Normal Intestinal Epithelial Cells Research Articles

Normal Mouse Intestinal Epithelial Cells as a Model for the in vitro Invasion of Trichinella spiralis Infective Larvae

It has been known for many years that Trichinella spiralis initiates infection by penetrating the columnar epithelium of the small intestine; however, the mechanisms used by the parasite in the establishment of its intramulticellular niche in the intestine are unknown. Although the previous observations indicated that invasion also occurs in vitro when the infective larvae are inoculated onto cultures of intestinal epithelial cells (e.g., human colonic carcinoma cell line Caco-2, HCT-8), a normal readily manipulated in vitro model has not been established because of difficulties in the culture of primary intestinal epithelial cells (IECs). In this study, we described a normal intestinal epithelial model in which T. spiralis infective larvae were shown to invade the monolayers of normal mouse IECs in vitro. The IECs derived from intestinal crypts of fetal mouse small intestine had the ability to proliferate continuously and express specific cytokeratins as well as intestinal functional cell markers. Furthermore, they were susceptible to invasion by T. spiralis. When inoculated onto the IEC monolayer, infective larvae penetrated cells and migrated through them, leaving trails of damaged cells heavily loaded with T. spiralis larval excretory-secretory (ES) antigens which were recognized by rabbit immune sera on immunofluorescence test. The normal intestinal epithelial model of invasion mimicking the natural environment in vivo will help us to further investigate the process as well as the mechanisms by which T. spiralis establishes its intestinal niche.

Adenovirus-mediated siRNA targeting Bcl-xL inhibits proliferation, reduces invasion and enhances radiosensitivity of human colorectal cancer cells

IntroductionBcl-xL, an important member of anti-apoptotic Bcl-2 family, plays critical roles in tumor progression and development. Previously, we have reported that overexpression of Bcl-xL was correlated with prognosis of colorectal cancer (CRC) patients. The aim of this study was to investigate the association of Bcl-xL expression with invasion and radiosensitivity of human CRC cells.MethodsRT-PCR and Western blot assays were performed to determine the expression of Bcl-xL mRNA and protein in CRC cells and normal human intestinal epithelial cell line. Then, adenovirus-mediated RNA interference technique was employed to inhibit the expression of Bcl-xL gene in CRC cells. The proliferation of CRC cells was analyzed by MTT and colony formation assay. The migration and invasion of CRC cells was determined by wound-healing and tranwell invasion assays. Additionally, the in vitro and in vivo radiosensitivity of CRC cells was determined by clonogenic cell survival assay and murine xnograft model, respectively.ResultsThe levels of Bcl-xL mRNA and protein expression were significantly higher in human CRC cells than in normal human intestinal epithelial cell line. Ad/shBcl-xL could significantly reduce the expression of Bcl-xL protein in CRC cells. Also, we showed that adenovirus-mediated siRNA targeting Bcl-xL could significantly inhibit proliferation and colony formation of CRC cells. Ad/shBcl-xL could significantly suppress migration and invasion of CRC cells. Moreover, Ad/shBcl-xL could enhance in vitro and in vivo radiosensitivity of CRC cells by increasing caspase-dependent apoptosis.ConclusionsTargeting Bcl-xL will be a promising strategy to inhibit the metastatic potential and reverse the radioresistance of human CRC.

Luminal-Applied Flagellin Is Internalized by Polarized Intestinal Epithelial Cells and Elicits Immune Responses via the TLR5 Dependent Mechanism

