Metformin Enhances 2-Aminoethyl Dihydrogen Phosphate-Induced Mitochondrial Dysfunction and Apoptosis in Melanoma...

Melanoma exhibits pronounced metabolic plasticity and mitochondrial dependency, contributing to therapeutic resistance and tumor progression. Targeting mitochondrial function therefore represents a promising anticancer strategy. 2-Aminoethyl dihydrogen phosphate (2-AEH₂P), a bioactive phosphomonoester, has demonstrated antiproliferative potential, while metformin, a clinically established antidiabetic drug, acts as a mitochondrial complex I inhibitor and metabolic modulator. This study investigated the cytotoxic and mechanistic effects of 2-AEH₂P and metformin hydrochloride, individually and in combination, in human (SK-MEL-28) and murine (B16-F10) melanoma models, using non-tumorigenic fibroblasts (FN1 and L929) as controls. Cell viability, proliferation dynamics, cell-cycle distribution, mitochondrial membrane potential (ΔΨm), and apoptosis-associated markers were evaluated by flow cytometry. 2-AEH₂P reduced melanoma cell viability and proliferation while inducing G2/M accumulation, DNA fragmentation, mitochondrial depolarization, increased cytochrome c release, caspase-3 and caspase-8 activation, upregulation of p53 and Bad, and downregulation of Bcl-2. Metformin alone exerted moderate cytotoxic and pro-apoptotic effects. Notably, combined treatment markedly potentiated mitochondrial depolarization and intrinsic apoptotic signaling in melanoma cells, significantly lowering IC₅₀ values and enhancing caspase activation and cytochrome c release. Bliss independence analysis demonstrated synergistic interaction in SK-MEL-28 and B16-F10 cells. Although interaction scores indicated synergy in one fibroblast model, absolute cytotoxicity remained lower than in melanoma cells. These findings demonstrate that metabolic co-targeting with metformin enhances mitochondrial dysfunction-associated apoptotic signaling in melanoma cells, supporting a drug repositioning strategy aimed at exploiting mitochondrial vulnerability in metabolically adaptable tumors.