Effects Of Apatinib And Its Mechanisms On Human Hepatocellular Carcinoma Multiple Drug-resistant Cell Line BEL-7402/5-FU

Objective:To investigate the effect of apatinib on human hepatocellular cell line BEL-7402/5-FU and its possible mechanisms.Methods:?1?The cell line BEL7402 was induced to form a multidrug resistant Sub-cell line BEL7402/5-FU through increasing fluorouracil with concentration gradient method and observed the cell morphology of two kinds of human hepatocellular cells.?2?The different concentrations of three chemotherapeutic drugs?fluorouracil,adriamycin and cisplatin?were selected to treat BEL-7402 cells and BEL-7402/5-FU cells for 24h,48h and 72h,the MTT method was used to determine the proliferation inhibition rate,half inhibition(IC₅₀)and calculate the drug resistance index?RI?;MTT assay was used to detect the changes of cell proliferation inhibition rate after apatinib intervened BEL-7402 cells for 24h,48h and 72h.The inhibition rate of apatinib on BEL-7402 cells for 48h and the index of drug resistance of BEL-7402/5-FU on apatinib were calculated.The IC₅₀ values of fluorouracil,adriamycin and cisplatin on BEL-7402/5-FU cells were selected to unite with the IC₅₀0 value of apatinib,the mixed drugs were used to treat BEL-7402/5-FU cells 24h,48h and 72h,and to observe the synergistic effect.?3?The IC₅₀ value of apatinib was selected to treat BEL-7402/5-FU cells for 48h and observe the morphological changes.?4?The different concentrations of apatinib treated BEL-7402/5-FU cells,the transwell assay was used to detect the migration and invasion ability of cells.?5?BEL-7402/5-FU cells were treated by different concentrations of apatinib for 48h,the cell cycle and apoptosis rate were analyzed by flow cytomete?FCM?.?6?Western blot method was used to detect the expression of the resistance protein MRP1,BCRP and P-gp between with BEL-7402 and BEL-7402/5-FU cells.After different concentrations of apatinib treated BEL-7402/5-FU cells for 48h,western blot method was used to detect the expression of the protein Bcl-2,Bax,caspase-3,MRP1,BCRP and P-gp in each group.Results:?1?Compared with BEL-7402 cells,BEL-7402/5-FU cells had slightly different morphologies,their long axis were shorter than BEL-7402 cells.?2?MTT:BEL-7402/5-FU cells showed different drug resistance to three drugs,among which fluorouracil had higest RI,about 10.715±1.363,while BEL-7402/5-FU cells showed no drug resistance to apatinib.Fluorouracil,adriamycin,cisplatin and apatinib were treated BEL-7402/5-FU cells for 24h,48h and 72h,all of them showed different degrees of cell proliferation inhibition.In the concentration range and within 72h,there were dose-effect and time-effect relationship.MTT assay results showed that the cells treated with fluorouracil,adriamycin,cisplatin and apatinib for 24h,48h and 72h later,they showed a significant synergistic enhancement effect?P<0.05?,and showed a time-effect and dose-dependent relationship.?3?After apatinib?20ug/ml?treated BEL-7402/5-FU cells for 48h,the morphologies of BEL-7402/5-FU cells were obviously changed,the number of cells decreased,the morphologies of cells were irregular,and the number of cells fragments increased.?4?The results of transwell assay showed that apatinib could inhibit the migration and invasion ability of BEL-7402/5-FU cells and the ability was significantly increased follow with the concentration?P<0.05?.?5?Flow cytometry assay results showed that the apoptotic rate of BEL-7402/5-FU cells in the experimental group increased significantly?P<0.05?,and the apoptotic rate of BEL-7402/5-FU cells increased with the increase of apatinib concentration?P<0.05?.The proportion of BEL-7402/5-FU cells in G1phase were significantly higher than that in control group?P<0.05?,and the percentage of cells in G1 phase was significantly increased with the increase of apatinib concentration?P<0.05?.?6?The results of western blot showed that the expression of the protein MRP1,BCRP and P-gp in resistant strain were higher than sensitive strain?P<0.05?.After apatinib treated BEL-7402/5-FU cells for 48h,the expression of the protein MRP1,BCRP,P-gp and Bcl-2 were decreased?P<0.05?,while the expression of the protein Bax and Caspase-3 were increased?P<0.05?.Conclusion:The cell line BEL7402/5-FU showed high drug resistance and multidrug resistance to a variety of chemotherapeutic drugs,but they did not exhibit significant resistance to apatinib.Apatinib could inhibit the growth of hepatoma cells and which had showed synergistic enhancement effect with various chemotherapeutic drugs.Apatinib could inhibit the ablity of BEL7402/5-FU cells migration,invasion and induce apoptosis.Its mechanism may be associated with upregulation protein of Bax,Caspase-3 and downregulation protein of Bcl-2,MRP1,BCRP and P-gp.