Axitinib,A Multi-target Tyrosine Kinase Inhibitor,Induces Growth Arrest In Human Hepatocellular Carcinoma SNU-423 Cells

Objective:1.To detect the effects of Axitinib on proliferation,cell cycle and apoptosis of hepatoma cells,and to reveal the complex molecular mechanisms.2.For clinical treatment of such liver cancer cells,Axitinib will use as a reference.At the same time,it provides experimental basis for the design and development of more effective molecular targeted drugs.Methods:1.Screening Axitinib-sensitive hepatocellular carcinoma cell lines by MTT assay.Axitinib with a certain concentration gradient was applied to BEL-7402 cells,SK-Hep1 cells,HepG2 cells and SNU-423 cells.The viability of various cells was detected by MTT assay.By analysis and comparison,the most sensitive cell line was determined..2.Cell proliferation assay,Hoechst staining and flow cytometry were used to detect the effects of Axitinib on the proliferation,apoptosis,necrosis and cell cycle of SNU-423 cells.3.The molecular mechanism of Axitinib affecting the survival and proliferation of SNU-423 cells line was initially explored by Western blotting.Results:1.The drug sensitive cell line SNU-423,which is the most sensitive one to the multi-target tyrosine kinase inhibitor Axitinib,was screened by drug sensitivity detection of four liver cancer cell lines.2.The result of cell cloning experiments showed that Axitinib had a significant inhibitory effect on the colony forming ability of SNU-423 cells,and this inhibition showed a good concentration-dependent relationship.Furthermore,it was shown that Axitinib has a strong inhibitory effect on the proliferation of SNU-423 cells.3.The result of apoptosis showed that the apoptotic SNU-423 cells increased with the increase of Axitinib concentrations.The detection of the expression of related proapoptotic proteins Cleaved Caspase-3 and Bcl-2 by Western blot showed that the expression of Cleaved Caspase-3 protein was positively correlated with the gray scale of the protein band with the increase of Axitinib concentration.The increase in Bcl-2protein was significantly reduced with increasing Axitinib concentration.Overall,Axitinib promoted apoptosis in SNU-423 cells,and cell apoptosis was dependent on drug concentrations.4.The flow cytometry and the Western blot analysis of the relevant cyclin CyclinD1 showed that the cycle kinetics of SNU-423 cells were significantly different after treatment with different concentrations of Axitinib,and the cells blocked by G1 phase accounted for The ratio increased with increasing Axitinib concentration.Therefore,Axitinib is able to cause G1 arrest in SNU-423 cells.5.Western blotting results showed that SNU-423 cells were treated with different concentrations of Axitinib,and the expression levels of intracellular VEGFR2,p38 MAPK,Src,P-Src(Tyr527),Akt,P-Akt(Thr308),MEK1/2 and Erk1/2 were not significantly affected,and the expression levels of these proteins remained relativelystable.The expression levels of P-p38 MAPK,P-Src(Tyr416),P-Akt(Ser473)and P-MEK1/2 were positively correlated with the concentrations of Axitinib.In the experimental group of 1 ?mol/L to 10 ?mol/L,Axitinib significantly inhibited the expression of P-VEGFR2 protein,and the phosphorylation of VEGFR2 was significantly decreased with increasing drug concentration.In the experimental group of 1 ?mol/L and5 ?mol/L drug concentration,the expression of P-Erk1/2 increased with the increase of drug concentration compared with the control group,while the experimental group of 10?mol/L drug concentration,The expression level of P-Erk1/2 remained relatively stable compared to the Control group.Conclusion:1.The multi-target tyrosine kinase inhibitor Axitinib has different inhibitory effects on the survival and proliferation of hepatocellular carcinoma cell lines(BEL-7402 cells,SK-Hep1 cells,HepG2 cells,SNU-423 cells).Among them,SNU-423 cells have the strongest inhibitory effect on survival and proliferation,and are the most sensitive to Axitinib.2.The multi-target tyrosine kinase inhibitor Axitinib inhibits the proliferation of SNU-423 cells in a concentration-dependent manner,promotes cell apoptosis,and induces G1 phase arrest.3.Axitinib can promote the phosphorylation of p38 MAPK protein through specific signal molecules,thereby activating p38 MAPK protein-related signaling pathway,and finally induces caspase-dependent apoptosis in SNU-423 cells.