4-n-butylresorcinol enhances proteolytic degradation of tyrosinase in B16F10 melanoma cells.
OBJECTIVE
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.


METHODS
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/PNGase F and E64/proteasome inhibitors, respectively.


RESULTS
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.


CONCLUSIONS
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.
Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul, 110-799, South Korea.
S-J
SJ
Lee
Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul, 110-799, South Korea.
Y H
YH
Son
Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul, 110-799, South Korea.
K B
KB
Lee
Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul, 110-799, South Korea.
J-H
JH
Lee
Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul, 110-799, South Korea.
H-J
HJ
Kim
Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul, 110-799, South Korea.|Institute of Human-Environment Interface Biology, Biomedical Research Institute, Seoul National University Hospital, Daehak-ro 101, Jongno-gu, Seoul 110-799, South Korea.
E M
EM
Jeong
Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul, 110-799, South Korea.
S C
SC
Park
Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul, 110-799, South Korea.|Institute of Human-Environment Interface Biology, Biomedical Research Institute, Seoul National University Hospital, Daehak-ro 101, Jongno-gu, Seoul 110-799, South Korea.
I-G
IG
Kim
D000818:Animals; D045744:Cell Line, Tumor; D006031:Glycosylation; D008546:Melanoma, Experimental / Q000201:enzymology* / Q000473:pathology; D051379:Mice; D014442:Monophenol Monooxygenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D059748:Proteolysis; D012118:Resorcinols / Q000494:pharmacology*
C508220:4-n-butylresorcinol; D012118:Resorcinols; D014442:Monophenol Monooxygenase
4-n-butylresorcinol; melanogenesis; skin physiology; tyrosinase