The Effects Of NVP-BEZ235 Combined With Autophagy Inhibitor On Proliferation And Apoptosis In Human Gastric Cancer Cells AGS

Objective:The aim of this study is to explore the effect of autophagy inhibitor 3-MA combined with novel doublel PI3K/mTOR inhibitor BEZ235 on the proliferation,apoptosis and autophagy of GC cells AGS to verify the hypothesis,so then,we can provide a reliable evidence for a new treatment strategy that is namely blockage of autophagy can augment the BEZ235's anticancer activity by promoting apoptosis and inhibiting cell proliferation.Methods:1.Add different concentrations of BEZ235(0?0.05?0.1?0.25?0.5?1?2?M)?3-MA(0 ? 0.5 ? 1 ? 1.25 ? 1.5 ? 2 ? 2.5mM)and Comb Group to DMEM high glucose medi?m(Including 10% fetal bovine serum),then,culture the GC cells AGS for 24 and 48ho?rs.The morphological changes were observed by inverted microscope,cell proliferation was detected by Counter Star counting and MTT.2.The cell apoptosis was detected by flow cytometry using Annexin V/PI.3.The protein changes including apoptosis-related,autophagy-related and the PI3K/AKT/mTOR pathway-related proteins were determined by Western Blot.Results:1.The AGS cells in the Control Group were in a good adherent status,by contrast,the AGS cells in the experimental group treated with BEZ235 or 3-MA showed obvious changes in morphology sich as distortion?pyknosis and partial sispension,a decrease in the number of cells as well;the proliferation of AGS cells was inhibited by BEZ235,showing apparent dose-effect and time-effect relationship;When the AGS cells were cultured in BEZ235 Group(P?0.003)?3-MA Group(P?0.006)or Comb Group(P?0.001)for 24 h and 48 h,the cell viability of the three groups were lower than the control group.2.Flow cytometry showed that compared with the Control Group,the BEZ235 Grouphad higher apoptosis rate(P=0.000);the cell apoptosis rate of the Comb Group was higher than that of any singe drug groups(P=0.000).3.Western Blot showed that the expression of cleaved caspase-3 and bax in the Comb Group was significantly higher than that in the BEZ235 Group(p<0.05);the expression of Bcl-2 in the Comb Group was lower than that in the BEZ235 Group(p<0.05);The ratio of Bcl-2 and Bax in the BEZ235 Group were significantly lower than that in the Control Group(P=0.000);the expression of LC3 II/Iand Beclin1 in the Comb Group was significantly lower than that in the BEZ235 Group(p<0.05);the expression of P62 in the Comb Group was higher than that in the BEZ235 Group(p<0.05);the expression of PTEN in the Comb Group was significantly higher than that in the BEZ235 Group(p<0.05);the expression of Akt?p-Akt(Thr308)? mTOR in the Comb Group was lower than that in the BEZ235Group(p<0.05).Conclusions:1.The proliferation of GC cells AGS can be inhibited significantly by PI3K/m TOR inhibitor BEZ235 in a time and concentration dependent way;autophagy inhibitor 3-MA can enhance BEZ235-induced apoptosis by inhibiting autophagy in GC cells AGS.2.PI3K/mTOR inhibitor BEZ235 can up-regulate the expression of cleaved caspase-3and bax,meanwhile,down-regulate the expression of Bcl-2.3.PI3K/mTOR inhibitor BEZ235 can induce the autophagy of the AGS cells,which increases the level of LC3II/I?Beclin 1 and decrease the level of P62.4.3-MA can inhibit the autophagy of the AGS cells induced by BEZ235,moreover,inhibiting autophagy can promote cell apoptosis.5.BEZ235 regulates the downstream molecules of the PI3K/Akt/mT0 R signaling pathway by inhibiting P13 K and mTOR.