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Sample records for normal colonic epithelial

  1. Cell surface glycopeptides from human intestinal epithelial cell lines derived from normal colon and colon adenocarcinomas

    SciTech Connect

    Youakim, A.; Herscovics, A.

    1985-11-01

    The cell surface glycopeptides from an epithelial cell line (CCL 239) derived from normal human colon were compared with those from three cell lines (HCT-8R, HCT-15, and CaCo-2) derived independently from human colonic adenocarcinomas. Cells were incubated with D-(2-TH)mannose or L-(5,6-TH)fucose for 24 h and treated with trypsin to release cell surface components which were then digested exhaustively with Pronase and fractionated on Bio-Gel P-6 before and after treatment with endo-beta-N-acetylglucosaminidase H. The most noticeable difference between the labeled glycopeptides from the tumor and CCL 239 cells was the presence in the former of an endo-beta-N-acetylglucosaminidase H-resistant high molecular weight glycopeptide fraction which was eluted in the void volume of Bio-Gel P-6. This fraction was obtained with both labeled mannose and fucose as precursors. However, acid hydrolysis of this fraction obtained after incubation with (2-TH)mannose revealed that as much as 60-90% of the radioactivity was recovered as fucose. Analysis of the total glycopeptides (cell surface and cell pellet) obtained after incubation with (2-TH)mannose showed that from 40-45% of the radioactivity in the tumor cells and less than 10% of the radioactivity in the CCL 239 cells was recovered as fucose. After incubation of the HCT-8R cells with D-(1,6-TH)glucosamine and L-(1- UC)fucose, strong acid hydrolysis of the labeled glycopeptide fraction excluded from Bio-Gel P-6 produced TH-labeled N-acetylglucosamine and N-acetylgalactosamine.

  2. Liver X receptor ligand cytotoxicity in colon cancer cells and not in normal colon epithelial cells depends on LXRβ subcellular localization.

    PubMed

    Courtaut, Flavie; Derangère, Valentin; Chevriaux, Angélique; Ladoire, Sylvain; Cotte, Alexia K; Arnould, Laurent; Boidot, Romain; Rialland, Mickaël; Ghiringhelli, François; Rébé, Cédric

    2015-09-29

    Increasing evidence indicates that Liver X Receptors (LXRs) have some anticancer properties. We recently demonstrated that LXR ligands induce colon cancer cell pyroptosis through an LXRβ-dependent pathway. In the present study, we showed that human colon cancer cell lines presented differential cytoplasmic localizations of LXRβ. This localization correlated with caspase-1 activation and cell death induction under treatment with LXR ligand. The association of LXRβ with the truncated form of RXRα (t-RXRα) was responsible for the sequestration of LXRβ in the cytoplasm in colon cancer cells. Moreover t-RXRα was not expressed in normal colon epithelial cells. These cells presented a predominantly nuclear localization of LXRβ and were resistant to LXR ligand cytotoxicity. Our results showed that predominant cytoplasmic localization of LXRβ, which occurs in colon cancer cells but not in normal colon epithelial cells, allowed LXR ligand-induced pyroptosis. This study strengthens the hypothesis that LXRβ could be a promising target in cancer therapy.

  3. Parallels between Global Transcriptional Programs of Polarizing Caco-2 Intestinal Epithelial Cells In Vitro and Gene Expression Programs in Normal Colon and Colon Cancer

    PubMed Central

    Sääf, Annika M.; Halbleib, Jennifer M.; Chen, Xin; Yuen, Siu Tsan; Leung, Suet Yi

    2007-01-01

    Posttranslational mechanisms are implicated in the development of epithelial cell polarity, but little is known about the patterns of gene expression and transcriptional regulation during this process. We characterized temporal patterns of gene expression during cell–cell adhesion-initiated polarization of cultured human Caco-2 cells, which develop structural and functional polarity resembling enterocytes in vivo. A distinctive switch in gene expression patterns occurred upon formation of cell–cell contacts. Comparison to gene expression patterns in normal human colon and colon tumors revealed that the pattern in proliferating, nonpolarized Caco-2 cells paralleled patterns seen in human colon cancer in vivo, including expression of genes involved in cell proliferation. The pattern switched in polarized Caco-2 cells to one more closely resembling that in normal colon tissue, indicating that regulation of transcription underlying Caco-2 cell polarization is similar to that during enterocyte differentiation in vivo. Surprisingly, the temporal program of gene expression in polarizing Caco-2 cells involved changes in signaling pathways (e.g., Wnt, Hh, BMP, FGF) in patterns similar to those during migration and differentiation of intestinal epithelial cells in vivo, despite the absence of morphogen gradients and interactions with stromal cells characteristic of enterocyte differentiation in situ. The full data set is available at http://microarray-pubs.stanford.edu/CACO2. PMID:17699589

  4. Effects of a Mediterranean Diet Intervention on Anti- and Pro-Inflammatory Eicosanoids, Epithelial Proliferation, and Nuclear Morphology in Biopsies of Normal Colon Tissue.

    PubMed

    Djuric, Zora; Turgeon, D Kim; Ren, Jianwei; Neilson, Andrew; Plegue, Missy; Waters, Ian G; Chan, Alexander; Askew, Leah M; Ruffin, Mack T; Sen, Ananda; Brenner, Dean E

    2015-01-01

    This randomized trial evaluated the effects of intervention with either a Healthy Eating or a Mediterranean diet on colon biomarkers in 120 healthy individuals at increased colon cancer risk. The hypothesis was that eicosanoids and markers of proliferation would be favorably affected by the Mediterranean diet. Colon epithelial biopsy tissues and blood samples were obtained at baseline and after 6 mo of intervention. Colonic eicosanoid concentrations were evaluated by HPLC-MS-MS, and measures of epithelial proliferation and nuclear morphology were evaluated by image analysis of biopsy sections. There was little change in proinflammatory eicosanoids and in plasma cytokine concentrations with either dietary intervention. There was, however, a 50% increase in colonic prostaglandin E3 (PGE3), which is formed from eicosapentanoic acid, in the Mediterranean arm. Unlike PGE2, PGE3, was not significantly affected by regular use of non-steroidal anti-inflammatory drugs at baseline, and normal weight subjects had significantly higher colon PGE3 than overweight or obese subjects. Increased proliferation in the colon at baseline, by Ki67 labeling, was associated with morphological features that defined smaller nuclei in the epithelial cells, lower colon leukotriene concentrations and higher plasma cytokine concentrations. Dietary intervention had little effect on measures of epithelial proliferation or of nuclear morphology. The increase in PGE3 with a Mediterranean diet indicates that in normal colon, diet might affect protective pathways to a greater extent than proinflammatory and proliferative pathways. Hence, biomarkers from cancer models might not be relevant in a true prevention setting.

  5. The Colonic Microbiome and Epithelial Transcriptome Are Altered in Rats Fed a High-Protein Diet Compared with a Normal-Protein Diet.

    PubMed

    Mu, Chunlong; Yang, Yuxiang; Luo, Zhen; Guan, Leluo; Zhu, Weiyun

    2016-03-01

    A high-protein diet (HPD) can produce hazardous compounds and reduce butyrate-producing bacteria in feces, which may be detrimental to gut health. However, information on whether HPD affects intestinal function is limited. The aim of this study was to determine the impact of an HPD on the microbiota, microbial metabolites, and epithelial transcriptome in the colons of rats. Adult male Wistar rats were fed either a normal-protein diet (20% protein, 56% carbohydrate) or an HPD (45% protein, 30% carbohydrate) for 6 wk (n = 10 rats per group, individually fed). After 6 wk, the colonic microbiome, microbial metabolites, and epithelial transcriptome were determined. Compared with the normal-protein diet, the HPD adversely altered the colonic microbiota by increasing (P < 0.05) Escherichia/Shigella, Enterococcus, Streptococcus, and sulfate-reducing bacteria by 54.9-fold, 31.3-fold, 5.36-fold, and 2.59-fold, respectively. However, the HPD reduced Ruminococcus (8.04-fold), Akkermansia (not detected in HPD group), and Faecalibacterium prausnitzii (3.5-fold) (P < 0.05), which are generally regarded as beneficial bacteria in the colon. Concomitant increases in cadaverine (4.88-fold), spermine (31.2-fold), and sulfide (4.8-fold) (P < 0.05) and a decrease in butyrate (2.16-fold) (P < 0.05) in the HPD rats indicated an evident shift toward the production of unhealthy microbial metabolites. In the colon epithelium of the HPD rats, transcriptome analysis identified an upregulation of genes (P < 0.05) involved in disease pathogenesis; these genes are involved in chemotaxis, the tumor necrosis factor signal process, and apoptosis. The HPD was also associated with a downregulation of many genes (P < 0.05) involved in immunoprotection, such as genes involved in innate immunity, O-linked glycosylation of mucin, and oxidative phosphorylation, suggesting there may be an increased disease risk in these rats. The abundance of Escherichia/Shigella, Enterococcus, and Streptococcus was

  6. ST6Gal-I protein expression is upregulated in human epithelial tumors and correlates with stem cell markers in normal tissues and colon cancer cell lines.

    PubMed

    Swindall, Amanda F; Londoño-Joshi, Angelina I; Schultz, Matthew J; Fineberg, Naomi; Buchsbaum, Donald J; Bellis, Susan L

    2013-04-01

    The ST6Gal-I sialyltransferase adds an α2-6-linked sialic acid to the N-glycans of certain receptors. ST6Gal-I mRNA has been reported to be upregulated in human cancer, but a prior lack of antibodies has limited immunochemical analysis of the ST6Gal-I protein. Here, we show upregulated ST6Gal-I protein in several epithelial cancers, including many colon carcinomas. In normal colon, ST6Gal-I localized selectively to the base of crypts, where stem/progenitor cells are found, and the tissue staining patterns were similar to the established stem cell marker ALDH1. Similarly, ST6Gal-I expression was restricted to basal epidermal layers in skin, another stem/progenitor cell compartment. ST6Gal-I was highly expressed in induced pluripotent stem (iPS) cells, with no detectable expression in the fibroblasts from which iPS cells were derived. On the basis of these observations, we investigated further an association of ST6Gal-I with cancer stem cells (CSC). Selection of irinotecan resistance in colon carcinoma cells led to a greater proportion of CSCs compared with parental cells, as measured by the CSC markers CD133 and ALDH1 activity (Aldefluor). These chemoresistant cells exhibited a corresponding upregulation of ST6Gal-I expression. Conversely, short hairpin RNA (shRNA)-mediated attenuation of ST6Gal-I in colon carcinoma cells with elevated endogenous expression decreased the number of CD133/ALDH1-positive cells present in the cell population. Collectively, our results suggest that ST6Gal-I promotes tumorigenesis and may serve as a regulator of the stem cell phenotype in both normal and cancer cell populations.

  7. ST6Gal-I protein expression is upregulated in human epithelial tumors and correlates with stem cell markers in normal tissues and colon cancer cell lines

    PubMed Central

    Swindall, Amanda F.; Londoño-Joshi, Angelina I.; Schultz, Matthew J.; Fineberg, Naomi; Buchsbaum, Donald J.; Bellis, Susan L.

    2014-01-01

    The ST6Gal-I sialyltransferase adds an α2–6-linked sialic acid to the N-glycans of certain receptors. ST6Gal-I mRNA has been reported to be upregulated in human cancer, but a prior lack of antibodies has limited immunochemical analysis of the ST6Gal-I protein. Here we show upregulated ST6Gal-I protein in several epithelial cancers, including many colon carcinomas. In normal colon, ST6Gal-I localized selectively to the base of crypts, where stem/progenitor cells are found, and the tissue staining patterns were similar to the established stem cell marker ALDH1. Similarly, ST6Gal-I expression was restricted to basal epidermal layers in skin, another stem/progenitor cell compartment. ST6Gal-I was highly expressed in induced pluripotent stem (iPS) cells, with no detectable expression in the fibroblasts from which iPS cells were derived. On the basis of these observations we investigated further an association of ST6Gal-I with cancer stem cells (CSCs). Selection of irinotecan resistance in colon carcinoma cells led to a greater proportion of CSCs compared with parental cells, as measured by the CSC markers CD133 and ALDH1 activity (Aldefluor). These chemoresistant cells exhibited a corresponding upregulation of ST6Gal-I expression. Conversely, shRNA-mediated attenuation of ST6Gal-I in colon carcinoma cells with elevated endogenous expression decreased the number of CD133/ALDH1-positive cells present in the cell population. Collectively, our results suggest that ST6Gal-I promotes tumorigenesis and may serve as a regulator of the stem cell phenotype in both normal and cancer cell populations. PMID:23358684

  8. Cell associated urokinase activity and colonic epithelial cells in health and disease.

    PubMed Central

    Gibson, P R; van de Pol, E; Doe, W F

    1991-01-01

    It is not known if urokinase-type plasminogen activator (uPA) is associated with normal colonic epithelial cells. The aims of this study were to determine if normal colonic epithelial cells have uPA activity and whether this is concentrated at the cell membrane. In addition, the contribution of colonic epithelial cell associated uPA activity to disease related pertubations of mucosal uPA activity were examined. A highly enriched population of colonic epithelial cells was isolated from resected colon or biopsy specimens by an enzymatic technique. uPA activity was measured in cell homogenates by a specific and sensitive colorimetric method and expressed relative to cellular DNA. In two experiments subcellular fractionation of colonic epithelial cells was performed by nitrogen cavitation followed by ultracentrifugation over a linear sucrose gradient. The fractions collected were analysed for uPA and organelle-specific enzyme activities. Normal colonic epithelial cells have cell associated uPA activity (mean (SEM) 5.6 (1.1) IU/mg, n = 18). This colocalised with fractions enriched for leucine-beta-naphthylamidase and 5'-nucleotidase, markers of plasma membrane. uPA activities in epithelial cells from cancerous colons (9.8 (3.1) n = 7) or from mucosa affected by inflammatory bowel disease (3.8 (0.7) n = 15) were not significantly different from normal (paired t test), while that in epithelial cells from greatly inflamed mucosa was similar to that from autologous normal or mildly inflamed areas (4.4 (1.2) v 5.9 (3.6), n = 9). Thus normal colonic epithelial cells have cell associated uPA activity which is concentrated on the plasma membranes, suggesting the presence of uPA receptors. Increased mucosal levels of uPA previously reported in patients with inflammatory bowel disease are not due to increased colonic epithelial cell associated uPA. PMID:1650741

  9. Ursodeoxycholic acid attenuates colonic epithelial secretory function

    PubMed Central

    Kelly, Orlaith B; Mroz, Magdalena S; Ward, Joseph B J; Colliva, Carolina; Scharl, Michael; Pellicciari, Roberto; Gilmer, John F; Fallon, Padraic G; Hofmann, Alan F; Roda, Aldo; Murray, Frank E; Keely, Stephen J

    2013-01-01

    Dihydroxy bile acids, such as chenodeoxycholic acid (CDCA), are well known to promote colonic fluid and electrolyte secretion, thereby causing diarrhoea associated with bile acid malabsorption. However, CDCA is rapidly metabolised by colonic bacteria to ursodeoxycholic acid (UDCA), the effects of which on epithelial transport are poorly characterised. Here, we investigated the role of UDCA in the regulation of colonic epithelial secretion. Cl− secretion was measured across voltage-clamped monolayers of T84 cells and muscle-stripped sections of mouse or human colon. Cell surface biotinylation was used to assess abundance/surface expression of transport proteins. Acute (15 min) treatment of T84 cells with bilateral UDCA attenuated Cl− secretory responses to the Ca2+ and cAMP-dependent secretagogues carbachol (CCh) and forskolin (FSK) to 14.0 ± 3.8 and 40.2 ± 7.4% of controls, respectively (n= 18, P < 0.001). Investigation of the molecular targets involved revealed that UDCA acts by inhibiting Na+/K+-ATPase activity and basolateral K+ channel currents, without altering their cell surface expression. In contrast, intraperitoneal administration of UDCA (25 mg kg−1) to mice enhanced agonist-induced colonic secretory responses, an effect we hypothesised to be due to bacterial metabolism of UDCA to lithocholic acid (LCA). Accordingly, LCA (50–200 μm) enhanced agonist-induced secretory responses in vitro and a metabolically stable UDCA analogue, 6α-methyl-UDCA, exerted anti-secretory actions in vitro and in vivo. In conclusion, UDCA exerts direct anti-secretory actions on colonic epithelial cells and metabolically stable derivatives of the bile acid may offer a new approach for treating intestinal diseases associated with diarrhoea. PMID:23507881

  10. Nuclear microscopy of rat colon epithelial cells

    NASA Astrophysics Data System (ADS)

    Ren, M.; Rajendran, Reshmi; Ng, Mary; Udalagama, Chammika; Rodrigues, Anna E.; Watt, Frank; Jenner, Andrew Michael

    2011-10-01

    Using Nuclear microscopy, we have investigated iron distributions in the colons of Sprague Dawley rats, in order to elucidate heme uptake. Four groups of five Sprague Dawley rats (mean weight 180 g) were fed different purified diets containing either heme diet (2.5% w/w hemoglobin), high fat diet (HFD) (18% w/w fat, 1% w/w cholesterol), 'western' diet (combination of hemoglobin 2.5% and 18% fat, 1% cholesterol) or control diet (7% w/w fat). After 4 weeks, animals were sacrificed by exsanguination after anaesthesia. Thin sections of frozen colon tissue were taken, freeze dried and scanned using nuclear microscopy utilising the techniques PIXE, RBS and STIM. The new data acquisition system (IonDaq) developed in CIBA was used to obtain high resolution images and line scans were used to map the iron distributions across the colon boundaries. The nuclear microscope results indicate that when HFD is given in addition to heme, the iron content of the epithelial cells that line the colon decreases, and the zinc in the smooth muscle wall increases. This implies that the level of heme and fat in diet has an important role in colon health, possibly by influencing epithelial cells directly or changing luminal composition such as bacterial flora or levels of metabolites and cytotoxins.

  11. Epithelial NAIPs protect against colonic tumorigenesis.

    PubMed

    Allam, Ramanjaneyulu; Maillard, Michel H; Tardivel, Aubry; Chennupati, Vijaykumar; Bega, Hristina; Yu, Chi Wang; Velin, Dominique; Schneider, Pascal; Maslowski, Kendle M

    2015-03-09

    NLR family apoptosis inhibitory proteins (NAIPs) belong to both the Nod-like receptor (NLR) and the inhibitor of apoptosis (IAP) families. NAIPs are known to form an inflammasome with NLRC4, but other in vivo functions remain unexplored. Using mice deficient for all NAIP paralogs (Naip1-6(Δ/Δ)), we show that NAIPs are key regulators of colorectal tumorigenesis. Naip1-6(Δ/Δ) mice developed increased colorectal tumors, in an epithelial-intrinsic manner, in a model of colitis-associated cancer. Increased tumorigenesis, however, was not driven by an exacerbated inflammatory response. Instead, Naip1-6(Δ/Δ) mice were protected from severe colitis and displayed increased antiapoptotic and proliferation-related gene expression. Naip1-6(Δ/Δ) mice also displayed increased tumorigenesis in an inflammation-independent model of colorectal cancer. Moreover, Naip1-6(Δ/Δ) mice, but not Nlrc4-null mice, displayed hyper-activation of STAT3 and failed to activate p53 18 h after carcinogen exposure. This suggests that NAIPs protect against tumor initiation in the colon by promoting the removal of carcinogen-elicited epithelium, likely in a NLRC4 inflammasome-independent manner. Collectively, we demonstrate a novel epithelial-intrinsic function of NAIPs in protecting the colonic epithelium against tumorigenesis.

  12. Commentary: acetaldehyde and epithelial-to-mesenchymal transition in colon.

    PubMed

    Rao, Radhakrishna K

    2014-02-01

    Elamin and colleagues in this issue report that acetaldehyde activates Snail, a transcription factor involved in epithelial-to-mesenchymal transition, in an intestinal epithelium. Snail mediates acetaldehyde-induced tight junction disruption and increase in paracellular permeability. Results of this study and other previous studies raise several important questions. This commentary addresses these questions by discussing the acetaldehyde concentration in colon, disruption of epical junctional complexes in the intestinal epithelium by acetaldehyde, and the consequence of long-term exposure to acetaldehyde on colonic epithelial regeneration, carcinogenesis, and metastases. The precise role of acetaldehyde in colonic epithelial modifications and promotion of colorectal cancers still remains to be understood.

  13. Hydrogen sulfide lowers proliferation and induces protective autophagy in colon epithelial cells.

    PubMed

    Wu, Ya C; Wang, Xiao J; Yu, Le; Chan, Francis K L; Cheng, Alfred S L; Yu, Jun; Sung, Joseph J Y; Wu, William K K; Cho, Chi H

    2012-01-01

    Hydrogen sulfide (H(2)S) is a gaseous bacterial metabolite that reaches high levels in the large intestine. In the present study, the effect of H(2)S on the proliferation of normal and cancerous colon epithelial cells was investigated. An immortalized colon epithelial cell line (YAMC) and a panel of colon cancer cell lines (HT-29, SW1116, HCT116) were exposed to H(2)S at concentrations similar to those found in the human colon. H(2)S inhibited normal and cancerous colon epithelial cell proliferation as measured by MTT assay. The anti-mitogenic effect of H(2)S was accompanied by G(1)-phase cell cycle arrest and the induction of the cyclin-dependent kinase inhibitor p21(Cip). Moreover, exposure to H(2)S led to features characteristic of autophagy, including increased formation of LC3B(+) autophagic vacuoles and acidic vesicular organelles as determined by immunofluorescence and acridine orange staining, respectively. Abolition of autophagy by RNA interference targeting Vps34 or Atg7 enhanced the anti-proliferative effect of H(2)S. Further mechanistic investigation revealed that H(2)S stimulated the phosphorylation of AMP-activated protein kinase (AMPK) and inhibited the phosphorylation of mammalian target of rapamycin (mTOR) and S6 kinase. Inhibition of AMPK significantly reversed H(2)S-induced autophagy and inhibition of cell proliferation. Collectively, we demonstrate that H(2)S inhibits colon epithelial cell proliferation and induces protective autophagy via the AMPK pathway.

  14. Potentiation of Colon Cancer Susceptibility in Mice by Colonic Epithelial PPAR-δ/β Overexpression

    PubMed Central

    2014-01-01

    Background The nuclear receptor peroxisome proliferator-activated receptor-δ/β (PPAR-d) is upregulated in human colorectal cancers, but its role in colonic tumorigenesis remains controversial. Methods We generated a novel mouse model of intestinally targeted PPAR-d overexpression to simulate PPAR-d upregulation in human colon carcinogenesis. Colon-specific PPAR-d overexpression was confirmed by real-time reverse transcription polymerase chain reaction, immunoblotting, and activity assays. Mice with and without targeted PPAR-d overexpression were tested for azoxymethane (AOM)–induced colonic tumorigenesis. Mouse whole-genome transcriptome microarray analyses were performed to identify PPAR-d target genes to promote tumorigenesis. We used linear models to test for PPAR-d overexpression trend effects on tumor multiplicity. All statistical tests were two-sided. Results Targeted PPAR-d overexpression markedly increased colonic tumor incidence (from 0 of 10 wild-type [WT] littermate mice to 9 of 10 mice [P < .001] in 2 FVB/N background mouse lines [villin-PPAR-d-1 and villin-PPAR-d-2] at a 5-mg/kg AOM dose) and multiplicity (number of tumors per mouse per mg/kg dose of AOM increased from 0.47 [95% confidence interval [CI] = 0.22 to 0.72] for the WT littermates to 2.15 [95% CI = 1.90 to 2.40] [P < .001] for the villin-PPAR-d-1 mice and from 0.44 [95% CI = 0.09 to 0.79] for the WT littermates to 1.91 [95% CI = 1.57 to 2.25] [P < .001] for the villin-PPAR-d-2 mice). PPAR-d overexpression reversed resistance to AOM-induced colonic tumorigenesis in C57BL/6 mice. PPAR-d overexpression modulated expression of several novel PPAR-d target genes in normal-appearing colonic epithelial cells of mice with PPAR-d overexpression in a pattern that matched the changes in colonic tumors. Conclusions Our finding that PPAR-d upregulation profoundly enhances susceptibility to colonic tumorigenesis should impact the development of strategies of molecularly targeting PPAR-d in cancer and

  15. Small molecule and RNAi induced phenotype transition of expanded and primary colonic epithelial cells.

    PubMed

    Sharbati, Jutta; Hanisch, Carlos; Pieper, Robert; Einspanier, Ralf; Sharbati, Soroush

    2015-07-30

    Recent progress in mammalian intestinal epithelial cell culture led to novel concepts of tissue modeling. Especially the development of phenotypically stable cell lines from individual animals enables an investigation of distinct intestinal loci and disease states. We here report primary and prolonged culture of normal porcine epithelial cells from colon for cell line development. In addition, a novel primary three-dimensional intestinal culture system is presented, which generated organoids composed of a highly polarized epithelial layer lining a core of subepithelial tissue. Cellular characterization of monolayer cell lines revealed epithelial identity and pointed to a proliferative crypt cell phenotype. We evaluated both RNAi and chemical approaches to induce epithelial differentiation in generated cell lines by targeting promoters of epithelial to mesenchymal transition (EMT). By in silico prediction and ectopic expression, miR-147b was proven to be a potent trigger of intestinal epithelial cell differentiation. Our results outline an approach to generate phenotypically stable cell lines expanded from primary colonic epithelial cultures and demonstrate the relevance of miR-147b and chemical inhibitors for promoting epithelial differentiation features.

  16. Small molecule and RNAi induced phenotype transition of expanded and primary colonic epithelial cells

    PubMed Central

    Sharbati, Jutta; Hanisch, Carlos; Pieper, Robert; Einspanier, Ralf; Sharbati, Soroush

    2015-01-01

    Recent progress in mammalian intestinal epithelial cell culture led to novel concepts of tissue modeling. Especially the development of phenotypically stable cell lines from individual animals enables an investigation of distinct intestinal loci and disease states. We here report primary and prolonged culture of normal porcine epithelial cells from colon for cell line development. In addition, a novel primary three-dimensional intestinal culture system is presented, which generated organoids composed of a highly polarized epithelial layer lining a core of subepithelial tissue. Cellular characterization of monolayer cell lines revealed epithelial identity and pointed to a proliferative crypt cell phenotype. We evaluated both RNAi and chemical approaches to induce epithelial differentiation in generated cell lines by targeting promoters of epithelial to mesenchymal transition (EMT). By in silico prediction and ectopic expression, miR-147b was proven to be a potent trigger of intestinal epithelial cell differentiation. Our results outline an approach to generate phenotypically stable cell lines expanded from primary colonic epithelial cultures and demonstrate the relevance of miR-147b and chemical inhibitors for promoting epithelial differentiation features. PMID:26223582

  17. Epithelial cell adhesion and gastrointestinal colonization of Lactobacillus in poultry.

    PubMed

    Spivey, Megan A; Dunn-Horrocks, Sadie L; Duong, Tri

    2014-11-01

    Administration of probiotic Lactobacillus cultures is an important alternative to the use of antibiotic growth promoters and has been demonstrated to improve animal health, growth performance, and preharvest food safety in poultry production. Whereas gastrointestinal colonization is thought to be critical to their probiotic functionality, factors important to Lactobacillus colonization in chickens are not well understood. In this study we investigate epithelial cell adhesion in vitro and colonization of Lactobacillusin vivo in broiler chickens. Adhesion of Lactobacillus cultures to epithelial cells was evaluated using the chicken LMH cell line. Lactobacillus cultures were able adhere effectively to LMH cells relative to Bacillus subtilis and Salmonella Typhimurium. Epithelial cell adhesion was similar for Lactobacillus crispatus TDCC 75, L. cristpatus TDCC 76, and Lactobacillus gallinarum TDCC 77, and all 3 were more adherent than L. gallinarum TDCC 78. However, when colonization was evaluated in the ileum and cecum of broiler chicks, L. crispatus TDCC 75 and L. gallinarum TDCC 77 were more persistent than L. crispatus TDCC 76 and L. gallinarum TDCC 78. The reduction of growth in medium supplemented with oxgal was greater for L. gallinarum TDCC 78 than L. gallinarum TDCC 77, suggesting that whereas adhesion was similar for the 2 strains, the difference in colonization between L. gallinarum strains may be due in part to their bile sensitivity. This study demonstrates that whereas adhesion to epithelial cells may be important in predicting gastrointestinal colonization, other factors including bile tolerance may also contribute to the colonization of Lactobacillus in poultry. Additionally, the chicken LMH cell line is expected to provide a platform for investigating mechanisms of Lactobacillus adhesion to epithelial tissue and evaluating the probiotic potential Lactobacillus in poultry.

  18. Normal morphogenesis of epithelial tissues and progression of epithelial tumors

    PubMed Central

    Wang, Chun-Chao; Jamal, Leen; Janes, Kevin A.

    2011-01-01

    Epithelial cells organize into various tissue architectures that largely maintain their structure throughout the life of an organism. For decades, the morphogenesis of epithelial tissues has fascinated scientists at the interface of cell, developmental, and molecular biology. Systems biology offers ways to combine knowledge from these disciplines by building integrative models that are quantitative and predictive. Can such models be useful for gaining a deeper understanding of epithelial morphogenesis? Here, we take inventory of some recurring themes in epithelial morphogenesis that systems approaches could strive to capture. Predictive understanding of morphogenesis at the systems level would prove especially valuable for diseases such as cancer, where epithelial tissue architecture is profoundly disrupted. PMID:21898857

  19. Normal morphogenesis of epithelial tissues and progression of epithelial tumors.

    PubMed

    Wang, Chun-Chao; Jamal, Leen; Janes, Kevin A

    2012-01-01

    Epithelial cells organize into various tissue architectures that largely maintain their structure throughout the life of an organism. For decades, the morphogenesis of epithelial tissues has fascinated scientists at the interface of cell, developmental, and molecular biology. Systems biology offers ways to combine knowledge from these disciplines by building integrative models that are quantitative and predictive. Can such models be useful for gaining a deeper understanding of epithelial morphogenesis? Here, we take inventory of some recurring themes in epithelial morphogenesis that systems approaches could strive to capture. Predictive understanding of morphogenesis at the systems level would prove especially valuable for diseases such as cancer, where epithelial tissue architecture is profoundly disrupted.

  20. Rebamipide promotes healing of colonic ulceration through enhanced epithelial restitution.

    PubMed

    Takagi, Tomohisa; Naito, Yuji; Uchiyama, Kazuhiko; Okuda, Toshimitsu; Mizushima, Katsura; Suzuki, Takahiro; Handa, Osamu; Ishikawa, Takeshi; Yagi, Nobuaki; Kokura, Satoshi; Ichikawa, Hiroshi; Yoshikawa, Toshikazu

    2011-09-07

    To investigate the efficacy of rebamipide in a rat model of colitis and restitution of intestinal epithelial cells in vitro. Acute colitis was induced with trinitrobenzene sulfonic acid (TNBS) in male Wistar rats. Rats received intrarectal rebamipide treatment daily starting on day 7 and were sacrificed on day 14 after TNBS administration. The distal colon was removed to evaluate the various parameters of inflammation. Moreover, wound healing assays were used to determine the enhanced restitution of rat intestinal epithelial (RIE) cells treated with rebamipide. Intracolonic administration of rebamipide accelerated TNBS-induced ulcer healing. Increases in the wet weight of the colon after TNBS administration were significantly inhibited by rebamipide. The wound assay revealed that rebamipide enhanced the migration of RIE cells through phosphorylation of extracellular signal-regulated kinase (ERK) and activation of Rho kinase. Rebamipide enema healed intestinal injury by enhancing restitution of RIE cells, via ERK activation. Rebamipide might be a novel therapeutic approach for inflammatory bowel disease.

  1. Serotonin disturbs colon epithelial tolerance of commensal E. coli by increasing NOX2-derived superoxide.

    PubMed

    Banskota, Suhrid; Regmi, Sushil Chandra; Gautam, Jaya; Gurung, Pallavi; Lee, Yu-Jeong; Ku, Sae Kwang; Lee, Jin-Hyung; Lee, Jintae; Chang, Hyeun Wook; Park, Sang Joon; Kim, Jung-Ae

    2017-02-17

    Adherent-invasive E. coli colonization and Toll-like receptor (TLR) expression are increased in the gut of inflammatory bowel disease (IBD) patients. However, the underlying mechanism of such changes has not been determined. In the current study, it was examined whether gut serotonin (5-hydroxytryptamine, 5-HT) can induce adherent-invasive E. coli colonization and increase TLR expression. In a co-culture system, commensal E. coli strain (BW25113, BW) adhered minimally to colon epithelial cells, but this was significantly enhanced by 5-HT to the level of a pathogenic strain (EDL933). Without inducing bacterial virulence, such as, biofilm formation, 5-HT enhanced BW-induced signaling in colon epithelial cells, that is, NADPH oxidase (NOX)-dependent superoxide production, the up-regulations of IL-8, TLR2, TLR4, and ICAM-1, and the down-regulations of E-cadherin and claudin-2. In a manner commensurate with these gene modulations, BW induced an increase in NF-κB and a decrease in GATA reporter signals in colon epithelial cells. However, 5-HT-enhanced BW adhesion and colon epithelial responses were blocked by knock-down of NOX2, TLR2, or TLR4. In normal mice, 5-HT induced the invasion of BW into gut submucosa, and the observed molecular changes were similar to those observed in vitro, except for significant increases in TNFα and IL-1β, and resulted in death. In dextran sulfate sodium-induced colitis mice (an IBD disease model), in which colonic 5-HT levels were markedly elevated, BW administration induced death in along with large amount of BW invasion into colon submucosa, and time to death was negatively related to the amount of BW injected. Taken together, our results demonstrate that 5-HT induces the invasion of commensal E. coli into gut submucosa by amplifying commensal bacteria-induced epithelial signaling (superoxide production and the inductions of NOX2 and TLR2/TLR4). The authors suggest that these changes may constitute the molecular basis for the

  2. TACE inhibition amplifies TNF-alpha-mediated colonic epithelial barrier disruption.

    PubMed

    Fréour, Thomas; Jarry, Anne; Bach-Ngohou, Kalyane; Dejoie, Thomas; Bou-Hanna, Chantal; Denis, Marc G; Mosnier, Jean-François; Laboisse, Christian L; Masson, Damien

    2009-01-01

    Inflammatory bowel diseases (IBD) are characterized by tumor necrosis factor alpha (TNF-alpha)-mediated epithelial barrier disruption. TNF-alpha production and the bioavailability of its receptors on the cell surface are regulated by TACE (TNF-alpha converting enzyme), a pleiotropic metalloprotease also known as ADAM17, and its specific inhibitor TIMP3. We therefore examined ADAM17 and TIMP3 expression in human intestinal epithelial cells (IEC) using immunohistochemistry on tissue microarrays and real-time PCR on preparations of IEC isolated from human normal and IBD colon. The effects of TACE inhibition by TIMP3 or a pharmacological inhibitor were assessed in inflammatory conditions on a TIMP3-deficient colonic cell line HT29-Cl.16E. Both TACE and TIMP3 were found to be constitutively expressed by intestinal epithelial cells in the normal and inflammatory human intestinal barrier. In the TIMP3-deficient cell line, the addition of recombinant human TIMP3 or of Tapi-2, a pharmacological ADAM17 inhibitor, i) sensitized the cells to TNF-alpha-mediated hyperpermeability, ii) down-regulated tight junction-associated protein expression and iii) inhibited TNFRI shedding. In conclusion, our data showed that TACE and TIMP3 were co-expressed in the human intestinal barrier and that TACE inhibition, either physiologically or pharmacologically, amplified TNF-alpha-mediated hyperpermeability. TIMP3 could thus play a major role in inflammatory conditions by creating an autocrine effect leading to amplified epithelial barrier hyperpermeability.

  3. Rebamipide promotes healing of colonic ulceration through enhanced epithelial restitution

    PubMed Central

    Takagi, Tomohisa; Naito, Yuji; Uchiyama, Kazuhiko; Okuda, Toshimitsu; Mizushima, Katsura; Suzuki, Takahiro; Handa, Osamu; Ishikawa, Takeshi; Yagi, Nobuaki; Kokura, Satoshi; Ichikawa, Hiroshi; Yoshikawa, Toshikazu

    2011-01-01

    AIM: To investigate the efficacy of rebamipide in a rat model of colitis and restitution of intestinal epithelial cells in vitro. METHODS: Acute colitis was induced with trinitrobenzene sulfonic acid (TNBS) in male Wistar rats. Rats received intrarectal rebamipide treatment daily starting on day 7 and were sacrificed on day 14 after TNBS administration. The distal colon was removed to evaluate the various parameters of inflammation. Moreover, wound healing assays were used to determine the enhanced restitution of rat intestinal epithelial (RIE) cells treated with rebamipide. RESULTS: Intracolonic administration of rebamipide accelerated TNBS-induced ulcer healing. Increases in the wet weight of the colon after TNBS administration were significantly inhibited by rebamipide. The wound assay revealed that rebamipide enhanced the migration of RIE cells through phosphorylation of extracellular signal-regulated kinase (ERK) and activation of Rho kinase. CONCLUSION: Rebamipide enema healed intestinal injury by enhancing restitution of RIE cells, via ERK activation. Rebamipide might be a novel therapeutic approach for inflammatory bowel disease. PMID:21987622

  4. Characterization of colonic dendritic cells in normal and colitic mice.

    PubMed

    Cruickshank, Sheena M; English, Nicholas R; Felsburg, Peter J; Carding, Simon R

    2005-10-28

    Recent studies demonstrating the direct involvement of dendritic cells (DC) in the activation of pathogenic T cells in animal models of inflammatory bowel disease identify DC as important antigen presenting cells in the colon. However, very little is known about the properties of colonic DC. Using immunohistochemistry, electron microscopy and flow cytometry we have characterized and compared colonic DC in the colon of healthy animals and interleukin-2-deficient (IL2(-/-)) mice that develop colitis. In the healthy colon, DC resided within the lamina propria and in close association with the basement membrane of colonic villi. Type 1 myeloid (CD11c(+), CD11b(+), B220(-), CD8alpha(-)) DC made up the largest (40-45%) population and all DC expressed low levels of CD80, CD86, and CD40, and had high endocytic activity consistent with an immature phenotype. In colitic IL2(-/-) mice, colonic DC numbers increased four- to five-fold and were localized within the epithelial layer and within aggregates of T and B cells. They were also many more DC in mesenteric lymph nodes (MLN). The majority (>85%) of DC in the colon and MLN of IL2(-/-) mice were type 1 myeloid, and expressed high levels of MHC class II, CD80, CD86, CD40, DEC 205, and CCR5 molecules and were of low endocytic activity consistent with mature DC. These findings demonstrate striking changes in the number, distribution and phenotype of DC in the inflamed colon. Their intimate association with lymphocytes in the colon and draining lymph nodes suggest that they may contribute directly to the ongoing inflammation in the colon.

  5. Characterization of colonic dendritic cells in normal and colitic mice

    PubMed Central

    Cruickshank, Sheena M; English, Nicholas R; Felsburg, Peter J; Carding, Simon R

    2005-01-01

    AIM: Recent studies demonstrating the direct involvement of dendritic cells (DC) in the activation of pathogenic T cells in animal models of inflammatory bowel disease identify DC as important antigen presenting cells in the colon. However, very little is known about the properties of colonic DC. METHODS: Using immunohistochemistry, electron microscopy and flow cytometry we have characterized and compared colonic DC in the colon of healthy animals and interleukin-2-deficient (IL2-/-) mice that develop colitis. RESULTS: In the healthy colon, DC resided within the lamina propria and in close association with the basement membrane of colonic villi. Type 1 myeloid (CD11c+, CD11b+, B220-, CD8α-) DC made up the largest (40-45%) population and all DC expressed low levels of CD80, CD86, and CD40, and had high endocytic activity consistent with an immature phenotype. In colitic IL2-/- mice, colonic DC numbers increased four- to five-fold and were localized within the epithelial layer and within aggregates of T and B cells. They were also many more DC in mesenteric lymph nodes (MLN). The majority (>85%) of DC in the colon and MLN of IL2-/- mice were type 1 myeloid, and expressed high levels of MHC class II, CD80, CD86, CD40, DEC 205, and CCR5 molecules and were of low endocytic activity consistent with mature DC. CONCLUSION: These findings demonstrate striking changes in the number, distribution and phenotype of DC in the inflamed colon. Their intimate association with lymphocytes in the colon and draining lymph nodes suggest that they may contribute directly to the ongoing inflammation in the colon. PMID:16419163

  6. The Toll-interleukin-1 receptor member SIGIRR regulates colonic epithelial homeostasis, inflammation, and tumorigenesis.

    PubMed

    Xiao, Hui; Gulen, Muhammet Fatih; Qin, Jinzhong; Yao, Jianhong; Bulek, Katarzyna; Kish, Danielle; Altuntas, Cengiz Zubeyir; Wald, David; Ma, Caixia; Zhou, Hang; Tuohy, Vincent K; Fairchild, Robert L; de la Motte, Carol; Cua, Daniel; Vallance, Bruce A; Li, Xiaoxia

    2007-04-01

    Despite constant contact with the large population of commensal bacteria, the colonic mucosa is normally hyporesponsive to these potentially proinflammatory signals. Here we report that the single immunoglobulin IL-1 receptor-related molecule (SIGIRR), a negative regulator for Toll-IL-1R signaling, plays a critical role in gut homeostasis, intestinal inflammation, and colitis-associated tumorigenesis by maintaining the microbial tolerance of the colonic epithelium. SIGIRR-deficient (Sigirr(-/-)) colonic epithelial cells displayed commensal bacteria-dependent homeostatic defects, as shown by constitutive upregulation of inflammatory genes, increased inflammatory responses to dextran sulfate sodium (DSS) challenge, and increased Azoxymethane (AOM)+DSS-induced colitis-associated tumorigenesis. Gut epithelium-specific expression of the SIGIRR transgene in the SIGIRR-deficient background reduced the cell survival of the SIGIRR-deficient colon epithelium, abrogated the hypersensitivity of the Sigirr(-/-) mice to DSS-induced colitis, and reduced AOM+DSS-induced tumorigenesis. Taken together, our results indicate that epithelium-derived SIGIRR is critical in controlling the homeostasis and innate immune responses of the colon to enteric microflora.

  7. Phenotypic plasticity in normal breast derived epithelial cells

    PubMed Central

    2014-01-01

    Background Normal, healthy human breast tissue from a variety of volunteer donors has become available for research thanks to the establishment of the Susan G. Komen for the Cure® Tissue Bank at the IU Simon Cancer Center (KTB). Multiple epithelial (K-HME) and stromal cells (K-HMS) were established from the donated tissue. Explant culture was utilized to isolate the cells from pieces of breast tissue. Selective media and trypsinization were employed to select either epithelial cells or stromal cells. The primary, non-transformed epithelial cells, the focus of this study, were characterized by immunohistochemistry, flow cytometry, and in vitro cell culture. Results All of the primary, non-transformed epithelial cells tested have the ability to differentiate in vitro into a variety of cell types when plated in or on biologic matrices. Cells identified include stratified squamous epithelial, osteoclasts, chondrocytes, adipocytes, neural progenitors/neurons, immature muscle and melanocytes. The cells also express markers of embryonic stem cells. Conclusions The cell culture conditions employed select an epithelial cell that is pluri/multipotent. The plasticity of the epithelial cells developed mimics that seen in metaplastic carcinoma of the breast (MCB), a subtype of triple negative breast cancer; and may provide clues to the origin of this particularly aggressive type of breast cancer. The KTB is a unique biorepository, and the normal breast epithelial cells isolated from donated tissue have significant potential as new research tools. PMID:24915897

  8. A Nasal Epithelial Receptor for Staphylococcus aureus WTA Governs Adhesion to Epithelial Cells and Modulates Nasal Colonization

    PubMed Central

    Faulstich, Manuela; Grau, Timo; Severin, Yannik; Unger, Clemens; Hoffmann, Wolfgang H.; Rudel, Thomas; Autenrieth, Ingo B.; Weidenmaier, Christopher

    2014-01-01

    Nasal colonization is a major risk factor for S. aureus infections. The mechanisms responsible for colonization are still not well understood and involve several factors on the host and the bacterial side. One key factor is the cell wall teichoic acid (WTA) of S. aureus, which governs direct interactions with nasal epithelial surfaces. We report here the first receptor for the cell wall glycopolymer WTA on nasal epithelial cells. In several assay systems this type F-scavenger receptor, termed SREC-I, bound WTA in a charge dependent manner and mediated adhesion to nasal epithelial cells in vitro. The impact of WTA and SREC-I interaction on epithelial adhesion was especially pronounced under shear stress, which resembles the conditions found in the nasal cavity. Most importantly, we demonstrate here a key role of the WTA-receptor interaction in a cotton rat model of nasal colonization. When we inhibited WTA mediated adhesion with a SREC-I antibody, nasal colonization in the animal model was strongly reduced at the early onset of colonization. More importantly, colonization stayed low over an extended period of 6 days. Therefore we propose targeting of this glycopolymer-receptor interaction as a novel strategy to prevent or control S. aureus nasal colonization. PMID:24788600

  9. Epithelial Hypoxia-Inducible Factor 2α Facilitates the Progression of Colon Tumors through Recruiting Neutrophils

    PubMed Central

    Triner, Daniel; Xue, Xiang; Schwartz, Andrew J.; Jung, Inkyung; Colacino, Justin A.

    2016-01-01

    ABSTRACT Inflammation is a significant risk factor for colon cancer. Recent work has demonstrated essential roles for several infiltrating immune populations in the metaplastic progression following inflammation. Hypoxia and stabilization of hypoxia-inducible factors (HIFs) are hallmark features of inflammation and solid tumors. Previously, we demonstrated an important role for tumor epithelial HIF-2α in colon tumors; however, the function of epithelial HIF-2α as a critical link in the progression of inflammation to cancer has not been elucidated. In colitis-associated colon cancer models, epithelial HIF-2α was essential in tumor growth. Concurrently, epithelial disruption of HIF-2α significantly decreased neutrophils in the colon tumor microenvironment. Intestinal epithelial HIF-2α-overexpressing mice demonstrated that neutrophil recruitment was a direct response to increased epithelial HIF-2α signaling. High-throughput RNA sequencing (RNA-seq) analysis of HIF-2α-overexpressing mice in conjunction with data mining from the Cancer Genome Atlas showed that the neutrophil chemokine CXCL1 gene was highly upregulated in colon tumor epithelium in a HIF-2α-dependent manner. Using selective peptide inhibitors of the CXCL1-CXCR2 signaling axis identified HIF-2α-dependent neutrophil recruitment as an essential mechanism to increase colon carcinogenesis. These studies demonstrate that HIF-2α is a novel regulator of neutrophil recruitment to colon tumors and that it is essential in shaping the protumorigenic inflammatory microenvironment in colon cancer. PMID:27956697

  10. Epithelial response to a high-protein diet in rat colon.

    PubMed

    Beaumont, Martin; Andriamihaja, Mireille; Armand, Lucie; Grauso, Marta; Jaffrézic, Florence; Laloë, Denis; Moroldo, Marco; Davila, Anne-Marie; Tomé, Daniel; Blachier, François; Lan, Annaïg

    2017-01-31

    High-protein diets (HPD) alter the large intestine microbiota composition in association with a metabolic shift towards protein degradation. Some amino acid-derived metabolites produced by the colon bacteria are beneficial for the mucosa while others are deleterious at high concentrations. The aim of the present work was to define the colonic epithelial response to an HPD. Transcriptome profiling was performed on colonocytes of rats fed an HPD or an isocaloric normal-protein diet (NPD) for 2 weeks. The HPD downregulated the expression of genes notably implicated in pathways related to cellular metabolism, NF-κB signaling, DNA repair, glutathione metabolism and cellular adhesion in colonocytes. In contrast, the HPD upregulated the expression of genes related to cell proliferation and chemical barrier function. These changes at the mRNA level in colonocytes were not associated with detrimental effects of the HPD on DNA integrity (comet assay), epithelium renewal (quantification of proliferation and apoptosis markers by immunohistochemistry and western blot) and colonic barrier integrity (Ussing chamber experiments). The modifications of the luminal environment after an HPD were associated with maintenance of the colonic homeostasis that might be the result of adaptive processes in the epithelium related to the observed transcriptional regulations.

  11. In colon cancer, normal colon tissue and blood cells have altered telomere lengths.

    PubMed

    Valls-Bautista, Cristina; Piñol-Felis, Carme; Reñé-Espinet, Josep M; Buenestado-García, Juan; Viñas-Salas, Joan

    2015-06-01

    Telomere length (TL) shortened occurs in colorectal carcinogenetic process. Our objective is to determine if it is only a local fact or there are alterations in normal colon cells and in other body cells. TL of tumoral and normal mucosa and leukocytes of 40 patients operated of colorectal cancer (CRC) and 40 control patients with normal colonoscopy were measured by Southern-blot. Groups were matched by the same localization as tumors, sex, and age. In CRC patients, TRFL (Telomere Repeat Factor Length) leukocytes mean was 8.84 kpb, normal colonic mucosa 7.97 kpb, and tumoral mucosa 7.33 kpb (P < 0.001). In the 40 normal control patients, mean TRFL of colonic mucosa was 7.76 kpb, while in blood cells was 7.01 kpb (P < 0.001). We observed an inverse correlation between leukocytes TRFL and age (r(2)  = 0.17, P = 0.008). Mucosa TRFL correlates significantly with patient's age (r(2)  = 0.138, P = 0.018). TRFL of controls colonic mucosa correlates with TRFL of their blood cells (r(2)  = 0.354, P < 0.001). Normal colonic mucosa and leukocytes in CCR patients presents telomere altered in respect to normal patients. Telomere length in normal leukocytes could be an initial marker for colorectal cancer. © 2015 Wiley Periodicals, Inc.

  12. A link between lipid metabolism and epithelial-mesenchymal transition provides a target for colon cancer therapy.

    PubMed

    Sánchez-Martínez, Ruth; Cruz-Gil, Silvia; Gómez de Cedrón, Marta; Álvarez-Fernández, Mónica; Vargas, Teodoro; Molina, Susana; García, Belén; Herranz, Jesús; Moreno-Rubio, Juan; Reglero, Guillermo; Pérez-Moreno, Mirna; Feliu, Jaime; Malumbres, Marcos; Ramírez de Molina, Ana

    2015-11-17

    The alterations in carbohydrate metabolism that fuel tumor growth have been extensively studied. However, other metabolic pathways involved in malignant progression, demand further understanding. Here we describe a metabolic acyl-CoA synthetase/stearoyl-CoA desaturase ACSL/SCD network causing an epithelial-mesenchymal transition (EMT) program that promotes migration and invasion of colon cancer cells. The mesenchymal phenotype produced upon overexpression of these enzymes is reverted through reactivation of AMPK signaling. Furthermore, this network expression correlates with poorer clinical outcome of stage-II colon cancer patients. Finally, combined treatment with chemical inhibitors of ACSL/SCD selectively decreases cancer cell viability without reducing normal cells viability. Thus, ACSL/SCD network stimulates colon cancer progression through conferring increased energetic capacity and invasive and migratory properties to cancer cells, and might represent a new therapeutic opportunity for colon cancer treatment.

  13. IL1{beta}-mediated Stromal COX-2 signaling mediates proliferation and invasiveness of colonic epithelial cancer cells

    SciTech Connect

    Zhu, Yingting; Zhu, Min; Lance, Peter

    2012-11-15

    COX-2 is a major inflammatory mediator implicated in colorectal inflammation and cancer. However, the exact origin and role of COX-2 on colorectal inflammation and carcinogenesis are still not well defined. Recently, we reported that COX-2 and iNOS signalings interact in colonic CCD18Co fibroblasts. In this article, we investigated whether activation of COX-2 signaling by IL1{beta} in primary colonic fibroblasts obtained from normal and cancer patients play a critical role in regulation of proliferation and invasiveness of human colonic epithelial cancer cells. Our results demonstrated that COX-2 level was significantly higher in cancer associated fibroblasts than that in normal fibroblasts with or without stimulation of IL-1{beta}, a powerful stimulator of COX-2. Using in vitro assays for estimating proliferative and invasive potential, we discovered that the proliferation and invasiveness of the epithelial cancer cells were much greater when the cells were co-cultured with cancer associated fibroblasts than with normal fibroblasts, with or without stimulation of IL1{beta}. Further analysis indicated that the major COX-2 product, prostaglandin E{sub 2}, directly enhanced proliferation and invasiveness of the epithelial cancer cells in the absence of fibroblasts. Moreover, a selective COX-2 inhibitor, NS-398, blocked the proliferative and invasive effect of both normal and cancer associate fibroblasts on the epithelial cancer cells, with or without stimulation of IL-1{beta}. Those results indicate that activation of COX-2 signaling in the fibroblasts plays a major role in promoting proliferation and invasiveness of the epithelial cancer cells. In this process, PKC is involved in the activation of COX-2 signaling induced by IL-1{beta} in the fibroblasts.

  14. Red meat and colon cancer: dietary haem-induced colonic cytotoxicity and epithelial hyperproliferation are inhibited by calcium.

    PubMed

    Sesink, A L; Termont, D S; Kleibeuker, J H; Van der Meer, R

    2001-10-01

    High intake of red meat is associated with increased colon cancer risk. We have shown earlier that this may be due to the high haem content of red meat, because dietary haem increased cytolytic activity of faecal water and colonic epithelial proliferation. Dietary calcium inhibits diet-induced epithelial hyperproliferation. Furthermore, it has been shown that supplemental calcium inhibited the recurrence of colorectal adenomas. Therefore, we studied whether dietary calcium phosphate can exert its protective effects by inhibiting the deleterious effects of haem. In vitro, calcium phosphate precipitated haem and inhibited the haem-induced cytotoxicity. Subsequently, rats were fed diets, differing in haem (0 or 1.3 micromol/g) and calcium phosphate content only (20 or 180 micromol/g). Faeces were collected for biochemical analyses. Cytolytic activity of faecal water was determined from the degree of lysis of erythrocytes by faecal water. Colonic epithelial proliferation was measured in vivo using [(3)H]thymidine incorporation. In rats fed low calcium diets, dietary haem increased cytolytic activity of faecal water (98 +/- 1 versus 1 +/- 1%, P < 0.001) and the concentration of cations in faeces (964 +/- 31 versus 254 +/- 20 micromol/g), when compared with controls. This indicates that dietary haem increased colonic mucosal exposure to luminal irritants. Colonic epithelial proliferation was increased compared with controls (70 +/- 4 versus 48 +/- 8 d.p.m./microg DNA, P < 0.001). This was accompanied by metabolism of the ingested haem and solubilization of haem compounds in the faecal water. A high calcium diet largely prevented this metabolism and solubilization. It also inhibited the haem-induced cytolytic activity of faecal water and increase in faecal cation concentration. In accordance, the haem-induced colonic epithelial hyperproliferation was prevented. We therefore suggest that dietary calcium phosphate acts as a chemopreventive agent in colon carcinogenesis by

  15. Colorectal Cancers Mimic Structural Organization of Normal Colonic Crypts

    PubMed Central

    Cernat, Laura; Blaj, Cristina; Jackstadt, Rene; Brandl, Lydia; Engel, Jutta; Hermeking, Heiko; Jung, Andreas; Kirchner, Thomas; Horst, David

    2014-01-01

    Colonic crypts are stereotypical structures with distinct stem cell, proliferating, and differentiating compartments. Colorectal cancers derive from colonic crypt epithelia but, in contrast, form morphologically disarrayed glands. In this study, we investigated to which extent colorectal cancers phenocopy colonic crypt architecture and thus preserve structural organization of the normal intestinal epithelium. A subset of colon cancers showed crypt-like compartments with high WNT activity and nuclear β-Catenin at the leading tumor edge, adjacent proliferation, and enhanced Cytokeratin 20 expression in most differentiated tumor epithelia of the tumor center. This architecture strongly depended on growth conditions, and was fully reproducible in mouse xenografts of cultured and primary colon cancer cells. Full crypt-like organization was associated with low tumor grade and was an independent prognostic marker of better survival in a collection of 221 colorectal cancers. Our findings suggest that full activation of preserved intestinal morphogenetic programs in colon cancer requires in vivo growth environments. Furthermore, crypt-like architecture was linked with less aggressive tumor biology, and may be useful to improve current colon cancer grading schemes. PMID:25111606

  16. Quantification of pancreatic secretory trypsin inhibitor in colonic carcinoma and normal adjacent colonic mucosa.

    PubMed Central

    Bohe, H; Bohe, M; Jönsson, P; Lindström, C; Ohlsson, K

    1992-01-01

    AIMS: To measure the content of immunoreactive human pancreatic secretory trypsin inhibitor (irPSTI) in colonic carcinoma and adjacent normal colonic mucosa. METHODS: From a stable hybridoma cell line producing monoclonal antibodies specific for human PSTI, a specific enzyme linked immunosorbent assay (ELISA) for human PSTI was developed. In a precipitation assay system these antibodies bound human PSTI in a dose-dependent manner. The specimens were obtained from resectional surgery. RESULTS: The content of irPSTI was 19.9 micrograms/g protein (0.55 micrograms/g tissue wet weight) in colonic carcinoma. In adjacent normal colonic mucosa 43.6 micrograms/g protein (1.12 micrograms/g tissue wet weight) was shown. CONCLUSIONS: The enzymatic degradation of surrounding tissue necessary for tumour cell invasion could be facilitated by this relative deficit of the inhibitor in infiltrative carcinoma. PMID:1479031

  17. Hydrogen sulfide and colonic epithelial metabolism: implications for ulcerative colitis.

    PubMed

    Jørgensen, J; Mortensen, P B

    2001-08-01

    Hydrogen sulfide (HS-) impairs the oxidation of butyrate in colonocytes and is found in excess in feces of patients with ulcerative colitis. The possible pathogenic role of HS- in ulcerative colitis was further investigated. To investigate the metabolic effect of free and bound fecal HS-, isolated rat colonocytes were incubated in the presence of butyrate without and with the addition of (1) HS- in water, (2) sterile filtrates of fecal homogenates supplemented and incubated with HS- and known sources of fecal HS- production, and (3) HS- incubated with fecal agents known to bind HS-. Oxidation rates were obtained by quantifying the production of CO2. Total and free HS-, as well as the fecal ability to bind HS-, were determined in health and ulcerative colitis. Compared to the production of CO2 by colonocytes incubated with 2 mmol/liter of butyrate, the further addition of 1.25 and 2.5 mmol/liter of HS- in water reduced the production of CO2 by 57.6+/-10.0 and 98.9+/-1.4%, respectively. However, when adding fecal filtrate of homogenate supplemented with HS- corresponding to 1.25 and 2.5 mmol/liter of HS- in water, the reduction of CO2 production was only 30.7+/-12.0 and 53.2+/-14.0%, respectively. Neither the fecal level of total or free HS- nor the remarkable fecal ability to bind HS- differed in health or quiescent and active ulcerative colitis. Bound HS- had no or little effect on CO2 production. Addition of fecal filtrate of nonsupplemented homogenate to colonocytes significantly reduced the oxidation of butyrate to CO2 about 25%, which could not be ascribed to fecal HS-. In conclusion, fecal HS- has little effect on butyrate oxidation in colonocytes and does not seem to play a pathogenic role for UC by impairing colonic epithelial metabolism. Other fecal agents seem to be more potent metabolic inhibitors than fecal HS-. The role of colonic contents in the pathogenesis of ulcerative colitis remains circumstantial.

  18. Distinct Transcriptional Changes and Epithelial-stromal Interactions are Altered in Early Stage Colon Cancer Development

    PubMed Central

    Mo, Allen; Jackson, Stephen; Varma, Kamini; Carpino, Alan; Giardina, Charles; Devers, Thomas J.; Rosenberg, Daniel W.

    2016-01-01

    While the progression of mutated colonic cells is dependent upon interactions between the initiated epithelium and surrounding stroma, the nature of these interactions is poorly understood. Here the development of an ultra-sensitive laser-capture microdissection (LCM)/RNA-seq approach for studying the epithelial and stromal compartments of aberrant crypt foci (ACF) is described. ACF are the earliest identifiable pre-neoplastic lesion found within the human colon and are detected using high-definition endoscopy with contrast dye-spray. The current analysis focused on the epithelium of ACF with somatic mutations to either KRAS, BRAF, or APC, with expression patterns compared to normal mucosa from each patient. By comparing gene expression patterns between groups, an increase in a number of pro-inflammatory NF-κB target genes were identified that were specific to ACF epithelium, including TIMP1, RELA and RELB. Distinct transcriptional changes associated with each somatic mutation were observed and a subset display a BRAFV600E-mediated senescence-associated transcriptome characterized by increased expression of CDKN2A. Finally, LCM-captured ACF-associated stroma was found to be transcriptionally distinct from normal stroma, with an up-regulation of genes related to immune cell infiltration and fibroblast activation. Immunofluorescence confirmed increased CD3+ T cells within the stromal microenvironment of ACF and an abundance of activated fibroblasts. Collectively, these results provide new insight into the cellular interplay that occurs at the earliest stages of colonic neoplasia, highlighting the important role of NF-kB, activated stromal fibroblasts and lymphocyte infiltration. Implications Fibroblasts and immune cells in the stromal microenvironment play an important role during the earliest stages of colon carcinogenesis. PMID:27353028

  19. Validation of methylation biomarkers that distinguish normal colon mucosa of cancer patients from normal colon mucosa of patients without cancer.

    PubMed

    Cesaroni, Matteo; Powell, Jasmine; Sapienza, Carmen

    2014-07-01

    We have validated differences in DNA methylation levels of candidate genes previously reported to discriminate between normal colon mucosa of patients with colon cancer and normal colon mucosa of individuals without cancer. Here, we report that CpG sites in 16 of the 30 candidate genes selected show significant differences in mean methylation level in normal colon mucosa of 24 patients with cancer and 24 controls. A support vector machine trained on these data and data for an additional 66 CpGs yielded an 18-gene signature, composed of ten of the validated candidate genes plus eight additional candidates. This model exhibited 96% sensitivity and 100% specificity in a 40-sample training set and classified all eight samples in the test set correctly. Moreover, we found a moderate-strong correlation (Pearson coefficients r = 0.253-0.722) between methylation levels in colon mucosa and methylation levels in peripheral blood for seven of the 18 genes in the support vector model. These seven genes, alone, classified 44 of the 48 patients in the validation set correctly and five CpGs selected from only two of the seven genes classified 41 of the 48 patients in the discovery set correctly. These results suggest that methylation biomarkers may be developed that will, at minimum, serve as useful objective and quantitative diagnostic complements to colonoscopy as a cancer-screening tool. These data also suggest that it may be possible to monitor biomarker methylation levels in tissues collected much less invasively than by colonoscopy.

  20. Validation of methylation biomarkers that distinguish normal colon mucosa from cancer patients from normal colon mucosa of patients without cancer

    PubMed Central

    Cesaroni, Matteo; Powell, Jasmine; Sapienza, Carmen

    2014-01-01

    We have validated differences in DNA methylation levels of candidate genes previously reported to discriminate between normal colon mucosa of colon cancer patients and normal colon mucosa of individuals without cancer. Here, we report that CpG sites in 16 of the 30 candidate genes selected show significant differences in mean methylation level in normal colon mucosa of 24 cancer patients and 24 controls. A support vector machine trained on these data and data for an additional 66 CpGs yielded an 18-gene signature, composed of 10 of the validated candidate genes plus eight additional candidates. This model exhibited 96% sensitivity and 100% specificity in a 40-sample training set and classified all eight samples in the test set correctly. Moreover, we found a moderate-strong correlation (Pearson coefficients r=0.253-0.722) between methylation levels in colon mucosa and methylation levels in peripheral blood for seven of the 18 genes in the support vector model. These seven genes, alone, classified 44 of the 48 patients in the validation set correctly and five CpGs selected from only two of the seven genes classified 41 of the 48 patients in the discovery set correctly. These results suggest that methylation biomarkers may be developed that will, at minimum, serve as useful objective and quantitative diagnostic complements to colonoscopy as a cancer-screening tool. These data also suggest that it may be possible to monitor biomarker methylation levels in tissues collected much less invasively than by colonoscopy. PMID:24806665

  1. Intestinal microbiota contributes to colonic epithelial changes in simulated microgravity mouse model.

    PubMed

    Shi, Junxiu; Wang, Yifan; He, Jian; Li, Pingping; Jin, Rong; Wang, Ke; Xu, Xi; Hao, Jie; Zhang, Yan; Liu, Hongju; Chen, Xiaoping; Wu, Hounan; Ge, Qing

    2017-08-01

    Exposure to microgravity leads to alterations in multiple systems, but microgravity-related changes in the gastrointestinal tract and its clinical significance have not been well studied. We used the hindlimb unloading (HU) mouse model to simulate a microgravity condition and investigated the changes in intestinal microbiota and colonic epithelial cells. Compared with ground-based controls (Ctrls), HU affected fecal microbiota composition with a profile that was characterized by the expansion of Firmicutes and decrease of Bacteroidetes. The colon epithelium of HU mice showed decreased goblet cell numbers, reduced epithelial cell turnover, and decreased expression of genes that are involved in defense and inflammatory responses. As a result, increased susceptibility to dextran sulfate sodium-induced epithelial injury was observed in HU mice. Cohousing of Ctrl mice with HU mice resulted in HU-like epithelial changes in Ctrl mice. Transplantation of feces from Ctrl to HU mice alleviated these epithelial changes in HU mice. Results indicate that HU changes intestinal microbiota, which leads to altered colonic epithelial cell homeostasis, impaired barrier function, and increased susceptibility to colitis. We further demonstrate that alteration in gastrointestinal motility may contribute to HU-associated dysbiosis. These animal results emphasize the necessity of evaluating astronauts' intestinal homeostasis during distant space travel.-Shi, J., Wang, Y., He, J., Li, P., Jin, R., Wang, K., Xu, X., Hao, J., Zhang, Y., Liu, H., Chen, X., Wu, H., Ge, Q. Intestinal microbiota contributes to colonic epithelial changes in simulated microgravity mouse model. © FASEB.

  2. Protective effects of Lactobacillus plantarum against epithelial barrier dysfunction of human colon cell line NCM460

    PubMed Central

    Liu, Zhi-Hua; Shen, Tong-Yi; Zhang, Peng; Ma, Yan-Lei; Moyer, Mary Pat; Qin, Huan-Long

    2010-01-01

    AIM: To investigate the effects of Lactobacillus plantarum (L. plantarum) in the intestinal permeability and expression of tight junction (TJ) using the normal human colon cell line NCM460. METHODS: Paracellular permeability of NCM460 monolayers was determined by transepithelial electrical resistance and dextran permeability. Expression of TJ proteins in NCM460 cell monolayers was detected by Western blotting and quantitative real-time polymerase chain reaction. RESULTS: L. plantarum played an important role in increasing transepithelial electrical resistance and decreasing the permeability to macromolecules of NCM460 monolayers against the disruption caused by enteropathogenic Escherichia coli (E. coli) or enteroinvasive E. coli. L. plantarum also prevented the decrease in the expression of TJ proteins and F-actin in NCM460 cells. CONCLUSION: L. plantarum can protect against dysfunction of NCM460 intestinal epithelial barrier caused by enteropathogenic E. coli or enteroinvasive E. coli, and thus can be a potential candidate of therapeutic agents for the treatment of intestinal diseases. PMID:21128328

  3. Dextran sodium sulphate-induced colitis perturbs muscarinic cholinergic control of colonic epithelial ion transport

    PubMed Central

    Sayer, Brooke; Lu, Jun; Green, Christina; Söderholm, Johan D; Akhtar, Mahmood; McKay, Derek M

    2002-01-01

    Neuronal cholinergic input is an important regulator of epithelial electrolyte transport and hence water movement in the gut. In this study, colitis was induced by treating mice with 4% (w v−1) dextran sodium-sulphate (DSS)-water for 5 days followed by 3 days of normal water. Mid-colonic segments were mounted in Ussing chambers and short-circuit current (Isc, indicates net ion movement) responses to the cholinergic agonist, carbachol (CCh; 10−4 M)±tetrodotoxin, atropine (ATR), hexamethonium (HEX), naloxone or phenoxybenzamine were assessed. Tissues from mice with DSS-induced colitis displayed a drop in Isc in response to CCh (−11.3±3.3 μA/cm2), while those from control mice showed a transient increase in Isc (76.3±13.0 μA/cm2). The ΔIsc in colon from DSS-treated mice was tetrodotoxin-sensitive, atropine-insensitive and was reversed by hexamethonium (HEX+CCh=16.7±7.8 μA/cm2), indicating involvement of a nicotinic receptor. CCh induced a drop in Isc in tissues from controls only when they were pretreated with the cholinergic muscarinic receptor blocker, atropine: ATR+CCh=−21.3±7.0 μA/cm2. Nicotine elicited a drop in Isc in Ussing-chambered colon from both control and DSS-treated mice that was TTX-sensitive. The drop in Isc evoked by CCh challenge of colonic tissue from DSS-treated mice or ATR+CCh challenge of control tissue was not significantly affected by blockade of opiate or α-adrenergic receptors by naloxone or phenoxybenzamine, respectively. The data indicate that DSS-colitis reveals a nicotinic receptor that becomes important in cholinergic regulation of ion transport. PMID:11934821

  4. Infrared spectroscopic characteristics of normal and malignant colonic epithelium

    NASA Astrophysics Data System (ADS)

    Krupnik, Eduardo; Jackson, Michael; Bird, Ranjana P.; Smith, Ian C. P.; Mantsch, Henry H.

    1998-04-01

    IR spectroscopy is being widely used to study the biochemical changes associated with cancer. In particular, based upon the hypothesis that biochemical changes associated with cancer precede morphological manifestations of the disease, IR spectroscopy is being evaluated as a potential early diagnostic and prognostic tool. In the current study, IR spectroscopy was applied to the study of colon tissue from rats treated with the specific colon carcinogen azoxymethane, to determine whether tumor induction was associated with identifiable spectroscopic changes in the colon. Characteristic spectra were found for each layer of the colon. Spectra of normal-appearing mucosa and tumors form treated animals then compared to spectra of control mucosa. Differences between tumors and control mucosa were apparent, indicating changes in cellular biochemistry associated with tumor development. In particular, differences in absorptions attributed to nucleic acids were seen, indicating alterations in the structure of cellular DNA in malignant and carcinogen treated tissues. Interestingly, spectra of carcinogen treated rates exhibit characteristics intermediate between those of normal mucosa and tumors. Application of multivariate analysis allowed non-subjective classification of the spectra into three distinct classes with and accuracy of 86.7 percent. The separate classification of control and treated mucosa suggests that IR spectroscopy, when combined with the appropriate classifier, can indeed detect biochemical changes in tissue before physical manifestation of the disease process.

  5. Colon epithelial proliferation and carcinogenesis in diet-induced obesity.

    PubMed

    Takahashi, Hirokazu; Hosono, Kunihiro; Endo, Hiroki; Nakajima, Atsushi

    2013-12-01

    Colorectal cancer is the third leading cause of cancer death in Japan and the United States and is strongly associated with obesity, especially visceral obesity. Several metabolic mediators, such as adiponectin, have been suspected to play a role in obesity-related carcinogenesis. In a previous human study, the existence of a significant correlation between the number of human dysplastic aberrant crypt foci (ACF) and the visceral fat area was demonstrated, and also that of a significant inverse correlation between the number of dysplastic ACF and the plasma adiponectin level. Other studies have investigated the effect of adiponectin under the normal and high-fat diet conditions in a mouse model of azoxymethane-induced colon cancer. Enhanced formation of both ACF and tumors was observed in the adiponectin-deficient mice, as compared with that in the wild-type, under the high-fat diet condition but not under the normal diet condition. Furthermore, that the 5'-AMP-activated kinase/mammalian target of rapamycin pathway is involved in the promotion of colorectal carcinogenesis in adiponectin-deficient mice under the high-fat diet condition was shown. Therefore, that the 5'-AMP-activated kinase/mammalian target of rapamycin signaling pathway may play an important role in colorectal carcinogenesis was speculated. Metformin, a biguanide derivative widely used in the treatment of diabetes mellitus, has been shown to exert a suppressive effect on ACF formation in both mouse models and humans. Therefore, metformin might be a promising candidate as a safe drug for chemoprevention of colorectal carcinogenesis. Further studies with high evidence levels, such as randomized, controlled studies, are needed to clarify these relationships. © 2013 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd.

  6. Extracellular calcium sensing receptor stimulation in human colonic epithelial cells induces intracellular calcium oscillations and proliferation inhibition.

    PubMed

    Rey, Osvaldo; Young, Steven H; Jacamo, Rodrigo; Moyer, Mary P; Rozengurt, Enrique

    2010-10-01

    The extracellular Ca(2+)-sensing receptor (CaR) is increasingly implicated in the regulation of multiple cellular functions in the gastrointestinal tract, including secretion, proliferation and differentiation of intestinal epithelial cells. However, the signaling mechanisms involved remain poorly defined. Here we examined signaling pathways activated by the CaR, including Ca(2+) oscillations, in individual human colon epithelial cells. Single cell imaging of colon-derived cells expressing the CaR, including SW-480, HT-29, and NCM-460 cells, shows that stimulation of this receptor by addition of aromatic amino acids or by an elevation of the extracellular Ca(2+) concentration promoted striking intracellular Ca(2+) oscillations. The intracellular calcium oscillations in response to extracellular Ca(2+) were of sinusoidal pattern and mediated by the phospholipase C/diacylglycerol/inositol 1,4,5-trisphosphate pathway as revealed by a biosensor that detects the accumulation of diacylglycerol in the plasma membrane. The intracellular calcium oscillations in response to aromatic amino acids were of transient type, that is, Ca(2+) spikes that returned to baseline levels, and required an intact actin cytoskeleton, a functional Rho, Filamin A and the ion channel TRPC1. Further analysis showed that re-expression and stimulation of the CaR in human epithelial cells derived from normal colon and from colorectal adenocarcinoma inhibits their proliferation. This inhibition was associated with the activation of the signaling pathway that mediates the generation of sinusoidal, but not transient, intracellular Ca(2+) oscillations. Thus, these results indicate that the CaR can function in two signaling modes in human colonic epithelial cells offering a potential link between gastrointestinal responses and food/nutrients uptake and metabolism.

  7. Vulnerability of Normal Human Mammary Epithelial Cells to Oncogenic Transformation

    DTIC Science & Technology

    2012-04-01

    algorithm for CpG-island detection. BMC Bioinformatics 7: 446. 17. Gardiner-Garden M, Frommer M (1987) CpG islands in vertebrate genomes. J Mol Biol...it does not have a CpG island according to the original criteria (Gardiner-Garden and Frommer 1987). H3K4me3 and H3Ac are present in miR-205...culture of normal human mammary epithelial cells. Cancer Res 69: 7557–7568. Gardiner-GardenM, Frommer M. 1987. CpG islands in vertebrate genomes. J Mol

  8. Growth Control in Colon Epithelial Cells: Gadolinium Enhances Calcium-Mediated Growth Regulation

    PubMed Central

    Attili, Durga; Jenkins, Brian; Aslam, Muhammad Nadeem; Dame, Michael K.

    2013-01-01

    Gadolinium, a member of the lanthanoid family of transition metals, interacts with calcium-binding sites on proteins and other biological molecules. The overall goal of the present investigation was to determine if gadolinium could enhance calcium-induced epithelial cell growth inhibition in the colon. Gadolinium at concentrations as low as 1–5 µM combined with calcium inhibits proliferation of human colonic epithelial cells more effectively than calcium alone. Gadolinium had no detectable effect on calcium-induced differentiation in the same cells based on change in cell morphology, induction of E-cadherin synthesis, and translocation of E-cadherin from the cytosol to the cell surface. When the colon epithelial cells were treated with gadolinium and then exposed to increased calcium concentrations, movement of extracellular calcium into the cell was suppressed. In contrast, gadolinium treatment had no effect on ionomycin-induced release of stored intracellular calcium into the cytoplasm. Whether these in vitro observations can be translated into an approach for reducing abnormal proliferation in the colonic mucosa (including polyp formation) is not known. These results do, however, provide an explanation for our recent findings that a multi-mineral supplement containing all of the naturally occurring lanthanoid metals including gadolinium are more effective than calcium alone in preventing colon polyp formation in mice on a high-fat diet. PMID:23008064

  9. Growth control in colon epithelial cells: gadolinium enhances calcium-mediated growth regulation.

    PubMed

    Attili, Durga; Jenkins, Brian; Aslam, Muhammad Nadeem; Dame, Michael K; Varani, James

    2012-12-01

    Gadolinium, a member of the lanthanoid family of transition metals, interacts with calcium-binding sites on proteins and other biological molecules. The overall goal of the present investigation was to determine if gadolinium could enhance calcium-induced epithelial cell growth inhibition in the colon. Gadolinium at concentrations as low as 1-5 μM combined with calcium inhibits proliferation of human colonic epithelial cells more effectively than calcium alone. Gadolinium had no detectable effect on calcium-induced differentiation in the same cells based on change in cell morphology, induction of E-cadherin synthesis, and translocation of E-cadherin from the cytosol to the cell surface. When the colon epithelial cells were treated with gadolinium and then exposed to increased calcium concentrations, movement of extracellular calcium into the cell was suppressed. In contrast, gadolinium treatment had no effect on ionomycin-induced release of stored intracellular calcium into the cytoplasm. Whether these in vitro observations can be translated into an approach for reducing abnormal proliferation in the colonic mucosa (including polyp formation) is not known. These results do, however, provide an explanation for our recent findings that a multi-mineral supplement containing all of the naturally occurring lanthanoid metals including gadolinium are more effective than calcium alone in preventing colon polyp formation in mice on a high-fat diet.

  10. MET signalling in primary colon epithelial cells leads to increased transformation irrespective of aberrant Wnt signalling

    PubMed Central

    Boon, E M J; Kovarikova, M; Derksen, P W B; van der Neut, R

    2005-01-01

    It has been shown that in hereditary and most sporadic colon tumours, components of the Wnt pathway are mutated. The Wnt target MET has been implicated in the development of colon cancer. Here, we show that overexpression of wild-type or a constitutively activated form of MET in colon epithelial cells leads to increased transformation irrespective of Wnt signalling. Fetal human colon epithelial cells without aberrant Wnt signalling were transfected with wild-type or mutated MET constructs. Expression of these constructs leads to increased phosphorylation of MET and its downstream targets PKB and MAPK. Upon stimulation with HGF, the expression of E-cadherin is downregulated in wild-type MET-transfected cells, whereas cells expressing mutated MET show low E-cadherin levels independent of stimulation with ligand. This implies a higher migratory propensity of these cells. Furthermore, fetal human colon epithelial cells expressing the mutated form of MET have colony-forming capacity in soft agar, while cells expressing wild-type MET show an intermediate phenotype. Subcutaneous injection of mutated MET-transfected cells in nude mice leads to the formation of tumours within 12 days in all mice injected. At this time point, mock-transfected cells do not form tumours, while wild-type MET-transfected cells form subcutaneous tumours in one out of five mice. We thus show that MET signalling can lead to increased transformation of colon epithelial cells independent of Wnt signalling and in this way could play an essential role in the onset and progression of colorectal cancer. PMID:15785735

  11. MET signalling in primary colon epithelial cells leads to increased transformation irrespective of aberrant Wnt signalling.

    PubMed

    Boon, E M J; Kovarikova, M; Derksen, P W B; van der Neut, R

    2005-03-28

    It has been shown that in hereditary and most sporadic colon tumours, components of the Wnt pathway are mutated. The Wnt target MET has been implicated in the development of colon cancer. Here, we show that overexpression of wild-type or a constitutively activated form of MET in colon epithelial cells leads to increased transformation irrespective of Wnt signalling. Fetal human colon epithelial cells without aberrant Wnt signalling were transfected with wild-type or mutated MET constructs. Expression of these constructs leads to increased phosphorylation of MET and its downstream targets PKB and MAPK. Upon stimulation with HGF, the expression of E-cadherin is downregulated in wild-type MET-transfected cells, whereas cells expressing mutated MET show low E-cadherin levels independent of stimulation with ligand. This implies a higher migratory propensity of these cells. Furthermore, fetal human colon epithelial cells expressing the mutated form of MET have colony-forming capacity in soft agar, while cells expressing wild-type MET show an intermediate phenotype. Subcutaneous injection of mutated MET-transfected cells in nude mice leads to the formation of tumours within 12 days in all mice injected. At this time point, mock-transfected cells do not form tumours, while wild-type MET-transfected cells form subcutaneous tumours in one out of five mice. We thus show that MET signalling can lead to increased transformation of colon epithelial cells independent of Wnt signalling and in this way could play an essential role in the onset and progression of colorectal cancer.

  12. Chemopreventive potential of in vitro fermented nuts in LT97 colon adenoma and primary epithelial colon cells.

    PubMed

    Schlörmann, Wiebke; Lamberty, Julia; Lorkowski, Stefan; Ludwig, Diana; Mothes, Henning; Saupe, Christian; Glei, Michael

    2017-05-01

    Due to their beneficial nutritional profile the consumption of nuts contributes to a healthy diet and might reduce colon cancer risk. To get closer insights into potential mechanisms, the chemopreventive potential of different in vitro fermented nut varieties regarding the modulation of genes involved in detoxification (CAT, SOD2, GSTP1, GPx1) and cell cycle (p21, cyclin D2) as well as proliferation and apoptosis was examined in LT97 colon adenoma and primary epithelial colon cells. Fermentation supernatants (FS) of nuts significantly induced mRNA expression of CAT (up to 4.0-fold), SOD2 (up to 2.5-fold), and GSTP1 (up to 2.3-fold), while GPx1 expression was significantly reduced by all nut FS (0.8 fold on average). Levels of p21 mRNA were significantly enhanced (up to 2.6-fold), whereas all nut FS significantly decreased cyclin D2 expression (0.4-fold on average). In primary epithelial cells, expression of CAT (up to 3.5-fold), GSTP1 (up to 3.0-fold), and GPx1 (up to 3.9-fold) was increased, whereas p21 and cyclin D2 levels were not influenced. Nut FS significantly inhibited growth of LT97 cells and increased levels of early apoptotic cells (8.4% on average) and caspase 3 activity (4.6-fold on average), whereas caspase 3 activity was not modulated in primary colon cells. The differential modulation of genes involved in detoxification and cell cycle together with an inhibition of proliferation and induction of apoptosis in adenoma cells might contribute to chemopreventive effects of nuts regarding colon cancer. © 2017 Wiley Periodicals, Inc.

  13. Immortalized epithelial cells derived from human colon biopsies express stem cell markers and differentiate in vitro.

