Melanin Synthesis In B16F10 Cells Research Articles

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

BackgroundSsanghwa-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.MethodCellular 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.ResultsSHT 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.ConclusionSHT 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.

P047 Role of IL-18 in the melanin synthesis of B16F10 melanoma cells

Introduction Interleukin-18 (IL-18), a member of the IL-1 cytokine super-family, has been known to interferon-g-inducing factor and plays a critical role in angiogenesis, bone metabolism, metabolic syndrome, inflammatory and immune response. However, the role of IL-18 in the melanin synthesis of B16F10 melanoma cells has not been investigated. Methods B16F10 cells were treated with IL-18 and/or for 5 days, and were then dissolved in 1N NaOH in 10% DMSO at 80 °C for 1 h. The relative melanin contents were determined by measuring the absorbance at 405 nm using an ELISA reader. Expression of IL-18, IL-18R and melanogenesis-related genes was examined by real time PCR. Results Here, we found that IL-18 significantly induced the melanin synthesis in B16F10 cells, and anti-IL-18 abrogated IL-18-induced melanin synthesis. We also observed that anti-IL-18 decreased gene and protein expression of microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein (TRP)-1, and dopachrome tautomerase (Dct) stimulated by IL-18. In addition, anti-IL-18 reduced IL-18-induced phosphorylation of mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK). Conclusion Taken together, these results suggest that IL-18 may be involved in regulation of the melanin synthesis in B16F10 cells via the ERK pathway.

Zeolite 4A, a Synthetic Silicate, Suppresses Melanogenesis through the Degradation of Microphthalmia-Associated Transcription Factor by Extracellular Signal-Regulated Kinase Activation in B16F10 Melanoma Cells

Zeolite 4A, synthetic silicate, has been shown to exhibit diverse biological activities such as anti-cancer and anti-oxidant activity. In the present study, we report that the zeolite 4A may improve skin-whitening. We found that zeolite 4A inhibited melanin production in a dose-dependent manner, which has not cytotoxicity. Zeolite 4A also inhibited alpha-melanocyte-stimulating hormone (alpha-MSH)-induced melanin synthesis in B16F10 cells. Interestingly, zeolite 4A decreased alpha-MSH-induced tyrosinase activity in B16F10 cells, which did not inhibit tyrosinase activity under cell-free conditions. The results of this study indicate that zeolite 4A may reduce pigmentation by way of an indirect nonenzymatic mechanism. We also found that zeolite 4A decreased alpha-MSH-induced microphthalmia-associated transcription factor (MITF) and tyrosinase expression and that zeolite 4A induced the activation of extracellular signal-regulated kinase (ERK). These results suggest that the depigmenting effect of zeolite 4A may result from the down-regulation of MITF and tyrosinase expression by increasing ERK activity. The results thus provide evidence that zeolite 4A can be used as a potential skin-whitening agent.

Inhibitory effect of p-coumaric acid by Rhodiola sachalinensis on melanin synthesis in B16F10 cells.

Rhodiola has been widely used in traditional Asian medicine. In this study, we tested the hypopigmentation effects of R. sachalinensis and its active compounds including catechin, chlorogenic acid, p-coumaric acid, and p-tyrosol. Results have shown that only p-coumaric acid inhibits melanin synthesis in B16F10 cells. However, p-coumaric acid did not inhibit tyrosinase activity when L-DOPA was used as a substrate. Instead, p-coumaric acid inhibited tyrosinase activity when L-tyrosine was used as a substrate. We further analyzed the changes of cAMP responsive element binding protein (CREB) phosphorylation and tyrosinase gene expression. The results indicate that p-coumaric acid does not affect CREB phosphorylation or tyrosinase protein production. In turn, these findings demonstrate that p-coumaric acid has no effect on the upstream regulation of tyrosinase gene expression, although p-coumaric acid showed a significant inhibitory effect on melanogenesis. Because p-coumaric acid showed different effects on tyrosinase activity according to different substrates, we tested whether tyrosinase can utilize p-coumaric acid as a substrate. Our findings revealed that competitive inhibition occurs between p-coumaric acid and tyrosine. Consequently, this finding could be a primary mechanism for the hypopigmenting action of p-coumaric acid.