Intestinal Cell Line Research Articles

Cellular differentiation of non-transformed intestinal epithelial cells is regulated by Lactobacillus rhamnosus and L. casei strains.

The aim of this study was to characterize an in vitro modulating effect of three commensal Lactobacillus strains on cellular differentiation of non-transformed crypt-like rat small intestinal cell line IEC-18. IEC-18 was grown on extracellular matrix, with or without presence of Lactobacillus strains. Gene expression of IEC-18 bacterial detection system - such as Toll-like receptors TLR-2, TLR-4, signal adapter MyD88, cytoplasmic NOD2 receptor, inflammatory cytokines IL-18, IL-1beta, chemokine IL-8 and enzyme caspase-1 - was evaluated using real-time PCR. Expression and localization of TLR-2, TLR-4, IL-18 and caspase-1 proteins was demonstrated by Western blotting and immunofluorescent staining. Secretion of IL-18 to apical and basolateral surfaces was assayed by ELISA. Our results suggested that L. casei LOCK0919 accelerated differentiation of IEC-18 by stimulating TLR-2, TLR-4, MyD88, IL-18, caspase-1 mRNAs and proteins. L. casei LOCK0919 increased expression and transfer of villin and beta-catenin from cytoplasm to cell membrane. Presence of L. rhamnosus LOCK0900 resulted in detachment of IEC-18 layer from extracellular matrix leading to induction of IL-1beta, of TLR-2 and IL-8 mRNAs and stimulation of MyD88, caspase-1 and cytosolic receptor NOD2 mRNAs. L. rhamnosus LOCK0908 was not recognized by TLR-2 or TLR-4 receptors. Lactobacilli-IEC-18 crosstalk enhanced immune and barrier mucosal functions.

INVESTIGATION INTO THE EFFECTS OF TESTOSTERONE ON DRUG METABOLIZING ENZYMES IN HEPATIC AND INTESTINAL HUMAN CELL LINES

IntroductionTestosterone is a well‐established probe substrate for assessing functional levels of cytochrome P450 (CYP) 3A4‐mediated metabolism. Parenteral testosterone replacement therapy is commonly prescribed for male hypogonadism, given the low oral bioavailability of oral testosterone. However, some investigators are exploring the oral delivery of testosterone or its esters using specialized formulations that might promote lymphatic intestinal absorption. Exogenous administration of hormones is known to down‐regulate endogenous production, which could lead to less than expected increases in exposure with repeated administration, but other factors such as self‐induction of metabolism could also contribute. There is little if any substantive information available that sheds light on whether or not testosterone has any inductive effects on CYP3A4 mediated metabolism, or on any other CYP isoforms. The intent of this study was to use intestinal and hepatic human cell lines to assess for possible induction of CYP3A4 and CYP1A1.MethodsHuman hepatic (HepG2) and colonic/intestinal (Lovo) cell lines were grown in 12 well plates and spiked with testosterone (300, 3000 or 30000 ng/dL once or twice daily for 3 days). Control cells were similarly treated with matching media minus testosterone. At the end of the 3 days of treatment, some cells were used to measure mRNA and protein for CYP1A1, CYP3A4, and multidrug resistance protein (MDR1). In addition, the effect of testosterone pretreatment on its own in vitro clearance, and CYP1A1–specific functional activity (using 7‐ethoxyin O‐deethylase activity) were also tested in the cell lines.ResultsBased on the mRNA and protein measures, there was no evidence of a testosterone‐related change in protein expression of the target genes or housekeeping genes. Functional studies indicated no change in CYP1A1 activity. Using a LC‐MS assay method, the cells lines displayed a rapid decrease in testosterone concentrations; it was established that there was no binding of testosterone to the plastic well plates used in the studies. HepG2 cells preexposed with 3000 ng/dL twice daily testosterone for 3 d had no associated changes in testosterone clearance after the testosterone preexposure phase. In the Lovo cells, testosterone pretreatment with 30000 ng/dL of testosterone was associated with an apparent inhibition in vitro of testosterone clearance. The testosterone concentrations exposed to intestinal cells were higher than liver cells because enterocyte exposure after oral administration would be expected to greatly exceed those in the liver.ConclusionThere was no evidence for induction of testosterone metabolism in vitro using human liver and intestinal cell lines. The decrease in testosterone clearance in the intestinal cells after preexposure might have been due to accumulation of metabolites, with a pursuant competitive or noncompetitive inhibition of testosterone metabolism.Support or Funding InformationFunding provided by TesoRx Pharma LLCThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A comparison of a Natural and Synthetic Stilbenoid, Arachadin‐3, on a Rotavirus Infected Human Intestinal Cell Line

