Laminin Receptor 37/67LR Regulates Adhesion and Proliferation of Normal Human Intestinal Epithelial Cells

Tumor-targeting therapy can be an efficacious way to cure a malignant tumor in clinical trials. Phage display is a molecular diversity technology that allows the presentation of a large number of peptides or proteins on the surface of filamentous phage for various applications. In this study, we report on using phage display to generate peptide libraries that bind to colon cancer tissues. To accomplish this, we developed a screening protocol that contained 3 rounds of in vitro positive panning on colon cancer cells (SW480) and 2 rounds of subtractive screening in vitro on normal human intestinal epithelial cells with a phage display-7 peptide library. After several rounds of panning, both phage titer and recovery efficiency were significantly improved. Through a cell-based enzyme-linked immunosorbent assay, immunofluorescence, in vivo binding assay, immunocytochemical staining, and immunohistochemical staining, peptide CP15 (VHLGYAT) was demonstrated to be the most effective peptide in targeting tumor cells (SW480 and HT29 cells) and tumor tissues but not the normal human intestinal epithelial cells and control colon tissue. These studies suggest that peptide CP15 may be a promising lead candidate in the development of a useful colon tumor diagnostic and targeted drug delivery agent. ( Journal of Biomolecular Screening 2007:429-435)

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Epigallocatechin-3-Gallate Regulates Anti-Inflammatory Action Through 67-kDa Laminin Receptor-Mediated Tollip Signaling Induction in Lipopolysaccharide-Stimulated Human Intestinal Epithelial Cells

Cellular Physiology and Biochemistry ◽

10.1159/000489447 ◽

2018 ◽

Vol 46 (5) ◽

pp. 2072-2081 ◽

Cited By ~ 11

Author(s):

Eui-Baek Byun ◽

Woo Sik Kim ◽

Nak-Yun Sung ◽

Eui-Hong Byun

Keyword(s):

Nitric Oxide ◽

Epithelial Cells ◽

Inflammatory Mediators ◽

Intestinal Epithelial Cells ◽

Laminin Receptor ◽

Intestinal Epithelial ◽

Tlr Signaling ◽

Egcg Treatment ◽

Human Intestinal Epithelial Cells ◽

Ht 29

Background/Aims: Inflammatory bowel disease (IBD) is a condition that involves chronic inflammation in all or part of the digestive tract. Often painful and debilitating, IBD can lead to life-threatening complications and increase the risk for colon cancer. In this study, we investigated the epigallocatechin-3-gallate (EGCG) mediated anti-inflammation response in lipopolysaccharide (LPS)-stimulated human colorectal cells through the negative regulator of Toll-like receptor (TLR) signaling. Methods: human intestinal epithelial cells (HT-29) were used in all experiments. Cell cytotoxicity and nitric oxide (NO) were evaluated by WST-1 and the Griess reagent. Western blot analysis and ELISA were used to determine inflammatory mediators and 67-kDa laminin receptor (67LR)-mediated Tollip signaling pathways. Results: Treatment of EGCG and LPS did not affect the cytotoxicity in HT-29 cells. LPS treatment dose-dependently increased the pro-inflammatory cytokine, such as interleukin (IL)-8, whereas EGCG significantly reduced the LPS-stimulated IL-8 production. Additionally, EGCG treatment markedly increased the Toll-interacting protein (Tollip) expression, which negatively regulates the TLR signaling in a dose and time-dependent manner. In particular, in the result from an RNA interference-mediated assay, our finding showed that silencing of Tollip resulted in abrogation of the inhibitory action of EGCG on LPS-induced production of pro-inflammatory mediators (inducible nitric oxide synthase-mediated NO/COX2, and IL-8) and activation of MAPKs and NF-κB signaling pathways. Interestingly, we also found that Tollip expression induced by EGCG could be modulated through 67LR expressed on the surface of HT-29 cells. Conclusions: Our novel finding indicates that 67LR and Tollip signaling activated by EGCG treatment is essential for inhibition of inflammation in human intestinal epithelial cells.

