Daphnane diterpene hirsein B downregulates melanogenesis in B16 murine melanoma cells by cAMP pathway inhibition

Background Skin pigmentation serves as protection against ultraviolet-induced skin damage through melanin’s optical and chemical filtering properties [1]. Although melanin plays and important role in skin protection, excessive melanin production or hyperpigmentation may lead to skin cancer. Recently, the inhibition of melanogenesis has been considered as a valid therapeutic target for the management of advanced melanotic melanomas [2] which increases the need for melanogenesis inhibitors that are of plant origin and are not cytotoxic to mammalian cells. The biosynthesis of the pigment melanin is catalyzed by the melanogenic enzymes tyrosinase, tyrosinase related protein 1 and the dopachrome tautomerase, the transcriptional regulation of which is being regulated by the microphthalmia associated transcription factor (Mitf) [3]. Previously, we have reported that hirsein B (HB) or 5b-hydroxyresiniferonol-6a,7a-epoxy-12b-coumaroyloxy-9,13,14-ortho-decanoate from Thymelaea hirsuta [4] has antimelanogenesis effect (without cytotoxicity) on B16 murine melanoma cells by downregulating the expressions of the Mitf gene and the melanogenic enzymes’ genes [5]. The exact mechanism by which hirsein B inhibited the Mitf gene expression, however, has not yet been determined. In melanogenesis, the Mitf gene expression can be regulated through the cAMP pathway or the Wnt signaling pathway. This study aimed to determine the mechanism underlying the inhibitory effect of HB on Mitf gene in B16 murine melanoma cells. Materials and methods Total RNA was isolated from B16 murine melanoma cells (Riken Cell Bank, Tsukuba, Japan) and used for DNA microarray analysis, using chips of 528 spots loaded with 265 genes prepared by GenopalTM (Mitsubishi Rayon Co., Ltd, Tokyo, Japan), to determine the expressions of genes for melanogenesis, membranebound receptors, tyrosine kinase regulation, melanosome transport, and other cell signal regulation-related genes (including the housekeeping and negative control genes). To validate the results, real-time PCR, using TaqMan FAST 7500 (Applied Biosystems, Foster City, CA, USA) and specific TaqMan primers (Applied Biosystems, Foster City, CA, USA) for the differentiallyexpressed genes, was performed.