CRME Enhances The Effect Of BAI On The Proliferation And Apoptosis Of SW982 Cells And Its Mechanism

Objective:To investigate the effects and mechanism of the combination of BAI and CRME on the proliferation and apoptosis of SW982 cells.Methods:1.The MTT assay was used to detect the proliferation of SW982 cells treated with different concentrations of CRME(0,20,40 and 60μg/m L),BAI(0,15,25,50,75 and 100μmol/L),and the combination of CRME+BAI(20μg/m L+25μmol/L,20μg/m L+50μmol/L,40μg/m L+25μmol/L and 40μg/m L+50μmol/L)for 24 h,48h,72 h,and 96 h,respectively,and to screen the optimal drug concentration;2.The plate cloning assay examined the colony formation of SW982 cells after 14 days of treatment with CRME,BAI and the combination of CRME+BAI;3.Flow cytometry Annexin PI single staining method was used to detect the cell cycle of SW982 cells after 48 h treatment with CRME,BAI and the combination of CRME+BAI;4.The scratch healing assay examined the lateral migration ability of SW982 cells after 48 h of treatment with CRME,BAI and the combination of CRME+BAI;5.Transwell migration assay was performed to detect the longitudinal migration ability of SW982 cells after 48 h treatment with CRME,BAI and the combination of CRME+BAI;6.Flow cytometry Annexin V-FITC/PI double staining method was used to detect the apoptosis of SW982 cells after 48 h treatment with CRME,BAI and the combination of CRME+BAI;7.The Western blot was performed to detect the expression of EMT,apoptosis and PI3K/AKT/m TOR signaling pathway-related proteins in SW982 cells treated with CRME,BAI and the combination of CRME+BAI for 48 h,respectively.Results:1.The results of MTT assay showed that:(i)Compared with the control group,BAI inhibited the proliferation of SW982 cells at 25,50,75,and 100μmol/L concentrations and in a time-and dose-dependent manner,especially at 25μmol/L and 50μmol/L(P<0.01).(ii)The cell proliferation rate was reduced in the CRME treatment group compared with the control group(P<0.01),but the inhibition rate of cells at different concentrations of CRME group at different times was not statistically significant.(iii)The combination of BAI and CRME significantly inhibited the viability of SW982 cells compared to the control and monotherapy groups(P<0.01).(iv)Combined treatment with 25μmol/L of BAI and 20μg/m L of CRME was screened.2.The plate colony formation experiment showed that BAI,CRME and the combined drug CRME20+BAI25 inhibited the colony forming ability of SW982 cells compared with the control group(P<0.01),and the combined drug group inhibited the colony forming ability of SW982 cell line more significantly than the drug group alone(P<0.01).3.The Annexin PI single-stain flow cytometry assay demonstrated that the percentage of SW982 cells in G0/G1 phase was 74.54%,79.76% and 84.28% in CRME20,BAI25 and combined drug CRME20+BAI25 treated groups,respectively,which was higher than that of untreated SW982 cells(62.11%),and the effect of combined drug group was more significant(P<0.05,P<0.01).4.The scratch healing experiment results showed that the lateral migration ability of SW982 cells was reduced in BAI,CRME and combined drug CRME20+BAI25 groups compared with the control group(P<0.01),and the reduction of migration ability was more significant in the combined drug group compared with the drug alone group(P<0.01).5.The results of Transwell cell migration test showed that the longitudinal invasion ability of SW982 cells was significantly reduced in the BAI,CRME and combined drug CRME20+BAI25 groups compared with the control group(P<0.01),and the reduction in the number of longitudinal migrating cells was more significant in the combined drug group compared with the drug alone group(P<0.01).6.The results of Annexin V-FITC/PI double-staining flow cytometry showed that the apoptosis ratio of SW982 cells was significantly increased in the BAI,CRME and combination drug CRME20+BAI25 groups compared with the control group(P<0.05,P<0.01),and the combination of BAI and CRME was more effective in increasing the number of apoptotic cells in SW982 cells(P<0.05,P<0.01).7.The results of Western blot demonstrated that:(i)Compared with the control group,the expression of Bax and the ratio of caspase3/Cleaved-caspase3,caspase9/Cleaved-caspase9 protein expression were significantly increased in the BAI,CRME and CRME20+BAI25 combination medication groups,while the expression of Bcl-2 and the ratio of Bcl-2/Bax protein expression showed a significant decrease trend(P<0.05,P<0.01),and the effect was more significant in the combination drug group compared with the drug administration alone(P<0.05,P<0.01).(ii)Compared with the control group,the expression levels of vimentin,Snail,MMP2,MMP9 proteins were significantly inhibited and decreased in SW982 cells in the BAI,CRME and combined drug administration CRME20+BAI25 groups(P<0.05,P<0.01),and the decrease effect was more significant in the combined drug administration group compared with the drug administration alone(P<0.01);Meanwhile,compared with the control group,the expression levels of E-cadherin protein were significantly increased in SW982 cells in the BAI,CRME and combined drug CRME+BAI groups(P<0.01),and the upregulation effect was more significant in the combined drug group compared with the drug alone group(P<0.01);(iii)Compared with the control group,the expression levels of p-PI3K、p-AKT 、p-m TOR and the p-PI3K/PI3 K,p-AKT/AKT and p-m TOR/m TOR ratios were significantly reduced in both the single drug group and the combination drug group(P<0.01),while the expression levels of total protein PI3 K,AKT,and m TOR did not show significant changes,and the effect was more significant in the combination drug group compared with the drug administration alone(P<0.05,P<0.01).Conclusion:1.The combination of BAI and CRME effectively inhibited the proliferation,colony formation,migration ability of SW982 cells,induced apoptosis and arrested the cell cycle at the G0/G1 phase in SW982 cell lines.2.The combination of BAI and CRME effectively inhibited the migration and induced apoptosis of SW982 cells,which was correlated with the combination of drugs regulating the expression levels of EMT,apoptosis and PI3K/AKT/m TOR signaling pathway-related proteins.