Decursin prevents melanogenesis by suppressing MITF expression through the regulation of PKA/CREB, MAPKs, and PI3K/Akt/GSK-3ß cascades.

Abnormal melanin synthesis upon UV exposure causes excessive oxidative stress, which leads to skin hyperpigmentation disorders such as freckles, melisma, and age spots. The present study investigated the anti-melanogenic effects of decursin and the underlying mechanism using multiple approaches. Decursin exhibited no cytotoxicity and significantly reduced intracellular tyrosinase activity and melanin content in B16F10 melanoma cells. Decursin also inhibited the expression of melanogenic enzymes such as tyrosinase and tyrosinase-related protein (TRP)- 1, but not TRP-2. Mechanistically, decursin suppressed melanin synthesis through cAMP-dependent protein kinase (PKA)/cAMP response element-binding protein (CREB)-dependent downregulation of microphthalmia-associated transcription factor (MITF), a master transcription factor in melanogenesis. Further, decursin exerted anti-melanogenic effects by downregulating the p38 signaling pathway and upregulating extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt)/glycogen synthesis kinase-3ß (GSK-3ß) cascades. in silico analysis showed that decursin formed specific interactions with residues of upstream regulators of MITF and exhibited optimal pharmacokinetic profiles, including permeability and skin sensitization. Finally, the anti-melanogenic effects of decursin were confirmed ex vivo in 3D human skin models, suggesting its applicability as a protective agent against hyperpigmentation.

Cajanin Suppresses Melanin Synthesis through Modulating MITF in Human Melanin-Producing Cells.

Despite its classification as a non-life-threatening disease, increased skin pigmentation adversely affects quality of life and leads to loss of self-confidence. Until now, there are no recommended remedies with high efficacy and human safety for hyperpigmentation. This study aimed to investigate anti-melanogenic activity and underlying mechanism of cajanin, an isoflavonoid extracted from Dalbergia parviflora Roxb. (Leguminosae) in human melanin-producing cells. Culture with 50 µM cajanin for 48-72 h significantly suppressed proliferation in human melanoma MNT1 cells assessed via MTT viability assay. Interestingly, cajanin also efficiently diminished melanin content in MNT1 cells with the half maximum inhibitory concentration (IC50) at 77.47 ± 9.28 µM. Instead of direct inactivating enzymatic function of human tyrosinase, down-regulated mRNA and protein expression levels of MITF and downstream melanogenic enzymes, including tyrosinase, TRP-1 and Dct (TRP-2) were observed in MNT1 cells treated with 50 µM cajanin for 24-72 h. Correspondingly, treatment with cajanin modulated the signaling pathway of CREB and ERK which both regulate MITF expression level. Targeted suppression on MITF-related proteins in human melanin-producing cells strengthens the potential development of cajanin as an effective treatment for human hyperpigmented disorders.

Novel Chemically Modified Curcumin (CMC) Derivatives Inhibit Tyrosinase Activity and Melanin Synthesis in B16F10 Mouse Melanoma Cells.