    PubMed

    Roig, Andres I; Eskiocak, Ugur; Hight, Suzie K; Kim, Sang Bum; Delgado, Oliver; Souza, Rhonda F; Spechler, Stuart J; Wright, Woodring E; Shay, Jerry W

    2010-03-01

    Long-term propagation of human colonic epithelial cells (HCEC) of adult origin has been a challenge; currently used HCEC lines are of malignant origin and/or contain multiple cytogenetic changes. We sought to immortalize human colon biopsy-derived cells expressing stem cell markers and retaining multilineage epithelial differentiation capability. We isolated and cultured cells from biopsy samples of 2 patients undergoing routine screening colonoscopy. Cells were immortalized by expression of the nononcogenic proteins cyclin-dependent kinase 4 (Cdk4) and the catalytic component of human telomerase (hTERT) and maintained for more than 1 year in culture. The actively proliferating HCECs expressed the mesenchymal markers vimentin and alpha-smooth muscle actin. Upon growth arrest, cells assumed a cuboidal shape, decreased their mesenchymal features, and expressed markers of colonic epithelial cells such as cytokeratin 18, zonula occludens-1, mucins-1 and -2, antigen A33, and dipeptidyl peptidase 4. Immortalized cells expressed stem cell markers that included LGR5, BMI1, CD29, and CD44. When placed in Matrigel in the absence of a mesenchymal feeder layer, individual cells divided and formed self-organizing, cyst-like structures; a subset of cells exhibited mucin-2 or polarized villin staining. We established immortalized HCECs that are capable of self-renewal and multilineage differentiation. These cells should serve as valuable reagents for studying colon stem cell biology, differentiation, and pathogenesis. Copyright 2010 AGA Institute. Published by Elsevier Inc. All rights reserved.

  14. Butyrate and bioactive proteolytic form of Wnt-5a regulate colonic epithelial proliferation and spatial development

    PubMed Central

    Uchiyama, Kazuhiko; Sakiyama, Toshio; Hasebe, Takumu; Musch, Mark W.; Miyoshi, Hiroyuki; Nakagawa, Yasushi; He, Tong-Chuan; Lichtenstein, Lev; Naito, Yuji; Itoh, Yoshito; Yoshikawa, Toshikazu; Jabri, Bana; Stappenbeck, Thaddeus; Chang, Eugene B.

    2016-01-01

    Proliferation and spatial development of colonic epithelial cells are highly regulated along the crypt vertical axis, which, when perturbed, can result in aberrant growth and carcinogenesis. In this study, two key factors were identified that have important and counterbalancing roles regulating these processes: pericrypt myofibroblast-derived Wnt-5a and the microbial metabolite butyrate. Cultured YAMC cell proliferation and heat shock protein induction were analzyed after butryate, conditioned medium with Wnt5a activity, and FrzB containing conditioned medium. In vivo studies to modulate Hsp25 employed intra-colonic wall Hsp25 encoding lentivirus. To silence Wnt-5a in vivo, intra-colonic wall Wnt-5a silencing RNA was used. Wnt-5a, secreted by stromal myofibroblasts of the lower crypt, promotes proliferation through canonical β-catenin activation. Essential to this are two key requirements: (1) proteolytic conversion of the highly insoluble ~40 kD Wnt-5a protein to a soluble 36 mer amino acid peptide that activates epithelial β-catenin and cellular proliferation, and (2) the simultaneous inhibition of butyrate-induced Hsp25 by Wnt-5a which is necessary to arrest the proliferative process in the upper colonic crypt. The interplay and spatial gradients of these factors insures that crypt epithelial cell proliferation and development proceed in an orderly fashion, but with sufficient plasticity to adapt to physiological perturbations including inflammation. PMID:27561676

  15. A Novel Role of Spred2 in the Colonic Epithelial Cell Homeostasis and Inflammation

    PubMed Central

    Takahashi, Sakuma; Yoshimura, Teizo; Ohkura, Takahiro; Fujisawa, Masayoshi; Fushimi, Soichiro; Ito, Toshihiro; Itakura, Junya; Hiraoka, Sakiko; Okada, Hiroyuki; Yamamoto, Kazuhide; Matsukawa, Akihiro

    2016-01-01

    Rapid and adequate mucosal healing is important for a remission of ulcerative colitis (UC) patients. Here, we examined whether Spred2, a member of the Sprouty-related EVH1-domain-containing proteins that inhibit the Ras/Raf/ERK pathway, plays a role in colonic mucosal homeostasis and inflammation by using Spred2 knockout (KO) mice. We first detected increased epithelial cell proliferation and cadherin 1 expression in the colon of naïve Spred2 KO mice compared to wild-type mice. Interestingly, Spred2 KO mice were resistant to dextran sulfate sodium (DSS)-induced acute colitis as indicated by lower levels of body weight loss and disease activity index. Histologically, epithelial cell injury and inflammation were milder in the colonic mucosa of Spred2 KO mice on day 3 and almost undetectable by day 8. Experiments with bone chimeric mice indicated that Spred2-deficiency in non-hematopoietic cells was responsible for the reduced sensitivity to DSS. Finally, Spred2 KO mice developed significantly fewer tumors in response to azoxymethane plus DSS. Taken together, our results demonstrate, for the first time, that Spred2 plays an important role in the regulation of colonic epithelial cell proliferation and inflammation by potentially down-regulating the activation of ERK. Thus, Spred2 may be a new therapeutic target for the treatment of UC. PMID:27869219

  16. Epithelial-specific A2B adenosine receptor signaling protects the colonic epithelial barrier during acute colitis

    PubMed Central

    Aherne, CM; Saeedi, B; Collins, CB; Masterson, JC; McNamee, EN; Perrenoud, L; Rapp, CR; Curtis, VF; Bayless, A; Fletcher, A; Glover, LE; Evans, CM; Jedlicka, P; Furuta, GT; de Zoeten, EF; Colgan, SP; Eltzschig, HK

    2015-01-01

    Central to inflammatory bowel disease (IBD) pathogenesis is loss of mucosal barrier function. Emerging evidence implicates extracellular adenosine signaling in attenuating mucosal inflammation. We hypothesized that adenosine-mediated protection from intestinal barrier dysfunction involves tissue-specific signaling through the A2B adenosine receptor (Adora2b) at the intestinal mucosal surface. To address this hypothesis, we combined pharmacologic studies and studies in mice with global or tissue-specific deletion of the Adora2b receptor. Adora2b−/− mice experienced a significantly heightened severity of colitis, associated with a more acute onset of disease and loss of intestinal epithelial barrier function. Comparison of mice with Adora2b deletion on vascular endothelial cells (Adora2bfl/flVeCadCre+) or intestinal epithelia (Adora2bfl/flVillinCre+) revealed a selective role for epithelial Adora2b signaling in attenuating colonic inflammation. In vitro studies with Adora2b knockdown in intestinal epithelial cultures or pharmacologic studies highlighted Adora2b-driven phosphorylation of vasodilator-stimulated phosphoprotein (VASP) as a specific barrier repair response. Similarly, in vivo studies in genetic mouse models or treatment studies with an Adora2b agonist (BAY 60-6583) recapitulate these findings. Taken together, our results suggest that intestinal epithelial Adora2b signaling provides protection during intestinal inflammation via enhancing mucosal barrier responses. PMID:25850656

  17. Malignant transformation of colonic epithelial cells by a colon-derived long noncoding RNA

    SciTech Connect

    Franklin, Jeffrey L.; Rankin, Carl R.; Levy, Shawn; Snoddy, Jay R.; Zhang, Bing; Washington, Mary Kay; Thomson, J. Michael; Whitehead, Robert H.; Coffey, Robert J.

    2013-10-11

    Highlights: •Non-coding RNAs are found in the colonic crypt progenitor compartment. •Colonocytes transformed by ncNRFR are highly invasive and metastatic. •ncNRFR has a region similar to the miRNA, let-7 family. •ncNRFR expression alters let-7 activity as measured by reporter construct. •ncNRFR expression upregulates let-7b targets. -- Abstract: Recent progress has been made in the identification of protein-coding genes and miRNAs that are expressed in and alter the behavior of colonic epithelia. However, the role of long non-coding RNAs (lncRNAs) in colonic homeostasis is just beginning to be explored. By gene expression profiling of post-mitotic, differentiated tops and proliferative, progenitor-compartment bottoms of microdissected adult mouse colonic crypts, we identified several lncRNAs more highly expressed in crypt bottoms. One identified lncRNA, designated non-coding Nras functional RNA (ncNRFR), resides within the Nras locus but appears to be independent of the Nras coding transcript. Stable overexpression of ncNRFR in non-transformed, conditionally immortalized mouse colonocytes results in malignant transformation, as determined by growth in soft agar and formation of highly invasive tumors in nude mice. Moreover, ncNRFR appears to inhibit the function of the tumor suppressor let-7. These results suggest precise regulation of ncNRFR is necessary for proper cell growth in the colonic crypt, and its misregulation results in neoplastic transformation.

  18. Normal Function of the Colon and Anorectal Area

    MedlinePlus

    ... it to be expelled as stool. This prolonged transit time is an important aspect of colonic function ... occur in adults. Nerves and muscles regulate the transit time of the colon. Derangements in either element ...

  19. Normal and Abnormal Epithelial Differentiation in the Female Reproductive Tract

    PubMed Central

    Kurita, Takeshi

    2011-01-01

    In mammals, the female reproductive tract (FRT) develops from a pair of paramesonephric or Müllerian ducts (MDs), which arise from coelomic epithelial cells of mesodermal origin. During development, the MDs undergo a dynamic morphogenetic transformation from simple tubes consisting of homogeneous epithelium and surrounding mesenchyme into several distinct organs namely the oviduct, uterus, cervix and vagina. Following the formation of anatomically distinctive organs, the uniform MD epithelium (MDE) differentiates into diverse epithelial cell types with unique morphology and functions in each organ. Classic tissue recombination studies, in which the epithelium and mesenchyme isolated from the newborn mouse FRT were recombined, have established that the organ specific epithelial cell fate of MDE is dictated by the underlying mesenchyme. The tissue recombination studies have also demonstrated that there is a narrow developmental window for the epithelial cell fate determination in MD-derived organs. Accordingly, the developmental plasticity of epithelial cells is mostly lost in mature FRT. If the signaling that controls epithelial differentiation is disrupted at the critical developmental stage, the cell fate of MD-derived epithelial tissues will be permanently altered and can result in epithelial lesions in adult life. A disruption of signaling that maintains epithelial cell fate can also cause epithelial lesions in the FRT. In this review, the pathogenesis of cervical/vaginal adenoses and uterine squamous metaplasia is discussed as examples of such incidences. PMID:21612855

  20. Determination of Normal Distribution of Distended Colon Volumes to Guide Performance of Colonic Imaging With Fluid Distention.

    PubMed

    Zheng, Karen S; Small, William C; Mittal, Pardeep K; Cai, Qingpo; Kang, Jian; Moreno, Courtney C

    2016-01-01

    The purpose was to determine the normal distribution of distended colon volumes as a guide for rectal contrast material administration protocols. All computed tomography colonography studies performed at Emory University Hospital, Atlanta, Georgia, between January 2009 and January 2015, were reviewed retrospectively. In total, 85 subjects were included in the analysis (64% [54 of 85] female and 36% [31 of 85] male). Mean patient age was 65 years (range: 42-86y). Distended colon volumes were determined from colon length and transaxial diameter measurements made using a 3-dimensional workstation. Age, sex, race, height, weight, and body mass index were recorded. The normal distributions of distended colon volumes and lengths were determined. Correlations between colonic volume and colonic length, and demographic variables were assessed. Mean colon volume was 2.1L (range: 0.7-4.4L). Nearly, 17% of patients had a distended colonic volume of >3L. Mean colon length was 197cm (range: 118-285cm). A weak negative correlation was found between age and colonic volume (r = -0.221; P = 0.04). A weak positive correlation was found between body mass index and colonic length (r = 0.368; P = 0.007). Otherwise, no significant correlations were found for distended colonic volume or length and demographic variables. In conclusion, an average of approximately 2L of contrast material may be necessary to achieve full colonic opacification. This volume is larger than previously reported volumes (0.8-1.5L) for rectal contrast material administration protocols. Copyright © 2015 Mosby, Inc. All rights reserved.

  1. Synergic production of neutrophil chemotactic activity by colonic epithelial cells and eosinophils.

    PubMed

    Dent, Gordon; Loweth, Sam C; Hasan, Anwar Matar; Leslie, Fiona M

    2014-10-01

    The presence of eosinophils in the lumen and mucosa of the intestine is characteristic of both ulcerative colitis (UC) and Crohn's disease (CD). There is evidence of eosinophil activation in the intestine during acute inflammatory episodes of these diseases; these episodes are also characterized by an influx of neutrophils, which have the potential to cause extensive tissue damage. We undertook a study to determine whether eosinophils in contact with colonic epithelial cells produce factors that may attract neutrophils in response to immunological stimulation. Neutrophil chemotactic activity (NCA) and concentrations of three neutrophil-attracting CXC chemokines - CXCL1 (Groα), CXCL5 (Ena78) and CXCL8 (IL8) - were measured in supernatants of T84 colonic epithelial cells and blood eosinophils or eosinophil-like myeloid leukaemia cells (AML14.3D10), alone or in combination. Cells were stimulated with serum-opsonized zymosan (OZ) particles. NCA (P<0.005) and CXCL5 levels (P<0.05) in the supernatants of OZ-stimulated epithelial/eosinophil co-cultures were significantly higher than in the supernatants of either cell type alone. Release of CXCL1 (P<0.05) and CXCL8 (P<0.01) from OZ-stimulated co-culture supernatants was significantly higher than from OZ-stimulated eosinophils but not higher than from OZ-stimulated epithelial cells. Eosinophils and colonic epithelial cells exhibit synergy in production of neutrophil chemoattractants in response to immunological stimulation. This may represent a mechanism for exaggerated recruitment of neutrophils to the intestine in response to acute infection in conditions that are characterized by the presence of eosinophils in the bowel.

  2. Evidence for the involvement of NOD2 in regulating colonic epithelial cell growth and survival.

    PubMed

    Cruickshank, Sheena-M; Wakenshaw, Louise; Cardone, John; Howdle, Peter-D; Murray, Peter-J; Carding, Simon-R

    2008-10-14

    To investigate the function of NOD2 in colonic epithelial cells (CEC). A combination of in vivo and in vitro analyses of epithelial cell turnover in the presence and absence of a functional NOD2 protein and, in response to enteric Salmonella typhimurium infection, were used. shRNA interference was also used to investigate the consequences of knocking down NOD2 gene expression on the growth and survival of colorectal carcinoma cell lines. In the colonic mucosa the highest levels of NOD2 expression were in proliferating crypt epithelial cells. Muramyl dipeptide (MDP), that is recognized by NOD2, promoted CEC growth in vitro. By contrast, the growth of NOD2-deficient CECs was impaired. In vivo CEC proliferation was also reduced and apoptosis increased in Nod2(-/-) mice, which were also evident following enteric Salmonella infection. Furthermore, neutralization of NOD2 mRNA expression in human colonic carcinoma cells by shRNA interference resulted in decreased survival due to increased levels of apoptosis. These findings are consistent with the involvement of NOD2 protein in promoting CEC growth and survival. Defects in proliferation by CECs in cases of CD may contribute to the underlying pathology of disrupted intestinal homeostasis and excessive inflammation.

  3. Evidence for the involvement of NOD2 in regulating colonic epithelial cell growth and survival

    PubMed Central

    Cruickshank, Sheena M; Wakenshaw, Louise; Cardone, John; Howdle, Peter D; Murray, Peter J; Carding, Simon R

    2008-01-01

    AIM: To investigate the function of NOD2 in colonic epithelial cells (CEC). METHODS: A combination of in vivo and in vitro analyses of epithelial cell turnover in the presence and absence of a functional NOD2 protein and, in response to enteric Salmonella typhimurium infection, were used. shRNA interference was also used to investigate the consequences of knocking down NOD2 gene expression on the growth and survival of colorectal carcinoma cell lines. RESULTS: In the colonic mucosa the highest levels of NOD2 expression were in proliferating crypt epithelial cells. Muramyl dipeptide (MDP), that is recognized by NOD2, promoted CEC growth in vitro. By contrast, the growth of NOD2-deficient CECs was impaired. In vivo CEC proliferation was also reduced and apoptosis increased in Nod2-/- mice, which were also evident following enteric Salmonella infection. Furthermore, neutralization of NOD2 mRNA expression in human colonic carcinoma cells by shRNA interference resulted in decreased survival due to increased levels of apoptosis. CONCLUSION: These findings are consistent with the involvement of NOD2 protein in promoting CEC growth and survival. Defects in proliferation by CECs in cases of CD may contribute to the underlying pathology of disrupted intestinal homeostasis and excessive inflammation. PMID:18855982

  4. Increased expression of chemokine receptor CCR3 and its ligands in ulcerative colitis: the role of colonic epithelial cells in in vitro studies.

    PubMed

    Manousou, P; Kolios, G; Valatas, V; Drygiannakis, I; Bourikas, L; Pyrovolaki, K; Koutroubakis, I; Papadaki, H A; Kouroumalis, E

    2010-11-01

    Human colonic epithelial cells express T helper type 1 (Th1)-associated chemoattractants, yet little is known about the production of Th2-associated chemoattractants. CCL11/eotaxin-1, CCL24/eotaxin-2 and CCL26/eotaxin-3 are known to attract CCR3-expressing, Th2-polarized lymphocytes. We studied constitutive and inflammation-induced expression and production of CCR3 together with its ligands in the colon and peripheral blood of patients with inflammatory bowel disease (IBD) by flow cytometry, reverse transcription–polymerase chain reaction (RT–PCR) and enzyme-linked immunosorbent assay (ELISA). We further defined the regulated expression of these chemokines by RT–PCR and ELISA using cultured human epithelial cell lines. A higher fraction of peripheral T lymphocytes were found to be positive for CCR3 in patients with ulcerative colitis (UC) compared to Crohn’s disease (CD), while almost no CCR3(+) T cells were found in normal controls (NC). Similarly, higher and more frequent expression of CCR3 was observed in colonic biopsies from patients with UC, regardless of the disease activity, when compared to CD or NCs. Serum CCL11/eotaxin-1 was increased significantly in UC (306 ± 87 pg/ml) and less so in CD (257 ± 43 pg/ml), whereas CCL24/eotaxin-2, and CCL26/eotaxin-3 were increased only in UC. Colonic expression of the three chemokines was minimal in NCs but high in inflammatory bowel diseases (especially UC) and was independent of disease activity. Th2, and to a lesser extent Th1, cytokines were able to induce expression and production of all three eotaxins from colonic epithelial cells in culture. CCR3 and ligands over-expression would appear to be a characteristic of UC. The production of CCR3 ligands by human colonic epithelial cells suggests further that epithelium can play a role in modulating pathological T cell-mediated mucosal inflammation.

  5. Characterisation of colonic dysplasia-like epithelial atypia in murine colitis

    PubMed Central

    Randall-Demllo, Sarron; Fernando, Ruchira; Brain, Terry; Sohal, Sukhwinder Singh; Cook, Anthony L; Guven, Nuri; Kunde, Dale; Spring, Kevin; Eri, Rajaraman

    2016-01-01

    AIM To determine if exacerbation of pre-existing chronic colitis in Winnie (Muc2 mutant) mice induces colonic dysplasia. METHODS Winnie mice and C57BL6 as a genotype control, were administered 1% w/v dextran sulphate sodium (DSS) orally, followed by drinking water alone in week-long cycles for a total of three cycles. After the third cycle, mice were killed and colonic tissue collected for histological and immunohistochemical evaluation. Inflammation and severity of dysplasia in the colonic mucosa were assessed in H&E sections of the colon. Epithelial cell proliferation was assessed using Ki67 and aberrant β-catenin signalling assessed with enzyme-based immunohistochemistry. Extracted RNA from colonic segments was used for the analysis of gene expression using real-time quantitative PCR. Finally, the distribution of Cxcl5 was visualised using immunohistochemistry. RESULTS Compared to controls, Winnie mice exposed to three cycles of DSS displayed inflammation mostly confined to the distal-mid colon with extensive mucosal hyperplasia and regenerative atypia resembling epithelial dysplasia. Dysplasia-like changes were observed in 100% of Winnie mice exposed to DSS, with 55% of these animals displaying changes similar to high-grade dysplasia, whereas high-grade changes were absent in wild-type mice. Occasional penetration of the muscularis mucosae by atypical crypts was observed in 27% of Winnie mice after DSS. Atypical crypts however displayed no evidence of oncogenic nuclear β-catenin accumulation, regardless of histological severity. Expression of Cav1, Trp53 was differentially regulated in the distal colon of Winnie relative to wild-type mice. Expression of Myc and Ccl5 was increased by DSS treatment in Winnie only. Furthermore, increased Ccl5 expression correlated with increased complexity in abnormal crypts. While no overall difference in Cxcl5 mucosal expression was observed between treatment groups, epithelial Cxcl5 protein appeared to be diminished in the

  6. Characterisation of colonic dysplasia-like epithelial atypia in murine colitis.

    PubMed

    Randall-Demllo, Sarron; Fernando, Ruchira; Brain, Terry; Sohal, Sukhwinder Singh; Cook, Anthony L; Guven, Nuri; Kunde, Dale; Spring, Kevin; Eri, Rajaraman

    2016-10-07

    To determine if exacerbation of pre-existing chronic colitis in Winnie (Muc2 mutant) mice induces colonic dysplasia. Winnie mice and C57BL6 as a genotype control, were administered 1% w/v dextran sulphate sodium (DSS) orally, followed by drinking water alone in week-long cycles for a total of three cycles. After the third cycle, mice were killed and colonic tissue collected for histological and immunohistochemical evaluation. Inflammation and severity of dysplasia in the colonic mucosa were assessed in H&E sections of the colon. Epithelial cell proliferation was assessed using Ki67 and aberrant β-catenin signalling assessed with enzyme-based immunohistochemistry. Extracted RNA from colonic segments was used for the analysis of gene expression using real-time quantitative PCR. Finally, the distribution of Cxcl5 was visualised using immunohistochemistry. Compared to controls, Winnie mice exposed to three cycles of DSS displayed inflammation mostly confined to the distal-mid colon with extensive mucosal hyperplasia and regenerative atypia resembling epithelial dysplasia. Dysplasia-like changes were observed in 100% of Winnie mice exposed to DSS, with 55% of these animals displaying changes similar to high-grade dysplasia, whereas high-grade changes were absent in wild-type mice. Occasional penetration of the muscularis mucosae by atypical crypts was observed in 27% of Winnie mice after DSS. Atypical crypts however displayed no evidence of oncogenic nuclear β-catenin accumulation, regardless of histological severity. Expression of Cav1, Trp53 was differentially regulated in the distal colon of Winnie relative to wild-type mice. Expression of Myc and Ccl5 was increased by DSS treatment in Winnie only. Furthermore, increased Ccl5 expression correlated with increased complexity in abnormal crypts. While no overall difference in Cxcl5 mucosal expression was observed between treatment groups, epithelial Cxcl5 protein appeared to be diminished in the atypical epithelium

  7. Polymorphonuclear leukocyte transmigration promotes invasion of colonic epithelial monolayer by Shigella flexneri.

    PubMed Central

    Perdomo, J J; Gounon, P; Sansonetti, P J

    1994-01-01

    In vivo and in vitro, Shigella flexneri, an invasive pathogen of the human colon, cannot invade epithelial cells through their apical pole. To identify ways by which it may reach the cellular basolateral domain in order to invade, we have established an assay using the human colonic T-84 cell line grown on permeable filters. Human PMN were added to the basal pole of the cells, and invasive shigellae to their apical pole. Apical addition of bacteria induced strong transmigration of PMN, reaching a maximum after 1 h of incubation. Transmigration depended on a receptor-specific interaction since it was inhibited by an anti-CD18 monoclonal antibody that antagonizes binding of MAC1 on its putative epithelial cell receptor. After 1 h of PMN transmigration, shigellae started to invade the monolayer in areas of intense PMN infiltration. Invasion was clearly dependant on PMN transmigration since it was also inhibited by addition of an anti-CD18 monoclonal antibody. This in vitro assay is consistent with in vivo observations showing early PMN efflux within colonic crypts in the course of shigellosis. PMN transmigration may therefore allow invasion in the colon by opening the paracellular pathway to invasive microorganisms. Images PMID:7906696

  8. Preparation and characterization of viable epithelial cells from rabbit distal colon

    SciTech Connect

    Kaunitz, J.D. Univ. of California, Los Angeles )

    1988-04-01

    A preparation of viable epithelial cells, suitable for transport studies, was prepared from rabbit distal colon. Enzymatic digestion of scraped mucosa liberated a population of intact colonic glands that were dissociated into cells by gentle agitation in culture medium. Metabolism of ({sup 14}C)glucose was constant over 2 h and was inhibited 70% by 1 mM ouabain. Cells were loaded with the trapped, pH-sensitive fluorescent dye 2{prime}-7{prime}-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxy methyl ester and leaked 0.1% of the dye per minute. Cell pH was 7.21 {+-} 0.03 in pH 7.4. Ringer medium. Intracellular potassium concentration, as measured by a nigericin null-point technique, was 128 {+-} 8 mM. Plasma membrane potential measured with the potential-sensitive fluorescent dye 3,3-dipropylthiadicarboxycanine iodide was -52 {+-} 2 mV. Recovery of intracellular pH in acid-loaded cells occurred after exposure to sodium-containing medium and was inhibited by 5 {times} 10{sup {minus}4} M amiloride. It is concluded that viable epithelial cells can be prepared from rabbit distal colon with relative simplicity and in high yield. These cells are suitable for measurement of intracellular pH and membrane potential and are thus a convenient model system for study of colonic cell physiology.

  9. Changes in the Luminal Environment of the Colonic Epithelial Cells and Physiopathological Consequences.

    PubMed

    Blachier, François; Beaumont, Martin; Andriamihaja, Mireille; Davila, Anne-Marie; Lan, Annaïg; Grauso, Marta; Armand, Lucie; Benamouzig, Robert; Tomé, Daniel

    2017-03-01

    Evidence, mostly from experimental models, has accumulated, indicating that modifications of bacterial metabolite concentrations in the large intestine luminal content, notably after changes in the dietary composition, may have important beneficial or deleterious consequences for the colonic epithelial cell metabolism and physiology in terms of mitochondrial energy metabolism, reactive oxygen species production, gene expression, DNA integrity, proliferation, and viability. Recent data suggest that for some bacterial metabolites, like hydrogen sulfide and butyrate, the extent of their oxidation in colonocytes affects their capacity to modulate gene expression in these cells. Modifications of the luminal bacterial metabolite concentrations may, in addition, affect the colonic pH and osmolarity, which are known to affect colonocyte biology per se. Although the colonic epithelium appears able to face, up to some extent, changes in its luminal environment, notably by developing a metabolic adaptive response, some of these modifications may likely affect the homeostatic process of colonic epithelium renewal and the epithelial barrier function. The contribution of major changes in the colonocyte luminal environment in pathological processes, like mucosal inflammation, preneoplasia, and neoplasia, although suggested by several studies, remains to be precisely evaluated, particularly in a long-term perspective.

  10. Aberrant, ectopic expression of VEGF and VEGF receptors 1 and 2 in malignant colonic epithelial cells. Implications for these cells growth via an autocrine mechanism

    SciTech Connect

    Ahluwalia, Amrita; Jones, Michael K.; Szabo, Sandor; Tarnawski, Andrzej S.

    2013-08-09

    Highlights: •Malignant colonic epithelial cells express VEGF and its receptors. •Cultured colon cancer cells secrete VEGF into the medium. •Inhibition of VEGF receptor significantly decreases colon cancer cell proliferation. •VEGF is critical for colon cancer cell growth. -- Abstract: Vascular endothelial growth factor A (referred to as VEGF) is implicated in colon cancer growth. Currently, the main accepted mechanism by which VEGF promotes colon cancer growth is via the stimulation of angiogenesis, which was originally postulated by late Judah Folkman. However, the cellular source of VEGF in colon cancer tissue; and, the expression of VEGF and its receptors VEGF-R1 and VEGF-R2 in colon cancer cells are not fully known and are subjects of controversy. Material and methods: We examined and quantified expression of VEGF, VEGF-R1 and VEGF-R2 in three different human colonic tissue arrays containing sections of adenocarcinoma (n = 43) and normal mucosa (n = 41). In human colon cancer cell lines HCT116 and HT29 and normal colon cell lines NCM356 and NCM460, we examined expression of VEGF, VEGF-R1 and VEGF-R2 mRNA and protein, VEGF production and secretion into the culture medium; and, the effect of a potent, selective inhibitor of VEGF receptors, AL-993, on cell proliferation. Results: Human colorectal cancer specimens had strong expression of VEGF in cancer cells and also expressed VEGF-R1 and VEGF-R2.In vitro studies showed that human colon cancer cell lines, HCT116 and HT29, but not normal colonic cell lines, express VEGF, VEGF-R1 and VEGF-R2 and secrete VEGF into the medium up to a concentration 2000 pg/ml within 48 h. Furthermore, we showed that inhibition of VEGF receptors using a specific VEGF-R inhibitor significantly reduced proliferation (by >50%) of cultured colon cancer cell lines. Conclusions: Our findings support the contention that VEGF generated by colon cancer cells stimulates their growth directly through an autocrine mechanism that is

  11. A high-affinity and specific carrier-mediated mechanism for uptake of thiamine pyrophosphate by human colonic epithelial cells.

    PubMed

    Nabokina, Svetlana M; Said, Hamid M

    2012-08-01

    All mammals require exogenous sources of thiamine (vitamin B1), as they lack the ability to synthesize the vitamin. These sources are dietary and bacterial (the latter is in reference to the vitamin, which is synthesized by the normal microflora of the large intestine). Bacterially generated thiamine exists in the free, as well as the pyrophosphorylated [thiamine pyrophosphate (TPP)], form. With no (or very little) phosphatase activity in the colon, we hypothesized that the bacterially generated TPP can also be taken up by colonocytes. To test this hypothesis, we examined [(3)H]TPP uptake in the human-derived, nontransformed colonic epithelial NCM460 cells and purified apical membrane vesicles isolated from the colon of human organ donors. Uptake of TPP by NCM460 cells occurred without metabolic alterations in the transported substrate and 1) was pH- and Na(+)-independent, but energy-dependent, 2) was saturable as a function of concentration (apparent K(m) = 0.157 ± 0.028 μM), 3) was highly specific for TPP and not affected by free thiamine (or its analogs) or by thiamine monophosphate and unrelated folate derivatives, 4) was adaptively regulated by extracellular substrate (TPP) level via what appears to be a transcriptionally mediated mechanism(s), and 5) appeared to be influenced by an intracellular Ca(2+)/calmodulin-mediated regulatory pathway. These findings suggest the involvement of a carrier-mediated mechanism for TPP uptake by colonic NCM460 cells, which was further confirmed by results from studies of native human colonic apical membrane vesicles. The results also suggest that the bacterially synthesized TPP in the large intestine is bioavailable and may contribute to overall body homeostasis of vitamin B1 and, especially, to the cellular nutrition of the local colonocytes.

  12. Preexisting epithelial diversity in normal human livers: a tissue-tethered cytometric analysis in portal/periportal epithelial cells.

    PubMed

    Isse, Kumiko; Lesniak, Andrew; Grama, Kedar; Maier, John; Specht, Susan; Castillo-Rama, Marcela; Lunz, John; Roysam, Badrinath; Michalopoulos, George; Demetris, Anthony J

    2013-04-01

    Routine light microscopy identifies two distinct epithelial cell populations in normal human livers: hepatocytes and biliary epithelial cells (BECs). Considerable epithelial diversity, however, arises during disease states when a variety of hepatocyte-BEC hybrid cells appear. This has been attributed to activation and differentiation of putative hepatic progenitor cells (HPC) residing in the canals of Hering and/or metaplasia of preexisting mature epithelial cells. A novel analytic approach consisting of multiplex labeling, high-resolution whole-slide imaging (WSI), and automated image analysis was used to determine if more complex epithelial cell phenotypes preexist in normal adult human livers, which might provide an alternative explanation for disease-induced epithelial diversity. "Virtually digested" WSI enabled quantitative cytometric analyses of individual cells displayed in a variety of formats (e.g., scatterplots) while still tethered to the WSI and tissue structure. We employed biomarkers specifically associated with mature epithelial forms (HNF4α for hepatocytes, CK19 and HNF1β for BEC) and explored for the presence of cells with hybrid biomarker phenotypes. The results showed abundant hybrid cells in portal bile duct BEC, canals of Hering, and immediate periportal hepatocytes. These bipotential cells likely serve as a reservoir for the epithelial diversity of ductular reactions, appearance of hepatocytes in bile ducts, and the rapid and fluid transition of BEC to hepatocytes, and vice versa. Novel imaging and computational tools enable increased information extraction from tissue samples and quantify the considerable preexistent hybrid epithelial diversity in normal human liver. This computationally enabled tissue analysis approach offers much broader potential beyond the results presented here. Copyright © 2012 American Association for the Study of Liver Diseases.

  13. Depth-cumulated epithelial redox ratio and stromal collagen quantity as quantitative intrinsic indicators for differentiating normal, inflammatory, and dysplastic epithelial tissues

    NASA Astrophysics Data System (ADS)

    Zhuo, Shuangmu; Zheng, Liqin; Chen, Jianxin; Xie, Shusen; Zhu, Xiaoqin; Jiang, Xingshan

    2010-10-01

    Multiphoton microscopy was used to isolate the intrinsic emission contribution of epithelial cellular origins and stromal collagen in normal, inflammatory, and dysplastic epithelial tissues, and quantify the depth-cumulated epithelial redox ratio and stromal collagen quantity. It was found that both inflammatory and dysplastic epithelial tissues display a large decrease in stromal collagen quantity but have very different epithelial redox ratio. These results suggest that probing differences in epithelial redox ratio in addition to stromal collagen quantity can serve as quantitative intrinsic indicators for differentiating normal, inflammatory, and dysplastic epithelial tissues.

  14. Reduced IL-37 Production Increases Spontaneous Chemokine Expressions in Colon Epithelial Cells.

    PubMed

    Günaltay, Sezin; Ghiboub, Mohammed; Hultgren, Olof; Hörnquist, Elisabeth Hultgren

    2017-05-01

    Microscopic colitis, comprising collagenous colitis and lymphocytic colitis, is a common cause of chronic diarrhea. Previously, we showed enhanced chemokine productions in microscopic colitis patients, indicating dysregulated immune cell chemotaxis in the immunopathogenesis. We also showed decreased mRNA of IL-37, mainly regarded as an anti-inflammatory cytokine, in the colonic mucosa of these patients, potentially an important factor for the chronicity of the colitis. Our aim in this study was to understand the possible role of IL-37 in chemokine production using a cell line model. A colon epithelial cell line, T84, was stimulated with the TLR5 ligand flagellin. IL-37 protein production was reduced 20% using the CRISPR/Cas9 system, and the changes in chemokine mRNA and protein expressions were compared to cells transfected with empty plasmid. The 20% reduction in IL-37 protein levels spontaneously increased CCL5, CXCL8, CXCL10, and CXCL11 mRNA and protein expressions. CCL2 mRNA and protein levels were enhanced upon TLR5 stimulation. CCL3, CCL20, and CX3CL1 mRNA expressions were increased either spontaneously or following TLR5 stimulation, whereas CCL4 and CCL22 mRNA expressions were significantly decreased. Even a minor decrease in the ability of colon epithelial cells to produce IL-37 results in altered chemokine expression, mainly an increase in the production of several chemokines. Our results indicate that a decreased IL-37 expression by colon epithelial cells may be an important factor for increasing the recruitment of immune cells and subsequently developing microscopic colitis.

  15. Epithelial vanin-1 controls inflammation-driven carcinogenesis in the colitis-associated colon cancer model.

    PubMed

    Pouyet, Laurent; Roisin-Bouffay, Céline; Clément, Aurélie; Millet, Virginie; Garcia, Stéphane; Chasson, Lionel; Issaly, Nathalie; Rostan, Agathe; Hofman, Paul; Naquet, Philippe; Galland, Franck

    2010-01-01

    Vanin-1 is an epithelial pantetheinase that provides cysteamine to tissue and regulates response to stress. Vanin-1 is expressed by enterocytes, and its absence limits intestinal epithelial cell production of proinflammatory signals. A link between chronic active inflammation and cancer is illustrated in patients with ulcerative colitis, who have an augmented risk of developing colorectal cancer. Indeed, sustained inflammation provides advantageous growth conditions to tumors. We examined whether epithelial cells affect tumorigenesis through vanin-1-dependent modulation of colonic inflammation. To vanin-1(-/-) mice, we applied the colitis-associated cancer (CAC) protocol, which combines injection of azoxymethane (AOM) with repeated administrations of dextran sodium sulfate (DSS). We numbered tumors and quantified macrophage infiltration and molecular markers of cell death and proliferation. We also tested DSS-induced colitis. We scored survival, tissue damages, proinflammatory cytokine production, and tissue regeneration. Finally, we explored activation pathways by biochemical analysis on purified colonic epithelial cells (CECs) and in situ immunofluorescence. Vanin-1(-/-) mice displayed a drastically reduced incidence of colorectal cancer in the CAC protocol and manifested mild clinical signs of DSS-induced colitis. The early impact of vanin-1 deficiency on tumor induction was directly correlated to the amount of inflammation and subsequent epithelial proliferation rather than cell death rate; all this was linked to the modulation of NF-kappaB pathway activation in CECs. These results emphasize the importance of the intestinal epithelium in the control of mucosal inflammation acting as a cofactor in carcinogenesis. This might lead to novel anti-inflammatory strategies useful in cancer therapy.

  16. Substance P induces CCN1 expression via histone deacetylase activity in human colonic epithelial cells.

    PubMed

    Koon, Hon Wai; Shih, David Q; Hing, Tressia C; Chen, Jeremy; Ho, Samantha; Zhao, Dezheng; Targan, Stephan R; Pothoulakis, Charalabos

    2011-11-01

    We have shown that substance P (SP) and its neurokinin-1 receptor (NK-1R) regulate intestinal angiogenesis by increasing expression of protein CYR61 (the cysteine-rich angiogenic inducer 61, or CCN1) in colonic epithelial cells. However, the mechanism involved in SP-induced CCN1 expression has not been studied, and the outcome of increased CCN1 expression in the development of colitis is not fully understood. Because histone deacetylase (HDAC) modulates transcription of several genes involved in inflammation, we investigated participation of HDAC in SP-induced CCN1 expression in human colonic epithelial NCM460 cells overexpressing NK-1R (NCM460-NK-1R) and in primary colonocytes. SP increased HDAC activity with deacetylation and dephosphorylation of nucleosome protein histone H3 in NCM460-NK-1R and/or primary colonocytes. Histone deacetylation and dephosphorylation was observed in colonic mucosa from irritable bowel disease patients. Similarly, colonic mucosal tissues from mice exposed to dextran sulfate sodium showed histone H3 deacetylation and dephosphorylation and increased HDAC activity that was reversed by the NK-1R antagonist CJ-12255. SP-induced increased CCN1 expression in NCM460-NK-1R cells was abolished by pharmacological HDAC inhibition. HDAC overexpression activated basal and SP-induced CCN1 promoter activity. Intracolonic CCN1 overexpression significantly ameliorated dextran sulfate sodium-induced colitis, with reduction of proinflammatory cytokine expression in mice. Thus, SP-mediated CCN1 expression in the inflamed human and mouse colon involves increased HDAC activity. Our results strongly suggest that increased CCN1 expression may be involved in mucosal healing during colitis.

  17. Role of reactive oxygen in bile salt stimulation of colonic epithelial proliferation.

    PubMed Central

    Craven, P A; Pfanstiel, J; DeRubertis, F R

    1986-01-01

    Our previous studies had suggested a link between bile salt stimulation of colonic epithelial proliferation and the release and oxygenation of arachidonate via the lipoxygenase pathway. In the present study, we examined the role of reactive oxygen versus end products of arachidonate metabolism via the cyclooxygenase and lipoxygenase pathways in bile salt stimulation of rat colonic epithelial proliferation. Intracolonic instillation of 5 mM deoxycholate increased mucosal ornithine decarboxylase activity and [3H]thymidine incorporation into DNA. Responses to deoxycholate were abolished by the superoxide dismutase mimetic CuII (3,5 diisopropylsalicylic acid)2 (CuDIPS), and by phenidone or esculetin, which inhibit both lipoxygenase and cyclooxygenase activities. By contrast, indomethacin potentiated the response. Phenidone and esculetin suppressed deoxycholate-induced increases in prostaglandin E2 (PGE2), leukotriene B4 (LTB4), and 5, 12, and 15-hydroxyeicosatetraenoic acid (HETE), whereas CuDIPS had no effect. Indomethacin suppressed only PGE2. Deoxycholate (0.5-5 mM) increased superoxide dismutase sensitive chemiluminescence 2-10-fold and stimulated superoxide production as measured by cytochrome c reduction in colonic mucosal scrapings or crypt epithelium. Bile salt-induced increases in chemiluminescence were abolished by CuDIPS, phenidone, and esculetin, but not by indomethacin. Intracolonic generation of reactive oxygen by xanthine-xanthine oxidase increased colonic mucosal ornithine decarboxylase activity and [3H]thymidine incorporation into DNA approximately twofold. These effects were abolished by superoxide dismutase. The findings support a key role for reactive oxygen, rather than more distal products of either the lipoxygenase or cyclooxygenase pathways, in the stimulation of colonic mucosal proliferation by bile salts. PMID:3005368

  18. Epithelial cell differentiation in normal and transgenic mouse intestinal isografts

    PubMed Central

    1991-01-01

    Transgenes consisting of segments of the rat liver fatty acid-binding protein (L-FABP) gene's 5' non-transcribed domain linked to the human growth hormone (hGH) gene (minus its regulatory elements) have provided useful tools for analyzing the mechanisms that regulate cellular and spatial differentiation of the continuously renewing gut epithelium. We have removed the jejunum from normal and transgenic fetal mice before or coincident with, cytodifferentiation of its epithelium. These segments were implanted into the subcutaneous tissues of young adult CBY/B6 nude mouse hosts to determine whether the bipolar, migration- dependent differentiation pathways of gut epithelial cells can be established and maintained in the absence of its normal luminal environment. Immunocytochemical analysis of isografts harvested 4-6 wk after implantation revealed that activation of the intact endogenous mouse L-FABP gene (fabpl) in differentiating enterocytes is perfectly recapitulated as these cells are translocated along the crypt-to-villus axis. Similarly, Paneth and goblet cells appear to appropriately differentiate as they migrate to the crypt base and villus tip, respectively. The enteroendocrine cell subpopulations present in intact 4-6-wk-old jejunum are represented in these isografts. Their precise spatial distribution along the crypt-to-villus axis mimics that seen in the intact gut. A number of complex interrelationships between enteroendocrine subpopulations are also recapitulated. In both "intact" and isografted jejunum, nucleotides -596 to +21 of the rat L-FABP gene were sufficient to direct efficient expression of the hGH reporter to enterocytes although precocious expression of the transgene occurred in cells located in the upper crypt, before their translocation to the villus base. Inappropriate expression of hGH occurred in a high percentage (greater than 80%) of secretin, gastrin, cholecystokinin, and gastric inhibitory peptide producing enteroendocrine cells present

  19. Entamoeba histolytica contains an occludin-like protein that can alter colonic epithelial barrier function.

    PubMed

    Goplen, Michael; Lejeune, Manigandan; Cornick, Steve; Moreau, France; Chadee, Kris

    2013-01-01

    The exact mechanism by which Entamoeba histolytica disrupts the human colonic epithelium and invades the mucosa has yet to be clearly elucidated. E. histolytica produces a diverse array of putative virulent factors such as glycosidase, cysteine proteinases and amebapore that can modulate and/or disrupt epithelial barrier functions. However, it is currently thought that E. histolytica produces numerous other molecules and strategies to disrupt colonic mucosal defenses. In this study, we document a putative mechanism whereby the parasite alters the integrity of human epithelium by expressing a cognate tight junction protein of the host. We detected this protein as "occludin-like" as revealed by immunoblotting and immunoprecipitation studies and visualization by confocal microscopy using antibodies highly specific for human occludin. We propose that E. histolytica occludin-like protein might displace mucosal epithelial occludin-occludin tight junction interactions resulting in epithelial disruption analogous to sub mucosal human dendritic cells sampling luminal contents. These results indicate that E. histolytica occludin is a putative virulent component that can play a role in the pathogenesis of intestinal amebiasis.

  20. Commensal Bacteria-Dependent Indole Production Enhances Epithelial Barrier Function in the Colon

    PubMed Central

    Shimada, Yosuke; Kinoshita, Makoto; Harada, Kazuo; Mizutani, Masafumi; Masahata, Kazunori; Kayama, Hisako; Takeda, Kiyoshi

    2013-01-01

    Microbiota have been shown to have a great influence on functions of intestinal epithelial cells (ECs). The role of indole as a quorum-sensing (QS) molecule mediating intercellular signals in bacteria has been well appreciated. However, it remains unknown whether indole has beneficial effects on maintaining intestinal barriers in vivo. In this study, we analyzed the effect of indole on ECs using a germ free (GF) mouse model. GF mice showed decreased expression of junctional complex molecules in colonic ECs. The feces of specific pathogen-free (SPF) mice contained a high amount of indole; however the amount was significantly decreased in the feces of GF mice by 27-fold. Oral administration of indole-containing capsules resulted in increased expression of both tight junction (TJ)- and adherens junction (AJ)-associated molecules in colonic ECs in GF mice. In accordance with the increased expression of these junctional complex molecules, GF mice given indole-containing capsules showed higher resistance to dextran sodium sulfate (DSS)-induced colitis. A similar protective effect of indole on DSS-induced epithelial damage was also observed in mice bred in SPF conditions. These findings highlight the beneficial role of indole in establishing an epithelial barrier in vivo. PMID:24278294

  1. Colonic epithelial cell proliferation in a rat model of nongenotoxin-induced colonic neoplasia.

    PubMed

    Wilcox, D K; Higgins, J; Bertram, T A

    1992-09-01

    The effect on colonic cell proliferation of poligeenan, a nongenotoxic polysaccharide that induces colon tumors in rats, was compared with guar gum and carrageenan. Fischer 344 rats were fed a basal diet supplemented with carrageenan and poligeenan fibers for up to 91 days. The quantitative levels of proliferation, location of the proliferating cells, and the ability of the mucosa to readapt by removing the experimental fibers from the diet were tested. The mucosal epithelium exhibited a 5-fold increase in thymidine kinase activity in both the carrageenan and poligeenan groups. Proliferating cells appeared at the luminal surface only in the poligeenan-treated rats, and the number of proliferating cells in the upper third of the crypt increased 35-fold. A second and third set of animals were fed one of the three test diets for either 28 or 64 days, followed by a 28-day recovery period. Proliferation in the guar- and carrageenan-treated groups returned to basal levels. In poligeenan-treated rats, thymidine kinase levels, and proliferating cells in the upper third of the crypt remained 2- and 11-fold, respectively, above controls. The difference in recovery time between the poligeenan group and the others, and the luminal location of proliferating cells may prove useful as markers in understanding early events in the carcinogenic process induced by a nongenotoxin.

  2. Targeted Colonic Claudin-2 Expression Renders Resistance to Epithelial Injury, Induces Immune Suppression and Protects from Colitis

    PubMed Central

    Ahmad, Rizwan; Chaturvedi, Rupesh; Olivares-Villagómez, Danyvid; Habib, Tanwir; Asim, Mohammad; Shivesh, Punit; Polk, Brent D.; Wilson, Keith T.; Washington, Mary K.; Van Kaer, Luc; Dhawan, Punita; Singh, Amar B.

    2014-01-01

    Expression of claudin-2, a tight junction protein, is highly upregulated during inflammatory bowel disease (IBD) and, due to its association with epithelial permeability, has been postulated to promote inflammation. Notably, claudin-2 has also been implicated in the regulation of intestinal epithelial proliferation. However, precise role of claudin-2 in regulating colonic homeostasis remains unclear. Here, we demonstrate, using Villin-Claudin-2 transgenic mice, that increased colonic claudin-2 expression augments mucosal permeability as well as colon and crypt length. Most notably, despite leaky colon, Cl-2TG mice were significantly protected against experimental colitis. Importantly, claudin-2 expression increased colonocyte proliferation and provided protection against colitis-induced colonocyte death in a PI-3Kinase/Bcl-2-dependent manner. However, Cl-2TG mice also demonstrated marked suppression of colitis-induced increases in immune activation and associated signaling, suggesting immune tolerance. Accordingly, colons from naïve Cl-2TG mice harbored significantly increased numbers of regulatory (CD4+Foxp3+) T-cells than WT-littermates. Furthermore, macrophages isolated from Cl-2TG mice colon exhibited immune anergy. Importantly, these immunosuppressive changes were associated with increased synthesis of the immunoregulatory cytokine TGF-β by colonic epithelial cells in Cl-2TG mice compared to WT-littermates. Taken together, our findings reveal a critical albeit complex role of claudin-2 in intestinal homeostasis by regulating epithelial permeability, inflammation and proliferation and suggest novel therapeutic opportunities. PMID:24670427

  3. Entamoeba histolytica induces cell death of HT29 colonic epithelial cells via NOX1-derived ROS.

    PubMed

    Kim, Kyeong Ah; Kim, Ju Young; Lee, Young Ah; Min, Arim; Bahk, Young Yil; Shin, Myeong Heon

    2013-02-01

    Entamoeba histolytica, which causes amoebic colitis and occasionally liver abscess in humans, is able to induce host cell death. However, signaling mechanisms of colon cell death induced by E. histolytica are not fully elucidated. In this study, we investigated the signaling role of NOX in cell death of HT29 colonic epithelial cells induced by E. histolytica. Incubation of HT29 cells with amoebic trophozoites resulted in DNA fragmentation that is a hallmark of apoptotic cell death. In addition, E. histolytica generate intracellular reactive oxygen species (ROS) in a contact-dependent manner. Inhibition of intracellular ROS level with treatment with DPI, an inhibitor of NADPH oxidases (NOXs), decreased Entamoeba-induced ROS generation and cell death in HT29 cells. However, pan-caspase inhibitor did not affect E. histolytica-induced HT29 cell death. In HT29 cells, catalytic subunit NOX1 and regulatory subunit Rac1 for NOX1 activation were highly expressed. We next investigated whether NADPH oxidase 1 (NOX1)-derived ROS is closely associated with HT29 cell death induced by E. histolytica. Suppression of Rac1 by siRNA significantly inhibited Entamoeba-induced cell death. Moreover, knockdown of NOX1 by siRNA, effectively inhibited E. histolytica-triggered DNA fragmentation in HT29 cells. These results suggest that NOX1-derived ROS is required for apoptotic cell death in HT29 colon epithelial cells induced by E. histolytica.

  4. Epithelial IL-22RA1-mediated fucosylation promotes intestinal colonization resistance to an opportunistic pathogen.

    PubMed

    Pham, Tu Anh N; Clare, Simon; Goulding, David; Arasteh, Julia M; Stares, Mark D; Browne, Hilary P; Keane, Jacqueline A; Page, Andrew J; Kumasaka, Natsuhiko; Kane, Leanne; Mottram, Lynda; Harcourt, Katherine; Hale, Christine; Arends, Mark J; Gaffney, Daniel J; Dougan, Gordon; Lawley, Trevor D

    2014-10-08

    Our intestinal microbiota harbors a diverse microbial community, often containing opportunistic bacteria with virulence potential. However, mutualistic host-microbial interactions prevent disease by opportunistic pathogens through poorly understood mechanisms. We show that the epithelial interleukin-22 receptor IL-22RA1 protects against lethal Citrobacter rodentium infection and chemical-induced colitis by promoting colonization resistance against an intestinal opportunistic bacterium, Enterococcus faecalis. Susceptibility of Il22ra1(-/-) mice to C. rodentium was associated with preferential expansion and epithelial translocation of pathogenic E. faecalis during severe microbial dysbiosis and was ameloriated with antibiotics active against E. faecalis. RNA sequencing analyses of primary colonic organoids showed that IL-22RA1 signaling promotes intestinal fucosylation via induction of the fucosyltransferase Fut2. Additionally, administration of fucosylated oligosaccharides to C. rodentium-challenged Il22ra1(-/-) mice attenuated infection and promoted E. faecalis colonization resistance by restoring the diversity of anaerobic commensal symbionts. These results support a model whereby IL-22RA1 enhances host-microbiota mutualism to limit detrimental overcolonization by opportunistic pathogens.

  5. Epithelial IL-22RA1-Mediated Fucosylation Promotes Intestinal Colonization Resistance to an Opportunistic Pathogen

    PubMed Central

    Pham, Tu Anh N.; Clare, Simon; Goulding, David; Arasteh, Julia M.; Stares, Mark D.; Browne, Hilary P.; Keane, Jacqueline A.; Page, Andrew J.; Kumasaka, Natsuhiko; Kane, Leanne; Mottram, Lynda; Harcourt, Katherine; Hale, Christine; Arends, Mark J.; Gaffney, Daniel J.; Dougan, Gordon; Lawley, Trevor D.

    2014-01-01

    Summary Our intestinal microbiota harbors a diverse microbial community, often containing opportunistic bacteria with virulence potential. However, mutualistic host-microbial interactions prevent disease by opportunistic pathogens through poorly understood mechanisms. We show that the epithelial interleukin-22 receptor IL-22RA1 protects against lethal Citrobacter rodentium infection and chemical-induced colitis by promoting colonization resistance against an intestinal opportunistic bacterium, Enterococcus faecalis. Susceptibility of Il22ra1−/− mice to C. rodentium was associated with preferential expansion and epithelial translocation of pathogenic E. faecalis during severe microbial dysbiosis and was ameloriated with antibiotics active against E. faecalis. RNA sequencing analyses of primary colonic organoids showed that IL-22RA1 signaling promotes intestinal fucosylation via induction of the fucosyltransferase Fut2. Additionally, administration of fucosylated oligosaccharides to C. rodentium-challenged Il22ra1−/− mice attenuated infection and promoted E. faecalis colonization resistance by restoring the diversity of anaerobic commensal symbionts. These results support a model whereby IL-22RA1 enhances host-microbiota mutualism to limit detrimental overcolonization by opportunistic pathogens. PMID:25263220

  6. Spectroscopic Microvascular Blood Detection from the Endoscopically Normal Colonic Mucosa: Biomarker for Neoplasia Risk

    PubMed Central

    Roy, Hemant K.; Gomes, Andrew; Turzhitsky, Vladimir; Goldberg, Michael J; Rogers, Jeremy; Ruderman, Sarah; L, Young Kim; Kromine, Alex; Brand, Randall E.; Jameel, Mohammed; Vakil, Parmede; Hasabou, Nahla; Backman, Vadim

    2012-01-01

    Background & Aims We have previously utilized a novel biomedical optics technology, four-dimensional elastically-scattered light fingerprinting, to demonstrate that in experimental colon carcinogenesis, the predysplastic epithelial microvascular blood content is markedly elevated. In order to assess the potential clinical translatability of this putative field effect marker, we characterized the early increase in blood supply (EIBS) in humans in vivo. Methods We developed a novel endoscopically-compatible polarization-gated spectroscopic probe that was capable of measuring oxygenated (Ohb) and deoxygenated (Dhb) hemoglobin specifically in the mucosal microcirculation through polarization-gating. Microvascular blood content was measured in 222 patients from the endoscopically-normal cecum, midtransverse colon and rectum. If polyp was present, readings were taken from the polyp tissue along with the normal mucosa 10 cm and 30 cm proximal and distal to the lesion. Results Tissue phantom studies demonstrated that the probe had outstanding accuracy for hemoglobin determination (r2=0.99). Augmentation of microvasculature blood content was most pronounced within the most superficial (~100µm) layer and dissipated in deeper layers (i.e. submucosa). EIBS was detectable within 30 cm from lesion and the magnitude mirrored adenoma proximity. This occurred for both OHb and DHb, with the effect size being slightly greater for DHb. EIBS correlated with adenoma size and was not engendered by non-neoplastic (hyperplastic) polyps. Conclusions We provide, herein, the first demonstration that in vivo microvascular blood content can be measured and provides an accurate marker of field carcinogenesis. This technological/biological advance has numerous potential applications in CRC screening such as improved polyp detection and risk-stratification. PMID:18722372

  7. Epigenetic differences in normal colon mucosa of cancer patients suggests altered dietary metabolic pathways

    PubMed Central

    Silviera, Matthew L.; Smith, Brian P.; Powell, Jasmine; Sapienza, Carmen

    2012-01-01

    We have compared DNA methylation in normal colon mucosa between colon cancer patients and patients without cancer. We identified significant differences in methylation between the two groups at 114 – 874 genes. The majority of the differences are in pathways involved in the metabolism of carbohydrates, lipids and amino acids. We also compared transcript levels of genes in the insulin-signaling pathway. We found that the mucosa of cancer patients had significantly higher transcript levels of several hormones regulating glucose metabolism and significantly lower transcript levels of a glycolytic enzyme and a key regulator of glucose and lipid homeostasis. The se differences suggest that the normal colon mucosa of cancer patients metabolizes dietary components differently than the colon mucosa of controls. Because the differences identified are present in morphologically normal tissue, they may be diagnostic of colon cancer and/or prognostic of colon cancer susceptibility. PMID:22300984

  8. Anthrax edema toxin inhibits Nox1-mediated formation of reactive oxygen species by colon epithelial cells.

    PubMed

    Kim, Jun-Sub; Bokoch, Gary M

    2009-01-01

    One major route of intoxication by Bacillus anthracis (anthrax) spores is via their ingestion and subsequent uptake by the intestinal epithelium. Anthrax edema toxin (ETx) is an adenylate cyclase that causes persistent elevation of cAMP in intoxicated cells. NADPH oxidase enzymes (Nox1-Nox5, Duox1 and 2) generate reactive oxygen species (ROS) as components of the host innate immune response to bacteria, including Nox1 in gastrointestinal epithelial tissues. We show that ETx effectively inhibits ROS formation by Nox1 in HT-29 colon epithelial cells. This inhibition requires the PKA-mediated phosphorylation of the Nox1-regulatory component, NoxA1, and the subsequent binding of 14-3-3zeta. Inhibition of Nox1-mediated ROS formation in the gut epithelium may be a mechanism used by B. anthracis to circumvent the innate immune response.

  9. HDAC inhibitors induce epithelial-mesenchymal transition in colon carcinoma cells.

    PubMed

    Ji, Meiying; Lee, Eun Jeoung; Kim, Ki Bae; Kim, Yangmi; Sung, Rohyun; Lee, Sang-Jeon; Kim, Don Soo; Park, Seon Mee

    2015-05-01

    The effects of histone deacetylase (HDAC) inhibitors on epithelial-mesenchymal transition (EMT) differ in various types of cancers. We investigated the EMT phenotype in four colon cancer cell lines when challenged with HDAC inhibitors trichostatin A (TSA) and valproic acid (VPA) with or without transforming growth factor-β1 (TGF-β1) treatment. Four colon cancer cell lines with different phenotypes in regards to tumorigenicity, microsatellite stability and DNA mutation were used. EMT phenotypes were assessed by the expression of E-cadherin and vimentin using western blot analysis, immunofluorescence, quantitative real-time RT-PCR following treatment with TSA (100 or 200 nM) or VPA (0.5 mM) with or without TGF-β1 (5 ng/ml) for 24 h. Biological EMT phenotypes were also evaluated by cell morphology, migration and invasion assays. TSA or VPA induced mesenchymal features in the colon carcinoma cells by a decrease in E-cadherin and an increase in vimentin expression at the mRNA and protein levels. Confocal microscopy revealed membranous attenuation or nuclear translocation of E-cadherin and enhanced expression of vimentin. These responses occurred after 6 h and increased until 24 h. Colon cancer cells changed from a round or rectangular shape to a spindle shape with increased migration and invasion ability following TSA or VPA treatment. The susceptibility to EMT changes induced by TSA or VPA was comparable in microsatellite stable (SW480 and HT29) and microsatellite unstable cells (DLD1 and HCT116). TSA or VPA induced a mesenchymal phenotype in the colon carcinoma cells and these effects were augmented in the presence of TGF-β1. HDAC inhibitors require careful caution before their application as new anticancer drugs for colon cancers.

  10. Increased production of BDNF in colonic epithelial cells induced by fecal supernatants from diarrheic IBS patients.

    PubMed

    Wang, Peng; Chen, Fei-Xue; Du, Chao; Li, Chang-Qing; Yu, Yan-Bo; Zuo, Xiu-Li; Li, Yan-Qing

    2015-05-22

    Colonic brain-derived neurotrophic factor (BDNF) plays an essential role in pathogenesis of abdominal pain in diarrhea-predominant irritable bowel syndrome (IBS-D), but regulation on its expression remains unclear. We investigated the role of fecal supernatants (FSN) from IBS-D patients on regulating BDNF expression in colonic epithelial cells of human and mice. Using human Caco-2 cells, we found that IBS-D FSN significantly increased BDNF mRNA and protein levels compared to control FSN, which were remarkably suppressed by the serine protease inhibitor. To further explore the potential mechanisms, we investigated the impact of protease-activated receptor-2 (PAR-2) on BDNF expression. We found a significant increase in PAR-2 expression in Caco-2 after IBS-D FSN stimulation. Knockdown of PAR-2 significantly inhibited IBS-D FSN-induced upregulation of BDNF. Moreover, we found that phosphorylation of p38 MAPK, not NF-κB p65, contributed to PAR-2-mediated BDNF overexpression. To confirm these results, we intracolonically infused IBS-D or control FSN in mice and found that IBS-D FSN significantly elevated colonic BDNF and visceral hypersensitivity in mice, which were both suppressed by the inhibitor of serine protease or antagonist of PAR-2. Together, our data indicate that activation of PAR-2 signaling by IBS-D FSN promotes expression of colonic BDNF, thereby contributing to IBS-like visceral hypersensitivity.

  11. Increased production of BDNF in colonic epithelial cells induced by fecal supernatants from diarrheic IBS patients

    PubMed Central

    Wang, Peng; Chen, Fei-Xue; Du, Chao; Li, Chang-Qing; Yu, Yan-Bo; Zuo, Xiu-Li; Li, Yan-Qing

    2015-01-01

    Colonic brain-derived neurotrophic factor (BDNF) plays an essential role in pathogenesis of abdominal pain in diarrhea-predominant irritable bowel syndrome (IBS-D), but regulation on its expression remains unclear. We investigated the role of fecal supernatants (FSN) from IBS-D patients on regulating BDNF expression in colonic epithelial cells of human and mice. Using human Caco-2 cells, we found that IBS-D FSN significantly increased BDNF mRNA and protein levels compared to control FSN, which were remarkably suppressed by the serine protease inhibitor. To further explore the potential mechanisms, we investigated the impact of protease-activated receptor-2 (PAR-2) on BDNF expression. We found a significant increase in PAR-2 expression in Caco-2 after IBS-D FSN stimulation. Knockdown of PAR-2 significantly inhibited IBS-D FSN-induced upregulation of BDNF. Moreover, we found that phosphorylation of p38 MAPK, not NF-κB p65, contributed to PAR-2-mediated BDNF overexpression. To confirm these results, we intracolonically infused IBS-D or control FSN in mice and found that IBS-D FSN significantly elevated colonic BDNF and visceral hypersensitivity in mice, which were both suppressed by the inhibitor of serine protease or antagonist of PAR-2. Together, our data indicate that activation of PAR-2 signaling by IBS-D FSN promotes expression of colonic BDNF, thereby contributing to IBS-like visceral hypersensitivity. PMID:25998025

  12. AMP-18 protects barrier function of colonic epithelial cells: role of tight junction proteins

    PubMed Central

    Walsh-Reitz, Margaret M.; Huang, Erick F.; Musch, Mark W.; Chang, Eugene B.; Martin, Terence E.; Kartha, Sreedharan; Toback, F. Gary

    2005-01-01

    AMP-18, a novel gastric antrum mucosal protein, and a synthetic peptide of amino acids 77-97, have mitogenic and motogenic properties for epithelial cells. The possibility that AMP-18 is also protective was evaluated in the colonic mucosa of mice and monolayer cultures of human colonic epithelial Caco2/bbe (C2) cells. Administration of AMP peptide to mice with dextran sulfate sodium (DSS)-induced colonic injury delayed the onset of bloody diarrhea, and reduced weight loss. Treatment of C2 cells with AMP peptide protected monolayers against decreases in transepithelial electrical resistance (TER) induced by the oxidant monochloramine, indomethacin, or DSS. A molecular mechanism for these barrier-protective effects was sought by asking if AMP peptide acted on specific tight junction (TJ) proteins. Immunoblots of detergent-insoluble fractions of C2 cells treated with AMP peptide exhibited increased accumulation of specific TJ proteins. Occludin immunoreactivity was also increased in detergent-insoluble fractions obtained from colonic mucosal cells of mice injected with AMP peptide. Laser scanning confocal microscopy (CF) supported the capacity of AMP peptide to enhance accumulation of occludin and ZO-1 in TJ domains of C2 cell monolayers, and together with immunoblot analysis showed that the peptide protected against loss of these TJ proteins following oxidant injury. AMP peptide also protected against a fall in TER during disruption of actin filaments by cytochalasin D, and stabilized perijunctional actin during oxidant injury when assessed by CF. These findings suggest that AMP-18 could protect the intestinal mucosal barrier by acting on specific TJ proteins and stabilizing perijunctional actin. PMID:15961882

  13. Effects of vasopressin on electrolyte transport across isolated colon from normal and dexamethasone-treated rats.

    PubMed Central

    Bridges, R J; Rummel, W; Wollenberg, P

    1984-01-01

    Vasopressin enhanced the absorption of Na+ and Cl- across the short-circuited colon descendens from normal rats. This effect of vasopressin results from an increase in the mucosal to serosal movement of Na+ and Cl- and a decrease in the serosal to mucosal movement of Cl- and was accompanied with a decrease in the short-circuit current (ISC). Neither the base-line absorption of Na+ and Cl-, the vasopressin-induced increase in Na+ and Cl- absorption nor the decrease in ISC were inhibited by amiloride in the colon from normal rats. Colon descendens from rats treated for 3 days with dexamethasone had remarkably higher transmural potential difference (p.d.), tissue conductance (Gt) and ISC. The absorption of Na+ across the short-circuited colon descendens from dexamethasone-treated rats was increased 3-fold when compared to colon from normal rats. The absorption of Cl- in normal rats was reversed to Cl- secretion in treated rats. Amiloride rapidly and reversibly decreased the p.d., Gt and ISC in colon from dexamethasone-treated rats. The transport of Na+ was nearly completely inhibited by amiloride in treated rats. In contrast to its enhancing effects on Na+ absorption in colon from normal rats vasopressin did not enhance Na+ absorption in colon from dexamethasone-treated rats. This enhancement of Cl- absorption by vasopressin was retained in colon from treated rats. This enhancement of Cl- transport was due solely to a decrease in the serosal to mucosal movement of Cl- and was accompanied with a decrease in ISC and Gt. The results support the hypothesis that vasopressin causes inhibition of the electrogenic secretion of Cl- in colon from dexamethasone-treated rats. Furthermore, the results suggest that the increase in the mucosal to serosal movement of Na+ and Cl- and the decrease in the serosal to mucosal movement of Cl- in colon from normal rats are caused by independent effects of vasopressin. PMID:6491990

  14. Blue dye injection does not induce dissemination of epithelial cells during SLN procedure in colon cancer patients.

    PubMed

    Larusson, Hannes J; von Holzen, Urs; Viehl, Carsten T; Rezaeian, Farid; Riehle, Hans-Martin; Oertli, Daniel; Guller, Ulrich; Zuber, Markus

    2014-06-01

    The sentinel lymph node (SLN) procedure for colon cancer patients has been increasingly performed over the past decade and has shown advantages regarding lymph node staging. However, there are concerns that the manipulation of the colon, particularly the blue dye injection, results in isolated tumor cell dissemination to lymph nodes. Therefore, the objective of the present study was to evaluate whether the blue dye injection during the SLN procedure for colon cancer induces epithelial cell dissemination to the regional lymph nodes using a fake SLN procedure as a model. One hundred seventy-four colon cancer patients underwent open oncologic colon resection and SLN procedure according to a standardized protocol. For the fake SLN procedure, blue dye was injected ex vivo, into the subserosa of a nontumor-bearing segment of the resected colon in 37 unselected patients. Three levels of each SLN were stained with H&E and with the pancytokeratin marker AE1/AE3 and were analyzed for the presence of cytokeratin positive cells. Identification of fake SLN was successful in 32 of the 37 patients (86 %). Seventy fake SLN were histologically confirmed. The median number of fake SLN was 2 per patient (range 1-8). None of the fake SLN showed any disseminated epithelial cells. The present prospective study provides compelling evidence that blue dye injection during sentinel lymph node procedure for colon cancer does not induce epithelial cell dissemination to the sentinel lymph nodes. Therefore, isolated tumor cells in sentinel lymph nodes result from a true metastatic process.

  15. Expression and Function of TLR2, TLR4, and Nod2 in Primary Canine Colonic Epithelial Cells

    PubMed Central

    Swerdlow, Mathew P.; Kennedy, Douglas R.; Kennedy, Jeffrey S.; Washabau, Robert J.; Henthorn, Paula S.; Moore, Peter F.; Carding, Simon R.; Felsburg, Peter J.

    2007-01-01

    The gut maintains a delicate balance between the downregulation of inflammatory reactions to commensal bacteria and the capacity to respond to pathogens with vigorous cellular and humoral immune responses. Intestinal epithelial cells, including colonic epithelial cells (CECs) possess many properties of cells of the innate immune system, in particular the ability to recognize and respond to microbial antigens. Recognition of microorganisms by CECs is based upon their recognition of signature molecules, called microbe-associated molecular patterns (MAMP), by pattern recognition receptors (PRR) including membrane Toll-like receptors (TLR) and cytosolic Nod2, an intracellular counterpart of TLRs. The purpose of this study was to determine whether primary CECs from normal dogs express a functional TLR2, TLR4 and Nod2 and whether they are regulated by inflammatory mediators. We show that canine primary CECs express TLR2, TLR4, and Nod2 that can be modulated in response to their respective MAMPs, lipopolysaccharides (LPS) or peptidoglycans (PGN). Furthermore, we demonstrate that these receptors are functional as evidenced by the induction of cytokine gene expression in response to LPS or PGN. PMID:17027090

  16. Mesenchymal Cancer Cell-Stroma Crosstalk Promotes Niche Activation, Epithelial Reversion, and Metastatic Colonization

    PubMed Central

    del Pozo Martin, Yaiza; Park, Danielle; Ramachandran, Anassuya; Ombrato, Luigi; Calvo, Fernando; Chakravarty, Probir; Spencer-Dene, Bradley; Derzsi, Stefanie; Hill, Caroline S.; Sahai, Erik; Malanchi, Ilaria

    2015-01-01

    Summary During metastatic colonization, tumor cells must establish a favorable microenvironment or niche that will sustain their growth. However, both the temporal and molecular details of this process remain poorly understood. Here, we found that metastatic initiating cells (MICs) exhibit a high capacity for lung fibroblast activation as a result of Thrombospondin 2 (THBS2) expression. Importantly, inhibiting the mesenchymal phenotype of MICs by blocking the epithelial-to-mesenchymal transition (EMT)-associated kinase AXL reduces THBS2 secretion, niche-activating ability, and, consequently, metastatic competence. Subsequently, disseminated metastatic cells revert to an AXL-negative, more epithelial phenotype to proliferate and decrease the phosphorylation levels of TGF-β-dependent SMAD2-3 in favor of BMP/SMAD1-5 signaling. Remarkably, newly activated fibroblasts promote this transition. In summary, our data reveal a crosstalk between cancer cells and their microenvironment whereby the EMT status initially triggers and then is regulated by niche activation during metastatic colonization. PMID:26670048

  17. Melanosis coli. A consequence of anthraquinone-induced apoptosis of colonic epithelial cells.

    PubMed Central

    Walker, N. I.; Bennett, R. E.; Axelsen, R. A.

    1988-01-01

    A condition closely resembling human melanosis coli was induced in the guinea pig large intestine by daily oral administration of the anthraquinone danthron. Each treatment caused a transient, dose-related wave of apoptosis of the colonic surface epithelial cells. Most of the resulting apoptotic bodies were phagocytosed by intraepithelial macrophages and carried by them through fenestrae in the epithelial basement membrane to the lamina propria. Here, the apoptotic bodies were transformed into typical lipofuscin pigment in macrophage heterolysosomes. Continued danthron administration caused progressive accumulation of pigmented macrophages in the bowel wall, whereas ongoing migration of pigmented macrophages to regional lymph nodes resulted, after danthron was ceased, in sequential loss of the pigmented cells from the superficial and deep lamina propria. Examination of colonic biopsies from patients with melanosis coli shows increased numbers of apoptotic bodies in the surface epithelium and lamina propria, suggesting implication of the same cellular processes in the formation of the pigment in man. Images Figure 1 Figure 3 Figure 4 Figure 5 PMID:3381879

  18. Effects of luminal thymol on epithelial transport in human and rat colon.

    PubMed

    Kaji, Izumi; Karaki, Shin-ichiro; Kuwahara, Atsukazu

    2011-06-01

    Gut lumen is continually exposed to a great variety of agents, including noxious compounds. Chemical receptors that detect the luminal environment are thought to play an important role as sensors and to modulate gastrointestinal functions. Recently, it has been reported that odorant receptors (ORs) are expressed in the small intestinal mucosa and that odorants stimulate serotonin secretion. However, ion transport in the responses to odorants has rarely been discussed, particularly in relation to the large intestine. In the present study, we examined the effects of the OR ligand thymol on ion transport in human and rat colonic epithelia using an Ussing chamber. In the mucosal-submucosal preparations, the mucosal addition of thymol evoked anion secretion concentration dependently. In addition, dextran (4 kDa) permeability was enhanced by the mucosal treatment with thymol. The response to thymol was not affected by tetrodotoxin (TTX) or piroxicam treatments in human or rat colon. Thymol-evoked electrogenic anion secretion was abolished under Ca(2+)-free conditions or mucosal treatment with transient receptor potential (TRP) A1 blocker (HC-030031). Pretreatment of thymol did not affect electrical field stimulation-evoked anion secretion but significantly attenuated short-chain fatty acid-evoked secretion in a concentration-dependent manner. OR1G1 and TRPA1 expression was investigated in isolated colonic mucosa by RT-PCR. The present results provide evidence that the OR ligand thymol modulates epithelial permeability and electrogenic anion secretion in human and rat colon. The anion secretion by luminal thymol is most likely mediated by direct activation of TRPA1 channel. We suggest that the sensing and responding to odorants in the colon also plays a role in maintaining intestinal homeostasis.

  19. Patterns of DNA methylation in the normal colon vary by anatomical location, gender, and age

    PubMed Central

    Kaz, Andrew M; Wong, Chao-Jen; Dzieciatkowski, Slavomir; Luo, Yanxin; Schoen, Robert E; Grady, William M

    2014-01-01

    Alterations in DNA methylation have been proposed to create a field cancerization state in the colon, where molecular alterations that predispose cells to transformation occur in histologically normal tissue. However, our understanding of the role of DNA methylation in field cancerization is limited by an incomplete characterization of the methylation state of the normal colon. In order to determine the colon’s normal methylation state, we extracted DNA from normal colon biopsies from the rectum, sigmoid, transverse, and ascending colon and assessed the methylation status of the DNA by pyrosequencing candidate loci as well as with HumanMethylation450 arrays. We found that methylation levels of repetitive elements LINE-1 and SAT-α showed minimal variability throughout the colon in contrast to other loci. Promoter methylation of EVL was highest in the rectum and progressively lower in the proximal segments, whereas ESR1 methylation was higher in older individuals. Genome-wide methylation analysis of normal DNA revealed 8388, 82, and 93 differentially methylated loci that distinguished right from left colon, males from females, and older vs. younger individuals, respectively. Although variability in methylation between biopsies and among different colon segments was minimal for repetitive elements, analyses of specific cancer-related genes as well as a genome-wide methylation analysis demonstrated differential methylation based on colon location, individual age, and gender. These studies advance our knowledge regarding the variation of DNA methylation in the normal colon, a prerequisite for future studies aimed at understanding methylation differences indicative of a colon field effect. PMID:24413027

  20. Interleukin-10 prevents epithelial cell apoptosis by regulating IFNγ and TNFα expression in rhesus macaque colon explants

    PubMed Central

    Pan, Diganta; Das, Arpita; Lala, Wendy; Kenway-Lynch, Carys S.; Liu, David X.; Veazey, Ronald S.; Pahar, Bapi

    2013-01-01

    Interleukin-10 (IL-10) is an important immunomodulatory cytokine that plays an obligate role in regulating inflammatory responses. Here we demonstrated the role of IL-10 in regulating crypts length and breadth as well as maintaining the survival of epithelial cells using rhesus colon explant cultures. Anti-IL-10 antibody treatment of colon explant cultures induced increased production of inflammatory cytokines/molecules like IFNγ, TNFα, CD107a and perforin as well as increased epithelial cell apoptosis compared to media controls tested. Our results suggest that IL-10 plays a crucial role in maintaining mucosal homeostasis by regulating mucosal IFNγ and TNFα cytokine production. PMID:23867612

  1. Epithelial transport pathways of rat colon determined in vivo by impulse response analysis.

    PubMed

    Edmonds, C J; Smith, T

    1979-11-01

    1. A method is described for studying transepithelial pathways for the movement of different solutes and water. Using the blood and the secretory curves of changing tracer activity following an intravenous bolus, the rate of transit of molecules together with their impulse response functions, which reflect the transfer processes can be examined. 2. Movements of Na, Cl, I, urea and water from blood to lumen across rat colonic epithelium were all consistent with simple diffusion through a paracellular route. Most of the secreted K, however, passed through a K selective route associated with a significant K epithelial pool. 3. Adding cyanide to the luminal solution caused a reversible fall of transepithelial potential difference associated with changes in the impulse response functions of water, urea and K indicating reduction of the restriction on diffusion. Cellular K content was unaffected. 4. K entered the bulk of the epithelial cellular K almost exclusively from the blood side. A small epithelial K pool, identified by studies with a miniature GM counter, had kinetic characteristics like those of the K selective pathway observed in the studies of impulse response functions.

  2. Gut microbiota facilitates dietary heme-induced epithelial hyperproliferation by opening the mucus barrier in colon.

    PubMed

    Ijssennagger, Noortje; Belzer, Clara; Hooiveld, Guido J; Dekker, Jan; van Mil, Saskia W C; Müller, Michael; Kleerebezem, Michiel; van der Meer, Roelof

    2015-08-11

    Colorectal cancer risk is associated with diets high in red meat. Heme, the pigment of red meat, induces cytotoxicity of colonic contents and elicits epithelial damage and compensatory hyperproliferation, leading to hyperplasia. Here we explore the possible causal role of the gut microbiota in heme-induced hyperproliferation. To this end, mice were fed a purified control or heme diet (0.5 μmol/g heme) with or without broad-spectrum antibiotics for 14 d. Heme-induced hyperproliferation was shown to depend on the presence of the gut microbiota, because hyperproliferation was completely eliminated by antibiotics, although heme-induced luminal cytotoxicity was sustained in these mice. Colon mucosa transcriptomics revealed that antibiotics block heme-induced differential expression of oncogenes, tumor suppressors, and cell turnover genes, implying that antibiotic treatment prevented the heme-dependent cytotoxic micelles to reach the epithelium. Our results indicate that this occurs because antibiotics reinforce the mucus barrier by eliminating sulfide-producing bacteria and mucin-degrading bacteria (e.g., Akkermansia). Sulfide potently reduces disulfide bonds and can drive mucin denaturation and microbial access to the mucus layer. This reduction results in formation of trisulfides that can be detected in vitro and in vivo. Therefore, trisulfides can serve as a novel marker of colonic mucolysis and thus as a proxy for mucus barrier reduction. In feces, antibiotics drastically decreased trisulfides but increased mucin polymers that can be lysed by sulfide. We conclude that the gut microbiota is required for heme-induced epithelial hyperproliferation and hyperplasia because of the capacity to reduce mucus barrier function.

  3. Gut microbiota facilitates dietary heme-induced epithelial hyperproliferation by opening the mucus barrier in colon

    PubMed Central

    Ijssennagger, Noortje; Belzer, Clara; Hooiveld, Guido J.; Dekker, Jan; van Mil, Saskia W. C.; Müller, Michael; Kleerebezem, Michiel; van der Meer, Roelof

    2015-01-01

    Colorectal cancer risk is associated with diets high in red meat. Heme, the pigment of red meat, induces cytotoxicity of colonic contents and elicits epithelial damage and compensatory hyperproliferation, leading to hyperplasia. Here we explore the possible causal role of the gut microbiota in heme-induced hyperproliferation. To this end, mice were fed a purified control or heme diet (0.5 μmol/g heme) with or without broad-spectrum antibiotics for 14 d. Heme-induced hyperproliferation was shown to depend on the presence of the gut microbiota, because hyperproliferation was completely eliminated by antibiotics, although heme-induced luminal cytotoxicity was sustained in these mice. Colon mucosa transcriptomics revealed that antibiotics block heme-induced differential expression of oncogenes, tumor suppressors, and cell turnover genes, implying that antibiotic treatment prevented the heme-dependent cytotoxic micelles to reach the epithelium. Our results indicate that this occurs because antibiotics reinforce the mucus barrier by eliminating sulfide-producing bacteria and mucin-degrading bacteria (e.g., Akkermansia). Sulfide potently reduces disulfide bonds and can drive mucin denaturation and microbial access to the mucus layer. This reduction results in formation of trisulfides that can be detected in vitro and in vivo. Therefore, trisulfides can serve as a novel marker of colonic mucolysis and thus as a proxy for mucus barrier reduction. In feces, antibiotics drastically decreased trisulfides but increased mucin polymers that can be lysed by sulfide. We conclude that the gut microbiota is required for heme-induced epithelial hyperproliferation and hyperplasia because of the capacity to reduce mucus barrier function. PMID:26216954

  4. Colon cancer releases alpha-tocopherol from its O-glycosides better than normal colon tissue.

    PubMed

    Knaś, Małgorzata; Szajda, Sławomir Dariusz; Snarska, Jadwiga; Zalewska-Szajda, Beata; Walejko, Piotr; Borzym-Kluczyk, Malgorzata; Knaś-Karaszewska, Katarzyna; Kepka, Alina; Chojnowska, Sylwia; Waszkiewicz, Napoleon; Zimnoch, Magdalena; Maj, Jadwiga; Hryniewicka, Agnieszka; Dudzik, Danuta; Witkowshi, Stanislaw; Puchalski, Zbigniew; Zwierz, Krzysztof

    2009-01-01

    Free radicals, in a colon, may damage DNA, make difficult DNA repair and change course of post-translational modifications of regulatory proteins, which promote tumor initiation and progression. Therefore risk of colon cancer is closely related to diet and other lifestyle factors. Dietary antioxidants, such as vitamin E, should reduce the levels of harmful oxidation products. However vitamin E is not soluble in water, which decreases its bioavailability. As O-glycosides of alpha-tocopherol are better soluble in water and penetrate to tissues easier than free alpha-tocopherol, the aim of our work was to investigate the rate of release the free tocopherol from its O-glycosides in colon cancer, in comparison to human healthy colon tissue. The activities of enzymes catalysing hydrolysis of alpha-tocopheryl glucoside (1a) and mannoside (1b) as well as p-nitrophenyl beta-glucoside (2a) and mannoside (2b) in cancer and healthy human colon tissues, were determined according to the modified method described by Zwierz et al. The alpha-tocopherol and p-nitrophenol were significantly better released from the respective glucosides and mannosides in cancer tissue than in "healthy" human colon tissues, with p = 0.000947 for la, p = 0.033024 for 1b; p = 0.0028 for 2a, and p = 0.0033 for 2b, respectively. Alpha-tocopherol and p-nitrophenol are released from the O-glycosides of glucose and mannose in significantly higher amount in colon cancer than in healthy tissues. The alpha-tocopherol O-glycosides can be considered as prodrugs in prevention and treatment of the colon cancer.

