Study On The Synergistic Anti-tumor Activity And Underlying Mechanism Of Tirapazamine And Topoisomerase I Inhibitors

Objective:Hepatocellular carcinoma (HCC) is a leading cause of cancer death worldwide, and is expected to become more prevalent in China over the next decade. Conventional chemotherapies are generally not prescribed for advanced HCC due to their low efficacies, thus it would be necessary to uncover the reason for the resistance to chemotherapy of HCC and develop effective combination strategies. topoisomerase I inhibitors are a class of anti-cancer drugs with a broad spectrum of clinical activity. However, they have limited efficacy in hepatocellular cancer. It’s worth noting that the topoisomerase I inhibitors has a very strong anti-tumor activity in vitro. The mechanism involved in the huge difference of activities between in vitro and in vivo is not clear. The blood supply is limited in HCC with cirrhosis, which ultimately leads to local hypoxia. In this study, it is presented in vitro and in vivo evidence that the high level of hypoxia-inducible factor-1α (HIF-1α) is directly linked to the resistance of topoisomerase I inhibitors in HCC. In a previous study conducted by our group, it is found that tirapazamine (TPZ), a hypoxic-selective anticancer drug, could downregulate HIF-1α expression through decreasing HIF-1α protein synthesis. I made this elementary research to investigate the anti-tumor effects by the combination of tirapazamine and topoisomerase I inhibitors in hepatocellular carcinoma in vivo and in vitro, and explore the anti-cancer mechanisms.Methods:(1) SRB method was used to detect anti-proliferation activity of tirapazamine and topoisomerase I inhibitors (SN-38/TPT/HCPT/MONCPT) on different human hepatocellular carcinoma cells and normal liver cells, and combination index (CI) were calculated.(2) Short hairpin RNA was introduced to silence related gene.(3) Western blotting assay were used to detect the protein expression.(4) Luciferase reporter gene assay was used to detect the transcription activity of transcription factor.(5) Clonogenic assay was performed to analyze the antitumor effect of the combination treatment of TPZ with the four topoisomerase I inhibitors.(6) Xenografted athymic mice model was introduced to investigate the in vivo anti-cancer activity of TPZ and CPT-11(the prodrug of SN-38).(7) DAPI stain was used to observe apoptotic bodies in Bel-7402cells.(8) Analysis of apoptosis was determined by propidium iodide staining or Annexin V/PI staining.(9) Mitochondrial membrane depolarization was determined by JC-1staining.(10) Immunofluorescence analysis was used to detect the distribution and expression of proteins in cells.(11) Real-Time reverse transcription PCR was used to detect the mRNA level.(12) Immunohistochemical analysis was used to detect the protein distribution in tumor issues.(13) Plasmid was introduced to overexpression of HIF-1α.Results:(1) The synergistic anti-tumor activity of tirapazamine and topoisomerase I inhibitors in hepatocellular carcinoma in vitro and in vivo:It is demonstrated that hypoxia can induce resistance to topoisomerase I inhibitors in HCC cell lines and HIF-1α play an important role in this phenomenon. TPZ is indeed inhibiting HIF-1α accumulation in HCC cell lines. The results demonstrated that the combination of tirapazamine and topoisomerase I inhibitors exerts strong synergistic antiproliferative effect against human hepatocellular carcinoma including HepG-2, Bel-7402, Hep3B, Huh7and SMMC-7721, with the combination index below0.7. However, in a normal liver cell line, the combination effect was minimal. Moreover, treatment of hepatocellular carcinoma Bel-7402-bearing nude mice with tirapazamine plus CPT-11resulted in more significant tumor growth inhibition, and caused no extended bodyweight loss to the animals.(2) The mechanism of the synergistic anti-tumor activity caused by the combination of tirapazamine and topoisomerase I inhibitors in hepatocellular carcinoma:It is demonstrated that the combination of TPZ and SN-38(the active metabolite of CPT-11) induces significant apoptosis in Hepatocellular Carcinoma. The enhanced apoptosis induced by TPZ plus SN-38was accompanied by mitochondrial membrane depolarization, caspases-3activation and PARP cleavage in the Bel-7402cells. The combination decreased the rate of HIF-loc protein synthesis, suppresses HIF-1α expression and transcriptional activity in vivo and in vitro. The suppression of HIF-loc by combination caused an inhibition of DNA repair, resulting in a larger population of persistently unrepaired DSBs. Rad51and phosphorylation of Chkl at Ser317were decreased in the combination treatment of TPZ and SN-38. The close correlation between loss of RAD51and DSB accumulation suggested that the inhibition of the RAD51-mediated HR repair pathway was likely responsible for the accumulation of DSBs in Bel-7402cells.Conclusion:(1) In this study, It was reported that HIF-1α is strongly correlated with HCC cell line resistance to topoisomerase I inhibitors. The combination of TPZ and topoisomerase I inhibitors significantly improved the anticancer activities of the drugs, as indicated by the synergistic inhibitory effects on cancer cell proliferation, the sensitized execution of apoptosis, and the enhanced in vivo antitumor efficiency. These results was attributed to the downregulation of HIF-1α.(2) The main mechanism of the combination of tirapazamine and topoisomerase I inhibitors was that through cooperative inhibition of HIF-1α accumulation, the combination of TPZ and SN-38inhibited the RAD51-mediated homologous recombination repair pathway, induced the accumulation of topoisomerase inhibitor-induced DNA damage, exhibited synergistic cytotoxicity and triggered significant apoptosis in HCC cell lines.