Bacteria release flagellin that elicits innate responses via Toll-like receptor 5 (TLR5). Here, we investigated the fate of apically administrated full length flagellin from virulent and avirulent bacteria, along with truncated recombinant flagellin proteins in intestinal epithelial cells and cellular responses. Flagellin was internalized by intestinal epithelial cell (IEC) monolayers of IEC-18. Additionally, apically applied flagellin was internalized by polarized human Caco-2BBe and T-84 cells in a TLR5 dependent mechanism. More, flagellin exposure did not affect the integrity of intestinal monolayers. With immunofluorescent staining, internalized flagellin was detected in both early endosomes as well as lysosomes. We found that apical exposure of polarized Caco-2BBe and T-84 to flagellin from purified Salmonella, Escherichia coli O83:H1 (isolate from Crohn’s lesion) or avirulent E. coli K12 induced comparable levels of basolateral IL-8 secretion. A recombinant protein representing the conserved amino (N) and carboxyl (C) domains (D) of the flagellin protein (ND1/2ECHCD2/1) induced IL-8 secretion from IEC similar to levels elicited by full-length flagellins. However, a recombinant flagellin protein containing only the D3 hypervariable region elicited no IL-8 secretion in both cell lines compared to un-stimulated controls. Silencing or blocking TLR5 in Caco-2BBe cells resulted in a lack of flagellin internalization and decreased IL-8 secretion. Furthermore, apical exposure to flagellin stimulated transepithelial migration of neutrophils and dendritic cells. The novel findings in this study show that luminal-applied flagellin is internalized by normal IEC via TLR5 and co-localizes to endosomal and lysosomal compartments where it is likely degraded as flagellin was not detected on the basolateral side of IEC cultures.

Anticancer activity and cDNA microarray studies of a ( RS)-1,2,3,5-tetrahydro-4,1-benzoxazepine-3-yl]-6-chloro-9 H-purine, and an acyclic ( RS)- O,N-acetalic 6-chloro-7 H-purine

Completing a SAR study, a series of ( RS)-6-substituted-7- or 9-(1,2,3,5-tetrahydro-4,1-benzoxazepine-3-yl)-7 H or 9 H-purines was previously prepared. The most potent antiproliferative agent against the MCF-7 adenocarcinoma cell line that belongs to the benzoxazepine O,N-acetalic family is ( RS)-9-[1-(9 H-fluorenyl-9-methoxycarbonyl)-1,2,3,5-tetrahydro-4,1-benzoxazepine-3-yl]-6-chloro-9 H-purine ( 16, IC 50 = 0.67 ± 0.18 μM), whilst ( RS)-7-{2-( N-hydroxymethylphenyl)-2-nitrobenzenesulfonamido]-1-methoxyethyl}-6-chloro-7 H-purine ( 37) shows the lowest IC 50 value between the family of acyclic O,N-acetals (IC 50 = 3.25 ± 0.23 μM). Moreover, 16 showed the better in vitro Therapeutic Index in breast cell lines (3.19), whilst 37 was found to be 3.69-fold more active against HT-29 human colon cancer cell line than versus IEC-6 normal rat intestinal epithelial cell line. The global apoptotic cells caused by 16 and 37 against MCF-7 were 80.08% and 54.85% of cell population after 48 h, respectively. cDNA microarray technology reveals potential drug targets, which are mainly centred on positive apoptosis regulatory pathway genes, and the repression of genes involved in carcinogenesis, proliferation and tumour invasion.