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Bcl10 mediates LPS-induced activation of NF-κB and IL-8 in human intestinal epithelial cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00149.2007 ◽

2007 ◽

Vol 293 (2) ◽

pp. G429-G437 ◽

Cited By ~ 32

Author(s):

Sumit Bhattacharyya ◽

Alip Borthakur ◽

Nitika Pant ◽

Pradeep K. Dudeja ◽

Joanne K. Tobacman

Keyword(s):

Epithelial Cells ◽

Confocal Microscopy ◽

Inflammatory Response ◽

Western Blot ◽

Intestinal Epithelial Cells ◽

Ex Vivo ◽

Intestinal Epithelial ◽

Human Intestinal Epithelial Cells ◽

Normal Human ◽

Interfering Rna

Lipopolysaccharide (LPS) is recognized as an inducer of the inflammatory response associated with gram-negative sepsis and systemic inflammatory response syndrome. LPS induction proceeds through Toll-like receptor (TLR) in immune cells and intestinal epithelial cells (IEC). This report presents the first identification of Bcl10 (B-cell CLL/lymphoma 10) as a mediator of the LPS-induced activation of IL-8 in human IEC. Bcl10 is a caspase-recruitment domain-containing protein, associated with constitutive activation of NF-κB in MALT (mucosa-associated lymphoid tissue) lymphomas. The normal human IEC line NCM460, normal primary human colonocytes, and ex vivo human colonic tissue were exposed to 10 ng/ml of LPS for 2–6 h. Effects on Bcl10, phospho-IκBα, NF-κB, and IL-8 were determined by Western blot, ELISA, immunohistochemistry, and confocal microscopy. Effects of Bcl10 silencing by small-interfering RNA (siRNA), TLR4 blocking antibody, TLR4 silencing by siRNA, and an IL-1 receptor-associated kinase (IRAK)-1/4 inhibitor on LPS-induced activation were examined. Following Bcl10 silencing, LPS-induced increases in NF-κB, IκBα, and IL-8 were significantly reduced ( P < 0.001). Increasing concentrations of LPS were associated with higher concentrations of Bcl10 protein when quantified by ELISA, and the association between LPS exposure and increased Bcl10 was also demonstrated by Western blot, immunohistochemistry, and confocal microscopy. Exposure to TLR4 antibody, TLR4 siRNA, or an IRAK-1/4 inhibitor eliminated the LPS-induced increases in Bcl10, NF-κB, and IL-8. Identification of Bcl10 as a mediator of LPS-induced activation of NF-κB and IL-8 in normal human IEC provides new insight into mechanisms of epithelial inflammation and new opportunities for therapeutic intervention.

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Carrageenan induces interleukin-8 production through distinct Bcl10 pathway in normal human colonic epithelial cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00380.2006 ◽

2007 ◽

Vol 292 (3) ◽

pp. G829-G838 ◽

Cited By ~ 85

Author(s):

Alip Borthakur ◽

Sumit Bhattacharyya ◽

Pradeep K. Dudeja ◽

Joanne K. Tobacman

Keyword(s):

Molecular Weight ◽

Epithelial Cells ◽

High Molecular Weight ◽

Intestinal Epithelial Cells ◽

Epithelial Cell Line ◽

Intestinal Epithelial ◽

Human Intestinal Epithelial Cells ◽

Normal Rat ◽

Normal Human ◽

Inflammatory Bowel

Carrageenan is a high molecular weight sulfated polygalactan used to improve the texture of commercial food products. Its use increased markedly during the last half century, although carrageenan is known to induce inflammation in rheumatological models and in intestinal models of colitis. We performed studies to determine its direct effects on human intestinal cells, including normal human intestinal epithelial cells from colonic surgeries, the normal intestinal epithelial cell line NCM460, and normal rat ileal epithelial cells. Cells were treated with high molecular weight λ-carrageenan at a concentration of 1 μg/ml for 1–96 h. IL-8, IL-8 promoter activity, total and nuclear NF-κB, IκBα, phospho-IκBα, and Bcl10 were assessed by immunohistochemistry, Western blot, ELISA, and cDNA microarray. Increased Bcl10, nuclear and cytoplasmic NF-κB, IL-8 promoter activation, and IL-8 secretion were detected following carrageenan exposure. Knockdown of Bcl10 by siRNA markedly reduced the increase in IL-8 that followed carrageenan exposure in the NCM460 cells. These results show, for the first time, that exposure of human intestinal epithelial cells to carrageenan triggers a distinct inflammatory pathway via activation of Bcl10 with NF-κB activation and upregulation of IL-8 secretion. Since Bcl10 contains a caspase-recruitment domain, similar to that found in NOD2/CARD15 and associated with genetic predisposition to Crohn's disease, the study findings may represent a link between genetic and environmental etiologies of inflammatory bowel disease. Because of the high use of carrageenan as a food additive in the diet, the findings may have clinical significance.

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