The Study Of Synergistic Anti-tumor Activity And Mechanism Of Irinotecan With Gefitinib On Hepatocellular Carcinoma

Objects: Liver cancer is a kind of malignant tumor with high morbidity and fatality, and its incidence is highest in China. Hepatocellular carcinoma(HCC) is the leading histological subtype of liver cancer, while its incidence is still increasing worldwide. The treatment options available for patients with HCC were surgery, transhepatic arterial chemotherapy embolization, early-stage radiofrequency ablation and chemotherapy. Surgical treatment is curative for patients with early stage-HCC but not for advanced HCC patients. Efficacy or response rate of radiofrequency ablation in advanced HCC were pretty low. Thus chemotherapy is the only or necessary choice in most of the HCC patients. For now, only Sorafenib is the standard systemic drug for advanced HCC. Unfortunately, limited survival benefits due to drug resistance and intolerance restricted the use of Sorafenib. Our previous study found that classical cytotoxic drug Topoisomerase-I inhibitor Irinotecan and EGFR-tyrosine kinase inhibitor Gefitinib have synergistic anticancer activity on HCC cell lines including Hep G-2 and BEL-7402. Based on this, we tried to investigate the synergistic effects both in vitro and in vivo and its mechanism.Methods: 1. Study the synergistic effects of Irinotecan(SN-38) with Gefitinib both in vitro and in vivo: SRB assay and clonogenic assay were employed to measure the survival of HCC cells lines(Hep G-2 and BEL-7402) after the treatment of SN-38 and/or Gefitnib. Hep G2 xenografts models were established to study the anti-tumor activity of the combination of Irinotecan and Gefitinib in vivo. 2. Investigate apoptosis level of HCC cells after the treatment of SN-38 and Gefitinib: The Annexin V-PI staining and flow cytometric assay was used to evaluate the apoptosis rate of HCC cells(Hep G-2 and BEL-7402) after the treatment of SN-38 and/or Gefitinib. Western Blot assay was applied to detect the expression level of apoptosis-related protein. DAPI staining was employed to observe the extensive nuclear condensation and cellular fragmentation in cells. 3. Determine the relationship between the synergistic effects and DNA damage response: Western Blot assay was used to detect the expression level of DNA damage response-related proteins in HCC cells(Hep G-2 and BEL-7402) treated with SN-38 and/or Gefitinib. Immunofluorescence was applied to detect DNA damage-related protein γ-H2 AX. Protein synthesis inhibitor CHX, lysosomal inhibitors chloroquine(CQ) and proteasome inhibitors MG132 were used to study the relationship between the degradation of Rad51 and synergistic anti-tumor activity of SN-38 with Gefitinib.Results: 1. Study the synergistic effects of Irinotecan and Gefitinib in HCC. In vitro, SRB assay showed that after exposed to SN38 and/or Gefitinib for 48 h, HCC cells(Hep G-2 and BEL-7402) were inhibited significantly. And the combination index(CI) indicated that SN-38 and Gefitinib have synergistic activity to suppress the growth of HCC cells. In additon, the result of clonogenic assay further verified that SN-38 plus Gefitinib have observably synergistic effect. In vivo, the Hep G2 xenograft model was used to test the efficacy of the combination of Irinotecan(1mg/kg) and Gefitinib(100mg/kg).Result showed that the combination of Irinotecan(1mg/kg) and Gefitinib(100mg/kg) significantly suppressed the tumor growth and such suppression was greater than that caused by Gefitinib or Irinotecan treatment alone, having synergistic treatment effect on HCC. 2. Study the mechanism of the synergistic anti-tumor activity caused by the combination of Irinotecan and Gefitinib in HCC.(1) Gefitinib enhances SN-38–triggered caspase-dependent apoptosis in hepatocellular carcinoma cells:(1) The Annexin V-PI staining and flow cytometric assay implied that the combination treatment of SN-38 and Gefitinb can lead to obvious apoptosis;(2)After DAPI staining, extensive nuclear condensation and cellular fragmentation can be observed in HCC cells treated with Gefitinib plus SN-38;(3) The result ofWestern Blot assay showd that combination treatment will increase the expression level of apoptosis-related proteins in HCC cells. And pre-treatment of pan-caspase inhibitor Z-VAD-FMK, apoptosis level decreased, which indicated that apoptosis was caspase-dependent.(2) The combination therapy resulted in apparent DNA damages:(1)Immunofluoresence assay showed that the drug combination apparently induced more γ-H2 AX nuclear foci formation, which means the augment of DNA damage;(2) Western Blot assay indicated the increase of DNA damagerelated proteins γ-H2 AX and p53 and confirm the aggravation of DNA damage.(3) Accumulation of DNA damages caused by the combination therapy was due to defects of homologous recombination:(1) Western bolt showed that after 6h drug combination, the phosphorylation level of DNA damage repair-related protein CHK1 and CHK2 increased, implying the activation of DNA damage repair, while the expression level of another DNA damage repair-related protein Rad51 decreased;(2)12h/24 h treatment of SN-38 and/or Gefitinib, the expression level of Rad51 in HCC cells of Gefitinib treatment and combination treatment further reduced, indicating that the downregulation of Rad51 may be a key reason for the defects of homologous recombination and accumulation of DNA damages.(4) The ubiquitin–proteasome systems were involved in gefitinib-induced suppression of Rad51 expression:(1)Results from Western Blot indicated that Gefitinib downregulated Rad51 in a both dose and time dependent manner in HCC cells;(2)Pre-treatment of protein synthesis inhibitor CHX could noxt block the reduction of Rad51. While lysosomal inhibitor chloroquine(CQ) had no effect on the gefitinib induced suppression of Rad51, proteasome inhibitor MG132 apparently reversed such reduction, indicating that Rad51 might be degraded through the ubiquitin–proteasome system.Conclusion:(1) The combination of Irinotecan and Gefitinib significantly ved the anti-HCC activities of the drugs, as indicated by the synergistic inhibitory effects on cancer cell proliferation and the tumor growth.(2) The degradation of Rad51 would impede homologous recombination repair, thus induced caspase-dependent apoptosis in hepatocellular carcinoma cells by the accumulation of DNA damages. Gefitinib synergizes with Irinotecan to suppress hepatocellular carcinoma by promoting proteasome-dependent degradation of Rad51.