  5. Colonic epithelial ion transport is not affected in patients with diverticulosis

    PubMed Central

    Osbak, Philip S; Bindslev, Niels; Poulsen, Steen S; Kaltoft, Nicolai; Tilotta, Maria C; Hansen, Mark B

    2007-01-01

    Background Colonic diverticular disease is a bothersome condition with an unresolved pathogenesis. It is unknown whether a neuroepithelial dysfunction is present. The aim of the study was two-fold; (1) to investigate colonic epithelial ion transport in patients with diverticulosis and (2) to adapt a miniaturized Modified Ussing Air-Suction (MUAS) chamber for colonic endoscopic biopsies. Methods Biopsies were obtained from the sigmoid part of the colon. 86 patients were included. All patients were referred for colonoscopy on suspicion of neoplasia and they were without pathological findings at colonoscopy (controls) except for diverticulosis in 22 (D-patients). Biopsies were mounted in MUAS chambers with an exposed area of 5 mm2. Electrical responses to various stimulators and inhibitors of ion transport were investigated together with histological examination. The MUAS chamber was easy to use and reproducible data were obtained. Results Median basal short circuit current (SCC) was 43.8 μA·cm-2 (0.8 – 199) for controls and 59.3 μA·cm-2 (3.0 – 177.2) for D-patients. Slope conductance was 77.0 mS·cm-2 (18.6 – 204.0) equal to 13 Ω·cm2 for controls and 96.6 mS·cm-2 (8.4 – 191.4) equal to 10.3 Ω·cm2 for D-patients. Stimulation with serotonin, theophylline, forskolin and carbachol induced increases in SCC in a range of 4.9 – 18.6 μA·cm-2, while inhibition with indomethacin, bumetanide, ouabain and amiloride decreased SCC in a range of 6.5 – 27.4 μA·cm-2, and all with no significant differences between controls and D-patients. Histological examinations showed intact epithelium and lamina propria before and after mounting for both types of patients. Conclusion We conclude that epithelial ion transport is not significantly altered in patients with diverticulosis and that the MUAS chamber can be adapted for studies of human colonic endoscopic biopsies. PMID:17888183

  6. Berberine promotes recovery of colitis and inhibits inflammatory responses in colonic macrophages and epithelial cells in DSS-treated mice

    PubMed Central

    Wang, Lihong; Shi, Yan; Cao, Hanwei; Liu, Liping; Washington, M. Kay; Chaturvedi, Rupesh; Israel, Dawn A.; Cao, Hailong; Wang, Bangmao; Peek, Richard M.; Wilson, Keith T.; Polk, D. Brent

    2012-01-01

    Inflammatory bowel disease (IBD) results from dysregulation of intestinal mucosal immune responses to microflora in genetically susceptible hosts. A major challenge for IBD research is to develop new strategies for treating this disease. Berberine, an alkaloid derived from plants, is an alternative medicine for treating bacterial diarrhea and intestinal parasite infections. Recent studies suggest that berberine exerts several other beneficial effects, including inducing anti-inflammatory responses. This study determined the effect of berberine on treating dextran sulfate sodium (DSS)-induced intestinal injury and colitis in mice. Berberine was administered through gavage to mice with established DSS-induced intestinal injury and colitis. Clinical parameters, intestinal integrity, proinflammatory cytokine production, and signaling pathways in colonic macrophages and epithelial cells were determined. Berberine ameliorated DSS-induced body weight loss, myeloperoxidase activity, shortening of the colon, injury, and inflammation scores. DSS-upregulated proinflammatory cytokine levels in the colon, including TNF, IFN-γ, KC, and IL-17 were reduced by berberine. Berberine decreased DSS-induced disruption of barrier function and apoptosis in the colon epithelium. Furthermore, berberine inhibited proinflammatory cytokine production in colonic macrophages and epithelial cells in DSS-treated mice and promoted apoptosis of colonic macrophages. Activation of signaling pathways involved in stimulation of proinflammatory cytokine production, including MAPK and NF-κB, in colonic macrophages and epithelial cells from DSS-treated mice was decreased by berberine. In summary, berberine promotes recovery of DSS-induced colitis and exerts inhibitory effects on proinflammatory responses in colonic macrophages and epithelial cells. Thus berberine may represent a new therapeutic approach for treating gastrointestinal inflammatory disorders. PMID:22173918

  7. Normalization of host intestinal mucus layers requires long-term microbial colonization

    PubMed Central

    Johansson, Malin E V; Jakobsson, Hedvig E; Holmén-Larsson, Jessica; Schütte, André; Ermund, Anna; Rodríguez-Piñeiro, Ana M; Arike, Liisa; Wising, Catharina; Svensson, Frida; Bäckhed, Fredrik; Hansson, Gunnar C

    2015-01-01

    SUMMARY The intestinal mucus layer provides a barrier limiting bacterial contact with the underlying epithelium. Mucus structure is shaped by intestinal location and the microbiota. To understand how commensals modulate gut mucus, we examined mucus properties under germ-free (GF) conditions and during microbial colonization. Although the colon mucus structure of GF mice was similar to conventionally raised (Convr) mice, the GF inner mucus layer was penetrable to bacteria-sized beads. During colonization, in which GF mice were gavaged with Convr microbiota, the small intestine mucus required five weeks to be normally detached and colonic inner mucus six weeks to become impenetrable. The composition of the small intestinal microbiota during colonization was similar to Convr donors until three weeks when Bacteroides increased, Firmicutes decreased, and segmented filamentous bacteria became undetectable. These findings highlight the dynamics of mucus layer development and indicate that studies of mature microbe-mucus interactions should be conducted weeks after colonization. PMID:26526499

  8. Normal Function of the Colon and Anorectal Area

    MedlinePlus

    ... What is Constipation Introduction: What is Constipation? Normal Function Common Questions & Mistaken Beliefs Signs & Symptoms Symptoms Overview ... What is Constipation Introduction: What is Constipation? Normal Function Common Questions & Mistaken Beliefs Signs & Symptoms Symptoms Overview ...

  9. Epigenetic Regulation of Normal and Transformed Breast Epithelial Cell Phenotype

    DTIC Science & Technology

    2009-06-01

    of nine cell lines corresponding to two different normal breast cell types isolated from three different individuals ( BPE 2, HME2, BPE3, HME3, BPE4...normal breast cell subtypes a ( BPE and HME) and their transformed derivatives (BPLER and HMLER) The results in Figure 1 indicate that the...process. 5 Table1 Karyotype analysis of two different normal breast cell subtypes a ( BPE and HME) and their

  10. Detection of elements and trace elements in endoscopy biopsies of colonic mucosa in normal and high risks colon cancer patients

    NASA Astrophysics Data System (ADS)

    Buoso, M. C.; Fazinic, S.; Galassini, S.; Lecis, P. E.; Moschini, G.; Makarewicz, M.; Naccarato, R.; Ogris, R.; Shao, H. R.; Sturniolo, G. C.; Valkovic, V.

    1991-05-01

    In the present study efforts are made to obtain a correlation between the trace element (TE) levels in patients and the presence of digestive cancer. The aim of the study is to detect selenium (Se), zinc (Zn) and other TE concentrations in different segments of colonic mucosa and to find out if there is any difference between the normal and pathological colonic mucosa. The concentration data (averages, standard deviations and ranges) obtained by neutron activation analysis and proton induced X-ray emission are presented and the data distribution is analyzed.

  11. Normal and keratoconic corneal epithelial thickness mapping using Fourier-domain optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Li, Yan; Tan, Ou; Huang, David

    2011-03-01

    The detection of early-stage keratoconus is one of the most important safety issues in screening candidates for corneal refractive surgeries. We propose to use epithelial thickness maps to assist the diagnosis of keratoconus. The corneal epithelial thickness in normal and keratoconic eyes was mapped with optical coherence tomography (OCT). A Fourier-domain OCT system capable of acquiring 26,000 axial-scans per second was used. It has an axial resolution of 5μm in cornea. A pachymetry scan pattern (8 radials, 1024 axial-scans each, 6mm diameter, repeat 3 times) centered at the pupil center was used to image the cornea. The 3 repeated radial scans on each meridian were registered and averaged. Then the anterior corneal, posterior corneal and epithelial boundaries were segmented automatically with a computer algorithm by increased signal intensity at corresponding boundaries. The epithelial thickness map was generated by interpolating epithelial thickness profile calculated from each meridian. Normal and keratoconic eyes (24 eyes each) were scanned 3 times. The central epithelial thickness in normal eyes was thicker than those of keratoconic eyes (mean difference 2.1 μm, t-test p=0.05). The epithelium was thinner superiorly than inferiorly in normal eyes (mean difference -1.4+/-1.1μm, p<0.001) while thicker superiorly than inferiorly in keratoconic eyes (2.0+/-4.1 μm, p=0.02).

  12. Bile acids modulate the Golgi membrane fission process via a protein kinase Ceta and protein kinase D-dependent pathway in colonic epithelial cells.

    PubMed

    Byrne, Anne-Marie; Foran, Eilis; Sharma, Ruchika; Davies, Anthony; Mahon, Ciara; O'Sullivan, Jacintha; O'Donoghue, Diarmuid; Kelleher, Dermot; Long, Aideen

    2010-04-01

    Deoxycholic acid (DCA) is a secondary bile acid that modulates signalling pathways in epithelial cells. DCA has been implicated in pathogenesis of colon carcinoma, particularly by activation of the protein kinase C (PKC) pathway. Ursodeoxycholic acid (UDCA), a tertiary bile acid, has been observed to have chemopreventive effects. The aim of this study was to investigate the effect of DCA and UDCA on the subcellular localization and activity of PKCeta and its downstream effects on Golgi structure in a colon cancer cell model. PKCeta expression was localized to the Golgi in HCT116 colon cancer cells. DCA induced fragmentation of the Golgi in these cells following activation of PKCeta and its downstream effector protein kinase D (PKD). Pretreatment of cells with UDCA or a glucocorticoid, dexamethasone, inhibited DCA-induced PKCeta/PKD activation and Golgi fragmentation. Knockdown of glucocorticoid receptor (GR) expression using small interfering RNA or inhibition using the GR antagonist mifepristone attenuated the inhibitory effect of UDCA on Golgi fragmentation. Elevated serum and faecal levels of DCA have been previously reported in patients with ulcerative colitis (UC) and colon cancer. Analysis of Golgi architecture in vivo using tissue microarrays revealed Golgi fragmentation in UC and colorectal cancer tissue. We have demonstrated that DCA can disrupt the structure of the Golgi, an organelle critical for normal cell function. Inhibition of this DCA-induced Golgi fragmentation by UDCA was mediated via the GR. This represents a potential mechanism of observed chemopreventive effects of UDCA in benign and malignant disease of the colon.

  13. Induction of the adenoma-carcinoma progression and Cdc25A-B phosphatases by the trefoil factor TFF1 in human colon epithelial cells.

    PubMed

    Rodrigues, S; Rodrigue, C M; Attoub, S; Fléjou, J F; Bruyneel, E; Bracke, M; Emami, S; Gespach, C

    2006-10-26

    TFF1 is overexpressed in inflammatory diseases and human cancers of the digestive and urogenital systems. To examine the transforming potential of TFF1 in human colon epithelial cells, premalignant PC/AA/C1 adenoma cells (PC) derived from a patient with familial adenomatous polyposis (FAP) were transformed by the TFF1 cDNA and used as a model of the adenoma-carcinoma transition. Constitutive expression of TFF1 increased anchorage-independent cell growth in soft agar, and induced or potentiated the growth of colon PC-TFF1 and kidney MDCKts.src-TFF1 tumor xenografts in athymic mice. This resulted in reduction of thapsigargin-induced apoptosis and promotion of collagen type I invasion through several oncogenic pathways. Using the differential display approach to identify TFF1 target genes, we found that the dual specific phosphatases Cdc25A and B implicated in cell cycle transitions are strongly upregulated under active forms in both PC-TFF1 and HCT8/S11-TFF1 colon cancer cells. Accordingly, TFF1 expression is absent in normal human colon crypts but is induced in correlation with Cdc25a and b transcript levels and tumor grade in familial and sporadic colon adenomas and carcinomas. We propose that TFF1 and Cdc25A-B cooperate with other dominant oncogenic pathways to induce the adenoma and adenocarcinoma transitions. Agents that target TFF1/Cdc25 signaling pathways may be useful for treating patients with TFF1-positive solid tumors.

  14. Differential gene expression in normal and transformed human mammary epithelial cells in response to oxidative stress

    PubMed Central

    Cortes, Diego F; Sha, Wei; Hower, Valerie; Blekherman, Greg; Laubenbacher, Reinhard; Akman, Steven; Torti, Suzy V; Shulaev, Vladimir

    2011-01-01

    Oxidative stress plays a key role in breast carcinogenesis. To investigate whether normal and malignant breast epithelial cells differ in their responses to oxidative stress, we examined the global gene expression profiles of three cell types, representing cancer progression from a normal to a malignant stage, under oxidative stress. Normal human mammary epithelial cells (HMEC), an immortalized cell line (HMLER-1), and a tumorigenic cell line (HMLER-5), were exposed to increased levels of reactive oxygen species (ROS) by treatment with glucose oxidase. Functional analysis of the metabolic pathways enriched with differentially expressed genes demonstrates that normal and malignant breast epithelial cells diverge substantially in their response to oxidative stress. While normal cells exhibit the up-regulation of antioxidant mechanisms, cancer cells are unresponsive to the ROS insult. However, the gene expression response of normal HMEC cells under oxidative stress is comparable to that of the malignant cells under normal conditions, indicating that altered redox status is persistent in breast cancer cells, which makes them resistant to increased generation of ROS. This study discusses some of the possible adaptation mechanisms of breast cancer cells under persistent oxidative stress that differentiate them from the response to acute oxidative stress in normal mammary epithelial cells. PMID:21397008

  15. Low glucose stress decreases cellular NADH and mitochondrial ATP in colonic epithelial cancer cells: Influence of mitochondrial substrates.

    PubMed

    Circu, Magdalena L; Maloney, Ronald E; Aw, Tak Yee

    2017-02-25

    In this study, we investigated how colonic epithelial cells maintained pyridine nucleotide (NADH/NAD(+)) redox homeostasis upon acute metabolic variation imposed by glucose deprivation or supplementation with mitochondrial substrates, succinate and malate/glutamate (M/G). Our results showed that low glucose caused cellular NADH/NAD(+) redox imbalance that diminished lactate dehydrogenase (LDH) activity and resulted in lower lactate contents. The concurrent activation of malic enzyme (ME) suggested a role for malate in preserving cellular pyruvate that remained unchanged at low glucose. Mitochondrial substrates restored cellular NADH/NAD(+) redox homeostasis at low glucose in association with specific compartmental catabolism of mitochondrial substrates. As compared with normal glucose, M/G and low glucose promoted glycolytic ATP production but inhibited mitochondrial-derived ATP generation in association with decreased glucose availability for mitochondrial respiration. At normal glucose, succinate and M/G enhanced mitochondrial respiratory activity, but had minimal impact on mitochondrial-derived ATP production. Collectively, these results are consistent with low glucose-induced NADH/NAD(+) redox imbalance in association with decreased aerobic glycolysis that is reversed by supplementation with M/G but not succinate. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Specific epidermal growth factor receptor autophosphorylation sites promote mouse colon epithelial cell chemotaxis and restitution.

    PubMed

    Yamaoka, Toshimitsu; Frey, Mark R; Dise, Rebecca S; Bernard, Jessica K; Polk, D Brent

    2011-08-01

    Upon ligand binding, epidermal growth factor (EGF) receptor (R) autophosphorylates on COOH-terminal tyrosines, generating docking sites for signaling partners that stimulate proliferation, restitution, and chemotaxis. Specificity for individual EGFR tyrosines in cellular responses has been hypothesized but not well documented. Here we tested the requirement for particular tyrosines, and associated downstream pathways, in mouse colon epithelial cell chemotactic migration. We compared these requirements to those for the phenotypically distinct restitution (wound healing) migration. Wild-type, Y992/1173F, Y1045F, Y1068F, and Y1086F EGFR constructs were expressed in EGFR(-/-) cells; EGF-induced chemotaxis or restitution were determined by Boyden chamber or modified scratch wound assay, respectively. Pharmacological inhibitors of p38, phospholipase C (PLC), Src, MEK, JNK/SAPK, phosphatidylinositol 3-kinase (PI 3-kinase), and protein kinase C (PKC) were used to block EGF-stimulated signaling. Pathway activation was determined by immunoblot analysis. Unlike wild-type EGFR, Y992/1173F and Y1086F EGFR did not stimulate colon epithelial cell chemotaxis toward EGF; Y1045F and Y1068F EGFR partially stimulated chemotaxis. Only wild-type EGFR promoted colonocyte restitution. Inhibition of p38, PLC, and Src, or Grb2 knockdown, blocked chemotaxis; JNK, PI 3-kinase, and PKC inhibitors or c-Cbl knockdown blocked restitution but not chemotaxis. All four EGFR mutants stimulated downstream signaling in response to EGF, but Y992/1173F EGFR was partially defective in PLCγ activation whereas both Y1068F and Y1086F EGFR failed to activate Src. We conclude that specific EGFR tyrosines play key roles in determining cellular responses to ligand. Chemotaxis and restitution, which have different migration phenotypes and physiological consequences, have overlapping but not identical EGFR signaling requirements.

  17. Comparison and correlation of candidal colonization in diabetic patients and normal individuals

    PubMed Central

    2014-01-01

    Background Diabetes mellitus is a common universal endocrine disorder with decreased host immunity towards infections. In these people the most common opportunistic infection is oral candidiasis. Oral candidiasis is most commonly caused by yeast like fungus Candida albicans. In healthy individuals these microorganisms are believed to be commensals but in diabetic patients, it forms severe colonization, even in the absence of any clinically evident oral candidiasis. This type of subclinical colonization can make them more prone to develop deeper mucosal colonization with further dissemination via blood. The aim of this study is to compare the frequency and severity of oral candidal colonization in diabetic patients with normal individuals through cytological method. Methods 30 cases of diabetic patients and 30 cases of normal healthy individuals were examined to determine the oral candidal colonization through oral exfoliative cytological methods. Statistical analysis was done using the Chi - square test. Results A statistically significant increase in the candidal colonization was observed in diabetic patients as compared to normal individuals. Conclusions Oral exfoliative cytological method is an easy and effective chair side technique to assess the oral candidal colonization in the diabetic group. PMID:24991533

  18. LINE-1 hypomethylation in normal colon mucosa is associated with poor survival in Chinese patients with sporadic colon cancer

    PubMed Central

    Wu, Yuchen; Li, Yiwei; Nie, Jia; Li, Dawei; Peng, Junjie; Lian, Peng; Li, Bin; Cai, Guoxiang; Li, Xinxiang; Cai, Sanjun

    2015-01-01

    Genetic and epigenetic pathways are not independent in colorectal cancer (CRC) carcinogenesis. We aimed to determine the influence of various molecular features on Chinese patients' colon cancer-specific survival (CCSS). Various genetic and epigenetic modifications were detected in paired tumor and normal mucosa tissue samples. The prognostic variables regarding patient CCSS were determined. Overall, 127 patients, including 83 males and 44 females, completed a median follow-up of 65 (3–85) months. A mean LINE-1 methylation rate of 64.62% (range, 9.45–86.93) was observed. Hypermethylation at the hMLH1 gene promoter was detected in 26 (20.47%) patients. KRAS was mutated in 52 (40.94%) patients. Sixteen (12.60%) patients were confirmed as microsatellite instability (MSI)-High, and 76 (59.84%) were found to have loss of heterozygosity at 18q. The LINE-1 methylation level, MSI status, perineural invasion and distant metastases were confirmed as independent prognostic factors for patient CCSS. A stratified survival analysis further revealed that certain subgroups of patients with LINE-1 hypomethylation had significantly worse survival (all p < 0.05). Our data revealed that both genetic and epigenetic abnormalities can concurrently exist during colonic tumorigenesis. As a global epigenetic change, LINE-1 hypomethylation in normal colon mucosa might be associated with a worse outcome in certain Chinese patients with colon cancer. PMID:26172297

  19. Multifactorial Patterns of Gene Expression in Colonic Epithelial Cells Predict Disease Phenotypes in Experimental Colitis

    PubMed Central

    Frantz, Aubrey L.; Bruno, Maria E.C.; Rogier, Eric W.; Tuna, Halide; Cohen, Donald A.; Bondada, Subbarao; Chelvarajan, R. Lakshman; Brandon, J. Anthony; Jennings, C. Darrell; Kaetzel, Charlotte S.

    2012-01-01

    Background The pathogenesis of inflammatory bowel disease (IBD) is complex and the need to identify molecular biomarkers is critical. Epithelial cells play a central role in maintaining intestinal homeostasis. We previously identified 5 “signature” biomarkers in colonic epithelial cells (CEC) that are predictive of disease phenotype in Crohn’s disease. Here we investigate the ability of CEC biomarkers to define the mechanism and severity of intestinal inflammation. Methods We analyzed expression of RelA, A20, pIgR, TNF and MIP-2 in CEC of mice with DSS acute colitis or T cell-mediated chronic colitis. Factor analysis was used to combine the 5 biomarkers into 2 multifactorial principal components (PCs). PC scores for individual mice were correlated with disease severity. Results For both colitis models, PC1 was strongly weighted toward RelA, A20 and pIgR, and PC2 was strongly weighted toward TNF and MIP-2, while the contributions of other biomarkers varied depending on the etiology of inflammation. Disease severity was correlated with elevated PC2 scores in DSS colitis and reduced PC1 scores in T cell transfer colitis. Down-regulation of pIgR was a common feature observed in both colitis models and was associated with altered cellular localization of pIgR and failure to transport IgA. Conclusions A multifactorial analysis of epithelial gene expression may be more informative than examining single gene responses in IBD. These results provide insight into the homeostatic and pro-inflammatory functions of CEC in IBD pathogenesis and suggest that biomarker analysis could be useful for evaluating therapeutic options for IBD patients. PMID:23070952

  20. ApoE deficiency promotes colon inflammation and enhances the inflammatory potential of oxidized-LDL and TNF-α in primary colon epithelial cells

    PubMed Central

    El-Bahrawy, Ali H.; Tarhuni, Abdelmetalab; Kim, Hogyoung; Subramaniam, Venkat; Benslimane, Ilyes; Elmajeed, Zakaria Y. Abd; Okpechi, Samuel C.; Ghonim, Mohamed A.; Hemeida, Ramadan A.M.; Abo-yousef, Amira M.; El-Sherbiny, Gamal A.; Abdel-Raheem, Ihab T.; Kim, Jong; Naura, Amarjit S.; Boulares, A. Hamid

    2016-01-01

    Although deficiency in Apolipoprotein E (ApoE) is linked to many diseases, its effect on colon homoeostasis remains unknown. ApoE appears to control inflammation by regulating nuclear factor-κB (NF-κB). The present study was designed to examine whether ApoE deficiency affects factors of colon integrity in vivo and given the likelihood that ApoE deficiency increases oxidized lipids and TNF-α, the present study also examined whether such deficiency enhances the inflammatory potential of oxidized-LDL (oxLDL) and TNF-α in colon epithelial cells (CECs), in vitro. Here we show that ApoE deficiency is associated with chronic inflammation systemically and in colonic tissues as assessed by TNF-α levels. Increased colon TNF-α mRNA coincided with a substantial increase in cyclooxygenase (COX)-2. ApoE deficiency enhanced the potential of oxLDL and TNF-α to induce COX-2 expression as well as several other inflammatory factors in primary CECs. Interestingly, oxLDL enhanced TGF-β expression only in ApoE−/−, but not in wild-type, epithelial cells. ApoE deficiency appears to promote COX-2 expression enhancement through a mechanism that involves persistent NF-κB nuclear localization and PI3 and p38 MAP kinases but independently of Src. In mice, ApoE deficiency promoted a moderate increase in crypt length, which was associated with opposing effects of an increase in cell proliferation and apoptosis at the bottom and top of the crypt respectively. Our results support the notion that ApoE plays a central role in colon homoeostasis and that ApoE deficiency may constitute a risk factor for colon pathologies. PMID:27538678

  1. The differences in colonic mucosal microbiota between normal individual and colon cancer patients by polymerase chain reaction-denaturing gradient gel electrophoresis.

    PubMed

    Huipeng, Wang; Lifeng, Gong; Chuang, Ge; Jiaying, Zhao; Yuankun, Cai

    2014-02-01

    The aim of this study was to analyze the differences in the intestinal composition between normal individuals and colon cancer patients. To establish the criteria for screening a normal individual for colon cancer, human colonic biopsies were obtained at routine colonoscopy. For patients with colon cancer, samples were obtained from cancerous regions. For normal individuals, colonic biopsies were taken from 3 sites of large intestine (descending, transverse, and ascending colon). Thereafter, a comparison of the microbiota structure by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) was carried out. At last, bacterial species were identified by sequencing special bands from DGGE gels and comparing data with sequence databases. With PCR-DGGE, we have discovered that the diversity and richness of the bacterial community from colon cancer patient's colonic mucosa were lower than that of the normal individual's sample. Then, a special DGGE band was found in the colon cancer patients. After sequencing, we confirmed that it had a high level of similarity with bacteroides. Colon cancers are closely related with the alteration of intestinal flora such as the reduction of biodiversity and richness of the bacterial community. Furthermore, the increase in proportion of bacteroides may be directly associated with colon cancer.

  2. Low power interstitial Nd YAG laser photocoagulation in normal and neoplastic rat colon.

    PubMed Central

    Matthewson, K; Barton, T; Lewin, M R; O'Sullivan, J P; Northfield, T C; Bown, S G

    1988-01-01

    The effects of low power (1-2 Watts), long exposure (30-400 seconds), interstitial Nd YAG laser therapy on dimethylhydrazine induced rat colonic neoplasms and normal rat colon have been studied. After a single exposure with appropriate laser parameters, dimethylhydrazine induced rat colonic neoplasms underwent coagulative necrosis, sloughed off over a four day period, and left an ulcer which healed within 28 days. Inadequate laser energy resulted in incomplete tumour necrosis whilst excessive laser power or energy increased the likelihood of perforation. Treatment of normal colon with 1 Watt for 30 seconds or longer resulted in coagulative damage which healed by granulation. Mean colonic bursting pressures were significantly decreased one hour after treatment with 1 Watt for 75 or 100 seconds compared with untreated colon (p less than 0.05 and p less than 0.001 respectively) but not in colon treated with 1 Watt for 30 or 50 seconds. In animals treated with 1 Watt for 100 seconds mean bursting pressures were significantly lower than untreated animals when the animals were killed two, four, and seven days after lasering (p less than 0.001 in each case) but not in animals killed at 11, 17, or 21 days. The technique may be of value in the treatment of some inoperable colorectal cancers and sessile polyps in man. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:3343010

  3. Lactic acid fermentation of germinated barley fiber and proliferative function of colonic epithelial cells in loperamide-induced rats.

    PubMed

    Jeon, Jeong Ryae; Choi, Joon Hyuk

    2010-08-01

    To develop a functional food from the dietary fiber fraction of germinated barley (Hordeum vulgare L.) (GBF), lactic acid fermentation was attempted using Lactobacillus acidophilus, Streptococcus thermophilus, and Bifidobacterium bifidus. The quality characteristics of the lactic acid-fermented product and its effect on gastrointestinal function in an animal model were examined. The anaerobic fermentation of 1% and 2% GBF yielded lactic acid bacteria at 8.9 +/- 1.0 x 10(8) and 1.6 +/- 0.2 x 10(9) colony-forming units/mL, and it was considered acceptable for consumption by sensory assessment. To determine the effect on gastrointestinal function, Sprague-Dawley rats were fed with three types of diets: a normal chow diet and chow diets supplemented with 10% lactic acid bacteria or a yogurt fermented with 2% GBF (GBFY). The rats fed GBFY for 6 weeks gained less body weight, excreted more fecal mass, and had improved gastrointestinal transit as examined with barium sulfate. The effect of GBFY on colonic epithelial proliferation was investigated through loperamide (LPM)-induced constipation in rats. The rats fed with GBFY for 6 weeks were intraperitoneally administered LPM twice daily for 7 days. GBFY supplementation decreased fecal excretion and moisture content in feces and depleted goblet cells as observed by hematoxylin and eosin stain. However, the rats supplemented with GBFY prior to the LPM administration had enhanced bowel movement, mucin secretion, and production of short-chain fatty acids compared with values for the LPM-alone group. Immunohistochemistry revealed that the GBFY supplement increased the numbers of nuclei stained positively for Ki-67 and extended from the base to the middle zone of crypts. These results indicate that GBFY alleviates constipation via the proliferation of the colonic crypts in LPM-administered rats.

  4. Phyllanthus emblica Fruit Extract Activates Spindle Assembly Checkpoint, Prevents Mitotic Aberrations and Genomic Instability in Human Colon Epithelial NCM460 Cells

    PubMed Central

    Guo, Xihan; Wang, Xu

    2016-01-01

    The fruit of Phyllanthus emblica Linn. (PE) has been widely consumed as a functional food and folk medicine in Southeast Asia due to its remarkable nutritional and pharmacological effects. Previous research showed PE delays mitotic progress and increases genomic instability (GIN) in human colorectal cancer cells. This study aimed to investigate the similar effects of PE by the biomarkers related to spindle assembly checkpoint (SAC), mitotic aberrations and GIN in human NCM460 normal colon epithelial cells. Cells were treated with PE and harvested differently according to the biomarkers observed. Frequencies of micronuclei (MN), nucleoplasmic bridge (NPB) and nuclear bud (NB) in cytokinesis-block micronucleus assay were used as indicators of GIN. Mitotic aberrations were assessed by the biomarkers of chromosome misalignment, multipolar division, chromosome lagging and chromatin bridge. SAC activity was determined by anaphase-to- metaphase ratio (AMR) and the expression of core SAC gene budding uninhibited by benzimidazoles related 1 (BubR1). Compared with the control, PE-treated cells showed (1) decreased incidences of MN, NPB and NB (p < 0.01); (2) decreased frequencies of all mitotic aberration biomarkers (p < 0.01); and (3) decreased AMR (p < 0.01) and increased BubR1 expression (p < 0.001). The results revealed PE has the potential to protect human normal colon epithelial cells from mitotic and genomic damages partially by enhancing the function of SAC. PMID:27598149

  5. Phyllanthus emblica Fruit Extract Activates Spindle Assembly Checkpoint, Prevents Mitotic Aberrations and Genomic Instability in Human Colon Epithelial NCM460 Cells.

    PubMed

    Guo, Xihan; Wang, Xu

    2016-09-03

    The fruit of Phyllanthus emblica Linn. (PE) has been widely consumed as a functional food and folk medicine in Southeast Asia due to its remarkable nutritional and pharmacological effects. Previous research showed PE delays mitotic progress and increases genomic instability (GIN) in human colorectal cancer cells. This study aimed to investigate the similar effects of PE by the biomarkers related to spindle assembly checkpoint (SAC), mitotic aberrations and GIN in human NCM460 normal colon epithelial cells. Cells were treated with PE and harvested differently according to the biomarkers observed. Frequencies of micronuclei (MN), nucleoplasmic bridge (NPB) and nuclear bud (NB) in cytokinesis-block micronucleus assay were used as indicators of GIN. Mitotic aberrations were assessed by the biomarkers of chromosome misalignment, multipolar division, chromosome lagging and chromatin bridge. SAC activity was determined by anaphase-to- metaphase ratio (AMR) and the expression of core SAC gene budding uninhibited by benzimidazoles related 1 (BubR1). Compared with the control, PE-treated cells showed (1) decreased incidences of MN, NPB and NB (p < 0.01); (2) decreased frequencies of all mitotic aberration biomarkers (p < 0.01); and (3) decreased AMR (p < 0.01) and increased BubR1 expression (p < 0.001). The results revealed PE has the potential to protect human normal colon epithelial cells from mitotic and genomic damages partially by enhancing the function of SAC.

  6. Short hairpin RNA screen indicates that Klotho beta/FGF19 protein overcomes stasis in human colonic epithelial cells.

    PubMed

    Kim, Jinyong; Eskiocak, Ugur; Stadler, Guido; Lou, Zhenjun; Kuro-o, Makoto; Shay, Jerry W; Wright, Woodring E

    2011-12-16

    Normal human colonic epithelial cells (HCECs) are not immortalized by telomerase alone but also require CDK4. Some human cell types growth-arrest due to stress- or aberrant signaling-induced senescence (stasis). Stasis represents the consequences of growth conditions culture that are inadequate to maintain long-term proliferation. Overexpressed CDK4 titers out p16 and allows cells to ignore the growth arrest signals produced by stasis. To identify factors contributing to the inadequate culture environment, we used a 62,000-member shRNA library to knock down factors cooperating with human telomerase reverse transcriptase (hTERT) in the immortalization of HCECs. Knockdown of Klotho gamma (KLG; also known as KLPH and LCTL) allowed hTERT to immortalize HCECs. KLG is one isoform of the Klotho family of factors that coordinate interaction between different FGF ligands and the FGF receptor. We also found that knockdown of KLG induced another member of the Klotho family, Klotho beta (KLB). Induction of KLB was maintained and could activate ERK1/2 in immortalized cells. Supplementation of the culture medium with the KLB ligand FGF19 had a similar effect on hTERT-expressing HCECs as knockdown of KLG regarding both immortalization and down-regulation of the tumor suppressor Klotho alpha. Together, these data suggest that KLB is an important regulator in the immortalization of HCECs by facilitating FGF19 growth factor signaling.

  7. Capsaicin induces NKCC1 internalization and inhibits chloride secretion in colonic epithelial cells independently of TRPV1

    PubMed Central

    Tang, Xu; Weber, Christopher R.; Shen, Le; Turner, Jerrold R.; Matthews, Jeffrey B.

    2013-01-01

    Colonic chloride secretion is regulated via the neurohormonal and immune systems. Exogenous chemicals (e.g., butyrate, propionate) can affect chloride secretion. Capsaicin, the pungent ingredient of the chili peppers, exerts various effects on gastrointestinal function. Capsaicin is known to activate the transient receptor potential vanilloid type 1 (TRPV1), expressed in the mesenteric nervous system. Recent studies have also demonstrated its presence in epithelial cells but its role remains uncertain. Because capsaicin has been reported to inhibit colonic chloride secretion, we tested whether this effect of capsaicin could occur by direct action on epithelial cells. In mouse colon and model T84 human colonic epithelial cells, we found that capsaicin inhibited forskolin-dependent short-circuit current (FSK-Isc). Using PCR and Western blot, we demonstrated the presence of TRPV1 in colonic epithelial cells. In T84 cells, TRPV1 localized at the basolateral membrane and in vesicular compartments. In permeabilized monolayers, capsaicin activated apical chloride conductance, had no effect on basolateral potassium conductance, but induced NKCC1 internalization demonstrated by immunocytochemistry and basolateral surface biotinylation. AMG-9810, a potent inhibitor of TRPV1, did not prevent the inhibition of the FSK-Isc by capsaicin. Neither resiniferatoxin nor N-oleoyldopamine, two selective agonists of TRPV1, blocked the FSK-Isc. Conversely capsaicin, resiniferatoxin, and N-oleoyldopamine raised intracellular calcium ([Ca2+]i) in T84 cells and AMG-9810 blocked the rise in [Ca2+]i induced by capsaicin and resiniferatoxin suggesting the presence of a functional TRPV1 channel. We conclude that capsaicin inhibits chloride secretion in part by causing NKCC1 internalization, but by a mechanism that appears to be independent of TRPV1. PMID:23139219

  8. DEMONSTRATION OF AN EPITHELIAL ANTIGEN IN COLON BY MEANS OF FLUORESCENT ANTIBODIES FROM CHILDREN WITH ULCERATIVE COLITIS

    PubMed Central

    Broberger, Ove; Perlmann, Peter

    1962-01-01

    Thirteen sera from children with ulcerative colitis were examined for antibodies reacting with constituents of human colonic tissue by means of immunofluorescent methods. 3 out of 10 sera reacted positively when tested by the direct staining method while 6 out of 13 reacted positively when tested by the indirect method with conjugates of rabbit anti-human gamma globulin. The specificity of the reactions could be confirmed by inhibition tests. 16 sera from healthy children and adults yielded completely negative results. The staining capacity of various sera was correlated to their hemagglutinating titer when they were tested with sheep erythrocytes, coated with phenol-water extract of human colon. Absorption experiments indicated that the stainable antigen was also present in the extracts used for the hemagglutination experiments. In unfixed tissue sections, fluorescent antibodies were adsorbed onto the epithelial cells of the mucosa. Adsorption on epithelial basement membranes could not be demonstrated. Fluorescent H agglutinins, isolated from eel serum, were adsorbed onto the same mucosal structures of human colon (blood group O) as the antibodies in the sera of patients with ulcerative colitis. However, any immunological relationship between H substance and the colonic antigen of ulcerative colitis could be ruled out by cross-inhibition and hemagglutination inhibition experiments. Fluorescent serum from patients with rheumatoid arthritis also stained sections of human colon but the localization of the stainable antigens was different from that visualized with the ulcerative colitis sera. Inhibition experiments indicated that the rheumatoid arthritis serum contained antibodies staining colon antigens different from those reacting with antibodies in the ulcerative colitis sera. Sera from patients with systemic lupus erythematosus or with the nephrotic syndrome, which all hemagglutinated erythrocytes coated with colon extract, did not stain the sections of the colon

  9. Biobanking of Fresh-Frozen Human Adenocarcinomatous and Normal Colon Tissues: Which Parameters Influence RNA Quality?

    PubMed Central

    Galissier, Thibaut; Schneider, Christophe; Nasri, Saviz; Kanagaratnam, Lukshe; Fichel, Caroline; Coquelet, Christelle; Diebold, Marie-Danièle; Kianmanesh, Reza; Bellon, Georges; Dedieu, Stéphane; Marchal Bressenot, Aude

    2016-01-01

    Medical research projects become increasingly dependent on biobanked tissue of high quality because the reliability of gene expression is affected by the quality of extracted RNA. Hence, the present study aimed to determine if clinical, surgical, histological, and molecular parameters influence RNA quality of normal and tumoral frozen colonic tissues. RNA Quality Index (RQI) was evaluated on 241 adenocarcinomas and 115 matched normal frozen colon tissues collected between October 2006 and December 2012. RQI results were compared to patients’ age and sex, tumor site, kind of surgery, anastomosis failure, adenocarcinoma type and grade, tumor cell percentage, necrosis extent, HIF-1α and cleaved caspase-3 immunohistochemistry, and BRAF, KRAS and microsatellites status. The RQI was significantly higher in colon cancer tissue than in matched normal tissue. RQI from left-sided colonic cancers was significantly higher than RQI from right-sided cancers. The RNA quality was not affected by ischemia and storage duration. According to histological control, 7.9% of the samples were unsatisfactory because of inadequate sampling. Biobanked tumoral tissues with RQI ≥5 had lower malignant cells to stromal cells ratio than samples with RQI <5 (p <0.05). Cellularity, necrosis extent and mucinous component did not influence RQI results. Cleaved caspase-3 and HIF-1α immunolabelling were not correlated to RQI. BRAF, KRAS and microsatellites molecular status did not influence RNA quality. Multivariate analysis revealed that the tumor location, the surgical approach (laparoscopy versus open colectomy) and the occurrence of anastomotic leakage were the only parameters influencing significantly RQI results of tumor samples. We failed to identify parameter influencing RQI of normal colon samples. These data suggest that RNA quality of colonic adenocarcinoma biospecimens is determined by clinical and surgical parameters. More attention should be paid during the biobanking procedure of

  10. Phenotype transformation of immortalized NCM460 colon epithelial cell line by TGF-β1 is associated with chromosome instability.

    PubMed

    Huang, Chao; Wen, Bin

    2016-10-01

    Transforming growth factor-β1 (TGF-β1) within tumor microenvironment has a pivotal function in cancer initiation and tumorigenesis, and hence this study was to observe the malignant transformation induced by TGF-β1 in an immortalized colon epithelial cell line NCM460 for better understanding the mechanisms of colon carcinogenesis. Immortalized colon epithelial cell line NCM460 was used as the model of this study, and was treated with different concentrations of TGF-β1 for different time. Then, immunofluorescence was performed to observe the change of phenotype hallmarks including adherent junction protein E-cadherin, cytoskeleton protein vimentin, and tight junction marker ZO-1, western blotting analysis was performed to detect the expression of the above three markers and two transcription factors (Snail and Slug) involved in the transformation by TGF-β1. In addition, chromosome instability (CHI) including analysis of DNA-ploid was detected by flow cytometry. Our results revealed significant loss or reduction of ZO-1 and E-cadherin, and robust emergence of vimentin in the cell line NCM460 after a 15-, 20-, and 25-day treatment with 10 ng/ml TGF-β1. Interestingly, 20 and 25 days after stimulation with 5 ng/ml TGF-β1, expression of E-cadherin and ZO-1 revealed a pattern roughly similar to that of 10 ng/ml TGF-β1, especially, both expressions was vanished and vimentin expression was dramatically increased at days 25 after TGF-β1 stimulation. After a stimulation with 10 ng/ml TGF-β1 for 15, 20, and 25 days, the levels of Snail and Slug expression in the cells were significantly up-regulated, compared with the cells treated with TGF-β1 inhibitor LY364947, PBS or balnk control (P < 0.01). Our results found that many abnormal mitotic patterns including lagging chromosomes and anaphase bridges in NCM460 cells were induced by TGF-β1 after its stimulation for 15, 20, and 25 days. Very few mitotic cells with treatment of PBS for 15, 20 and 25 days were

  11. Tissue-specific patterns of gene expression in the epithelium and stroma of normal colon in healthy individuals in an aspirin intervention trial.

    PubMed

    Thomas, Sushma S; Makar, Karen W; Li, Lin; Zheng, Yingye; Yang, Peiying; Levy, Lisa; Rudolph, Rebecca Y; Lampe, Paul D; Yan, Min; Markowitz, Sanford D; Bigler, Jeannette; Lampe, Johanna W; Potter, John D

    2015-12-01

    Regular aspirin use reduces colon adenoma and carcinoma incidence. UDP-glucuronosyltransferases (UGT) are involved in aspirin metabolism and clearance, and variant alleles in UGT1A6 have been shown to alter salicylic acid metabolism and risk of colon neoplasia. In a randomized, cross-over, placebo-controlled trial of 44 healthy men and women, homozygous for UGT1A6*1 or UGT1A6*2, we explored differences between global epithelial and stromal expression, using Affymetrix U133 + 2.0 microarrays and tested effects of 60-day aspirin supplementation (325 mg/d) on epithelial and stromal gene expression and colon prostaglandin E2 (PGE2) levels. We conducted a comprehensive study of differential gene expression between normal human colonic epithelium and stroma from healthy individuals. Although no statistically significant differences in gene expression were observed in response to aspirin or UGT1A6 genotype, we have identified the genes uniquely and reproducibly expressed in each tissue type and have analyzed the biologic processes they represent. Here we describe in detail how the data, deposited in the Gene Expression Omnibus (GEO) - accession number GSE71571 - was generated including the basic analysis as contained in the manuscript published in BMC Medical Genetics with the PMID 25927723 (Thomas et al., 2015 [9]).

  12. Normal cell phenotypes of breast epithelial cells provide the foundation of a breast cancer taxonomy.

    PubMed

    Santagata, Sandro; Ince, Tan A

    2014-12-01

    The current classification system for breast cancer is based on expression of empirical prognostic and predictive biomarkers. As an alternative, we propose a hypothesis-based ontological breast cancer classification modeled after the taxonomy of species in evolutionary biology. This approach uses normal breast epithelial cell types and differentiation lineages as the gold standard to classify tumors. We show that there are at least eleven previously undefined normal cell types in human breast epithelium and that each breast carcinoma is related to one of these normal cell types. We find that triple negative breast cancers do not have a 'basal-like' phenotype. Normal breast epithelial cells conform to four novel hormonal differentiation states and almost all human breast tumors duplicate one of these hormonal differentiation states which have significant survival differences. This ontological classification scheme provides actionable treatment strategies and provides an alternative approach for understanding tumor biology with wide-ranging implications for tumor taxonomy.

  13. Different cytokine response of primary colonic epithelial cells to commensal bacteria.

    PubMed

    Lan, Jing-Gang; Cruickshank, Sheena-Margaret; Singh, Joy-Carmelina-Indira; Farrar, Mark; Lodge, James-Peter-Alan; Felsburg, Peter-John; Carding, Simon-Richard

    2005-06-14

    To determine if primary murine colonic epithelial cells (CEC) respond to commensal bacteria and discriminate between different types of bacteria. A novel CEC: bacteria co-culture system was used to compare the ability of the colonic commensal bacteria, Bacteroides ovatus, E. coli (SLF) and Lactobacillus rhamnosus (LGG) to modulate production of different cytokines (n = 15) by primary CEC. Antibody staining and flow cytometry were used to investigate Toll-like receptor (TLR) expression by CEC directly ex vivo and TLR responsiveness was determined by examining the ability of TLR ligands to influence CEC cytokine production. Primary CEC constitutively expressed functional TLR2 and TLR4. Cultured in complete medium alone, CEC secreted IL-6, MCP-1 and IP-10 the levels of which were significantly increased upon addition of the TLR ligands peptidoglycan (PGN) and lipopolysaccharide (LPS). Exposure to the commensal bacteria induced or up-regulated different patterns of cytokine production and secretion. E. coli induced production of MIP-1alpha/beta and betadefensin3 whereas B. ovatus and L. rhamnosus exclusively induced MCP-1 and MIP-2alpha expression, respectively. TNFalpha, RANTES and MEC were induced or up-regulated in response to some but not all of the bacteria whereas ENA78 and IP-10 were up-regulated in response to all bacteria. Evidence of bacterial interference and suppression of cytokine production was obtained from mixed bacterial: CEC co-cultures. Probiotic LGG suppressed E. coli- and B. ovatus-induced cytokine mRNA accumulation and protein secretion. These observations demonstrate the ability of primary CEC to respond to and discriminate between different strains of commensal bacteria and identify a mechanism by which probiotic bacteria (LGG) may exert anti-inflammatory effects in vivo.

  14. Different cytokine response of primary colonic epithelial cells to commensal bacteria

    PubMed Central

    Lan, Jing-Gang; Cruickshank, Sheena Margaret; Singh, Joy Carmelina Indira; Farrar, Mark; Lodge, James Peter Alan; Felsburg, Peter John; Carding, Simon Richard

    2005-01-01

    AIM: To determine if primary murine colonic epithelial cells (CEC) respond to commensal bacteria and discriminate between different types of bacteria. METHODS: A novel CEC: bacteria co-culture system was used to compare the ability of the colonic commensal bacteria, Bacteroides ovatus, E. coli (SLF) and Lactobacillus rhamnosus (LGG) to modulate production of different cytokines (n = 15) by primary CEC. Antibody staining and flow cytometry were used to investigate Toll-like receptor (TLR) expression by CEC directly ex vivo and TLR responsiveness was determined by examining the ability of TLR ligands to influence CEC cytokine production. RESULTS: Primary CEC constitutively expressed functional TLR2 and TLR4. Cultured in complete medium alone, CEC secreted IL-6, MCP-1 and IP-10 the levels of which were significantly increased upon addition of the TLR ligands peptidoglycan (PGN) and lipopolysaccharide (LPS). Exposure to the commensal bacteria induced or up-regulated different patterns of cytokine production and secretion. E. coli induced production of MIP-1α/β and β defensin3 whereas B. ovatus and L. rhamnosus exclusively induced MCP-1 and MIP-2α expression, respectively. TNFα, RANTES and MEC were induced or up-regulated in response to some but not all of the bacteria whereas ENA78 and IP-10 were up-regulated in response to all bacteria. Evidence of bacterial interference and suppression of cytokine production was obtained from mixed bacterial: CEC co-cultures. Probiotic LGG suppressed E. coli- and B. ovatus-induced cytokine mRNA accumulation and protein secretion. CONCLUSION: These observations demonstrate the ability of primary CEC to respond to and discriminate between different strains of commensal bacteria and identify a mechanism by which probiotic bacteria (LGG) may exert anti-inflammatory effects in vivo. PMID:15948242

  15. Serpin B1 protects colonic epithelial cell via blockage of neutrophil elastase activity and its expression is enhanced in patients with ulcerative colitis.

    PubMed

    Uchiyama, Kazuhiko; Naito, Yuji; Takagi, Tomohisa; Mizushima, Katsura; Hirai, Yasuko; Hayashi, Natsuko; Harusato, Akihito; Inoue, Ken; Fukumoto, Kohei; Yamada, Shinya; Handa, Osamu; Ishikawa, Takeshi; Yagi, Nobuaki; Kokura, Satoshi; Yoshikawa, Toshikazu

    2012-05-15

    Serpin B1 is a monocyte neutrophil elastase (NE) inhibitor and is one of the most efficient inhibitors of NE. In the present study, we investigated the role of serpin B1 in the pathogenesis of ulcerative colitis by using clinical samples and an experimental model. The colonic expression of serpin B1 was determined by real-time polymerase chain reaction (PCR), Western blot analysis, and immunohistological studies in both normal and inflamed mucosa from patients with ulcerative colitis. Serpin B1 mRNA expression was determined by real-time PCR in the mouse dextran sodium sulfate (DSS)-induced colitis model. Young adult mouse colonic epithelial (YAMC) cells were used to determine the role of serpin B1. Serpin B1 gene transfected YAMC cells were treated with H(2)O(2) to measure cell viability. The expression of NE was determined in YAMC cells treated with H(2)O(2). NE-silenced YAMC cells were also treated with H(2)O(2) and then measured for viability. Upregulated expression of serpin B1 in colonic mucosa was confirmed from patients with active ulcerative colitis. Immunohistochemical studies showed that serpin B1 expression was localized not only in inflammatory infiltration cells but also in epithelial cells. Serpin B1 mRNA expression was also increased in colonic mucosa of mouse DSS-induced colitis. Serpin B1-transfected YAMC cells were resistant against the treatment of H(2)O(2). H(2)O(2) treatment significantly induced NE in YAMC cells, and NE-silenced YAMC cells were also resistant against the treatment of H(2)O(2). These results suggest that serpin B1 may be a novel marker of active ulcerative colitis and may play an important role in the pathogenesis of inflammatory bowel disease.

  16. Effect of resveratrol and zinc on intracellular zinc status in normal human prostate epithelial cells

    USDA-ARS?s Scientific Manuscript database

    To evaluate the influence of resveratrol on cellular zinc status, normal human prostate epithelial (NHPrE) cells were treated with 6 levels of resveratrol (0, 0.5, 1, 2.5, 5 and 10 microM) and 4 levels of zinc [0, 4, 16, and 32 microM for zinc-deficient (ZD), zinc-normal (ZN), zinc-adequate (ZA), an...

  17. α-Synuclein in the colon and premotor markers of Parkinson disease in neurologically normal subjects.

    PubMed

    Kim, Joong-Seok; Park, In-Seok; Park, Hyung-Eun; Kim, Su-Young; Yun, Jung A; Jung, Chan Kwon; Sung, Hye-Young; Lee, Jin-Kwon; Kang, Won-Kyung

    2017-01-01

    Extranigral non-motor signs precede the first motor manifestations of Parkinson's disease by many years in some patients. The presence of α-synuclein deposition within colon tissues in patients with Parkinson's disease can aid in identifying early neuropathological changes prior to disease onset. In the present study, we evaluated the roles of non-motor symptoms and signs and imaging biomarkers of nigral neuronal changes and α-synuclein accumulation in the colon. Twelve subjects undergoing colectomy for primary colon cancer were recruited for this study. Immunohistochemical staining for α-synuclein in normal and phosphorylated forms was performed in normally appearing colonic tissue. We evaluated 16 candidate premotor risk factors in this study cohort. Among them, ten subjects showed positive immunostaining with normal- and phosphorylated-α-synuclein. An accumulation of premotor markers in each subject was accompanied with positive normal- and phosphorylated-α-synuclein immunostaining, ranging from 2 to 7 markers per subject, whereas the absence of Lewy bodies in the colon was associated with relative low numbers of premotor signs. A principal component analysis and a cluster analysis of these premotor markers suggest that urinary symptoms were commonly clustered with deposition of peripheral phosphorylated-α-synuclein. Among other premotor marker, color vision abnormalities were related to non-smoking. This mathematical approach confirmed the clustering of premotor markers in preclinical stage of Parkinson's disease. This is the first report showing that α-synuclein in the colon and other premotor markers are related to each other in neurologically normal subjects.

  18. Cigarette smoke extracts induced the colon cancer migration via regulating epithelial mesenchymal transition and metastatic genes in human colon cancer cells.

    PubMed

    Kim, Cho-Won; Go, Ryeo-Eun; Lee, Hae-Miru; Hwang, Kyung-A; Lee, Kyuhong; Kim, Bumseok; Lee, Moo-Yeol; Choi, Kyung-Chul

    2017-02-01

    There was considerable evidence that exposure to cigarette smoke is associated with an increased risk for colon cancer. Nevertheless, the mechanism underlying the relationship between cigarette smoking and colon cancer remains unclear. Moreover, there were only a few studies on effects of complexing substance contained in cigarette smoke on colon cancer. Thus, we further investigated whether cigarette smoke extract (CSE) affects the cell cycle, apoptosis and migration of human metastatic colon cancer cells, SW-620. MTT assay revealed that SW-620 cell proliferation was significantly inhibited following treatments with all CSEs, 3R4F, and two-domestic cigarettes, for 9 days in a concentration-dependent manner. Moreover, CSE treatments decreased cyclin D1 and E1, and increased p21 and p27 proteins by Western blot analysis in SW-620 cells. Additionally, the treatment of the cells with CSE contributed to these effects expressing by apoptosis-related proteins. An increased migration or invasion ability of SW-620 cells following CSE treatment was also confirmed by a scratch or fibronectin invasion assay in vitro. In addition, the protein levels of E-cadherin as an epithelial maker were down-regulated, while the mesenchymal markers, N-cadherin, snail, and slug, were up-regulated in a time-dependent manner. A metastatic marker, cathepsin D, was also down-regulated by CSE treatment. Taken together, these results indicate that CSE exposure in colon cancer cells may deregulate the cell growth by altering the expression of cell cycle-related proteins and pro-apoptotic protein, and stimulate cell metastatic ability by altering epithelial-mesenchymal transition (EMT) markers and cathepsin D expression. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 690-704, 2017.

  19. L-arginine uptake by cationic amino acid transporter 2 is essential for colonic epithelial cell restitution

    PubMed Central

    Singh, Kshipra; Coburn, Lori A.; Barry, Daniel P.; Boucher, Jean-Luc; Chaturvedi, Rupesh

    2012-01-01

    Restoration of the colonic epithelial barrier is an important response during colitis. L-arginine (L-Arg) is a semiessential amino acid that reduces murine colitis induced by Citrobacter rodentium. Cationic amino acid transporter (CAT) proteins increase L-Arg uptake into cells. L-Arg is utilized to produce nitric oxide (NO), by inducible NO synthase (iNOS), or L-ornithine (L-Orn) by arginase (Arg) enzymes. The latter is followed by generation of polyamines by ornithine decarboxylase (ODC) and L-proline (L-Pro) by ornithine aminotransferase (OAT). We show that L-Arg enhanced epithelial restitution in conditionally immortalized young adult mouse colon (YAMC) cells in a wound repair model, and in isolated mouse colonic epithelial cells (CECs), using a cell migration assay. Restitution was impaired by C. rodentium. Wounding induced CAT2, and inhibition of L-Arg uptake by the competitive inhibitor L-lysine (L-Lys) or by CAT2 shRNA, but not CAT1 shRNA, decreased restitution. Migration was impaired in CECs treated with L-Lys or from CAT2−/− mice. Wounding increased Arg1 expression, and inhibition of arginase with S-(2-boronoethyl)-L-cysteine (BEC) or Arg1 shRNA inhibited restitution in YAMC cells; cell migration in CECs was also impaired by BEC. Inhibition of ODC or iNOS did not alter restitution. L-Orn or L-Pro restored restitution in cells treated with BEC or Arg1 shRNA, whereas the polyamine putrescine had no benefit. Wounding increased OAT levels, OAT shRNA inhibited restitution, and L-Pro restored restitution in cells with OAT knockdown. Uptake of L-Arg, and its metabolism by Arg1 to L-Orn and conversion to L-Pro by OAT is essential for colonic epithelial wound repair. PMID:22361732

  20. Titanium dioxide nanoparticles activate IL8-related inflammatory pathways in human colonic epithelial Caco-2 cells

    NASA Astrophysics Data System (ADS)

    Krüger, Kristin; Cossais, François; Neve, Horst; Klempt, Martin

    2014-05-01

    Nanosized titanium dioxide (TiO2) particles are widely used as food additive or coating material in products of the food and pharmaceutical industry. Studies on various cell lines have shown that TiO2 nanoparticles (NPs) induced the inflammatory response and cytotoxicity. However, the influences of TiO2 NPs' exposure on inflammatory pathways in intestinal epithelial cells and their differentiation have not been investigated so far. This study demonstrates that TiO2 NPs with particle sizes ranging between 5 and 10 nm do not affect enterocyte differentiation but cause an activation of inflammatory pathways in the human colon adenocarcinoma cell line Caco-2. 5 and 10 nm NPs' exposures transiently induce the expression of ICAM1, CCL20, COX2 and IL8, as determined by quantitative PCR, whereas larger particles (490 nm) do not. Further, using nuclear factor (NF)-κB reporter gene assays, we show that NP-induced IL8 mRNA expression occurs, in part, through activation of NF-κB and p38 mitogen-activated protein kinase pathways.

  1. Okadaic Acid Toxin at Sublethal Dose Produced Cell Proliferation in Gastric and Colon Epithelial Cell Lines

    PubMed Central

    del Campo, Miguel; Toledo, Héctor; Lagos, Néstor

    2013-01-01

    The aim of this study was to analyze the effect of Okadaic Acid (OA) on the proliferation of gastric and colon epithelial cells, the main target tissues of the toxin. We hypothesized that OA, at sublethal doses, activates multiple signaling pathways, such as Erk and Akt, through the inhibition of PP2A. To demonstrate this, we carried out curves of doses and time response against OA in AGS, MKN-45 and Caco 2 cell lines, and found an increase in the cell proliferation at sublethal doses, at 24 h or 48 h exposure. Indeed, cells can withstand high concentrations of the toxin at 4 h exposure, the time chosen considering the maximum time before total gastric emptying. We have proved that this increased proliferation is due to an overexpression of Cyclin B, a cyclin that promotes the passage from G2 to mitosis. In addition, we have demonstrated that OA induces activation of Akt and Erk in the three cells lines, showing that OA can activate pathways involved in oncogenesis. In conclusion, this study contributes to the knowledge about the possible effects of chronic OA consumption. PMID:24317467

  2. A Neu differentiation factor (NDF) domain essential for proliferation and alterations in morphology of colonic epithelial cells in vitro.

    PubMed

    Whoriskey, J S; Pekar, S K; Elliott, G S; Hara, S; Liu, N; Lenz, D M; Zamborelli, T; Mayer, J P; Tarpley, J E; Lacey, D L; Ratzkin, B; Yoshinaga, S K

    1998-01-01

    The Neu Differentiation Factors (NDFs, also termed "heregulins") are a family of proteins that were first isolated as ligands for the HER2 (ergB2, or p185neu) receptor protein tyrosine kinase. Here we show that NDF acts to stimulate the proliferation and alter the cellular morphology of colonic epithelial cells in culture. Dramatic NDF-induced changes in cellular morphology were noted in the colonic epithelial cell line, LIM 1215. In addition, the expression of specific cell proteins, such as carcinoembryonic antigen and integrin beta 4, was induced in LIM 1215 cells by NDF. These effects were more pronounced with the beta isoform than with the alpha isoform of NDF. The EGF-homology domain of NDF beta was sufficient to stimulate the proliferation and alteration in cell morphology. The use of chemically synthesized chimeric NDF alpha and NDF beta proteins enabled use to identify a region of seven amino acids in the EGF-homology domain of NDF beta that is required for both activities. These in vitro experiments suggest that NDF may act as a regulator of growth and differentiation of colonic epithelial cells in vivo.

  3. HLA-A, -B, -C expression in colon carcinoma mimics that of the normal colonic mucosa and is prognostically relevant.

    PubMed

    Benevolo, Maria; Mottolese, Marcella; Piperno, Giulia; Sperduti, Isabella; Cione, Antonio; Sibilio, Leonardo; Martayan, Aline; Donnorso, Raffaele Perrone; Cosimelli, Maurizio; Giacomini, Patrizio

    2007-01-01

    Whether human leukocyte antigen (HLA)-A, -B, -C expression has any predictive value on the prognosis of human malignancies remains controversial. Herein, monoclonal antibodies with preferential reactivity for HLA-A, HLA-B, and HLA-C (HCA2, HC10, and L31) were used to stain an archival collection of 291 formalin-fixed/paraffin-embedded tissues, comprising neoplastic lesions from stages II and III colon carcinoma patients (n=165), and the uninvolved, morphologically normal mucosae from a subset (n=126) of these patients. Marked staining variability was detected not only in the tumors as in previous studies, but also in the normal paired mucosae. HLA-A, -B, -C expression was similar in approximately two thirds of the available 126 normal/neoplastic pairs, confirming in vivo our previous observation that most tumor cells mimic the HLA phenotypes of their normal counterparts. Both up and down-regulation occurred in the remaining third of the pairs, but did not coincide with high and low expression, respectively, conventionally evaluated on the tumor lesion only. Remarkably, a "paired" evaluation, but not high or low expression in the tumor, was predictive of the clinical outcome. Deviations from the expression in the normal paired mucosa (both increases and decreases) of HCA2-reactive class I molecules (possibly HLA-A), and down-regulation of L31-reactive class I molecules (possibly HLA-C), particularly in tumors from stage II patients, correlated with poor 5-year overall and disease-free survival, hazard risk ranging from 2 to 6, approximately. Thus, a paired immunohistochemical comparison reveals a novel immune evasion strategy that may impact on the prognosis of colon carcinoma.

  4. Normal human epithelial cells regulate the size and morphology of tissue-engineered capillaries.

    PubMed

    Rochon, Marie-Hélène; Fradette, Julie; Fortin, Véronique; Tomasetig, Florence; Roberge, Charles J; Baker, Kathleen; Berthod, François; Auger, François A; Germain, Lucie

    2010-05-01

    The survival of thick tissues/organs produced by tissue engineering requires rapid revascularization after grafting. Although capillary-like structures have been reconstituted in some engineered tissues, little is known about the interaction between normal epithelial cells and endothelial cells involved in the in vitro angiogenic process. In the present study, we used the self-assembly approach of tissue engineering to examine this relationship. An endothelialized tissue-engineered dermal substitute was produced by adding endothelial cells to the tissue-engineered dermal substitute produced by the self-assembly approach. The latter consists in culturing fibroblasts in the medium supplemented with serum and ascorbic acid. A network of tissue-engineered capillaries (TECs) formed within the human extracellular matrix produced by dermal fibroblasts. To determine whether epithelial cells modify TECs, the size and form of TECs were studied in the endothelialized tissue-engineered dermal substitute cultured in the presence or absence of epithelial cells. In the presence of normal keratinocytes from skin, cornea or uterine cervix, endothelial cells formed small TECs (cross-sectional area estimated at less than 50 microm(2)) reminiscent of capillaries found in the skin's microcirculation. In contrast, TECs grown in the absence of epithelial cells presented variable sizes (larger than 50 microm(2)), but the addition of keratinocyte-conditioned media or exogenous vascular endothelial growth factor induced their normalization toward a smaller size. Vascular endothelial growth factor neutralization inhibited the effect of keratinocyte-conditioned media. These results provide new direct evidence that normal human epithelial cells play a role in the regulation of the underlying TEC network, and advance our knowledge in tissue engineering for the production of TEC networks in vitro.

  5. Antioxidant macromolecules in the epithelial lining fluid of the normal human lower respiratory tract.

    PubMed Central

    Cantin, A M; Fells, G A; Hubbard, R C; Crystal, R G

    1990-01-01

    We hypothesized that the alveolar structures may contain extracellular macromolecules with antioxidant properties to defend against oxidants. To evaluate this 51Cr-labeled human lung fibroblasts (HFL-1) and cat lung epithelial cells (AKD) were exposed to a H2O2-generating system and alveolar epithelial lining fluid (ELF) from healthy nonsmokers was tested for its ability to protect the lung cells from H2O2-mediated injury. The ELF provided marked antioxidant protection, with most from a H2O-soluble fraction in the 100-300-kD range. Plasma proteins with anti-H2O2 properties were in insufficient concentrations to provide the antioxidant protection observed. However, catalase, a normal intracellular antioxidant, was present in sufficient concentration to account for most of the observed anti-H2O2 properties of ELF. Depletion of ELF with an anticatalase antibody abolished the anti-H2O2 macromolecular defenses of ELF. Since catalase is not normally released by cells, a likely explanation for its presence in high concentrations in normal ELF is that it is released by lung inflammatory and parenchymal cells onto the epithelial surface of the lower respiratory tract during their normal turnover and collects there due to the slow turnover of ELF. It is likely that catalase in the ELF of normal individuals plays a role in protecting lung parenchymal cells against oxidants present in the extracellular milieu. Images PMID:2394842

  6. Epidermal growth factor stimulates Rac activation through Src and phosphatidylinositol 3-kinase to promote colonic epithelial cell migration.

    PubMed

    Dise, Rebecca S; Frey, Mark R; Whitehead, Robert H; Polk, D Brent

    2008-01-01

    Regulated intestinal epithelial cell migration plays a key role in wound healing and maintenance of a healthy gastrointestinal tract. Epidermal growth factor (EGF) stimulates cell migration and wound closure in intestinal epithelial cells through incompletely understood mechanisms. In this study we investigated the role of the small GTPase Rac in EGF-induced cell migration using an in vitro wound-healing assay. In mouse colonic epithelial (MCE) cell lines, EGF-stimulated wound closure was accompanied by a doubling of the number of cells containing lamellipodial extensions at the wound margin, increased Rac membrane translocation in cells at the wound margin, and rapid Rac activation. Either Rac1 small interfering (si)RNA or a Rac1 inhibitor completely blocked EGF-stimulated wound closure. Whereas EGF failed to activate Rac in colon cells from EGF receptor (EGFR) knockout mice, stable expression of wild-type EGFR restored EGF-stimulated Rac activation and migration. Pharmacological inhibition of either phosphatidylinositol 3-kinase (PI3K) or Src family kinases reduced EGF-stimulated Rac activation. Cotreatment of cells with both inhibitors completely blocked EGF-stimulated Rac activation and localization to the leading edge of cells and lamellipodial extension. Our results present a novel mechanism by which the PI3K and Src signaling cascades cooperate to activate Rac and promote intestinal epithelial cell migration downstream of EGFR.

  7. Empirical comparison of color normalization methods for epithelial-stromal classification in H and E images

    PubMed Central

    Sethi, Amit; Sha, Lingdao; Vahadane, Abhishek Ramnath; Deaton, Ryan J.; Kumar, Neeraj; Macias, Virgilia; Gann, Peter H.

    2016-01-01

    Context: Color normalization techniques for histology have not been empirically tested for their utility for computational pathology pipelines. Aims: We compared two contemporary techniques for achieving a common intermediate goal – epithelial-stromal classification. Settings and Design: Expert-annotated regions of epithelium and stroma were treated as ground truth for comparing classifiers on original and color-normalized images. Materials and Methods: Epithelial and stromal regions were annotated on thirty diverse-appearing H and E stained prostate cancer tissue microarray cores. Corresponding sets of thirty images each were generated using the two color normalization techniques. Color metrics were compared for original and color-normalized images. Separate epithelial-stromal classifiers were trained and compared on test images. Main analyses were conducted using a multiresolution segmentation (MRS) approach; comparative analyses using two other classification approaches (convolutional neural network [CNN], Wndchrm) were also performed. Statistical Analysis: For the main MRS method, which relied on classification of super-pixels, the number of variables used was reduced using backward elimination without compromising accuracy, and test - area under the curves (AUCs) were compared for original and normalized images. For CNN and Wndchrm, pixel classification test-AUCs were compared. Results: Khan method reduced color saturation while Vahadane reduced hue variance. Super-pixel-level test-AUC for MRS was 0.010–0.025 (95% confidence interval limits ± 0.004) higher for the two normalized image sets compared to the original in the 10–80 variable range. Improvement in pixel classification accuracy was also observed for CNN and Wndchrm for color-normalized images. Conclusions: Color normalization can give a small incremental benefit when a super-pixel-based classification method is used with features that perform implicit color normalization while the gain is

  8. Differential expression in normal-adenoma-carcinoma sequence suggests complex molecular carcinogenesis in colon.

    PubMed

    Lee, Seungkoo; Bang, Seunghyun; Song, Kyuyoung; Lee, Inchul

    2006-10-01

    The majority of colon cancers develop from pre-existing adenomas. We analyzed the expression profiles in the sequence of normal colon crypts, adenomas and early-stage carcinomas using microdissected cells from tubular adenomas with foci of malignant transformation. Differentially expressed genes were detected between normal-adenoma and adenoma-carcinoma, and were grouped according to the patterns of expression changes in the sequence. Down-regulated genes in the sequence included PLA2G2A, TSPAN1, PDCD4, FCGBP, AATK, EPLIN, FABP1, AGR2, MTUS1, TSC1, galectin 4 and MT1F. PLA2G2A has been shown to suppress colon tumorigenesis in mice, but the pathobiological role in humans has been controversial. Our data showed continuous down-regulation of PLA2G2A in the sequence supporting an implication in human colon cancer. Tumor suppressor and/ or proapoptotic activities have also been reported in other genes. Up-regulated genes included ribosomal proteins, IER3 and TPR. TGF-beta2 and matrix metalloproteinase 23B were up-regulated in carcinoma but not in adenoma, supporting the pathobiological roles in malignant transformation. Differentially expressed genes partly coincided with those in the adenoma-carcinoma sequence of the stomach, which was published previously, suggesting a partial overlap between the adenoma-carcinoma sequences of the colon and stomach.

  9. Ex vivo characterization of normal and adenocarcinoma colon samples by Mueller matrix polarimetry

    NASA Astrophysics Data System (ADS)

    Ahmad, Iftikhar; Ahmad, Manzoor; Khan, Karim; Ashraf, Sumara; Ahmad, Shakil; Ikram, Masroor

    2015-05-01

    Mueller matrix polarimetry along with polar decomposition algorithm was employed for the characterization of ex vivo normal and adenocarcinoma human colon tissues by polarized light in the visible spectral range (425-725 nm). Six derived polarization metrics [total diattenuation (DT), retardance (RT), depolarization (ΔT), linear diattenuation (DL), retardance (δ), and depolarization (ΔL)] were compared for normal and adenocarcinoma colon tissue samples. The results show that all six polarimetric properties for adenocarcinoma samples were significantly higher as compared to the normal samples for all wavelengths. The Wilcoxon rank sum test illustrated that total retardance is a good candidate for the discrimination of normal and adenocarcinoma colon samples. Support vector machine classification for normal and adenocarcinoma based on the four polarization properties spectra (ΔT, ΔL, RT,and δ) yielded 100% accuracy, sensitivity, and specificity, while both DT and D showed 66.6%, 33.3%, and 83.3% accuracy, sensitivity, and specificity, respectively. The combination of polarization analysis and given classification methods provides a framework to distinguish the normal and cancerous tissues.

  10. Ex vivo characterization of normal and adenocarcinoma colon samples by Mueller matrix polarimetry.

    PubMed

    Ahmad, Iftikhar; Ahmad, Manzoor; Khan, Karim; Ashraf, Sumara; Ahmad, Shakil; Ikram, Masroor

    2015-05-01

    Mueller matrix polarimetry along with polar decomposition algorithm was employed for the characterization of ex vivo normal and adenocarcinoma human colon tissues by polarized light in the visible spectral range (425-725 nm). Six derived polarization metrics [total diattenuation (DT ), retardance (RT ), depolarization(ΔT ), linear diattenuation (DL), retardance (δ), and depolarization (ΔL)] were compared for normal and adenocarcinoma colon tissue samples. The results show that all six polarimetric properties for adenocarcinoma samples were significantly higher as compared to the normal samples for all wavelengths. The Wilcoxon rank sum test illustrated that total retardance is a good candidate for the discrimination of normal and adenocarcinoma colon samples. Support vector machine classification for normal and adenocarcinoma based on the four polarization properties spectra (ΔT , ΔL, RT ,and δ) yielded 100% accuracy, sensitivity, and specificity, while both DTa nd DL showed 66.6%, 33.3%, and 83.3% accuracy, sensitivity, and specificity, respectively. The combination of polarization analysis and given classification methods provides a framework to distinguish the normal and cancerous tissues.

  11. Human Normal Bronchial Epithelial Cells: A Novel In Vitro Cell Model for Toxicity Evaluation

    PubMed Central

    Huang, Haiyan; Xia, Bo; Liu, Hongya; Li, Jie; Lin, Shaolin; Li, Tiyuan; Liu, Jianjun; Li, Hui

    2015-01-01

    Human normal cell-based systems are needed for drug discovery and toxicity evaluation. hTERT or viral genes transduced human cells are currently widely used for these studies, while these cells exhibited abnormal differentiation potential or response to biological and chemical signals. In this study, we established human normal bronchial epithelial cells (HNBEC) using a defined primary epithelial cell culture medium without transduction of exogenous genes. This system may involve decreased IL-1 signaling and enhanced Wnt signaling in cells. Our data demonstrated that HNBEC exhibited a normal diploid karyotype. They formed well-defined spheres in matrigel 3D culture while cancer cells (HeLa) formed disorganized aggregates. HNBEC cells possessed a normal cellular response to DNA damage and did not induce tumor formation in vivo by xenograft assays. Importantly, we assessed the potential of these cells in toxicity evaluation of the common occupational toxicants that may affect human respiratory system. Our results demonstrated that HNBEC cells are more sensitive to exposure of 10~20 nm-sized SiO2, Cr(VI) and B(a)P compared to 16HBE cells (a SV40-immortalized human bronchial epithelial cells). This study provides a novel in vitro human cells-based model for toxicity evaluation, may also be facilitating studies in basic cell biology, cancer biology and drug discovery. PMID:25861018

  12. [Raman spectral characteristics of oral squamous cell carcinoma, epithelial dysplasia and normal mucosa].

    PubMed

    Xue, Lili; Li, Yi; Cai, Qiaoling; Sun, Pei; Luo, Xianyang; Yan, Bing

    2015-01-01

    To investigate the Raman spectral characteristics of oral squamous cell carcinoma, high-grade epithelial dysplasia and normal mucosa. Fifty- six fresh samples of oral carcinoma, 50 of high-grade epithelial dysplasia and 32 of normal mucosa were collected. The i-Raman spectrometer with an optical fiber tube was applied to acquire Raman spectrum. The diagnostic model established by principle component analysis (PCA) and discriminant function analysis (DFA) was used to analyze and classify the spectra of different samples. There were significant differences among the Raman spectra of these samples. Compared with the spectra of normal mucosa, the spectra of oral carcinoma and dysplasia showed strong peaks which were contributed to nucleic acids, proteins and lipids. The diagnostic models established by PCA-DFA could successfully classify these Raman spectra of different samples with a high accuracy of 96.4% (133/138). The model was evaluated by 'Leave one out' cross-validation and reached a high accuracy of 92.8% (128/138). The proliferation and metabolism of oral squamous cell carcinoma and epithelial high-grade dysplasia are more active than normal mucosa. The diagnostic models established by PCA-DFA can classify these Raman spectra of different samples with a high accuracy.

  13. Detection of Keratoconus in Clinically and Algorithmically Topographically Normal Fellow Eyes Using Epithelial Thickness Analysis

    PubMed Central

    Reinstein, Dan Z.; Archer, Timothy J.; Urs, Raksha; Gobbe, Marine; RoyChoudhury, Arindam; Silverman, Ronald H.

    2017-01-01

    PURPOSE To assess the effectiveness of a keratoconus-detection algorithm derived from Artemis very high-frequency (VHF) digital ultrasound (ArcScan Inc., Morrison, CO) epithelial thickness maps in the fellow eye from a series of patients with unilateral keratoconus. METHODS The study included 10 patients with moderate to advanced keratoconus in one eye but a clinically and algorithmically topographically normal fellow eye. VHF digital ultrasound epithelial thickness data were acquired and a previously developed classification model was applied for identification of keratoconus to the clinically normal fellow eyes. Pentacam (Oculus Optikgeräte, Wetzlar, Germany) Belin-Ambrósio Display (BAD) data (5 of 10 eyes), and Orbscan (Bausch & Lomb, Rochester, NY) SCORE data (9 of 10 eyes) were also evaluated. RESULTS Five of the 10 fellow eyes were classified as keratoconic by the VHF digital ultrasound epithelium model. Five of 9 fellow eyes were classified as keratoconic by the SCORE model. For the 5 fellow eyes with Pentacam and VHF digital ultrasound data, one was classified as keratoconic by the VHF digital ultrasound model, one (different) eye by a combined VHF digital ultrasound and Pentacam model, and none by BAD-D alone. CONCLUSIONS Under the assumption that keratoconus is a bilateral but asymmetric disease, half of the ‘normal’ fellow eyes could be found to have keratoconus using epithelial thickness maps. The Orbscan SCORE or the combination of topographic BAD criteria with epithelial maps did not perform better. PMID:26544561

  14. Notch signaling is required for normal prostatic epithelial cell proliferation and differentiation.

    PubMed

    Wang, Xi-De; Leow, Ching Ching; Zha, Jiping; Tang, Zhijun; Modrusan, Zora; Radtke, Freddy; Aguet, Michel; de Sauvage, Frederic J; Gao, Wei-Qiang

    2006-02-01

    Notch pathway is crucial for stem/progenitor cell maintenance, growth and differentiation in a variety of tissues. Using a transgenic cell ablation approach, we found in our previous study that cells expressing Notch1 are crucial for prostate early development and re-growth. Here, we further define the role of Notch signaling in regulating prostatic epithelial cell growth and differentiation using biochemical and genetic approaches in ex vivo or in vivo systems. Treatment of developing prostate grown in culture with inhibitors of gamma-secretase/presenilin, which is required for Notch cleavage and activation, caused a robust increase in proliferation of epithelial cells co-expressing cytokeratin 8 and 14, lack of luminal/basal layer segregation and dramatically reduced branching morphogenesis. Using conditional Notch1 gene deletion mouse models, we found that inactivation of Notch1 signaling resulted in profound prostatic alterations, including increased tufting, bridging and enhanced epithelial proliferation. Cells within these lesions co-expressed both luminal and basal cell markers, a feature of prostatic epithelial cells in predifferentiation developmental stages. Microarray analysis revealed that the gene expression in a number of genetic networks was altered following Notch1 gene deletion in prostate. Furthermore, expression of Notch1 and its effector Hey-1 gene in human prostate adenocarcinomas were found significantly down-regulated compared to normal control tissues. Taken together, these data suggest that Notch signaling is critical for normal cell proliferation and differentiation in the prostate, and deregulation of this pathway may facilitate prostatic tumorigenesis.

  15. Fluticasone Induces Epithelial Injury and Alters Barrier Function in Normal Subjects

    PubMed Central

    MacRedmond, Ruth E.; Singhera, Gurpreet K.; Wadsworth, Samuel J.; Attridge, Susan; Bahzad, Mohammed; Williams, Kristy; Coxson, Harvey O.; White, Steven R.; Dorscheid, Delbert R.

    2014-01-01

    Objective The airway epithelium has a number of roles pivotal to the pathogenesis of asthma, including provision of a physical and immune barrier to the inhaled environment. Dysregulated injury and repair responses in asthma result in loss of airway epithelial integrity. Inhaled corticosteroids are a corner stone of asthma treatment. While effective in controlling asthma symptoms, they fail to prevent airway remodeling. Direct cytopathic effects on the airway epithelium may contribute to this. Methods This study examined the effects of a 4-week treatment regimen of inhaled fluticasone 500 μg twice daily in healthy human subjects. Induced sputum was collected for cell counts and markers of inflammation. Barrier function was examined by diethylenetriaminepentacetic acid (DTPA) clearance measured by nuclear scintillation scan, and albumin concentration in induced sputum. Results Steroid exposure resulted in epithelial injury as measured by a significant increase in the number of airway epithelial cells in induced sputum. There was no change in airway inflammation by induced sputum inflammatory cell counts or cytokine levels. Epithelial shedding was associated with an increase in barrier function, as measured by both a decrease in DTPA clearance and decreased albumin in induced sputum. This likely reflects the normal repair response. Conclusion Inhaled corticosteroids cause injury to normal airway epithelium. These effects warrant further evaluation in asthma, where the dysregulated repair response may contribute to airway remodeling. PMID:25324978

  16. Discrimination between normal and malignant gastric epithelial cells by computer image analysis.

    PubMed

    Weinreb, M; Zajicek, G; Levij, I S

    1984-09-01

    The nuclei of 21 normal and 23 malignant epithelial cells from gastric smears obtained by brushing were analyzed by a black-and-white video camera under computer control. Each nucleus was digitized and its relief smoothed and displayed. A Sobel operator determined the nuclear boundaries and nuclear core area. Eighteen nuclear parameters (form descriptors and gray-value descriptors) were extracted for each nucleus and used as variables for discriminant analysis. Nine of these parameters proved useful for discrimination between normal and malignant gastric epithelial cells, with a correct classification rate of 100%. Of these, the best discriminating variables were the maximal gray value in the core, the maximal horizontal and vertical diameters, the core area and the mean derivative value in the core.

  17. Screening of the residual normal ovarian tissue adjacent to orthotopic epithelial ovarian carcinomas in nude mice.

    PubMed

    Zhu, G H; Wang, S T; Yao, M Z; Cai, J H; Chen, C Y; Yang, Z X; Hong, L; Yang, S Y

    2014-04-16

    The objective of this study was to explore the feasibility and methods of screening the residual normal ovarian tissue adjacent to orthotopic ovarian carcinomas in nude mice. Human epithelial ovarian cancer cells (OVCAR3) were subcutaneously implanted for a tumor source and ovarian orthotopic transplantation. The cancer tissue, proximal paraneoplastic tissue, middle paraneoplastic tissue, remote paraneoplastic tissue, and normal ovarian tissue were removed. CK-7, CA125, p53, survivin, MMP-2, and TIMP-2 expression was detected by reverse transcription polymerase chain reaction. We obtained 35 paraneoplastic residual ovarian tissues with normal biopsies from 40 cases of an orthotopic epithelial ovarian carcinoma model (87.5%). CK-7, CA125, p53, survivin, MMP-2, and TIMP-2 expression was lower in proximal paraneoplastic tissue than in cancer tissue (P < 0.05) and higher than in middle and remote paraneoplastic tissue (P < 0.01). There was no statistically significant difference between the expression of these genes in middle and proximal paraneoplastic tissue as well as among residual normal ovarian tissues with different severity (P > 0.05). In ovarian tissues of 20 normal nude mice, the expression of CK- 7, CA125, p53, survivin, MMP-2, and TIMP-2 was negative. Overall, the expression levels of CK-7, CA125, p53, survivin, MMP-2, TIMP-2, and other molecular markers showed a decreasing trend in the non-cancer tissue direction. The expression levels can be used as standards to screen residual normal ovarian tissue. We can obtain relatively safe normal ovarian tissues adjacent to epithelial ovarian cancer.

