Colon cancer cell apoptosis is induced by combined exposure to the n-3 fatty acid docosahexaenoic acid and butyrate through promoter methylation

Abstract

DNA methylation and histone acetylation contribute to the transcriptional regulation of genes involved in apoptosis. We have demonstrated that docosahexaenoic acid (DHA, 22:6 n-3) and butyrate enhance colonocyte apoptosis. To determine if DHA and/or butyrate elevate apoptosis through epigenetic mechanisms thereby restoring the transcription of apoptosis-related genes, we examined global methylation; gene-specific promoter methylation of 24 apoptosis-related genes; transcription levels of Cideb, Dapk1, and Tnfrsf25; and global histone acetylation in the HCT-116 colon cancer cell line. Cells were treated with combinations of (50 µM) DHA or linoleic acid (18:2 n-6), (5 mM) butyrate or an inhibitor of DNA methyltransferases, and 5-aza-2'-deoxycytidine (5-Aza-dC, 2 µM). Among highly methylated genes, the combination of DHA and butyrate significantly reduced methylation of the proapoptotic Bcl2l11, Cideb, Dapk1, Ltbr, and Tnfrsf25 genes compared to untreated control cells. DHA treatment reduced the methylation of Cideb, Dapk1, and Tnfrsf25. These data suggest that the induction of apoptosis by DHA and butyrate is mediated, in part, through changes in the methylation state of apoptosis-related genes.

Keywords: DNA methylation; apoptosis; butyrate; docosahexaenoic acid; epigenetics.

Figure 1

Design of cell culture experiments

Figure 2

Global DNA methylation status (percent of the negative control) in HCT-116 cells cultured with 5-Aza-dC (48 h), butyrate (12 h), DHA (72 h), LA (72 h), or co-treatment (see “Experiment I” section for details of Experiment I). Values are means ± SEM. Values not sharing common letters are significantly different, P <0.05, n = 6 per treatment. Negative controls represent cells incubated in media only. DHA: docosahexaenoic acid; LA: linoleic acid

Figure 3

Time-course analysis of Tnfrsf25 (Panel a) and Dapk1 (Panel b) transcript levels (expressed as percentage of the negative control) in HCT-116 cells cultured with 5-Aza-dC, butyrate, DHA, LA, or co-treatment for 48, 72, or 96 h (see “Experiment II” section for details of Experiment II). Values are means ± SEM. *Different from the negative control sample measured at the same time point, P <0.05, n = 3 per treatment. The negative controls were cells incubated in media only

Figure 4

H3 and H4 histone acetylation (expressed as percentage of the negative control) in HCT-116 cells incubated with 5-Aza-dC (48 h), butyrate (12 h), DHA (72 h), LA (72 h), or co-treatment (see “Experiment I” section for details of Experiment I). Values are means ± SEM. *Different from the negative control, P <0.05, n = 3 per treatment. The negative controls were cells incubated in media only