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Sample records for adenocarcinoma caco-2 cells

  1. Carbon metabolism of enterobacterial human pathogens growing in epithelial colorectal adenocarcinoma (Caco-2 cells.

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    Andreas Götz

    Full Text Available Analysis of the genome sequences of the major human bacterial pathogens has provided a large amount of information concerning their metabolic potential. However, our knowledge of the actual metabolic pathways and metabolite fluxes occurring in these pathogens under infection conditions is still limited. In this study, we analysed the intracellular carbon metabolism of enteroinvasive Escherichia coli (EIEC HN280 and EIEC 4608-58 and Salmonella enterica Serovar Typhimurium (Stm 14028 replicating in epithelial colorectal adenocarcinoma cells (Caco-2. To this aim, we supplied [U-(13C(6]glucose to Caco-2 cells infected with the bacterial strains or mutants thereof impaired in the uptake of glucose, mannose and/or glucose 6-phosphate. The (13C-isotopologue patterns of protein-derived amino acids from the bacteria and the host cells were then determined by mass spectrometry. The data showed that EIEC HN280 growing in the cytosol of the host cells, as well as Stm 14028 replicating in the Salmonella-containing vacuole (SCV utilised glucose, but not glucose 6-phosphate, other phosphorylated carbohydrates, gluconate or fatty acids as major carbon substrates. EIEC 4608-58 used C(3-compound(s in addition to glucose as carbon source. The labelling patterns reflected strain-dependent carbon flux via glycolysis and/or the Entner-Doudoroff pathway, the pentose phosphate pathway, the TCA cycle and anapleurotic reactions between PEP and oxaloacetate. Mutants of all three strains impaired in the uptake of glucose switched to C(3-substrate(s accompanied by an increased uptake of amino acids (and possibly also other anabolic monomers from the host cell. Surprisingly, the metabolism of the host cells, as judged by the efficiency of (13C-incorporation into host cell amino acids, was not significantly affected by the infection with either of these intracellular pathogens.

  2. Use of the human colorectal adenocarcinoma (Caco-2) cell line for isolating respiratory viruses from nasopharyngeal aspirates.

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    Chan, K H; Yan, M K; To, K K W; Lau, S K; Woo, P C; Cheng, V C C; Li, W S; Chan, J F W; Tse, H; Yuen, K Y

    2013-05-01

    The human colorectal adenocarcinoma-derived Caco-2 cell line was evaluated as a means isolating common respiratory viruses from nasopharyngeal aspirates for the diagnosis of respiratory diseases. One hundred eighty-nine direct immunofluorescence positive nasopharyngeal aspirates obtained from patients with various viral respiratory diseases were cultured in the presence of Caco-2 cells or the following conventional cell lines: LLC-MK2, MDCK, HEp-2, and A549. Caco-2 cell cultures effectively propagated the majority (84%) of the viruses present in nasopharyngeal aspirate samples compared with any positive cultures obtained using the panel cells (78%) or individual cell line MDCK (38%), HEp-2 (21%), LLC-MK2 (27%), or A549 (37%) cell lines. The differences against individual cell line were statistically significant (P = viruses which were not cultivated in conventional cell lines. By contrast, 80% (24/30) of viruses not cultivated in Caco-2 cells were isolated using the conventional panel. The findings indicated that Caco-2 cells were sensitive to a wide range of viruses and can be used to culture a broad range of respiratory viruses.

  3. In vitro cytotoxicity of silver nanoparticles and zinc oxide nanoparticles to human epithelial colorectal adenocarcinoma (Caco-2) cells

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    Song, Yijuan [Zhejiang Provincial Key Laboratory of Biometrology and Inspection and Quarantine, China Jiliang University, Hangzhou 310018 (China); Guan, Rongfa, E-mail: rongfaguan@163.com [Zhejiang Provincial Key Laboratory of Biometrology and Inspection and Quarantine, China Jiliang University, Hangzhou 310018 (China); Lyu, Fei [Department of Food Science and Technology, Zhejiang University of Technology, Hangzhou 310014 (China); Kang, Tianshu; Wu, Yihang [Zhejiang Provincial Key Laboratory of Biometrology and Inspection and Quarantine, China Jiliang University, Hangzhou 310018 (China); Chen, Xiaoqiang [Hubei University of Technology, Wuhan 430068 (China)

    2014-11-15

    Highlights: • The characterization of Ag NPs and ZnO NPs. • The various morphologies of Caco-2 cells stained with AO/EB. • The viability of Caco-2 cells after Ag NPs and ZnO NPs exposure. • The cytotoxicity of Ag NPs and ZnO NPs on Caco-2 cells by oxidative stress assays. - Abstract: With the increasing applications of silver nanoparticles (Ag NPs) and zinc oxide nanoparticles (ZnO NPs) in foods and cosmetics, the concerns about the potential toxicities to human have been raised. The aims of this study are to observe the cytotoxicity of Ag NPs and ZnO NPs to human epithelial colorectal adenocarcinoma (Caco-2) cells in vitro, and to discover the toxicity mechanism of nanoparticles on Caco-2 cells. Caco-2 cells were exposed to 10, 25, 50, 100, 200 μg/mL of Ag NPs and ZnO NPs (90 nm). AO/EB double staining was used to characterize the morphology of the treated cells. The cell counting kit-8 (CCK-8) assay was used to detect the proliferation of the cells. Reactive oxygen species (ROS), superoxide dismutase (SOD) and glutathione (GSH) assay were used to explore the oxidative damage of Caco-2 cells. The results showed that Ag NPs and ZnO NPs (0–200 μg/mL) had highly significant effect on the Caco-2 cells activity. ZnO NPs exerted higher cytotoxicity than Ag NPs in the same concentration range. ZnO NPs have dose-depended toxicity. The LD{sub 50} of ZnO NPs in Caco-2 cells is 0.431 mg/L. Significant depletion of SOD level, variation in GSH level and release of ROS in cells treated by ZnO NPs were observed, which suggests that cytotoxicity of ZnO NPs in intestine cells might be mediated through cellular oxidative stress. While Caco-2 cells treated with Ag NPs at all experimental concentrations showed no cellular oxidative damage. Moreover, the cells’ antioxidant capacity increased, and reached the highest level when the concentration of Ag NPs was 50 μg/mL. Therefore, it can be concluded that Ag NPs are safer antibacterial material in food packaging materials

  4. Anti-proliferative effect of rhein, an anthraquinone isolated from Cassia species, on Caco-2 human adenocarcinoma cells.

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    Aviello, Gabriella; Rowland, Ian; Gill, Christopher I; Acquaviva, Angela Maria; Capasso, Francesco; McCann, Mark; Capasso, Raffaele; Izzo, Angelo A; Borrelli, Francesca

    2010-07-01

    In recent years, the use of anthraquinone laxatives, in particular senna, has been associated with damage to the intestinal epithelial layer and an increased risk of developing colorectal cancer. In this study, we evaluated the cytotoxicity of rhein, the active metabolite of senna, on human colon adenocarcinoma cells (Caco-2) and its effect on cell proliferation. Cytotoxicity studies were performed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), neutral red (NR) and trans-epithelial electrical resistance (TEER) assays whereas (3)H-thymidine incorporation and Western blot analysis were used to evaluate the effect of rhein on cell proliferation. Moreover, for genoprotection studies Comet assay and oxidative biomarkers measurement (malondialdehyde and reactive oxygen species) were used. Rhein (0.1-10 microg/ml) had no significant cytotoxic effect on proliferating and differentiated Caco-2 cells. Rhein (0.1 and 1 microg/ml) significantly reduced cell proliferation as well as mitogen-activated protein (MAP) kinase activation; by contrast, at high concentration (10 microg/ml) rhein significantly increased cell proliferation and extracellular-signal-related kinase (ERK) phosphorylation. Moreover, rhein (0.1-10 microg/ml): (i) did not adversely affect the integrity of tight junctions and hence epithelial barrier function; (ii) did not induce DNA damage, rather it was able to reduce H(2)O(2)-induced DNA damage and (iii) significantly inhibited the increase in malondialdehyde and reactive oxygen species (ROS) levels induced by H(2)O(2)/Fe(2+). Rhein was devoid of cytotoxic and genotoxic effects in colon adenocarcinoma cells. Moreover, at concentrations present in the colon after a human therapeutic dosage of senna, rhein inhibited cell proliferation via a mechanism that seems to involve directly the MAP kinase pathway. Finally, rhein prevents the DNA damage probably via an anti-oxidant mechanism.

  5. Pharmacological study of Buthus martensii Karsch venom on human colon adenocarcinoma cell line Caco-2 in vitro%东亚钳蝎毒素对Caco-2人结肠癌细胞的体外药理学研究

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    梁琼娟; 胡吉林; 肖凯夫; 王智

    2014-01-01

    The effects of Buthus martensii Karsch venom on human colon adenocarcinoma cell line Caco-2 were investigated in this study. The test was assayed by means of MTT assay in vitro cell culture to observe the effects of different concentrations(10, 20, 40μg/mL) of the Buthus martensii Karsch venom on the proliferation of Caco-2 cells in varing action time (24, 48 h). The mechanism of venom on Caco-2 cells could be measured by the test of lymphocyte transformation and lactate dehydrogenase release ratio. The data indicated that Buthus martensii Karsch venom could inhibit the Caco-2 cells proliferation and promote the lymphocyte transformation. Besides, the activity is also closely related to the treatment time and venom concentration.%研究东亚钳蝎毒素对人结肠癌细胞Caco-2增殖的影响。以不同浓度的东亚钳蝎(Buthus martensii Karsch)毒素(10、20、40滋g/mL)干预体外培养的Caco-2细胞,分别于24 h、48 h后,用四甲基偶氮唑盐(MTT)比色法,观察毒素对Caco-2细胞的增殖抑制作用。运用淋巴细胞转化实验和乳酸脱氢酶(LDH)释放实验检测蝎毒素对Caco-2细胞的作用途径。结果表明:东亚钳蝎毒不仅能抑制Caco-2细胞的增殖而且能促进淋巴细胞转化,毒素对Caco-2细胞增殖的抑制作用与浓度和作用时间密切相关。

  6. Maslinic Acid, a Natural Triterpene, Induces a Death Receptor-Mediated Apoptotic Mechanism in Caco-2 p53-Deficient Colon Adenocarcinoma Cells

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    Reyes-Zurita, Fernando J.; Rufino-Palomares, Eva E.; García-Salguero, Leticia; Peragón, Juan; Medina, Pedro P.; Parra, Andrés; Cascante, Marta; Lupiáñez, José A.

    2016-01-01

    Maslinic acid (MA) is a natural triterpene present in high concentrations in the waxy skin of olives. We have previously reported that MA induces apoptotic cell death via the mitochondrial apoptotic pathway in HT29 colon cancer cells. Here, we show that MA induces apoptosis in Caco-2 colon cancer cells via the extrinsic apoptotic pathway in a dose-dependent manner. MA triggered a series of effects associated with apoptosis, including the cleavage of caspases -8 and -3, and increased the levels of t-Bid within a few hours of its addition to the culture medium. MA had no effect on the expression of the Bax protein, release of cytochrome-c or on the mitochondrial membrane potential. This suggests that MA triggered the extrinsic apoptotic pathway in this cell type, as opposed to the intrinsic pathway found in the HT29 colon-cancer cell line. Our results suggest that the apoptotic mechanism induced in Caco-2 may be different from that found in HT29 colon-cancer cells, and that in Caco-2 cells MA seems to work independently of p53. Natural antitumoral agents capable of activating both the extrinsic and intrinsic apoptotic pathways could be of great use in treating colon-cancer of whatever origin. PMID:26751572

  7. Maslinic Acid, a Natural Triterpene, Induces a Death Receptor-Mediated Apoptotic Mechanism in Caco-2 p53-Deficient Colon Adenocarcinoma Cells.

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    Fernando J Reyes-Zurita

    Full Text Available Maslinic acid (MA is a natural triterpene present in high concentrations in the waxy skin of olives. We have previously reported that MA induces apoptotic cell death via the mitochondrial apoptotic pathway in HT29 colon cancer cells. Here, we show that MA induces apoptosis in Caco-2 colon cancer cells via the extrinsic apoptotic pathway in a dose-dependent manner. MA triggered a series of effects associated with apoptosis, including the cleavage of caspases -8 and -3, and increased the levels of t-Bid within a few hours of its addition to the culture medium. MA had no effect on the expression of the Bax protein, release of cytochrome-c or on the mitochondrial membrane potential. This suggests that MA triggered the extrinsic apoptotic pathway in this cell type, as opposed to the intrinsic pathway found in the HT29 colon-cancer cell line. Our results suggest that the apoptotic mechanism induced in Caco-2 may be different from that found in HT29 colon-cancer cells, and that in Caco-2 cells MA seems to work independently of p53. Natural antitumoral agents capable of activating both the extrinsic and intrinsic apoptotic pathways could be of great use in treating colon-cancer of whatever origin.

  8. Caco-2 cells, biopharmaceutics classification system (BCS) and biowaiver.

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    Smetanová, Libuse; Stĕtinová, Vĕra; Svoboda, Zbynek; Kvetina, Jaroslav

    2011-01-01

    Almost all orally administered drugs are absorbed across the intestinal mucosa. The Caco-2 monolayers are used as an in vitro model to predict drug absorption in humans and to explore mechanism of drug absorption. The Caco-2 cells are derived from a human colon adenocarcinoma and spontaneously differentiate to form confluent monolayer of polarized cells structurally and functionally resembling the small intestinal epithelium. For studying drug permeability, Caco-2 cells are seeded onto the Transwell inserts with semipermeable membrane and grown to late confluence (21 days). After determination of cell viability, the integrity of monolayer is checked by phenol red permeability and by 14C-mannitol permeability. The transport from apical to basolateral (AP-BL) and basolateral to apical (BL-AP) is studied by adding the diluted drug on the apical or basolateral side and withdrawing the samples from the opposite compartment, respectively, for HPLC analysis or liquid scintillation spectrometry. Ca2+ -free transport medium is used to determine paracellular component of the drug transport. On the basis of permeability and solubility, drugs can be categorized into four classes of Biopharmaceutics Classification System (BCS). For certain drugs, the BCS-based biowaiver approach can be used which enables to reduce in vivo bioequivalence studies.

  9. Competition of Lactobacillus paracasei with Salmonella enterica for Adhesion to Caco-2 Cells

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    Alicja Jankowska

    2008-01-01

    Full Text Available Competition of commensal and probiotic bacteria with pathogens for adhesion and colonization is one of the important protective mechanisms of gastrointestinal tract. In this study, we examined the ability of Lactobacillus paracasei to inhibit the adhesion of pathogenic Salmonella enterica to human colon adenocarcinoma Caco-2 cells. Caco-2 cells were grown for 6 or 21 days to obtain nondifferentiated or well-differentiated cells, respectively. In adhesion experiments, bacteria were added to the cells for 2 or 4 hours. The number of attached bacteria was expressed as colony-forming units (CFUs, Caco-2 cells were counted in hematocytometer. Both bacterial strains used adhered better to well-differentiated than to nondifferentiated Caco-2 cells, however, the amount of Salmonella adhered to Caco-2 after 2 hours of contact was 12-fold higher in comparison to . paracasei and almost 27-fold higher after 4 hours of contact. Two types of experiments were done: coincubation (both bacteria were added to Caco-2 cells simultaneously, and preincubation (. paracasei was incubated with Caco-2 cells first, and then . enterica was added. In coincubation experiment, the presence of . paracasei decreased . enterica adhesion by 4-fold and in preincubation experiment even 7-fold. Generally, Lactobacillus spent culture supernatants (SCSs acted weaker as inhibitors of Salmonella adhesion in comparison to the whole . paracasei culture in coincubation experiment. In conclusion, the displacement of pathogens by lactic acid bacteria and its secretions showed here depends on the time of bacteria-epithelial cell contact, and also on the stage of Caco-2 differentiation.

  10. Heme oxygenase-1 promotes Caco-2 cell proliferation and migration by targeting CTNND1

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    ZHANG Li; LIU Yu-lin; CHEN Guang-xiang; CUI Bin; WANG Jin-shen; SHI Yu-long; LI Le-ping

    2013-01-01

    Background Heme oxygenase-1 (HO-1) can be induced by inflammatory cytokines,oxidation,ischemia,hypoxia,and endotoxins.As a "graft survival protective gene," HO-1 is a hot spot in organ transplantation research.However,the role of HO-1 gene expression in the function of human colon adenocarcinoma cell line (Caco-2) cells has not been reported previously.Methods The role of HO-1 in the proliferation and migration of Caco-2 cells was analyzed using a stable HO-1 expression plasmid.We constructed a recombinant adeno-associated virus plasmid containing the HO-1 gene,heme oxygenase 1 (HMOX1),which was transfected into Caco-2 intestinal cells.We identified a number of target genes by global microarray analysis combined with real-time polymerase chain reaction (PCR) and chromatin immunoprecipitation assay.Results Our results showed that significant HO-1 upregulation was demonstrated in the Caco-2 cells after HO-1 transfection.Restoration of HO-1 expression promoted proliferation and invasion in vitro.The CTNND1 gene,a member of the armadillo protein family,was identified as a direct HO-1 target gene.Conclusion Overexpression of HO-1 promotes Caco-2 cell proliferation and migration by targeting the CTNND1 gene.

  11. Absorption and Metabolism Characteristics of Rutin in Caco-2 Cells

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    Xiaofang Zhang

    2013-01-01

    Full Text Available The intestinal absorption and metabolism characteristics of the potentially beneficial polyphenol rutin were studied by measuring the intracellular accumulation and transport of rutin into Caco-2 cells with the sensitive and reliable analytical method of HPLC-coupled tandem mass spectrometry. Rutin and glucuronidated rutin were absorbed differently by the basolateral and apical membranes, and rutin showed differential permeability through the apical and basolateral sides. Approximately 33% of the rutin was metabolized to glucuronidated rutin, and the intracellular concentration of glucuronidated rutin was much lower than that of parent rutin. P-glycoprotein and multidrug-resistant proteins 2 and 3 were involved in the transmembrane transport and intracellular accumulation of rutin by Caco-2 cells. These results suggest that a specific transport system mediates rutin movement across the apical membrane in Caco-2 cells and that metabolic enzymes are important for this process.

  12. Cytotoxicity of monodispersed chitosan nanoparticles against the Caco-2 cells

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    Loh, Jing Wen [Laboratory for Drug Delivery, Pharmacy, Characterisation and Analysis, University of Western Australia (Australia); Saunders, Martin [Centre for Microscopy, Characterisation and Analysis, University of Western Australia (Australia); Lim, Lee-Yong, E-mail: lee.lim@uwa.edu.au [Laboratory for Drug Delivery, Pharmacy, Characterisation and Analysis, University of Western Australia (Australia); School of Biomedical, Biomolecular and Chemical Sciences, 35 Stirling Hwy, Crawley 6009 (Australia)

    2012-08-01

    Published toxicology data on chitosan nanoparticles (NP) often lack direct correlation to the in situ size and surface characteristics of the nanoparticles, and the repeated NP assaults as experienced in chronic use. The aim of this paper was to breach these gaps. Chitosan nanoparticles synthesized by spinning disc processing were characterised for size and zeta potential in HBSS and EMEM at pHs 6.0 and 7.4. Cytotoxicity against the Caco-2 cells was evaluated by measuring the changes in intracellular mitochondrial dehydrogenase activity, TEER and sodium fluorescein transport data and cell morphology. Cellular uptake of NP was observed under the confocal microscope. Contrary to established norms, the collective data suggest that the in vitro cytotoxicity of NP against the Caco-2 cells was less influenced by positive surface charges than by the particle size. Particle size was in turn determined by the pH of the medium in which the NP was dispersed, with the mean size ranging from 25 to 333 nm. At exposure concentration of 0.1%, NP of 25 ± 7 nm (zeta potential 5.3 ± 2.8 mV) was internalised by the Caco-2 cells, and the particles were observed to inflict extensive damage to the intracellular organelles. Concurrently, the transport of materials along the paracellular pathway was significantly facilitated. The Caco-2 cells were, however, capable of recovering from such assaults 5 days following NP removal, although a repeat NP exposure was observed to produce similar effects to the 1st exposure, with the cells exhibiting comparable resiliency to the 2nd assault. -- Highlights: ► Chitosan nanoparticles reduced mitochondrial dehydrogenase activity. ► Cellular uptake of chitosan nanoparticles was observed. ► Chitosan nanoparticles inflicted extensive damage to the cell morphology. ► The transport of materials along the paracellular pathway was facilitated.

  13. Titanium Dioxide Particle Type and Concentration Influence the Inflammatory Response in Caco-2 Cells.

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    Tada-Oikawa, Saeko; Ichihara, Gaku; Fukatsu, Hitomi; Shimanuki, Yuka; Tanaka, Natsuki; Watanabe, Eri; Suzuki, Yuka; Murakami, Masahiko; Izuoka, Kiyora; Chang, Jie; Wu, Wenting; Yamada, Yoshiji; Ichihara, Sahoko

    2016-04-16

    Titanium dioxide (TiO₂) nanoparticles are widely used in cosmetics, sunscreens, biomedicine, and food products. When used as a food additive, TiO₂ nanoparticles are used in significant amounts as white food-coloring agents. However, the effects of TiO₂ nanoparticles on the gastrointestinal tract remain unclear. The present study was designed to determine the effects of five TiO₂ particles of different crystal structures and sizes in human epithelial colorectal adenocarcinoma (Caco-2) cells and THP-1 monocyte-derived macrophages. Twenty-four-hour exposure to anatase (primary particle size: 50 and 100 nm) and rutile (50 nm) TiO₂ particles reduced cellular viability in a dose-dependent manner in THP-1 macrophages, but in not Caco-2 cells. However, 72-h exposure of Caco-2 cells to anatase (50 nm) TiO₂ particles reduced cellular viability in a dose-dependent manner. The highest dose (50 µg/mL) of anatase (100 nm), rutile (50 nm), and P25 TiO₂ particles also reduced cellular viability in Caco-2 cells. The production of reactive oxygen species tended to increase in both types of cells, irrespective of the type of TiO₂ particle. Exposure of THP-1 macrophages to 50 µg/mL of anatase (50 nm) TiO₂ particles increased interleukin (IL)-1β expression level, and exposure of Caco-2 cells to 50 µg/mL of anatase (50 nm) TiO₂ particles also increased IL-8 expression. The results indicated that anatase TiO₂ nanoparticles induced inflammatory responses compared with other TiO₂ particles. Further studies are required to determine the in vivo relevance of these findings to avoid the hazards of ingested particles.

  14. Titanium Dioxide Particle Type and Concentration Influence the Inflammatory Response in Caco-2 Cells

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    Saeko Tada-Oikawa

    2016-04-01

    Full Text Available Titanium dioxide (TiO2 nanoparticles are widely used in cosmetics, sunscreens, biomedicine, and food products. When used as a food additive, TiO2 nanoparticles are used in significant amounts as white food-coloring agents. However, the effects of TiO2 nanoparticles on the gastrointestinal tract remain unclear. The present study was designed to determine the effects of five TiO2 particles of different crystal structures and sizes in human epithelial colorectal adenocarcinoma (Caco-2 cells and THP-1 monocyte-derived macrophages. Twenty-four-hour exposure to anatase (primary particle size: 50 and 100 nm and rutile (50 nm TiO2 particles reduced cellular viability in a dose-dependent manner in THP-1 macrophages, but in not Caco-2 cells. However, 72-h exposure of Caco-2 cells to anatase (50 nm TiO2 particles reduced cellular viability in a dose-dependent manner. The highest dose (50 µg/mL of anatase (100 nm, rutile (50 nm, and P25 TiO2 particles also reduced cellular viability in Caco-2 cells. The production of reactive oxygen species tended to increase in both types of cells, irrespective of the type of TiO2 particle. Exposure of THP-1 macrophages to 50 µg/mL of anatase (50 nm TiO2 particles increased interleukin (IL-1β expression level, and exposure of Caco-2 cells to 50 µg/mL of anatase (50 nm TiO2 particles also increased IL-8 expression. The results indicated that anatase TiO2 nanoparticles induced inflammatory responses compared with other TiO2 particles. Further studies are required to determine the in vivo relevance of these findings to avoid the hazards of ingested particles.

  15. Catechins Variously Affect Activities of Conjugation Enzymes in Proliferating and Differentiated Caco-2 Cells

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    Kateřina Lněničková

    2016-09-01

    Full Text Available The knowledge of processes in intestinal cells is essential, as most xenobiotics come into contact with the small intestine first. Caco-2 cells are human colorectal adenocarcinoma that once differentiated, exhibit enterocyte-like characteristics. Our study compares activities and expressions of important conjugation enzymes and their modulation by green tea extract (GTE and epigallocatechin gallate (EGCG using both proliferating (P and differentiated (D caco-2 cells. The mRNA levels of the main conjugation enzymes were significantly elevated after the differentiation of Caco-2 cells. However, no increase in conjugation enzymes’ activities in differentiated cells was detected in comparison to proliferating ones. GTE/EGCG treatment did not affect the mRNA levels of any of the conjugation enzymes tested in either type of cells. Concerning conjugation enzymes activities, GTE/EGCG treatment elevated glutathione S-transferase (GST activity by approx. 30% and inhibited catechol-O-methyltransferase (COMT activity by approx. 20% in differentiated cells. On the other hand, GTE as well as EGCG treatment did not significantly affect the activities of conjugation enzymes in proliferating cells. Administration of GTE/EGCG mediated only mild changes of GST and COMT activities in enterocyte-like cells, indicating a low risk of GTE/EGCG interactions with concomitantly administered drugs. However, a considerable chemo-protective effect of GTE via the pronounced induction of detoxifying enzymes cannot be expected as well.

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

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

    2004-12-01

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

  17. Vitamin D-regulated calcium transport in Caco-2 cells: unique in vitro model.

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    Giuliano, A R; Wood, R J

    1991-02-01

    The human colon adenocarcinoma cell line Caco-2 is the only intestinal cell line to differentiate spontaneously in culture exhibiting structural and biochemical characteristics of mature enterocytes and to possess a vitamin D receptor in the fully differentiated state. Transepithelial calcium transport was characterized in differentiated Caco-2 cells grown on permeable filters supports to assess the potential utility of this cell line as an in vitro model to study 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-induced calcium transport. Calcium transport was increased in a dose-dependent manner by 1,25(OH)2D3. Total calcium transport at different calcium concentrations could be fitted to a modified Michaelis-Menten equation containing a linear transport component. The maximum rate of saturable calcium transport was increased by 4.3-fold (P less than 0.005) in cells treated with 10(-8) M 1,25(OH)2D3. This treatment also increased the apparent buffer calcium concentration that results in half-maximal velocity from 0.4 to 1.3 mM but had no significant effect on nonsaturable calcium transport. Caco-2 cells grown on permeable filter supports provide a unique in vitro human cell culture model to study the mechanism of vitamin D-regulated transepithelial intestinal calcium transport.

  18. [Effect of Siwu decoction on function and expression of P-glycoprotein in Caco-2 cells].

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    Jiang, Yi; Ma, Zeng-chun; Huang, Xian-ju; You, Qing; Tan, Hong-ling; Wang, Yu-guang; Liang, Qian-de; Tang, Xiang-lin; Xiao, Cheng-rong; Gao, Yue

    2015-03-01

    To study the effect of Siwu decoction on the function and expression of P-glycoprotein (P-gp) in Caco-2 cells. The Real-time quantitative poly-merase chain reaction (Q-PCR) was used to analyze the mRNA expression of MDR1 gene in Caco-2 cells. Flow cytometer was used to study the effect of Siwu decoction on the uptake of Rhodamine 123 in Caco-2 cells, in order to evaluate the efflux function of P-gp. Western blotting method was used to detect the effect of Siwu decoction on the P-gp protein expression of Caco-2 cells. Compared with the blank control group, after Caco-2 incubation with Siwu decoction at concentrations of 3.3, 5.0, 10.0 g x L(-1) for 24, 48, 72 h, the mRNA expression of MDR1 was up-regulated, suggesting the effect of Siwu decoction in inducing the expression of MDR1. After the administration with Siwu decoction in Caco-2 cells for 48 h, the uptake of Rhodamine 123 in Caco-2 cells decreased by respectively 16.6%, 22.1% (P P P-gp efflux function of Caco-2 cells. After the incubation of Caco-2 cells with Siwu decoction for 48 h, the P-gp protein expression on Caco-2 cell emebranes, demonstrating the effect of Siwu decoction in inducing the protein expression of P-gp.

  19. Modulatory effects of quercetin on proliferation and differentiation of the human colorectal cell line Caco-2

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    Dihal, A.A.; Woutersen, R.A.; Ommen, B.v.; Rietjens, I.M.C.M.; Stierum, R.H.

    2006-01-01

    The effect of the dietary flavonoid quercetin was investigated on proliferation and differentiation of the human colon cancer cell line Caco-2. Confluent Caco-2 monolayers exposed to quercetin showed a biphasic effect on cell proliferation and a decrease in cell differentiation (0.001

  20. Transepithelial transport of putrescine across monolayers of the human intestinal epithelial cell line, Caco-2

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    Vladan Milovic; Lyudmila Turchanowa; Jurgen Stein; Wolfgang F. Caspary

    2001-01-01

    AIM To study the transepithelial transport characteristics of the polyamine putrescine in human intestinal Caco-2 cell monolayers to elucidate the mechanisms of the putrescine intestinal absorption.METHODS The transepithelial transport and the cellular accumulation of putrescine was measured using Caco 2 cell monolayers grown on permeable filters.RESULTS Transepithelial transport of putrescine in physiological concentrations (>0.5 mM)from the apical to basolateral side was linear. Intracellular accumulation of putrescine was higher in confluent than in fully differentiated Caco-2 cells, but still negligible (less than 0.5%) of the overall transport across the monolayers in apical-to-basolateral direction. EGF enhanced putrescine accumulation in Caco-2 cells by four-fold, as well as putrescine conversion to spermidine and spermine by enhancing the activity of Sadenosylmethionine decarboxylase. However,EGF did not have any significant influence on putrescine flux across the Caco-2 cell monolayers. Excretion of putrescine from Caco-2cells into the basolateral medium did not exceed 50 picomoles, while putrescine passive flux from the apical to the basolateral chamber,contributed hundreds of micromoles polyamines to the basolateral chamber.CONCLUSION Transepithelial transport of putrescine across Caco-2 cell monolayers occurs in passive diffusion, and is not influenced when epithelial cells are stimulated to proliferate by a potent mitogen such as EGF.

  1. HNF1 alpha activates the aminopeptidase N promoter in intestinal (Caco-2) cells

    DEFF Research Database (Denmark)

    Olsen, Jørgen; Laustsen, Lotte; Troelsen, J

    1994-01-01

    The importance of HNF1 binding proteins for intestinal aminopeptidase N expression was investigated using the Caco-2 cell-line. Aminopeptidase N promoter activity in Caco-2 cells depends on the HNF1 element (positions -85 to -58) and co-transfection with an HNF1 alpha expression vector demonstrates...... a direct activation of the promoter by HNF1 alpha through this element. Electrophoretic mobility shift assays using nuclear extracts from Caco-2 cells show the presence of high amounts of HNF1 binding proteins irrespective of their state of differentiation....

  2. Engineered Nanoparticles as Potential Food Contaminants and Their Toxicity to Caco-2 Cells.

    Science.gov (United States)

    Mao, Xiaomo; Nguyen, Trang H D; Lin, Mengshi; Mustapha, Azlin

    2016-08-01

    Engineered nanoparticles (ENPs), such as metallic or metallic oxide nanoparticles (NPs), have gained much attention in recent years. Increasing use of ENPs in various areas may lead to the release of ENPs into the environment and cause the contamination of agricultural and food products by ENPs. In this study, we selected two important ENPs (zinc oxide [ZnO] and silver [Ag] NPs) as potential food contaminants and investigated their toxicity via an in vitro model using Caco-2 cells. The physical properties of ENPs and their effects on Caco-2 cells were characterized by electron microscopy and energy dispersive X-ray spectroscopic (EDS) techniques. Results demonstrate that a significant inhibition of cell viability was observed after a 24-h of exposure of Caco-2 cells to 3-, 6-, and 12-mM ZnO NPs or 0.5-, 1.5-, and 3-mM Ag NPs. The noticeable changes of cells include the alteration in cell shape, abnormal nuclear structure, membrane blebbing, and cytoplasmic deterioration. The toxicity of ZnO NPs, but not that of Ag NPs after exposure to simulated gastric fluid, significantly decreased. Scanning transmission electron microscopy shows that ZnO and Ag NPs penetrated the membrane of Caco-2 cells. EDS results also confirm the presence of NPs in the cytoplasm of the cells. This study demonstrates that ZnO and Ag NPs have cytotoxic effects and can inhibit the growth of Caco-2 cells.

  3. Design of 3D printed insert for hanging culture of Caco-2 cells.

    Science.gov (United States)

    Shen, Chong; Meng, Qin; Zhang, Guoliang

    2014-12-17

    A Caco-2 cell culture on Transwell, an alternative testing to animal or human testing used in evaluating drug intestinal permeability, incorrectly estimated the absorption of actively transported drugs due to the low expression of membrane transporters. Similarly, three-dimensional (3D) cultures of Caco-2 cells, which have been recommended to be more physiological relevant, were not superior to the Transwell culture in either accuracy or convenience in drug permeability testing. Using rapid 3D printing prototyping techniques, this study proposed a hanging culture of Caco-2 cells that performed with high accuracy in predicting drug permeability in humans. As found, hanging cultured Caco-2 cells formed a confluent monolayer and maintained high cell viability on the 3D printed insert. Compared with the normal culture on Transwell, the Caco-2 cells on the 3D printed insert presented ∼30-100% higher brush border enzyme activity and ∼2-7 folds higher activity of P-glycoprotein/multidrug resistance-associated protein 2 during 21 days of incubation. For the eight membrane transporter substrates, the predictive curve of the 3D printing culture exhibited better linearity (R(2) = 0.92) to the human oral adsorption than that of the Transwell culture (R(2) = 0.84), indicating better prediction by the 3D printing culture. In this regard, the 3D printed insert for hanging culture could be potentially developed as a convenient and low-cost tool for testing drug oral absorption.

  4. Cytotoxicity of folic acid conjugated hollow silica nanoparticles toward Caco2 and 3T3 cells, with and without encapsulated DOX.

    Science.gov (United States)

    Patel, Kunal; Sundara Raj, Behin; Chen, Yan; Lou, Xia

    2016-04-01

    Hollow silica nanoparticles of two sizes with and without a folic acid targeting ligand were synthesized. Fickian diffusion of the antitumor drug doxorubicin hydrochloride (DOX) was demonstrated by the produced nanoparticles, achieving a cumulative release of 73% and 45% for 215 nm and 430 nm particles respectively over a period of 500 h. The hollow silica nanoparticles presented a time and dose dependent toxicity, selective to human epithelial colorectal adenocarcinoma (Caco2) cells, over mouse embryonic fibroblast (3T3) cells. At 24h Caco2 cell viability was reduced to 66% using pure hollow silica at a concentration of 50 μg mL(-1), while that of 3T3 cells remained at 94% under the same conditions. The selective cytotoxicity of hollow silica nanoparticles was further enhanced by conjugation of folic acid and incorporation of DOX: at 24h and an equivalent DOX concentration of 0.5 μg mL(-1), viable Caco2 cells were reduced to 45% while 3T3 cells were reduced to 83%. Interestingly the equivalent dose of free DOX was more toxic to 3T3 than to Caco2 cells, reducing the 3T3 viability to 72% and the Caco2 viability to 80%, which is likely due to the presence of the p-glycoprotein pumps in Caco2 cells. Folic acid conjugation served to enhance the viability of both cell lines in this work. Careful optimization of the folate content should further improve the cell specificity of the hollow silica nanoparticles, thus providing a viable targeting platform for cancer therapy.

  5. Comparative evaluation of nano-CuO crossing Caco-2 cell monolayers and cellular uptake

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Gao; Lianqin, Zhu, E-mail: lianqinz1963@163.com; Fenghua, Zhu [Qingdao Agricultural University, College of Animal Science and Veterinary Medicine (China); Fang, Zheng [Dezhou University, College of Agriculture (China); Mingming, Song; Kai, Huang [Qingdao Agricultural University, College of Animal Science and Veterinary Medicine (China)

    2015-04-15

    Different concentrations of CuSO{sub 4}, micro-CuO, and nano-CuO were added to Caco-2 cell monolayers to study the absorption and transport characteristics in this epithelial cell model. Nano-CuO nanoparticles had a diameter of 10–20 nm. Inhibitors of endocytosis were used to explore whether nano-CuO could enter the Caco-2 cell in the form of nanoparticles, and to ascertain the endocytotic pathway that is involved in the transport process. The apparent permeability coefficient (P{sub app}) of CuSO{sub 4} and nano-CuO increased with the Cu concentration in the culture medium (p < 0.05). The micro-CuO of different concentrations had no significant impact on the P{sub app} value of Caco-2 cells (p > 0.05). When the Cu concentration in the culture medium was in the range 31.25–500 μM, the P{sub app} value of Caco-2 cells incubated with nano-CuO was significantly higher than that obtained with CuSO{sub 4}. The latter was also significantly higher than that when cells were incubated with micro-CuO (p < 0.05). The amount of Cu transport increased with the increase of CuSO{sub 4} concentration in the culture medium. After 90 min, the amount of transport began to saturate, and the transport rate of Cu declined with the increase of CuSO{sub 4} concentration. For the cells incubated with nano-CuO, the amount of Cu transport increased with the increase of nano-CuO concentration, but did not show an obvious saturation with the extension of transport time. Nano-CuO could enter the Caco-2 cell in the form of nanoparticles, and were found in the cytoplasm, vesicles, lysosomes, and cell nuclei. Several inhibitors of endocytosis effectively prevented the entry of nano-CuO into the Caco-2 cells. It was concluded that nano-CuO particles can enter the Caco-2 cells through several cellular endocytotic pathways.

  6. Interaction of Aeromonas strains with lactic acid bacteria via Caco-2 cells.

    Science.gov (United States)

    Hatje, E; Neuman, C; Katouli, M

    2014-01-01

    The genus Aeromonas includes some species that have now been identified as human pathogens of significant medical importance. We investigated the ability of 13 selected Aeromonas strains belonging to nine species isolated from clinical cases (n = 5), environmental waters (n = 5), and fish (n = 3) to adhere to and translocate Caco-2 cells in the absence and presence of two lactic acid bacteria (LAB), i.e., Lactobacillus acidophilus and Bifidobacterium breve. Aeromonas isolates were also assessed for their cytotoxicity, the presence of virulence genes, and hemolysin production. Among the clinical isolates, one strain of Aeromonas veronii biovar veronii and two strains of Aeromonas hydrophila carried cytotoxin (act), heat-labile toxin (alt), hemolysin (hlyA), and aerolysin (aerA) genes, were cytotoxic to Vero cells, produced hemolysin, and showed higher adherence to Caco-2 cells. In contrast, this was seen in only one environmental strain, a strain of A. veronii biovar sobria. When Aeromonas strains were coinoculated with LAB onto Caco-2 cells, their level of adhesion was reduced. However, their rate of translocation in the presence of LAB increased and was significantly (P Aeromonas and LAB strains could have a detrimental effect on the Caco-2 cells, allowing the Aeromonas to translocate more readily, or the presence of the LAB stimulated the Aeromonas strains to produce more toxins and/or increase their translocation rate.

  7. Adherence and viability of intestinal bacteria to differentiated Caco-2 cells quantified by flow cytometry.

    Science.gov (United States)

    Grootaert, Charlotte; Boon, Nico; Zeka, Fjoralba; Vanhoecke, Barbara; Bracke, Marc; Verstraete, Willy; Van de Wiele, Tom

    2011-07-01

    Recent developments in host-microbe research give rise to a growing demand for rapid and accurate methods to quantify bacterial adhesion to epithelial cells. Here, we describe a new flow cytometric method to determine the amount and viability of gut bacteria, adhered to a monolayer of differentiated cells. The latter is a more relevant epithelium model than the suspended eukaryotic cells currently used in flow cytometric protocols. During the development of the method, we monitored the adhesion potential of six bacterial species and an intestinal microbial community to Caco-2 cells. The combination of SYBR Green I/propidium iodide was more efficient than carboxyfluorescein diacetate to stain the bacterial cells. In addition, a better separation between the Caco-2 background signal and viable and dead bacteria was obtained. A precise amount of Triton X-100 was used to detach adhered bacteria from Caco-2 cells and cell debris. Yet, a limited decrease in viability was observed for the intestinal microbial community treated with Triton X-100. The flow cytometric lower detection limit for pure bacterial cultures was 3.0-4.0log/mL, whereas a 5.0-5.5log/mL detection limit was obtained in the presence of Caco-2 cell background. The latter was sufficient to quantify adhered bacteria. To the best of our knowledge, this is the first description of a flow cytometric protocol that quantifies adhesion of both pure and mixed gut microbial cultures to a differentiated monolayer of Caco-2 cells and that allows to distinguish between viable and dead adhered bacteria.

  8. Palmitic acid and linoleic acid metabolism in Caco-2 cells: Different triglyceride synthesis and lipoprotein secretion

    NARCIS (Netherlands)

    van Greevenbroek, M.M.J.; Voorhout, W.F.; Erkelens, D.W.; van Meer, G.; de Bruin, T.W.A.

    1995-01-01

    Polarized monolayers of intestinal Caco-2 cells were used to study the effects of saturated palmitic acid (16:0) and polyunsaturated linoleic acid (18:2) on triglyceride synthesis and lipoprotein secretion. Monolayers were incubated for 24 h, at the apical or lumenal side, with palmitic acid (16:0)

  9. Application of Caco-2 Cell Line in Herb-Drug Interaction Studies: Current Approaches and Challenges

    Science.gov (United States)

    Awortwe, C.; Fasinu, P.S.; Rosenkranz, B.

