Microphthalmia-associated Transcription Factor Protein Expression Research Articles

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.

Ginsenoside Re inhibits melanogenesis and melanoma growth by downregulating microphthalmia-associated transcription factor

Panax ginseng, also known as Korean ginseng, is a traditional remedy widely used in Asian countries. Its major active compounds are ginsenosides, specifically triterpenoid saponins. Among them, one notable ginsenoside called Re has shown various biological effects, including anti-cancer and anti-inflammatory properties. However, the potential beneficial effects of Re on melanogenesis and skin cancer remain poorly understood. To investigate this, we conducted a comprehensive study using biochemical assays, cell-based models, a zebrafish pigment formation model, and a tumor xenograft model. Our results revealed that Re effectively inhibited melanin biosynthesis in a dose-dependent manner by competitively inhibiting the activity of tyrosinase, an enzyme involved in melanin production. Moreover, Re significantly reduced the mRNA expression levels of microphthalmia-associated transcription factor (MITF), a key regulator of melanin biosynthesis and melanoma growth. Furthermore, Re decreased the protein expression of MITF and its target genes, including tyrosinase, TRP-1, and TRP-2, through a partially ubiquitin-dependent proteasomal degradation mechanism, mediated by the AKT and ERK signaling pathways. These findings indicate that Re exerts its hypopigmentary effects by directly inhibiting tyrosinase activity and suppressing its expression via MITF. Additionally, Re demonstrated inhibitory effects on skin melanoma growth and induced tumor vascular normalization in our in vivo experiments. This study represents the first evidence of Re-mediated inhibition of melanogenesis and skin melanoma, shedding light on the underlying mechanisms. These promising preclinical findings warrant further investigation to determine the suitability of Re as a natural agent for treating hyperpigmentation disorders and skin cancer.

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.

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.

Hydrogen Sulfide Promotes Cell Proliferation and Melanin Synthesis in Primary Human Epidermal Melanocytes

Background/Aim: Hydrogen sulfide (H₂S) has been found to act as a physiological intercellular messenger to regulate cell survival. In this study, we evaluated whether H₂S could promote cell proliferation and melanin synthesis in human epidermal melanocytes (HEMs). Methods: Primary HEMs were cocultured with sodium hydrosulfide (NaHS, the most widely used H₂S donor) or endogenously overexpressed with cystathionine-γ-lyase (CSE) gene, which is the most predominant H₂S-producing enzyme. Then, cell viability, intracellular melanin content, tyrosinase (TYR) activity, and expression of microphthalmia-associated transcription factor (MITF), TYR, together with TYR-related protein 1 (TRP-1) in both transcript and protein levels, were detected. Results: We first confirmed that NaHS (10–100 μm) increased cell viability, intracellular melanin content, and TYR activity in a dose-dependent manner. Then, we found that endogenous H₂S production also promoted cell proliferation, intracellular melanin content, and TYR activity. In addition, we observed the mRNA and protein expression of MITF, TYR, and TRP-1 was significantly up-regulated after NaHS treatment and CSE gene transfection. Conclusions: This study demonstrates that H₂S promotes cell proliferation and melanin synthesis in HEMs, which indicates pharmacologic regulation of H₂S may be potential treatment for skin disorders caused by loss of melanocytes or dysfunction of melanogenesis.

Signaling Cascades Activated by UVB in Human Melanocytes Lead to the Increased Expression of Melanocyte Receptors, Endothelin B Receptor and c-KIT.

A single exposure of normal human melanocytes (NHMs) to ultraviolet B (UVB) radiation induces a distinct increase in the expression of c-KIT and endothelin B receptor (EDNRB) and upregulates the expression of microphthalmia-associated transcription factor (MITF). In this review, we clarify the signaling mechanisms by which UVB stimulates the expression of MITF in NHMs, thus leading to upregulation of those two important melanogenic receptors. The increased expression of MITF in UVB-exposed NHMs is accompanied by a markedly stimulated and prolonged phosphorylation of p38/CREB. The UVB-stimulated expression of c-KIT and EDNRB could be completely abolished by a p38 inhibitor concomitant with a reduced phosphorylation of CREB and a downregulation of MITF expression. The UVB exposure of NHMs stimulates the phosphorylation of p38 and c-jun N-terminal kinase, but not ERK, followed by the increased phosphorylation of MSK1 and subsequently CREB. Postirradiation treatment with the MSK1 inhibitor H89 significantly downregulates the increased mRNA and protein expression of MITF, EDNRB and c-KIT in UVB-exposed NHMs. Our findings indicate for the first time that the increased expression of MITF that leads to the upregulation of melanocyte-specific proteins in UVB-exposed NHMs is mediated via activation of the p38/MSK1/CREB axis but not the ERK/RSK/CREB axis.

