Mitochondrial uncoupling sensitizes gastric cancer cells to elesclomol-induced cuproptosis via FDX1/DLAT upregulation.

Gastric cancer treatment remains challenging due to tumor heterogeneity and drug resistance. Cuproptosis, a novel form of cell death induced by excessive copper accumulation in mitochondria, is regulated by FDX1 and involves cytotoxic aggregation of lipoylated proteins such as DLAT. Given the markedly elevated copper levels observed in gastric cancer tissues, triggering cuproptosis represents a promising therapeutic strategy. Since cuproptosis strongly relies on active mitochondrial respiration, we hypothesized that mitochondrial uncoupling-which dissociates electron transport from oxidative phosphorylation by dissipating the proton gradient across the inner mitochondrial membrane, thereby enhancing respiratory activity-could sensitize gastric cancer cells to cuproptosis. In this study, we first established a cuproptosis model in gastric cancer cells using elesclomol combined with copper. We then systematically evaluated the enhancing effect of mitochondrial uncoupling on this process. Using the classical uncoupler FCCP and clinically relevant agents (niclosamide, nitazoxanide, and oxyclozanide), we confirmed mitochondrial uncoupling through membrane potential depolarization, an increased NAD/NADH ratio, elevated oxygen consumption, and decreased ATP levels. Importantly, mitochondrial uncoupling significantly potentiated the cytotoxic effect of elesclomol-copper ion treatment in gastric cancer cells, which was closely associated with increased DLAT oligomerization. Mechanistic investigations revealed that mitochondrial uncoupling promotes cuproptosis susceptibility by upregulating DLAT and FDX1 protein expression and remodeling cellular metabolism. In summary, this study highlights the key role of mitochondrial uncoupling in amplifying cuproptosis and proposes a novel combination strategy based on metabolic intervention. These findings offer a new theoretical foundation and potential clinical translation prospects for gastric cancer treatment.