    2015-01-01

    The Caco-2 model is employed in pre-clinical investigations to predict the likely gastrointestinal permeability of drugs because it expresses cytochrome P450 enzymes, transporters, microvilli and enterocytes of identical characteristics to the human small intestine. The FDA recommends this model as integral component of the Biopharmaceutics Classification System (BCS). Most dedicated laboratories use the Caco-2 cell line to screen new chemical entities through prediction of its solubility, bioavailability and the possibility of drug-drug or herb-drug interactions in the gut lumen. However, challenges in the inherent characteristics of Caco-2 cell and inter-laboratory protocol variations have resulted to generation of irreproducible data. These limitations affect the extrapolation of data from pre-clinical research to clinical studies involving drug-drug and herb-drug interactions. This review addresses some of these caveats and enumerates the plausible current and future approaches to reduce the anomalies associated with Caco-2 cell line investigations focusing on its application in herb-drug interactions. PMID:24735758

  10. Pseudomonas aeruginosa serA Gene Is Required for Bacterial Translocation through Caco-2 Cell Monolayers

    Science.gov (United States)

    Yasuda, Masashi; Nagata, Syouya; Yamane, Satoshi; Kunikata, Chinami; Kida, Yutaka; Kuwano, Koichi; Suezawa, Chigusa; Okuda, Jun

    2017-01-01

    To specify critical factors responsible for Pseudomonas aeruginosa penetration through the Caco-2 cell epithelial barrier, we analyzed transposon insertion mutants that demonstrated a dramatic reduction in penetration activity relative to P. aeruginosa PAO1 strain. From these strains, mutations could be grouped into five classes, specifically flagellin-associated genes, pili-associated genes, heat-shock protein genes, genes related to the glycolytic pathway, and biosynthesis-related genes. Of these mutants, we here focused on the serA mutant, as the association between this gene and penetration activity is yet unknown. Inactivation of the serA gene caused significant repression of bacterial penetration through Caco-2 cell monolayers with decreased swimming and swarming motilities, bacterial adherence, and fly mortality rate, as well as repression of ExoS secretion; however, twitching motility was not affected. Furthermore, L-serine, which is known to inhibit the D-3-phosphoglycerate dehydrogenase activity of the SerA protein, caused significant reductions in penetration through Caco-2 cell monolayers, swarming and swimming motilities, bacterial adherence to Caco-2 cells, and virulence in flies in the wild-type P. aeruginosa PAO1 strain. Together, these results suggest that serA is associated with bacterial motility and adherence, which are mediated by flagella that play a key role in the penetration of P. aeruginosa through Caco-2 cell monolayers. Oral administration of L-serine to compromised hosts might have the potential to interfere with bacterial translocation and prevent septicemia caused by P. aeruginosa through inhibition of serA function. PMID:28046014

  11. Altered global gene expression profiles in human gastrointestinal epithelial Caco2 cells exposed to nanosilver

    Directory of Open Access Journals (Sweden)

    Saura C. Sahu

    2016-01-01

    Full Text Available Extensive consumer exposure to food- and cosmetics-related consumer products containing nanosilver is of public safety concern. Therefore, there is a need for suitable in vitro models and sensitive predictive rapid screening methods to assess their toxicity. Toxicogenomic profile showing subtle changes in gene expressions following nanosilver exposure is a sensitive toxicological endpoint for this purpose. We evaluated the Caco2 cells and global gene expression profiles as tools for predictive rapid toxicity screening of nanosilver. We evaluated and compared the gene expression profiles of Caco-2 cells exposed to 20 nm and 50 nm nanosilver at a concentration 2.5 μg/ml. The global gene expression analysis of Caco2 cells exposed to 20 nm nanosilver showed that a total of 93 genes were altered at 4 h exposure, out of which 90 genes were up-regulated and 3 genes were down-regulated. The 24 h exposure of 20 nm silver altered 15 genes in Caco2 cells, out of which 14 were up-regulated and one was down-regulated. The most pronounced changes in gene expression were detected at 4 h. The greater size (50 nm nanosilver at 4 h exposure altered more genes by more different pathways than the smaller (20 nm one. Metallothioneins and heat shock proteins were highly up-regulated as a result of exposure to both the nanosilvers. The cellular pathways affected by the nanosilver exposure is likely to lead to increased toxicity. The results of our study presented here suggest that the toxicogenomic characterization of Caco2 cells is a valuable in vitro tool for assessing toxicity of nanomaterials such as nanosilver.

  12. Purified glycosaminoglycans from cooked haddock may enhance Fe uptake via endocytosis in a Caco-2 cell culture model

    Science.gov (United States)

    This study aims to understand the enhancing effect of glycosaminoglycans (GAGs), such as chondroitin/dermatan structures, on Fe uptake to Caco-2 cells. High sulfated GAGs were selectively purified from cooked haddock. An in vitro digestion/Caco-2 cell culture model was used to evaluate Fe uptake (ce...

  13. Proteome analysis reveals novel proteins associated with proliferation and differentiation of the colorectal cancer cell line Caco-2

    NARCIS (Netherlands)

    Stierum, R.; Gaspari, M.; Dommels, Y.; Ouatas, T.; Pluk, H.; Jespersen, S.; Vogels, J.; Verhoeckx, K.; Groten, J.; Ommen, B. van

    2003-01-01

    Here, we describe a proteomics approach to study protein expression changes in differentiating Caco-2 cells. Caco-2 is a colorectal carcinoma cell line, which upon differentiation loses its tumorigenic phenotype and displays characteristics of mature enterocytes, including brush borders with microvi

  14. Milk peptides increase iron solubility in water but do not affect DMT-1 expression in Caco-2 cells

    Science.gov (United States)

    In vitro digestion of milk produces peptide fractions that enhance iron uptake by Caco-2 cells. Our objectives were to investigate whether these fractions a) exert their effect by increasing relative gene expression of DMT-1 in Caco-2 cells b) enhance iron dialyzability when added in meals. Peptid...

  15. Anti-inflammatory activity of the basolateral fraction of Caco-2 cells exposed to a rosemary supercritical extract

    NARCIS (Netherlands)

    Arranz, E.; Mes, J.J.; Wichers, H.J.; Jaime, L.; Reglero, G.; Santoyo, S.

    2015-01-01

    The anti-inflammatory activity of the basolateral fraction of Caco-2 cells exposed to a rosemary supercritical extract was examined. Uptake of rosemary extract fractions was tested on Caco-2 cell monolayers (2–12 h incubation times) and the quantification of carnosic acid and carnosol was performed

  16. Surface charge-specific interactions between polymer nanoparticles and ABC transporters in Caco-2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Bhattacharjee, Sourav, E-mail: sourav.bhattacharjee@wur.nl [Wageningen University, Laboratory of Organic Chemistry (Netherlands); Opstal, Edward J. van; Alink, Gerrit M. [Wageningen University, Division of Toxicology (Netherlands); Marcelis, Antonius T. M.; Zuilhof, Han [Wageningen University, Laboratory of Organic Chemistry (Netherlands); Rietjens, Ivonne M. C. M. [Wageningen University, Division of Toxicology (Netherlands)

    2013-06-15

    The surface charge-dependent transport of polymeric nanoparticles (PNPs) across Caco-2 monolayers grown on transwell culture systems as an in vitro model for intestinal transport was tested. The transport of well-characterized, monodisperse, and fluorescent tri-block copolymer nanoparticles (TCNPs/size {approx}45 nm) and polystyrene nanoparticles (PSNPs/size {approx}50 nm), with different surface charges (positive and negative), was quantified. The positive PNPs showed a higher intracellular uptake and flux across the Caco-2 monolayers than the negative PNPs. Multidrug resistance/P-glycoprotein (MDR1/P-gp), a specific ATP-binding cassette (ABC) transporter, was found to play a major role in the cellular efflux of positive PNPs, whereas the multidrug resistance protein 1 took part in the efflux of negative PNPs from Caco-2 cells. The positive PNPs also caused an increased cellular uptake and apical to basolateral transport of the carcinogen PhIP across the Caco-2 monolayer. The flavonoid quercetin, which is known to interact with ABC transporters, promoted the intracellular uptake of different PNPs and interfered with the normal distribution patterns of PNPs in the transwell system. These results indicate that PNPs display surface charge-specific interactions with ABC transporters and can even affect the bioavailability of toxic food-borne compounds (like pro-carcinogens).

  17. Characterization of adhesive molecule with affinity to Caco-2 cells in Lactobacillus acidophilus by proteome analysis.

    Science.gov (United States)

    Ashida, Nobuhisa; Yanagihara, Sae; Shinoda, Tadashi; Yamamoto, Naoyuki

    2011-10-01

    The adhesive activities of eight Lactobacillus acidophilus strains toward intestinal epithelial Caco-2 cells were studied to understand the probiotic characteristics of the L. acidophilus L-92 strain. Most of the strains, including L-92, showed high adhesive activity; CP23 showed the lowest adhesive activity. CP23 was selected for comparative analysis of cell wall-associated proteins versus the L-92 strain. Cell wall-associated proteins extracted from L-92 and CP23 were subjected to two-dimensional electrophoresis, and major spots observed in the former were compared to the corresponding spots in the latter. To understand the effects of key components of L-92 on its adhesion to Caco-2 cells, 18 spots with stronger signals in L-92 than those in CP23 were identified by a MALDI-TOF/TOF of Ultraflex analysis. Among the identified proteins of L-92, surface-layer protein A (SlpA) was considered strongly involved in adhesion in the eight L. acidophilus strains. To study the importance of SlpA in the adhesion of L. acidophilus, the amounts of SlpA proteins in LiCl extracts of the eight strains were compared by SDSpolyacrylamide gel electrophoresis. As a result, the adhesive abilities of L. acidophilus strains to Caco-2 cells correlated closely to the amount of SlpA in the cells and the productivity of IL-12, an inflammatory cytokine, in all eight strains. These results strongly suggested that SlpA in L. acidophilus might play a key role in its attachment to Caco-2 cells and in the release of IL-12 from dendritic cells.

  18. Biphasic effect of falcarinol on caco-2 cell proliferation, DNA damage, and apoptosis.

    Science.gov (United States)

    Young, Jette F; Duthie, Susan J; Milne, Lesley; Christensen, Lars P; Duthie, Garry G; Bestwick, Charles S

    2007-02-07

    The polyacetylene falcarinol, isolated from carrots, has been shown to be protective against chemically induced colon cancer development in rats, but the mechanisms are not fully understood. In this study CaCo-2 cells were exposed to falcarinol (0.5-100 microM) and the effects on proliferation, DNA damage, and apoptosis investigated. Low-dose falcarinol exposure (0.5-10 microM) decreased expression of the apoptosis indicator caspase-3 concomitantly with decreased basal DNA strand breakage. Cell proliferation was increased (1-10 microM), whereas cellular attachment was unaffected by falcarinol. At concentrations above 20 microM falcarinol, proliferation of CaCo-2 cells decreased and the number of cells expressing active caspase-3 increased simultaneously with increased cell detachment. Furthermore, DNA single-strand breakage was significantly increased at concentrations above 10 microM falcarinol. Thus, the effects of falcarinol on CaCo-2 cells appear to be biphasic, inducing pro-proliferative and apoptotic characteristics at low and high concentrations of falcarinol, respectively.

  19. Evaluation of Iron Bioavailability in Caco-2 cell Culture Model: Modification of the Original Method

    Directory of Open Access Journals (Sweden)

    Bahareh Nikooyeh

    2016-07-01

    Full Text Available Background and Objectives: In the original in vitro method for evaluation of iron bioavailability using caco-2 cell model, ferritin formation by cells is considered as a functional indicator of both absorption and utilization of iron. However, as iron is measured in the tested material and then ferritin concentration is measured in caco-2 cell lysate, it is almost impossible to have a concrete measure of iron bioavailability because the percent of absorbable iron cannot be calculated by the concentration of iron in caco-2 cell lysate. To overcome this problem, the original method was modified by using ferrous sulfate tablet as a standard and also including blank well in the cell culture plate to subtract the baseline ferritin concentration from the ferritin concentration of other wells. Materials and Methods: To evaluate iron bioavailability in this model, one kind of Iranian traditional breads (Sangak was used. Standard curve was plotted using iron concentration and ferritin/protein ratio in the X and Y axes, respectively. Results: The standard curve showed a linear equation: Ferritin (ng/protein (mg = (5.96 × bread Fe+ 11.55, R2=0.989. Using this equation, the bioavailability of iron from the tested bread was 79.76 ± 11.5% of ferrous sulfate. Considering that the reported iron bioavailability from ferrous sulfate is about 10%, bioavailability of iron from the bread could be regarded 8%. Conclusions: This data can be potentially usable to adjust the amount of absorbable iron in the target population for fortification programs. Keywords: Iron, Bioavailability, Caco-2 cell

  20. A Caco-2 cell-based quantitative antioxidant activity assay for antioxidants.

    Science.gov (United States)

    Wan, Hongxia; Liu, Dong; Yu, Xiangying; Sun, Haiyan; Li, Yan

    2015-05-15

    A Caco-2 cell-based antioxidant activity (CAA) assay for quantitative evaluation of antioxidants was developed by optimizing seeding density and culture time of Caco-2 cells, incubation time and concentration of fluorescent probe (2',7'-dichlorofluorescin diacetate, DCFH-DA), incubation way and incubation time of antioxidants (pure phytochemicals) and DCFH-DA with cells, and detection time of fluorescence. Results showed that the CAA assay was of good reproducibility and could be used to evaluate the antioxidant activity of antioxidants at the following conditions: seeding density of 5 × 10(4)/well, cell culture time of 24h, co-incubation of 60 μM DCFH-DA and pure phytochemicals with Caco-2 cells for 20 min and fluorescence recorded for 90 min. Additionally, a significant correlation was observed between CAA values and rat plasma ORAC values following the intake of antioxidants for selected pure phytochemicals (R(2) = 0.815, p < 0.01), demonstrating the good biological relevance of CAA assay.

  1. miRNAs modified by dietary lipids in Caco-2 cells. A microarray screening

    Directory of Open Access Journals (Sweden)

    Lidia Daimiel

    2015-09-01

    Full Text Available We performed a screening of miRNAs regulated by dietary lipids in a cellular model of enterocytes, Caco-2 cells. Our aim was to describe new lipid-modified miRNAs with an implication in lipid homeostasis and cardiovascular disease [1,2]. For that purpose, we treated differentiated Caco-2 cells with micelles containing the assayed lipids (cholesterol, conjugated linoleic acid and docosahexaenoic acid and the screening of miRNAs was carried out by microarray using the μParaflo®Microfluidic Biochip Technology of LC Sciences (Huston, TX, USA. Experimental design, microarray description and raw data have been made available in the GEO database with the reference number of GSE59153. Here we described in detail the experimental design and methods used to obtain the relative expression data.

  2. Isoflavones in food supplements: chemical profile, label accordance and permeability study in Caco-2 cells.

    Science.gov (United States)

    Almeida, I M C; Rodrigues, F; Sarmento, B; Alves, R C; Oliveira, M B P P

    2015-03-01

    Consumers nowadays are playing an active role in their health-care. A special case is the increasing number of women, who are reluctant to use exogenous hormone therapy for the treatment of menopausal symptoms and are looking for complementary therapies. However, food supplements are not clearly regulated in Europe. The EFSA has only recently begun to address the issues of botanical safety and purity regulation, leading to a variability of content, standardization, dosage, and purity of available products. In this study, isoflavones (puerarin, daidzin, genistin, daidzein, glycitein, genistein, formononetin, prunetin, and biochanin A) from food supplements (n = 15) for menopausal symptoms relief are evaluated and compared with the labelled information. Only four supplements complied with the recommendations made by the EC on the tolerable thresholds. The intestinal bioavailability of these compounds was investigated using Caco-2 cells. The apparent permeability coefficients of the selected isoflavonoids across the Caco-2 cells were affected by the isoflavone concentration and product matrix.

  3. Studies on Absorption and Tansport of Limoninoids from Fructus Evodiae in Caco-2 Cell Monolayer Model

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Objective To study the intestinal absorption and transepithelial transport of three limoninoids: evodol (EVO), limonin (LIM), and shihulimonin A (SHIA), isolated from Fructus Evodiae [the unripe fruit of Evodia rutaecarpa and Evodia rutaecarpa var. bodinieri] in the human intestine. Methods The in vitro cultured human colon carcinoma cell line, Caco-2 cell monolayer model, was applied to studying the absorption and transepithelial transport of the three limoninoids from apical (AP) to basolateral (BL) side and from BL to AP side. The three limoninoids were measured by reversed-phase high performance liquid chromatography coupled with ultraviolet absorption detector. Transport parameters and apparent permeability coefficients (Papp) were then calculated and compared with those of Propranolol as a control substance of high permeability and Atenolol as a control substance of poor permeability. Results The Papp value of EVO and LIM from AP to BL side for absorption and transport were 1.78 × 10-5 cm/s and 1.16 × 10-5 cm/s, respectively, which was comparable to that of Propranolol with Papp 2.18 × 10-5 cm/s. Conclusion The absorption and transport of both EVO and LIM are main passive diffusion as the dominating process in Caco-2 cell monolayer model, and they were estimated to be high absorbed compounds. SHIA in Caco-2 cell monolayer model may be involved in metabolism in the transport processes.

  4. Anthocyanin Absorption and Metabolism by Human Intestinal Caco-2 Cells--A Review.

    Science.gov (United States)

    Kamiloglu, Senem; Capanoglu, Esra; Grootaert, Charlotte; Van Camp, John

    2015-09-08

    Anthocyanins from different plant sources have been shown to possess health beneficial effects against a number of chronic diseases. To obtain any influence in a specific tissue or organ, these bioactive compounds must be bioavailable, i.e., effectively absorbed from the gut into the circulation and transferred to the appropriate location within the body while still maintaining their bioactivity. One of the key factors affecting the bioavailability of anthocyanins is their transport through the gut epithelium. The Caco-2 cell line, a human intestinal epithelial cell model derived from a colon carcinoma, has been proven to be a good alternative to animal studies for predicting intestinal absorption of anthocyanins. Studies investigating anthocyanin absorption by Caco-2 cells report very low absorption of these compounds. However, the bioavailability of anthocyanins may be underestimated since the metabolites formed in the course of digestion could be responsible for the health benefits associated with anthocyanins. In this review, we critically discuss recent findings reported on the anthocyanin absorption and metabolism by human intestinal Caco-2 cells.

  5. Caco-2 cell acquisition of dietary iron(III invokes a nanoparticulate endocytic pathway.

    Directory of Open Access Journals (Sweden)

    Dora I A Pereira

    Full Text Available Dietary non-heme iron contains ferrous [Fe(II] and ferric [Fe(III] iron fractions and the latter should hydrolyze, forming Fe(III oxo-hydroxide particles, on passing from the acidic stomach to less acidic duodenum. Using conditions to mimic the in vivo hydrolytic environment we confirmed the formation of nanodisperse fine ferrihydrite-like particles. Synthetic analogues of these (~ 10 nm hydrodynamic diameter were readily adherent to the cell membrane of differentiated Caco-2 cells and internalization was visualized using transmission electron microscopy. Moreover, Caco-2 exposure to these nanoparticles led to ferritin formation (i.e., iron utilization by the cells, which, unlike for soluble forms of iron, was reduced (p=0.02 by inhibition of clathrin-mediated endocytosis. Simulated lysosomal digestion indicated that the nanoparticles are readily dissolved under mildly acidic conditions with the lysosomal ligand, citrate. This was confirmed in cell culture as monensin inhibited Caco-2 utilization of iron from this source in a dose dependent fashion (p<0.05 whilet soluble iron was again unaffected. Our findings reveal the possibility of an endocytic pathway for acquisition of dietary Fe(III by the small intestinal epithelium, which would complement the established DMT-1 pathway for soluble Fe(II.

  6. Intestinal Transportations of Main Chemical Compositions of Polygoni Multiflori Radix in Caco-2 Cell Model

    Directory of Open Access Journals (Sweden)

    Jie Yu

    2014-01-01

    Full Text Available Context. Polygoni Multiflori Radix (PMR is originated from the root of Polygonum multiflorum Thunb. and used in oriental countries for centuries. However, little researches pay close attention to the absorption of its major constituents. Objective. Transepithelial transport of TSG, RL, PL, and four anthraquinones is carried out. Materials and Methods. Caco-2 cell monolayer, which represented a well-established model for the study of intestinal transport of nutrients and xenobiotics, was used in this paper. Results. The apparent permeability coefficients (Papp in the Caco-2 cell monolayers were TSG (2.372 × 10−9 < EG (2.391 × 10−9 < EN (2.483 × 10−9 < PL (4.917 × 10−9 < RN (1.707 × 10−8 < RL (1.778 × 10−8 < AE (1.952 × 10−8. Thus, RN, RL, and AE were considered partly absorbed, while other constituents were hardly absorbed. Discussion and Conclusion. Glycosides showed poor permeabilities than aglycones. In the meantime, TSG and EN gave out poor recovery rates in this assay, which indicated that TSG and EN may accumulate or metabolise in the Caco-2 cells. In silico prediction indicated that Gibbs energy (r=0.751, p<0.05 and heat of form (r=0.701, p<0.05 were strongly positively correlated with Papp.

  7. Betalains increase vitexin-2-O-xyloside cytotoxicity in CaCo-2 cancer cells.

    Science.gov (United States)

    Farabegoli, F; Scarpa, E S; Frati, A; Serafini, G; Papi, A; Spisni, E; Antonini, E; Benedetti, S; Ninfali, P

    2017-03-01

    Vitexin-2-O-xyloside (XVX) from Beta vulgaris var. cicla L. (BVc) seeds, betaxanthin (R1) and betacyanin (R2) fractions from Beta vulgaris var. rubra L. (BVr) roots were combined and tested for cytotoxicity in CaCo-2 colon cancer cells. XVX was the most cytotoxic molecule, but the combination of XVX with R1 and R2 significantly prolonged its cytotoxicity. Cytotoxicity was mediated by the intrinsic apoptotic pathway, as shown by an increase in Bcl2-like protein 4, cleaved Poly ADP-Ribosyl Polymerase 1 and cleaved Caspase 3 levels with a parallel decrease in anti-apoptotic protein B-cell leukemia/lymphoma 2 levels. R1 and R2, used alone or in combination, reduced oxidative stress triggered by H2O2 in CaCo-2 cells. Betalains dampened cyclooxygenase-2 and interleukin-8 mRNA expression after lipopolysaccharide induction in CaCo-2, showing an anti-inflammatory action. Our results support the use of a cocktail of R1, R2 and XVX as a chemopreventive tool against colon cancer.

  8. Sensing Small Changes in Protein Abundance: Stimulation of Caco-2 Cells by Human Whey Proteins.

    Science.gov (United States)

    Cundiff, Judy K; McConnell, Elizabeth J; Lohe, Kimberly J; Maria, Sarah D; McMahon, Robert J; Zhang, Qiang

    2016-01-04

    Mass spectrometry (MS)-based proteomic approaches have largely facilitated our systemic understanding of cellular processes and biological functions. Cutoffs in protein expression fold changes (FCs) are often arbitrarily determined in MS-based quantification with no demonstrable determination of small magnitude changes in protein expression. Therefore, many biological insights may remain veiled due to high FC cutoffs. Herein, we employ the intestinal epithelial cell (IEC) line Caco-2 as a model system to demonstrate the dynamicity of tandem-mass-tag (TMT) labeling over a range of 5-40% changes in protein abundance, with the variance controls of ± 5% FC for around 95% of TMT ratios when sampling 9-12 biological replicates. We further applied this procedure to examine the temporal proteome of Caco-2 cells upon exposure to human whey proteins (WP). Pathway assessments predict subtle effects due to WP in moderating xenobiotic metabolism, promoting proliferation and various other cellular functions in differentiating enterocyte-like Caco-2 cells. This demonstration of a sensitive MS approach may open up new perspectives in the system-wide exploration of elusive or transient biological effects by facilitating scrutiny of narrow windows of proteome abundance changes. Furthermore, we anticipate this study will encourage more investigations of WP on infant gastrointestinal tract development.

  9. Apomorphine and its esters: Differences in Caco-2 cell permeability and chylomicron affinity.

    Science.gov (United States)

    Borkar, Nrupa; Chen, Zhizhong; Saaby, Lasse; Müllertz, Anette; Håkansson, Anders E; Schönbeck, Christian; Yang, Mingshi; Holm, René; Mu, Huiling

    2016-07-25

    Oral delivery of apomorphine via prodrug principle may be a potential treatment for Parkinson's disease. The purpose of this study was to investigate the transport and stability of apomorphine and its esters across Caco-2 cell monolayer and their affinity towards chylomicrons. Apomorphine, monolauroyl apomorphine (MLA) and dilauroyl apomorphine (DLA) were subjected to apical to basolateral (A-B) and basolateral to apical (B-A) transport across Caco-2 cell monolayer. The stability of these compounds was also assessed by incubation at intestinal pH and physiological pH with and without Caco-2 cells. Molecular dynamics (MD) simulations were performed to understand the stability of the esters on a molecular level. The affinity of the compounds towards plasma derived chylomicrons was assessed. The A-B transport of intact DLA was about 150 times lower than the transport of apomorphine. In contrast, MLA was highly unstable in the aqueous media leading to apomorphine appearance basolaterally. MD simulations possibly explained the differences in hydrolysis susceptibilities of DLA and MLA. The affinity of apomorphine diesters towards plasma derived chylomicrons provided an understanding of their potential lymphatic transport. The intact DLA transport is not favorable; therefore, the conversion of DLA to MLA is an important step for intestinal apomorphine absorption.

  10. Mechanistic studies of the transport of peimine in the Caco-2 cell model

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    Lihua Chen

    2016-03-01

    Full Text Available Fritillaria thunbergii Miq. has been widely used in traditional Chinese medicine for its expectorant, antitussive, antiinflammatory and analgesic properties. Moreover, modern pharmacological studies have demonstrated that F. thunbergii Miq. has efficacy in the treatment of leukemia and cancers of the liver and cervix. Although the alkaloid, peimine, is largely responsible for these pharmacological effects, it has very low oral bioavailability. The aim of this study was to investigate the intestinal absorption of peimine in Caco-2 cell monolayers. Having demonstrated that peimine is non-toxic to Caco-2 cells at concentrations <200 μmol/L, the effect of peimine concentration, pH, temperature, efflux transport protein inhibitors and EDTA-Na2 on peimine transport were studied. The results show that peimine transport is concentration-dependent; that at pH 6.0 and 7.4, the Papp(AP-BL of peimine is not significantly different but the Papp(BL-AP is; that both Papp(AP-BL and Papp(BL-AP at 4 °C are significantly higher than their corresponding values at 37 °C; that the P-glycoprotein (P-gp inhibitors, verapamil and cyclosporin A, increase absorption of peimine; and that EDTA-Na2 has no discernible effect. In summary, the results demonstrate that the intestinal absorption of peimine across Caco-2 cell monolayers involves active transport and that peimine is a substrate of P-gp.

  11. Influence of Polyphenol Extract from Evening Primrose (Oenothera Paradoxa Seeds on Proliferation of Caco-2 Cells and on Expression, Synthesis and Activity of Matrix Metalloproteinases and Their Inhibitors

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    Szewczyk Karolina

    2014-09-01

    Full Text Available Evening primrose (Oenothera paradoxa Hudziok seeds are a rich source of not only a valuable oil containing an essential fatty acid - ᵧ-linolenic acid (GLA - but also polyphenols which can be obtained from the biomass remaining after oil pressing. The aim of our studies was to evaluate the influence of a polyphenol extract from defatted seeds of evening primrose on human colorectal adenocarcinoma Caco-2 cell proliferation and matrix metalloproteinases (MMPs synthesis and activity. To assess the effect of evening primrose extract on Caco-2 cell proliferation, crystal violet staining and sulforhodamine B (SRB assays were used whereas mRNA expression and activity of MMPs were evaluated by RT-PCR and gelatin zymography.

  12. Peanut Allergens Alter Intestinal Barrier Permeability and Tight Junction Localisation in Caco-2 Cell Cultures1

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    Dwan B. Price

    2014-05-01

    Full Text Available Background/Aims: Allergen absorption by epithelia may play an important role in downstream immune responses. Transport mechanisms that can bypass Peyer's patches include transcellular and paracellular transport. The capacity of an allergen to cross via these means can modulate downstream processing of the allergen by the immune system. The aim of this study was to investigate allergen-epithelial interactions of peanut allergens with the human intestinal epithelium. Methods: We achieved this using the human Caco-2 cell culture model, exposed to crude peanut extract. Western and immunofluorescence analysis were used to identify the cellular and molecular changes of peanut extract on the intestinal epithelium. Results: Following exposure of Caco-2 cells to peanut extract, binding of the peanut allergens Ara h 1 and Ara h 2 to the apical cellular membrane and transcytosis across the monolayers were observed. Additionally, the co-localisation of the transmembrane tight junction proteins occludin, JAM-A and claudin-1, with the intracellular adhesion protein ZO-1 was modified. Conclusion: Disruption of Caco-2 barrier integrity through tight junction disruption may enable movement of peanut proteins across the intestinal epithelium. This accounts for peanut's increased allergenicity, compared to other food allergens, and provides an explanation for the potency of peanut allergens in immune response elicitation.

  13. Effect of cationized gelatins on the paracellular transport of drugs through caco-2 cell monolayers.

    Science.gov (United States)

    Seki, Toshinobu; Kanbayashi, Hiroshi; Nagao, Tomonobu; Chono, Sumio; Tabata, Yasuhiko; Morimoto, Kazuhiro

    2006-06-01

    Cationized gelatins, candidate absorption enhancers, were prepared by addition of ethylenediamine or spermine to gelatin and the effects of the resulting ethylenediaminated gelatin (EG) and sperminated gelatin (SG) on the paracellular transport of 5(6)-carboxyfluorescein (CF), FITC-dextran-4 (FD4), and insulin through caco-2 cell monolayers were examined. The Renkin function was used for characterization of the paracellular pathway and changes in the pore radius (R) and pore occupancy/length ratio (epsilon/L) calculated from the apparent permeability coefficients (P(app)) of CF and FD4 are discussed. Ethylenediaminetetraacetic acid (EDTA) increased the R of the caco-2 cell monolayer and the P(app) of all compounds examined was markedly increased by the addition of EDTA. On the other hand, EG and SG did not increase R and their enhancing effects were not as strong as those of EDTA. The increase in epsilon/L could be the enhancing mechanism for the cationized gelatins. The number of pathways for water-soluble drugs, such as CF and FD4, in the caco-2 monolayers could be increased by the addition of the cationized gelatins. The ratios of the permeability coefficients of insulin (observed/calculated based on the Renkin function) suggest that insulin undergoes enzymatic degradation during transport which is not inhibited by enhancers.

  14. Modulation of cytokine release by differentiated CACO-2 cells in a compartmentalized coculture model with mononuclear leucocytes and nonpathogenic bacteria

    DEFF Research Database (Denmark)

    Parlesak, Alexandr; Haller, D.; Brinz, S.

    2004-01-01

    To further investigate the interaction between human mononuclear leucocytes [peripheral blood mononuclear cells (PBMC)] and enterocytes, the effect of a confluent layer of differentiated CACO-2 cells on cytokine kinetics during challenge with bacteria in a compartmentalized coculture model...... cells when leucocytes were stimulated directly with bacteria. This suppression was not paralleled by changes in the production of IL-10, IL-6 and transforming growth factor (TGF)-beta. When the bacteria were applied apically to the CACO-2 cell layer, the production of TNF-alpha, IL-12, IL-1beta, IL-8...... analysis revealed that IL-8 gene expression was equally induced in both CACO-2 and PBMC after apical stimulation with bacteria. Of note, bacteria-stimulated CACO-2 cells produced little or no cytokines in the absence of leucocytes, supporting the concept of leucocyte-epithelial cell cross...

  15. Investigation of the cytotoxic effects of titanate nanotubes on Caco-2 cells.

    Science.gov (United States)

    Fenyvesi, Ferenc; Kónya, Zoltán; Rázga, Zsolt; Vecsernyés, Miklós; Kása, Péter; Pintye-Hódi, Klára; Bácskay, Ildikó

    2014-08-01

    Titanate nanotubes can be used as drug delivery systems, but limited information is available on their interactions with intestinal cells. In this study, we investigated the cytotoxicity and cellular uptake of titanate nanotubes on Caco-2 monolayers and found that up to 5 mg/ml concentration, these nanotubes are not cytotoxic and not able to permeate through the intestinal cell layer. Transmission electron microscopic experiments showed that titanate nanotubes are not taken up by cells, only caused a high-density granulation on the surface of the endoplasmic reticulum. According to these results, titanate nanotubes are suitable systems for intestinal drug delivery.

  16. Cacao polyphenols influence the regulation of apolipoprotein in HepG2 and Caco2 cells.

    Science.gov (United States)

    Yasuda, Akiko; Natsume, Midori; Osakabe, Naomi; Kawahata, Keiko; Koga, Jinichiro

    2011-02-23

    Cocoa powder is rich in polyphenols, such as catechins and procyanidins, and has been shown to inhibit low-density lipoprotein (LDL) oxidation and atherogenesis in a variety of models. Human studies have also shown daily intake of cocoa increases plasma high-density lipoprotein (HDL) and decreases LDL levels. However, the mechanisms responsible for these effects of cocoa on cholesterol metabolism have yet to be fully elucidated. The present study investigated the effects of cacao polyphenols on the production of apolipoproteins A1 and B in human hepatoma HepG2 and intestinal Caco2 cell lines. The cultured HepG2 cells or Caco2 cells were incubated for 24 h in the presence of cacao polyphenols such as (-)-epicatechin, (+)-catechin, procyanidin B2, procyanidin C1, and cinnamtannin A2. The concentration of apolipoproteins in the cell culture media was quantified using an enzyme-linked immunoassay, and the mRNA expression was quantified by RT-PCR. Cacao polyphenols increased apolipoprotein A1 protein levels and mRNA expression, even though apolipoprotein B protein and the mRNA expression were slightly decreased in both HepG2 cells and Caco2 cells. In addition, cacao polyphenols increased sterol regulatory element binding proteins (SREBPs) and activated LDL receptors in HepG2 cells. These results suggest that cacao polyphenols may increase the production of mature form SREBPs and LDL receptor activity, thereby increasing ApoA1 and decreasing ApoB levels. These results elucidate a novel mechanism by which HDL cholesterol levels become elevated with daily cocoa intake.

  17. Hormonal regulation of dipeptide transporter (PepT1) in Caco-2 cells with normal and anoxia/reoxygenation management

    Institute of Scientific and Technical Information of China (English)

    Bing-Wei Sun; Xiao-Chen Zhao; Guang-Ji Wang; Ning Li; Jie-Shou Li

    2003-01-01

    AIM: To determine the regulation effects of recombinant human growth hormone (rhGH) on dipeptide transporter (PepT1) in Caco-2 cells with normal culture and anoxia/reoxygenation injury.METHODS: A human intestinal cell monolayer (Caco-2) was used as the in vitro model of human small intestine and cephalexin as the model substrate for dipeptide transporter (PepT1). Caco-2 cells grown on Transwell membrane filters were preincubated in the presence of rhGH in the culture medium for 4 d, serum was withdrawn from monolayers for 24 h before each experiment. The transport experiments of cephalexin across apical membromes were then conducted;Caco-2 cells grown on multiple well dishes (24 pore) with normal culture or anoxia/reoxygenation injury were preincubated with rhGH as above and uptake of cephalexin was then measured.RESULTS: The transport and uptake of cephelaxin across apical membranes of Caco-2 cells after preincubation with rhGH were significantly increased compared with controls (P=0.045, 0.0223). Also, addition of rhGH at physiological concentration (34 nM) to incubation medium greatly stimulates cephalexin uptake by anoxia/reoxygenation injuried Caco-2 cells (P=0.0116), while the biological functions of PepT1 in injured Caco-2 cells without rhGH were markedly downregulated. Northem blot analysis showed that the level of PepT1 mRNA of rhGH-treated injured Caco-2cells was greatly increased compared to controls.CONCLUSION: The present results of rhGH stimulating the uptake and transport of cephalexin indicated that rhGH greatly upregulates the physiological effects of dipeptide transporters of Caco-2 cells. The alteration in the gene expression may be a mechanism of regulation of PepT1. In addition, Caco-2 cells take up cephalexin by the Proton-dependent dipeptide transporters that closely resembles the transporters present in the intestine. Caco-2 cells represent an ideal cellular model for future studies of the dipeptide transporter.

  18. Binding of the hop (Humulus lupulus L.) chalcone xanthohumol to cytosolic proteins in Caco-2 intestinal epithelial cells.

    Science.gov (United States)

    Pang, Yan; Nikolic, Dejan; Zhu, Dongwei; Chadwick, Lucas R; Pauli, Guido F; Farnsworth, Norman R; van Breemen, Richard B

    2007-07-01

    Used in the brewing of beer, hops (Humulus lupulus L.) contain the prenylated chalcone xanthohumol, which is under investigation as a cancer chemoprevention agent and as a precursor for the estrogenic flavanones isoxanthohumol and 8-prenylnaringenin. The uptake, transport and accumulation of xanthohumol were studied using the human intestinal epithelial cell line Caco-2 to help understand the poor bioavailability of this chalcone. Studies were carried out using Caco-2 cell monolayers 18-21 days after seeding. The apparent K(m) and V(max) values of xanthohumol accumulation in Caco-2 cells were determined, and the protein binding of xanthohumol in sub-cellular fractions of Caco-2 cells was investigated. Approximately 70% of xanthohumol added to the apical side of Caco-2 cells accumulated inside the cells, while 93% of the intracellular xanthohumol was localized in the cytosol. Xanthohumol accumulation was temperature dependent and saturable with an apparent K(m )value of 26.5 +/- 4.66 muM and an apparent V(max) of 0.215 +/- 0.018 nmol/mg protein/min. Facilitated transport was not responsible for the uptake of xanthohumol, instead, accumulation inside the Caco-2 cells was apparently the result of specific binding to cytosolic proteins. These data suggest that specific binding of xanthohumol to cytosolic proteins in intestinal epithelial cells contributes to the poor oral bioavailability observed previously in vivo.

  19. Modulatory Effect of Gliadin Peptide 10-mer on Epithelial Intestinal CACO-2 Cell Inflammatory Response.

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    Antonella Capozzi

    Full Text Available Celiac Disease (CD is a chronic inflammatory enteropathy, triggered in genetically susceptible individuals by dietary gluten. Gluten is able to elicit proliferation of specific T cells and secretion of inflammatory cytokines in the small intestine. In this study we investigated the possibility that p10-mer, a decapeptide from durum wheat (QQPQDAVQPF, which was previously shown to prevent the activation of celiac peripheral lymphocytes, may exert an inhibitory effect on peptic-tryptic digested gliadin (PT-Gly-stimulated intestinal carcinoma CACO-2 cells. In these cells, incubated with PT-Gly or p31-43 α-gliadin derived peptide in the presence or in the absence of p10-mer, IRAK1 activation and NF-kB, ERK1/2 and p38 MAPK phosphorylation were measured by immunoblotting, Cyclooxigenase 2 (COX-2 activity by PGE-2 release assay, and production of cytokines in the cell supernatants by ELISA. Our results showed that pre-treatment of CACO-2 cells with p10-mer significantly inhibited IRAK1 activation and NF-kB, ERK1/2 and p38 MAPK phosphorylation, as well as COX-2 activity (i.e. PGE-2 release and production of the IL-6 and IL-8 pro-inflammatory cytokines, induced by gliadin peptides. These findings demonstrate the inhibitory effect of the p10-mer peptide on inflammatory response in CACO-2 cells. The results of the present study show that this p10-mer peptide can modulate "in vitro" the inflammatory response induced by gliadin peptides, allowing to move towards new therapeutic strategies. Turning off the inflammatory response, may in fact represent a key target in the immunotherapy of celiac disease.

  20. Comparative Cellular Toxicity of Hydrophilic and Hydrophobic Microcystins on Caco-2 Cells

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    Jussi A. O. Meriluoto

    2012-10-01

    Full Text Available Microcystins (MC, cyanobacterial peptide hepatotoxins, comprise more than 100 different variants. They are rather polar molecules but some variants contain hydrophobic amino acid residues in the highly variable parts of the molecule. In MC-LF and MC-LW, the more hydrophobic phenylalanine (F and tryptophan (W, respectively, have replaced arginine (R in MC-LR. Depending on the structure, microcystins are expected to have different in vivo toxicity and bioavailability, but only a few studies have considered the toxic properties of the more hydrophobic variants. The present study shows that MC-LF and MC-LW have more pronounced cytotoxic effects on Caco-2 cells as compared to those of MC-LR. Treatment of Caco-2 cells with MC-LW and especially MC-LF showed clear apoptotic features including shrinkage and blebbing, and the cell–cell adhesion was lost. An obvious reduction of cell proliferation and viability, assessed as the activity of mitochondrial dehydrogenases, was observed with MC-LF, followed by MC-LW and MC-LR. Cytotoxicity was quantified by measuring lactate dehydrogenase leakage. The more hydrophobic MC-LW and MC-LF induced markedly enhanced lactate dehydrogenase leakage compared to controls and MC-LR, indicating that the plasma membrane was damaged. All of the three toxins examined inhibited protein phosphatase 1, with MC-LF and MC-LW to a weaker extent compared to MC-LR. The higher toxic potential of the more hydrophobic microcystins could not be explained by the biophysical experiments performed. Taken together, our data show that the more hydrophobic microcystin variants induce higher toxicity in Caco-2 cells.

