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

OBJECTIVE: Cisplatin-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. METHODS: Clinical 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. RESULTS: Clinical 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. CONCLUSIONS: Our 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.