Rotavirus (RV) causes severe diarrhea in young children and immunocompromised individuals. Several effective vaccines have been developed to immunize children between 2 to 8 months of age, but there are no vaccines or antiviral therapeutics for RV‐infected immunocompromised patients of any age. Our laboratory has shown a decrease in the number of infectious RV particles produced in HT29.f8 cells with the addition of the stilbenoid, arachidin‐3 (A3). Additionally, the viral protein, NSP4, that is important for viral replication is decreased, suggesting an effect on virus replication. Also, ultrastructural changes were observed with TEM studies that imply a connection with the apoptosis and autophagy pathways which is supported by qRT PCR data. Recently, a synthetic A3 (sA3) has been developed. This study compared the effects of the natural A3 and sA3 on HT29.f8 cell viability, production of infectious RV particles, production of structural and nonstructural viral proteins, ultrastructural changes and the gene regulation of apoptosis and autophagy transcripts. From the data collected, the viability of the cells was not affected with the introduction of either natural or sA3, but the amount of infectious virus particles produced was significantly decreased by 50‐fold, respectively. The TEM images depicted changes in ultrastructures of RV‐infected HT29.f8 cells that appear to show signs of apoptosis while RV‐infected cells with the addition of either the natural or synthetic A3 showed a more normal appearance like the negative controls. The nucleus to cytoplasm ratios of TEM micrographs of RV‐infected cells was greater than 1.0 whereas the arachidin treated RV‐infected cells was about 0.5 which was like the control cells. To validate this data, whole cell staining of the nucleus and cytoplasm showed similar changes with the addition of both the A3 and sA3. qRT PCR analysis of transcripts for genes that are important in regulating the apoptosis and autophagy pathways regulations corroborated the TEM observation, and suggests a cross talk between the two pathways during an RV infection that is regulated with the addition of the arachidins. Our data suggests that both the natural A3 and sA3 have anti‐RV activity that implies a potential therapeutic application.Support or Funding InformationThis work was supported by: 1) The Faculty‐Student Collaborative Research Program of The Office of Research and Sponsored Programs at Stephen F. Austin State University and 2) The NSF‐EPSCoR (grant# EPS‐ 0701890; Center for Plant‐Powered Production‐P3).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inflammatory response of gliadin protein isolated from various wheat varieties on human intestinal cell line

Wheat protein contributes a significant part in human diet, apart from its well-known nutritional values, wheat gluten/gliadin proteins are also responsible for the many allergic/inflammatory diseases and chronic inflammation in the small intestine may cause diarrhea and malabsorption, in a specific population of individuals. In the present study, the antigenic characteristics of twelve wheat varieties of diverse origin namely C273, C281, C286, C306, C518, C591, Agra Local, 9D, 8A, Raj4229, HD3027, NP824 released during 1920–2012 were evaluated. Gliadin proteins from these varieties were tested on human colon cancer cell line HCT116 to assess their effect on inflammation, oxidative and nitrosative stress, pro-inflammatory cytokines. The results show that these wheat varieties induced high levels of ROS/RNS and MPO activity which was further supported by the increase in the mRNA levels of a cytokine such as IL-1β and IL-15. It can be concluded that gliadin from these wheat varieties is suggested to act as a potential antigen by enhancing the level of inflammation irrespective of their year of release and origin which if not controlled may lead to the initiation of celiac disease in genetically susceptible individuals or may be responsible for other wheat protein intolerance associated diseases.

