Capsaicin reverses the inhibitory effect of licochalcone A/β-Arbutin on tyrosinase expression in b16 mouse melanoma cells

Abstract

Introduction: Melanin is synthesized by melanocytes, which are located in the basal layer of the skin. After synthesis, melanin is further deposited on the surface of the skin to form black spots or chloasma. Tyrosinase is a rate-limiting enzyme that plays an important role in melanogenesis. Currently, there are many drugs that inhibit tyrosinase expression to further reduce melanogenesis. Nevertheless, some of these could reverse the pharmacological effect of other drugs, when used simultaneously.

Materials and methods: B16 mouse melanoma cells were treated with the tyrosinase inhibitors licochalcone A and β-arbutin, alone or in combination with capsaicin, an alkaloid found in peppers. Cytotoxicity, melanin content, and tyrosinase activity and expression were determined.

Results: Licochalcone A/β-arbutin inhibited tyrosinase expression and further hindered melanin synthesis when applied individually to B16 mouse melanoma cells. However, licochalcone A/β-arbutin combined with 50 μmol/L capsaicin enhanced the expression of tyrosinase in these cells and further increased melanin content.

Conclusion: Our data implied that capsaicin could reverse the inhibitory effect of licochalcone A/β-arbutin on tyrosinase expression in B16 mouse melanoma cells.

Summary: B16 mouse melanoma cells were treated with the tyrosinase inhibitors licochalcone A and β-arbutin, alone or in combination with capsaicin, an alkaloid found in peppers. Cytotoxicity, melanin content, and tyrosinase activity and expression were determined. Licochalcone A/β-arbutin inhibited tyrosinase expression and further hindered melanin synthesis when applied individually to B16 mouse melanoma cells. However, licochalcone A/β-arbutin combined with 50 μmol/L capsaicin enhanced the expression of tyrosinase in these cells and further increased melanin content. Our research implied that capsaicin could reverse the inhibitory effect of licochalcone A/β-arbutin on tyrosinase expression in B16 mouse melanoma cells. Abbreviations used: B16: B16 mouse melanoma cells; L-DOPA: 3, 4-L-dihydroxyphenylalanine; TYR: Tyrosinase; USP: United States Pharmacopeia; FBS: Fetal bovine serum; EDTA: Ethylenediaminetetraacetic acid; DMSO: Dimethyl sulfoxide; RPMI: Roswell Park Memorial Institute; MTT3: 4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, NaOH: Sodium hydroxide; PBS: Phosphate-buffered saline; RIPA: Radio-immunoprecipitation assay; PMSF: Phenylmethanesulfonyl fluoride or phenylmethylsulfonyl fluoride; SDS: Sodium dodecyl sulfate, sodium salt; PVDF: Polyvinylidene fluoride; ECL: Enhanced chemiluminescence.

Keywords: Capsaicin; licochalcone A; melanin; melanocyte; tyrosinase; β-arbutin.

Figure 1

Effects of licochalcone A (a) or β-arbutin (b) on melanogenesis in B16 mouse melanoma cells simultaneously treated with or without 50 μmol/L capsaicin. Melanin production was assessed by performing a NaOH assay, and cells were treated for 48 h. All data are shown as mean ± standard deviation (n = 3). *P < 0.05, ** P < 0.001, compared to the control group

Figure 2

Effects of licochalcone A (a) or β-arbutin (b) on tyrosinase activity in B16 mouse melanoma cells simultaneously treated with or without 50 μmol/L capsaicin. Tyrosinase activity was assessed by performing an L-DOPA-oxidation-assay 48 h after treatment. All data are shown as mean ± SD (n = 3); *P < 0.05, **P < 0.001, compared to the control group

Figure 3

Western blot showing tyrosinase expression in B16 mouse melanoma cells after treatment with licochalcone A or β-arbutin simultaneously with or without 50 μmol/L capsaicin. (1): The blank control group, (2): licochalcone A and capsaicin, (3): licochalcone A, (4): capsaicin, (5): β-arbutin and capsaicin, (6): β-arbutin