The functional property of royal jelly 10-hydroxy-2-decenoic acid as a melanogenesis inhibitor

BackgroundIt has been reported that royal jelly would reduce melanin synthesis and inhibit the expression of melanogensis related proteins and genes. In this study, we evaluate the anti-melanogenic and depigmenting activity of 10-hydroxy-2-decenoic acid (10-HDA) from royal jelly of Apis mellifera.MethodsIn this study, we assesses the 10-HDA whitening activity in comparison with the changes in the intracellular tyrosinase activity, melanin content and melanin production related protein levles in B16F1 melanoma cells after treating with 10-HDA. Furthermore, the skin whitening effect was evaluated by applying a cream product containing with 0.5%, 1% and 2% of 10-HDA onto the skin of mice (C57BL/6 J) for 3 week to observe the effect of DL*-values.ResultsThe results showed that 10-HDA inhibited the MITF protein expression (IC50 0.86 mM) in B16F1 melanoma cells. Western blot analysis revealed that 10-HDA inhibited the activity of tyrosinase and the expression of tyrosinase-related protein 1 (TRP-1), TRP-2, and microphthalmia-associated transcription factor (MITF) in B16F1 melanoma cells. In addition, the 10-HDA was applied on the skin of mice show significantly increased the average skin-whitening index (L value).ConclusionsThe validation data indicated the potential of 10-HDA for use in suppressing skin pigmentation. The 10-HDA is proposed as a candidate to inhibit melanogenesis, thus it could be developed as cosmetics skin care products.

Melanogenesis inhibition activity of floralginsenoside A from Panax ginseng berry

BackgroundPanax ginseng is a traditional herb used for medicinal purposes in eastern Asia. P. ginseng contains various ginsenosides with pharmacological effects. In this study, floralginsenoside A (FGA), ginsenoside Rd (GRD), and ginsenoside Re (GRE) were purified from P. ginseng berry. MethodsChemical structures of FGA, GRD, and GRE were determined based on spectroscopic methods, including fast atom bombardment mass spectroscopy, ID-nuclear magnetic resonance, and infrared spectroscopy. Inhibitory activities of these compounds on melanogenesis were studied by measuring the expression of protein and melanin content in the melan-a cell line. This inhibitory activity was confirmed by observing pigmentation and tyrosinase activities of zebrafish. ResultsGRD, GRE, and FGA were not cytotoxic at concentrations less than 20μM, 80μM, and 160μM in melan-a cells, respectively. GRD, GRE, and FGA inhibited melanin biosynthesis in melan-a cells by 15.2%, 22.9%, and 23.9% at 20μM, 80μM, and 160μM, respectively. FGA was observed to display the most potent inhibitory effect. In addition, FGA decreased microphthalmia-associated transcription factor protein expression in a dose-dependent manner. Moreover, FGA induced extracellular signal-regulated kinase phosphorylation level in melan-a cells. In addition, melanin pigment content and tyrosinase activity in zebrafish treated with FGA at160μM were reduced. ConclusionFGA showed the most potent inhibition of melanogenesis in both in vitro and in vivo studies. This study suggests that FGA purified from P. ginseng may be an effective melanogenesis inhibitor.