Evaluating Human Intestinal Cell Lines for Studying Dietary Protein Absorption.

With the global population rising, the need for sustainable and resource-efficiently produced proteins with nutritional and health promoting qualities has become urgent. Proteins are important macronutrients and are involved in most, if not all, biological processes in the human body. This review discusses these absorption mechanisms in the small intestine. To study intestinal transport and predict bioavailability, cell lines are widely applied as screening models and often concern Caco-2, HT-29, HT-29/MTX and T84 cells. Here, we provide an overview of the presence and activities of peptide- and amino acid transporters in these cell models. Further, inter-laboratory differences are discussed as well as the culture micro-environment, both of which may influence cell culture phenotype and performance. Finally, the value of new developments in the field, including culturing cells in 3-dimensional systems under shear stress (i.e., gut-on-chips), is highlighted. In particular, their suitability in screening novel food proteins and prediction of the nutritional quality needed for inclusion in the human diet of the future is addressed.

Rare Variants in the ABCG2 Promoter Modulate In Vivo Activity.

ABCG2 encodes the breast cancer resistance protein (BCRP), an efflux membrane transporter important in the detoxification of xenobiotics. In the present study, the basal activity of the ABCG2 promoter in liver, kidney, intestine, and breast cell lines was examined using luciferase reporter assays. The promoter activities of reference and variant ABCG2 sequences were compared in human hepatocellular carcinoma cell (HepG2), human embryonic kidney cell (HEK293T), human colorectal carcinoma cell (HCT116), and human breast adenocarcinoma cell (MCF-7) lines. The ABCG2 promoter activity was strongest in the kidney and intestine cell lines. Four variants in the basal ABCG2 promoter (rs76656413, rs66664036, rs139256004, and rs59370292) decreased the promoter activity by 25%-50% in at least three of the four cell lines. The activity of these four variants was also examined in vivo using the hydrodynamic tail vein assay, and two single nucleotide polymorphisms (rs76656413 and rs59370292) significantly decreased in vivo liver promoter activity by 50%-80%. Electrophoretic mobility shift assays confirmed a reduction in nuclear protein binding to the rs59370292 variant probe, whereas the rs76656413 probe had a shift in transcription factor binding specificity. Although both rs59370292 and rs76656413 are rare variants in all populations, they could contribute to patient-level variation in ABCG2 expression in the kidney, liver, and intestine.

Evaluating the probiotic potential and technological characteristics of yeasts implicated in cv. Kalamata natural black olive fermentation

In the present study, 49 yeast strains previously isolated from cv. Kalamata table olive fermentation were assessed for their probiotic potential and technological characteristics. The probiotic assays included the in vitro survival in simulated gastric and pancreatic digestions, surface adhesion to the intestinal cell line Caco-2, hydrophobicity, autoaggregation and haemolytic activity. The technological features of the strains were also elucidated in terms of enzymatic activity and susceptibility to diverse salt levels (0–250 g/L) and pH values (3.5, 5.0, and 6.5). The obtained results indicated that during the simulated gastric and pancreatic digestions, 42 out of the 49 yeast strains presented overall survival rate higher than 50%, while 24 strains showed survival percentage higher than 70% at the end of the digestions. Furthermore, the majority of the assayed strains presented hydrophobicity percentage higher than 75%, while the autoaggregation ability ranged between 72 and 91%. None of the strains showed haemolytic activity. The majority of the strains presented high tolerance to salt with some strains exhibiting tolerance even at salt concentrations higher than 200 g/L. Concerning the enzymatic activity, 45 strains presented valine and cystine arylamidase activity, while positive reactions for the enzymes β- and α-glucosidase were observed for 27 and 14 strains, respectively. Moreover, 11 strains presented α-galactosidase and alkaline phosphatase activity. From the total number of studied yeasts, the strain Y34 belonging to Saccharomyces cerevisiae presented positive results in the majority of both probiotic and technological assays and thus it could be considered a potential starter either as single or as combined culture with lactic acid bacteria in the fermentation of Greek-style natural black olives.

