A 7-Hydroxy 4-Methylcoumarin Enhances Melanogenesis in B16-F10 Melanoma Cells

Abstract

The objectives of this study were to investigate the melanogenetic potentials of the naturally occurring 7-hydroxy coumarin derivatives 7-hydroxy 5,6-dimethoxycoumarin (7H-5,6DM), 7-hydroxy 6,8-dimethoxycoumarin (7H-6,8DM), 7-hydroxy 6-methoxycoumarin (7H-6M), and 7-hydroxy 4-methylcoumarin (7H-4M) in the melanogenic cells model for murine B16F10 melanoma cells. The initial results indicated that melanin production and intracellular tyrosinase activity were significantly stimulated by 7H-4M but not by 7H-5,6DM, 7H-6,8DM, or 7H-6M. Therefore, our present study further investigated the melanogenic effects of 7H-4M in B16-F10 cells, as well as its mechanisms of action. In a concentration-dependent manner, 7H-4M increased intracellular tyrosinase activity, leading to the accumulation of melanin without affecting the viability of B16-F10 cells. Our study further investigated the effects of 7H-4M on melanogenesis, including its ability to promote tyrosinase activity, increase melanin content, and activate molecular signaling pathways. The results indicate that 7H-4M effectively stimulated tyrosinase activity and significantly increased the expression of melanin synthesis-associated proteins, such as microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein-1 (TRP1), and TRP2. Based on our findings, we can conclude that 7H-4M has the ability to activate the melanogenesis process through the upregulation of cAMP-dependent protein kinase (PKA) and the cAMP response element-binding protein (CREB). Additionally, our study showed that 7H-4M induced melanogenic effects by downregulating the extracellular signal-regulated kinase (ERK) and the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt)/glycogen synthesis kinase-3β (GSK-3β) cascades, while upregulating the JNK and p38 signaling pathways. Finally, the potential of using 7H-4M in topical applications was tested through primary human skin irritation tests. During these tests, no adverse reactions were induced by 7H-4M. In summary, our results indicate that 7H-4M regulates melanogenesis through various signaling pathways such as GSK3β/β-catenin, AKT, PKA/CREB, and MAPK. These findings suggest that 7H-4M has the potential to prevent the development of pigmentation diseases.

Keywords: 7-hydroxy 4-methycoumarin; B16-F10; hypopigmentary; melanogenesis; signaling pathway.

Figure 1

The structure of the 7-hydroxy coumarin derivatives: (a) 7-hydroxy-5,6-dimethoxycoumarin, (b) 7-hydroxy-6,8-dimethoxycoumarin, (c) 7-hydroxy-4-methlycoumarin, and (d) 7-hydroxy-6-methoxycoumarin.

Figure 2

The effect of 7H-5,6DM (a), 7H-6,8DM (b), 7H-4M (c), and 7H-6M (d) on the viability of B16-F10 melanoma cells. The cells were plated in 24-well plates (8 × 10³ cells/well), incubated for 24 h, and treated with 7H-5,6DM (25, 50, 100, and 200 µM), 7H-6,8DM (25, 50, 100, and 200 µM), 7H-4M (25, 50, 100, and 200 µM), 7H-6M (25, 50, 100, and 200 µM), and α-MSH (100 nM) for 72 h. The cytotoxicity of 7H-5,6DM, 7H-6,8DM, 7H-4M, and 7H-6M was evaluated using MTT assays. The results are presented as the mean ± SD of independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the unstimulated control group.

Figure 2

The effect of 7H-5,6DM (a), 7H-6,8DM (b), 7H-4M (c), and 7H-6M (d) on the viability of B16-F10 melanoma cells. The cells were plated in 24-well plates (8 × 10³ cells/well), incubated for 24 h, and treated with 7H-5,6DM (25, 50, 100, and 200 µM), 7H-6,8DM (25, 50, 100, and 200 µM), 7H-4M (25, 50, 100, and 200 µM), 7H-6M (25, 50, 100, and 200 µM), and α-MSH (100 nM) for 72 h. The cytotoxicity of 7H-5,6DM, 7H-6,8DM, 7H-4M, and 7H-6M was evaluated using MTT assays. The results are presented as the mean ± SD of independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the unstimulated control group.

Figure 3

The effect of 7H-5,6DM, 7H-6,8DM, 7H-4M, and 7H-6M on the production of melanin (a) and tyrosinase activity (b) in B16-F10 melanoma cells. The cells were plated in 6-well plates (5 × 10⁴ cells/well), incubated for 24 h, and treated with 7H-5,6DM (25, 50, and 100 μM), 7H-6,8DM (25, 50, and 100 μM), 7H-4M (25, 50, and 100 μM), 7H-6M (25, 50, and 100 μM), and α-MSH (100 nM) for 72 h. α-MSH was used as the negative control. The results are presented as the mean ± SD from independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the control group.

