Abstract 163: Common food additive carrageenan activates wnt signaling pathway in mouse colon and human colonic epithelial cells

Abstract

Abstract The Wnt-β-catenin signaling pathway plays a pivotal role in development and disease. It is crucial for basic developmental processes, such as cell-fate specification, progenitor-cell proliferation and the control of asymmetric cell division. Recent studies suggest that self-renewal of stem/progenitor cells by the canonical Wnt cascade is subverted in cancer cells to allow malignant proliferation in intestine. Carrageenans are highly sulfated polysaccharides, which are widely used as food additives to improve the texture of processed foods, and are consumed at levels of about 250 mg/day in the average diet. Reports of many investigators have demonstrated that carrageenan induces inflammation, colonic ulcerations, polyps, and colorectal tumors. We have reported distinct pathways by which carrageenan induces the activation of NF-κB and secretion of IL-8, mediated through TLR4-BCL10 or reactive oxygen species (ROS) in colonic epithelial cells. Our previous work also indicated that carrageenan exposure caused activation of the Wnt-β-catenin pathway in colonic epithelial cells. This investigation was undertaken to elucidate the Wnt signaling mechanisms activated by carrageenan exposure, using NCM460 (INCELL) human colonic epithelial cells exposed to carrageenan (1 μg/ml) x 24 h and colonic tissue from ten-week old C57BL/6J mice exposed to carrageenan (10 - 100 μg/ml) in their water for four weeks. Nuclear β-catenin in NCM460 cells and mouse colonic tissue was determined by Western blot and ELISA and increased to two times the baseline level following carrageenan. This increase in nuclear β-catenin in NCM460 cells was inhibited by the ROS quencher Tempol, but not by TLR4 or BCL10 siRNA. TCF/Lef-1 reporter assay demonstrated increased nuclear binding post carrageenan treatment in the mouse colon (to 4x baseline) and in the NCM460 cells (to 6.3x baseline). Also, mRNA expression of cyclin D1 and wnt9a was increased more than two-fold, and BMP4 expression was decreased. In the NCM460 cells, these responses to carrageenan were inhibited by Tempol, but not BCL10 silencing. Also, carrageenan-induced increases in ROS and lipid peroxidation were inhibited by Tempol. Since Wnt signaling is affected by free dishevelled 2 (dsh2), and dsh2 binding to nucleoredoxin (NRX) is greater when NRX is in its reduced state, we tested whether carrageenan exposure could impact upon the Wnt signaling pathway by modifying the redox status of NRX. The ratio of free NRX to NRX bound to dsh2 increased to six times baseline following carrageenan, and was reversed by Tempol. These profound effects of carrageenan on the Wnt signaling pathway in colonic cells can help to explain how diet can contribute to development of colonic neoplasia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 163. doi:1538-7445.AM2012-163