Soluble uric acid increases PDZK1 and ABCG2 expression in human intestinal cell lines via the TLR4-NLRP3 inflammasome and PI3K/Akt signaling pathway

BackgroundIn addition to the kidney, the intestine is one of the most important organs involved in uric acid excretion. However, the mechanism of urate excretion in the intestine remains unclear. Therefore, the relationship between soluble uric acid and the gut excretion in human intestinal cells was explored. The relevant signaling molecules were then also examined.MethodsHT-29 and Caco-2 cell lines were stimulated with soluble uric acid. Western blotting and qRT-PCR were used to measure protein and mRNA levels. Subcellular fractionation methods and immunofluorescence were used to quantify the proteins in different subcellular compartments. Flow cytometry experiments examined the function of ATP-binding cassette transporter, subfamily G, member 2 (ABCG2). Small interfering RNA transfection was used to assess the interaction between ABCG2 and PDZ domain-containing 1 (PDZK1).ResultsSoluble uric acid increased the expression of PDZK1 and ABCG2. The stimulation of soluble uric acid also facilitated the translocation of ABCG2 from the intracellular compartment to the plasma membrane and increased its transport activity. Moreover, the upregulation of PDZK1 and ABCG2 by soluble uric acid was partially decreased by either TLR4-NLRP3 inflammasome inhibitors or PI3K/Akt signaling inhibitors. Furthermore, PDZK1 knockdown significantly inhibited the expression and transport activity of ABCG2 regardless of the activation by soluble uric acid, demonstrating a pivotal role for PDZK1 in the regulation of ABCG2.ConclusionsThese findings suggest that urate upregulates the expression of PDZK1 and ABCG2 for excretion in intestinal cells via activating the TLR4-NLRP3 inflammasome and PI3K/Akt signaling pathway.

Biotransformation of Benzo[ a]pyrene by Three Rainbow Trout ( Onchorhynchus mykiss) Cell Lines and Extrapolation To Derive a Fish Bioconcentration Factor.

Permanent fish cell lines constitute a promising complement or substitute for fish in the environmental risk assessment of chemicals. We demonstrate the potential of a set of cell lines originating from rainbow trout ( Oncorhynchus mykiss) to aid in the prediction of chemical bioaccumulation in fish, using benzo[ a]pyrene (BaP) as a model chemical. We selected three cell lines from different tissues to more fully account for whole-body biotransformation in vivo: the RTL-W1 cell line, representing the liver as major site of biotransformation, and the RTgill-W1 (gill) and RTgutGC (intestine) cell lines, as important environment-organism interfaces, which likely influence chemical uptake. All three cell lines were found to effectively biotransform BaP. However, rates of in vitro clearance differed, with the RTL-W1 cell line being most efficient, followed by RTgutGC. Co-exposures with α-naphthoflavone as potent inhibitor of biotransformation, assessment of CYP1A catalytic activity, and the progression of cellular toxicity upon prolonged BaP exposure revealed that BaP is handled differently in the RTgill-W1 compared to the other two cell lines. Application of the cell-line-derived in vitro clearance rates into a physiology-based toxicokinetic model predicted a BaP bioconcentration factor (BCF) of 909-1057 compared to 920 reported for rainbow trout in vivo.

The estrogenic hormone effect in gastric cancer.

