Study On The Relationship Between The Promoter Methylation Of EGFR Gene And The Susceptibility To Gefitinib In Human Non - Small Cell Lung Cancer Cells

Objective:1. To investigate the effect of growth ihhibition rates on human non-small cell lung cancer (NSCLC) cell line HCC827, H1650, H1975, H1299, H358, A549which contains different EGFR mutation status.2. To evaluate the correlativity between the therapeutic effect of gefitinib and EGFR mutation status in non-small cell lung cancer cell line.Methods:l.Cell counting Kit-8(CCK-8) was used to assess the cytotoxicity of gefitinib (0.01μmol/L、0.1μmol/L、1μmol/L、10μmol/L、100μmol/L、200μmol/L) on the NSCLC cell line HCC827,H1650,H1975,H1299,H358,A549.2.SPSS software was used to analyzed the correlativity between the therapeutic effect of gefitinib and EGFR mutation status in regarding non-small cell lung cancer cell lines according to their50%inhibition concentration (IC50).Results:1.CCK-8showed that HCC827、H358cells are comparatively sensitive to gefitinib than H1650、H1975、H1299、A549cells, according to the50%inhibition concentration (IC50)(P<0.05). And their IC50are as follows:HCC827(0.214±0.015)μmol/L、H1650(15.964±1.614) μmol/L、H1975(263.491±15.112) μmol/L、H358(5.039±0.573) μmol/L、H1299(26.275±2.250) μmol/L、A549(21.292±1.525) μmol/L.2. HCC827, H1650cells both containing EGFR mutation (del E746-A750), but only HCC827cells are sensitive to gefitinib. Except for H358, gefitinib had no effect on the proliferation of EGFR mutation wide-type cells such as A549, H1299. Conclusion:Both EGFR mutation (del E746-A750) cells HCC827and wide-type cells H358shows sensitivity to gefitinib, this may suggest that using only EGFR mutation status to predictive gefitinib sensitivity in non small cell lung cancer may not always work correctly. Objective:To examine the relationship between EGFR gene promoter methylation and the therapeutic effect of gefitinib in non-small cell lung cancer cell lines HCC827,H1650,H1975,H1299,H358,A549, and investigate the possible mechanism preliminarily.Methods:1.The methylation of EGFR gene promoter region in NSCLC cell lines HCC827,H1650,H1975,H1299,H358,A549were examined by methylation-specific PCR.2. Expression of EGFR mRNA of all NSCLC cell lines wre detected by Real-time quantitative PCR.3. Expression of EGFR protein of all cell lines were also detected by western blot assay.Results:EGFR gene promoter region is unmethylated in HCC827cells and partly methylated in H358cells, while in other four cells are all methylated. The expression of EGFR protein and mRNA are relatively higher in HCC827,H358cells comparing with other four cell lines(P<0.05).Conclusion:These results suggest that EGFR gene promoter methylation may contribute to downregulation of EGFR gene and consequently affect the sensitivity of gefitinib in non-small cell lung cancer cell lines. Analysis of methylation status of EGFR gene promoter may have definite value in predicting the therapeutic response of gefitinib.