Curcumol overcomes cisplatin resistance and rewires glycolysis-H3K9la-ORC6 axis to trigger ferroptosis in bladder cancer

ObjectiveCisplatin-based chemotherapy is a cornerstone for bladder cancer treatment, but the development of resistance remains a major clinical challenge. Curcumol, a bioactive sesquiterpenoid derived from Curcumae Rhizoma, has shown anti-tumor potential. This study investigated the efficacy of curcumol in overcoming cisplatin resistance and elucidated its underlying molecular mechanisms in bladder cancer progression. MethodsClinical correlation was assessed in patients receiving neoadjuvant chemotherapy with or without Curcumae Rhizoma. The anti-tumor effects of curcumol were evaluated in both cisplatin-sensitive and cisplatin-resistant bladder cancer cells. Multi-omics approaches, including RNA sequencing, proteomics and metabolomics, were employed. Key mechanisms involving H3K9 lactylation (H3K9la) were explored via Western blotting, immunohistochemistry, and cleavage under targets and tagmentation (CUT&Tag) assays. The role of the identified target ORC6 was validated through genetic knockout and overexpression. Finally, ferroptosis was confirmed by measuring lipid peroxidation [malondialdehyde (MDA)], total iron levels, and ferroptosis-related protein markers in vitro. ResultsClinical data indicated that patients administered Curcumae Rhizoma exhibited enhanced responses to neoadjuvant chemotherapy. In addition, curcumol suppressed the proliferation, migration, and invasion of both bladder cancer cells and cisplatin-resistant cells. Mechanistically, proteomic analysis and non-targeted metabolomics revealed that curcumol suppresses glycolysis and lactate production. Subsequently, Western blotting analysis demonstrated a marked reduction in H3K9la levels in both T24 and 5637 cells following curcumol treatment. This decrease in H3K9la was also observed in patient tumor tissues via immunohistochemistry staining. CUT&Tag analysis identified that H3K9la is enriched with the highest number of reads at the ORC6 promoter region. Combined in vitro and in vivo experiments indicated that OCR6 exerted a tumor-promoting effect on bladder cancer. Its knockout induced G0/G1 phase arrest and enhanced apoptosis, while its expression contributed to cancer progression by enhancing invasive and migratory capabilities. Furthermore, ORC6 overexpression correlated with ferroptosis scores and ferroptosis-related genes. In vitro, OCR6 knockout promoted ferroptosis via DNA damage, characterized by elevated MDA content, decreased expression of core ferroptosis-related proteins (GPX4 and SLC7A11), increased percentage of γH2AX-positive cells and longer DNA tails. Finally, we performed rescue experiments using a ferroptosis inhibitor in ORC6 knockout cells, which indicated that ferroptosis inhibitor could weaken the effect of ORC6 knockout on the invasive, migratory, and proliferative capacities. ConclusionsOur findings demonstrated that curcumol effectively counteracted cisplatin resistance and inhibited bladder cancer progression by targeting the glycolysis-H3K9la-ORC6 axis to induce ferroptosis. This study established a critical link between metabolic reprogramming, histone lactylation, and ferroptosis, providing a novel therapeutic avenue for treating chemoresistant bladder cancer.