Research On Apatinib As A Chemotherapy Drug For Hepatocellular Carcinoma

Objective:The inhibitory effect of Apatinib which is oral small molecule anti-tumor angiogenesis drug was investigated on Hep3B cell in vitro and in vivo,and its mechanism of inhibiting the proliferation of hepatoma cells was explored.It is provided a theory and experimental basis for the clinical study of Apatinib as a chemotherapy drug for liver cancer.Methods:1.In vitro:（1）The cell lines were screened for experiments by CCK8 method;（2）The viabilities of Apatinib on Hep3B cell were detected by CCK8 method;（3）The apoptosis rates of Apatinib on Hep3B cell were examinated by flow cytometry;（4）The change of mitochondrial membrane potential of Apatinib on Hep3B cell was observed by JC-1 staining;（5）The expression levels of protein USP22,Parkin and MCL1 were tested on Hep3B cell treated with Apatinib by western blot.2.In vivo:（1）The animal model of human Hepatocellular carcinoma Hep3B cell transplanted in nude was estabilished to test anticancer effect of Apatinib;（2）HE staining was used to observe the effect of tumor and normal liver tissue cell morphology in untreated and Apatinib-treated group;（3）The expression levels of the protein USP22,Parkin and MCL1 were detected by immunohistochemistry experiment in untreated and Apatinib-treated group.Sorafenib was used as a contrast drug in the experiment.Results:1.Hep3B cell was the lowest viability cell treated with Apatinib（30μmol/L）for 24 h.It was used as the research object of this experiment.2.Apatinib had good inhibitory effect on Hep3B cell.The inhibition rates were positively correlated with time and dose.It discovered that IC₅₀ values of Hep3B cell were exceeded 25μmol/L,18.23±0.89μmol/L,9.87±0.57μmol/L for 24 h,48 h,72 h treated with Apatinib,respectively.And the IC₅₀ values of Hep3B cell were 13.17±1.23μmol/L,5.11±0.98μmol/L,less than 5μmol/L for 24 h,48 h,72 h treated with Sorafenib,respectively.3.Apatinib or Sorafenib could promote the Hep3B cell apoptosis.The apoptosis rates were increased in a dose-dependent manner.The apoptotics rates of Apatinib（0μmol/L,15μmol/L,20μmol/L,25μmol/L）were 5.73±0.8%,7.83±0.76%,16.9±2.62%,21.1±1.08%,and the apoptosis rates of Sorafenib（0μmol/L,5μmol/L,10μmol/L,15μmol/L）were 5.9±0.46%,7.3±0.89%,14.5±1.14%,29.83±5.37%,respectively.4.Apatinib or Sorafenib could decrease the mitochondrial membrane potential of Hep3B cell.The mitochondrial membrane potential also decreased in a dose-dependent manner.5.Apatinib or Sorafenib could down-regulated the expression levels of protein USP22,Parkin and MCL1 protein in a time-and dose-dependent manner on Hep3B cell.6.Apatinib or Sorafenib could inhibit the growth of liver cancer transplanted tumors.The anti-tumor effect of Apatinib（50 mg/kg）was the most obvious,which of Sorafenib and Apatinib were closer in the dose of 30 mg/kg.7.The numbers of cells were decreased,and the cell gaps were significantly increased.There were more vacuoles between the cells in the Apatinib or Sorafenib-treated group.The morphological changes of Apatinib（50 mg/kg）were the most obvious.8.The expression levels of protein USP22,Parkin and MCL1 were down-regulated in the Apatinib or Sorafenib-treated group,which of Apatinib（50mg/kg）was the most obvious.The protein USP22,Parkin and MCL1 expression levels of Apatinib or Sorafenib in the dose of30mg/kg were equivalent.Conclusions:（1）Apatinib or Sorafenib inhibited the proliferation of human liver cancer cells and tumors in a time-and dose-dependent manner;（2）Apatinib or Sorafenib inhibited liver cancer cells proliferation may through the USP22/Parkin/MCL1single pathway.