회화나무(Sophora japonica L.) 꽃(괴화)과 열매(괴각)의 미백활성 평가

회화나무는 한의학에서 간과 혈관계 질환의 치료에 사용되어온 약용 식물로, 그 꽃과 열매를 괴화와 괴각이라 한다. 괴화와 괴각은 메탄올 추출물로부터 에틸아세테이트 분획물을 얻어 시료로 사용하였고, B16 F10 세포를 이용하여 Western blot을 실행하였다. 미백효과를 확인하기 위해, tyrosinase, TRP-1, TRP-2, MITF 단백질을 확인하였다. 그 결과, 괴화와 괴각 추출물은 세포 생존율에는 영향이 미미하였으며, 모두tyrosinase, TRP-1, TRP-2, MITF 단백질의 발현 억제효과를 나타냈다. 또한 농도 의존적으로 억제효과를 보였으며, 이를 통해 전통적으로 사용되어 왔던 괴화와 괴각은 기능성 화장품의 원료와 같은 천연식물 유래 자원으로 상당한 가치를 나타냈다. Sophora japonica has been used for treatment of liver and blood-related diseases in herbology. We evaluated whitening activities of Sophorae Flos and Fructus. Sophorae Flos and Fructus were extracted with methanol (MeOH) and divided into petroleum ether, ethyl acetate(EtOAC) and water fraction. For whitening effect by western blot in B16 F10 cells, we analyzed it by investigating the effect of tyrosinase, TRP-l (tyrosinase-related protein 1), TRP-2 (tyrosinase-related protein 2) and MITF (microphthalmia-associated transcription factor) protein expression. The protein expressions of tyrosinase, TRP-1, TRP-2 and MITF in B16 F10 cells treated with Sophorae Flos and Fructus extract were reduced in a dose-dependant manner. Therefore, these results suggest that Sophorae Flos and Fructus have useful plant resource to be developed as functional cosmetic.

Pimecrolimus increases the melanogenesis and migration of melanocytes in vitro.

Vitiligo is an intriguing depigmentary disorder and is notoriously difficult to be treated. The ultimate goal of vitiligo treatment is to replenish the lost melanocytes by immigration from hair follicle and to restore the normal function of melanogenesis by residual melanocytes. There are two types of topical calcineurin inhibitors called tacrolimus and pimecrolimus, and are recommended as the first-line treatments in vitiligo. Although pimecrolimus is efficacious for the repigmentation of vitiligo, its intrinsic mechanisms have never been investigated in vitro. This research aimed to study the ability of pimecrolimus on stimulating melanogenesis, melanocyte migration and MITF (microphthalmia associated transcription factor) protein expression. Results showed that pimecrolimus at the dosages of 1, 10, 102 nM were neither mitogenic nor cytotoxic to melanocytes. The addition of pimecrolimus at 10, 102 and 103 nM significantly increased intracellular tyrosinase activity, which was consistent with the elevated content of melanin content at the same concentrations. The peak effect was seen at 72 h in response to 102 nM pimecrolimus. Results of the wound scratch assay and Transwell assays indicate that pimecrolimus is effective in facilitating melanocyte migration on a collagen IV-coated surface. In addition, MITF protein yield reached the highest by pimecrolimus at 102 nM. In brief, pimecrolimus enhances melanin synthesis as well as promotes migration of melanocytes directly, possibly via their effects on MITF protein expression.

Inhibitory effect of Gastrodia elata Blume extract on alpha-melanocyte stimulating hormone-induced melanogenesis in murine B16F10 melanoma.

BACKGROUND/OBJECTIVESGastrodia elata Blume (GEB), a traditional herbal medicine, has been used to treat a wide range of neurological disorders (e.g., paralysis and stroke) and skin problems (e.g., atopic dermatitis and eczema) in oriental medicine. This study was designed to investigate whether GEB extract inhibits melanogenesis activity in murine B16F10 melanoma.MATERIALS/METHODMurine B16F10 cells were treated with 0-5 mg/mL of GEB extract or 400 µg/mL arbutin (a positive control) for 72 h after treatment with/without 200 nM alpha-melanocyte stimulating hormone (α-MSH) for 24 h. Melanin concentration, tyrosinase activity, mRNA levels, and protein expression of microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein (Trp)1, and Trp2 were analyzed in α-MSH-untreated and α-MSH-treated B16F10 cells.RESULTSTreatment with 200 nM α-MSH induced almost 2-fold melanin synthesis and tyrosinase activity along with increased mRNA levels and protein expression of MITF, tyrosinase, Trp1 and Trp2. Irrespective of α-MSH stimulation, GEB extract at doses of 0.5-5 mg/mL inhibited all these markers for skin whitening in a dose-dependent manner. While lower doses (0.5-1 mg/mL) of GEB extract generally had a tendency to decrease melanogenesis, tyrosinase activity, and mRNA levels and protein expression of MITF, tyrosinase, Trp1, and Trp2, higher doses (2-5 mg/mL) significantly inhibited all these markers in α-MSH-treated B16F10 cells in a dose-dependent manner. These inhibitory effects of the GEB extract at higher concentrations were similar to those of 400 µg/mL arbutin, a well-known depigmenting agent.CONCLUSIONSThese results suggest that GEB displays dose-dependent inhibition of melanin synthesis through the suppression of tyrosinase activity as well as molecular levels of MITF, tyrosinase, Trp1, and Trp2 in murine B16F10 melanoma. Therefore, GEB may be an effective and natural skin-whitening agent for application in the cosmetic industry.

