Complanatoside A disrupts copper homeostasis and induces cuproptosis via directly targeting ATOX1 in prostate cancer

Complanatoside A (CA), a flavonoid derived from the Chinese medicinal herb Semen Astragali Complanati, exhibits anticancer activity. However, its effects on prostate cancer (PCa) remain unclear. We aimed to elucidate the anti-PCa effects and underlying mechanisms of action of CA, both in vitro and in vivo. MTT, transwell, wound-healing, and flow cytometry assays were used to detect PCa cell growth, invasion, migration, and apoptosis and cell cycle progression after CA treatment, respectively. The target of CA was predicted to be antioxidant 1 copper chaperone (ATOX1), and its expression levels were determined using immunoblotting analysis. As ATOX1 acts as copper carrier to maintain copper homeostasis and disrupted copper homeostasis leads to oxidative stress in mitochondria and cuproptosis, the concentration of copper, ATP，pyruvate, α-ketoglutamic acid, malondialdehyde, and glutathione were determined and markers of cuproptosis were analyzed by immunoblotting analysis. The copper chelator tetrathiomolybdate was used to identify CA-induced cuproptosis of PCa cells. Finally, a subcutaneous mouse model was constructed, and tumor growth, oxidative stress, and carcinogenesis were analyzed in vivo. Our investigation revealed that CA inhibited PCa cell growth, invasion, migration, and cell cycle progression and induced apoptosis. ATOX1 was downregulated by CA and promoted Cu ion accumulation, which inhibited mitochondrial activity and induced cuproptosis in PCa cells. In addition, CA markedly suppressed tumor growth in vivo and induced cuproptosis in tumor tissues. In conclusion, CA exerts its anti-PCa effects by reducing ATOX1 protein levels and promoting Cu ion accumulation, which in turn, inhibits mitochondrial activity and induces cuproptosis.