Abstract 1505: The role of cathepsin B in colorectal tumorigenesis

Abstract Many cancers express elevated protease levels which contribute to certain aspects of tumor behavior such as growth and metastatic spread. Specifically, elevation of the cysteine protease cathepsin B has been shown to correlate with malignant progression of tumors. The majority of reports on cathepsin B expression in tumors have focused on measurements of activity or protein staining. However, multiple transcripts species have been detected resulting from alternative splicing in the 5’- or 3-untranslated regions, and possibly the use of alternative promoter regions. The specific role of these variants remains to be established. Methods: Expression of cathepsin B was analyzed in normal human intestinal epithelial cells (HIEC) and colorectal cancer cell lines (Caco-2/15, HCT116, DLD1, HT29, SW480, T84, Colo205) by qPCR and Western blot. The role of cathepsin B was examined in HT29 and DLD1 cell lines through recombinant lentiviruses encoding anti-cathepsin B short hairpin RNA (shRNA). Results: RT-PCR analyses demonstrated that the levels of the full transcript for cathepsin B containing exons 1-12 (CB) were similar between normal and cancerous cell lines. By contrast, the splice variant lacking exons 2 and 3 (CB-2,3) was expressed only in cancer cells and in biopsies from human colorectal tumors when compared to the adjacent healthy tissues. Immunofluorescence studies showed that this novel tumor form of cathepsin B (CB-2,3) was associated with nuclei and mitrochondria while the full-length cathepsin B protein (CB) was found in the lysosomes. Western blot analyses on culture media showed a strong secretion of various cathepsin B isoforms by cancer cell lines. The lentiviral infection of a shRNA which specifically knocked-down cathepsin B expression, while decreasing enzyme activity in cell lysates, did not influence colorectal cancer cell proliferation under anchorage-dependent conditions. By contrast, decrease of cathepsin B expression in HT29 and DLD1 colon cancer cell lines resulted in a marked reduction of their capacity to grow in soft agar. Levels of β-catenin protein and phosphorylated ERK1/2 were not affected following cathepsin B silencing in contrast to phosphorylated Akt which was significantly reduced. Further, migration and invasion through Matrigel was reduced by 60% in cells expressing the shRNA against cathepsin B compared to cells expressing a control shRNA. Conclusion: These results suggest that the alternative splicing of cathepsin B could contribute to the aberrant intracellular trafficking and function of cathepsin B in colorectal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1505. doi:10.1158/1538-7445.AM2011-1505

Intestinal epithelial cells express galectin-9 in patients with food allergy that plays a critical role in sustaining allergic status in mouse intestine

Mechanisms in sustaining the allergic hypersensitivity status in the body are unclear. Galectin-9 (Gal-9) has strong immune regulatory capacity. The present study aims to elucidate the role of Gal-9 in sustaining allergic status in the intestine. Duodenal biopsies were obtained from 20 patients with peptic ulcer and food allergy (FA). The expression of Gal-9 in intestinal tissue was examined at both protein level and mRNA level. Two coculture systems with intestinal epithelial cells (IEC) and mast cells, or dendritic cells (DC) and T cells were established to investigate the source of Gal-9 in the intestine and the mechanism by which Gal-9 modulated DC's phenotyping and sustained the T helper 2 polarization. Normal IEC showed mild expression of Gal-9 that was markedly enhanced in patients with FA. Mast cells had the capability to induce IEC to produce Gal-9 via releasing tryptase that activated the proteinase-activated receptor 2 on IEC. Gal-9 activated DC to produce TIM4 (T-cell immunoglobulin mucin domain) via ligating TIM3 on DC via activating the cyclic guanosine monophosphate (cGMP) pathway. In a mouse FA model, blocking Gal-9 inhibited the allergic hypersensitivity status and the antigen-specific Th2 response in the intestine. IEC-derived Gal-9 contributes to sustaining the allergic status in the intestine.

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.

Sodium Selenite Radiosensitizes Hormone-Refractory Prostate Cancer Xenograft Tumors but Not Intestinal Crypt Cells In Vivo

