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Inhibition of melanogenesis by jineol from Scolopendra subspinipes mutilans via MAP-Kinase mediated MITF downregulation and the proteasomal degradation of tyrosinase

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ABSTRACT

In this study, the authors investigated the anti-melanogenic effects of 3,8-dihydroxyquinoline (jineol) isolated from Scolopendra subspinipes mutilans, the mechanisms responsible for its inhibition of melanogenesis in melan-a cells, and its antioxidant efficacy. Mushroom tyrosinase activities and melanin contents were determined in melan-a cells, and the protein and mRNA levels of MITF, tyrosinase, TYRP-1, and TYRP-2 were assessed. Jineol exhibited significant, concentration-dependent antioxidant effects as determined by DPPH, ABTS, CUPRAC, and FRAP assays. Jineol significantly inhibited mushroom tyrosinase activity by functioning as an uncompetitive inhibitor, and markedly inhibited melanin production and intracellular tyrosinase activity in melan-a cells. In addition, jineol abolished the expressions of tyrosinase, TYRP-1, TYRP-2, and MITF, thereby blocking melanin production and interfering with the phosphorylations of ERK1/2 and p38. Furthermore, specific inhibitors of ERK1/2 and p38 prevented melanogenesis inhibition by jineol, and the proteasome inhibitor (MG-132) prevented jineol-induced reductions in cellular tyrosinase levels. Taken together, jineol was found to stimulate MAP-kinase (ERK1/2 and p38) phosphorylation and the proteolytic degradation pathway, which led to the degradations of MITF and tyrosinase, and to suppress the productions of melanin.

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Effect of jineol on the levels of melanogenesis-related mRNA and proteins in melan-a cells.(A) Cells (5 × 105 cells/ml) were cultured for 24 h, and the medium was then replaced with fresh medium containing the indicated concentrations of jineol or arbutin for 24 h. mRNA was extracted using TRIzol and mRNA expressions was analyzed by RT PCR. (B) Cells (1 × 105 cells/ml) were cultured for 24 h; medium was replaced with fresh medium containing the indicated concentrations of jineol or arbutin for 3 days. Total cell lysates were extracted and assayed by western blotting using antibodies against tyrosinase, TYRP-1, TYRP-2, and MITF. Equal protein loadings were confirmed using β-actin. Arb: Arbutin. (C) Statistical analysis of the band intensity of tyrosinase, TYRP-1, TYRP-2, and MITF obtained by western blot analysis. *P < 0.05, **P < 0.01, versus non-treated controls, Student’s t-test. (D) Cells (1 × 105 cells/ml) were cultured for 24 h; the medium was replaced with fresh medium containing the indicated concentrations of jineol for the indicated time interval. Total cell lysates were extracted and assayed by western blotting using antibodies against tyrosinase, and MITF. Equal protein loadings were confirmed using β-actin.
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f5: Effect of jineol on the levels of melanogenesis-related mRNA and proteins in melan-a cells.(A) Cells (5 × 105 cells/ml) were cultured for 24 h, and the medium was then replaced with fresh medium containing the indicated concentrations of jineol or arbutin for 24 h. mRNA was extracted using TRIzol and mRNA expressions was analyzed by RT PCR. (B) Cells (1 × 105 cells/ml) were cultured for 24 h; medium was replaced with fresh medium containing the indicated concentrations of jineol or arbutin for 3 days. Total cell lysates were extracted and assayed by western blotting using antibodies against tyrosinase, TYRP-1, TYRP-2, and MITF. Equal protein loadings were confirmed using β-actin. Arb: Arbutin. (C) Statistical analysis of the band intensity of tyrosinase, TYRP-1, TYRP-2, and MITF obtained by western blot analysis. *P < 0.05, **P < 0.01, versus non-treated controls, Student’s t-test. (D) Cells (1 × 105 cells/ml) were cultured for 24 h; the medium was replaced with fresh medium containing the indicated concentrations of jineol for the indicated time interval. Total cell lysates were extracted and assayed by western blotting using antibodies against tyrosinase, and MITF. Equal protein loadings were confirmed using β-actin.

Mentions: To elucidate the mechanism of melanogenesis, we examined the expressions of tyrosinase, TYRP-1, TYRP-2, and MITF in melan-a cells after jineol treatment (12.5, 25, or 50 μM) for 4 days. Jineol was observed to suppress the mRNA expressions of MITF and of its downstream genes, tyrosinase, TYRP-1 and TYRP-2 (Fig. 5A). In addition, jineol at 50 μM significantly and dose-dependently reduced tyrosinase, TYRP-1, TYRP-2, and MITF protein levels versus untreated controls (Fig. 5B). Furthermore, to observe the onset and durability of the effect of jineol on melanogenesis-related proteins, time-dependent expression of tyrosinase and MITF in melan-a cells after jineol (50 μM) treatment was performed. As expected, jineol mitigated the expression of tyrosianse and MITF from 3 to 24 h with a peak level 12 h after jineol treatment (Fig. 5D). These results suggest jineol inhibits melanogenesis by suppressing the expressions of tyrosinase-related genes and of the MITF gene.