  1. Comparative cellular toxicity of hydrophilic and hydrophobic microcystins on Caco-2 cells.

    Science.gov (United States)

    Vesterkvist, Pia S M; Misiorek, Julia O; Spoof, Lisa E M; Toivola, Diana M; Meriluoto, Jussi A O

    2012-10-25

    Microcystins (MC), cyanobacterial peptide hepatotoxins, comprise more than 100 different variants. They are rather polar molecules but some variants contain hydrophobic amino acid residues in the highly variable parts of the molecule. In MC-LF and MC-LW, the more hydrophobic phenylalanine (F) and tryptophan (W), respectively, have replaced arginine (R) in MC-LR. Depending on the structure, microcystins are expected to have different in vivo toxicity and bioavailability, but only a few studies have considered the toxic properties of the more hydrophobic variants. The present study shows that MC-LF and MC-LW have more pronounced cytotoxic effects on Caco-2 cells as compared to those of MC-LR. Treatment of Caco-2 cells with MC-LW and especially MC-LF showed clear apoptotic features including shrinkage and blebbing, and the cell–cell adhesion was lost. An obvious reduction of cell proliferation and viability, assessed as the activity of mitochondrial dehydrogenases, was observed with MC-LF, followed by MC-LW and MC-LR. Cytotoxicity was quantified by measuring lactate dehydrogenase leakage. The more hydrophobic MC-LW and MC-LF induced markedly enhanced lactate dehydrogenase leakage compared to controls and MC-LR, indicating that the plasma membrane was damaged. All of the three toxins examined inhibited protein phosphatase 1, with MC-LF and MC-LW to a weaker extent compared to MC-LR. The higher toxic potential of the more hydrophobic microcystins could not be explained by the biophysical experiments performed. Taken together, our data show that the more hydrophobic microcystin variants induce higher toxicity in Caco-2 cells.

  2. Effects of Lactic Acid Bacteria on APRIL and IL-10 Secretion in Caco-2 Cells%乳酸菌对Caco-2细胞分泌APRIL和IL-10的影响

    Institute of Scientific and Technical Information of China (English)

    黄怡; 黄琴; 李雅丽; 崔志文; 李卫芬; 余东游

    2012-01-01

    [Objective] The study was designed to evaluate the immunological effects of probiotic strains Enterococcus faecium and Lactococcus lactis on production of pro-inflammatory cytokine of APRIL and anti-inflammatory cytokine interleukin-10 in intestinal epithelial cells. [Method] Human colon adenocarcinoma cell line, Caco-2 cells were incubated with PBS (Group CT, negative control), Escherichia coli K88 (Group EC, positive control), and Enterococcus faecium (Group EF), and Lactococcus lactis (Group LL) for 2 h, respectively. Besides, Caco-2 cells were firstly treated with E. Faecium and L. Lactis for 1 h, respectively, and then followed by incubation with E. Coli K.88 for an additional 2 h (Group EF-EC and Group LL-EC). Culture supematants were collected for analyzing the contents ofAPRIL and IL-10 by ELISA method. [Result] Results showed that E.faecium and L. Lactis induced an increased release ofAPRIL and IL-10. Pre-culture of Caco-2 cells with E. Faecium and L. Lactis was capable of markedly up-regulating production of IL-10 while decreasing APRIL secretion following co-culture with E. Coli K.88. [Conclusion] These findings demonstrated that E. Faecium and L. Lactis could activate immunity of intestinal epithelial cells by inducing APRIL and IL-10 secretion. Moreover, these two strains of lactic acid bacteria also exhibited anti-inflammatory properties via modulating the immune response in Caco-2 cells when they were infected with E. Coli K88.%[目的]通过检测促炎细胞因子APRIL和抗炎细胞因子IL-10的分泌水平来观察屎肠球菌和乳酸乳球菌对肠上皮细胞先天性免疫应答的调节作用.[方法]Caco-2细胞分别和PBS(CT组,阴性对照组)、Escherichia coli K88(EC组,阳性对照组),Enterococcus faecium(EF组)或Lactococcus Iactis(LL组)共孵育2h,以及先分别和Enterococcus faecium或Lactococcus lactis共孵育1h,再和Escherichia coli K88共孵育2h(EF-EC组和LL-EC组).试验结束时,用ELISA方法检

  3. Oxidative stress and mitochondrial functions in the intestinal Caco-2/15 cell line.

    Directory of Open Access Journals (Sweden)

    Rame Taha

    Full Text Available BACKGROUND: Although mitochondrial dysfunction and oxidative stress are central mechanisms in various pathological conditions, they have not been extensively studied in the gastrointestinal tract, which is known to be constantly exposed to luminal oxidants from ingested foods. Key among these is the simultaneous consumption of iron salts and ascorbic acid, which can cause oxidative damage to biomolecules. METHODOLOGY/PRINCIPAL FINDINGS: The objective of the present work was to evaluate how iron-ascorbate (FE/ASC-mediated lipid peroxidation affects mitochondrion functioning in Caco-2/15 cells. Our results show that treatment of Caco-2/15 cells with FE/ASC (0.2 mM/2 mM (1 increased malondialdehyde levels assessed by HPLC; (2 reduced ATP production noted by luminescence assay; (3 provoked dysregulation of mitochondrial calcium homeostasis as evidenced by confocal fluorescence microscopy; (4 upregulated the protein expression of cytochrome C and apoptotic inducing factor, indicating exaggerated apoptosis; (5 affected mitochondrial respiratory chain complexes I, II, III and IV; (6 elicited mtDNA lesions as illustrated by the raised levels of 8-OHdG; (7 lowered DNA glycosylase, one of the first lines of defense against 8-OHdG mutagenicity; and (8 altered the gene expression and protein mass of mitochondrial transcription factors (mtTFA, mtTFB1, mtTFB2 without any effects on RNA Polymerase. The presence of the powerful antioxidant BHT (50 microM prevented the occurrence of oxidative stress and most of the mitochondrial abnormalities. CONCLUSIONS/SIGNIFICANCE: Collectively, our findings indicate that acute exposure of Caco-2/15 cells to FE/ASC-catalyzed peroxidation produces harmful effects on mitochondrial functions and DNA integrity, which are abrogated by the powerful exogenous BHT antioxidant. Functional derangements of mitochondria may have implications in oxidative stress-related disorders such as inflammatory bowel diseases.

  4. Transport of Aflatoxin M1 in human intestinal Caco-2/TC7 cells

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    Francesca eCaloni

    2012-06-01

    Full Text Available Aflatoxin M1 (AFM1 is a hydroxylated metabolite of aflatoxin B1 (AFB1. After it is formed, it is secreted in the milk of mammals.Despite the potential risk of human exposure to AFM1, data reported in literature on the metabolism, toxicity and bioavailability of this molecule are limited and out of date. The aim of the present research was to study the absorption profile of AFM1 and possible damage to tight junctions of the intestinal Caco-2/TC7 clone grown on microporous filter supports. These inserts allowed for the separation of the apical and basolateral compartments which correspond to the in vivo lumen and the interstitial space/vascular systems of intestinal mucosa respectively.In this study, the Caco-2/TC7 cells were treated with different AFM1 concentrations (10-10,000 ng/kg for short (40 minutes and long periods of time (48 hours. The AFM1 influx/efflux transport and effects on tight junctions were evaluated by measuring trans-epithelial electrical resistance and observing tight junction protein (Zonula occludens-1 and occludin localization.The results showed that: i when introduced to the apical and basolateral compartments, AFM1 was poorly absorbed by the Caco-2/TC7 cells but its transport across the cell monolayer occurred very quickly (Papp value of 105.10 ± 7.98 cm/s x 10-6. ii The integrity of tight junctions was not permanently compromised after exposure to the mycotoxin. Viability impairment or barrier damage did not occur either.The present results contribute to the evaluation of human risk exposure to AFM1, although the AFM1 transport mechanism need to be clarified.

  5. Biphasic regulation of P-glycoprotein function and expression by NO donors in Caco-2 cells

    Institute of Scientific and Technical Information of China (English)

    Ru DUAN; Nan HU; Hai-yan LIU; Jia LI; Hai-fang GUO; Can LIU; Li LIU; Xiao-dong LIU

    2012-01-01

    Aim:To investigate the effects of nitric oxide (NO) donors on the function and expression of P-glycoprotein (P-gp) in Caco-2 cells.Methods:Caco-2 cells were exposed to NO donors for designated times.P-gp function and expression were assessed using Rhodamine123 uptake assay and Western blotting,respectively.Intracellular reactive oxygen species (iROS) and intracellular reactive nitrogen species (iRNS) levels were measured using ROS and RNS assay kits,respectively.Results:Exposure of Caco-2 cells to 0.1 or 2 mmol/L of sodium nitroprusside (SNP) affected the function and expression of P-gp in concentration- and time-dependent manners.A short-term (4 h) exposure reduced P-gp function and expression accompanied with significantly increased levels of iROS and iRNS.In contrast,a long-term (24 h) exposure stimulated the P-gp function and expression.The stimulatory effects of 2 mmol/L SNP was less profound as compared to those caused by 0.1 mmol/L SNP.The other NO donors SIN-1 and SNAP showed similar effects.Neither the NO scavenger PTIO (2 mmol/L) nor soluble guanylate cyclase inhibitor ODQ (50 μmol/L) reversed the SNP-induced alteration of P-gp function.On the other hand,free radical scavengers ascorbate,glutathione and uric acid (2 mmol/L for each),PKC inhibitor chelerythrine (5 μmol/L),PI3K/Akt inhibitor wortmannin (1 pmol/L) and p38 MAPK inhibitor SB203580 (10 μmol/L) reversed the upregulation of P-gp function by the long-term exposure to SNP,but these agents had no effect on the impaired P-gp function following the short-term exposure to SNP.Conclusion:NO donors time-dependently regulate P-gp function and expression in Caco-2 cells:short-term exposure impairs P-gp function and expression,whereas long-term exposure stimulates P-gp function and expression.The regulation occurs via a NO-independent mechanism.

  6. Bovine and soybean milk bioactive compounds: Effects on inflammatory response of human intestinal Caco-2 cells.

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    Calvello, Rosa; Aresta, Antonella; Trapani, Adriana; Zambonin, Carlo; Cianciulli, Antonia; Salvatore, Rosaria; Clodoveo, Maria Lisa; Corbo, Filomena; Franchini, Carlo; Panaro, Maria Antonietta

    2016-11-01

    In this study the effects of commercial bovine and soybean milks and their bioactive compounds, namely genistein, daidzein and equol, on the inflammatory responses induced by lipopolysaccharide (LPS) treatment of human intestinal Caco-2 cells were examined, in terms of nitric oxide (NO) release and inducible nitric oxide synthetase (iNOS) expression. Both milks and their bioactive compounds significantly inhibited, dose-dependently, the expression of iNOS mRNA and protein, resulting in a decreased NO production. The NF-κB activation in LPS-stimulated intestinal cells was also examined. In all cases we observed that cell pre-treatment before LPS activation inhibited the IkB phosphorylation. Accordingly, quantification of bioactive compounds by solid phase microextraction coupled with liquid chromatography has shown that they were absorbed, metabolized and released by Caco-2 cells in culture media. In conclusion, we demonstrated that milks and compounds tested are able to reduce LPS-induced inflammatory responses from intestinal cells, interfering with NF-kB dependent molecular mechanisms.

  7. Selenium is critical for cancer-signaling gene expression but not cell proliferation in human colon Caco-2 cells.

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    Zeng, Huawei; Botnen, James H

    2007-01-01

    Selenium (Se) is a potential anticarcinogenic nutrient, and the essential role of Se in cell growth is well recognized but certain cancer cells appear to have acquired a survival advantage under conditions of Se-deficiency. To understand the molecular basis of Se-anticancer effects at nutritional doses (nmol/L) for cultured cells, we generated Se-deficient colon Caco-2 cells by gradually reducing serum in media because serum contains a trace amount of Se. The glutathione peroxidase (GPx) activity of Se-deficient Caco-2 cells was 10.8 mU/mg protein compared to 133.6 approximately 146.3 mU/mg protein in Caco-2 cells supplemented with 500 nmol/L selenite, SeMSC or SeMet (three tested Se-chemical forms) after 7-d culture in serum free media. Interestingly, there were no detectable differences in cell growth, cell cycle progression between Se-deficient cells and cells supplemented with 500 nmol/L Se. To examine differential cancer signaling-gene expression between Se-deficient and Se-supplemented cells, we employed a cancer signal pathway-specific array assay coupled with the real time PCR analysis. Our data demonstrate that although Caco-2 cells are resistant to Se deprivation, Se may exert its anticancer property through increasing the expression of humoral defense gene (A2M) and tumor suppressor-related genes (IGFBP3, HHIP) while decreasing pro-inflammatory gene (CXC L9, HSPB2) expression.

  8. Endocytosis of fluorescent cyclodextrins by intestinal Caco-2 cells and its role in paclitaxel drug delivery.

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    Réti-Nagy, Katalin; Malanga, Milo; Fenyvesi, Éva; Szente, Lajos; Vámosi, György; Váradi, Judit; Bácskay, Ildikó; Fehér, Pálma; Ujhelyi, Zoltán; Róka, Eszter; Vecsernyés, Miklós; Balogh, György; Vasvári, Gábor; Fenyvesi, Ferenc

    2015-12-30

    Cyclodextrins are widely used excipients in pharmaceutical formulations. They are mainly utilized as solubilizers and absorption enhancers, but recent results revealed their effects on cell membranes and pharmacological barriers. In addition to the growing knowledge on their interaction with plasma membranes, it was confirmed that cyclodextrins are able to enter cells by endocytosis. The number of the tested cyclodextrins was limited, and the role of this mechanism in drug absorption and delivery is not known. Our aim was to examine the endocytosis of fluorescently labeled hydroxypropyl-β-cyclodextrin, random methyl-β-cyclodextrin and soluble β-cyclodextrin polymer, and the cellular uptake of the fluorescent paclitaxel derivative-random methyl-β-cyclodextrin complex. The studied cyclodextrin derivatives were able to enter Caco-2 intestinal cells and localized in vesicles in the cytoplasm, while their permeability was very limited through Caco-2 monolayers. We demonstrated for the first time that the fluorescent paclitaxel derivative and rhodamine-labeled random methyl-β-cyclodextrin were detected in the same intracellular vesicles after treating cells with their inclusion complex. These results indicate that the endocytosis of cyclodextrin complexes can contribute to drug absorption processes.

  9. Effect of linear alkylbenzene sulfonate (LAS) on human intestinal Caco-2 cells at non cytotoxic concentrations.

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    Bradai, Mohamed; Han, Junkyu; Omri, Abdelfatteh El; Funamizu, Naoyuki; Sayadi, Sami; Isoda, Hiroko

    2016-08-01

    Linear alkylbenzene sulfonate (LAS) is a cytotoxic synthetic anionic surfactant widely present in the environment due to its large-scale production and intensive use in the detergency field. In this study, we investigated the effect of LAS (CAS No. 25155-30-0) at non cytotoxic concentrations on human intestinal Caco-2 cells using different in vitro bioassays. As results, LAS increased Caco-2 cell proliferation at concentrations ranging from 1 to 15 ppm, more significantly for shorter exposure time (24 h), confirmed using flow cytometry and trypan blue exclusion methods. Moreover, proteomics analysis revealed that this effect was associated with an over-expression of elongation factor 2 and dipeptidyl peptidase 3, and a down-regulation of 14-3-3 protein theta, confirmed at mRNA level using real-time PCR. These findings suggest that LAS at non cytotoxic concentrations, similar to those observed at wastewater treatment plants outlets, increases the growth rate of colon cancer cells, raising thereby its tumor promotion effect potential.

  10. Evaluation of the expression of P-glycoprotein in propoxur-resistant Caco-2 cells.

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    Shabnam Yazdian

    2014-10-01

    Full Text Available There is a great concern about the effect of propoxur, as one of the more common N-methyl carbamate pesticides, on human health due to its extensive use in agricultural and non-agricultural applications. Caco-2 cells became resistant to propoxur, and the resistance was confirmed through MTT assay. Then the cell membrane integrity and P-glycoprotein expression were measured by LDH assay and western blot analysis, respectively and compared to the parent cells.  Contrary to what was expected, the expression of P-glycoprotein in propoxur resistant cells was lower than parent cells.This study indicates that the resistance to propoxur may not be related to P-glycoprotein expression directly, since P-glycoprotein expression has decreased in these cells.

  11. Modulation of tight junctions does not predict oral absorption of hydrophilic compounds: use of Caco-2 and Calu-3 cells.

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    Kamath, Amrita V; Morrison, Richard A; Mathias, Neil R; Dando, Sandra A; Marino, Anthony M; Chong, Saeho

    2007-08-01

    Permeability estimates using Caco-2 cells do not accurately predict the absorption of hydrophilic drugs that are primarily absorbed via the paracellular pathway. The objective of this study was to investigate whether modulation of tight junctions would help differentiation of paracellularly absorbed compounds. Tight junctions in Caco-2 cell monolayers were manipulated using calcium depletion approaches to decrease the transepithelial electrical resistance (TEER) of the monolayers, and permeability of hydrophilic compounds were measured under these conditions. Permeability of these compounds were also measured in Calu-3 cells, which have tighter junctions than Caco-2 cells. Calcium depletion loosened the tight junctions of Caco-2 cells to varying levels as measured by the decrease in TEER values of the monolayers. While the absolute permeability of all the model compounds increased as the tight junctions were loosened, the ratios of their permeability relative to mannitol permeability were similar. The permeability of these compounds in the tighter Calu-3 cells were also found to be similar to each other. Altering the tight junctions of Caco-2 cells to obtain leakier cell monolayers, or using a cell line with tighter junctions like Calu-3 cells, did not improve differentiation between well absorbed and poorly absorbed hydrophilic drugs. Mere manipulation of the tight junctions to increase or decrease transepithelial electrical resistance does not appear to be a viable approach to predict human absorption for hydrophilic compounds that are primarily absorbed via the paracellular pathway.

  12. Arctigenin from Fructus Arctii (Seed of Burdock Reinforces Intestinal Barrier Function in Caco-2 Cell Monolayers

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    Hee Soon Shin

    2015-01-01

    Full Text Available Fructus Arctii is used as a traditional herbal medicine to treat inflammatory diseases in oriental countries. This study aimed to investigate effect of F. Arctii extract on intestinal barrier function in human intestinal epithelial Caco-2 cells and to reveal the active component of F. Arctii. We measured transepithelial electrical resistance (TEER value (as an index of barrier function and ovalbumin (OVA permeation (as an index of permeability to observe the changes of intestinal barrier function. The treatment of F. Arctii increased TEER value and decreased OVA influx on Caco-2 cell monolayers. Furthermore, we found that arctigenin as an active component of F. Arctii increased TEER value and reduced permeability of OVA from apical to the basolateral side but not arctiin. In the present study, we revealed that F. Arctii could enhance intestinal barrier function, and its active component was an arctigenin on the functionality. We expect that the arctigenin from F. Arctii could contribute to prevention of inflammatory, allergic, and infectious diseases by reinforcing intestinal barrier function.

  13. Arctigenin from Fructus Arctii (Seed of Burdock) Reinforces Intestinal Barrier Function in Caco-2 Cell Monolayers.

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    Shin, Hee Soon; Jung, Sun Young; Back, Su Yeon; Do, Jeong-Ryong; Shon, Dong-Hwa

    2015-01-01

    Fructus Arctii is used as a traditional herbal medicine to treat inflammatory diseases in oriental countries. This study aimed to investigate effect of F. Arctii extract on intestinal barrier function in human intestinal epithelial Caco-2 cells and to reveal the active component of F. Arctii. We measured transepithelial electrical resistance (TEER) value (as an index of barrier function) and ovalbumin (OVA) permeation (as an index of permeability) to observe the changes of intestinal barrier function. The treatment of F. Arctii increased TEER value and decreased OVA influx on Caco-2 cell monolayers. Furthermore, we found that arctigenin as an active component of F. Arctii increased TEER value and reduced permeability of OVA from apical to the basolateral side but not arctiin. In the present study, we revealed that F. Arctii could enhance intestinal barrier function, and its active component was an arctigenin on the functionality. We expect that the arctigenin from F. Arctii could contribute to prevention of inflammatory, allergic, and infectious diseases by reinforcing intestinal barrier function.

  14. Lactobacillus paracasei CBA L74 interferes with gliadin peptides entrance in Caco-2 cells.

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    Sarno, Marco; Lania, Giuliana; Cuomo, Marialaura; Nigro, Federica; Passannanti, Francesca; Budelli, Andrea; Fasano, Francesca; Troncone, Riccardo; Auricchio, Salvatore; Barone, Maria Vittoria; Nigro, Roberto; Nanayakkara, Merlin

    2014-12-01

    Several recent reports describe a role of probiotics as a therapeutic approach for celiac disease (CD). Two undigested A-gliadin peptides, P31-43 and P57-68, are central to CD pathogenesis, inducing an innate and an adaptive immune response, respectively. They enter enterocytes and localize to vesicular compartment to induce their toxic/immunogenics effects. In this article, we tested the effect of probiotic Lactobacillus paracasei (LP) CBA L74 (International Depository Accession Number LMG P-24778), its supernatant and LP-fermented cereals on gliadin peptides, P31-43 and P57-68, entrance in Caco-2 cells. Both LP CBA L74 and its supernatant inhibit P31-43 (intensity of fluorescence; FI: 75%) and P57-68 (FI: 50%) entrance in Caco2 cells, indicating that this biological effect is due to some product included in LP CBA L74 supernatant. This effect was present also after fermentation of cereals. This study describes a novel effect of probiotics in the prevention of undigested gliadin peptides toxic effects.

  15. Tectorigenin inhibits Caco-2 human colon cells via NF-κB pathway suppression

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    Xiao-Yu Dai

    2015-12-01

    Full Text Available Effective immunomodulator and pro-inflammatory cytokine, Tumor necrosis factor-α (TNF-α are considered to be responsible for connecting autoimmune pathological process and contagious diseases. TNF-α stimulates the C-X-C motif chemokine 10 (CXCL10 expression that is participated in migration of tumor, metastasis, and invasion. Tectorigenin, an o-methylated isoflavone, is present as a major portion in the Iris tectorum rhizomes. In this study, we investigated the tectorigenin effects as an anticancer drug. The obtained results showed that tectorigenin hinders the invasion of human colon cancer cells (Caco-2. We used reverse transcription PCR, q-PCR and enzyme linked immunosorbent assays to test whether the tectorigenin is involved in the inhibition of TNF-α induced CXCL-10 expression in the Caco-2 cell lines. Further, we tested TNF-α induced NF-κB activity using the tectorigenin. Collective results showed that tectorigenin inhibits the CXCL10 production by using TNF-α via NF-κB inhibition.

  16. Lycopene Modulates THP1 and Caco2 Cells Inflammatory State through Transcriptional and Nontranscriptional Processes

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    Njock Makon-Sébastien

    2014-01-01

    Full Text Available We revisited the action of a carotenoid, the lycopene, on the expression of proinflammatory genes, reactive oxygen species (ROS production, and metalloprotease (MMP9 activity. THP1 and Caco2 cell lines were used as in vitro models for the two main cell types found in intestine tissue, that is, monocytes and epithelial cells. Proinflammatory condition was induced using either phorbol ester acetate (PMA, lipopolysaccharide (LPS or tumor necrosis factor (TNF. In THP1 cells, short term pretreatment (2 h with a low concentration (2 μM of lycopene reinforce proinflammatory gene expression. The extent of the effect of lycopene is dependent on the proinflammtory stimulus (PMA, LPS or TNF used. Lycopene enhanced MMP9 secretion via a c-AMP-dependent process, and reduced ROS production at higher concentrations than 2 μM. Cell culture media, conditioned by PMA-treated monocytes and then transferred on CaCo-2 epithelial cells, induced a proinflammatory state in these cells. The extent of this inflammatory effect was reduced when cells has been pretreated (12 h with lycopene. At low concentration (2 μM or less, lycopene appeared to promote an inflammatory state not correlated with ROS modulation. At higher concentration (5 μM–20 μM, an anti-inflammatory effect takes place as a decrease of ROS production was detected. So, both concentration and time have to be considered in order to define the exact issue of the effect of carotenoids present in meals.

  17. Sinomenine sensitizes multidrug-resistant colon cancer cells (Caco-2 to doxorubicin by downregulation of MDR-1 expression.

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    Zhen Liu

    Full Text Available Chemoresistance in multidrug-resistant (MDR cells over expressing P-glycoprotein (P-gp encoded by the MDR1 gene, is a major obstacle to successful chemotherapy for colorectal cancer. Previous studies have indicated that sinomenine can enhance the absorption of various P-gp substrates. In the present study, we investigated the effect of sinomenine on the chemoresistance in colon cancer cells and explored the underlying mechanism. We developed multidrug-resistant Caco-2 (MDR-Caco-2 cells by exposure of Caco-2 cells to increasing concentrations of doxorubicin. We identified overexpression of COX-2 and MDR-1 genes as well as activation of the NF-κB signal pathway in MDR-Caco-2 cells. Importantly, we found that sinomenine enhances the sensitivity of MDR-Caco-2 cells towards doxorubicin by downregulating MDR-1 and COX-2 expression through inhibition of the NF-κB signaling pathway. These findings provide a new potential strategy for the reversal of P-gp-mediated anticancer drug resistance.

  18. Mn bioavailability by polarized Caco-2 cells: comparison between Mn gluconate and Mn oxyprolinate

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    Fulgenzi Alessandro

    2011-07-01

    Full Text Available Abstract Background Micronutrient inadequate intake is responsible of pathological deficiencies and there is a need of assessing the effectiveness of metal supplementation, frequently proposed to rebalance poor diets. Manganese (Mn is present in many enzymatic intracellular systems crucial for the regulation of cell metabolism, and is contained in commercially available metal supplements. Methods We compared the effects of two different commercial Mn forms, gluconate (MnGluc and oxyprolinate (MnOxP. For this purpose we used the polarized Caco-2 cells cultured on transwell filters, an established in vitro model of intestinal epithelium. Since micronutrient deficiency may accelerate mitochondrial efficiency, the mitochondrial response of these cells, in the presence of MnGluc and MnOxP, by microscopy methods and by ATP luminescence assay was used. Results In the presence of both MnOxP and MnGluc a sustained mitochondrial activity was shown by mitoTraker labeling (indicative of mitochondrial respiration, but ATP intracellular content remained comparable to untreated cells only in the presence of MnOxP. In addition MnOxP transiently up-regulated the antioxidant enzyme Mn superoxide dismutase more efficiently than MnGluc. Both metal treatments preserved NADH and βNADPH diaphorase oxidative activity, avoided mitochondrial dysfunction, as assessed by the absence of a sustained phosphoERK activation, and were able to maintain cell viability. Conclusions Collectively, our data indicate that MnOxP and MnGluc, and primarily the former, produce a moderate and safe modification of Caco-2 cell metabolism, by activating positive enzymatic mechanisms, thus could contribute to long-term maintenance of cell homeostasis.

  19. Identification of transport pathways for citric acid cycle intermediates in the human colon carcinoma cell line, Caco-2.

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    Weerachayaphorn, Jittima; Pajor, Ana M

    2008-04-01

    Citric acid cycle intermediates are absorbed from the gastrointestinal tract through carrier-mediated mechanisms, although the transport pathways have not been clearly identified. This study examines the transport of citric acid cycle intermediates in the Caco-2 human colon carcinoma cell line, often used as a model of small intestine. Inulin was used as an extracellular volume marker instead of mannitol since the apparent volume measured with mannitol changed with time. The results show that Caco-2 cells contain at least three distinct transporters, including the Na+-dependent di- and tricarboxylate transporters, NaDC1 and NaCT, and one or more sodium-independent pathways, possibly involving organic anion transporters. Succinate transport is mediated mostly by Na+-dependent pathways, predominantly by NaDC1, but with some contribution by NaCT. RT-PCR and functional characteristics verified the expression of these transporters in Caco-2 cells. In contrast, citrate transport in Caco-2 cells occurs by a combination of Na+-independent pathways, possibly mediated by an organic anion transporter, and Na+-dependent mechanisms. The non-metabolizable dicarboxylate, methylsuccinate, is also transported by a combination of Na+-dependent and -independent pathways. In conclusion, we find that multiple pathways are involved in the transport of di- and tricarboxylates by Caco-2 cells. Since many of these pathways are not found in human intestine, this model may be best suited for studying Na+-dependent transport of succinate by NaDC1.

  20. Celiac Anti-Type 2 Transglutaminase Antibodies Induce Phosphoproteome Modification in Intestinal Epithelial Caco-2 Cells

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    Marabotti, Anna; Lepretti, Marilena; Salzano, Anna Maria; Scaloni, Andrea; Vitale, Monica; Zambrano, Nicola; Sblattero, Daniele; Esposito, Carla

    2013-01-01

    Background Celiac disease is an inflammatory condition of the small intestine that affects genetically predisposed individuals after dietary wheat gliadin ingestion. Type 2-transglutaminase (TG2) activity seems to be responsible for a strong autoimmune response in celiac disease, TG2 being the main autoantigen. Several studies support the concept that celiac anti-TG2 antibodies may contribute to disease pathogenesis. Our recent findings on the ability of anti-TG2 antibodies to induce a rapid intracellular mobilization of calcium ions, as well as extracellular signal-regulated kinase phosphorylation, suggest that they potentially act as signaling molecules. In line with this concept, we have investigated whether anti-TG2 antibodies can induce phosphoproteome modification in an intestinal epithelial cell line. Methods and Principal Findings We studied phosphoproteome modification in Caco-2 cells treated with recombinant celiac anti-TG2 antibodies. We performed a two-dimensional electrophoresis followed by specific staining of phosphoproteins and mass spectrometry analysis of differentially phosphorylated proteins. Of 14 identified proteins (excluding two uncharacterized proteins), three were hypophosphorylated and nine were hyperphosphorylated. Bioinformatics analyses confirmed the presence of phosphorylation sites in all the identified proteins and highlighted their involvement in several fundamental biological processes, such as cell cycle progression, cell stress response, cytoskeletal organization and apoptosis. Conclusions Identification of differentially phosphorylated proteins downstream of TG2-antibody stimulation suggests that in Caco-2 cells these antibodies perturb cell homeostasis by behaving as signaling molecules. We hypothesize that anti-TG2 autoantibodies may destabilize the integrity of the intestinal mucosa in celiac individuals, thus contributing to celiac disease establishment and progression. Since several proteins here identified in this study

  1. Celiac anti-type 2 transglutaminase antibodies induce phosphoproteome modification in intestinal epithelial Caco-2 cells.

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    Gaetana Paolella

    Full Text Available BACKGROUND: Celiac disease is an inflammatory condition of the small intestine that affects genetically predisposed individuals after dietary wheat gliadin ingestion. Type 2-transglutaminase (TG2 activity seems to be responsible for a strong autoimmune response in celiac disease, TG2 being the main autoantigen. Several studies support the concept that celiac anti-TG2 antibodies may contribute to disease pathogenesis. Our recent findings on the ability of anti-TG2 antibodies to induce a rapid intracellular mobilization of calcium ions, as well as extracellular signal-regulated kinase phosphorylation, suggest that they potentially act as signaling molecules. In line with this concept, we have investigated whether anti-TG2 antibodies can induce phosphoproteome modification in an intestinal epithelial cell line. METHODS AND PRINCIPAL FINDINGS: We studied phosphoproteome modification in Caco-2 cells treated with recombinant celiac anti-TG2 antibodies. We performed a two-dimensional electrophoresis followed by specific staining of phosphoproteins and mass spectrometry analysis of differentially phosphorylated proteins. Of 14 identified proteins (excluding two uncharacterized proteins, three were hypophosphorylated and nine were hyperphosphorylated. Bioinformatics analyses confirmed the presence of phosphorylation sites in all the identified proteins and highlighted their involvement in several fundamental biological processes, such as cell cycle progression, cell stress response, cytoskeletal organization and apoptosis. CONCLUSIONS: Identification of differentially phosphorylated proteins downstream of TG2-antibody stimulation suggests that in Caco-2 cells these antibodies perturb cell homeostasis by behaving as signaling molecules. We hypothesize that anti-TG2 autoantibodies may destabilize the integrity of the intestinal mucosa in celiac individuals, thus contributing to celiac disease establishment and progression. Since several proteins here

  2. Functionalized carbon nanomaterials: exploring the interactions with Caco-2 cells for potential oral drug delivery

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    Coyuco JC

    2011-10-01

    Full Text Available Jurja C Coyuco, Yuanjie Liu, Bee-Jen Tan, Gigi NC ChiuDepartment of Pharmacy, Faculty of Science, National University of Singapore, SingaporeAbstract: Although carbon nanomaterials (CNMs have been increasingly studied for their biomedical applications, there is limited research on these novel materials for oral drug delivery. As such, this study aimed to explore the potential of CNMs in oral drug delivery, and the objectives were to evaluate CNM cytotoxicity and their abilities to modulate paracellular transport and the P-glycoprotein (P-gp efflux pump. Three types of functionalized CNMs were studied, including polyhydroxy small-gap fullerenes (OH-fullerenes, carboxylic acid functionalized single-walled carbon nanotubes (fSWCNT-COOH and poly(ethylene glycol functionalized single-walled carbon nanotubes (fSWCNT-PEG, using the well-established Caco-2 cell monolayer to represent the intestinal epithelium. All three CNMs had minimum cytotoxicity on Caco-2 cells, as demonstrated through lactose dehydrogenase release and 3-(4,5-dimethyliazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assays. Of the three CNMs, fSWCNT-COOH significantly reduced transepithelial electrical resistance and enhanced transport of Lucifer Yellow across the Caco-2 monolayer. Confocal fluorescence microscopy showed that fSWCNT-COOH treated cells had the highest perturbation in the distribution of ZO-1, a protein marker of tight junction, suggesting that fSWCNT-COOH could enhance paracellular permeability via disruption of tight junctions. This modulating effect of fSWCNT-COOH can be reversed over time. Furthermore, cellular accumulation of the P-gp substrate, rhodamine-123, was significantly increased in cells treated with fSWCNT-COOH, suggestive of P-gp inhibition. Of note, fSWCNT-PEG could increase rhodamine-123 accumulation without modifying the tight junction. Collectively, these results suggest that the functionalized CNMs could be useful as modulators for oral drug

  3. Impact Assessment of Cadmium Toxicity and Its Bioavailability in Human Cell Lines (Caco-2 and HL-7702

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    Rukhsanda Aziz

    2014-01-01

    Full Text Available Cadmium (Cd is a widespread environmental toxic contaminant, which causes serious health-related problems. In this study, human intestinal cell line (Caco-2 cells and normal human liver cell line (HL-7702 cells were used to investigate the toxicity and bioavailability of Cd to both cell lines and to validate these cell lines as in vitro models for studying Cd accumulation and toxicity in human intestine and liver. Results showed that Cd uptake by both cell lines increased in a dose-dependent manner and its uptake by Caco-2 cells (720.15 µg mg−1 cell protein was significantly higher than HL-7702 cells (229.01 µg mg−1 cell protein at 10 mg L−1. A time- and dose-dependent effect of Cd on cytotoxicity assays (LDH release, MTT assay was observed in both Cd-treated cell lines. The activities of antioxidant enzymes and differentiation markers (SOD, GPX, and AKP of the HL-7702 cells were higher than those of Caco-2 cells, although both of them decreased significantly with raising Cd levels. The results from the present study indicate that Cd above a certain level inhibits cellular antioxidant activities and HL-7702 cells are more sensitive to Cd exposure than Caco-2 cells. However, Cd concentrations <0.5 mg L−1 pose no toxic effects on both cell lines.

  4. Lycium barbarum L. Polysaccharide (LBP Reduces Glucose Uptake via Down-Regulation of SGLT-1 in Caco2 Cell

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    Huizhen Cai

    2017-02-01

    Full Text Available Lycium barbarum L. polysaccharide (LBP is prepared from Lycium barbarum L. (L. barbarum, which is a traditional Chinese medicine. LPB has been shown to have hypoglycemic effects. In order to gain some mechanistic insights on the hypoglycemic effects of LBP, we investigated the uptake of LBP and its effect on glucose absorption in the human intestinal epithelial cell line Caco2 cell. The uptake of LBP through Caco2 cell monolayer was time-dependent and was inhibited by phloridzin, a competitive inhibitor of SGLT-1. LPB decreased the absorption of glucose in Caco2 cell, and down-regulated the expression of SGLT-1. These results suggest that LBP might be transported across the human intestinal epithelium through SGLT-1 and it inhibits glucose uptake via down-regulating SGLT-1.

  5. Thiolated chitosan nanoparticles: transfection study in the Caco-2 differentiated cell culture

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    Martien, Ronny [Department of Pharmaceutical Technology, Institute of Pharmacy, Leopold-Franzens-University of Innsbruck, Innrain 52, Josef Moeller Haus, A-6020 Innsbruck (Austria); Loretz, Brigitta [Department of Pharmaceutical Technology, Institute of Pharmacy, Leopold-Franzens-University of Innsbruck, Innrain 52, Josef Moeller Haus, A-6020 Innsbruck (Austria); Sandbichler, Adolf Michael [Institute of Zoology, Leopold-Franzens-University of Innsbruck, Technikerstrasse 25, A-6020 Innsbruck (Austria); Schnuerch, Andreas Bernkop [Department of Pharmaceutical Technology, Institute of Pharmacy, Leopold-Franzens-University of Innsbruck, Innrain 52, Josef Moeller Haus, A-6020 Innsbruck (Austria)

    2008-01-30

    The aim of this study was to monitor the expression of secreted protein in differentiated Caco-2 cells after transfection with nanoparticles, in order to improve gene delivery. Based on unmodified chitosan and thiolated chitosan conjugates, nanoparticles with the gene reporter pSEAP (recombinant Secreted Alkaline Phosphatase) were generated at pH 4.0. Transfection studies of thiolated chitosan in Caco-2 cells during the exponential growth phase and differentiation growth phase of the cells led to a 5.0-fold and 2.0-fold increase in protein expression when compared to unmodified chitosan nanoparticles. The mean particle size for both unmodified chitosan and cross-linked thiolated chitosan nanoparticles is 212.2 {+-} 86 and 113.6 {+-} 40 nm, respectively. The zeta potential of nanoparticles was determined to be 7.9 {+-} 0.38 mV for unmodified chitosan nanoparticles and 4.3 {+-} 0.74 mV for cross-linked thiolated chitosan nanoparticles. Red blood cell lysis evaluation was used to evaluate the membrane damaging properties of unmodified and thiolated chitosan nanoparticles and led to 4.61 {+-} 0.36% and 2.29 {+-} 0.25% lysis, respectively. Additionally, cross-linked thiolated chitosan nanoparticles were found to exhibit higher stability toward degradation in gastric juices. Furthermore the reversible effect of thiolated chitosan on barrier properties was monitored by measuring the transepithelial electrical resistance (TEER) and is supported by immunohistochemical staining for the tight junction protein claudin. According to these results cross-linked thiolated chitosan nanoparticles have the potential to be used as a non-viral vector system for gene therapy.

  6. Lysophosphatidylcholine enhances carotenoid uptake from mixed micelles by Caco-2 human intestinal cells.

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    Sugawara, T; Kushiro, M; Zhang, H; Nara, E; Ono, H; Nagao, A

    2001-11-01

    Despite the interest in the beneficial roles of dietary carotenoids in human health, little is known about their solubilization from foods to mixed bile micelles during digestion and the intestinal uptake from the micelles. We investigated the absorption of carotenoids solubilized in mixed micelles by differentiated Caco-2 human intestinal cells, which is a useful model for studying the absorption of dietary compounds by intestinal cells. The micelles were composed of 1 micromol/L carotenoids, 2 mmol/L sodium taurocholate, 100 micromol/L monoacylglycerol, 33.3 micromol/L fatty acid and phospholipid (0-200 micromol/L). The phospholipid content of micelles had profound effects on the cellular uptake of carotenoids. Uptake of micellar beta-carotene and lutein was greatly suppressed by phosphatidylcholine (PC) in a dose-dependent manner, whereas lysophosphatidylcholine (lysoPC), the lipolysis product of PC by phospholipase A2 (PLA2), markedly enhanced both beta-carotene and lutein uptake. The addition of PLA2 from porcine pancreas to the medium also enhanced the uptake of carotenoids from micelles containing PC. Caco-2 cells could take up 15 dietary carotenoids, including epoxy carotenoids, such as violaxanthin, neoxanthin and fucoxanthin, from micellar carotenoids, and the uptakes showed a linear correlation with their lipophilicity, defined as the distribution coefficient in 1-octanol/water (log P(ow)). These results suggest that pancreatic PLA2 and lysoPC are important in regulating the absorption of carotenoids in the digestive tract and support a simple diffusion mechanism for carotenoid absorption by the intestinal epithelium.

  7. Proteomic responses of human intestinal Caco-2 cells exposed to silver nanoparticles and ionic silver.

    Science.gov (United States)

    Oberemm, Axel; Hansen, Ulf; Böhmert, Linda; Meckert, Christine; Braeuning, Albert; Thünemann, Andreas F; Lampen, Alfonso

    2016-03-01

    Even although quite a number of studies have been performed so far to demonstrate nanoparticle-specific effects of substances in living systems, clear evidence of these effects is still under debate. The present study was designed as a comparative proteomic analysis of human intestinal cells exposed to a commercial silver nanoparticle reference material and ions from AgNO3. A two-dimensional gel electrophoresis/MALDI mass spectrometry (MS)-based proteomic analysis was conducted after 24-h incubation of differentiated Caco-2 cells with non-cytotoxic and low cytotoxic silver concentrations (2.5 and 25 µg ml(-1) nanosilver, 0.5 and 5 µg ml(-1) AgNO3). Out of an overall number of 316 protein spots differentially expressed at a fold change of ≥ 1.4 or ≤ -1.4 in all treatments, 169 proteins could be identified. In total, 231 spots were specifically deregulated in particle-treated groups compared with 41 spots, which were limited to AgNO3-treatments. Forty-four spots (14 %) were commonly deregulated by both types of treatment. A considerable fraction of the proteins differentially expressed after treatment with nanoparticles is related to protein folding, synthesis or modification of proteins as well as cellular assembly and organization. Overlays of networks obtained for particulate and ionic treatments showed matches, indicating common mechanisms of combined particle and ionic silver exposure and exclusive ionic silver treatment. However, proteomic responses of Caco-2 cells treated with higher concentrations of silver species also showed some differences, for example regarding proteins related to fatty acid and energy metabolism, suggesting an induction of also some different molecular mechanisms for particle exposure and ionic treatment.