  18. Prostaglandin E(2) couples through EP(4) prostanoid receptors to induce IL-8 production in human colonic epithelial cell lines.

    PubMed

    Dey, I; Giembycz, M A; Chadee, K

    2009-02-01

    Prostaglandin (PG) E(2) and interleukin (IL)-8 are simultaneously increased during the inflammation that characterizes numerous pathologies such as inflammatory bowel disease. IL-8 is a potent neutrophil chemo-attractant and activator, and can initiate and/or exacerbate tissue injury. PGE(2) signals principally through prostanoid receptors of the EP(2) and/or EP(4) subtypes to promote cAMP-dependent cellular functions. The aim of this study was to identify the role of the EP(2) and EP(4) receptor subtype(s) on two human colonic epithelial cell lines (Caco-2 and T84), in regulating PGE(2)-induced IL-8 production. To identify the causative receptor, we knocked-down and over-expressed EP(2) and EP(4) receptor subtypes in colonic epithelial cells and studied the effect of several selective EP(2)/EP(4) receptor agonists and antagonists. The inductions of IL-8 and EP receptor mRNA and protein expression were determined by real-time PCR and western blot analysis. The affinity of PGE(2) and Bmax values for the EP(2) and EP(4) receptor on colonic epithelial cells were determined by radioligand-binding assays with [(3)H]PGE(2). PGE(2) had the highest affinity for the EP(4) receptor subtype and promoted a robust stimulation of cAMP-dependent IL-8 synthesis. This effect was mimicked by a selective EP(4) receptor agonist, ONO-AE1-329, and abolished by silencing the EP(4) receptor gene by using siRNA techniques, a selective EP(4) receptor antagonist (ONO-AE3-208) and a selective inhibitor (Rp-cAMP) of cAMP-dependent protein kinase. These findings suggest that initiation and progression of colonic inflammation induced by IL-8 could be mediated, at least in part, by PGE(2) acting via the EP(4) receptor subtype.

  19. Aloe vera gel facilitates re-epithelialization of corneal alkali burn in normal and diabetic rats

    PubMed Central

    Atiba, Ayman; Wasfy, Tamer; Abdo, Walied; Ghoneim, Ahmed; Kamal, Tarek; Shukry, Mustafa

    2015-01-01

    Purpose To investigate the efficacy of topical applied aloe vera (AV) and to facilitate the repair of the standardized alkaline corneal ulcer in normal and diabetic rats. Materials and methods The corneal alkali-burn injury model was established unilaterally in Wistar rats by filter paper saturated with 0.01 M NaOH contacting the eyes for 45 seconds. Rats were divided into four groups: normal control (NC), normal AV (NAV), diabetic control (DC), and diabetic AV (DAV). NAV and DAV groups were treated with AV gel eye drops four times daily, and NC and DC groups were treated with normal saline for 3 days. Corneal epithelial wound closure and degree of edema were recorded using slit lamp and optical coherence tomography at 0, 24, 48, and 72 hours postwounding. Histological examination was conducted to evaluate the degree of inflammation and the healing effect. Results Corneal epithelial wound healing was better in the NAV group than in the NC group, and it was significantly higher in the DAV group than in the DC group (P<0.05). In comparison to the DC group, DAV treated with AV demonstrated a marked reduction in edema at 48 and 72 hours. Histologically, corneal re-epithelialization was complete and higher in DAV group than that in DC group; moreover, the inflammatory cells were increased in DC group than DAV group (P<0.05). Conclusion This study demonstrated the efficacy of AV for enhanced corneal re-epithelialization, as well as reduced inflammatory response after alkali burn in rats; therefore, it could be useful as a therapy for diabetic keratopathy. PMID:26604672

  20. Keratins as markers that distinguish normal and tumor-derived mammary epithelial cells

    SciTech Connect

    Trask, D.K.; Band, V.; Zajchowski, D.A.; Yaswen, P.; Suh, T.; Sager, R. )

    1990-03-01

    Keratin 5 (K5) mRNA and protein are shown to be expressed in normal mammary epithelial cells in culture and are absent from tumor-derived dell lines. To extend these findings, the full complements of keratins in normal, immortalized, and tumor cells were compared. It is shown here that normal cells produce keratins K5, K6, K7, K14, and K17, whereas tumor cells produce mainly keratins K8, K18, and K19. In immortalized cells, which are preneoplastic or partially transformed, the levels of K5 mRNA and protein are lower than in normal cells, whereas the amount of K18 is increased. Thus, K5 is an important marker in the tumorigenic process, distinguishing normal from tumor cells, and decrease K5 expression correlates with tumorigenic progression.

  1. Ultrasonic differentiation of normal versus malignant breast epithelial cells in monolayer cultures

    PubMed Central

    Doyle, Timothy E.; Goodrich, Jeffrey B.; Ambrose, Brady J.; Patel, Hemang; Kwon, Soonjo; Pearson, Lee H.

    2010-01-01

    Normal and malignant mammary epithelial cells were studied using laboratory measurements, wavelet analysis, and numerical simulations of monolayer cell cultures to determine whether microscopic breast cancer can be detected in vitro with high-frequency ultrasound. Pulse-echo waveforms were acquired by immersing a broadband, unfocused 50-MHz transducer in the growth media of cell culture well plates and collecting the first reflection from the well bottoms. The simulations included a multilayer pulse-reflection model and a model of two-dimensional arrays of spherical cells and nuclei. The results show that normal and malignant cells produce time-domain signals and spectral features that are significantly different. PMID:21110531

  2. Inhibition of TGF-β signaling in normal lung epithelial cells confers resistance to ionizing radiation

    PubMed Central

    Reeves, Anna; Zagurovskaya, Marianna; Gupta, Seema; Shareef, Mohammed M.; Mohiuddin, Mohammed; Ahmed, Mansoor M.

    2007-01-01

    Purpose To address the functional role of radiation-induced TGF-β signaling in normal epithelial background, we selected spontaneously immortalized lung epithelial cell line derived from the normal lung tissue of dominant-negative mutant of TGF-β RII (ΔRII) transgenic mouse that expressed conditionally ΔRII under the control of metallothionein promoter (MT-1) and assessed it's impact on radio-sensitivity. Method and Materials Spontaneously immortalized lung epithelial cell culture (SILECC) was established and all analyses were performed within 50 passages. Colony-forming and TUNEL assays were used to assess the clonogenic inhibition and apoptosis respectively. Western blot analysis was performed to assess the kinetics of p21, bax and RII proteins. TGF-β responsive promoter activity was measured using dual-luciferase reporter assay. Results Exposure to ZnSO4 inhibited TGF-β signaling induced either by recombinant TGF-β1 or ionizing radiation. SILECC treated either with ZnSO4 or neutralizing antibody against TGF-β showed a significant increase in radio-resistance when compared to untreated cells. Furthermore, the expression of the ΔRII inhibited the radiation-induced up-regulation of the TGF-β effector gene p21waf1/cip1.. Conclusions Our findings imply that inhibition of radiation-induced TGF-β signaling via abrogation of RII function enhances radio-resistance of the normal lung epithelial cells, and this can be directly attributed to the loss of TGF-β signaling function. PMID:17448872

  3. Epidermal growth factor-mediated proliferation and sodium transport in normal and PKD epithelial cells

    PubMed Central

    Zheleznova, Nadezhda N.; Wilson, Patricia D.; Staruschenko, Alexander

    2010-01-01

    Members of the epidermal growth factor (EGF)-family bind to ErbB (EGFR)-family receptors which play an important role in the regulation of various fundamental cell processes including cell proliferation and differentiation. The normal rodent kidney has been shown to express at least three members of the ErbB receptor family and is a major site of EGF ligand synthesis. Polycystic kidney disease (PKD) is a group of diseases caused by mutations in single genes and is characterized by enlarged kidneys due to the formation of multiple cysts in both kidneys. Tubule cells proliferate, causing segmental dilation, in association with the abnormal deposition of several proteins. One of the first abnormalities described in cell biological studies of PKD pathogenesis was the abnormal mislocalization of the EGFR in cyst lining epithelial cells. The kidney collecting duct (CD) is predominantly an absorptive epithelium where electrogenic Na+ entry is mediated by the epithelial Na+ channel (ENaC). ENaC-mediated sodium absorption represents an important ion transport pathway in the CD that might be involved in the development of PKD. A role for EGF in the regulation of ENaC-mediated sodium absorption has been proposed. However, several investigations have reported contradictory results indicating opposite effects of EGF and its related factors on ENaC activity and sodium transport. Recent advances in understanding how proteins in the EGF-family regulate the proliferation and sodium transport in normal and PKD epithelial cells are discussed here. PMID:20959142

  4. Nitric oxide regulation of colonic epithelial ion transport: a novel role for enteric glia in the myenteric plexus

    PubMed Central

    MacEachern, Sarah J; Patel, Bhavik A; McKay, Derek M; Sharkey, Keith A

    2011-01-01

    Abstract Enteric glia are increasingly recognized as important in the regulation of a variety of gastrointestinal functions. Here we tested the hypothesis that nicotinic signalling in the myenteric plexus results in the release of nitric oxide (NO) from neurons and enteric glia to modulate epithelial ion transport. Ion transport was assessed using full-thickness or muscle-stripped segments of mouse colon mounted in Ussing chambers. The cell-permeant NO-sensitive dye DAR-4M AM and amperometry were utilized to identify the cellular sites of NO production within the myenteric plexus and the contributions from specific NOS isoforms. Nicotinic receptors were localized using immunohistochemistry. Nicotinic cholinergic stimulation of colonic segments resulted in NO-dependent changes in epithelial active electrogenic ion transport that were TTX sensitive and significantly altered in the absence of the myenteric plexus. Nicotinic stimulation of the myenteric plexus resulted in NO production and release from neurons and enteric glia, which was completely blocked in the presence of nitric oxide synthase (NOS) I and NOS II inhibitors. Using the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), neuronal and enteric glial components of NO production were demonstrated. Nicotinic receptors were identified on enteric neurons, which express NOS I, and enteric glia, which express NOS II. These data identify a unique pathway in the mouse colon whereby nicotinic cholinergic signalling in myenteric ganglia mobilizes NO from NOS II in enteric glia, which in coordinated activity with neurons in the myenteric plexus modulates epithelial ion transport, a key component of homeostasis and innate immunity. PMID:21558161

  5. Nitric oxide regulation of colonic epithelial ion transport: a novel role for enteric glia in the myenteric plexus.

    PubMed

    MacEachern, Sarah J; Patel, Bhavik A; McKay, Derek M; Sharkey, Keith A

    2011-07-01

    Enteric glia are increasingly recognized as important in the regulation of a variety of gastrointestinal functions.Here we tested the hypothesis that nicotinic signalling in the myenteric plexus results in the release of nitric oxide (NO) from neurons and enteric glia to modulate epithelial ion transport. Ion transport was assessed using full-thickness or muscle-stripped segments of mouse colon mounted in Ussing chambers. The cell-permeant NO-sensitive dye DAR-4M AM and amperometry were utilized to identify the cellular sites of NO production within the myenteric plexus and the contributions from specific NOS isoforms. Nicotinic receptors were localized using immunohistochemistry. Nicotinic cholinergic stimulation of colonic segments resulted in NO-dependent changes in epithelial active electrogenic ion transport that were TTX sensitive and significantly altered in the absence of the myenteric plexus. Nicotinic stimulation of the myenteric plexus resulted in NO production and release from neurons and enteric glia, which was completely blocked in the presence of nitric oxide synthase (NOS) I and NOS II inhibitors. Using the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), neuronal and enteric glial components of NO production were demonstrated. Nicotinic receptors were identified on enteric neurons, which express NOS I, and enteric glia, which express NOS II. These data identify a unique pathway in the mouse colon whereby nicotinic cholinergic signalling in myenteric ganglia mobilizes NO from NOS II in enteric glia, which in coordinated activity with neurons in the myenteric plexus modulates epithelial ion transport, a key component of homeostasis and innate immunity.

  6. Progesterone facilitates chromosome instability (aneuploidy) in p53 null normal mammary epithelial cells

    NASA Technical Reports Server (NTRS)

    Goepfert, T. M.; McCarthy, M.; Kittrell, F. S.; Stephens, C.; Ullrich, R. L.; Brinkley, B. R.; Medina, D.

    2000-01-01

    Mammary epithelial cells from p53 null mice have been shown recently to exhibit an increased risk for tumor development. Hormonal stimulation markedly increased tumor development in p53 null mammary cells. Here we demonstrate that mammary tumors arising in p53 null mammary cells are highly aneuploid, with greater than 70% of the tumor cells containing altered chromosome number and a mean chromosome number of 56. Normal mammary cells of p53 null genotype and aged less than 14 wk do not exhibit aneuploidy in primary cell culture. Significantly, the hormone progesterone, but not estrogen, increases the incidence of aneuploidy in morphologically normal p53 null mammary epithelial cells. Such cells exhibited 40% aneuploidy and a mean chromosome number of 54. The increase in aneuploidy measured in p53 null tumor cells or hormonally stimulated normal p53 null cells was not accompanied by centrosome amplification. These results suggest that normal levels of progesterone can facilitate chromosomal instability in the absence of the tumor suppressor gene, p53. The results support the emerging hypothesis based both on human epidemiological and animal model studies that progesterone markedly enhances mammary tumorigenesis.

  7. Progesterone facilitates chromosome instability (aneuploidy) in p53 null normal mammary epithelial cells

    NASA Technical Reports Server (NTRS)

    Goepfert, T. M.; McCarthy, M.; Kittrell, F. S.; Stephens, C.; Ullrich, R. L.; Brinkley, B. R.; Medina, D.

    2000-01-01

    Mammary epithelial cells from p53 null mice have been shown recently to exhibit an increased risk for tumor development. Hormonal stimulation markedly increased tumor development in p53 null mammary cells. Here we demonstrate that mammary tumors arising in p53 null mammary cells are highly aneuploid, with greater than 70% of the tumor cells containing altered chromosome number and a mean chromosome number of 56. Normal mammary cells of p53 null genotype and aged less than 14 wk do not exhibit aneuploidy in primary cell culture. Significantly, the hormone progesterone, but not estrogen, increases the incidence of aneuploidy in morphologically normal p53 null mammary epithelial cells. Such cells exhibited 40% aneuploidy and a mean chromosome number of 54. The increase in aneuploidy measured in p53 null tumor cells or hormonally stimulated normal p53 null cells was not accompanied by centrosome amplification. These results suggest that normal levels of progesterone can facilitate chromosomal instability in the absence of the tumor suppressor gene, p53. The results support the emerging hypothesis based both on human epidemiological and animal model studies that progesterone markedly enhances mammary tumorigenesis.

  8. Cadmium malignantly transforms normal human breast epithelial cells into a basal-like phenotype.

    PubMed

    Benbrahim-Tallaa, Lamia; Tokar, Erik J; Diwan, Bhalchandra A; Dill, Anna L; Coppin, Jean-François; Waalkes, Michael P

    2009-12-01

    Breast cancer has recently been linked to cadmium exposure. Although not uniformly supported, it is hypothesized that cadmium acts as a metalloestrogenic carcinogen via the estrogen receptor (ER). Thus, we studied the effects of chronic exposure to cadmium on the normal human breast epithelial cell line MCF-10A, which is ER-negative but can convert to ER-positive during malignant transformation. Cells were continuously exposed to low-level cadmium (2.5 muM) and checked in vitro and by xenograft study for signs of malignant transformation. Transformant cells were molecularly characterized by protein and transcript analysis of key genes in breast cancer. Over 40 weeks of cadmium exposure, cells showed increasing secretion of matrix metalloproteinase-9, loss of contact inhibition, increased colony formation, and increasing invasion, all typical for cancer cells. Inoculation of cadmium-treated cells into mice produced invasive, metastatic anaplastic carcinoma with myoepithelial components. These cadmium-transformed breast epithelial (CTBE) cells displayed characteristics of basal-like breast carcinoma, including ER-alpha negativity and HER2 (human epidermal growth factor receptor 2) negativity, reduced expression of BRCA1 (breast cancer susceptibility gene 1), and increased CK5 (cytokeratin 5) and p63 expression. CK5 and p63, both breast stem cell markers, were prominently overexpressed in CTBE cell mounds, indicative of persistent proliferation. CTBE cells showed global DNA hypomethylation and c-myc and k-ras overexpression, typical in aggressive breast cancers. CTBE cell xenograft tumors were also ER-alpha negative. Cadmium malignantly transforms normal human breast epithelial cells-through a mechanism not requiring ER-alpha-into a basal-like cancer phenotype. Direct cadmium induction of a malignant phenotype in human breast epithelial cells strongly fortifies a potential role in breast cancer.

  9. Inhibition of microRNA-31-5p protects human colonic epithelial cells against ionizing radiation

    NASA Astrophysics Data System (ADS)

    Kim, Sang Bum; Zhang, Lu; Barron, Summer; Shay, Jerry W.

    2014-04-01

    MicroRNAs (miRNAs), endogenous non-coding small RNAs, are sensitive to environmental changes, and their differential expression is important for adaptation to the environment. However, application of miRNAs as a clinical prognostic or diagnostic tool remains unproven. In this study we demonstrate a chronic/persistent change of miRNAs from the plasma of a colorectal cancer susceptible mouse model (CPC;Apc) about 250 days after exposure to a simulated solar particle event (SPE). Differentially expressed miRNAs were identified compared to unirradiated control mice, including miR-31-5p, which we investigated further. To address the cellular function of miR-31-5p, we transfected a miR-31-5p mimic (sense) or inhibitor (antisense) into immortalized human colonic epithelial cells followed by gamma-irradiation. A miR-31-5p mimic sensitized but a miR-31-5p inhibitor protected colonic epithelial cells against radiation induced killing. We found that the miR-31-5p mimic inhibited the induction of hMLH1 expression after irradiation, whereas the miR-31-5p inhibitor increased the basal level of hMLH1 expression. The miR-31-5p inhibitor failed to modulate radiosensitivity in an hMLH1-deficient HCT116 colon cancer cell line but protected HCT116 3-6 and DLD-1 (both hMLH1-positive) colon cancer cell lines. Our findings demonstrate that miR-31-5p has an important role in radiation responses through regulation of hMLH1 expression. Targeting this pathway could be a promising therapeutic strategy for future personalized anti-cancer radiotherapy.

  10. Four carcinoembryonic antigen subfamily members, CEA, NCA, BGP and CGM2, selectively expressed in the normal human colonic epithelium, are integral components of the fuzzy coat.

    PubMed

    Frängsmyr, L; Baranov, V; Hammarström, S

    1999-01-01

    To elucidate which of the seven transcriptionally active genes of the carcinoembryonic antigen (CEA) subfamily are expressed in human colon, we first examined mRNA expression using reverse transcriptase PCR. The result showed the CEA, nonspecific crossreacting antigen 50/90 (NCA), biliary glycoprotein (BGP), and carcinoembryonic antigen gene family member 2 (CGM2) mRNAs were expressed in the colon. To determine the cellular sources of these members within normal colonic mucosa, in situ hybridization and immunocytochemistry were then performed. CEA and NCA mRNAs were clearly detectable in the cytoplasm of columnar and goblet cells at the free luminal surface and the upper crypts with low hybridization in the mid crypt and the crypt base. In contrast, BGP and CGM2 mRNAs were restricted only to columnar cells at the upper third of the crypts and the luminal surface. Colon epithelium expression of CEA, NCA, BGP and CGM2 coincided with that of corresponding mRNAs. Ultrastructurally, CEA, NCA, BGP and CGM2 were localized mainly to the apical surface glycocalyx, the fuzzy coat, of columnar cells. Interestingly, these molecules were localized in different microdomains within the fuzzy coat. Furthermore, BGP was highly expressed in the fuzzy coat of cryptal caveolated cells. As integral components of the fuzzy coat, CEA, NCA, BGP and CGM2 can hardly function as intercellular adhesion molecules; they possibly play an important role in epithelial-microbial interactions.

  11. The expression of CD44v6 in colon: from normal to malignant.

    PubMed

    Afify, Alaa; Durbin-Johnson, Blythe; Virdi, Avnit; Jess, Heidi

    2016-02-01

    CD44v6, an integral transmembrane protein belonging to a family of adhesion molecule receptors, plays an important role in tumor growth, progression and metastasis. The purpose of this study was to evaluate the expression of CD44v6 in normal, hyperplastic, adenomatous, and malignant colonic epithelium and to determine its correlation with tumor pathologic stage and lymph node metastasis. We examined the immunohistochemical expression of CD44v6 in normal colonic tissue (n = 25), hyperplastic polyps (n = 45), tubular adenomas (n = 57), tubulovillous adenomas (n = 25), villous adenomas (n = 9), adenocarcinomas stage I (n = 26), adenocarcinomas stage III (n = 26), and lymph node metastasis (n = 26). The percentage of positive cells and the staining intensity were assessed and scored. Statistical analysis was performed using logistic regression and McNemar test. All normal colonic tissue and hyperplastic polyps showed CD44v6 staining confined to the base of the crypt. In tubular adenomas, the dysplastic surface adenomatous epithelium expressed CD44v6 in 49 (86%) cases. CD44v6 was expressed in the glandular areas of tubulovillous adenomas in 21 (84%) cases and in the villous portion in 18 (72%) cases. All villous adenomas expressed CD44v6. CD44v6 was expressed in 23 (88%) cases of stage I adenocarcinomas, in 24 (92%) cases of stage III adenocarcinomas, and in 9 (35%) cases of metastatic adenocarcinomas. We concluded that the gain of CD44v6 expression in premalignant and malignant colonic lesions suggests that CD44v6 may be functionally involved in the adenoma-to-carcinoma progression. CD44v6 did not correlate to tumor pathologic stage and is lost during the acquisition of migratory function by metastatic tumor cells.

  12. Diets High in Heat-Treated Soybean Meal Reduce the Histamine-Induced Epithelial Response in the Colon of Weaned Piglets and Increase Epithelial Catabolism of Histamine

    PubMed Central

    Kröger, Susan; Pieper, Robert; Schwelberger, Hubert G.; Wang, Jing; Villodre Tudela, Carmen; Aschenbach, Jörg R.; Van Kessel, Andrew G.; Zentek, Jürgen

    2013-01-01

    We examined the influence of dietary fermentable protein (fCP) and fermentable carbohydrates (fCHO) on the colonic epithelial response to histamine in pigs. Thirty-two weaned piglets were fed 4 diets in a 2 × 2 factorial design with low fCP/low fCHO, low fCP/high fCHO, high fCP/low fCHO and high fCP/high fCHO. After 21-23 days, the pigs were killed and tissue from the proximal colon was stimulated with carbachol, histamine, PGE2 or sodium hydrogen sulphide in Ussing chambers. Changes in short-circuit current and tissue conductance were measured. Diamine oxidase, histamine N-methyltransferase, stem cell growth factor receptor, Fc-epsilon receptor I and cystic fibrosis transmembrane conductance regulator gene expression was determined. Activities of diamine oxidase and histamine N-methyltransferase and numbers of colonic mast cells were measured. The change in the short-circuit current in response to histamine was lower (P = 0.002) and tended to be lower for PGE2 (P = 0.053) in high fCP groups compared to low fCP groups, irrespective of fCHO. Additionally, the change in tissue conductance after the application of histamine was lower (P = 0.005) in the high fCP groups. The expression of histamine N-methyltransferase mRNA (P = 0.033) and the activities of diamine oxidase (P = 0.001) and histamine N-methyltransferase (P = 0.006) were higher with high fCP in comparison with low fCP. The expression of mast cell markers, stem cell growth factor receptor (P = 0.005) and Fc-epsilon receptor I (P = 0.049) was higher with high fCP diets compared to diets low in fCP, whereas the mast cell count did not differ between groups. The expression of the cystic fibrosis transmembrane conductance regulator was reduced (P = 0.001) with high fCP diets compared to low fCP diets. The lower epithelial response to histamine and PGE2 and elevated epithelial histamine inactivation suggests an adaptation to high fCP diets. PMID:24260435

  13. Changes in telomerase activity, expression and splicing in response to differentiation of normal and carcinoma colon cells.

    PubMed

    Fajkus, Jirí; Borsky, Marek; Kunická, Zuzana; Kovaríková, Martiná; Dvoráková, Dana; Hofmanová, Jirina; Kozubík, Alois

    2003-01-01

    Telomerase, a ribonucleoprotein complex catalysing synthesis of telomeric DNA, is an essential cellular immortalizing factor whose activation is a critical step in the progression to malignancy. An important agent maintaining the balance between proliferation, differentiation and apoptosis of intestinal epithelial cells in crypts is butyrate, which is formed in the gastrointestinal tract by anaerobic bacterial fermentation. It inhibits cell growth, induces differentiation and triggers apoptosis in neoplastic colonocytes. In this study, the responses of adenocarcinoma (HT-29) and fetal (FHC) human colon cells to 5 mM sodium butyrate (NaBt) have been compared. Despite the similar general response of both cell lines to NaBt, i.e., G0/G1 arrest, decrease of growth rate and increase of differentiation (as indicated by alkaline phosphatase activity), they differ in the level and dynamics of the measured parameters. Telomerase activity and the level of mRNA for its catalytic subunit (hTERT) decline significantly after 48 hours, reaching a complete inhibition after 144 hours. While both cell lines show similar kinetics of hTERT transcriptional silencing, the down-regulation of telomerase activity is faster in FHC cells. Correspondingly, we show that a candidate posttranscriptional regulation step, differential splicing of hTERT mRNA, may be involved in the faster loss of telomerase activity in FHC cells. Differences in hTERT mRNA splicing may represent a useful marker of telomere metabolism in normal and malignant colon cells and that these changes may be connected with different cytokinetic patterns of these cells.

  14. Cdx2 modulates proliferation in normal human intestinal epithelial crypt cells

    SciTech Connect

    Escaffit, Fabrice; Pare, Frederic; Gauthier, Remy; Rivard, Nathalie; Boudreau, Francois; Beaulieu, Jean-Francois . E-mail: Jean-Francois.Beaulieu@USherbrooke.ca

    2006-03-31

    The homeobox gene Cdx2 is involved in the regulation of the expression of intestine specific markers such as sucrase-isomaltase and lactase-phlorizin hydrolase. Previous studies performed with immortalized or transformed intestinal cell lines have provided evidence that Cdx2 can promote morphological and functional differentiation in these experimental models. However, no data exist concerning the implication of this factor in normal human intestinal cell physiology. In the present work, we have investigated the role of Cdx2 in normal human intestinal epithelial crypt (HIEC) cells that lack this transcription factor. The establishment of HIEC cells expressing Cdx2 in an inducible manner shows that forced expression of Cdx2 significantly alters the proliferation of intestinal crypt cells and stimulates dipeptidylpeptidase IV expression but is not sufficient to trigger intestinal terminal differentiation. These observations suggest that Cdx2 requires additional factors to activate the enterocyte differentiation program in normal undifferentiated cells.

  15. Can Villin be Used to Identify Malignant and Undifferentiated Normal Digestive Epithelial Cells?

    NASA Astrophysics Data System (ADS)

    Robine, S.; Huet, C.; Moll, R.; Sahuquillo-Merino, C.; Coudrier, E.; Zweibaum, A.; Louvard, D.

    1985-12-01

    We have investigated the presence of villin (a Ca2+-regulated actin binding protein) in various tissues (normal or malignant) and in established cell lines by using sensitive immunochemical techniques on cell extracts and immunofluorescence analysis on frozen sections. Our results show that villin is a marker that can be used to distinguish normal differentiated epithelial cells from the simple epithelia lining the gastrointestinal tract and renal tubules. Villin is found in the absorptive cells of the small and large intestines, in the duct cells of pancreas and biliary system, and in the cells of kidney proximal tubules. Furthermore, undifferentiated normal and tumoral cells of intestinal origin in vivo and in cell culture express villin. Therefore, expression of villin is seen in cells that do not necessarily display the morphological features characteristic of their terminally differentiated state, such as the microvilli-lined brush border. We suggest the possible clinical implications of using villin as a marker in the diagnosis of metastatic adenocarcinomas.

  16. Localization of tumor-associated glycoprotein DF3 in normal, inflammatory, and neoplastic lesions of the colon.

    PubMed

    Andrews, C W; Jessup, J M; Goldman, H; Hayes, D F; Kufe, D W; O'Hara, C J; Steele, G D

    1993-12-01

    The expression of DF3 was assessed by a monoclonal antibody in normal, inflammatory, and neoplastic conditions in the large bowel. Using immunohistochemistry, expression was examined in formalin-fixed paraffin-embedded biopsy and resection samples of 19 normal colonic mucosal specimens, 49 inflammatory lesions, 34 adenomas, and 38 primary colonic adenocarcinomas. In addition, Western blots of normal colonic mucosa and adenocarcinoma were examined. DF3 expression was detected in 84% of the adenocarcinomas with coarse membrane staining, intense positivity of luminal secretions, and focal cytoplasmic and intracytoplasmic vacuole staining. Nine of 32 areas of transitional mucosa revealed reactivity along apical membranes in crypt cells. Five adenomas containing carcinoma revealed DF3 positivity in the malignant areas only, whereas the remaining 29 were negative. Staining was membrane, luminal, and intracytoplasmic. Two examples of active ulcerative colitis revealed focal reactivity along the apical membrane of crypt cells. No other areas of staining were noted, including 12 cases containing dysplasia. Four of 10 other inflammatory lesions also revealed similar membrane reactivity in crypt cells. Normal colonic mucosa was nonreactive. Examples of normal colonic mucosa were negative for DF3 by Western blot analysis, whereas two carcinoma samples that reacted immunohistochemically were positive. DF3 is not detectable in normal colonic tissues. It is expressed focally and predominantly along the apical membrane of crypt cells in some inflammatory lesions and in the transitional mucosa of primary adenocarcinomas. Most adenocarcinomas of the colon and adenomas with foci of invasive carcinoma demonstrate reactivity in the cytoplasm and luminal secretions.

  17. CDDO-Me protects normal lung and breast epithelial cells but not cancer cells from radiation.

    PubMed

    El-Ashmawy, Mariam; Delgado, Oliver; Cardentey, Agnelio; Wright, Woodring E; Shay, Jerry W

    2014-01-01

    Although radiation therapy is commonly used for treatment for many human diseases including cancer, ionizing radiation produces reactive oxygen species that can damage both cancer and healthy cells. Synthetic triterpenoids, including CDDO-Me, act as anti-inflammatory and antioxidant modulators primarily by inducing the transcription factor Nrf2 to activate downstream genes containing antioxidant response elements (AREs). In the present series of experiments, we determined if CDDO-Me can be used as a radioprotector in normal non-cancerous human lung and breast epithelial cells, in comparison to lung and breast cancer cell lines. A panel of normal non-cancerous, partially cancer progressed, and cancer cell lines from both lung and breast tissue was exposed to gamma radiation with and without pre-treatment with CDDO-Me. CDDO-Me was an effective radioprotector when given ∼18 hours before radiation in epithelial cells (average dose modifying factor (DMF) = 1.3), and Nrf2 function was necessary for CDDO-Me to exert these radioprotective effects. CDDO-Me did not protect cancer lines tested from radiation-induced cytotoxicity, nor did it protect experimentally transformed human bronchial epithelial cells (HBECs) with progressive oncogenic manipulations. CDDO-Me also protected human lymphocytes against radiation-induced DNA damage. A therapeutic window exists in which CDDO-Me protects normal cells from radiation by activating the Nrf2 pathway, but does not protect experimentally transformed or cancer cell lines. This suggests that use of this oral available, non-toxic class of drug can protect non-cancerous healthy cells during radiotherapy, resulting in better outcomes and less toxicity for patients.

  18. CDDO-Me Protects Normal Lung and Breast Epithelial Cells but Not Cancer Cells from Radiation

    PubMed Central

    El-Ashmawy, Mariam; Delgado, Oliver; Cardentey, Agnelio; Wright, Woodring E.; Shay, Jerry W.

    2014-01-01

    Although radiation therapy is commonly used for treatment for many human diseases including cancer, ionizing radiation produces reactive oxygen species that can damage both cancer and healthy cells. Synthetic triterpenoids, including CDDO-Me, act as anti-inflammatory and antioxidant modulators primarily by inducing the transcription factor Nrf2 to activate downstream genes containing antioxidant response elements (AREs). In the present series of experiments, we determined if CDDO-Me can be used as a radioprotector in normal non-cancerous human lung and breast epithelial cells, in comparison to lung and breast cancer cell lines. A panel of normal non-cancerous, partially cancer progressed, and cancer cell lines from both lung and breast tissue was exposed to gamma radiation with and without pre-treatment with CDDO-Me. CDDO-Me was an effective radioprotector when given ∼18 hours before radiation in epithelial cells (average dose modifying factor (DMF) = 1.3), and Nrf2 function was necessary for CDDO-Me to exert these radioprotective effects. CDDO-Me did not protect cancer lines tested from radiation-induced cytotoxicity, nor did it protect experimentally transformed human bronchial epithelial cells (HBECs) with progressive oncogenic manipulations. CDDO-Me also protected human lymphocytes against radiation-induced DNA damage. A therapeutic window exists in which CDDO-Me protects normal cells from radiation by activating the Nrf2 pathway, but does not protect experimentally transformed or cancer cell lines. This suggests that use of this oral available, non-toxic class of drug can protect non-cancerous healthy cells during radiotherapy, resulting in better outcomes and less toxicity for patients. PMID:25536195

  19. Analysis of differential protein expression in normal and neoplastic human breast epithelial cell lines

    SciTech Connect

    Williams, K.; Chubb, C.; Huberman, E.; Giometti, C.S.

    1997-07-01

    High resolution two dimensional get electrophoresis (2DE) and database analysis was used to establish protein expression patterns for cultured normal human mammary epithelial cells and thirteen breast cancer cell lines. The Human Breast Epithelial Cell database contains the 2DE protein patterns, including relative protein abundances, for each cell line, plus a composite pattern that contains all the common and specifically expressed proteins from all the cell lines. Significant differences in protein expression, both qualitative and quantitative, were observed not only between normal cells and tumor cells, but also among the tumor cell lines. Eight percent of the consistently detected proteins were found in significantly (P < 0.001) variable levels among the cell lines. Using a combination of immunostaining, comigration with purified protein, subcellular fractionation, and amino-terminal protein sequencing, we identified a subset of the differentially expressed proteins. These identified proteins include the cytoskeletal proteins actin, tubulin, vimentin, and cytokeratins. The cell lines can be classified into four distinct groups based on their intermediate filament protein profile. We also identified heat shock proteins; hsp27, hsp60, and hsp70 varied in abundance and in some cases in the relative phosphorylation levels among the cell lines. Finally, we identified IMP dehydrogenase in each of the cell lines, and found the levels of this enzyme in the tumor cell lines elevated 2- to 20-fold relative to the levels in normal cells.

  20. Single stage management of a unique variant of congenital pouch colon with triplet fistula and normal anus.

    PubMed

    Pandey, Vaibhav; Gangopadhyay, Ajay Narayan; Gupta, Dinesh Kumar; Sharma, Shiv Prasad

    2015-01-01

    Congenital pouch colon (CPC) in the female patient presents with highly variable and anomalous anatomy. We herein report the first case of CPC with uterus didelphys having normal anal opening, H-type vestibular fistula, two other fistulous communications between pouch colon and two vagina managed in a single stage with excellent postoperative outcome.

  1. Transfection of normal human bronchial epithelial cells with the bcl-2 oncogene

    SciTech Connect

    Kennedy, C.H.; Kenyon, K.D.; Tesfaigzi, J.

    1995-12-01

    In vitro, studies examining the transformation of virus-immortalized human bronchial epithelial (HBE) cells after exposure to chemical and physical carcinogens have contributed to our understanding of the mechanisms that underlie the development of lung cancer. Virus-immortalized HBE cells have been used because of both the limited life span of normal human bronchial epithelial (NHBE) cells in culture (approximately 30-35 population doublins) and their resistance to in vitro malignant transformation. For example, human papillomavirus (HPV)-immortalized HBE cells have been used to study the genetic changes that occur after exposure to {alpha}-particles in vitro. Although this model may prove to be useful for studying the 18% or less of bronchogenic carcinomas found to contain HPV sequences, it is not an appropriate model for studying the majority of lung epithelial malignancies in which HPV DNA is not detected. This view is supported by the fact that HPV-immortalized cell lines commonly exhibit aneuploidy. This results of this study suggest that: (1) NHBE cells can be transiently transfected with the pCMV{Beta} vector; and (2) the antibiotic hygromycin-resistant transfected cells.

  2. Human papilloma virus DNAs immortalize normal human mammary epithelial cells and reduce their growth factor requirements

    SciTech Connect

    Band, V.; Zajchowski, D.; Kulesa, V.; Sager, R. )

    1990-01-01

    Human papilloma virus (HPV) types 16 and 18 are most commonly associated with cervical carcinoma in patients and induce immortalization of human keratinocytes in culture. HPV has not been associated with breast cancer. This report describes the immortalization of normal human mammary epithelial cells (76N) by plasmid pHPV18 or pHPV16, each containing the linearized viral genome. Transfectants were grown continuously for more than 60 passages, whereas 76N cells senesce after 18-20 passages. The transfectants also differ from 76N cells in cloning in a completely defined medium called D2 and growing a minimally supplemented defined medium (D3) containing epidermal growth factor. All transfectant tested contain integrated HPV DNA, express HPV RNA, and produce HPV E7 protein. HPV transfectants do not form tumors in a nude mouse assay. It is concluded that products of the HPV genome induce immortalization of human breast epithelial cells and reduce their growth factor requirements. This result raises the possibility that HPV might be involved in breast cancer. Furthermore, other tissue-specific primary epithelial cells that are presently difficult to grown and investigate may also be immortalized by HPV.

  3. Propagation of normal human epithelial cell populations using an in vivo culture system. Description and applications.

    PubMed Central

    Klein-Szanto, A. J.; Terzaghi, M.; Mirkin, L. D.; Martin, D.; Shiba, M.

    1982-01-01

    A new model using xenotransplanted human epithelia was developed for the study of toxic and carcinogenic effects of chemicals. Epithelial cells from the respiratory tract of 4 male and 3 female premature and fullterm fetuses were enzymatically removed and inoculated into deepithelialized rat tracheas. These were sealed at both ends and transplanted subcutaneously into nude mice. After 3-4 weeks, a normal mucociliary epithelium covered the tracheal lumen. At this stage the epithelial cells could be isolated again and transplanted into new denuded rat tracheas. This passaging could be repeated up to six times, each permitting an amplification factor of approximately 3. Tracheal transplants containing cells of human origin (in vivo Passages 2-4) were treated with 7,12-dimethylbenz(a)anthracene. Hyperplasias, squamous metaplasias, and dysplasias were seen 1-8 weeks after initiation of treatment, indicating that the responses of human and rodent epithelial cells to polycyclic aromatic hydrocarbons are similar. Initial experiments with skin and esophageal epithelia suggest that other covering epithelia could also be used in this fashion for evaluation of toxicants and carcinogens that are likely to come into contact with these tissues. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:6821529

  4. R-spondin1 is required for normal epithelial morphogenesis during mammary gland development.

    PubMed

    Chadi, Sead; Buscara, Laurine; Pechoux, Christine; Costa, José; Laubier, Johann; Chaboissier, Marie-Christine; Pailhoux, Eric; Vilotte, Jean-Luc; Chanat, Eric; Le Provost, Fabienne

    2009-12-18

    The R-spondin (Rspo) proteins constitute a novel class of ligands that induce Wnt signalling. Rspo1 knockout XX mice were previously shown to be sex-reversed, but some remain sub-fertile. These last were unable to feed their pups for some unknown reason. Using these mice and transplanted mammary tissues from Rspo1(-/-) virgin mice in nude mice, we report that the lack of Rspo1 expression results in the absence of duct side-branching development and subsequent alveolar formation, explaining the above mentioned phenotype. Our data demonstrate that local epithelial Rspo1 signalling is required for normal development of the mammary gland.

  5. Wild-type and IL10-null mice have differential colonic epithelial gene expression responses to dietary supplementation with synbiotic Bifidobacterium animalis subspecies lactis and inulin.

    PubMed

    Kuo, Shiu-Ming; Chan, Wan-Chun; Hu, Zihua

    2014-03-01

    Prebiotic plus probiotic (synbiotic) supplementations promote fermentation and have shown anti-inflammatory activity in colonic epithelium. However, in many instances, patients with inflammatory bowel disease (IBD) have demonstrated adverse effects after prebiotic supplementation at a dose well tolerated by normal individuals. To test the hypothesis that the host inflammation affects the colonic epithelial response to increased fermentation, the gene expression of colonic epithelium was analyzed. In a 1-way experimental design to test the effect of supplements in wild-type mice using the standard diet formulated by the American Institute of Nutrition (AIN-93G) as the control diet, fermentable fiber inulin (5%) in the absence or presence of the probiotic Bifidobacterium animalis subspecies lactis (Bb12) (10(8) CFU/kg diet) showed limited effects on gene expression as determined by whole-genome microarray. Bb12 supplementation alone was known not to increase fermentation and here instead significantly upregulated genes in nucleic acid metabolic processes. The effects of the synbiotic diet were then determined in mice exposed to LPS-induced inflammation in a 2-way experimental design testing the effect of diet and LPS. The microarray and quantitative reverse transcription-polymerase chain reaction analyses on the wild-type mice revealed that LPS-induced changes in the colonic epithelium were 4- to 10-fold less in the synbiotic diet group compared with the control diet group. Unlike the wild-type mice, anti-inflammatory cytokine interleukin 10 (IL10)-null mice (susceptible to IBD) given the synbiotic diet, compared with those given the control diet, had 3- to 40-fold increased expression of inflammation-related genes such as Cxcl1 (chemokine C-X-C motif ligand 1) and S100a9 (S100 calcium binding protein A9) in the absence and presence of LPS exposure. These contrasting intestinal epithelial responses to increased fermentation in wild-type and IL10-null mice are similar

  6. Mitochondria sustain store-operated currents in colon cancer cells but not in normal colonic cells: reversal by non-steroidal anti-inflammatory drugs.

    PubMed

    Hernández-Morales, Miriam; Sobradillo, Diego; Valero, Ruth A; Muñoz, Eva; Ubierna, Daniel; Moyer, Mary P; Núñez, Lucía; Villalobos, Carlos

    2017-08-15

    Tumor cells undergo a critical remodeling of intracellular Ca(2+) homeostasis that contribute to important cancer hallmarks. Store-operated Ca(2+) entry (SOCE), a Ca(2+) entry pathway modulated by mitochondria, is dramatically enhanced in colon cancer cells. In addition, most cancer cells display the Warburg effect, a metabolic switch from mitochondrial metabolism to glycolysis that provides survival advantages. Accordingly, we investigated mitochondria control of store-operated currents (SOCs) in two cell lines previously selected for representing human normal colonic cells and colon cancer cells. We found that, in normal cells, mitochondria are important for SOCs activity but they are unable to prevent current inactivation. In contrast, in colon cancer cells, mitochondria are dispensable for SOCs activation but are able to prevent the slow, Ca(2+)-dependent inactivation of SOCs. This effect is associated to increased ability of tumor cell mitochondria to take up Ca(2+) due to increased mitochondrial potential (ΔΨ) linked to the Warburg effect. Consistently with this view, selected non-steroidal anti-inflammatory drugs (NSAIDs) depolarize mitochondria, inhibit mitochondrial Ca(2+) uptake and promote SOC inactivation, leading to inhibition of both SOCE and cancer cell proliferation. Thus, mitochondria sustain store-operated currents in colon cancer cells but not in normal colonic cells and this effect is counteracted by selected NSAIDs providing a mechanism for cancer chemoprevention.

  7. Mitochondria sustain store-operated currents in colon cancer cells but not in normal colonic cells: reversal by non-steroidal anti-inflammatory drugs

    PubMed Central

    Hernández-Morales, Miriam; Sobradillo, Diego; Valero, Ruth A.; Muñoz, Eva; Ubierna, Daniel; Moyer, Mary P.; Núñez, Lucía; Villalobos, Carlos

    2017-01-01

    Tumor cells undergo a critical remodeling of intracellular Ca2+ homeostasis that contribute to important cancer hallmarks. Store-operated Ca2+ entry (SOCE), a Ca2+ entry pathway modulated by mitochondria, is dramatically enhanced in colon cancer cells. In addition, most cancer cells display the Warburg effect, a metabolic switch from mitochondrial metabolism to glycolysis that provides survival advantages. Accordingly, we investigated mitochondria control of store-operated currents (SOCs) in two cell lines previously selected for representing human normal colonic cells and colon cancer cells. We found that, in normal cells, mitochondria are important for SOCs activity but they are unable to prevent current inactivation. In contrast, in colon cancer cells, mitochondria are dispensable for SOCs activation but are able to prevent the slow, Ca2+-dependent inactivation of SOCs. This effect is associated to increased ability of tumor cell mitochondria to take up Ca2+ due to increased mitochondrial potential (ΔΨ) linked to the Warburg effect. Consistently with this view, selected non-steroidal anti-inflammatory drugs (NSAIDs) depolarize mitochondria, inhibit mitochondrial Ca2+ uptake and promote SOC inactivation, leading to inhibition of both SOCE and cancer cell proliferation. Thus, mitochondria sustain store-operated currents in colon cancer cells but not in normal colonic cells and this effect is counteracted by selected NSAIDs providing a mechanism for cancer chemoprevention. PMID:28903423

  8. TRAF-4 expression in epithelial progenitor cells. Analysis in normal adult, fetal, and tumor tissues.

    PubMed Central

    Krajewska, M.; Krajewski, S.; Zapata, J. M.; Van Arsdale, T.; Gascoyne, R. D.; Berern, K.; McFadden, D.; Shabaik, A.; Hugh, J.; Reynolds, A.; Clevenger, C. V.; Reed, J. C.

    1998-01-01

    TRAF-4 was discovered because of its expression in breast cancers and is a member of the tumor necrosis factor (TNF) receptor-associated factor (TRAF) family of putative signal-transducing proteins. In vitro binding assays demonstrated that TRAF-4 interacts with the cytosolic domain of the lymphotoxin-beta receptor (LT beta R) and weakly with the p75 nerve growth factor receptor (NGFR) but not with TNFR1, TNFR2, Fas, or CD40. Immunofluorescence analysis of TRAF-4 in transfected cells demonstrated localization to cytosol but not nucleus. Immunohistochemical assays of normal human adult tissues revealed prominent cytosolic immunostaining in thymic epithelial cells and lymph node dendritic cells but not in lymphocytes or thymocytes, paralleling the reported patterns of LT beta R expression. The basal cell layer of most epithelia in the body was very strongly TRAF-4 immunopositive, including epidermis, nasopharynx, respiratory tract, salivary gland, and esophagus. Similar findings were obtained in 12- to 18-week human fetal tissue, indicating a highly restricted pattern of expression even during development in the mammary gland, epithelial cells of the terminal ducts were strongly TRAF-4 immunopositive whereas myoepithelial cells and most of the mammary epithelial cells lining the extralobular ducts were TRAF-4 immunonegative. Of 84 primary breast cancers evaluated, only 7 expressed TRAF-4. Ductal carcinoma in situ (DCIS) lesions were uniformly TRAF-4 immunonegative (n = 21). In the prostate, the basal cells were strongly immunostained for TRAF-4, whereas the secretory epithelial cells were TRAF-4 negative. Basal cells in prostate hypertrophy (n = 6) and prostatic intraepithelial neoplasia (PIN; n = 6) were strongly TRAF-4 positive, but none of the 32 primary and 16 metastatic prostate cancer specimens examined contained TRAF-4-positive malignant cells. Although also expressed in some types of mesenchymal cells, these findings suggest that TRAF-4 is a marker of normal

  9. Differential effects of vitamin D on normal human prostate epithelial and stromal cells in primary culture.

    PubMed

    Krill, D; Stoner, J; Konety, B R; Becich, M J; Getzenberg, R H

    1999-07-01

    Because epidemiologic evidence has demonstrated that vitamin D may play a role in the etiology of prostate cancer, we tested the inhibitory effect of the biologically active form of vitamin D (1,25-D) on the cell proliferation of human prostate epithelial and stromal cells in a chemically defined situation in the presence and absence of dihydrotestosterone (DHT). We also tested the effect of 1,25-D in castrated rats in the presence and absence of flutamide, an androgen receptor blocker. Prostate stromal and epithelial cells were isolated from freshly collected human prostatectomy specimens, and cell proliferation was measured with the MTT assay. Immunohistochemistry was performed to detect the presence of 1,25-D receptors, androgen receptors, smooth muscle actin, and E-cadherin. For in vivo analysis of 1,25-D, male Sprague-Dawley rats were castrated, then treated with either 1,25-D, 1,25-D with flutamide, or vehicle control. Incubation of primary cultures of prostate epithelial cells with 1,25-D at a concentration of 10(-8) M reduced cell proliferation by 40% of controls. The inhibition of growth by 1,25-D was maintained in the presence of DHT. Conversely, the effect of a similar dose of 1,25-D on stromal cell exposure was increased proliferation. In vivo, 1,25-D increased the prostatic weight of castrated rats that had serum testosterone levels below the detectable limit. The addition of flutamide did not alter this effect. These results confirm that vitamin D may be an effective antiproliferative agent of epithelial cells in prostate cancer therapy and support in vivo studies performed in the normal rat prostate.

  10. Antiapoptotic effects of estrogen in normal and cancer human cervical epithelial cells.

    PubMed

    Wang, Qifang; Li, Xin; Wang, Liqin; Feng, Ying-Hong; Zeng, Robin; Gorodeski, George

    2004-12-01

    The present study investigated the antiapoptotic effects of estrogen in normal and cancer human cervical cells and the mechanisms involved. Baseline apoptosis in human cervical epithelial cells is mediated predominantly by P2X7-receptor-induced, Ca(2+)-dependent activation of the mitochondrial (caspase-9) pathway. Treatment with 10 nM 17beta-estradiol blocked apoptosis induced by the P2X7-receptor ligands ATP and 2',3'-0-(4-benzoylbenzoyl)-ATP in normal human cervical epithelial cells (hECEs) and attenuated the effect in hECEs immortalized with human papillomavirus-16 (ECE16-1) and the cancer cervical cells HT3 and CaSki. Diethylstilbestrol and to a lesser degree estrone could mimic the effects of 17beta-estradiol, whereas actinomycin-D and cycloheximide attenuated the response. The antiapoptotic effect of estrogen did not depend on cell cycle phase, and in both normal and cancer cervical cells, it involved attenuation of activation of caspase-9 and the terminal caspase-3. However, involvement of cascades upstream to the caspase-9 differed in normal vs. cancer cervical cells. In the normal hECEs estrogen blocked P2X7-receptor-induced calcium influx. In contrast, in the cancer CaSki cells, estrogen up-regulated expression of Bcl-2 and attenuated Ca(2+)-induced mitochondrial swelling (i.e. formation of mitochondrial permeability transition pores). Estrogen had no effect on P2X7-receptor-induced apoptosis in the anaplastic SiHa and Hela cells. These results point to a novel antiapoptotic effect of estrogen in the cervix that is independent of its mitogenic function. The results also suggest that cancer cervical cells evolved antiapoptotic mechanisms that enable the cells to evade apoptosis and could therefore promote tumor progression.

  11. Bile acids deoxycholic acid and ursodeoxycholic acid differentially regulate human β-defensin-1 and -2 secretion by colonic epithelial cells.

    PubMed

    Lajczak, Natalia K; Saint-Criq, Vinciane; O'Dwyer, Aoife M; Perino, Alessia; Adorini, Luciano; Schoonjans, Kristina; Keely, Stephen J

    2017-09-01

    Bile acids and epithelial-derived human β-defensins (HβDs) are known to be important factors in the regulation of colonic mucosal barrier function and inflammation. We hypothesized that bile acids regulate colonic HβD expression and aimed to test this by investigating the effects of deoxycholic acid (DCA) and ursodeoxycholic acid on the expression and release of HβD1 and HβD2 from colonic epithelial cells and mucosal tissues. DCA (10-150 µM) stimulated the release of both HβD1 and HβD2 from epithelial cell monolayers and human colonic mucosal tissue in vitro In contrast, ursodeoxycholic acid (50-200 µM) inhibited both basal and DCA-induced defensin release. Effects of DCA were mimicked by the Takeda GPCR 5 agonist, INT-777 (50 μM), but not by the farnesoid X receptor agonist, GW4064 (10 μM). INT-777 also stimulated colonic HβD1 and HβD2 release from wild-type, but not Takeda GPCR 5(-/-), mice. DCA stimulated phosphorylation of the p65 subunit of NF-κB, an effect that was attenuated by ursodeoxycholic acid, whereas an NF-κB inhibitor, BMS-345541 (25 μM), inhibited DCA-induced HβD2, but not HβD1, release. We conclude that bile acids can differentially regulate colonic epithelial HβD expression and secretion and discuss the implications of our findings for intestinal health and disease.-Lajczak, N. K., Saint-Criq, V., O'Dwyer, A. M., Perino, A., Adorini, L., Schoonjans, K., Keely, S. J. Bile acids deoxycholic acid and ursodeoxycholic acid differentially regulate human β-defensin-1 and -2 secretion by colonic epithelial cells. © FASEB.

  12. Exposure of colonic epithelial cells to oxidative and endoplasmic reticulum stress causes rapid potassium efflux and calcium influx.

    PubMed

    Shabala, Lana; Walker, Emma J; Eklund, Annelie; Randall-Demllo, Sarron; Shabala, Sergey; Guven, Nuri; Cook, Anthony L; Eri, Rajaraman D

    2013-10-01

    Endoplasmic reticulum (ER) stress and oxidative stress have recently been linked to the pathogenesis of inflammatory bowel diseases. Under physiological conditions, intestinal epithelial cells are exposed to ER and oxidative stress affecting the cellular ionic homeostasis. However, these altered ion flux 'signatures' during these stress conditions are poorly characterized. We investigated the kinetics of K(+) , Ca(2+) and H(+) ion fluxes during ER and oxidative stress in a colonic epithelial cell line LS174T using a non-invasive microelectrode ion flux estimation technique. ER and oxidative stress were induced by cell exposure to tunicamycin (TM) and copper ascorbate (CuAsc), respectively, from 1 to 24 h. Dramatic K(+) efflux was observed following acute ER stress with peak K(+) efflux being -30·6 and -138·7 nmolm(-2)  s(-1) for 10 and 50 µg ml(-1) , respectively (p < 0·01). TM-dependent Ca(2+) uptake was more prolonged with peak values of 0·85 and 2·68 nmol m(-2)  s(-1) for 10 and 50 µg ml(-1) TM, respectively (p < 0·02). Ion homeostasis was also affected by the duration of ER stress. Increased duration of TM treatment from 0 to 18 h led to increases in both K(+) efflux and Ca(2+) uptake. While K(+) changes were significantly higher at each time point tested, Ca(2+) uptake was significantly higher only after prolonged treatment (18 h). CuAsc also led to an increased K(+) efflux and Ca(2+) uptake. Functional assays to investigate the effect of inhibiting K(+) efflux with tetraethylammonium resulted in increased cell viability. We conclude that ER/oxidative stress in colonic epithelial cells cause dramatic K(+) , Ca(2+) and H(+) ion flux changes, which may predispose this lineage to poor stress recovery reminiscent of that seen in inflammatory bowel diseases.

  13. Lipoxin A4 prevents tight junction disruption and delays the colonization of cystic fibrosis bronchial epithelial cells by Pseudomonas aeruginosa.

    PubMed

    Higgins, Gerard; Fustero Torre, Coral; Tyrrell, Jean; McNally, Paul; Harvey, Brian J; Urbach, Valerie

    2016-06-01

    The specialized proresolution lipid mediator lipoxin A4 (LXA4) is abnormally produced in cystic fibrosis (CF) airways. LXA4 increases the CF airway surface liquid height and stimulates airway epithelial repair and tight junction formation. We report here a protective effect of LXA4 (1 nM) against tight junction disruption caused by Pseudomonas aeruginosa bacterial challenge together with a delaying action against bacterial invasion in CF airway epithelial cells from patients with CF and immortalized cell lines. Bacterial invasion and tight junction integrity were measured by gentamicin exclusion assays and confocal fluorescence microscopy in non-CF (NuLi-1) and CF (CuFi-1) bronchial epithelial cell lines and in primary CF cultures, grown under an air/liquid interface, exposed to either a clinical or laboratory strains of P. aeruginosa LXA4 delayed P. aeruginosa invasion and transepithelial migration in CF and normal bronchial epithelial cell cultures. These protective effects of LXA4 were inhibited by the ALX/FPR2 lipoxin receptor antagonist BOC-2. LXA4 prevented the reduction in mRNA biosynthesis and protein abundance of the tight junction protein ZO-1 and reduced tight junction disruption induced by P. aeruginsosa inoculation. In conclusion, LXA4 plays a protective role in bronchial epithelium by stimulating tight junction repair and by delaying and reducing the invasion of CF bronchial epithelial cells by P. aeruginsosa. Copyright © 2016 the American Physiological Society.

  14. Expression of epithelial cell-derived cytokine genes in the duodenal and colonic mucosae of dogs with chronic enteropathy

    PubMed Central

    OSADA, Hironari; OGAWA, Misato; HASEGAWA, Ayana; NAGAI, Makoto; SHIRAI, Junsuke; SASAKI, Kazuaki; SHIMODA, Minoru; ITOH, Hiroshi; KONDO, Hirotaka; OHMORI, Keitaro

    2016-01-01

    It remains unclear whether epithelial cell-derived cytokines, including interleukin (IL)-25, IL-33 and thymic stromal lymphopoietin (TSLP), contribute to development of canine chronic enteropathy (CE), which includes antibiotic-responsive enteropathy (ARE), food-responsive enteropathy (FRE) and inflammatory bowel disease (IBD). In the present study, we examined mRNA expression of il-25, il-33 and tslp in the duodenal and colonic mucosae of dogs with ARE, FRE and IBD. Real-time PCR analysis revealed that mRNA expression of il-33 was significantly lower in the duodenum in dogs with FRE than in healthy dogs. The results suggest that epithelial cell-derived cytokines may not be an inducer of Th2-type immunity in the gut of dogs with CE, and decreased expression of IL-33 may be involved in induction of FRE. Further studies are required to clarify roles of epithelial cell-derived cytokines, especially IL-33, in the pathogenesis of canine CE. PMID:28049868

  15. Expression of epithelial cell-derived cytokine genes in the duodenal and colonic mucosae of dogs with chronic enteropathy.

    PubMed

    Osada, Hironari; Ogawa, Misato; Hasegawa, Ayana; Nagai, Makoto; Shirai, Junsuke; Sasaki, Kazuaki; Shimoda, Minoru; Itoh, Hiroshi; Kondo, Hirotaka; Ohmori, Keitaro

    2017-02-28

    It remains unclear whether epithelial cell-derived cytokines, including interleukin (IL)-25, IL-33 and thymic stromal lymphopoietin (TSLP), contribute to development of canine chronic enteropathy (CE), which includes antibiotic-responsive enteropathy (ARE), food-responsive enteropathy (FRE) and inflammatory bowel disease (IBD). In the present study, we examined mRNA expression of il-25, il-33 and tslp in the duodenal and colonic mucosae of dogs with ARE, FRE and IBD. Real-time PCR analysis revealed that mRNA expression of il-33 was significantly lower in the duodenum in dogs with FRE than in healthy dogs. The results suggest that epithelial cell-derived cytokines may not be an inducer of Th2-type immunity in the gut of dogs with CE, and decreased expression of IL-33 may be involved in induction of FRE. Further studies are required to clarify roles of epithelial cell-derived cytokines, especially IL-33, in the pathogenesis of canine CE.

  16. Tumor necrosis factor (TNF)-neuropeptide Y (NPY) crosstalk regulates inflammation, epithelial barrier functions and colonic motility

    PubMed Central

    Chandrasekharan, Bindu; Jeppsson, Sabrina; Pienkowski, Stefan; Belsham, Denise D; Sitaraman, Shanthi V.; Merlin, Didier; Kokkotou, Efi; Nusrat, Asma; Tansey, Malu G.; Srinivasan, Shanthi

    2014-01-01

    Background Neuro-immune interactions play a significant role in regulating the severity of inflammation. Our previous work demonstrated that neuropeptide Y (NPY) is up regulated in the enteric nervous system (ENS) during murine colitis, and that NPY knockout mice exhibit reduced inflammation. Here we investigated if NPY expression during inflammation is induced by tumor necrosis factor (TNF), the main pro-inflammatory cytokine. Methods Utilizing primary enteric neurons and colon explant cultures from WT and NPY knockout (NPY−/−) mice, we determined if NPY knockdown modulates TNF release and epithelial permeability. Further we assessed if NPY expression is inducible by TNF in enteric neuronal cells and mouse model of experimental colitis, utilizing the TNF inhibitors-etanercept (blocks transmembrane and soluble TNF) and XPro1595 (blocks soluble TNF only). Results We found that enteric neurons express TNF receptors (TNFR1 and R2). Primary enteric neurons from NPY−/− mice produced less TNF compared to WT. Further, TNF activated NPY promoter in enteric neurons via phospho-c-jun. NPY−/− mice had decreased intestinal permeability. In vitro, NPY increased epithelial permeability via phosphatidyl inositol-3-kinase (PI3-K)-induced pore-forming claudin-2. TNF inhibitors attenuated NPY expression in vitro and in vivo. TNF-inhibitor-treated colitic mice exhibited reduced NPY expression and inflammation, reduced oxidative stress, enhanced neuronal survival and improved colonic motility. XPro1595 had more protective effects on neuronal survival and motility compared to etanercept. Conclusions We demonstrate a novel TNF-NPY cross talk that modulates inflammation, barrier functions and colonic motility during inflammation. It is also suggested that selective blocking of soluble TNF maybe a better therapeutic option than using anti-TNF antibodies. PMID:24108115

  17. Targeting normal and neoplastic tissues in the rat jejunum and colon with boronated, cationic acrylamide copolymers.

    PubMed

    Azab, Abdel-Kareem; Srebnik, Morris; Doviner, Victoria; Rubinstein, Abraham

    2005-08-18

    A series of boronated cationic copolymers, composed of different ratios of acrylamide, N-acryloyl-3-aminophenylboronic acid and N-acryloyl-diaminoethane (the cationic moiety), were prepared with the intention of localizing boron neutron capture therapy (BNCT) in experimentally induced polyps on the luminal side of the gut of the rat. The goals of this study were to: (a) test the effect of cationization of the boronated copolymers on their uptake by polyps and normal adjacent epithelium; (b) compare the whole rat body distribution of aminophenylboronic acid (APB) and polymeric APB after local application; (c) measure the effect of micro-environmental parameters such as pH, the presence of mucin and cations on the interaction between the APB-copolymers and the epithelium of the rat intestines. Direct analysis of tissue boron levels showed that polymeric APB-uptake was higher in the colonic polyps than in the surrounding normal tissues. Free APB, however, was found in similar quantities in both. When tested in the normal jejunum and colon of the rat, polymeric APB uptake was directly proportional to the molar content of the cationic monomer in the copolymers. The presence of magnesium ions, free boron cationic monomer and mucin interfered with this uptake in a concentration-dependent manner. The uptake was pH-independent at pH 5, 7 and 10. APB accumulation in the colon polyps was inversely proportional to the cationic monomer content in the copolymers, suggesting an increased amount of mucus around the polyps, which probably impeded the electrostatic attachment of the polymer to the malignant tissue. The use polymeric APB for targeting BNCT in perioperative treatment of colorectal carcinoma is suggested, especially in the cases of microscopic residual disease after curative resection.

  18. Label-free imaging of basement membranes differentiates normal, precancerous, and cancerous colonic tissues by second-harmonic generation microscopy.

    PubMed

    Zhuo, Shuangmu; Yan, Jun; Chen, Gang; Shi, Hong; Zhu, Xiaoqin; Lu, Jianping; Chen, Jianxin; Xie, Shusen

    2012-01-01

    Since changes in the basement membranes are the critical indicators for differentiating normal, precancerous, and cancerous colonic tissues, direct visualization of these warning signs is essential for the early diagnosis and treatment of colonic cancer. Here, we present that second harmonic generation (SHG) microscopy can probe the changes of basement membranes in different colonic cancer stages. Our results also show the capability of using the quantitative analyses of images for quantifying these changes in different cancer stages. These results suggest that SHG microscopy has the potential in label-freely imaging the changes of basement membranes for effectively distinguishing between normal, precancerous, and cancerous colonic tissues. To our knowledge, this is the first demonstration of the dynamics of basement membrane changes in different colonic cancer stages using entirely intrinsic source of contrast.

  19. Surface modification of microparticles causes differential uptake responses in normal and tumoral human breast epithelial cells

    NASA Astrophysics Data System (ADS)

    Patiño, Tania; Soriano, Jorge; Barrios, Lleonard; Ibáñez, Elena; Nogués, Carme

    2015-06-01

    The use of micro- and nanodevices as multifunctional systems for biomedical applications has experienced an exponential growth during the past decades. Although a large number of studies have focused on the design and fabrication of new micro- and nanosystems capable of developing multiple functions, a deeper understanding of their interaction with cells is required. In the present study, we evaluated the effect of different microparticle surfaces on their interaction with normal and tumoral human breast epithelial cell lines. For this, AlexaFluor488 IgG functionalized polystyrene microparticles (3 μm) were coated with Polyethyleneimine (PEI) at two different molecular weights, 25 and 750 kDa. The effect of microparticle surface properties on cytotoxicity, cellular uptake and endocytic pathways were assessed for both normal and tumoral cell lines. Results showed a differential response between the two cell lines regarding uptake efficiency and mechanisms of endocytosis, highlighting the potential role of microparticle surface tunning for specific cell targeting.

  20. The cinnamon-derived dietary factor cinnamic aldehyde activates the Nrf2-dependent antioxidant response in human epithelial colon cells.

    PubMed

    Wondrak, Georg Thomas; Villeneuve, Nicole F; Lamore, Sarah D; Bause, Alexandra S; Jiang, Tao; Zhang, Donna D

    2010-05-07

    Colorectal cancer (CRC) is a major cause of tumor-related morbidity and mortality worldwide. Recent research suggests that pharmacological intervention using dietary factors that activate the redox sensitive Nrf2/Keap1-ARE signaling pathway may represent a promising strategy for chemoprevention of human cancer including CRC. In our search for dietary Nrf2 activators with potential chemopreventive activity targeting CRC, we have focused our studies on trans-cinnamic aldehyde (cinnamaldeyde, CA), the key flavor compound in cinnamon essential oil. Here we demonstrate that CA and an ethanolic extract (CE) prepared from Cinnamomum cassia bark, standardized for CA content by GC-MS analysis, display equipotent activity as inducers of Nrf2 transcriptional activity. In human colon cancer cells (HCT116, HT29) and non-immortalized primary fetal colon cells (FHC), CA- and CE-treatment upregulated cellular protein levels of Nrf2 and established Nrf2 targets involved in the antioxidant response including heme oxygenase 1 (HO-1) and gamma-glutamyl-cysteine synthetase (gamma-GCS, catalytic subunit). CA- and CE-pretreatment strongly upregulated cellular glutathione levels and protected HCT116 cells against hydrogen peroxide-induced genotoxicity and arsenic-induced oxidative insult. Taken together our data demonstrate that the cinnamon-derived food factor CA is a potent activator of the Nrf2-orchestrated antioxidant response in cultured human epithelial colon cells. CA may therefore represent an underappreciated chemopreventive dietary factor targeting colorectal carcinogenesis.

  1. The Cinnamon-derived Dietary Factor Cinnamic Aldehyde Activates the Nrf2-dependent Antioxidant Response in Human Epithelial Colon Cells

    PubMed Central

    Wondrak, Georg T.; Villeneuve, Nicole F.; Lamore, Sarah D.; Bause, Alexandra S.; Jiang, Tao; Zhang, Donna D.

    2011-01-01

    Colorectal cancer (CRC) is a major cause of tumor-related morbidity and mortality worldwide. Recent research suggests that pharmacological intervention using dietary factors that activate the redox sensitive Nrf2/Keap1-ARE signaling pathway may represent a promising strategy for chemoprevention of human cancer including CRC. In our search for dietary Nrf2 activators with potential chemopreventive activity targeting CRC, we have focused our studies on trans-cinnamic aldehyde (cinnamaldeyde, CA), the key flavor compound in cinnamon essential oil. Here we demonstrate that CA and an ethanolic extract (CE) prepared from Cinnamomum cassia bark, standardized for CA content by GC-MS analysis, display equipotent activity as inducers of Nrf2 transcriptional activity. In human colon cancer cells (HCT116, HT29) and non-immortalized primary fetal colon cells (FHC), CA- and CE-treatment upregulated cellular protein levels of Nrf2 and established Nrf2 targets involved in the antioxidant response including heme oxygenase 1 (HO-1) and γ-glutamylcysteine synthetase (γ-GCS, catalytic subunit). CA- and CE-pretreatment strongly upregulated cellular glutathione levels and protected HCT116 cells against hydrogen peroxide-induced genotoxicity and arsenic-induced oxidative insult. Taken together our data demonstrate that the cinnamon-derived food factor CA is a potent activator of the Nrf2-orchestrated antioxidant response in cultured human epithelial colon cells. CA may therefore represent an underappreciated chemopreventive dietary factor targeting colorectal carcinogenesis. PMID:20657484

  2. Campylobacter protein oxidation influences epithelial cell invasion or intracellular survival as well as intestinal tract colonization in chickens.

    PubMed

    Lasica, A M; Wyszynska, A; Szymanek, K; Majewski, P; Jagusztyn-Krynicka, E K

    2010-01-01

    The Dsb family of redox proteins catalyzes disulfide bond formation and isomerization. Since mutations in dsb genes change the conformation and stability of many extracytoplasmic proteins, and since many virulence factors of pathogenic bacteria are extracytoplasmic, inactivation of dsb genes often results in pathogen attenuation. This study investigated the role of 2 membrane-bound oxidoreductases, DsbB and DsbI, in the Campylobacter jejuni oxidative Dsb pathway. Campylobacter mutants, lacking DsbB or DsbI or both, were constructed by allelic replacement and used in the human intestinal epithelial T84 cell line for the gentamicin protection assay (invasion assay) and chicken colonization experiments. In C. coli strain 23/1, the inactivation of the dsbB or dsbI gene separately did not significantly affect the colonization process. However, simultaneous disruption of both membrane-bound oxidoreductase genes significantly decreased the strain’s ability to colonize chicken intestines. Moreover, C. jejuni strain 81-176 with mutated dsbB or dsbI genes showed reduced invasion/intracellular survival abilities. No cells of the double mutants (dsbB⁻ dsbI⁻) of C. jejuni 81-176 were recovered from human cells after 3 h of invasion.

  3. Constipation-Predominant Irritable Bowel Syndrome Females Have Normal Colonic Barrier and Secretory Function.

    PubMed

    Peters, Stephanie A; Edogawa, Shoko; Sundt, Wendy J; Dyer, Roy B; Dalenberg, Daniel A; Mazzone, Amelia; Singh, Ravinder J; Moses, Natalie; Smyrk, Thomas C; Weber, Christopher; Linden, David R; MacNaughton, Wallace K; Turner, Jerrold R; Camilleri, Michael; Katzka, David A; Farrugia, Gianrico; Grover, Madhusudan

    2017-06-01

    The objective of this study was to determine whether constipation-predominant irritable bowel syndrome (IBS-C) is associated with changes in intestinal barrier and secretory function. A total of 19 IBS-C patients and 18 healthy volunteers (all females) underwent saccharide excretion assay (0.1 g (13)C mannitol and 1 g lactulose), measurements of duodenal and colonic mucosal barrier (transmucosal resistance (TMR), macromolecular and Escherichia coli Bio-Particle translocation), mucosal secretion (basal and acetylcholine (Ach)-evoked short-circuit current (Isc)), in vivo duodenal mucosal impedance, circulating endotoxins, and colonic tight junction gene expression. There were no differences in the in vivo measurements of barrier function between IBS-C patients and healthy controls: cumulative excretion of (13)C mannitol (0-2 h mean (s.e.m.); IBS-C: 12.1 (0.9) mg vs. healthy: 13.2 (0.8) mg) and lactulose (8-24 h; IBS-C: 0.9 (0.5) mg vs. healthy: 0.5 (0.2) mg); duodenal impedance IBS-C: 729 (65) Ω vs. healthy: 706 (43) Ω; plasma mean endotoxin activity level IBS-C: 0.36 (0.03) vs. healthy: 0.35 (0.02); and in colonic mRNA expression of occludin, zonula occludens (ZO) 1-3, and claudins 1-12 and 14-19. The ex vivo findings were consistent, with no group differences: duodenal TMR (IBS-C: 28.2 (1.9) Ω cm(2) vs. healthy: 29.8 (1.9) Ω cm(2)) and colonic TMR (IBS-C: 19.1 (1.1) Ω cm(2) vs. healthy: 17.6 (1.7) Ω cm(2)); fluorescein isothiocyanate (FITC)-dextran (4 kDa) and E. coli Bio-Particle flux. Colonic basal Isc was similar, but duodenal basal Isc was lower in IBS-C (43.5 (4.5) μA cm(-2)) vs. healthy (56.9 (4.9) μA cm(-2)), P=0.05. Ach-evoked ΔIsc was similar. Females with IBS-C have normal colonic barrier and secretory function. Basal duodenal secretion is decreased in IBS-C.

  4. Vibrio fischeri Outer Membrane Protein OmpU Plays a Role in Normal Symbiotic Colonization

    PubMed Central

    Aeckersberg, F.; Lupp, C.; Feliciano, B.; Ruby, E. G.

    2001-01-01

    The nascent light-emitting organ of newly hatched juveniles of the Hawaiian sepiolid squid Euprymna scolopes is specifically colonized by cells of Vibrio fischeri that are obtained from the ambient seawater. The mechanisms that promote this specific, cooperative colonization are likely to require a number of bacterial and host-derived factors and activities, only some of which have been described to date. A characteristic of many host-pathogen associations is the presence of bacterial mechanisms that allow attachment to specific tissues. These mechanisms have been well characterized and often involve bacterial fimbriae or outer membrane proteins (OMPs) that act as adhesins, the expression of which has been linked to virulence regulators such as ToxR in Vibrio cholerae. Analogous or even homologous mechanisms are probably operative in the initiation and persistence of cooperative bacterial associations, although considerably less is known about them. We report the presence in V. fischeri of ompU, a gene encoding a 32.5-kDa protein homolog of two other OMPs, OmpU of V. cholerae (50.8% amino acid sequence identity) and OmpL of Photobacterium profundum (45.5% identity). A null mutation introduced into the V. fischeri ompU resulted in the loss of an OMP with an estimated molecular mass of about 34 kDa; genetic complementation of the mutant strain with a DNA fragment containing only the ompU gene restored the production of this protein. The expression of the V. fischeri OmpU was not significantly affected by either (i) iron or phosphate limitation or (ii) a mutation that renders V. fischeri defective in the synthesis of a homolog of the OMP-regulatory protein ToxR. The ompU mutant grew normally in complex nutrient media but was more susceptible to growth inhibition in the presence of either anionic detergents or the antimicrobial peptide protamine sulfate. Interestingly, colonization experiments showed that the ompU null mutant initiated a symbiotic association with

  5. Expression Profiles of miRNA Subsets Distinguish Human Colorectal Carcinoma and Normal Colonic Mucosa

    PubMed Central

    Pellatt, Daniel F; Stevens, John R; Wolff, Roger K; Mullany, Lila E; Herrick, Jennifer S; Samowitz, Wade; Slattery, Martha L

    2016-01-01

    OBJECTIVES: MicroRNAs (miRNAs) are small, non-protein-coding RNA molecules that are commonly dysregulated in colorectal tumors. The objective of this study was to identify smaller subsets of highly predictive miRNAs. METHODS: Data come from population-based studies of colorectal cancer conducted in Utah and the Kaiser Permanente Medical Care Program. Tissue samples were available for 1,953 individuals, of which 1,894 had carcinoma tissue and 1,599 had normal mucosa available for statistical analysis. Agilent Human miRNA Microarray V.19.0 was used to generate miRNA expression profiles; validation of expression levels was carried out using quantitative PCR. We used random forest analysis and verified findings with logistic modeling in separate data sets. Important microRNAs are identified and bioinformatics tools are used to identify target genes and related biological pathways. RESULTS: We identified 16 miRNAs for colon and 17 miRNAs for rectal carcinoma that appear to differentiate between carcinoma and normal mucosa; of these, 12 were important for both colon and rectal cancer, hsa-miR-663b, hsa-miR-4539, hsa-miR-17-5p, hsa-miR-20a-5p, hsa-miR-21-5p, hsa-miR-4506, hsa-miR-92a-3p, hsa-miR-93-5p, hsa-miR-145-5p, hsa-miR-3651, hsa-miR-378a-3p, and hsa-miR-378i. Estimated misclassification rates were low at 4.83% and 2.5% among colon and rectal observations, respectively. Among independent observations, logistic modeling reinforced the importance of these miRNAs, finding the primary principal components of their variation statistically significant (P<0.001 among both colon and rectal observations) and again producing low misclassification rates. Repeating our analysis without those miRNAs initially identified as important identified other important miRNAs; however, misclassification rates increased and distinctions between remaining miRNAs in terms of classification importance were reduced. CONCLUSIONS: Our data support the hypothesis that while many miRNAs are

  6. Global DNA Hypomethylation (LINE-1) in the Normal Colon and Lifestyle Characteristics, Dietary and Genetic Factors

    PubMed Central

    Figueiredo, Jane C.; Grau, Maria V.; Wallace, Kristin; Levine, A. Joan; Shen, Lanlan; Hamdan, Randala; Chen, Xinli; Bresalier, Robert S.; McKeown-Eyssen, Gail; Haile, Robert W.; Baron, John A.; Issa, Jean-Pierre J.

    2009-01-01

    Background Global loss of methylated cytosines in DNA, thought to predispose to chromosomal instability and aneuploidy, has been associated with an increased risk of colorectal neoplasia. Little is known about the relationships between global hypomethylation and lifestyle, demographics, dietary measures and genetic factors. Methods Our data were collected as part of a randomized clinical trial testing the efficacy of aspirin and folic acid for the prevention of colorectal adenomas. At a surveillance colonoscopy approximately three years after the qualifying exam, we obtained two biopsies of the normal-appearing mucosa from the right colon and two from the left colon. Specimens were assayed for global hypomethylation using a pyrosequencing assay for LINE-1 (long interspersed nucleotide elements) repeats. Results The analysis included data from 388 subjects. There was relatively little variability in LINE methylation overall. Mean LINE-1 methylation levels in normal mucosa from the right bowel were significantly lower than those on the left side (p<0.0001). No significant associations were found between LINE-1 methylation and folate treatment, age, sex, body-mass-index, smoking status, alcohol use, dietary intake or circulating levels of B-vitamins, homocysteine, or selected genotypes. Race, dietary folic acid and plasma B6 showed associations with global methylation that differed between the right and left bowel. The effect of folic acid on risk of adenomas did not differ according to extent of LINE-1 methylation and we found no association between LINE-1 methylation and risk of adenomas. Conclusions LINE-1 methylation is not influenced by folic acid supplementation, but differs by colon subsite. PMID:19336559

  7. [Bioinformatic analysis of adenoma-normal mucosa SSH library of colon].

    PubMed

    Lü, Bing-Jian; Cui, Jing; Xu, Jing; Zhang, Hao; Luo, Min-Jie; Zhu, Yi-Min; Lai, Mao-De

    2006-04-01

    We established a colonic adenoma-normal mucosa suppressive subtraction hybridization (SSH) library in 1999. In this study, we wanted to explore the expression profile of all candidate genes in this library. We developed an EST pipeline which contained two in-house software packages, nucleic acid analytical software and GetUni. The nucleic acid analytical software, an integrator of the universal bioinformatics tools including phred, phd2fasta, cross_match, repeatmasker and blast2.0, can blast sequences of differential clones with the downloaded non-redundant nucleotide (NR) database. GetUni can cluster these NR sequences into Unigene via matching with the downloaded Homo Sapiens UniGene database. Sixty-two candidate genes in A-N library were obtained via the high throughput automatic gene expression bioinformatics pipeline. Gene Ontology online analysis revealed that ribosome genes and immunity-regulating genes were the two most common categories in the KEGG or Biocarta Pathway. We also detected the expression of 2 genes with highest hits, Reg4 and FAM46A, by semi-quantitative RT-PCR. Both genes were up-regulated in 10 or 9 out of 10 adenomas in comparison with the paired normal mucosa, respectively. The candidate genes in A-N library would be of great significance in disclosing the molecular mechanism underlying in colonic adenoma initiation and progression.

  8. Aluminium chloride promotes tumorigenesis and metastasis in normal murine mammary gland epithelial cells

    PubMed Central

    Tenan, Mirna; Ferrari, Paolo; Sappino, André‐Pascal

    2016-01-01

    Aluminium salts, present in many industrial products of frequent use like antiperspirants, anti‐acid drugs, food additives and vaccines, have been incriminated in contributing to the rise in breast cancer incidence in Western societies. However, current experimental evidence supporting this hypothesis is limited. For example, no experimental evidence that aluminium promotes tumorigenesis in cultured mammary epithelial cells exists. We report here that long‐term exposure to concentrations of aluminium—in the form of aluminium chloride (AlCl3)—in the range of those measured in the human breast, transform normal murine mammary gland (NMuMG) epithelial cells in vitro as revealed by the soft agar assay. Subcutaneous injections into three different mouse strains with decreasing immunodeficiency, namely, NOD SCID gamma (NSG), NOD SCID or nude mice, revealed that untreated NMuMG cells form tumors and metastasize, to a limited extent, in the highly immunodeficient and natural killer (NK) cell deficient NSG strain, but not in the less permissive and NK cell competent NOD SCID or nude strains. In contrast, NMuMG cells transformed in vitro by AlCl3 form large tumors and metastasize in all three mouse models. These effects correlate with a mutagenic activity of AlCl3. Our findings demonstrate for the first time that concentrations of aluminium in the range of those measured in the human breast fully transform cultured mammary epithelial cells, thus enabling them to form tumors and metastasize in well‐established mouse cancer models. Our observations provide experimental evidence that aluminium salts could be environmental breast carcinogens. PMID:27541736

  9. Vitiligo patient-derived keratinocytes exhibit characteristics of normal wound healing via epithelial to mesenchymal transition.

