Antimelanogenesis Effect of Methyl Gallate through the Regulation of PI3K/Akt and MEK/ERK in B16F10 Melanoma Cells

Abstract

Methyl gallate is a polyphenolic compound found in many plants, and its antioxidant, antitumor, antibacterial, and anti-inflammatory effects have been extensively studied. More recently, antidepressant-like effects of methyl gallate have been demonstrated in some studies. In the present study, we examined the effects of methyl gallate on melanogenesis, including the tyrosinase inhibitory effect, the melanin content, and the molecular signaling pathways involved in this inhibition. The results showed that methyl gallate inhibited tyrosinase activity and significantly downregulated the expressions of melanin synthesis-associated proteins, including microphthalmia-associated transcription factor (MITF), tyrosinase, dopachrome tautomerase (Dct), and tyrosinase-related protein-1 (TRP1). In conclusion, our findings indicated that activation of MEK/ERK and PI3K/Akt promoted by methyl gallate caused downregulation of MITF and triggered its downstream signaling pathway, thereby inhibiting the production of melanin. In summary, methyl gallate showed significant inhibitory activity against melanin formation, implying that it may be a potential ingredient for application in skin-whitening cosmetics.

Figure 1

Cell viability of methyl gallate-treated B16F10 cells. (a) B16F10 cells were treated with methyl gallate of a series of concentrations, and the cell viability was determined by MTT assay. (b) The morphological change of B16F10 cells after methyl gallate treatment. (100× magnification).

Figure 2

Effects of methyl gallate on tyrosinase activity and melanin production in B16F10 cells. Cells were incubated with 5, 10, 50, 100, 200, and 400 μM methyl gallate for 24 h, and the tyrosinase activity and melanin content in the cells were measured. (a) Tyrosinase enzyme activity assay, (b) activity of tyrosinase detected in polyacrylamide electrophoresis gel after incubation with 3,4-dihydroxyphenylalanine, (c) melanin levels in cells after methyl gallate treatment. Data presented as percentage of the control. ^#p < 0.05, ^∗p < 0.01.

Figure 3

Western blotting analysis of melanogenesis-associated proteins in B16F10 cells treated with methyl gallate. (a) Antibodies against a series of melanogenesis-associated proteins as indicated were utilized to detect the protein expression levels in cells treated with 50, 100, and 200 μM methyl gallate. An antibody against β-actin was used as the protein loading control. (b) Quantification of western blot bar graphs with β-actin (C control). ^#p < 0.05, ^∗p < 0.01.

Figure 4

The recovery effect of inhibition of PI3K/Akt or MEK/ERK on tyrosinase activity, melanin content, and melanogenesis-associated protein expressions in B16F10 cells treated with methyl gallate. B16F10 cells were pretreated with/without 20 μM PD98059 or LY294002 for 1 hr, followed by incubation with 200 μM methyl gallate for 24 hr. (a) Melanin content and tyrosinase activity in the cells. Data are presented as percentage of the control, and the results were obtained from a representative of three independent experiments. An antibody against β-actin was used as the protein loading control. (b) Western blotting analysis of melanogenesis-associated protein expressions. (c) Quantification of western blot bar graphs with β-actin (C control). ^#p < 0.05, ^∗p < 0.01.