We have previously shown that sodium selenite (SSE) increases radiation-induced cell killing of human prostate carcinoma cells in vitro. In this study we further evaluated the in vivo radiosensitizing effect of SSE in prostate cancer xenograft tumors and normal radiosensitive intestinal crypt cells. Immunodeficient (SCID) mice with hormone-independent LAPC-4 (HI-LAPC-4) and PC-3 xenograft tumors (approximately 200 mm(3)) were divided into four groups: control (untreated), radiation therapy (XRT, local irradiation), SSE (2 mg/kg, intraperitoneally, 3 times/week), and XRT plus SSE. The XRT was given at the beginning of the regimen as a single dose of 5 Gy for HI-LAPC-4 tumors and a single dose of 7 Gy followed by a fractional dose of 3 Gy/d for 5 days for PC-3 tumors. The tumor volume was measured 3 times per week. The radiosensitizing effect of SSE on normal intestinal epithelial cells was assessed by use of a crypt cell microcolony assay. In the efficacy study, SSE alone significantly inhibited the tumor growth in HI-LAPC-4 tumors but not PC-3 tumors. Sodium selenite significantly enhanced the XRT-induced tumor growth inhibition in both HI-LAPC-4 and PC-3 tumors. In the toxicity study, SSE did not affect the intestinal crypt cell survival either alone or in combination with XRT. Sodium selenite significantly enhances the effect of radiation on well-established hormone-independent prostate tumors and does not sensitize the intestinal epithelial cells to radiation. These results suggest that SSE may increase the therapeutic index of XRT for the treatment of prostate cancer.

Oncogenic engagement of the Met receptor is sufficient to evoke angiogenic, tumorigenic, and metastatic activities in rat intestinal epithelial cells

The deregulation of Met/hepatocyte growth factor (HGF) receptor tyrosine kinase signaling constitutes a common event in colorectal cancers. However, the physiopathological functions of such a deregulation remain poorly understood. In the present study, we investigated the role of the deregulation of Met receptor in the neoplastic transformation of intestinal epithelial cells. To do so, the normal, well-established and characterized rat intestinal epithelial IEC-6 cells were transduced with a retrovirus carrying the oncogenic constitutive active form of Met receptor, Tpr-Met. Herein, we show that compared with control IEC-6 cells, Tpr-Met-IEC-6 cells exhibit enhanced proliferation, loss of growth-contact inhibition, cell morphological alterations, actin cytoskeletal reorganization, loss of E-cadherin expression and anchorage-independent growth. Moreover, Tpr-Met-IEC-6 cells are conferred the capacity to produce the proangiogenic factor VEGF and to reduce the potent antiangiogenic factor thrombospondin-1. Of significance, Tpr-Met-IEC-6 cells are endowed with the ability to elicit angiogenic responses and to form tumors and metastases in vivo. Hence, our study demonstrates for the first time that the sole oncogenic engagement of Met receptor in normal intestinal epithelial cells is sufficient to induce a wide array of cancerous biological processes that are fundamental to the initiation and malignant progression of colorectal cancers.

Atonal Homolog 1 Is Required for Growth and Differentiation Effects of Notch/γ-Secretase Inhibitors on Normal and Cancerous Intestinal Epithelial Cells

The atonal homolog 1 (Atoh1) transcription factor is required for intestinal secretory (goblet, Paneth, enteroendocrine) cell differentiation. Notch/gamma-secretase inhibitors (GSIs) block proliferation and induce secretory cell differentiation in the intestine. We used genetic analyses of mice to determine whether Atoh1 mediates the effects of GSIs in normal and cancerous intestinal epithelia. We studied mice with intestine-specific disruption of Atoh1 (Atoh1(Deltaintestine)), the adenomatosis polyposis coli (APC)(min) mutation, both mutations (Atoh1(Deltaintestine); APC(min)), or littermate controls; mice were given GSI or vehicle. Colorectal cancer (CRC) cell lines were treated with GSI or vehicle and with small hairpin RNAs to reduce ATOH1. Differentiation and homeostasis were assessed by protein, RNA, and histologic analyses. GSIs failed to induce secretory cell differentiation or apoptosis or decrease proliferation of Atoh1-null progenitor cells, compared with wild-type cells. Exposure of APC(min) adenomas to GSIs decreased proliferation and increased secretory cell numbers in an Atoh1-dependent manner. In CRC cells treated with GSI, ATOH1 levels were correlated inversely with proliferation. ATOH1 was required for secretory cell gene expression in cell lines and in mice. ATOH1 is required for all effects of GSIs in intestinal crypts and adenomas; Notch has no unique function in intestinal progenitors and cancer cells other than to regulate ATOH1 expression. Reducing ATOH1 activity might mitigate intestinal toxicity from systemic GSI therapy for nonintestinal diseases. Among gastrointestinal malignancies, ATOH1 mediates the effects of GSIs, so ATOH1 expression levels might predict responses to these inhibitors. We propose that only the subset of CRCs that retain ATOH1 expression will respond to GSIs.

