Antitumoral Activity of Diffractaic Acid in A375 Human Melanoma Cells: Induction of Apoptosis and Inhibition of Cell Viability

Melanoma is one of the most aggressive forms of skin cancer due to its high metastatic potential and resistance to conventional therapies. Apoptosis resistance represents a key mechanism contributing to melanoma progression and therapeutic failure. Natural products have recently attracted increasing attention as potential sources of novel anticancer agents. Lichen-derived secondary metabolites, in particular, possess diverse biological activities, including cytotoxic and antiproliferative effects against various cancer types. Diffractaic acid, a depside compound isolated from several lichen species, has demonstrated anticancer potential in multiple tumor cell lines; however, its effects on malignant melanoma remain insufficiently explored. Therefore, this study aimed to investigate the cytotoxic and pro-apoptotic effects of diffractaic acid on A375 human melanoma cells. A375 human melanoma cells were cultured under standard conditions and treated with different concentrations of diffractaic acid (10–100 μg/mL). Cell viability was evaluated after 24 and 48 h using the MTT assay to determine the half-maximal inhibitory concentration (IC₅₀). Based on these results, three concentrations corresponding to ½×IC₅₀, IC₅₀, and 2×IC₅₀ were selected for further analyses. Apoptotic activity was assessed after 48 h of treatment by measuring the levels of apoptosis-related proteins, including Bax, Bcl-2, caspase-3, caspase-8, caspase-9, cytochrome c, apoptotic protease activating factor-1 (Apaf-1), and p53 using ELISA-based assays. Statistical analyses were performed using one-way ANOVA followed by Tukey’s post-hoc test. Diffractaic acid significantly reduced the viability of A375 melanoma cells in a dose- and time-dependent manner. The IC₅₀ values were determined as 59.5 μg/mL and 55.7 μg/mL after 24 and 48 h of treatment, respectively. Because stronger cytotoxic effects were observed at 48 h, this incubation period was selected for apoptosis analyses. Treatment with diffractaic acid at moderate and high concentrations significantly increased the levels of several pro-apoptotic markers, including caspase-3, caspase-9, cytochrome c, and Apaf-1, indicating activation of the mitochondrial apoptotic pathway. Additionally, caspase-8 levels increased at the highest concentration, suggesting involvement of the extrinsic apoptotic pathway. A significant elevation in p53 levels was also observed at the highest dose, indicating activation of p53-mediated apoptotic signaling. The findings demonstrate that diffractaic acid exerts significant cytotoxic and pro-apoptotic effects on A375 melanoma cells. The compound appears to induce apoptosis through activation of both intrinsic mitochondrial and extrinsic apoptotic pathways. These results suggest that diffractaic acid may represent a promising natural compound for further investigation as a potential therapeutic agent against malignant melanoma.