Activation of Notch-1 enhances epithelial-mesenchymal transition in gefitinib-acquired resistant lung cancer cells
10.3760/cma.j.issn.1006-9801.2015.05.003
- VernacularTitle:Notch信号通路体外介导肺癌细胞表皮生长因子受体酪氨酸激酶抑制剂获得性耐药的分子机制研究
- Author:
Mian XIE
;
Chaosheng HE
;
Shenhai WEI
- Publication Type:Journal Article
- Keywords:
Notch;
Epidermal growth factor receptor;
Drug resistance;
Epithelial-mesenchymal transition
- From:
Cancer Research and Clinic
2015;27(5):298-304
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate whether the Notch-1 signaling pathway is involved in the acquisition of the epithelial-mesenchymal transition (EMT) phenotype of gefitinib-acquired resistant lung cancer cells.Methods The PC9 cell line (harboring EGFR exon 19 deletion) and PC9/AB2 cells (gefitinibacquired resistant PC9 cells) were used.siRNA targeting Notch-1 eukaryotic expression vector (siNotch-1) was constructed and PC9/AB2 cells were transfected with siNotch-1.The protein expression of EGFR,Akt,Erk,Notch receptors and ligands,TGF-β receptors,E-cadherin,Vimentin,and Snail were detected by Western blot assay.DNA sequencing and fluorescence in situ hybridization (FISH) analysis were processed to detect mutation of EGFR exon 20 and MET amplification,respectively.For cytotoxicity assay,cell viability was assessed by Cell Counting Kit 8.Results Gefitinib resistant cell line PC9/AB2 had no evidence of MET amplification or EGFR T790M mutation.The expression of Notch-1 was upregulated in gefitinib resistant PC9/AB2 cells compared with that in gefitinib-sensitive PC9 cells.There were no significant protein expression differences of other Notch receptors,Notch ligands or TGF-β receptors between both paired cell lines.Western blot results showed that protein expression of E-cadherin was greatly reduced in PC9/AB2 cells,while elevated levels of Vimentin and Snail were observed.A significant reduction of the expression of Snail and Vimentin in Notch-1 siRNA transfected PC9/AB2 cells with increased E-cadherin expression was found by Western blot assay.PC9/AB2 cells displayed a round like cell morphology after Notch-1 siRNA transfection.Silence of Notch-1 decreased colony-formation ability but enhanced sensitivity of gefitinib on PC9/AB2 cells.Conclusion Notch-1 might play a novel role in acquired resistance to gefitinib,which could be reversed by inhibiting Notch-l.
