosinase activity; and levels of melanogenic proteins, i.e., microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related protein 1 (TYRP1), and signaling proteins, i.e, Akt, glycogen synthase kinase 3β (GSK3β), and β-catenin, were analyzed. Change in cell morphology was also observed. THC-Treatment caused a significant and dose-dependent decrease in total melanin content compared to control cells treated with α-MSH alone. At the early stage of melanogenesis, when melanin secretion was minimal, THC-treatment caused decreases in the activity of tyrosinase and the levels of tyrosinase and MITF proteins. Conversely, at the late stage of melanogenesis, when melanin secretion was evidently exhibited in control cells, remarkable increases in the intracellular levels of tyrosinase and TYRP1, the melanosomal proteins, were observed in THC-treated cells. THC-treatment caused decreases in Akt and GSK3β phosphorylation and β-catenin content. A significant alteration of cell morphology, i.e. elongation and loss of dendritic structure, was also observed. The present study demonstrated that THC-treatment inhibited α-MSH-induced melanin synthesis in B16F10 cells by downregulating MITF and tyrosinase proteins and decreasing tyrosinase activity. In addition, THC inhibited secretion of melanosomes, which appears to be associated with cell morphology alteration accompanying a loss of dendritic structure. THC caused decreases in the phosphorylation of Akt and GSK3β and the content of β-catenin, suggesting involvement of GSK3β activation and subsequent attenuation of β-catenin signaling in the anti-melanogenic effects of THC.

Antimelanoma activity of perphenazine and prochlorperazine in human COLO829 and C32 cell lines

Cutaneous melanoma is least common (only about 1% of skin cancers) but is the deadliest malignant tumor. Moreover, amelanotic types of melanoma are very difficult for clinical diagnosis. The standard therapy can cause a lot of side effects, e.g., nausea, vomiting, and headaches, which means that novel and effective strategies are required. Interestingly, phenothiazine derivatives possess sedative, antiemetic, and anticancer activity. Our goal was to determine the effect of perphenazine and prochlorperazine on cell viability, motility, microphthalmia-associated transcription factor (MITF) and tyrosinase content in melanotic and amelanotic melanoma cells. The viability of C32 and COLO829 melanoma cells was evaluated by the WST-1 colorimetric assay; impact on motility of human melanoma was performed by wound-healing assay, while tyrosinase and MITF content were determined by Western blot. In the present study, we explore the anticancer effect of perphenazine and prochlorperazine in human melanotic (COLO829) and amelanotic (C32) melanoma cells concluding that prochlorperazine inhibits cell viability in a concentration-dependent manner, impairs motility, and decreases tyrosinase and MITF amounts. Moreover, the analyzed drugs decrease/increase MITF amount depending on the type of melanoma. We demonstrated that the decrease of MITF and tyrosinase protein induces motility inhibition of C32 cells, which suggests the ability of those drugs to restore cancer cell sensitivity to treatment. The ability of prochlorperazine to contain the spread of the amelanotic melanoma in vivo may be helpful in the development of a new and effective antimelanoma therapies.

ROS-scavenging and Anti-tyrosinase Properties of Crocetin on B16F10 Murine Melanoma Cells.

Crocus sativus (Iridaceae) has been traditionally used in the Iranian folk medicine and as a culinary additive. Major components of the plant that are responsible for biological properties are saffranal, crocin, picrocrocin and crocetin. Although the level of crocetin is not high, some of the important activities of saffron such as antioxidant activity have been attributed to crocetin. In the present study, we investigated the effects of crocetin on melanogenesis in B16 melanoma cells. The effect of crocetin on intracellular and mushroom tyrosinase activity and the content of melanin was evaluated spectrophotometrically. Tyrosinase and Microphthalmia-Associated Transcription Factor (MITF) protein levels were compared between Crocetin-treated and control cells after western blot analysis. The antioxidative activity of crocetin was also investigated. Crocetin could inhibit mushroom tyrosinase activity and lower the amount of melanin in B16 melanoma cells. Protein levels of tyrosinase and MITF were also decreased by crocetin. Crocetin also showed antioxidant activity and depleted cellular Reactive Oxygen Species (ROS) content but had no cytotoxicity in alamarBlue® assay. Taken together, decreased tyrosinase activity, melanin content, tyrosinase and MITF proteins levels, and ROS production showed the inhibition of melanogenesis in B16F10 cells by crocetin. Hence, crocetin could be suggested as a potential dermatological whitening agent in skin care products.