Skin hyperpigmentation disorders arise due to excessive production of the macromolecular pigment melanin catalyzed by the enzyme tyrosinase. Recently, the therapeutic use of curcumin for inhibiting tyrosinase activity and production of melanin have been recognized, but poor stability and solubility have limited its use, which has inspired synthesis of curcumin analogs. Here, we investigated four novel chemically modified curcumin (CMC) derivatives (CMC2.14, CMC2.5, CMC2.23 and CMC2.24) and compared them to the parent compound curcumin (PC) for inhibition of in vitro tyrosinase activity using two substrates for monophenolase and diphenolase activities of the enzyme and for diminution of cellular melanogenesis. Enzyme kinetics were analyzed using Lineweaver-Burk and Dixon plots and nonlinear curve-fitting to determine the mechanism for tyrosinase inhibition. Copper chelating activity, using pyrocatechol violet dye indicator assay, and antioxidant activity, using a DPPH radical scavenging assay, were also conducted. Next, the capacity of these derivatives to inhibit tyrosinase-catalyzed melanogenesis was studied in B16F10 mouse melanoma cells and the mechanisms of inhibition were elucidated. Inhibition mechanisms were studied by measuring intracellular tyrosinase activity, cell-free and intracellular α-glucosidase enzyme activity, and effects on MITF protein level and cAMP maturation factor. Our results showed that CMC2.24 showed the greatest efficacy as a tyrosinase inhibitor of all the CMCs and was better than PC as well as a popular tyrosinase inhibitor-kojic acid. Both CMC2.24 and CMC2.23 inhibited tyrosinase enzyme activity by a mixed mode of inhibition with a predominant competitive mode. In addition, CMC2.24 as well as CMC2.23 showed a comparable robust efficacy in inhibiting melanogenesis in cultured melanocytes. Furthermore, after removal of CMC2.24 or CMC2.23 from the medium, we could demonstrate a partial recovery of the suppressed intracellular tyrosinase activity in the melanocytes. Our results provide a proof-of-principle for the novel use of the CMCs that shows them to be far superior to the parent compound, curcumin, for skin depigmentation.

l-tyrosine induces melanocyte differentiation in novel pink-eyed dilution castaneus mouse mutant showing age-related pigmentation.

BACKGROUND: The mouse pink-eyed dilution (oculocutaneous albinism II; p/Oca2(p)) locus is known to control tyrosinase activity, melanin content, and melanosome development in melanocytes. Pink-eyed dilution castaneus (p(cas)/Oca2(p-cas)) is a novel mutant allele on mouse chromosome 7 that arose spontaneously in Indonesian wild mice, Mus musculus castaneus. Mice homozygous for Oca2(p-cas) usually exhibit pink eyes and beige-colored coat on nonagouti C57BL/6 (B6) background. Recently, a novel spontaneous mutation occurred in the progeny between this mutant and B6 mice. The eyes of this novel mutant progressively become black from pink and the coat becomes dark gray from beige with aging. OBJECTIVE: The aim of this study is to clarify whatever differences exist in melanocyte proliferation and differentiation between the ordinary (pink-eyed) and novel (black-eyed) mutant using serum-free primary culture system. METHODS: The characteristics of melanocyte proliferation and differentiation were investigated by serum-free primary culture system using melanocyte-proliferation medium (MDMD). RESULTS: The proliferation of melanoblasts in MDMD did not differ between the two mice. However, when the epidermal cell suspensions were cultured with MDMD supplemented with l-tyrosine (Tyr), the differentiation of black-eyed melanocytes was greatly induced in a concentration-dependent manner compared with pink-eyed melanocytes. Immunocytochemistry demonstrated that the expression of tyrosinase and tyrosinase-related protein-1 (Tyrp1) was greatly induced or stimulated both in pink-eyed and black-eyed melanocytes, whereas the expression of microphthalmia-associated transcription factor (Mitf) was stimulated only in black-eyed melanocytes. CONCLUSION: These results suggest that the age-related coat darkening in black-eyed mutant may be caused by the increased ability of melanocyte differentiation dependent on l-Tyr through the upregulation of tyrosinase, Tyrp1, and Mitf. This mutant mouse may be useful for animal model to clarify the mechanisms of age-related pigmentation in human skin, such as melasma and solar lentigines.

MITF Modulates Therapeutic Resistance through EGFR Signaling.

Response to targeted therapies varies significantly despite shared oncogenic mutations. Nowhere is this more apparent than in BRAF (V600E)-mutated melanomas where initial drug response can be striking and yet relapse is commonplace. Resistance to BRAF inhibitors have been attributed to the activation of various receptor tyrosine kinases (RTKs), although the underlying mechanisms have been largely uncharacterized. Here, we found that EGFR-induced vemurafenib resistance is ligand dependent. We employed whole-genome expression analysis and discovered that vemurafenib resistance correlated with the loss of microphthalmia-associated transcription factor (MITF), along with its melanocyte lineage program, and with the activation of EGFR signaling. An inverse relationship between MITF, vemurafenib resistance, and EGFR was then observed in patient samples of recurrent melanoma and was conserved across melanoma cell lines and patients' tumor specimens. Functional studies revealed that MITF depletion activated EGFR signaling and consequently recapitulated the resistance phenotype. In contrast, forced expression of MITF in melanoma and colon cancer cells inhibited EGFR and conferred sensitivity to BRAF/MEK inhibitors. These findings indicate that an "autocrine drug resistance loop" is suppressed by melanocyte lineage signal(s), such as MITF. This resistance loop modulates drug response and could explain the unique sensitivity of melanomas to BRAF inhibition.