  8. Bovine Muc1 inhibits binding of enteric bacteria to Caco-2 cells.

    Science.gov (United States)

    Parker, Phillip; Sando, Lillian; Pearson, Roger; Kongsuwan, Kritaya; Tellam, Ross L; Smith, Stuart

    2010-01-01

    Inhibition of bacterial adhesion to intestinal epithelial receptors by the consumption of natural food components is an attractive strategy for the prevention of microbial related gastrointestinal illness. We hypothesised that Muc1, a highly glycosylated mucin present in cows' milk, may be one such food component. Purified bovine Muc1 was tested for its ability to inhibit binding of common enteric bacterial pathogens to Caco-2 cells grown in vitro. Muc1 caused dose-dependent binding inhibition of Escherichia coli, Salmonella enterica serovar Typhimurium (S. Typhimurium), Staphylococcus aureus and Bacillus subtilis. This inhibition was more pronounced for the Gram negative compared with Gram positive bacteria. It was also demonstrated that Muc1, immobilised on a membrane, bound all these bacterial species in a dose-dependent manner, although there was greater interaction with the Gram negative bacteria. A range of monosaccharides, representative of the Muc1 oligosaccharide composition, were tested for their ability to prevent binding of E. coli and S. Typhimurium to Caco-2 cells. Inhibition was structure dependent with sialic acid, L(-) fucose and D(+) mannose significantly inhibiting binding of both Gram negative species. N-acetylglucosamine and N-acetylgalactosamine significantly inhibited binding of E. coli whilst galactose, one of the most abundant Muc1 monosaccharides, showed the strongest inhibition against S. Typhimurium. Treatment with sialidase significantly decreased the inhibitory properties of Muc1, demonstrating the importance of sialic acid in adhesion inhibition. It is concluded that bovine Muc1 prevents binding of bacteria to human intestinal cells and may have a role in preventing the binding of common enteropathogenic bacteria to human intestinal epithelial surfaces.

  9. Milk peptides increase iron dialyzability in water but do not affect DMT-1 expression in Caco-2 cells.

    Science.gov (United States)

    Argyri, Konstantina; Tako, Elad; Miller, Dennis D; Glahn, Raymond P; Komaitis, Michael; Kapsokefalou, Maria

    2009-02-25

    In vitro digestion of milk produces peptide fractions that enhance iron uptake by Caco-2 cells. The objectives of this study were to investigate whether these fractions (a) exert their effect by increasing relative gene expression of DMT-1 in Caco-2 cells and (b) enhance iron dialyzability when added in meals. Two milk peptide fractions that solubilize iron were isolated by Sephadex G-25 gel filtration of a milk digest. These peptide fractions did not affect relative gene expression of DMT-1 when incubated with Caco-2 cells for 2 or 48 h. Dialyzability was measured after in vitro simulated gastric and pancreatic digestion. Both peptide fractions enhanced the dialyzability of iron from ferric chloride added to PIPES buffer, but had no effect on dialyzability from milk or a vegetable or fruit meal after in vitro simulated gastric and pancreatic digestion. However, dialyzability from milk was enhanced by the addition of a more concentrated lyophilized peptide fraction.

  10. Differential modulation of enterocyte-like Caco-2 cells after exposure to short-chain fatty acids

    NARCIS (Netherlands)

    Malago, J.J.; Koninkx, J.F.J.G.; Douma, P.M.; Dirkzwager, A.; Veldman, K.T.; Hendriks, H.G.C.J.M.; Dijk, van J.E.

    2003-01-01

    The response of intestinal epithelial cells to short-chain fatty acids, which are increasingly used as food additives, was investigated. Human small intestinal epithelial cell model Caco-2 cells were exposed to formate, propionate and butyrate to assess their effect on cellular growth, metabolism, d

  11. Identification of TNF-alpha-Responsive Promoters and Enhancers in the Intestinal Epithelial Cell Model Caco-2

    DEFF Research Database (Denmark)

    Boyd, Mette; Coskun, Mehmet; Lilje, Berit;

    2014-01-01

    promoters. As a case example, we characterize an enhancer regulating the laminin-5 γ2-chain (LAMC2) gene by nuclear factor (NF)-κB binding. This report is the first to present comprehensive TSS and enhancer maps over Caco-2 cells, and highlights many novel inflammation-specific promoters and enhancers....... genome-wide maps of active transcription start sites (TSSs), and active enhancers in Caco-2 cells with or without tumour necrosis factor (TNF)-α stimulation to mimic an inflammatory state. We found 520 promoters that significantly changed their usage level upon TNF-α stimulation; of these, 52...

  12. Influence of charge on FITC-BSA-loaded chondroitin sulfate-chitosan nanoparticles upon cell uptake in human Caco-2 cell monolayers

    Directory of Open Access Journals (Sweden)

    Hu CS

    2012-09-01

    Full Text Available Chieh-shen Hu,1 Chiao-hsi Chiang,2 Po-da Hong,1,4,* Ming-kung Yeh1–3,*1Biomedical Engineering Program, Graduate Institute of Applied Science and Technology, National Taiwan University of Science and Technology; 2School of Pharmacy, National Defence Medical Center; 3Bureau of Pharmaceutical Affairs, Ministry of National Defence Medical Affairs Bureau; 4Department of Materials Science and Engineering, National Taiwan University of Science and Technology, Taiwan, Republic of China*These authors contributed equally to this workBackground and methods: Chondroitin sulfate-chitosan (ChS-CS nanoparticles and positively and negatively charged fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA-loaded ChS-CS nanoparticles were prepared and characterized. The properties of ChS-CS nanoparticles, including cellular uptake, cytotoxicity, and transepithelial transport, as well as findings on field emission-scanning electron microscopy, transmission electron microscopy, and confocal laser scanning microscopy were evaluated in human epithelial colorectal adenocarcinoma (Caco-2 fibroblasts. ChS-CS nanoparticles with a mean particle size of 250 nm and zeta potentials ranging from –30 to +18 mV were prepared using an ionic gelation method.Results: Standard cell viability assays demonstrated that cells incubated with ChS-CS and FITC-BSA-loaded ChS-CS nanoparticles remained more than 95% viable at particle concentrations up to 0.1 mg/mL. Endocytosis of nanoparticles was confirmed by confocal laser scanning microscopy and measured by flow cytometry. Ex vivo transepithelial transport studies using Caco-2 cells indicated that the nanoparticles were effectively transported into Caco-2 cells via endocytosis. The uptake of positively charged FITC-BSA-loaded ChS-CS nanoparticles across the epithelial membrane was more efficient than that of the negatively charged nanoparticles.Conclusion: The ChS-CS nanoparticles fabricated in this study were

  13. Curcumin inhibits the proliferation of a human colorectal cancer cell line Caco-2 partially by both apoptosis and G2/M cell cycle arrest

    Directory of Open Access Journals (Sweden)

    Yohko Fujimoto

    2014-06-01

    Full Text Available The aim of this study was to assess the possible roles of the phytochemical compounds, curcumin, quercetin and resveratrol in the proliferation of human colorectal cancer cell line Caco-2. All three phytochemical compounds inhibited Caco-2 cell proliferation, with curcumin being more effective than quercetin and resveratrol. Investigations concerning DNA fragmentation in the nucleus, Bax and Bcl-2 mRNA expression levels, and caspase-3/7 activity indicated that curcumin induced apoptosis in Caco-2 cells through an increase in the Bax/Bcl-2 ratio and activation of caspase-3/7. Furthermore, the analysis of flow-cytometry showed that curcumin caused an arrest of G2/M phase in Caco-2 cells. These results suggest that curcumin suppresses Caco-2 proliferation partially via activation of the mitochondrial apoptotic pathway and cell cycle retardation.

  14. Availability and toxicity of Fe(Ⅱ) and Fe(Ⅲ) in Caco-2 cells

    Institute of Scientific and Technical Information of China (English)

    Wan-ling HE; Ying FENG; Xiao-li LI; Yan-yan WEI; Xiao-e YANG

    2008-01-01

    The objective of the present study was to compare the toxicity and availability of Fe(Ⅱ) and Fe(Ⅲ) to Caco-2 cells.Cellular damage was studied by measuring cell proliferation and lactate dehydrogenase (LDH) release. The activities of two major antioxidative enzymes [superoxide dismutase (SOD) and glutathione peroxidase (GPx)] and differentiation marker (alkaline phosphatase) were determined after the cells were exposed to different levels of iron salts. The cellular iron concentration was investigated to evaluate iron bioavailability. The results show that iron uptake of the cells treated with Fe(Ⅱ) is significantly higher than that of the cells treated with Fe(Ⅲ) (P<0.05). Fe(Ⅱ) at a concentration>1.5 mmol/L was found to be more effective in reducing cellular viability than Fe(Ⅲ). LDH release investigation suggests that Fe(Ⅱ) can reduce stability of the cell membrane. The activities of SOD and GPx of the cells treated with Fe(Ⅱ) were higher than those of the cells treated with Fe(Ⅲ), although both of them increased with raising iron supply levels. The results indicate that both Fe(Ⅱ) and Fe(Ⅲ) could reduce the cellular antioxidase gene expression at high levels.

  15. Polarized secretion of newly synthesized lipoproteins by the Caco-2 human intestinal cell line.

    Science.gov (United States)

    Traber, M G; Kayden, H J; Rindler, M J

    1987-11-01

    Lipoprotein secretion by Caco-2 cells, a human intestinal cell line, was studied in cells grown on inserts containing a Millipore filter (0.45 micron), separating secretory products from the apical and basolateral membranes into separate chambers. Under these conditions, as observed by electron microscopy, the cells formed a monolayer of columnar epithelial cells with microvilli on the apical surface and tight junctions between cells. The electrical resistances of the cell monolayers were 250-500 ohms/cm2. Both 14C-labeled lipids and 35S-labeled proteins were used to assess lipoprotein secretion. After a 24-hr incubation with [14C]oleic acid, 60-80% of the secreted triglyceride (TG) was in the basolateral chamber; 40% of the TG was present in the d less than 1.006 g/ml (chylomicron + VLDL) fraction and 50% in the 1.006 less than d less than 1.063 g/ml (LDL) fraction. After a 4-hr incubation with [35S]methionine, apolipoproteins were found to be major secretory products with 75-100% secreted to the basolateral chamber. Apolipoproteins B-100, B-48, E, A-I, A-IV, and C-III were identified by immunoprecipitation. The d less than 1.006 g/ml fraction was found to contain all of the major apolipoproteins, while the LDL fraction contained primarily apoB-100 and apoE; the HDL (1.063 less than d less than 1.21 g/ml) fraction principally contained apoA-I and apoA-IV. Mn-heparin precipitated all of the [35S]methionine-labeled apoB-100 and B-48 and a majority of the other apolipoproteins, and 80% of the [14C]oleic acid-labeled triglyceride, but only 15% of the phospholipid, demonstrating that Caco-2 cells secrete triglyceride-rich lipoproteins containing apoB. Secretion of lipoproteins was dependent on the lipid content of the medium; prior incubation with lipoprotein-depleted serum specifically reduced the secretion of lipoproteins, while addition of both LDL and oleic acid to the medium maintained the level of apoB-100, B-48, and A-IV secretion to that observed in the control

  16. 盐酸度洛西汀对Caco-2细胞P-糖蛋白功能及表达的影响%Effects of duloxetine on p-glycoprotein function and expression in Caco-2 cells

    Institute of Scientific and Technical Information of China (English)

    陈芳; 赵瑞柯

    2013-01-01

    Objective To investigate the effects of duloxetine(DLX) on P-glycoprotein(P-gp) function and expression in Caco-2 cells. Methods Caco-2 cells were incubated in vitro and divided into three groups of A( negative controls) .B( treated with verapamil 10 μmol/L as positive controls) and C (treated with different concentrations of DLX 0.2, 5, 10 μmol/L). The fluorescence intensity of intracellular rhodamine-123 and P-gp expression in cell membrane after DLX intervention were detected by flow cytometry. Results Compared with group A, DLX 0. 2, 5,10 μmol/L significantly inhibited the efflux of rhodamine-123 from Caco-2 cells(96. 3 ± 4. 5 vs. 129. 5 ± 14. 5, 131. 2± 7. 0, 135. 6 ± 17. l)(P0. 05). Conclusion DLX can inhibit P-gp function and decrease the efflux of rhodamine-123, but does not affect P-gp expression.%目的 研究盐酸度洛西汀(DLX)对Caco-2细胞上P-糖蛋白(P-gp)功能和表达的影响.方法 将Caco-2细胞分为阴性对照组、维拉帕米10 μmol/L阳性对照组(VER组)和DLX 0.2、5、10 μmol/L不同浓度处理组.采用流式细胞术检测DLX干预后Caco-2细胞内罗丹明-123的荧光强度和细胞膜上P-gP的表达量.结果 与阴性对照组相比,DLX 0.2、5、10 μmol/L均显著抑制罗丹明-123从Caco-2细胞的外排(96.3±4.5 vs.129.5±14.5、131.2±7.0、135.6±17.1)(P<0.05),而对P-gp阳性率无明显改变(90% vs.94%、91%、89%)(P>0.05).结论 DLX能抑制Caco-2细胞上P-gp的功能,降低罗丹明-123从Caco-2细胞中的外排,但并不影响P-gp的表达.

  17. Absorption Mechanism of Palmatine Chloride Across Caco-2 Cell Monolayer Mode%盐酸巴马汀在Caco-2细胞中的吸收机制

    Institute of Scientific and Technical Information of China (English)

    谢社平; 谭晓婧; 毕开顺; 张蕾; 廖琼峰

    2011-01-01

    Objective: To investigate the absorption mechanism of palmatine chloride by using Caco-2 monolayer model. Method: A Caco-2 cell monolayer model was used to investigate the bi-directional transport of palmatine chloride. The effect of time, drug concentration, temperature, pH and inhibitors on the absorption of palmatine chloride was studied. Apparent permeability coefficient (Papp) of palmatine was calculated based on quantitation by HPLC. Result: Directional flux studies with palmatine chloride suggested transport with the permeability in the apical to basolateral direction higher than that in the basolateral to apical direction. Transport of palmatine chloride could be of saturation with time and concentration, and it was also effected by P-glycoprotein inhibitor, pH and temperature. Conclusion: The absorption of palmatine chloride in Caco-2 cell model was an active transportation mediated by transporter, which mainly located in the apical side of Caco-2 cell monolayer.%目的:研究盐酸巴马汀在Caco-2细胞模型中的吸收机制.方法:用Caco-2细胞单层模型研究盐酸巴马汀的双向转运,并考察时间、药物浓度、抑制剂、pH及温度对盐酸巴马汀吸收的影响.用高效液相色谱法检测药物浓度,计算其表观渗透系数.结果:盐酸巴马汀在Caco-2细胞模型中,从单层细胞层顶端到基底端的转运大于基底端到顶端的转运,随时间和浓度的增加,药物吸收呈饱和趋势,且P-糖蛋白(P-glycoprotein,P-gp)抑制剂维拉帕米、pH和温度对它的转运有影响.结论:盐酸巴马汀在Caco-2细胞模型中的吸收主要是由载体介导的主动转运,且该主动转运的载体位于Caco-2细胞单层的顶端.

  18. Caco-2 cells cytotoxicity of nifuroxazide derivatives with potential activity against Methicillin-resistant Staphylococcus aureus (MRSA).

    Science.gov (United States)

    Fernandes, Mariane B; Gonçalves, José E; Scotti, Marcus T; de Oliveira, Alex A; Tavares, Leoberto C; Storpirtis, Sílvia

    2012-04-01

    It is important to determine the toxicity of compounds and co-solvents that are used in cell monolayer permeability studies to increase confidence in the results obtained from these in vitro experiments. This study was designed to evaluate the cytotoxicity of new nifuroxazide derivatives with potential activity against Methicillin-resistant Staphylococcus aureus (MRSA) in Caco-2 cells to select analogues for further in vitro permeability analyses. In this study, nitrofurantoin and nifuroxazide, in addition to 6 furanic and 6 thiophenic nifuroxazide derivatives were tested at 2, 4, 6, 8 and 10 μg/mL. In vitro cytotoxicity assays were performed according to the MTT (methyl tetrazolium) assay protocol described in ISO 10993-5. The viability of treated Caco-2 cells was greater than 83% for all tested nitrofurantoin concentrations, while those treated with nifuroxazide at 2, 4 and 6 μg/mL had viabilities greater than 70%. Treatment with the nifuroxazide analogues resulted in viability values greater than 70% at 2 and 4 μg/mL with the exception of the thiophenic methyl-substituted derivative, which resulted in cell viabilities below 70% at all tested concentrations. Caco-2 cells demonstrated reasonable viability for all nifuroxazide derivatives, except the thiophenic methyl-substituted compound. The former were selected for further permeability studies using Caco-2 cells.

  19. Effect of heat stress on intestinal barrier function of human intestinal epithelial Caco-2 cells

    Directory of Open Access Journals (Sweden)

    Gui-zhen XIAO

    2013-07-01

    Full Text Available Objective To investigate the heat stress-induced dysfunction of intestinal barrier including intestinal tight junction and apoptosis of epithelial cells. Methods Human intestinal epithelial Caco-2 cell monolayers, serving as the intestinal barrier model, were exposed to different temperature (37-43℃ for designated time. Transepithelial electrical resistance (TEER and horseradish peroxidase (HRP flux permeability were measured to evaluate barrier integrity. Level of tight junction (TJ protein occludin was analyzed by Western blotting. Cell apoptosis rate was determined using Annexin V-FITC/PI kit by flow cytometry. Results Compared with the 37℃ group, TEER lowered and the permeability for HRP increased significantly after heat exposure (P<0.01 in 39℃, 41℃ and 43℃ groups. The expression of occludin increased when the temperature was elevated from 37℃ to 41℃, and it reached the maximal level at 41℃. However, its expression gradually decreased with passage of time at 43℃. Cell apoptosis was enhanced with elevation of the temperature (P<0.05 or P<0.01. Conclusion Heat stress can induce damage to tight junction and enhance apoptosis of epithelial cells, thus causing dysfunction of intestinal epithelial barrier.

  20. Interaction of citrinin and resveratrol and their effect on Caco-2 cell growth

    Directory of Open Access Journals (Sweden)

    Ivana BOVDISOVA

    2016-12-01

    Full Text Available The aim of this study was to use Caco-2 cells as an in vitro model of human intestinal barrier. This model was employed to investigate interaction of citrinin and resveratrol and to determine the doses that affect cell number. Citrinin (CTN is a toxic secondary metabolite produced by fungi of the genera Penicillium, Aspergillus and Monascus. It is contaminant of cereals, grains, food and feed products. Previous studies have shown that CTN has teratogenic, nephrotoxic, hepatotoxic and embryotoxic effects. Resveratrol (RES is polyphenol belongs to the group of stilbenes. At lower doses it has positive effects on human health and higher doses may induce apoptosis. The results of study indicate a significant (P < 0.05 decrease in cell number in all conditions: A (RES 20 μM, B (CTN 100 μg*ml-1, C (CTN 250 μg*ml-1, D (CTN 100 μg*ml-1 + RES 20 μM, E (CTN 250 μg*ml-1 + RES 20 μM in comparison with the control group of non-treated cells (NT and control group with ethanol (Ce. A significant (P <0.05 decrease in the cells number was observed between groups A - E, B - C and D - E. Citrinin effect seems to be dose-dependent.

  1. Hypertonic stress induces VEGF production in human colon cancer cell line Caco-2: inhibitory role of autocrine PGE₂.

    Directory of Open Access Journals (Sweden)

    Luciana B Gentile

    Full Text Available Vascular Endothelial Growth Factor (VEGF is a major regulator of angiogenesis. VEGF expression is up regulated in response to micro-environmental cues related to poor blood supply such as hypoxia. However, regulation of VEGF expression in cancer cells is not limited to the stress response due to increased volume of the tumor mass. Lipid mediators in particular arachidonic acid-derived prostaglandin (PGE₂ are regulators of VEGF expression and angiogenesis in colon cancer. In addition, increased osmolarity that is generated during colonic water absorption and feces consolidation seems to activate colon cancer cells and promote PGE₂ generation. Such physiological stimulation may provide signaling for cancer promotion. Here we investigated the effect of exposure to a hypertonic medium, to emulate colonic environment, on VEGF production by colon cancer cells. The role of concomitant PGE₂ generation and MAPK activation was addressed by specific pharmacological inhibition. Human colon cancer cell line Caco-2 exposed to a hypertonic environment responded with marked VEGF and PGE₂ production. VEGF production was inhibited by selective inhibitors of ERK 1/2 and p38 MAPK pathways. To address the regulatory role of PGE₂ on VEGF production, Caco-2 cells were treated with cPLA₂ (ATK and COX-2 (NS-398 inhibitors, that completely block PGE₂ generation. The Caco-2 cells were also treated with a non selective PGE₂ receptor antagonist. Each treatment significantly increased the hypertonic stress-induced VEGF production. Moreover, addition of PGE₂ or selective EP₂ receptor agonist to activated Caco-2 cells inhibited VEGF production. The autocrine inhibitory role for PGE₂ appears to be selective to hypertonic environment since VEGF production induced by exposure to CoCl₂ was decreased by inhibition of concomitant PGE₂ generation. Our results indicated that hypertonicity stimulates VEGF production in colon cancer cell lines. Also PGE

  2. Protective effect of polyphenols from Glycyrrhiza glabra against oxidative stress in Caco-2 cells.

    Science.gov (United States)

    D'Angelo, Stefania; Morana, Alessandra; Salvatore, Anna; Zappia, Vincenzo; Galletti, Patrizia

    2009-12-01

    In the present article, we have investigated the antioxidant properties of methanolic liquorice polyphenol extracts (LPE(s)). Polyphenol extraction was performed with 60% and 100% methanol. Analysis of LPE(s) by thin-layer chromatography revealed that a higher amount of polyphenols was recovered by extraction with 60% methanol. Antioxidant activity measurement of the reducing power, scavenging effect on 2,2'-diphenyl-1-picrylhydrazyl free radical, and hydrogen peroxide scavenging capability have been taken as the parameters for assessment of antioxidant potential of LPE(s). Results have been compared with both natural and synthetic antioxidants. All experimental data have indicated that LPE(s) possess strong antioxidant power proportional to their o-diphenolic and total polyphenolic content, independently from the assay used. Therefore, the LPE(s) antioxidant property was examined against the cytotoxic effects of reactive oxygen species in human colon carcinoma cells. Pretreatment of Caco-2 cells with liquorice polyphenolic extracts provided a remarkable protection against oxidative damage induced by H(2)O(2). The highest oxidative stress protection (72% of cell vitality) was measured in cells pretreated with 0.54 mM polyphenols. This effect seems to be associated to the antioxidant activity of liquorice polyphenolic compounds. Our data suggest that polyphenols from Glycyrrhiza glabra could exert a beneficial action in the prevention of intestinal pathologies related to production of reactive oxygen species.

  3. Effects of red wine on ochratoxin A toxicity in intestinal Caco-2/TC7 cells.

    Science.gov (United States)

    Ranaldi, G; Mancini, E; Ferruzza, S; Sambuy, Y; Perozzi, G

    2007-03-01

    Ochratoxin A (OTA) is found in a variety of foods and beverages, including red wine. OTA was reported to be nephrotoxic, immunotoxic, hepatotoxic and a potential carcinogen, with yet uncharacterized mechanisms. Consumption of contaminated wines might contribute up to 13% of OTA daily human intake. Potentially chronic exposure has therefore raised public health concern. OTA toxicity in the presence of de-alcoholated red wine was investigated in human intestinal Caco-2/TC7 cells, differentiated on filter supports, by measuring tight junction (TJ) permeability, morphological alterations of TJ proteins and occurrence of apoptosis. Cells were treated with OTA, in the presence of de-alcoholated red wine, for 48h and the ability to recover from the effects of OTA was evaluated after 24h in complete medium. OTA treatment increased TJ permeability and caused intracellular redistribution of claudin-4. However, cells were able to restore permeability and correct localization of claudin-4 following 24h recovery. Conversely, in the presence of red wine, OTA produced faster and irreversible increase in TJ permeability, intracellular delocalization of claudin-4 and extensive apoptosis. Our results point at a possible synergy between OTA and some red wine components, such as polyphenols, in the induction of apoptotic cell death.

  4. In vitro digestion and lactase treatment influence uptake of quercetin and quercetin glucoside by the Caco-2 cell monolayer

    Directory of Open Access Journals (Sweden)

    Brown Dan

    2005-01-01

    Full Text Available Abstract Background Quercetin and quercetin glycosides are widely consumed flavonoids found in many fruits and vegetables. These compounds have a wide range of potential health benefits, and understanding the bioavailability of flavonoids from foods is becoming increasingly important. Methods This study combined an in vitro digestion, a lactase treatment and the Caco-2 cell model to examine quercetin and quercetin glucoside uptake from shallot and apple homogenates. Results The in vitro digestion alone significantly decreased quercetin aglycone recovery from the shallot digestate (p p > 0.05. Digestion increased the Caco-2 cell uptake of shallot quercetin-4'-glucoside by 2-fold when compared to the non-digested shallot. Despite the loss of quercetin from the digested shallot, the bioavailability of quercetin aglycone to the Caco-2 cells was the same in both the digested and non-digested shallot. Treatment with lactase increased quercetin recovery from the shallot digestate nearly 10-fold and decreased quercetin-4'-glucoside recovery by more than 100-fold (p p Conclusions The increase in quercetin uptake following treatment with lactase suggests that dietary supplementation with lactase may increase quercetin bioavailability in lactose intolerant humans. Combining the digestion, the lactase treatment and the Caco-2 cell culture model may provide a reliable in vitro model for examining flavonoid glucoside bioavailability from foods.

  5. Effects of saturated, mono-, and polyunsaturated fatty acids on the secretion of apo B containing lipoproteins by Caco-2 cells

    NARCIS (Netherlands)

    van Greevenbroek, M.M.J.; van Meer, G.; Erkelens, D.W.; de Bruin, T.W.A.

    1996-01-01

    We studied the effects of addition of physiological concentrations (0.5 mM) of fatty acids i.e., palmitic (16:0), stearic (18:0), oleic (18:1) and linoleic acid (18:2) on lipoprotein secretion by polarized Caco-2 cells. With saturated fatty acids, secreted lipoproteins were at IDL/LDL density, 1.009

  6. Listeria monocytogenes efficiently invades caco-2 cells after low-temperature storage in broth and on deli meat

    DEFF Research Database (Denmark)

    Larsen, Marianne Halberg; Koch, Anette Granly; Ingmer, Hanne

    2010-01-01

    The objective of this study was to investigate how various growth conditions influence the virulence of Listeria monocytogenes monitored by its ability to invade the epithelial cell lines Caco-2 and INT-407. The growth conditions examined were modified atmosphere-packaged deli meat and brain heart...

  7. The Potential Health Benefits of Polyphenol-Rich Extracts from Cichorium intybus L. Studied on Caco-2 Cells Model.

    Science.gov (United States)

    Azzini, Elena; Maiani, Giuseppe; Garaguso, Ivana; Polito, Angela; Foddai, Maria S; Venneria, Eugenia; Durazzo, Alessandra; Intorre, Federica; Palomba, Lara; Rauseo, Maria L; Lombardi-Boccia, Ginevra; Nobili, Fabio

    2016-01-01

    Phytochemicals can exert their bioactivity without reaching the systemic circulation; scarcely absorbed antioxidants might reach the large bowel contributing to protection from oxidative damage-induced gastrointestinal diseases. In the present work, we aimed to study the relationship between potential activity of polyphenol-rich extracts from Cichorium intybus L. and changes in morphological characteristics on Caco-2 cells. Phytochemicals content (carotenoids and flavonoids) and total antioxidant activity of Red Chicory of Treviso and Variegated Chicory of Castelfranco were evaluated. The bioactivity of polyphenol-rich extracts from chicories was studied in in vitro Caco-2 cell monolayers model. Morphological characteristics changes to test the antioxidant and/or prooxidant effect were verified by histological analysis and observed by Electronic Scansion Microscopy (SEM). On Caco-2 cell model, the polyphenols fractions from chicories have indicated a moderate antioxidant behavior until 17 μM concentration, while 70 μM and 34 μM exert cytotoxic effects for Treviso's and Castelfranco's Chicory, respectively, highlighted by TEER decreasing, increased permeability, and alteration of epithelium. Our findings support the beneficial effects of these products in counteracting the oxidative stress and cellular damage, induced in vitro on Caco-2 cell model, through interaction with the mucopolysaccharide complexes in the glycocalyx, maintaining in vivo a healthy and effective intestinal barrier.

  8. Availability of polychlorinated biphenyls (PCBs) and lindane for uptake by intestinal Caco-2 cells.

    Science.gov (United States)

    Oomen, A G; Tolls, J; Kruidenier, M; Bosgra, S S; Sips, A J; Groten, J P

    2001-07-01

    Children may ingest contaminated soil from hand to mouth. To assess this exposure route, we need to know the oral bioavailability of the contaminants. Two determining steps in bioavailability of soil-borne contaminants are mobilization from soil during digestion, which is followed by intestinal absorption. The first step has been investigated in previous studies that showed that a substantial fraction of PCBs and lindane is mobilized from soil during artificial digestion. Furthermore, almost all contaminants are sorbed to constituents of artificial human small intestinal fluid (i.e., chyme), whereas only a small fraction is freely dissolved. In this study, we examine the second step using intestinal epithelial Caco-2 cells. The composition of the apical exposure medium was varied by addition of artificial chyme, bile, or oleic acid at similar or increasing total contaminant concentrations. The uptake curves were described by rate constants. The uptake flux seemed to be dose-dependent. Furthermore, different exposure media with similar total contaminant concentrations resulted in various uptake rates. This can be attributed to different freely dissolved concentrations and carrier effects. In addition, the large fractions of contaminants in the cells indicate that PCBs and lindane sorbed to bile, oleic acid, and digestive proteins contributed to the uptake flux toward the cells. These results can be extrapolated qualitatively to in vivo conditions. Because the sorbed contaminants should be considered available for absorption, the first step of mobilization from soil is the most important step for oral bioavailability of the presently investigated soil-borne contaminants.

  9. Regulation of Intestinal Epithelial Calcium Transport Proteins by Stanniocalcin-1 in Caco2 Cells

    Directory of Open Access Journals (Sweden)

    Jinmei Xiang

    2016-07-01

    Full Text Available Stanniocalcin-1 (STC1 is a calcium and phosphate regulatory hormone. However, the exact molecular mechanisms underlying how STC1 affects Ca2+ uptake remain unclear. Here, the expression levels of the calcium transport proteins involved in transcellular transport in Caco2 cells were examined following over-expression or inhibition of STC1. These proteins include the transient receptor potential vanilloid members (TRPV 5 and 6, the plasma membrane calcium ATPase 1b (PMCA1b, the sodium/calcium exchanger (NCX1, and the vitamin D receptor (VDR. Both gene and protein expressions of TRPV5 and TRPV6 were attenuated in response to over-expression of STC1, and the opposite trend was observed in cells treated with siRNASTC1. To further investigate the ability of STC1 to influence TRPV6 expression, cells were treated with 100 ng/mL of recombinant human STC1 (rhSTC1 for 4 h following pre-transfection with siRNASTC1 for 48 h. Intriguingly, the increase in the expression of TRPV6 resulting from siRNASTC1 was reversed by rhSTC1. No significant effect of STC1 on the expression of PMCA1b, NCX1 or VDR was observed in this study. In conclusion, the effect of STC1 on calcium transport in intestinal epithelia is due to, at least in part, its negative regulation of the epithelial channels TRPV5/6 that mediate calcium influx.

  10. Among plant lignans, pinoresinol has the strongest antiinflammatory properties in human intestinal Caco-2 cells.

    Science.gov (United States)

    During, Alexandrine; Debouche, Céline; Raas, Thomas; Larondelle, Yvan

    2012-10-01

    Dietary lignans show some promising health benefits, but little is known about their fate and activities in the small intestine. The purpose of this study was thus to investigate whether plant lignans are taken up by intestinal cells and modulate the intestinal inflammatory response using the Caco-2 cell model. Six lignan standards [secoisolariciresinol diglucoside (SDG), secoisolariciresinol (SECO), pinoresinol (PINO), lariciresinol, matairesinol (MAT), and hydroxymatairesinol] and their colonic metabolites [enterolactone (ENL) and enterodiol] were studied. First, differentiated cells were exposed to SDG, SECO, PINO, or ENL at increasing concentrations for 4 h, and their cellular contents (before and after deconjugation) were determined by HPLC. Second, in IL-1β-stimulated confluent and/or differentiated cells, lignan effects were tested on different soluble proinflammatory mediators quantified by enzyme immunoassays and on the NF-κB activation pathway by using cells transiently transfected. SECO, PINO, and ENL, but not SDG, were taken up and partly conjugated by cells, which is a saturable conjugation process. PINO was the most efficiently conjugated (75% of total in cells). In inflamed cells, PINO significantly reduced IL-6 by 65% and 30% in confluent and differentiated cells, respectively, and cyclooxygenase (COX)-2-derived prostaglandin E(2) by 62% in confluent cells. In contrast, MAT increased significantly COX-2-derived prostaglandin E(2) in confluent cells. Moreover, PINO dose-dependently decreased IL-6 and macrophage chemoattractant protein-1 secretions and NF-κB activity. Our findings suggest that plant lignans can be absorbed and metabolized in the small intestine and, among the plant lignans tested, PINO exhibited the strongest antiinflammatory properties by acting on the NF-κB signaling pathway, possibly in relation to its furofuran structure and/or its intestinal metabolism.

  11. Effects of extracellular iron concentration on calcium absorption and relationship between Ca2+ and cell apoptosis in Caco-2 cells

    Institute of Scientific and Technical Information of China (English)

    Li Wang; Qing Li; Xiang-Lin Duan; Yan-Zhong Chang

    2005-01-01

    AIM: To determine the method of growing small intestinal epithelial cells in short-term primary culture and to investigate the effect of extracellular iron concentration ([Fe3+]) on calcium absorption and the relationship between the rising intracellular calcium concentration ([Ca2+]i) and cell apoptosis in human intestinal epithelial Caco-2 cells. METHODS: Primary culture was used for growing small intestinal epithelial cells. [Ca2+]i was detected by a confocal laser scanning microscope. The changes in [Ca2+]i were represented by fluorescence intensity (FI). The apoptosis was evaluated by flow cytometry.RESULTS: Isolation of epithelial cells and preservation of its three-dimensional integrity were achieved using the digestion technique of a mixture of collagenase Ⅺ and dispase Ⅰ. Purification of the epithelial cells was facilitated by using a simple differential sedimentation method. The results showed that proliferation of normal gut epithelium in vitro was initially dependent upon the maintenance of structural integrity of the tissue. If 0.25% trypsin was used for digestion, the cells were severely damaged and very difficult to stick to the Petri dish for growing. The Fe3+ chelating agent desferrioxamine (100, 200 and 300 μmol/L) increased the FI of Caco-2 cells from 27.50±13.18 (control,n = 150) to 35.71±13.99 (n = 150, P<0.01), 72.19±35.40 (n = 150, P<0.01) and 211.34±29.03 (n = 150, P<0.01) in a concentration-dependent manner. There was a significant decrease in the FI of Caco-2 cells treated by ferric ammonium citrate (FAC, a Fe3+ donor; 10, 50 and 100 μmol/L). The FIvalue of Caco-2 cells treated by FAC was 185.85±33.77 (n = 150, P<0.01), 122.73±58.47 (n = 150, P<0.01), and 53.29±19.82 (n = 150, P<0.01), respectively, suggesting that calcium absorption was influenced by [Fe3+]. Calcium ionophore A23187 (0.1, 1.0 and 10 μmol/L) increased the FI of Caco-2 cells from 40.45±13.95 (control, n = 150) to 45.19±21.95 (n = 150, P<0

  12. Vitamin D Receptor-Mediated Upregulation of CYP3A4 and MDR1 by Quercetin in Caco-2 cells.

    Science.gov (United States)

    Chae, Yoon-Jee; Cho, Kwan Hyung; Yoon, In-Soo; Noh, Chi-Kyoung; Lee, Hyo-Jong; Park, Yohan; Ji, Eunhee; Seo, Min-Duk; Maeng, Han-Joo

    2016-01-01

    To examine whether quercetin interacts with vitamin D receptor, we investigated the effects of quercetin on vitamin D receptor activity in human intestinal Caco-2 cells. The effects of quercetin on the expression of the vitamin D receptor target genes, vitamin D3 24-hydroxylase, cytochrome P450 3A4, multidrug resistance protein 1, and transient receptor potential vanilloid type 6 were measured using quantitative polymerase chain reaction. The vitamin D receptor siRNA was used to assess the involvement of the vitamin D receptor. Vitamin D receptor activation using a vitamin D responsive element-mediated cytochrome P450 3A4 reporter gene assay was investigated in Caco-2 cells transfected with human vitamin D receptor. We also studied the magnitude of the vitamin D receptor activation and/or synergism between 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] and quercetin-like flavonoids. Slight but significant increases in the mRNA expression of cytochrome P450 3A4, vitamin D3 24-hydroxylase, multidrug resistance protein 1, and transient receptor potential vanilloid type 6 were observed after 3 days of continual quercetin treatment. The silencing effect of vitamin D receptor by vitamin D receptor siRNA in Caco-2 cells significantly attenuated the induction of the vitamin D receptor target genes. Moreover, quercetin significantly enhanced cytochrome P450 3A4 reporter activity in Caco-2 cells in a dose-dependent manner, and the expression of exogenous vitamin D receptor further stimulated the vitamin D receptor activity. Quercetin-like flavonoids such as kaempferol stimulated the vitamin D receptor activity in a manner similar to that seen with quercetin. Taken together, the data indicates that quercetin upregulates cytochrome P450 3A4 and multidrug resistance protein 1 expression in Caco-2 cells likely via a vitamin D receptor-dependent pathway.

  13. Gastric digestion of pea ferritin and modulation of its iron bioavailability by ascorbic and phytic acids in caco-2 cells

    Institute of Scientific and Technical Information of China (English)

    Satyanarayana Bejjani; Raghu Pullakhandam; Ravinder Punjal; K Madhavan Nair

    2007-01-01

    AIM: To understand the digestive stability and mechanism of release and intestinal uptake of pea ferritin iron in caco-2 cell line model.METHODS: Pea seed ferritin was purified using salt fractionation followed by gel filtration chromatography.The bioavailability of ferritin iron was assessed using coupled in vitro digestion/Caco-2 cell model in the presence or absence of ascorbic acid and phytic acid.Caco-2 cell ferritin formation was used as a surrogate marker of iron uptake. Structural changes of pea ferritin under simulated gastric pH were characterized using electrophoresis, gel filtration and circular dichroism spectroscopy.RESULTS: The caco-2 cell ferritin formation was significantly increased (P < 0.001) with FeSO4 (19.3±9.8 ng/mg protein) and pea ferritin (13.9 ± 6.19 ng/mg protein) compared to the blank digest (3.7 ± 1.8 ng/mg protein). Ascorbic acid enhanced while phytic acid decreased the pea ferritin iron bioavailability. However,either in the presence or absence of ascorbic acid, the ferritin content of caco-2 cells was significantly less with pea ferritin than with FeSO4. At gastric pH, no band corresponding to ferritin was observed in the presence of pepsin either on native PAGE or SDS-PAGE. Gel filtration chromatography and circular dichroism spectroscopy revealed a pH dependent loss of quaternary and secondary structure.CONCLUSION: Under gastric conditions, the iron core of pea ferritin is released into the digestive medium due to acid induced structural alterations and dissociation of protein. The released iron interacts with dietary factors leading to modulation of pea ferritin iron bioavailability,resembling the typical characteristics of non-heme iron.

  14. In Situ Perfusion Model in Rat Colon for Drug Absorption Studies: Comparison with Small Intestine and Caco-2 Cell Model.

    Science.gov (United States)

    Lozoya-Agullo, Isabel; González-Álvarez, Isabel; González-Álvarez, Marta; Merino-Sanjuán, Matilde; Bermejo, Marival

    2015-09-01

    Our aim is to develop and to validate the in situ closed loop perfusion method in rat colon and to compare with small intestine and Caco-2 cell models. Correlations with human oral fraction absorbed (Fa) and human colon fraction absorbed (Fa_colon) were developed to check the applicability of the rat colon model for controlled release (CR) drug screening. Sixteen model drugs were selected and their permeabilities assessed in rat small intestine and colon, and in Caco-2 monolayers. Correlations between colon/intestine/Caco-2 permeabilities versus human Fa and human Fa_colon have been explored to check model predictability and to apply a BCS approach in order to propose a cut off value for CR screening. Rat intestine perfusion with Doluisio's method and single-pass technique provided a similar range of permeabilities demonstrating the possibility of combining data from different laboratories. Rat colon permeability was well correlated with Caco-2 cell-4 days model reflecting a higher paracellular permeability. Rat colon permeabilities were also higher than human colon ones. In spite of the magnitude differences, a good sigmoidal relationship has been shown between rat colon permeabilities and human colon fractions absorbed, indicating that rat colon perfusion can be used for compound classification and screening of CR candidates.

  15. A mucus adhesion promoting protein, MapA, mediates the adhesion of Lactobacillus reuteri to Caco-2 human intestinal epithelial cells.

    Science.gov (United States)

    Miyoshi, Yukihiro; Okada, Sanae; Uchimura, Tai; Satoh, Eiichi

    2006-07-01

    Lactobacillus reuteri is one of the dominant lactobacilli found in the gastrointestinal tract of various animals. A surface protein of L. reuteri 104R, mucus adhesion promoting protein (MapA), is considered to be an adhesion factor of this strain. We investigated the relation between MapA and adhesion of L. reuteri to human intestinal (Caco-2) cells. Quantitative analysis of the adhesion of L. reuteri strains to Caco-2 cells showed that various L. reuteri strains bind not only to mucus but also to intestinal epithelial cells. In addition, purified MapA bound to Caco-2 cells, and this binding inhibited the adhesion of L. reuteri in a concentration-dependent manner. Based on these observations, the adhesion of L. reuteri appears due to the binding of MapA to receptor-like molecules on Caco-2 cells. Further, far-western analysis indicated the existence of multiple receptor-like molecules in Caco-2 cells.

