ABSTRACT
BACKGROUND: In mammals, UV radiation induces melanin synthesis in melanocyte for protecting their skin through the stimulation of α-melanocyte stimulating hormone (α-MSH) from keratinocytes. In this study, the inhibitory effects of dehydroglyasperin C (DGC), an useful component of Glycyrrhiza uralensis (G. uralensis), was investigated on melanogenesis induced by α-melanocyte stimulating hormone (α-MSH) and its mechanisms. METHODS: Melanogenesis suppression effect of DGC on α-MSH induced B16F1 melanoma cells. The cell viability was measured by MTT assay. Expression and phosphorylation of melanogeic protein were conducted using western blot. cAMP acceleration was measured by cAMP immunoassay kit. To investigate whitening mechanism, we used ERK inhibitor (PD98059). RESULTS: DGC decreased intra cellular tyrosinase (TYR) activity and expression of melanin synthesis related proteins (TYR and TRP-1) in a dose-dependent manner on α-MSH induced melanogenesis. In addition, DGC induced the downregulation of MITF (melanocyte-specific transcription factor) through suppression of cAMP-CREB pathway. Also, phosphorylation of extracellular signal regulated kinase (ERK) decreased MITF by DGC treatment. CONCLUSION: Therefore, DGC could be used as a whitening ingredient in skin and clinical usage against hyperpigmentation.
Subject(s)
Benzopyrans/pharmacology , Cyclic AMP/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Melanins/biosynthesis , Melanoma, Experimental/drug therapy , Melanoma, Experimental/pathology , Microphthalmia-Associated Transcription Factor/metabolism , Animals , Cell Line, Tumor , Cell Survival/drug effects , Dose-Response Relationship, Drug , Down-Regulation/drug effects , Enzyme Activation/drug effects , Flavonoids/pharmacology , Melanoma, Experimental/enzymology , Melanoma, Experimental/metabolism , Membrane Glycoproteins/metabolism , Mice , Monophenol Monooxygenase/metabolism , Oxidoreductases/metabolism , alpha-MSH/antagonists & inhibitors , alpha-MSH/pharmacologyABSTRACT
BACKGROUND: Microphthalmia-associated transcription factor (MITF) is regulated by expression and/or degradation pathway, controlling to the expression of melanogenic enzymes for melanin synthesis. Methyl-2-acetylamino-3-(4-hydroxyl-3,5-dimethoxybenzoylthio)propanoate (MAHDP) is reported to anti-melanogenesis effect but its mechanism remain unclear. OBJECTIVE: To investigate the effects of MAHDP on melanogenesis and elucidate its mechanism. METHODS: Tyrosinase activity, melanogenic proteins and gene expression levels were measured with MAHDP treatment in B16F1 cells, human melanocytes, reconstructed skin and clinical trial. RESULTS: MAHDP attenuated melanin production in α-MSH (melanocyte stimulating hormone) stimulated-B16F1 cells. MAHDP decreased the expression of tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2). But, MADPH did not affect the phosphorylation of p38 MAPK, JNK and AKT, which are associated with the regulation of MITF expression. These results suggest that MITF downstream is regulated not transcriptionally but translationally. Treatment of MG132 (a proteasomal degradation inhibitor) almost abolished the decrease of MITF protein levels by MAHDP. Phosphorylation and ubiquitination of MITF for proteasomal degradation were increased by treatment of MAHDP. Treatment of PD98059 (an ERK phosphorylation inhibitor) abrogated ERK phosphorylation, downregulation of MITF and tyrosinase as well as the decrease of melanin contents by MAHDP. Therefore, the degradation of MITF proteins by MAHDP is regulated to the ERK signaling. Finally, MAHDP improved the pigmentation in human epidermal melanocytes, a UVB-irradiated the reconstructed skin model and clinical trial without cytotoxicity and skin irritation. CONCLUSION: These results clearly demonstrate that MAHDP suppresses the expression of melanogenic enzymes through ERK phosphorylation-mediated MITF proteasomal degradation, and suggest that MAHDP may be efficient as a therapeutic agent for hyperpigmentation.
