Grape Extract Promoted α-MSH-Induced Melanogenesis in B16F10 Melanoma Cells, Which Was Inverse to Resveratrol

Abstract

Melanin is a natural pigment produced by cells to prevent damage caused by ultraviolet radiation. Previously, resveratrol was shown to reduce melanin synthesis. As a natural polyphenol with various biological activities, resveratrol occurs in a variety of beverages and plant foods, such as grapes. Therefore, we investigated whether grape extracts containing resveratrol also had the ability to regulate melanin synthesis. In this study, we used mouse B16F10 melanoma cells as a model for melanin synthesis with the melanogenesis-inducing α-melanocyte-stimulating hormone (α-MSH) as a positive control. Our results confirmed previous reports that resveratrol reduces melanin synthesis by reducing the activity of the rate-limiting enzyme tyrosinase. In contrast, the grape extract could not reduce melanin synthesis, and in fact promoted melanogenesis in the presence of α-MSH. The expression of genes related to melanin synthesis, such as tyrosinase, tyrosinase-related protein-1, tyrosinase-related protein-2, and microphthalmia-associated transcription factor, also supports these phenomena, which means that even in the presence of resveratrol, grape extract will strengthen the function of α-MSH in promoting melanin synthesis. Therefore, these results also provide a point of view for research on cosmetics.

Keywords: B16F10 melanoma cells; grape extract; melanin synthesis; resveratrol; tyrosinase.

Figure 1

Effect of resveratrol (Res; A) and grape extract (GE) on B16F10 melanoma cell viability. (B) Cell viability after 72 h of treatment with Res (10, 20, 50, or 100 μM) or 10 nM α-MSH, measured using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. (C) Cell survival rate (live cell count/total cell count) after 72 h of treatment with Res (10, 20, or 50 μM). Trypan blue staining was used to count the live and dead cells. (D) Cell viability and (E) survival rate after 72 h of treatment with GE (50, 75, or 100 μg/mL), measured using an MTT assay and trypan blue staining, respectively. The results are presented as the mean ± standard deviation; n ≥ 3; *** p < 0.001 vs. Ct (non-treatment group). n.s. means non-significant. Dimethyl sulfoxide (DMSO) at a safe concentration (0.1% v/v) was the solvent for Res, GE, and α-MSH.

Figure 2

Effect of 72 h treatment with resveratrol (Res; 10 μM or 20 μM) and grape extract (GE; 50 μg/mL or 100 μg/mL) on melanogenesis in B16F10 melanoma cells. The positive control for melanin synthesis was α-melanocyte-stimulating hormone (α-MSH). The absorbance at a wavelength of 405 nm was measured after 72 h. The relative melanin content was normalized to the total protein content of the cell. (A) The melanin content in cells without α-MSH stimulation. (B) The melanin content in cells stimulated with α-MSH (10 μM). The results are indicated as the mean ± standard deviation, n ≥ 3. * p < 0.05 vs. Ct (non-treatment), # p < 0.05, ## p < 0.01 vs. α-MSH.

Figure 3

Effect of 72 h treatment with resveratrol (Res; 20 μM) and grape extract (GE; 100 μg/mL) on intracellular tyrosinase activity in B16F10 melanoma cells. The positive control for melanin synthesis was α-melanocyte-stimulating hormone (α-MSH). After 72 h, L-DOPA was added to the cell lysate, and the tyrosinase activity was expressed in terms of the amount of melanin synthesized after 20 min, normalized to the total protein content of the cell. (A) Intracellular tyrosinase activity in melanocytes without α-MSH treatment. (B) Intracellular tyrosinase activity in melanocytes stimulated with α-MSH (10 µM). The results are expressed as the mean ± standard deviation, n ≥ 3, ** p < 0.01 vs. Ct (non-treatment); # p < 0.05, ## p < 0.01 vs. α-MSH.

Figure 4

Effect of 72 h treatment with resveratrol (Res; 20 μM) and grape extract (GE; 100 μg/mL) on melanogenesis-related gene expression in B16F10 melanocytes. The positive control was α-melanocyte-stimulating hormone (α-MSH; 10 nM). Western blotting was performed to evaluate the expression of tyrosinase (90 kDa), tyrosinase-related protein-1 (TRP-1) (70 kDa), tyrosinase-related protein-2 (TRP-2) (60–80 kDa), and microphthalmia-associated transcription factor (MITF) (60–80 kDa) following treatment with (A) Res and (B) GE. The internal control was β-actin. The results are presented as the mean ± standard deviation, n ≥ 3, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Ct (non-treatment); # p < 0.05 vs. α-MSH.