  16. Transepithelial transport of ambroxol hydrochloride across human intestinal Caco-2 cell monolayers.

    Science.gov (United States)

    Stetinová, Vera; Smetanová, Libuse; Kholová, Dagmar; Svoboda, Zbynek; Kvetina, Jaroslav

    2009-09-01

    This study aimed i) to characterize the transepithelial transport of the mucolytic agent ambroxol hydrochloride across the intestinal barrier, ii) to classify the ambroxol according to Biopharmaceutics Classification System (BCS) and iii) to predict ambroxol absorption in humans. Transport of ambroxol (100, 300 and 1000 micromol/l) was studied in a human colon carcinoma cell line Caco-2 in apical to basolateral and basolateral to apical direction, under iso-pH 7.4 and pH-gradient (6 vs. 7.4) conditions. The relative contribution of the paracellular route was estimated using Ca2+-free transport medium. Ambroxol samples from receiver compartments were analysed by HPLC with UV detection (242 nm). Results showed that ambroxol transport is linear with time, pH-dependent and direction-independent, displays non-saturable (first-order) kinetics. Thus, the transport seems to be transcellular mediated by passive diffusion. Estimated high solubility and high permeability (P(app) = 45 x 10(-6) cm/s) of ambroxol rank it among well absorbed compounds and class I of BCS. It can be expected that the oral dose fraction of ambroxol absorbed in human intestine is high.

  17. Iron bioavailability of maize hemoglobin in a Caco-2 cell culture model.

    Science.gov (United States)

    Bodnar, Anastasia L; Proulx, Amy K; Scott, M Paul; Beavers, Alyssa; Reddy, Manju B

    2013-07-31

    Maize ( Zea mays ) is an important staple crop in many parts of the world but has low iron bioavailability, in part due to its high phytate content. Hemoglobin is a form of iron that is highly bioavailable, and its bioavailability is not inhibited by phytate. It was hypothesized that maize hemoglobin is a highly bioavailable iron source and that biofortification of maize with iron can be accomplished by overexpression of maize globin in the endosperm. Maize was transformed with a gene construct encoding a translational fusion of maize globin and green fluorescent protein under transcriptional control of the maize 27 kDa γ-zein promoter. Iron bioavailability of maize hemoglobin produced in Escherichia coli and of stably transformed seeds expressing the maize globin-GFP fusion was determined using an in vitro Caco-2 cell culture model. Maize flour fortified with maize hemoglobin was found to have iron bioavailability that is not significantly different from that of flour fortified with ferrous sulfate or bovine hemoglobin but is significantly higher than unfortified flour. Transformed maize grain expressing maize globin was found to have iron bioavailability similar to that of untransformed seeds. These results suggest that maize globin produced in E. coli may be an effective iron fortificant, but overexpressing maize globin in maize endosperm may require a different strategy to increase bioavailable iron content in maize.

  18. Uptake and transport of a novel anticancer drug-delivery system: lactosyl-norcantharidin-associated N-trimethyl chitosan nanoparticles across intestinal Caco-2 cell monolayers

    Directory of Open Access Journals (Sweden)

    Zhang Q

    2012-04-01

    Full Text Available Min Guan1, Qiao-Ling Zhu1, Yang Liu1, Yong-Yan Bei1, Zong-Lin Gu1, Xue-Nong Zhang1, Qiang Zhang21Department of Pharmaceutics, College of Pharmaceutical Science, Soochow University, Suzhou, People's Republic of China; 2Department of Pharmaceutics, School of Pharmaceutical Science, Peking University, Beijing, People's Republic of ChinaAbstract: In this paper, novel liver-targeting nanoparticles (NPs, lactosyl-norcantharidin (Lac-NCTD-associated N-trimethyl chitosan (TMC NPs (Lac-NCTD-TMC-NPs, were prepared using ionic cross-linkage. The physical properties, particle size, and encapsulation efficiency of the nanoparticles were then investigated. The continuous line of heterogeneous human epithelial colorectal adenocarcinoma cells (Caco-2 cell monolayer model was used to study the transport mechanism of Lac-NCTD, and the effects of factors such as time, temperature, pH level, drug concentration, enhancers, and inhibitors. This model was also used to indicate the differences among Lac-NCTD, Lac-NCTD-associated chitosan NPs (Lac-NCTD-CS-NPs, and Lac-NCTD-TMC-NPs in the absorption and transportation of membranes. Drug concentration levels were measured using high-performance liquid chromatography. Active transport and paracellular transport were suggested to be both the primary and secondary mechanisms for Lac-NCTD absorption, respectively. Lac-NCTD uptake and absorption were not controlled by pH levels, but were positively correlated to uptake time, and negatively correlated to temperature. The basolateral to apical apparent permeability coefficients (Papps were higher than those of the apical to basolateral values. The inhibitor of P-glycoprotein and the multidrug resistance-associated protein 2 significantly enhanced the uptake amount of Lac-NCTD. Compared with Lac-NCTD, Lac-NCTD-CS-NPs and Lac-NCTD-TMC-NPs significantly enhanced drug absorption. Additionally, the latter exhibited stronger action. Lac-NCTD-NPs could penetrate the plasma membrane of

  19. Electrostatic properties of confluent Caco-2 cell layer correlates to their microvilli growth and determines underlying transcellular flow.

    Science.gov (United States)

    Vandrangi, P; Lo, D D; Kozaka, R; Ozaki, N; Carvajal, N; Rodgers, V G J

    2013-10-01

    Recently, Rajapaksa et al. (2010) showed that the rate of uptake of potential vaccine delivery nanoparticles in the mucosal layer is a function of the electrostatic properties of the corresponding solvent. This fundamentally implies that the dominant driving forces that may be capitalized on for mucosal vaccine strategies are electrostatic in nature. We hypothesize that the driving force normal to the cell (in the direction from apical to basolateral across the cell) is of particular importance. In addition, it has been theoretically shown that the electrostatic properties of mucosal cells are directly related to their development of brush border. Here we correlate the development of brush border on a human mucosal epithelial model (Caco-2) cultured in DMEM on 3.0 µm pore sized polycarbonate membranes to their corresponding electrostatic properties characterized by measuring their normal zeta potential. Properties of normal streaming potential, hydraulic permeability, and brush border development (as determined by size and number) were monitored for 2, 6, and 16 days (after cells were confluent). Human endothelial cells (HECs), which lack brush border, were used as the control. Our results demonstrate that normal zeta potential of Caco-2 cells significantly changed from -5.7 ± 0.11 mV to -3.4 ± 0.11 mV for a period between 2 and 16 days, respectively. The zeta potential of the control cell line, HECs, stayed constant (statistically not different, P > 0.05) for the duration of the experiments. Our results show that the calculated increase in surface area of the Caco-2 cells with microvilli from 6 to 16 days was directly proportional to the corresponding measured zeta potential difference. These results imply that microvilli alter the electrostatic local environment around Caco-2 cells and, hence, enhance the normal electrostatic selective transport of solute across the mucosal barrier.

  20. Upregulation of 25-hydroxyvitamin D3-1α-hydroxylase by butyrate in Caco-2 cells

    Institute of Scientific and Technical Information of China (English)

    Oliver Schr(o)der; Sinan Turak; Carolin Daniel; Tanja Gaschott; Jürgen Stein

    2005-01-01

    AIM: To investigate the possible involvement of 25-hydroxyvitamin D3-1α-hydroxylase [1α-25(OH)2D3] in butyrate-induced differentiation in human intestinal cell line Caco-2 cells.METHODS: Caco-2 cells were incubated either with 3 mmol/L butyrate and 1 μmol/L 25(OH)2D3 or with 1μmol/L 1α-25(OH)2D3 for various time intervals ranging from 0 to 72 h. Additionally, cells were co-incubated with butyrate and either 25(OH)2D3 or 1α-25(OH)2D3.1α-25(OH)2D3 mRNA was determined semi-quantitatively using the fluorescent dye PicoGreen. Immunoblotting was used for the detection of 1α-25(OH)2D3 protein.Finally, enzymatic activity was measured by ELISA.RESULTS: Both butyrate and 1α-25(OH)2D3 stimulated differentiation of Caco-2 cells after a 48 h incubation period, while 25(OH)2D3 had no impact on cell differentiation. Synergistic effects on differentiation were observed when cells were co-incubated with butyrate and vitamin D metabolite. Butyrate transiently upregulated 1α-25(OH)2D3 mRNA followed by a timely delayed protein upregulation. Coincidently, enzymatic activity was enhanced significantly. The induction of the enzyme allowed for comparable differentiating effects of both vitamin D metabolites.CONCLUSION: Our experimental data pr ovide a further mechanism for the involvement of the vitamin D signaling pathway in colonic epithelial cell differentiation by butyrate. The enhancement of 1α-25(OH)2D3 followed by antiproliferative effects of the vitamin D prohormone in the Caco-2 cell line suggest that 25(OH)2D3 in combination with butyrate may offer a new therapeutic approach for the treatment of colon cancer.

  1. Iron repletion relocalizes hephaestin to a proximal basolateral compartment in polarized MDCK and Caco2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Seung-Min [Department of Biological Sciences, University of Columbia, NY (United States); Department of Nutritional Science and Toxicology, University of California, Berkeley, CA (United States); Attieh, Zouhair K. [Department of Laboratory Science and Technology, American University of Science and Technology, Ashrafieh (Lebanon); Department of Nutritional Science and Toxicology, University of California, Berkeley, CA (United States); Son, Hee Sook [Department of Food Science and Human Nutrition, College of Human Ecology, Chonbuk National University (Korea, Republic of); Department of Nutritional Science and Toxicology, University of California, Berkeley, CA (United States); Chen, Huijun [Medical School, Nanjing University, Nanjing 210008, Jiangsu Province (China); Department of Nutritional Science and Toxicology, University of California, Berkeley, CA (United States); Bacouri-Haidar, Mhenia [Department of Biology, Faculty of Sciences (I), Lebanese University, Hadath (Lebanon); Department of Nutritional Science and Toxicology, University of California, Berkeley, CA (United States); Vulpe, Chris D., E-mail: vulpe@berkeley.edu [Department of Nutritional Science and Toxicology, University of California, Berkeley, CA (United States)

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer Hephaestin localizes in the perinuclear space in non-polarized cells. Black-Right-Pointing-Pointer Hephaestin localizes in the perinuclear space in iron deficient and polarized cells. Black-Right-Pointing-Pointer Hephaestin with apical iron moves near to basolateral membrane of polarized cells. Black-Right-Pointing-Pointer Peri-basolateral location of hephaestin is accessible to the extracellular space. Black-Right-Pointing-Pointer Hephaestin is involved in iron mobilization from the intestine to circulation. -- Abstract: While intestinal cellular iron entry in vertebrates employs multiple routes including heme and non-heme routes, iron egress from these cells is exclusively channeled through the only known transporter, ferroportin. Reduced intestinal iron export in sex-linked anemia mice implicates hephaestin, a ferroxidase, in this process. Polarized cells are exposed to two distinct environments. Enterocytes contact the gut lumen via the apical surface of the cell, and through the basolateral surface, to the body. Previous studies indicate both local and systemic control of iron uptake. We hypothesized that differences in iron availability at the apical and/or basolateral surface may modulate iron uptake via cellular localization of hephaestin. We therefore characterized the localization of hephaestin in two models of polarized epithelial cell lines, MDCK and Caco2, with varying iron availability at the apical and basolateral surfaces. Our results indicate that hephaestin is expressed in a supra-nuclear compartment in non-polarized cells regardless of the iron status of the cells and in iron deficient and polarized cells. In polarized cells, we found that both apical (as FeSO{sub 4}) and basolateral iron (as the ratio of apo-transferrin to holo-transferrin) affect mobilization of hephaestin from the supra-nuclear compartment. We find that the presence of apical iron is essential for relocalization of hephaestin to a

  2. Surface charge-specific interactions between polymer nanoparticles and ABC transporters in Caco-2 cells

    NARCIS (Netherlands)

    Bhattacharjee, S.; Opstal, van E.J.; Alink, G.M.; Marcelis, A.T.M.; Zuilhof, H.; Rietjens, I.M.C.M.

    2013-01-01

    The surface charge-dependent transport of polymeric nanoparticles (PNPs) across Caco-2 monolayers grown on transwell culture systems as an in vitro model for intestinal transport was tested. The transport of well-characterized, monodisperse, and fluorescent tri-block copolymer nanoparticles (TCNPs/s

  3. Noni (Morinda citrifolia L.) Fruit Extracts Improve Colon Microflora and Exert Anti-Inflammatory Activities in Caco-2 Cells.

    Science.gov (United States)

    Huang, Hsin-Lun; Liu, Cheng-Tzu; Chou, Ming-Chih; Ko, Chien-Hui; Wang, Chin-Kun

    2015-06-01

    Intestinal microflora and inflammation are associated with the risk of inflammatory bowel diseases. Noni (Morinda citrifolia L.) has various bioactivities, but its effect on colon health remains unknown. This study focused on the effects of fermented noni fruit extracts on colon microflora and inflammation of colon epithelial cells. The anti-inflammatory activities of ethanol and ethyl acetate extracts on Caco-2 cells were evaluated including interleukin-8 (IL-8) and cyclooxygenase-2 (COX-2). The growth of Lactobacillus and Bifidobacterium species was promoted by ethanol extract. Ethyl acetate extract decreased intracellular reactive oxygen species and significantly suppressed COX-2, IL-8, and prostaglandin E2 production and neutrophil chemotaxis by suppressing the translocation of the p65 subunit. Quercetin was the main contributor to the anti-inflammatory activity. The fermented noni fruit promoted probiotic growths and downregulated the intracellular oxidation and inflammation in Caco-2 cells. These results suggest that fermented noni fruit might protect against inflammatory diseases of the colon.

  4. Lactobacillus acidophilus S-layer protein-mediated inhibition of Salmonella-induced apoptosis in Caco-2 cells.

    Science.gov (United States)

    Li, Pengcheng; Yin, Yinyan; Yu, Qinghua; Yang, Qian

    2011-05-27

    Surface layer (S-layer) proteins are crystalline arrays of proteinaceous subunits present as the outermost component of the cell wall in several Lactobacillus species. The underlying mechanism for how S-layer proteins inhibit pathogen infections remains unclear. To gain insights into the mechanism of the antimicrobial activity of Lactobacillus S-layer proteins, we examined how Lactobacillus S-layer proteins impact Salmonella Typhimurium-induced apoptosis in vitro in Caco-2 human colon epithelial cells. When Caco-2 cells infected with Salmonella Typhimurium SL1344, we found that apoptosis was mediated by activation of caspase-3, but not caspase-1. When Salmonella Typhimurium SL1344 and S-layer proteins were coincubated simultaneously, Caco-2 cell apoptosis was markedly decreased and the cell damage was modified, as evaluated by flow cytometry and microscopy. Detailed analyses showed that the S-layer proteins inhibited the caspase-3 activity and activated the extracellular signal-regulated kinases 1 and 2 (ERK1/2) signaling pathway. Taken together, these findings suggest that Lactobacillus S-layer proteins protected against Salmonella-induced apoptosis through reduced caspase-3 activation. In addition, Salmonella-induced apoptotic cell damage was modified by S-layer proteins through the ERK1/2 signaling pathway. This mechanism may represent a novel approach for antagonizing Salmonella infection.

  5. Solid lipid nanoparticles for hydrophilic biotech drugs: optimization and cell viability studies (Caco-2 & HEPG-2 cell lines).

    Science.gov (United States)

    Severino, Patrícia; Andreani, Tatiana; Jäger, Alessandro; Chaud, Marco V; Santana, Maria Helena A; Silva, Amélia M; Souto, Eliana B

    2014-06-23

    Insulin was used as model protein to developed innovative Solid Lipid Nanoparticles (SLNs) for the delivery of hydrophilic biotech drugs, with potential use in medicinal chemistry. SLNs were prepared by double emulsion with the purpose of promoting stability and enhancing the protein bioavailability. Softisan(®)100 was selected as solid lipid matrix. The surfactants (Tween(®)80, Span(®)80 and Lipoid(®)S75) and insulin were chosen applying a 2(2) factorial design with triplicate of central point, evaluating the influence of dependents variables as polydispersity index (PI), mean particle size (z-AVE), zeta potential (ZP) and encapsulation efficiency (EE) by factorial design using the ANOVA test. Therefore, thermodynamic stability, polymorphism and matrix crystallinity were checked by Differential Scanning Calorimetry (DSC) and Wide Angle X-ray Diffraction (WAXD), whereas the effect of toxicity of SLNs was check in HepG2 and Caco-2 cells. Results showed a mean particle size (z-AVE) width between 294.6 nm and 627.0 nm, a PI in the range of 0.425-0.750, ZP about -3 mV, and the EE between 38.39% and 81.20%. After tempering the bulk lipid (mimicking the end process of production), the lipid showed amorphous characteristics, with a melting point of ca. 30 °C. The toxicity of SLNs was evaluated in two distinct cell lines (HEPG-2 and Caco-2), showing to be dependent on the concentration of particles in HEPG-2 cells, while no toxicity in was reported in Caco-2 cells. SLNs were stable for 24 h in in vitro human serum albumin (HSA) solution. The resulting SLNs fabricated by double emulsion may provide a promising approach for administration of protein therapeutics and antigens.

  6. Transcriptome Profiling of Caco-2 Cancer Cell Line following Treatment with Extracts from Iodine-Biofortified Lettuce (Lactuca sativa L..

    Directory of Open Access Journals (Sweden)

    Aneta A Koronowicz

    Full Text Available Although iodization of salt is the most common method used to obtain iodine-enriched food, iodine deficiency disorders are still a global health problem and profoundly affect the quality of human life. Iodine is required for the synthesis of thyroid hormones, which are crucial regulators of human metabolism, cell growth, proliferation, apoptosis and have been reported to be involved in carcinogenesis. In this study, for the first time, we evaluated the effect of iodine-biofortified lettuce on transcriptomic profile of Caco-2 cancer cell line by applying the Whole Human Genome Microarray assay. We showed 1326 differentially expressed Caco-2 transcripts after treatment with iodine-biofortified (BFL and non-fortified (NFL lettuce extracts. We analysed pathways, molecular functions, biological processes and protein classes based on comparison between BFL and NFL specific genes. Iodine, which was expected to act as a free ion (KI-NFL or at least in part to be incorporated into lettuce macromolecules (BFL, differently regulated pathways of numerous transcription factors leading to different cellular effects. In this study we showed the inhibition of Caco-2 cells proliferation after treatment with BFL, but not potassium iodide (KI, and BFL-mediated induction of mitochondrial apoptosis and/or cell differentiation. Our results showed that iodine-biofortified plants can be effectively used by cells as an alternative source of this trace element. Moreover, the observed differences in action of both iodine sources may suggest a potential of BFL in cancer treatment.

  7. Olive oil hydroxytyrosol reduces toxicity evoked by acrylamide in human Caco-2 cells by preventing oxidative stress.

    Science.gov (United States)

    Rodríguez-Ramiro, Ildefonso; Martín, María Ángeles; Ramos, Sonia; Bravo, Laura; Goya, Luis

    2011-10-09

    Humans are exposed to dietary acrylamide (AA) during their lifetime, it is therefore necessary to investigate the mechanisms associated with AA-induced toxic effects. Accumulating evidence indicates that oxidative stress contributes to AA cytotoxicity, thus, dietary antioxidants might have a protective role in colonic cells against AA toxicity. We have recently reported that hydroxytyrosol (HTy), a natural antioxidant abundant in olive oil, is able to enhance the cellular antioxidant defence capacity, thereby protecting cells from oxidative stress. In this study, we evaluate the protective role of HTy on alterations of the redox balance induced by AA in Caco-2 intestinal cells. AA cytotoxicity was counteracted by HTy by powerfully reducing ROS generation, recovering the excited enzyme antioxidant defences and decreasing phospho-Jun kinase concentration and caspase-3 activity induced by AA. Therefore, AA-induced cytotoxicity and apoptosis are closely related to oxidative stress in Caco-2 cells and the olive oil natural dietary antioxidant HTy was able to contain AA toxicity by improving the redox status of Caco-2 cells and by partly restraining the apoptotic pathway activated by AA.

  8. Transcriptome Profiling of Caco-2 Cancer Cell Line following Treatment with Extracts from Iodine-Biofortified Lettuce (Lactuca sativa L.).

    Science.gov (United States)

    Koronowicz, Aneta A; Kopeć, Aneta; Master, Adam; Smoleń, Sylwester; Piątkowska, Ewa; Bieżanowska-Kopeć, Renata; Ledwożyw-Smoleń, Iwona; Skoczylas, Łukasz; Rakoczy, Roksana; Leszczyńska, Teresa; Kapusta-Duch, Joanna; Pysz, Mirosław

    2016-01-01

    Although iodization of salt is the most common method used to obtain iodine-enriched food, iodine deficiency disorders are still a global health problem and profoundly affect the quality of human life. Iodine is required for the synthesis of thyroid hormones, which are crucial regulators of human metabolism, cell growth, proliferation, apoptosis and have been reported to be involved in carcinogenesis. In this study, for the first time, we evaluated the effect of iodine-biofortified lettuce on transcriptomic profile of Caco-2 cancer cell line by applying the Whole Human Genome Microarray assay. We showed 1326 differentially expressed Caco-2 transcripts after treatment with iodine-biofortified (BFL) and non-fortified (NFL) lettuce extracts. We analysed pathways, molecular functions, biological processes and protein classes based on comparison between BFL and NFL specific genes. Iodine, which was expected to act as a free ion (KI-NFL) or at least in part to be incorporated into lettuce macromolecules (BFL), differently regulated pathways of numerous transcription factors leading to different cellular effects. In this study we showed the inhibition of Caco-2 cells proliferation after treatment with BFL, but not potassium iodide (KI), and BFL-mediated induction of mitochondrial apoptosis and/or cell differentiation. Our results showed that iodine-biofortified plants can be effectively used by cells as an alternative source of this trace element. Moreover, the observed differences in action of both iodine sources may suggest a potential of BFL in cancer treatment.

  9. The flavanone homoeriodictyol increases SGLT-1-mediated glucose uptake but decreases serotonin release in differentiated Caco-2 cells

    Science.gov (United States)

    Hoi, Julia Katharina; Holik, Ann-Katrin; Geissler, Katrin; Hans, Joachim; Friedl, Barbara; Liszt, Kathrin; Krammer, Gerhard E.; Ley, Jakob P.; Somoza, Veronika

    2017-01-01

    Flavanoids and related polyphenols, among them hesperitin, have been shown to modulate cellular glucose transport by targeting SGLT-1 and GLUT-2 transport proteins. We aimed to investigate whether homoeriodictyol, which is structurally related to hesperitin, affects glucose uptake in differentiated Caco-2 cells as a model for the intestinal barrier. The results revealed that, in contrast to other polyphenols, the flavanon homoeriodictyol promotes glucose uptake by 29.0 ± 3.83% at a concentration of 100 μM. The glucose uptake stimulating effect was sensitive to phloridzin, but not to phloretin, indicating an involvement of the sodium-coupled glucose transporter SGLT-1, but not of sodium-independent glucose transporters (GLUT). In addition, in contrast to the increased extracellular serotonin levels by stimulation with 500 mM D-(+)-glucose, treatment with 100 μM homoeriodictyol decreased serotonin release by –48.8 ± 7.57% in Caco-2 cells via a phloridzin-sensitive signaling pathway. Extracellular serotonin levels were also reduced by –57.1 ± 5.43% after application of 0.01 μM homoeriodictyol to human neural SH-SY5Y cells. In conclusion, we demonstrate that homoeriodictyol affects both the glucose metabolism and the serotonin system in Caco-2 cells via a SGLT-1-meditated pathway. Furthermore, the results presented here support the usage of Caco-2 cells as a model for peripheral serotonin release. Further investigations may address the value of homoeriodictyol in the treatment of anorexia and malnutrition through the targeting of SGLT-1. PMID:28192456

  10. Identification of TNF-α-responsive promoters and enhancers in the intestinal epithelial cell model Caco-2

    DEFF Research Database (Denmark)

    Boyd, Mette; Coskun, Mehmet; Lilje, Berit;

    2014-01-01

    promoters. As a case example, we characterize an enhancer regulating the laminin-5 γ2-chain (LAMC2) gene by nuclear factor (NF)-κB binding. This report is the first to present comprehensive TSS and enhancer maps over Caco-2 cells, and highlights many novel inflammation-specific promoters and enhancers....... genome-wide maps of active transcription start sites (TSSs), and active enhancers in Caco-2 cells with or without tumour necrosis factor (TNF)-α stimulation to mimic an inflammatory state. We found 520 promoters that significantly changed their usage level upon TNF-α stimulation; of these, 52......% are not annotated. A subset of these has the potential to confer change in protein function due to protein domain exclusion. Moreover, we locate 890 transcribed enhancer candidates, where ∼50% are changing in usage after TNF-α stimulation. These enhancers share motif enrichments with similarly responding gene...

  11. Identification of TNF-α-responsive promoters and enhancers in the intestinal epithelial cell model Caco-2

    DEFF Research Database (Denmark)

    Boyd, Mette; Coskun, Mehmet; Lilje, Berit

    2014-01-01

    genome-wide maps of active transcription start sites (TSSs), and active enhancers in Caco-2 cells with or without tumour necrosis factor (TNF)-α stimulation to mimic an inflammatory state. We found 520 promoters that significantly changed their usage level upon TNF-α stimulation; of these, 52......The Caco-2 cell line is one of the most important in vitro models for enterocytes, and is used to study drug absorption and disease, including inflammatory bowel disease and cancer. In order to use the model optimally, it is necessary to map its functional entities. In this study, we have generated......% are not annotated. A subset of these has the potential to confer change in protein function due to protein domain exclusion. Moreover, we locate 890 transcribed enhancer candidates, where ∼50% are changing in usage after TNF-α stimulation. These enhancers share motif enrichments with similarly responding gene...

  12. Evaluation of the intestinal permeability of rosemary (Rosmarinus officinalis L.) extract polyphenols and terpenoids in Caco-2 cell monolayers

    Science.gov (United States)

    Arráez-Román, David; González-Álvarez, Isabel; Ibáñez, Elena; Segura-Carretero, Antonio; Bermejo, Marival; Micol, Vicente

    2017-01-01

    Rosemary (Rosmarinus officinalis) is grown throughout the world and is widely used as a medicinal herb and to season and preserve food. Rosemary polyphenols and terpenoids have attracted great interest due to their potential health benefits. However, complete information regarding their absorption and bioavailability in Caco-2 cell model is scarce. The permeation properties of the bioactive compounds (flavonoids, diterpenes, triterpenes and phenylpropanoids) of a rosemary extract (RE), obtained by supercritical fluid extraction, was studied in Caco-2 cell monolayer model, both in a free form or liposomed. Compounds were identified and quantitated by liquid chromatography coupled to quadrupole time-of-flight with electrospray ionization mass spectrometry analysis (HPLC-ESI-QTOF-MS), and the apparent permeability values (Papp) were determined, for the first time in the extract, for 24 compounds in both directions across cell monolayer. For some compounds, such as triterpenoids and some flavonoids, Papp values found were reported for the first time in Caco-2 cells.Our results indicate that most compounds are scarcely absorbed, and passive diffusion is suggested to be the primary mechanism of absorption. The use of liposomes to vehiculize the extract resulted in reduced permeability for most compounds. Finally, the biopharmaceutical classification (BCS) of all the compounds was achieved according to their permeability and solubility data for bioequivalence purposes. BCS study reveal that most of the RE compounds could be classified as classes III and IV (low permeability); therefore, RE itself should also be classified into this category. PMID:28234919

  13. Listeria monocytogenes efficiently invades Caco-2 cells after low-temperature storage in broth and on deli meat.

    Science.gov (United States)

    Larsen, Marianne Halberg; Koch, Anette Granly; Ingmer, Hanne

    2010-09-01

    The objective of this study was to investigate how various growth conditions influence the virulence of Listeria monocytogenes monitored by its ability to invade the epithelial cell lines Caco-2 and INT-407. The growth conditions examined were modified atmosphere-packaged deli meat and brain heart infusion broth (BHI) with and without salt. Five strains of L. monocytogenes were selected to investigate their invasiveness and all strains invaded Caco-2 cells at higher levels than INT-407 cells. Further, the clinical strains (3443 and 3734) were more invasive (p 0.05) in invasiveness after 7 days at 10 degrees C in BHI broth or on sausage, whereas a slight increase (p < 0.05) was observed after incubation on ham for 2 and 4 weeks compared to that in BHI broth. Most importantly, our results show that L. monocytogenes efficiently invade Caco-2 cells even after 4 weeks of storage at chilled temperature. This is highly relevant for safety assessment of this organism in food as these conditions reflect storage of ready-to-eat food products in domestic refrigerators.

  14. [Establishment of Caco-2 cell monolayer model with collagen coating 6-well plates for study of traditional Chinese medicine prescription].

    Science.gov (United States)

    Yang, Yan-Fang; Wu, Ni; Yang, Xiu-Wei

    2014-02-01

    Caco-2 cell monolayer model is widely utilized in drug absorption study and 12-well transwellTM plates were commonly used to study the absorption of different kinds of natural products. To establish a stable method for the study of traditional Chinese medicine prescription, 6-well plates were chosen because of the larger well volumes than 12-well plates. To study the impacts of collagen kinds, coating density as well as coating time on the cell culture, the transepithelial electrical resistance of Caco-2 cell monolayers grown on different collagen coating transwells was determined, and the permeations of propranolol and atenolol as standard markers were detected with HPLC. The results showed that the kinds of collagen, the different coating densities and coating time of rat tail collagen had no significant influences on the Caco-2 cell monolayer integrality and absorption capacity. 6-well plates coated with 2 micro g Scm-2 rat tail collagen for 1 hour were enough reliable and suitable for the study of traditional Chinese medicine prescription in vitro.

  15. Protective effect of simple phenols from extravirgin olive oil against lipid peroxidation in intestinal Caco-2 cells.

    Science.gov (United States)

    Deiana, Monica; Corona, Giulia; Incani, Alessandra; Loru, Debora; Rosa, Antonella; Atzeri, Angela; Paola Melis, M; Assunta Dessì, M

    2010-10-01

    Complex polyphenols present in extravirgin olive oil are not directly absorbed, but undergo gastrointestinal biotransformation, increasing the relative amount of tyrosol (TYR) and hydroxytyrosol (HT) entering the small and large intestine. We investigated the capacity of TYR and HT to inhibit the insult of dietary lipid hydroperoxydes on the intestinal mucosa, using cultures of Caco-2, a cell line with enterocyte-like features, and studying the effect of tert-butyl hydroperoxide (TBH) treatment on specific cell membrane lipid targets. The effect of homovanillic alcohol (HVA), metabolite of HT in humans and detected as metabolite of HT in Caco-2 cells, was also evaluated. Exposure to TBH induced a significant increase of the level of MDA, the formation of fatty acid hydroperoxides and 7-ketocholesterol and the loss of α-tocopherol. Pretreatment with both HT and HVA protected Caco-2 cells from oxidative damage: there was no significant detection of oxidation products and the level of α-tocopherol was preserved. Noteworthy, TYR also exerted a protective action against fatty acids degradation. In vitro trials, where the simple phenols were tested during linoleic acid and cholesterol oxidation, gave evidence of a direct scavenging of peroxyl radicals and suggested a hydrogen atom-donating activity.

  16. Vitamin D increases tight-junction conductance and paracellular Ca2+ transport in Caco-2 cell cultures.

    Science.gov (United States)

    Chirayath, M V; Gajdzik, L; Hulla, W; Graf, J; Cross, H S; Peterlik, M

    1998-02-01

    We investigated the effects of 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] on paracellular intestinal Ca2+ absorption by determination of transepithelial electric resistance (TEER), as a measure of tight-junction ion permeability and bidirectional transepithelial 45Ca2+ fluxes in confluent Caco-2 cell cultures. The rise of TEER to steady-state levels of approximately 2,000 omega.cm2 was significantly attenuated by 1,25(OH)2D3 (by up to 50%) in a dose-dependent fashion between 10(-11) and 10(-8) M. Synthetic analogs of 1,25(OH)2D3, namely, 1 alpha,25-dihydroxy-16-ene,23-yne-vitamin D3 and 1 alpha,25-dihydroxy-26,27-hexafluoro-16-ene,23-yne-vitamin D3, exhibited similar biopotency, whereas their genomically inactive 1-deoxy congeners were only marginally effective. Enhancement of transepithelial conductance of Caco-2 cell monolayers by vitamin D was accompanied by a significant increase in bidirectional transepithelial 45Ca2+ fluxes. Although 1,25(OH)2D3 also induced cellular 45Ca2+ uptake from the apical aspect of Caco-2 cell layers and upregulated the expression of calbindin-9kDa mRNA, no significant contribution of the Ca(2+)-adenosinetriphosphatase-mediated transcellular pathway to transepithelial Ca2+ transport could be detected. Therefore stimulation of Ca2+ fluxes across confluent Caco-2 cells very likely results from a genomic effect of vitamin D sterols on assembly and permeability of tight-junctional complexes.

  17. The expression of efflux and uptake transporters are regulated by statins in Caco-2 and hepG2 cells

    Institute of Scientific and Technical Information of China (English)

    Alice Cristina RODRIGUES; Rui CURI; Fabiana Dalla Vecchia GENVIGIR; Mario Hiroyuki HIRATA; Rosario Dominguez Crespo HIRATA

    2009-01-01

    Aim:Statin disposition and response are greatly determined by the activities of drug metabolizing enzymes and effiux/uptake transporters.There is little information on the regulation of these proteins in human ceils after statin therapy.In this study,the effects of atorvastatin and simvastatin on mRNA expression of efflux (ABCB1,ABCG2 and ABCC2) and uptake (SLCO1B1,SLCO2B1 and SLC22A1) drug transporters in Caco-2 and HepG2 cells were investigated.Methods:Quantitative real-time PCR was used to measure mRNA levels after exposure of HepG2 and Caco-2 cells to statins Results:Differences in mRNA basal levels of the transporters were as follows:ABCC2>ABCG2>ABCB1>SLCOIB1>>>SLC22A1>SLC 02B1 for HepG2 cells,and SLCO2B1>>ABCC2>ABCB1>ABCG2>>>SLC22A1 for Caco-2 cells.While for HepG2 cells,ABCC2,ABCG2 and SLCO2B1 mRNA levels were significantly up-regulated at 1,10 and 20 μmol/L after 12 or 24 h treatment,in Caco-2 cells,only the efflux transporter ABCB1 was significantly down-regulated by two-fold following a 12 h treatment with atorvastatin.Interestingly,whereas treatment with simvastatin had no effect on mRNA levels of the transporters in HepG2 cells,in Caco-2 cells the statin signifi cantly down-regulated ABCB1,ABCC2,SLC22A1,and SLCO2B1 mRNA levels after 12 or 24 h treatment.Conclusion:These findings reveal that statins exhibits differential effects on mRNA expression of drug transporters,and this effect depends on the cell type.Furthermore,alterations in the expression levels of drug transporters in the liver and/or intestine may contribute to the variability in oral disposition of statins.

  18. Sucrose esters increase drug penetration, but do not inhibit p-glycoprotein in caco-2 intestinal epithelial cells.

    Science.gov (United States)

    Kiss, Lóránd; Hellinger, Éva; Pilbat, Ana-Maria; Kittel, Ágnes; Török, Zsolt; Füredi, András; Szakács, Gergely; Veszelka, Szilvia; Sipos, Péter; Ózsvári, Béla; Puskás, László G; Vastag, Monika; Szabó-Révész, Piroska; Deli, Mária A

    2014-10-01

    Sucrose fatty acid esters are increasingly used as excipients in pharmaceutical products, but few data are available on their toxicity profile, mode of action, and efficacy on intestinal epithelial models. Three water-soluble sucrose esters, palmitate (P-1695), myristate (M-1695), laurate (D-1216), and two reference absorption enhancers, Tween 80 and Cremophor RH40, were tested on Caco-2 cells. Caco-2 monolayers formed a good barrier as reflected by high transepithelial resistance and positive immunostaining for junctional proteins claudin-1, ZO-1, and β-catenin. Sucrose esters in nontoxic concentrations significantly reduced resistance and impedance, and increased permeability for atenolol, fluorescein, vinblastine, and rhodamine 123 in Caco-2 monolayers. No visible opening of the tight junctions was induced by sucrose esters assessed by immunohistochemistry and electron microscopy, but some alterations were seen in the structure of filamentous actin microfilaments. Sucrose esters fluidized the plasma membrane and enhanced the accumulation of efflux transporter ligands rhodamine 123 and calcein AM in epithelial cells, but did not inhibit the P-glycoprotein (P-gp)-mediated calcein AM accumulation in MES-SA/Dx5 cell line. These data indicate that in addition to their dissolution-increasing properties sucrose esters can enhance drug permeability through both the transcellular and paracellular routes without inhibiting P-gp.

  19. Phenylpyrazole insecticides induce cytotoxicity by altering mechanisms involved in cellular energy supply in the human epithelial cell model Caco-2.

    Science.gov (United States)

    Vidau, Cyril; Brunet, Jean-Luc; Badiou, Alexandra; Belzunces, Luc P

    2009-06-01

    Phenylpyrazoles are relatively new insecticides designed to manage problematic insect resistance and public health hazards encountered with older pesticide families. In vitro cytotoxicity induced by the phenylpyrazole insecticides, Ethiprol and Fipronil, and Fipronil metabolites, sulfone and sulfide, was studied in Caco-2 cells. This cellular model was chosen because it made possible to mimic the primary site of oral exposure to xenobiotics, the intestinal epithelium. Assessment of the barrier function of Caco-2 epithelium was assessed by TEER measurement and showed a major loss of barrier integrity after exposure to Fipronil and its metabolites, but not to Ethiprol. The disruption of the epithelial barrier was attributed to severe ATP depletion independent of cell viability, as revealed by LDH release. The origin of energetic metabolism failure was investigated and revealed a transient enhancement of tetrazolium salt reduction and an increase in lactate production by Caco-2 cells, suggesting an increase in glucose metabolism by pesticides. Cellular symptoms observed in these experiments lead us to hypothesize that phenylpyrazole insecticides interacted with mitochondria.

  20. Beta-cryptoxanthin from citrus juices: assessment of bioaccessibility using an in vitro digestion/Caco-2 cell culture model.

    Science.gov (United States)

    Dhuique-Mayer, Claudie; Borel, Patrick; Reboul, Emmanuelle; Caporiccio, Bertrand; Besancon, Pierre; Amiot, Marie-Josèphe

    2007-05-01

    Beta-Cryptoxanthin (beta-CX), a provitaminic carotenoid of potential interest for health, is found principally in Citrus fruit in both free and esterified forms. Little is known about the intestinal absorption of beta-CX especially with regard to the esterified forms. The aim of this study was to evaluate the absorption of free and esterified beta-CX using simulated digestion coupled with the Caco-2 model. Bioaccessibility was investigated by measuring the transfer of carotenoids from different citrus juices into micelles using an in vitro digestion system. Then, carotenoid uptake was evaluated by adding carotenoid-rich micelles (from the in vitro digestion) or synthetic micelles (made from synthetic lipids and carotenoids purified from citrus juice) to human intestinal cells (Caco-2 TC7 clone). Our results showed that beta-cryptoxanthin esters (beta-CXE) were partially hydrolysed during the in vitro digestion. The bioaccessibility of free beta-CX measured was significantly higher (40 (SD 1.05) %) than that of beta-carotene (30 (SD 1.9) %) and beta-CXE (16 (SD 1.5) %). In the same way, the incorporation of free beta-CX (27 (SD 1.01) %) into synthetic micelles exceeded (Pdigestion, the uptake of beta-carotene, free beta-CX and beta-CXE forms by Caco-2 cells was 14.3 (SD 1.8), 3.9 (SD 1.3), and 0.7 (SD 0.08) % respectively. These results showed a preferential uptake by Caco-2 cells of beta-carotene and free beta-CX compared with the two esters of beta-CX.

  1. Effect of PSC 833 liposomes and Intralipid on the transport of epirubicin in Caco-2 cells and rat intestines.

    Science.gov (United States)

    Lo, Y; Liu, F; Cherng, J

    2001-09-11

    Clinical applications of first-generation multidrug resistance (MDR) modulators, such as cyclosporin A (CsA) have been hampered because of their severe side effects in vivo. Recent investigations have led to the development of a more potent and less toxic modulator, PSC 833, which is a nonimmunosuppressive analogue of CsA. However, adverse pharmacokinetic interactions between anticancer drugs and PSC 833 have resulted in increased toxicity as compared to the individual toxicity. Our study evaluated the MDR reversing effect of PSC 833 in free, liposomal or Intralipid formulations on the uptake and transport of epirubicin in Caco-2 cells and everted gut sacs of rats. The results showed that PSC 833 in free or liposomal formulations significantly enhanced the intracellular accumulation of epirubicin in a dose-related manner in Caco-2 cells. The optimum in enhancement was observed at the concentration of 2 microM PSC 833. These formulations markedly increased the apical to basolateral absorption of epirubicin in Caco-2 cells and substantially improved the mucosal to serosal absorption of epirubicin in rat jejunum and ileum. PSC 833 in free, liposomal or Intralipid formulations all significantly reduced basolateral to apical efflux of epirubicin across Caco-2 monolayers. However, PSC 833 in liposomes showed greater enhancement than other formulations. In conclusion, PSC 833 and PSC 833 liposomes have the function as MDR reversing agents for the inhibition of intestinal P-glycoprotein. Liposomal preparations of PSC833 may provide a useful alternative dosage form for intravenous administration of PSC 833 to be combined with anticancer drugs to circumvent drug resistance in cancer chemotherapy.

