Claudin-2 is a unique member of the claudin family of transmembrane proteins as its manifestation is restricted to the leaky epithelium correlates with epithelial leakiness = 7/group). of 5-FU Forced claudin-2 manifestation significantly increased tumorigenicity of both cell types under study. Since, claudin-2 manifestation varies with changes in cell proliferation (Guillemot and Citi 2006; Buchert, Papin et al. 2010), we further decided effects of claudin-2 overexpression upon cell proliferation using MTT assay. As SID 26681509 manufacture shown in Fig. 3, we observed significant increases in cell proliferation in both HCT116Claudin-2 and SW480Claudin-2 cells compared to respective controls [Fig. 3A & 3B; p<0.05]. Unchecked proliferation is usually central to tumorigenesis, including colon malignancy, and is usually a key aspect of the resistance to the malignancy treatment drugs. Therefore, we further examined effects of 5-FU, a colon malignancy treatment drug (Ansfield, Klotz et al. 1977), upon HCT116Claudin-2 and control cells. Cells were uncovered to gradually increasing concentrations of 5-FU and cell viability was decided at 24, 48, and 72 hours after exposure to 5-FU. As expected, 5-FU decreased cell viability in a time- and dose-dependent manner, however HCT116Claudin-2 cells were significantly (p<0.001 at 50 or 100 M and p<0.01 at 200 M) protected from the effects of 5-FU compared to the control cells (Fig. 3C). We further decided specific contribution of proliferation apoptosis to the 5-FU-dependent switch in cell viability. As shown in supplementary fig. 4, 5-FU-treatment inhibited proliferation (p<0.001) while increased apoptosis (p<0.05) in HCT116control cells. However, HCT116claudin-2 cells were largely resistant to the effects of 5-FU upon proliferation as well as apoptosis. Physique 3 Forced claudin-2 manifestation increased proliferation of colon malignancy cells and guarded from the effects of 5-FU Co-culture of colon malignancy cells with colonic fibroblasts increases claudin-2 manifestation in an EGFR-dependent manner In colon, a close association exists between the crypt epithelial cells and pericryptal fibroblasts. Emerging evidence supports the importance of the stromal rules of intestinal tumors (Cutler, Graves-Deal et al. 2003). As explained, claudin-2 is uniquely, compared to other colonic claudins, expressed in the crypt base (Holmes, Van Itallie et al. 2006). Therefore, we further decided the potential role of the tissue microenvironment in the rules of colonic claudin-2 manifestation. In this regard, we performed co-culture of colonic fibroblasts (Fig. 4A) with Caco-2 colon malignancy cells that express endogenous claudin-2 protein. Effect of co-culture upon claudin-2 and claudin-4 manifestation was examined. Co-culture with either HCF-27 (normal pericryptal fibroblasts) or HNPCC (fibroblasts from HNPCC individuals) (Cutler, Graves-Deal et al. 2003) cells had no appreciable effect upon claudin-4 manifestation, however Rabbit Polyclonal to Gab2 (phospho-Tyr452) claudin-2 manifestation was significantly increased (Fig. 4B). This fibroblast-dependent increase in claudin-2 manifestation was higher in cells co-cultured with the HNPCC (p<0.001) compared to the HCF-27 (p<0.01) fibroblasts (Fig. 4B). Importantly, this increase in claudin-2 manifestation was inhibited upon use of PD153035, an EGFR tyrosine kinase specific inhibitor [(Bos, Mendelsohn et al. 1997); Fig. 4C] or EGFR blocking antibody [Clone#528, 20 g/ml; (Supplementary fig. 5)]. Together, our data supported the role of the colonic stroma in the rules of claudin-2 manifestation in an EGFR-dependent manner. Physique 4 Co-culture of colon malignancy cells with colonic fibroblasts induced claudin-2 manifestation in an EGFR-dependent manner Exogenous EGF increases claudin-2 manifestation in colon malignancy cells The SID 26681509 manufacture data from the co-culture studies suggested a potential role of EGFR activation in the fibroblast-dependent induction of claudin-2 manifestation in Caco-2 cells. This obtaining SID 26681509 manufacture was in sharp contrast to our earlier statement SID 26681509 manufacture that in renal epithelial (MDCK-II) cells, EGFR-activation decreases claudin-2 manifestation (Singh and Harris 2004). Therefore, we further decided effect of the exogenous EGF upon claudin-2 manifestation in the colon produced cells. Quiescent and polarized Caco-2 cells were uncovered to EGF (100 ng/ml) and effect upon TER, paracellular permeability and claudins manifestation was decided. Particularly, EGF treatment resulted in a time-dependent and significant decrease in TER.