ogression of CAC. Given these results, we hypothesized that the increased protumorigenic cytokines produced by the infiltrating cells in the inflamed tissues may be associated with the disrupted epithelial tight junctions, which results in bacterial translocation into the lamina propria. In addition, we have also observed that TNFR2 expression was specifically up-regulated in the inflamed epithelia. In this regard, it has been reported by Wang et al. that the upregulated MLCK in human intestinal cells is required for TNFinduced collapse of epithelial TJ by TNFR2 signaling. Moreover, it has been reported by two groups that MLCK promoter activity is mediated by NF-kB activation. 
Therefore, we addressed the epithelial MLCK expression in the CAC model. As seen in Fig. 1C, Western blotting BAY-41-2272 manufacturer revealed upregulated MLCK expression in the epithelia from non-tumor area, and further up-regulation in that of tumor, in association with the up-regulated TNFR2 and p65. However, up-regulation of TNFR1 was not remarkable compared to that of TNFR2. These results indicate that the development of CAC may be associated with the disrupted TJ and elevated pro-tumorigenic cytokines, which are essentially induced by TNFR2 signaling and MLCK expression in the epithelia. MLCK Expression in the Epithelial Cells is Up-regulated by the Presence of Pro-inflammatory Cytokines in vitro Given the up-regulated TNFR2 and MLCK expressions in the epithelial cells associated with pro-tumorigenic and pro-inflammatory PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19645691 cytokines expressions in the mucosal tissues from chronic colitis and CAC model, it was surmised that each of these complicated phenomena may be associated with the mechanisms by which tumorigenesis is induced in the context of IBD. Especially, previous reports suggested that stimulation of human intestinal epithelial cell lines with IFN-c results in the regulation of TNF receptors. Therefore, a murine colonic epithelial cell line derived from colonic epithelial cell was used to study the specific roles of TNFR2 and MLCK expressions in the epithelial cells in the context of inflammation. MOC1 cells were cultured in the presence of pro-inflammatory cytokines, rIFN-c and rTNF. As seen in Fig. 2A, the up-regulations of TNFR1 and 2 were induced by rIFN-c alone, and this is consistent with previous observations with human T84 and Caco2 cells. However, the increased TNFR2 level was more remarkable compared to that of TNFR1, and such TNFR2 expression in MOC1 cells was maximal at the concentration of 1.0 ng/ml rIFNc. Next, MOC1 cells were stimulated with different concentrations of rTNF together with rIFN-c at 1.0 ng/ml. Western blotting showed that phosphorylations of p65 and IkBa were induced in these cells by the presence of rTNF in a dose-dependent manner. Moreover, the expressions of MLCK as well as TNFR2, but not much of TNFR1, were further up-regulated by the addition of rTNF in a dose-dependent fashion in collaboration with constant concentration of rIFN-c, and these expressions were maximal at 2.5 ng/ml of rTNF. These results suggest that the pro-inflammatory cytokines, especially TNF, may be Statistical Analysis The results are expressed as the mean 6 standard error of the mean. Statistical significance was determined using nonparametric Mann-Whitney U-test, and differences were considered to be statistically significant with p,0.05. Results Secretion of Cytokines that Support Tumor Growth is Associated with Epithelial MLCK Expression in the CAC Model We previous