Abstract: We obtained metastasized melanoma tissue from a primary acral lentiginous melanoma (ALM) patient and established a melanoma cell line named primary culture of melanoma cell derived from lymph node (PML)-1. PML-1 cells had a light brown color and decreased the expression of melanogenesis markers, including tyrosinase (TYR), microphthalmia-associated transcription factor, and tyrosinase-related protein-1. To identify genes differentially regulated in PML-1 melanoma cells, we performed DNA microarray and two-dimensional matrix-assisted laser desorption ionization-time of flight mass spectrometry analyses. Among the candidate genes identified, we chose NAD(P)H:quinone oxidoreductase-1 (NQO1) for further study. Reverse transcription-PCR and western blot analyses showed that NQO1 was markedly decreased in PML-1 cells and in several amelanotic melanoma cell lines. To investigate whether NQO1 affects the melanogenesis, we treated the cultured normal human melanocytes (NHMC) and zebrafish with NQO1 inhibitors, ES936 and dicoumarol. Interestingly, melanogenesis was significantly decreased by the addition of NQO1 inhibitors in both NHMC and zebrafish models. In contrast, overexpression of NQO1 using a recombinant adenovirus clearly induced melanogenesis, concomitantly with an increase of TYR protein level. These results suggest that NQO1 is a positive regulator of the pigmentation process.
Abstract: Haginin A, an isoflav-3-ens isolated from the branch of Lespedeza cyrtobotrya, is almost unknown. Here, we report that haginin A exhibits a strong hypopigmentary effect in Melan-a cells and significantly inhibits melanin synthesis. Haginin A shows potent inhibitory effects with an IC(50) (half-maximal inhibitory concentration) value of 5.0 microM on mushroom tyrosinase activity, and functioned as a noncompetitive inhibitor. Also, haginin A decreased microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related protein-1 (TRP-1) protein production. To identify the signaling pathway of haginin A, the ability of haginin A to influence extracellular signal-regulated protein kinase (ERK) and Akt/protein kinase B (PKB) activation was investigated. Apparently, haginin A induced ERK and Akt/PKB in a dose-dependent manner. In addition, the specific inhibition of the ERK and the Akt/PKB signaling pathways by PD98059 and LY294002, respectively, increased melanin synthesis. Furthermore, haginin A decreased UV-induced skin pigmentation in brown guinea-pigs. Also, haginin A presented remarkable inhibition on the body pigmentation in the zebrafish model system and decreased tyrosinase activity. Together, haginin A is an effective inhibitor of hyperpigmentation caused by UV irradiation or by pigmented skin disorders through downregulation via ERK and Akt/PKB activation, MITF, and also by the subsequent downregulation of tyrosinase and TRP-1 production.
Abstract: Although many hypo-pigmenting agents are currently available, the demand for novel whitening agents is increasing, in part due to the weak effectiveness and unwanted side effects of currently available compounds. To screen for novel hypo-pigmenting agents, many methodologies such as cell culture and enzymatic assays are routinely used. However, these models have disadvantages in terms of physiological and economic relevance. In this study, we validated zebrafish as a whole-animal model for phenotype-based screening of melanogenic inhibitors or stimulators. We used both the well-known melanogenic inhibitors (1-phenyl-2-thiourea, arbutin, kojic acid, 2-mercaptobenzothiazole) and newly developed small molecule compounds (haginin, YT16i). All the tested compounds produced inhibitory effects on the pigmentation of zebrafish, most likely due to their inhibitory potential on tyrosinase activity. In simultaneous in vivo toxicity tests, a newly developed melanogenic inhibitor YT16i showed massive abnormalities in terms of deformed morphologies and cardiac function. Together, these results provide a rationale in screening and evaluating the putative melanogenic regulatory compounds. We suggest that the zebrafish system is a novel alternative to mammalian models, with several advantages including the rapidity, cost-effectiveness, and physiological relevance.