173 Engagement of Shc-Dependent Signals by RTK Promotes In Vivo Tumorigenic and Metastatic Behaviors in Normal Intestinal Epithelial Cells

173 Engagement of Shc-Dependent Signals by RTK Promotes In Vivo Tumorigenic and Metastatic Behaviors in Normal Intestinal Epithelial Cells

268 Atonal Homolog 1 (ATOH1) is Essential for Growth and Differentiation Effects of Notch/γ Secretase Inhibitors on Normal and Cancerous Intestinal Epithelial Cells

Background: The Atonal Homolog 1 (Atoh1 or Math1) transcription factor is required for intestinal secretory (goblet, Paneth, enteroendocrine) cell differentiation. Notch/γ secretase inhibitors (GSIs) block proliferation and induce secretory cell differentiation in the intestine. Here, we genetically determined whether Atoh1 mediates the effects of GSIs in both normal and cancerous intestinal epithelia. Methods: Mice with intestine-specific Atoh1 mutation (Atoh1Δintestine), APCmin mutation, double mutation [Atoh1Δintestine; APCmin], and littermate controls, were treated with GSI or vehicle. Colorectal cancer (CRC) cell lines were treated with GSI or vehicle, and with shRNAs to reduce ATOH1. Differentiation and homeostasis was assessed by quantitative gene expression and histologic analysis. Results: Unlike wild type controls, GSIs failed to induce secretory cell differentiation or apoptosis, or decrease proliferation of Atoh1-null progenitor cells. Similarly, GSI treatment of APCmin adenomas decreased proliferation and increased secretory cells in an Atoh1-dependent fashion. In CRC cells treated with GSI, ATOH1 was inversely correlated with proliferation and was required for secretory cell gene expression. Conclusions:We find that ATOH1 is critical for all effects of GSIs in intestinal crypts and adenomas, suggesting that Notch has no unique function in intestinal progenitors and cancer cells other than regulating appropriate ATOH1 expression. Reducing or circumventing ATOH1may mitigate intestinal toxicity from systemic GSI therapy for non-intestinal diseases. Among gastrointestinal malignancies, ATOH1 is a critical mediator of GSI effects, and ATOH1 expression status may predict response to GSI. We suggest that only the subset of CRCs which retain ATOH1 expression will respond to GSI therapy.

T1186 Loss of RKIP Expression is Associated With Metastasis in Esophageal Adenocarcinoma

The PTEN phosphatase acts on phosphatidylinositols resulting from phosphatidylinositol 3-kinase (PI3K) activation. Germinal inactive mutations of PTEN have been identified in Cowden syndrome, a disease characterized by the development of hamartomatous polyps in the digestive tract and associated with an increased risk of cancer. However, little is known about the specific cellular role of PTEN in human intestinal epithelial cells. Objective. To investigate the role of PTEN in human colorectal cancer cells. Methods. PTEN expression was analyzed by Western blot in human colorectal tumours, in normal epithelial crypt cells (HIEC) and in many colorectal cancer cell lines: Caco-2/15, DLD-1, HCT116, HT29, LoVo, Colo205 and SW480. Caco-2/15, HCT116 and CT-26 cells were infected with recombinant lentiviruses expressing a shRNA that specifically knocked-down PTEN. The impact of PTEN downregulation was analyzed on cell migration (wound assay), invasion (matrigel-coated transwells) and on tumour and metastasis formation in mice. Results. 1PTEN expression is decreased in approximately 60% of all colon cancer tumours of all grades. 2PTEN is expressed in all cancer cell lines but at a lower level than normal human intestinal epithelial cells. 3The lentiviral infection of PTEN shRNA significantly increases spreading, migration and invasion capacities of Caco-2/15 and HCT116 cells. 4PTEN downregulation in both Caco-2/15 and HCT116 cells increases tumour size following subcutaneous injection of these cells in nude mice. 5Loss of PTEN expression in CT-26 (mouse colorectal cancer cells) leads to an increase in their metastatic potential following tail-vein and intrasplenic injections in BALB/c mice. Conclusions. Altogether, these results indicate that PTEN is involved in the control of migration/invasion capacity and tumorigenic/metastatic potential of human colon cancer cells. Supported by CIHR Team grant.

