Nontransformed Human Intestinal Epithelial Cells Research Articles

Exposure to dietary fatty acids oleic and palmitic acid alters structure and mechanotransduction of intestinal cells in vitro

Intestinal cells are continuously exposed to food constituents while adapting to peristaltic movement and fluid shear stress. Oleic acid (OA) and palmitic acid (PA) are among the most prevalent fatty acids with respect to dietary lipids. Despite the central importance of dietary lipids for a balanced diet, awareness about potential detrimental effects related to excessive consumption is increasing; this includes toxicity, metabolic deregulation, and, particularly for cancer cells, a benefit from the uptake of fatty acids related to promotion of metastasis. Expanding on this, we started elucidating the effects of OA and PA (25–500 µM) on non-transformed human intestinal epithelial cells (HCEC-1CT) in comparison to colon carcinoma cells (HCT116), with regard to the mechanosensory apparatus. Hence, intestinal cells’ motility is on the one side essential to ensure adaption to peristaltic movement and barrier function, but also to enable metastatic progression. Incubation with both OA and PA (≥ 25 µM) significantly decreased membrane fluidity of HCT116 cells, whereas the effect on HCEC-1CT was more limited. Application of rhodamine-labelled PA demonstrated that the fatty acid is incorporated into the plasma membrane of HCT116, which could not be observed in the non-tumorigenic cell line. Down-streaming into the intracellular compartment, a pronounced rearrangement of actin cytoskeleton was evident in both cell lines (OA and PA; 25 and 100 µM). This was accompanied by a variation of translocation efficiency of the mechanosensitive co-transcription factor YAP1, albeit with a stronger effect seen for PA and the cancer cells. Untargeted proteomic analysis confirmed that exposure to OA and PA could alter the response capacity of HCT116 cells to fluid shear stress. Taken together, OA and PA were able to functionally modulate the mechanosensory apparatus of intestinal cells, implying a novel role for dietary fatty acids in the regulation of intestinal pathophysiology.

Global Transcriptional Analysis of Nontransformed Human Intestinal Epithelial Cells (FHs 74 Int) after Exposure to Selected Drinking Water Disinfection By-Products.

Background:Drinking water disinfection inadvertently leads to the formation of numerous disinfection by-products (DBPs), some of which are cytotoxic, mutagenic, genotoxic, teratogenic, and potential carcinogens both in vitro and in vivo.Objectives:We investigated alterations to global gene expression (GE) in nontransformed human small intestine epithelial cells (FHs 74 Int) after exposure to six brominated and two chlorinated DBPs: bromoacetic acid (BAA), bromoacetonitrile (BAN), 2,6-dibromo-p-benzoquinone (DBBQ), bromoacetamide (BAM), tribromoacetaldehyde (TBAL), bromate (), trichloroacetic acid (TCAA), and trichloroacetaldehyde (TCAL).Methods:Using whole-genome cDNA microarray technology (Illumina), we examined GE in nontransformed human cells after exposure to DBPs at predetermined equipotent concentrations, identified significant changes in gene expression (), and investigated the relevance of these genes to specific toxicity pathways via gene and pathway enrichment analysis.Results:Genes related to activation of oxidative stress–responsive pathways exhibited fewer alterations than expected based on prior work, whereas all DBPs induced notable effects on transcription of genes related to immunity and inflammation.Discussion:Our results suggest that alterations to genes associated with immune and inflammatory pathways play an important role in the potential adverse health effects of exposure to DBPs. The interrelationship between these pathways and the production of reactive oxygen species (ROS) may explain the common occurrence of oxidative stress in other studies exploring DBP toxicity. Finally, transcriptional changes and shared induction of toxicity pathways observed for all DBPs caution of additive effects of mixtures and suggest further assessment of adverse health effects of mixtures is warranted. https://doi.org/10.1289/EHP4945

Human Cell Toxicogenomic Analysis Linking Reactive Oxygen Species to the Toxicity of Monohaloacetic Acid Drinking Water Disinfection Byproducts

Chronic exposure to drinking water disinfection byproducts has been linked to adverse health risks. The monohaloacetic acids (monoHAAs) are generated as byproducts during the disinfection of drinking water and are cytotoxic, genotoxic, mutagenic, and teratogenic. Iodoacetic acid toxicity was mitigated by antioxidants, suggesting the involvement of oxidative stress. Other monoHAAs may share a similar mode of action. Each monoHAA generated a significant concentration-response increase in the expression of a β-lactamase reporter under the control of the antioxidant response element (ARE). The monoHAAs generated oxidative stress with a rank order of iodoacetic acid (IAA) > bromoacetic acid (BAA) ≫ chloroacetic acid (CAA); this rank order was observed with other toxicological end points. Toxicogenomic analysis was conducted with a nontransformed human intestinal epithelial cell line (FHs 74 Int). Exposure to the monoHAAs altered the transcription levels of multiple oxidative stress responsive genes, indicating that each exposure generated oxidative stress. The transcriptome profiles showed an increase in thioredoxin reductase 1 (TXNRD1) and sulfiredoxin (SRXN1), suggesting peroxiredoxin proteins had been oxidized during monoHAA exposures. Three possible sources of reactive oxygen species were identified, the hypohalous acid generating peroxidase enzymes lactoperoxidase (LPO) and myeloperoxidase (MPO), nicotinamide adenine dinucleotide phosphate (NADPH)-dependent oxidase 5 (NOX5), and PTGS2 (COX-2) mediated arachidonic acid metabolism. Each monoHAA exposure caused an increase in COX-2 mRNA levels. These data provide a functional association between monoHAA exposure and adverse health outcomes such as oxidative stress, inflammation, and cancer.

