Study On The Role Of Autophagy In Liver X Receptor (LXR) Agonists In Inhibiting Lung Cancer Cells' Secondary Resistance To Gefitinib

Objection: To investigate the role and possible mechanism of autophagy in inhibiting proliferation of PC9/GR cells by LXR receptor agonist(GW3965)combined with gefitinib.Material and methods: The effect of LXR agonist(GW3965)combined with gefitinib on PC9/GR cell proliferation was determined by MTT assay.Changes in Beclin1 and LC3 expression of autophagocyte-related proteins in PC9/GR cells were detected by Western blot analysis of LXR agonist(GW3965)and gfitinib.The changes of PC9/GR autophagosome in LXR receptor agonist(GW3965)and gefitinib were observed by fluorescence microscopy using acridine orange(AO)staining method.Flow cytometry was used to detect the effect of LXR agonist(GW3965)combined with gefitinib on the apoptosis of PC9/GR cells.Western blot analysis of the expression of autophagy signaling pathway related proteins: AKT and p-AKT,m TOR and p-m TOR,AMPK and p-AMPK,and 70s6k1 and p-70s6k1 in PC9/GR cells treated with LXR agonist(GW3965)and gfitinib.Results: 1.Both gefitinib and GW3965 showed a certain inhibitory effect on PC9/GR cell proliferation,which was more obvious when the concentration of single drug was 20 μM(P<0.05);The inhibition of GW3965 combined with gefitinib on PC9/GR cell proliferation was significantly better than that of gefitinib treatment alone(P<0.05)and GW3965(P<0.05,1μM and 10μM: P<0.01).2.Compared with the control group,the expression of Beclin1 and the proportion of LC3 II/I of PC9/GR cells were not significantly affected by gfetinib and GW3965 alone(P >0.05),but the combination of the two drugs promoted their increase(P<0.01).Compared to gefitinib or GW3965,Beclin1 expression(P<0.01)and the proportion of LC3 II/I increased as well(P<0.05).3.Compared with the control group,gefitinib monotherapy had no significant effect on the production of autophagosome of PC9/GR cells(P >0.05),while GW3965 monotherapy promoted the production of autophagosome of PC9/GR cells(P<0.05).Whether compared with the control group(P<0.01),gefitinib(P<0.01)or GW3965 single drug(P<0.05),the effect of combination drug on PC9/GR cells forming autophagosome was more obvious.4.Both gefitinib and GW3965 promoted the apoptosis of PC9/GR cells(P<0.001),and the combination of the two drugs further increased the apoptosis of PC9/GR cells(P<0.01).5.The combination of gefitinib and GW3965 decreased the expression of p-AKT and p-m TOR proteins and increased the p-AMPK protein levels,both in comparison with the control group and in comparison with gefitinib or GW3965 alone.Conclusion: LXR agonist(GW3965)may induce autophagy to inhibit the proliferation of PC9/GR cells combined with gefitinib,which is related to AKT/m TOR and AMPK cell signaling pathways.