Synergistic Effect Of AZT Combined With As₂O₃ On Hepatocellular Carcinoma In Mice And Its Mechanism

Objective:To explore the effects of Zidovudine（AZT）combined with Arsenic trioxide（As₂O₃）on the proliferation,apoptosis,invasion and metastasis of hepatocellular carcinoma cells through synergistic effects in the complex tumor microenvironment of C57BL/6J tumor bearing mice.To further explore the role of Egr-1 in hepatocellular carcinoma and provide theoretical support for further study.Methods:（1）Cultured hepatocellular carcinoma cells were in situ modeled in C57BL/6J mice,which were successfully divided into 12 groups（n=10）.They were blank control group,negative control group,AZT（5,10,15,20 mg/kg）group,As₂O₃（0.5,1.0,1.5,2.0 mg/kg）group,combined administration(AZT 10 mg/kg+As₂O₃1.0 mg/kg,（AZT 15 mg/kg+As₂O₃1.5 mg/kg）group,different groups of mice were treated with medication intervention and feeding.（2）The inhibitory rate of drug intervention on liver cancer in tumor-bearing mice and the synergistic index of the combination of two drugs were measured.And using the method of real-time fluorescent quantitative PCR（RT-qPCR）and Western blot method（Western blot）respectively to detect Cyclin D1 and CDK4,Caspase-3,E cadherin,q,Egr 1 m RNA and protein expression level.RESULTS:（1）The optimal concentration of tumor inhibition in AZT group was10mg/kg,and the tumor inhibition rate was 46.42%.The optimal tumor inhibition concentration in As₂O₃group was 1.5mg/kg,and the tumor inhibition rate was 50.98%.When AZT（10mg/kg）combined with As₂O₃（1.0mg/kg）was used to intervene with tumor bearing mice,the tumor inhibition rate was 58.36%.The synergistic index of AZT（10mg/kg）combined with As₂O₃（1.0mg/kg）is 0.234,showing strong synergistic effect.（2）RT-qPCR and Western blot detection showed that after successful modeling of C57BL/6J mice,Cyclin D1,CDK4 and Snail gene and protein expression levels in negative control group were significantly increased compared with blank control group.The expression levels of Caspase-3,E-cadherin and Egr-1 genes and proteins were significantly decreased.（3）RT-qPCR and Western blot detection showed that after drug intervention,the gene and protein expression levels of Caspase-3,E-cadherin and Egr-1 in mouse liver cancer tissues were significantly up-regulated compared with those in the negative control group.In addition,the drug effect of AZT combined with As₂O₃was significantly higher than that of AZT and As₂O₃alone,while the expression levels of Cyclin D1,CDK4 and Snail were significantly down-regulated.The drug effect of AZT combined with As₂O₃was significantly lower than that of AZT and As₂O₃alone.Conclusion:（1）AZT combined with As₂O₃inhibited the development of hepatocellular carcinoma in tumor bearing mice,and the two drugs had synergistic effect;（2）AZT combined with As₂O₃inhibited the proliferation of hepatocellular carcinoma cells in tumor-bearing mice,which may be related to the blocking of G1/S phase of cell cycle;（3）AZT combined with As₂O₃promoted apoptosis of hepatoma cells in tumor-bearing mice,which may be related to the activation of Caspase-3 pathway.（4）AZT combined with As₂O₃inhibited the invasion and metastasis of hepatocellular carcinoma cells in tumor-bearing mice,which may be related to the inhibition of EMT process.