Abstract 528: Leukotrienes modulate expression and activity of matrix metalloproteinases (MMPs) in colorectal cancer cells

Abstract Background: Pro-inflammatory cysteinyl leukotrienes (CysLTs) have been implicated to play a role in colon cancer. Previously, we have shown that leukotriene D4 (LTD4) signaling via the CysLT1 receptor is linked to tumorigenic processes. Elevated CysLT1R expression in tumoral tissue correlates with a poor prognosis in colon cancer patients; and CysLT1R signaling promotes intestinal epithelial cells’ proliferation, survival and migration. Tumor-associated macrophages (TAMs) have been shown to promote cancers’ progression and invasion. These activated macrophages (CD68+) are not only a major source of CysLTs in tumoral tissues, but also have been shown to promote cancer cells’ invasion, indicating that TAMs are part of the mechanism whereby inflammation promotes cancers’ progression. In addition to releasing pro-inflammatory mediators, TAMs also can produce protease enzymes at primary tumoral sites, both of which are tumor-promoting factors. Matrix metalloproteinase (MMPs), a family of zinc-dependent proteases, are proteolytic enzymes intimately linked to tumors’ invasion and progression. Although additional functions of MMPs have emerged recently, they are well known for their ability to degrade the extracellular matrix and basement membranes, facilitating tumoral cells’ invasion and tumors’ metastasis. However, little is known about a possible relation between pro-inflammatory mediators such as leukotrienes and MMPs’ expression and activities in colon cancer. Aim: The study's overall objective is to explore a possible correlation between TAM-derived pro-inflammatory leukotrienes and MMPs and their inhibitors in colon cancer. Methods: Using different human colon cancer cell lines and normal human embryonic intestinal epithelial cells, we explored MMPs’ expression in the absence or presence of leukotrienes. We employed various techniques such as RT-PCR, Immuno-histochemical, -fluorescent staining, Western blotting, ELISA and cell migration assays. Results: We first examined selected MMPs’ expression level in non-transformed human intestinal epithelial cells; Int 407 cells; and the human colon cancer cell lines Caco-2, HT-29 and CysLT1R-transfected Caco-2 cells at the mRNA level. We detected significant increases in the expression of MMP-2, MMP-3 and MMP-7 after 12 and 24 hours of LTD4 stimulation. In addition to these increases in MMPs’ expression, we observed an enhanced invasion capacity of CysLT1R-transfected Caco-2 cells compared to empty vector-transfected Caco-2 cells in the presence of LTD4. Conclusion: These preliminary in vitro findings indicate that TAM-derived leukotrienes can modulate MMPs’ expression in colon cancer, and thus their invading capacity. Further investigations will address how leukotrienes affect MMPs’ expression and activities in colon cancer cells and tumors. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 528.

DNA damage and toxicogenomic analyses of hydrogen sulfide in human intestinal epithelial FHs 74 Int cells

Hydrogen sulfide (H(2)S), a metabolic end product of sulfate-reducing bacteria, represents a genotoxic insult to the colonic epithelium, which may also be linked with chronic disorders such as ulcerative colitis and colorectal cancer. This study defined the early (30 min) and late (4 hr) response of nontransformed human intestinal epithelial cells (FHs 74 Int) to H(2)S. The genotoxicity of H(2)S was measured using the single-cell gel electrophoresis (comet) assay. Changes in gene expression were analyzed after exposure to a genotoxic, but not cytotoxic, concentration of H(2)S (500 muM H(2)S) using pathway-specific quantitative RT-PCR gene arrays. H(2)S was genotoxic in a concentration range from 250 to 2,000 microM, which is similar to concentrations found in the large intestine. Significant changes in gene expression were predominantly observed at 4 hr, with the greatest responses by PTGS2 (COX-2; 7.92-fold upregulated) and WNT2 (7.08-fold downregulated). COX-2 was the only gene upregulated at both 30 min and 4 hr. Overall, the study demonstrates that H(2)S modulates the expression of genes involved in cell-cycle progression and triggers both inflammatory and DNA repair responses. This study confirms the genotoxic properties of H(2)S in nontransformed human intestinal epithelial cells and identifies functional pathways by which this bacterial metabolite may perturb cellular homeostasis and contribute to the onset of chronic intestinal disorders.

