Biphasic pro-melanogenic and pro-apoptotic effects of all-trans-retinoic acid (ATRA) on human melanocytes: Time-course study

Background: The effects of retinoids on melanogenesis and their mechanism as depigmenting agents in topical therapy have not been fully elucidated. Conflicting data about their impact on melanogenic pathways have been reported. Objective: To investigate the effects of all-trans-retinoic acid (ATRA) on normal human melanocytes from Caucasian subjects. Methods: We assessed ATRA's cytotoxicity by measuring viability with a cell proliferation assay, and apoptotic effects using Annexin V and gamma-H(2)AX markers. ATRA's melanogenic activity was investigated based on spectrophotometric measurement of melanin content and tyrosinase enzymatic activity. Tyrosinase expression was assessed by Western blotting. We tested the antioxidant activity of superoxide dismutase (SOD) and catalase (CAT) in melanocytes using a spectrophotometric assay. Results: Of the concentrations tested in this 72 h time-course study, the 1.0 mu M ATRA had a well-defined two-stage pro-melanogenic and pro-apoptotic effect on melanocytes. In the first 6 h, treated cells showed significant increase (p <= 0.01) of melanin content, tyrosinase, SOD, and CAT activities compared to the controls. While overall tyrosinase expression was not affected by ATRA, all other tested parameters decreased progressively beyond the short-term point of 6 h. ATRA treatment of over 6 h induced melanocyte apoptosis, as shown by the time-dependent decrease in cell viability, coupled with significant increase in Annexin V positive cells and nuclear accumulation of gamma-H(2)AX foci. Conclusion: The results obtained using this testing platform show a biphasic ATRA action: immediate pro-melanogenic effect and longer-term exposure pro-apoptotic activity. These data qualify ATRA as a potent tool to better understand the mechanisms that regulate the pigmentary system. (C) 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.