Abstract:

Objective To explore the relationship between death associated protein kinase（DAPK） promoter methylation and epidermal growth factor receptortyrosine kinase inhibitor（EGFR-TKI） resistance in human lung cancer cell lines. Methods Two EGFR-mutation lung cancer cells（H1650， PC9） and two wild type lung cancer cells（A549， H520） were treated with 5-aza-CdR（1μmol/L） plus gefitinib at different concentrations. The cell proliferation was determined by CCK-8 assay. Apoptosis of cells was observed using flow cytometry. The mRNA expression level of DAPK was detected by RT-PCR. The methylation of DAPK gene promoter region was examined by methylationspecific PCR. Results Gefitinib had proliferative inhibition on the 4 lung cancer cell lines at different extent in a dose dependent manner. CCK-8 assay showed that inhibitive effect of H1650 and H520 cells after 5-aza-CdR demethylation was significantly stronger than before（P＜0.05）. Flow cytometry results showed that the apoptosis rate of H1650 and H520 cells treated with 5-aza-CdR was significantly higher than that of them untreated with 5-aza-CdR（P＜0.05）. In lung cancer cell lines without the processing of 5-aza-CdR， the gefitinib sensitive PC9 and A549 cell lines displayed high expression of DAPK mRNA， with the gene promoter in a non-methylation state， while in drug-resistant type（H1650 and H520 cell lines）， the DAPK mRNA expression level was low with DAPK promoter in high methylation state. Removing the methylation of DAPK gene promoter region in H1650 and H520 cell lines by 5-aza-CdR， DAPK gene expression and gefitinib sensitivity in H1650 and H520 cell lines apparently increased compared to the previous（P＜0.05）， but the drug sensitivity in PC9 and A549 cell lines did not change（P＞0.05）. Conclusion High promoter methylation of tumor suppressor gene DAPK can lead to down-regulation of DAPK gene expression， and reduce the sensitivity of gefitinib on NSCLC. It may provide a new method for predicting clinical curative effect to develop DAPK gene methylation state detection.