    PubMed

    Banerjee, Poulomi; Venkatachalam, Sandhyaa; Mamidi, Murali Krishna; Bhonde, Ramesh; Shankar, Krupa; Pal, Rajarshi

    2015-05-01

    Vitiligo is an autoimmune disorder that leads to depigmentation of skin via melanocyte dysfunction. Keratinocyte-induced toxicity is one among the several etiological factors implicated for vitiligo, and hence, autologous keratinocyte grafting is projected as one of the primary mode of treatment for vitiligo. However, reports indicate that perilesional keratinocytes not only display signatures of apoptosis but also could secrete cytokines and mediators which have antagonistic effect on proliferation or survival. Therefore, we investigated how vitiligo patients' derived keratinocytes respond to surplus amounts of inflammatory cytokines and whether they recapitulate events that take place during conventional wound healing. The primary objective of our study was to determine whether keratinocytes isolated from a vitiligo patient would undergo epithelial-mesenchymal transition similar to their normal counterparts upon induction with inflammatory cytokines such as TGF-b1 and EGF. We found that these keratinocytes undergo EMT during wound repair accompanied with increase in the levels of mesenchymal markers and ECM proteins; decrease in the levels of epithelial markers and enhanced migratory ability. Besides, we also demonstrated that EMT induction leads to activation of SMAD and MAPK pathways via Ras, Raf, PAI 1, Snail, Slug and ZO1. To our knowledge, this is the first report on the characterization of primary keratinocytes isolated from vitiligo patients with respect to their wound healing capacity.

  10. Application of HPLC for determination of phytic acid in the colonic epithelial Caco-2 cells.

    PubMed

    Weglarz, Ludmiła; Parfiniewicz, Beata; Bat, Beata; Orchel, Arkadiusz; Dzierzewicz, Zofia; Wilczok, Tadeusz

    2004-12-01

    Phytic acid (myo-inositol hexaphosphate, IP6) is currently receiving a considerable interest because of its anticancer (preventive and therapeutic) potential against colon tumors and the need for methods of its determination. The aim of this study was to analyze the uptake of IP6 by human colon adenocarcinoma cells (Caco-2 cell line) and to evaluate the method of its intracellular quantification with the use of high performance liquid chromatography (HPLC) technique. Chromatographic analysis revealed a rapid uptake of IP6 by cells. The intracellular accumulation of IP6 was saturable at 0.5 h and it did not change with the prolongation of incubation up to 72 h.

  11. Listeriolysin O affects barrier function and induces chloride secretion in HT-29/B6 colon epithelial cells.

    PubMed

    Richter, Jan F; Gitter, Alfred H; Günzel, Dorothee; Weiss, Siegfried; Mohamed, Walid; Chakraborty, Trinad; Fromm, Michael; Schulzke, Jörg D

    2009-06-01

    Listeria monocytogenes is a food-borne pathogen, which is able to induce diarrhea when residing in the intestine. We studied the effect of listeriolysin O (LLO), an extracellular virulence factor of L. monocytogenes, on intestinal transport and barrier function in monolayers of HT-29/B6 human colon cells using the Ussing technique to understand the pathomechanisms involved. Mucosal addition of LLO, but not a LLO mutant, induced a dose- and pH-dependent increase in short-circuit current (I(SC)). Sodium and chloride tracer flux and DIDS sensitivity studies revealed that I(SC) was mainly due to electrogenic chloride secretion. Barrier function was impaired by LLO, as assessed by transepithelial resistance (R(t)) and mannitol flux measurements. Intracellular signal transduction occurred through Ca(2+) release from intracellular stores and PKC activation. In conclusion, listeriolysin induces chloride secretion and perturbs epithelial barrier function, thus potentially contributing to Listeria-induced diarrhea.

  12. Immortalization of normal human kidney epithelial cells by nickel(II)

    SciTech Connect

    Tveito, G.; Hansteen, I.L.; Dalen, H.; Haugen, A.

    1989-04-01

    The occupational and environmental hazards of nickel exposure are of great concern in environmental medicine. Nickel workers have increased risk of cancer of the nose, lung, larynx, and possibly the kidney. In the present investigation we have studied the effects of nickel ions on fetal human kidney cortex explants. The explants were continuously exposed to 5 micrograms/ml NiSO4. After 70-100 days in culture foci of phenotypically altered cells appeared. Immortalized cell lines were established and demonstrated to be of human epithelial origin. Tumorigenicity was not induced, but the cells demonstrated decreased requirement for serum, increased plating efficiency and saturation density, and formation of colonies in soft agar. Chromosome changes in the treated cells were observed. Worth mentioning are change in ploidy (3n) and abnormalities of chromosomes 1, 7, 9, 11, 13, 14 and 20; increased numbers of chromosome 17; and loss of normal chromosomes 20 and 22.

  13. Epithelial-to-Mesenchymal Transition in the Female Reproductive Tract: From Normal Functioning to Disease Pathology

    PubMed Central

    Bilyk, Olena; Coatham, Mackenzie; Jewer, Michael; Postovit, Lynne-Marie

    2017-01-01

    Epithelial-to-mesenchymal transition (EMT) is a physiological process that is vital throughout the human lifespan. In addition to contributing to the development of various tissues within the growing embryo, EMT is also responsible for wound healing and tissue regeneration later in adulthood. In this review, we highlight the importance of EMT in the development and normal functioning of the female reproductive organs (the ovaries and the uterus) and describe how dysregulation of EMT can lead to pathological conditions, such as endometriosis, adenomyosis, and carcinogenesis. We also summarize the current literature relating to EMT in the context of ovarian and endometrial carcinomas, with a particular focus on how molecular mechanisms and the tumor microenvironment can govern cancer cell plasticity, therapy resistance, and metastasis. PMID:28725636

  14. Chemical carcinogen-induced decreases in genomic 5-methyldeoxycytidine content of normal human bronchial epithelial cells

    SciTech Connect

    Wilson, V.L.; Smith, R.A.; Longoria, J.; Liotta, M.A.; Harper, C.M.; Harris, C.C.

    1987-05-01

    The genomic content of DNA 5-methyldeoxycytidine (m/sup 5/dC) was measured in dividing normal human bronchial epithelial cells treated with a broad range of chemical carcinogens. At noncytotoxic concentrations, all of the carcinogenic agents tested significantly reduced cellular DNA m/sup 5/dC content whereas the weakly carcinogenic and noncarcinogenic agents, benzo(e)pyrene and phenanthrene (respectively), did not. These reductions varied from 8% to 31% depending on the agent and the donor cells. The reduction is genomic m/sup 5/dC levels were concentration dependent for the carcinogenic polycyclic aromatic hydrocarbon benzo(a)pyrene. The authors speculate that carcinogen-induced perturbation of DNA m/sup 5/dC patterns may lead to heritable changes in gene expression and contribute to the molecular alterations involved in the initiation and the subsequent steps of the carcinogenesis process.

  15. Expression of VLA-alpha 2, VLA-alpha 6, and VLA-beta 1 chains in normal mucosa and adenomas of the colon, and in colon carcinomas and their liver metastases.

    PubMed Central

    Koretz, K.; Schlag, P.; Boumsell, L.; Möller, P.

    1991-01-01

    'Very late antigen' (VLA) proteins are members of the integrin superfamily with cell-surface receptor function and are involved in the cell-cell matrix interaction. They are heterodimers with a common beta 1 chain and different alpha chains counted through VLA-1 to VLA-6. The VLA-2 complex (alpha 2/beta 1) was found to act as collagen receptor on platelets and the VLA-6 complex (alpha 6/beta 1) as laminin receptor. Using monoclonal antibodies and an indirect immunoperoxidase method, we investigated the expression of VLA-alpha 2, VLA-alpha 6, and VLA-beta 1 chains in 20 normal colonic mucosa samples, in 20 colonic adenomas, and in 96 carcinomas together with 10 accompanying liver metastases. All three proteins were expressed throughout the colonic epithelium, except for VLA-alpha 2, which was present in the cryptic gland but was absent on the mucosal surface in some cases. In general, adenomas were strongly positive for the VLA proteins but 3 of 20 cases showed focal VLA-alpha 2-negative areas. The carcinomas revealed considerable heterogeneity of VLA-alpha 2 expression; ie, 59 tumors were completely positive, 35 tumors revealed a focal loss of antigen, and 2 cases were negative. This reduced antigen expression was statistically associated with Dukes' stage C/D (P = 0.003). VLA-alpha 6 was expressed throughout in all tumors. VLA-beta 1 was found extensively expressed in 77 carcinomas, partially expressed in 17 carcinomas, and was absent in 2 carcinomas. As compared to their primary tumors, liver metastases showed roughly corresponding patterns of antigen expression. The down regulation/loss of VLA proteins in a subset of epithelial colon tumors might cause a disturbed cell-cell/cell-matrix interaction that might augment the invasive property of their cells. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:2000944

  16. Chlorpheniramine Increases Paracellular Permeability to Marker Fluorescein Lucifer Yellow Mediated by Internalization of Occludin in Murine Colonic Epithelial Cells.

    PubMed

    Manabe, Aya; Furukawa, Chisa; Endo, Satoshi; Marunaka, Kana; Nishiyama, Tsubasa; Fujii, Naoko; Tabuchi, Yoshiaki; Matsunaga, Toshiyuki; Ikari, Akira

    2017-01-01

    Ions, small molecules, and drugs are absorbed in the intestinal epithelium mediated by transcellular and paracellular pathways. The function of various transporters expressing in the apical and basolateral membranes of intestinal epithelial cells has been well characterized. In contrast, claudins and occludin, components of the tight junctions (TJs), determine the paracellular permeability to ions and low molecular weight compounds, but the properties for permeability has not been clarified in detail. In the present study, we examined the effects of anti-histamine drugs, chlorpheniramine and diphenhydramine, on transepithelial electrical resistance (TER) and permeability to lucifer yellow (LY), a marker of paracellular permeability, using murine colonic MCE301 cells. Chlorpheniramine significantly decreased the steady state of TER and increased permeability to LY, whereas the effects of diphenhydramine were not significant. The mRNAs of occludin and claudin-1-claudin-8 except for claudin-5 were expressed in MCE301 cells. Both anti-histamine drugs did not change solubility of claudins to 0.5% Triton X-100 solution. In contrast, the detergent solubility and intracellular localization of occludin were significantly increased by chlorpheniramine. These results indicate that occludin is dissociated from the TJs by chlorpheniramine. Chlorpheniramine increased protein phosphatase-2A (PP-2A) activity, which was inhibited by cantharidin, a potent PP-2A inhibitor. Furthermore, the changes of TER, permeability to LY, and de-phosphorylation and tight junctional localization of occludin caused by chlorpheniramine were recovered by cantharidin. These results suggest that chlorpheniramine could increase paracellular permeability to low molecular weight compounds mediated by the activation of PP-2A and internalization of occludin in the colonic epithelial cells.

  17. Prostaglandin E2 modulates IL-8 expression through formation of a multiprotein enhanceosome in human colonic epithelial cells.

    PubMed

    Srivastava, Vikas; Dey, Indranil; Leung, Pearl; Chadee, Kris

    2012-04-01

    Gastrointestinal inflammation is mediated by the pro-inflammatory mediators interleukin-8 (IL-8) and prostaglandin E(2) (PGE(2) ). PGE(2) binding and coupling through EP2/4 receptor subtypes on colonic epithelial cells stimulates cyclic adenosine monophosphate (cAMP) and IL-8 production. Here we determined the mechanisms whereby PGE(2) regu-lates IL-8 in Caco2 colonic epithelial cells and in cells over-expressing the EP2/4 receptors (EP2S/EP4S). PGE(2) coupling through EP2 activated the transcription factor inducible cAMP early repressor (ICER), whereas coupling through EP4 receptors activated the cyclic AMP-responsive element-binding protein (CREB). Activation of CREB in Caco2/EP2S was protein kinase A (PKA) dependent, whereas in EP4S cells, activation of CREB occurred through the PKA and phosphatidylinositol 3-kinase pathways. Since ICER lacks the transactivation domain, it functions as a transcription repressor as opposed to CREB. PGE(2) coupling through EP2/4 receptors can therefore acts in an opposing manner to either decrease (EP2) or promote IL-8 expression by recruiting CREB-binding protein (CBP) (EP4), which formed a multiprotein IL-8 enhanceosome. A novel half CRE (167CRE) and a composite NFAT1-AP1-like site in the IL-8 promoter participated in binding and complex formation as confirmed by mutagenesis and expression studies. These data unravel the mechanisms by which expression of IL-8 is controlled by different signalling pathways that are activated by PGE(2) but acting through different EP receptors. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Antiproliferative and apoptotic effects of tocopherols and tocotrienols on normal mouse mammary epithelial cells.

    PubMed

    McIntyre, B S; Briski, K P; Tirmenstein, M A; Fariss, M W; Gapor, A; Sylvester, P W

    2000-02-01

    Studies were conducted to determine the comparative effects of tocopherols and tocotrienols on normal mammary epithelial cell growth and viability. Cells isolated from midpregnant BALB/c mice were grown within collagen gels and maintained on serum-free media. Treatment with 0-120 microM alpha- and gamma-tocopherol had no effect, whereas 12.5-100m microM tocotrienol-rich fraction of palm oil (TRF), 100-120 microM delta-tocopherol, 50-60 microM alpha-tocotrienol, and 8-14 microM gamma- or delta-tocotrienol significantly inhibited cell growth in a dose-responsive manner. In acute studies, 24-h exposure to 0-250 microM alpha-, gamma-, and delta-tocopherol had no effect, whereas similar treatment with 100-250 microM TRF, 140-250 microM alpha-, 25-100 microM gamma- or delta-tocotrienol significantly reduced cell viability. Growth-inhibitory doses of TRF, delta-tocopherol, and alpha-, gamma-, and delta-tocotrienol were shown to induce apoptosis in these cells, as indicated by DNA fragmentation. Results also showed that mammary epithelial cells more easily or preferentially took up tocotrienols as compared to tocopherols, suggesting that at least part of the reason tocotrienols display greater biopotency than tocopherols is because of greater cellular accumulation. In summary, these findings suggest that the highly biopotent gamma- and delta-tocotrienol isoforms may play a physiological role in modulating normal mammary gland growth, function, and remodeling.

  19. Role of activation of protein kinase C in the stimulation of colonic epithelial proliferation and reactive oxygen formation by bile acids.

    PubMed Central

    Craven, P A; Pfanstiel, J; DeRubertis, F R

    1987-01-01

    Deoxycholate (DOC), chenodeoxycholate, 12-O-tetradecanoyl phorbol-13-acetate (TPA), or 1-oleoyl-2-acetyl-glycerol (OAG) activated colonic epithelial protein kinase C as reflected by translocation from the soluble to the particulate cell fraction. Activation of protein kinase C was correlated with stimulation of enhanced proliferative activity of colonic mucosa and reactive oxygen production. TPA and OAG, but not DOC, directly activated soluble protein kinase C in vitro. However, DOC rapidly increased labeled inositol phosphate and diacylglycerol accumulation in colonic epithelial cells. Retinoic acid inhibited protein kinase C activity and suppressed DOC-, TPA-, and OAG-induced increases in reactive oxygen production. The results support a role for protein kinase C in the stimulation of colonic epithelial proliferative activity and reactive oxygen production induced by bile acids, TPA and OAG. In contrast to TPA and OAG, which activate protein kinase C directly, bile acids appear to activate protein kinase C indirectly by increasing the diacylglycerol content of colonic epithelium. PMID:3027128

  20. HIN-1, a putative cytokine highly expressed in normal but not cancerous mammary epithelial cells

    PubMed Central

    Krop, Ian E.; Sgroi, Dennis; Porter, Dale A.; Lunetta, Kathryn L.; LeVangie, Rebbecca; Seth, Pankaj; Kaelin, Carolyn M.; Rhei, Esther; Bosenberg, Marcus; Schnitt, Stuart; Marks, Jeffrey R.; Pagon, Zrinka; Belina, Drazen; Razumovic, Jasminka; Polyak, Kornelia

    2001-01-01

    To identify molecular alterations implicated in the initiating steps of breast tumorogenesis, we compared the gene expression profiles of normal and ductal carcinoma in situ (DCIS) mammary epithelial cells by using serial analysis of gene expression (SAGE). Through the pair-wise comparison of normal and DCIS SAGE libraries, we identified several differentially expressed genes. Here, we report the characterization of one of these genes, HIN-1 (high in normal-1). HIN-1 expression is significantly down regulated in 94% of human breast carcinomas and in 95% of preinvasive lesions, such as ductal and lobular carcinoma in situ. This decrease in HIN-1 expression is accompanied by hypermethylation of its promoter in the majority of breast cancer cell lines (>90%) and primary tumors (74%). HIN-1 is a putative cytokine with no significant homology to known proteins. Reintroduction of HIN-1 into breast cancer cells inhibits cell growth. These results indicate that HIN-1 is a candidate tumor suppressor gene that is inactivated at high frequency in the earliest stages of breast tumorogenesis. PMID:11481438

  1. Three-dimensional telomere architecture of esophageal squamous cell carcinoma: comparison of tumor and normal epithelial cells.

    PubMed

    Sunpaweravong, S; Sunpaweravong, P; Sathitruangsak, C; Mai, S

    2016-05-01

    Telomeres are repetitive nucleotide sequences (TTAGGG)n located at the ends of chromosomes that function to preserve chromosomal integrity and prevent terminal end-to-end fusions. Telomere loss or dysfunction results in breakage-bridge-fusion cycles, aneuploidy, gene amplification and chromosomal rearrangements, which can lead to genomic instability and promote carcinogenesis. Evaluating the hypothesis that changes in telomeres contribute to the development of esophageal squamous cell carcinoma (ESCC) and to determine whether there are differences between young and old patients, we compared the three-dimensional (3D) nuclear telomere architecture in ESCC tumor cells with that of normal epithelial cells obtained from the same patient. Patients were equally divided by age into two groups, one comprising those less than 45 years of age and the other consisting of those over 80 years of age. Tumor and normal epithelial cells located at least 10 cm from the border of the tumor were biopsied in ESCC patients. Hematoxylin and eosin staining was performed for each sample to confirm and identify the cancer and normal epithelial cells. This study was based on quantitative 3D fluorescence in situ hybridization (Q-FISH), 3D imaging and 3D analysis of paraffin-embedded slides. The 3D telomere architecture data were computer analyzed using 100 nuclei per slide. The following were the main parameters compared: the number of signals (number of telomeres), signal intensity (telomere length), number of telomere aggregates, and nuclear volume. Tumor and normal epithelial samples from 16 patients were compared. The normal epithelial cells had more telomere signals and higher intensities than the tumor cells, with P-values of P < 0.0001 and P = 0.0078, respectively. There were no statistically significant differences in the numbers of telomere aggregates or the nuclear volumes between the tumor and normal epithelial cells. Secondary analyses examined the effects of age on 3D telomere

  2. Normal Human Lung Epithelial Cells Inhibit Transforming Growth Factor-β Induced Myofibroblast Differentiation via Prostaglandin E2

    PubMed Central

    Epa, Amali P.; Thatcher, Thomas H.; Pollock, Stephen J.; Wahl, Lindsay A.; Lyda, Elizabeth; Kottmann, R. M.; Phipps, Richard P.; Sime, Patricia J.

    2015-01-01

    Introduction Idiopathic pulmonary fibrosis (IPF) is a chronic progressive disease with very few effective treatments. The key effector cells in fibrosis are believed to be fibroblasts, which differentiate to a contractile myofibroblast phenotype with enhanced capacity to proliferate and produce extracellular matrix. The role of the lung epithelium in fibrosis is unclear. While there is evidence that the epithelium is disrupted in IPF, it is not known whether this is a cause or a result of the fibroblast pathology. We hypothesized that healthy epithelial cells are required to maintain normal lung homeostasis and can inhibit the activation and differentiation of lung fibroblasts to the myofibroblast phenotype. To investigate this hypothesis, we employed a novel co-culture model with primary human lung epithelial cells and fibroblasts to investigate whether epithelial cells inhibit myofibroblast differentiation. Measurements and Main Results In the presence of transforming growth factor (TGF)-β, fibroblasts co-cultured with epithelial cells expressed significantly less α-smooth muscle actin and collagen and showed marked reduction in cell migration, collagen gel contraction, and cell proliferation compared to fibroblasts grown without epithelial cells. Epithelial cells from non-matching tissue origins were capable of inhibiting TGF-β induced myofibroblast differentiation in lung, keloid and Graves’ orbital fibroblasts. TGF-β promoted production of prostaglandin (PG) E2 in lung epithelial cells, and a PGE2 neutralizing antibody blocked the protective effect of epithelial cell co-culture. Conclusions We provide the first direct experimental evidence that lung epithelial cells inhibit TGF-β induced myofibroblast differentiation and pro-fibrotic phenotypes in fibroblasts. This effect is not restricted by tissue origin, and is mediated, at least in part, by PGE2. Our data support the hypothesis that the epithelium plays a crucial role in maintaining lung homeostasis

  3. Surface modification of microparticles causes differential uptake responses in normal and tumoral human breast epithelial cells

    PubMed Central

    Patiño, Tania; Soriano, Jorge; Barrios, Lleonard; Ibáñez, Elena; Nogués, Carme

    2015-01-01

    The use of micro- and nanodevices as multifunctional systems for biomedical applications has experienced an exponential growth during the past decades. Although a large number of studies have focused on the design and fabrication of new micro- and nanosystems capable of developing multiple functions, a deeper understanding of their interaction with cells is required. In the present study, we evaluated the effect of different microparticle surfaces on their interaction with normal and tumoral human breast epithelial cell lines. For this, AlexaFluor488 IgG functionalized polystyrene microparticles (3 μm) were coated with Polyethyleneimine (PEI) at two different molecular weights, 25 and 750 kDa. The effect of microparticle surface properties on cytotoxicity, cellular uptake and endocytic pathways were assessed for both normal and tumoral cell lines. Results showed a differential response between the two cell lines regarding uptake efficiency and mechanisms of endocytosis, highlighting the potential role of microparticle surface tunning for specific cell targeting. PMID:26068810

  4. 3D-fibroblast tissues constructed by a cell-coat technology enhance tight-junction formation of human colon epithelial cells.

    PubMed

    Matsusaki, Michiya; Hikimoto, Daichi; Nishiguchi, Akihiro; Kadowaki, Koji; Ohura, Kayoko; Imai, Teruko; Akashi, Mitsuru

    2015-02-13

    Caco-2, human colon carcinoma cell line, has been widely used as a model system for intestinal epithelial permeability because Caco-2 cells express tight-junctions, microvilli, and a number of enzymes and transporters characteristic of enterocytes. However, the functional differentiation and polarization of Caco-2 cells to express sufficient tight-junctions (a barrier) usually takes over 21 days in culture. This may be due to the cell culture environment, for example inflammation induced by plastic petri dishes. Three-dimensional (3D) sufficient cell microenvironments similar to in vivo natural conditions (proteins and cells), will promote rapid differentiation and higher functional expression of tight junctions. Herein we report for the first time an enhancement in tight-junction formation by 3D-cultures of Caco-2 cells on monolayered (1L) and eight layered (8L) normal human dermal fibroblasts (NHDF). Trans epithelial electric resistance (TEER) of Caco-2 cells was enhanced in the 3D-cultures, especially 8L-NHDF tissues, depending on culture times and only 10 days was enough to reach the same TEER value of Caco-2 monolayers after a 21 day incubation. Relative mRNA expression of tight-junction proteins of Caco-2 cells on 3D-cultures showed higher values than those in monolayer structures. Transporter gene expression patterns of Caco-2 cells on 3D-constructs were almost the same as those of Caco-2 monolayers, suggesting that there was no effect of 3D-cultures on transporter protein expression. The expression correlation between carboxylesterase 1 and 2 in 3D-cultures represented similar trends with human small intestines. The results of this study clearly represent a valuable application of 3D-Caco-2 tissues for pharmaceutical applications. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. E-Cigarette Affects the Metabolome of Primary Normal Human Bronchial Epithelial Cells.

    PubMed

    Aug, Argo; Altraja, Siiri; Kilk, Kalle; Porosk, Rando; Soomets, Ursel; Altraja, Alan

    2015-01-01

    E-cigarettes are widely believed to be safer than conventional cigarettes and have been even suggested as aids for smoking cessation. However, while reasonable with some regards, this judgment is not yet supported by adequate biomedical research data. Since bronchial epithelial cells are the immediate target of inhaled toxicants, we hypothesized that exposure to e-cigarettes may affect the metabolome of human bronchial epithelial cells (HBEC) and that the changes are, at least in part, induced by oxidant-driven mechanisms. Therefore, we evaluated the effect of e-cigarette liquid (ECL) on the metabolome of HBEC and examined the potency of antioxidants to protect the cells. We assessed the changes of the intracellular metabolome upon treatment with ECL in comparison of the effect of cigarette smoke condensate (CSC) with mass spectrometry and principal component analysis on air-liquid interface model of normal HBEC. Thereafter, we evaluated the capability of the novel antioxidant tetrapeptide O-methyl-l-tyrosinyl-γ-l-glutamyl-l-cysteinylglycine (UPF1) to attenuate the effect of ECL. ECL caused a significant shift in the metabolome that gradually gained its maximum by the 5th hour and receded by the 7th hour. A second alteration followed at the 13th hour. Treatment with CSC caused a significant initial shift already by the 1st hour. ECL, but not CSC, significantly increased the concentrations of arginine, histidine, and xanthine. ECL, in parallel with CSC, increased the content of adenosine diphosphate and decreased that of three lipid species from the phosphatidylcholine family. UPF1 partially counteracted the ECL-induced deviations, UPF1's maximum effect occurred at the 5th hour. The data support our hypothesis that ECL profoundly alters the metabolome of HBEC in a manner, which is comparable and partially overlapping with the effect of CSC. Hence, our results do not support the concept of harmlessness of e-cigarettes.

  6. Annexin II binds progastrin and gastrin-like peptides, and mediates growth factor effects of autocrine and exogenous gastrins on colon cancer and intestinal epithelial cells.

    PubMed

    Singh, P; Wu, H; Clark, C; Owlia, A

    2007-01-18

    We and others have reported the presence of novel progastrin (PG)/gastrin receptors on normal and cancerous intestinal cells. We had earlier reported the presence of 33-36 kDa gastrin-binding proteins on cellular membranes of colon cancer cells. The goal of the current study was to identify the protein(s) in the 33-36 kDa band, and analyse its functional significance. A carbodiimide crosslinker was used for crosslinking radio-labeled gastrins to membrane proteins from gastrin/PG responsive cell lines. Native membrane proteins, crosslinked to the ligand, were solubulized and enriched by >1000-fold, and analysed by surface-enhanced laser desorption/ionization-time of flight-mass spectrometry. The peptide masses were researched against the NCBInr database using the ProFound search engine. Annexin II (ANX II) was identified, and confirmed by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry. As HCT-116 cells express autocrine PG, the in situ association of PG with ANX II was demonstrated in pulldown assays. Direct binding of PG with ANX II was confirmed in an in vitro binding assay. In order to confirm a functional importance of these observations, sense and anti-sense (AS) ANX II RNA-expressing clones of intestinal epithelial (IEC-18) and human colon cancer (HCT-116) cell lines were generated. AS clones demonstrated a significant loss in the growth response to exogenous (IEC-18) and autocrine (HCT-116) PG. We have thus discovered that membrane-associated ANX II binds PG/gastrins, and partially mediates growth factor effects of the peptides.

  7. Reciprocal Paracrine Interactions Between Normal Human Epithelial and Mesenchymal Cells Protect Cellular DNA from Radiation-Induced Damage

    SciTech Connect

    Nakazawa, Yuka; Saenko, Vladimir Rogounovitch, Tatiana; Suzuki, Keiji; Mitsutake, Norisato; Matsuse, Michiko; Yamashita, Shunichi

    2008-06-01

    Purpose: To explore whether interactions between normal epithelial and mesenchymal cells can modulate the extent of radiation-induced DNA damage in one or both types of cells. Methods and Materials: Human primary thyrocytes (PT), diploid fibroblasts BJ, MRC-5, and WI-38, normal human mammary epithelial cells (HMEC), and endothelial human umbilical cord vein endothelial cells (HUV-EC-C), cultured either individually or in co-cultures or after conditioned medium transfer, were irradiated with 0.25 to 5 Gy of {gamma}-rays and assayed for the extent of DNA damage. Results: The number of {gamma}-H2AX foci in co-cultures of PT and BJ fibroblasts was approximately 25% lower than in individual cultures at 1 Gy in both types of cells. Reciprocal conditioned medium transfer to individual cultures before irradiation resulted in approximately a 35% reduction of the number {gamma}-H2AX foci at 1 Gy in both types of cells, demonstrating the role of paracrine soluble factors. The DNA-protected state of cells was achieved within 15 min after conditioned medium transfer; it was reproducible and reciprocal in several lines of epithelial cells and fibroblasts, fibroblasts, and endothelial cells but not in epithelial and endothelial cells. Unlike normal cells, human epithelial cancer cells failed to establish DNA-protected states in fibroblasts and vice versa. Conclusions: The results imply the existence of a network of reciprocal interactions between normal epithelial and some types of mesenchymal cells mediated by soluble factors that act in a paracrine manner to protect DNA from genotoxic stress.

  8. Electroporation-assisted penetration of zinc oxide nanoparticles in ex vivo normal and cancerous human colon tissue

    NASA Astrophysics Data System (ADS)

    Zhou, L. P.; Wu, G. Y.; Wei, H. J.; Guo, Z. Y.; Yang, H. Q.; He, Y. H.; Xie, S. S.

    2015-11-01

    In this study, we presented the research of the penetration of zinc oxide nanoparticles (ZnO NPs) (30 and 90 nm), and electroporation (EP) assisted penetration of the ZnO NPs in the human normal colon (NC) and adenomatous colon (AC) tissues studied with optical coherence tomography (OCT) and diffuse reflectance (DR) measurement. The results have shown that the attenuation coefficient of colon tissue after the application of 30 or 90 nm ZnO NPs alone decreased approximately by 28% and 14% for NC tissue, 35% and 22% for AC tissue, respectively; while the attenuation coefficient of colon tissue after combined application of 30 or 90 nm ZnO NPs/EP decreased approximately by 46% and 30% for NC tissue, and 53% and 42% for AC tissue, respectively. The results illustrate EP can significantly increase the penetration of ZnO NPs in the colon tissue, especially in AC tissue. Through the analysis of attenuation coefficient and reflectance intensity of the colon tissue, we find that the accumulation of the ZnO NPs in the colon tissue greatly influenced the tissue optical properties.

  9. Zinc Induced G2/M Blockage is p53 and p21 Dependent in Normal Human Bronchial Epithelial Cells

    USDA-ARS?s Scientific Manuscript database

    The involvement of the p53 and p21 signal pathway in the G2/M cell cycle progression of zinc supplemented normal human bronchial epithelial (NHBE) cells was examined using the siRNA approach. Cells were cultured for one passage in different concentrations of zinc: <0.4 microM (ZD) as zinc-deficient;...

  10. Extensive quantitative remodeling of the proteome between normal colon tissue and adenocarcinoma

    PubMed Central

    Wiśniewski, Jacek R; Ostasiewicz, Paweł; Duś, Kamila; Zielińska, Dorota F; Gnad, Florian; Mann, Matthias

    2012-01-01

    We report a proteomic analysis of microdissected material from formalin-fixed and paraffin-embedded colorectal cancer, quantifying >7500 proteins between patient matched normal mucosa, primary carcinoma, and nodal metastases. Expression levels of 1808 proteins changed significantly between normal and cancer tissues, a much larger fraction than that reported in transcript-based studies. Tumor cells exhibit extensive alterations in the cell-surface and nuclear proteomes. Functionally similar changes in the proteome were observed comparing rapidly growing and differentiated CaCo-2 cells. In contrast, there was minimal proteomic remodeling between primary cancer and metastases, suggesting that no drastic proteome changes are necessary for the tumor to propagate in a different tissue context. Additionally, we introduce a new way to determine protein copy numbers per cell without protein standards. Copy numbers estimated in enterocytes and cancer cells are in good agreement with CaCo-2 and HeLa cells and with the literature data. Our proteomic data set furthermore allows mapping quantitative changes of functional protein classes, enabling novel insights into the biology of colon cancer. PMID:22968445

  11. Analysis of CUL-5 expression in breast epithelial cells, breast cancer cell lines, normal tissues and tumor tissues

    PubMed Central

    Fay, Michael J; Longo, Kenneth A; Karathanasis, George A; Shope, David M; Mandernach, Craig J; Leong, Jason R; Hicks, Alfred; Pherson, Kenneth; Husain, Amyna

    2003-01-01

    Background The chromosomal location of CUL-5 (11q 22-23) is associated with LOH in breast cancer, suggesting that CUL-5 may be a tumor suppressor. The purpose of this research was to determine if there is differential expression of CUL-5 in breast epithelial cells versus breast cancer cell lines, and normal human tissues versus human tumors. The expression of CUL-5 in breast epithelial cells (HMEC, MCF-10A), and breast cancer cells (MCF-7, MDA-MB-231) was examined using RT-PCR, Northern blot analysis, and Western blot analysis. The expression of mRNA for other CUL family members (CUL-1, -2, -3, -4A, and -4B) in these cells was evaluated by RT-PCR. A normal human tissue expression array and a cancer profiling array were used to examine CUL-5 expression in normal human tissues and matched normal tissues versus tumor tissues, respectively. Results CUL-5 is expressed at the mRNA and protein levels by breast epithelial cells (HMEC, MCF-10A) and breast cancer cells (MCF-7, MDA-MB-231). These cells also express mRNA for other CUL family members. The normal human tissue expression array revealed that CUL-5 is widely expressed. The cancer profiling array revealed that 82% (41/50) of the breast cancers demonstrated a decrease in CUL-5 expression versus the matched normal tissue. For the 50 cases of matched breast tissue there was a statistically significant ~2.2 fold decreased expression of CUL-5 in tumor tissue versus normal tissue (P < 0.0001). Conclusions The data demonstrate no apparent decrease in CUL-5 expression in the breast cancer cell lines (MCF-7, MDA-MB-231) versus the breast epithelial cells (HMEC, MCF-10A). The decrease in CUL-5 expression in breast tumor tissue versus matched normal tissue supports the hypothesis that decreased expression of CUL-5 may play a role in breast tumorigenesis. PMID:14641918

  12. SD-OCT analysis of regional epithelial thickness profiles in keratoconus, postoperative corneal ectasia, and normal eyes.

    PubMed

    Rocha, Karolinne Maia; Perez-Straziota, Claudia E; Perez-Straziota, E; Stulting, R Doyle; Randleman, J Bradley

    2013-03-01

    To assess corneal microarchitecture and regional epithelial thickness profile in eyes with keratoconus, postoperative corneal ectasia (ectasia), and normal unoperated eyes (controls) using spectral-domain optical coherence tomography (SD-OCT). Regional corneal epithelial thickness profiles were measured with anterior segment SD-OCT (Optovue RTVue-100, Optovue Inc., Fremont, CA). Epithelial thickness was assessed at 21 points, 0.5 mm apart, across the central 6-mm of the corneal apex in the horizontal and vertical meridians. One hundred twenty eyes were evaluated, including 49 eyes from 29 patients with keratoconus, 32 eyes from 16 patients with ectasia, and 39 eyes from 21 control patients. Average epithelial thickness at the corneal apex was 41.18 ± 6.47 μm (range: 30 to 51 μm) for keratoconus, 46.5 ± 6.72 μm for ectasia (range: 34 to 60 μm), and 50.45 ± 3.92 μm for controls (range: 42 to 55 μm). Apical epithelial thickness was significantly thinner in eyes with keratoconus (P < .0001) and ectasia (P = .0007) than in controls. Epithelial thickness ranges in all other areas varied widely for keratoconus (range: 21 to 101 μm) and ectasia (range: 30 to 82 μm) compared to controls (range: 43 to 64) (P = .0063). SD-OCT demonstrated significant central and regional epithelial thickness profile differences between keratoconus, ectasia, and control eyes, with significant variability and unpredictability in ectatic eyes. This regional irregularity may necessitate direct epithelial thickness measurement for treatments where underlying stromal variations may be clinically relevant, including corneal collagen cross-linking or topography-guided ablations. Copyright 2013, SLACK Incorporated.

  13. The enhancement of phase 2 enzyme activities by sodium butyrate in normal intestinal epithelial cells is associated with Nrf2 and p53.

    PubMed

    Yaku, Keisuke; Enami, Yuka; Kurajyo, Chika; Matsui-Yuasa, Isao; Konishi, Yotaro; Kojima-Yuasa, Akiko

    2012-11-01

    Dietary fiber fermentation by the colonic bacterial flora produces short-chain fatty acids, acetate, propionate and butyrate. Among them, butyrate is considered to be the major energy substrate for colonocytes and, at least in rats, seems to protect against colonic carcinogenesis. In this study, we examined the effect and the mechanisms of short-chain fatty acids on the activity of phase 2 enzymes. Sodium butyrate increased phase 2 enzyme activities in normal rat small intestine epithelial cells, Glutathione S-transferase and NAD(P)H:quinone oxidoreductase (NQO) in a dose-dependent manner(;) however, other short-chain fatty acids did not increase them. The mechanism of the induction of phase 2 enzymes with sodium butyrate sodium butyrate, but not other short-chain fatty acids was related to the increase of NF-E2-related factor 2 (Nrf2) nuclear translocation and the decrease in the levels of nuclear fraction p53. Sodium butyrate also caused enhancement of Nrf2 mRNA levels and suppression of p53 mRNA levels. Sodium butyrate enhances the activities of phase 2 enzymes via an increase in the Nrf2 protein levels in the nucleus and a decrease in the mRNA and protein levels of p53.

  14. CCR6 expression in colon cancer is associated with advanced disease and supports epithelial-to-mesenchymal transition.

    PubMed

    Kapur, Neeraj; Mir, Hina; Clark Iii, Clarence E; Krishnamurti, Uma; Beech, Derrick J; Lillard, James W; Singh, Shailesh

    2016-06-14

    Adjuvant chemotherapy offered to treat colon cancer is based on the TNM staging system, which often fails due to molecular heterogeneity and undefined molecular mechanisms independent of TNM. Therefore, identification of markers to better predict therapeutic option and outcome is needed. In this study we have characterised the clinical association of CCR6 with colon cancer and defined CCR6-mediated molecular pathway. Immunohistochemistry, RT-qPCR, western blot and FACS were used to determine expression of CCR6 and/or EMT markers in colon tissues/cells. BrdU assay and trans-well system were used to determine cell proliferation, migration and invasion in response to CCL20. CCR6 was higher in cancer cases compared to normal adjacent tissue and expression was associated with nodal status and distant metastasis. Similarly, CCR6 expression was higher in cells derived from node-positive cases and highest expression was in cells derived from metastatic cases. Significant changes in EMT markers, that is, E-cadherin, vimentin, β-catenin, N-cadherin, α-SMA, SNAILl and ZEB1 were observed in response to CCL20 along with decreased proliferation, increased migratory and invasive potential. Results suggest CCR6 as a potential therapeutic target as well as biomarker in addition to nodal status for predicting therapeutic option.

  15. CCR6 expression in colon cancer is associated with advanced disease and supports epithelial-to-mesenchymal transition

    PubMed Central

    Kapur, Neeraj; Mir, Hina; Clark III, Clarence E; Krishnamurti, Uma; Beech, Derrick J; Lillard, James W; Singh, Shailesh

    2016-01-01

    Background: Adjuvant chemotherapy offered to treat colon cancer is based on the TNM staging system, which often fails due to molecular heterogeneity and undefined molecular mechanisms independent of TNM. Therefore, identification of markers to better predict therapeutic option and outcome is needed. In this study we have characterised the clinical association of CCR6 with colon cancer and defined CCR6-mediated molecular pathway. Methods: Immunohistochemistry, RT-qPCR, western blot and FACS were used to determine expression of CCR6 and/or EMT markers in colon tissues/cells. BrdU assay and trans-well system were used to determine cell proliferation, migration and invasion in response to CCL20. Results: CCR6 was higher in cancer cases compared to normal adjacent tissue and expression was associated with nodal status and distant metastasis. Similarly, CCR6 expression was higher in cells derived from node-positive cases and highest expression was in cells derived from metastatic cases. Significant changes in EMT markers, that is, E-cadherin, vimentin, β-catenin, N-cadherin, α-SMA, SNAILl and ZEB1 were observed in response to CCL20 along with decreased proliferation, increased migratory and invasive potential. Conclusions: Results suggest CCR6 as a potential therapeutic target as well as biomarker in addition to nodal status for predicting therapeutic option. PMID:27149649

  16. Simultaneous evaluation of tear turnover and corneal epithelial permeability by fluorophotometry in normal subjects and patients with keratoconjunctivitis sicca (KCS).

    PubMed Central

    Nelson, J D

    1995-01-01

    PURPOSE: To simultaneously determine the tear turn over rate (TT) and corneal epithelial permeability (Pdc) in normal subjects and patients with KCS by a single-drop fluorophotometric technique using disodium fluorescein (DSF) or carboxyfluorescein (CF). METHODS: DSF was instilled in one eye chosen at random and CF in the fellow eye of 13 normal subjects and in 13 patients with KCS. TT and Pdc were determined using a single-drop technique on a Fluorotron Master. RESULTS: In normals and KCS subjects, TT was found to be independent of age and sex and essentially identical for DSF and CF. TT was approximately 42% lower in KCS subjects than normals (Table). TT was independent of Schirmer's I and Pdc. [table: see text] Pdc values were similar for DSF and CF in normals and increased with age. In KCS subjects, Pdc was 3-4 times higher compared to normal subjects (p < 0.01) and was directly correlated with the severity of corneal punctate staining. Pdc was independent of TT. CONCLUSION: Patients with KCS are more susceptible to the therapeutic and toxic effects of topical medications and preservatives due to increased corneal epithelial permeability. With the decreased TT in KCS patients, there is also slower elimination of substances from the tear film. This, combined with the increase in epithelial permeability, puts the KCS eye doubly at risk for the toxic effects of topically applied substances. PMID:8719697

  17. Transporter function and cyclic AMP turnover in normal colonic mucosa from patients with and without colorectal neoplasia.

    PubMed

    Kleberg, Karen; Jensen, Gerda Majgaard; Christensen, Dan Ploug; Lundh, Morten; Grunnet, Lars Groth; Knuhtsen, Svend; Poulsen, Steen Seier; Hansen, Mark Berner; Bindslev, Niels

    2012-06-26

    The pathogenesis of colorectal neoplasia is still unresolved but has been associated with alterations in epithelial clearance of xenobiotics and metabolic waste products. The aim of this study was to functionally characterize the transport of cyclic nucleotides in colonic biopsies from patients with and without colorectal neoplasia. Cyclic nucleotides were used as model substrates shared by some OATP- and ABC-transporters, which in part are responsible for clearance of metabolites and xenobiotics from the colonic epithelium. On colonic biopsies from patients with and without colorectal neoplasia, molecular transport was electrophysiologically registered in Ussing-chamber set-ups, mRNA level of selected transporters was quantified by rt-PCR, and subcellular location of transporters was determined by immunohistochemistry. Of four cyclic nucleotides, dibuturyl-cAMP induced the largest short circuit current in both patient groups. The induced short circuit current was significantly lower in neoplasia-patients (p = 0.024). The observed altered transport of dibuturyl-cAMP in neoplasia-patients could not be directly translated to an observed increased mRNA expression of OATP4A1 and OATP2B1 in neoplasia patients. All other examined transporters were expressed to similar extents in both patient groups. OATP1C1, OATP4A1, OATP4C1 seem to be involved in the excretory system of human colon. ABCC4 is likely to be involved from an endoplasmic-Golgi complex and basolateral location in goblet cells. ABCC5 might be directly involved in the turnover of intracellular cAMP at the basolateral membrane of columnar epithelial cells, while OATP2B1 is indirectly related to the excretory system. Colorectal neoplasia is associated with lower transport or sensitivity to cyclic nucleotides and increased expression of OATP2B1 and OATP4A1 transporters, known to transport PGE(2).

  18. Transporter function and cyclic AMP turnover in normal colonic mucosa from patients with and without colorectal neoplasia

    PubMed Central

    2012-01-01

    Background The pathogenesis of colorectal neoplasia is still unresolved but has been associated with alterations in epithelial clearance of xenobiotics and metabolic waste products. The aim of this study was to functionally characterize the transport of cyclic nucleotides in colonic biopsies from patients with and without colorectal neoplasia. Methods Cyclic nucleotides were used as model substrates shared by some OATP- and ABC-transporters, which in part are responsible for clearance of metabolites and xenobiotics from the colonic epithelium. On colonic biopsies from patients with and without colorectal neoplasia, molecular transport was electrophysiologically registered in Ussing-chamber set-ups, mRNA level of selected transporters was quantified by rt-PCR, and subcellular location of transporters was determined by immunohistochemistry. Results Of four cyclic nucleotides, dibuturyl-cAMP induced the largest short circuit current in both patient groups. The induced short circuit current was significantly lower in neoplasia-patients (p = 0.024). The observed altered transport of dibuturyl-cAMP in neoplasia-patients could not be directly translated to an observed increased mRNA expression of OATP4A1 and OATP2B1 in neoplasia patients. All other examined transporters were expressed to similar extents in both patient groups. Conclusions OATP1C1, OATP4A1, OATP4C1 seem to be involved in the excretory system of human colon. ABCC4 is likely to be involved from an endoplasmic-Golgi complex and basolateral location in goblet cells. ABCC5 might be directly involved in the turnover of intracellular cAMP at the basolateral membrane of columnar epithelial cells, while OATP2B1 is indirectly related to the excretory system. Colorectal neoplasia is associated with lower transport or sensitivity to cyclic nucleotides and increased expression of OATP2B1 and OATP4A1 transporters, known to transport PGE2. PMID:22734885

  19. Bacteria penetrate the normally impenetrable inner colon mucus layer in both murine colitis models and patients with ulcerative colitis.

    PubMed

    Johansson, Malin E V; Gustafsson, Jenny K; Holmén-Larsson, Jessica; Jabbar, Karolina S; Xia, Lijun; Xu, Hua; Ghishan, Fayez K; Carvalho, Frederic A; Gewirtz, Andrew T; Sjövall, Henrik; Hansson, Gunnar C

    2014-02-01

    The inner mucus layer in mouse colon normally separates bacteria from the epithelium. Do humans have a similar inner mucus layer and are defects in this mucus layer a common denominator for spontaneous colitis in mice models and ulcerative colitis (UC)? The colon mucus layer from mice deficient in Muc2 mucin, Core 1 O-glycans, Tlr5, interleukin 10 (IL-10) and Slc9a3 (Nhe3) together with that from dextran sodium sulfate-treated mice was immunostained for Muc2, and bacterial localisation in the mucus was analysed. All murine colitis models revealed bacteria in contact with the epithelium. Additional analysis of the less inflamed IL-10(-/-) mice revealed a thicker mucus layer than wild-type, but the properties were different, as the inner mucus layer could be penetrated both by bacteria in vivo and by fluorescent beads the size of bacteria ex vivo. Clear separation between bacteria or fluorescent beads and the epithelium mediated by the inner mucus layer was also evident in normal human sigmoid colon biopsy samples. In contrast, mucus on colon biopsy specimens from patients with UC with acute inflammation was highly penetrable. Most patients with UC in remission had an impenetrable mucus layer similar to that of controls. Normal human sigmoid colon has an inner mucus layer that is impenetrable to bacteria. The colon mucus in animal models that spontaneously develop colitis and in patients with active UC allows bacteria to penetrate and reach the epithelium. Thus colon mucus properties can be modulated, and this suggests a novel model of UC pathophysiology.

  20. Bacteria penetrate the normally impenetrable inner colon mucus layer in both murine colitis models and patients with ulcerative colitis

    PubMed Central

    Johansson, Malin E V; Gustafsson, Jenny K; Holmén-Larsson, Jessica; Jabbar, Karolina S; Xia, Lijun; Xu, Hua; Ghishan, Fayez K; Carvalho, Frederic A; Gewirtz, Andrew T; Sjövall, Henrik; Hansson, Gunnar C

    2014-01-01

    Objective The inner mucus layer in mouse colon normally separates bacteria from the epithelium. Do humans have a similar inner mucus layer and are defects in this mucus layer a common denominator for spontaneous colitis in mice models and ulcerative colitis (UC)? Methods and results The colon mucus layer from mice deficient in Muc2 mucin, Core 1 O-glycans, Tlr5, interleukin 10 (IL-10) and Slc9a3 (Nhe3) together with that from dextran sodium sulfate-treated mice was immunostained for Muc2, and bacterial localisation in the mucus was analysed. All murine colitis models revealed bacteria in contact with the epithelium. Additional analysis of the less inflamed IL-10−/− mice revealed a thicker mucus layer than wild-type, but the properties were different, as the inner mucus layer could be penetrated both by bacteria in vivo and by fluorescent beads the size of bacteria ex vivo. Clear separation between bacteria or fluorescent beads and the epithelium mediated by the inner mucus layer was also evident in normal human sigmoid colon biopsy samples. In contrast, mucus on colon biopsy specimens from patients with UC with acute inflammation was highly penetrable. Most patients with UC in remission had an impenetrable mucus layer similar to that of controls. Conclusions Normal human sigmoid colon has an inner mucus layer that is impenetrable to bacteria. The colon mucus in animal models that spontaneously develop colitis and in patients with active UC allows bacteria to penetrate and reach the epithelium. Thus colon mucus properties can be modulated, and this suggests a novel model of UC pathophysiology. PMID:23426893

  1. Spectral-Domain OCT Analysis of Regional Epithelial Thickness Profiles in Keratoconus, Postoperative Corneal Ectasia, and Normal Eyes

    PubMed Central

    Rocha, Karolinne Maia; Straziota, Claudia Perez; Stulting, R. Doyle; Randleman, J. Bradley

    2014-01-01

    PURPOSE To assess corneal microarchitecture and regional epithelial thickness profile in eyes with keratoconus, postoperative corneal ectasia, and normal unoperated eyes using spectral-domain optical coherence tomography (SD OCT). METHODS Regional corneal epithelial thickness profiles of eyes with keratoconus (KC) and postoperative corneal ectasia (Ectasia) were measured with anterior segment SC OCT (Optovue RTVue-100, Optovue Inc., Fremont, CA) and compared retrospectively to those of normal eyes (Control). Epithelial thickness was assessed at 21 points, 0.5 mm apart, across the central 6-mm of the corneal apex in the horizontal and vertical meridians. RESULTS One hundred twenty eyes were evaluated, including 49 eyes from 29 patients with KC, 32 eyes from 16 patients with Ectasia, and 39 eyes from 21 control patients. Average epithelial thickness at the corneal apex was 41.18±6.47μm (range 30 to 51 μm) in eyes with KC, 46.5±6.72μm in eyes with ectasia (range 34 to 60 μm), and 50.45±3.92 μm in normal eyes (range 42 to 55 μm). Apical epithelial thickness was significantly thinner in eyes with KC (p <.0001) and ectasia (p=.0007) than it was in controls. Epithelial thickness ranges in all other areas varied widely for KC (SD, range 21 to 101 μm) and ectasia (SD, range 30 to 82 μm) compared to controls (SD, range 43 to 64), p = .0063 CONCLUSION Central epithelial thickness was, on average, significantly thinner in ectatic corneas compared to controls; however, both central and regional epithelial thickness was highly irregular and variable in corneas with keratoconus and postoperative corneal ectasia. These thickness variations may alter preoperative topographic features and measurements in unpredictable ways, especially steepest K values. Regional epithelial thickness cannot be assumed to be uniform in ectatic corneas and therefore may require direct measurement when considering treatments for which underlying stromal thickness is particularly important

  2. Influence of diet or intrarectal bile acid injections on colon epithelial cell proliferation in rats previously injected with 1,2-dimethylhydrazine

    SciTech Connect

    Glauert, H.P.; Bennink, M.R.

    1983-03-01

    The effects of varying colon bile acid concentrations on rat colon epithelial cell proliferation were studied. Bile acid concentrations were altered by intrarectally injecting either deoxycholic or lithocholic acid for 4 weeks or by increasing the dietary fat or fiber (wheat bran, agar, or carrageenan) intake for 4 weeks. 1,2-Dimethylhydrazine (DMH) was s.c. injected into half of the rats 1 week before treatments began. Colon epithelial cell proliferation was measured by (/sup 3/H)thymidine autoradiography of colon crypts. Rats injected with DMH had more DNA-synthesizing cells per crypt. Neither bile acid injection nor any of the diets altered the number of DNA-synthesizing cells per crypt. DMH injections, deoxycholic and lithocholic acid intrarectal injections, and dietary agar and wheat bran all increased the total number of cells per crypt. High fat diets and dietary carrageenan did not affect cell number. All diets containing fiber lowered total fecal bile acid concentrations, but increasing the fat content of the diet did not affect them. These results indicate that the bile acid injections and dietary agar and wheat bran induce a slight hyperplasia in the colon.

  3. Overexpression of forkhead Box C2 promotes tumor metastasis and indicates poor prognosis in colon cancer via regulating epithelial-mesenchymal transition.

    PubMed

    Li, Qingguo; Wu, Jitao; Wei, Ping; Xu, Ye; Zhuo, Changhua; Wang, Yuwei; Li, Dawei; Cai, Sanjun

    2015-01-01

    Forkhead box protein C2 (FOXC2) plays a vital role in carcinogenesis; however, its significance and prognostic value in colon cancer remain unclear. In this study, FOXC2 expression was analyzed in a tissue microarray (TMA) containing 185 samples of primary colon cancer tumor samples and in human colon cancer cell lines. The effect of FOXC2 on cell proliferation, tumorigenesis, and metastasis was examined in vitro and in vivo. FOXC2 was overexpressed in human colon cancer cells and tissues, and correlated with colon cancer progression and patient survival. Functional study demonstrated that FOXC2 promoted cell growth, cell migration, and tumor formation in nude mice, whereas knockdown of FOXC2 by short hairpin RNA (shRNAs) significantly suppressed cell growth, cell migration and tumor formation. Further study found that FOXC2 enhanced AKT activity with subsequent GSK-3β phosphorylation and Snail stabilization, and then induced epithelial-mesenchymal transition (EMT) and promoted tumor invasion and metastasis. Collectively, FOXC2 promotes colon cancer metastasis by facilitating EMT and acts as a potential prognostic factor and therapeutic target in colon cancer.

  4. Molecular Mechanisms of Malignant Transformation by Low Dose Cadmium in Normal Human Bronchial Epithelial Cells

    PubMed Central

    Kluz, Thomas; Cohen, Lisa; Shen, Steven S.; Costa, Max

    2016-01-01

    Cadmium is a carcinogenic metal, the mechanisms of which are not fully understood. In this study, human bronchial epithelial cells were transformed with sub-toxic doses of cadmium (0.01, 0.05, and 0.1 μM) and transformed clones were characterized for gene expression changes using RNA-seq, as well as other molecular measurements. 440 genes were upregulated and 47 genes were downregulated in cadmium clones relative to control clones over 1.25-fold. Upregulated genes were associated mostly with gene ontology terms related to embryonic development, immune response, and cell movement, while downregulated genes were associated with RNA metabolism and regulation of transcription. Several embryonic genes were upregulated, including the transcription regulator SATB2. SATB2 is critical for normal skeletal development and has roles in gene expression regulation and chromatin remodeling. Small hairpin RNA knockdown of SATB2 significantly inhibited growth in soft agar, indicating its potential as a driver of metal-induced carcinogenesis. An increase in oxidative stress and autophagy was observed in cadmium clones. In addition, the DNA repair protein O6-methylguanine-DNA-methyltransferase was depleted by transformation with cadmium. MGMT loss caused significant decrease in cell viability after treatment with the alkylating agent temozolomide, demonstrating diminished capacity to repair such damage. Results reveal various mechanisms of cadmium-induced malignant transformation in BEAS-2B cells including upregulation of SATB2, downregulation of MGMT, and increased oxidative stress. PMID:27186882

  5. Molecular Mechanisms of Malignant Transformation by Low Dose Cadmium in Normal Human Bronchial Epithelial Cells.

    PubMed

    Cartularo, Laura; Kluz, Thomas; Cohen, Lisa; Shen, Steven S; Costa, Max

    2016-01-01

    Cadmium is a carcinogenic metal, the mechanisms of which are not fully understood. In this study, human bronchial epithelial cells were transformed with sub-toxic doses of cadmium (0.01, 0.05, and 0.1 μM) and transformed clones were characterized for gene expression changes using RNA-seq, as well as other molecular measurements. 440 genes were upregulated and 47 genes were downregulated in cadmium clones relative to control clones over 1.25-fold. Upregulated genes were associated mostly with gene ontology terms related to embryonic development, immune response, and cell movement, while downregulated genes were associated with RNA metabolism and regulation of transcription. Several embryonic genes were upregulated, including the transcription regulator SATB2. SATB2 is critical for normal skeletal development and has roles in gene expression regulation and chromatin remodeling. Small hairpin RNA knockdown of SATB2 significantly inhibited growth in soft agar, indicating its potential as a driver of metal-induced carcinogenesis. An increase in oxidative stress and autophagy was observed in cadmium clones. In addition, the DNA repair protein O6-methylguanine-DNA-methyltransferase was depleted by transformation with cadmium. MGMT loss caused significant decrease in cell viability after treatment with the alkylating agent temozolomide, demonstrating diminished capacity to repair such damage. Results reveal various mechanisms of cadmium-induced malignant transformation in BEAS-2B cells including upregulation of SATB2, downregulation of MGMT, and increased oxidative stress.

  6. LncRNAs expression profiling in normal ovary, benign ovarian cyst and malignant epithelial ovarian cancer

    PubMed Central

    Wang, Huan; Fu, Ziyi; Dai, Chencheng; Cao, Jian; Liu, Xiaoguang; Xu, Juan; Lv, Mingming; Gu, Yun; Zhang, Jingmin; Hua, Xiangdong; Jia, Genmei; Xu, Sujuan; Jia, Xuemei; Xu, Pengfei

    2016-01-01

    Long noncoding RNA (lncRNA) has been recognized as a regulator of gene expression, and the dysregulation of lncRNAs is involved in the progression of many types of cancer, including epithelial ovarian cancer (EOC). To explore the potential roles of lncRNAs in EOC, we performed lncRNA and mRNA microarray profiling in malignant EOC, benign ovarian cyst and healthy control tissues. In this study, 663 transcripts of lncRNAs were found to be differentially expressed in malignant EOC compared with benign and normal control tissues. We also selected 18 altered lncRNAs to confirm the validity of the microarray analysis using quantitative real-time PCR (qPCR). Pathway and Gene Ontology (GO) analyses demonstrated that these altered transcripts were involved in multiple biological processes, especially the cell cycle. Furthermore, Series Test of Cluster (STC) and lncRNA-mRNA co-expression network analyses were conducted to predict lncRNA expression trends and the potential target genes of lncRNAs. We also determined that two antisense lncRNAs (RP11-597D13.9 and ADAMTS9-AS1) were associated with their nearby coding genes (FAM198B, ADAMTS9), which participated in cancer progression. This study offers helpful information to understand the initiation and development mechanisms of EOC. PMID:27941916

  7. Intestinal Microbial Dysbiosis and Colonic Epithelial Cell Hyperproliferation by Dietary α-Mangostin is Independent of Mouse Strain

    PubMed Central

    Gutierrez-Orozco, Fabiola; Thomas-Ahner, Jennifer M.; Galley, Jeffrey D.; Bailey, Michael T.; Clinton, Steven K.; Lesinski, Gregory B.; Failla, Mark L.

    2015-01-01

    Beverages and supplements prepared from mangosteen fruit are claimed to support gut health and immunity, despite the absence of supporting evidence from clinical trials. We recently reported that α-mangostin (α-MG), the most abundant xanthone in mangosteen fruit, altered the intestinal microbiome, promoted dysbiosis, and exacerbated colitis in C57BL/6J mice. The objective of this study was to determine whether induction of dysbiosis by dietary α-MG is limited to the C57BL/6J strain or represents a more generic response to chronic intake of the xanthone on the gut microbiota of mice. C3H, Balb/c, Nude FoxN1nu, and C57BL/6J mice, each demonstrating unique microbiomes, were fed standard diet or diet containing 0.1% α-MG for four weeks. Dietary α-MG significantly altered the cecal and colonic microbiota in all four strains of mice, promoting a reduction in generally assumed beneficial bacterial groups while increasing the abundance of pathogenic bacteria. Consumption of α-MG was associated with reduced abundance of Firmicutes and increased abundance of Proteobacteria. The abundance of Lachnospiraceae, Ruminococcaceae, and Lactobacillaceae was reduced in α-MG-fed mice, while that of Enterobacteriaceae and Enterococcaceae was increased. Dietary α-MG also was associated with increased proliferation of colonic epithelial cells, infiltration of immune cells, infiltration of immune cells and increased fluid content in stool. These results suggest that ingestion of pharmacologic doses of xanthones in mangosteen-containing supplements may adversely alter the gut microbiota and should be used with caution. PMID:25621505

  8. Estrogens regulate the expression of NHERF1 in normal colon during the reproductive cycle of Wistar rats.

    PubMed

    Cuello-Carrión, F Darío; Troncoso, Mariana; Guiñazu, Elina; Valdez, Susana R; Fanelli, Mariel A; Ciocca, Daniel R; Kreimann, Erica L

    2010-12-01

    In breast cancer cell lines, the Na(+)/H(+) exchanger regulator factor 1 (NHERF1) gene is regulated at the transcriptional level by estrogens, the protein expression levels correlate with the presence of estrogen receptors and the effect is blocked by anti-estrogens. However, there is limited information regarding the regulation of NHERF1 by estrogens in normal colon tissue. The NHERF1 protein has an important role in the maintenance of the intestine ultrastructure. NHERF1-deficient mice showed defects in the intestinal microvilli as well as molecular alterations in brush border membrane proteins. Here, we have studied the expression of NHERF1 in normal rat colon and uterus during the reproductive cycle of Wistar rats. We found that NHERF1 expression in rat colon during the estral cycle is modified by estrogen levels: higher expression of NHERF1 was observed during the proestrous and estrous stages and lower expression in diestrous 1 when estrogen levels decreased. In uterus, NHERF1 was expressed in the apical region of the luminal epithelium and glands in all stages of the estral cycle, and in both colon and uterus, the expression was independent of the proliferation status. Our results show that NHERF1 expression is regulated by estrogens in colon during the rat estral cycle.

  9. The antifungal antibiotic, clotrimazole, inhibits Cl- secretion by polarized monolayers of human colonic epithelial cells.

    PubMed Central

    Rufo, P A; Jiang, L; Moe, S J; Brugnara, C; Alper, S L; Lencer, W I

    1996-01-01

    Clotrimazole (CLT) prevents dehydration of the human HbSS red cell through inhibition of Ca++-dependent (Gardos) K+ channels in vitro (1993. J. Clin Invest. 92:520-526.) and in patients (1996. J. Clin Invest. 97:1227-1234.). Basolateral membrane K+ channels of intestinal crypt epithelial cells also participate in secretagogue-stimulated Cl- secretion. We examined the ability of CLT to block intestinal Cl- secretion by inhibition of K+ transport. Cl- secretion was measured as short-circuit current (Isc) across monolayers of T84 cells. CLT reversibly inhibited Cl- secretory responses to both cAMP- and Ca2+-dependent agonists with IC50 values of approximately 5 microM. Onset of inhibition was more rapid when CLT was applied to the basolateral cell surface. Apical Cl- channel and basolateral NaK2Cl cotransporter activities were unaffected by CLT treatment as assessed by isotopic flux measurement. In contrast, CLT strongly inhibited basolateral 86Rb efflux. These data provide evidence that CLT reversibly inhibits Cl- secretion elicited by cAMP-, cGMP-, or Ca2+-dependent agonists in T84 cells. CLT acts distal to the generation of cAMP and Ca2+ signals, and appears to inhibit basolateral K+ channels directly. CLT and related drugs may serve as novel antidiarrheal agents in humans and animals. PMID:8903326

  10. Intracellular pH regulates basolateral K+ and Cl- conductances in colonic epithelial cells by modulating Ca2+ activation

    PubMed Central

    1991-01-01

    The role of intracellular pH as a modulator of basolateral K+ and Cl- conductances in epithelial cells was studied using digitonin- permeabilized colonic cell layers so that cytosolic pH could be clamped at specific values, while basolateral K+ and Cl- conductances were activated by stepwise increases in intracellular free Ca2+. Increasing the intracellular pH from 6.6 to 8.0 enhanced the sensitivity of both ionic conductances to intracellular Ca2+, but changing extracellular pH had no effect. Maximal K+ and Cl- currents activated by Ca2+ were not affected by changes in intracellular pH, suggesting that protons do not alter the conduction properties of the channels. Hill analysis of the Ca2+ activation process revealed that raising the cytosolic pH from 6.6 to 8.0 reduced the K1/2 for Ca2+ activation. In the absence of Ca2+, changes in intracellular pH did not have a significant effect on the basolateral K+ and Cl- conductances. These results are consistent with the notion that changes in cytosolic pH can modulate basolateral conductances by modifying the action of calcium, perhaps by acting at or near the activation site to provide a mechanism of variable "gain control." PMID:1719125

  11. Anti-inflammatory and immunomodulatory activities of stevioside and steviol on colonic epithelial cells.

    PubMed

    Boonkaewwan, Chaiwat; Burodom, Anyanee

    2013-12-01

    Stevioside is a natural non-caloric sweetener isolated from Stevia rebaudiana Bertoni leaves. We have proposed its effect on attenuation of tumour necrosis factor α (TNF-α) and interleukin 1β (IL-1β) release in lipopolysaccharide (LPS)-stimulated monocytes. In this study, the anti-inflammatory and immunomodulatory activities of stevioside and its metabolite, steviol, on human colon carcinoma cell line (Caco-2) were evaluated. Stevioside and steviol, in the doses used in this study, had no cytotoxicity on Caco-2 cells. Anti-inflammatory activities of these two compounds were observed by potentially suppressed LPS-mediated TNF-α, IL-1β and IL-6 release. In addition, stevioside and steviol showed immunomodulatory effects on IκBα activation and nuclear factor kappa B (NF-κB) suppression in western blotting. Stevioside and steviol attenuate LPS-induced pro-inflammatory cytokine productions by affecting cytokine gene expression via IκBα/NF-κB signalling pathway. © 2013 Society of Chemical Industry.

  12. Detoxification of H(2)S by differentiated colonic epithelial cells: implication of the sulfide oxidizing unit and of the cell respiratory capacity.

    PubMed

    Mimoun, Sabria; Andriamihaja, Mireille; Chaumontet, Catherine; Atanasiu, Calina; Benamouzig, Robert; Blouin, Jean Marc; Tomé, Daniel; Bouillaud, Frédéric; Blachier, François

    2012-07-01

    Sulfide is released in the large intestine lumen by the microbiota and is an inhibitor of mitochondrial respiration and a genotoxic agent in colonocytes when present in excess. Deciphering how colonocytes metabolize sulfide is an important issue. In this study, using the human colonic epithelial HT-29 Glc(-/+) cells, we found that 50 μM sodium hydrogen sulfide represents the threshold of concentration above which respiration is decreased. The capacity of HT-29 Glc(-/+) cells to oxidize lower concentration of sulfide was associated with the expression of transcripts corresponding to the enzymes of the sulfide oxidizing unit (SOU), that is, sulfide quinone reductase (SQR), dioxygenase ethylmalonic encephalopathy, and thiosulfate sulfur transferase (TST). Inhibition of cell O(2) consumption by sulfide was reverted by zinc but not by calcium and iron. When the cells undergo either spontaneous or butyrate-induced differentiation, their capacity to oxidize sulfide was significantly increased. The expression levels of the genes corresponding to the enzymes of the SOU were not increased, whereas increased cellular maximal respiratory capacity and oxygen consumption by the dioxygenase were both measured. In human biopsies recovered from various parts of the large intestine, the three enzymes of the SOU were expressed. SOU and cell respiratory capacity are crucial for sulfide detoxification in colonocytes. Sulfide oxidative capacity in the colonic mucosa is higher in differentiated than in proliferative epithelial cells. The cell respiratory capacity and SOU activity appear to represent major determinants allowing sulfide detoxification in colonic epithelial cells.

  13. Vitamin D Transport Proteins Megalin and Disabled-2 Are Expressed in Prostate and Colon Epithelial Cells and Are Induced and Activated by All-Trans-Retinoic Acid

    PubMed Central

    Ternes, Shantel B.; Rowling, Matthew J.

    2014-01-01

    Megalin and disabled-2 (Dab2) are essential for uptake of the 25-hydroxycholecalciferol (25D3)-vitamin D binding protein (DBP) complex in tissues. In the kidney, this mechanism regulates serum 25D3 levels and production of 1,25-dihydroxycholecalciferol (1,25D3) by CYP27B1 for systemic use. Previously, we showed that mammary epithelial cells expressing CYP27B1 express megalin and Dab2 and internalize DBP by endocytosis, indicating 25D3 was accessible for conversion to 1,25D3 in extra-renal tissues. Moreover, induction of megalin and Dab2 (protein and mRNA abundance) by all-trans-retinoic acid (RA) enhanced DBP uptake. This suggests megalin and Dab2 play a central role in uptake of vitamin D and may predict actions of vitamin D in extra-renal tissues. Here, we characterized megalin and Dab2 expression and uptake of DBP in transformed human prostate and colon epithelial cells. Megalin and Dab2 were expressed in prostate and colon epithelial cells, which was markedly enhanced following treatment with RA. Furthermore, DBP uptake was stimulated by low-dose RA supplementation in LNCaP, PC-3, and Caco-2 cells. Taken together, these are the first studies to our knowledge that have demonstrated modulated expression of megalin and Dab2, as well as an association between increased expression of endocytic proteins with DBP uptake in prostate and colon cells. PMID:23909735

  14. BRCA1 promoter methylation of normal breast epithelial cells as a possible precursor for BRCA1-methylated breast cancer

    PubMed Central

    Otani, Yoko; Miyake, Tomohiro; Kagara, Naofumi; Shimoda, Masafumi; Naoi, Yasuto; Maruyama, Naomi; Shimomura, Atsuhi; Shimazu, Kenzo; Kim, Seung Jin; Noguchi, Shinzaburo

    2014-01-01

    The breast cancer susceptibility gene 1 (BRCA1) and glutathione S-transferase P1 (GSTP1) promoters are reportedly often methylated in breast cancer tissues. Their methylation status in surrounding normal breast tissues has not been examined thoroughly although this may well be important for a better understanding of breast carcinogenesis. In this study, BRCA1 and GSTP1 promoter methylation was examined by methylation-specific PCR assay. Patients with BRCA1-methylated (n = 15) or BRCA1-unmethylated (n = 15) tumors and those with GSTP1-methylated (n = 9) or GSTP1-unmethylated (n = 11) tumors were included in the present study. Methylation status of manually micro-dissected normal epithelial cells from the formalin-fixed paraffin-embedded sections of normal breast tissues adjacent to and distant from the tumors was examined at multiple sites (n = 1–5). Of the 15 patients with BRCA1-methylated tumors, 9 harbored BRCA1 promoter methylation in at least one site of the normal breast tissues. However, no BRCA1 promoter methylation was observed at any site of the normal tissues of the 15 patients with BRCA1-unmethylated tumors. No GSTP1 promoter methylation was observed in the normal tissues regardless of the methylation status of the tumors. The presence of BRCA1 promoter methylation in the normal tissues was confirmed in the epithelial cells enriched with the magnetic-activated cell sorting method. Our findings suggest that a small proportion of normal breast epithelial cells with BRCA1 promoter methylation can be precursor cells from which BRCA1-methylated breast tumors may originate. This does not apply to GSTP1 promoter methylation. PMID:25155055

  15. Cytokine changes in gastric and colonic epithelial cell in response to Planta ovata extract.

    PubMed

    Yakoob, Javed; Jafri, Wasim; Mehmood, Malik Hassan; Abbas, Zaigham; Tariq, Kanwal

    2017-03-22

    Background Psyllium (Planta ovata, Ispaghul) seed and husk are used for treatment of altered bowel habit, i. e. constipation and diarrhea. We studied the effect of Ispaghul extract on secretion of interleukin-1 beta (IL-1β) by AGS (ATCC CRL 1739) and SW480 (ATCC CCL-227) epithelial cell lines and determined whether Ispaghul extract has an effect on IL-1β secretion by Helicobacter pylori (H. pylori)-stimulated AGS cell and Escherichia coli K-12 (E. coli K-12)-stimulated SW480 cells in vitro. Methods The AGS cells and SW480 cells were pretreated with Ispaghul extract in concentrations, i. e. 3.5 and 7 μg/mL prior to infection with H. pylori and E. coli K-12. Results DNA fragmentation in AGS and SW480 cells treated with Ispaghul extract was not significant (2.3±0.8 %) compared with untreated cells (2.2±0.6 %). Ispaghul extract decreased the H. pylori-stimulated secretion of IL-1β by AGS cell (p<0.0001). This effect did not increase as the concentration of extract was increased. Ispaghul extract also decreased E. coli K-12-stimulated IL-1β secretion by SW480 cell (p<0.0001). This effect increased as the concentration of extracts was increased. Conclusions Ispaghul extract had an effect on stimulated secretion of IL-1β by the AGS and SW480 cell. It decreased pro-inflammatory reaction from both cell lines stimulated by bacteria.

  16. The Insect Peptide CopA3 Increases Colonic Epithelial Cell Proliferation and Mucosal Barrier Function to Prevent Inflammatory Responses in the Gut*

    PubMed Central

    Kim, Dae Hong; Hwang, Jae Sam; Lee, Ik Hwan; Nam, Seung Taek; Hong, Ji; Zhang, Peng; Lu, Li Fang; Lee, Junguee; Seok, Heon; Pothoulakis, Charalabos; Lamont, John Thomas; Kim, Ho

    2016-01-01

    The epithelial cells of the gut form a physical barrier against the luminal contents. The collapse of this barrier causes inflammation, and its therapeutic restoration can protect the gut against inflammation. EGF enhances mucosal barrier function and increases colonocyte proliferation, thereby ameliorating inflammatory responses in the gut. Based on our previous finding that the insect peptide CopA3 promotes neuronal growth, we herein tested whether CopA3 could increase the cell proliferation of colonocytes, enhance mucosal barrier function, and ameliorate gut inflammation. Our results revealed that CopA3 significantly increased epithelial cell proliferation in mouse colonic crypts and also enhanced colonic epithelial barrier function. Moreover, CopA3 treatment ameliorated Clostridium difficile toxin As-induced inflammation responses in the mouse small intestine (acute enteritis) and completely blocked inflammatory responses and subsequent lethality in the dextran sulfate sodium-induced mouse model of chronic colitis. The marked CopA3-induced increase of colonocyte proliferation was found to require rapid protein degradation of p21Cip1/Waf1, and an in vitro ubiquitination assay revealed that CopA3 directly facilitated ubiquitin ligase activity against p21Cip1/Waf1. Taken together, our findings indicate that the insect peptide CopA3 prevents gut inflammation by increasing epithelial cell proliferation and mucosal barrier function. PMID:26655716

  17. The Insect Peptide CopA3 Increases Colonic Epithelial Cell Proliferation and Mucosal Barrier Function to Prevent Inflammatory Responses in the Gut.

    PubMed

    Kim, Dae Hong; Hwang, Jae Sam; Lee, Ik Hwan; Nam, Seung Taek; Hong, Ji; Zhang, Peng; Lu, Li Fang; Lee, Junguee; Seok, Heon; Pothoulakis, Charalabos; Lamont, John Thomas; Kim, Ho

    2016-02-12

    The epithelial cells of the gut form a physical barrier against the luminal contents. The collapse of this barrier causes inflammation, and its therapeutic restoration can protect the gut against inflammation. EGF enhances mucosal barrier function and increases colonocyte proliferation, thereby ameliorating inflammatory responses in the gut. Based on our previous finding that the insect peptide CopA3 promotes neuronal growth, we herein tested whether CopA3 could increase the cell proliferation of colonocytes, enhance mucosal barrier function, and ameliorate gut inflammation. Our results revealed that CopA3 significantly increased epithelial cell proliferation in mouse colonic crypts and also enhanced colonic epithelial barrier function. Moreover, CopA3 treatment ameliorated Clostridium difficile toxin As-induced inflammation responses in the mouse small intestine (acute enteritis) and completely blocked inflammatory responses and subsequent lethality in the dextran sulfate sodium-induced mouse model of chronic colitis. The marked CopA3-induced increase of colonocyte proliferation was found to require rapid protein degradation of p21(Cip1/Waf1), and an in vitro ubiquitination assay revealed that CopA3 directly facilitated ubiquitin ligase activity against p21(Cip1/Waf1). Taken together, our findings indicate that the insect peptide CopA3 prevents gut inflammation by increasing epithelial cell proliferation and mucosal barrier function.

  18. Carcinogen-induced histone alteration in normal human mammary epithelial cells.

    PubMed

    Bradley, Chastity; van der Meer, Riet; Roodi, Nady; Yan, Heping; Chandrasekharan, Mahesh B; Sun, Zu-Wen; Mernaugh, Ray L; Parl, Fritz F

    2007-10-01

    Little is known about early carcinogen-induced protein alterations in mammary epithelium. Detection of early alterations would enhance our understanding of early-stage carcinogenesis. Here, normal human mammary epithelial cells (HMECs) were exposed to dietary and environmental carcinogens [2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine (PhIP), 4-aminobiphenyl (ABP), benzo[a]pyrene, 2,3,7,8-tetrachlorodibenzo-p-dioxin] individually or in combination. A phage display library of single-chain variable fragment antibodies was used to screen protein targets altered by the treatment. In combination with matrix-assisted laser desorption time of flight, we identified histone H3 as a target antigen. Although histone H3 total protein remained unchanged in control and treated HMEC, the methylation of lysine 4 was altered. A reduction in mono-methyl histone H3 (Lys 4) was observed in treated HMEC compared with control HMEC. This alteration was shown to be dependent on carcinogen concentration and specific for PhIP and ABP. To characterize potential histone demethylation mechanisms, localization and protein expression patterns of lysine-specific demethylase 1 (LSD1) were analyzed. In control HMEC, LSD1 was present at the nuclear periphery. However, following 72 h carcinogen treatment, LSD1 localized within the nucleus. Within 48 h after treatment, mono-methyl histone H3 (Lys 4) was restored and LSD1 localization was reversed. Protein expression levels of LSD1 were also increased in treated HMEC compared with control HMEC. Our data suggest that the induction of a single enzyme, LSD1, represents an early response to carcinogen exposure, which leads to the demethylation of histone H3 (Lys 4), which, in turn, may influence the expression of multiple genes critical in early-stage mammary carcinogenesis.

  19. IL-32θ inhibits stemness and epithelial-mesenchymal transition of cancer stem cells via the STAT3 pathway in colon cancer

    PubMed Central

    Bak, Yesol; Kwon, Taeho; Bak, In seon; Hong, Jintae; Yu, Dae-Yeul; Yoon, Do-Young

    2016-01-01

    Interleukin (IL)-32 is a well-known cytokine associated with inflammation, virus infections and cancer. IL-32θ is a newly identified isoform of IL-32, whose function has yet to be elucidated. In this study, we investigated IL-32θ function in colon cancer stem cells. Using samples from colon cancer patients, we found that the expression of IL-32θ mRNAs was significantly suppressed in tumor regions. We investigated the effects of IL-32θ on colon cancer. Ectopic expression of IL-32θ attenuated invasion, migration in vitro and in vivo tumorigenicity of colon cancer cells. IL-32θ inhibited epithelial-mesenchymal transition (EMT), resulting in the suppression of their migratory and invasive capabilities of HT29 colon cancer cells. In addition, IL-32θ altered various properties of CSCs, including sphere formation and expression of stemness related genes. IL-32θ directly bound to STAT3 and inhibited its nuclear translocation, leading to inhibited transcription of downstream factors, including Bmi1 and ZEB1. We showed that IL-32θ inhibited the STAT3-ZEB1 pathway and consequently inhibited key factors of stemness and EMT. Taken together, our findings reveal that IL-32θ can be a tumor suppressor, indicating that IL-32θ could possibly be used in therapies for colon cancer. PMID:26824417

  20. Identification and Characterization of a Diamine Exporter in Colon Epithelial Cells*S⃞

    PubMed Central

    Uemura, Takeshi; Yerushalmi, Hagit F.; Tsaprailis, George; Stringer, David E.; Pastorian, Kirk E.; Hawel, Leo; Byus, Craig V.; Gerner, Eugene W.

    2008-01-01

    SLC3A2, a member of the solute carrier family, was identified by proteomics methods as a component of a transporter capable of exporting the diamine putrescine in the Chinese hamster ovary (CHO) cells selected for resistance to growth inhibition by high exogenous concentrations of putrescine. Putrescine transport was increased in inverted plasma membrane vesicles prepared from cells resistant to growth inhibition by putrescine compared with transport in inverted vesicles prepared from non-selected cells. Knockdown of SLC3A2 in human cells, using short hairpin RNA, caused an increase in putrescine uptake and a decrease in arginine uptake activity. SLC3A2 knockdown cells accumulated higher polyamine levels and grew faster than control cells. The growth of SLC3A2 knockdown cells was inhibited by high concentrations of putrescine. Knockdown of SLC3A2 reduced export of polyamines from cells. Expression of SLC3A2 was suppressed in human HCT116 colon cancer cells, which have an activated K-RAS, compared with their isogenic clone, Hkh2 cells, which lack an activated K-RAS allele. Spermidine/spermine N1-acetyltransferase (SAT1) was co-immunoprecipitated by an anti-SLC3A2 antibody as was SLC3A2 with an anti-SAT1 antibody. SLC3A2 and SAT1 colocalized on the plasma membrane. These data provide the first molecular characterization of a polyamine exporter in animal cells and indicate that the diamine putrescine is exported by an arginine transporter containing SLC3A2, whose expression is negatively regulated by K-RAS. The interaction between SLC3A2 and SAT1 suggests that these proteins may facilitate excretion of acetylated polyamines. PMID:18660501

  1. Acquisition of 5-fluorouracil resistance induces epithelial-mesenchymal transitions through the Hedgehog signaling pathway in HCT-8 colon cancer cells

    PubMed Central

    LIU, YANJUN; DU, FANGFANG; ZHAO, QIANNAN; JIN, JIAN; MA, XIN; LI, HUAZHONG

    2015-01-01

    Colon cancer has a high incidence in individuals >60-years-old. The commonly used chemotherapeutic agent, 5-fluorouracil (5-FU), has gradually lost its potency in treating colorectal cancer following the acquisition of resistance. Drug resistance is usually associated with epithelial-mesenchymal transitions (EMTs) in cancer cells. In the present study, the EMT phenotypes of two colon cancer cell lines, wild-type (HCT-8/WT) and 5-FU-resistant (HCT-8/5-FU), were characterized following the analysis of cellular migration, proliferation, morphology and molecular changes. In order to further clarify the mechanism of EMT in HCT-8/5-FU cells, the effect of EMT pathway inhibitors upon drug sensitivity was investigated. The results revealed that the Hedgehog signaling pathway inhibitor, GDC0449, reversed drug resistance. Therefore, inhibition of the Hedgehog pathway may provide a novel chemotherapeutic strategy for the treatment of patients with 5-FU-resistant colon cancer. PMID:26137127

  2. Long-term exposure of MCF-12A normal human breast epithelial cells to ethanol induces epithelial mesenchymal transition and oncogenic features.