Ssanghwa-tang, an oriental herbal cocktail, exerts anti-melanogenic activity by suppression of the p38 MAPK and PKA signaling pathways in B16F10 cells.

BACKGROUND: Ssanghwa-tang (SHT) is a widely used medication for the treatment of fatigue, pain, inflammation, hypothermia, erectile dysfunction, cancer, and osteoporosis in Asia, however, role of SHT on the melanin synthesis has not been checked previously. Thus, the present study was designed to determine the effect of SHT on α-melanocyte stimulating hormone (α-MSH)-induced melanogensis and its mechanisms of action in murine B16F10 melanoma cells. METHOD: Cellular melanin content and tyrosinase activity in murine B16F10 melanoma cells were determined after α-MSH stimulation with or without pre-treatment of SHT at the concentration of 250 and 500 µg/ml. Expression level of tyrosinase, tyrosinase-related protein 1 (TRP-1), TRP-2, microphthalmia-associated transcription factor (MITF), and activation of c-AMP-dependent protein kinase (PKA), c-AMP-related element binding protein (CREB), and mitogen-activated protein kinases (MAPKs) were examined by Western blot analysis. RESULTS: SHT significantly inhibited α-MSH-induced melanin synthesis and tyrosinase activity, and also decreased α-MSH-induced expression of MITF, tyrosinase, and TRP-1. In addition, SHT remarkably suppressed tyrosinase, CRE, and MITF luciferase reporter activity in a resting state as well as in α-MSH-stimulating condition. Phosphorylation of p38 MAPK by α-MSH stimulation was efficiently blocked by SHT pre-treatment. Moreover, SHT as an herbal cocktail showed synergistic anti-melanogenic effect compared with that of each single constituent herb. CONCLUSION: SHT efficiently inhibited c-AMP-induced melanin synthesis in B16F10 cells via suppression of PKA and p38 MAPK signaling pathways and subsequently decreased the level of CREB phosphorylation, MITF, and melanogenic enzymes. These results indicate that SHT may be useful as herbal medicine for treating hyperpigmentation and cosmetics as a skin-whitening agent.

Antimelanogenic effect of c-phycocyanin through modulation of tyrosinase expression by upregulation of ERK and downregulation of p38 MAPK signaling pathways.

BACKGROUND: Pigmentation is one of the essential defense mechanisms against oxidative stress or UV irradiation; however, abnormal hyperpigmentation in human skin may pose a serious aesthetic problem. C-phycocyanin (Cpc) is a phycobiliprotein from spirulina and functions as an antioxidant and a light harvesting protein. Though it is known that spirulina has been used to reduce hyperpigmentation, little literature addresses the antimelanogenic mechanism of Cpc. Herein, we investigated the rationale for the Cpc-induced inhibitory mechanism on melanin synthesis in B16F10 melanoma cells. METHODS: Cpc-induced inhibitory effects on melanin synthesis and tyrosinase expression were evaluated. The activity of MAPK pathways-associated molecules such as MAPK/ERK and p38 MAPK, were also examined to explore Cpc-induced antimelanogenic mechanisms. Additionally, the intracellular localization of Cpc was investigated by confocal microscopic analysis to observe the migration of Cpc. RESULTS: Cpc significantly (P < 0.05) reduced both tyrosinase activity and melanin production in a dose-dependent manner. This phycobiliprotein elevated the abundance of intracellular cAMP leading to the promotion of downstream ERK1/2 phosphorylation and the subsequent MITF (the transcription factor of tyrosinase) degradation. Further, Cpc also suppressed the activation of p38 causing the consequent disturbed activation of CREB (the transcription factor of MITF). As a result, Cpc negatively regulated tyrosinase gene expression resulting in the suppression of melanin synthesis. Moreover, the entry of Cpc into B16F10 cells was revealed by confocal immunofluorescence localization and immunoblot analysis. CONCLUSIONS: Cpc exerted dual antimelanogenic mechanisms by upregulation of MAPK/ERK-dependent degradation of MITF and downregulation of p38 MAPK-regulated CREB activation to modulate melanin formation. Cpc may have potential applications in biomedicine, food, and cosmetic industries.

