Normal Rat Intestinal Epithelial Cells Research Articles

Compound hydrogels derived from gelatin and gellan gum regulates the release of anthocyanins in simulated digestion

Anthocyanins need be absorbed in the small intestine for prevention and therapy of intestinal diseases. However, they are usually degraded in the stomach during digestion process. Therefore, it is necessary to protect anthocyanins from degradation and maximize the release of anthocyanins in the small intestine. In this study, we encapsulated anthocyanins with Gelatin/Gellan gum in five proportions (Gelatin/Gellan gum: 10/0, 9/1, 8/2, 7/3, 6/4, w/w). The results of embedding efficiency, morphology and gel strength of the Gelatin/Gellan gum-Anthocyanins (Gel/Gen-Ans) system showed that Gelatin/Gellan gum = 8/2 (H 2) had the most compact and stable structure, as proved by the distribution analysis and structural characterization of anthocyanins. In vitro simulated digestion experiments demonstrated that anthocyanins were mainly retained inside Gel/Gen-Ans in the stomach, and subsequently released in the intestine. Moreover, biocompatibility results showed that compound hydrogels did not inhibit the proliferation of normal rat intestinal epithelial cells (IEC-6), which indicated the safety of this compound hydrogels. Taken together, this system was an effective and safe method to ensure the target release of anthocyanins.

Malic Enzyme 1 (ME1) is pro-oncogenic in ApcMin/+ mice

Cytosolic Malic Enzyme (ME1) provides reduced NADP for anabolism and maintenance of redox status. To examine the role of ME1 in tumor genesis of the gastrointestinal tract, we crossed mice having augmented intestinal epithelial expression of ME1 (ME1-Tg mice) with ApcMin/+ mice to obtain male ApcMin/+/ME1-Tg mice. ME1 protein levels were significantly greater within gut epithelium and adenomas of male ApcMin/+/ME1-Tg than ApcMin/+ mice. Male ApcMin/+/ME1-Tg mice had larger and greater numbers of adenomas in the small intestine (jejunum and ileum) than male ApcMin/+ mice. Male ApcMin/+/ME1-Tg mice exhibited greater small intestine crypt depth and villus length in non-adenoma regions, correspondent with increased KLF9 protein abundance in crypts and lamina propria. Small intestines of male ApcMin/+/ME1-Tg mice also had enhanced levels of Sp5 mRNA, suggesting Wnt/β-catenin pathway activation. A small molecule inhibitor of ME1 suppressed growth of human CRC cells in vitro, but had little effect on normal rat intestinal epithelial cells. Targeting of ME1 may add to the armentarium of therapies for cancers of the gastrointestinal tract.

COX-2/EP2-EP4/β-catenin signaling regulates patulin-induced intestinal cell proliferation and inflammation

Patulin (PAT), a mycotoxin, is a natural contaminant that is produced by certain species of Penicillium, Aspergillus and Byssochlamys. The major contamination of PAT is in apple and apple based products. PAT is known to cause glutathione depletion, oxidative DNA damage and cell proliferation. Recently, in vitro studies have indicated that PAT can also increase the intestinal epithelial permeability, modulate tight junctions and decrease transepithelial electrical resistance. Nonetheless, no previous study has evaluated the mechanisms responsible for PAT-induced intestinal toxicity or its relevance to the in vivo situation. Here, Wistar rats were orally treated with 100 μg/kg body weight (b.wt.) of PAT, either alone or along with 100 mg/kg b. wt. of celecoxib for 3 days. We found that PAT exposure led to significantly higher levels of PGE2 in serum and intestinal tissue and high expression of COX-2 and Ki-67 compared to controls. Interestingly, our results showed that celecoxib treatment could decrease the PAT-induced PGE2 and reduce the PAT-induced intestinal damage. To study the mechanistic aspect, normal rat intestinal epithelial cells (IEC-6) were treated with non-toxic concentrations (100 nM, 250 nM and 500 nM) of PAT for 6 h. It was observed that PAT exposure caused enhanced proliferation, higher expression of COX-2, and EP2 and EP4 receptors, along with increased PGE2 secretion. Additionally, PAT exposure caused enhanced Akt expression, which in turn inhibits GSK-3β and stabilizes β-catenin. Overall, our study suggests that the COX-2/EP2-EP4/β-catenin signaling cascades are involved in the regulation of PAT-induced intestinal cell proliferation and inflammation.

