4‐n‐butylresorcinol is a competitive inhibitor of tyrosinase and has been used as an antimelanogenic agent. However, its inhibition mechanism in intact cells is not fully understood. To elucidate the cellular mechanism, we compared in vitro and in vivo inhibitory effects of 4‐n‐butylresorcinol on tyrosinase activity.
B16F10 melanoma cells were cultured in media containing α ‐MSH in the presence or absence of 4‐n‐butylresorcinol. Tyrosinase mRNA levels, protein levels and activity in B16F10 cells were compared by real‐time PCR , immunostaining combined with western blot and colorimetric analysis, respectively. Melanin concentration was measured by colorimetry both in the cells and in the media. Tyrosinase glycosylation and proteolytic degradation were analysed by immunoblotting after cells were treated with Endo H/PNG ase F and E64/proteasome inhibitors, respectively.
4‐n‐butylresorcinol inhibited tyrosinase activity and melanin synthesis more effectively in intact cells than in cell lysates. Western blotting and real‐time RT ‐PCR showed that 4‐n‐butylresorcinol reduced protein levels, but not mRNA levels, of tyrosinase in B16F10 cells. 4‐n‐butylresorcinol showed no effect on the processing of tyrosinase glycosylation or on trafficking to melanosomes. However, treatment of B16F10 cells with E64 or proteasome inhibitor abrogated the 4‐n‐butylresorcinol‐induced decrease of tyrosinase. Moreover, 4‐n‐butylresorcinol activated p38 MAPK , resulting in increased ubiquitination of tyrosinase.
4‐n‐butylresorcinol inhibits melanogenesis by enhancing proteolytic degradation of tyrosinase as well as competitive binding to tyrosinase. These findings will help to develop new, effective and safe chemicals for the treatment of hyperpigmentation disorders.
This article describes a new hypopigmenting mechanism of 4‐n‐butylresorcinol, a derivative of resorcinol. 4‐n‐butylresorcinol is known to inhibit tyrosinase and tyrosinase‐related protein‐1 in a substrate‐competitive manner. In this study, 4‐n‐butylresorcinol inhibits melanogenesis by enhancing proteolytic degradation of tyrosinase as well as competitive binding to tyrosinase.
Figure 1 4-n-butylresorcinol inhibits tyrosinase activity more effectively in intact cells than in cell lysates. (A) Mushroom tyrosinase was incubated with various concentrations of 4-n-butylresorcinol at 37°C for 30 min, and then, tyrosinase activity was measured. (B) B16F10 cell lysates were incubated with 5 nM a-MSH and various concentrations of 4-n-butylresorcinol at 37°C for 30 min, and then, tyrosinase activity was measured. (C–E) B16F10 cells were cultured with 5 nM a-MSH in the presence or absence of 10 lM 4-n-butylresorcinol for 48 h. After cell lysis, tyrosinase activity was measured (C), concentration of secreted melanin was determined in cultured media (D) and concentration of intracellular melanin was measured in the cell lysates (E). Results are presented as the mean S.D. Data are representative of at least three experiments. (F) Microscopy images showed melanin deposition in 4-n-butyl-resorcinol-treated B16F10 cells. Magnification 9100, ***P < 0.001 compared with control.
Published in International journal of cosmetic science 2017 (https://doi.org/10.1111/ics.12368)