Diethylstilbestrol enhances melanogenesis via cAMP-PKA-mediating up-regulation of tyrosinase and MITF in mouse B16 melanoma cells.

BACKGROUND: It is well known that melanin synthesis in melanoma cells is controlled by melanogenic enzymes, which regulate the cAMP-PKA signaling pathway. Estrogen was previously reported to upregulate melanogenesis that is associated with human skin pigmentation. OBJECTIVE: To investigate the influence and mechanism of diethylstilbestrol (DES) on melanogenesis in mouse B16 melanoma cells. METHODS: The effects of diethylstilbestrol on cell viability, melanin content, tyrosinase activity, cAMP level, expression of the tyrosinase family and microphthalmia related transcription factor (MITF) were measured in B16 melanoma. Estrogen receptor (ER) expression were detected in B16 melanoma and A375 melanoma. Diethylstilbestrol-induced melanin synthesis were evaluated in the presence and absence of H89 (a PKA-specific inhibitor) and ICI182, 780 (a pure ER antagonist). Tyrosinase activity, the mRNA levels of tyrosinase and MITF were evaluated in the presence and absence of H89. RESULTS: In B16 cells, diethylstilbestrol increased cell proliferation, melanin synthesis, tyrosinase activity and expression of the tyrosinase family and MITF. ER expression have not difference in human and mouse melanoma. When ER were inhibited by ICI182, 780, DES-induced melanogenesis was significantly reduced. Diethylstilbestrol enhanced the level of cAMP. The upregulation of melanin content and tyrosinase activity stimulated by diethylstilbestrol was significantly attenuated in the presence of H89. Further, diethylstilbestrol-induced upregulation of tyrosinase and MITF were significantly attenuated when the PKA pathway was blocked. CONCLUSIONS: Diethylstilbestrol can enhance melanin synthesis in melanoma cells. This effect is associated with activation of the cAMP-PKA pathway and upregulation of expression and activity of the melanogenesis-related enzyme tyrosinase and MITF.

Subcellular localization of Mitf in monocytic cells.

Microphthalmia-associated transcription factor (Mitf) is a transcription factor that plays an important role in regulating the development of several cell lineages. The subcellular localization of Mitf is dynamic and is associated with its transcription activity. In this study, we examined factors that affect its subcellular localization in cells derived from the monocytic lineage since Mitf is present abundantly in these cells. We identified a domain encoded by Mitf exon 1B1b to be important for Mitf to commute between the cytoplasm and the nucleus. Deletion of this domain disrupts the shuttling of Mitf to the cytoplasm and results in its retention in the nucleus. M-CSF and RANKL both induce nuclear translocation of Mitf. We showed that Mitf nuclear transport is greatly influenced by ratio of M-CSF/Mitf protein expression. In addition, cell attachment to a solid surface also is needed for the nuclear transport of Mitf.

Hypopigmentary action of dihydropyranocoumarin D2, a decursin derivative, as a MITF-degrading agent.

