Mechanistic Insights into the Stimulatory Effect of Melanogenesis of 4-Methylcoumarin Derivatives in B16F10 Melanoma Cells

Abstract

Vitiligo is a skin condition characterized by the loss of pigment, resulting in white patches on various parts of the body. It occurs when melanocytes, the cells that are responsible for producing skin pigment, are destroyed or stop functioning. This study aimed to investigate the melanogenic potential of various 4-methylcoumarin (4MC) derivatives, including 6-methoxy-4-methylcoumarin (6M-4MC), 7-methoxy-4-methylcoumarin (7M-4MC), 7-amino-4-methylcoumarin (7A-4MC), 6,7-dihydroxy-4-methylcoumarin (6,7DH-4MC), 7,8-dihydroxy-4-methylcoumarin (7,8DH-4MC), and 6,7-dimethoxy-4-methylcoumarin (6,7DM-4MC), in B16F10 melanoma cells. Our findings revealed that, while 4MC, 7A-4MC, 6,7DH-4MC, and 7,8DH-4MC did not exhibit any effect on melanin production, significant stimulation of melanogenesis was observed with 6M-4MC, 7M-4MC, and 6,7DM-4MC, with 6M-4MC demonstrating the most pronounced effect. 6M-4MC significantly stimulated melanin production and tyrosinase activity in a concentration-dependent manner in B16F10 cells. A Western blot analysis revealed that 6M-4MC increased the expression levels of microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2). Further mechanistic studies showed that 6M-4MC inhibited extracellular signal-regulated kinase (ERK) and protein kinase B (AKT), which led to the upregulation of MITF and TRP proteins and subsequent activation of melanin synthesis. Additionally, 6M-4MC activated GSK3β phosphorylation, reduced β-catenin phosphorylation, and stimulated melanogenesis via the GSK3β/β-catenin pathway. Moreover, a primary skin irritation test was conducted on the upper backs of 32 healthy female volunteers to assess the potential irritation or sensitization from 6M-4MC when applied topically at concentrations of 50 µM and 100 µM. The test results showed no adverse effects on the skin. Collectively, these findings suggest that 6M-4MC may be a promising pigmentation stimulator for use in cosmetics and in the medical treatment of hypopigmentation disorders, particularly in the treatment of skin conditions such as vitiligo.