Purpose: Several clinical trials in human neoplasms have demonstrated the effectiveness of combination therapy with 5-fluorouracil (FUra), cisplatin (CDDP), and leucovorin (LV). Thymidylate synthase (TS), the target enzyme of FUra, and dihydropyrimidine dehydrogenase (DPD), the rate-limiting catabolic enzyme of pyrimidines, have both been reported to be predictors of the response to FUra-based chemotherapies. Therefore, we aimed to clarify the effects of a combination of the three drugs against hepatoma cells and to determine the role of these two enzymes using in vitro models.
Methods: Five human hepatoma cell lines (Hep3B, HepG2, HuH7, PLC/PRF/5 and Chang) were used. Cytotoxicity was determined after exposure to various concentrations and combinations of antitumor agents. The combination effects of FUra and CDDP in terms of synergy, additivity or antagonism were evaluated by median effect analysis. The mRNA levels of TS and DPD were measured by quantitative real-time PCR. Expression of TS and DPD proteins was also investigated.
Results: LV alone did not show any cytotoxicity, although it enhanced the cytotoxicity of FUra, but not that of CDDP. Synergistic enhancement was observed with the combination of FUra and CDDP against all cells. The median combination index at fraction 0.5 was 0.554 (range 0.273-0.616). All cells expressed TS and DPD with median relative quantities of mRNA normalized to that of HuH7 cells of 1.04 (range 1.00-1.32) and 1.18 (range 0.88-1.55), respectively. A strong correlation was found between the IC(50) of FUra and the mRNA level of DPD (r=0.912, P=0.0295).
Conclusions: LV and CDDP enhanced the cytotoxicity of FUra, which provided a rationale for the regimen combining the three drugs for the treatment of hepatocellular carcinoma. DPD plays an important role in the sensitivity to FUra, and the DPD mRNA expression level may be used to predict the response to FUra-based chemotherapy for HCC.