In this study, the decursin derivative dihydropyranocoumarin D2 (1) was selected for its effects on melanogenesis using a spontaneously immortalized mouse melanocyte cell line (Mel-Ab). The results showed that 1 effectively inhibited melanin synthesis in a concentration-dependent manner, but that it did not inhibit tyrosinase in a cell-free system. In addition, the changes in ERK, Akt, and microphthalmia-associated transcription factor (MITF) in response to treatment with 1 were assessed. The results revealed that ERK was dramatically up-regulated and MITF was down-regulated in response to treatment with 1, but that Akt was unchanged. Therefore, the effects of 1 on melanogenesis were examined in the absence or presence of PD98059 (a specific inhibitor of the ERK pathway). PD98059 restored hypopigmentation and the down-regulation of MITF induced by 1. Finally, MITF down-regulation by 1 was clearly restored by both chloroquine, a lysosomal proteolysis inhibitor, and MG132, a proteasome inhibitor.

The hypopigmentary action of KI-063 (a new tyrosinase inhibitor) combined with terrein.

Resorcinol derivatives are known to inhibit melanin synthesis. In this study, resorcinol derivatives were synthesized and screened for their activity on melanogenesis. KI-063 (a tyrosinase inhibitor) was examined for its effects on melanogenesis using a spontaneously immortalized mouse melanocyte cell line (Mel-Ab). In a cell-free system, KI-063 directly inhibited tyrosinase, the rate-limiting melanogenic enzyme. Moreover, in a cell system, it inhibited melanin synthesis in a concentration-dependent manner. In addition, KI-063 inhibited the activity of cellular tyrosinase. Thus, this study examined the effects of a combination of KI-063 with terrein, an agent that down-regulates microphthalmia-associated transcription factor. The data suggest that KI-063 has an additive effect in combination with terrein. Thus, the suppression of tyrosinase activity by KI-063 and the inhibition of tyrosinase production by terrein appear to be an optimal combination for skin whitening.

Overload of the heat-shock protein H11/HspB8 triggers melanoma cell apoptosis through activation of transforming growth factor-beta-activated kinase 1.

Molecular therapeutics is a recognized promising approach for melanoma, but relevant target genes remain elusive. We report that overload of the recently cloned H11/HspB8 induces apoptosis in 55% of examined melanoma cultures. Apoptosis was determined by activation of caspases-9 and -3 and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL), and was not seen in normal melanocytes. It was associated with H11/HspB8 complexation with transforming growth factor-beta-activated kinase (TAK) 1 and activation of TAK1 and p38 mitogen activated protein 3 kinases. TAK1 was not bound, nor activated by the H11/HspB8 mutant W51C, which has dominant antiapoptotic activity. beta-Catenin was phosphorylated by activated TAK1, inhibiting its nuclear accumulation and mictophthalmia-associated transcription factor and cyclin dependent kinase 2 expression. The dominant-negative TAK1 mutant K63W inhibited beta-catenin phosphorylation and caspase activation. The data indicate that H11/HspB8 overload causes melanoma growth arrest and apoptosis through TAK1 activation and suggest that H11/HspB8 is a promising molecular therapy target.

Inhibitory effects of 4-n-butylresorcinol on tyrosinase activity and melanin synthesis.

In this study, we investigated the effects of 4-n-butylresorcinol on melanogenesis in a spontaneously immortalized mouse melanocyte cell line, Mel-Ab. Our results show that 4-n-butylresorcinol significantly inhibits melanin synthesis in a concentration-dependent manner. In addition, it was also found to inhibit the activity of tyrosinase, the rate-limiting melanogenic enzyme. Several reports have indicated that the activation of extracellular signal-regulated kinase (ERK) or of Akt reduces melanin synthesis via microphthalmia-associated transcription factor (MITF) down-regulation. Accordingly, we examined the effects of 4-n-butylresorcinol on the ERK and Akt signaling pathways. 4-n-Butylresorcinol did not induce ERK, Akt activation, or MITF degradation, and had no effect on cAMP response element binding protein (CREB) phosphorylation, which stimulates MITF expression. In contrast, 4-n-butylresorcinol strongly reduced tyrosinase activity in a cell-free system. Moreover, 4-n-butylresorcinol showed an additive effect in combination with hinokitiol, which reduces MITF expression. These results show that the hypopigmentary effect of 4-n-butylresorcinol results from its direct inhibition of tyrosinase.