An Experimental Study On Quercetin Stimulates Endoplasmic Reticulum Stress To Enhance Sensitivity Of NSCLC A549 Cells To Cisplatin Chemotherapy

Objective: To investigate the effect of quercetin combined with cisplatin on human lung adenocarcinoma A549 cells in proliferation,apoptosis,cell cycle arrest and its potential molecular mechanism.Methods:1.A549 cells were exposed to different concentrations of quercetin(0,20,40,80,160,320 μmol/L)and cisplatin(0,20,40,80,160,320 μM)for different times(12h,24 h,48h,72h).CCK-8 was used to detect the cell proliferation rate of each treatment group,and the appropriate time and concentration were selected.2.Draw cell proliferation curve according to CCK-8 experimental results,select the concentration of cisplatin which has killing effect on A549 cells,and select the concentration of quercetin which has not killing effect on A549 cells for follow-up experiments.3.Observe the effects of different treatment groups(control group,alone quercetin group,alone cisplatin group and quercetin combined with cisplatin group)on cell proliferation,apoptosis and cycle arrest at different time.The apoptosis rate and cycle arrest were detected by flow cytometry.4.Observe the effects of each treatment group on the levels of apoptosis protein BCL-2,Bax and endoplasmic reticulum stress-related protein GRP78,CHOP,caspase-12,PERK,ATF6,IRE1 in experimental cell lines.The protein level was detected by Western Blot.Results:1.Quercetin has a time-and dose-dependent killing effect on A549.With the increasing concentration,the cell proliferation rate gradually decreases.At the same time,with the increasing time,the cell proliferation rate of the same concentration gradually decreased;Similarly,cisplatin also has a time-and dose-dependent effect on A549 cells.The difference was statistically significant(P <0.05).2.In the quercetin group,cell proliferation was inhibited at quercetin ≥160 μM at 12 h and Qu ≥40 μM at 24 h and 48 h.However,cell proliferation was significantly inhibited at quercetin 20 μM in 72 h,whose toxic effect is greater than 80%.In the cisplatin group,cell growth was inhibited at 320 μM at 12 h and was significantly inhibited at quercetin concentration ≥80 μM at 24 h and 48 h,and only at 20 μM in 72 h.Hoechst 33342 fluorescence staining showed that with the increasing concentration of quercetin and cisplatin,the number of cells gradually decreased,the cell morphology became rounder,and the proportion of apoptotic cells increased,which indicated that quercetin and cisplatin had obvious anti-tumor effect on A549 cells.Therefore,in order to eliminate the interference of quercetin on cytotoxicity,quercetin 20μM,cisplatin 80μM,24 h and 48 h were selected for follow-up test.3.The cells were exposed to quercetin(20 μM),cisplatin(80 μM),and quercetin combined with cisplatin for 24 h and 48 h.Quercetin combined with cisplatin significantly reduced the proliferation rate of A549 cells,and the effect was more significant at 48 h,which indicated that quercetin combined with cisplatin could increase the cytotoxicity.The apoptosis rate was detected by flow cytometry.The results showed that compared with the control group,quercetin combined with cisplatin significantly increased the apoptosis rate,and the effect was obvious after 48 hours.The cell cycle was detected by flow cytometry.Compared with the control group,the ratio of S phase and G2/M phase in quercetin combined with cisplatin group increased,but G0/G1 phase decreased,especially in 48 hours treatment group.This result shows that quercetin combined with cisplatin can arrest the cell cycle in S and G2/M phases,hence inhibiting the cell proliferation.4.Western blot showed that compared with the control group,quercetin combined with cisplatin significantly reduced the level of BCL-2 protein and increased the expression of Bax protein.At the same time,quercetin combined with cisplatin increased the protein levels of endoplasmic reticulum stress marker proteins GRP78,CHOP,caspase-12.In addition,quercetin combined with cisplatin also increased the levels of ATF6 and IRE1 protein,but had no obvious effect on PERK protein.Conclusion:1.Quercetin and cisplatin have significant anti-tumor effects on A549 cells of non-small cell lung cancer,and low-dose quercetin combined with cisplatin can enhance the sensitivity of A549 cells to cisplatin chemotherapy.2.Quercetin induced endoplasmic reticulum stress enhances the sensitivity of A549 cells to cisplatin,and its mechanism may involve ATF6/IRE1 signal transduction pathway.