Abstract 4420: Serum regulates reovirus-mediated cytopathy in K-Ras activated colorectal cancer and intestinal epithelial cell lines

Abstract Background: The oncolytic potency of replication competent REOvirus has been demonstrated in various cancers. The activity is most pronounced in Kras mutant cancer cells, which account for 30-40 % of all cancers. Herein we simultaneously utilized isogenic human derived colorectal cancer cell lines that differ only by the presence of mutant Kras and normal rat intestinal epithelial cells with inducible Kras to evaluate whether the presence of oncogenic Kras alters the sensitivity of colon cancer cells to reovirus. Methods: Reovirus was obtained from Oncolytics Biotech Inc. Rat IEC's harboring IPTG inducible mutant Ras oncogene (IEC-iKras) were treated with reovirus ± IPTG. The infectivity was maintained at a multiplicity of infection (MOI) of 2. MTT assay was performed at 72 hours to determine cell vability (CV). Similar experiments were also performed with HCT116 (Kras mut) and its isogenic derivative Hke3 [(Kras wild type (WT)]. Cells were cultured and treated in both serum rich (SR) and serum free (SF) media to determine the effect of growth factors on sensitivity to reovirus. Alterations in gene expression were determined by real time PCR and protein expression by western blot. Results: The activity of reovirus was observed in all cell lines studied. Reduction in CV was greater in Kras mutant HCT116 compared to WT Hke3 cells. Consistently, induction of Kras in IEC cells increased the potency of reovirus. Furthermore, this effect was more prominent in cells cultured in SF suggesting that growth factors in the serum may attenuate the effect of mutant Kras on reovirus sensitivity. Expression of the cell cycle regulator, p21 was preferentially increased upon reovirus infection in Kras mutant compared to WT cells, and in cells cultured in SF compared to SR conditions. Conclusion: Oncogenic Kras mutations increase the sensitivity of colon cancer cells to Reovirus. Clinical trials are underway testing reovirus in patients with Kras mutant metastatic colorectal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4420. doi:1538-7445.AM2012-4420

The serine protease inhibitor serpinE2 is a novel target of ERK signaling involved in human colorectal tumorigenesis

BackgroundAmong the most harmful of all genetic abnormalities that appear in colorectal cancer (CRC) development are mutations of KRAS and its downstream effector BRAF as they result in abnormal extracellular signal-related kinase (ERK) signaling. In a previous report, we had shown that expression of a constitutive active mutant of MEK1 (caMEK) in normal rat intestinal epithelial cells (IECs) induced morphological transformation associated with epithelial to mesenchymal transition, growth in soft agar, invasion and metastases in nude mice. Results from microarrays comparing control to caMEK-expressing IECs identified the gene encoding for serpinE2, a serine protease inhibitor, as a potential target of activated MEK1.Results1- RT-PCR and western blot analyses confirmed the strong up-regulation of serpinE2 expression and secretion by IECs expressing oncogenic MEK, Ras or BRAF. 2- Interestingly, serpinE2 mRNA and protein were also markedly enhanced in human CRC cells exhibiting mutation in KRAS and BRAF. 3- RNAi directed against serpinE2 in caMEK-transformed rat IECs or in human CRC cell lines HCT116 and LoVo markedly decreased foci formation, anchorage-independent growth in soft agarose, cell migration and tumor formation in nude mice. 4- Treatment of CRC cell lines with U0126 markedly reduced serpinE2 mRNA levels, indicating that expression of serpinE2 is likely dependent of ERK activity. 5- Finally, Q-PCR analyses demonstrated that mRNA levels of serpinE2 were markedly increased in human adenomas in comparison to healthy adjacent tissues and in colorectal tumors, regardless of tumor stage and grade.ConclusionsOur data indicate that serpinE2 is up-regulated by oncogenic activation of Ras, BRAF and MEK1 and contributes to pro-neoplastic actions of ERK signaling in intestinal epithelial cells. Hence, serpinE2 may be a potential therapeutic target for colorectal cancer treatment.

