| Objective: 1.To clarify whether polyphyllin I can improve Gefitinib resistance in lung adenocarcinoma.2.To elucidate the related signaling pathway of polyphyllin I improving Gefitinib resistance,in order to provide a scientific basis for the treatment of lung adenocarcinoma with polyphyllin I in Gefitinib resistance.Methods: 1.Human lung adenocarcinoma cell line PC9 was exposed to increasing concentrations gradually or intermittent high doses of Gefitinib,which established the Gefitinib-resistance cell line(PC9/GR).Analysis of cell proliferation and drug resistance by CCK8 assay;assessment of cell invasion and migration ability by scratch and trans-well assays.Using Western blot to detect the expression of Ecadherin,N-cadherin,and Vimentin.2.The phosphorylation antibody chip(RTK)was performed to screen the targets of PC9/GR and the intervention targets of polyphyllin I,using quantitative real-time PCR(q RT-PCR)and Western blot to further verify the expression levels of related proteins.The changes in the fluorescence intensity of vascular endothelial growth factor(VEGF)and p38 mitogen-activated protein kinase(p38MAPK)were observed by fluorescence microscope.The tube formation assay was performed to observe the effect of drugs on the ability of human umbilical vein endothelial cells(HUVEC)to form lumen structures;the protein expression contents of VEGF,HIF-1α,and p38MAPK were validated by Western blot.3.Hypoxia was induced by the hypoxia mimetic agent cobalt chloride(Co Cl2)to observe the effects of polyphyllin I on HIF-1α,VEGF,and p38 MAPK proteins under hypoxia or normoxia conditions.Results: 1.The IC50 of Gefitinib in PC9 and PC9/GR were 4.75±1.17μM,17.09±2.22μM(P<0.05)respectively,and the drug resistance index was 3.6,indicating the Gefitinib-resistant cell line PC9/GR was successfully constructed and used in subsequent cell experiments.CCK8 assay revealed Polyphyllin I effectively restrain the proliferation of PC9 and PC9/GR in a dose-dependent and time-dependent manner.The results of the scratch healing assay indicated that the scratch healing rate of the cells in the combo group was considerably lower compared with that in the control group and drug-alone groups;the results of the transwell assay suggested that the number of cells that migrated and crossed Matrigel were greatly inhibited in the combo group(P<0.001).Western blot results uncovered the expression level of E-cadherin protein was significantly upregulated in the combo group,while N-cadherin and Vimentin were decreased(P<0.001).2.RTK assay showed the protein levels of phosphorylated VEGFR2 and VEGFR3 in PC9/GR were significantly higher than those in PC9 while decreasing after being treated with polyphyllin I(P<0.01).The results of q RT-PCR and WB were consistent with RTK analysis,indicating VEGFR may be the intervention target of polyphyllin I.Western blot results discovered the phosphorylation level of p38 MAPK protein was significantly decreased in PC9/GR after treatment with polyphyllin I(P<0.05).The fluorescence intensities of VEGF and phosphorylation level of p38 MAPK in the combo group were dramatically depressed.Tube formation assay showed that polyphyllin I inhibited the tube formation ability of HUVEC cells in a concentration-dependent manner,and the inhibitory effect was enhanced when polyphyllin I was combined with Gefitinib.Western blot results demonstrated the expression levels of VEGF,HIF-1α,and phosphorylation p38 MAPK were decreased in the combo group(P<0.001).The results of Western blot verified polyphyllin I inhibited hypoxia-induced upregulation of VEGF,HIF-1α,and phosphorylated p38 MAPK protein(P<0.05).Conclusions :1.Polyphyllin I can improve Gefitinib resistance.2.The mechanism of polyphyllin I improving Gefitinib resistance in lung adenocarcinoma is related to VEGFR2/p38 MAPK signaling pathway... |
PDF Full Text Request