Effect Of γ-secretase Inhibitor Combined With Autophagy Inhibitor On Proliferation And Apoptosis Of Gastric Cancer MKN-45 Cells

ObjectiveTo observe the effect of Notch signaling pathway inhibitor DAPT on autophagic signaling pathway in gastric cancer MKN-45 cells,observe the effect of autophagy inhibitor CQ on Notch signaling pathway in gastric cancer MKN-45 cells,and preliminary study on the proliferation of MKN-45 cells in gastric cancer with DAPT combined with CQ The effect of apoptosis.MethodsThe cells were divided into 4 groups: the control group(cultivated with RPMI-1640 complete medium without intervention drug),abbreviated as Control group;CQ group(cultured with RPMI-1640 complete medium containing 40 ummol/L CQ);DAPT group(in RPMI-1640 complete medium containing 80 ummol/L DAPT);combination group(in RPMI-1640 complete medium containing 40 ummol/L CQ and 80 ummol/L DAPT),hereinafter referred to as Comb group.MTT assay was used to detect cell proliferation.Flow cytometry was used to detect cell apoptosis.Transmission electron microscope,Western Blot and RT-PCR were used to detect the level of autophagy.Western blot was used to detect the expression of Notch signaling pathway.Results1.The proliferative capacity of gastric cancer MKN-45 cells decreased gradually with the increase of DAPT and CQ concentration and the prolongation of the action time.It showed a significant dose-effect and time-dependent relationship.The proliferation of gastric cancer cells treated with DAPT was significantly lower than that of DAPT group.In the Control group,the difference was statistically significant(t=17.971,P=0.000).The cell proliferation ability of the CQ group was significantly lower than that of the Control group,and the difference was statistically significant(t=27.882,P=0.000).The cell proliferation of the Comb group was significant.The ability was significantly lower than the DAPT group and the CQ group.The difference was statistically significant(t=18.514,P=0.000;t=7.185,P=0.002).2.The apoptosis rate of DAPT group in flow cytometry was significantly higher than that in Control group(t=12.470,P=0.000).The apoptosis rate in CQ group was significantly higher than that in Control group,and the difference was statistically significant(2).t=12.794,P=0.000),the apoptosis rate of Comb group was significantly higher than that of DAPT group and CQ group,the difference was statistically significant(t=7.339,P=0.002;t=11.415,P=0.000).3.TEM observation of the Control group showed a small amount of autophagic bodies and a small amount of autophagic lysosomes,DAPT group can see more autophagic bodies and more autophagic lysosomes,CQ group see more autophagic bodies And a small amount of autophagic lysosomes,a large number of autophagosomes and a small amount of autophagic lysosomes were observed in the Comb group.4.When DAPT concentration was 80umol/L,which was effected on gastric cancer MKN-45 cells for 24 h,the relative expression of autophagy-associated egg LC3 II DAPT group was significantly higher than that of the Control group when DAPT concentration was 80umol/L(t=14.893,P= 0.004)The relative expression of P62 in the DAPT group was significantly lower than that in the Control group.The difference was statistically significant(t=5.290,P=0.006).The relative expression of Beclin1 in the DAPT group was significantly higher than that in the Control group,and the difference was statistically significant(t= 6.695,P=0.003);RT-PCR detection of ATG7 m RNA relative expression level DAPT group was significantly higher than the Control group,the difference was statistically significant(t = 12.57,P = 0.000),ATG12 m RNA relative expression level DAPT group was significantly higher In the Control group,the difference was statistically significant(t=22.446,P=0.000).5.When the concentration of CQ was 40umol/L,which was effected on gastric cancer MKN-45 cells for 24 h,the relative expression of Notch1 in the Notch signaling pathway in Western Blot was significantly higher in the CQ group than in the Control group(t=11.590,P=0.000),the relative expression of Glut1 in the CQ group was significantly higher than that in the Control group(t=8.728,P=0.001).6.When 80umol/L DAPT was combined with 40umol/L CQ for 24 h in gastric cancer MKN-45 cells,the relative expression of autophagy-associated egg LC3 II in the Western Blot was significantly higher than that in the DAB and CQ groups.t=76.603,P=0.000;t=42.238,P=0.000),P62 relative expression level in Comb group was significantly higher than DAPT group and CQ group,the difference was statistically significant(t=48.765,P=0.000;t=5.837,P=0.004),the relative expression of Beclin1 in the Comb group was significantly lower than that in the DAPT group but higher than that in the CQ group(t=4.212,P=0.014;t=6.569,P=0.003);see ATG7 for RT-PCR.The relative expression of m RNA in the Comb group was significantly lower than that in the DAPT group but higher than that in the CQ group(t=3.371,P=0.028;t=17.9,P55=0.000).The m RNA expression level of ATG12 was significantly higher in the Comb group.The difference was statistically significant between the DAPT group and the CQ group(t=5.369,P=0.006;t=4.820,P=0.009).In Western Blot,the relative expression level of Notch1 in Notch channel-associated eggs was significantly higher than that in DAPT group and CQ group(t=11.936,P=0.000;t=7.779,P=0.001).Glut1 relative expression The amount of Comb was significantly higher than that of DAPT but lower than that of CQ.The difference was statistically significant(t=7.507,P=0.002;t=5.335,P=0.006).Conclusions1.DAPT can induce autophagy in gastric cancer MKN-45 cells.2.CQ can induce the expression of Notch signaling pathway in gastric cancer MKN-45 cells.3.DAPT combined with CQ can make the proliferation of gastric cancer MKN-45 cells decreased,apoptotic rate increased,and enhance the treatment of gastric cancer cells.