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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.

No MeSH data available.


Effect of jineol on the lysosomal and proteosomal degradations of tyrosinase in melan-a cells.(A) Cells (3 × 105 cells/ml) were pretreated with 25 μg/ml of cycloheximide (a protein synthesis inhibitor) for 1 h. Separately, cells were pretreated with 10 μM MG-132 (a proteasomal inhibitor) or 50 μM chloroquine (lysosomal degradation inhibitor) for 1 h. These cycloheximide, MG-132, or chloroquine pretreated cells were then treated with jineol for 6 h. Whole cell lysates were subjected to western blot analysis using anti-tyrosinase antibodies. Equal protein loadings were confirmed using β-actin antibodies. (B) Melanin contents were determined in triplicate, and results are presented as means ± SDs. *P < 0.05, versus non-treated controls, Student’s t-test.
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f7: Effect of jineol on the lysosomal and proteosomal degradations of tyrosinase in melan-a cells.(A) Cells (3 × 105 cells/ml) were pretreated with 25 μg/ml of cycloheximide (a protein synthesis inhibitor) for 1 h. Separately, cells were pretreated with 10 μM MG-132 (a proteasomal inhibitor) or 50 μM chloroquine (lysosomal degradation inhibitor) for 1 h. These cycloheximide, MG-132, or chloroquine pretreated cells were then treated with jineol for 6 h. Whole cell lysates were subjected to western blot analysis using anti-tyrosinase antibodies. Equal protein loadings were confirmed using β-actin antibodies. (B) Melanin contents were determined in triplicate, and results are presented as means ± SDs. *P < 0.05, versus non-treated controls, Student’s t-test.

Mentions: Balance between the synthesis and degradation of tyrosinase are tightly coupled to its regulation of melanin biosynthesis, and the proteasomal and lysosomal degradations of tyrosinase have been reported to involve in the turnover of tyrosinase9. Thus, to investigate whether the jineol-induced downregulation of tyrosinase is associated with its post-translation degradation, we used MG-132 (a proteasome inhibitor) and/or chloroquine (a lysosomal proteolysis inhibitor). To inhibit protein synthesis, melan-a cells were pretreated with cycloheximide and MG-132 and/or chloroquine for 1 h, and this was then followed by 6 h of treatment with jineol. Western blot analysis was performed to analysis tyrosinase levels. Jineol-induced decreases in tyrosinase level were prevented by MG-132, whereas chloroquine had no effect (Fig. 7A). Intriguingly, MG-132 pretreatment prevented the suppression of melanin by jineol (Fig. 7B). Taken together, these findings demonstrated that jineol may mitigate melanin synthesis by downregulating tyrosinase by proteasomal degradation rather than by lysosomal degradation.


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 lysosomal and proteosomal degradations of tyrosinase in melan-a cells.(A) Cells (3 × 105 cells/ml) were pretreated with 25 μg/ml of cycloheximide (a protein synthesis inhibitor) for 1 h. Separately, cells were pretreated with 10 μM MG-132 (a proteasomal inhibitor) or 50 μM chloroquine (lysosomal degradation inhibitor) for 1 h. These cycloheximide, MG-132, or chloroquine pretreated cells were then treated with jineol for 6 h. Whole cell lysates were subjected to western blot analysis using anti-tyrosinase antibodies. Equal protein loadings were confirmed using β-actin antibodies. (B) Melanin contents were determined in triplicate, and results are presented as means ± SDs. *P < 0.05, versus non-treated controls, Student’s t-test.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5385534&req=5

f7: Effect of jineol on the lysosomal and proteosomal degradations of tyrosinase in melan-a cells.(A) Cells (3 × 105 cells/ml) were pretreated with 25 μg/ml of cycloheximide (a protein synthesis inhibitor) for 1 h. Separately, cells were pretreated with 10 μM MG-132 (a proteasomal inhibitor) or 50 μM chloroquine (lysosomal degradation inhibitor) for 1 h. These cycloheximide, MG-132, or chloroquine pretreated cells were then treated with jineol for 6 h. Whole cell lysates were subjected to western blot analysis using anti-tyrosinase antibodies. Equal protein loadings were confirmed using β-actin antibodies. (B) Melanin contents were determined in triplicate, and results are presented as means ± SDs. *P < 0.05, versus non-treated controls, Student’s t-test.
Mentions: Balance between the synthesis and degradation of tyrosinase are tightly coupled to its regulation of melanin biosynthesis, and the proteasomal and lysosomal degradations of tyrosinase have been reported to involve in the turnover of tyrosinase9. Thus, to investigate whether the jineol-induced downregulation of tyrosinase is associated with its post-translation degradation, we used MG-132 (a proteasome inhibitor) and/or chloroquine (a lysosomal proteolysis inhibitor). To inhibit protein synthesis, melan-a cells were pretreated with cycloheximide and MG-132 and/or chloroquine for 1 h, and this was then followed by 6 h of treatment with jineol. Western blot analysis was performed to analysis tyrosinase levels. Jineol-induced decreases in tyrosinase level were prevented by MG-132, whereas chloroquine had no effect (Fig. 7A). Intriguingly, MG-132 pretreatment prevented the suppression of melanin by jineol (Fig. 7B). Taken together, these findings demonstrated that jineol may mitigate melanin synthesis by downregulating tyrosinase by proteasomal degradation rather than by lysosomal degradation.

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.