Activation of cPLA 2 is required for leukotriene D 4 -induced proliferation in colon cancer cells

It is well documented that prolonged inflammatory conditions, particularly those relating to the colon, have been shown to induce cancer. We have previously demonstrated that the pro-inflammatory mediator leukotriene D(4) (LTD(4)) induces survival and proliferation in intestinal cells and that its receptor, CysLT(1), is upregulated in human colon cancer tissue. Here we demonstrate, for the first time that in both Int 407 (a non-transformed human intestinal epithelial cell line) and Caco-2 cells (a human colorectal carcinoma cell line), cytosolic phospholipase A(2)alpha (cPLA(2)alpha) is activated and translocates to the nucleus upon LTD(4) stimulation via a calcium-dependent mechanism that involves activation of protein kinase C (PKC), and the mitogen-activated protein kinases ERK1/2 and p38. We also show with a cPLA(2)alpha promoter luciferase assay, that LTD(4) induces an increase in the transcriptional activity of cPLA(2)alpha via activation of cPLA(2)alpha and the transcription factor NFkappaB. Interestingly we demonstrate here that both the basal and the LTD(4)-induced cPLA(2)alpha activity is elevated approximately 3-fold in Caco-2 colon cancer cells compared with Int 407 cells. The difference in basal activity was confirmed in human colon tumor samples by the finding of a similar increase in cPLA(2)alpha activity when compared with normal colon tissue. A functional role of the increased cPLA(2)alpha activity in tumor cells was revealed by our findings that inhibition of this enzyme reduced both basal and LTD(4)-induced proliferation, the effects being most pronounced in Caco-2 tumor cells. The present data reveal that cPLA(2)alpha, an important intracellular signal activated by inflammatory mediators, is an important regulator of colon tumor growth.

Cytokine regulation of human intestinal primary epithelial cell susceptibility to Fas-mediated apoptosis.

The regulatory mechanisms of nontransformed intestinal epithelial cell apoptosis have not been thoroughly investigated. We determined the susceptibility and mechanism of Fas-mediated apoptosis in nontransformed human intestinal epithelial cells (HIPEC) in the presence and absence of inflammatory cytokines. Despite ample expression of Fas, HIPEC were relatively insensitive to Fas-mediated apoptosis in that agonist anti-Fas antibody (CH11) induced a <25% increase in HIPEC apoptosis. Pretreatment of HIPEC with interferon (IFN)-gamma, but not tumor necrosis factor-alpha or granulocyte-macrophage colony-stimulating factor, significantly increased CH11-induced apoptosis of these cells without increasing Fas expression. Increased apoptosis correlated with increased caspase 3 activation but not expression of procaspase 3. Also, there was a significant delay in the onset of Fas-mediated apoptosis in HIPEC, which correlated with the generation of an activated caspase 3 p22/20 subunit. HIPEC required both initiator caspases 8 and 9 activity but expressed significantly less of the zymogen form of these caspases than did control cells. IFN-gamma-mediated sensitization of HIPEC occurred upstream of caspase 9 activation and correlated with a small increase in procaspase 8 expression (<1-fold increase) and a significant increase in expression of an intermediate form (p35) of caspase 4 (3.3-fold increase).

Susceptibility to FAS-induced apoptosis in human nontumoral enterocytes: role of costimulatory factors.

FAS-FAS ligand interaction has been implicated in increased enterocyte apoptosis seen in immune-mediated bowel injury. However, scant information exists on the role of FAS in physiological enterocyte turnover. In the present study, the regulation of enterocyte FAS and FAS ligand expression by cytokines and its functional role in human intestinal epithelial cell apoptosis and proliferation were analyzed with two different models: a nontransformed human intestinal epithelial cell line (HIEC) and normal colonic explant cultures. HIEC constitutively expressed FAS, as analyzed by flow cytometry. However, stimulation with agonistic anti-FAS antibody (1-500 ng/ml) did not induce HIEC apoptosis. In contrast, in the presence of tumor necrosis factor alpha (TNFalpha) and/or interferon gamma (IFNgamma), HIEC became highly susceptible to FAS-induced apoptosis. The sensitizing effect to FAS-induced apoptosis was mediated via TNFalpha- and IFNgamma-induced upregulation of FAS expression (maximally 348%). Receptor studies showed that the effect of TNFalpha on FAS was mediated via the p55 TNF receptor. In colonic organ cultures, IFNgamma and TNFalpha also enhanced colonocyte FAS expression, resulting in a markedly increased apoptotic response to stimulation of this receptor, as shown by in situ terminal deosyuridine triphosphate nick-end staining. Neither FAS ligand expression nor its induction by cytokines was observed in HIEC or colonic explants. Proliferation studies showed that FAS is not implicated in regulating HIEC growth. These findings suggest that, despite the fact that normal human enterocytes express FAS, costimulatory factors, such as TNFalpha or IFNgamma, abundantly secreted under inflammatory conditions, are necessary to sensitize intestinal epithelial cells to FAS-induced apoptosis by upregulating this receptor.