Cinobufotalin Induced Hepatocellular Carcinoma Cell Apoptosis Through Inhibition Of HIF-1?-Mediated Warburg Effect

Objective:Hepatocellular carcinoma(HCC)is one of the deadliest forms of cancer worldwide and the most common primary liver cancer to present in the clinic.Recent research suggests that the energy metabolism alternation is one of the hallmarks of cancer cells.Compared with normal cells,the increase of glucose uptake rate in liver cancer cells and the aerobic glycolysis enhancement,which are also known as the Warburg effect.A change in energy metabolism often correlates with tumor aggressiveness and poor patient prognosis in HCC.Therefore,it is essential to inhibit the glucose uptake and glycolysis of HCC cells.Cinobufotalin is a butadiene lactone found in toad venom,several studies have identified its anticancer activity.This study was to investigate whether Cinobufotalin affects glucose uptake and aerobic glycolysis in liver cancer? What is the molecular mechanism? The results of this thesis will provide a new strategy for the clinical treatment of liver cancer.Methods:Firstly,to verify the effects of Cinobufotalin on liver cancer cells,we performed CCK-8 assays in Hep G2 and SNU-739 cells with different concentrations of Cinobufotalin for 48 h.Apoptosis of Hep G2 and SNU-739 cells treated with Cinobufotalin were evaluated by Flow cytometer.Western blot assays were used to evaluate the protein levels of BCL-2,Cleaved Caspase-3/9,and PARP in HCC cells after treated with Cinobufotalin for 48?h.Also,we analyze the protein of HIF-1?,GLUT1,HK2 and PKM2 in HCC cells incubated with Cinobufotalin by Western blotting.HIF-1? was knock-down in Hep G2 and SNU-739 cells by transient transfection of sh RNA-HIF-1?,and then the influence of Cinobufotalin on protein expression of HK2,GLUT1,Cleaved Caspase-3 and Cleaved Caspase-9 was evaluated using these cell models.Based on the above experimental results,in order to determine whether Cinobufotalin could enhance doxorubicin sensibility in doxorubicin-resistant HCC cells,the typical doxorubicin-resistant HCC cells(Hep G2/ADM),were treated with Cinobufotalin and doxorubicin,we want to know whether Cinobufotalin enhanced doxorubicin sensibility in doxorubicin-resistance HCC cells.Furthermore,a xenografted tumor model was established using male BALB/c mice and used to measure the anti-tumor efficacy of Cinobufotalin and its combination with doxorubicin in vivo.HE and IHC assays were performed to evaluate the effect of Cinobufotalin on apoptosis.Meanwhile,the variation of HIF-1? and GLUT1 protein expression levels in tumor were measured through IHC assays.Results:1.CCK-8 assays results clearly showed that Cinobufotalin inhibited cell viability in Hep G2 and SNU-739 cells treated with different concentrations of Cinobufotalin in a dose-dependent manner.2.Cinobufotalin significantly induced apoptosis in Hep G2 and SNU-739 cells in a dose-dependent manner.3.The expression of Cleaved Caspase-3,Cleaved Caspase-9,BCL-2 and Cleaved-PARP were significantly increased in Hep G2 and SNU-739 cells after treated with Cinobufotalin.4.When treated with Cinobufotalin for 48?h,the protein expression levels of HIF-1?,HK2,PKM2 and GLUT1 in Hep G2 and SNU-739 cells were observably decreased in a dose-dependent manner.5.Cinobufotalin could induce the apoptosis of Hep G2/ADM cells and inhibit glycolysis in Hep G2/ADM cells.In addition,the results of CCK-8 assays indicated that Cinobufotalin enhanced doxorubicin-induced cell growth inhibition in Hep G2/ADM cells.6.Based on the tumor model of BALB/c mice,we observed that the-growth of solid tumors were remarkably suppressed in mice by given Cinobufotalin.HE and IHC results have shown that Cinobufotalin could interfere with the glycolysis process in solid tumors,and induce apoptosis in hepatocellular carcinoma cells.In addition,the combination of Cinobufotalin and doxorubicin therapy is more effective.Conclusion:Cinobufotalin induced hepatocellular carcinoma cell apoptosis through inhibition of HIF-1?-mediated Warburg effect.