| Objective: To study the effects of Cx26 on EMT and acquired gefitinib resistance of NSCLC and its mechanism.Methods:1.The expression levels of Cx26,Cx32,Cx31.1 and Cx43 in the NSCLC HCC827,PC9,A549,H1299 cell lines were explored by RT-PCR and WB.2.HCC827 GR and PC9 GR,two gefitinib-resistant(GR)NSCLC cell lines from their parental cells were established by gradually increasing the concentration of Gefitinib.The IC50 values of the above cells were measured using the MTT method.3.The functional GJIC of HCC827,PC9,HCC827 GR,PC9 GR cells was measured by parachute assay.The cellular localization of Cx26 protein was evaluated by IF staining.4.Overexpression or knockdown the Cx26 expression of NSCLC cells was stably both transfected by lentivirus.Overexpression the Akt expression of NSCLC cells was transfected by pcDNA3.1-Akt plasmid.Inhibition the Akt expression of NSCLC cells was inhibited by LY294002.The expressions of Cx26,E-cadherin,vimentin,slug,Akt and p-Akt in the NSCLC cell lines were investigated by WB.The migration and invasion abilities of NSCLC cell lines were detected by transwell assays.5.BALB/C nude mice which were randomly selected of HCC827,HCC827 GR and HCC827 GR-shCx26 were injected 200 ?L single cell suspension(cells above 1×107)in forelimb armpit,when tumor volumes(calculated as(length × width2)/2)reached ~ 100 mm3,mice were randomly allocated into groups to receive gefitinib or LY294002,or a combination of gefitinib and LY294002.All mice were killed on day 15 after their tumor size had been measured.Results:1.Western blotting and RT-PCR analyses showed that four major Cx isoforms,Cx26,Cx32,Cx31.1 and Cx43 were differentially expressed in four NSCLC cell lines(HCC827,PC9,A549 and H1299).In particular,Cx26 was the predominant Cx isoform expressed in various NSCLC cell lines.Moreover,the level of Cx26 was markedly higher in gefitinib-insensitive A549 and H1299 cell lines than that in gefitinib-sensitive HCC827 and PC9 cell lines.2.Gefitinib showed less cytotoxicity in established HCC827 GR and PC9 GR cells than that in their parental cells with IC50 of 12.17 ± 0.18 ?M versus 0.06 ± 0.11 ?M and 16.51 ± 0.17 ?M versus 0.25 ± 0.07 ?M,respectively.HCC827 GR and PC9 GR cells were sufficient to induce elongated mesenchymal-like morphology transition,consistent with decreased expression of E-cadherin while increased expression of vimentin and slug,and enhanced migratory and invasive potential of HCC827 GR and PC9 GR cells.Moreover,the levels of Cx26 were increased in HCC827 GR and PC9 GR cells.3.Parachute assay revealed that GJIC in primarily human foreskin fibroblasts(HFFs)as positive control was confirmed,and treatment of these cells with RA(a welldefined GJIC enhancer)significantly enhanced GJIC among these cells.Moreover,no detectable GJIC was found in HCC827,PC9,and their GR cells and no enhancement of GJIC in these cells incubated with RA.Immunofluorescence staining showed that Cx26 protein accumulated in the cytoplasm of HCC827,PC9 cells,and their GR cells.4.Overexpression of Cx26 expression in gefitinib sensitive NSCLC HCC827 and PC9 cells was sufficient to induce elongated mesenchymal-like morphology transition,consistent with decreased expression of E-cadherin while increased expression of vimentin and slug,and enhanced migratory and invasive potential.MTT assay showed that Cx26 overexpression exerted obvious gefitinib insensitivity in these cells.Besides,the in vivo data showed that compared with vehicle groups,tumor volume was increased significantly in Cx26 overexpression HCC827 and PC9 xenografts.5.Knockdown of Cx26 expression in gefitinib resistance NSCLC HCC827 GR and PC9 GR cells significantly restored the rounded epithelial-like appearance,enhanced E-cadherin expression while reduced vimentin and slug expression,and meanwhile strongly inhibited migratory and invasive potential.MTT assay showed that Cx26 inhibition exerted obvious gefitinib insensitivity in these cells.Besides,the in vivo data showed that compared with vehicle groups,tumor volume was decreased significantly in Cx26 knockdown HCC827 GR and PC9 GR xenografts.6.WB assay showed that expression of p-Akt was significantly increased in Cx26 overexpression HCC827 and PC9 cells and significantly decreased in Cx26 knockdown HCC827 GR and PC9 GR cells.Treatment of HCC827-Cx26 and PC9-Cx26 cells with a specific PI3K/Akt pathway inhibitor LY294002 could reverse the Mesenchymal-like appearance,enhanced E-cadherin expression while reduced vimentin and slug expression,and meanwhile strongly inhibited migratory and invasive potential of Cx26 overexpression HCC827 and PC9 cells.MTT assay showed that LY294002 also could reverse the effect of Cx26 inhibition exerted obvious gefitinib insensitivity in these cells.Besides,the in vivo data showed that compared with HCC827-mock and PC9-mock groups,tumor volume was decreased in HCC827-Cx26 and PC9-Cx26 xenografts after treatment with LY294002.7.Overexpression of Akt was sufficient to induce elongated mesenchymal-like morphology transition,consistent with decreased expression of E-cadherin while increased expression of vimentin and slug,and enhanced migratory and invasive potential of HCC827 and PC9 cells.MTT assay showed that Akt overexpression exerted obvious gefitinib insensitivity in these cells.Besides,Cx26 overexpression strengthened Akt-facilitated EMT and gefitinib resistance,whereas Cx26 depletion rendered impaired Aktpromoted effects in these cells.Furthermore,treatment with LY294002 for caused a significant reduced Cx26 expression both in HCC827,PC9 cells,and their GR cells.Moreover,ectopic expression of Akt significantly increased Cx26 expression in these cells.Conclusion: Cx26 upregulation was positively correlated with NSCLC gefitinib acquired resistance.The reciprocal positive interplay between Cx26 and PI3K/Akt pathway contributes to acquired gefitinib resistance in NSCLC cells via an GJIC-independent manner and induction of EMT. |
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