Effects of aqueous extracts of Ecliptae herba, Polygoni multiflori radix praeparata and Rehmanniae radix praeparata on melanogenesis and the migration of human melanocytes

Ethnopharmacological relevancePolygoni multiflori radix praeparata (PMRP), Ecliptae herba (EH) and Rehmanniae radix praeparata (RRP) are the most frequently-used herbs by Traditional Chinese Medicine practitioners for the treatment of vitiligo. Their abilities to stimulate melanogenesis, melanocyte migration and MITF (microphthalmia associated transcription factor) protein expression were evaluated in this study. Materials and methodsThe effects of aqueous extracts of PMRP, EH and RRP on human melanocytes in vitro were examined by MTT assay, tyrosinase activity, melanin synthesis, migration assay and Western blot. ResultsTreatment with EH (at 100μg/ml and 400μg/ml) significantly increased intracellular tyrosinase activity in accordance with the elevation of melanin content at the same concentrations. Treatment with RRP (at 100μg/ml and 400μg/ml) promoted melanin production but had no stimulatory effect on tyrosinase activity. Treatment with PMRP and EH (at 100μg/ml) promoted the migration of human melanocytes in a type IV collagen-coated transwell migration assay. Western blot analysis showed MITF protein expression was elevated by PMRP, EH and RRP (at 100μg/ml). ConclusionAn aqueous extract of EH has a synergistic effect on melanocytes by up-regulating tyrosinase activity, enhancing melanin synthesis and promoting melanocyte migration as well as elevating MITF protein expression. RRP exhibits a significant stimulating effect on melanogenesis and MITF protein expression. These results suggest that EH and RRP contain substances with direct enhancing effects on melanogenesis and migration, possibly via their effects on MITF protein expression.

Emodin isolated from Polygoni Multiflori Ramulus inhibits melanogenesis through the liver X receptor-mediated pathway

Melanogenesis is a physiological process that results in the synthesis of melanin pigments, which play a crucial protective role against skin photocarcinogenesis. We investigated the effects of a Polygoni Multiflori Ramulus extract on melanogenesis and isolated emodin from Polygoni Multiflori as an active compound. In addition, the possible mechanisms of action were examined. We found that emodin inhibited both melanin content and tyrosinase activity concentration and time dependently. Tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2 mRNA levels decreased following emodin treatment. However, while the mRNA levels of microphthalmia-associated transcription factor (MITF) were not affected by emodin, emodin reduced MITF protein levels. Furthermore, expression of the liver X-receptor (LXR) α gene, but not the LXR β gene was upregulated by emodin. Moreover, emodin regulated melanogenesis by promoting degradation of the MITF protein by upregulating the LXR α gene. The emodin effects on MITF was found to be mediated by phosphorylation of p42/44 MAPK. Taken together, these findings indicate that the inhibition of melanogenesis by emodin occurs through reduced MITF protein expression, which is mediated by upregulation of the LXR α gene and suggest that emodin may be useful as a hyperpigmentation inhibitor.

윈터체리 추출물의 항산화 및 미백 개선 효과

Withania somnifera has been used in folk medicine to treat various ailments for centuries. In this studies to investigate the whitening effect of Withania somnifera extracts as an active ingredient for whitening cosmet- ics, the antioxidant capacity and the effects of Withania somnifera extracts on melanogenesis in B16-F10 melanoma cells were identified. Withania somnifera extracts significantly reduced both tyrosinase activity and melanin content in a concentration-dependent manner. Furthermore, it was found that Withania somnifera extracts decreased α-MSH (melanocyte-stimulating hormone)-induced tyrosinase activity and MITF(microphthalmia associated transcription factor) protein expression. These data indicate that Withania somnifera extracts attenuate α-MSH-stimulated melanin synthesis by modulating MITF expression and that they may be a useful therapeutic agent for treating hyperpigmen- tation and an ingredient of whitening cosmetics.

Angiotensin II stimulates melanogenesis via the protein kinase C pathway.

