| Objective: To observe the proliferation effect of apatinib respectively and combined with octreotide on human hepatocellular cells line HepG2 and normal cell line L02 in vitro,and to explore the possible mechanism.Methods: Using different concentrations of apatinib and octreotide respectively in human hepatocellular carcinoma cell line HepG2 and normal cell line L02 in 24 h,48h,72 h,respectively.Using CCK-8 to detect the OD value,and calculate the inhibition rate of cell proliferation in each treatment group;And through the IC50 result,select the best dose,concentration and time of the two drugs,combined with the concentration gradient of drugs.The detection effect of each treatment group after the treatment with 48 h on the cell cycle by flow cytometry;the apoptosis of cells were determined by flow cytometry after48 h treatment.;and using immunocytochemical assay to detect expressions of VEGFR-2,Hic-5,p-ERK in each group cells;After 48 hours of treatment,the expression changes of Mcl-1,ERK,p-ERK,Hic-5,VEGFR-2,TGF-?1 in each cell group were measured by Western blotting.Results:1.Apatinib on HepG2 cell 24 h,48h,72 h,were measured by CCK-8,and half inhibitory concentration(IC50)were 17.588 ?M,10.523 ?M,9.324 ?Mrespectively;after octreotide affects on HepG2 cell groups 24 h,48h,72 h,IC50were 0.428 ?M,0.068 ?M,0.061 ?M.In the same time,compared with the control group,octreotide group of different concentration on HepG2 cell proliferation inhibition rate was significantly higher(P<0.05),and increased with the increase of drug concentration.And two kinds of medicine affect L02 the same concentration and time was not significantat,and all less than 3%.2.According to the results of CCK-8,selected 0.01 ?M octreotide combined different concentration gradient of apatinib 48 hours,and 10 ?M apatinib respectively with different concentrations of octreotide 48 hours showed inhibitory effects on HepG2,combined effect was significantly higher than that of the single drug group(P<0.05).3.HepG2 cell cycle distribution was detected by flow cytometry(FCM),apatinib(10 ?M)group,octreotide group(0.01 ?M),apatinib(10 ?M)conbined octreotide(0.01 ?M)group were treated after 48 hours,there are significant increase of the proportion of G1 phase after treatment than the control group(P<0.05),and after combination therapy further increased(P<0.05)in the S phase after treatment compared with the control group decreased(P<0.05),and further reduce the combined treatment(P<0.05).4.Apoptosis of HepG2 cells was detected by flow cytometry(FCM),apatinib and octreotide alone group were significantly higher than those in control group induced apoptosis(P<0.05),the comparative analysis found the combination group was better than that of single drug group(P<0.05).5.The results of immunocytochemistry,after 48 hours of treatment,the expression of VEGFR-2,Hic-5 and p-ERK in HepG2 cells was lower than that in control group(P<0.05),and the combination group was more obvious than that of the single drug(P<0.05)6.Western blot result,apatinib(10?M)treatment group,octreotide treatment group(0.01 ?M),apatinib(10?M)conbined octreotide(0.01 ?M)treatment group were treated for 48 hours,showed the expression of Mcl-1,TGF-?1,Hic-5,p-ERK,VEGFR-2 were lower compared with the control(P<0.05),combination group was lower than single group(P<0.05).While the ERK relative expression level between the various approaches,the difference has no statistical significance(P>0.05).Conclusion: Apatinib and octreotide monotherapy had significant effects on HepG2 cell proliferation inhibition and induced its apoptosis,depengding on concentration;the effect is more obvious in combined treatment group.The main effect of octreotide and apatinib through stagnating cells in G1 phase.Single drug effect of anti apoptotic protein Mcl-1,Hic-5,TGF-?1,p-ERK,VEGFR-2 expression decreased,apatinib combined treatment with octreotide,drugs,decreased more significantly,thus we can inference that it may through these pathways to inhibit HepG2 proliferation. |
PDF Full Text Request