  2. 姜黄素抑制Caco-2细胞胆固醇吸收的作用及机制研究%THE INHIBITORY EFFECT OF CURCUMIN ON Caco-2 CELLS CHOLESTEROL ABSORPTION AND THE MECHANISM

    Institute of Scientific and Technical Information of China (English)

    冯丹; 凌文华

    2011-01-01

    Objective To investigate the effects of curcumin on intestinal cholesterol absorption and the possible mechanisms. Method The delipidized fetal bovine serum and the micelles composed of bile salt, monoolein, and 14C-cholesterol were prepared. Caco-2 cells were pretreated with 25, 50, 100 μmol/L curcumin or Niemann-Pick C1-Like 1 (NPC1L1) inhibitor ezetimibe for 24h or 2h respectively, and then incubated with cholesterol micelle solution for anther 2h. The absorption of cholesterol in Caco-2 cells was determined by liquid scintillation counting. The mRNA and protein expression of NPC1L1 was analyzed by qPCR and Western blot respectively. Results The absorption of radioactive cholesterol in Caco-2 cells was inhibited by ezetimibe in dose-dependent manner. The absorption of cholesterol was mediated by NPC1L1. The cells were pretreated with 25-100 umol/L curcumin for 24 h and cholesterol absorption was 40% inhibited dose-dependently by 100 umol/L curcumin. In addition, the curcumin induced inhibition of cholesterol absorption was associated with significant decrease in the levels of NPC1L1 mRNA and NPC1L1 protein. Conclusion Curcumin inhibits cholesterol absorption by suppression of NPC1L1 expression. [ACTA NUTRIMENTA SIN1CA, 201133(5): 488-49l]%目的 探讨姜黄素对Caco-2细胞胆固醇吸收的影响以及可能机制.方法 建立Caco-2细胞单层模型,分别用25、50、100 μmol/L姜黄素或胆固醇吸收转运蛋白尼曼-匹克C1型类似蛋白1(NPC1L1)的抑制剂依折麦布孵育细胞24h或2h后,再用[14C]-胆固醇微胶溶液孵育细胞2h.用液闪计数仪检测细胞胆固醇吸收量,荧光定量PCR和Western blot检测NPC1L1的基因和蛋白表达量.结果 Caco-2细胞[14C]-胆固醇吸收可被依折麦布呈浓度依赖性地抑制,表明本研究建立的单层Caco-2细胞模型的胆固醇吸收是由NPC1L1所介导的.姜黄素能有效的下调NPC1L1的基因及蛋白表达水平,降低Caco-2细胞胆固醇吸收,100

  3. 肝部分切除术促人结肠癌Caco-2细胞肝转移及相关机制%Partial hepatectomy promotes Caco-2 human colon cancer cells liver metastases and corresponding mechanism

    Institute of Scientific and Technical Information of China (English)

    任春振; 杨贺庆; 刘艳良

    2011-01-01

    Objective To investigate the effects of partial hepatectomy to the liver metastases of Caco-2 human coloncancer cells and discover its mechanism. Methods In order to constructing the colorectal liver metastases nude mice model, the Caco-2 human colon cells were injected into nude mice through portal vein trunk to imitate the liver metastases pathway of colon cancer cells. Meanwhile, the partial hepatectomy (PH ) was performed, and the Sham operation was set to control. Then the siRNA-Snail was imported into Caco-2 cells to interfere the expression of Snail mRNA; and area of the liver metastases, the expression of Snail and MMP-2 was detected. Results The metastases area, the expression of snail and MMP-2 of PH group was significantly higher than Sham group and the siRNA-Snail-PH group. Conclusion PH may promote the liver metastases of colorectal cancer, this closely correlates with up-regulating Snail expression and increasing its nucleus displacement and thus up-regulating MMP-2 expression.%目的 研究肝部分切除术对人结肠癌Caco-2细胞肝转移的影响及其相关机制.方法 应用人结肠癌Caco-2细胞系,构建结肠癌肝转移裸小鼠模型,同时行肝部分切除术(PH),假手术(Sham)设为对照,并对Caco-2细胞行siRNA-Snail干扰.观察肝转移灶面积,肿瘤组织Snail和基质金属蛋白酶2蛋白(MMP-2)表达.结果 PH组转移灶面积、Snail蛋白和MMP-2表达均显著大于Sham组和siRNA-Snail-PH组.结论 PH可促进结肠癌发生肝转移,该作用与促进Snail表达及上调Snail调控的MMP-2表达相关.

  4. Lactobacillus delbrueckii ssp. bulgaricus B-30892 can inhibit cytotoxic effects and adhesion of pathogenic Clostridium difficile to Caco-2 cells

    Directory of Open Access Journals (Sweden)

    Banerjee Pratik

    2009-04-01

    Full Text Available Abstract Background Probiotic microorganisms are receiving increasing interest for use in the prevention, treatment, or dietary management of certain diseases, including antibiotic-associated diarrhea (AAD. Clostridium difficile is the most common cause of AAD and the resulting C. difficile – mediated infection (CDI, is potentially deadly. C. difficile associated diarrhea (CDAD is manifested by severe inflammation and colitis, mostly due to the release of two exotoxins by C. difficile causing destruction of epithelial cells in the intestine. The aim of this study was to determine the effect of probiotic bacteria Lactobacillus delbrueckii ssp. bulgaricus B-30892 (LDB B-30892 on C. difficile-mediated cytotoxicity using Caco-2 cells as a model. Methods Experiments were carried out to test if the cytotoxicity induced by C. difficile-conditioned-medium on Caco-2 cells can be altered by cell-free supernatant (CFS from LDB B-30892 in different dilutions (1:2 to 1:2048. In a similar experimental setup, comparative evaluations of other probiotic strains were made by contrasting the results from these strains with the results from LDB B-30892, specifically the ability to affect C. difficile induced cytotoxicity on Caco-2 monolayers. Adhesion assays followed by quantitative analysis by Giemsa staining were conducted to test if the CFSs from LDB B-30892 and other probiotic test strains have the capability to alter the adhesion of C. difficile to the Caco-2 monolayer. Experiments were also performed to evaluate if LDB B-30892 or its released components have any bactericidal effect on C. difficile. Results and discussion Co-culturing of LDB B-30892 with C. difficile inhibited the C. difficile-mediated cytotoxicity on Caco-2 cells. When CFS from LDB B-30892-C. difficile co-culture was administered (up to a dilution of 1:16 on Caco-2 monolayer, there were no signs of cytotoxicity. When CFS from separately grown LDB B-30892 was mixed with the cell-free toxin

  5. Benzotropolone moiety in theaflavins is responsiblefor inhibitingpeptide-transport and activating AMP-activated protein kinase in Caco-2 cells

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    Ha-Young Park

    2013-05-01

    Full Text Available ABSTRACTObjective:In the small intestine, peptide transporter 1 (PEPT1 plays a role in the transport of di- and tri-peptides. Recently, we found that theaflavins (TFs, dimeric catechins, inhibitedthe transport of di-peptides across Caco-2 monolayersby suppressingthe expression of PEPT1 through AMP-activated protein kinase (AMPK activation. In this study, we investigated the structural requirement of theaflavinsfor the effect, and the mechanism(sunderling theaflavin-induced AMPK activation.Methods:Theaflavin-3’-O-gallate (TF3’G was used forthis study, since it possessed the most potent inhibition power for peptide-transport among theaflavins. Absorption ability was measured with Caco-2 cell monolayers treated with or without 20 M sample (TF3’G or its related compounds in an Ussing Chamber. The amountof Gly-Sar (a model of PEPT1-transporing peptide transportat fixed time-pointsto 60min wasdeterminedby fluorescent naphthalene-2,3-dicarboxaldehyde-derivatized assay(Ex/Em: 405 nm/460 nm. The apparent permeability coefficient(Papp wasused to evaluate the permeability. Expression of PEPT1 protein in Caco-2 cells treated with or without 20 M TF3’G in the presence or absence of inhibitor (10 μM compound C as AMPK inhibitor or 25 μMSTO-609 as CaMKK inhibitor wasevaluated by Western blot.Results:The Pappvalue of Gly-Sar significantly (P<0.05 decreasedin 20 μM purprogallin-treated Caco-2 cellsas well asin TF3’G-treated cells, together with the reduction of PEPT1 expression, while their monomeric catechins did not show any Pappreduction. In TF3’G-treated Caco-2 cells, the recovery of the reduced PEPT1 expression was found by 10 μM compound C,but not STO-609.Conclusion:The study demonstrated that the benzotropolone moiety in theaflavins was a crucial structural requirement for exerting the inhibition of intestinal peptide-transport,and the suppression of PEPT1 expression by theaflavins would be caused by activating LKB1/AMPK pathway

  6. Comparative Study of Domoic Acid and Okadaic Acid Induced - Chromosomal Abnormalities in the CACO-2 Cell Line

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    Edmond E. Creppy

    2006-03-01

    Full Text Available Okadaic Acid (OA the major diarrheic shellfish poisoning (DSP toxin is known as a tumor promoter and seems likely implicated in the genesis of digestive cancer. Little is known regarding genotoxicity and carcinogenicity of Domoic Acid (DA, the major Amnesic Shellfish Poisoning (ASP toxin. Both OA and DA occur in seafood and are of human health concerns. Micronuclei (MN arise from abnormalities in nuclear division during mitosis due to a failure of the mitotic spindle or by complex chromosomal configurations that pose problems during anaphase. In order to evaluate the ability of okadaic acid (OA and domoic acid (DA to induce DNA damage we performed the micronucleus assay using the Caco-2 cell line. To discriminate between a clastogenic or aneugenic effect of OA and DA, the micronucleus assay was conducted by cytokinesis-block micronucleus assay using cytochalasin B with Giemsa staining and/or acridine orange staining, in parallel to fluorescence in situ hybridization (FISH using a concentrated human pan-centromeric chromosome paint probe. Our results showed that OA and DA significantly increased the frequency of MN in Caco-2 cells. The MN caused by OA are found in mononucleated cells and binucleated cells, whereas those caused by DA are mainly in binucleated cells. The results of FISH analysis showed that OA induced centromere-positive micronuclei and DA increased the percentage of MN without a centromeric signal. In conclusion, both OA and DA bear mutagenic potential as revealed in Caco-2 cells by induction of MN formation. Moreover, OA induced whole chromosome loss suggesting a specific aneugenic potential, whereas DA seems simply clastogenic. At present, one cannot rule out possible DNA damage of intestinal cells if concentrations studied are reached in vivo, since this may happen with concentrations of toxins just below regulatory limits in case of frequent consumption of contaminated shell fishes.

  7. Comparison of invasiveness among surface-adherent variants of Listeria monocytogenes in Caco-2 cell culture assays.

    Science.gov (United States)

    Kushwaha, Kalpana; Muriana, Peter M

    2010-03-31

    The persistence of Listeria monocytogenes in meat processing plants is believed to be partly linked with their adherence properties to abiotic surfaces that may allow contamination of ready-to-eat (RTE) foods. Isolates of raw meats, RTE meats and environmental samples from RTE meat processing facilities that demonstrated differences in adherence to abiotic surfaces (glass, plastic, stainless steel, and rubber) were tested for cellular adherence and invasion in the Caco-2 human cell line. Strains of L. monocytogenes were classified into strong (CW50, 99-38, CW77, SM5 and CW62) and weakly adherent (CW34, CW35, CW72, SM3, J7 and J126) based on a microplate adherence assay using the fluorescing substrate, 5,6-CFDA. These strains were tested for adherence and invasion in cell culture assays with Caco-2 cells. At long incubation time (2h) and high multiplicity of infection (MOI, 100:1) we observed equivalent cellular adherence and invasiveness. After optimizing conditions for time of infection (15-, 30-, 60-, 90-, and 120-min) and MOI (100:1, 10:1, 1:1, and 0.1:1), we found that under the conditions of 15-min infection time at an MOI of 10:1 (Listeria:Caco-2 cells), we again observed equivalent cellular adherence by the two distinct surface-adherent groups, but we only recovered invading Listeria from the group that was strongly adherent to environmental surfaces. The data indicate that a surface factor that provides for strong environmental surface adherence may also be involved with internal cellular virulence and survival and implicates strongly adherent strains as possibly being of greater invasiveness than weakly adherent strains.

  8. DNA-fragments are transcytosed across CaCo-2 cells by adsorptive endocytosis and vesicular mediated transport.

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    Lene E Johannessen

    Full Text Available Dietary DNA is degraded into shorter DNA-fragments and single nucleosides in the gastrointestinal tract. Dietary DNA is mainly taken up as single nucleosides and bases, but even dietary DNA-fragments of up to a few hundred bp are able to cross the intestinal barrier and enter the blood stream. The molecular mechanisms behind transport of DNA-fragments across the intestine and the effects of this transport on the organism are currently unknown. Here we investigate the transport of DNA-fragments across the intestinal barrier, focusing on transport mechanisms and rates. The human intestinal epithelial cell line CaCo-2 was used as a model. As DNA material a PCR-fragment of 633 bp was used and quantitative real time PCR was used as detection method. DNA-fragments were found to be transported across polarized CaCo-2 cells in the apical to basolateral direction (AB. After 90 min the difference in directionality AB vs. BA was >10(3 fold. Even undegraded DNA-fragments of 633 bp could be detected in the basolateral receiver compartment at this time point. Transport of DNA-fragments was sensitive to low temperature and inhibition of endosomal acidification. DNA-transport across CaCo-2 cells was not competed out with oligodeoxynucleotides, fucoidan, heparin, heparan sulphate and dextrane sulphate, while linearized plasmid DNA, on the other hand, reduced transcytosis of DNA-fragments by a factor of approximately 2. Our findings therefore suggest that vesicular transport is mediating transcytosis of dietary DNA-fragments across intestinal cells and that DNA binding proteins are involved in this process. If we extrapolate our findings to in vivo conditions it could be hypothesized that this transport mechanism has a function in the immune system.

  9. Effect of dephytinization on bioavailability of iron, calcium and zinc from infant cereals assessed in the Caco-2 cell model

    Institute of Scientific and Technical Information of China (English)

    Carmen Frontela; Maria Laura Scarino; Simonetta Ferruzza; Gaspar Ros; Carmen Martínez

    2009-01-01

    AIM: To test the effect of the dephytinization of three different commercial infant cereals on iron, calcium,and zinc bioavailability by estimating the uptake, retention,and transport by Caco-2 cells.METHODS: Both dephytinized (by adding an exogenous phytase) and non-dephytinized infant cereals were digested using an in vitro digestion protocol adapted to the gastrointestinal conditions of infants younger than 6 mo. Mineral cell retention, transport,and uptake from infant cereals were measured using the soluble fraction of the simulated digestion and the Caco-2 cells.RESULTS: Dephytinization of infant cereals significantly increased ( P < 0.05) the cell uptake efficiency (from 0.66%-6.05% to 3.93%-13%), retention (from 6.04%-16.68% to 14.75%-20.14%) and transport efficiency (from 0.14%-2.21% to 1.47%-6.02%), of iron, and the uptake efficiency (from 5.0%-35.4% to 7.3%-41.6%) and retention (from 4.05%-20.53% to 14.45%-61.3%) of zinc, whereas calcium only cell uptake showed a significant increase ( P < 0.05) after removing phytate from most of the samples analyzed.A positive relationship ( P < 0.05) between mineral solubility and the cell uptake and transport efficiencies was observed.

  10. 槲皮素及其糖苷衍生物在Caco-2单层细胞上的吸收特征%ABSORPTION OF QUERCETIN AND ITS GLYCOSIDES BY CACO-2 CELL MONOLAYER

    Institute of Scientific and Technical Information of China (English)

    李素云; 李峥; 李敬来; 高蔚娜; 张振清; 郭长江

    2012-01-01

    目的 探讨槲皮素及其糖苷衍生物在人小肠吸收模型Caco-2细胞单层上的吸收特征.方法 采用Caco-2单层细胞模型,研究槲皮素、槲皮苷、异槲皮苷的肠道吸收特征,以LC-MS法测定槲皮素、槲皮苷、异槲皮苷、异鼠李亭.结果 槲皮素及其糖苷衍生物孵育30~150min后,均能在透过侧检测到槲皮素、槲皮苷和异槲皮苷以及槲皮素的甲基化代谢产物之一异鼠李亭;透过的槲皮素及其代谢产物异鼠李亭含量在150min孵育时间内呈先升后降的趋势特征;而槲皮苷和异槲皮苷的透过量则随孵育时间呈持续升高的趋势,在孵育后期可检测到微量异鼠李亭.结论 槲皮素、槲皮苷和异槲皮苷可以完整的分子形式被Caco-2细胞单层吸收,其吸收特征有显著差异,并在吸收过程中伴有广泛的代谢转化.%Objective To investigate the intestinal absorption of quercetin and its glycosides. Method The absorption of quercetin, quercitrin, isoquercitrin and isorhamnetin were studied using Caco-2 cell monolayer by liquid chromatography-mass spectrometry (HPLC-MS) method. Results Quercetin, quercitrin, isoquercitrin and isorhamnetin were found in the receiver sides throughout 150 min incubation period. The contents of quercetin and isorhamnetin in the receiver sides reached to the peak volume at 120 min, and then decreased gradually. Meanwhile, the contents of quercitrin and isoquercitrin rose up in a linear manner during incubation. The isorhamnetin could also be detected. Conclusion Quercetin and its glycosides can transport wholly across the Caco-2 cell monolayer and subject to an extensive metabolism, but there is remarkable difference between them.

  11. Inhibition of tea extracts on glucose uptaking by polarized intestinal Caco-2 cells%茶叶提取物对小肠Caco-2细胞糖吸收的抑制作用

    Institute of Scientific and Technical Information of China (English)

    倪德江; 陈玉琼; 余志; 彼得 R.埃利斯; 克里斯托弗 P.科尔佩

    2013-01-01

    The green tea extract (GT), oolong tea extract (OT), black tea extract (BT) ,Qingzhuan tea extract (QT) and Pu-erh tea extract (PT) were prepared by using water and the effect of them on intestinal glucose uptaking was investigated using polarized Caco-2 intestinal cells. The extracts (0. 5 mg/mL) inhibited glucose uptaking into the intestinal Caco-2 cells under sodium-dependent conditions in the order:GT>OT>BT>QT>PT. Compared with control,GT,OT,BT and QT inhibited significantly glucose-uptaking (P0. 05). Catechin may be the predominant inhibitor in tea and its conversion during manufacturing could affect the glucose-uptake inhibitory activity.%利用水提法制备绿茶、乌龙茶、红茶、青砖茶和普洱茶提取物,研究5种提取物对体外小肠Caco-2细胞吸收葡萄糖的抑制效果.结果表明:在相同质量浓度(0.5 mg/mL)条件下,5种茶叶提取物均能抑制Caco-2细胞吸收葡萄糖的活性,抑制能力大小依次为绿茶>乌龙茶>红茶>青砖茶>普洱茶;与对照相比,绿茶、乌龙茶、红茶和青砖茶提取物极显著抑制葡萄糖的吸收,但普洱茶提取物的抑制效果并未达到显著水平(P>0.05).茶叶提取物抑制小肠Caco-2细胞吸收葡萄糖的能力可能与加工过程中儿茶素的转化有关.

  12. The absorptive flux of the anti-epileptic drug substance vigabatrin is carrier-mediated across Caco-2 cell monolayers

    DEFF Research Database (Denmark)

    Nøhr, Martha Kampp; Hansen, Steen Honoré; Brodin, Birger;

    2014-01-01

    Vigabatrin is an anti-epileptic drug substance. The oral bioavailability of vigabatrin is high (60-70%), however, little is known about the mechanism(s) mediating the intestinal absorption. The aim of the present study was to identify which solute carrier(s) are involved in the absorption...... of the human proton-coupled amino acid transporter (hPAT1) to the apical solution. The present study indicates that the transepithelial A-B flux of vigabatrin is mainly mediated by hPAT1 in Caco-2 cells at dose-relevant concentrations....

  13. Evaluation of the Cytotoxicity of α-Cyclodextrin Derivatives on the Caco-2 Cell Line and Human Erythrocytes

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    Eszter Róka

    2015-11-01

    Full Text Available Cyclodextrins, even the 6-membered α-cyclodextrin, are approved in the various pharmacopoeias as pharmaceutical excipients for solubilizing and stabilizing drugs as well as for controlling drug release. Recently α-cyclodextrin has also been marketed as health food with beneficial effects on blood lipid profiles. However, the concentration of α-cyclodextrin used may be very high in these cases, and its toxic attributes have to be seriously considered. The objective of this study was to investigate the cytotoxicity of various, differently substituted α-cyclodextrin derivatives and determine relationship between the structures and cytotoxicity. Three different methods were used, viability tests (MTT assay and Real Time Cell Electronic Sensing on Caco-2 cells as well as hemolysis test on human red blood cells. The effect of α-cyclodextrin derivatives resulted in concentration-dependent cytotoxicity, so the IC50 values have been determined. Based on our evaluation, the Real Time Cell Electronic Sensing method is the most accurate for describing the time and concentration dependency of the observed toxic effects. Regarding the cytotoxicity on Caco-2 cells, phosphatidylcholine extraction may play a main role in the mechanism. Our results should provide help in selecting those α-cyclodextrin derivatives which have the potential of being used safely in medical formulations.

  14. HT-29 and Caco-2 Reporter Cell Lines for Functional Studies of Nuclear Factor Kappa B Activation

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    Giuliana Mastropietro

    2015-01-01

    Full Text Available The NF-κB is a transcription factor which plays a key role in regulating biological processes. In response to signals, NF-κB activation occurs via phosphorylation of its inhibitor, which dissociates from the NF-κB dimer allowing the translocation to the nucleus, inducing gene expression. NF-κB activation has direct screening applications for drug discovery for several therapeutic indications. Thus, pathway-specific reporter cell systems appear as useful tools to screen and unravel the mode of action of probiotics and natural and synthetic compounds. Here, we describe the generation, characterization, and validation of human epithelial reporter cell lines for functional studies of NF-κB activation by different pro- and anti-inflammatory agents. Caco-2 and HT-29 cells were transfected with a pNF-κB-hrGFP plasmid which contains the GFP gene under the control of NF-κB binding elements. Three proinflammatory cytokines (TNF-α, IL-1β, and LPS were able to activate the reporter systems in a dose-response manner, which corresponds to the activation of the NF-κB signaling pathway. Finally, the reporter cell lines were validated using lactic acid bacteria and a natural compound. We have established robust Caco-2-NF-κB-hrGFP and HT-29-NF-κB-hrGFP reporter cell lines which represent a valuable tool for primary screening and identification of bacterial strains and compounds with a potential therapeutic interest.

  15. Dietary flavanols exert different effects on antioxidant defenses and apoptosis/proliferation in Caco-2 and SW480 colon cancer cells.

    Science.gov (United States)

    Ramos, Sonia; Rodríguez-Ramiro, Ildefonso; Martín, María Angeles; Goya, Luis; Bravo, Laura

    2011-12-01

    Flavanols intake has been associated with reduced risk of cancer. In this study, the anticarcinogenic effects of the flavanols epicatechin (EC), epicatechin-gallate (ECG) and procyanidin B2 (PB2) on Caco-2 and SW480 colon cancer cells were investigated. Catechins showed different cytotoxicity depending on the cell line. ECG displayed strong growth inhibitory effects against SW480 cells, but was ineffective on Caco-2 cells. In contrast, PB2 did not affect Caco-2 cells, whereas promoted cell growth in SW480 cells and EC had no obvious effects on any cell line. Exposure of SW480 cells to ECG led to apoptosis as determined by caspase-3 activity, imbalance among Bcl-2 anti- and pro-apoptotic protein levels, ERK activation and AKT inhibition, whereas PB2 treatment enhanced phospho-AKT and phospho-ERK levels. Incubation of Caco-2 cells with ECG increased glutathione levels without affecting the expression of pro- and anti-apoptotic Bcl-2 proteins, AKT or ERK. The results suggest that the different cytotoxicity of flavanols is caused by their different activity and the degree of differentiation of the colon cancer cell line. Thus, ECG induced apoptosis in SW480 cells and contributed to the cytotoxic effect, whereas ECG enhanced the antioxidant potential in Caco-2 cells. PB2 activated cell proliferation and survival/proliferation pathways in SW480 cells.

  16. Effects of soyasaponin I and soyasaponins-rich extract on the alternariol-induced cytotoxicity on Caco-2 cells.

    Science.gov (United States)

    Vila-Donat, Pilar; Fernández-Blanco, Celia; Sagratini, Gianni; Font, Guillermina; Ruiz, María-José

    2015-03-01

    Alternariol (AOH) is a mycotoxin produced by Alternaria spp. Soyasaponin I (Ss-I) is present naturally in legumes, and it has antioxidant properties. Cytotoxic and genotoxic effects of AOH have been demonstrated previously in vitro. In the present study, the cytotoxicity of AOH, Ss-I, and soyasaponins-rich extract from lentils was investigated; as well as, the cytoprotective effects of Ss-I and lentil extracts against AOH induced-cytotoxicity on Caco-2 cells. Cytotoxicity was carried out using MTT and PC assays (AOH: 3.125-100 µM, Ss-I: 3.125-50 µM, and lentil extracts: 1:0-1:32) during 24 h of exposure. Only AOH showed cytotoxic effect. The reduction in cell proliferation ranged from 25% to 47%. Simultaneous combination of Ss-I with AOH (1:1) increased cell proliferation (35%) compared to AOH tested alone. The Ss-I and extracts showed synergistic cytoprotective effects against cytotoxicity induced by AOH on Caco-2 cells. Food commodities containing Ss-I could contribute to diminish the toxicological risk that natural contaminant as AOH in diet can produce to humans.

  17. Gliadins induce TNFalpha production through cAMP-dependent protein kinase A activation in intestinal cells (Caco-2).

    Science.gov (United States)

    Laparra Llopis, José Moisés; Sanz Herranz, Yolanda

    2010-06-01

    Celiac disease is an autoimmune enteropathy caused by a permanent intolerance to gliadins. In this study the effects of two gliadin-derived peptides (PA2, PQPQLPYPQPQLP and PA9, QLQPFPQPQLPY) on TNFalpha production by intestinal epithelial cells (Caco-2) and whether these effects were related to protein kinase A (PKA) and/or -C (PKC) activities have been evaluated. Caco-2 cell cultures were challenged with several sets of gliadin peptides solutions (0.25 mg/mL), with/without different activators of PKA or PKC, bradykinin (Brdkn) and pyrrolidine dithiocarbamate (PDTC). The gliadin-derived peptides assayed represent the two major immunodominant epitopes of the peptide 33-mer of alpha-gliadin (56-88) (LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF). Both peptides induced the TNFalpha production triggering the inflammatory cell responses, the PA2 being more effective. The addition of the peptides in the presence of dibutyril cyclic AMP (cAMP), Brdkn or PDTC, inhibited the TNFalpha production. The PKC-activator phorbol 12-myristate 13-diacetate additionally increased the PA2- and PA9-induced TNFalpha production. These results link the gliadin-derived peptides induced TNFalpha production through cAMP-dependent PKA activation, where ion channels controlling calcium influx into cells could play a protective role, and requires NF-kappaB activation.

  18. Cytotoxic effect of linear alkylbenzene sulfonate on human intestinal Caco-2 cells: associated biomarkers for risk assessment.

    Science.gov (United States)

    Bradai, Mohamed; Han, Junkyu; El Omri, Abdelfatteh; Funamizu, Naoyuki; Sayadi, Sami; Isoda, Hiroko

    2014-09-01

    Linear alkylbenzene sulfonate (LAS) is a synthetic anionic surfactant widely present in the environment due to its intensive production and use in the detergency field. Admitting that current procedure of risk assessment has limits in providing realistic risk assessment data and predicting the cumulative effect of the toxicant mixtures, the incorporation of information regarding the mode of action and cell response mechanism seems to be a potential solution to overcome these limits. In this regard, we investigated in this study the LAS cytotoxicity on human intestinal Caco-2 cells, trying to unveil the protein actors implicated in the cell response using proteomics approach in order to give a better understanding of the toxicological effect and allow the identification of appropriate biomarkers reflecting the mode of action associated with LAS. As results, we demonstrated that LAS induces a time- and dose-dependent cytotoxicity in Caco-2 cells accompanied by an induction of oxidative stress followed by an excessive increase of intracellular calcium level. Proteomics approach helped in discovering three informative biomarkers of effect associated with LAS cytotoxic effect, reported for the first time: calreticulin, thioredoxin, and heat shock cognate 71 (HSP7C), confirmed by real-time PCR and western blot analysis. These biomarkers could serve for more reliable future risk assessment studies that consider the toxicants mode of action in order to help in the prediction of potential cumulative effects of environmentally coexisting contaminants.

  19. The Appearance of a Leptin Effect on Glucose Absorption in Caco2 Cells Depends on Their Differentiation Level

    Directory of Open Access Journals (Sweden)

    Ola El-Zein

    2015-08-01

    Full Text Available Backdround/Aims: The aim of this work was to study the effect and mechanism of action of leptin added apically, on glucose absorption, using Caco-2 cells as a model. Methods: Cells were grown on inserts and treated with leptin, at different time points after confluence. Radiolabelled glucose was added to the upper chamber and samples from the lower chamber were collected and assayed for radioactivity. Results: Glucose absorption increased with an increase in the level of differentiation and was associated with an increase in the protein expression level of glucose transporters. Leptin reduced glucose absorption only by day 16 after confluence, the time at which apical leptin receptors started appearing. This inhibitory effect became higher the longer the post confluence period, and was prominent on day 23. The hormone effect was found to be mediated via a decrease in the number of glucose transporters (SGLT1 and GLUT2 and a decrease in the activity of the Na+/K+ ATPase which was assayed by measuring the amount of inorganic phosphate liberated in presence and absence of enzyme activators. Conclusion: It was concluded that by day 23 post confluence, Caco-2 cells are differentiated and are appropriate to use as a model for intestinal transport studies.

  20. Almond milk fermented with different potentially probiotic bacteria improves iron uptake by intestinal epithelial (Caco-2 cells

    Directory of Open Access Journals (Sweden)

    Neus Bernat

    2015-04-01

    Full Text Available New fermented almond milks were developed, using different potentially probiotic bacteria, in order to meet the current demand for healthy, versatile non-dairy products. An in vitro digestion/Caco-2 cell model was used to evaluate the effect of both non-fermented and fermented almond milks on the mitochondrial enzymatic activities of enterocytes. Moreover, macrophages were challenged with the in-vitro digested samples and the production of pro-inflammatory biomarkers TNF-a and IL-6 was quantified. Enzymatic activities of cell cultures seemed to be stimulated by the exposure to both fermented and non-fermented almond milks. Both biomarkers decreased (p< 0.05 in fermented almond milks with either B. bifidum or B. longum. Results showed that fermented almond products favored the energetic metabolism of enterocytes and had a lower inflammatory response than non-fermented almond milk, suggesting its benefits for the management of allergies/intolerances. Moreover, the fermentation process enhanced the uptake of iron by Caco-2 cells, especially when using L. rhamnosus and either B. bifidum or B. longum as starters, thus improving the product bioactivity. Therefore, new non-dairy fermented products with functional properties were developed, which might be positioned as alternatives to cow-milk products for sensitized groups of population (allergic and/or intolerant to cow milk or anemic population, among others.

  1. Fluorescently labeled methyl-beta-cyclodextrin enters intestinal epithelial Caco-2 cells by fluid-phase endocytosis.

    Directory of Open Access Journals (Sweden)

    Ferenc Fenyvesi

    Full Text Available Cyclodextrins are widely used excipients for increasing the bioavailability of poorly water-soluble drugs. Their effect on drug absorption in the gastrointestinal tract is explained by their solubility- and permeability-enhancement. The aims of this study were to investigate penetration properties of fluorescently labeled randomly methylated-beta-cyclodextrin (FITC-RAMEB on Caco-2 cell layer and examine the cellular entry of cyclodextrins on intestinal cells. The permeability of FITC-RAMEB through Caco-2 monolayers was very limited. Using this compound in 0.05 mM concentration the permeability coefficient was 3.35±1.29×10(-8 cm/s and its permeability did not change in the presence of 5 mM randomly methylated-beta-cyclodextrin. Despite of the low permeability, cellular accumulation of FITC-RAMEB in cytoplasmic vesicles was significant and showed strong time and concentration dependence, similar to the characteristics of the macropinocytosis marker Lucifer Yellow. The internalization process was fully inhibited at 0°C and it was drastically reduced at 37°C applying rottlerin, an inhibitor of macropinocytosis. Notably, FITC-RAMEB colocalized with the early endosome organizer Rab5a. These results have revealed that FITC-RAMEB is able to enter intestinal epithelial cells by fluid-phase endocytosis from the apical side. This mechanism can be an additional process which helps to overcome the intestinal barrier and contributes to the bioavailability enhancement of cyclodextrins.

  2. Bioactive dietary polyphenols decrease heme iron absorption by decreasing basolateral iron release in human intestinal Caco-2 cells.

    Science.gov (United States)

    Ma, Qianyi; Kim, Eun-Young; Han, Okhee

    2010-06-01

    Because dietary polyphenolic compounds have a wide range of effects in vivo and vitro, including chelation of metals such as iron, it is prudent to test whether the regular consumption of dietary bioactive polyphenols impair the utilization of dietary iron. Because our previous study showed the inhibitory effect of (-) -epigallocatechin-3-gallate (EGCG) and grape seed extract (GSE) on nonheme iron absorption, we investigated whether EGCG and GSE also affect iron absorption from heme. The fully differentiated intestinal Caco-2 cells grown on microporous membrane inserts were incubated with heme (55)Fe in uptake buffer containing EGCG or GSE in the apical compartment for 7 h. Both EGCG and GSE decreased (P heme-derived iron. However, apical heme iron uptake was increased (P heme (55)Fe, the transfer of iron across the intestinal basolateral membrane was extremely low, indicating that basolateral export was impaired by GSE. In contrast, EGCG moderately decreased the cellular assimilation of heme (55)Fe, but the basolateral iron transfer was extremely low, suggesting that the basolateral efflux of heme iron was also inhibited by EGCG. Expression of heme oxygenase, ferroportin, and hephaestin protein was not changed by EGCG and GSE. The apical uptake of heme iron was temperature dependent and saturable in fully differentiated Caco-2 cells. Our data show that bioactive dietary polyphenols inhibit heme iron absorption mainly by reducing basolateral iron exit rather than decreasing apical heme iron uptake in intestinal cells.

  3. Caco-2 cells permeability evaluation of nifuroxazide derivatives with potential activity against methicillin-resistant Staphylococcus aureus (MRSA).

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    B Fernandes, Mariane; Gonçalves, José E; C Tavares, Leoberto; Storpirtis, Sílvia

    2015-01-01

    Throughout the period of evaluation and selection in drug development, the assessment of the permeability potential of a compound to achieve an efficient refinement of the molecular structure has been widely appraised by the transport of substances across cell monolayers. This study aims to develop in vitro assays through Caco-2 cells in order to analyze the permeability of 5-nitro-heterocyclic compounds analogues to nifuroxazide with antimicrobial activity, especially showing promising activity against multidrug-resistant Staphylococcus aureus (MRSA). Caco-2 cell monolayers cultivated for 21 days in Transwell® plates were used for the in vitro permeability assays. The quantification of the nifuroxazide derivatives in the basolateral chambers was performed by a validated high performance liquid chromatography with UV (HPLC-UV) method. Apparent permeability values (Papp) show that these compounds can be considered as new drug candidates with the potential to present high absorption in vivo, according to the classifications of Yee and Biganzoli. The thiophenic derivatives showed permeability values higher than the furanic ones, being AminoTIO the compound with the greatest potential for the development of a new drug against MRSA, since it showed the best cytotoxicity, permeability and solubility ratio among all the derivatives.

  4. 黄芪多糖对Caco-2细胞P-gp功能、表达的影响%Effect of Astragalus Polysaccharide on the Function and Expression of P-glycoprotein in Caco-2 cells

    Institute of Scientific and Technical Information of China (English)

    陈琳瑶; 阮丹; 王跃鑫; 黄巧玲

    2015-01-01

    Objective To investigate the effect of astragalus polysaccharide(APS) on the function and expression of p-glycoprotein (P-gp) in Caco-2 cells. Methods The cytotoxicity of different drug concentrations was measured by MTT assay, in order to confirm the maximum non-toxic concentration of the drug. Flow cytometry was used to detect the fluorescence of rhodamine 123 concentration and antibody concentration to evaluate the effect of APS on the function and expression of P-gp. The Caco-2 cell monolayer was built to evaluate the effect of APS on the bi-directional transports of Rh-123. Results The cell viability of Caco-2 cells was higher than 90% when the concentration of APS was between 0.1 and 100μg/mL with MTT. Compared with control groups, APS increased the cellular Rh-123 concentration, showing an inhibitory effect on P-gp function. When the cells treated with APS at 50 and 100μg/mL concentrations, respectively, the expression levels of P-gp in Caco-2 cells were decreased by 11.23% and 16.41% to 26.72±2.43 and 23.97±2.08 compared to that in control group (30.10±1.28, P<0.05). Rh-123 bi-directional transport experiment showed that the p-gp level was obviously inhibited after 72 h incubation with 100 μg/mL concentration APS. Conclusion APS can inhibit the function and expression of P-gp in Caco-2 cells, and can also reduce the bi-directional transport of Rh-123 when it is at a high concentration.%目的:观察黄芪多糖(APS)对Caco-2细胞P糖蛋白(P-gp)功能、表达的影响。方法采用MTT法考察药物对细胞的毒性,确定药物最大无毒浓度;流式细胞仪测定Caco-2细胞P-gp底物罗丹明-123(Rh-123)和抗体的荧光强度,评价APS对P-gp功能和表达的影响;建立Caco-2细胞单层模型,观察APS对Rh-123跨膜转运的影响。结果细胞毒性实验结果显示,APS在0.1~100μg/mL浓度范围内对Caco-2细胞无明显毒性,细胞存活率跃90%;不同浓度的APS作用后,增加细胞内Rh-123

  5. Transport and metabolism of flavonoids from Chinese herbal remedy Xi-aochaihu-tang across human intestinal Caco-2 cell monolayers

    Institute of Scientific and Technical Information of China (English)

    Jie-yu DAI; Jun-ling YANG; Chuan LI

    2008-01-01

    Aim:To investigate the limiting factors for oral bioavailability of flavonoids derived from the Chinese herbal remedy Xiaochaihu-tang. The investi-gational flavonoids included baicalin, wogonoside, oroxylin-A 7-O-β-D-glucopyranosiduronide, liquiritin, liquiritin apioside, isoliquiritin, and isoliquiri-tin apioside, as well as their aglycones baicalein, wogonin, oroxylin-A, liquir-itigenin, and isoliquiritigenin. Methods: Caco-2 cell monolayers were used and the apparent permeability both in apical to basolateral and basolateral to apical directions was measured for each investigational compound. Meanwhile, chemoinformatics was carried out to provide insight into the mechanism govern-ing the permeability. In addition, carrier-mediated transport with or without in-hibitors, as well as the metabolism by conjugation, was also examined with Ca-co-2 cell monolayers for the flavonoids. Results: The investigational flavonoid aglycones exhibited favorable membrane permeability, but efficient glucuronida-tion and/or sulfation by the enterocytes may limit their bioavailability. For the flavonoid glycosides, their poor membrane permeability was found to be caused by high hydrogen-bonding potential. Among the glycosides, oroxylin-A 7-O-β-D-glucopyranosiduronide, isoliquiritin, and isoliquiritin apioside were transport-ed under the mediation of the efflux transporters multidrug resistance-associated protein and/or P-glycoprotein. Conclusion: The limiting factors of oral bioavail-ability for the flavonoids derived from Xiaochaihu-tang appeared to include poor membrane permeability, significant effiux, and efficient intestinal metabolism by conjugation.

  6. Nutriomic analysis of fresh and processed fruit products. 2. During in vitro simultaneous molecular passages using Caco-2 cell monolayers.

    Science.gov (United States)

    Epriliati, Indah; D'Arcy, Bruce; Gidley, Mike

    2009-04-22

    Many studies have used Caco-2 cell monolayers as human intestinal absorption models. However, only a few studied digested foods, instead of pure standard compounds. Moreover, beneficial and nutritional molecules (nutriome) have not been investigated simultaneously. The present study explored nutriome passages from digest solution of fresh, dried, and juiced tomato, mango, and papaya using Caco-2 cell monolayers in apical-->basolateral directions. A validation method using complementary TEER and P(app) values or internal standard caffeine is recommended because physiologically passive diffusion is unlikely to happen. Sugars were transported into basolateral sides, resulting in potential glucose equivalent bioavailability of 2.26-75 mg h(-1)/100 g (WB). Using sugar passage rates (DB) of juices as 100% references, the rate order was tomato (49.8% dried; 89.5% fresh) > mango (56.8% dried; 22.8% fresh) > papaya (18.7% dried; 36.7% fresh). Major indications that phytochemical absorption does not occur in the small intestine were obtained from the bioassay condition selected. Apical organic acid levels decreased, which occasionally were transported into basolateral sides, whereas the disappearances of apical carotenoids and phenolics were not. Pectin substances were predicted to be responsible for the disappearances of bioactive compounds in those pectin-rich fruits. Further investigations on the role of pectin substances in intestinal passages are recommended.

  7. The effect of phytic acid on tight junctions in the human intestinal Caco-2 cell line and its mechanism.

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    Fu, Qingxue; Wang, Huizhen; Xia, Mengxin; Deng, Bing; Shen, Hongyi; Ji, Guang; Li, Guowen; Xie, Yan

    2015-12-01

    This study investigated the effect of phytic acid (IP6), a potential absorption enhancer of flavonoid components, on tight junction (TJ) integrity in Caco-2 cell monolayers and its possible mechanisms. Transepithelial electrical resistance (TEER) across the monolayers decreased rapidly, and the flux of fluorescein sodium (a paracellular marker) increased after treating with IP6 in a concentration-dependent manner. Confocal microscopy results showed that IP6 produced a concentration-dependent attenuation in the distribution of occludin, ZO-1, and claudin-1. Immunoblot analysis revealed that IP6 could down-regulate the expression level of these TJ proteins, which resulted in the opening of TJ. Additionally, the divalent cations Ca(2+) and Mg(2+) influenced the IP6-induced distribution of occludin, ZO-1, and claudin-1 in different directions, which enhanced barrier function. In conclusion, IP6 can decrease the integrity of Caco-2 cell monolayers by modulating the TJ proteins' localization and down-regulating the expression levels of TJ proteins including claudin-1, occludin, and ZO-1; the reduction effects of divalent cations such as Ca(2+) and Mg(2+) on the regulation of TJ induced by IP6 should be addressed. The present work will offer some useful guidance for the application of IP6 in drug delivery area.