871 Eosinophilic Esophagitis (EoE)-Associated Eosinophil Products and Cytokines Reduce Neurogenic Esophageal Smooth Muscle Contractility

In the intestinal epithelium, the Cdx, GATA and HNF transcription factor families are responsible for the expression of differentiation markers such as sucrase-isomaltase. Although previous studies have shown that Cdx-2 can induce differentiation in rat intestinal IEC-6 cells, no data are available concerning the direct implication of transcription factors on differentiation in human normal intestinal epithelial cell types. We investigated the role of Cdx-2, GATA-4 and HNF-1α using the undifferentiated human intestinal epithelial crypt cell line HIEC. These transcription factors were tested on proliferation and expression of polarization and differentiation markers. Ectopic expression of Cdx-2 or HNF-1α, alone and in combination, altered cell proliferation abilities through the regulation of cyclin D1 and p27 expression. HNF-1α and GATA-4 together induced morphological modifications of the cells towards polarization resulting in the appearance of functional features such as microvilli. HNF-1α was also sufficient to induce the expression of cadherins and dipeptidylpeptidase while in combination with Cdx-2 it allowed the expression of the late differentiation marker sucrase-isomaltase. Large scale analysis of gene expression confirmed the cooperative effects of these factors. Finally, while DCAMKL1 and Musashi-1 expression were downregulated in differentiated HIEC, other intestinal stem cell markers, such as Bmi-1 and Lgr5, were unaffected. These observations show that, cooperatively with Cdx2, HNF-1α acts as a key factor on human intestinal cells to trigger the onset of their functional differentiation program whereas GATA-4 initiates the establishment of cell polarity.

Abstract 4258: Role of β2SP in asymmetric division and differentiation of intestine epithelial cells

Abstract The adult intestinal epithelium of the gastrointestinal tract has a well-defined organizational structure. The epithelium can be divided into two regions, a functional region that houses differentiated cells (villi) and a proliferative region (crypts of Lieberkühn) that represents the epithelial stem cell niche. In the small intestine, finger-like projections protrude from the floor of the intestine, forming villi. Multiple proliferative crypts surround each villus base. All epithelial cells that populate the villus originate from these crypts. Multipotent epithelial stem cells reside in the crypts and give rise to four principal epithelial lineages: absorptive enterocytes, mucin secreting-goblet cells, peptide hormone secreting enteroendocrine cells, and Paneth cells. Three of these cell lineages differentiate as they migrate up and out of the crypts onto the adjacent villus. The fourth lineage, Paneth cells, differentiates with a downward migration to reside at the base of the crypt. It is believed that these different cell types are generated by asymmetric cell divisions of stem/progenitor cells. However, the molecular mechanisms of how stem cells differentiate into four different cell types are only partially understood. Aim: in the current study, we evaluated the role of β2SP (β2 spectrin) in the differentiation and asymmetric division of stem/progenitor cells in normal intestine epithelium. Results: Our preliminary data showed that the number of secreting-goblet cells and peptide hormone secreting enteroendocrine cells was reduced in β2SP+/− mice compared to the wt mice. The number of cells undergo asymmetric division was also 3 folds lower in β2SP+/− mice than in wt mice. In in vitro studies, polarization of Par3 and PKCζ was impaired in β2SP−/− MEF cells. The coimmunoprecipitation assay demonstrated that β2SP directly bound to Par3 and actin in normal intestine epithelial cells, indicating that β2SP was involved in the spindle orientation and positioning during the asymmetric division of intestine epithelial cells. Conclusion: β2SP interacts with Par3 and regulates spindle orientation and polarization in the process of cell division, thereby contributing to the differentiation and asymmetric division of intestinal stem/progenitor cells. 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 4258.