85 Background: Estrogen receptors (ERs) are steroid hormone receptors that regulate cellular activities in many physiological and pathological processes in different tissues. A few of studies have examined the expression of ER in gastric cancer. However, considerable controversy is raised as to the expression level of ER and its prognostic value in gastric cancer. In the present study, the expression profile of ERα, ERβ was determined in gastric cancer cell lines according Lauren classification and evaluate that the treatment effect of selective estrogen receptor modulator. Methods: Using four cell lines established from human gastric carcinomas according Lauren classification, check endogenous ERα, ERβ expression levels with RT-PCR. The SERM treatment effect were detected MTT test. Using immunohistochemical detection, the present study analyzed the clinical relevance of ERα, ERβ expression in tumor cells in 197 patients who underwent curative radical surgery and who were observed on long-term follow-up. Results: Endogenous ERα was high expression not intestinal cancer cell lines but in diffuse cancer cell line. Endogenous ERβ was high expression both type cancer cell line than normal gastrointestinal cell lines. According MTT assay, only raloxifene among SERM was significant treatment effect. In immonohistochemial study of gastric tissue, ERα negative and ERβ positive was associated with good prognosis. Conclusions: ERβ may be partly involved in gastric carcinogenesis and ERβ antagonist might be new therapeutic drug for gastric cancer.

Influence of in vitro digestion process on polyphenolic profile of skin grape (cv. Italia) and on antioxidant activity in basal or stressed conditions of human intestinal cell line (HT-29)

White table grape cv. Italia is a typical component of the Mediterranean diet and a source of phenolic compounds, particularly abundant in the skin portion. The aim of this study was to characterize the phenolic profile of the table grape skin and to assess its stability after the in vitro digestion process. The main phenolic compounds identified by the HPLC-DAD analysis were: procyanidin B1, caftaric acid, catechin, coutaric acid, quercetin 3-glucuronide and quercetin 3-glucoside. All compounds showed a good stability after in vitro digestion (from 43 to 80%). Moreover, the influence of grape skin polyphenols on the modulation of ROS and GSH levels was evaluated in basal and in stressed conditions on human intestinal cells (HT-29). In basal conditions, a higher polyphenol concentrations exerted pro-oxidant effect corresponding to high ROS level and low GSH content. This effect was probably due to the polyphenolic oxidation in cell culture condition with consequent production of hydrogen peroxide. Otherwise, in stressed conditions, grape skin polyphenols exerted antioxidant effects up to 1.3 × 10−6 μg/g and restored the stress-related GSH reduction. The in vitro digestion process attenuated the biological effect of grape skin polyphenols on intestinal cell line (HT-29). In conclusion, grape skin polyphenols showed different behavior in relation to their concentrations and to the intracellular ROS levels.

Cytotoxicological evaluation of semi-purified extracts of some dye yielding plants of the Kashmir Valley on Normal Intestinal Cell Line (IEC-6) by MTT assay

Plant extracts are widely used in many fields and there is a need to evaluate their cytotoxic effect to determine their non-cytotoxic concentration at which they can be used in a safe manner. Keeping this in view, the present study was designed to evaluate the in vitro toxicity of Celosia argentia L. var plumosa (Cockscomb), Calendula officinalis L. (Pot Marigold), Indigofera heterantha Wall. (Himalayan Indigo) and Rubia cordifolia L. (Indian Madder) on Normal Intestinal Cell Line (IEC-6) by MTT assay to test their feasibility for natural edible dye extraction. The experimental material, comprised of inflorescence of Celosia argentia L. var plumose, petals of the two varieties of Calendula officinalis L., leaves of Indigofera heterantha Wall. and leaves and roots of the Rubia cordifolia L. Cell line was exposed to 1, 4, 16, 64 and 256µg/ml concentrations of plant extracts for 24, 48, and 72hr at 37oC. Results revealed that both the varieties of Calendula officinalis L. var. Gitana Orange and Gitana Yellow did not show any cytotoxic effect on IEC-6 cell line while as Celosia argentia L. var plumose, Indigofera heterantha Wall. and Rubia cordifolia L. showed cytotoxicity. From the present study it was concluded that the extracts of the both varieties of Calendula officinalis L. var. Gitana Orange and Gitana Yellow extracts are non-toxic in nature, thus can be utilized for the extraction of natural edible dye while as the extracts of Celosia argentia L. var plumose, Indigofera heterantha Wall. and Rubia cordifolia L. had potent in vitro cytotoxic activity thus they cannot be used for extraction of natural edible food colour. However, to better evaluate the cytotoxic effect of these plant extracts, in vivo experiments on laboratory animal followed by histological analysis should be done.

