Effect And Mechanism Of Quercetin On Inhibiting Proliferation Of A549 Cells Via Induction Of Ferroptosis

Objective:To investigate the effects of quercetin on the growth,proliferation,apoptosis and ferroptosis of human non-small cell lung cancer cells and its molecular mechanism at the cellular and molecular levels.Methods:1.CCK-8(cell counting kit-8)method was used to detect the effects of quercetin on the proliferation ability of A549 cell,to explore the IC50 inhibitory concentration(IC50)of quercetin on A549 cell.According to the results,the concentration of subsequent medications is determined.2.Colony formation assay was used to detect the effects of proliferation ability and the rate clones.3.Glutathione(GSH)level was detected by kit;4.Western blot was used to detect the expression of ferroptosis-related proteins P53、SLC7A11、GPX4 and apoptosis-related proteins Caspase-9、Caspase-3、Bcl-2、Bax、Cyt c.5.Mitochondrial reactive oxygen species(mtROS),lipid peroxide level and apoptosis were detected by flow cytometry.6.Fluorescence microscopy was used to observe the changes in the content of ferrous ions.7.The expression of cell viability,GSH and ferroptosis-related proteins were measured by plate photometry as well as Western blot assay after the combination of ferroptosis inhibitor(Ferrostatin-1)or ROS production inhibitor(NAC).Results:1.CCK-8 assay showed that Compared with control group,quercetin significantly inhibited the survival rate of A549 cells,with a time-dose correlation(P<0.01).2.The results of colony formation assay showed that the number of clones formed after quercetin treatment was significantly reduced when compared with the control group(P<0.01),and the rate of colonies was positively correlated with drug concentration.3.The results of glutathione detection kit showed that compared with the control group,the content of GSH in A549 cells was decreased after quercetin treatment(P<0.01),and the concentration of quercetin was dependent.4.Western blot results showed that in quercetin-treated A549 cells,the expression of ferroptosis related protein p53(P<0.05,0.01),and inhibited the expressions of GPX4 and SLC7A11(P<0.01);Quercetin significantly promoted expressions of mitochondrial apoptosis related proteins such as Caspase-3,Caspase-9,cytochrome c(Cyt C)and Bax(P<0.05,0.01),and inhibited the protein expression of anti-apoptotic factor Bcl-2(P<0.01).5.Flow cytometry assay showed that after treated with quercetin,Quercetin up-regulated mtROS and lipid peroxide levels in A549 cells(P<0.05,0.01),and induced apoptosis(P<0.01).6.Fluorescence microscope results showed that,compared with the control group,after quercetin treatment,the content of ferrous ions in A549 cells increased(P<0.01),and the concentration of quercetin was dependent.7.The results of the plate photometry showed that compared to the quercetin single addition group,both the quercetin+Ferrostatin-1 group and the quercetin+NAC group restored the quercetin-induced decrease in cell viability(P<0.05,0.001),and the quercetin+Ferrostatin-1 group could regress the intracellular GSH expression level(P<0.05).8.Western blot experiments showed that quercetin+Ferrostatin-1 group significantly upregulated GPX4 and SLC7A11 protein expression levels compared with quercetin single addition group(P<0.05).Conclusion:Quercetin can inhibit the viability and proliferation of A549 cells,and the mechanism of action is related to the induction of ferroptosis,and can lead to apoptosis,thus exerting anti-tumor effects,which provides a new idea to investigate the mechanism of action for the treatment of non-small cell lung cancer.