Mechanistic Insights into the Ameliorating Effect of Melanogenesis of Psoralen Derivatives in B16F10 Melanoma Cells

Abstract

The objectives of this study were to investigate the melanogenetic potential of the psoralen derivatives 5-hydroxypsoralen, 5-methoxypsoralen, 8-hydroxypsoralen, 8-methoxypsoralen, and 5,8-dimethoxypsoralen in B16F10 melanoma cells. The results indicated that melanin production is significantly stimulated in B16F10 melanoma cells with 5-methoxypsoralen, 8-methoxypsoralen, and 5,8-dimethoxypsoralen, especially for 5-methoxypsoralen (bergapten), as reported previously. In addition, Western blot results showed that the protein levels of microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2) increase after bergapten treatment for the first time. The results also showed that bergapten promotes the phosphorylation of Akt at Ser 473 and glycogen synthase kinase-3β at Ser 9. Moreover, bergapten increased the content of β-catenin in the cell cytoplasm and nucleus by reducing the phosphorylated β-catenin (p-β-catenin) content. The results also indicated that bergapten regulates melanogenesis by upregulating the phosphorylation of p38 and JNK-mitogen-activated protein kinase. Taken together, these findings suggest that the regulation of melanogenesis by bergapten may be mediated by the β-catenin and MAPK signaling pathways and that bergapten might provide new insights into the pathogenesis of pigmented diseases.

Keywords: 5,8-dimethoxypsoralen; 5-hydroxypsoralen; 5-methoxypsoralen; 8-hydroxypsoralen; 8-methoxypsoralen; MAPK; β-catenin.

Figure 1

Structure of psoralen derivatives. (a) Psoralen, (b) 5-hydroxypsoralen (bergaptol), (c) 5-methoxypsoralen (bergapten), (d) 8-hydroxypsoralen (xanthotoxol), (e) 8-methoxypsoralen (xanthotoxin), and (f) 5,8-dimethoxypsoralen (isopimpinellin).

Figure 2

Effects of psoralen derivatives on cell viability in B16F10 melanoma cells. The cells were treated with psoralen derivatives (12.5, 25, 50, 100, 200, and 400 μM) and α-MSH (100 nM) for 72 h. Cell viability of B16F10 cells subjected to (a) 5-methoxypsoralen (bergapten), (b) 8-methoxypsoralen (xanthotoxin), (c) 5,8-dimethoxypsoralen (isopimpinellin), (d) 5-hydroxypsoralen (bergaptol), and (e) 8-hydroxypsoralen (xanthotoxol) was measured by MTT assay. The results are presented as the mean ± SD of three independent experiments. *** p < 0.001, ** p < 0.01 vs. the untreated group.

Figure 3

Effects of psoralen derivatives on the melanin content in B16F10 melanoma cells. The cells were treated with psoralen derivatives (3.13, 6.25, 12.5, and 25 μM) and α-MSH (100 nM) for 72 h. Melanin content of B16F10 cells subjected to (a) 5-methoxypsoralen (bergapten), (b) 8-methoxypsoralen (xanthotoxin), (c) 5,8-methoxypsoralen (isopimpinellin), (d) 5-hydroxypsoralen (bergaptol), and (e) 8-hydroxypsoralen (xanthotoxol). The results are presented as the mean ± SD of three independent experiments. * p < 0.05; ** p < 0.01 vs. the untreated group.

Figure 4

Effects of bergapten on tyrosinase activity in B16F10 melanoma cells. The cells were treated with bergapten (6.25, 12.5, and 25 μM) and α-MSH (100 nM) for 72 h. α-MSH was used as a positive control. The results are presented as the mean ± SD of three independent experiments. ** p < 0.01 vs. the untreated group.

Figure 5

Effects of bergapten on the protein expression level of tyrosinase, TRP-1, TRP-2, and MITF in B16F10 melanoma cells. The cells were treated with bergapten (6.25, 12.5, and 25 μM) for 48 h. (a) Western blotting results and protein expression of (b) tyrosinase, (c) TRP-1, (d) TRP-2, and (e) MITF. β-actin was used as a loading control. The results are presented as the mean ± SD of three independent measurements using Image J. ** p < 0.01 vs. untreated group.

Figure 6

Effects of bergapten on the protein expression level of β-catenin, p-GSK3β, and p-β-catenin in B16F10 melanoma cells. The cells were treated with bergapten (6.25, 12.5, and 25 μM) for 24 h. (a) Western blotting results and protein expression of (b) β-catenin, (c) P-GSK3β/T-GSK3β, and (d) P-β-catenin. β-actin was used as a loading control. The results are presented as the mean ± SD of three independent measurements using Image J. ** p < 0.01 vs. the untreated group.

Figure 7

Effects of bergapten on the phosphorylation level of AKT in B16F10 melanoma cells. The cells were treated with bergapten (6.25, 12.5, and 25 μM) for 4 h. (a) Western blotting results and protein expression of (b) P-AKT/T-AKT. β-actin was used as a loading control. The results are presented as the mean ± SD of three independent measurements using Image J. ** p < 0.01 vs. the untreated group.

Figure 8

Effects of bergapten on the phosphorylation level of PKA in B16F10 melanoma cells. The cells were treated with bergapten (6.25, 12.5, and 25 μM) for 20 h. (a) Western blotting results and protein expression of (b) P-PKA/T-PKA. β-actin was used as a loading control. The results are presented as the mean ± SD of three independent measurements using Image J. ** p < 0.01 vs. the untreated group.

Figure 9

Effects of bergapten on the phosphorylation level of MAPKs in B16F10 melanoma cells. The cells were treated with bergapten (6.25, 12.5, and 25 μM) for 4 h. (a) Western blotting results and protein expression of (b) P-ERK/T-ERK, (c) P-JNK/T-JNK, and (d) P-p38/T-p38. β-actin was used as a loading control. The results are presented as the mean ± SD of three independent measurements using Image J. ** p < 0.01 vs. the untreated group.