Abstract 816: Anti-inflammatory effects of black raspberries in ulcerative colitis are associated with demethylation of genes in the Wnt signaling and protective modulation of toll-like receptor pathway

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Ulcerative Colitis (UC) causes pervasive, chronic inflammation of the colon and is a potential precursor to colon cancer. Reduced Interleukin 10 (IL10), an anti-inflammatory cytokine, is associated with an increased risk of developing UC in rodents and humans. IL10 knockout (KO) mice develop UC spontaneously with subsequent development of colon tumors. Toll-like receptor and Wnt pathways are deregulated in UC patients and IL10 KO mice. Toll-like receptor pathway plays critical roles in the inflammatory response partly by mediating the Wnt pathway. We previously showed that 5-10% dietary freeze-dried black raspberries (BRBs) reduced chronic inflammation and tumor development in IL10 KO mice; aberrant promoter methylation of negative Wnt regulators, e.g., dkk2, dkk3, and sfrp1, were observed in colon from these animals. The goals of the current study were to determine if the anti-inflammatory effects of BRBs on IL10 KO mice are associated with modulation of the Wnt pathway through DNA demethylation, with alteration of enzymes regulating DNA methylation, and with modulation of toll-like receptor pathway. IL10 KO mice were fed control or 5% BRB diet for 4 wks. Cells from bone marrow and spleen as well as colon tissue were collected. Promoter methylation of dkk2, dkk3, and sfrp1 was quantified by Pyrosequencing. Protein expression of enzymes regulating DNA methylation, e.g., histone deacetylase 1, 2 (HDAC1, 2), methyl binding domain 2 (MBD2), and DNA methyltransferase 3B (DNMT3B), and β-catenin were assessed using quantitative immunohistochemistry. mRNA expression of genes associated with the Wnt and toll-like receptor pathways were quantified using real-time PCR based arrays, each of which contained 84 genes. Increased promoter methylation of dkk2, dkk3, and sfrp1 was observed in colon from KO mice when compared with colon from wild-type mice. The methylation levels of these genes in bone marrow and spleen were lower than those in colon. BRBs significantly decreased promoter methylation of dkk2 in colon and dkk3 in spleen. This was associated with decreased protein expression of HDAC1, HDAC2, MBD2, and DNMT3B. The expression of 84 genes in both pathways was different in colon of IL10 KO mice when compared to colon of wild-type mice. BRBs modulated 80% (67/84) and 95% (80/84) of differentially expressed genes toward normal levels of expression in the Wnt and toll-like receptor pathways, respectively. Protein expression of nuclear β-catenin was decreased by BRBs. In summary, berries are capable of reversing aberrant promoter methylation of genes in the Wnt pathway presumably through inhibition of proteins regulating DNA methylation. These changes could result in protective modulation of the Wnt pathway and/or its interaction with the toll-like receptor pathway which, in turn, reduce inflammation in ulcerative colitis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 816. doi:10.1158/1538-7445.AM2011-816