    PubMed

    Gelfand, Robert; Vernet, Dolores; Bruhn, Kevin; Vadgama, Jaydutt; Gonzalez-Cadavid, Nestor F

    2016-06-01

    Alcoholism is associated with breast cancer incidence and progression, and moderate chronic consumption of ethanol is a risk factor. The mechanisms involved in alcohol's oncogenic effects are unknown, but it has been speculated that they may be mediated by acetaldehyde. We used the immortalized normal human epithelial breast cell line MCF-12A to determine whether short- or long-term exposure to ethanol or to acetaldehyde, using in vivo compatible ethanol concentrations, induces their oncogenic transformation and/or the acquisition of epithelial mesenchymal transition (EMT). Cultures of MCF-12A cells were incubated with 25 mM ethanol or 2.5 mM acetaldehyde for 1 week, or with lower concentrations (1.0-2.5 mM for ethanol, 1.0 mM for acetaldehyde) for 4 weeks. In the 4-week incubation, cells were also tested for anchorage-independence, including isolation of soft agar selected cells (SASC) from the 2.5 mM ethanol incubations. Cells were analyzed by immunocytofluorescence, flow cytometry, western blotting, DNA microarrays, RT/PCR, and assays for miRs. We found that short-term exposure to ethanol, but not, in general, to acetaldehyde, was associated with transcriptional upregulation of the metallothionein family genes, alcohol metabolism genes, and genes suggesting the initiation of EMT, but without related phenotypic changes. Long-term exposure to the lower concentrations of ethanol or acetaldehyde induced frank EMT changes in the monolayer cultures and in SASC as demonstrated by changes in cellular phenotype, mRNA expression, and microRNA expression. This suggests that low concentrations of ethanol, with little or no mediation by acetaldehyde, induce EMT and some traits of oncogenic transformation such as anchorage-independence in normal breast epithelial cells.

  3. Long-term exposure of MCF-12A normal human breast epithelial cells to ethanol induces epithelial mesenchymal transition and oncogenic features

    PubMed Central

    GELFAND, ROBERT; VERNET, DOLORES; BRUHN, KEVIN; VADGAMA, JAYDUTT; GONZALEZ-CADAVID, NESTOR F.

    2016-01-01

    Alcoholism is associated with breast cancer incidence and progression, and moderate chronic consumption of ethanol is a risk factor. The mechanisms involved in alcohol's oncogenic effects are unknown, but it has been speculated that they may be mediated by acetaldehyde. We used the immortalized normal human epithelial breast cell line MCF-12A to determine whether short- or long-term exposure to ethanol or to acetaldehyde, using in vivo compatible ethanol concentrations, induces their oncogenic transformation and/or the acquisition of epithelial mesenchymal transition (EMT). Cultures of MCF-12A cells were incubated with 25 mM ethanol or 2.5 mM acetaldehyde for 1 week, or with lower concentrations (1.0–2.5 mM for ethanol, 1.0 mM for acetaldehyde) for 4 weeks. In the 4-week incubation, cells were also tested for anchorage-independence, including isolation of soft agar selected cells (SASC) from the 2.5 mM ethanol incubations. Cells were analyzed by immunocytofluorescence, flow cytometry, western blotting, DNA microarrays, RT/PCR, and assays for miRs. We found that short-term exposure to ethanol, but not, in general, to acetaldehyde, was associated with transcriptional upregulation of the metallothionein family genes, alcohol metabolism genes, and genes suggesting the initiation of EMT, but without related phenotypic changes. Long-term exposure to the lower concentrations of ethanol or acetaldehyde induced frank EMT changes in the monolayer cultures and in SASC as demonstrated by changes in cellular phenotype, mRNA expression, and microRNA expression. This suggests that low concentrations of ethanol, with little or no mediation by acetaldehyde, induce EMT and some traits of oncogenic transformation such as anchorage-independence in normal breast epithelial cells. PMID:27035792

  4. Ex vivo 2D and 3D HSV-2 infection model using human normal vaginal epithelial cells.

    PubMed

    Zhu, Yaqi; Yang, Yan; Guo, Juanjuan; Dai, Ying; Ye, Lina; Qiu, Jianbin; Zeng, Zhihong; Wu, Xiaoting; Xing, Yanmei; Long, Xiang; Wu, Xufeng; Ye, Lin; Wang, Shubin; Li, Hui

    2017-02-28

    Herpes simplex virus type 2 (HSV-2) infects human genital mucosa and establishes life-long latent infection. It is unmet need to establish a human cell-based microphysiological system for virus biology and anti-viral drug discovery. One of barriers is lacking of culture system of normal epithelial cells in vitro over decades. In this study, we established human normal vaginal epithelial cell (HNVEC) culture using co-culture system. HNVEC cells were then propagated rapidly and stably in a defined culture condition. HNVEC cells exhibited a normal diploid karyotype and formed the well-defined and polarized spheres in matrigel three-dimension (3D) culture, while malignant cells (HeLa) formed disorganized and nonpolar solid spheres. HNVEC cells had a normal cellular response to DNA damage and had no transforming property using soft agar assays. HNVEC expressed epithelial marker cytokeratin 14 (CK14) and p63, but not cytokeratin 18 (CK18). Next, we reconstructed HNVEC-derived 3D vaginal epithelium using air-liquid interface (ALI) culture. This 3D vaginal epithelium has the basal and apical layers with expression of epithelial markers as its originated human vaginal tissue. Finally, we established an HSV-2 infection model based on the reconstructed 3D vaginal epithelium. After inoculation of HSV-2 (G strain) at apical layer of the reconstructed 3D vaginal epithelium, we observed obvious pathological effects gradually spreading from the apical layer to basal layer with expression of a viral protein. Thus, we established an ex vivo 2D and 3D HSV-2 infection model that can be used for HSV-2 virology and anti-viral drug discovery.

  5. Influence of acid and bile acid on ERK activity, PPARγ expression and cell proliferation in normal human esophageal epithelial cells

    PubMed Central

    Jiang, Zhi-Ru; Gong, Jun; Zhang, Zhen-Ni; Qiao, Zhe

    2006-01-01

    AIM: To observe the effects of acid and bile acid exposure on cell proliferation and the expression of extracellular signal-regulated protein kinase (ERK) and peroxisome proliferator-activated receptor γ (PPARγ) in normal human esophageal epithelial cells in vitro. METHODS: In vitro cultured normal human esophageal epithelial cells were exposed to acidic media (pH 4.0 - 6.5), media containing different bile acid (250 μmol/L), media containing acid and bile acid, respectively. Cell proliferation was assessed using MTT and flow cytometry. The expressions of phosphorylated ERK1/2 and PPARγ protein were determined by the immunoblotting technique. RESULTS: Acid-exposed (3 min) esophageal cells exhibited a significant increase in proliferation ratio, S phase of the cell cycle (P < 0.05) and the level of phosphorylated ERK1/2 protein. When the acid-exposure period exceeded 6 min, we observed a decrease in proliferation ratio and S phase of the cell cycle, with an increased apoptosis ratio (P < 0.05). Bile acid exposure (3-12 min) also produced an increase in proliferation ratio, S phase of the cell cycle (P < 0.05) and phosphorylated ERK1/2 expression. On the contrary, deoxycholic acid (DCA) exposure (> 20 min) decreased proliferation ratio. Compared with bile acid exposure (pH 7.4), bile acid exposure (pH 6.5, 4) significantly decreased proliferation ratio (P < 0.05). There was no expression of PPARγ in normal human esophageal epithelial cells. CONCLUSION: The rapid stimuli of acid or bile acid increase proliferation in normal human esophageal epithelial cells by activating the ERK pathway. PMID:16688842

  6. Progastrin Represses the Alternative Activation of Human Macrophages and Modulates Their Influence on Colon Cancer Epithelial Cells

    PubMed Central

    Hernández, Carlos; Barrachina, María Dolores; Cosín-Roger, Jesús; Ortiz-Masiá, Dolores; Álvarez, Ángeles; Terrádez, Liria; Nicolau, María Jesús; Alós, Rafael; Esplugues, Juan Vicente; Calatayud, Sara

    2014-01-01

    Macrophage infiltration is a negative prognostic factor for most cancers but gastrointestinal tumors seem to be an exception. The effect of macrophages on cancer progression depends on their phenotype, which may vary between M1 (pro-inflammatory, defensive) to M2 (tolerogenic, pro-tumoral). Gastrointestinal cancers often become an ectopic source of gastrins and macrophages present receptors for these peptides. The aim of the present study is to analyze whether gastrins can affect the pattern of macrophage infiltration in colorectal tumors. We have evaluated the relationship between gastrin expression and the pattern of macrophage infiltration in samples from colorectal cancer and the influence of these peptides on the phenotype of macrophages differentiated from human peripheral monocytes in vitro. The total number of macrophages (CD68+ cells) was similar in tumoral and normal surrounding tissue, but the number of M2 macrophages (CD206+ cells) was significantly higher in the tumor. However, the number of these tumor-associated M2 macrophages correlated negatively with the immunoreactivity for gastrin peptides in tumor epithelial cells. Macrophages differentiated from human peripheral monocytes in the presence of progastrin showed lower levels of M2-markers (CD206, IL10) with normal amounts of M1-markers (CD86, IL12). Progastrin induced similar effects in mature macrophages treated with IL4 to obtain a M2-phenotype or with LPS plus IFNγ to generate M1-macrophages. Macrophages differentiated in the presence of progastrin presented a reduced expression of Wnt ligands and decreased the number and increased cell death of co-cultured colorectal cancer epithelial cells. Our results suggest that progastrin inhibits the acquisition of a M2-phenotype in human macrophages. This effect exerted on tumor associated macrophages may modulate cancer progression and should be taken into account when analyzing the therapeutic value of gastrin immunoneutralization. PMID:24901518

  7. Progastrin represses the alternative activation of human macrophages and modulates their influence on colon cancer epithelial cells.

    PubMed

    Hernández, Carlos; Barrachina, María Dolores; Cosín-Roger, Jesús; Ortiz-Masiá, Dolores; Álvarez, Ángeles; Terrádez, Liria; Nicolau, María Jesús; Alós, Rafael; Esplugues, Juan Vicente; Calatayud, Sara

    2014-01-01

    Macrophage infiltration is a negative prognostic factor for most cancers but gastrointestinal tumors seem to be an exception. The effect of macrophages on cancer progression depends on their phenotype, which may vary between M1 (pro-inflammatory, defensive) to M2 (tolerogenic, pro-tumoral). Gastrointestinal cancers often become an ectopic source of gastrins and macrophages present receptors for these peptides. The aim of the present study is to analyze whether gastrins can affect the pattern of macrophage infiltration in colorectal tumors. We have evaluated the relationship between gastrin expression and the pattern of macrophage infiltration in samples from colorectal cancer and the influence of these peptides on the phenotype of macrophages differentiated from human peripheral monocytes in vitro. The total number of macrophages (CD68+ cells) was similar in tumoral and normal surrounding tissue, but the number of M2 macrophages (CD206+ cells) was significantly higher in the tumor. However, the number of these tumor-associated M2 macrophages correlated negatively with the immunoreactivity for gastrin peptides in tumor epithelial cells. Macrophages differentiated from human peripheral monocytes in the presence of progastrin showed lower levels of M2-markers (CD206, IL10) with normal amounts of M1-markers (CD86, IL12). Progastrin induced similar effects in mature macrophages treated with IL4 to obtain a M2-phenotype or with LPS plus IFNγ to generate M1-macrophages. Macrophages differentiated in the presence of progastrin presented a reduced expression of Wnt ligands and decreased the number and increased cell death of co-cultured colorectal cancer epithelial cells. Our results suggest that progastrin inhibits the acquisition of a M2-phenotype in human macrophages. This effect exerted on tumor associated macrophages may modulate cancer progression and should be taken into account when analyzing the therapeutic value of gastrin immunoneutralization.

  8. Expression and Function of the Protein Tyrosine Phosphatase Receptor J (PTPRJ) in Normal Mammary Epithelial Cells and Breast Tumors

    PubMed Central

    Smart, Chanel E.; Askarian Amiri, Marjan E.; Wronski, Ania; Dinger, Marcel E.; Crawford, Joanna; Ovchinnikov, Dmitry A.; Vargas, Ana Cristina; Reid, Lynne; Simpson, Peter T.; Song, Sarah; Wiesner, Christiane; French, Juliet D.; Dave, Richa K.; da Silva, Leonard; Purdon, Amy; Andrew, Megan; Mattick, John S.; Lakhani, Sunil R.

    2012-01-01

    The protein tyrosine phosphatase receptor J, PTPRJ, is a tumor suppressor gene that has been implicated in a range of cancers, including breast cancer, yet little is known about its role in normal breast physiology or in mammary gland tumorigenesis. In this paper we show that PTPRJ mRNA is expressed in normal breast tissue and reduced in corresponding tumors. Meta-analysis revealed that the gene encoding PTPRJ is frequently lost in breast tumors and that low expression of the transcript associated with poorer overall survival at 20 years. Immunohistochemistry of PTPRJ protein in normal human breast tissue revealed a distinctive apical localisation in the luminal cells of alveoli and ducts. Qualitative analysis of a cohort of invasive ductal carcinomas revealed retention of normal apical PTPRJ localization where tubule formation was maintained but that tumors mostly exhibited diffuse cytoplasmic staining, indicating that dysregulation of localisation associated with loss of tissue architecture in tumorigenesis. The murine ortholog, Ptprj, exhibited a similar localisation in normal mammary gland, and was differentially regulated throughout lactational development, and in an in vitro model of mammary epithelial differentiation. Furthermore, ectopic expression of human PTPRJ in HC11 murine mammary epithelial cells inhibited dome formation. These data indicate that PTPRJ may regulate differentiation of normal mammary epithelia and that dysregulation of protein localisation may be associated with tumorigenesis. PMID:22815804

  9. Expression and function of the protein tyrosine phosphatase receptor J (PTPRJ) in normal mammary epithelial cells and breast tumors.

    PubMed

    Smart, Chanel E; Askarian Amiri, Marjan E; Wronski, Ania; Dinger, Marcel E; Crawford, Joanna; Ovchinnikov, Dmitry A; Vargas, Ana Cristina; Reid, Lynne; Simpson, Peter T; Song, Sarah; Wiesner, Christiane; French, Juliet D; Dave, Richa K; da Silva, Leonard; Purdon, Amy; Andrew, Megan; Mattick, John S; Lakhani, Sunil R; Brown, Melissa A; Kellie, Stuart

    2012-01-01

    The protein tyrosine phosphatase receptor J, PTPRJ, is a tumor suppressor gene that has been implicated in a range of cancers, including breast cancer, yet little is known about its role in normal breast physiology or in mammary gland tumorigenesis. In this paper we show that PTPRJ mRNA is expressed in normal breast tissue and reduced in corresponding tumors. Meta-analysis revealed that the gene encoding PTPRJ is frequently lost in breast tumors and that low expression of the transcript associated with poorer overall survival at 20 years. Immunohistochemistry of PTPRJ protein in normal human breast tissue revealed a distinctive apical localisation in the luminal cells of alveoli and ducts. Qualitative analysis of a cohort of invasive ductal carcinomas revealed retention of normal apical PTPRJ localization where tubule formation was maintained but that tumors mostly exhibited diffuse cytoplasmic staining, indicating that dysregulation of localisation associated with loss of tissue architecture in tumorigenesis. The murine ortholog, Ptprj, exhibited a similar localisation in normal mammary gland, and was differentially regulated throughout lactational development, and in an in vitro model of mammary epithelial differentiation. Furthermore, ectopic expression of human PTPRJ in HC11 murine mammary epithelial cells inhibited dome formation. These data indicate that PTPRJ may regulate differentiation of normal mammary epithelia and that dysregulation of protein localisation may be associated with tumorigenesis.

  10. Cell competition with normal epithelial cells promotes apical extrusion of transformed cells through metabolic changes.

    PubMed

    Kon, Shunsuke; Ishibashi, Kojiro; Katoh, Hiroto; Kitamoto, Sho; Shirai, Takanobu; Tanaka, Shinya; Kajita, Mihoko; Ishikawa, Susumu; Yamauchi, Hajime; Yako, Yuta; Kamasaki, Tomoko; Matsumoto, Tomohiro; Watanabe, Hirotaka; Egami, Riku; Sasaki, Ayana; Nishikawa, Atsuko; Kameda, Ikumi; Maruyama, Takeshi; Narumi, Rika; Morita, Tomoko; Sasaki, Yoshiteru; Enoki, Ryosuke; Honma, Sato; Imamura, Hiromi; Oshima, Masanobu; Soga, Tomoyoshi; Miyazaki, Jun-Ichi; Duchen, Michael R; Nam, Jin-Min; Onodera, Yasuhito; Yoshioka, Shingo; Kikuta, Junichi; Ishii, Masaru; Imajo, Masamichi; Nishida, Eisuke; Fujioka, Yoichiro; Ohba, Yusuke; Sato, Toshiro; Fujita, Yasuyuki

    2017-05-01

    Recent studies have revealed that newly emerging transformed cells are often apically extruded from epithelial tissues. During this process, normal epithelial cells can recognize and actively eliminate transformed cells, a process called epithelial defence against cancer (EDAC). Here, we show that mitochondrial membrane potential is diminished in RasV12-transformed cells when they are surrounded by normal cells. In addition, glucose uptake is elevated, leading to higher lactate production. The mitochondrial dysfunction is driven by upregulation of pyruvate dehydrogenase kinase 4 (PDK4), which positively regulates elimination of RasV12-transformed cells. Furthermore, EDAC from the surrounding normal cells, involving filamin, drives the Warburg-effect-like metabolic alteration. Moreover, using a cell-competition mouse model, we demonstrate that PDK-mediated metabolic changes promote the elimination of RasV12-transformed cells from intestinal epithelia. These data indicate that non-cell-autonomous metabolic modulation is a crucial regulator for cell competition, shedding light on the unexplored events at the initial stage of carcinogenesis.

  11. The molecular and cellular response of normal and progressed human bronchial epithelial cells to HZE particles

    NASA Astrophysics Data System (ADS)

    Story, Michael; Ding, Liang-Hao; Minna, John; Park, Seong-mi; Larsen, Jill

    We have used a model of non-oncogenically immortalized normal human bronchial epithelial cells to determine the response of such cells to particles found outside the protection of the earth’s electromagnetic field. We have identified an enhanced frequency of cellular transformation, as measured by growth in soft agar, for both 56Fe and 28Si (1 GeV/n) that is maximal (4-6 fold) at 0.25 Gy and 0.40 Gy, respectively. At 4 months post-irradiation 38 individual soft agar clones were isolated. These clones were characterized extensively for cellular and molecular changes. Gene expression analysis suggested that these clones had down-regulated several genes associated with anti-oxidant pathways including GLS2, GPX1 and 4, SOD2, PIG3, and NQO1 amongst others. As a result, many of these transformed clones were exposed to high levels of intracellular radical oxygen species (ROS), although there appeared not to be any enhanced mitochondrial ROS. DNA repair pathways associated with ATM/ATR signaling were also upregulated. However, these transformants do not develop into tumors when injected into immune-compromised mice, suggesting that they have not progressed sufficiently to become oncogenic. Therefore we chose 6 soft agar clones for continuous culture for an additional 14 months. Amongst the 6 clones, only one clone showed any significant change in phenotype. Clone 3kt-ff.2a, propagated for 18 months, were 2-fold more radioresistant, had a shortened doubling time and the background rate of transformation more than doubled. Furthermore, the morphology of transformed clones changed. Clones from this culture are being compared to the original clone as well as the parental HBEC3KT and will be injected into immune-compromised mice for oncogenic potential. Oncogenically progressed HBECs, HBEC3KT cells that overexpress a mutant RAS gene and where p53 has been knocked down, designated HBEC3KTR53, responded quite differently to HZE particle exposure. First, these cells are more

  12. Identification and Characterization of Mesenchymal-Epithelial Progenitor-Like Cells in Normal and Injured Rat Liver

    PubMed Central

    Liu, Daqing; Yovchev, Mladen I.; Zhang, Jinghang; Alfieri, Alan A.; Tchaikovskaya, Tatyana; Laconi, Ezio; Dabeva, Mariana D.

    2016-01-01

    In normal rat liver, thymocyte antigen 1 (Thy1) is expressed in fibroblasts/myofibroblasts and in some blood progenitor cells. Thy1-expressing cells also accumulate in the liver during impaired liver regeneration. The origin and nature of these cells are not well understood. By using RT-PCR analysis and immunofluorescence microscopy, we describe the presence of rare Thy1+ cells in the liver lobule of normal animals, occasionally forming small collections of up to 20 cells. These cells constitute a small portion (1.7% to 1.8%) of nonparenchymal cells and reveal a mixed mesenchymal-epithelial phenotype, expressing E-cadherin, cytokeratin 18, and desmin. The most potent mitogens for mesenchymal-epithelial Thy1+ cells in vitro are the inflammatory cytokines interferon γ, IL-1, and platelet-derived growth factor-BB, which are not produced by Thy1+ cells. Thy1+ cells express all typical mesenchymal stem cell and hepatic progenitor cell markers and produce growth factor and cytokine mRNA (Hgf, Il6, Tgfa, and Tweak) for proteins that maintain oval cell growth and differentiation. Under appropriate conditions, mesenchymal-epithelial cells differentiate in vitro into hepatocyte-like cells. In this study, we show that the adult rat liver harbors a small pool of endogenous mesenchymal-epithelial cells not recognized previously. In the quiescent state, these cells express both mesenchymal and epithelial cell markers. They behave like hepatic stem cells/progenitors with dual phenotype, exhibiting high plasticity and long-lasting proliferative activity. PMID:25447047

  13. One-hit effects in cancer: Altered proteome of morphologically normal colon crypts in Familial Adenomatous Polyposis

    PubMed Central

    Yeung, Anthony T.; Patel, Bhavinkumar B.; Li, Xin-Ming; Seeholzer, Steven H.; Coudry, Renata A.; Cooper, Harry S.; Bellacosa, Alfonso; Boman, Bruce M.; Zhang, Tao; Litwin, Samuel; Ross, Eric A.; Conrad, Peggy; Crowell, James A.; Kopelovich, Levy; Knudson, Alfred

    2008-01-01

    We studied patients with Familial Adenomatous Polyposis (FAP), because they are virtually certain to develop colon cancer, and because much is known about the causative APC gene. We hypothesized that the inherited heterozygous mutation itself leads to changes in the proteome of morphologically normal crypts and the proteins that changed may represent targets for preventive and therapeutic agents. We determined the differential protein expression of morphologically normal colon crypts of FAP patients versus those of individuals without the mutation, using two-dimensional gel electrophoresis, mass spectrometry and validation by 2D gel Western blotting. Approximately 13% of 1,695 identified proteins were abnormally expressed in the morphologically normal crypts of APC mutation carriers, indicating that a colon crypt cell under the one-hit state is already abnormal. Many of the expression changes affect pathways consistent with the function of the APC protein, including apoptosis, cell adhesion, cell motility, cytoskeletal organization and biogenesis, mitosis, transcription and oxidative stress response. Thus, heterozygosity for a mutant APC tumor suppressor gene alters the proteome of normal-appearing crypt cells in a gene-specific manner, consistent with a detectable one-hit event. These changes may represent the earliest biomarkers of colorectal cancer development, potentially leading to the identification of molecular targets for cancer prevention. PMID:18794146

  14. Colon luminal content and epithelial cell morphology are markedly modified in rats fed with a high-protein diet.

    PubMed

    Andriamihaja, Mireille; Davila, Anne-Marie; Eklou-Lawson, Mamy; Petit, Nathalie; Delpal, Serge; Allek, Fadhila; Blais, Anne; Delteil, Corine; Tomé, Daniel; Blachier, François

    2010-11-01

    Hyperproteic diets are used in human nutrition to obtain body weight reduction. Although increased protein ingestion results in an increased transfer of proteins from the small to the large intestine, there is little information on the consequences of the use of such diets on the composition of large intestine content and on epithelial cell morphology and metabolism. Rats were fed for 15 days with either a normoproteic (NP, 14% protein) or a hyperproteic isocaloric diet (HP, 53% protein), and absorptive colonocytes were observed by electron microscopy or isolated for enzyme activity studies. The colonic luminal content was recovered for biochemical analysis. Absorbing colonocytes were characterized by a 1.7-fold reduction in the height of the brush-border membranes (P = 0.0001) after HP diet consumption when compared with NP. This coincided in the whole colon content of HP animals with a 1.8-fold higher mass content (P = 0.0020), a 2.2-fold higher water content (P = 0.0240), a 5.2-fold higher protease activity (P = 0.0104), a 5.5-fold higher ammonia content (P = 0.0008), and a more than twofold higher propionate, valerate, isobutyrate, and isovalerate content (P < 0.05). The basal oxygen consumption of colonocytes was similar in the NP and HP groups, but ammonia was found to provoke a dose-dependent decrease of oxygen consumption in the isolated absorbing colonocytes. The activity of glutamine synthetase (which condenses ammonia and glutamate) was found to be much higher in colonocytes than in small intestine enterocytes and was 1.6-fold higher (P = 0.0304) in colonocytes isolated from HP animals than NP. Glutaminase activity remained unchanged. Thus hyperproteic diet ingestion causes marked changes both in the luminal environment of colonocytes and in the characteristics of these cells, demonstrating that hyperproteic diet interferes with colonocyte metabolism and morphology. Possible causal relationships between energy metabolism, reduced height of colonocyte

  15. Growth of Normal Mouse Vaginal Epithelial Cells in and on Collagen Gels

    NASA Astrophysics Data System (ADS)

    Iguchi, Taisen; Uchima, Francis-Dean A.; Ostrander, Patricia L.; Bern, Howard A.

    1983-06-01

    Sustained growth in primary culture of vaginal epithelial cells from ovariectomized adult BALB/cCrg1 mice embedded within or seeded on collagen gel matrix was achieved in a serum-free medium composed of Ham's F-12 medium/Dulbecco's modified Eagle's medium, 1:1 (vol/vol), supplemented with insulin, bovine serum albumin fraction V, epidermal growth factor, cholera toxin, and transferrin. Three-dimensional growth of vaginal epithelial cells occurred inside the collagen gel matrix. Cell numbers increased 4- to 8-fold in collagen gel and about 4-fold on collagen gel after 9-10 days in culture. The effect of 17β -estradiol (0.00018-180 nM in gel or 0.018-180 nM on gel) and diethylstilbestrol (DES; 0.0186-186 nM in gel) on the growth of vaginal epithelial cells was examined. The addition of estrogen did not enhance the growth of vaginal epithelial cells during this time period either in the complete medium or in a suboptimal medium. Cultures on floating collagen gels in the serum-free medium are composed of 1-3 cell layers with superficial cornification. Estrogen does not appear to be a direct mitogen for vaginal epithelial cells, at least in this system.

  16. PAC exhibits potent anti-colon cancer properties through targeting cyclin D1 and suppressing epithelial-to-mesenchymal transition.

    PubMed

    Al-Qasem, Abeer; Al-Howail, Huda A; Al-Swailem, Mashael; Al-Mazrou, Amer; Al-Otaibi, Basem; Al-Jammaz, Ibrahim; Al-Khalaf, Huda H; Aboussekhra, Abdelilah

    2016-03-01

    Colorectal cancer (CRC) is a major cause of cancer morbidity and mortality worldwide. Although response rates and overall survival have been improved in recent years, resistance to multiple drug combinations is inevitable. Therefore, the development of more efficient drugs, with fewer side effects is urgently needed. To this end, we have investigated in the present report the effect of PAC, a novel cucumin analogue, on CRC cells both in vitro and in vivo. We have shown that PAC induces apoptosis, mainly via the internal mitochondrial route, and inhibits cell proliferation through delaying the cell cycle at G2/M phase. Interestingly, the pro-apoptotic effect was mediated through STAT3-dependent down-regulation of cyclin D1 and its downstream target survivin. Indeed, change in the expression level of cyclin D1 modulated the expression of survivin and the response of CRC cells to PAC. Furthermore, using the ChIP assay, we have shown PAC-dependent reduction in the binding of STAT3 to the cyclin D1 promoter in vivo. Additionally, PAC suppressed the epithelial-to-mesenchymal process through down-regulating the mesenchymal markers (N-cadherin, vimentin and Twist1) and inhibiting the invasion/migration abilities of the CRC cells via repressing the pro-migration/invasion protein kinases AKT and ERK1/2. In addition, PAC inhibited tumor growth and repressed the JAK2/STAT3, AKT/mTOR and MEK/ERK pathways as well as their common downstream effectors cyclin D1 and survivin in humanized CRC xenografts. Collectively, these results indicate that PAC has potent anti-CRC effects, and therefore could constitute an effective alternative chemotherapeutic agent, which may consolidate the adjuvant treatment of colon cancer.

  17. Metabolism of aflatoxin B1 by normal human bronchial epithelial cells.

    PubMed

    Van Vleet, T R; Klein, P J; Coulombe, R A

    2001-08-10

    Aflatoxin B, (AFB1) is a potent hepatocarcinogen in animal models and a suspected carcinogen in humans. High concentrations of AFB, have been found in respirable grain dusts, and may therefore be a risk factor for human lung cancer in certain occupations. To study the potential for AFB, activation in human lung, cytochrome P-450 (CYP)-mediated activation and glutathione S-transferase (GST)-mediated detoxification of AFB1 were examined in cultured normal human bronchial epithelial (NHBE) cells. Cells were exposed to 0. 15 microM or 1.5 microM AFB, for 48 h and media was collected for metabolite analysis by high-performance liquid chromatography (HPLC). At 0. 15 microM, AFB1 was metabolized only to the detoxified metabolite aflatoxin Q1 (AFQ1). At 1.5 microM AFB1, both aflatoxin M1 (AFM1), and AFQ1 were produced. Cells pretreated with 50 degrees M 3-methylcholanthrene (3MC), a CYP 1A inducer, for 72 h prior to 0.15 microM AFB1, produced the activated AFB1 8,9-epoxide (AFBO). Similarly, microsomes prepared from 3MC-pretreated cells formed AFBO, but microsomes from noninduced cells did not. While AFB1-DNA adducts were not detected at low AFB1 concentrations in untreated NHBE, 3MC induction caused the production of AFB1-DNA adducts at 0.015 and 0.15 microM AFB1. Western immunoblots showed that the primary CYP isoforms responsible for AFB1 activation in the liver, 1A and 3A4, to be constitutively expressed in NHBE cells. Expression of CYP 1A was significantly increased in 3MC-pretreated cells, while CYP 3A4 expression increased slightly, but not to the extent of the 1A isoforms. The principal AFBO detoxifying enzyme, glutathione S-transferase (GST), was constitutively expressed in NHBE cells, and was increased approximately twofold by 3MC pretreatment. Cytosolic fractions from neither control nor 3MC-induced NHBE had measurable AFBO conjugating activity, indicating that these cells may lack AFB1-relevant GST activity. From these data, it appears that NHBE cells activate

  18. A lung cancer risk classifier comprising genome maintenance genes measured in normal bronchial epithelial cells.

    PubMed

    Yeo, Jiyoun; Crawford, Erin L; Zhang, Xiaolu; Khuder, Sadik; Chen, Tian; Levin, Albert; Blomquist, Thomas M; Willey, James C

    2017-05-02

    Annual low dose CT (LDCT) screening of individuals at high demographic risk reduces lung cancer mortality by more than 20%. However, subjects selected for screening based on demographic criteria typically have less than a 10% lifetime risk for lung cancer. Thus, there is need for a biomarker that better stratifies subjects for LDCT screening. Toward this goal, we previously reported a lung cancer risk test (LCRT) biomarker comprising 14 genome-maintenance (GM) pathway genes measured in normal bronchial epithelial cells (NBEC) that accurately classified cancer (CA) from non-cancer (NC) subjects. The primary goal of the studies reported here was to optimize the LCRT biomarker for high specificity and ease of clinical implementation. Targeted competitive multiplex PCR amplicon libraries were prepared for next generation sequencing (NGS) analysis of transcript abundance at 68 sites among 33 GM target genes in NBEC specimens collected from a retrospective cohort of 120 subjects, including 61 CA cases and 59 NC controls. Genes were selected for analysis based on contribution to the previously reported LCRT biomarker and/or prior evidence for association with lung cancer risk. Linear discriminant analysis was used to identify the most accurate classifier suitable to stratify subjects for screening. After cross-validation, a model comprising expression values from 12 genes (CDKN1A, E2F1, ERCC1, ERCC4, ERCC5, GPX1, GSTP1, KEAP1, RB1, TP53, TP63, and XRCC1) and demographic factors age, gender, and pack-years smoking, had Receiver Operator Characteristic area under the curve (ROC AUC) of 0.975 (95% CI: 0.96-0.99). The overall classification accuracy was 93% (95% CI 88%-98%) with sensitivity 93.1%, specificity 92.9%, positive predictive value 93.1% and negative predictive value 93%. The ROC AUC for this classifier was significantly better (p < 0.0001) than the best model comprising demographic features alone. The LCRT biomarker reported here displayed high accuracy and ease

  19. JS-K, a nitric oxide-releasing prodrug, induces breast cancer cell death while sparing normal mammary epithelial cells.

    PubMed

    McMurtry, Vanity; Saavedra, Joseph E; Nieves-Alicea, René; Simeone, Ann-Marie; Keefer, Larry K; Tari, Ana M

    2011-04-01

    Targeted therapy with reduced side effects is a major goal in cancer research. We investigated the effects of JS-K, a nitric oxide (NO) prodrug designed to release high levels of NO when suitably activated, on human breast cancer cell lines, on non-transformed human MCF-10A mammary cells, and on normal human mammary epithelial cells (HMECs). Cell viability assay, flow cytometry, electron microscopy, and Western blot analysis were used to study the effects of JS-K on breast cancer and on mammary epithelial cells. After a 3-day incubation, the IC50s of JS-K against the breast cancer cells ranged from 0.8 to 3 µM. However, JS-K decreased the viability of the MCF-10A cells by only 20% at 10-µM concentration, and HMECs were unaffected by 10 µM JS-K. Flow cytometry indicated that JS-K increased the percentages of breast cancer cells under-going apoptosis. Interestingly, flow cytometry indicated that JS-K increased acidic vesicle organelle formation in breast cancer cells, suggesting that JS-K induced autophagy in breast cancer cells. Electron microscopy confirmed that JS-K-treated breast cancer cells underwent autophagic cell death. Western blot analysis showed that JS-K induced the expression of microtubule light chain 3-II, another autophagy marker, in breast cancer cells. However, JS-K did not induce apoptosis or autophagy in normal human mammary epithelial cells. These data indicate that JS-K selectively induces programmed cell death in breast cancer cells while sparing normal mammary epithelial cells under the same conditions. The selective anti-tumor activity of JS-K warrants its further investigation in breast tumors.

  20. Inhibition of Transforming Growth Factor-{beta} Signaling in Normal Lung Epithelial Cells Confers Resistance to Ionizing Radiation

    SciTech Connect

    Reeves, Anna; Zagurovskaya, Marianna; Gupta, Seema; Shareef, Mohammed M.; Mohiuddin, Mohammed; Ahmed, Mansoor M. . E-mail: mmahmed@geisinger.edu

    2007-05-01

    Purpose: To address the functional role of radiation-induced transforming growth factor-{beta} (TGF-{beta}) signaling in a normal epithelial background, we selected a spontaneously immortalized lung epithelial cell line derived from the normal lung tissue of a dominant-negative mutant of the TGF-{beta} RII ({delta}RII) transgenic mouse that conditionally expressed {delta}RII under the control of the metallothionein promoter (MT-1), and assessed this cell line's response to radiation. Methods and Materials: A spontaneously immortalized lung epithelial cell culture (SILECC) was established and all analyses were performed within 50 passages. Colony-forming and terminal transferase dUPT nick end labeling (TUNEL) assays were used to assess clonogenic inhibition and apoptosis, respectively. Western-blot analysis was performed to assess the kinetics of p21, bax, and RII proteins. Transforming growth factor-{beta}-responsive promoter activity was measured using dual-luciferase reporter assay. Results: Exposure to ZnSO{sub 4} inhibited TGF-{beta} signaling induced either by recombinant TGF-{beta}1 or ionizing radiation. The SILECC, treated with either ZnSO{sub 4} or neutralizing antibody against TGF-{beta}, showed a significant increase in radio-resistance compared to untreated cells. Furthermore, the expression of {delta}RII inhibited the radiation-induced up-regulation of the TGF-{beta} effector gene p21{sup waf1/cip1}. Conclusions: Our findings imply that inhibition of radiation-induced TGF-{beta} signaling via abrogation of the RII function enhances the radio-resistance of normal lung epithelial cells, and this can be directly attributed to the loss of TGF-{beta} signaling function.

  1. Classification and risk assessment of individuals with familial polyposis, Gardner's syndrome, and familial non-polyposis colon cancer from (/sup 3/H)thymidine labeling patterns in colonic epithelial cells

    SciTech Connect

    Lipkin, M.; Blattner, W.A.; Gardner, E.J.; Burt, R.W.; Lynch, H.; Deschner, E.; Winawer, S.; Fraumeni, J.F. Jr.

    1984-09-01

    A probabilistic analysis has been developed to assist the binary classification and risk assessment of members of familial colon cancer kindreds. The analysis is based on the microautoradiographic observation of (/sup 3/H)thymidine-labeled epithelial cells in colonic mucosa of the kindred members. From biopsies of colonic mucosa which are labeled with (/sup 3/H)thymidine in vitro, the degree of similarity of each subject's cell-labeling pattern measured over entire crypts was automatically compared to the labeling patterns of high-risk and low-risk reference populations. Each individual was then presumptively classified and assigned to one of the reference populations, and a degree of risk for the classification was provided. In carrying out the analysis, a linear score was calculated for each individual relative to each of the reference populations, and the classification was based on the polarity of the score difference; the degree of risk was then quantitated from the magnitude of the score difference. When the method was applied to kindreds having either familial polyposis or familial non-polyposis colon cancer, it effectively segregated individuals affected with disease from others at low risk, with sensitivity and specificity ranging from 71 to 92%. Further application of the method to asymptomatic family members believed to be at 50% risk on the basis of pedigree evaluation revealed a biomodal distribution to nearly zero or full risk. The accuracy and simplicity of this approach and its capability of revealing early stages of abnormal colonic epithelial cell development indicate potential for preclinical screening of subjects at risk in cancer-prone kindreds and for assisting the analysis of modes of inheritance.

  2. Human Antibodies to PhtD, PcpA, and Ply Reduce Adherence to Human Lung Epithelial Cells and Murine Nasopharyngeal Colonization by Streptococcus pneumoniae

    PubMed Central

    Kaur, Ravinder; Surendran, Naveen; Ochs, Martina

    2014-01-01

    Streptococcus pneumoniae adherence to human epithelial cells (HECs) is the first step in pathogenesis leading to infections. We sought to determine the role of human antibodies against S. pneumoniae protein vaccine candidates PhtD, PcpA, and Ply in preventing adherence to lung HECs in vitro and mouse nasopharyngeal (NP) colonization in vivo. Human anti-PhtD, -PcpA, and -Ply antibodies were purified and Fab fragments generated. Fabs were used to test inhibition of adherence of TIGR4 and nonencapsulated strain RX1 to A549 lung HECs. The roles of individual proteins in adherence were tested using isogenic mutants of strain TIGR4. Anti-PhtD, -PcpA, and -Ply human antibodies were assessed for their ability to inhibit NP colonization in vivo by passive transfer of human antibody in a murine model. Human antibodies generated against PhtD and PcpA caused a decrease in adherence to A549 cells (P < 0.05). Anti-PhtD but not anti-PcpA antibodies showed a protective role against mouse NP colonization. To our surprise, anti-Ply antibodies also caused a significant (P < 0.05) reduction in S. pneumoniae colonization. Our results support the potential of PhtD, PcpA, and Ply protein vaccine candidates as alternatives to conjugate vaccines to prevent non-serotype-specific S. pneumoniae colonization and invasive infection. PMID:25245804

  3. Immunohistochemical and Western blot analysis of two protein tyrosine phosphatase receptors, R and Z1, in colorectal carcinoma, colon adenoma and normal colon tissues.

    PubMed

    Woźniak, Marta; Gamian, Elżbieta; Łaczmańska, Izabela; Sąsiadek, Maria M; Duś-Szachniewicz, Kamila; Ziółkowski, Piotr

    2014-05-01

    Two classes of proteins, namely tyrosine kinases (PTK) and phosphatases (PTP), play an important role in cell proliferation and differentiation, thus leading to an acceleration or inhibition of tumour growth. The role of the above proteins in colorectal carcinoma (CRC) growth is a well-known event. In this study we carried out immunohistochemical and Western blot analysis of colorectal carcinoma, adenoma and normal colon tissue in relation to two protein tyrosine phosphatase receptors, R and Z1. Twenty-five cases of CRC were analyzed and the results were compared with similar data obtained in non-malignant tissues. High expression of both PTP receptors was observed in all examined cases of CRC, adenoma and normal colon tissue in this study. These results are not in line with recently published data, showing that genetic coding for PTPRR and PTPRZ1 were hypermethylated in CRC's. We presume that the protein tyrosine phosphatase overexpression in colorectal carcinoma is not enough to protect from the progression of disease.

  4. Oestradiol decreases colonic permeability through oestrogen receptor β-mediated up-regulation of occludin and junctional adhesion molecule-A in epithelial cells

    PubMed Central

    Braniste, Viorica; Leveque, Mathilde; Buisson-Brenac, Claire; Bueno, Lionel; Fioramonti, Jean; Houdeau, Eric

    2009-01-01

    Oestradiol modulates paracellular permeability and tight junction (TJ) function in endothelia and reproductive tissues, but whether the ovarian hormones and cycle affect the paracellular pathway in the intestinal epithelium remains unclear. Oestrogen receptors (ERs) are expressed in intestinal epithelial cells, and oestradiol regulates epithelium formation. We examined the effects of oestrous cycle stage, oestradiol benzoate (EB), and progesterone (P) on colonic paracellular permeability (CPP) in the female rat, and whether EB affects expression of the TJ proteins in the rat colon and the human colon cell line Caco-2. In cyclic rats, CPP was determined through lumen-to-blood 51Cr-labelled EDTA clearance, and in Ussing chambers for dextran permeability. CPP was also examined in ovariectomized (OVX) rats treated with P or EB, with and without the ER antagonist ICI 182,780, or with the selective agonists for ERα (propyl pyrazole triol; PPT) or ERβ (diarylpropionitrile; DPN). In oestrus rats, CPP was reduced (P < 0.01) relative to dioestrus. In OVX rats, EB dose-dependently decreased CPP, an effect mimicked by DPN and blocked by ICI 182,780, whereas P had no effect. Oestradiol increased occludin mRNA and protein in the colon (P < 0.05), but not zona occludens (ZO)-1. Further, EB and DPN enhanced occludin and junctional adhesion molecule (JAM)-A expression in Caco-2 cells without change in ZO-1, an effect blocked by ICI 182,780. These data show that oestrogen reinforces intestinal epithelial barrier through ERβ-mediated up-regulation of the transmembrane proteins occludin and JAM-A determining paracellular spaces. These findings highlight the importance of the ERβ pathway in the control of colonic paracellular transport and mucosal homeostasis. PMID:19433574

  5. Oxalobacter formigenes colonization normalizes oxalate excretion in a gastric bypass model of hyperoxaluria.

    PubMed

    Canales, Benjamin K; Hatch, Marguerite

    2017-07-01

    Hyperoxaluria and oxalate kidney stones frequently develop after Roux-en-Y gastric bypass (RYGB). Oxalobacter formigenes can degrade ingested oxalate. Examine the effect of O. formigenes wild rat strain (OXWR) colonization on urinary oxalate excretion and intestinal oxalate transport in a hyperoxaluric RYGB model. Basic Science Laboratory, United States. At 21 weeks of age, 28 obese male Sprague-Dawley rats survived Sham (n = 10) or RYGB (n = 18) surgery and were maintained on a 1.5% potassium oxalate, 40% fat diet. At 12 weeks postoperatively, half the animals in each group were gavaged with OXWR. At 16 weeks, percent dietary fat content was lowered to 10%. Urine and stool were collected weekly to determine oxalate and colonization status, respectively. At week 20, [14 C]-oxalate fluxes and electrical parameters were measured in vitro across isolated distal colon and jejunal (Roux limb) tissue mounted in Ussing Chambers. RYGB animals lost 22% total weight while Shams gained 5%. On a moderate oxalate diet, urinary oxalate excretion was 4-fold higher in RYGB than Sham controls. OXWR colonization, obtained in all gavaged animals, reduced urinary oxalate excretion 74% in RYGB and 39% in Sham and was further augmented by lowering the percentage of dietary fat. Finally, OXWR colonization significantly enhanced basal net colonic oxalate secretion in both groups. In our model, OXWR lowered urinary oxalate by luminal oxalate degradation in concert with promotion of enteric oxalate elimination. Trials of O. formigenes colonization and low-fat diet are warranted in calcium oxalate stone formers with gastric bypass and resistant hyperoxaluria. Copyright © 2017 American Society for Bariatric Surgery. Published by Elsevier Inc. All rights reserved.

  6. Control of growth and squamous differentiation in normal human bronchial epithelial cells by chemical and biological modifiers and transferred genes.

    PubMed Central

    Pfeifer, A M; Lechner, J F; Masui, T; Reddel, R R; Mark, G E; Harris, C C

    1989-01-01

    The majority of human lung cancers arise from bronchial epithelial cells. The normal pseudostratified bronchial epithelium is composed of basal, mucous, and ciliated cells. This multi-differentiated epithelium usually responds to xenobiotics and physical injury by undergoing basal cell hyperplasia, mucous cell hyperplasia, and squamous metaplasia. One step of the multistage process of carcinogenesis is thought to involve aberrations in control of the squamous metaplastic processes. Decreased responsiveness to regulators of terminal squamous differentiation may confer a selective clonal expansion advantage to an initiated cell. We studied the effects of endogenous [e.g., transforming growth factor beta 1 (TGF-beta 1) and serum] and exogenous [e.g., 12-O-tetradecanoyl-13-phorbol-acetate (TPA), tobacco smoke condensate, and aldehydes] modifiers of normal human bronchial epithelial (NHBE) cell in a serum-free culture system. NHBE cells are growth inhibited by all of these compounds and induced to undergo squamous differentiation by TGF-beta 1 or TPA. In contrast, lung carcinoma cell lines are relatively resistant to inducers of terminal squamous differentiation which may provide them with a selective growth advantage. Chemical agents and activated protooncogenes (ras,raf,myc) altered the response to endogenous and exogenous inducers of squamous differentiation and caused extended cellular lifespan, aneuploidy, and/or tumorigenicity. The data suggest a close relationship between dysregulation of terminal differentiation pathways and neoplastic transformation of human bronchial epithelial cells. PMID:2538323

  7. GENE EXPRESSION PROFILING OF NORMAL HUMAN BRONCHIAL EPITHELIAL CELLS EXPOSED TO TRIVALENT ARSENICALS AND DIMETHYLTHIOARSINIC ACID

    EPA Science Inventory

    Lung is a major target for arsenic carcinogenesis in humans. However, the carcinogenic mode of action of arsenicals is unknown. We investigated, in human bronchial epithelial (BEAS2B) cells, the effects of inorganic arsenic (iAsIII), monomethylarsonous acid (MMAIII), dimethylarsi...

  8. GENE EXPRESSION PROFILING OF NORMAL HUMAN BRONCHIAL EPITHELIAL CELLS EXPOSED TO TRIVALENT ARSENICALS AND DIMETHYLTHIOARSINIC ACID

    EPA Science Inventory

    Lung is a major target for arsenic carcinogenesis in humans. However, the carcinogenic mode of action of arsenicals is unknown. We investigated, in human bronchial epithelial (BEAS2B) cells, the effects of inorganic arsenic (iAsIII), monomethylarsonous acid (MMAIII), dimethylarsi...

  9. Resveratrol and some glucosyl, glucosylacyl, and glucuronide derivatives reduce Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes Scott A adhesion to colonic epithelial cell lines.

    PubMed

    Selma, María V; Larrosa, Mar; Beltrán, David; Lucas, Ricardo; Morales, Juan C; Tomás-Barberán, Francisco; Espín, Juan C

    2012-08-01

    The efficacy of resveratrol and some glucosyl, glucosylacyl, and glucuronide derivatives in inhibiting the adhesion of Salmonella Typhimurium, Escherichia coli O157:H7, and Listeria monocytogenes Scott A to Caco-2 and HT-29 colonic cells was investigated. The three bacteria strains were capable of adhering to both colonic epithelial cell lines, which responded by producing the pro-inflammatory interleukin 8 (IL-8). Adhesion inhibition of E. coli O157:H7 and S. Typhimurium to colonic cells was ≥60 and ≥40%, respectively, when resveratrol and most of the resveratrol derivatives were applied. Lower adhesion inhibition was observed for the bacteria with higher adherence potential, L. monocytogenes (≥20%). Resveratrol-3-O-(6'-O-butanoyl)-β-D-glucopyranoside (BUT) (50 and 100 μM) and resveratrol-3-O-(6'-O-octanoyl)-β-D-glucopyranoside (OCT) (50 μM) reduced IL-8 secretion by 100%. These results suggest that one mechanism for the beneficial attributes of resveratrol and especially the derivatives BUT and OCT could be the ability to reduce the adhesion and consequent pro-inflammatory cytokine production in intestinal epithelial cells in response to pathogen adhesion. The potential use of these compounds in the prevention of foodborne infections, intestinal homeostasis loss, and inflammatory bowel diseases could be another step in finding coadjuvants or alternatives to antibiotic treatments.

  10. Fluorescence lifetime spectroscopy of tissue autofluorescence in normal and diseased colon measured ex vivo using a fiber-optic probe.

    PubMed

    Coda, Sergio; Thompson, Alex J; Kennedy, Gordon T; Roche, Kim L; Ayaru, Lakshmana; Bansi, Devinder S; Stamp, Gordon W; Thillainayagam, Andrew V; French, Paul M W; Dunsby, Chris

    2014-02-01

    We present an ex vivo study of temporally and spectrally resolved autofluorescence in a total of 47 endoscopic excision biopsy/resection specimens from colon, using pulsed excitation laser sources operating at wavelengths of 375 nm and 435 nm. A paired analysis of normal and neoplastic (adenomatous polyp) tissue specimens obtained from the same patient yielded a significant difference in the mean spectrally averaged autofluorescence lifetime -570 ± 740 ps (p = 0.021, n = 12). We also investigated the fluorescence signature of non-neoplastic polyps (n = 6) and inflammatory bowel disease (n = 4) compared to normal tissue in a small number of specimens.

  11. Fluorescence lifetime spectroscopy of tissue autofluorescence in normal and diseased colon measured ex vivo using a fiber-optic probe

    PubMed Central

    Coda, Sergio; Thompson, Alex J.; Kennedy, Gordon T.; Roche, Kim L.; Ayaru, Lakshmana; Bansi, Devinder S.; Stamp, Gordon W.; Thillainayagam, Andrew V.; French, Paul M. W.; Dunsby, Chris

    2014-01-01

    We present an ex vivo study of temporally and spectrally resolved autofluorescence in a total of 47 endoscopic excision biopsy/resection specimens from colon, using pulsed excitation laser sources operating at wavelengths of 375 nm and 435 nm. A paired analysis of normal and neoplastic (adenomatous polyp) tissue specimens obtained from the same patient yielded a significant difference in the mean spectrally averaged autofluorescence lifetime −570 ± 740 ps (p = 0.021, n = 12). We also investigated the fluorescence signature of non-neoplastic polyps (n = 6) and inflammatory bowel disease (n = 4) compared to normal tissue in a small number of specimens. PMID:24575345

  12. Differential expression of IGF-1 mRNA isoforms in colorectal carcinoma and normal colon tissue.

    PubMed

    Kasprzak, Aldona; Szaflarski, Witold; Szmeja, Jacek; Andrzejewska, Małgorzata; Przybyszewska, Wiesława; Kaczmarek, Elżbieta; Koczorowska, Maria; Kościński, Tomasz; Zabel, Maciej; Drews, Michał

    2013-01-01

    RNA decreased as compared to the normal colon tissues, although however, with conservation of both gene promoter activities and with the continued principal splicing IGF-1 mRNA isoforms.

  13. p120 Catenin is required for normal tubulogenesis but not epithelial integrity in developing mouse pancreas.

    PubMed

    Hendley, Audrey M; Provost, Elayne; Bailey, Jennifer M; Wang, Yue J; Cleveland, Megan H; Blake, Danielle; Bittman, Ross W; Roeser, Jeffrey C; Maitra, Anirban; Reynolds, Albert B; Leach, Steven D

    2015-03-01

    The intracellular protein p120 catenin aids in maintenance of cell-cell adhesion by regulating E-cadherin stability in epithelial cells. In an effort to understand the biology of p120 catenin in pancreas development, we ablated p120 catenin in mouse pancreatic progenitor cells, which resulted in deletion of p120 catenin in all epithelial lineages of the developing mouse pancreas: islet, acinar, centroacinar, and ductal. Loss of p120 catenin resulted in formation of dilated epithelial tubules, expansion of ductal epithelia, loss of acinar cells, and the induction of pancreatic inflammation. Aberrant branching morphogenesis and tubulogenesis were also observed. Throughout development, the phenotype became more severe, ultimately resulting in an abnormal pancreas comprised primarily of duct-like epithelium expressing early progenitor markers. In pancreatic tissue lacking p120 catenin, overall epithelial architecture remained intact; however, actin cytoskeleton organization was disrupted, an observation associated with increased cytoplasmic PKCζ. Although we observed reduced expression of adherens junction proteins E-cadherin, β-catenin, and α-catenin, p120 catenin family members p0071, ARVCF, and δ-catenin remained present at cell membranes in homozygous p120(f/f) pancreases, potentially providing stability for maintenance of epithelial integrity during development. Adult mice homozygous for deletion of p120 catenin displayed dilated main pancreatic ducts, chronic pancreatitis, acinar to ductal metaplasia (ADM), and mucinous metaplasia that resembles PanIN1a. Taken together, our data demonstrate an essential role for p120 catenin in pancreas development. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. A molecular signature of normal breast epithelial and stromal cells from Li-Fraumeni syndrome mutation carriers

    PubMed Central

    Herbert, Brittney-Shea; Chanoux, Rebecca A.; Liu, Yunlong; Baenziger, Peter H.; Goswami, Chirayu P.; McClintick, Jeanette N.; Edenberg, Howard J.; Pennington, Robert E.; Lipkin, Steven M.; Kopelovich, Levy

    2010-01-01

    Specific changes in gene expression during cancer initiation should enable discovery of biomarkers for risk assessment, early detection and targets for chemoprevention. It has been previously demonstrated that altered mRNA and proteome signatures of morphologically normal cells bearing a single inherited “hit” in a tumor suppressor gene parallel many changes observed in the corresponding sporadic cancer. Here, we report on the global gene expression profile of morphologically normal, cultured primary breast epithelial and stromal cells from Li-Fraumeni syndrome (LFS) TP53 mutation carriers. Our analyses identified multiple changes in gene expression in both morphologically normal breast epithelial and stromal cells associated with TP53 haploinsufficiency, as well as interlocking pathways. Notably, a dysregulated p53 signaling pathway was readily detectable. Pharmacological intervention with the p53 rescue compounds CP-31398 and PRIMA-1 provided further evidence in support of the central role of p53 in affecting these changes in LFS cells and treatment for this cancer. Because loss of signaling mediated by TP53 is associated with the development and survival of many human tumors, identification of gene expression profiles in morphologically normal cells that carry “one-hit” p53 mutations may reveal novel biomarkers, enabling the discovery of potential targets for chemoprevention of sporadic tumors as well. PMID:21311097

  15. Scribble is required for normal epithelial cell–cell contacts and lumen morphogenesis in the mammalian lung

    PubMed Central

    Yates, Laura L.; Schnatwinkel, Carsten; Hazelwood, Lee; Chessum, Lauren; Paudyal, Anju; Hilton, Helen; Romero, M. Rosario; Wilde, Jonathan; Bogani, Debora; Sanderson, Jeremy; Formstone, Caroline; Murdoch, Jennifer N.; Niswander, Lee A.; Greenfield, Andy; Dean, Charlotte H.

    2013-01-01

    During lung development, proper epithelial cell arrangements are critical for the formation of an arborized network of tubes. Each tube requires a lumen, the diameter of which must be tightly regulated to enable optimal lung function. Lung branching and lumen morphogenesis require close epithelial cell–cell contacts that are maintained as a result of adherens junctions, tight junctions and by intact apical–basal (A/B) polarity. However, the molecular mechanisms that maintain epithelial cohesion and lumen diameter in the mammalian lung are unknown. Here we show that Scribble, a protein implicated in planar cell polarity (PCP) signalling, is necessary for normal lung morphogenesis. Lungs of the Scrib mouse mutant Circletail (Crc) are abnormally shaped with fewer airways, and these airways often lack a visible, ‘open’ lumen. Mechanistically we show that Scrib genetically interacts with the core PCP gene Vangl2 in the developing lung and that the distribution of PCP pathway proteins and Rho mediated cytoskeletal modification is perturbed in ScribCrc/Crc lungs. However A/B polarity, which is disrupted in Drosophila Scrib mutants, is largely unaffected. Notably, we find that Scrib mediates functions not attributed to other PCP proteins in the lung. Specifically, Scrib localises to both adherens and tight junctions of lung epithelia and knockdown of Scrib in lung explants and organotypic cultures leads to reduced cohesion of lung epithelial cells. Live imaging of Scrib knockdown lungs shows that Scrib does not affect bud bifurcation, as previously shown for the PCP protein Celsr1, but is required to maintain epithelial cohesion. To understand the mechanism leading to reduced cell–cell association, we show that Scrib associates with β-catenin in embryonic lung and the sub-cellular distribution of adherens and tight junction proteins is perturbed in mutant lung epithelia. Our data reveal that Scrib is required for normal lung epithelial organisation and lumen

  16. The Pathophysiology of Epithelial-Mesenchymal Transition Induced by Transforming Growth Factor-β in Normal and Malignant Mammary Epithelial Cells

    PubMed Central

    Taylor, Molly A.; Parvani, Jenny G.; Schiemann, William P.

    2013-01-01

    Epithelial-mesenchymal transition (EMT) is an essential process that drives polarized, immotile mammary epithelial cells (MECs) to acquire apolar, highly migratory fibroblastoid-like features. EMT is an indispensable process that is associated with normal tissue development and organogenesis, as well as with tissue remodeling and wound healing. In stark contrast, inappropriate reactivation of EMT readily contributes to the development of a variety of human pathologies, particularly those associated with tissue fibrosis and cancer cell invasion and metastasis, including that by breast cancer cells. Although metastasis is unequivocally the most lethal aspect of breast cancer and the most prominent feature associated with disease recurrence, the molecular mechanisms whereby EMT mediates the initiation and resolution of breast cancer metastasis remains poorly understood. Transforming growth factor-β (TGF-β) is a multifunctional cytokine that is intimately involved in regulating numerous physiological processes, including cellular differentiation, homeostasis, and EMT. In addition, TGF-β also functions as a powerful tumor suppressor in MECs, whose neoplastic development ultimately converts TGF-β into an oncogenic cytokine in aggressive late-stage mammary tumors. Recent findings have implicated the process of EMT in mediating the functional conversion of TGF-β during breast cancer progression, suggesting that the chemotherapeutic targeting of EMT induced by TGF-β may offer new inroads in ameliorating metastatic disease in breast cancer patients. Here we review the molecular, cellular, and microenvironmental factors that contribute to the pathophysiological activities of TGF-β during its regulation of EMT in normal and malignant MECs. PMID:20467795

  17. Expression of a TGF-{beta} regulated cyclin-dependent kinase inhibitor in normal and immortalized airway epithelial cells

    SciTech Connect

    Tierney, L.A.; Bloomfield, C.; Johnson, N.F.

    1995-12-01

    Tumors arising from epithelial cells, including lung cancers are frequently resistant to factors that regulate growth and differentiation in normal in normal cells. Once such factor is transforming growth factor-{Beta} (TGF-{Beta}). Escape from the growth-inhibitory effects of TGF-{Beta} is thought to be a key step in the transformation of airway epithelial cells. most lung cancer cell lines require serum for growth. In contrast, normal human bronchial epithelial (NHBE) cells are exquisitely sensitive to growth-inhibitory and differentiating effects of TGF-{Beta}. The recent identification of a novel cyclin-dependent kinase inhibitor, p15{sup INK4B}, which is regulated by TGF-{Beta}, suggests a mechanism by which TGF-{Beta} mediates growth arrest in NHBE cells. The purpose of this study was two-fold: (1) to determine if p15{sup INK4B} is induced by TGF-{Beta} in NHBE cells or immortalized bronchial epithelial (R.1) cells and if that induction corresponds to a G1/S cell-cycle arrest; (2) to determine the temporal relationship between p15{sup INK4B} induction, cell-cycle arrest, and the phosphorylation state of the pRB because it is thought that p15{sup INK4B} acts indirectly by preventing phosphorylation of the RB gene product. In this study, expression of p15{sup INK4B} was examined in NHBE cells and R.1 cells at different time intervals following TGF-{Beta} treatment. The expression of this kinase inhibitor and its relationship to the cell and the pRb phosphorylation state were examined in cells that were both sensitive (NHBE) and resistant (R.1) to the effects of TGF-{Beta}. These results suggest that the cyclin-dependent kinase inhibitor, p15{sup INK4B}, is involved in airway epithelial cell differentiation and that loss or reduction of expression plays a role in the resistance of transformed or neoplastic cells to the growth-inhibitory effects of TGF-{Beta}.

  18. MGSA/GRO transcription is differentially regulated in normal retinal pigment epithelial and melanoma cells.

    PubMed Central

    Shattuck, R L; Wood, L D; Jaffe, G J; Richmond, A

    1994-01-01

    We have characterized constitutive and cytokine-regulated MGSA/GRO alpha, -beta, and -gamma gene expression in normal retinal pigment epithelial (RPE) cells and a malignant melanoma cell line (Hs294T) to discern the mechanism for MGSA/GRO constitutive expression in melanoma. In RPE cells, constitutive MGSA/GRO alpha, -beta, and -gamma mRNAs are not detected by Northern (RNA) blot analysis although nuclear runoff experiments show that all three genes are transcribed. In Hs294T cells, constitutive MGSA/GRO alpha expression is detectable by Northern blot analysis, and the level of basal MGSA/GRO alpha transcription is 8- to 30-fold higher than in RPE cells. In contrast, in Hs294T cells, basal MGSA/GRO beta and -gamma transcription is only twofold higher than in RPE cells and no beta or gamma mRNA is detected by Northern blot. These data suggest that the constitutive MGSA/GRO alpha mRNA in Hs294T cells is due to increased basal MGSA/GRO alpha gene transcription. The cytokines interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha) significantly increase the mRNA levels for all three MGSA/GRO isoforms in Hs294T and RPE cells, and both transcriptional and posttranscriptional mechanisms are operational. Nuclear runoff assays indicate that in RPE cells, a 1-h IL-1 treatment induces a 10- to 20-fold increase in transcription of MGSA/GRO alpha, -beta and -gamma but only a 2-fold increase in Hs294T cells. Similarly, chloramphenicol acetyltransferase (CAT) reporter gene analysis using the MGSA/GRO alpha, -beta, and -gamma promoter regions demonstrates that IL-1 treatment induces an 8- to 14-fold increase in CAT activity in RPE cells but only a 2-fold increase in Hs294T cells. The effect of deletion or mutation of the MGSA/GRO alpha NF-kappa B element, combined with data from gel mobility shift analyses, indicates that the NF-kappa B p50/p65 heterodimer in RPE cells plays an important role in IL-1- and TNF alpha-enhanced gene transcription. In Hs294T cells, gel shift

  19. Expression of four CEA family antigens (CEA, NCA, BGP and CGM2) in normal and cancerous gastric epithelial cells: up-regulation of BGP and CGM2 in carcinomas.

    PubMed

    Kinugasa, T; Kuroki, M; Takeo, H; Matsuo, Y; Ohshima, K; Yamashita, Y; Shirakusa, T; Matsuoka, Y

    1998-03-30

    Four human carcinoembryonic antigen (CEA) family members, CEA (CD66e), non-specific cross-reacting antigen (NCA, CD66c), biliary glycoprotein (BGP, CD66a) and CEA gene-family member 2 (CGM2), are expressed in normal mucosal epithelia of the colon. Expression of BGP and CGM2 has recently been demonstrated to be down-regulated in colorectal adenocarcinomas. We have now investigated the expression of the 4 CEA family antigens in gastric adenocarcinoma and carcinoma cell lines in comparison with adjacent normal gastric mucosa. The transcripts of the CEA, NCA and BGP genes evaluated by reverse transcription-polymerase chain reaction were detectable at various levels in all the gastric adenocarcinoma cell lines tested, while CGM2 mRNA was detectable in the cell lines of poorly differentiated but not of well-differentiated carcinomas. The levels of CEA mRNA in normal gastric mucosa were variable but mostly increased in adenocarcinomas. The sparse expression of NCA observed in the normal tissues was markedly up-regulated in the carcinomas. In contrast to previous findings on normal and cancerous colonic tissues, the transcripts of CGM2 were totally undetectable and those of BGP were recognized only marginally, if at all, in normal gastric mucosa, while both messages were detected at significant levels in most of the gastric adenocarcinomas. This was confirmed by in situ hybridization. Our findings indicate that expression of the CEA family antigens, particularly that of BGP and CGM2, is differently regulated in epithelial cells of the colon and the stomach.

  20. Detachment-mediated resistance to TRAIL-induced apoptosis is associated with stimulation of the PI3K/Akt pathway in fetal and adenocarcinoma epithelial colon cells.

    PubMed

    Kočí, Lenka; Hýžd'alová, Martina; Vaculová, Alena; Hofmanová, Jiřina; Kozubík, Alois

    2011-07-01

    The resistance of transformed epithelial cells to a detachment-induced apoptosis (anoikis) can significantly affect their susceptibility to anticancer therapy. We showed that detachment of both fetal (FHC) and adenocarcinoma (HT-29) human colon epithelial cells resulted in the activation of the pro-survival Akt pathway, and significant changes in integrin-linked kinase (ILK) and focal adhesive kinase (FAK) phosphorylation. We demonstrated a detachment-induced and PI3K/Akt-mediated resistance to apoptotic effects of TRAIL, which was not associated with any changes in the cell surface TRAIL death receptor levels. Instead, a modulation of downstream intracellular signaling events was suggested to be involved. Our results may have important implications for optimization of new strategies in treatment of cancers at different stages of development. Copyright © 2011 Elsevier Ltd. All rights reserved.

  1. Exploring processes of organization of normal and neoplastic epithelial tissues in gradient culture.

    PubMed

    Leighton, J

    1994-09-01

    The biology of animal cells in culture is often studied in individual cells or in sheets of cells. The relevance of such studies to the intact animal is unclear, since the spatial conditions encountered by cells in animals is one of dense three-dimensional masses of cells, with limits to migration, and with gradients both of diffusion of metabolites and of morphologic maturation. These spatial requisites have gradually been met in culture. A brief account describes sponge matrix culture for three-dimensional growth and unilaminar, bilaminar, and radial histophysiologic gradient cultures. Some of the common neoplastic abnormalities of surface epithelial tissues are considered. Proposals for investigating the histokinetic mechanisms regulating some epithelial tissue processes are suggested. In the most recent development of gradient culture methods, a thin permeable transparent collagen membrane is intrinsically strengthened by producing a waffle membrane pattern for histophysiologic gradient culture.

  2. Oral epithelial stem cells – implications in normal development and cancer metastasis

    PubMed Central

    Papagerakis, Silvana; Pannone, Giuseppe; Zheng, Li; About, Imad; Taqi, Nawar; Nguyen, Nghia P.T.; Matossian, Margarite; McAlpin, Blake; Santoro, Angela; McHugh, Jonathan; Prince, Mark E.; Papagerakis, Petros

    2014-01-01

    Oral mucosa is continuously exposed to environmental forces and has to be constantly renewed. Accordingly, the oral mucosa epithelium contains a large reservoir of epithelial stem cells necessary for tissue homeostasis. Despite considerable scientific advances in stem cell behavior in a number of tissues, fewer studies have been devoted to the stem cells in the oral epithelium. Most of oral mucosa stem cells studies are focused on identifying cancer stem cells (CSC) in oral squamous cell carcinomas (OSCCs) among other head and neck cancers. OSCCs are the most prevalent epithelial tumors of the head and neck region, marked by their aggressiveness and invasiveness. Due to their highly tumorigenic properties, it has been suggested that CSC may be the critical population of cancer cells in the development of OSCC metastasis. This review presents a brief overview of epithelium stem cells with implications in oral health, and the clinical implications of the CSC concept in OSCC metastatic dissemination. PMID:24803391

  3. An Assessment of Database-Validated microRNA Target Genes in Normal Colonic Mucosa: Implications for Pathway Analysis.

    PubMed

    Slattery, Martha L; Herrick, Jennifer S; Stevens, John R; Wolff, Roger K; Mullany, Lila E

    2017-01-01

    Determination of functional pathways regulated by microRNAs (miRNAs), while an essential step in developing therapeutics, is challenging. Some miRNAs have been studied extensively; others have limited information. In this study, we focus on 254 miRNAs previously identified as being associated with colorectal cancer and their database-identified validated target genes. We use RNA-Seq data to evaluate messenger RNA (mRNA) expression for 157 subjects who also had miRNA expression data. In the replication phase of the study, we replicated associations between 254 miRNAs associated with colorectal cancer and mRNA expression of database-identified target genes in normal colonic mucosa. In the discovery phase of the study, we evaluated expression of 18 miR-NAs (those with 20 or fewer database-identified target genes along with miR-21-5p, miR-215-5p, and miR-124-3p which have more than 500 database-identified target genes) with expression of 17 434 mRNAs to identify new targets in colon tissue. Seed region matches between miRNA and newly identified targeted mRNA were used to help determine direct miRNA-mRNA associations. From the replication of the 121 miRNAs that had at least 1 database-identified target gene using mRNA expression methods, 97.9% were expressed in normal colonic mucosa. Of the 8622 target miRNA-mRNA associations identified in the database, 2658 (30.2%) were associated with gene expression in normal colonic mucosa after adjusting for multiple comparisons. Of the 133 miRNAs with database-identified target genes by non-mRNA expression methods, 97.2% were expressed in normal colonic mucosa. After adjustment for multiple comparisons, 2416 miRNA-mRNA associations remained significant (19.8%). Results from the discovery phase based on detailed examination of 18 miRNAs identified more than 80 000 miRNA-mRNA associations that had not previously linked to the miRNA. Of these miRNA-mRNA associations, 15.6% and 14.8% had seed matches for CRCh38 and CRCh37

  4. Mitochondria organelle transplantation: introduction of normal epithelial mitochondria into human cancer cells inhibits proliferation and increases drug sensitivity.

    PubMed

    Elliott, R L; Jiang, X P; Head, J F

    2012-11-01

    Mitochondrial dysfunction of cancer cells includes increased aerobic glycolysis, elevated levels of ROS, decreased apoptosis, and resistance to chemotherapeutic agents. We hypothesized that the introduction of normal mitochondria into cancer cells might restore mitochondrial function and inhibit cancer cell growth, and reverse chemoresistance. First, in the present study, we tested if mitochondria of immortalized, untransformed mammary epithelial MCF-12A cells could enter into human cancer cell lines. Second, if introducing normal mitochondria into cancer cells would inhibit proliferation. And third, would the addition of normal mitochondria increase the sensitivity of human breast cancer MCF-7 cells to chemotherapy. We found that JC-1-stained mitochondria of immortalized, untransformed mammary epithelial MCF-12A cells can enter into the cancer cell lines MCF-7, MDA-MB-231, and NCI/ADR-Res, but cannot enter immortalized, untransformed MCF-12A cells. The normal mitochondria from immortalized, untransformed MCF-12A cells suppressed the proliferation of MCF-7 and NCI/ADR-Res cells in a dose-dependent pattern, but did not affect the proliferation of immortalized, untransformed MCF-12A cells. The normal mitochondria from immortalized, untransformed MCF-12A cells increased the sensitivity of human breast cancer MCF-7 cells to doxorubicin, Abraxane, and carboplatin. In conclusion, the introduction of normal mammary mitochondria into human breast cancer cells inhibits cancer cell proliferation and increases the sensitivity of the MCF-7 human breast cancer cell line to doxorubicin, Abraxane, and carboplatin. These results support the role of mitochondrial dysfunction in cancer and suggest the possible use of targeted mitochondria for cancer therapeutics.

  5. Secondhand smoke inhibits both Cl- and K+ conductances in normal human bronchial epithelial cells

    PubMed Central

    2009-01-01

    Secondhand smoke (SHS) exposure is an independent risk factor for asthma, rhinosinusitis, and more severe respiratory tract infections in children and adults. Impaired mucociliary clearance with subsequent mucus retention contributes to the pathophysiology of each of these diseases, suggesting that altered epithelial salt and water transport may play an etiological role. To test the hypothesis that SHS would alter epithelial ion transport, we designed a system for in vitro exposure of mature, well-differentiated human bronchial epithelial cells to SHS. We show that SHS exposure inhibits cAMP-stimulated, bumetanide-sensitive anion secretion by 25 to 40% in a time-dependent fashion in these cells. Increasing the amount of carbon monoxide to 100 ppm from 5 ppm did not increase the amount of inhibition, and filtering SHS reduced inhibition significantly. It was determined that SHS inhibited cAMP-dependent apical membrane chloride conductance by 25% and Ba2+-sensitive basolateral membrane potassium conductance by 50%. These data confirm previous findings that cigarette smoke inhibits chloride secretion in a novel model of smoke exposure designed to mimic SHS exposure. They also extend previous findings to demonstrate an effect on basolateral K+ conductance. Therefore, pharmacological agents that increase either apical membrane chloride conductance or basolateral membrane potassium conductance might be of therapeutic benefit in patients with diseases related to SHS exposure. PMID:19943936

  6. Secondhand smoke inhibits both Cl- and K+ conductances in normal human bronchial epithelial cells.

    PubMed

    Savitski, Amy N; Mesaros, Clementina; Blair, Ian A; Cohen, Noam A; Kreindler, James L

    2009-11-27

    Secondhand smoke (SHS) exposure is an independent risk factor for asthma, rhinosinusitis, and more severe respiratory tract infections in children and adults. Impaired mucociliary clearance with subsequent mucus retention contributes to the pathophysiology of each of these diseases, suggesting that altered epithelial salt and water transport may play an etiological role. To test the hypothesis that SHS would alter epithelial ion transport, we designed a system for in vitro exposure of mature, well-differentiated human bronchial epithelial cells to SHS. We show that SHS exposure inhibits cAMP-stimulated, bumetanide-sensitive anion secretion by 25 to 40% in a time-dependent fashion in these cells. Increasing the amount of carbon monoxide to 100 ppm from 5 ppm did not increase the amount of inhibition, and filtering SHS reduced inhibition significantly. It was determined that SHS inhibited cAMP-dependent apical membrane chloride conductance by 25% and Ba2+-sensitive basolateral membrane potassium conductance by 50%. These data confirm previous findings that cigarette smoke inhibits chloride secretion in a novel model of smoke exposure designed to mimic SHS exposure. They also extend previous findings to demonstrate an effect on basolateral K+ conductance. Therefore, pharmacological agents that increase either apical membrane chloride conductance or basolateral membrane potassium conductance might be of therapeutic benefit in patients with diseases related to SHS exposure.

  7. Regulating effect of TongXie-YaoFang on colonic epithelial secretion via Cl- and HCO3- channel

    PubMed Central

    Yang, Cheng; Xiong, Ying; Zhang, Sheng-Sheng; An, Fang-Mei; Sun, Jing; Zhang, Qing-Lin; Zhan, Qiang

    2016-01-01

    AIM To investigate the pharmacological effect of TongXie-YaoFang (TXYF) formula, a Chinese herbal formula, on Diarrhea-predominant irritable bowel syndrome (D-IBS) rats. METHODS In a neonatal maternal separation plus restraint stress (NMS + RS) model of D-IBS, male Sprague Dawley rats were randomly divided into two groups (NMS + RS group and TXYF-formula group) with no handlings were used as controls (NH group). Starting from postnatal day 60, rats in TXYF-formula group were administered TXYF-formula (4.92 g/100 g bodyweight) orally twice a day for 14 consecutive days while NH group and NMS + RS group were given distilled water. Using short-circuit current technology, we observed 5-HT-induced changes of current across ion channels, such as cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel, epithelial Na+ channel (ENaC), Ca2+-dependent Cl- channel (CACC), Na+-K+-2Cl- co-transporter (NKCC), and Na+-HCO3- co-transporter (NBC), in the colonic epithelium of three groups after exposure to drugs and specific blockers with a Power Lab System (AD Instruments International). RESULTS Under basal conditions, the changes of short-circuit current (∆Isc, µA/cm2) induced by 5-HT were similar in NH group and TXYF-formula group, and both higher than NMS + RS group (70.86 µA/cm2 ± 12.32 µA/cm2, 67.67 µA/cm2 ± 11.68 µA/cm2 vs 38.8 µA/cm2 ± 7.25 µA/cm2, P < 0.01, respectively). When CACC was blocked by 4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid, 5-HT-induced ∆Isc was smaller in NMS + RS group than in NH group and TXYF-formula group, respectively (48.41 µA/cm2 ± 13.15 µA/cm2 vs 74.62 µA/cm2 ± 10.73 µA/cm2, 69.22 µA/cm2 ± 11.7 µA/cm2, P < 0.05, respectively). The similar result could be obtained when ENaC was blocked by Amiloride (44.69 µA/cm2 ± 12.58 µA/cm2 vs 62.05 µA/cm2 ± 11.26 µA/cm2, 62.11 µA/cm2 ± 12.01 µA/cm2, P < 0.05, respectively). However, when CFTR Cl- channel was blocked by 1,1-dimethyl piperidinium chloride

  8. A Helicobacter pylori Homolog of Eukaryotic Flotillin Is Involved in Cholesterol Accumulation, Epithelial Cell Responses and Host Colonization.

    PubMed

    Hutton, Melanie L; D'Costa, Kimberley; Rossiter, Amanda E; Wang, Lin; Turner, Lorinda; Steer, David L; Masters, Seth L; Croker, Ben A; Kaparakis-Liaskos, Maria; Ferrero, Richard L

    2017-01-01

    The human pathogen Helicobacter pylori acquires cholesterol from membrane raft domains in eukaryotic cells, commonly known as "lipid rafts." Incorporation of this cholesterol into the H. pylori cell membrane allows the bacterium to avoid clearance by the host immune system and to resist the effects of antibiotics and antimicrobial peptides. The presence of cholesterol in H. pylori bacteria suggested that this pathogen may have cholesterol-enriched domains within its membrane. Consistent with this suggestion, we identified a hypothetical H. pylori protein (HP0248) with homology to the flotillin proteins normally found in the cholesterol-enriched domains of eukaryotic cells. As shown for eukaryotic flotillin proteins, HP0248 was detected in detergent-resistant membrane fractions of H. pylori. Importantly, H. pylori HP0248 mutants contained lower levels of cholesterol than wild-type bacteria (P < 0.01). HP0248 mutant bacteria also exhibited defects in type IV secretion functions, as indicated by reduced IL-8 responses and CagA translocation in epithelial cells (P < 0.05), and were less able to establish a chronic infection in mice than wild-type bacteria (P < 0.05). Thus, we have identified an H. pylori flotillin protein and shown its importance for bacterial virulence. Taken together, the data demonstrate important roles for H. pylori flotillin in host-pathogen interactions. We propose that H. pylori flotillin may be required for the organization of virulence proteins into membrane raft-like structures in this pathogen.

  9. EZH2 is regulated by ERK/AKT and targets integrin alpha2 gene to control Epithelial-Mesenchymal Transition and anoikis in colon cancer cells.

    PubMed

    Ferraro, Angelo; Mourtzoukou, Despoina; Kosmidou, Vivian; Avlonitis, Spiros; Kontogeorgos, George; Zografos, George; Pintzas, Alexander

    2013-02-01

    Epithelial-Mesenchymal Transition is a good example of cell plasticity. In tumorigenesis, this process has been associated with metastasis. Overexpression of EZH2 has been detected in most malignant human tumors, including colorectal carcinomas. Herein, we provide evidence supporting the idea that oncogenic Epithelial-Mesenchymal Transition in colon cancer cell models is partially controlled by epigenetic factors such as the transcription regulator EZH2. Evaluation of EZH2 mRNA and protein levels revealed overexpression in cell lines with metastatic traits. Analysis of EZH2 mRNA expression was expanded in clinical samples of colon cancer, and high level of EZH2 correlates with appearance of metastasis. Furthermore, inhibition of ERK and AKT pathways in metastatic colon cancer cell lines attenuates EZH2 overexpression. EZH2 promoter analysis illustrates presence of putative AP-1 binding sites and occupancy of transcription factors such as FRA-1 and C-JUN is demonstrated here on EZH2 promoter. Abrogation of EZH2 expression impairs the ability of colon cancer cells to move associated with anoikis in three-dimensional environment. Integrin alpha2 was identified to be a novel EZH2 target by chromatin immunoprecipitation and short hairpin RNA analysis. This study proposes that activation of ERK/AKT pathways and FRA1/C-JUN induce EZH2 overexpression, which results in Integrin alpha2 silencing. Our results show how deregulation of epigenetic factors and mechanisms can affect cancer cell aggressiveness and propose EZH2 as a potential metastasis marker and/or therapeutic target for colorectal cancer treatment. Copyright © 2012 Elsevier Ltd. All rights reserved.

  10. Characterization of Epithelial Progenitors in Normal Human Palatine Tonsils and Their HPV16 E6/E7-Induced Perturbation

    PubMed Central

    Kang, Sung Yoon Catherine; Kannan, Nagarajan; Zhang, Lewei; Martinez, Victor; Rosin, Miriam P.; Eaves, Connie J.

