Depigmenting Effect of Resveratrol Is Dependent on FOXO3a Activation without SIRT1 Activation

Abstract

Resveratrol exhibits not only anti-melanogenic property by inhibiting microphthalmia-associated transcription factor (MITF), but also anti-aging property by activating sirtuin-1 (SIRT1). In this study, the relationship between depigmenting effect of resveratrol and SIRT1/forkhead box O (FOXO) 3a activation and was investigated. Resveratrol suppressed melanogenesis by the downregulation of MITF and tyrosinase via ERK pathway. Results showed that the expression of both SIRT1 and FOXO3a were increased. It is reported that SIRT1 is critical regulator of FOXO-mediated transcription in response to oxidative stress. However in our study, FOXO3a activation appeared earlier than that of SIRT1. Furthermore, the effect of resveratrol on the levels of MITF and tyrosinase was suppressed when melanocytes were pre-treated with SP600125 (JNK inhibitor). However, pre-treatment with SIRT1 inhibitor (EX527, or sirtinol) did not affect the levels of MITF and tyrosinase. Therefore, resveratrol inhibits melanogenesis through the activation of FOXO3a but not by the activation of SIRT1. Although SIRT1 activation by resveratrol is a well-known mechanism of resveratrol-induced antiaging effects, our study showed that not SIRT1 but FOXO3a activation is involved in depigmenting effects of resveratrol.

Keywords: extracellular signal-regulate kinase; forkhead box O; melanogenesis; resveratrol; sirtuin-1.

Figure 1

Fluorescent microscopic observation. After resveratrol (50 µM, overnight) treatment, cells then treated by hydrogen peroxide (50 µM, overnight). Fluorescent microscopic examination was done after H₂DCF-DA (10 µM) staining.

Figure 2

(a) ELISA was carried after adding L-DOPA to normal human melanocytes which were treated with resveratrol for three days to measure tyrosinase activity; (b) Cells were treated with resveratrol for 72 h and amount of melanin was measured; (c) Direct inhibition of tyrosinase by resveratrol was studied using a cell-free system.

Figure 3

Normal human melanocytes were treated with 10 to 100 μM of resveratrol for 24 h. Then, the levels of ERK, MITF, tyrosinase, SIRT1, and FOXO3a were investigated.

Figure 4

Time-dependent changes of SIRT1 and FOXO3a were investigated after treating normal human melanocytes with 50 μM of resveratrol.

Figure 5

Normal human melanocytes were treated with resveratrol (50 μM) after pre-treated with EX527 (SIRT1 inhibitor) and SP600125 (JNK inhibitor).

Figure 6

Normal human melanocytes were treated with resveratrol (50 μM) with or without pre-treatment with sirtinol (SIRT1 inhibitor).

Figure 7

Fluorescent microscopic observation of SIRT1, FOXO3a in resveratrol-treated normal human melanocytes. (a) After resveratrol treatment, cells were stained at different time points (0, 6, 16, and 24 h); (b) Staining intensity of SIRT1 and FOXO3a after resveratrol treatment at different time points (0, 6, 16, and 24 h). Scale bar = 50 μm.