Inhibition of melanogenesis by jineol from Scolopendra subspinipes mutilans via MAP-Kinase mediated MITF downregulation and the proteasomal degradation of tyrosinase
Effect of jineol on the levels of melanogenesis-related mRNA and proteins in melan-a cells.(A) Cells (5 × 105 cells/ml) were cultured for 24 h, and the medium was then replaced with fresh medium containing the indicated concentrations of jineol or arbutin for 24 h. mRNA was extracted using TRIzol and mRNA expressions was analyzed by RT PCR. (B) Cells (1 × 105 cells/ml) were cultured for 24 h; medium was replaced with fresh medium containing the indicated concentrations of jineol or arbutin for 3 days. Total cell lysates were extracted and assayed by western blotting using antibodies against tyrosinase, TYRP-1, TYRP-2, and MITF. Equal protein loadings were confirmed using β-actin. Arb: Arbutin. (C) Statistical analysis of the band intensity of tyrosinase, TYRP-1, TYRP-2, and MITF obtained by western blot analysis. *P < 0.05, **P < 0.01, versus non-treated controls, Student’s t-test. (D) Cells (1 × 105 cells/ml) were cultured for 24 h; the medium was replaced with fresh medium containing the indicated concentrations of jineol for the indicated time interval. Total cell lysates were extracted and assayed by western blotting using antibodies against tyrosinase, and MITF. Equal protein loadings were confirmed using β-actin.
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f5: Effect of jineol on the levels of melanogenesis-related mRNA and proteins in melan-a cells.(A) Cells (5 × 105 cells/ml) were cultured for 24 h, and the medium was then replaced with fresh medium containing the indicated concentrations of jineol or arbutin for 24 h. mRNA was extracted using TRIzol and mRNA expressions was analyzed by RT PCR. (B) Cells (1 × 105 cells/ml) were cultured for 24 h; medium was replaced with fresh medium containing the indicated concentrations of jineol or arbutin for 3 days. Total cell lysates were extracted and assayed by western blotting using antibodies against tyrosinase, TYRP-1, TYRP-2, and MITF. Equal protein loadings were confirmed using β-actin. Arb: Arbutin. (C) Statistical analysis of the band intensity of tyrosinase, TYRP-1, TYRP-2, and MITF obtained by western blot analysis. *P < 0.05, **P < 0.01, versus non-treated controls, Student’s t-test. (D) Cells (1 × 105 cells/ml) were cultured for 24 h; the medium was replaced with fresh medium containing the indicated concentrations of jineol for the indicated time interval. Total cell lysates were extracted and assayed by western blotting using antibodies against tyrosinase, and MITF. Equal protein loadings were confirmed using β-actin.
Mentions: To elucidate the mechanism of melanogenesis, we examined the expressions of tyrosinase, TYRP-1, TYRP-2, and MITF in melan-a cells after jineol treatment (12.5, 25, or 50 μM) for 4 days. Jineol was observed to suppress the mRNA expressions of MITF and of its downstream genes, tyrosinase, TYRP-1 and TYRP-2 (Fig. 5A). In addition, jineol at 50 μM significantly and dose-dependently reduced tyrosinase, TYRP-1, TYRP-2, and MITF protein levels versus untreated controls (Fig. 5B). Furthermore, to observe the onset and durability of the effect of jineol on melanogenesis-related proteins, time-dependent expression of tyrosinase and MITF in melan-a cells after jineol (50 μM) treatment was performed. As expected, jineol mitigated the expression of tyrosianse and MITF from 3 to 24 h with a peak level 12 h after jineol treatment (Fig. 5D). These results suggest jineol inhibits melanogenesis by suppressing the expressions of tyrosinase-related genes and of the MITF gene.

View Article: PubMed Central - PubMed

ABSTRACT

In this study, the authors investigated the anti-melanogenic effects of 3,8-dihydroxyquinoline (jineol) isolated from Scolopendra subspinipes mutilans, the mechanisms responsible for its inhibition of melanogenesis in melan-a cells, and its antioxidant efficacy. Mushroom tyrosinase activities and melanin contents were determined in melan-a cells, and the protein and mRNA levels of MITF, tyrosinase, TYRP-1, and TYRP-2 were assessed. Jineol exhibited significant, concentration-dependent antioxidant effects as determined by DPPH, ABTS, CUPRAC, and FRAP assays. Jineol significantly inhibited mushroom tyrosinase activity by functioning as an uncompetitive inhibitor, and markedly inhibited melanin production and intracellular tyrosinase activity in melan-a cells. In addition, jineol abolished the expressions of tyrosinase, TYRP-1, TYRP-2, and MITF, thereby blocking melanin production and interfering with the phosphorylations of ERK1/2 and p38. Furthermore, specific inhibitors of ERK1/2 and p38 prevented melanogenesis inhibition by jineol, and the proteasome inhibitor (MG-132) prevented jineol-induced reductions in cellular tyrosinase levels. Taken together, jineol was found to stimulate MAP-kinase (ERK1/2 and p38) phosphorylation and the proteolytic degradation pathway, which led to the degradations of MITF and tyrosinase, and to suppress the productions of melanin.

No MeSH data available.