Melanogenesis is a physiological process that results in the synthesis of melanin pigments, which serve a crucial function in hyperpigmentation. The aim of the present study was to determine the effects of angiotensin II (Ang II) on melanogenesis and to elucidate the molecular events of Ang II-induced melanogenesis. Experiments were performed on human melanocytes to elucidate the pigmenting effect of Ang II and the underlying mechanisms. The elements involved in melanogenesis, including melanin content, tyrosinase (TYR) activity, and microphthalmia-associated transcription factor (MITF) and TYR expression at the mRNA and protein levels were evaluated. Melanin content and TYR activity increased in response to Ang II treatment in a concentration-dependent manner. MITF and TYR mRNA and protein expression levels were increased significantly in response to Ang II in a concentration-dependent manner. The Ang II-induced increase in melanin synthesis was reduced significantly in response to co-treatment with Ro-32-0432, a protein kinase C (PKC) inhibitor, whereas co-treatment with H-89, a PKA inhibitor, did not attenuate the Ang II-induced increase in melanin levels. These results suggest that PKC is required for Ang II-induced pigmentation in human melanocytes and that the mechanism involves the PKC pathway and MITF upregulation.

Molecular characterization of two candidate genes associated with coat color in Tibetan sheep (Ovis arise)

Coat color is a key economic trait in sheep. Some candidate genes associated with animal's coat color were found. Particularly, v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) and microphthalmia-associated transcription factor (MITF) play a key role in the modulation of hair pigmentation in mammals. This study investigated those two candidate genes' mutations and expressions associated with wool color in Tibetan sheep. First, the gene polymorphisms of those two genes were analyzed, and then, relative mRNA expression levels of those two genes in skin tissue with different coat colors were compared. Thirdly, KIT and MITF protein expression levels were detected through Western blot and immune-histochemical. Allele C was predominant allele in the white coat color Tibetan sheep population of the MITF coding region g. 1548 C/T loci. The relative MITF mRNA expression in black coat skin tissue was significantly higher than white (P<0.01). However, no significant differences were detected in the KIT gene's mRNA expression of these two different coat color skin tissues (P>0.05), while the level of KIT protein expression in skin tissues of white and black coats was also roughly equivalent. Our study observed that, the level of MITF protein expression in black coat skin tissue was significantly higher than that in white coat skin tissue, and positive staining for MITF protein expression was detected mainly in the epidermis and the dermal papilla, bulb, and outer root sheath of hair follicles. We conclude that the black coat of Tibetan sheep is related to high MITF expression in the hair follicles, and MITF may be important for coat color formation of Tibetan sheep.

연자육 추출물의 멜라닌 합성 저해효과

In order to develop new skin whitening agents, we prepared the CH₂Cl₂ layer (NGC) and BuOH layer (NGB) of 75% EtOH extract of the Nelumbinis nucifera Gaertner. We measured their tyrosinase inhibitory activity in vitro and melanin synthesis inhibitory activity in B16-F1 melanoma cells. They did not show inhibitory activity against mushroom tyrosinase but showed melanin synthesis inhibitory activity in a dose-dependent manner. In a melanin synthesis inhibition assay, NGC and NGB suppressed melanin production up to 52% and 46% at a concentration of 100 μg/mL, respectively. To elucidate the mechanism of the inhibitory effects of NGC and NGB on melanogenesis, we measured the expression of melanogenesis-related proteins by western blot assay. As a result, NGC suppressed the expression of tyrosinase, tyrosinase related protein 1 (TRP-1), tyrosinase related protein 2 (TRP-2), phosphorylated cAMP responsive element binding (p-CREB) protein, and microphthalmia associated transcription factor (MITF). And NGB inhibited the protein expression of tyrosinase and MITF, but had no significant effect on TRP-1, TRP-2, and p-CREB expression. Moreover, NGB increased the expression of phosphorylated extracellular signal-regulated kinase (p-ERK). In addition, we examined the inhibitory effect on the glycosylation of tyrosinase. As a result, NGC and NGB inhibited the activity of α-glucosidase in vitro and the glycosylation of tyrosinase in B16-F1 melanoma cells. From these results, we concluded that NGC and NGB could be used as active ingredients for skin whitening.

