The Effects Of Metformin Combined With Apatinib On Proliferation,Migration And Apoptosis Of Gastric Cancer Cells

ObjectiveTo investigate the effects of metformin combined with apatinib on the proliferation,migration,apoptosis and cell cycle of gastric cancer(BGC-823,SGC-7901)cell lines and the possible mechanism,and provide laboratory data for the treatment of gastric cancer.Methods1.MTT assay was used to detect the cell proliferation inhibition rate.Gastric cancer BGC-823 cells and SGC-7901 cells were treated with metformin(0-30 mmol/L),apatinib(0-60 μmol/L),and the combination of the two drugs for 24 h,48 h,72 h,the cell proliferation inhibition rate was detected by MTT assay.2.The crystal violet staining method was used to detect the colony formation ability.Gastric cancer BGC-823 cells and SGC-7901 cells were treated with 5 mmol/L metformin,20 μmol/L apatinib,and the combination of the two drugs for 7 d,the effect of the drug on cell colony formation ability was detected by crystal violet staining.3.Cell scratch repair experiments were used to detect cell migration ability.The gastric cancer BGC-823 cells were treated with 5 mmol/L metformin,20 μmol/L apatinib,and the combination of the two drugs for 48 h,the effect of the drug on the migration capacity of gastric cancer cells was observed by microscope.4.Flow cytometry was used to detect the apoptosis rate and cell cycle.The gastric cancer BGC-823 cells were treated with 5 mmol/L metformin,20 μmol/L apatinib,and the combination of the two drugs for 48 h,the effects of the drugs on apoptosis and cell cycle were examined by flow cytometry.5.Western blot was used to detect protein expression.The gastric cancer BGC-823 cells were treated with 5 mmol/L metformin,20 μmol/L apatinib,and the combination of the two drugs for 48 h.the expression levels of related proteins of the vascular endothelial growth factor signaling pathway and migration,apoptosis,cell cycle was detected by Western blot.Results1.MTT results showed that compared with the control group,the cell proliferation inhibition rate of gastric cancer BGC-823 cells and SGC-7901 cells increased after being treated by metformin,or apatinib alone,which in a doseand time-dependent manner.Furthermore,metformin combined with apatinib could inhibit the cell proliferation of gastric cancer obviously than that of the single drug group(P<0.05).2.The resulsts of cell colony formation experiments showed that the formation colonies of gastric cancer cells(BGC-823 cells and SGC-7901 cell)were decreased no matter single treatment or combined one.The number of cell colonies in the combined drug group was less than that in the single drug group(P <0.05).3.The results of cell scratch repair experiments showed that the cell migration of gastric cancer BGC-823 cell was decreased no matter single treatment or combined one.Furthermore,metformin combined with apatinib had a more significant inhibitory effect(P <0.05).4.Flow cytometry results showed that compared with the control group,both metformin and apatinib could induce apoptosis of gastric cancer BGC-823 cells(P <0.05),and the combined application has enhanced apoptosis.The cell cycle results showed that the use of metformin combined with apatinib blocked the cell cycle in G0/G1 phase.5.Western blot showed that after treatment by metformin combined with apatinib,the protein expression levels of VEGFR2,PI3 K,AKT,Bcl-2,Cyclin D1,CDK4,CDK6 and MMP9 were decreased significantly,and PI3 K and AKT protein phosphorylation levels were decreased significantly,and the expression level of Bax and Cleaved-Casepase3 were increased in the group treated with the combinations of drugs(P<0.05).ConclusionMetformin combined with apatinib can inhibit the proliferation and colony formation of gastric cancer BGC-823 and SGC-7901 cells in vitro.Moreover.this combination therapy can inhibit the migration of gastric cancer cells,induce apoptosis,and block the cell cycle.The mechanism may be related to the inhibition of VEGF / VEGFR2 / PI3 K / AKT signaling pathway.