title: Effect of 5-aza-2'-deoxycytidine on cell proliferation of non- small cell lung cancer cell line A549 cells and expression of the TFPI-2 gene.
abstract: OBJECTIVE
  The present study employed 5-aza-2'-deoxycytidine (5-Aza-CdR) to treat non-small cell lung cancer (NSCLC) cell line A549 to investigate the effects on proliferation and expression of the TFPI-2 gene.
  
  
  METHODS
  Proliferation was assessed by MTT assay after A549 cells were treated with 0, 1, 5, 10 μmol/L 5-Aza-CdR, a specific demethylating agent, for 24 ,48 and 72h. At the last time point cells were also analyzed by flow cytometry (FCM) to identify any change in their cell cycle profiles. Methylation-specific polymerase chain reaction (MSPCR), real time polymerase chain reaction(real-time PCR) and western blotting were carried out to determine TFPI-2 gene methylation status, mRNA expression and protein expression.
  
  
  RESULTS
  MTT assay showed that the growth of A549 cells which were treated with 5-Aza-CdR was significantly suppressed as compared with the control group (0 μmol/L 5-Aza-CdR). After treatment with 0, 1, 5, 10 μmol/L 5-Aza-CdR for 72h, FCM showed their proportion in G0/G1 was 69.7±0.99%, 76.1±0.83%, 83.8±0.35%, 95.5±0.55% respectively (P<0.05), and the proportion in S was 29.8±0.43%, 23.7±0.96%, 15.7±0.75%, 1.73±0.45%, respectively (P<0.05), suggesting 5-Aza-CdR treatment induced G0/G1 phase arrest. MSPCR showed that hypermethylation in the promoter region of TFPI-2 gene was detected in control group (0 μmol/L 5-Aza-CdR), and demethylation appeared after treatment with 1, 5, 10 μmol/L 5-Aza-CdR for 72h. Real-time PCR showed that the expression levels of TFPI-2 gene mRNA were 1±0, 1.49±0.14, 1.86±0.09 and 5.80±0.15 (P<0.05) respectively. Western blotting analysis showed the relative expression levels of TFPI-2 protein were 0.12±0.01, 0.23±0.02, 0.31±0.02, 0.62±0.03 (P<0.05). TFPI-2 protein expression in A549 cells was gradually increased significantly with increase in the 5-Aza-CdR concentration.
  
  
  CONCLUSIONS
  TFPI-2 gene promoter methylation results in the loss of TFPI-2 mRNA and protein expression in the non-small cell lung cancer cell line A549, and 5-Aza-CdR treatment could induce the demethylation of TFPI-2 gene promoter and restore TFPI-2 gene expression. These findings provide theoretic evidence for clinical treatment of advanced non-small cell lung cancer with the demethylation agent 5-Aza-CdR. TFPI-2 may be one molecular marker for effective treatment of advanced non-small cell lung cancer with 5-Aza-CdR.
authors.affiliation: Department of Cardio-Thoracic Surgery, Wuhan General Hospital of Guangzhou Military Command, Wuhan, China.
authors.forename: Yong-Qiang
authors.initials: YQ
authors.lastname: Dong
authors.forename: Jiang-Shui
authors.initials: JS
authors.lastname: Liang
authors.forename: Shui-Bo
authors.initials: SB
authors.lastname: Zhu
authors.forename: Xiao-Ming
authors.initials: XM
authors.lastname: Zhang
authors.forename: Tao
authors.initials: T
authors.lastname: Ji
authors.forename: Jia-Hang
authors.initials: JH
authors.lastname: Xu
authors.forename: Gui-Lin
authors.initials: GL
authors.lastname: Yin
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D001374:Azacitidine / Q000494:pharmacology*; D015153:Blotting, Western; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D002453:Cell Cycle / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects*; D005434:Flow Cytometry; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
chemical_list: D000964:Antimetabolites, Antineoplastic; D006023:Glycoproteins; D012333:RNA, Messenger; C021240:tissue-factor-pathway inhibitor 2; D001374:Azacitidine