Effect of 5-Aza-2'-Deoxycytidine in Comparison to Valproic Acid and Trichostatin A on Histone Deacetylase 1, DNA Methyltransferase 1, and CIP/KIP Family (p21, p27, and p57) Genes Expression, Cell Growth Inhibition, and Apoptosis Induction in Colon Cancer SW480 Cell Line

Abstract

Background: Cancer initiation and progression depends on genetic and epigenetic alterations such as DNA methylation and histone modifications. Hypermethylation and deacetylation of the CIP/KIP family (p21, p27, and p57) lead to tumorigenesis. Our previous study indicated that DNA methyltransferase (DNMT) inhibitor and histone deacetylase (HDAC) inhibitors can inhibit cell growth and induce apoptosis. The aim of the present study was to investigate the effect of 5-Aza-2'-deoxycytidine (5-Aza-CdR) in comparison to valproic acid (VPA) and trichostatin A (TSA) on HDAC1, DNMT1, and CIP/KIP family (p21, p27, and p57) genes expression, cell growth inhibition, and apoptosis induction in colon cancer SW480 cell line.

Materials and methods: The effect of the compounds on the cell viability was measured by MTT assay. The expression of HDAC1, DNMT1, and CIP/KIP family (p21, p27, and p57) genes was evaluated by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). For the detection of cell apoptosis, apoptotic cells were examined by the Annexin V-FITC/PI detection kit.

Results: The results of MTT assay indicated that 5-Aza-CdR, VPA, and TSA significantly inhibited cell growth (P < 0.002, P < 0.001, and P < 0.001, respectively). The results of real-time RT-PCR demonstrated that all compounds significantly down-regulated DNMT1 and HDAC1, and up-regulated p21, p27, and p57 genes expression. The result of flow cytometry assay revealed that all agents induced apoptosis significantly.

Conclusion: 5-Aza-CdR, VPA, and TSA can significantly downregulate DNMT1 and HDAC1 and up-regulate p21, p27, and p57 genes expression through which enhance cell apoptosis and cell growth inhibition in colon cancer.

Keywords: 5-Aza-2'-deoxycytidine; cancer; trichostatin A; valproic acid.

Figure 1

In vitro effects of 5-Aza-2'-deoxycytidine (0, 0.5, 1, 5, 10, and 20 μM), valproic acid (0, 0.5, 1, 5, 10, and 20 μM), and trichostatin A (0, 0.5, 1, 5, 10, and 20 μM) on colon cancer SW480 cell viability tested by MTT assay at different times (24 and 48 h). As shown in the figure, the first column of each group belongs to untreated cells (control group), and the others belong to treated cells with the compounds (5-Aza-2'-deoxycytidine, valproic acid, and trichostatin A) at a concentration of 0.5, 1, 5, 10, and 20 μM. Values are means of three experiments in triplicate. Standard errors were <5%. Asterisks (*) indicate significant differences between 5-Aza-2'-deoxycytidine, valproic acid, and trichostatin A treated and the control groups

Figure 2

Relative expression level of DNA methyltransferase 1 and histone deacetylase 1 in the experimental groups treated with 5-Aza-2'-deoxycytidine (2.5 μM), valproic acid (5 μM), and trichostatin A (1.5 μM) versus control groups at 24 and 48 h. The first column of each group belongs to untreated cells (control group), and the others belong to treated cells with the compounds with the mentioned concentrations at 24 and 48 h. Asterisks (*) indicate significant differences between 5-Aza-2'-deoxycytidine, valproic acid and trichostatin A treated and the control groups

Figure 3

Relative expression level of P21, P27, and P57 in the experimental groups treated with 5-Aza-2'-deoxycytidine (2.5 μM), valproic acid (5 μM), and trichostatin A (1.5 μM) versus control groups at 24 and 48 h. The first column of each group belongs to untreated cells (control group), and the others belong to treated cells with the compounds with the mentioned concentrations at 24 and 48 h. Asterisks (*) indicate significant differences between 5-Aza-2'-deoxycytidine, valproic acid and trichostatin A treated and the control groups

Figure 4

The apoptotic effects of 5-Aza-2'-deoxycytidine (2.5 μM), valproic acid (5 μM), and trichostatin A (1.5 μM) versus control groups at 24 and 48 h on colon cancer SW480 cell apoptosis. The cells were treated with the compounds for 24, and 48 h and the apoptosis-inducing the effect of the agents was investigated by flow cytometric analysis. The upper right quadrant shows the percentage of cells in late apoptosis, the lower right quadrant shows the percentage of cells in early apoptosis, the upper left quadrant shows the percentage of necrotic cells, and the lower left quadrant shows the percentage of viable cells. Results were obtained from three independent experiments and were expressed as mean ± standard error of the mean

Figure 5

The comparative effects of 5-Aza-2'-deoxycytidine (2.5 μM), valproic acid (5 μM), and trichostatin A (1.5 μM) on colon cancer SW480 cell apoptosis at 24 and 48 h. The first column of each group belongs to untreated cells (control group) and the others belong to treated cells with the compounds with the mentioned concentrations at 24 and 48 h. Asterisks (*) indicate significant differences between 5-Aza-2'-deoxycytidine, valproic acid and trichostatin A treated and the control groups. As shown above, trichostatin A indicated a more significant apoptotic effect in comparison to other agents