title: 5-Aza-CdR can reverse gefitinib resistance caused by DAPK gene promoter methylation in lung adenocarcinoma cells.
abstract: To explore the relationship between death associated protein kinase (DAPK) gene promoter methylation and gefitinib resistance in Lung adenocarcinoma cell lines. EGFR-mutation lung adenocarcinoma cell lines PC9 and the gefitinib-resistant with T790M Mutation cell lines PC9/GR were chosen as cell models, and PC9/GR were treated with 5-aza-CdR (1 μmol/L). The experiments were divided into three groups: PC9 group, PC9/GR group and PC9/GR with 5-Aza-CdR pretreatment group. Treat three groups cell with different concentrations gefitinib, the cell proliferation was determined by MTT assay. The apoptotic rates were detected by flow cytometry. The methylation of DAPK gene promoter region was examined by methylation-specific PCR (MSP). The expressions of DAPK protein were detected by Western blot. MTT results showed that the half maximal inhibitory concentration (IC50) of PC9 and PC9/GR cell lines increase from 0.12 μmol/L to 8.52 μmol/L. But after treated with 5-aza-CdR, the IC50 of PC9/GR cell lines decrease to 4.35 μmol/L, and the resistance index (RI) decrease from 71 to 36 (P<0.05). Flow cytometry results showed that the apoptosis rate were 24.80% ± 0.28%, 12.70% ± 0.31%, 19.8% ± 0.15% respectively. MSP results showed that DAPK gene promoter region was un-methylated in PC9 cells and methylated in PC9/GR cells, when treated with 5-aza-CdR, DAPK gene promoter region was partly methylated in PC9/GR cells (P<0.05). Western blot results showed that the levels of DAPK protein were reduced significantly in PC9/GR cell lines compared with PC9, and after treated with 5-aza-CdR, the expression levels of DAPK protein in PC9/GR were increased (P<0.05). In conclusion, DAPK gene promoter methylation may contribute to the downregulation of DAPK gene and protein, and consequently affect the sensitivity of gefitinib in lung adenocarcinoma lines, induced gefitinib resistance. But 5-Aza-CdR can reverse gefitinib resistance by demethylation of DAPK gene promoter.
authors.affiliation: Department of Thoracic Surgery, The First Bethune Hospital of Jilin University Changchun 13000, Jilin, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Yang
authors.affiliation: Department of Thoracic Surgery, The First Bethune Hospital of Jilin University Changchun 13000, Jilin, China.
authors.forename: Zhi-Guang
authors.initials: ZG
authors.lastname: Yang
authors.affiliation: Department of Thoracic Surgery, The First Bethune Hospital of Jilin University Changchun 13000, Jilin, China.
authors.forename: Bao
authors.initials: B
authors.lastname: Gao
authors.affiliation: Department of Thoracic Surgery, The First Bethune Hospital of Jilin University Changchun 13000, Jilin, China.
authors.forename: Guo-Guang
authors.initials: GG
authors.lastname: Shao
authors.affiliation: Department of Thoracic Surgery, The First Bethune Hospital of Jilin University Changchun 13000, Jilin, China.
authors.forename: Guang-Hu
authors.initials: GH
authors.lastname: Li
mesh_terms: D000230:Adenocarcinoma / Q000235:genetics*; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D019175:DNA Methylation / Q000187:drug effects*; D064550:Death-Associated Protein Kinases / Q000235:genetics; D019008:Drug Resistance, Neoplasm / Q000187:drug effects* / Q000235:genetics; D005434:Flow Cytometry; D000077156:Gefitinib; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D011799:Quinazolines / Q000494:pharmacology
chemical_list: D000970:Antineoplastic Agents; D011799:Quinazolines; D064550:Death-Associated Protein Kinases; D001374:Azacitidine; D000077156:Gefitinib
keywords: DAPK gene; Lung adenocarcinoma; drug resistance; gefitinib; methylation