Syndecan-2 overexpression regulates adhesion and migration through cooperation with integrin α2

Syndecan-2, a transmembrane heparan sulfate proteoglycan, is known to serve as an adhesion receptor, but details of the regulatory mechanism governing syndecan-2 cell adhesion and migration remain unclear. Here, we examined this regulatory mechanism, showing that overexpression of syndecan-2 enhanced collagen adhesion, cell migration and invasion of normal rat intestinal epithelial cells (RIE1), and increased integrin α2 expression levels. Interestingly, RIE1 cells transfected with either syndecan-2 or integrin α2 showed similar adhesion and migration patterns, and a function-blocking anti-integrin α2 antibody abolished syndecan-2-mediated adhesion and migration. Consistent with these findings, transfection of integrin α2 siRNA diminished syndecan-2-induced cell migration in HCT116 human colon cancer cells. Taken together, these results demonstrate a novel cooperation between syndecan-2 and integrin α2β1 in adhesion-mediated cell migration and invasion. This interactive dynamic might be a possible mechanism underlying the tumorigenic activities of colon cancer cells.

W1269 HIV Protease Inhibitors Induce ER Stress and Disrupt Barrier Function in Intestinal Epithelial Cells

HIV protease inhibitor (PI)-induced diarrhea is one of the most frequent adverse side effects of these drugs and occurs in 16 to 62% of patients. Recently it has been reported that HIV PIs induce apoptosis and decrease barrier function in intestinal epithelial cells. However, the underlying cellular/molecular mechanisms remain unclear.We have previously demonstrated that HIV PIs induce endoplasmic reticulum (ER) stress, activate the unfolded protein response (UPR), and promote apoptosis in macrophages and hepatocytes. In the present study, we examined whether HIV PI-induced ER stress also plays a role in HIV PI-induced apoptosis and disruption of barrier function in intestinal epithelial cells. Methods: In Vitro studies were conducted in IEC6 cells (normal rat intestinal epithelial cells), differentiated IEC6 (stable Cdx2-transfected, IEC6-Cdx2L1), and stable SEAP-transfected IEC6 cells. HIV PIinduced ER stress was measured by decrease of SEAP activity. The mRNA and protein expression levels of ER stress-related genes were determined by real-time RT-PCR and western blot analysis, respectively. The paracellular permeability was measured by paracellular tracer flux assay and membrane-impermeable molecule, [14C]-mannitol, served as tracer. Lentiviral CHOP siRNA was used to knock down CHOP expression in IEC6 cells. Both C57/B6 wild type and CHOP knock out mice were used in In Vivo studies. Mice were gavaged with individual HIV PI (50 mg/kg) for three weeks. The pathological changes in intestine and colon were determined by histological evaluation. Results: Both ritonavir and lopinavir significantly induced ER stress, increased the expression of GRP78, CHOP, XBP-1 and ATF4, and promoted apoptosis in IEC6 and IEC6-Cdx2L1 cells. Paracellular permeability was increased by ~ 2.5 to 5 fold after cells were treated with 15 μM of ritonavir or lopinavir for 24 h. In contrast, amprenavir neither induced ER stress, nor increased paracellular permeability. Down-regulation of CHOP expression in IEC6 cells reduced HIV PI-induced apoptosis and disruption of barrier function. In Vivo studies demonstrated that ritonavir and lopinavir also significantly induced CHOP, XBP-1 and ATF4 expression in the intestine and colon of wild type mice. Furthermore, histological assessment indicated that ritonavir and lopinavir significantly induced intestinal epithelial damage in wild type mice, but had less effect in CHOP knock out mice. Conclusions: These findings indicate that HIV PI-induced ER stress and subsequent activation of the UPR represents an important cellular/molecular mechanism underlying HIV PI-induced disruption of intestinal barrier function.