Figure 4

The effect of 7H-4M on the melanogenesis-related protein expression of B16-F10 cells. The cells were treated with various concentrations of 7H-4M for 72 h and were collected for protein extraction. The expression levels of tyrosinase (TYR), TRP-1, TRP-2, and MITF were evaluated using a Western blot experiment. (a,b) Western blotting results and (c) tyrosinase, (d) TRP-1, (e) TRP-2 and (f) MITF protein expression. The protein bands were quantified using ImageJ software, and the intensity values were normalized to the loading control. The untreated cells were considered as 100%, and the values obtained from treated cells were compared to the control group. The results are presented as mean ± SD of at least three independent experiments. Statistical analysis showed that there were significant differences between the control group and the 7H-4M-treated groups at concentrations of 25, 50, and 100 μM for TYR, TRP-1, TRP-2, and MITF. The significance levels were *** p < 0.001 and ** p < 0.01.

Figure 5

The effect of 7H-4M on the melanogenesis-related protein expression of B16-F10 cells. The experimental protocol involved treating B16-F10 cells with 7H-4M at varying concentrations for 24 h. The cells were then collected and protein extracts were prepared from each group. The expression levels of P-GSK3β, P-β-catenin, and β-catenin were assessed through Western blot analysis. The relative intensity of the protein bands was measured using ImageJ software, and the values were normalized to the corresponding loading control. The untreated cells were considered as having 100% expression. (a) Western blotting results and (b) β-catenin, (c) P-GSK3, and (d) P-β-catenin protein expression. The reported values are the mean ± SD of at least three independent experiments, and the significance level is shown as ** p < 0.01 and *** p < 0.001 compared to the control group.

Figure 6

The effect of 7H-4M on the melanogenesis-related protein expression of B16-F10 cells. The cells were treated with 7H-4M at the indicated concentrations for 24 h and were then harvested. Protein extracts were prepared from each treatment group to evaluate the expression of P-AKT. The relative intensity of the protein band was quantified using ImageJ software, and the value was normalized to that of the corresponding loading control. The untreated cells were considered as 100%. (a) Western blotting results and (b) P-AKT protein expression. The values are presented as the mean ± SD of at least three independent experiments. Statistical analysis was performed using *** p < 0.001 compared to the control group.

Figure 7

The effect of 7H-4M on the melanogenesis-related protein expression of B16-F10 cells. The cells were treated with 7H-4M at specified concentrations for 24 h, followed by harvesting. Protein extracts were then prepared from each treatment group to evaluate the expressions of P-PKA and P-CREB. Using ImageJ software, the relative intensity of the protein band was quantified and normalized to that of the corresponding loading control. The untreated cells were considered as 100%. (a) Western blotting results and (b) P-PKA, and (c) P-CREB protein expression. The results are shown as mean ± SD of at least three independent experiments, with *** p < 0.001, ** p < 0.01, and * p < 0.05 indicating significance compared to the control group.

Figure 8

The effect of 7H-4M on the melanogenesis-related protein expression of B16-F10 cells. The experiment involved treating cells with various concentrations of 7H-4M for 24 h, followed by harvesting the cells. Protein extracts were prepared from each treatment group and the expressions of MAPKs (P-ERK, P-p38, and P-JNK) were evaluated. The relative intensity of the protein band was quantified using ImageJ software and the value was normalized to that of the corresponding loading control. Untreated cells were used as the control group and were considered to have a value of 100%. (a) Western blotting results and (b) P-ERK, (c) P-p38, and (d) P-JNK protein expression. The results were shown as the mean ± SD of at least three independent experiments, with statistical significance indicated by *** p < 0.001.

Figure 9

Effect of 7H-4M on production of melanin in a B16F10 melanoma cells. To evaluate the role of ERK, AKT, and PKA in melanin synthesis induced by 7H-4M, B16F10 cells were co-treated with specific inhibitors PD98059 (ERK inhibitor, 1 μM), LY294002 (AKT inhibitor, 1 μM), and H-89 (PKA inhibitor, 1 μM) for 72 h. Melanin content was measured and compared to untreated cells, which were regarded as 100%. The results showed that PD98059 (a), LY294002 (b), and H-89 (c) significantly decreased melanin content induced by 7H-4M. Values are shown as the mean ± SD of at least three independent experiments. *** p < 0.001 compared to the control group. ### p < 0.001 compared to the 7H-4M group without specific inhibitor.

Figure 10

Schematic diagram of the proposed mechanism regulating action of 7H-4M on melanogenesis.