Sensitive Screening Of SMKI And Research On Anti-hepatoma Mechanism Of RDEA119 And HG6-64-1

Objective:1.To screen out sensitive drugs for BEL-7402 cells.2.To investigate the effect of MEK kinase inhibitor RDEA119 and B-Raf kinase inhibitor HG6-64-1 on the proliferation and apoptosis of hepatoma cells,and its mechanism.Methods:1.The effects of 16 drugs targeting different signaling pathways on the activity of BEL-7402 cells were detected by MTT.2.CCK8 assay,plate cloning and flow cytometry were employed to detect the effect of the cell proliferation and apoptosis which treated by different concentrations(0 ?M,0.1 ?M,0.5 ?M,1 ?M)of RDEA119 or HG6-64-1.3.Western blot was used to detected the chang of B-Raf-MEK-ERK pathway related protein,G0/G1 phase protein CyclinD,and anti-apoptotic protein Bcl-2expression of the BEL-7402 cells which treated by different concentrations of RDEA119 or HG6-64-1.Results:1.The IC50 of RDEA119(MEK inhibitor)and HG6-64-1(B-Raf inhibitor)to BEL-7402 cells were only 0.41 ?M and 0.66 ?M,respectively.2.After 72 h of RDEA119 treatment in BEL-7402 cells,the CCK8 results showed that the survival rate of BEL-7402 cells was reduced in dose dependence.The formation of flat clones showed that the number of clones in the cells decreased in a dose-dependent manner,and the flow cytometry showed that the percentage of cells in G0/ G1 phase in the drug group was significantly higher than that in the control group(P<0.05).There was no significant difference in the percentage of cells in G0/G1 phase between different concentrations(P>0.05).The flow cytometry showed that the apoptosis rate of the cells in the drug group was higher than that of the control group,and was dose-dependent(P<0.05).Western blot results showed that RDEA119 could inhibit the expression of p-ERK,CyclinD and Bcl-2 at low concentration(0.1 uM),and had a dose-dependent manner,and had no effect on the expression of ERK protein.3.After BEL-7402 cells treated by different concentrations of HG6-64-1,the CCK8 results showed that the survival rate of BEL-7402 cells was reduced in dose dependence.The formation of flat clones showed that the number of clones in the cells decreased in a dose-dependent manner,and the flow cytometry showed that the percentage of cells in G0/G1 phase in the drug group was significantly higher than that in the control group(P<0.05).There was no significant difference in the percentage of cells in G0/G1 phase between different concentrations(P>0.05).The flow cytometry showed that the apoptosis rate of the cells in the drug group was higher than that of the control group,and was dose-dependent(P<0.05).Western blot results showed that HG6-64-1 could inhibit the expression of p-MEK,p-ERK,CyclinD and Bcl-2 at low concentration(0.1 uM),and had a dose-dependent manner,and had no effect on the expression of MEK and ERK protein.Conclusions:1.RDEA119 and HG6-64-1 have strong anti BEL-7402 ability,which can inhibit the proliferation of BEL-7402,induce G0/G1 phase arrest and induce apoptosis of BEL-7402 cells at a low concentration.2.Preliminary mechanism study shows that RDEA119 and HG6-64-1 may inhibit G0/G1 arrest,promote cell apoptosis and inhibit cell proliferation by inhibiting phosphorylation of B-Raf/MEK/ERK pathway related protein and inhibiting Bcl-2and CyclinD expression.