BackgroundSkin pigmentation serves as protection against ultravio-let-induced skin damage through melanin’s optical andchemical filtering properties [1]. Although melanin playsand important role in skin protection, excessive melaninproduction or hyperpigmentation may lead to skin can-cer. Recently, the inhibition of melanogenesis has beenconsidered as a valid therapeutic target for the manage-ment of advanced melanotic melanomas [2] whichincreases the need for melanogenesis inhibitors that areof plant origin and are not cytotoxic to mammaliancells. The biosynthesis of the pigment melanin is cata-lyzed by the melanogenic enzymes tyrosinase, tyrosinaserelated protein 1 and the dopachrome tautomerase, thetranscriptional regulation of which is being regulated bythe microphthalmia associated transcription factor(Mitf) [3]. Previously, we have reported that hirsein B(HB) or 5b-hydroxyresiniferonol-6a,7a-epoxy-12b-cou-maroyloxy-9,13,14-ortho-decanoate from Thymelaea hir-suta [4] has antimelanogenesis effect (withoutcytotoxicity) on B16 murine melanoma cells by downre-gulating the expressions of the Mitf gene and the mela-nogenic enzymes’ genes [5]. The exact mechanism bywhich hirsein B inhibited the Mitf gene expression, how-ever, has not yet been determined. In melanogenesis, theMitf gene expression can be regulated through thecAMP pathway or the Wnt signaling pathway. Thisstudy aimed to determine the mechanism underlyingthe inhibitory effect of HB onMitf gene in B16 murinemelanoma cells.Materials and methodsTotal RNA was isolated from B16 murine melanomacells (Riken Cell Bank, Tsukuba, Japan) and used forDNA microarray analysis, using chips of 528 spotsloaded with 265 genes prepared by Genopal™ (Mitsu-bishi Rayon Co., Ltd, Tokyo, Japan), to determine theexpressions of genes for melanogenesis, membrane-bound receptors, tyrosine kinase regulation, melanosometransport, and other cell signal regulation-related genes(including the housekeeping and negative controlgenes). To validate the results, real-time PCR, usingTaqMan FAST 7500 (Applied Biosystems, Foster City,CA, USA) and specific TaqMan primers (Applied Bio-systems, Foster City, CA, USA) for the differentially-expressed genes, was performed.ResultsResults showed that the expressions of theMitf geneand the melanogenic enzymes’ genes were downregu-lated, verifying our previous report [5]. In addition, theexpression of the gene for melanocortin 1 receptor(Mc1r) of the cAMP pathway was downregulated whilemost of the genes that were upregulated are thoseinvolved in the Wnt signaling pathway (Table 1).In mouse, peptide hormones from the pituitary glandbind to the MC1R and stimulate melanin productionthrough the cAMP/PKA signalling pathway [6], by indu-cing changes in the protein phosphorylation and geneexpression, through the MITF gene product.ConclusionsThe results obtained suggest that the significant antime-lanogenesis effect of hirsein B is through the inhibitionof the expression of theMc1r gene of the cAMP path-way. HB may therefore be used as a treatment for
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