Inhibition of melanogenesis by gallic acid: possible involvement of the PI3K/Akt, MEK/ERK and Wnt/β-catenin signaling pathways in B16F10 cells

Abstract

Gallic acid is one of the major flavonoids found in plants. It acts as an antioxidant, and seems to have anti-inflammatory, anti-viral, and anti-cancer properties. In this study, we investigated the effects of gallic acid on melanogenesis, including the activation of melanogenesis signaling pathways. Gallic acid significantly inhibited both melanin synthesis and tyrosinase activity in a dose- and time-dependent manner, and decreased the expression of melanogenesis-related proteins, such as microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein-1 (TRP1), and dopachrome tautomerase (Dct). In addition, gallic acid also acts by phosphorylating and activating melanogenesis inhibitory proteins such as Akt and mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK). Using inhibitors against PI3K/Akt (LY294002) or MEK/ERK-specific (PD98059), the hypopigmentation effect was suppressed, and the gallic acid-initiated activation of MEK/ERK and PI3K/Akt was also revoked. Gallic acid also increased GSK3β and p-β-catenin expression but down-regulated p-GSK3β. Moreover, GSK3β-specific inhibitor (SB216763) restored gallic acid-induced melanin reduction. These results suggest that activation of the MEK/ERK, PI3K/Akt, and inhibition of Wnt/β-catenin signaling pathways is involved in the melanogenesis signaling cascade, and that activation by gallic acid reduces melanin synthesis via down-regulation of MITF and its downstream signaling pathway. In conclusion, gallic acid may be a potentially agent for the treatment of certain skin conditions.

Figure 1

Effect of gallic acid on the cell viability of B16F10 cells. B16F10 melanoma cells were treated with various concentrations of gallic acid for 24 h, and cell viability was determined by 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The data presented are from three independent experiments (^#p < 0.05 compared with the control).

Figure 2

Tyrosinase activity and melanin synthesis in B16F10 cells with gallic acid treatment. Cells were treated with 0–400 μM gallic acid to evaluate the cellular tyrosinase activity and cellular melanin content. (A) Cellular tyrosinase activity stain; (B) Cellular tyrosinase activity assay; (C) Cellular melanin content. The data presented are from three independent experiments (^#p < 0.05, * p < 0.001 compared with the control).

Figure 3

Expressions of melanogenesis-related proteins in B16F10 cells with gallic acid treatment. Western blotting data show the changes in MC1R, MITF, p-MITF, tyrosinase, TRP1, and Dct expressions in B16F10 melanoma cells treated with gallic acid at different concentrations (0–200 μM) for 24 h and treated with 200 μM of gallic acid at different times. β-Actin was used as the protein loading control. Statistical results represented as Means ± SEM (n = 3) by ANOVA with the Tukey-Kramer test (* p < 0.001 compared with the control).

Figure 4

Intracellular cAMP concentration and the expressions of melanogenesis-related proteins in B16F10 melanoma cells with gallic acid treatment. (A) Cells were treated with 0~400 μM gallic acid for the indicated durations. Intracellular cAMP levels were measured using a cAMP ELISA kit. The results were representative of three independent experiments (^#p < 0.05, * p < 0.001 compared with the control); (B) The western blot assay results showed the changes in p-MEK, MEK, p-ERK, ERK, p-Akt, Akt, p-RSK1, RSK1, p-CREB, and CREB expressions in B16F10 melanoma cells treated with different concentrations of gallic acid (0–200 μM) for 24 h and treated with 200 μM gallic acid at different times. β-Actin was used as the protein loading control. Statistical results represented as Means ± SEM (n = 3) by ANOVA with the Tukey-Kramer test (^#p < 0.05, * p < 0.001 compared with the control).

Figure 5

Recovery effect of co-treatment with PI3K/Akt or MEK/ERK specific inhibitors and gallic acid on cellular melanin content, cellular tyrosinase activity and melanogenesis-related proteins expression in B16F10 cells. Cells were pretreated with (or without) 20 μM MEK/ERK inhibitor (PD98059) or PI3K/Akt inhibitor (LY294002) for 1 h and further incubated with 200 μM gallic acid for 24 h. (A) The cellular melanin content and cellular tyrosinase activity were evaluated. The results shown are representative of three independent experiments (^#p < 0.05, * p < 0.001 compared with the control); (B) Melanogenesis-related proteins expressions were analyzed by western blotting. β-Actin was used as the protein loading control. (GA: gallic acid). Statistical results represented as Means ± SEM (n = 3) performed by ANOVA with the Tukey-Kramer test (^#p < 0.05, * p < 0.001 compared with the control).

Figure 5

Recovery effect of co-treatment with PI3K/Akt or MEK/ERK specific inhibitors and gallic acid on cellular melanin content, cellular tyrosinase activity and melanogenesis-related proteins expression in B16F10 cells. Cells were pretreated with (or without) 20 μM MEK/ERK inhibitor (PD98059) or PI3K/Akt inhibitor (LY294002) for 1 h and further incubated with 200 μM gallic acid for 24 h. (A) The cellular melanin content and cellular tyrosinase activity were evaluated. The results shown are representative of three independent experiments (^#p < 0.05, * p < 0.001 compared with the control); (B) Melanogenesis-related proteins expressions were analyzed by western blotting. β-Actin was used as the protein loading control. (GA: gallic acid). Statistical results represented as Means ± SEM (n = 3) performed by ANOVA with the Tukey-Kramer test (^#p < 0.05, * p < 0.001 compared with the control).

Figure 6

The expressions of Wnt/β-catenin related-proteins in B16F10 melanoma cells treated with gallic acid. (A) The Western blot assay results showed the changes in GSK3β, p-GSK3β, β-catenin, and p-β-catenin expressions in B16F10 melanoma cells treated with different concentrations of gallic acid (0–200 μM) for 24 h and treated with 200 μM of gallic acid at different times. β-Actin was used as the protein loading control. Statistical results represented as Means ± SEM (n = 3) performed by ANOVA with the Tukey-Kramer test (^#p < 0.05, * p < 0.001 compared with the control); (B) The cellular melanin content was evaluated. These data are representative of three independent experiments (^#p < 0.05 compared with the control).