    2015-01-01

    Summary Human palatine tonsils are oropharyngeal lymphoid tissues containing multiple invaginations (crypts) in which the continuity of the outer surface epithelium is disrupted and the isolated epithelial cells intermingle with other cell types. We now show that primitive epithelial cells detectable in vitro in 2D colony assays and in a 3D culture system are CD44+NGFR+ and present in both surface and crypt regions. Transcriptome analysis indicated a high similarity between CD44+NGFR+ cells in both regions, although those isolated from the crypt contained a higher proportion of the most primitive (holo)clonogenic cells. Lentiviral transduction of CD44+NGFR+ cells from both regions with human papillomavirus 16-encoded E6/E7 prolonged their growth in 2D cultures and caused aberrant differentiation in 3D cultures. Our findings therefore reveal a shared, site-independent, hierarchical organization, differentiation potential, and transcriptional profile of normal human tonsillar epithelial progenitor cells. They also introduce a new model for investigating the mechanisms of their transformation. PMID:26527383

  11. Exposure to meat-derived carcinogens and bulky DNA adduct levels in normal-appearing colon mucosa.

    PubMed

    Ho, Vikki; Brunetti, Vanessa; Peacock, Sarah; Massey, Thomas E; Godschalk, Roger W L; van Schooten, Frederik J; Ashbury, Janet E; Vanner, Stephen J; King, Will D

    2017-09-01

    Meat consumption is a risk factor for colorectal cancer. This research investigated the relationship between meat-derived carcinogen exposure and bulky DNA adduct levels, a biomarker of DNA damage, in colon mucosa. Least squares regression was used to examine the relationship between meat-derived carcinogen exposure (PhIP and meat mutagenicity) and bulky DNA adduct levels in normal-appearing colon tissue measured using (32)P-postlabelling among 202 patients undergoing a screening colonoscopy. Gene-diet interactions between carcinogen exposure and genetic factors relevant to biotransformation and DNA repair were also examined. Genotyping was conducting using the MassARRAY(®) iPLEX(®) Gold SNP Genotyping assay. PhIP and higher meat mutagenicity exposures were not associated with levels of bulky DNA adducts in colon mucosa. The XPC polymorphism (rs2228001) was found to associate with bulky DNA adduct levels, whereby genotypes conferring lower DNA repair activity were associated with higher DNA adduct levels than the normal activity genotype. Among individuals with genotypes associated with lower DNA repair (XPD, rs13181 and rs1799179) or detoxification activity (GSTP1, rs1695), higher PhIP or meat mutagenicity exposures were associated with higher DNA adduct levels. Significant interactions between the XPC polymorphism (rs2228000) and both dietary PhIP and meat mutagenicity on DNA adduct levels was observed, but associations were inconsistent with the a priori hypothesized direction of effect. Exposure to meat-derived carcinogens may be associated with increased DNA damage occurring directly in the colon among genetically susceptible individuals. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. [Colon adenoma detection using Kubelka-Munk spectral function of DNA and protein bands].

    PubMed

    Wei, Hua-Jiang; Guo, Zhou-Yi; Xie, Shu-Sen; He, Bo-Hua; Li, Li-Bo; Chen, Xue-Mei; Wu, Guo-Yong; Lu, Jian-Jun

    2009-06-01

    Differential diagnosis of human colon adenoma was studied using the Kubelka-Munk spectral function of the DNA and protein absorption bands at 260 and 280 nm in vitro. Diffuse reflectance spectra of tissue were measured using a spectrophotometer with an integrating sphere attachment. The results of measurement showed that for the spectral range from 590 to 1 064 nm pathological changes of colon epithelial tissues were induced so that there were significant differences in the averaged values of the Kubelka-Munk function f(r infinity) and logarithmic Kubelka-Munk function log [f(r infinity)] of the DNA absorption bands at 260 nm between normal and adenomatous colon epithelial tissues, and the differences were 218% (p < 0.05) and 68.5% (p < 0.05) respectively. Pathological changes of colon epithelial tissues were induced so that there were significant differences in the averaged values of the Kubelka-Munk function f(r infinity) and logarithmic Kubelka-Munk function log [f(r infinity)] of the protein absorption bands at 280 nm between normal and adenomatous colon epithelial tissues, and the differences were 208% (p < 0.05) and 59.0% (p < 0.05) respectively. Pathological changes of colon epithelial tissues were induced so that there were significant differences in the averaged values of the Kubelka-Munk function f(r infinity) and logarithmic Kubelka-Munk function log [f(r infinity)] of the beta-carotene absorption bands at 480 nm between normal and adenomatous colon epithelial tissues, and the differences were 41.7% (p < 0.05) and 32.9% (p < 0.05) respectively. Obviously, pathological changes of colon epithelial tissues were induced so that there were significant changes in the contents of the DNA, protein and beta-carotene of colon epithelial tissues. The conclusion can be applied to rapid, low-cost and noninvasive optical biopsy of colon adenoma, and provides a useful reference.

  13. Aging phenotypes in cultured normal human mammary epithelial cells are correlated with decreased telomerase activity independent of telomere length

    PubMed Central

    2013-01-01

    Background Shortening of telomeres, which are essential for maintenance of genomic integrity, is a mechanism commonly associated with the aging process. Here we ascertained whether changes in telomere lengths or telomerase activity correlated with age in normal human mammary epithelial cells (HMEC), or with phenotypes of aging in breast. Accordingly, flow cytometry fluorescence in situ hybridization (flowFISH) was used to determine relative telomere lengths (RTL), and telomerase activity was measured by the telomeric repeat amplification protocol (TRAP), in a collection of 41 primary HMEC strains established from women aged 16 to 91 years. Results RTL measurements of HMEC strains that were heterogeneous with respect to lineage composition revealed no significant associations between telomere length with age, maximum observed population doublings, or with lineage composition of the strains. However, within strains, luminal epithelial and cKit-expressing epithelial progenitor cells that were flow cytometry-enriched from individual HMEC strains exhibited significantly shorter telomeres relative to isogenic myoepithelial cells (P < 0.01). In unsorted strains, detectable telomerase activity did not correlate with RTL. Telomerase activity declined with age; the average age of strains that exhibited TRAP activity was 29.7 ± 3.9y, whereas the average age of strains with no detectable TRAP activity was 49.0 ± 4.9y (P < 0.01). Non-detectable TRAP activity also was correlated with phenotypes of aging previously described in HMEC strains; increased proportions of CD227-expressing luminal epithelial cells (P < 0.05) and cKit-expressing progenitor cells (P < 0.05). Conclusions Telomere shortening did not correlate with the chronological ages of HMEC strains, whereas decreased telomerase activity correlated with age and with lineage distribution phenotypes characteristic of aging. PMID:23718190

  14. Two-component sensor required for normal symbiotic colonization of euprymna scolopes by Vibrio fischeri.

    PubMed

    Visick, K L; Skoufos, L M

    2001-02-01

    The light organ of the squid Euprymna scolopes is specifically colonized to a high density by the marine bacterium Vibrio fischeri. To date, only a few factors contributing to the specificity of this symbiosis have been identified. Using a genetic screen for random transposon mutants defective in initiating the symbiotic association or in colonizing the light organ to high density, we identified a mutant of V. fischeri that exhibited an apparent defect in symbiosis initiation. This mutant was not defective in motility, luminescence, or growth in minimal medium, suggesting that it lacks an essential, previously unidentified symbiotic function. By sequence analysis, we showed that the locus inactivated in this mutant encodes a predicted 927-amino-acid protein with a high degree of similarity to the sensor component of hybrid two-component regulatory systems. We have therefore designated this locus rscS, for regulator of symbiotic colonization-sensor. Sequence analysis revealed two hydrophobic regions which may result in the formation of a periplasmic loop involved in signal recognition; PhoA fusion data supported this proposed membrane topology. We have investigated the start site of rscS transcription by primer extension and identified a putative promoter region. We hypothesize that RscS recognizes a signal associated with the light organ environment and responds by stimulating a putative response regulator that controls protein function or gene expression to coordinate early colonization events. Further studies on RscS, its cognate response regulator, and the signaling conditions will provide important insight into the interaction between V. fischeri and E. scolopes.

  15. The effects of cyclo-oxygenase inhibitors on bile-injured and normal equine colon.

    PubMed

    Campbell, N B; Jones, S L; Blikslager, A T

    2002-07-01

    A potential adverse effect of cyclo-oxygenase (COX) inhibitors (nonsteroidal anti-inflammatory drugs [NSAIDs]) in horses is colitis. In addition, we have previously shown an important role for COX-produced prostanoids in recovery of ischaemic-injured equine jejunum. It was hypothesised that the nonselective COX inhibitor flunixin would retard repair of bile-injured colon by preventing production of reparative prostaglandins, whereas the selective COX-2 inhibitor, etodolac would not inhibit repair as a result of continued COX-1 activity. Segments of the pelvic flexure were exposed to 1.5 mmol/l deoxycholate for 30 min, after which they were recovered for 4 h in Ussing chambers. Contrary to the proposed hypothesis, recovery of bile-injured colonic mucosa was not affected by flunixin or etodolac, despite significantly depressed prostanoid production. However, treatment of control tissue with flunixin led to increases in mucosal permeability, whereas treatment with etodolac had no significant effect. Therefore, although recovery from bile-induced colonic injury maybe independent of COX-elaborated prostanoids, treatment of control tissues with nonselective COX inhibitors may lead to marked increases in permeability. Alternatively, selective inhibition of COX-2 may reduce the incidence of adverse effects in horses requiring NSAID therapy.

  16. Epithelial and Mesenchymal Cells in the Bovine Colonic Mucosa Differ in Their Responsiveness to Escherichia coli Shiga Toxin 1

    USDA-ARS?s Scientific Manuscript database

    Cells in the depth of the crypts in the bovine colon express CD77 molecules that potentially act as receptors for Shiga toxins (Stx). The implication of this finding for the intestinal colonization 25 of cattle with human pathogenic Stx-producing Escherichia coli (STEC) remains undefined. We used f...

  17. Spectral Slope from the Endoscopically-Normal Mucosa Predicts Concurrent Colonic Neoplasia: A Pilot Ex-Vivo Clinical Study

    PubMed Central

    Roy, Hemant K.; Turzhitsky, Vladimir; Kim, Young L.; Goldberg, Michael J.; Muldoon, Joseph P.; Liu, Yang; Brand, Randall E.; Hall, Curtis; Hasabou, Nahla; Jameel, Mohammed; Backman, Vadim

    2008-01-01

    Purpose We previously reported that analysis of histologically normal intestinal epithelium for spectral slope, a marker for aberrations in nanoscale tissue architecture, had outstanding accuracy in identifying field carcinogenesis in preclinical colorectal cancer models. In this study, we assessed the translatability of spectral slope analysis to human colorectal cancer screening. Methods Subjects (n=127) undergoing colonoscopy had spectral slope determined from two endoscopically normal midtransverse colonic biopsies using four dimensional elastic light-scattering fingerprinting and correlated with clinical findings. Results Four dimensional elastic light-scattering fingerprinting analysis showed the submicron particles size progressively shifted towards larger sizes in subjects with harboring neoplasia. There was a corresponding decrease in spectral slope values from the endoscopically normal mucosa in subjects harboring adenomas (n=41) and advanced adenomas (n=10), compared to neoplasia-free subjects (p<0.00001). These factors did not appear to be confounded by either age or adenoma location. For detecting advanced adenomas, spectral slope had a negative and positive predictive value of 95 percent and 50 percent respectively. Conclusions We demonstrate, for the first time, that spectral slope in “normal” mucosa accurately risk-stratify patients for colonic neoplasia. This proof of concept study serves to underscore the promise of four-dimensional elastic light-scattering fingerprinting analysis for colorectal cancer screening. PMID:18536963

  18. HMGA1 silencing restores normal stem cell characteristics in colon cancer stem cells by increasing p53 levels

    PubMed Central

    Puca, Francesca; Colamaio, Marianna; Federico, Antonella; Gemei, Marica; Tosti, Nadia; Bastos, André Uchimura; Vecchio, Luigi Del; Pece, Salvatore; Battista, Sabrina; Fusco, Alfredo

    2014-01-01

    High-mobility group A1 (HMGA1) proteins are architectural chromatinic proteins, abundantly expressed during embryogenesis and in most cancer tissues, but expressed at low levels or absent in normal adult tissues. Several studies have demonstrated that HMGA1 proteins play a causal role in neoplastic cell transformation. The aim of this study was to investigate the role of these proteins in the control of cancer stem cells (CSCs), which have emerged as a preferred target in cancer therapy, because of their role in cancer recurrence. We observed that HMGA1 is overexpressed in colon tumour stem cell (CTSC) lines compared to normal and colon cancer tissues. We demonstrated that HMGA1 silencing in CTSCs increases stem cell quiescence and reduces self-renewal and sphere-forming efficiency (SFE). The latter, together with the upregulation and asymmetric distribution of NUMB, is indicative of the recovery of an asymmetric division pattern, typical of normal stem cells. We further found that HMGA1 transcriptionally regulates p53, which is known to control the balance between symmetric and asymmetric divisions in CSCs. Therefore, our data indicate a critical role for HMGA1 in regulating both self-renewal and the symmetric/asymmetric division ratio in CSCs, suggesting that blocking HMGA1 function may be an effective anti-cancer therapy. PMID:24833610

  19. Monochloramine induces reorganization of nuclear speckles and phosphorylation of SRp30 in human colonic epithelial cells: role of protein kinase C.

    PubMed

    Zhu, Ya-Qin; Lu, Yu; Tan, Xiao-Di

    2003-11-01

    Intestinal epithelial cells are constantly stimulated by reactive oxidant metabolites (ROMs) in inflamed mucosa. Monochloramine (NH2Cl), a cell-permeant ROM, is particularly relevant to the pathogenesis of inflammation in the gastrointestinal tract. Nuclear speckles, a unique nuclear subcompartment, accumulate a family of proteins, namely, serine- and arginine-rich (SR) proteins. They play important roles in regulation of pre-mRNA splicing. Currently, little is known about the link between inflammatory stimulation and the pre-mRNA splicing process, although gene expression is changed in inflamed tissues. The present study was designed to investigate whether stimulation of human colonic epithelial cells (HT-29 and Caco-2 cell lines) with NH2Cl affects nuclear speckles and their components. By indirect immunofluorescence, nuclear speckles have been shown to undergo rapid aggregation after NH2Cl stimulation. By utilizing Western blotting, SRp30 (a subset of SR proteins) in intestinal epithelial cells was found to be phosphorylated after NH2Cl treatment, whereas other SR proteins were not responsive to NH2Cl stimulation. The cytotoxic effect of NH2Cl was excluded by both negative lactate dehydrogenase assay and propidium iodide staining. Therefore, NH2Cl-induced morphological changes on nuclear speckles and phosphorylated SRp30 do not result from intestinal epithelial injury. Furthermore, the effect of NH2Cl on nuclear speckles and SRp30 was blocked by bisindolylmaleimide I, a selective PKC inhibitor. Together, the available data suggest that stimulation of intestinal epithelial cells with NH2Cl results in a consequent change on pre-mRNA splicing machinery via a distinctive signal pathway involving activation of PKC. This effect may contribute to oxidant-induced pathophysiological changes in the gastrointestinal tract.

  20. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptosis in normal prostate epithelial cells.

    PubMed

    Nesterov, Alexandre; Ivashchenko, Yuri; Kraft, Andrew S

    2002-02-07

    TRAIL is a pro-apoptotic cytokine believed to selectively kill cancer cells without harming normal ones. However, we found that in normal human prostate epithelial cells (PrEC) TRAIL is capable of inducing apoptosis as efficiently as in some tumor cell lines. At the same time, TRAIL did not cause apoptosis in several other human primary cell lines: aorta smooth muscle cells, foreskin fibroblasts, and umbilical vein endothelial cells. Compared to these primary cells, PrEC were found to contain significantly fewer TRAIL receptors DcR1 and DcR2 which are not capable of conducting the apoptotic signal. This result suggests that the unusual sensitivity of PrEC to TRAIL may result from their deficiency in anti-apoptotic decoy receptors. The protein synthesis inhibitor cycloheximide significantly enhanced TRAIL toxicity toward PrEC as measured by tetrazolium conversion but had little or no effect on other TRAIL-induced apoptotic responses. Although cycloheximide did not further accelerate the processing of caspases 3 and 8, it significantly enhanced cleavage of the caspase 3 substrate gelsolin, indicating that in PrEC a protein(s) with a short half-life may inhibit the activity of the executioner caspases toward specific substrates. As the majority of prostate cancers are derived from epithelial cells, our data suggest the possibility that TRAIL could be a useful treatment for the early stages of prostate cancer.

  1. Normal human mammary epithelial cells spontaneously escape senescence and acquire genomic changes

    NASA Technical Reports Server (NTRS)

    Romanov, S. R.; Kozakiewicz, B. K.; Holst, C. R.; Stampfer, M. R.; Haupt, L. M.; Tlsty, T. D.

    2001-01-01

    Senescence and genomic integrity are thought to be important barriers in the development of malignant lesions. Human fibroblasts undergo a limited number of cell divisions before entering an irreversible arrest, called senescence. Here we show that human mammary epithelial cells (HMECs) do not conform to this paradigm of senescence. In contrast to fibroblasts, HMECs exhibit an initial growth phase that is followed by a transient growth plateau (termed selection or M0; refs 3-5), from which proliferative cells emerge to undergo further population doublings (approximately 20-70), before entering a second growth plateau (previously termed senescence or M1; refs 4-6). We find that the first growth plateau exhibits characteristics of senescence but is not an insurmountable barrier to further growth. HMECs emerge from senescence, exhibit eroding telomeric sequences and ultimately enter telomere-based crisis to generate the types of chromosomal abnormalities seen in the earliest lesions of breast cancer. Growth past senescent barriers may be a pivotal event in the earliest steps of carcinogenesis, providing many genetic changes that predicate oncogenic evolution. The differences between epithelial cells and fibroblasts provide new insights into the mechanistic basis of neoplastic transformation.

  2. DNA methylation signatures of the AIRE promoter in thymic epithelial cells, thymomas and normal tissues.

    PubMed

    Kont, Vivian; Murumägi, Astrid; Tykocinski, Lars-Oliver; Kinkel, Sarah A; Webster, Kylie E; Kisand, Kai; Tserel, Liina; Pihlap, Maire; Ströbel, Philipp; Scott, Hamish S; Marx, Alexander; Kyewski, Bruno; Peterson, Pärt

    2011-12-01

    Mutations in the AIRE gene cause autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), which is associated with autoimmunity towards several peripheral organs. The AIRE protein is almost exclusively expressed in medullary thymic epithelial cells (mTEC) and CpG methylation in the promoter of the AIRE gene has been suggested to control its tissue-specific expression pattern. We found that in human AIRE-positive medullary and AIRE-negative cortical epithelium, the AIRE promoter is hypomethylated, whereas in thymocytes, the promoter had high level of CpG methylation. Likewise, in mouse mTECs the AIRE promoter was uniformly hypomethylated. In the same vein, the AIRE promoter was hypomethylated in AIRE-negative thymic epithelial tumors (thymomas) and in several peripheral tissues. Our data are compatible with the notion that promoter hypomethylation is necessary but not sufficient for tissue-specific regulation of the AIRE gene. In contrast, a positive correlation between AIRE expression and histone H3 lysine 4 trimethylation, an active chromatin mark, was found in the AIRE promoter in human and mouse TECs. Copyright © 2011 Elsevier Ltd. All rights reserved.

  3. Normal human mammary epithelial cells spontaneously escape senescence and acquire genomic changes

    NASA Technical Reports Server (NTRS)

    Romanov, S. R.; Kozakiewicz, B. K.; Holst, C. R.; Stampfer, M. R.; Haupt, L. M.; Tlsty, T. D.

    2001-01-01

    Senescence and genomic integrity are thought to be important barriers in the development of malignant lesions. Human fibroblasts undergo a limited number of cell divisions before entering an irreversible arrest, called senescence. Here we show that human mammary epithelial cells (HMECs) do not conform to this paradigm of senescence. In contrast to fibroblasts, HMECs exhibit an initial growth phase that is followed by a transient growth plateau (termed selection or M0; refs 3-5), from which proliferative cells emerge to undergo further population doublings (approximately 20-70), before entering a second growth plateau (previously termed senescence or M1; refs 4-6). We find that the first growth plateau exhibits characteristics of senescence but is not an insurmountable barrier to further growth. HMECs emerge from senescence, exhibit eroding telomeric sequences and ultimately enter telomere-based crisis to generate the types of chromosomal abnormalities seen in the earliest lesions of breast cancer. Growth past senescent barriers may be a pivotal event in the earliest steps of carcinogenesis, providing many genetic changes that predicate oncogenic evolution. The differences between epithelial cells and fibroblasts provide new insights into the mechanistic basis of neoplastic transformation.

  4. Detection and Antibiotic Treatment of Mycoplasma arginini Contamination in a Mouse Epithelial Cell Line Restore Normal Cell Physiology

    PubMed Central

    Resnick, Andrew

    2014-01-01

    Mycoplasma contamination of cultured cell lines is difficult to detect by routine observation. Infected cells can display normal morphology and the slow growth rate of mycoplasma can delay detection for extended periods of time, compromising experimental results. Positive identification of mycoplasma typically requires cells to be either fixed and stained for DNA or processed with PCR. We present a method to detect mycoplasma using live-cell optical microscopy typically used for routine observation of cell cultures. Images of untreated mycoplasma-infected epithelial cells alongside images of infected cells treated with Plasmocin, a commercially available antibiotic targeted to mycoplasma, are shown. We found that optical imaging is an effective screening tool for detection of mycoplasma contamination. Importantly, we found that cells regained normal function after the contamination was cleared. In conclusion, we present a technique to diagnose probable mycoplasma infections in live cultures without fixation, resulting in faster response times and decreased loss of cell material. PMID:24772428

  5. Detection and antibiotic treatment of Mycoplasma arginini contamination in a mouse epithelial cell line restore normal cell physiology.

    PubMed

    Boslett, Brianna; Nag, Subhra; Resnick, Andrew

    2014-01-01

    Mycoplasma contamination of cultured cell lines is difficult to detect by routine observation. Infected cells can display normal morphology and the slow growth rate of mycoplasma can delay detection for extended periods of time, compromising experimental results. Positive identification of mycoplasma typically requires cells to be either fixed and stained for DNA or processed with PCR. We present a method to detect mycoplasma using live-cell optical microscopy typically used for routine observation of cell cultures. Images of untreated mycoplasma-infected epithelial cells alongside images of infected cells treated with Plasmocin, a commercially available antibiotic targeted to mycoplasma, are shown. We found that optical imaging is an effective screening tool for detection of mycoplasma contamination. Importantly, we found that cells regained normal function after the contamination was cleared. In conclusion, we present a technique to diagnose probable mycoplasma infections in live cultures without fixation, resulting in faster response times and decreased loss of cell material.

  6. Markers of fibrosis and epithelial to mesenchymal transition demonstrate field cancerization in histologically normal tissue adjacent to breast tumors

    PubMed Central

    Trujillo, Kristina A.; Heaphy, Christopher M.; Mai, Minh; Vargas, Keith M.; Jones, Anna C.; Vo, Phung; Butler, Kimberly S.; Joste, Nancy E.; Bisoffi, Marco; Griffith, Jeffrey K

    2011-01-01

    Previous studies have shown that a field of genetically altered but histologically normal tissue extends 1 cm or more from the margins of human breast tumors. The extent, composition and biological significance of this field are only partially understood, but the molecular alterations in affected cells could provide mechanisms for limitless replicative capacity, genomic instability and a microenvironment that supports tumor initiation and progression. We demonstrate by microarray, qRT-PCR and immunohistochemistry a signature of differential gene expression that discriminates between patient-matched, tumor-adjacent histologically normal breast tissues located 1 cm and 5 cm from the margins of breast adenocarcinomas (TAHN-1 and TAHN-5, respectively). The signature includes genes involved in extracellular matrix remodeling, wound healing, fibrosis and epithelial to mesenchymal transition (EMT). Myofibroblasts, which are mediators of wound healing and fibrosis, and intra-lobular fibroblasts expressing MMP2, SPARC, TGF-β3, which are inducers of EMT, were both prevalent in TAHN-1 tissues, sparse in TAHN-5 tissues, and absent in normal tissues from reduction mammoplasty. Accordingly, EMT markers S100A4 and vimentin were elevated in both luminal and myoepithelial cells, and EMT markers α-smooth muscle actin and SNAIL were elevated in luminal epithelial cells of TAHN-1 tissues. These results identify cellular processes that are differentially activated between TAHN-1 and TAHN-5 breast tissues, implicate myofibroblasts as likely mediators of these processes, provide evidence that EMT is occurring in histologically normal tissues within the affected field and identify candidate biomarkers to investigate whether or how field cancerization contributes to the development of primary or recurrent breast tumors. PMID:21105047

  7. Anti-inflammatory Effects of Herbal Preparations STW5 and STW5-II in Cytokine-Challenged Normal Human Colon Cells

    PubMed Central

    Schneider, Mathias; Efferth, Thomas; Abdel-Aziz, Heba

    2016-01-01

    Inflammatory bowel diseases (IBD) are chronic relapsing intestinal disorders characterized by up-regulation of pro-inflammatory cytokines followed by invasion of immune cells to the intestinal lamina propria. Standard therapies consist of anti-inflammatory or immunosuppressive drugs. Since clinical efficiency is not satisfactory and the established drugs have massive side effects, new strategies to treat IBD are required. Herein, we investigate the protective effect of the fixed combination herbal preparations STW5 and STW5-II and the contribution of the corresponding single components in an in vitro inflammation model. The normal human colon epithelial cell line, NCM460, was treated with STW5, STW5-II or their single components for 4 h followed by experimental conditions comparable to induction of colitis. A pro-inflammatory cytokine cocktail consisting of TNF-α, IL-β, and IFN-γ was used to simulate inflammatory stimuli normally caused by immune cells. The effects on NCM460 cells were investigated by enzyme-linked immunoassay and Proteome Profiler®. Levels of IP-10, MCP-1, I-TAC, Groα, and IL-8 were elevated in chemokine-treated cells compared to untreated cells, but significantly reduced upon pretreatment with STW5 or STW5-II. However, the single compounds revealed only little effects on protein expression. Furthermore, we investigated the effect of both combination preparations on pro-inflammatory transcription factors of the STAT family using Western blot. In addition, we tested the effects on upstream MAPK p38. Both, STW5 and STW5-II did not show any effect on MAPK p38, but were effective in reducing phosphorylated levels of STAT1. In conclusion, both combination preparations act in an anti-inflammatory manner by influencing cytokine secretion via reduced activity of the JAK/STAT1 pathway. Relevant differences between STW5 and STW5-II were not found indicating similar efficacies. PMID:27833553

  8. Metabolic and morphological differences between rapidly proliferating cancerous and normal breast epithelial cells.

    PubMed

    Meadows, Adam L; Kong, Becky; Berdichevsky, Marina; Roy, Siddhartha; Rosiva, Rosiva; Blanch, Harvey W; Clark, Douglas S

    2008-01-01

    The metabolic and morphological characteristics of two human epithelial breast cell populations--MCF7 cells, a cancerous cell line, and 48R human mammary epithelial cells (48R HMECs), a noncancerous, finite lifespan cell strain--were compared at identical growth rates. Both cell types were induced to grow rapidly in nutrient-rich media containing 13C-labeled glucose, and the isotopic enrichment of cellular metabolites was quantified to calculate metabolic fluxes in key pathways. Despite their similar growth rates, the cells exhibited distinctly different metabolic and morphological profiles. MCF7 cells have an 80% smaller exposed surface area and contain 26% less protein per cell than the 48R cells. Surprisingly, rapidly proliferating 48R cells exhibited a 225% higher per-cell glucose consumption rate, a 250% higher per-cell lactate production rate, and a nearly identical per-cell glutamine consumption rate relative to the cancer cell line. However, when fluxes were considered on the basis of exposed area, the cancer cells were observed to have higher glucose, lactate, and glutamine fluxes, demonstrating superior transport capabilities per unit area of cell membrane. MCF7 cells also consumed amino acids at rates much higher than are generally required for protein synthesis, whereas 48R cells generally did not. Pentose phosphate pathway activity was higher in MCF7 cells, and the flux of glutamine to glutamate was less reversible. Energy efficiency was significantly higher in MCF7 cells, as a result of a combination of their smaller size and greater reliance on the TCA cycle than the 48R cells. These observations support evolutionary models of cancer cell metabolism and suggest targets for metabolic drugs in metastatic breast cancers.

  9. Clostridium difficile-derived membrane vesicles induce the expression of pro-inflammatory cytokine genes and cytotoxicity in colonic epithelial cells in vitro.

    PubMed

    Nicholas, Asiimwe; Jeon, Hyejin; Selasi, Gati Noble; Na, Seok Hyeon; Kwon, Hyo Il; Kim, Yoo Jeong; Choi, Chi Won; Kim, Seung Il; Lee, Je Chul

    2017-03-09

    Clostridium difficile is the most common etiological agent of antibiotic-associated diarrhea in hospitalized and non-hospitalized patients. This study investigated the secretion of membrane vesicles (MVs) from C. difficile and determined the expression of pro-inflammatory cytokine genes and cytotoxicity of C. difficile MVs in epithelial cells in vitro. C. difficile ATCC 43255 and two clinical isolates secreted spherical MVs during in vitro culture. Proteomic analysis revealed that MVs of C. difficile ATCC 43255 contained a total of 262 proteins. Translation-associated proteins were the most commonly identified in C. difficile MVs, whereas TcdA and TcdB toxins were not detected. C. difficile ATCC 43255-derived MVs stimulated the expression of pro-inflammatory cytokine genes, including interleukin (IL)-1β, IL-6, IL-8, and monocyte chemoattractant protein-1 in human colorectal epithelial Caco-2 cells. Moreover, these extracellular vesicles induced cytotoxicity in Caco-2 cells. In conclusion, C. difficile MVs are important nanocomplexes that elicit a pro-inflammatory response and induce cytotoxicity in colonic epithelial cells, which may contribute, along with toxins, to intestinal mucosal injury during C. difficile infection.

  10. The Innate Immune Responses of Colonic Epithelial Cells to Trichuris muris Are Similar in Mouse Strains That Develop a Type 1 or Type 2 Adaptive Immune Response

    PubMed Central

    deSchoolmeester, Matthew L.; Manku, Harinder; Else, Kathryn J.

    2006-01-01

    Trichuris muris resides in intimate contact with its host, burrowing within cecal epithelial cells. However, whether the enterocyte itself responds innately to T. muris is unknown. This study investigated for the first time whether colonic intestinal epithelial cells (IEC) produce cytokines or chemokines following T. muris infection and whether divergence of the innate response could explain differentially polarized adaptive immune responses in resistant and susceptible mice. Increased expression of mRNA for the proinflammatory cytokines gamma interferon (IFN-γ) and tumor necrosis factor and the chemokine CCL2 (MCP-1) were seen after infection of susceptible and resistant strains, with the only difference in expression being a delayed increase in CCL2 in BALB/c IEC. These increases were ablated in MyD88−/− mice, and NF-κB p65 was phosphorylated in response to T. muris excretory/secretory products in the epithelial cell line CMT-93, suggesting involvement of the MyD88-NF-κB signaling pathway in IEC cytokine expression. These data reveal that IEC respond innately to T. muris. However, the minor differences identified between resistant and susceptible mice are unlikely to underlie the subsequent development of a susceptible type 1 (IFN-γ-dominated) or resistant type 2 (interleukin-4 [IL-4]/IL-13-dominated) adaptive immune response. PMID:17057095

  11. The innate immune responses of colonic epithelial cells to Trichuris muris are similar in mouse strains that develop a type 1 or type 2 adaptive immune response.

    PubMed

    deSchoolmeester, Matthew L; Manku, Harinder; Else, Kathryn J

    2006-11-01

    Trichuris muris resides in intimate contact with its host, burrowing within cecal epithelial cells. However, whether the enterocyte itself responds innately to T. muris is unknown. This study investigated for the first time whether colonic intestinal epithelial cells (IEC) produce cytokines or chemokines following T. muris infection and whether divergence of the innate response could explain differentially polarized adaptive immune responses in resistant and susceptible mice. Increased expression of mRNA for the proinflammatory cytokines gamma interferon (IFN-gamma) and tumor necrosis factor and the chemokine CCL2 (MCP-1) were seen after infection of susceptible and resistant strains, with the only difference in expression being a delayed increase in CCL2 in BALB/c IEC. These increases were ablated in MyD88-/- mice, and NF-kappaB p65 was phosphorylated in response to T. muris excretory/secretory products in the epithelial cell line CMT-93, suggesting involvement of the MyD88-NF-kappaB signaling pathway in IEC cytokine expression. These data reveal that IEC respond innately to T. muris. However, the minor differences identified between resistant and susceptible mice are unlikely to underlie the subsequent development of a susceptible type 1 (IFN-gamma-dominated) or resistant type 2 (interleukin-4 [IL-4]/IL-13-dominated) adaptive immune response.

  12. Ginger Compound [6]-Shogaol and Its Cysteine-Conjugated Metabolite (M2) Activate Nrf2 in Colon Epithelial Cells in Vitro and in Vivo

    PubMed Central

    2015-01-01

    In this study, we identified Nrf2 as a molecular target of [6]-shogaol (6S), a bioactive compound isolated from ginger, in colon epithelial cells in vitro and in vivo. Following 6S treatment of HCT-116 cells, the intracellular GSH/GSSG ratio was initially diminished but was then elevated above the basal level. Intracellular reactive oxygen species (ROS) correlated inversely with the GSH/GSSG ratio. Further analysis using gene microarray showed that 6S upregulated the expression of Nrf2 target genes (AKR1B10, FTL, GGTLA4, and HMOX1) in HCT-116 cells. Western blotting confirmed upregulation, phosphorylation, and nuclear translocation of Nrf2 protein followed by Keap1 decrease and upregulation of Nrf2 target genes (AKR1B10, FTL, GGTLA4, HMOX1, and MT1) and glutathione synthesis genes (GCLC and GCLM). Pretreatment of cells with a specific inhibitor of p38 (SB202190), PI3K (LY294002), or MEK1 (PD098059) attenuated these effects of 6S. Using ultra-high-performance liquid chromatography–tandem mass spectrometry, we found that 6S modified multiple cysteine residues of Keap1 protein. In vivo 6S treatment induced Nrf2 nuclear translocation and significantly upregulated the expression of MT1, HMOX1, and GCLC in the colon of wild-type mice but not Nrf2–/– mice. Similar to 6S, a cysteine-conjugated metabolite of 6S (M2), which was previously found to be a carrier of 6S in vitro and in vivo, also activated Nrf2. Our data demonstrated that 6S and its cysteine-conjugated metabolite M2 activate Nrf2 in colon epithelial cells in vitro and in vivo through Keap1-dependent and -independent mechanisms. PMID:25148906

  13. Vibrio fischeri flagellin A is essential for normal motility and for symbiotic competence during initial squid light organ colonization.

    PubMed

    Millikan, Deborah S; Ruby, Edward G

    2004-07-01

    The motile bacterium Vibrio fischeri is the specific bacterial symbiont of the Hawaiian squid Euprymna scolopes. Because motility is essential for initiating colonization, we have begun to identify stage-specific motility requirements by creating flagellar mutants that have symbiotic defects. V. fischeri has six flagellin genes that are uniquely arranged in two chromosomal loci, flaABCDE and flaF. With the exception of the flaA product, the predicted gene products are more similar to each other than to flagellins of other Vibrio species. Immunoblot analysis indicated that only five of the six predicted proteins were present in purified flagella, suggesting that one protein, FlaF, is unique with respect to either its regulation or its function. We created mutations in two genes, flaA and flaC. Compared to a flaC mutant, which has wild-type flagellation, a strain having a mutation in the flaA gene has fewer flagella per cell and exhibits a 60% decrease in its rate of migration in soft agar. During induction of light organ symbiosis, colonization by the flaA mutant is impaired, and this mutant is severely outcompeted when it is presented to the animal as a mixed inoculum with the wild-type strain. Furthermore, flaA mutant cells are preferentially expelled from the animal, suggesting either that FlaA plays a role in adhesion or that normal motility is an advantage for retention within the host. Taken together, these results show that the flagellum of V. fischeri is a complex structure consisting of multiple flagellin subunits, including FlaA, which is essential both for normal flagellation and for motility, as well as for effective symbiotic colonization.

  14. Proteomics demonstration that normal breast epithelial cells can induce apoptosis of breast cancer cells through insulin-like growth factor-binding protein-3 and maspin.

    PubMed

    Toillon, Robert-Alain; Lagadec, Chann; Page, Adeline; Chopin, Valérie; Sautière, Pierre-Eric; Ricort, Jean-Marc; Lemoine, Jérôme; Zhang, Ming; Hondermarck, Hubert; Le Bourhis, Xuefen

    2007-07-01

    Normal breast epithelial cells are known to exert an apoptotic effect on breast cancer cells, resulting in a potential paracrine inhibition of breast tumor development. In this study we purified and characterized the apoptosis-inducing factors secreted by normal breast epithelial cells. Conditioned medium was concentrated by ultrafiltration and separated on reverse phase Sep-Pak C18 and HPLC. The proapoptotic activity of eluted fractions was tested on MCF-7 breast cancer cells, and nano-LC-nano-ESI-MS/MS allowed the identification of insulin-like growth factor-binding protein-3 (IGFBP-3) and maspin as the proapoptotic factors produced by normal breast epithelial cells. Western blot analysis of conditioned media confirmed the specific secretion of IGFBP-3 and maspin by normal cells but not by breast cancer cells. Immunodepletion of IGFBP-3 and maspin completely abolished the normal cell-induced apoptosis of cancer cells, and recombinant proteins reproduced the effect of normal cell-conditioned medium on apoptosis of breast cancer cells. Together our results indicated that normal breast epithelial cells can induce apoptosis of breast cancer cells through IGFBP-3 and maspin. These findings provide a molecular hypothesis for the long observed inhibitory effect of normal surrounding cells on breast cancer development.

  15. GPER mediates estrogen-induced signaling and proliferations in human breast epithelial cells, and normal and malignant breast

    PubMed Central

    Scaling, Allison L.

    2014-01-01

    17β-estradiol (estrogen), through receptor binding and activation, is required for mammary gland development. Estrogen stimulates epithelial proliferation in the mammary gland, promoting ductal elongation and morphogenesis. In addition to a developmental role, estrogen promotes proliferation in tumorigenic settings, particularly breast cancer. The proliferative effects of estrogen in the normal breast and breast tumors are attributed to estrogen receptor α. Although in vitro studies have demonstrated that the G protein-coupled estrogen receptor (GPER, previously called GPR30) can modulate proliferation in breast cancer cells both positively and negatively depending on cellular context, its role in proliferation in the intact normal or malignant breast remains unclear. Estrogen-induced GPER-dependent proliferation was assessed in the immortalized non-tumorigenic human breast epithelial cell line, MCF10A, and an ex vivo organ culture model employing human breast tissue from reduction mammoplasty or tumor resections. Stimulation by estrogen and the GPER-selective agonist G-1 increased the mitotic index in MCF10A cells and proportion of cells in the cell cycle in human breast and breast cancer explants, suggesting increased proliferation. Inhibition of candidate signaling pathways that may link GPER activation to proliferation revealed a dependence on Src, epidermal growth factor receptor transactivation by heparin-bound EGF and subsequent ERK phosphorylation. Proliferation was not dependent on matrix metalloproteinase cleavage of membrane bound pro-HB-EGF. The contribution of GPER to estrogen-induced proliferation in MCF10A cells and breast tissue was confirmed by the ability of GPER-selective antagonist G36 to abrogate estrogen- and G-1-induced proliferation, and the ability of siRNA knockdown of GPER to reduce estrogen- and G-1-induced proliferation in MCF10A cells. This is the first study to demonstrate GPER-dependent proliferation in primary normal and malignant

  16. Flow cytometric determination of stem/progenitor content in epithelial tissues: an example from nonsmall lung cancer and normal lung.

    PubMed

    Donnenberg, Vera S; Landreneau, Rodney J; Pfeifer, Melanie E; Donnenberg, Albert D

    2013-01-01

    Single cell analysis and cell sorting has enabled the study of development, growth, differentiation, repair and maintenance of "liquid" tissues and their cancers. The application of these methods to solid tissues is equally promising, but several unique technical challenges must be addressed. This report illustrates the application of multidimensional flow cytometry to the identification of candidate stem/progenitor populations in non-small cell lung cancer and paired normal lung tissue. Seventeen paired tumor/normal lung samples were collected at the time of surgical excision and processed immediately. Tissues were mechanically and enzymatically dissociated into single cell suspension and stained with a panel of antibodies used for negative gating (CD45, CD14, CD33, glycophorin A), identification of epithelial cells (intracellular cytokeratin), and detection of stem/progenitor markers (CD44, CD90, CD117, CD133). DAPI was added to measure DNA content. Formalin fixed paraffin embedded tissue samples were stained with key markers (cytokeratin, CD117, DAPI) for immunofluorescent tissue localization of populations detected by flow cytometry. Disaggregated tumor and lung preparations contained a high proportion of events that would interfere with analysis, were they not eliminated by logical gating. We demonstrate how inclusion of doublets, events with hypodiploid DNA, and cytokeratin+ events also staining for hematopoietic markers reduces the ability to quantify epithelial cells and their precursors. Using the lung cancer/normal lung data set, we present an approach to multidimensional data analysis that consists of artifact removal, identification of classes of cells to be studied further (classifiers) and the measurement of outcome variables on these cell classes. The results of bivariate analysis show a striking similarity between the expression of stem/progenitor markers on lung tumor and adjacent tumor-free lung.

  17. Thymosin beta-4 knockdown in IEC-6 normal intestinal epithelial cells induces DNA re-replication via downregulating Emi1.

    PubMed

    Chao, Ta-Chung; Chen, Ke-Jay; Tang, Mei-Chuan; Chan, Li-Chuan; Chen, Po-Min; Tzeng, Cheng-Hwai; Su, Yeu

    2014-11-01

    Thymosin β4 (Tβ4 ) is a multifunctional protein already used clinically to treat various diseases; however, the promoting effect of this protein on tumor malignancy should not be neglected. Here, we assessed whether Tβ4 alteration influences normal intestinal epithelial cells because Tβ4 is deemed a novel target for treating colorectal cancer (CRC). For this purpose, we examined the consequences of shRNA-mediated knockdown of Tβ4 in IEC-6 normal rat small intestinal cells and found that inhibiting Tβ4 expression significantly suppressed their growth and induced apoptosis in some cells. Flow cytometric analysis further revealed a marked decrease of G0/G1 population but a drastic increase of polyploid ones in these cells. The increase of polyploidy likely resulted from DNA re-replication because not only the de novo DNA synthesis was greatly increased but also the expression levels of Cdc6 (a replication-licensing factor), cyclin A, and phosphorylated-checkpoint kinase 1 were all dramatically elevated. Moreover, marked reductions in both RNA and protein levels of Emi1 (early mitotic inhibitor 1) were also detected in Tβ4 -downregulated IEC-6 cells which might be accounted by the downregulation of E2F1, a transcription factor capable of inducing Emi1 expression, mediated by glycogen synthase-3β (GSK-3β). To our best knowledge, this is the first report showing that inhibiting Tβ4 expression triggers DNA re-replication in normal intestinal epithelial cells, suggesting that this G-actin sequester may play a crucial role in maintaining genome stability in these cells. More importantly, clinical oncologists should take this novel activity into consideration when design CRC therapy based on targeting Tβ4 . © 2014 Wiley Periodicals, Inc.

  18. Enteric oxalate elimination is induced and oxalate is normalized in a mouse model of primary hyperoxaluria following intestinal colonization with Oxalobacter

    PubMed Central

    Gjymishka, Altin; Salido, Eduardo C.; Allison, Milton J.; Freel, Robert W.

    2011-01-01

    Oxalobacter colonization of rat intestine was previously shown to promote enteric oxalate secretion and elimination, leading to significant reductions in urinary oxalate excretion (Hatch et al. Kidney Int 69: 691–698, 2006). The main goal of the present study, using a mouse model of primary hyperoxaluria type 1 (PH1), was to test the hypothesis that colonization of the mouse gut by Oxalobacter formigenes could enhance enteric oxalate secretion and effectively reduce the hyperoxaluria associated with this genetic disease. Wild-type (WT) mice and mice deficient in liver alanine-glyoxylate aminotransferase (Agxt) exhibiting hyperoxalemia and hyperoxaluria were used in these studies. We compared the unidirectional and net fluxes of oxalate across isolated, short-circuited large intestine of artificially colonized and noncolonized mice. In addition, plasma and urinary oxalate was determined. Our results demonstrate that the cecum and distal colon contribute significantly to enteric oxalate excretion in Oxalobacter-colonized Agxt and WT mice. In colonized Agxt mice, urinary oxalate excretion was reduced 50% (to within the normal range observed for WT mice). Moreover, plasma oxalate concentrations in Agxt mice were also normalized (reduced 50%). Colonization of WT mice was also associated with marked (up to 95%) reductions in urinary oxalate excretion. We conclude that segment-specific effects of Oxalobacter on intestinal oxalate transport in the PH1 mouse model are associated with a normalization of plasma oxalate and urinary oxalate excretion in otherwise hyperoxalemic and hyperoxaluric animals. PMID:21163900

  19. Enteric oxalate elimination is induced and oxalate is normalized in a mouse model of primary hyperoxaluria following intestinal colonization with Oxalobacter.

    PubMed

    Hatch, Marguerite; Gjymishka, Altin; Salido, Eduardo C; Allison, Milton J; Freel, Robert W

    2011-03-01

    Oxalobacter colonization of rat intestine was previously shown to promote enteric oxalate secretion and elimination, leading to significant reductions in urinary oxalate excretion (Hatch et al. Kidney Int 69: 691-698, 2006). The main goal of the present study, using a mouse model of primary hyperoxaluria type 1 (PH1), was to test the hypothesis that colonization of the mouse gut by Oxalobacter formigenes could enhance enteric oxalate secretion and effectively reduce the hyperoxaluria associated with this genetic disease. Wild-type (WT) mice and mice deficient in liver alanine-glyoxylate aminotransferase (Agxt) exhibiting hyperoxalemia and hyperoxaluria were used in these studies. We compared the unidirectional and net fluxes of oxalate across isolated, short-circuited large intestine of artificially colonized and noncolonized mice. In addition, plasma and urinary oxalate was determined. Our results demonstrate that the cecum and distal colon contribute significantly to enteric oxalate excretion in Oxalobacter-colonized Agxt and WT mice. In colonized Agxt mice, urinary oxalate excretion was reduced 50% (to within the normal range observed for WT mice). Moreover, plasma oxalate concentrations in Agxt mice were also normalized (reduced 50%). Colonization of WT mice was also associated with marked (up to 95%) reductions in urinary oxalate excretion. We conclude that segment-specific effects of Oxalobacter on intestinal oxalate transport in the PH1 mouse model are associated with a normalization of plasma oxalate and urinary oxalate excretion in otherwise hyperoxalemic and hyperoxaluric animals.

  20. Glutathione-dependent and -independent oxidative stress-control mechanisms distinguish normal human mammary epithelial cell subsets.

    PubMed

    Kannan, Nagarajan; Nguyen, Long V; Makarem, Maisam; Dong, Yifei; Shih, Kingsley; Eirew, Peter; Raouf, Afshin; Emerman, Joanne T; Eaves, Connie J

    2014-05-27

    Mechanisms that control the levels and activities of reactive oxygen species (ROS) in normal human mammary cells are poorly understood. We show that purified normal human basal mammary epithelial cells maintain low levels of ROS primarily by a glutathione-dependent but inefficient antioxidant mechanism that uses mitochondrial glutathione peroxidase 2. In contrast, the matching purified luminal progenitor cells contain higher levels of ROS, multiple glutathione-independent antioxidants and oxidative nucleotide damage-controlling proteins and consume O2 at a higher rate. The luminal progenitor cells are more resistant to glutathione depletion than the basal cells, including those with in vivo and in vitro proliferation and differentiation activity. The luminal progenitors also are more resistant to H2O2 or ionizing radiation. Importantly, even freshly isolated "steady-state" normal luminal progenitors show elevated levels of unrepaired oxidative DNA damage. Distinct ROS control mechanisms operating in different subsets of normal human mammary cells could have differentiation state-specific functions and long-term consequences.

  1. Protein profiling of isolated leukocytes, myofibroblasts, epithelial, Basal, and endothelial cells from normal, hyperplastic, cancerous, and inflammatory human prostate tissues.

    PubMed

    Khamis, Zahraa I; Iczkowski, Kenneth A; Sahab, Ziad J; Sang, Qing-Xiang Amy

    2010-06-15

    In situ neoplastic prostate cells are not lethal unless they become invasive and metastatic. For cells to become invasive, the prostate gland must undergo degradation of the basement membrane and disruption of the basal cell layer underneath the luminal epithelia. Although the roles of proteinases in breaking down the basement membrane have been well-studied, little is known about the factors that induce basal cell layer disruption, degeneration, and its eventual disappearance in invasive cancer. It is hypothesized that microenvironmental factors may affect the degradation of the basal cell layer, which if protected may prevent tumor progression and invasion. In this study, we have revealed differential protein expression patterns between epithelial and stromal cells isolated from different prostate pathologies and identified several important epithelial and stromal proteins that may contribute to inflammation and malignant transformation of human benign prostate tissues to cancerous tissues using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and proteomics methods. Cellular retinoic acid-binding protein 2 was downregulated in basal cells of benign prostate. Caspase-1 and interleukin-18 receptor 1 were highly expressed in leukocytes of prostate cancer. Proto-oncogene Wnt-3 was downregulated in endothelial cells of prostatitis tissue and tyrosine phosphatase non receptor type 1 was only found in normal and benign endothelial cells. Poly ADP-ribose polymerase 14 was downregulated in myofibroblasts of prostatitis tissue. Interestingly, integrin alpha-6 was upregulated in epithelial cells but not detected in myofibroblasts of prostate cancer. Further validation of these proteins may generate new strategies for the prevention of basal cell layer disruption and subsequent cancer invasion.

  2. Reg4+ deep crypt secretory cells function as epithelial niche for Lgr5+ stem cells in colon

    PubMed Central

    Sasaki, Nobuo; Sachs, Norman; Wiebrands, Kay; Ellenbroek, Saskia I. J.; Fumagalli, Arianna; Lyubimova, Anna; Begthel, Harry; van den Born, Maaike; van Es, Johan H.; Karthaus, Wouter R.; Li, Vivian S. W.; López-Iglesias, Carmen; Peters, Peter J.; van Rheenen, Jacco; van Oudenaarden, Alexander; Clevers, Hans

    2016-01-01

    Leucine-rich repeat-containing G-protein coupled receptor 5-positive (Lgr5+) stem cells reside at crypt bottoms of the small and large intestine. Small intestinal Paneth cells supply Wnt3, EGF, and Notch signals to neighboring Lgr5+ stem cells. Whereas the colon lacks Paneth cells, deep crypt secretory (DCS) cells are intermingled with Lgr5+ stem cells at crypt bottoms. Here, we report regenerating islet-derived family member 4 (Reg4) as a marker of DCS cells. To investigate a niche function, we eliminated DCS cells by using the diphtheria-toxin receptor gene knocked into the murine Reg4 locus. Ablation of DCS cells results in loss of stem cells from colonic crypts and disrupts gut homeostasis and colon organoid growth. In agreement, sorted Reg4+ DCS cells promote organoid formation of single Lgr5+ colon stem cells. DCS cells can be massively produced from Lgr5+ colon stem cells in vitro by combined Notch inhibition and Wnt activation. We conclude that Reg4+ DCS cells serve as Paneth cell equivalents in the colon crypt niche. PMID:27573849

  3. Ectopic runx2 expression in mammary epithelial cells disrupts formation of normal acini structure: implications for breast cancer progression.

    PubMed

    Pratap, Jitesh; Imbalzano, Karen M; Underwood, Jean M; Cohet, Nathalie; Gokul, Karthiga; Akech, Jacqueline; van Wijnen, Andre J; Stein, Janet L; Imbalzano, Anthony N; Nickerson, Jeffrey A; Lian, Jane B; Stein, Gary S

    2009-09-01

    The transcription factor Runx2 is highly expressed in breast cancer cells compared with mammary epithelial cells and contributes to metastasis. Here we directly show that Runx2 expression promotes a tumor cell phenotype of mammary acini in three-dimensional culture. Human mammary epithelial cells (MCF-10A) form polarized, growth-arrested, acini-like structures with glandular architecture. The ectopic expression of Runx2 disrupts acini formation, and electron microscopic ultrastructural analysis revealed the absence of lumens. Characterization of the disrupted acini structures showed increased cell proliferation (Ki-67 positive cells), decreased apoptosis (Bcl-2 induction), and loss of basement membrane formation (absence of beta(4) integrin expression). In complementary experiments, inhibition of Runx2 function in metastatic MDA-MB-231 breast cancer cells by stable expression of either short hairpin RNA-Runx2 or a mutant Runx2 deficient in subnuclear targeting resulted in reversion of acini to more normal structures and reduced tumor growth in vivo. These novel findings provide direct mechanistic evidence for the biological activity of Runx2, dependent on its subnuclear localization, in promoting early events of breast cancer progression and suggest a molecular therapeutic target.

  4. Rapid Akt activation by nicotine and a tobacco carcinogen modulates the phenotype of normal human airway epithelial cells.

    PubMed

    West, Kip A; Brognard, John; Clark, Amy S; Linnoila, Ilona R; Yang, Xiaowei; Swain, Sandra M; Harris, Curtis; Belinsky, Steven; Dennis, Phillip A

    2003-01-01

    Tobacco-related diseases such as lung cancer cause over 4.2 million deaths annually, with approximately 400,000 deaths per year occurring in the US. Genotoxic effects of tobacco components have been described, but effects on signaling pathways in normal cells have not been described. Here, we show activation of the serine/threonine kinase Akt in nonimmortalized human airway epithelial cells in vitro by two components of cigarette smoke, nicotine and the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Activation of Akt by nicotine or NNK occurred within minutes at concentrations achievable by smokers and depended upon alpha(3)-/alpha(4)-containing or alpha(7)-containing nicotinic acetylcholine receptors, respectively. Activated Akt increased phosphorylation of downstream substrates such as GSK-3, p70(S6K), 4EBP-1, and FKHR. Treatment with nicotine or NNK attenuated apoptosis caused by etoposide, ultraviolet irradiation, or hydrogen peroxide and partially induced a transformed phenotype manifest as loss of contact inhibition and loss of dependence on exogenous growth factors or adherence to ECM. In vivo, active Akt was detected in airway epithelial cells and lung tumors from NNK-treated A/J mice, and in human lung cancers derived from smokers. Redundant Akt activation by nicotine and NNK could contribute to tobacco-related carcinogenesis by regulating two processes critical for tumorigenesis, cell growth and apoptosis.

  5. Magnetic Nanodrug Delivery Through the Mucus Layer of Air-Liquid Interface Cultured Primary Normal Human Tracheobronchial Epithelial Cells

    PubMed Central

    Economou, E. C.; Marinelli, S.; Smith, M. C.; Routt, A. A.; Kravets, V. V.; Chu, H. W.; Spendier, K.; Celinski, Z. J.

    2016-01-01

    Superparamagnetic iron oxide (Fe3O4) and highly anisotropic barium hexaferrite (BaFe12O19) nanoparticles were coated with an anti-inflammatory drug and magnetically transported through mucus produced by primary human airway epithelial cells. Using wet planetary ball milling, dl-2-amino-3-phosphonopropionic acid-coated BaFe12O19 nano-particles (BaNPs) of 1–100 nm in diameter were prepared in water. BaNPs and conventional 20–30-nm Fe3O4 nanoparticles (FeNPs) were then encased in a polymer (PLGA) loaded with dexamethasone (Dex) and tagged for imaging. PLGA-Dex-coated BaNPs and FeNPs were characterized using dynamic light scattering (DLS), transmission electron microscopy (TEM), and superconducting quantum interference device (SQUID) magnetometry. Both PLGA-Dex-coated BaNPs and FeNPs were transferred to the surface of a ~100-μm thick mucus layer of air-liquid interface cultured primary normal human tracheobronchial epithelial (NHTE) cells. Within 30 min, the nanoparticles were pulled successfully through the mucus layer by a permanent neodymium magnet. The penetration time of the nanomedicine was monitored using confocal microscopy and tailored by varying the thickness of the PLGA-Dex coating around the particles. PMID:27774374

  6. Validation of Normal Human Bronchial Epithelial Cells as a Model for Influenza A Infections in Human Distal Trachea

    PubMed Central

    Davis, A. Sally; Chertow, Daniel S.; Moyer, Jenna E.; Suzich, Jon; Sandouk, Aline; Dorward, David W.; Logun, Carolea; Shelhamer, James H.

    2015-01-01

    Primary normal human bronchial/tracheal epithelial (NHBE) cells, derived from the distal-most aspect of the trachea at the bifurcation, have been used for a number of studies in respiratory disease research. Differences between the source tissue and the differentiated primary cells may impact infection studies based on this model. Therefore, we examined how well-differentiated NHBE cells compared with their source tissue, the human distal trachea, as well as the ramifications of these differences on influenza A viral pathogenesis research using this model. We employed a histological analysis including morphological measurements, electron microscopy, multi-label immunofluorescence confocal microscopy, lectin histochemistry, and microarray expression analysis to compare differentiated NHBEs to human distal tracheal epithelium. Pseudostratified epithelial height, cell type variety and distribution varied significantly. Electron microscopy confirmed differences in cellular attachment and paracellular junctions. Influenza receptor lectin histochemistry revealed that α2,3 sialic acids were rarely present on the apical aspect of the differentiated NHBE cells, but were present in low numbers in the distal trachea. We bound fluorochrome bioconjugated virus to respiratory tissue and NHBE cells and infected NHBE cells with human influenza A viruses. Both indicated that the pattern of infection progression in these cells correlated with autopsy studies of fatal cases from the 2009 pandemic. PMID:25604814

  7. Magnetic Nanodrug Delivery Through the Mucus Layer of Air-Liquid Interface Cultured Primary Normal Human Tracheobronchial Epithelial Cells.

    PubMed

    Economou, E C; Marinelli, S; Smith, M C; Routt, A A; Kravets, V V; Chu, H W; Spendier, K; Celinski, Z J

    2016-09-01

    Superparamagnetic iron oxide (Fe3O4) and highly anisotropic barium hexaferrite (BaFe12O19) nanoparticles were coated with an anti-inflammatory drug and magnetically transported through mucus produced by primary human airway epithelial cells. Using wet planetary ball milling, dl-2-amino-3-phosphonopropionic acid-coated BaFe12O19 nano-particles (BaNPs) of 1-100 nm in diameter were prepared in water. BaNPs and conventional 20-30-nm Fe3O4 nanoparticles (FeNPs) were then encased in a polymer (PLGA) loaded with dexamethasone (Dex) and tagged for imaging. PLGA-Dex-coated BaNPs and FeNPs were characterized using dynamic light scattering (DLS), transmission electron microscopy (TEM), and superconducting quantum interference device (SQUID) magnetometry. Both PLGA-Dex-coated BaNPs and FeNPs were transferred to the surface of a ~100-μm thick mucus layer of air-liquid interface cultured primary normal human tracheobronchial epithelial (NHTE) cells. Within 30 min, the nanoparticles were pulled successfully through the mucus layer by a permanent neodymium magnet. The penetration time of the nanomedicine was monitored using confocal microscopy and tailored by varying the thickness of the PLGA-Dex coating around the particles.

  8. Malignant transformation of human colon epithelial cells by benzo[c]phenanthrene dihydrodiolepoxides as well as 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine

    SciTech Connect

    Herbst, Uta; Fuchs, Judith Iris; Teubner, Wera; Steinberg, Pablo . E-mail: steinber@rz.uni-potsdam.de

    2006-04-15

    Polycyclic aromatic hydrocarbons (PAHs) and heterocyclic aromatic amines (HCAs) ingested with food have repeatedly been suggested to be involved in the malignant transformation of colon epithelial cells. In order to test this hypothesis, HCEC cells (SV40 large T antigen-immortalized human colon epithelial cells) were incubated with a racemic mixture of benzo[c]phenanthrene dihydrodiol epoxides (B[c]PhDE), extremely potent carcinogenic PAH metabolites in vivo, or with 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP), the N-hydroxylated metabolite of the most abundant HCA in cooked meat. First, it was shown that HCEC cells express sulfotransferase 1A1, which is needed to metabolize N-OH-PhIP to the corresponding N-sulfonyloxy derivative, the direct precursor molecule of genotoxic nitrenium ions. Thereafter, exponentially growing HCEC cells were exposed five times to 0.1 {mu}g (0.37 nmol) B[c]PhDE/ml for 30 min or 0.72 {mu}g (3 nmol) N-OH-PhIP/ml for 24 h. Chemically treated HCEC cells showed an enhanced saturation density and grew faster than the corresponding solvent-treated cell cultures. After five treatment cycles, HCEC{sup B[c]PhDE} as well as HCEC {sup N-OH-PhIP} cells lost cell-cell contact inhibition and started piling up and forming foci in the culture flasks. Furthermore, HCEC{sup B[c]PhDE} and HCEC {sup N-OH-PhIP} cells were injected i.m. into SCID mice. Within 6 weeks after injection, eight animals out of eight injected with HCEC{sup B[c]PhDE} or HCEC {sup N-OH-PhIP} cells developed tumors at the site of injection, thus demonstrating the high tumorigenic potential of the HCEC{sup B[c]PhDE} and HCEC {sup N-OH-PhIP} cell cultures. Taken together, we show for the first time that the abovementioned active PAH metabolites as well as N-OH-PhIP are indeed able to malignantly transform human colon epithelial cells in vitro.

  9. Detection of Epstein-Barr virus genome and latent infection gene expression in normal epithelia, epithelial dysplasia, and squamous cell carcinoma of the oral cavity.

    PubMed

    Kikuchi, Kentaro; Noguchi, Yoshihiro; de Rivera, Michelle Wendoline Garcia-Niño; Hoshino, Miyako; Sakashita, Hideaki; Yamada, Tsutomu; Inoue, Harumi; Miyazaki, Yuji; Nozaki, Tadashige; González-López, Blanca Silvia; Ide, Fumio; Kusama, Kaoru

    2016-03-01

    A relationship between Epstein-Barr virus (EBV) infection and cancer of lymphoid and epithelial tissues such as Burkitt's lymphoma, Hodgkin's disease, nasopharyngeal carcinoma (NPC), gastric carcinoma, and oral cancer has been reported. EBV is transmitted orally and infects B cells and epithelial cells. However, it has remained uncertain whether EBV plays a role in carcinogenesis of oral mucosal tissue. In the present study, we detected the EBV genome and latent EBV gene expression in normal mucosal epithelia, epithelial dysplasia, and oral squamous cell carcinoma (OSCC) to clarify whether EBV is involved in carcinogenesis of the oral cavity. We examined 333 formalin-fixed, paraffin-embedded tissue samples (morphologically normal oral mucosa 30 samples, gingivitis 32, tonsillitis 17, oral epithelial dysplasia 83, OSCC 150, and NPC 21). EBV latent infection genes (EBNA-2, LMP-1) were detected not only in OSCC (50.2 %, 10.7 %) but also in severe epithelial dysplasia (66.7 %, 44.4 %), mild to moderate epithelial dysplasia (43.1 %, 18.5 %), gingivitis (78.1 %, 21.9 %), and normal mucosa (83.3 %, 23.3 %). Furthermore, the intensity of EBV latent infection gene expression (EBER, LMP-1) was significantly higher in severe epithelial dysplasia (94.4 %, 72.2 %) than in OSCC (34.7 %, 38.7 %). These results suggest that EBV latent infection genes and their increased expression in severe epithelial dysplasia might play an important role in the dysplasia-carcinoma sequence in the oral cavity.

  10. Exogenous normal mammary epithelial mitochondria suppress glycolytic metabolism and glucose uptake of human breast cancer cells.

    PubMed

    Jiang, Xian-Peng; Elliott, Robert L; Head, Jonathan F

    2015-10-01

    We hypothesized that normal mitochondria inhibited cancer cell proliferation and increased drug sensitivity by the mechanism of suppression of cancer aerobic glycolysis. To demonstrate the mechanism, we used real-time PCR and glycolysis cell-based assay to measure gene expression of glycolytic enzymes and glucose transporters, and extracellular lactate production of human breast cancer cells. We found that isolated fluorescent probe-stained mitochondria of MCF-12A (human mammary epithelia) could enter into human breast cancer cell lines MCF-7, T47D, and MDA-MB-231, confirmed by fluorescent and confocal microscopy. Mitochondria from the untransformed human mammary epithelia increased drug sensitivity of MCF-7 cells to paclitaxel. Real-time PCR showed that exogenous normal mitochondria of MCF-12A suppressed gene expression of glycolytic enzymes, lactate dehydrogenase A, and glucose transporter 1 and 3 of MCF-7 and MDA-MB-231 cells. Glycolysis cell-based assay revealed that normal mitochondria significantly suppressed lactate production in culture media of MCF-7, T47D, and MDA-MB-231 cells. In conclusion, normal mitochondria suppress cancer proliferation and increase drug sensitivity by the mechanism of inhibition of cancer cell glycolysis and glucose uptake.

  11. Quantification of the global and local complexity of the epithelial-connective tissue interface of normal, dysplastic, and neoplastic oral mucosae using digital imaging.

    PubMed

    Abu Eid, Rasha; Landini, Gabriel

    2003-01-01

    This study aimed at quantifying the complexity of the epithelial-connective tissue interface (ECTI) in human normal mucosa, premalignant, and malignant lesions using fractal geometry. Two approaches were used to describe the complexity of 377 oral mucosa ECTI profiles. The box counting method was used to estimate their global fractal dimension, while local fractal dimensions were estimated using the mass radius relation at various local scales. The ECTI complexity significantly increased from normal through premalignant to malignant profiles in both global and local (over 283 microm) scales. Normal mucosa samples from different sites of the oral cavity also had different degrees of global complexity. Fractal geometry is a useful morphological marker of tissue complexity changes taking place during epithelial malignancy and premalignancy, and we propose it as a quantitative marker of epithelial complexity.

  12. Acupuncture at heterotopic acupoints facilitates distal colonic motility via activating M3 receptors and somatic afferent C-fibers in normal, constipated, or diarrhoeic rats.

    PubMed

    Gao, X; Qin, Q; Yu, X; Liu, K; Li, L; Qiao, H; Zhu, B

    2015-12-01

    Previous studies have demonstrated the efficacy of somatic stimulation for patients with gastrointestinal motility disorders. However, little effort has been made to investigate the effects of acupuncture on colonic motility, particularly in pathological conditions. The precise mechanism employed in the regulation of acupuncture on colonic motility still remains unclear. We assessed the effect of acupuncture at heterotopic acupoints on distal colonic motility using a warm-water-filled manometric balloon inserted 5-6 cm into the rectum of anesthetized normal rats or rats with diarrhea or constipation. Choline chloride, 4-DAMP, cobra venom and capsaicin were separately applied to investigate the role of M3 receptors in the regulation of distal colonic motility by acupuncture at heterotopic acupoints, and whether Aδ- and/or C-fibers are required for triggering distal colonic motility by acupuncture. Acupuncture at heterotopic acupoints increased distal colonic motility not only in normal rats but also in rats with constipation or diarrhea. M3 receptors play an important role in the facilitation of distal colonic motility triggered by acupuncture at heterotopic acupoints. Afferent nerve Aδ- and C-fibers mediate the transduction of the acupuncture signal and C-fibers are essential for enhancing the effect of acupuncture at the heterotopic acupoint on distal colonic motility. Our results reveal that acupuncture at heterotopic acupoints increases distal colonic motility regardless of normal or pathological conditions via predominately activating C-fibers of somatic afferent nerve and M3 receptors. © 2015 The Authors.Neurogastroenterology & Motility published by John Wiley & Sons Ltd.

  13. Basement-membrane heparan sulphate with high affinity for antithrombin synthesized by normal and transformed mouse mammary epithelial cells.

    PubMed Central

    Pejler, G; David, G

    1987-01-01

    Basement-membrane proteoglycans, biosynthetically labelled with [35S]sulphate, were isolated from normal and transformed mouse mammary epithelial cells. Proteoglycans synthesized by normal cells contained mainly heparan sulphate and, in addition, small amounts of chondroitin sulphate chains, whereas transformed cells synthesized a relatively higher proportion of chondroitin sulphate. Polysaccharide chains from transformed cells were of lower average Mr and of lower anionic charge density compared with chains isolated from the untransformed counterparts, confirming results reported previously [David & Van den Berghe (1983) J. Biol. Chem. 258, 7338-7344]. A large proportion of the chains isolated from normal cells bound with high affinity to immobilized antithrombin, and the presence of 3-O-sulphated glucosamine residues, previously identified as unique markers for the antithrombin-binding region of heparin [Lindahl, Bäckström, Thunberg & Leder (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 6551-6555], could be demonstrated. A significantly lower proportion of the chains derived from transformed cells bound with high affinity to antithrombin, and a corresponding decrease in the amount of incorporated 3-O-sulphate was observed. PMID:2963617

  14. AFM method to detect differences in adhesion of silica bids to cancer and normal epithelial cells

    NASA Astrophysics Data System (ADS)

    Sokolov, Igor; Iyer, Swaminathan; Gaikwad, Ravi; Woodworth, Craig

    2009-03-01

    To date, the methods of detection of cancer cells have been mostly based on traditional techniques used in biology, such as visual identification of malignant changes, cell growth analysis, specific ligand-receptor labeling, or genetic tests. Despite being well developed, these methods are either insufficiently accurate or require a lengthy complicated analysis. A search for alternative methods for the detection of cancer cells may be a fruitful approach. Here we describe an AFM study that may result in a new method for detection of cancer cells in vitro. Here we use atomic force microscopy (AFM) to study adhesion of single silica beads to malignant and normal cells cultured from human cervix. We found that adhesion depends on the time of contact, and can be statistically different for malignant and normal cells. Using these data, one could develop an optical method of cancer detection based on adhesion of various silica beads.

  15. Expression of nuclear membrane proteins in normal, hyperplastic, and neoplastic thyroid epithelial cells.

    PubMed

    Wang, Jieying; Kondo, Tetsuo; Yamane, Tetsu; Nakazawa, Tadao; Oish, Naoki; Mochizuki, Kunio; Katoh, Ryohei

    2015-10-01

    Emerin, lamin A/C, lamin B, and lamin-associated polypeptide 2 (LAP2) are nuclear membrane proteins that play an important role in maintaining nuclear structure and coordinating cell activity. We studied the expression and significance of nuclear membrane proteins in neoplastic thyroid cells by immunohistochemistry, RT-PCR, and real-time PCR. In papillary carcinomas (PCs), the nuclear proteins most frequently expressed at high levels were emerin (82 % positive), lamin A/C (64 %), and LAP2 (82 %). Follicular carcinomas (FCs) most frequently expressed lamin B, while none of the undifferentiated carcinomas (UCs) showed strong expression of emerin or lamin A/C. In all medullary carcinomas (MCs), intermediate to high levels of expression of lamin A/C and LAP2 were found. By RT-PCR analysis, messenger RNA (mRNA) expression of all nuclear membrane proteins except emerin was higher in PC than in normal tissue. Real-time PCR analysis showed that mRNA expression of nuclear membrane protein varied between cell lines. Our findings suggest that expression of nuclear membrane proteins may be related to follicular function in normal and hyperplastic follicles, and we hypothesize that they are also involved in the proliferation and differentiation of neoplastic thyroid cells. We suggest that they reflect the biological nature and/or function of normal, hyperplastic, and neoplastic thyroid cells and may have some value in diagnosing thyroid tumors.

  16. Assessing the Toxicities of Regulated and Unregulated Disinfection By-products in Normal Human Colon Cells.

    EPA Science Inventory

    The presence of over six hundred disinfection by-products (DBPs) and less than half of the total organic halides present in finished water has created a need for short-term in vitro assays to address toxicities that might be associated with human exposure. . We are using a normal...

  17. Assessing the Toxicities of Regulated and Unregulated Disinfection By-products in Normal Human Colon Cells.

    EPA Science Inventory

    The presence of over six hundred disinfection by-products (DBPs) and less than half of the total organic halides present in finished water has created a need for short-term in vitro assays to address toxicities that might be associated with human exposure. . We are using a normal...

  18. ADA3 regulates normal and tumor mammary epithelial cell proliferation through c-MYC.

    PubMed

    Griffin, Nicolas I; Sharma, Gayatri; Zhao, Xiangshan; Mirza, Sameer; Srivastava, Shashank; Dave, Bhavana J; Aleskandarany, Mohammed; Rakha, Emad; Mohibi, Shakur; Band, Hamid; Band, Vimla

    2016-11-16

    We have established the critical role of ADA3 as a coactivator of estrogen receptor (ER), as well as its role in cell cycle progression. Furthermore, we showed that ADA3 is predominantly nuclear in mammary epithelium, and in ER+, but is cytoplasmic in ER- breast cancers, the latter correlating with poor survival. However, the role of nuclear ADA3 in human mammary epithelial cells (hMECs), and in ER+ breast cancer cells, as well as the importance of ADA3 expression in relation to patient prognosis and survival in ER+ breast cancer have remained uncharacterized. We overexpressed ADA3 in hMECs or in ER+ breast cancer cells and assessed the effect on cell proliferation. The expression of ADA3 was analyzed then correlated with the expression of various prognostic markers, as well as survival of breast cancer patients. Overexpression of ADA3 in ER- hMECs as well as in ER+ breast cancer cell lines enhanced cell proliferation. These cells showed increased cyclin B and c-MYC, decreased p27 and increased SKP2 levels. This was accompanied by increased mRNA levels of early response genes c-FOS, EGR1, and c-MYC. Analysis of breast cancer tissue specimens showed a significant correlation of ADA3 nuclear expression with c-MYC expression. Furthermore, nuclear ADA3 and c-MYC expression together showed significant correlation with tumor grade, mitosis, pleomorphism, NPI, ER/PR status, Ki67 and p27 expression. Importantly, within ER+ cases, expression of nuclear ADA3 and c-MYC also significantly correlated with Ki67 and p27 expression. Univariate Kaplan Meier analysis of four groups in the whole, as well as the ER+ patients showed that c-MYC and ADA3 combinatorial phenotypes showed significantly different breast cancer specific survival with c-MYC-high and ADA3-Low subgroup had the worst outcome. Using multivariate analyses within the whole cohort and the ER+ subgroups, the significant association of ADA3 and c-MYC expression with patients' outcome was independent of tumor grade

  19. Live cell imaging reveals extensive intracellular cytoplasmic colonization of banana by normally non-cultivable endophytic bacteria.

    PubMed

    Thomas, Pious; Sekhar, Aparna Chandra

    2014-01-01

    colonization in bananas by normally non-cultivable endophytic bacteria in two niches, namely cytoplasmic and periplasmic, designated as 'Cytobacts' and 'Peribacts', respectively. The integral intracellular association with their clonal perpetuation suggests a mutualistic relationship between endophytes and the host.

  20. Innate mechanisms for Bifidobacterium lactis to activate transient pro-inflammatory host responses in intestinal epithelial cells after the colonization of germ-free rats

    PubMed Central

    Ruiz, Pedro A; Hoffmann, Micha; Szcesny, Silke; Blaut, Michael; Haller, Dirk

    2005-01-01

    Bifidobacteria comprise a dominant microbial population group in the human intestinal tract with purported beneficial health effects on the host. In this study, we characterized the molecular mechanisms for the initial interaction of probiotic Bifidobacterium lactis strain BB12 with native and intestinal epithelial cell (IEC) lines. We showed that B. lactis-monoassociated Fisher F344 rats transiently induce phosphorylation/activation of the NF-κB transcriptionally active subunit RelA and the mitogen-activated protein kinase (MAPK) p38 in native IEC at day 5 after initial bacterial colonization. In addition, Interleukin 6 (IL-6) gene expression was significantly increased at day 5, demonstrating the physiological relevance of transient transcription factor activation in IEC. In contrast, Bacteroides vulgatus-monoassociated Fisher rats revealed RelA but not p38 MAPK phosphorylation and failed to trigger significant IL-6 gene expression in native IEC. Moreover, we demonstrated that B. lactis triggers NF-κB RelA and p38 MAPK phosphorylation in IEC lines. Adenoviral delivery of mutant IKK-β (Ad5dnIKKβ) and inhibition of the p38 MAPK pathway through the pharmacological inhibitor SB203580 significantly blocked B. lactis-induced IL-6 gene expression in IEC, suggesting that B. lactis triggers NF-κB and MAPK signaling to induce gene expression in the intestinal epithelium. Regarding the mechanisms of bacteria epithelial cell cross-talk, B. lactis-induced IL-6 gene expression was completely inhibited in TLR2 deficient mouse embryogenic fibroblasts (MEF TLR2−/−) as well as TLR2ΔTIR transfected Mode-K cells. In conclusion, we demonstrated that probiotic bacteria transiently trigger innate signal transduction and pro-inflammatory gene expression in the intestinal epithelium at early stages of bacterial colonization. PMID:16011513

  1. Dietary Soy Protein Inhibits DNA Damage and Cell Survival of Colon Epithelial Cells through Attenuated Expression of Fatty Acid Synthase

    USDA-ARS?s Scientific Manuscript database

    Dietary intake of soy protein decreases tumor incidence in rat models of chemically induced colon cancer. We hypothesized that decreased expression of Fatty Acid Synthase (FASN) underlies, in part, the tumor preventive effects of soy protein, since FASN over-expression characterizes early tumorigene...

  2. Colonic mucosal gene expression and genotype in irritable bowel syndrome patients with normal or elevated fecal bile acid excretion.

    PubMed

    Camilleri, Michael; Carlson, Paula; Acosta, Andres; Busciglio, Irene

    2015-07-01

    The mucosal gene expression in rectosigmoid mucosa (RSM) in irritable bowel syndrome with diarrhea (IBS-D) is unknown. Our objectives were, first, to study mRNA expression [by RT(2) PCR of 19 genes pertaining to tight junctions, immune activation, intestinal ion transport and bile acid (BA) homeostasis] in RSM in IBS-D patients (n = 47) and healthy controls (n = 17) and study expression of a selected protein (PDZD3) in 10 IBS-D patients and 4 healthy controls; second, to assess RSM mRNA expression according to genotype and fecal BA excretion (high ≥ 2,337 μmol/48 h); and third, to determine whether genotype or mucosal mRNA expression is associated with colonic transit or BA parameters. Fold changes were corrected for false detection rate for 19 genes studied (P < 0.00263). In RSM in IBS-D patients compared with controls, mRNA expression of GUC2AB, PDZD3, and PR2Y4 was increased, whereas CLDN1 and FN1 were decreased. One immune-related gene was upregulated (C4BP4) and one downregulated (CCL20). There was increased expression of a selected ion transport protein (PDZD3) on immunohistochemistry and Western blot in IBS-D compared with controls (P = 0.02). There were no significant differences in mucosal mRNA in 20 IBS-D patients with high compared with 27 IBS-D patients with normal BA excretion. GPBAR1 (P < 0.05) was associated with colonic transit. We concluded that mucosal ion transport mRNA (for several genes and PDZD3 protein) is upregulated and barrier protein mRNA downregulated in IBS-D compared with healthy controls, independent of genotype. There are no differences in gene expression in IBS-D with high compared with normal fecal BA excretion.

  3. Anti-Epithelial Cell Adhesion Molecule Antibodies and the Detection of Circulating Normal-Like Breast Tumor Cells

    PubMed Central

    Kraan, Jaco; Bolt, Joan; van der Spoel, Petra; Elstrodt, Fons; Schutte, Mieke; Martens, John W. M.; Gratama, Jan-Willem; Sleijfer, Stefan; Foekens, John A.

    2009-01-01

    Identification of specific subtypes of circulating tumor cells in peripheral blood of cancer patients can provide information about the biology of metastasis and improve patient management. However, to be effective, the method used to identify circulating tumor cells must detect all tumor cell types. We investigated whether the five subtypes of human breast cancer cells that have been defined by global gene expression profiling—normal-like, basal, HER2-positive, and luminal A and B—were identified by CellSearch, a US Food and Drug Administration–approved test that uses antibodies against the cell surface–expressed epithelial cell adhesion molecule (EpCAM) to isolate circulating tumor cells. We used global gene expression profiling to determine the subtypes of a well-defined panel of 34 human breast cancer cell lines (15 luminal, nine normal-like, five basal-like, and five Her2-positive). We mixed 50-150 cells from 10 of these cell lines with 7.5 mL of blood from a single healthy human donor, and the mixtures were subjected to the CellSearch test to isolate the breast cancer cells. We found that the CellSearch isolation method, which uses EpCAM on the surface of circulating tumor cells for cell isolation, did not recognize, in particular, normal-like breast cancer cells, which in general have aggressive features. New tests that include antibodies that specifically recognize normal-like breast tumor cells but not cells of hematopoietic origin are needed. PMID:19116383

  4. Cl- secretion induced by bile salts. A study of the mechanism of action based on a cultured colonic epithelial cell line.

    PubMed Central

    Dharmsathaphorn, K; Huott, P A; Vongkovit, P; Beuerlein, G; Pandol, S J; Ammon, H V

    1989-01-01

    When applied to the basolateral (serosal) side of the T84 colonic epithelial monolayer, taurodeoxycholate caused net Cl- secretion in a dose-dependent manner with a threshold effect observed at 0.2 mM. In contrast, when applied to the apical (luminal) surface, concentrations of taurodeoxycholate below 1 mM had little or no effect. Only when the concentration of taurodeoxycholate present on the apical side was greater than or equal to 1 mM did apical addition results in an electrolyte transport effect. This apical effect on electrolyte transport was associated with an abrupt increase in the permeability of the monolayer. Cyclic AMP and cyclic GMP in the T84 monolayers were not increased by the bile salt, but in the presence of extracellular Ca2+, free cytosolic Ca2+ increased with a graded dose effect and time course that corresponded approximately to the changes in short circuit current (Isc). The results suggest that luminal bile salts at a relatively high concentration (greater than or equal to 1 mM) increase tight junction permeability. Once tight junction permeability increases, luminal bile salts could reach the basolateral membrane of the epithelial cells where they act to increase free cytosolic Ca2+ from extracellular sources. The resulting increases in free cytosolic Ca2+, rather than in cyclic nucleotides, appear to be involved in transcellular Cl- secretion. PMID:2547841

  5. Histochemical Detection of Collagen Fibers by Sirius Red/Fast Green Is More Sensitive than van Gieson or Sirius Red Alone in Normal and Inflamed Rat Colon

    PubMed Central

    Antonioli, Luca; Pellegrini, Carolina; Blandizzi, Corrado; Dolfi, Amelio; Bernardini, Nunzia

    2015-01-01

    Collagen detection in histological sections and its quantitative estimation by computer-aided image analysis represent important procedures to assess tissue localization and distribution of connective fibers. Different histochemical approaches have been proposed to detect and quantify collagen deposition in paraffin slices with different degrees of satisfaction. The present study was performed to compare the qualitative and quantitative efficiency of three histochemical methods available for collagen staining in paraffin sections of colon. van Gieson, Sirius Red and Sirius Red/Fast Green stainings were carried out for collagen detection and quantitative estimation by morphometric image analysis in colonic specimens from normal rats or animals with 2,4-dinitrobenzenesulfonic acid (DNBS) induced colitis. Haematoxylin/eosin staining was carried out to assess tissue morphology and histopathological lesions. Among the three investigated methods, Sirius Red/Fast Green staining allowed to best highlight well-defined red-stained collagen fibers and to obtain the highest quantitative results by morphometric image analysis in both normal and inflamed colon. Collagen fibers, which stood out against the green-stained non-collagen components, could be clearly appreciated, even in their thinner networks, within all layers of normal or inflamed colonic wall. The present study provides evidence that, as compared with Sirius Red alone or van Gieson staining, the Sirius Red/Fast Green method is the most sensitive, in terms of both qualitative and quantitative evaluation of collagen fibers, in paraffin sections of both normal and inflamed colon. PMID:26673752

  6. TLR4-mediated galectin-1 production triggers epithelial-mesenchymal transition in colon cancer cells through ADAM10- and ADAM17-associated lactate production.