P096 RADIATION AND IDO1-SELECTIVE INHIBITOR EPACADOSTAT EXHIBIT THERAPEUTIC SYNERGY IN COLITIS ASSOCIATED CANCER

We have previously demonstrated that gene deletion of the immunosuppressive enzyme IDO1 (indoleamine-2,3-dioxygenase 1) reduces but does not eliminate tumor burden in a mouse model of colitis associated cancer (CAC). In the current study, we test the hypothesis that a new highly specific IDO1 inhibitor epacadostat (Incyte Corp.) will synergize with cytotoxic radiation therapy (RT) as a novel treatment strategy for CAC. CAC was modeled in vivo and in vitro using IDO1 expressing colorectal cancer (CRC) cell lines from mice (CT26, MC38) and humans (HT29, HCT116, and DLD1) cell lines, as well as normal human primary epithelial colonoids and normal colon and small intestinal (SI) cell lines (FHC and IEC6, respectively). Standard molecular biology assays were used to assay gene expression, proliferation (WST), cell death (LDH) and apoptosis (TUNEL/Caspase). CAC was modeled with heterotopic implants with treatment groups of control, epacadostat (300mg/kg/day), focal RT, and epacadostat+ focal RT (n=7/group). Tumor infiltrating lymphocytes (TIL) were examined by flow cytometry and cytokine multiplex assays. In Vitro and in vivo: RT enhanced IDO1 gene expression in CRC cells, but not in normal colon or SI cells. The combination of epacadostat and RT decreased CRC proliferation and colony formation (p<0.01) more than either monotherapy in vitro. In vivo, the combinationalso reduced the tumor growth and size than either treatment alone (Figure 1a, b). Impressively, unilateral RT also enhanced anti-tumor effect of epacadostat in the contralateral unirradiated tumor indicating an enhancement of the recently described abscopal effect. Consistent with this, TILs and cytokine profiles demonstrated enhanced anti-tumor immune environment. These data identify a therapeutic synergy of IDO1 inhibition and RT in CAC/CRC. A Phase 1 clinical study examining this combination strategy is being initiated.

P026 The impact of trophic effects of multi strain probiotic preparation on paracellular permeability

Abstract Background Probiotics are defined as live, non-pathogenic bacteria that confer health benefits beyond their nutritional value. Particularly VSL#3, a probiotic mix containing four strains of Lactobacilli (L. paracasei, L. plantarum, L. acidophilus, and L. delbrueckii subsp. bulgaricus*), three strains of Bifidobacteria (B. longum**, B. infantis**, B. breve) and Streptococcus thermophilus, has demonstrated efficacy in the management of diseases characterised by increased intestinal permeability such as irritable bowel syndrome and ulcerative colitis. The aim of the present study was to evaluate the mechanism of action of VSL#3 to reduce intestinal permeability, and its effect in modulating the activity on tight junctions, focusing on the effects of secreted bioactive factors. *Recently reclassified as L. helveticus. **Recently reclassified as B. animalis subsp. lactis. Methods Two different lots of VSL#3 (Manufacturer: Nutrilinea Srl, Gallarate (VA), Italy) were used. Caco2 and HT29 cell lines were treated with a conditioning media (CM) prepared using 1g of probiotic formula grown in D-MEM cell culture medium (free of serum and antibiotics) at 37°C for 48 h without shaking and in anaerobic conditions. The effects of probiotic on proliferations of cells were evaluated by cell growth curve and MTT assay. The effect on apoptosis will be analysed by cytometry using double staining with Annexin V-FITC and Propidium Iodide, as well as it is well established. Furthermore, the effect on expression of tight junctions, in particular claudin-2, occludin, and ZO-1, will be investigated by western blot analysis. Results The multistrain probiotic formula, in particular the more concentrate CM (1:10) increase Caco2 and HT29 cells proliferation and the percentage of metabolic cell activity (up 20%), together with a decrease of 90% of apoptotic cells in the presence of CM. These trophic effects may have an impact on paracellular permeability. The expression of tight junction proteins increase in 24 h for claudin and ZO-1, and after 48 h of treatment in case of occludin. Also the densitometry and the ratio between of relative density of protein and endogenous control (β-actin) confirms these data. The results are the same using two different lots. Conclusions These preliminary data in vitro could be able to explain how VSL#3 works at intestinal mucosa level, in particular by secretion of factors that enhances barrier integrity. The proliferative stimuli and the increase of expression of tight junction proteins are consistent with an effect on mucosa regeneration and re-epithelization. Other experiments, also on other intestinal cell line, are necessary to deeply understand the mechanism of action of this kind of probiotic formula.

