Effects of 5-Aza-2'-deoxycytidine on biological behaviors and the RASSF1A gene expression of human lung carcinoma cell lines H460

Abstract

Objective To investigate the effects of demethylating agent 5-Aza-2'-deoxycytidine (5-Aza-CdR) on the biological behaviors and the expression of RASSF1A of lung carcinoma cells H460. Methods Lung carcinoma cell line H460 were treated with 5-Aza-CdR. Cell proliferation was determined by MTT assay and colony forming test. The methylation status of RASSF1A gene was detected by PCR. The expression of RASSF1A protein was measured by Western blotting, and the cell cycle was analyzed by flow cytometry. Results With 5-Aza-CdR treatment, the proliferation speed of H460 lung carcinoma cells was slowed down and the colony formation rate of H460 cells was decreased significantly compared with control group (38.5 %, 27.5 % and 60.5 % in 5-Aza-CdR 5 and 10 μmol/L groups and control group, respectively, P <0.05). The methylation degree in the promoter of RASSF1A gene was decreased and the expression of RASSF1A protein was detected after 5-Aza-CdR treatment. 5-Aza-CdR induced G1 phase arrest of the H460 cells. Conclusion The hypermethylation of CpG island in the promoter of RASSF1A gene results in the loss of RASSF1A protein expression in human Lung carcinoma cell line. The demethylating agent 5-Aza-CdR could restore RASSF1A gene expression.These findings provide theoretic evidence for clinical treatment of human lung carcinoma with demethylation agent 5-Aza-CdR. Key words: Lung neoplasms; DNA methylation; Deoxycytidine