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. 2013

Yong-Qiang Dong, and Jiang-Shui Liang, and Shui-Bo Zhu, and Xiao-Ming Zhang, and Tao Ji, and Jia-Hang Xu, and Gui-Lin Yin
Department of Cardio-Thoracic Surgery, Wuhan General Hospital of Guangzhou Military Command, Wuhan, China.

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.

UI MeSH Term Description Entries
D008175 Lung Neoplasms Tumors or cancer of the LUNG. Cancer of Lung,Lung Cancer,Pulmonary Cancer,Pulmonary Neoplasms,Cancer of the Lung,Neoplasms, Lung,Neoplasms, Pulmonary,Cancer, Lung,Cancer, Pulmonary,Cancers, Lung,Cancers, Pulmonary,Lung Cancers,Lung Neoplasm,Neoplasm, Lung,Neoplasm, Pulmonary,Pulmonary Cancers,Pulmonary Neoplasm
D002289 Carcinoma, Non-Small-Cell Lung A heterogeneous aggregate of at least three distinct histological types of lung cancer, including SQUAMOUS CELL CARCINOMA; ADENOCARCINOMA; and LARGE CELL CARCINOMA. They are dealt with collectively because of their shared treatment strategy. Carcinoma, Non-Small Cell Lung,Non-Small Cell Lung Cancer,Non-Small Cell Lung Carcinoma,Non-Small-Cell Lung Carcinoma,Nonsmall Cell Lung Cancer,Carcinoma, Non Small Cell Lung,Carcinomas, Non-Small-Cell Lung,Lung Carcinoma, Non-Small-Cell,Lung Carcinomas, Non-Small-Cell,Non Small Cell Lung Carcinoma,Non-Small-Cell Lung Carcinomas
D002453 Cell Cycle The complex series of phenomena, occurring between the end of one CELL DIVISION and the end of the next, by which cellular material is duplicated and then divided between two daughter cells. The cell cycle includes INTERPHASE, which includes G0 PHASE; G1 PHASE; S PHASE; and G2 PHASE, and CELL DIVISION PHASE. Cell Division Cycle,Cell Cycles,Cell Division Cycles,Cycle, Cell,Cycle, Cell Division,Cycles, Cell,Cycles, Cell Division,Division Cycle, Cell,Division Cycles, Cell
D005434 Flow Cytometry Technique using an instrument system for making, processing, and displaying one or more measurements on individual cells obtained from a cell suspension. Cells are usually stained with one or more fluorescent dyes specific to cell components of interest, e.g., DNA, and fluorescence of each cell is measured as it rapidly transverses the excitation beam (laser or mercury arc lamp). Fluorescence provides a quantitative measure of various biochemical and biophysical properties of the cell, as well as a basis for cell sorting. Other measurable optical parameters include light absorption and light scattering, the latter being applicable to the measurement of cell size, shape, density, granularity, and stain uptake. Cytofluorometry, Flow,Cytometry, Flow,Flow Microfluorimetry,Fluorescence-Activated Cell Sorting,Microfluorometry, Flow,Cell Sorting, Fluorescence-Activated,Cell Sortings, Fluorescence-Activated,Cytofluorometries, Flow,Cytometries, Flow,Flow Cytofluorometries,Flow Cytofluorometry,Flow Cytometries,Flow Microfluorometries,Flow Microfluorometry,Fluorescence Activated Cell Sorting,Fluorescence-Activated Cell Sortings,Microfluorimetry, Flow,Microfluorometries, Flow,Sorting, Fluorescence-Activated Cell,Sortings, Fluorescence-Activated Cell
D006023 Glycoproteins Conjugated protein-carbohydrate compounds including MUCINS; mucoid, and AMYLOID glycoproteins. C-Glycosylated Proteins,Glycosylated Protein,Glycosylated Proteins,N-Glycosylated Proteins,O-Glycosylated Proteins,Glycoprotein,Neoglycoproteins,Protein, Glycosylated,Proteins, C-Glycosylated,Proteins, Glycosylated,Proteins, N-Glycosylated,Proteins, O-Glycosylated
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000964 Antimetabolites, Antineoplastic Antimetabolites that are useful in cancer chemotherapy. Antineoplastic Antimetabolites
D001374 Azacitidine A pyrimidine analogue that inhibits DNA methyltransferase, impairing DNA methylation. It is also an antimetabolite of cytidine, incorporated primarily into RNA. Azacytidine has been used as an antineoplastic agent. Azacytidine,5-Azacytidine,NSC-102816,Vidaza,5 Azacytidine,NSC 102816,NSC102816
D012333 RNA, Messenger RNA sequences that serve as templates for protein synthesis. Bacterial mRNAs are generally primary transcripts in that they do not require post-transcriptional processing. Eukaryotic mRNA is synthesized in the nucleus and must be exported to the cytoplasm for translation. Most eukaryotic mRNAs have a sequence of polyadenylic acid at the 3' end, referred to as the poly(A) tail. The function of this tail is not known for certain, but it may play a role in the export of mature mRNA from the nucleus as well as in helping stabilize some mRNA molecules by retarding their degradation in the cytoplasm. Messenger RNA,Messenger RNA, Polyadenylated,Poly(A) Tail,Poly(A)+ RNA,Poly(A)+ mRNA,RNA, Messenger, Polyadenylated,RNA, Polyadenylated,mRNA,mRNA, Non-Polyadenylated,mRNA, Polyadenylated,Non-Polyadenylated mRNA,Poly(A) RNA,Polyadenylated mRNA,Non Polyadenylated mRNA,Polyadenylated Messenger RNA,Polyadenylated RNA,RNA, Polyadenylated Messenger,mRNA, Non Polyadenylated
D014407 Tumor Cells, Cultured Cells grown in vitro from neoplastic tissue. If they can be established as a TUMOR CELL LINE, they can be propagated in cell culture indefinitely. Cultured Tumor Cells,Neoplastic Cells, Cultured,Cultured Neoplastic Cells,Cell, Cultured Neoplastic,Cell, Cultured Tumor,Cells, Cultured Neoplastic,Cells, Cultured Tumor,Cultured Neoplastic Cell,Cultured Tumor Cell,Neoplastic Cell, Cultured,Tumor Cell, Cultured

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