Gellan Gum/Pectin Beads Are Safe and Efficient for the Targeted Colonic Delivery of Resveratrol.

This work addresses the establishment and characterization of gellan gum:pectin (GG:P) biodegradable mucoadhesive beads intended for the colon-targeted delivery of resveratrol (RES). The impact of the polymer carrier system on the cytotoxicity and permeability of RES was evaluated. Beads of circular shape (circularity index of 0.81) with an average diameter of 914 μm, Span index of 0.29, and RES entrapment efficiency of 76% were developed. In vitro drug release demonstrated that beads were able to reduce release rates in gastric media and control release for up to 48 h at an intestinal pH of 6.8. Weibull’s model correlated better with release data and b parameter (0.79) indicated that the release process was driven by a combination of Fickian diffusion and Case II transport, indicating that both diffusion and swelling/polymer chains relaxation are processes that contribute equally to control drug release rates. Beads and isolated polymers were observed to be safe for Caco-2 and HT29-MTX intestinal cell lines. RES encapsulation into the beads allowed for an expressive reduction of drug permeation in an in vitro triple intestinal model. This feature, associated with low RES release rates in acidic media, can favor targeted drug delivery from the beads in the colon, a promising behavior to improve the local activity of RES.

Expression and transcriptional as well as epigenetic regulation of metabolizing enzymes and transporter proteins in intestinal cell lines

Expression and transcriptional as well as epigenetic regulation of metabolizing enzymes and transporter proteins in intestinal cell lines

P096 RADIATION AND IDO1-SELECTIVE INHIBITOR EPACADOSTAT EXHIBIT THERAPEUTIC SYNERGY IN COLITIS ASSOCIATED CANCER

We have previously demonstrated that gene deletion of the immunosuppressive enzyme IDO1 (indoleamine-2,3-dioxygenase 1) reduces but does not eliminate tumor burden in a mouse model of colitis associated cancer (CAC). In the current study, we test the hypothesis that a new highly specific IDO1 inhibitor epacadostat (Incyte Corp.) will synergize with cytotoxic radiation therapy (RT) as a novel treatment strategy for CAC. CAC was modeled in vivo and in vitro using IDO1 expressing colorectal cancer (CRC) cell lines from mice (CT26, MC38) and humans (HT29, HCT116, and DLD1) cell lines, as well as normal human primary epithelial colonoids and normal colon and small intestinal (SI) cell lines (FHC and IEC6, respectively). Standard molecular biology assays were used to assay gene expression, proliferation (WST), cell death (LDH) and apoptosis (TUNEL/Caspase). CAC was modeled with heterotopic implants with treatment groups of control, epacadostat (300mg/kg/day), focal RT, and epacadostat+ focal RT (n=7/group). Tumor infiltrating lymphocytes (TIL) were examined by flow cytometry and cytokine multiplex assays. In Vitro and in vivo: RT enhanced IDO1 gene expression in CRC cells, but not in normal colon or SI cells. The combination of epacadostat and RT decreased CRC proliferation and colony formation (p<0.01) more than either monotherapy in vitro. In vivo, the combinationalso reduced the tumor growth and size than either treatment alone (Figure 1a, b). Impressively, unilateral RT also enhanced anti-tumor effect of epacadostat in the contralateral unirradiated tumor indicating an enhancement of the recently described abscopal effect. Consistent with this, TILs and cytokine profiles demonstrated enhanced anti-tumor immune environment. These data identify a therapeutic synergy of IDO1 inhibition and RT in CAC/CRC. A Phase 1 clinical study examining this combination strategy is being initiated.