Anti-Melanogenesis Effect ofGlechoma hederaceaL. Extract on B16 Murine Melanoma Cells

Glechoma hederacea L. (Labiatae) has been used in folk medicine to treat various ailments for centuries. We investigated the effects of G. hederacea extract on melanogenesis in B16 melanoma cells. It significantly reduced both the cellular melanin content and tyrosinase activity in a concentration-dependent manner. An MTT assay did not reveal any obvious cytotoxicity. Furthermore, we found that G. hederacea extract decreased tyrosinase and microphthalmia-associated transcription factor protein expression, but did not inhibit tyrosinase-related protein-1 and tyrosinase-related protein-2 expression. RT-PCR analysis indicated that the antimelanogenic effect of G. hederacea extract might be due to inhibition of tyrosinase gene transcription. Moreover, this effect is regulated via suppression of microphthalmia-associated transcription factor protein expression. Our data indicate that G. hederacea extract inhibits melanin synthesis in B16 melanoma cells but is not cytotoxic. Hence it might prove a useful therapeutic agent for treating hyperpigmentation and an effective component of whitening cosmetics.

The role of 8q24 copy number gains and c-MYC expression in amelanotic cutaneous melanoma

Cutaneous melanomas may be quite heterogeneous in their clinical, histological and molecular findings. Correlating these features may help identify distinctive subgroups of melanomas and improve our overall understanding and prognostication of melanoma. We recently identified a subgroup of melanomas with increased chromosomal copy number gains in 8q24 at MYC having several distinctive clinical and histopathological characteristics, including an aggressive clinical course and an amelanotic clinical and histological appearance. It has been postulated that oncogenes such as MYC may have regulatory effects on genes critical to melanin pigment synthesis, specifically microphthalmia-associated transcription factor (MITF), which is known to have a key role in regulating the expression of tyrosinase (TYR), an important enzyme in the production of melanin pigment. We investigated the possible mechanism underlying the amelanotic appearance of melanomas with gains in 8q24 by evaluating the relationship between melanomas with and without 8q24 copy number gains and c-MYC, MITF and TYR protein expression. Immunohistochemical analysis of c-MYC, MITF and TYR was performed on 36 melanomas with gains in 8q24 and 40 melanomas without gains in 8q24. The melanomas with gains of 8q24 correlated with elevated c-MYC protein expression and melanomas without gains in 8q24 showed significantly decreased c-MYC protein expression. A direct relationship between the presence of gains in 8q24 and decreased MITF expression, as well as between c-MYC and TYR protein expression was also observed. Our results suggest that MYC can have a role in the pigmentary pathway of melanoma. In amelanotic melanomas with gains in 8q24, downregulation of TYR and other melanocyte-specific genes may be mediated by MYC leading to transcriptional suppression of MITF. As MITF is a frequently used marker to establish melanocytic lineage in melanoma, our study also raises the important clinical consideration that amelanotic melanomas, especially those with gains in 8q24 may lack expression of MITF.

Alcohol extract from Vernonia anthelmintica (L.) willd seed enhances melanin synthesis through activation of the p38 MAPK signaling pathway in B16F10 cells and primary melanocytes

Ethnopharmacological relevanceVernonia anthelmintica (L.) willd has been used in folk medicine to treat leukoderma in China for centuries. The promoting activities of its extract (AVE) in melanogenesis and possible signaling pathways were investigated in this article. Materials and methodsThe improving activities of AVE were examined by melanin synthesis, tyrosinase activity assay and Western blot in B16F10 mouse melanoma cells and normal human primary melanocytes (NHMC). ResultsAVE increased the tyrosinase activity and melanin content in a dosage-dependent manner at concentrations of 1–40μg/ml and treatment with 20μg/ml AVE enhanced the expression of tyrosinase time-dependently in both B16F10 cells and NHMC. Whether AVE affects the expression of microphthalmia-associated transcription factor (MITF), which is required for tyrosinase expression, was investigated. Our results showed that AVE induced MITF protein expression up-regulation. Besides, Western blot analysis revealed that AVE promoted the level of phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) markedly at 0–6h, while the level of phosphorylation of CREB at 0–2h. The special p38 MAPK inhibitor, SB203580, and protein kinase A (PKA) inhibitor, H89, both attenuated the AVE-induced up-regulation of MITF and tyrosinase expression in B16F10 cells and NHMC. However, SB203580 could significantly decrease the melanin biosynthesis induced by AVE, but not H89. ConclusionAVE exerts its improving effect on melanogenesis mainly by p38 MAPK activation and MITF induction of tyrosinase.