Metronomic Capecitabine Triggers Ferroptosis in Hepatocellular Carcinoma Cells Through Inhibition of <scp>TYMS</scp>

OBJECTIVE: Capecitabine (CAP) is an orally administered prodrug of fluorouracil, predominantly utilized in the treatment of solid tumors. Triggering tumor ferroptosis is an important mechanism for the treatment of hepatocellular carcinoma (HCC). However, the potential of CAP to induce ferroptosis in HCC, along with the underlying mechanisms, remains unknown. METHODS: In this study, a subcutaneous HCC model was constructed using the syngeneic Hepa1-6 cell line in C57BL/6 mice, followed by treatment with metronomic CAP (mCAP). The anti-tumor effects of mCAP were evaluated by monitoring tumor volume, performing pathological staining, and evaluating tumor oxidative stress levels. In vitro, various thymidylate synthase (TYMS) inhibitors were used to treat both mouse and human HCC cell lines. Furthermore, TYMS-overexpressing plasmids were transfected into mouse and human HCC cell lines to directly investigate their impact on intracellular oxidative stress. Intracellular oxidative stress and ferroptosis-related markers were detected using flow cytometry, transmission electron microscopy, and Western blotting. RESULTS: expression, activated nicotinamide adenine dinucleotide phosphate hydrogen oxidase (NOX), induced reactive oxygen species production, and increased ferrous ion accumulation. Electron microscopy revealed mitochondrial alterations characteristic of ferroptosis. Raltitrexed, another TYMS inhibitor, also induced ferroptosis in hepatocellular carcinoma cells. In vivo, the anti-tumor effect of mCAP was antagonized by co-treatment with an NOX inhibitor. CONCLUSION: CAP targets TYMS to induce ferroptosis by activating NOX, thereby inhibiting HCC progression.