]. Cosmet. Sci. 1 59, 117-125 (March/April 2008) A dual mechanism of 4-hydroxy-5-methyl-3[2H]-furanone inhibiting cellular melanogenesis HYO JUNG KIM, SANG MI AN and YONG CHOOL BOO, Department of Molecular Medicine (H.].K., S.M.A., Y.C.B.) and Cell and Matrix Research Institute (Y.C.B.), Kyungpook National University School of Medicine, 101 Dongindong-2-ga, Junggu, Daegu, 700-422, Republic of Korea. Accepted for publication November 28, 2007. Synopsis In previous studies, 4-hydroxy-5-methyl-3[2H}-furanone (HMF) was shown to have potent antioxidative and antimelanogenic effects, suggesting its potential use as a depigmenting agent. The present study investigated its mechanism of action on murine melanoma B16F10 cells stimulated by theophylline, an activator of the cyclic AMP/protein kinase A signaling leading to tyrosinase gene expression. When the cells were stimulated with theophylline, there were dose-dependent increases in cellular tyrosinase protein content and melanin formation, as expected. HMF inhibited the theophylline-stimulated melanin formation as effectively as arbutin, one of the most widely used depigmenting agents in cosmetics. HMF appeared to reduce tyrosinase mRNA and protein content in the cells stimulated by theophylline, indicating it inhibited tyrosinase gene expression. HMF also effectively inhibited tyrosinase-catalyzed melanin formation from dihydroxyphenylalanine in the cells as well as in vitro. Therefore, the antimelanogenic effects of HMF were best explained by a dual mechanism inhibiting tyrosinase gene expression and the enzyme activity of pre-existing tyrosinase. INTRODUCTION In a classical model of melanogenesis, a-melanocyte-stimulating hormone (a-MSH) stimulates adenylate cyclase, producing cyclic AMP through the G protein-coupled melanocortin 1 receptor (1). Cyclic AMP activates protein kinase A and CREB (cAMP responsive element binding protein) transcription factor, leading to an increase of MITF (microphthalmia associated transcription factor) (2). Binding of MITF to the gene promoters of melanogenic enzymes induces their gene expression (3). Newly synthesized tyrosinase protein undergoes maturation and activation by multiple mechanisms includ- ing copper binding, glycosylation, and phosphorylation (4-6). The active tyrosinase then Hyo Jung Kim and Sang Mi An contributed equally to this work. Address all correspondence to Yong Chool Boo. 117

118 JOURNAL OF COSMETIC SCIENCE catalyzes the oxidation of tyrosine and dihydroxyphenylalanine (DOPA), subsequently leading to the formation of melanin (7). Skin pigmentation triggered by pathophysiological or environmental factors has a great cosmetic relevance and has prompted the screening of effective natural or chemical agents for decreasing melanogenesis (8). The potential depigmenting agents so far identified include inhibitors of tyrosinase gene expression (9, 10) and inhibitors of ty- rosinase enzyme activity (11-13). In our previous studies, 4-hydroxy-5-methyl-3[2H}- furanone (HMF) has been isolated from pine needles as an antioxidant component and has been shown to inhibit tyrosinase activity, suggesting its potential use as a depig- menting agent (14, 15 ). In an attempt to clarify the action mechanism of HMF, the present study investigates its effects on melanin formation and tyrosinase expression in the murine melanoma B16F10 cells stimulated with theophylline, an activator of cyclic AMP/protein kinase A signaling, leading to tyrosinase gene expression. It also examines if HMF affects tyros- inase-catalyzed DOPA oxidation in vitro and in situ in the cells. The results suggest that HMF may inhibit cellular melanogenesis through a dual mechanism inhibiting tyros- inase gene expression and enzyme activity of the pre-existing tyrosinase. MATERIALS AND METHODS REAGENTS HMF, arbutin, theophylline, and DOPA were purchased from Sigma-Aldrich (St. Louis, MO). Antibodies for tyrosinase and �-actin were purchased from Santa Cruz Biotech (Santa Cruz, CA) and Sigma-Aldrich, respectively. CELL CULTURE AND MELANIN MEASUREMENT Murine melanoma Bl6F10 cells (CRL-6475) were obtained from American Type Cul- ture Collection (Manassas, VA). The cells were cultured in Dulbecco's modified Eagle's medium that contained 10% fetal bovine serum, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 µg/ml amphotericin B at 3 7°C in a humidified atmosphere containing 5% CO2 and 95% air. Typically, cells cultured in 24-well culture plates were treated with HMF or arbutin at 50 ~ 400 µM for 60 minutes prior to stimulation with 1.0 mM theophylline for 72 hours. After incubation, the extracellular melanin content in the cell culture media was observed at 490 nm using a multi-well scanning spectro- photometer. Cell morphology and pigmentation were examined under an inverted-phase microscope (Eclipse TSlO0) from Nikon (Melville, NY). Cell viability was assayed using 3-[ 4, 5-dimethy l thiazol-2-y 1)-2, 5-di pheny 1 tetrazoli um bromide (MTT). PREPARATION OF CELL L YSA TES AND WESTERN BLOTTING After treatment, cells were washed twice with ice-cold PBS and yielded into a lysis buffer (10 mM Tris-Cl, pH 7.4, 120 mM NaCl, 25 mM KCl, 2 mM EGTA, 1 mM EDTA, and 0.5% Triton X-100 and protease inhibitor cocktail) in a pre-cooled tube. After 45 minutes of lysis on ice, the homogenate was centrifuged at 14,000 x g for 15 minutes