Empirical Study Of Autophagy And Apoptosis In HeLa Cells Induced By Quercetin

PART I Role of Autophagy in Cervical Cancer Cell Line Induced by QuercetinObjective:To detect the inhibitory effect of HeLa cells'proliferation with treatment of different concentrations of quercetin,to observe the changes of autophagy level during the action of quercetin on HeLa cells,and to explore the possible mechanism of autophagy.Methods?MTT was used to detect the growth inhibition rate of HeLa cells with treatment of different concentrations of quercetin,and MDC staining was used to examine the autophagosome.Western bolt was used to detect the conversion level of LC3-I to LC3-? and protein expression of Beclin 1?active caspase 3 and p-S6Kl, transmission electron microscopy(TEM) was used to observe the ultrastructure of autophagy.Results:Quercetin could obviously inhibit the proliferation of HeLa cells, and the inhibitory effect was concentration dependent.MDC staining showed that after being treated by different concentrations of quercetin,green fluorescent reached its maximum at 50?mol/L,and then decreased.the results of western bolt showed that quercetin concentrations below 50?mol/L,LC3 I to the LC3 ? conversion level and Beclin 1 protein expression level increased gradually,and decreased on 100?mol/L,active caspase 3 protein levels increased gradually with the increased of the concentration of quercetin,The phosphorylation level of S6K1 decreased with the increased of autophagy.Ultrastructural observation by transmission electron microscope found that autophagy lysosomal reached its maximum after treated by quercetin in concentration of 50?mol/L.Conclusion:Quercetin can strongly inhibit proliferation of HeLa cells, quercetin can induce autophagy in HeLa cells,with the increased of concentration of quercetin, Autophagy was strongest in 50?mol/L quercetin and then decreased in the concentration of 100?mol/L.The mechanism may be related to the pathway of mTORC1.PART II The Relationship between Autophagy and Apoptosis in HeLa Cells Induced by QuercetinObjective:To explore the relationship between autophagy and apoptosis in cervical cancer HeLa cells induced by quercetin,to investigate the effect on cell apoptosis after inhibition of autophagy.Methods:HeLa cells treated by quercetin,after down-regulation of autophagy in HeLa cells by an autophagy inhibitor 3-methyla-denine(3-MA),the proliferation inhibition rate of HeLa cells with quercetin was evaluated by using the MTT assay.Apoptosis rate of HeLa cells was detected by AnnexinV-FITC/PI flow cytometry.Western bolt was used to detect the conversion level of LC3-I to LC3-II and protein expression of active caspase 3.Results:After inhibiting autophagy with 3-MA,the proliferation inhibition rate of quercetin on HeLa cells increased,the rate of apoptosis increased,the conversion rate of LC3-? to LC3-? decreased,suggesting that the autophagy process could be inhibited by 3-MA.and the expression of active caspase 3 protein increased,it showed that the inhibition of autophagy promoted the apoptosis of cells.Conclusion:Inhibition of autophagy could increase the inhibitory effect of quercetin on cervical cancer HeLa cells,and the apoptosis rate of the cells increased At low concentrations of quercetin,autophagy plays a protective role,down-regulation of autophagy enhanced the sensitivity of HeLa cells to the treatment of quercetin.PART ? Effects of autophagy on the growth of transplanted tumor in nude miceObjective:To study the effect of autophagy on quercetin's inhibition to the growth of transplanted cervical cancer in nude mice.Methods:The animal models of cervical cancer in nude mice were established, 3-MA and quercetin were injected into the animals,The growth of cervical cancer were observed by its size.The pathologic manifestations were detected by HE-staining,the expression of Beclin 1 and LC3-? in the three groups were detected by immunohistochemical staining.Results:There were significant differences among the control group, quercetin group and 3-MA+quercetin group.quercetin could be able to effectively inhibit tumor growth in nude mice,the inhibitory effect of 3-MA+quercetin on the transplanted tumor in nude mice was increased compared with that of quercetin alone group,And 3-MA could enhance the sensitivity of quercetin,tumor growth was further inhibited.HE-staining showed that there were clear areas of necrosis in the transplanted tumor tissue in 3-MA+quercetin group,the area of necrosis was seen in the quercetin group,but not in the control group,the expression of Beclin 1 and LC3-? were lower in 3-MA+quercetin group than the other two groups.Conclusion:Quercetin could inhibit the growth of transplanted tumor in nude mice,inhibition of autophagy could enhance the inhibitory effect of quercetin on the growth of transplanted tumor in nude mice.