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.

Abstract 2130: Genomic profiling identifies CDX2 as a lineage-dependent oncogene amplified in colorectal cancer

Abstract Somatic DNA alteration underlies tumor development and progression, and gives rise to tumors with diverse genetic contexts. Characterizing these contexts helps identify driving genetic lesions and potential therapeutic vulnerabilities. Here, we surveyed a collection of 29 colorectal cancer cell lines for DNA copy number alterations and identified in a subset of samples gain/amplification on chromosome 13, which was well represented among the genomic profiles of 344 primary colorectal tumors examined, but not in other tumor types. A minimal region of high-level amplification on 13q12.2, containing the caudal type homeobox transcription factor CDX2, was identified in one cell line and 7 of 123 (6%) primary colorectal tumors. A tissue microarray consisting of an independent set of 83 colorectal tumors confirmed the presence of a subset of tumors harboring copy number gain/amplification of 13q with increased CDX2 protein expression. In the context of genomic amplification, RNA interference experiments showed CDX2 is required for proliferation of colorectal cancer cells and promotes cell cycle progression without an effect on apoptosis. CDX2 is a known transcriptional regulator of normal gut development and intestinal epithelial cell differentiation and maintenance. Here we have shown that for a subset of lineage-derived colorectal tumors, cell proliferation is dependent on the abnormal amplification and overexpression of CDX2. Thus, we nominate CDX2 as a novel lineage-dependent oncogene deregulated in colorectal cancer. 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 2130.

Development of a Novel Self-Microemulsifying Drug Delivery System for Reducing HIV Protease Inhibitor-Induced Intestinal Epithelial Barrier Dysfunction

The development of HIV protease inhibitors (PIs) has been one of the most significant advances of the past decade in controlling HIV infection. Unfortunately, the benefits of HIV PIs are compromised by serious side effects. One of the most frequent and deleterious side effects of HIV PIs is severe gastrointestinal (GI) disorders including mucosal erosions, epithelial barrier dysfunction, and leak-flux diarrhea, which occurs in 16-62% of patients on HIV PIs. Although the underlying mechanisms behind HIV PI-associated serious adverse side effects remain to be identified, our recent studies have shown that activation of endoplasmic reticulum (ER) stress response plays a critical role in HIV PI-induced GI complications. The objective of this study was to develop a novel self-microemulsifying drug delivery system (SMEDDS) using various antioxidants as surfactants and cosurfactants to reduce the GI side effects of the most commonly used HIV PI, ritonavir. The biological activities of this SMSDDS of ritonavir were compared with that of Norvir, which is currently used in the clinic. Rat normal intestinal epithelial cells (IEC-6) and mouse Raw 264.7 macrophages were used to examine the effect of new SMEDDS of ritonavir on activation of ER stress and oxidative stress. Sprague-Dawley rats and C57/BL6 mice were used for pharmacokinetic studies and in vivo studies. The intracellular and plasma drug concentrations were determined by HPLC analysis. Activation of ER stress was detected by Western blot analysis and secreted alkaline phosphatase (SEAP) reporter assay. Reactive oxygen species (ROS) was measured using dichlorodihydrofluorescein diacetate as a probe. Cell viability was determined by Roche's cell proliferation reagent WST-1. Protein levels of inflammatory cytokines (TNF-alpha and IL-6) were determined by enzyme-linked immunosorbent assays (ELISA). The intestinal permeability was assessed by luminal enteral administration of fluorescein isothiocyanate conjugated dextran (FITC-dextran, 4 kDa). The pathologic changes in intestine were determined by histological examination. The results indicated that incorporation of antioxidants in this new SMEDDS not only significantly reduced ritonavir-induced ER stress activation, ROS production and apoptosis in intestinal epithelial cells and macrophages, but also improved the solubility, stability and bioavailability of ritonavir, and significantly reduced ritonavir-induced disruption of intestinal barrier function in vivo. In conclusion, this new SMEDDS of ritonavir has less GI side effects compared to Norvir. This new SMEDDS can be used for other HIV PIs and any insoluble antiviral drug with serious GI side effects.