Gene expression patterns of human colon tops and basal crypts and BMP antagonists as intestinal stem cell niche factors

Human colonic epithelial cell renewal, proliferation, and differentiation are stringently controlled by numerous regulatory pathways. To identify genetic programs of human colonic epithelial cell differentiation in vivo as well as candidate marker genes that define colonic epithelial stem/progenitor cells and the stem cell niche, we applied gene expression analysis of normal human colon tops and basal crypts by using expression microarrays with 30,000 genes. Nine hundred and sixty-nine cDNA clones were found to be differentially expressed between human colon crypts and tops. Pathway analysis revealed the differential expression of genes involved in cell cycle maintenance and apoptosis, as well as genes in bone morphogenetic protein (BMP), Notch, Wnt, EPH, and MYC signaling pathways. BMP antagonists gremlin 1, gremlin 2, and chordin-like 1 were found to be expressed by colon crypts. In situ hybridization and RT-PCR confirmed that these BMP antagonists are expressed by intestinal cryptal myofibroblasts and smooth muscle cells at the colon crypt. In vitro analysis demonstrated that gremlin 1 partially inhibits Caco-2 cell differentiation upon confluence and activates Wnt signaling in normal rat intestinal epithelial cells. Collectively, the expression data set provides a comprehensive picture of human colonic epithelial cell differentiation. Our study also suggests that BMP antagonists are candidate signaling components that make up the intestinal epithelial stem cell niche.

MEK Activation Suppresses CPT11-Induced Apoptosis in Rat Intestinal Epithelial Cells Through a COX-2-Dependent Mechanism

Resistance to chemotherapeutic agents is one of the distinct features of cancer cells. We evaluate the role of activated MEK-ERK signaling in Camptotecin/irinotecan (CPT-11)-induced cell death using constitutively activated MEK1-transfected normal rat intestinal epithelial cells (IEC-caMEK cells). A CPT-11-induced inhibitory concentration of 50% was determined by WST assay. Apoptosis was evaluated by DNA staining and fragmented DNA analysis. Protein expressions were analyzed by western blotting. We also examined the role of cyclooxygenase-2 in the cell systems. IEC-caMEK cells possessed survival advantages compared to control cells. Apoptosis was remarkably suppressed in IEC-caMEK cells. Western blot analysis revealed increased expression of Bcl-2, Bcl-xL, Mcl-1, and COX-2 and decreased expression of Bak in IEC-caMEK cells. The COX-2 selective inhibitor ameliorated the antiapoptotic nature of IEC-caMEK cells. MEK activation suppressed CPT-11-induced apoptosis in IEC-caMEK cells via a COX-2- dependent mechanism. Therefore, MEK-ERK signaling may contribute to the drug-resistant nature of cancer cells.

The Motogenic Effects of Cyclic Mechanical Strain on Intestinal Epithelial Monolayer Wound Closure Are Matrix Dependent

Complex deformation during normal digestion due to peristalsis or villous motility may be trophic for the intestinal mucosa. Because tissue fibronectin is increased in inflammatory states that may accompany mucosal injury, we evaluated the effects of cyclic mechanical strain and fibronectin on intestinal epithelial monolayer wound closure in Caco-2 and IEC-6 intestinal epithelial cells. Wounds created in intestinal epithelial monolayers were subjected to cyclic deformation. Wound closure was assessed by morphometry using microscopic imaging. Cell signals were assessed by Western blot and confocal microscopy. Mechanical strain stimulated wound closure on fibronectin but inhibited closure on collagen in Caco-2 and IEC-6 cells. The effect was independent of proliferation or cell spreading. Myosin light chain (MLC) and extracellular signal-regulated kinase (ERK) were phosphorylated in response to strain in confluent monolayers on both collagen and fibronectin. Blocking MLC or ERK phosphorylation inhibited the motogenic effect of strain on fibronectin. Although phosphorylated MLC was redistributed to the leading edge of migrating cells following 6 hours of strain on collagen and fibronectin, phosphorylated ERK was redistributed to the lamellipodial edge only on fibronectin. Strain promotes intestinal epithelial wound closure by a pathway requiring ERK and MLC kinase. Fibronectin-dependent ERK redistribution in response to strain in confluent migrating cells may explain the matrix dependence of the motogenic effect. Repetitive deformation stimulates intestinal epithelial proliferation on a collagen substrate, but not fibronectin. Deformation may exert matrix-dependent effects on intestinal epithelial cells, promoting epithelial restitution in fibronectin-rich tissue and proliferation in fibronectin-poor mucosa.

