Enhanced Gefitinib Antitumor Activity In Lung Cancer Cell H1650Induced By5-aza-CdR

Objective:1. To investigate the effect of growth ihhibition rates on human non-smallcell lung cancer (NSCLC) cell line H1650, which contains EGFR mutation (delE746-A750) treated by gefitinib.2. To detect the cell cycle on H1650cells treated bygefitinib.3. To evaluate EGFR mRNA and protein expression on H1650cells treatedby gefitinib.Methods:1.Cell counting Kit-8(CCK-8) was used to assay the cytotoxicity ofgefitinib（0.01μmol/L、0.1μmol/L、1μmol/L、10μmol/L）on the NSCLC cell lineH1650.2.Cell cycle treated with gefitinib（0.01μmol/L、0.1μmol/L、1μmol/L、10μmol/L）was analyzed by flow cytometry.3.Expression of EGFR mRNA treatedwith gefitinib（0.01μmol/L、0.1μmol/L、1μmol/L、10μmol/L）was detected byReal-time quantitative PCR.4.Expression of EGFR protein treated with gefitinib（0.01μmol/L、0.1μmol/L、1μmol/L、10μmol/L）was detected by western blot assay.Results:1.CCK-8showed gefitinib had no effect on the proliferation of H1650cellswhen compared to the control group, there was no significant difference (P＞0.05).2.Larger proportion of H1650cells were in G1phase of the cell cycle.3.Real-timequantitative PCR analysis indicated that the expression of EGFR mRNA had nochang when compared to control group (P＞0.05).4. Western blot analysis indicatedthat the expression of EGFR protein had no chang when compared to control group(P＞0.05).Conclusion: Although H1650contains EGFR mutation (del E746-A750), it is less sensitive to gefitinib. Objective: To investigate the correlation between EGFR gene promoter methylationand the therapeutic effect of gefitinib on human non-small cell lung cancer (NSCLC)cell line H1650.Methods:1.The methylation of EGFR gene promoter region was examined bymethylation-specific PCR before and after（5-Aza-2’-deoxycyti-dine,5-aza-CdR）treatment.2. NSCLC cell line H1650cells were treated with gefitinib and/or5-aza-CdR.Counting Kit-8(CCK-8) assay was used to evaluate the growth inhibitionrates.3. Apoptosis rate of H1650cells treated with gefitinib and/or5-aza-CdR wasdetermined by Annexin V/PI double staining flow cytometry.4. Expression of EGFRmRNA treated with gefitinib and/or5-aza-CdR was detected by Real-timequantitative PCR.5. Expression of EGFR protein treated with gefitinib and/or5-aza-CdR was detected by western blot assay.Results:1.The promoter region of the EGFR gene in H1650cells was methylatedand5-aza-CdR could induce the demethylation of EGFR gene promoter region.2.Cells were treated with medicine for72h, the50%inhibition concentration (IC50)in the combination treated group（2.93±0.95）μmol/L was significantly decreasedthan gefitinib group（14.53±1.13）μmol/L (P＜0.05).3.The apoptosis rate in thecombination treated group（25.73±2.9）％was more significant than gefitinib group（10.91±3.9）％(P＜0.05). The inhibitive effect of gefitinib and5-aza-CdR wassignificantly stronger than that of gefitinib alone.4.The expression of EGFR mRNA significantly decreased in the combination treated group compared with gefitinibgroup(P＜0.05).5.The expression of EGFR protein significantly decreased in thecombination treated group compared with gefitinib group(P＜0.05).ConclusionThese results suggest that blockade of methylation may enhance the antitumoreffects of gefitinib in NSCLC cell. Thus, EGFR gene promoter methylation may be apotential mechanism for the resistance to gefitinib.