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. 2013 Apr 17:6:411-7.
doi: 10.2147/OTT.S43744. Print 2013.

RUNX3 gene promoter demethylation by 5-Aza-CdR induces apoptosis in breast cancer MCF-7 cell line

Affiliations

RUNX3 gene promoter demethylation by 5-Aza-CdR induces apoptosis in breast cancer MCF-7 cell line

Hua-Feng Kang et al. Onco Targets Ther. .

Abstract

Runt-related transcription factor 3 (RUNX3) is a tumor suppressor gene, its inactivation due to hypermethylation related to carcinogenesis. The aim of this study was to investigate the effects of 5-aza-2'-deoxycytidine (5-Aza-CdR) on cell proliferation and apoptosis by demethylation of the promoter region and restoring the expression of RUNX3 in the breast cancer MCF-7 cell line. MCF-7 cells were cultured with different concentrations (0.4-102.4 μmol/L) of 5-Aza-CdR in vitro. MTT assay was used to determine the proliferation of MCF-7 cells. Flow cytometry and Hoechst staining were used for analyzing cell apoptosis. The methylation status and expression of RUNX3 in mRNA and protein levels were measured by methylation-specific polymerase chain reaction (PCR [MSP]), reverse transcription (RT)-PCR, and Western blot. It was shown that the RUNX3 gene downregulated and hypermethylated in MCF-7 cells. 5-Aza-CdR induced demethylation, upregulated the expression of RUNX3 on both mRNA and protein levels in cancer cells, and induced growth suppression and apoptosis in vitro in a dose- and time-dependent manner. The results demonstrate that RUNX3 downregulation in breast cancer is frequently due to hypermethylation, and that 5-Aza-CdR can inhibit cell proliferation and induce apoptosis by eliminating the methylation status of RUNX3 promoter and restoring its expression.

Keywords: RUNX3; apoptosis; breast cancer; methylation.

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Figures

Figure 1
Figure 1
Growth-inhibiting effects of 5-Aza-CdR on MCF-7 cells. Notes: MCF-7 cells were treated with different concentrations of 5-Aza-CdR for 0–72 hours. Cell viability was determined by MTT assay, performed in triplicate. Dose- and time-dependent inhibition of cell growth could be observed after 72 hours (P < 0.05, one-way ANOVA). Abbreviations: 5-Aza-CdR, 5-aza-2′-deoxycytidine; ANOVA, analysis of variance; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; h, hours.
Figure 2
Figure 2
FCM analysis for apoptosis after treatment by annexin V-FITC and PI staining on MCF-7 cells. After treatment with different doses of 5-Aza-CdR for 48 hours, apoptosis induction was observed. Apoptotic cells were differentiated from viable or necrotic ones by combined application of annexin V-FITC and PI. This assay was performed in triplicate. (A) Blank control group; (B) 0.4 μmol/L 5-Aza-CdR group; (C) 1.6 μmol/L 5-Aza-CdR group; (D) 6.4 μmol/L 5-Aza-CdR group; (E) 25.6 μmol/L 5-Aza-CdR group; (F) 102.4 μmol/L 5-Aza-CdR group. (G) comparison of all groups. Notes: *P < 0.05; **P < 0.01 versus control group. Abbreviations: 5-Aza-CdR, 5-aza-2′-deoxycytidine; FCM, flow cytometry; FITC, fluorescein isothiocyanate; PI, propidium iodide.
Figure 3
Figure 3
Cell apoptosis observed by Hoechst 33258 staining using a fluorescence microscope (200×). Values represent mean ± SD from three independent experiments. (A) Blank control group; (B) 0.4 μmol/L 5-Aza-CdR group; (C) 6.4 μmol/L 5-Aza-CdR group; (D) 102.4 μmol/L 5-Aza-CdR group. Notes: The arrows represent the apoptotic cells. Abbreviations: 5-Aza-CdR 5-aza-2′-deoxycytidine; SD, standard deviation.
Figure 4
Figure 4
Effects of 5-Aza-CdR on the cell cycle of MCF-7 cells by FCM. Notes: The cell cycle distributions in MCF-7 cells were determined by PI staining and FCM analysis after treatment with 0.4–102.4 μmol/L 5-Aza-CdR for 48 hours. A = Blank control group; B = 0.4 μmol/L 5-Aza-CdR group; C = 1.6 μmol/L 5-Aza-CdR group; D = 6.4 μmol/L 5-Aza-CdR group; E = 25.6 μmol/L 5-Aza-CdR group; F = 102.4 μmol/L 5-Aza-CdR group. Results presented were representative of three independent experiments. *P < 0.05; **P < 0.01 versus control group. Abbreviations: 5-Aza-CdR 5-aza-2′-deoxycytidine; FCM, flow cytometry; G0/G1, gap0/gap1 phase. G2/M, gap2/mitosis phase; PI, propidium iodide; S, synthesis phase.
Figure 5
Figure 5
Effect of 5-Aza-CdR on RUNX3 demethylation in MCF-7 cells. MCF-7 cells were treated with various concentrations of 5-Aza-CdR (0.4, 1.6, 6.4, 25.6, and 102.4 μmol/L) for 48 hours, and then RUNX3 methylation was identified by MSP assay. This assay was done in triplicate. (A) RUNX3 methylation in each group. (B) Relative expression level of RUNX3 methylation. A = Control group; B = 0.4 μmol/L 5-Aza-CdR group; C = 1.6 μmol/L 5-Aza-CdR group; D = 6.4 μmol/L 5-Aza-CdR group; E = 25.6 μmol/L 5-Aza-CdR group; F = 102.4 μmol/L 5-Aza-CdR group. Notes: Values represent means ± SEM. *P < 0.05; **P < 0.01 versus blank control group. Abbreviations: 5-Aza-CdR, 5-aza-2′-deoxycytidine; MSP, methylation-specific polymerase chain reaction; RUNX3, runt-related transcription factor 3; SEM, standard error of the mean.
Figure 6
Figure 6
Effects of 5-Aza-CdR on RUNX3 mRNA and protein expression in MCF-7 cells. MCF-7 cells were treated with various concentrations of 5-Aza-CdR (0.4, 1.6, 6.4, 25.6, and 102.4 μmol/L) for 48 hours. The mRNA and protein expression of RUNX3 were determined by RT-PCR (A) and Western blot analysis (B) respectively. These assays were done in triplicate. 1 = Control group; 2 = 0.4 μmol/L 5-Aza-CdR group; 3 = 1.6 μmol/L 5-Aza-CdR group; 4 = 6.4 μmol/L 5-Aza-CdR group; 5 = 25.6 μmol/L 5-Aza-CdR group; 6 = 102.4 μmol/L 5-Aza-CdR group. (C). Relative expression level of RUNX3. Values represent means ± SEM. Notes: *P < 0.05; **P < 0.01 versus blank control group. Abbreviations: 5-Aza-CdR, 5-aza-2′-deoxycytidine; bp, base pair; GAPDH, phosphoglyceraldehyde dehydrogenase; RT-PCR, reverse transcription polymerase chain reaction; RUNX3, runt-related transcription factor 3; SEM, standard error of the mean.

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