Delineating the effect of demethylating agent 5-aza-2'-deoxycytidine on human Caco-2 colonic carcinoma cells

Abstract

Aberrant epigenetic changes are known to contribute to various phases of tumor development. The gene function loss caused by aberrant methylation is analogous to genetic mutations. Unlike genetic mutations, epigenetic alterations can be reversed. 5-Aza-2'-deoxycytidine (5-aza-CdR) has been approved by the Food and Drug Administration for the treatment of certain types of cancer, such as MDS and leukemia. The aim of the present study was to determine whether 5-aza-CdR has the potential to be used in the treatment of colon cancer using a human Caco-2 colonic carcinoma cell line. The effect of 5-aza-CdR on cell proliferation, cell cycle, apoptosis and reversal of aberrant methylation of the Ras association domain family 1A (RASSF1A) gene was also examined. The 5-aza-CdR was prepared at different concentrations in sterile tri-distilled water at 0.4, 1.6, 6.4, 25.6 and 102.4 µmol/l and employed to treat the human Caco-2 colonic carcinoma cells. An MTT assay was used to detect the effect of 5-aza-CdR on cell proliferation. Flow cytometry was used to examine the cell cycle and apoptosis. The RASSF1A mRNA transcript level was examined by reverse transcription-polymerase chain reaction. The results showed that 5-aza-CdR inhibited the proliferation of Caco-2 cells in a time- and concentration-dependent manner (p<0.01). The 5-aza-CdR treatment affected the cell cycle and caused accumulation of cells in the G0/G1 phase and this effect was concentration-dependent (p<0.05). 5-aza-CdR treatment caused an increase in the number of cells undergoing apoptosis and reactivated the RASSF1A tumor suppressor gene that was silenced by hypermethylation in Caco-2 cells. In conclusion, 5-aza-CdR inhibited growth and promoted apoptosis in Caco-2 cells by upregulating the epigenetically silenced tumor suppressor RASSF1A gene.

Keywords: 5-aza-2′-deoxycytidine; Caco-2; Ras association domain family 1A; aberrant methylation; colon cancer; demethylation.

Figure 1.

(A and B) Caco-2 cell morphology following treatment with 6.4 µmol/l 5-aza-2′-deoxycytidine for 3 days. A decrease in volume and density was identified.

Figure 1.

(A and B) Caco-2 cell morphology following treatment with 6.4 µmol/l 5-aza-2′-deoxycytidine for 3 days. A decrease in volume and density was identified.

Figure 2.

Caco-2 cell morphology without treatment.

Figure 3.

Cell growth curve at different concentrations of 5-aza-2′-deoxycytidine.

Figure 4.

Ras association domain family 1A expression in 5-aza-2′-deoxycytidine (5-aza-CdR)-treated cells. Lane M, DNA molecular weight marker; lane 1, GAPDH; lane 2, 0.4 µmol/l (5-aza-CdR); lane 3, 1.6 µmol/l; lane 4, 6.4 µmol/l; and lane 5, 25.6 µmol/l.