ABSTRACT
BACKGROUND: The ultraviolet B (UVB) from solar radiation increases the generation of reactive oxygen species (ROS), which mediate the production of matrix metalloproteinases (MMPs), and acts mainly on the epidermis layer of the skin. This study was aimed at assessing the anti-photoaging effects of dehydroglyasperin C isolated from Glycyrrhiza uralensis Fisch on MMPs levels in HaCaT human keratinocytes and to elucidate the underlying mechanism. METHODS: The cell viability was measured by MTT assay. Expression, phosphorylation and enzymatic activity of the protein were examined using ELISA, Western blot or gelatin zymography. Intracellular ROS measurement was evaluated by fluorescent ELISA and 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA) assay. RESULTS: In the present study, we found that dehydroglyasperin C markedly inhibited UVB-mediated expression of collagenase (MMP-1) and gelatinase (MMP-9) by inhibiting ROS generation. Dehydroglyasperin C treatment also decreased the UVB irradiation-mediated activation of mitogen-activated protein kinase (MAPK), c-Jun phosphorylation, and c-Fos expression. In addition, the down-regulation of UVB-induced c-Jun phosphorylation caused by dehydroglyasperin C treatment was more than the down-regulation of c-Fos expression in the HaCaT human keratinocytes. CONCLUSION: Our results indicated that dehydroglyasperin C may function as a potential anti-photoaging agent by inhibiting UVB-mediated MMPs expression via suppression of MAPK and AP-1 signaling.
Subject(s)
Benzopyrans/pharmacology , Keratinocytes/drug effects , Reactive Oxygen Species/metabolism , Ultraviolet Rays , Benzopyrans/isolation & purification , Cell Line , Cell Survival/drug effects , Cell Survival/radiation effects , Enzyme-Linked Immunosorbent Assay , Glycyrrhiza uralensis/chemistry , Humans , Keratinocytes/radiation effects , Matrix Metalloproteinase 1/drug effects , Matrix Metalloproteinase 1/metabolism , Matrix Metalloproteinase 1/radiation effects , Matrix Metalloproteinase 9/drug effects , Matrix Metalloproteinase 9/metabolism , Matrix Metalloproteinase 9/radiation effects , Phosphorylation/drug effects , Proto-Oncogene Proteins c-fos/metabolism , Reactive Oxygen Species/radiation effects , Signal Transduction/drug effects , Signal Transduction/radiation effects , Transcription Factor AP-1/metabolismABSTRACT
AIMS: To report the effects of a novel syringic-acid derivative, (R)-ethyl-2-acetamido-3-(4-hydroxy-3,5-dimethoxybenzoylthio)propanoate (EABTO), on melanin synthesis and to identify its mechanism of action in B16F1 melanoma cells. METHODS: The effects of EABTO on melanin synthesis in B16F1 cells and human epidermal melanocytes and the influence on cell-free tyrosinase activity were evaluated. EABTO-induced cellular signaling cascades were studied by western blotting. KEY FINDINGS: EABTO effectively decreased melanin synthesis in a dose-dependent manner but had no effect on cell-free tyrosinase activity. EABTO significantly decreased the expression of melanogenic enzymes such as tyrosinase, tyrosinase-related protein 1 (TRP-1), and TRP-2. EABTO decreased the amounts of phosphorylated cAMP response element-binding protein (CREB) and cyclic adenosine monophosphate (cAMP), thereby inhibiting expression of microphthalmia-associated transcription factor (MITF). Moreover, EABTO upregulated phosphorylated ERK. A specific ERK pathway inhibitor, PD98059, reduced EABTO-induced ERK phosphorylation and restored the expression of MITF and melanin content. SIGNIFICANCE: EABTO inhibits melanogenesis in B16F1 melanoma cells via suppression of the cAMP-CREB pathway and activation of ERK, thus decreasing expression of MITF and of melanogenic enzymes.