Translational Regulation of CUG‐BP1 by Polyamines through the RNA‐binding Protein HuR

All mammalian cells depend on polyamines for growth and division, but the exact roles of polyamines at the molecular level remain unclear. CUG‐BP1 protein interacts with CG‐rich element of many labile mRNAs and regulates decay and translation of its target transcripts. HuR modulates mRNA turnover and translation and its activity is tightly regulated by cellular polyamines. This study tests if polyamines regulate CUG‐BP1 expression through HuR in normal intestinal epithelial cells (IEC‐6 line).MethodsPolyamine levels were depleted by inhibiting ODC (key enzyme for polyamine biosynthesis) with DFMO but increased by overexpressing ODC gene. CUG‐BP1 expression was examined by newly translated protein and reporter assay using chimeric CUG‐BP1 3′‐UTR. RNA‐binding was examined by biotin pull‐down and RNP‐IP assays.ResultsDecreased polyamines by DFMO increased CUG‐BP1 protein, whereas elevated polyamines through ODC overexpression reduced CUG‐BP1 protein, neither intervention changed CUG‐BP1 mRNA levels. Polyamine depletion also increased CUG‐BP1 translation, but it failed to alter CUG‐BP1 protein stability. HuR bound the CUG‐BP1 mRNA and this association increased following polyamine depletion. HuR silencing repressed CUG‐BP1 translation in polyamine‐deficient cells, thus reducing CUG‐BP1 expression levels.ConclusionsThese results indicate that polyamines repress CUG‐BP1 translation by reducing HuR interaction with the CUG‐BP1 mRNA.

Cooperation between HNF-1α, Cdx2, and GATA-4 in initiating an enterocytic differentiation program in a normal human intestinal epithelial progenitor cell line

In the intestinal epithelium, the Cdx, GATA, and HNF transcription factor families are responsible for the expression of differentiation markers such as sucrase-isomaltase. Although previous studies have shown that Cdx2 can induce differentiation in rat intestinal IEC-6 cells, no data are available concerning the direct implication of transcription factors on differentiation in human normal intestinal epithelial cell types. We investigated the role of Cdx2, GATA-4, and HNF-1alpha using the undifferentiated human intestinal epithelial crypt cell line HIEC. These transcription factors were tested on proliferation and expression of polarization and differentiation markers. Ectopic expression of Cdx2 or HNF-1alpha, alone or in combination, altered cell proliferation abilities through the regulation of cyclin D1 and p27 expression. HNF-1alpha and GATA-4 together induced morphological modifications of the cells toward polarization, resulting in the appearance of functional features such as microvilli. HNF-1alpha was also sufficient to induce the expression of cadherins and dipeptidylpeptidase, whereas in combination with Cdx2 it allowed the expression of the late differentiation marker sucrase-isomaltase. Large-scale analysis of gene expression confirmed the cooperative effect of these factors. Finally, although DcamKL1 and Musashi-1 expression were downregulated in differentiated HIEC, other intestinal stem cell markers, such as Bmi1, were unaffected. These observations show that, in cooperation with Cdx2, HNF-1alpha acts as a key factor on human intestinal cells to trigger the onset of their functional differentiation program whereas GATA-4 appears to promote morphological changes.