Dietary factors and cancer cell epigenetics
Date of Completion
Biology, Genetics|Health Sciences, Nutrition|Health Sciences, Oncology
We have performed experiments using human colon cancer cell lines to enhance our understanding of these epigenetic mechanisms in the suppression of growth regulatory genes.^ Use of an improved siRNA transfection method identifies involvement of HDAC 1 and 3, respectively, in RARβ2 and p21 repression. The role of nonspecific histone deacetylase (HDAC) inhibitors in achieving re-expression of various growth regulatory genes and inducing apoptosis in cancer cells has been well established. In order to explore the potential value of selectively targeting individual HDACs for inhibition in cancer treatment, we developed an improved method for siRNA transfection of colon cancer cells. While the suppression of individual HDACs did not result in nearly as robust an activation of expression as that attained in response to treatment with the nonspecific class I and II HDAC inhibitor butyrate, siRNA knockdown of individual class I and class II HDACs did provide evidence that HDACs 1 and 3, respectively, are involved with the repression of RARβ2 and p21 growth regulatory genes.^ The short chain fatty acid butyrate induces promoter demethylation and reactivation of RARβ2 in colon cancer cells. It has been proposed that cancer prevention results from multiple dietary agents acting together as "action packages". Here we obtained evidence that butyrate, which is generated from dietary fiber, enhances the responsiveness of colon cancer cells to all-trans retinoic acid (ATRA). Evidence was obtained that this interaction depends on HDAC 1 inhibition by butyrate and RARα activation by ATRA. The enhancement of RARβ2 activation was accompanied by a rapid demethylation of the RARβ2 promoter. This demethylation could be achieved by butyrate alone, and it differed from that triggered by the DNA methyltransferase (DNMT) inhibitor 5-Aza-2'deoxycytidine in that it was: (1) sporadic on the RARβ2 promoter; (2) not genome-wide, and (3) independent of extensive DNA replication. An analysis of inter-methylated sites (AIMS) assay indicated that only a few percent of loci analyzed showed reduced methylation. In colon cancer cells that were particularly resistant to RARβ2 reactivation, the actions of butyrate could be further enhanced by the soy isoflavone genistein, which has also been reported to work through an epigenetic mechanism. These data suggest that dietary compounds that modulate epigenetic programming are likely to function best in the presence of retinoids and other cancer-preventing compounds that are sensitive to a cell's epigenetic state.^ Histone reacetylation is followed with a progressive demethylation of RARβ2 in colon cancer cells treated with butyrate. Until only recently, it was commonly believed that administration of a DNMT inhibitor was necessary in order to establish re-expression of genes silenced through CpG methylation. However, work in our lab as well as that of others has demonstrated the ability for HDAC inhibitors like sodium butyrate to induce the re-expression of some aberrantly methylated genes in cancer cells. Through a time-course analysis of the RARβ2 promoter following butyrate treatment, we broaden our understanding of the crosstalk which can occur between reacetylation and demethylation events. We find evidence that histone re-acetylation preceeds a seemingly progressive demethylation that initiates upstream of the promoter RARE and TATA-box region. Additionally, use of siRNA knockdown of RARα and RARγ, implicates RARγ involvement in maintaining CpG methylation of the RARβ2 promoter. Interestingly, following a re-methylation event of the promoter, we find the CpG dinucleotide immediately 5' to the RARE escapes methylation. Use of the transcription factor binding site prediction program, TFSITESCAN, identified a binding site for the orphan receptor COUP-TF 19 bases upstream of this CpG. We propose an interaction between COUP-TF and RARα binding to the RARE may serve to protect this CpG from methylation.^ Conclusions. These results call our attention to a number of important aspects involved with the aberrant epigenetic silencing of growth regulatory genes in cancer. In particular, our results emphasize the importance of dietary factors in maintaining and acquiring re-expression of growth regulatory genes. Additionally, these data provide important insight into the cross-talk between the mechanisms of acetylation and demethylation. This information may be exploitable in the future for the development of improved chemotherapeutics. (Abstract shortened by UMI.)^
Spurling, Colleen C, "Dietary factors and cancer cell epigenetics" (2008). Doctoral Dissertations. AAI3345208.