IL-6 Protects enterocytes from hypoxia-induced apoptosis by induction of bcl-2 mRNA and reduction of fas mRNA

Interleukin-6 (IL-6) has been shown to rescue enterocytes from hypoxia-induced apoptosis when given orally following hemorrhagic shock. In vitro models using an intestinal epithelial cell line (IEC-6) cultured with lipopolysaccharide (LPS) under low O 2 conditions, to mimic intestinal conditions, show that these cells also undergo apoptosis, which can be reduced by subsequent culture with IL-6. To examine further the mechanisms of rescue, we cultured normal rat intestinal epithelial cells (IEC-6) under both normoxic and hypoxic conditions and analyzed their responses to LPS and IL-6. We showed that IEC-6 expressed IL-6 receptor on its surface. Further, IEC-6 cells could be rescued from hypoxia-induced apoptosis by co-culture with IL-6. RNase protection assay (RPA) examination revealed that under hypoxic conditions, IEC-6 cells that were resistant to apoptosis showed reduced fas expression and increased bcl-2 expression after co-culture with LPS + IL-6.

Up-regulation of the enzymes involved in prostacyclin synthesis via Ras induces vascular endothelial growth factor

The constitutive activation of Ras is an important step in the development and progression of several different cancers and is known to increase the level of cyclooxygenase 2 (COX-2). Prostaglandins are the downstream bioactive lipid mediators produced by the COX-2 enzyme. We sought to determine the role of Ras-induced up-regulation of the enzymes involved in prostacyclin biosynthesis in nontransformed rat intestinal epithelial cells (IECs). Messenger RNA (mRNA) and protein expression were analyzed by Northern and Western analysis, respectively, to determine the level of enzymes induced by Ras. In vitro assays were used to determine the production of vascular endothelial growth factor (VEGF) and prostaglandins as well as the promoter and enzymatic activation of the rate-limiting enzyme in prostaglandin production (phospholipase A(2) [cPLA(2)]). The inducible expression of Ha-Ras(V12) increased the production of prostaglandin (PG)F(2alpha) and prostacyclin by 2- and 13-fold, respectively. The induction of Ha-Ras(V12) also up-regulated the mRNA and protein levels of cPLA(2), COX-2, and prostacyclin synthase, as well as the promoter and enzyme activity of cPLA(2). Furthermore, oncogenic Ras increased the production of the pro-angiogenic factor VEGF. The increase of VEGF was abolished after treatment with celecoxib, a selective COX-2 inhibitor. The addition of PGI 2 alone also induced the expression of VEGF. Inducible Ha-Ras(V12) increases the production of PGI(2) through the coordinate up-regulation of cPLA(2), COX-2, and prostacyclin synthase (PGIS). The production of PGI(2) leads to an increase in the level of the pro-angiogenic factor VEGF, which is known to play a crucial role in the regulation of tumor-associated angiogenesis.

Growth and propagation of normal rat intestinal epithelial cells.

A combination of mild proteolytic digestion and selective growth stimulation has been used to isolate and propagate adult rat intestinal epithelial cells with a finite life span. Growth of these cells on a variety of matrices and on mesenchymal cells has resulted in the expression of brush border enzymes including sucrase-isomaltase, aminopeptidase N, and alkaline phosphatase. Examination of the cells at the electron microscopic level has revealed that although these cells express key brush border enzymes, they do not have a fully formed brush border. These findings suggest that the expression of brush border enzymes and structural proteins represent distinct stages of enterocyte differentiation that are under separate transcriptional and temporal control.

Response of Cultured IEC-17 Normal Rat Intestinal Epithelial Cells to X Radiation

The growth parameters and radiosensitivity of normal rat intestinal epithelial cells, IEC-17, were studied. The cells were cultured by standard methods and exposed to an array of doses (1-12 Gy) of 250 kVp X rays. The survival curves generated exhibited no initial shoulder and were bimodal. The Do of the first component was about 0.2 Gy and the second component. 5.0 Gy. The ability of this cell line to repair sublethal lesions was examined by fractionation studies; repair was completed within 60 min after the first dose. When Chinese hamster ovary (CHO) cells were grown under the same conditions used for the IEC-17 cells and then irradiated with single doses, a typical survival curve with a Do of 1.4 Gy was obtained. The survival curves obtained for the IEC-17 cell line are consistent with the response of a morphologically distinct single population containing two functionally separate types of cells.