    PubMed

    Park, Ga Bin; Kim, Daejin

    2017-01-01

    Toll-like receptor 4 (TLR4) activation is a key contributor to the carcinogenesis of colon cancer. Overexpression of galectin-1 (Gal-1) also correlates with increased invasive activity of colorectal cancer. Lactate production is a critical predictive factor of risk of metastasis, but the functional relationship between intracellular lactate and Gal-1 expression in TLR4-activated colon cancer remains unknown. In this study, we investigated the underlying mechanism and role of Gal-1 in metastasis and invasion of colorectal cancer (CRC) cells after TLR4 stimulation. Exposure to the TLR4 ligand lipopolysaccharide (LPS) increased expression of Gal-1, induced EMT-related cytokines, triggered the activation of glycolysis-related enzymes, and promoted lactate production. Gene silencing of TLR4 and Gal-1 in CRC cells inhibited lactate-mediated epithelial-mesenchymal transition (EMT) after TLR4 stimulation. Gal-1-mediated activation of a disintegrin and metalloproteinase 10 (ADAM10) and ADAM 17 increased the invasion activity and expression of mesenchymal characteristics in LPS-activated CRC cells. Conversely, inhibition of ADAM10 or ADAM17 effectively blocked the generation of lactate and the migration capacity of LPS-treated CRC cells. Thus, the TLR4/Gal-1 signaling pathway regulates lactate-mediated EMT processes through the activation of ADAM10 and ADAM17 in CRC cells.

  7. Tumor necrosis factor alpha activates transcription of the NADPH oxidase organizer 1 (NOXO1) gene and upregulates superoxide production in colon epithelial cells.

    PubMed

    Kuwano, Yuki; Tominaga, Kumiko; Kawahara, Tsukasa; Sasaki, Hidekazu; Takeo, Keiko; Nishida, Kensei; Masuda, Kiyoshi; Kawai, Tomoko; Teshima-Kondo, Shigetada; Rokutan, Kazuhito

    2008-12-15

    NADPH oxidase 1 (Nox1) is a multicomponent enzyme consisting of p22(phox), Nox organizer 1 (NOXO1), Nox1 activator 1, and Rac1. Interleukin-1beta, flagellin, interferon-gamma, and tumor necrosis factor alpha (TNF-alpha) similarly induced Nox1 in a colon cancer cell line (T84), whereas only TNF-alpha fully induced NOXO1 and upregulated superoxide-producing activity by ninefold. This upregulation was canceled by knockdown of NOXO1 with small interfering RNAs. TNF-alpha rapidly phosphorylated p38 mitogen-activated protein kinase and c-Jun N-terminal kinase 1/2, followed by phosphorylation of c-Jun and c-Fos and appearance of an AP-1 binding activity within 30 min. We cloned the 5' flank of the human NOXO1 gene (-3888 to +263 bp), and found that the region between -585 and -452 bp, which contains consensus elements of YY-1, AP-1, and Ets, and the GC-rich region encoding three putative binding sites for SP-1, was crucial for TNF-alpha-dependent promoter activity. Serial mutation analysis of the elements identified an AP-1 binding site (from -561 to -551 bp, agtAAGtcatg) as a crucial element for TNF-alpha-stimulated transcription of the human NOXO1 gene, which was also confirmed by the AP-1 decoy experiments. Thus, TNF-alpha acts as a potent activator of Nox1-based oxidase in colon epithelial cells, suggesting a potential role of this oxidase in inflammation of the colon.

  8. Comparison study of distinguishing cancerous and normal prostate epithelial cells by confocal and polarization diffraction imaging

    NASA Astrophysics Data System (ADS)

    Jiang, Wenhuan; Lu, Jun Qing; Yang, Li V.; Sa, Yu; Feng, Yuanming; Ding, Junhua; Hu, Xin-Hua

    2016-07-01

    Accurate classification of malignant cells from benign ones can significantly enhance cancer diagnosis and prognosis by detection of circulating tumor cells (CTCs). We have investigated two approaches of quantitative morphology and polarization diffraction imaging on two prostate cell types to evaluate their feasibility as single-cell assay methods toward CTC detection after cell enrichment. The two cell types have been measured by a confocal imaging method to obtain their three-dimensional morphology parameters and by a polarization diffraction imaging flow cytometry (p-DIFC) method to obtain image texture parameters. The support vector machine algorithm was applied to examine the accuracy of cell classification with the morphology and diffraction image parameters. Despite larger mean values of cell and nuclear sizes of the cancerous prostate cells than the normal ones, it has been shown that the morphologic parameters cannot serve as effective classifiers. In contrast, accurate classification of the two prostate cell types can be achieved with high classification accuracies on measured data acquired separately in three measurements. These results provide strong evidence that the p-DIFC method has the potential to yield morphology-related "fingerprints" for accurate and label-free classification of the two prostate cell types.

  9. Comparison study of distinguishing cancerous and normal prostate epithelial cells by confocal and polarization diffraction imaging.

    PubMed

    Jiang, Wenhuan; Lu, Jun Qing; Yang, Li V; Sa, Yu; Feng, Yuanming; Ding, Junhua; Hu, Xin-Hua

    2016-07-01

    Accurate classification of malignant cells from benign ones can significantly enhance cancer diagnosis and prognosis by detection of circulating tumor cells (CTCs). We have investigated two approaches of quantitative morphology and polarization diffraction imaging on two prostate cell types to evaluate their feasibility as single-cell assay methods toward CTC detection after cell enrichment. The two cell types have been measured by a confocal imaging method to obtain their three-dimensional morphology parameters and by a polarization diffraction imaging flow cytometry (p-DIFC) method to obtain image texture parameters. The support vector machine algorithm was applied to examine the accuracy of cell classification with the morphology and diffraction image parameters. Despite larger mean values of cell and nuclear sizes of the cancerous prostate cells than the normal ones, it has been shown that the morphologic parameters cannot serve as effective classifiers. In contrast, accurate classification of the two prostate cell types can be achieved with high classification accuracies on measured data acquired separately in three measurements. These results provide strong evidence that the p-DIFC method has the potential to yield morphology-related “fingerprints” for accurate and label-free classification of the two prostate cell types.

  10. DEK Proto-Oncogene Expression Interferes with the Normal Epithelial Differentiation Program

    PubMed Central

    Wise-Draper, Trisha M.; Morreale, Richard J.; Morris, Teresa A.; Mintz-Cole, Rachael A.; Hoskins, Elizabeth E.; Balsitis, Scott J.; Husseinzadeh, Nader; Witte, David P.; Wikenheiser-Brokamp, Kathryn A.; Lambert, Paul F.; Wells, Susanne I.

    2009-01-01

    Overexpression of the DEK gene is associated with multiple human cancers, but its specific roles as a putative oncogene are not well defined. DEK transcription was previously shown to be induced by the high-risk human papillomavirus (HPV) E7 oncogene via E2F and Rb pathways. Transient DEK overexpression was able to inhibit both senescence and apoptosis in cultured cells. In at least the latter case, this mechanism involved the destabilization of p53 and the decreased expression of p53 target genes. We show here that DEK overexpression disrupts the normal differentiation program in a manner that is independent of either p53 or cell death. DEK expression was distinctly repressed upon the differentiation of cultured primary human keratinocytes, and stable DEK overexpression caused epidermal thickening in an organotypic raft model system. The observed hyperplasia involved a delay in keratinocyte differentiation toward a more undifferentiated state, and expansion of the basal cell compartment was due to increased proliferation, but not apoptosis. These phenotypes were accompanied by elevated p63 expression in the absence of p53 destabilization. In further support of bona fide oncogenic DEK activities, we report here up-regulated DEK protein levels in both human papilloma virus-positive hyperplastic murine skin and a subset of human squamous cell carcinomas. We suggest that DEK up-regulation may contribute to carcinoma development at least in part through increased proliferation and retardation of differentiation. PMID:19036808

  11. The proliferative activity of mammary epithelial cells in normal tissue predicts breast cancer risk in premenopausal women

    PubMed Central

    Huh, Sung Jin; Oh, Hannah; Peterson, Michael A.; Almendro, Vanessa; Hu, Rong; Bowden, Michaela; Lis, Rosina L.; Cotter, Maura B.; Loda, Massimo; Barry, William T.; Polyak, Kornelia; Tamimi, Rulla M.

    2016-01-01

    The frequency and proliferative activity of tissue-specific stem and progenitor cells are suggested to correlate with cancer risk. In this study, we investigated the association between breast cancer risk and the frequency of mammary epithelial cells expressing p27, estrogen receptor (ER), and Ki67 in normal breast tissue. We performed a nested case-control study of 302 women (69 breast cancer cases, 233 controls) who had been initially diagnosed with benign breast disease according to the Nurses’ Health Studies. Immunofluorescence for p27, ER, and Ki67 was performed on tissue microarrays constructed from benign biopsies containing normal mammary epithelium and scored by computational image analysis. We found that the frequency of Ki67+ cells was positively associated with breast cancer risk among premenopausal women (odds ratio [OR]=10.1, 95% confidence interval [CI]=2.12–48.0). Conversely, the frequency of ER+ or p27+ cells was inversely, but not significantly, associated with subsequent breast cancer risk (ER+: OR=0.70, 95% CI=0.33–1.50; p27+: OR=0.89, 95% CI=0.45–1.75). Notably, high Ki67+/low p27+ and high Ki67+/low ER+ cell frequencies were significantly associated with a 5-fold higher risk of breast cancer compared to low Ki67+/low p27+ and low Ki67+/low ER+ cell frequencies, respectively, among premenopausal women (Ki67hi/p27lo: OR=5.08, 95% CI=1.43–18.1; Ki67hi/ERlo: OR=4.68, 95% CI=1.63–13.5). Taken together, our data suggest that the fraction of actively cycling cells in normal breast tissue may represent a marker for breast cancer risk assessment, which may therefore impact the frequency of screening procedures in at-risk women. PMID:26941287

  12. T-cell receptor expression in intestinal intra-epithelial lymphocyte subpopulations of normal and athymic mice.

    PubMed Central

    Viney, J L; MacDonald, T T; Kilshaw, P J

    1989-01-01

    Intra-epithelial lymphocytes (IEL) in murine small intestine were analysed for the presence of cell-surface antigens and T-cell receptor allotype in normal and athymic BALB/c mice by immunoperoxidase histochemistry on frozen sections and immunofluorescence on isolated IEL. In frozen sections, IEL of normal mice were 97.7% CD45+, 93.5% CD3+, 46.2% Thy-1+, 91.1% CD8+, 10.7% CD4+ and 21.1% KJ16+ (V beta 8.1 and 8.2). FACS analysis of isolated IEL confirmed the level of KJ16 expression and also demonstrated that 25% of IEL were F23.1+ (V beta 8.1-8.3). Immunofluorescent double-staining revealed a skewed distribution of T-cell receptor (TcR) expression on Thy-1+ and Thy-1- IEL. KJ16 and F23.1 were expressed on 25.9% and 32.7% of Thy-1+ IEL, respectively; however, the frequency of V beta 8 expression was diminished on Thy-1- IEL (4.1% KJ16+ and 12.1% F23.1+). IEL are present in athymic mice, but at reduced levels. In frozen sections these cells were 91.9% CD45+, 69.5% CD3+, less than 1% Thy-1+, 83.6% CD8+, less than 1% CD4+ and less than 1% KJ16+. Thus it appears that in normal mice there may be two distinct lineages of IEL, a thymus-dependent Thy-1+ population which utilizes the alpha beta T-cell receptor and a thymus-independent Thy-1- population (represented in athymic mice), which may possibly utilize the alternative gamma delta TcR. Images Figure 1 PMID:2565884

  13. Quantitation of chemopreventive synergism between (-)-epigallocatechin-3-gallate and curcumin in normal, premalignant and malignant human oral epithelial cells.

    PubMed

    Khafif, A; Schantz, S P; Chou, T C; Edelstein, D; Sacks, P G

    1998-03-01

    An in vitro model for oral cancer was used to examine the growth inhibitory effects of chemopreventive agents when used singly and in combination. The model consists of primary cultures of normal oral epithelial cells, newly established cell lines derived from dysplastic leukoplakia and squamous cell carcinoma. Two naturally occurring substances, (-)-epigallocatechin-3-gallate (EGCG) from green tea and curcumin from the spice turmeric were tested. Cells were treated singly and in combination and effects on growth determined in 5-day growth assays and by cell cycle analysis. Effective dose 50s and the combination index were calculated with the computerized Chou-Talalay method which is based on the median-effect principle. Agents were shown to differ in their inhibitory potency. EGCG was less effective with cell progression; the cancer cells were more resistant than normal or dysplastic cells. In contrast, curcumin was equally effective regardless of the cell type tested. Cell cycle analysis indicated that EGCG blocked cells in G1, whereas curcumin blocked cells in S/G2M. The combination of both agents showed synergistic interactions in growth inhibition and increased sigmoidicity (steepness) of the dose-effect curves, a response that was dose and cell type dependent. Combinations allowed for a dose reduction of 4.4-8.5-fold for EGCG and 2.2-2.8-fold for curcumin at ED50s as indicated by the dose reduction index (DRI). Even greater DRI values were observed above ED50 levels. Our results demonstrate that this model which includes normal, premalignant and malignant oral cells can be used to analyse the relative potential of various chemopreventive agents. Two such naturally-occurring agents, EGCG and curcumin, were noted to inhibit growth by different mechanisms, a factor which may account for their demonstrable interactive synergistic effect.

  14. Endogenous optical biomarkers of normal and human papillomavirus immortalized epithelial cells.

    PubMed

    Mujat, Claudia; Greiner, Cherry; Baldwin, Amy; Levitt, Jonathan M; Tian, Fenghua; Stucenski, Lee A; Hunter, Martin; Kim, Young L; Backman, Vadim; Feld, Michael; Münger, Karl; Georgakoudi, Irene

    2008-01-15

    Cellular transformation is associated with a number of phenotypic, cell biological, biochemical and metabolic alterations. The detection and classification of morphological cellular abnormalities represents the foundation of classical histopathology and remains an important mainstay in the clinic. More recently, significant effort is being expended towards the development of noninvasive modalities for the detection of cancer at an early stage, when therapeutic interventions are highly successful. Methods that rely on the detection of optical signatures represent one class of such approaches that have yielded promising results. In our study, we have applied two spectroscopic imaging approaches to systematically identify in a quantitative manner the fluorescence and light scattering signatures of subcellular abnormalities that are associated with cellular transformation. Notably, we find that tryptophan images reveal not only intensity but also localization differences between normal and human papillomavirus immortalized cells, possibly originating from changes in the expression, 3D packing and organization of proteins and protein-rich subcellular organelles. Additionally, we detect alterations in cellular metabolism through quantitative evaluation of the NADH, FAD fluorescence and the corresponding redox ratio. Finally, we use light scattering spectroscopy to identify differences in nuclear morphology and subcellular organization that occur from the nanometer to the micrometer scale. Thus, these optical approaches provide complementary biomarkers based on endogenous fluorescence and scattering cellular changes that occur at the molecular, biochemical and morphological level. Since they obviate the need for staining and tissue removal and can be easily combined, they provide desirable options for further clinical development and assessment. Copyright 2007 Wiley-Liss, Inc.

  15. Nuclear location of tumor suppressor protein maspin inhibits proliferation of breast cancer cells without affecting proliferation of normal epithelial cells

    PubMed Central

    2014-01-01

    Background Maspin, which is classified as a tumor suppressor protein, is downregulated in many types of cancer. Several studies have suggested potential anti-proliferative activity of maspin as well as sensitizing activity of maspin for therapeutic cytotoxic agents in breast cancer tissue culture and animal models. All of the experimental data gathered so far have been based on studies with maspin localized cytoplasmically, while maspin in breast cancer tumor cells may be located in the cytoplasm, nucleus or both. In this study, the effect of maspin cytoplasmic and nuclear location and expression level on breast cancer proliferation and patient survival was studied. Methods Tissue sections from 166 patients with invasive ductal breast cancer were stained by immunohistochemistry for maspin and Ki-67 protein. The localization and expression level of maspin were correlated with estimated patient overall survival and percent of Ki-67-positive cells. In further studies, we created constructs for transient transfection of maspin into breast cancer cells with targeted cytoplasmic and nuclear location. We analyzed the effect of maspin location in normal epithelial cell line MCF10A and three breast cancer cell lines - MCF-7, MDA-MB-231 and SKBR-3 - by immunofluorescence and proliferation assay. Results We observed a strong positive correlation between moderate and high nuclear maspin level and survival of patients. Moreover, a statistically significant negative relationship was observed between nuclear maspin and Ki-67 expression in patients with invasive ductal breast cancer. Spearman’s correlation analysis showed a negative correlation between level of maspin localized in nucleus and percentage of Ki-67 positive cells. No such differences were observed in cells with cytoplasmic maspin. We found a strong correlation between nuclear maspin and loss of Ki-67 protein in breast cancer cell lines, while there was no effect in normal epithelial cells from breast. The anti

  16. Effects of supplemental vitamin D and calcium on normal colon tissue and circulating biomarkers of risk for colorectal neoplasms.

    PubMed

    Bostick, Roberd M

    2015-04-01

    This brief review, based on an invited presentation at the 17th Workshop on Vitamin D, is to summarize a line of the author's research that has been directed at the intertwined missions of clarifying and/or developing vitamin D and calcium as preventive agents against colorectal cancer in humans, understanding the mechanisms by which these agents may reduce risk for the disease, and developing 'treatable' biomarkers of risk for colorectal cancer. The biological plausibility and observational and clinical trial evidence for vitamin D and calcium in reducing risk for colorectal neoplasms, the development of pre-neoplastic biomarkers of risk for colorectal neoplasms, and the clinical trial findings from the author's research group on the efficacy of vitamin D and calcium in modulating these biomarkers are summarized. Regarding the latter, we tested the efficacy of 800 IU (20μg) of vitamin D3 and 2.0g of calcium daily, alone and combined vs. placebo over 6 months on modulating normal colon tissue and circulating hypothesis-based biomarkers of risk for colorectal neoplasms in a randomized, double-blind, placebo-controlled, 2×2 factorial design clinical trial (n=92). The tissue-based biomarkers were measured in biopsies of normal-appearing rectal mucosa using immunohistochemistry with quantitative image analysis, and a panel of circulating inflammation markers was measured using enzyme-linked immunoassays (ELISA). Statistically significant proportional tissue increases in the vitamin D group relative to the placebo group were found in bax (51%), p21 (141%), APC (48%), E-cadherin (78%), MSH2 (179%), the CaSR (39%), and CYP27B1 (159%). In blood, there was a 77% statistically significant decrease in a summary inflammation z-score. The findings for calcium were similar to those for vitamin D. These findings indicate that supplemental vitamin D3 or calcium can favorably modulate multiple normal colon tissue and circulating hypothesis-based biomarkers of risk for colorectal

  17. Effects of Supplemental Vitamin D and Calcium on Normal Colon Tissue and Circulating Biomarkers of Risk for Colorectal Neoplasms

    PubMed Central

    Bostick, Roberd M.

    2015-01-01

    This brief review, based on an invited presentation at the 17th Workshop on Vitamin D, is to summarize a line of the author’s research that has been directed at the intertwined missions of clarifying and/or developing vitamin D and calcium and as preventive agents against colorectal cancer in humans, understanding the mechanisms by which these agents may reduce risk for the disease, and developing ‘treatable’ biomarkers of risk for colorectal cancer. The biological plausibility and observational and clinical trial evidence for vitamin D and calcium in reducing risk for colorectal neoplasms, the development of pre-neoplastic biomarkers of risk for colorectal neoplasms, and the clinical trial findings from the author’s research group on the efficacy of vitamin D and calcium in modulating these biomarkers are summarized. Regarding the latter, we tested the efficacy of 800 IU (20 µg) of vitamin D3 and 2.0g of calcium daily, alone and combined vs. placebo over 6 months on modulating normal colon tissue and circulating hypothesis-based biomarkers of risk for colorectal neoplasms in a randomized, double-blind, placebo-controlled, 2×2 factorial design clinical trial (n = 92). The tissue-based biomarkers were measured in biopsies of normal-appearing rectal mucosa using immunohistochemistry with quantitative image analysis, and a panel of circulating inflammation markers was measured using enzyme-linked immunoassays (ELISA). Statistically significant proportional tissue increases in the vitamin D group relative to the placebo group were found in bax (51%), p21 (141%), APC (48%), E-cadherin (78%), MSH2 (179%), the CaSR (39%), and CYP27B1 (159%). In blood, there was a 77% statistically significant decrease in a summary inflammation z-score. The findings for calcium were similar to those for vitamin D. These findings indicate that supplemental vitamin D3 or calcium can favorably modulate multiple normal colon tissue and circulating hypothesis-based biomarkers of risk

  18. Newly Diagnosed Colonic Adenocarcinoma: The Presenting Sign in a Young Woman with Undiagnosed Crohn's Disease in the Absence of Primary Sclerosing Cholangitis and a Normal Microsatellite Instability Profile

    PubMed Central

    2017-01-01

    Ulcerative colitis has long been linked with an increased risk for colonic adenocarcinoma, whereas Crohn's disease (CD) has recently been reported to pose a similar increased risk. We report a 33-year-old healthy female with no family history who presented with abdominal pain and a colon mass. Histopathology revealed a moderately differentiated adenocarcinoma extending through the muscularis propria with metastatic lymph nodes and intact mismatch repair proteins by immunohistochemical expression and gene sequencing. The nonneoplastic grossly uninvolved background mucosa showed marked crypt distortion, crypt abscesses, CD-like lymphoid hyperplasia, transmural inflammation, and reactive epithelial atypia. Additional patient questioning revealed frequent loose stools since she was a teenager leading to diagnosis of a previously undiagnosed CD without primary sclerosing cholangitis (PSC). The adenocarcinoma is suspected to be related to the underlying CD. Newly diagnosed adenocarcinoma in a young female as the presenting sign for CD in the absence of PSC is extremely rare. PMID:28255489

  19. SRC is dephosphorylated at tyrosine 530 in human colon carcinomas.

    PubMed

    Zhu, Shudong; Bjorge, Jeffrey D; Fujita, Donald J

    2011-09-01

    Src is a protein tyrosine kinase that plays important roles in cancer development, and Src kinase activity has been found to be elevated in several types of cancers. However, the cause of the elevation of Src kinase activity in the majority of human colon carcinomas is still largely unknown. We aim at finding the cause of elevated Src kinase activity in human colon carcinomas. We employed normal colon epithelial FHC cells and examined Src activation in human colon carcinoma specimens from 8 patients. Protein expression levels were determined by Western blotting, and the activity of Src kinase by kinase assay. Actin levels were different between tumor and normal tissues, demonstrating the complexities and inhomogeneities of the tissue samples. Src kinase activities were increased in the majority of the colon carcinomas as compared with normal colon epithelial cells (range 13-29). Src protein levels were reduced in the colon carcinomas. Src Y530 phosphorylation levels were reduced to a higher extent than protein levels in the carcinomas. The results suggest that Src specific activities were highly increased in human colon carcinomas; phosphorylation at Src Y530 was reduced, contributing to the highly elevated Src specific activity and Src kinase activity.

  20. Effects of high levels of dietary zinc oxide on ex vivo epithelial histamine response and investigations on histamine receptor action in the proximal colon of weaned piglets.

    PubMed

    Kröger, S; Pieper, R; Aschenbach, J R; Martin, L; Liu, P; Rieger, J; Schwelberger, H G; Neumann, K; Zentek, J

    2015-11-01

    The aim of the study was to identify the effect of high dietary zinc oxide (ZnO) levels on the histamine-induced secretory-type response and histamine metabolism in the porcine proximal colon. After weaning at d 26, 3 diets with low (LZn), normal (NZn), and high (HZn) concentrations of zinc (57, 164, or 2,425 mg/kg) were fed to a total of 120 piglets. Digesta and tissue samples were taken from the ascending colon after 7 ± 1, 14 ± 1, 21 ± 1, and 28 ± 1 d. Partially stripped tissue was mounted in Ussing chambers, and histamine was applied either to the serosal or mucosal compartments. Tissue was pretreated with or without aminoguanidine and amodiaquine to block the histamine-degrading enzymes diamine oxidase (DAO) and histamine -methyltransferase (HMT), respectively. Gene expression and catalytic activity of DAO and HMT in the tissue were analyzed. The numbers of mast cells were determined in tissue samples, and histamine concentration was measured in the colon digesta. Colon tissue from another 12 piglets was used for functional studies on histamine H and H receptors by using the neuronal conduction blocker tetrodotoxin (TTX) and the H and H receptor blocker chloropyramine and famotidine, respectively. After serosal histamine application to colonic tissue in Ussing chambers, the change of short-circuit current (Δ) was not affected by pretreatment and was not different between Zn feeding groups. The Δ after mucosal histamine application was numerically lower ( = 0.168) in HZn compared to LZn and NZn pigs. Mast cell numbers increased from 32 to 46 d of life ( < 0.05). Further studies elucidated that the serosal histamine response was partly inhibited by chloropyramine or famotidine ( < 0.01). The response to mucosal histamine tended to be decreased when chloropyramine but not famotidine was applied from either the serosal or the mucosal side ( = 0.055). Tetrodotoxin alone or in combination with chloropyramine resulted in a similar reduction in the mucosal

  1. [Influence of moxibustion with different duration on colonic epithelial structure, serum inflammatory cytokines, and intestinal mucosa inflammatory cell signal transduction pathways].

    PubMed

    Ma, Tie-Ming; Han, Yang; Ma, Xian-De; Zeng, Xiao-Xia; Ge, Wei

    2014-02-01

    H. E. staining, and by electron microscope, respectively.The colonic mucosal structure was observed by light microscope after H. E. staining, and by electron microscope, respectively. In comparison with the blank control group, the Disease Activity Index (DAI), serum IL-8 content, colonic TLR-9 andResults - In comparison with the blank control group, the Disease Activity Index (DAI), serum IL-8 content, colonic TLR-9 and NE-KB p 65 protein expression levels were significantly increased in the model group ( P<0. 05), and serum IL-la content wasNF-mB p 65 protein expression levels were significantly increased in the model group ( P < 0.05), and serum IL-10 content was notably decreased in the model group (P < 0.05). While in comparison with the model group, the DAI, serum IL-8 content, co-notably decreased in the model group (P<0.05). While in comparison with the model group, the DAI, serum IL-8 content, coIonic TLR-9 and NE-kappaB p 65 protein expression levels in the 3-cones-M, 6-cones-M and 9-cones-M groups were remarkably down-lonic TLR-9 and NF-mB p 65 protein expression levels in the 3-cones-M. 6-cones-M and 9-cones-M groups were remarkably down- regulated (P < 0.05), and serum IL-10 contents considerably up-regulated in the three moxibustion groups (P < 0.05). No significant differences were found among the three moxibustion groups in the DAI (P > 0.05). The serum IL-8 contents were significantly lower and serum IL-10 contents were considerably higher in the 6-cones-M and 9-cones-M groups than in the 3-cones-M group (P < 0.05). The changes of colonic TLR-9 and NF-kappaB p 65 protein expression were more remarkable in the 9-cones-M group than in the 3-cones-M and 6-cones-M groups (P < 0.05). Results of H.E. staining and electron microscopy showed that in the model group, mucosal injury, partial disorganization of the glandular organ, edema and congestion and inflammatory cell infiltration, mucosal epithelial microvili injury with disordered arrangement, etc

  2. Three-Dimensional Organotypic Co-Culture Model of Intestinal Epithelial Cells and Macrophages to Study "Salmonella Enterica" Colonization Patterns

    NASA Technical Reports Server (NTRS)

    Ott, Mark; Yang, J; Barilla, J.; Crabbe, A.; Sarker, S. F.; Liu, Y.

    2017-01-01

    Three-dimensional/3-D organotypic models of human intestinal epithelium mimic the differentiated form and function of parental tissues often not exhibited by 2-D monolayers and respond to Salmonella in ways that reflect in vivo infections. To further enhance the physiological relevance of 3-D models to more closely approximate in vivo intestinal microenvironments during infection, we developed and validated a novel 3-D intestinal co-culture model containing multiple epithelial cell types and phagocytic macrophages, and applied to study enteric infection by different Salmonella pathovars.

  3. Ionizing Irradiation Not Only Inactivates Clonogenic Potential in Primary Normal Human Diploid Lens Epithelial Cells but Also Stimulates Cell Proliferation in a Subset of This Population

    PubMed Central

    Fujimichi, Yuki; Hamada, Nobuyuki

    2014-01-01

    Over the past century, ionizing radiation has been known to induce cataracts in the crystalline lens of the eye, but its mechanistic underpinnings remain incompletely understood. This study is the first to report the clonogenic survival of irradiated primary normal human lens epithelial cells and stimulation of its proliferation. Here we used two primary normal human cell strains: HLEC1 lens epithelial cells and WI-38 lung fibroblasts. Both strains were diploid, and a replicative lifespan was shorter in HLEC1 cells. The colony formation assay demonstrated that the clonogenic survival of both strains decreases similarly with increasing doses of X-rays. A difference in the survival between two strains was actually insignificant, although HLEC1 cells had the lower plating efficiency. This indicates that the same dose inactivates the same fraction of clonogenic cells in both strains. Intriguingly, irradiation enlarged the size of clonogenic colonies arising from HLEC1 cells in marked contrast to those from WI-38 cells. Such enhanced proliferation of clonogenic HLEC1 cells was significant at ≥2 Gy, and manifested as increments of ≤2.6 population doublings besides sham-irradiated controls. These results suggest that irradiation of HLEC1 cells not only inactivates clonogenic potential but also stimulates proliferation of surviving uniactivated clonogenic cells. Given that the lens is a closed system, the stimulated proliferation of lens epithelial cells may not be a homeostatic mechanism to compensate for their cell loss, but rather should be regarded as abnormal. This is because these findings are consistent with the early in vivo evidence documenting that irradiation induces excessive proliferation of rabbit lens epithelial cells and that suppression of lens epithelial cell divisions inhibits radiation cataractogenesis in frogs and rats. Thus, our in vitro model will be useful to evaluate the excessive proliferation of primary normal human lens epithelial cells that

  4. Enhanced TLR4 Expression on Colon Cancer Cells After Chemotherapy Promotes Cell Survival and Epithelial-Mesenchymal Transition Through Phosphorylation of GSK3β.

    PubMed

    Chung, Yoon Hee; Kim, Daejin

    2016-07-01

    Phosphorylation of glycogen synthase kinase 3β (GSK3β) by phosphatidyl-inositide 3-kinase (PI3K)/protein kinase B (AKT) or inhibition of GSK3β with small-molecule inhibitor attenuates cell survival and proliferation and increases apoptosis in most cancer cell lines. In this study, we investigated the role of phosphorylated GSK3β activated by enhanced toll-like receptor 4 (TLR4) expression in drug-treated colon cancer cells as a model of post-chemotherapy cancer cells. The effect of TLR4 stimulation on metastasis and apoptosis in drug-exposed colon cancer cells was determined by real-time polymerase chain reaction (PCR) and immunoblotting. Despite the induction of apoptosis after treatment with oxaliplatin and 5-fluorouracil, lipopolysaccharide (LPS) stimulation via increased TLR4 in drug-treated cancer cells effectively inhibited apoptosis through up-regulation of expression of anti-apoptosis-related B-cell lymphoma 2 (BCL2) family proteins [X-linked inhibitor of apoptosis protein (XIAP), BCL2, and survivin] and drug-resistance proteins [multidrug-resistance protein 1 (MDR1), multidrug resistance-associated protein (MRP)1/2/3]. LPS-mediated signaling in drug-treated cancer cells elevated the expression of phosphorylated GSK3β, extracellular signal-regulated kinase (ERK), and the p65 subunit of nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB). Pharmacological inhibition of GSK3β (using SB216763) reduced phosphorylation of GSK3β, re-activated caspase-dependent apoptosis, and blocked the expression of cancer stem cell markers and invasive characteristics in LPS-stimulated drug-treated cells. In addition, the ERK-specific inhibitor, PD98059, triggered the apoptosis of TLR4-activated drug-exposed colon cancer cells, whereas there was no effect on the expression of epithelial-mesenchymal transition markers or GSK3β phosphorylation. These results suggest that TLR4-induced GSK3β and ERK phosphorylation independently controls cancer cell

  5. Actin stress fiber disruption and tropomysin isoform switching in normal thyroid epithelial cells stimulated by thyrotropin and phorbol esters

    SciTech Connect

    Roger, P.P.; Rickaert, F.; Lamy, F.; Authelet, M.; Dumont, J.E. )

    1989-05-01

    Thyrotropin (TSH), through cyclic AMP, promotes both proliferation and differentiation expression in dog thyroid epithelial cells in primary culture, whereas the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) also stimulates proliferation but antagonizes differentiating effects of TSH. In this study, within 20 min both factors triggered the disruption of actin-containing stress fibers. This process preceded distinct morphological changes: cytoplasmic retraction and arborization in response to TSH and cyclic AMP, cell shape distortion, and increased motility in response to TPA and diacylglycerol. TSH and TPA also induced a marked decrease in the synthesis of three high M{sub r} tropomyosin isoforms, which were not present in dog thyroid tissue but appeared in culture during cell spreading and stress fiber formation. The tropomyosin isoform switching observed here closely resembled similar processes in various cells transformed by oncogenic viruses. However, it did not correlate with differentiation or mitogenic activation. Contrasting with current hypothesis on this process in transformed cells, tropomyosin isoform switching in normal thyroid cells was preceded and thus might be caused by early disruption of stress fibers.

  6. Hypoxia primes human normal prostate epithelial cells and cancer cell lines for the NLRP3 and AIM2 inflammasome activation

    PubMed Central

    Panchanathan, Ravichandran; Liu, Hongzhu; Choubey, Divaker

    2016-01-01

    The molecular mechanisms by which hypoxia contributes to prostatic chronic inflammation (PCI) remain largely unknown. Because hypoxia stimulates the transcriptional activity of NF-κB, which “primes” cells for inflammasome activation by inducing the expression of NLRP3 or AIM2 receptor and pro-IL-1β, we investigated whether hypoxia could activate the NLRP3 and AIM2 inflammasome in human normal prostate epithelial cells (PrECs) and cancer cell lines. Here we report that hypoxia (1% O2) treatment of PrECs, prostate cell lines, and a macrophage cell line (THP-1) increased the levels of NLRP3, AIM2, and pro-IL-1β. Further, hypoxia in cells potentiated activation of the NLRP3 and AIM2 inflammasome activity. Notably, hypoxia “primed” cells for NLRP3 and AIM2 inflammasome activation through stimulation of the NF-κB activity. Our observations support the idea that hypoxia in human prostatic tumors contributes to PCI, in part, by priming cells for the activation of NLRP3 and AIM2 inflammasome. PMID:27058421

  7. Simian virus 40 large tumor antigen-immortalized normal human liver epithelial cells express hepatocyte characteristics and metabolize chemical carcinogens.

    PubMed Central

    Pfeifer, A M; Cole, K E; Smoot, D T; Weston, A; Groopman, J D; Shields, P G; Vignaud, J M; Juillerat, M; Lipsky, M M; Trump, B F

    1993-01-01

    Normal human liver tissue and cultured human hepatocytes are valuable models to study xenobiotic metabolism and toxicity, but they only have a limited in vitro life-span and are not readily available. This report describes the establishment of replicative cultures of human adult liver epithelial cells in serum-free medium. The longevity of three of these cultures, derived from different donors, was extended by introduction of the simian virus 40 large T antigen gene. Two cell lines, THLE-2 and -3, established with a recombinant simian virus 40 large T antigen virus have undergone > 100 population doublings, are nontumorigenic when injected into athymic nude mice, have near-diploid karyotypes, and do not express alpha-fetoprotein. The cells express cytokeratin 18 and albumin in early passage, whereas higher-passage cells in logarithmic-phase growth also express cytokeratin 19. THLE-2 and -3 cells metabolize benzo[a]pyrene, N-nitrosodimethylamine, and aflatoxin B1 to their ultimate carcinogenic metabolites that adduct DNA, which indicates functional cytochrome P450 pathways. Other enzymes involved in metabolism of chemical carcinogens, such as epoxide hydrolase, NADPH cytochrome P450 reductase, superoxide dismutase, catalase, glutathione S-transferases, and glutathione peroxidase are also retained by THLE cells. Thus, these immortalized human liver cells constitute an in vitro model for pharmacotoxicological studies and for the investigation of etiology and pathogenesis of human hepatocellular carcinoma. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7685115

  8. Respiratory Syncytial Virus Inhibits Ciliagenesis in Differentiated Normal Human Bronchial Epithelial Cells: Effectiveness of N-Acetylcysteine

    PubMed Central

    Mata, Manuel; Sarrion, Irene; Armengot, Miguel; Carda, Carmen; Martinez, Isidoro; Melero, Jose A.; Cortijo, Julio

    2012-01-01

    Persistent respiratory syncytial virus (RSV) infections have been associated with the exacerbation of chronic inflammatory diseases, including chronic obstructive pulmonary disease (COPD). This virus infects the respiratory epithelium, leading to chronic inflammation, and induces the release of mucins and the loss of cilia activity, two factors that determine mucus clearance and the increase in sputum volume. These alterations involve reactive oxygen species-dependent mechanisms. The antioxidant N-acetylcysteine (NAC) has proven useful in the management of COPD, reducing symptoms, exacerbations, and accelerated lung function decline. NAC inhibits RSV infection and mucin release in human A549 cells. The main objective of this study was to analyze the effects of NAC in modulating ciliary activity, ciliagenesis, and metaplasia in primary normal human bronchial epithelial cell (NHBEC) cultures infected with RSV. Our results indicated that RSV induced ultrastructural abnormalities in axonemal basal bodies and decreased the expression of β-tubulin as well as two genes involved in ciliagenesis, FOXJ1 and DNAI2. These alterations led to a decrease in ciliary activity. Furthermore, RSV induced metaplastic changes to the epithelium and increased the number of goblet cells and the expression of MUC5AC and GOB5. NAC restored the normal functions of the epithelium, inhibiting ICAM1 expression, subsequent RSV infection through mechanisms involving nuclear receptor factor 2, and the expression of heme oxygenase 1, which correlated with the restoration of the antioxidant capacity, the intracellular H2O2 levels and glutathione content of NHBECs. The results presented in this study support the therapeutic use of NAC for the management of chronic respiratory diseases, including COPD. PMID:23118923

  9. Respiratory syncytial virus inhibits ciliagenesis in differentiated normal human bronchial epithelial cells: effectiveness of N-acetylcysteine.

    PubMed

    Mata, Manuel; Sarrion, Irene; Armengot, Miguel; Carda, Carmen; Martinez, Isidoro; Melero, Jose A; Cortijo, Julio

    2012-01-01

    Persistent respiratory syncytial virus (RSV) infections have been associated with the exacerbation of chronic inflammatory diseases, including chronic obstructive pulmonary disease (COPD). This virus infects the respiratory epithelium, leading to chronic inflammation, and induces the release of mucins and the loss of cilia activity, two factors that determine mucus clearance and the increase in sputum volume. These alterations involve reactive oxygen species-dependent mechanisms. The antioxidant N-acetylcysteine (NAC) has proven useful in the management of COPD, reducing symptoms, exacerbations, and accelerated lung function decline. NAC inhibits RSV infection and mucin release in human A549 cells. The main objective of this study was to analyze the effects of NAC in modulating ciliary activity, ciliagenesis, and metaplasia in primary normal human bronchial epithelial cell (NHBEC) cultures infected with RSV. Our results indicated that RSV induced ultrastructural abnormalities in axonemal basal bodies and decreased the expression of β-tubulin as well as two genes involved in ciliagenesis, FOXJ1 and DNAI2. These alterations led to a decrease in ciliary activity. Furthermore, RSV induced metaplastic changes to the epithelium and increased the number of goblet cells and the expression of MUC5AC and GOB5. NAC restored the normal functions of the epithelium, inhibiting ICAM1 expression, subsequent RSV infection through mechanisms involving nuclear receptor factor 2, and the expression of heme oxygenase 1, which correlated with the restoration of the antioxidant capacity, the intracellular H(2)O(2) levels and glutathione content of NHBECs. The results presented in this study support the therapeutic use of NAC for the management of chronic respiratory diseases, including COPD.

  10. Intrinsic resistance triggered under acid loading within normal esophageal epithelial cells: NHE1- and ROS-mediated survival.

    PubMed

    Park, Sun Young; Lee, Yeon Joo; Cho, Eun Jeong; Shin, Chang Yell; Sohn, Uy Dong

    2015-07-01

    The transition to a pathological phenotype such as Barrett's esophagus occurs via induction of resistance upon repeated contact with gastric refluxate in esophagus. This study examined the molecular changes within normal esophageal epithelial cells (EECs) under short-term acid loading and the role of these changes in defensive resistance against acidic cytotoxicity. After primary cultured EECs were exposed to pH 4-acidified medium (AM4), cell viability was determined by the MTT assay. Reactive oxygen species (ROS) and NAD(P)H oxidase (NOX) activity were measured. Activation of the mitogen-activated protein kinases (MAPKs) MEK/ERK1/2, p38 and JNK; phosphoinositol-3-kinase (PI3K)/Akt, and nuclear factor-kappa B (NF-κB) were detected by Western blot analysis or immunofluorescence staining. AM4 incubation induced intracellular ROS generation accompanied by increase in NOX activity, which was further increased by Na(+) /H(+) exchange-1 (NHE1)-dependent inhibition but was prevented by inhibition of NOX or mitochondria complex I. AM4 also induced phosphorylation of MEK/ERK1/2, p38 MAPK, PI3K/Akt, and nuclear translocation of NF-κB, and all these effects, except for p38 MAPK phosphorylation, were abolished by inhibition of ROS. ROS-dependent PI3K/Akt activation, which mediates NF-κB nuclear translocation, was inhibited by protein tyrosine kinase (PTK) inhibitors and NHE1-specific inhibitor. All inhibitors of NHE, ROS, PTK, PI3K, or NF-κB further decreased AM4-induced cell viability. Acid loading in the presence of NHE1-dependent protection induced ROS generation by activating NOX and mitochondria complex I, which stimulated PTK/PI3K/Akt/NF-κB-dependent survival in EEC. Our data indicate that normal EEC initially respond to acid loading through intrinsic survival activation.

  11. Identification of a developmental gene expression signature, including HOX genes, for the normal human colonic crypt stem cell niche: overexpression of the signature parallels stem cell overpopulation during colon tumorigenesis.

    PubMed

    Bhatlekar, Seema; Addya, Sankar; Salunek, Moreh; Orr, Christopher R; Surrey, Saul; McKenzie, Steven; Fields, Jeremy Z; Boman, Bruce M

    2014-01-15

    Our goal was to identify a unique gene expression signature for human colonic stem cells (SCs). Accordingly, we determined the gene expression pattern for a known SC-enriched region--the crypt bottom. Colonic crypts and isolated crypt subsections (top, middle, and bottom) were purified from fresh, normal, human, surgical specimens. We then used an innovative strategy that used two-color microarrays (∼18,500 genes) to compare gene expression in the crypt bottom with expression in the other crypt subsections (middle or top). Array results were validated by PCR and immunostaining. About 25% of genes analyzed were expressed in crypts: 88 preferentially in the bottom, 68 in the middle, and 131 in the top. Among genes upregulated in the bottom, ∼30% were classified as growth and/or developmental genes including several in the PI3 kinase pathway, a six-transmembrane protein STAMP1, and two homeobox (HOXA4, HOXD10) genes. qPCR and immunostaining validated that HOXA4 and HOXD10 are selectively expressed in the normal crypt bottom and are overexpressed in colon carcinomas (CRCs). Immunostaining showed that HOXA4 and HOXD10 are co-expressed with the SC markers CD166 and ALDH1 in cells at the normal crypt bottom, and the number of these co-expressing cells is increased in CRCs. Thus, our findings show that these two HOX genes are selectively expressed in colonic SCs and that HOX overexpression in CRCs parallels the SC overpopulation that occurs during CRC development. Our study suggests that developmental genes play key roles in the maintenance of normal SCs and crypt renewal, and contribute to the SC overpopulation that drives colon tumorigenesis.

  12. HO-1 inhibits IL-13-induced goblet cell hyperplasia associated with CLCA1 suppression in normal human bronchial epithelial cells.

    PubMed

    Mishina, Kei; Shinkai, Masaharu; Shimokawaji, Tadasuke; Nagashima, Akimichi; Hashimoto, Yusuke; Inoue, Yoriko; Inayama, Yoshiaki; Rubin, Bruce K; Ishigatsubo, Yoshiaki; Kaneko, Takeshi

    2015-12-01

    Mucus hypersecretion and goblet cell hyperplasia are common features that characterize asthma. IL-13 increases mucin (MUC) 5AC, the major component of airway mucus, in airway epithelial cells. According to the literature, IL-13 receptor activation leads to STAT6 activation and consequent induction of chloride channel accessory 1 (CLCA1) gene expression, associated with the induction of MUC5AC. Heme oxygenase-1 (HO-1) is an enzyme that catalyzes oxidation of heme to biliverdin, and has anti-inflammatory and anti-oxidant properties. We examined the effects of HO-1 on mucin production and goblet cell hyperplasia induced by IL-13. Moreover, we assessed the cell signaling intermediates that appear to be responsible for mucin production. Normal human bronchial epithelial (NHBE) cells were grown at air liquid interface (ALI) in the presence or absence of IL-13 and hemin, a HO-1 inducer, for 14 days. Protein concentration was analyzed using ELISA, and mRNA expression was examined by real-time PCR. Histochemical analysis was performed using HE staining, andWestern blotting was performed to evaluate signaling transduction pathway. Hemin (4 μM) significantly increased HO-1 protein expression (p b 0.01) and HO-1 mRNA expression (p b 0.001). IL-13 significantly increased goblet cells, MUC5AC protein secretion (p b 0.01) and MUC5AC mRNA (p b 0.001), and these were decreased by hemin by way of HO-1. Tin protoporphyrin (SnPP)-IX, a HO-1 inhibitor, blocked the effect of hemin restoring MUC5AC protein secretion (p b 0.05) and goblet cell hyperplasia. Hemin decreased the expression of CLCA1 mRNA (p b 0.05) and it was reversed by SnPP-IX, but could not suppress IL-13-induced phosphorylation of STAT6 or SAM pointed domain-containing ETS transcription factor (SPDEF) and Forkhead box A2 (FOXA2) mRNA expression. In summary, HO-1 overexpression suppressed IL-13-induced goblet cell hyperplasia and MUC5AC production, and involvement of CLCA1 in the mechanism was suggested.

  13. MicroRNA profiles in colorectal carcinomas, adenomas and normal colonic mucosa: variations in miRNA expression and disease progression.

    PubMed

    Slattery, Martha L; Herrick, Jennifer S; Pellatt, Daniel F; Stevens, John R; Mullany, Lila E; Wolff, Erica; Hoffman, Michael D; Samowitz, Wade S; Wolff, Roger K

    2016-03-01

    MiRNAs are small, non-protein-coding RNA molecules that regulate gene expression either by post-transcriptionally suppressing mRNA translation or by mRNA degradation. We examine differentially expressed miRNAs in colorectal carcinomas, adenomas and normal colonic mucosa. Data come from population-based studies of colorectal cancer conducted in Utah and the Kaiser Permanente Medical Care Program. A total of 1893 carcinoma/normal-paired samples and 290 adenoma tissue samples were run on the Agilent Human miRNA Microarray V19.0 which contained 2006 miRNAs. We tested for significant differences in miRNA expression between paired carcinoma/adenoma/normal colonic tissue samples. Fewer than 600 miRNAs were expressed in >80% of people for colonic tissue; of these 86.5% were statistically differentially expressed between carcinoma and normal colonic mucosa using a false discovery rate of 0.05. Roughly half of these differentially expressed miRNAs showed a progression in levels of expression from normal to adenoma to carcinoma tissue. Other miRNAs appeared to be altered at the normal to adenoma stage, while others were only altered at the adenoma to carcinoma stage or only at the normal to carcinoma stage. Evaluation of the Agilent platform showed a high degree of repeatability (r = 0.98) and reasonable agreement with the NanoString platform. Our data suggest that miRNAs are highly dysregulated in colorectal tissue among individuals with colorectal cancer; the pattern of disruption varies by miRNA as tissue progresses from normal to adenoma to carcinoma.

  14. The putative human stem cell marker, Rex-1 (Zfp42): structural classification and expression in normal human epithelial and carcinoma cell cultures.

    PubMed

    Mongan, Nigel P; Martin, Kisha M; Gudas, Lorraine J

    2006-12-01

    Human Rex-1 (hRex-1) (also referred to as zinc-finger protein-42, Zfp42) encodes a zinc finger protein expression of which is believed to be characteristic of pluripotent stem cells. We have applied bioinformatics to classify the relationship of human, rat, and mouse REX1 proteins in the C2H2 family of zinc finger proteins and demonstrate that REX1 is a member of the YY1 sub-family of transcription factors, which includes the Drosophila pleiohomeotic (Pho) protein. We have generated a molecular model of the human REX1 zinc finger domains based on the crystal structure of the YY1 transcription factor. To date, expression of hRex-1 and its extensively studied mouse homolog mRex-1, has been reported only in embryonic and adult stem cells and in differentiated spermatocytes. In this study, reverse transcription-PCR and Western analysis were employed to assay for hRex-1 expression in cultured normal human epithelial cells and human carcinoma cell lines. Expression of hRex-1 mRNA was detected in normal human epidermal keratinocytes, normal prostate epithelial cells (PrEC), bronchial, and small airway lung epithelial cells. Other stem cell markers, such as Oct 4, DAB2, and cMyc were also detected in normal human epidermal keratinocyte cultures. Expression of hRex-1 was also detected in some human tumor cell lines including MDA-MB-468 mammary carcinoma, SCC-15 head and neck squamous cell carcinoma, and N-TERA2 human teratocarcinoma cells. Western analyses confirmed expression of the human REX1 (ZFP42) protein in MDA-MB-468 cells and normal human keratinocytes. This research has identified model human cell culture systems, in addition to embryonic stem (ES) cells, in which Rex-1 is expressed, and this should enable the characterization of REX1 functions in normal adult epithelial cells and tumorigenic stem cells.

  15. Pien Tze Huang inhibits hypoxia-induced epithelial-mesenchymal transition in human colon carcinoma cells through suppression of the HIF-1 pathway.

    PubMed

    Chen, Hongwei; Shen, Aling; Zhang, Yuchen; Chen, Youqin; Lin, Jiumao; Lin, Wei; Sferra, Thomas; Peng, Jun

    2014-05-01

    Hypoxia-induced activation of the hypoxia-inducible factor 1 (HIF-1) signaling pathway is frequently observed in solid tumors and is strongly associated with numerous pathophysiological processes, including the induction of epithelial-mesenchymal transition (EMT), which result in cancer progression and metastasis. Thus, inhibiting EMT through the suppression of the HIF-1 pathway may be a promising strategy for anticancer chemotherapy. Pien Tze Huang (PZH), a well-established traditional Chinese medicine has been prescribed for >450 years and has been used for centuries to clinically treat various types of human cancer. We previously reported that PZH suppresses multiple intracellular signaling pathways and thereby promotes the apoptosis of cancer cells and the inhibition of cell proliferation and tumor angiogenesis. In the present study, to further explore the mechanisms underlying the antitumor action of PZH, HCT-8 human colon carcinoma cells were cultured under hypoxic conditions and the effect of PZH on hypoxia-induced EMT was assessed. Hypoxia was found to induce EMT-associated morphological changes in HCT-8 cells, including loss of cell adhesion and the development of spindle-shaped fibroblastoid-like morphology. In addition, hypoxia was observed to reduce the expression of the epithelial marker E-cadherin, but increase that of the mesenchymal marker N-cadherin. In addition, hypoxia significantly enhanced HCT-8 cell migration and invasion and induced the activation of the HIF-1 pathway. However, treatment of the HCT-8 cells with PZH significantly inhibited the hypoxia-mediated EMT and HIF-1 signaling. These findings suggest that PZH inhibits hypoxia-induced cancer EMT through the suppression of the HIF-1 pathway, which may be one of the molecular mechanisms by which PZH exerts its antitumor activity.

  16. Pien Tze Huang inhibits hypoxia-induced epithelial-mesenchymal transition in human colon carcinoma cells through suppression of the HIF-1 pathway

    PubMed Central

    CHEN, HONGWEI; SHEN, ALING; ZHANG, YUCHEN; CHEN, YOUQIN; LIN, JIUMAO; LIN, WEI; SFERRA, THOMAS; PENG, JUN

    2014-01-01

    Hypoxia-induced activation of the hypoxia-inducible factor 1 (HIF-1) signaling pathway is frequently observed in solid tumors and is strongly associated with numerous pathophysiological processes, including the induction of epithelial-mesenchymal transition (EMT), which result in cancer progression and metastasis. Thus, inhibiting EMT through the suppression of the HIF-1 pathway may be a promising strategy for anticancer chemotherapy. Pien Tze Huang (PZH), a well-established traditional Chinese medicine has been prescribed for >450 years and has been used for centuries to clinically treat various types of human cancer. We previously reported that PZH suppresses multiple intracellular signaling pathways and thereby promotes the apoptosis of cancer cells and the inhibition of cell proliferation and tumor angiogenesis. In the present study, to further explore the mechanisms underlying the antitumor action of PZH, HCT-8 human colon carcinoma cells were cultured under hypoxic conditions and the effect of PZH on hypoxia-induced EMT was assessed. Hypoxia was found to induce EMT-associated morphological changes in HCT-8 cells, including loss of cell adhesion and the development of spindle-shaped fibroblastoid-like morphology. In addition, hypoxia was observed to reduce the expression of the epithelial marker E-cadherin, but increase that of the mesenchymal marker N-cadherin. In addition, hypoxia significantly enhanced HCT-8 cell migration and invasion and induced the activation of the HIF-1 pathway. However, treatment of the HCT-8 cells with PZH significantly inhibited the hypoxia-mediated EMT and HIF-1 signaling. These findings suggest that PZH inhibits hypoxia-induced cancer EMT through the suppression of the HIF-1 pathway, which may be one of the molecular mechanisms by which PZH exerts its antitumor activity. PMID:24940418

  17. Cooperation between the bacterial-derived short-chain fatty acid butyrate and interleukin-22 detected in human Caco2 colon epithelial/carcinoma cells.

    PubMed

    Bachmann, Malte; Meissner, Carlotta; Pfeilschifter, Josef; Mühl, Heiko

    2017-03-01

    By generating biologically active factors luminal microbiota shape the intestinal micro-milieu thereby regulating pathological processes such as inflammation and carcinogenesis. Preclinical data suggest that bacterial-derived butyrate and the signal transducer and activator of transcription (STAT)-3 activating cytokine interleukin (IL)-22 display concordant protective properties at the inflamed colonic epithelium. Herein, biochemical cooperation between the short-chain fatty acid butyrate and IL-22 was investigated by focusing on human Caco2 colon epithelial/carcinoma cells. We report that physiological levels of butyrate enhance IL-22 signaling thereby enforcing expression of the prototypic STAT3-downstrean target genes α1-antichymotrypsin and suppressor of cytokine signaling (SOCS)-3. A dual mode of butyrate action on the IL-22/STAT3 axis was identified. Butyrate acted by upregulating IL-22R1, the decisive chain of the heterodimeric IL-22 receptor, and, independent from that, has the potential to directly amplify STAT3-mediated gene activation as detected by chromatin immunoprecipitation analysis of STAT3 binding to the SOCS3 promoter. Since trichostatin A acted similarly, inhibition of histone deacetylases is likely at the root of these butyrate biological properties. The mutual benefit gained from interactions between the host and commensal intestinal bacteria-derived factors is an expanding field of research beginning to affect clinical practice. Data presented herein propose a supportive and fine-tuning role for butyrate in IL-22 signaling that might be therapeutically exploited by local butyrate administration or by increasing its bacterial production in the context of a fiber-rich diet. © 2016 BioFactors, 43(2):283-292, 2017. © 2016 International Union of Biochemistry and Molecular Biology.

  18. Obesity and intestinal epithelial deletion of the insulin receptor, but not the IGF 1 receptor, affect radiation-induced apoptosis in colon

    PubMed Central

    Santoro, M. Agostina; Blue, R. Eric; Andres, Sarah F.; Mah, Amanda T.; Van Landeghem, Laurianne

    2015-01-01

    Current views suggest that apoptosis eliminates genetically damaged cells that may otherwise form tumors. Prior human studies link elevated insulin and reduced apoptosis to risk of colorectal adenomas. We hypothesized that hyperinsulinemia associated with obesity would lead to reduced colon epithelial cell (CEC) apoptosis after radiation and that this effect would be altered by deletion of the insulin-like growth factor (IGF) 1 receptor (IGF1R) or the insulin receptor (IR). Mice with villin-Cre-mediated IGF1R or IR deletion in CECs and floxed littermates were fed a high-fat diet to induce obesity and hyperinsulinemia or control low-fat chow. Mice were exposed to 5-Gy abdominal radiation to induce DNA damage and euthanized 4 h later for evaluation of apoptosis by localization of cleaved caspase-3. Obese mice exhibited decreased apoptosis of genetically damaged CECs. IGF1R deletion did not affect CEC apoptosis in lean or obese animals. In contrast, IR loss increased CEC apoptosis in both diet groups but did not prevent antiapoptotic effects of obesity. Levels of p53 protein were significantly reduced in CECs of obese mice with intact IR but increased in both lean and obese mice without IR. Levels of mRNAs encoding proapoptotic Perp and the cell cycle inhibitor Cdkn1b/p27 were reduced in CECs of obese mice and increased in lean mice lacking IR. Together, our studies provide novel evidence for antiapoptotic roles of obesity and IR, but not IGF1R, in colonic epithelium after DNA damage. However, neither IR nor IGF1R deletion prevented a reduction in radiation-induced CEC apoptosis during obesity and hyperinsulinemia. PMID:26251471

  19. The effect of acute hypoxia on short-circuit current and epithelial resistivity in biopsies from human colon.

    PubMed

    Carra, Graciela E; Ibáñez, Jorge E; Saraví, Fernando D

    2013-09-01

    In isolated colonic mucosa, decreases in short-circuit current (ISC) and transepithelial resistivity (RTE) occur when hypoxia is either induced at both sides or only at the serosal side of the epithelium. We assessed in human colon biopsies the sensitivity to serosal-only hypoxia and mucosal-only hypoxia and whether Na, K-ATPase blockade with ouabain interacts with hypoxia. Biopsy material from patients undergoing colonoscopy was mounted in an Ussing chamber for small samples (1-mm2 window). In a series of experiments we assessed viability and the electrical response to the mucolytic, dithiothreitol (1 mmol/l). In a second series, we explored the effect of hypoxia without and with ouabain. In a third series, we evaluated the response to a cycle of hypoxia and reoxygenation induced at the serosal or mucosal side while keeping the oxygenation of the opposite side. 1st series: Dithiothreitol significantly decreased the unstirred layer and ISC but increased RTE. 2nd series: Both hypoxia and ouabain decreased ISC, but ouabain increased RTE and this effect on RTE prevailed even during hypoxia. 3rd series: Mucosal hypoxia caused lesser decreases of ISC and RTE than serosal hypoxia; in the former, but not in the latter, recovery was complete upon reoxygenation. In mucolytic concentration, dithiothreitol modifies ISC and RTE. Oxygen supply from the serosal side is more important to sustain ISC and RTE in biopsy samples. The different effect of hypoxia and Na, K-ATPase blockade on RTE suggests that their depressing effect on ISC involves different mechanisms.

  20. Establishment of the epithelial-specific transcriptome of normal and malignant human breast cells based on MPSS and array expression data

    PubMed Central

    Grigoriadis, Anita; Mackay, Alan; Reis-Filho, Jorge S; Steele, Dawn; Iseli, Christian; Stevenson, Brian J; Jongeneel, C Victor; Valgeirsson, Haukur; Fenwick, Kerry; Iravani, Marjan; Leao, Maria; Simpson, Andrew JG; Strausberg, Robert L; Jat, Parmjit S; Ashworth, Alan; Neville, A Munro; O'Hare, Michael J

    2006-01-01

    Introduction Diverse microarray and sequencing technologies have been widely used to characterise the molecular changes in malignant epithelial cells in breast cancers. Such gene expression studies to identify markers and targets in tumour cells are, however, compromised by the cellular heterogeneity of solid breast tumours and by the lack of appropriate counterparts representing normal breast epithelial cells. Methods Malignant neoplastic epithelial cells from primary breast cancers and luminal and myoepithelial cells isolated from normal human breast tissue were isolated by immunomagnetic separation methods. Pools of RNA from highly enriched preparations of these cell types were subjected to expression profiling using massively parallel signature sequencing (MPSS) and four different genome wide microarray platforms. Functional related transcripts of the differential tumour epithelial transcriptome were used for gene set enrichment analysis to identify enrichment of luminal and myoepithelial type genes. Clinical pathological validation of a small number of genes was performed on tissue microarrays. Results MPSS identified 6,553 differentially expressed genes between the pool of normal luminal cells and that of primary tumours substantially enriched for epithelial cells, of which 98% were represented and 60% were confirmed by microarray profiling. Significant expression level changes between these two samples detected only by microarray technology were shown by 4,149 transcripts, resulting in a combined differential tumour epithelial transcriptome of 8,051 genes. Microarray gene signatures identified a comprehensive list of 907 and 955 transcripts whose expression differed between luminal epithelial cells and myoepithelial cells, respectively. Functional annotation and gene set enrichment analysis highlighted a group of genes related to skeletal development that were associated with the myoepithelial/basal cells and upregulated in the tumour sample. One of the most

  1. Growth hormone is permissive for neoplastic colon growth.

    PubMed

    Chesnokova, Vera; Zonis, Svetlana; Zhou, Cuiqi; Recouvreux, Maria Victoria; Ben-Shlomo, Anat; Araki, Takako; Barrett, Robert; Workman, Michael; Wawrowsky, Kolja; Ljubimov, Vladimir A; Uhart, Magdalena; Melmed, Shlomo

    2016-06-07

    Growth hormone (GH) excess in acromegaly is associated with increased precancerous colon polyps and soft tissue adenomas, whereas short-stature humans harboring an inactivating GH receptor mutation do not develop cancer. We show that locally expressed colon GH is abundant in conditions predisposing to colon cancer and in colon adenocarcinoma-associated stromal fibroblasts. Administration of a GH receptor (GHR) blocker in acromegaly patients induced colon p53 and adenomatous polyposis coli (APC), reversing progrowth GH signals. p53 was also induced in skin fibroblasts derived from short-statured humans with mutant GHR. GH-deficient prophet of pituitary-specific positive transcription factor 1 (Prop1)(-/-) mice exhibited induced colon p53 levels, and cross-breeding them with Apc(min+/-) mice that normally develop intestinal and colon tumors resulted in GH-deficient double mutants with markedly decreased tumor number and size. We also demonstrate that GH suppresses p53 and reduces apoptosis in human colon cell lines as well as in induced human pluripotent stem cell-derived intestinal organoids, and confirm in vivo that GH suppresses colon mucosal p53/p21. GH excess leads to decreased colon cell phosphatase and tensin homolog deleted on chromosome 10 (PTEN), increased cell survival with down-regulated APC, nuclear β-catenin accumulation, and increased epithelial-mesenchymal transition factors and colon cell motility. We propose that GH is a molecular component of the "field change" milieu permissive for neoplastic colon growth.

  2. Heat-killed probiotic bacteria differentially regulate colonic epithelial cell production of human β-defensin-2: dependence on inflammatory cytokines.

    PubMed

    Habil, N; Abate, W; Beal, J; Foey, A D

    2014-12-01

    The inducible antimicrobial peptide human β-defensin-2 (hBD-2) stimulated by pro-inflammatory cytokines and bacterial products is essential to antipathogen responses of gut epithelial cells. Commensal and probiotic bacteria can augment such mucosal defences. Probiotic use in the treatment of inflammatory bowel disease, however, may have adverse effects, boosting inflammatory responses. The aim of this investigation was to determine the effect of selected probiotic strains on hBD-2 production by epithelial cells induced by pathologically relevant pro-inflammatory cytokines and the role of cytokine modulators in controlling hBD-2. Caco-2 colonic intestinal epithelial cells were pre-incubated with heat-killed probiotics, i.e. Lactobacillus casei strain Shirota (LcS) or Lactobacillus fermentum strain MS15 (LF), followed by stimulation of hBD-2 by interleukin (IL)-1β and tumour necrosis factor alpha (TNF-α) in the absence or presence of exogenous IL-10 or anti-IL-10 neutralising antibody. Cytokines and hBD-2 mRNA and protein were analysed by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. LcS augmented IL-1β-induced hBD-2, whereas LF enhanced TNF-α- and suppressed IL-1β-induced hBD-2. LF enhanced TNF-α-induced TNF-α and suppressed IL-10, whereas augmented IL-1β-induced IL-10. LcS upregulated IL-1β-induced TNF-α mRNA and suppressed IL-10. Endogenous IL-10 differentially regulated hBD-2; neutralisation of IL-10 augmented TNF-α- and suppressed IL-1β-induced hBD-2. Exogenous IL-10, however, suppressed both TNF-α- and IL-1β-induced hBD-2; LcS partially rescued suppression in TNF-α- and IL-1β-stimulation, whereas LF further suppressed IL-1β-induced hBD-2. It can be concluded that probiotic strains differentially regulate hBD-2 mRNA expression and protein secretion, modulation being dictated by inflammatory stimulus and resulting cytokine environment.

  3. Proteolytic cleavage and truncation of NDRG1 in human prostate cancer cells, but not normal prostate epithelial cells

    PubMed Central

    Ghalayini, Mohammad K.; Dong, Qihan; Richardson, Des R.; Assinder, Stephen J.

    2013-01-01

    NDRG1 (N-myc downstream regulated gene-1) is a metastasis suppressor that is down-regulated in prostate cancer. NDRG1 phosphorylation is associated with inhibition of metastasis and Western blots indicate two bands at ~41 and ~46 kDa. Previous investigations by others suggest the higher band is due to NDRG1 phosphorylation. However, the current study using a dephosphorylation assay and the Phos-tag (phosphate-binding tag) SDS/PAGE assay, demonstrated that the 46 kDa NDRG1 protein band was not due to phosphorylation. Further experiments showed that the NDRG1 protein bands were not affected upon glycosidase treatment, despite marked effects of these enzymes on the glycosylated protein, fetuin. Analysis using RT–PCR (reverse transcriptase–PCR) demonstrated only a single amplicon, and thus, the two bands could not result from an alternatively spliced NDRG1 transcript. Western-blot analysis of prostate cancer cell lysates identified the 41 kDa band to be a truncated form of NDRG1, with MS confirming the full and truncated proteins to be NDRG1. Significantly, this truncated protein was not present in normal human PrECs (prostate epithelial cells). Western-blot analysis using anti-NDRG1 raised to its N-terminal sequence failed to detect the truncated protein, suggesting that it lacked N-terminus amino acids (residues 1–49). Sequence analysis predicted a pseudotrypsin protease cleavage site between Cys49–Gly50. Such cleavage of NDRG1 in cancer cells may result in loss of NDRG1 tumour suppressive activity. PMID:23634903

  4. Kefir extracts suppress in vitro proliferation of estrogen-dependent human breast cancer cells but not normal mammary epithelial cells.

    PubMed

    Chen, Chujian; Chan, Hing Man; Kubow, Stan

    2007-09-01

    Anti-tumorigenic effects have been demonstrated in animal studies from the intake of kefir, a traditional fermented milk product believed to originate from the Caucasian mountains of Russia. In the present study, the antiproliferative effects of extracts of kefir, yogurt, and pasteurized cow's milk on human mammary cancer cells (MCF-7) and normal human mammary epithelial cells (HMECs) was investigated at doses of 0.31%, 0.63%, 1.25%, 2.5%, 5%, and 10% (vol/vol). After 6 days of culture, extracts of kefir-fermented milk depressed MCF-7 cell growth in a dose-dependent manner, showing 29% inhibition of proliferation at a concentration as low as 0.63%, whereas yogurt extracts began to show dose-dependent antiproliferative effects only at the 2.5% dose. Moreover, at the 2.5% dose, kefir extracts decreased the MCF-7 cell numbers by 56%, while yogurt extracts decreased MCF-7 cell proliferation by only 14%. No antiproliferative effects of kefir extracts were observed in the HMECs, while the yogurt extracts exerted antiproliferative effects on HMECs at the 5% and 10% doses. Unfermented milk extracts stimulated proliferation of MCF-7 cells and HMECs at concentrations above 0.31%. Peptide content and capillary electrophoresis analyses showed that kefir-mediated milk fermentation led to an increase in peptide concentrations and a change in peptide profiles relative to milk or yogurt. The present findings suggest that kefir extracts contain constituents that specifically inhibit the growth of human breast cancer cells, which might eventually be useful in the prevention or treatment of breast cancer.

  5. A Network Biology Approach Identifies Molecular Cross-Talk between Normal Prostate Epithelial and Prostate Carcinoma Cells.

    PubMed

    Trevino, Victor; Cassese, Alberto; Nagy, Zsuzsanna; Zhuang, Xiaodong; Herbert, John; Antczak, Philipp; Clarke, Kim; Davies, Nicholas; Rahman, Ayesha; Campbell, Moray J; Guindani, Michele; Bicknell, Roy; Vannucci, Marina; Falciani, Francesco

    2016-04-01

    The advent of functional genomics has enabled the genome-wide characterization of the molecular state of cells and tissues, virtually at every level of biological organization. The difficulty in organizing and mining this unprecedented amount of information has stimulated the development of computational methods designed to infer the underlying structure of regulatory networks from observational data. These important developments had a profound impact in biological sciences since they triggered the development of a novel data-driven investigative approach. In cancer research, this strategy has been particularly successful. It has contributed to the identification of novel biomarkers, to a better characterization of disease heterogeneity and to a more in depth understanding of cancer pathophysiology. However, so far these approaches have not explicitly addressed the challenge of identifying networks representing the interaction of different cell types in a complex tissue. Since these interactions represent an essential part of the biology of both diseased and healthy tissues, it is of paramount importance that this challenge is addressed. Here we report the definition of a network reverse engineering strategy designed to infer directional signals linking adjacent cell types within a complex tissue. The application of this inference strategy to prostate cancer genome-wide expression profiling data validated the approach and revealed that normal epithelial cells exert an anti-tumour activity on prostate carcinoma cells. Moreover, by using a Bayesian hierarchical model integrating genetics and gene expression data and combining this with survival analysis, we show that the expression of putative cell communication genes related to focal adhesion and secretion is affected by epistatic gene copy number variation and it is predictive of patient survival. Ultimately, this study represents a generalizable approach to the challenge of deciphering cell communication networks

  6. Proteolytic cleavage and truncation of NDRG1 in human prostate cancer cells, but not normal prostate epithelial cells.

    PubMed

    Ghalayini, Mohammad K; Dong, Qihan; Richardson, Des R; Assinder, Stephen J

    2013-06-11

    NDRG1 (N-myc downstream regulated gene-1) is a metastasis suppressor that is down-regulated in prostate cancer. NDRG1 phosphorylation is associated with inhibition of metastasis and Western blots indicate two bands at ~41 and ~46 kDa. Previous investigations by others suggest the higher band is due to NDRG1 phosphorylation. However, the current study using a dephosphorylation assay and the Phos-tag (phosphate-binding tag) SDS/PAGE assay, demonstrated that the 46 kDa NDRG1 protein band was not due to phosphorylation. Further experiments showed that the NDRG1 protein bands were not affected upon glycosidase treatment, despite marked effects of these enzymes on the glycosylated protein, fetuin. Analysis using RT-PCR (reverse transcriptase-PCR) demonstrated only a single amplicon, and thus, the two bands could not result from an alternatively spliced NDRG1 transcript. Western-blot analysis of prostate cancer cell lysates identified the 41 kDa band to be a truncated form of NDRG1, with MS confirming the full and truncated proteins to be NDRG1. Significantly, this truncated protein was not present in normal human PrECs (prostate epithelial cells). Western-blot analysis using anti-NDRG1 raised to its N-terminal sequence failed to detect the truncated protein, suggesting that it lacked N-terminus amino acids (residues 1-49). Sequence analysis predicted a pseudotrypsin protease cleavage site between Cys49-Gly50. Such cleavage of NDRG1 in cancer cells may result in loss of NDRG1 tumour suppressive activity.

  7. Overexpression of RGPR-p117 enhances regucalcin gene expression in cloned normal rat kidney proximal tubular epithelial cells.

    PubMed

    Sawada, Natsumi; Yamaguchi, Masayoshi

    2005-12-01

    A novel protein RGPR-p117 was discovered as a regucalcin gene promoter region-related protein that binds to the TTGGC motif using a yeast one-hybrid system. Whether overexpression of RGPR-p117 can modulate gene expression in the cloned normal rat kidney proximal tubular epithelial NRK52E cells was investigated. NRK52E cells (wild-type) or HA-RGPR-p117/phCMV2-transfected NRK52E cells were cultured in Dulbecco's minimum essential medium (DMEM) containing 5% bovine serum (BS). Proliferation of NRK52E cells (wild-type) was not significantly altered by overexpression of HA-RGPR-p117. The expression of rat regucalcin, alpha-fetoprotein, albumin, glucokinase, 11beta-hydroxy-steroid dehydrogenase, phosphoenolpyruvate carboxykinase, which contains TTGGC motif in the promoter region of their genes, was seen in NRK52E cells (wild-type) by using reverse transcription-polymerase chain reaction (RT-PCR). Of these genes, regucalcin mRNA levels were significantly enhanced in transfectants. The expression of p21 or glycero-aldehyde-3-phosphate dehydrogenase mRNA was not significantly changed in transfectants. The results of Western blot analysis showed that regucalcin protein was significantly increased in transfectants. The enhancement of regucalcin mRNA expression in transfectants was significantly suppressed in the presence of staurosporine (10(-10) M), an inhibitor of protein kinase C. This enhancement was not significantly changed in the presence of dibucaine (10(-8) M), PD98059 (10(-8) M) or vanadate (10(-6) M). This study demonstrates that overexpression of RGPR-p117 enhances the expression of regucalcin mRNA and its protein level in NRK52E cells. RGPR-p117 may play a role as a transcriptional factor.

  8. A Network Biology Approach Identifies Molecular Cross-Talk between Normal Prostate Epithelial and Prostate Carcinoma Cells

    PubMed Central

    Trevino, Victor; Cassese, Alberto; Nagy, Zsuzsanna; Zhuang, Xiaodong; Herbert, John; Antzack, Philipp; Clarke, Kim; Davies, Nicholas; Rahman, Ayesha; Campbell, Moray J.; Bicknell, Roy; Vannucci, Marina; Falciani, Francesco

    2016-01-01

    Abstract The advent of functional genomics has enabled the genome-wide characterization of the molecular state of cells and tissues, virtually at every level of biological organization. The difficulty in organizing and mining this unprecedented amount of information has stimulated the development of computational methods designed to infer the underlying structure of regulatory networks from observational data. These important developments had a profound impact in biological sciences since they triggered the development of a novel data-driven investigative approach. In cancer research, this strategy has been particularly successful. It has contributed to the identification of novel biomarkers, to a better characterization of disease heterogeneity and to a more in depth understanding of cancer pathophysiology. However, so far these approaches have not explicitly addressed the challenge of identifying networks representing the interaction of different cell types in a complex tissue. Since these interactions represent an essential part of the biology of both diseased and healthy tissues, it is of paramount importance that this challenge is addressed. Here we report the definition of a network reverse engineering strategy designed to infer directional signals linking adjacent cell types within a complex tissue. The application of this inference strategy to prostate cancer genome-wide expression profiling data validated the approach and revealed that normal epithelial cells exert an anti-tumour activity on prostate carcinoma cells. Moreover, by using a Bayesian hierarchical model integrating genetics and gene expression data and combining this with survival analysis, we show that the expression of putative cell communication genes related to focal adhesion and secretion is affected by epistatic gene copy number variation and it is predictive of patient survival. Ultimately, this study represents a generalizable approach to the challenge of deciphering cell communication

  9. Claudin-4 undergoes age-dependent change in cellular localization on pig jejunal villous epithelial cells, independent of bacterial colonization.

    PubMed

    Pasternak, J Alex; Kent-Dennis, Coral; Van Kessel, Andrew G; Wilson, Heather L

    2015-01-01

    Newborn piglets are immunologically naïve and must receive passive immunity via colostrum within 24 hours to survive. Mechanisms by which the newborn piglet gut facilitates uptake of colostral cells, antibodies, and proteins may include FcRn and pIgR receptor-mediated endocytosis and paracellular transport between tight junctions (TJs). In the present study, FcRn gene (FCGRT) was minimally expressed in 6-week-old gut and newborn jejunum but it was expressed at significantly higher levels in the ileum of newborn piglets. pIgR was highly expressed in the jejunum and ileum of 6-week-old animals but only minimally in neonatal gut. Immunohistochemical analysis showed that Claudin-5 localized to blood vessel endothelial cells. Claudin-4 was strongly localized to the apical aspect of jejunal epithelial cells for the first 2 days of life after which it was redistributed to the lateral surface between adjacent enterocytes. Claudin-4 was localized to ileal lateral surfaces within 24 hours after birth indicating regional and temporal differences. Tissue from gnotobiotic piglets showed that commensal microbiota did not influence Claudin-4 surface localization on jejunal or ileal enterocytes. Regulation of TJs by Claudin-4 surface localization requires further investigation. Understanding the factors that regulate gut barrier maturation may yield protective strategies against infectious diseases.

  10. Claudin-4 Undergoes Age-Dependent Change in Cellular Localization on Pig Jejunal Villous Epithelial Cells, Independent of Bacterial Colonization

    PubMed Central

    Van Kessel, Andrew G.; Wilson, Heather L.

    2015-01-01

    Newborn piglets are immunologically naïve and must receive passive immunity via colostrum within 24 hours to survive. Mechanisms by which the newborn piglet gut facilitates uptake of colostral cells, antibodies, and proteins may include FcRn and pIgR receptor-mediated endocytosis and paracellular transport between tight junctions (TJs). In the present study, FcRn gene (FCGRT) was minimally expressed in 6-week-old gut and newborn jejunum but it was expressed at significantly higher levels in the ileum of newborn piglets. pIgR was highly expressed in the jejunum and ileum of 6-week-old animals but only minimally in neonatal gut. Immunohistochemical analysis showed that Claudin-5 localized to blood vessel endothelial cells. Claudin-4 was strongly localized to the apical aspect of jejunal epithelial cells for the first 2 days of life after which it was redistributed to the lateral surface between adjacent enterocytes. Claudin-4 was localized to ileal lateral surfaces within 24 hours after birth indicating regional and temporal differences. Tissue from gnotobiotic piglets showed that commensal microbiota did not influence Claudin-4 surface localization on jejunal or ileal enterocytes. Regulation of TJs by Claudin-4 surface localization requires further investigation. Understanding the factors that regulate gut barrier maturation may yield protective strategies against infectious diseases. PMID:25948883

  11. Carbohydrate-binding motif in Chitinase 3-like 1 (CHI3L1/YKL-40) specifically activates Akt signaling pathway in colonic epithelial cells

    PubMed Central

    Chen, Chun-Chuan; Llado, Victoria; Eurich, Katrin; Tran, Hoa T.; Mizoguchi, Emiko

    2011-01-01

    Host-microbial interactions play a key role during the development of colitis. We have previously shown that chinase 3-like 1 (CHI3L1) is an inducible molecule overexpressed in colonic epithelial cells (CECs) under inflammatory conditions. In this study, we found that chitin-binding motif (CBM) of CHI3L1 is specifically associated with the CHI3L1-mediated activation of the Akt-signaling in CEC by transfecting the CBM-mutant CHI3L1 vectors in SW480 CECs. Downstream, CHI3L1 enhanced the secretion of IL-8 and TNFα in a dose-dependent manner. We previously show that 325 through 339 amino-acids in CBM are crucial for the biological function of CHI3L1. Here we demonstrated that 325th–339th residues of CBM in CHI3L1 is a critical region for the activation of Akt, IL-8 production, and for a specific cellular localization of CHI3L1. In conclusion, CBM region of CHI3L1 is critical in activating Akt signaling in CECs, and the activation may be associated with the development of chronic colitis. PMID:21546314

  12. IL-17A Signaling in Colonic Epithelial Cells Inhibits Pro-Inflammatory Cytokine Production by Enhancing the Activity of ERK and PI3K

    PubMed Central

    Xiao, Yan; Zhou, Tingting; Guo, Yueling; Wang, Renxi; Zhao, Zhi; Xiao, He; Hou, Chunmei; Ma, Lingyun; Lin, Yanhua; Lang, Xiaoling; Feng, Jiannan; Chen, Guojiang; Shen, Beifen; Han, Gencheng; Li, Yan

    2014-01-01

    Our previous data suggested that IL-17A contributes to the inhibition of Th1 cell function in the gut. However, the underlying mechanisms remain unclear. Here we demonstrate that IL-17A signaling in colonic epithelial cells (CECs) increases TNF-α-induced PI3K-AKT and ERK phosphorylation and inhibits TNF-α induced expression of IL-12P35 and of a Th1 cell chemokine, CXCL11 at mRNA level. In a co-culture system using HT-29 cells and PBMCs, IL-17A inhibited TNF-ãinduced IL-12P35 expression by HT-29 cells and led to decreased expression of IFN-γ and T-bet by PBMCs. Finally, adoptive transfer of CECs from mice with Crohn's Disease (CD) led to an enhanced Th1 cell response and exacerbated colitis in CD mouse recipients. The pathogenic effect of CECs derived from CD mice was reversed by co-administration of recombinant IL-17A. Our data demonstrate a new IL-17A-mediated regulatory mechanism in CD. A better understanding of this pathway might shed new light on the pathogenesis of CD. PMID:24586980

  13. Sulforaphane Regulates NFE2L2/Nrf2-Dependent Xenobiotic Metabolism Phase II and Phase III Enzymes Differently in Human Colorectal Cancer and Untransformed Epithelial Colon Cells.

    PubMed

    Lubelska, Katarzyna; Wiktorska, Katarzyna; Mielczarek, Lidia; Milczarek, Małgorzata; Zbroińska-Bregisz, Ilona; Chilmonczyk, Zdzisław

    2016-01-01

    Sulforaphane (SFN), a naturally occurring chemopreventive and anticancer agent, is a nuclear factor, erythroid 2-like 2 (NFE2L2/Nrf2) inducer. Nrf2 plays a critical role in coordinating the cell defense system by initiating the transcription of cytoprotective genes, including detoxification enzymes such as NAD(P)H quinone dehydrogenase 1 (NQO1) and transport proteins such as ATP-binding cassette, subfamily C (CFTR/MRP). Recently, the essential role of Nrf2 in tumor development and progression and in the development of multidrug resistance in cancer cells has been highlighted. The aim of this study was to compare the effect of SFN on the Nrf2 system and the Nrf2-target enzymes NQO1 and MRP in human untransformed epithelial colon CRL-1790 cells and in HT-29 and Caco-2 colorectal cancer cells to elucidate the role of SFN in cancer prevention and treatment. We have demonstrated that SFN has excellent cytoprotective properties in CRL-1790 cells, as it induced Nrf2-dependent expression of MRP1 and NQO1. SFN induced Nrf2 target enzyme activity in HT-29 and Caco-2 cancer cells but regulated the Nrf2/ARE signaling pathway differently in cancer and untransformed cells.

  14. Immortalization of Fetal Bovine Colon Epithelial Cell