  8. Influence of gastrointestinal system conditions on adhesion of exopolysaccharide-producing Lactobacillus delbrueckii subsp. bulgaricus strains to caco-2 cells

    Directory of Open Access Journals (Sweden)

    Derya Onal Darilmaz

    2011-10-01

    Full Text Available This study aimed to assess the transit tolerance of potential probiotic dairy Lactobacillus strains in human uppergastrointestinal tract in vitro, and to evaluate the effect of EPS production on the viability and adhesion of these strains. Survival and adhesion of two exopolysaccharide (EPS-producing L. delbrueckii subsp. bulgaricus strains (B3 and B2 and E. coli ATCC11229 were assessed after the exposure of different pH (gastric juice and gastric plus pancreatic juice challenges. In the artificial gastric juice (pH 2, both the viability of the strain B3 and B2 was decreased. Artificial juice treatments significantly reduced the adhesion to caco-2 cells (P< 0.05. High EPS-producing B3 survived better in the adverse gastrointestinal conditions and showed better ability of adhesion to Caco-2 cells when assessed for competition with E. coli ATCC 11229 compared to low EPS-producing B2. This investigation showed that EPS production could be affected or be involved in the viability, adherence and competition of L. delbrueckii subsp. bulgaricus strains and support the potential of B3 strain for development of new probiotic products.

  9. Alanylglutamine dipeptide and growth hormone maintain PepT1-mediated transport in oxidatively stressed Caco-2 cells.

    Science.gov (United States)

    Alteheld, B; Evans, M E; Gu, L H; Ganapathy, V; Leibach, F H; Jones, D P; Ziegler, T R

    2005-01-01

    Reactive oxygen species (ROS) produced by gut mucosal cells during conditions such as inflammatory bowel disease (IBD) may impair mucosal repair and nutrient transport/absorptive function. Absorption of di- and tripeptides in the small intestine and colon is mediated by the H(+)-dependent transporter PepT1, but effects of oxidative stress on di- and tripeptide transport are unknown. We assessed whether exposure to hydrogen peroxide (H(2)O(2)) influences dipeptide transport in human colonic epithelial (Caco-2) cells. Uptake of [(14)C]glycylsarcosine (Gly-Sar) was used to evaluate PepT1-mediated dipeptide transport. Exposure to 1-5 mmol/L H(2)O(2) for 24 h caused a dose-dependent decrease in Gly-Sar transport, which was associated with decreased PepT1 transport velocity (V(max)). Treatment with alanylglutamine (Ala-Gln) or growth hormone (GH) did not alter Caco-2 Gly-Sar transport in the absence of H(2)O(2). However, both Ala-Gln and GH prevented the decrease in dipeptide transport observed with 1 mmol/L H(2)O(2) treatment. Ala-Gln, but not GH, maintained cellular glutathione and prevented the decrease in PepT1 protein expression. Thus, these agents should be further investigated as potential therapies to improve absorption of small peptides in disorders associated with oxidative injury to the gut mucosa.

  10. Fatty acids affect micellar properties and modulate vitamin D uptake and basolateral efflux in Caco-2 cells.

    Science.gov (United States)

    Goncalves, Aurélie; Gleize, Béatrice; Roi, Stéphanie; Nowicki, Marion; Dhaussy, Amélie; Huertas, Alain; Amiot, Marie-Josèphe; Reboul, Emmanuelle

    2013-10-01

    We have recently shown that vitamin D3 (cholecalciferol) absorption is not a simple passive diffusion but involves cholesterol transporters. As free fatty acids (FAs) modulate cholesterol intestinal absorption and metabolism, we hypothesized that FAs may also interact with vitamin D absorption. Effects of FAs were evaluated at different levels of cholecalciferol intestinal absorption. First, the physicochemical properties of micelles formed with different FAs were analyzed. The micelles were then administered to human Caco-2 cells in culture to evaluate FA effects on (i) cholecalciferol uptake and basolateral efflux and (ii) the regulation of genes coding proteins involved in lipid absorption process. Micellar electric charge was correlated with both FA chain length and degree of unsaturation. Long-chain FAs at 500 μM in mixed micelles decreased cholecalciferol uptake in Caco-2 cells. This decrease was annihilated as soon as the long-chain FAs were mixed with other FAs. Oleic acid significantly improved cholecalciferol basolateral efflux compared to other FAs. These results were partly explained by a modulation of genes coding for lipid transport proteins such as Niemann-pick C1-like 1 and scavenger receptor class B type I. The data reported here show for the first time that FAs can interact with cholecalciferol intestinal absorption at different key steps of the absorption process. Cholecalciferol intestinal absorption may thus be optimized according to oil FA composition.

  11. Study on Absorption of Theaflavin and EGCG in Caco-2 Cell Model%TF1与EGCG在Caco-2细胞中的吸收规律研究

    Institute of Scientific and Technical Information of China (English)

    孟庆; 屠幼英

    2015-01-01

    为了探究茶黄素单体(TF1)与表没食子儿茶素没食子酸酯(EGCG)在生物体中的吸收规律,本实验建立了体外 Caco-2单层细胞模型,模拟小肠对 TF1与 EGCG的吸收,并研究了浓度和时间对吸收的影响。结果表明,在10~100µmol·L-1范围内,TF1与 EGCG 在 Caco-2单层细胞模型中的吸收呈现表观渗透系数 Papp随浓度增加而上升的规律。但两者的外排率也呈现同样的规律,并且外排率上升的幅度均大于二者吸收率上升的幅度。由于 TF1与 EGCG 在细胞单层模型中的 Papp 均小于1×10-6 cm·s -1,说明两者都属于难吸收的药物,但是TF1在Caco-2细胞模型中的吸收率高于EGCG。因其外排比均大于2,说明两者在细胞模型上的外排是被动转运。从吸收时间看,TF1的外排规律与EGCG一致。%In order to explore the absorption regularity of TF1 and EGCG in the organism. This research used the Caco-2 cell monolayer model in vitro to simulate the absorption of TF1 and EGCG in the small intestine. The influences of concentration and time on the absorption regularity of TF1 and EGCG in Caco-2 cell monolayer model were investigated. The results illustrated that the absorption of TF1 and EGCG in Caco-2 model showed that the apparent permeability coefficient raised with the increasing of concentrations of two compound in the range of 10~100 µ mol·L-1. The efflux rates of the TF1 and EGCG showed the same rules as absorption. However, the increasing range of efflux rate was higher than that of absorption rate. The values of Papp about TF 1 and EGCG in the cell model were lower than 1×10-6 cm·s -1, which indicated that both of them belonged to the kind of drugs which were difficult to absorb. However, with comparing the absorption rate, TF1 was higher than EGCG in this model. Both of efflux transport showed the passive process because the efflux rates of TF 1 and EGCG were larger than 2 and higher than in cell

  12. Curcumin inhibits cholesterol uptake in Caco-2 cells by down-regulation of NPC1L1 expression

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    Duan Rui-Dong

    2010-04-01

    Full Text Available Abstract Background Curcumin is a polyphenol and the one of the principle curcuminoids of the spice turmeric. Its antioxidant, anti-cancer and anti-inflammatory effects have been intensively studied. Previous in vivo studies showed that administration of curcumin also decreased cholesterol levels in the blood, and the effects were considered to be related to upregulation of LDL receptor. However, since plasma cholesterol levels are also influenced by the uptake of cholesterol in the gut, which is mediated by a specific transporter Niemann-Pick Cl-like 1 (NPC1L1 protein, the present study is to investigate whether curcumin affects cholesterol uptake in the intestinal Caco-2 cells. Methods Caco-2 cells were cultured to confluence. The micelles composed of bile salt, monoolein, and 14C-cholesterol were prepared. We first incubated the cells with the micelles in the presence and absence of ezetimibe, the specific inhibitor of NPC1L1, to see whether the uptake of the cholesterol in the cells was mediated by NPC1L1. We then pretreated the cells with curcumin at different concentrations for 24 h followed by examination of the changes of cholesterol uptake in these curcumin-treated cells. Finally we determined whether curcumin affects the expression of NPC1L1 by both Western blot analysis and qPCR quantification. Results We found that the uptake of radioactive cholesterol in Caco-2 cells was inhibited by ezetimibe in a dose-dependent manner. The results indicate that the uptake of cholesterol in this study was mediated by NPC1L1. We then pretreated the cells with 25-100 μM curcumin for 24 h and found that such a treatment dose-dependently inhibited cholesterol uptake with 40% inhibition obtained by 100 μM curcumin. In addition, we found that the curcumin-induced inhibition of cholesterol uptake was associated with significant decrease in the levels of NPC1L1 protein and NPC1L1 mRNA, as analyzed by Western blot and qPCR, respectively. Conclusion

  13. Toxicological Effects of Caco-2 Cells Following Short-Term and Long-Term Exposure to Ag Nanoparticles

    Science.gov (United States)

    Chen, Ni; Song, Zheng-Mei; Tang, Huan; Xi, Wen-Song; Cao, Aoneng; Liu, Yuanfang; Wang, Haifang

    2016-01-01

    Extensive utilization increases the exposure of humans to Ag nanoparticles (NPs) via the oral pathway. To comprehensively address the action of Ag NPs to the gastrointestinal systems in real situations, i.e., the long-term low-dose exposure, we evaluated and compared the toxicity of three Ag NPs (20–30 nm with different surface coatings) to the human intestine cell Caco-2 after 1-day and 21-day exposures, using various biological assays. In both the short- and long-term exposures, the variety of surface coating predominated the toxicity of Ag NPs in a descending order of citrate-coated Ag NP (Ag-CIT), bare Ag NP (Ag-B), and poly (N-vinyl-2-pyrrolidone)-coated Ag NP (Ag-PVP). The short-term exposure induced cell growth inhibition and death. The cell viability loss appeared after cells were exposed to 0.7 μg/mL Ag-CIT, 0.9 μg/mL Ag-B or >1.0 μg/mL Ag-PVP for 24 h. The short-term and higher-dose exposure also induced reactive oxygen species (ROS) generation, mitochondrial damage, cell membrane leakage, apoptosis, and inflammation (IL-8 level). The long-term exposure only inhibited the cell proliferation. After 21-day exposure to 0.4 μg/mL Ag-CIT, the cell viability dropped to less than 50%, while cells exposed to 0.5 μg/mL Ag-PVP remained normal as the control. Generally, 0.3 μg/mL is the non-toxic dose for the long-term exposure of Caco-2 cells to Ag NPs in this study. However, cells presented inflammation after exposure to Ag NPs with the non-toxic dose in the long-term exposure. PMID:27338357

  14. Toxicological Effects of Caco-2 Cells Following Short-Term and Long-Term Exposure to Ag Nanoparticles

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    Ni Chen

    2016-06-01

    Full Text Available Extensive utilization increases the exposure of humans to Ag nanoparticles (NPs via the oral pathway. To comprehensively address the action of Ag NPs to the gastrointestinal systems in real situations, i.e., the long-term low-dose exposure, we evaluated and compared the toxicity of three Ag NPs (20–30 nm with different surface coatings to the human intestine cell Caco-2 after 1-day and 21-day exposures, using various biological assays. In both the short- and long-term exposures, the variety of surface coating predominated the toxicity of Ag NPs in a descending order of citrate-coated Ag NP (Ag-CIT, bare Ag NP (Ag-B, and poly (N-vinyl-2-pyrrolidone-coated Ag NP (Ag-PVP. The short-term exposure induced cell growth inhibition and death. The cell viability loss appeared after cells were exposed to 0.7 μg/mL Ag-CIT, 0.9 μg/mL Ag-B or >1.0 μg/mL Ag-PVP for 24 h. The short-term and higher-dose exposure also induced reactive oxygen species (ROS generation, mitochondrial damage, cell membrane leakage, apoptosis, and inflammation (IL-8 level. The long-term exposure only inhibited the cell proliferation. After 21-day exposure to 0.4 μg/mL Ag-CIT, the cell viability dropped to less than 50%, while cells exposed to 0.5 μg/mL Ag-PVP remained normal as the control. Generally, 0.3 μg/mL is the non-toxic dose for the long-term exposure of Caco-2 cells to Ag NPs in this study. However, cells presented inflammation after exposure to Ag NPs with the non-toxic dose in the long-term exposure.

  15. Absorption Mechanism of Dauricine based on Caco-2 Cell Line%基于Caco-2细胞模型研究蝙蝠葛碱的跨膜吸收机制

    Institute of Scientific and Technical Information of China (English)

    高秀蓉; 蒋学华; 杜青青

    2012-01-01

    Objective: To investigate the absorption mechanism of dauricine based on Caco-2 cell line. Method; Bidirectional transport of dauricine in Caco-2 cell model was used; apparent permeability coefficients (Papp) , efflux ratio (ER) and accumulation transport amount were calculated; the effect of drug concentration , pH gradient and EGTA on the bidirectional transport of dauricine were studied. Result: At high, middle and low concentrations the efflux ratio ( ER) was 1.11, 4.49, 7.24 respectively, so there was significant polar phenomenon for transport of dauricine; at apical lateral when pH was 7. 4 and 6. 5, ER were 3. 95 and 9. 38 respectively, so pH gradient could reduce the absorption of dauricine; chelator EGTA couldn't affect the Pa , ER and accumulation transport amount of dauricine. Conclusion; The active transport was included in the absorption mechanism of dauricine, pH gradient could increase the polar phenomenon, transmembrane pathway is the main absorption route for dauricine.%目的:研究蝙蝠葛碱在Caco-2细胞模型中的跨膜转运机制.方法:采用Caco-2细胞模型,进行A-B和B-A双向转运实验,计算表观渗透系数(Papp)、外排比率(ER)和累积转运量,考察蝙蝠葛碱不同质量浓度、细胞两侧pH梯度和螫合剂EGTA对蝙蝠葛碱转运的影响.结果:高、中、低浓度下的ER分别为1.11,4.49和7.24,即中、低浓度下转运有明显极化现象;顶侧pH 7.4和6.5时,ER值分别为3.95和9.38,即pH梯度存在时,蝙蝠葛碱吸收减少,外排增加;加入螯合剂EGTA后,Papp,ER和累积转运量均无显著改变.结论:蝙蝠葛碱的吸收有主动转运机制存在;pH梯度能驱动了蝙蝠葛碱的外排转运,偏碱性环境较酸性环境易于吸收;其吸收途径主要为跨细胞通道转运.

  16. Effect of calcium, tannic acid, phytic acid and pectin over iron uptake in an in vitro Caco-2 cell model.

    Science.gov (United States)

    Andrews, M; Briones, L; Jaramillo, A; Pizarro, F; Arredondo, M

    2014-04-01

    Calcium, phytic acid, polyphenols and fiber are major inhibitors of iron absorption and they could be found in excess in some diets, thereby altering or modifying the iron nutrition status. The purpose of this study is to evaluate the effect of calcium, tannic acid, phytic acid, and pectin over iron uptake, using an in vitro model of epithelial cells (Caco-2 cell line). Caco-2 cells were incubated with iron (10-30 μM) with or without CaCl2 (500 and 1,000 μM) for 24 h. Then, cells were challenged with phytic acid (50-150 μM); pectin (50-150 nM) or tannic acid (100-500 μM) for another 24 h. Finally, (55)Fe (10 μM) uptake was determined. Iron dialyzability was studied using an in vitro digestion method. Iron uptake in cells pre-incubated with 20 and 30 μM Fe was inhibited by CaCl2 (500 μM). Iron uptake decreased in cells cultured with tannic acid (300 μM) and CaCl2 (500-1,000 μM) (two-way ANOVA, p = 0.002). Phytic acid also decreased iron uptake mainly when cells were treated with CaCl2 (1,000 μM) (two-way ANOVA; p phytic or tannic acid (T test p Phytic acid combined with calcium is a strong iron uptake inhibitor. Pectin slightly decreased iron uptake with or without calcium. Tannic acid showed an unexpected behavior, inducing an increase on iron uptake, despite its low Fe dialyzability.

  17. Sub-emetic toxicity of Bacillus cereus toxin cereulide on cultured human enterocyte-like Caco-2 cells.

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    Rajkovic, Andreja; Grootaert, Charlotte; Butorac, Ana; Cucu, Tatiana; De Meulenaer, Bruno; van Camp, John; Bracke, Marc; Uyttendaele, Mieke; Bačun-Družina, Višnja; Cindrić, Mario

    2014-08-04

    Cereulide (CER) intoxication occurs at relatively high doses of 8 µg/kg body weight. Recent research demonstrated a wide prevalence of low concentrations of CER in rice and pasta dishes. However, the impact of exposure to low doses of CER has not been studied before. In this research, we investigated the effect of low concentrations of CER on the behavior of intestinal cells using the Caco-2 cell line. The MTT (mitochondrial 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and the SRB (sulforhodamine B) reactions were used to measure the mitochondrial activity and cellular protein content, respectively. Both assays showed that differentiated Caco-2 cells were sensitive to low concentrations of CER (in a MTT reaction of 1 ng/mL after three days of treatment; in an SRB reaction of 0.125 ng/mL after three days of treatment). Cell counts revealed that cells were released from the differentiated monolayer at 0.5 ng/mL of CER. Additionally, 0.5 and 2 ng/mL of CER increased the lactate presence in the cell culture medium. Proteomic data showed that CER at a concentration of 1 ng/mL led to a significant decrease in energy managing and H2O2 detoxification proteins and to an increase in cell death markers. This is amongst the first reports to describe the influence of sub-emetic concentrations of CER on a differentiated intestinal monolayer model showing that low doses may induce an altered enterocyte metabolism and membrane integrity.

  18. Sub-Emetic Toxicity of Bacillus cereus Toxin Cereulide on Cultured Human Enterocyte-Like Caco-2 Cells

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    Andreja Rajkovic

    2014-08-01

    Full Text Available Cereulide (CER intoxication occurs at relatively high doses of 8 µg/kg body weight. Recent research demonstrated a wide prevalence of low concentrations of CER in rice and pasta dishes. However, the impact of exposure to low doses of CER has not been studied before. In this research, we investigated the effect of low concentrations of CER on the behavior of intestinal cells using the Caco-2 cell line. The MTT (mitochondrial 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide and the SRB (sulforhodamine B reactions were used to measure the mitochondrial activity and cellular protein content, respectively. Both assays showed that differentiated Caco-2 cells were sensitive to low concentrations of CER (in a MTT reaction of 1 ng/mL after three days of treatment; in an SRB reaction of 0.125 ng/mL after three days of treatment. Cell counts revealed that cells were released from the differentiated monolayer at 0.5 ng/mL of CER. Additionally, 0.5 and 2 ng/mL of CER increased the lactate presence in the cell culture medium. Proteomic data showed that CER at a concentration of 1 ng/mL led to a significant decrease in energy managing and H2O2 detoxification proteins and to an increase in cell death markers. This is amongst the first reports to describe the influence of sub-emetic concentrations of CER on a differentiated intestinal monolayer model showing that low doses may induce an altered enterocyte metabolism and membrane integrity.

  19. Inhibitory action of soybean beta-conglycinin hydrolysates on Salmonella typhimurium translocation in Caco-2 epithelial cell monolayers.

    Science.gov (United States)

    Yang, Baichong; Lv, Ying; Chen, Yang; Wang, Jin; Tang, Wuxia; Guo, Shuntang

    2008-08-27

    Soybean protein hydrolysates are widely used as functional foods as they have antioxidative properties able to enhance immune responses in humans. The alcalase enzymatic hydrolysates of beta-conglycinin were fractionated by ultrafiltration, and two main fractions, SP1 (0.05 g/L of SP2 improved epithelial cell growth and that a concentration of 0.5 g/L of SP2 increased intercellular tight junction formation, which resulted in increased of transepithelial monolayer resistance (TER) values. Moreover, a lower S. typhimurium count compared to control was obtained when Caco-2 cells were grown in 0.05 and 0.5 g/L of SP2. These results show that beta-conglycinin hydrolysates play an important role in resisting S. typhimurium penetration into intestinal epithelial cells and that high molecular mass peptides (10-20 kDa) were more effective overall than low molecular mass peptides.

  20. Acetylcholinesterase inhibition, antioxidant activity and toxicity of Peumus boldus water extracts on HeLa and Caco-2 cell lines.

    Science.gov (United States)

    Falé, P L; Amaral, F; Amorim Madeira, P J; Sousa Silva, M; Florêncio, M H; Frazão, F N; Serralheiro, M L M

    2012-08-01

    This work aimed to study the inhibition on acetylcholinesterase activity (AChE), the antioxidant activity and the toxicity towards Caco-2 and HeLa cells of aqueous extracts of Peumus Boldus. An IC(50) value of 0.93 mg/mL, for AChE inhibition, and EC(50) of 18.7 μg/mL, for the antioxidant activity, was determined. This activity can be attributed to glycosylated flavonoid derivatives detected, which were the main compounds, although boldine and other aporphine derivatives were also present. No changes in the chemical composition or the biochemical activities were found after gastrointestinal digestion. Toxicity of P. boldus decoction gave an IC(50) value 0.66 mg/mL for HeLa cells, which caused significant changes in the cell proteome profile.

  1. ROCK activity affects IL-1-induced signaling possibly through MKK4 and p38 MAPK in Caco-2 cells.

    Science.gov (United States)

    Banerjee, Sayantan; McGee, Dennis W

    2016-09-01

    Elevated levels of interleukin-1 (IL-1) accompany inflammatory bowel disease. IL-1-stimulated intestinal epithelial cells can secrete potent chemokines like CXCL8 to exacerbate inflammation. Previously, we found that inhibiting the Rho-associated kinase (ROCK) could inhibit IL-1- or TNF-α-induced CXCL8 secretion by the Caco-2 colonic epithelial cell line. This ROCK inhibition did not affect IκBα phosphorylation and degradation, but suppressed the phosphorylation of c-Jun N-terminal kinase (JNK). Therefore, ROCK must play an important role in epithelial cell CXCL8 responses through an effect on the JNK signaling pathway. Here, we extend these studies by showing that inhibiting ROCK suppressed the IL-1-induced phosphorylation of MKK4, a known activator of JNK, but not MKK7. Yet, ROCK inhibition had no significant effect on the IL-1-induced phosphorylation of extracellular-signal-regulated kinase (ERK) 1/2. Inhibiting ROCK also suppressed the phosphorylation of p38 MAPK after IL-1 stimulation, but this inhibition had no significant effect on the stability of CXCL8 messenger RNA (mRNA) after IL-1 stimulation. These results suggest that ROCK may be important in IL-1-induced signaling through MKK4 to JNK and the activation of p38 MAPK. Finally, inhibiting ROCK in IL-1 and TNF-α co-stimulated Caco-2 cells also resulted in a significant suppression of CXCL8 secretion and mRNA levels suggesting that inhibiting ROCK may be a mechanism to inhibit the overall response of epithelial cells to both cytokines. These studies indicate a novel signaling event, which could provide a target for suppressing intestinal epithelial cells (IEC) chemokine responses involved in mucosal inflammation.

  2. Inhibitory Effect of Flavonoids on the Efflux of -Acetyl 5-Aminosalicylic Acid Intracellularly Formed in Caco-2 Cells

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    Shin Yoshimura

    2009-01-01

    Full Text Available -acetyl 5-aminosalicylic acid (5-AcASA that was intracellularly formed from 5-aminosalicylic acid (5-ASA at 200 M was discharged 5.3, 7.1, and 8.1-fold higher into the apical site than into the basolateral site during 1, 2, and 4-hour incubations, respectively, in Caco-2 cells grown in Transwells. The addition of flavonols (100 M such as fisetin and quercetin with 5-ASA remarkably decreased the apically directed efflux of 5-AcASA. When 5-ASA (200 M was added to Caco-2 cells grown in tissue culture dishes, the formation of 5-AcASA decreased, and, in addition, the formed 5-AcASA was found to be accumulated within the cells in the presence of such flavonols. Thus, the decrease in 5-AcASA efflux by such flavonols was attributed not only to the inhibition of -acetyl-conjugation of 5-ASA but to the predominant cellular accumulation of 5-AcASA. Various flavonoids also had both of the effects with potencies that depend on their specific structures. The essential structure of flavonoids was an absence of a hydroxyl substitution at the C5 position on the A-ring of flavone structure for the inhibitory effect on the -acetyl-conjugation of 5-ASA, and a presence of hydroxyl substitutions at the C3 or C4 position on the B-ring of flavone structure for the promoting effect on the cellular accumulation of 5-AcASA. Both the decrease in 5-AcASA apical efflux and the increase in 5-AcASA cellular accumulation were also caused by MK571 and indomethacin, inhibitors of MRPs, but not by quinidine, cyclosporin A, P-glycoprotein inhibitors, and mitoxantrone, a BCRP substrate. These results suggest that certain flavonoids suppress the apical efflux of 5-AcASA possibly by inhibiting MRPs pumps located on apical membranes in Caco-2 cells.

  3. Unsaturated fatty acids and phytosterols regulate cholesterol transporter genes in Caco-2 and HepG2 cell lines.

    Science.gov (United States)

    Park, Youngki; Carr, Timothy P

    2013-02-01

    Dietary consumption of phytosterols and certain fatty acids has been shown to reduce cholesterol absorption and plasma cholesterol concentrations. However, it has not been fully elucidated whether phytosterols or fatty acids can alter the expression of cholesterol transporters by functioning as signaling molecules. This study tested the hypothesis that various fatty acids and phytosterols commonly found in the food supply can modulate the expression of transporters including Niemann-Pick C1-like 1, low-density lipoprotein receptor, and scavenger receptor class B type I and 3-hydroxy-3-methylglutaryl-coenzyme A reductase in the intestine and liver. Caco-2 cells were used as models of enterocytes, and HepG2 cells were used as a model of hepatocytes. The cells were treated for 18 hours with 100 μmol/L of a fatty acid, or for 24 hours with 10 μmol/L of 25α-hydroxycholesterol, or 100 μmol/L of cholesterol, sitosterol, and stigmasterol to measure expression of genes involved in cholesterol transport using quantitative real-time polymerase chain reaction. Polyunsaturated fatty acids in Caco-2 cells and sterols in HepG2 cells significantly reduced the messenger RNA expression levels of Niemann-Pick C1-like 1, scavenger receptor class B type I, low-density lipoprotein receptor, and 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Importantly, sitosterol and stigmasterol reduced the messenger RNA levels of genes to a similar extent as cholesterol. The data support the hypothesis that unsaturated fatty acid and phytosterols can act as signaling molecules and alter the expression of genes involved in cholesterol transport and metabolism.

  4. Hispidin derived from Phellinus linteus affords protection against acrylamide-induced oxidative stress in Caco-2 cells.

    Science.gov (United States)

    Chen, Wei; Shen, Yang; Su, Hongming; Zheng, Xiaodong

    2014-08-05

    Acrylamide (AA), a well-known toxicant, has attracted numerous attentions for its presumably carcinogenesis, neurotoxicity and cytotoxicity. Oxidative stress was considered to be associated with acrylamide cytotoxicity, but the link between oxidative stress and acrylamide cytotoxicity is still unclear. In the present study, hispidin produced from the edible fungus Phellinus linteus displayed dramatically antioxidant activities against DPPH radicals, ABTS radicals, ferric reducing and hydroxyl radicals, as well as superoxide anion radicals. Moreover, the cytoprotective effect of hispidin against AA-induced oxidative stress was verified upon Caco-2 cells according to evaluate the cell viability, intracellular ROS, mitochondrial membrane potential (MMP) and glutathione (GSH) in the presence or absence of AA (5 mM) in a dose-dependent manner. Collectively, our results demonstrated for the first time that hispidin was able to inhibit AA-induced oxidative stress, which might have implication for the dietary preventive application.

  5. A selenium-deficient Caco-2 cell model for assessing differential incorporation of chemical or food selenium into glutathione peroxidase.

    Science.gov (United States)

    Zeng, Huawei; Botnen, James H; Johnson, Luann K

    2008-01-01

    Assessing the ability of a selenium (Se) sample to induce cellular glutathione peroxidase (GPx) activity in Se-deficient animals is the most commonly used method to determine Se bioavailability. Our goal is to establish a Se-deficient cell culture model with differential incorporation of Se chemical forms into GPx, which may complement the in vivo studies. In the present study, we developed a Se-deficient Caco-2 cell model with a serum gradual reduction method. It is well recognized that selenomethionine (SeMet) is the major nutritional source of Se; therefore, SeMet, selenite, or methylselenocysteine (SeMSC) was added to cell culture media with different concentrations and treatment time points. We found that selenite and SeMSC induced GPx more rapidly than SeMet. However, SeMet was better retained as it is incorporated into proteins in place of methionine; compared with 8-, 24-, or 48-h treatment, 72-h Se treatment was a more sensitive time point to measure the potential of GPx induction in all tested concentrations. Based on induction of GPx activity, the cellular bioavailability of Se from an extract of selenobroccoli after a simulated gastrointestinal digestion was comparable with that of SeMSC and SeMet. These in vitro data are, for the first time, consistent with previous published data regarding selenite and SeMet bioavailability in animal models and Se chemical speciation studies with broccoli. Thus, Se-deficient Caco-2 cell model with differential incorporation of chemical or food forms of Se into GPx provides a new tool to study the cellular mechanisms of Se bioavailability.

  6. Evaluation of the Availability and Antioxidant Capacity of Maillard Compounds Present in Bread Crust: Studies in Caco-2 Cells

    Science.gov (United States)

    de la Cueva, Silvia Pastoriza; Seiquer, Isabel; Mesías, Marta; Rufián-Henares, José Ángel; Delgado-Andrade, Cristina

    2017-01-01

    Bread crust is one of the major contributors to the intake of Maillard reaction products (MRP). MRP improve the organoleptic properties of foods and can provide biological actions such as antioxidant properties. The transport and availability of Amadori compounds (measured as furosine) and hydroxymethylfurfural (HMF)—early and intermediary MRP—from enzymatically digested bread crust (BC) and from its soluble low-molecular weight (LMW) and high-molecular weight (HMW) fractions were investigated in the Caco-2 cell line. The absorption of the early and final MRP pool was tested by measuring the absorbance recovery (280 and 420 nm). The ability of soluble BC or its fractions to lessen the production of reactive oxygen species (ROS) was examined. Amadori compounds (furosine) were transported across Caco-2 cell monolayers from the soluble BC in percentages ranging between 40% and 56%; the lower amount of the compound supplied, the higher transport rate. However, HMF transport rate (35%) was unaffected by the initial amount of the compound. Amadori compounds and HMF contained in the LMW fraction were more efficiently transported than those present in the HMW fraction, suggesting improved absorption when supplied as free forms or linked to LMW compounds. Absorbance recovery at 280 nm was higher from the LMW fraction, whereas higher recovery was detected for the HMW fraction at 420 nm. The digested BC—but not its isolated fractions—was able to significantly reduce ROS production at basal conditions and after subjecting cells to an oxidant. A clear positive action of BC on the antioxidant defence is manifested, seemingly attributable to the combined presence of soluble LMW and HMW products. PMID:28231083

  7. Evaluation of the Availability and Antioxidant Capacity of Maillard Compounds Present in Bread Crust: Studies in Caco-2 Cells

    Directory of Open Access Journals (Sweden)

    Silvia Pastoriza de la Cueva

    2017-01-01

    Full Text Available Bread crust is one of the major contributors to the intake of Maillard reaction products (MRP. MRP improve the organoleptic properties of foods and can provide biological actions such as antioxidant properties. The transport and availability of Amadori compounds (measured as furosine and hydroxymethylfurfural (HMF—early and intermediary MRP—from enzymatically digested bread crust (BC and from its soluble low-molecular weight (LMW and high-molecular weight (HMW fractions were investigated in the Caco-2 cell line. The absorption of the early and final MRP pool was tested by measuring the absorbance recovery (280 and 420 nm. The ability of soluble BC or its fractions to lessen the production of reactive oxygen species (ROS was examined. Amadori compounds (furosine were transported across Caco-2 cell monolayers from the soluble BC in percentages ranging between 40% and 56%; the lower amount of the compound supplied, the higher transport rate. However, HMF transport rate (35% was unaffected by the initial amount of the compound. Amadori compounds and HMF contained in the LMW fraction were more efficiently transported than those present in the HMW fraction, suggesting improved absorption when supplied as free forms or linked to LMW compounds. Absorbance recovery at 280 nm was higher from the LMW fraction, whereas higher recovery was detected for the HMW fraction at 420 nm. The digested BC—but not its isolated fractions—was able to significantly reduce ROS production at basal conditions and after subjecting cells to an oxidant. A clear positive action of BC on the antioxidant defence is manifested, seemingly attributable to the combined presence of soluble LMW and HMW products.

  8. Olive Oil-Supplemented Lipid Emulsion Induces CELF1 Expression and Promotes Apoptosis in Caco-2 Cells

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    Jun-Kai Yan

    2017-02-01

    Full Text Available Background and Aims: Parenterally-administered lipid emulsion (LE is a key cause of enterocyte apoptosis under total parenteral nutrition, yet the pathogenesis has not been fully understood. CUGBP, Elav-like family member 1 (CELF1 has been recently identified as a crucial modulator of apoptosis, and thus this study sought to investigate its role in the LE-induced apoptosis in vitro. Methods: Caco-2 cells were used as an in vitro model. The cells were treated with varying LEs derived from soybean oil, olive oil or fish oil, and changes in the apoptosis and CELF1 expression were assessed. Rescue study was performed using transient knockdown of CELF1 with specific siRNA prior to LE treatment. Regulation of CELF1 by LE treatment was studied using quantitative real-time PCR and Western blotting. Results: All the LEs up-regulated CELF1expression and induced apoptosis, but only olive oil-supplemented lipid emulsion (OOLE-induced apoptosis was attenuated by depletion of CELF1. Up-regulation of apoptosis-inducing factor (AIF was involved in OOLE-induced CELF1 dependent apoptosis. The protein expression of CELF1 was up-regulated by OOLE in a dose- and time-dependent manner, but the mRNA expression of CELF1 was unchanged. Analysis by polysomal profiling and nascent protein synthesis revealed that the regulation of CELF1 by OOLE treatment was mediated by directly accelerating its protein translation. Conclusion: OOLE-induces apoptosis in Caco-2 cells partially through up-regulation of CELF1.

  9. Transport characteristics of isorhamnetin across intestinal Caco-2 cell monolayers and the effects of transporters on it.

    Science.gov (United States)

    Duan, Jingze; Xie, Yan; Luo, Huilin; Li, Guowen; Wu, Tao; Zhang, Tong

    2014-04-01

    Flavonoid isorhamnetin occurs in various plants and herbs, and demonstrates various biological effects in humans. This work will clarify the isorhamnetin absorption mechanism using the Caco-2 monolayer cell model. The isorhamnetin transport characteristics at different concentrations, pHs, temperatures, tight junctions and potential transporters were systemically investigated. Isorhamnetin was poorly absorbed by both passive diffusion and active transport mechanisms. Both trans- and paracellular pathways were involved during isorhamnetin transport. Active transport under an ATP-dependent transport mechanism was mediated by the organic anion transporting peptide (OATP); isorhamnetin's permeability from the apical to the basolateral side significantly decreased after estrone-3-sulfate was added (pisorhamnetin transport process. Among them, the MRPs (especially MRP2) were the main efflux transporters for isorhamnetin; transport from the apical to the basolateral side increased 10.8-fold after adding an MRP inhibitor (MK571). This study details isorhamnetin's cellular transport and elaborates isorhamnetin's absorption mechanisms to provide a foundation for further studies.

  10. Permeability of Rosmarinic acid in Prunella vulgaris and Ursolic acid in Salvia officinalis Extracts across Caco-2 Cell Monolayers

    Science.gov (United States)

    Qiang, Zhiyi; Ye, Zhong; Hauck, Cathy; Murphy, Patricia A.; McCoy, Joe-Ann; Widrlechner, Mark P.; Reddy, Manju B.; Hendrich, Suzanne

    2011-01-01

    Ethnopharmacological relevance Rosmarinic acid (RA), a caffeic acid-related compound found in high concentrations in Prunella vulgaris (self-heal), and ursolic acid (UA), a pentacyclic triterpene acid concentrated in Salvia officinalis (sage), have been traditionally used to treat inflammation in the mouth, and may also be beneficial for gastrointestinal health in general. Aim of the study To investigate the permeabilities of RA and UA as pure compounds and in P. vulgaris and S. officinalis ethanol extracts across human intestinal epithelial Caco-2 cell monolayers. Materials and methods The permeabilities and Phase II biotransformation of RA and UA as pure compounds and in herbal extracts were compared using Caco-2 cells with HPLC detection. Results The apparent permeability coefficient (Papp) for RA and RA in P. vulgaris extracts was 0.2 ± 0.05 × 10−6 cm/s, significantly increased to 0.9 ± 0.2 × 10−6 cm/s after β-glucuronidase/sulfatase treatment. Papp for UA and UA in S. officinalis extract was 2.7 ± 0.3 × 10−6 cm/s and 2.3 ± 0.5 × 10−6 cm/s before and after β-glucuronidase/sulfatase treatment, respectively. Neither compound was affected in permeability by the herbal extract matrix. Conclusion RA and UA in herbal extracts had similar uptake as that found using the pure compounds, which may simplify the prediction of compound efficacy, but the apparent lack of intestinal glucuronidation/sulfation of UA is likely to further enhance the bioavailability of that compound compared with RA. PMID:21798330

  11. Pharmacokinetics of amino acid ester prodrugs of acyclovir after oral administration: interaction with the transporters on Caco-2 cells.

    Science.gov (United States)

    Katragadda, Suresh; Jain, Ritesh; Kwatra, Deep; Hariharan, Sudharshan; Mitra, Ashim K

    2008-10-01

    In vivo systemic absorption of the amino acid prodrugs of acyclovir (ACV) after oral administration was evaluated in rats. Stability of the prodrugs, L-alanine-ACV (AACV), L-serine-ACV (SACV), L-isoleucine-ACV (IACV), gamma-glutamate-ACV (EACV) and L-valine-ACV (VACV) was evaluated in various tissues. Interaction of these prodrugs with the transporters on Caco-2 cells was studied. In vivo systemic bioavailability of these prodrugs upon oral administration was evaluated in jugular vein cannulated rats. The amino acid ester prodrugs showed affinity towards various amino acid transporters as well as the peptide transporter on the Caco-2 cells. In terms of stability, EACV was most enzymatically stable compared to other prodrugs especially in liver homogenate. In oral absorption studies, ACV and AACV showed high terminal elimination rate constants (lambda(z)). SACV and VACV exhibited approximately five-fold increase in area under the curve (AUC) values relative to ACV (pACV. C(last(T)) (concentration at the last time point) of SACV was observed to be 0.18+/-0.06 microM in plasma which is two times better than VACV and three times better than ACV. Amino acid ester prodrugs of ACV were absorbed at varying amounts (C(max)) and eliminated at varying rates (lambda(z)) thereby leading to varying extents (AUC). The amino acid ester prodrug SACV owing to its enhanced stability, higher AUC and better concentration at last time point seems to be a promising candidate for the oral treatment of herpes infections.

  12. In vitro studies on the stability in the proximal gastrointestinal tract and bioaccessibility in Caco-2 cells of chlorogenic acids from spent coffee grounds.

    Science.gov (United States)

    Monente, Carmen; Ludwig, Iziar A; Stalmach, Angelique; de Peña, Maria Paz; Cid, Concepción; Crozier, Alan

    2015-01-01

    Spent coffee grounds are a potential commercial source of substantial amounts of chlorogenic acids (CGAs). The aim of this study was to evaluate the stability of spent coffee CGAs using in vitro simulated gastroduodenal digestion and to investigate their potential absorption using an in vitro Caco-2 model of human small intestinal epithelium. During in vitro digestion, lactones were partially degraded while caffeoylquinic and feruloylquinic acids were much more stable. Transport and metabolism studies showed that 1% of the total CGAs were absorbed and transported from the apical to the basolateral side of a Caco-2 cell monolayer after 1 h. Lactones and coumaroylquinic acids showed the rate of highest absorption. Caco-2 cells possessed low metabolic activity. In conclusion, spent coffee extracts contain large amounts of CGAs, which remained bioaccessible across the intestinal barrier, albeit to a relatively low degree.

  13. Influence of 5-amino-3-methyl-4-isoxazolecarbohydrazide on selective gene expression in Caco-2 cultured cells.

    Science.gov (United States)

    Płoszaj, Paulina; Regiec, Andrzej; Ryng, Stanisław; Piwowar, Agnieszka; Kruzel, Marian L

    2016-10-17

    The 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide (HIX) is a synthetic isoxazole derivative with a potential for development as an anti-inflammatory drug candidate. The goal of this study was to explore in vitro autoimmune and inflammatory gene modulation by HIX in human Caco-2 cultured cells. The effect of low dose of HIX was tested on the expression level of RNA in 24h Caco-2 cultures using the QIAGEN Th17 for Autoimmunity & Inflammation RT(2) Profiler PCR Array. We choose the PCR technology as the most reliable and sensitive gene expression profiling method for analyzing specific gene regulatory networks. In all experiments, Leflunomide (5-methyl-N-[4-(trifluoromethyl)phenyl]-4-isoxazolecarboxamide), an immuno-suppressive disease-modifying antirheumatic drug was used, as a reference to clinical utility of the isoxazole derivatives. Changes in RNA levels were analyzed and differentially expressed genes with at least 2-fold change were identified. For the majority of genes tested, the effects of HIX and Leflunomide were similar, including up-regulation of CX3CL1 and IL-17F, and down-regulation of IL-10 and TLR4. However twelve genes were were differently regulated by the two compounds: interleukins (IL) IL-1B, IL-6 and a chemokine CCL22 were upregulated by HIX and significantly supressed by Leflunomide. In contrary, IL-2 and IL-27 were upregulated by Leflunomide and suppressed by HIX. The network search by Ingenuity Pathway Analysis showed, that majority of differentially expressed genes were involved in cellular inflammatory responses. These results suggest that 5-amino-3-methyl-4-isoxazolecarbohydrazide has a potential for future clinical developments with structure modification as a disease modifying agent in different than Leflunomide applications.

