Metronomic Capecitabine Triggers Ferroptosis in Hepatocellular Carcinoma Cells Through Inhibition of TYMS

  • Cancer Med. 2026 May;15(5):e71898. doi: 10.1002/cam4.71898.
Ruining Yang  1  2 ,  Yuan Chang  1  2 ,  Zhan Feng  3 ,  Jiaowen Yang  1  2 ,  Qian Sun  1  2 ,  Hao Wang  1  2 ,  Dejun Kong  1  2 ,  Jinliang Duan  1  2 ,  Shaofeng Chen  1  2 ,  Lei Cao  4 ,  Jiashu Ren  1  2 ,  Sei Yoshida  5 ,  Zhenglu Wang  2  5  6  7 ,  Hong Zheng  2  5  6  8
Affiliations
  • 1. Nankai University School of Medicine, Tianjin, China.
  • 2. Tianjin Organ Transplantation Research Center, Tianjin, Tianjin, China.
  • 3. Department of Immunology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China.
  • 4. National Health Commission's Key Laboratory for Critical Care Medicine, Tianjin, China.
  • 5. Research Institute of Transplant Medicine, Nankai University, Tianjin, China.
  • 6. Key Laboratory of Transplant Medicine, Chinese Academy of Medical Sciences, Tianjin, China.
  • 7. Biological Sample Resource Sharing Center, Tianjin First Central Hospital, Nankai University School of Medicine, Tianjin, China.
  • 8. Tianjin Key Laboratory for Organ Transplantation, Tianjin First Central Hospital, Tianjin, China.
PMID: 42108395 DOI: 10.1002/cam4.71898
Abstract

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: 5-FU, an active metabolite of CAP, as well as TYMS-specific inhibitors, suppressed the proliferation of Hepa1-6 and HepG2 cells. These treatments promoted p67phox 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.

Keywords
capecitabine; ferroptosis; hepatocellular carcinoma; thymidylate synthase.
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