Abstract 5734: Prolonged activation of NFκB (RelA and RelB), BCL10, and IL-8 following exposure to common food additive carrageenan in human colonic epithelial cells may be attributable to NFκB binding site in BCL10 promoter

Abstract

Abstract The common food additive carrageenan (CGN) is a sulfated polysaccharide that resembles the naturally occurring sulfated glycosaminoglycans (sGAG), particularly chondroitin sulfate and keratan sulfate. Unlike the naturally occurring sGAG, CGN contains alpha-1,3-galactosidic linkages that are immunogenic in humans. Previously, we reported that CGN activation of a TLR4-MyD88 mediated inflammatory cascade proceeds through BCL10 and NFκB (p65), leading to increased secretion of IL-8 in human colonic epithelial cells. Translocations involving BCL10 and MALT1 are found in mucosa-associated lymphoid tissue (MALT) lymphomas, associated with constitutive activation of NFκB. Since CGN has been widely used to induce colitis in animal models of inflammatory bowel disease, we addressed whether CGN exposure can lead to sustained activation of BCL10 and NFκB, consistent with a model of constitutive activation of inflammation and possible pre-malignant changes. Experiments were performed with NCM460 cells, a human colonic epithelial cell line derived from normal colon, in M3:10A medium (Incell) with λCGN (1μg/ml). Triplicate cell preparations and spent media from untreated control cell preparations, cells treated with CGN for only the initial 12 hour time interval, and cells treated for six 12-hour periods were harvested every 12 hours over 72 hours, and media, with or without CGN was replenished. IL-8 in the spent media, and BCL10 in the cytosolic preparations and NFκB [RelA (p65) and RelB] in the nuclear preparations were measured at successive 12-hour periods. IL-8 remained significantly increased for an additional 36 hours following withdrawal of CGN. BCL10 and RelA remained significantly increased for an additional 24 hours, and RelB was significantly increased for an additional 12 hours. These findings are consistent with previous data that demonstrate that CGN activates both canonical and non-canonical pathways of NFκB. The presence of a sequence with high homology to the established NFκB consensus oligonucleotide sequence in the BCL10 promoter may be responsible for an ongoing process of activation following withdrawal of the CGN exposure. In vitro testing of binding of RelA and RelB to the NFκB consensus oligonucleotide, the homologous BCL10 promoter sequence, a mutated BCL10 promoter sequence, and a scrambled NFκB consensus sequence indicates over 50% binding of either RelA or RelB to the BCL10 promoter sequence, compared to 100% to the established NFκB consensus sequence. Hence, the study findings demonstrate that an environmental exposure to a common food additive (CGN) can trigger prolonged activation of an inflammatory loop involving BCL10, RelA, RelB, and IL-8 in human colonic epithelial cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5734.