PARP-1 Inhibition Rescues Short Lifespan in Hyperglycemic C. Elegans And Improves GLP-1 Secretion in Human Cells.

TCF7L2 is located at one of the most strongly associated type 2 diabetes loci reported to date. We previously reported that the most abundant member of a specific protein complex to bind across the presumed causal variant at this locus, rs7903146, was poly [ADP-ribose] polymerase type 1 (PARP-1). We analyzed the impact of PARP-1 inhibition on C. elegans health in the setting of hyperglycemia and on glucose-stimulated GLP-1 secretion in human intestinal cells. Given that high glucose concentrations progressively shorten the lifespan of C. elegans, in part by impacting key well-conserved insulin-modulated signaling pathways, we investigated the effect of PARP-1 inhibition with Olaparib on the lifespan of C. elegans nematodes under varying hyperglycemic conditions. Subsequently, we investigated whether Olaparib treatment had any effect on glucose-stimulated GLP-1 secretion in the human NCI-H716 intestinal cell line, a model system for the investigation of enteroendocrine function. Treatment with 100uM Olaparib in nematodes exposed to high concentrations of glucose led to significant lifespan rescue. The beneficial lifespan effect of Olaparib appeared to require both PARP-1 and TCF7L2, since treatment had no effect in hyperglycemic conditions in knock-out worm strains for either of these homologs. Further investigation using the NCI-H716 cells revealed that Olaparib significantly enhanced secretion of the incretin, GLP-1, plus the gene expression of TCF7L2, GCG and PC1. These data from studies in both C. elegans and a human cell line suggest that PARP-1 inhibition offers a novel therapeutic avenue to treat type 2 diabetes.

Lactobacillus reuteri strains protect epithelial barrier integrity of IPEC-J2 monolayers from the detrimental effect of enterotoxigenic Escherichia coli.

Lactobacillus reuteri is an inhabitant of the gastrointestinal (GI) tract of mammals and birds and several strains of this species are known to be effective probiotics. The mechanisms by which L. reuteri confers its health‐promoting effects are far from being fully understood, but protection of the mucosal barrier is thought to be important. Leaky gut is a state of abnormal intestinal permeability with implications for the pathophysiology of various gastrointestinal disorders. Enterotoxigenic Escherichia coli (ETEC) can invade the intestinal mucosa and induce changes in barrier function by producing enterotoxin or by direct invasion of the intestinal epithelium. Our hypothesis was that L. reuteri can protect the mucosal barrier, and the goal of the study was to challenge this hypothesis by monitoring the protective effect of L. reuteri strains on epithelial dysfunction caused by ETEC. Using an infection model based on the porcine intestinal cell line IPEC‐J2, it was demonstrated that pretreatment of the cells with human‐derived L. reuteri strains (ATCC PTA 6475, DSM 17938 and 1563F) and a rat strain (R2LC) reduced the detrimental effect of ETEC in a dose‐dependent manner, as monitored by permeability of FITC‐dextran and transepithelial electrical resistance (TEER). Moreover, the results revealed that ETEC upregulated proinflammatory cytokines IL‐6 and TNF α and decreased expression of the shorter isoform of ZO‐1 (187 kDa) and E‐cadherin. In contrast, pretreatment with L. reuteri DSM 17938 and 1563F downregulated expression of IL‐6 and TNF α, and led to an increase in production of the longer isoform of ZO‐1 (195 kDa) and maintained E‐cadherin expression. Interestingly, expression of ZO‐1 (187 kDa) was preserved only when the infected cells were pretreated with strain 1563F. These findings demonstrate that L. reuteri strains exert a protective effect against ETEC‐induced mucosal integrity disruption.

Global proteomic analysis of transporter proteins in the human small intestine and established intestinal cell lines

Global proteomic analysis of transporter proteins in the human small intestine and established intestinal cell lines