  14. Evaluation of metallothionein formation as a proxy for zinc absorption in an in vitro digestion/caco-2 cell culture model

    Science.gov (United States)

    Caco-2 cell metallothionein (MT) formation was studied to determine if MT could be used as a proxy for zinc (Zn) absorption in a cell culture model. MT intracellular concentration was determined by using a cadmium/hemoglobin affinity assay. Cellular Zn uptake was determined in acid digests (5% HNO3)...

  15. Evaluation of the antioxidant capacity, furan compounds and cytoprotective/cytotoxic effects upon Caco-2 cells of commercial Colombian coffee.

    Science.gov (United States)

    Bedoya-Ramírez, Daniel; Cilla, Antonio; Contreras-Calderón, José; Alegría-Torán, Amparo

    2017-03-15

    Antioxidant capacity (AC), total phenolics (TPs), furan compounds (HMF and furfural F) and cytoprotective/cytotoxic effects upon Caco-2 cells (MTT, cell cycle and reactive oxygen species (ROS)) were evaluated in Colombian coffee (2 ground and 4 soluble samples). The AC (ABTS and FRAP), TPs and HMF ranged between 124-722, 95-802μmoles Trolox/g, 21-100mg gallic acid/g and 69-2900mg/kg, respectively. Pretreatment of cells for 24h with lyophilized coffee infusions at the highest dose without cytotoxic effects (500μg/mL) significantly prevented the decrease in cell viability compared to control stress with H2O2 (5mM/2h), recovering viability to values between 34% and 45% and restoring the control values without stress induction in the G1 phase of cell cycle. After exposure to stress, four extracts decreased ROS values significantly to 22.5-24.9%. The coffee samples exerted a cytoprotective effect against oxidative stress, with improvement in cell viability and a reduction of intracellular ROS.

  16. Ethanol Extract of Abnormal Savda Munziq, a Herbal Preparation of Traditional Uighur Medicine, Inhibits Caco-2 Cells Proliferation via Cell Cycle Arrest and Apoptosis

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    Abdiryim Yusup

    2012-01-01

    Full Text Available Aims. Study the effect of Abnormal Savda Munziq (ASMq ethanol extract on the proliferation, apoptosis, and correlative gene, expression in colon cancer cells (Caco-2 to elucidate the molecular mechanisms responsible for the anticancer property of Abnormal Savda Munziq. Materials and Methods. ASMq ethanol extract was prepared by a professional pharmacist. Caco-2 cells were treated with different concentration of ASMq ethanol extract (0.5–7.5 mg/mL for different time intervals (48 and 72 h. Antiproliferative effect of ASMq ethanol extract was determined by MTT assay; DNA fragmentation was determined by gel electrophoresis assay; cell cycle analysis was detected by flow cytometer; apoptosis-related gene expression was detected by RT-PCR assay. Results. ASMq ethanol extract possesses an inhibition effect on Caco-2 cells proliferation, induction of cell apoptosis, cell cycle arrest in sub-G1 phase, and downregulation of bcl-2 and upregulation of Bax gene expression. Conclusion. The anticancer mechanism of ASMq ethanol extract may be involved in antiproliferation, induction of apoptosis, cell cycle arrest, and regulation of apoptosis-related gene expression such as bcl-2 and Bax activity pathway.

  17. Absorption of hydroxycamptothecin nanosuspension across Caco-2 cells monolayer%羟基喜树碱纳米混悬剂在Caco-2细胞模型中的吸收特性

    Institute of Scientific and Technical Information of China (English)

    蒲晓辉; 孙进; 张鹏; 王永军; 何仲贵

    2013-01-01

    目的 研究羟基喜树碱纳米混悬剂在小肠内潜在的吸收特性.方法 采用Caco-2细胞模型研究不同质量浓度的羟基喜树碱纳米混悬剂的细胞摄取与跨膜转运.结果 羟基喜树碱纳米混悬剂在Caco-2细胞模型上显示出很好的吸收特性,且细胞摄取量随着制剂质量浓度的增加而增加,如:50、100和200 mg· L-13个试验质量浓度制剂的细胞蛋白摄取量分别为11.684 6、26.452 4和30.903 9 mg·g-1,试验质量浓度为100 mg·L-1制剂的细胞转运渗透系数Papp,AP→BL为(27.8±1.37×10-7)cm·s-1.结论 试验结果证明,纳米混悬剂能有效地增加羟基喜树碱的细胞摄取量和跨膜转运速率.因此,纳米混悬剂可能有助于提高羟基喜树碱的口服生物利用度.%Objective To study the potential absorption properties of hydroxycamptothecin(HCPT)nanosus-pension in the small bowel. Methods The uptake and the transport across cells for different concentration of HCPT nanosuspension were performed with Caco-2 cells. Results The test data indicated that HCPT nano-suspension had a good absorption characteristic in the Caco-2 cells. The results showed the uptake of 50 mg·L-1, 100 mg·L-1, 200 mg·L-1 HCPT nanosuspension were 11.684 6 mg·g-1 protein, 26. 452 4 mg·g-1 protein,30. 903 9 mg·g-1 protein,respectively. The membrane permeability coefficient of 100 mg·L-1 HCPT nanosuspension was (27. 8 ±1. 37×10-7)cm·s-1. Conclusions The results demonstrated that the uptake and the transport ratio increased with increasing concentration of HCPT nanosuspension. Therefore,nanosuspension could effectively increase the uptake and transport rate of HCPT.

  18. Transportation of Schizandrin B in Caco-2 Cell Model%五味子乙素在 Caco-2细胞模型中的转运研究

    Institute of Scientific and Technical Information of China (English)

    李维亮; 宋建军; 辛华雯

    2016-01-01

    Objective:To investigate the effects of schizandrin B on P- glycoprotein by a classical in vitro cell model. Methods:Caco-2 cell model was used as the carrier,and rhodamine 123 and cyclosporin A were employed as the P-gp substrates, the transmembrane transportation of schizandrin B,rodamine 123 and cyclosporin A were detected by HPLC and a liquid scintillation counting assay,and the apparent permeability coefficient and permeability directional ratio were calculated. Results:The bidirectional transportation rates of schizandrin B(20 μg·ml -1 ,40 μg·ml -1 and 80μg·ml-1 )were similar,and showed non-selective difference. Schizandrin B(20-160 μg ·ml -1 )significantly inhibited the BL → AP directional transportation of rhodamine 123 and cyclosporine A in Caco-2 cell model(P < 0. 05) in a concentration-dependent manner. Conclusion:Schizandrin B is a P-gp inhibitor,while it isn’t a P-gp substrate.%目的:通过经典的体外细胞模型探讨五味子乙素对 P-糖蛋白的影响。方法:以 Caco-2细胞模型为载体,选取罗丹明123和环孢素-为 P-gp 底物,采用高效液相色谱法(HPLC)和液体闪烁计数法检测不同条件下五味子乙素、罗丹明123及环孢素-跨膜转运的变化,计算表观渗透系数、表观渗透率等参数。结果:五味子乙素浓度在20,40,80μg·ml-1时双向跨膜转运的速率相似,无选择性差异。五味子乙素浓度为20~160μg·ml -1时可显著抑制罗丹明123和环孢素 A 在 Caco-2细胞模型中BL → AP 定向转运(P <0.05),且呈剂量相关性。结论:五味子乙素为 P-gp 抑制药,但不是 P-gp 底物。

  19. Low molecular weight procyanidins from grape seeds enhance the impact of 5-Fluorouracil chemotherapy on Caco-2 human colon cancer cells.

    Directory of Open Access Journals (Sweden)

    Ker Y Cheah

    Full Text Available OBJECTIVE: Grape seed procyanidins (PC are flavan-3-ol oligomers and polymers known for their biological activity in the gut. Grape seed extract (GSE have been reported to reduce intestinal injury in a rat model of mucositis. We sought to investigate effects of purified PC fractions differing in mean degree of polymerization (mDP combined with 5-Fluorouracil (5-FU chemotherapy on the viability of colon cancer cells (Caco-2. DESIGN: SixPC fractions (F1-F6 were isolated from Cabernet Sauvignon seeds at two ripeness stages: pre-veraison unripe (immature and ripe (mature, utilizing step gradient, low-pressure chromatography on a Sephadex LH-20 resin. Fractions were tested on Caco-2 cells, alone and in combination with 5-FU. Eluted fractions were characterized by phloroglucinolysis and gel permeation chromatography. Cell viability was determined by the 3-(4,5-Dimethylthiazol-2yl-2,5-diphenyl-tetrazolium bromide (MTT assay. RESULTS: All isolated fractions significantly reduced Caco-2 cell viability compared to the control (P<0.05, but F2 and F3 (mDP 2-6 were the most active fractions (immature F2 = 32% mDP 2.4, F3 = 35% mDP 5.8 and mature F2 = 13% mDP 3.6 and F3 = 17% mDP 5.9; percentage of viable cells remaining on Caco-2 cells. When combined with 5-FU, immature fractions F1-F3 enhanced the cell toxicity effects of 5-FU by 27-73% (P<0.05. Mature seed PC fractions (F1-F4 significantly enhanced the toxicity of 5-FU by 60-83% against Caco-2 cells (P<0.05. Moreover, some fractions alone were more potent at decreasing viability in Caco-2 cells (P<0.05; immature fractions = 65-68% and mature fractions = 83-87% compared to 5-FU alone (37%. CONCLUSIONS: PCs of mDP 2-6 (immature F1-F3 and mature F1 and F4not only enhanced the impact of 5-FU in killing Caco-2 cells, but also surpassed standard 5-FU chemotherapy as an anti-cancer agent.The bioactivity of PC is therefore attributed primarily to lower molecular weight PCs.

  20. Clostridium botulinum Type E Toxins Bind to Caco-2 Cells by a Different Mechanism from That of Type A Toxins

    Directory of Open Access Journals (Sweden)

    Zhang,Kai

    2012-06-01

    Full Text Available Cultured Clostridium botulinum strains produce progenitor toxins designated as 12S, 16S, and 19S toxins. The 12S toxin consists of a neurotoxin (NTX, 7S and a non-toxic non-hemagglutinin (NTNH. The 16S and 19S toxins are formed by conjugation of the 12S toxin with hemagglutinin (HA, and the 19S toxin is a dimer of the 16S toxin. Type A cultures produce all 3 of these progenitor toxins, while type E produces only the 12S toxin. The 7S toxin is cleaved into heavy (H and light (L chains by a protease(s in some strains, and the H chain has 2 domains, the N-terminus (Hn and C-terminus (Hc. It has been reported that type A toxins bind to the intestinal cells or cultured cells via either HA or Hc. In this study, we investigated the binding of type A and E toxins to Caco-2 cells using Western blot analysis. Both the type E 7S and 12S toxins bound to the cells, with the 7S toxin binding more strongly, whereas, in the type A strain, only the 16S/19S toxins showed obvious binding. Pre-incubation of the type E 7S toxin with IgG against recombinant type E Hc significantly inhibited the 7S toxin binding, indicating that Hc might be a main binding domain of the type E toxin.

  1. Impact of anatase and rutile titanium dioxide nanoparticles on uptake carriers and efflux pumps in Caco-2 gut epithelial cells.

    Science.gov (United States)

    Dorier, M; Brun, E; Veronesi, G; Barreau, F; Pernet-Gallay, K; Desvergne, C; Rabilloud, T; Carapito, C; Herlin-Boime, N; Carrière, M

    2015-04-28

    TiO2 microparticles are widely used in food products, where they are added as a white food colouring agent. This food additive contains a significant amount of nanoscale particles; still the impact of TiO2 nanoparticles (TiO2-NPs) on gut cells is poorly documented. Our study aimed at evaluating the impact of rutile and anatase TiO2-NPs on the main functions of enterocytes, i.e. nutrient absorption driven by solute-liquid carriers (SLC transporters) and protection against other xenobiotics driven by efflux pumps from the ATP-binding cassette (ABC) family. We show that acute exposure of Caco-2 cells to both anatase (12 nm) and rutile (20 nm) TiO2-NPs induce early upregulation of a battery of efflux pumps and nutrient transporters. In addition they cause overproduction of reactive oxygen species and misbalance redox repair systems, without inducing cell mortality or DNA damage. Taken together, these data suggest that TiO2-NPs may increase the functionality of gut epithelial cells, particularly their property to form a protective barrier against exogenous toxicants and to absorb nutrients.

  2. Assesing potential effects of inulin and probiotic bacteria on Fe bioavailability from common beans (Phaseolus vulgaris L.) to Caco-2 cells

    Science.gov (United States)

    Inulin, a prebiotic, may enhance intestinal Fe absorption. Our objective was to assess the effects of supplemental inulin and two probiotic bacteria (B. infantis and L.acidophillus) on Fe availability to Caco-2 cells from common white and red beans (Phaseolus vulgaris L.). Cooked beans were mixed o...

  3. Inulin affects iron dialyzability from FeSO4 and FeEDTA solutions but does not alter Fe uptake by Caco-2 cells

    Science.gov (United States)

    The in vitro effects of inulin on the fluxes of Fe (FFe), uptake by Caco-2 cells from FeSO4 and FeEDTA which are commonly used for food fortification, were evaluated. For an element to be absorbed it is necessary that it should be soluble in the gastrointestinal tract, thus, changes in FFe diffussio...

  4. Characterization of loxoprofen transport in Caco-2 cells: the involvement of a proton-dependent transport system in the intestinal transport of loxoprofen.

    Science.gov (United States)

    Narumi, Katsuya; Kobayashi, Masaki; Kondo, Ayuko; Furugen, Ayako; Yamada, Takehiro; Takahashi, Natsuko; Iseki, Ken

    2016-11-01

    Loxoprofen, a propionate non-steroidal anti-inflammatory drug (NSAID), is used widely in East Asian countries. However, little is known about the transport mechanisms contributing to its intestinal absorption. The objectives of this study were to characterize the intestinal transport of loxoprofen using the human intestinal Caco-2 cell model. The transport of loxoprofen was investigated in cellular uptake studies. The uptake of loxoprofen into Caco-2 cells was pH- and concentration-dependent, and was described by a Michaelis-Menten equation with passive diffusion (Km : 4.8 mm, Vmax : 142 nmol/mg protein/30 s, and Kd : 2.2 μl/mg protein/30 s). Moreover, the uptake of loxoprofen was inhibited by a typical monocarboxylate transporter (MCT) inhibitor as well as by various monocarboxylates. The uptake of [(14) C] l-lactic acid, a typical MCT substrate, in Caco-2 cells was saturable with relatively high affinity for MCT. Because loxoprofen inhibited the uptake of [(14) C] l-lactic acid in a noncompetitive manner, it was unlikely that loxoprofen uptake was mediated by high-affinity MCT(s). Our results suggest that transport of loxoprofen in Caco-2 cells is, at least in part, mediated by a proton-dependent transport system. Copyright © 2016 John Wiley & Sons, Ltd.

  5. Seed coat removal improves Fe bioavailability in cooked lentils: studies using an in vitro digestion/Caco-2 cell culture model

    Science.gov (United States)

    This study examined the range of Fe concentration and relative Fe bioavailability of 24 varieties of cooked lentils, as well as the impact of seed coat removal on lentil Fe nutritional quality. Relative Fe bioavailability was assessed by the in vitro/Caco-2 cell culture method. While Fe concentrat...

  6. Effects of Ascorbic Acid, Phytic Acid and Tannic Acid on Iron Bioavailability from Reconstituted Ferritin Measured by an In Vitro Digestion/Caco-2 Cell Model

    Science.gov (United States)

    The effects of ascorbic acid, phytate and tannic acid on Fe bioavailability from Fe supplied as ferritin was compared to FeSO4 using an in vitro digestion/Caco-2 cell model. Horse spleen ferritin (HSF) was chemically reconstituted into a plant-type ferritin (P-HSF). In the presence of ascorbic acid...

  7. Sodium cromoglycate inhibits absorption of the major soybean allergen, Gly m Bd 30K, in mice and human intestinal Caco-2 cells.

    Science.gov (United States)

    Weangsripanaval, Thanakorn; Murota, Kaeko; Murakami, Yoko; Kominami, Masaru; Kusudo, Tatsuya; Moriyama, Tatsuya; Ogawa, Tadashi; Kawada, Teruo

    2006-11-01

    Our previous data showed that Gly m Bd 30K was absorbed from the gastrointestinal tract and circulated in blood in mice. This study was conducted to determine the mechanism and identify the inhibitor of such absorption. Using sandwich ELISA and immunoblotting, we found that intact Gly m Bd 30K was absorbed from apical to basolateral solutions and intracellularly accumulated by Caco-2 cells in a dose- and time-dependent manner. The absorption and intracellular accumulation of Gly m Bd 30K were significantly suppressed when Caco-2 cells were treated with sodium cromoglycate (SCG) (0-50 mmol/L) in a dose-dependent manner. In 24-d-old mice orally treated with SCG (10-1000 mg/kg body weight), plasma Gly m Bd 30K concentration decreased significantly 30-120 min after Gly m Bd 30K (2000 mg/kg body weight) administration. Moreover, inhibitors that suppress the clathrin-dependent endocytosis dansylcadaverine, the caveolae-dependent endocytosis nystatin and clathrin, and the caveolae-dependent endocytosis methyl-beta-cyclodextrin had inhibitory effects on the absorption and intracellular accumulation of Gly m Bd 30K by Caco-2 cells. These data indicate that Gly m Bd 30K is absorbed and intracellularly accumulated in Caco-2 cells via clathrin- or caveolae-dependent endocytosis. We propose that the absorption and intracellular accumulation of Gly m Bd 30K are inhibited by SCG via clathrin- or caveolae-dependent endocytosis.

  8. Genistein and Glyceollin Effects on ABCC2 (MRP2 and ABCG2 (BCRP in Caco-2 Cells

    Directory of Open Access Journals (Sweden)

    Chandler Schexnayder

    2015-12-01

    Full Text Available The goal of the present study was to determine the effects of glyceollins on intestinal ABCC2 (ATP Binding Cassette C2, multidrug resistance protein 2, MRP2 and ABCG2 (ATP Binding Cassette G2, breast cancer resistance protein, BCRP function using the Caco-2 cell intestinal epithelial cell model. Glyceollins are soy-derived phytoestrogens that demonstrate anti-proliferative activity in several sources of cancer cells. 5 (and 6-carboxy-2′,7′-dichloroflourescein (CDF was used as a prototypical MRP2 substrate; whereas BODIPY-prazosin provided an indication of BCRP function. Comparison studies were conducted with genistein. Glyceollins were shown to inhibit MRP2-mediated CDF transport, with activity similar to the MRP2 inhibitor, MK-571. They also demonstrated concentration-dependent inhibition BCRP-mediated efflux of BODIPY-prazosin, with a potency similar to that of the recognized BCRP inhibitor, Ko143. In contrast, genistein did not appear to alter MRP2 activity and even provided a modest increase in BCRP efflux of BODIPY-prazosin. In particular, glyceollin inhibition of these two important intestinal efflux transporters suggests the potential for glyceollin to alter the absorption of other phytochemicals with which it might be co-administered as a dietary supplement, as well as alteration of the absorption of pharmaceuticals that may be administered concomitantly.

  9. Remodeling of Tight Junctions and Enhancement of Barrier Integrity of the CACO-2 Intestinal Epithelial Cell Layer by Micronutrients.

    Science.gov (United States)

    Valenzano, Mary Carmen; DiGuilio, Katherine; Mercado, Joanna; Teter, Mimi; To, Julie; Ferraro, Brendan; Mixson, Brittany; Manley, Isabel; Baker, Valerissa; Moore, Beverley A; Wertheimer, Joshua; Mullin, James M

    2015-01-01

    The micronutrients zinc, quercetin, butyrate, indole and berberine were evaluated for their ability to induce remodeling of epithelial tight junctions (TJs) and enhance barrier integrity in the CACO-2 gastrointestinal epithelial cell culture model. All five of these chemically very diverse micronutrients increased transepithelial electrical resistance (Rt) significantly, but only berberine also improved barrier integrity to the non-electrolyte D-mannitol. Increases of Rt as much as 200% of untreated controls were observed. Each of the five micronutrients also induced unique, signature-like changes in TJ protein composition, suggesting multiple pathways (and TJ arrangements) by which TJ barrier function can be enhanced. Decreases in abundance by as much as 90% were observed for claudin-2, and increases of over 300% could be seen for claudins -5 and -7. The exact effects of the micronutrients on barrier integrity and TJ protein composition were found to be highly dependent on the degree of differentiation of the cell layer at the time it was exposed to the micronutrient. The substratum to which the epithelial layer adheres was also found to regulate the response of the cell layer to the micronutrient. The implications of these findings for therapeutically decreasing morbidity in Inflammatory Bowel Disease are discussed.

  10. Distinctive toxicity of TiO2 rutile/anatase mixed phase nanoparticles on Caco-2 cells.

    Science.gov (United States)

    Gerloff, Kirsten; Fenoglio, Ivana; Carella, Emanuele; Kolling, Julia; Albrecht, Catrin; Boots, Agnes W; Förster, Irmgard; Schins, Roel P F

    2012-03-19

    Titanium dioxide has a long-standing use as a food additive. Micrometric powders are, e.g., applied as whiteners in confectionary or dairy products. Possible hazards of ingested nanometric TiO(2) particles for humans and the potential influence of varying specific surface area (SSA) are currently under discussion. Five TiO(2)-samples were analyzed for purity, crystallinity, primary particle size, SSA, ζ potential, and aggregation/agglomeration. Their potential to induce cytotoxicity, oxidative stress, and DNA damage was evaluated in human intestinal Caco-2 cells. Only anatase-rutile containing samples, in contrast to the pure anatase samples, induced significant LDH leakage or mild DNA damage (Fpg-comet assay). Evaluation of the metabolic competence of the cells (WST-1 assay) revealed a highly significant correlation between the SSA of the anatase samples and cytotoxicity. The anatase/rutile samples showed higher toxicity per unit surface area than the pure anatase powders. However, none of the samples affected cellular markers of oxidative stress. Our findings suggest that both SSA and crystallinity are critical determinants of TiO(2)-toxicity toward intestinal cells.

  11. Chemical form of selenium affects its uptake, transport, and glutathione peroxidase activity in the human intestinal Caco-2 cell model.

    Science.gov (United States)

    Zeng, Huawei; Jackson, Matthew I; Cheng, Wen-Hsing; Combs, Gerald F

    2011-11-01

    Determining the effect of selenium (Se) chemical form on uptake, transport, and glutathione peroxidase activity in human intestinal cells is critical to assess Se bioavailability at nutritional doses. In this study, we found that two sources of L-selenomethionine (SeMet) and Se-enriched yeast each increased intracellular Se content more effectively than selenite or methylselenocysteine (SeMSC) in the human intestinal Caco-2 cell model. Interestingly, SeMSC, SeMet, and digested Se-enriched yeast were transported at comparable efficacy from the apical to basolateral sides, each being about 3-fold that of selenite. In addition, these forms of Se, whether before or after traversing from apical side to basolateral side, did not change the potential to support glutathione peroxidase (GPx) activity. Although selenoprotein P has been postulated to be a key Se transport protein, its intracellular expression did not differ when selenite, SeMSC, SeMet, or digested Se-enriched yeast was added to serum-contained media. Taken together, our data show, for the first time, that the chemical form of Se at nutritional doses can affect the absorptive (apical to basolateral side) efficacy and retention of Se by intestinal cells; but that, these effects are not directly correlated to the potential to support GPx activity.

  12. ADME Properties Evaluation in Drug Discovery: Prediction of Caco-2 Cell Permeability Using a Combination of NSGA-II and Boosting.

    Science.gov (United States)

    Wang, Ning-Ning; Dong, Jie; Deng, Yin-Hua; Zhu, Min-Feng; Wen, Ming; Yao, Zhi-Jiang; Lu, Ai-Ping; Wang, Jian-Bing; Cao, Dong-Sheng

    2016-04-25

    The Caco-2 cell monolayer model is a popular surrogate in predicting the in vitro human intestinal permeability of a drug due to its morphological and functional similarity with human enterocytes. A quantitative structure-property relationship (QSPR) study was carried out to predict Caco-2 cell permeability of a large data set consisting of 1272 compounds. Four different methods including multivariate linear regression (MLR), partial least-squares (PLS), support vector machine (SVM) regression and Boosting were employed to build prediction models with 30 molecular descriptors selected by nondominated sorting genetic algorithm-II (NSGA-II). The best Boosting model was obtained finally with R(2) = 0.97, RMSEF = 0.12, Q(2) = 0.83, RMSECV = 0.31 for the training set and RT(2) = 0.81, RMSET = 0.31 for the test set. A series of validation methods were used to assess the robustness and predictive ability of our model according to the OECD principles and then define its applicability domain. Compared with the reported QSAR/QSPR models about Caco-2 cell permeability, our model exhibits certain advantage in database size and prediction accuracy to some extent. Finally, we found that the polar volume, the hydrogen bond donor, the surface area and some other descriptors can influence the Caco-2 permeability to some extent. These results suggest that the proposed model is a good tool for predicting the permeability of drug candidates and to perform virtual screening in the early stage of drug development.

  13. Screening of Cytotoxic B. cereus on Differentiated Caco-2 Cells and in Co-Culture with Mucus-Secreting (HT29-MTX) Cells.

    Science.gov (United States)

    Castiaux, Virginie; Laloux, Laurie; Schneider, Yves-Jacques; Mahillon, Jacques

    2016-11-05

    B. cereus is an opportunistic foodborne pathogen able to cause diarrhoea. However, the diarrhoeal potential of a B. cereus strain remains difficult to predict, because no simple correlation has yet been identified between the symptoms and a unique or a specific combination of virulence factors. In this study, 70 B. cereus strains with different origins (food poisonings, foods and environment) have been selected to assess their enterotoxicity. The B. cereus cell-free supernatants have been tested for their toxicity in vitro, on differentiated (21 day-old) Caco-2 cells, using their ATP content, LDH release and NR accumulation. The genetic determinants of the main potential enterotoxins and virulence factors (ces, cytK, entFM, entS, hbl, nhe, nprA, piplC and sph) have also been screened by PCR. This analysis showed that none of these genes was able to fully explain the enterotoxicity of B. cereus strains. Additionally, in order to assess a possible effect of the mucus layer in vitro, a cytotoxicity comparison between a monoculture (Caco-2 cells) and a co-culture (Caco-2 and HT29-MTX mucus-secreting cells) model has been performed with selected B. cereus supernatants. It appeared that, in these conditions, the mucus layer had no notable influence on the cytotoxicity of B. cereus supernatants.

  14. Intestinal absorption of forsythoside A in in situ single-pass intestinal perfusion and in vitro Caco-2 cell models

    Institute of Scientific and Technical Information of China (English)

    Wei ZHOU; Liu-qing DI; Juan WANG; Jin-jun SHAN; Shi-jia LIU; Wen-zheng JU; Bao-chang CAI

    2012-01-01

    Aim:To investigate the mechanisms underlying the intestinal absorption of the major bioactive component forsythoside A (FTA) extracted from Forsythiae fructus.Methods:An in vitro Caco-2 cell model and a single-pass intestinal perfusion in situ model in SD rats were used.Results:In the in vitro Caco-2 cell model,the mean apparent permeability value (Papp-value) was 4.15x 107 cm/s in the apical-tobasolateral (AP-BL) direction.At the concentrations of 2.6-10.4 μg/mL,the efflux ratio of FTA in the bi-directional transport experiments was approximately 1.00.After the transport,>96% of the apically loaded FTA was retained on the apical side,while >97% of the basolaterally loaded FTA was retained on the basolateral side.The Papp-values of FTA were inversely correlated with the transepithelial electrical resistance.The paracellular permeability enhancers sodium caprate and EDTA,the P-gp inhibitor verapamil and the multidrug resistance related protein (MRP) inhibitors cyclosporine and MK571 could concentration-dependently increase the Papp-values,while the uptake (OATP) transporter inhibitors diclofenac sodium and indomethacin could concentration-dependently decrease the Papp-values.The intake transporter SGLT1 inhibitor mannitol did not cause significant change in the Papp-values.In the in situ intestinal perfusion model,both the absorption rate constant (Ka) and the effective permeability (Peff-values) following perfusion of FTA 2.6,5.2,and 10.4 μg/mL via the duodenum,jejunum and ileum had no significant difference,although the values were slightly higher for the duodenum as compared to those in the jejunum and ileum.The low,medium and high concentrations of verapamil caused the largest increase in the Peff-values for duodenum,jejunum and ileum,respectively.Sodium caprate,EDTA and cyclosporine resulted in concentration-dependent increase in the Peff-values.Diclofenac sodium and indomethacin caused concentration-dependent decrease in the Peff-values.Mannitol did

  15. Induction of spermidine/spermine N1-acetyltransferase (SSAT) by aspirin in Caco-2 colon cancer cells.

    Science.gov (United States)

    Babbar, Naveen; Gerner, Eugene W; Casero, Robert A

    2006-02-15

    Epidemiological, experimental and clinical results suggest that aspirin and other NSAIDs (non-steroidal anti-inflammatory drugs) inhibit the development of colon cancer. It has been shown that the NSAID sulindac induces apoptosis and suppresses carcinogenesis, in part, by a mechanism leading to the transcriptional activation of the gene encoding SSAT (spermidine/spermine N1-acetyltransferase), a rate-limiting enzyme in polyamine catabolism. In the present study, we show that a variety of NSAIDs, including aspirin, sulindac, ibuprofen and indomethacin, can induce SSAT gene expression in Caco-2 cells. Aspirin, at physiological concentrations, can induce SSAT mRNA via transcriptional initiation mechanisms. This induction leads to increased SSAT protein levels and enzyme activity. Promoter deletion analysis of the 5' SSAT promoter-flanking region led to the identification of two NF-kappaB (nuclear factor kappaB) response elements. Electrophoretic mobility-shift assays showed binding of NF-kappaB complexes at these sequences after aspirin treatment. Aspirin treatment led to the activation of NF-kappaB signalling and increased binding at these NF-kappaB sites in the SSAT promoter, hence providing a potential mechanism for the induction of SSAT by aspirin in these cells. Aspirin-induced SSAT ultimately leads to a decrease in cellular polyamine content, which has been associated with decreased carcinogenesis. These results suggest that activation of SSAT by aspirin and different NSAIDs may be a common property of NSAIDs that plays an important role in their chemopreventive actions in colorectal cancer.

  16. New insights into the carrier-mediated transport of estrone-3-sulfate in the Caco-2 cell model

    DEFF Research Database (Denmark)

    Grandvuinet, Anne Sophie; Gustavsson, Lena; Steffansen, Bente

    2013-01-01

    from the "Deutsche sammlung von mikroorganismen und zellkulturen" (DSMZ) exhibit extensive and consistent carrier-mediated uptake of [3H]-E1S after a culture period of 11-13 days. The kinetic characterization, the inhibitory profile and the pH dependence for the initial linear uptake permeability (PUP...... the efflux of radio labeled substance in the absence or presence of BCRP or OST α/β inhibitors. Similar effluxes of [ 3H]-E1S was observed across the apical and basolateral membrane, and the apical efflux was greatly decreased in the presence of the BCRP inhibitor fumitremorgin C. In contrast, efflux of [3H...... at the basolateral membrane, neither for [ 3H]-E1S nor for [3H]-TCA. These results highlight the importance of transporter interplay for E1S and drug compounds in Caco-2 cells and emphasize the importance of identifying the basolateral transporters in these cells. © 2013 American Chemical Society....

  17. Effect of Apple, Baobab, Red-Chicory, and Pear Extracts on Cellular Energy Expenditure and Morphology of Caco-2 Cells using Transepithelial Electrical Resistance (TEER) and Scanning Electron Microscopy (SEM)

    Science.gov (United States)

    The present study investigated the effects of four food extracts on the Caco-2 intestinal cell line using a new transepithelial electrical resistance method (TEER) concurrent with electron microscopy (SEM). Caco-2 cells are widely used in transepithelial studies because they can be cultured to creat...

  18. Hydrogen sulfide improves colonic barrier integrity in DSS-induced inflammation in Caco-2 cells and mice.

    Science.gov (United States)

    Zhao, Hongyu; Yan, Rui; Zhou, Xiaogang; Ji, Fang; Zhang, Bing

    2016-10-01

    Intestinal barrier involves in the pathogeny of inflammatory bowel disease (IBD) and hydrogen sulfide (H2S) has been reported to improve intestinal barrier integrity. Thus, this study investigated the effects of GYY4137, a slow-release H2S donor, on DSS-induced inflammation and intestinal dysfunction. In vitro model, cellular permeability was significantly increased and expression of tight junctions (ZO-1, Cauldin4, and Occludin) was downregulated in Caco-2 cells. GYY4137 treatment markedly attenuated DSS-induced inflammation and barrier dysfunction. Cystathionine β-synthase (CBS)-siRNA transfection further demonstrated that endogenous H2S system involves in DSS-induced inflammation and mediates barrier function. In vivo model, DSS exposure caused colonic inflammation and injury in mice and GYY4137 injection alleviated inflammatory response and improved intestinal barrier via reducing intestinal permeability and upregulating of tight junctions. In conclusion, endogenous H2S system involves in DSS-induced inflammation and H2S addition alleviated inflammation and intestinal dysfunction in vitro and in vivo.

  19. Novel Peptide with Specific Calcium-Binding Capacity from Schizochytrium sp. Protein Hydrolysates and Calcium Bioavailability in Caco-2 Cells

    Science.gov (United States)

    Cai, Xixi; Lin, Jiaping; Wang, Shaoyun

    2016-01-01

    Peptide-calcium can probably be a suitable supplement to improve calcium absorption in the human body. In this study, a specific peptide Phe-Tyr (FY) with calcium-binding capacity was purified from Schizochytrium sp. protein hydrolysates through gel filtration chromatography and reversed phase HPLC. The calcium-binding capacity of FY reached 128.77 ± 2.57 μg/mg. Results of ultraviolet spectroscopy, fluorescence spectroscopy, and infrared spectroscopy showed that carboxyl groups, amino groups, and amido groups were the major chelating sites. FY-Ca exhibited excellent thermal stability and solubility, which were beneficial to be absorbed and transported in the basic intestinal tract of the human body. Moreover, the calcium bioavailability in Caco-2 cells showed that FY-Ca could enhance calcium uptake efficiency by more than three times when compared with CaCl2, and protect calcium ions against dietary inhibitors, such as tannic acid, oxalate, phytate, and Zn2+. Our findings further the progress of algae-based peptide-calcium, suggesting that FY-Ca has the potential to be developed as functionally nutraceutical additives. PMID:28036002

  20. Investigation of transport of genistein, daidzein and their inclusion complexes prepared with different cyclodextrins on Caco-2 cell line.

    Science.gov (United States)

    Daruházi, Agnes Emma; Kiss, Tímea; Vecsernyés, Miklós; Szente, Lajos; Szőke, Eva; Lemberkovics, Eva

    2013-10-01

    Isoflavonoids are widespread constituents in medical plants especially in legumes (Fabaceae), but occur in other different plant families as well (Rosaceae, Iridaceae, Amaranthaceae). Their antioxidant, estrogen-like, anti-inflammatory and analgesic effects make them promising compounds in therapy of important disorders especially in estrogen related diseases. Poor solubility in aqueous system of genistein and daidzein needs a solubility enhancement for pharmaceutical use. These compounds are suitable guest molecules for inclusion complex formation with cyclodextrins (CDs) considering matching their size and polarity. The molecular encapsulation with beta-cyclodextrin (β-CD), gamma-cyclodextrin (γ-CD), hydroxypropyl-beta-cyclodextrin (HP-β-CD) and random methyl-beta cyclodextrin (RAMEB-CD) results in a solid, molecularly dispersed form and in a significantly improved aqueous solubility of genistein and daidzein. Determining enhancement in solubility and bioavailability we investigated the transport of these inclusion complexes across Caco-2 cell line comparing that of the pure compounds and found significant improving effect of the different CD derivatives on membrane permeation of the two isoflavone aglycons.

  1. Extraction of Antioxidant Components from Bidens pilosa Flowers and Their Uptake by Human Intestinal Caco-2 Cells

    Directory of Open Access Journals (Sweden)

    Charng-Cherng Chyau

    2013-01-01

    Full Text Available Bidens pilosa L. var. radiata (BPR, Asteraceae is a commonly used folk medicine for treating various disorders such as diabetes, inflammation and hypertension. Recent studies to determine its chemical composition have revealed three di-O-caffeoylquinic acids (DiCQAs and three polyacetylene glucosides (PGAs to be among the major bioactive markers. To obtain the major compounds of these two chemical classes, the ethyl acetate fraction (EM obtained using liquid-liquid partition from the methanol extract resulted in a fraction with the highest total phenolic and total flavonoid contents and antioxidant activities in radical scavenging and ferric reducing power assays. To assess the bioavailability of EM, we examined the in vitro uptake using the Caco-2 human colonic cell line. The apparent permeability coefficient (Papp for each of the compounds within PGAs measured in both apical (AP to basolateral (BL and BL to AP was found to preferentially appear BL to AP direction, indicated that a basolateral to apical efflux system was detected in the study. DiCQAs had a lower efflux ratio than those from PGAs (2.32–3.67 vs. 6.03–78.36. Thus, it strongly implies that most of the DiCQAs are better absorbed than the PGAs.

  2. Aflatoxin M1 cytotoxicity against human intestinal Caco-2 cells is enhanced in the presence of other mycotoxins.

    Science.gov (United States)

    Gao, Y N; Wang, J Q; Li, S L; Zhang, Y D; Zheng, N

    2016-10-01

    Aflatoxin M1 (AFM1), a class 2B human carcinogen, is the only mycotoxin with established maximum residue limits (MRLs) in milk. Toxicological data for other mycotoxins in baby food, containing cereals and milk, either in isolation or in combination with AFM1, are sparse. The aim of this study was to investigate the cytotoxicity of AFM1, ochratoxin A (OTA), zearalenone (ZEA), and α-zearalenol (α-ZOL), individually and in combinations, in human Caco-2 cells. The tetrazolium salt (MTT) assay demonstrated that (i) OTA and AFM1 had similar cytotoxicity, which was higher than that of ZEA and α-ZOL, after a 72 h exposure; and (ii) the quaternary combination had the highest cytotoxicity, followed by tertiary and binary combinations and individual mycotoxins. Isobologram analysis indicated that the presence of OTA, ZEA, and/or α-ZOL with AFM1 led to additive and synergistic cytotoxicity in most combinations. The cytotoxicity of OTA was similar to that of AFM1, suggesting that OTA in food poses a health risk to consumers. Furthermore, AFM1 cytotoxicity increased dramatically in the presence of OTA, ZEA, and/or α-ZOL (p mycotoxins in baby food which contains milk and cereals.

  3. Novel Peptide with Specific Calcium-Binding Capacity from Schizochytrium sp. Protein Hydrolysates and Calcium Bioavailability in Caco-2 Cells

    Directory of Open Access Journals (Sweden)

    Xixi Cai

    2016-12-01

    Full Text Available Peptide-calcium can probably be a suitable supplement to improve calcium absorption in the human body. In this study, a specific peptide Phe-Tyr (FY with calcium-binding capacity was purified from Schizochytrium sp. protein hydrolysates through gel filtration chromatography and reversed phase HPLC. The calcium-binding capacity of FY reached 128.77 ± 2.57 μg/mg. Results of ultraviolet spectroscopy, fluorescence spectroscopy, and infrared spectroscopy showed that carboxyl groups, amino groups, and amido groups were the major chelating sites. FY-Ca exhibited excellent thermal stability and solubility, which were beneficial to be absorbed and transported in the basic intestinal tract of the human body. Moreover, the calcium bioavailability in Caco-2 cells showed that FY-Ca could enhance calcium uptake efficiency by more than three times when compared with CaCl2, and protect calcium ions against dietary inhibitors, such as tannic acid, oxalate, phytate, and Zn2+. Our findings further the progress of algae-based peptide-calcium, suggesting that FY-Ca has the potential to be developed as functionally nutraceutical additives.

  4. The transport mechanism of integrin αvβ3 receptor targeting nanoparticles in Caco-2 cells.

    Science.gov (United States)

    Xu, Yining; Xu, Juan; Shan, Wei; Liu, Min; Cui, Yi; Li, Lian; Liu, Chong; Huang, Yuan

    2016-03-16

    As for the existence of epithelium barrier, accelerating the transport remains huge challenges for orally delivered protein and peptide drugs into blood circulation. Modifying nanopaticles (NPs) with targeting peptides can enhance the intestinal absorption of loaded macromolecular drugs. However, the transport process, which mainly means how the NPs pass through the apical membrane and the basolateral side and then enter into blood circulation, is needed comprehensive investigation. In this study, we systemically studied the transport mechanisms in Caco-2 cell model of trimethyl chitosan based NPs (TMC NPs) before and after modification of FQS, an integrin αvβ3 receptor targeting peptide. Our results showed FQS peptide mediated multiple endocytosis pathways and could activate integrin αvβ3 receptor by interacting with FAK and Src-family kinases to induce receptor-mediated endocytosis of the NPs. Then, both endocytosed NPs could transport from early endosome to lysososmes via late endosomes/lysosome pathway, as well as to recycling endosomes and Golgi apparatus through early endosome/recycling endosomes and Golgi apparatus/recycling endosomes/plasma membrane pathways, respectively. After FQS peptide modification, the endocytosis subpathways of NPs have been changed, and more pathways are involved in exocytosis process for FQS-modified NPs compared with non-modified NPs. Our study indicated the ligand modification could enhance the uptake and transport by altering some pathways in whole transport process of NPs.

  5. Flow cytometric evaluation of the contribution of ionic silver to genotoxic potential of nanosilver in human liver HepG2 and colon Caco2 cells.

    Science.gov (United States)

    Sahu, Saura C; Njoroge, Joyce; Bryce, Steven M; Zheng, Jiwen; Ihrie, John

    2016-04-01

    Exposure to nanosilver found in food- and cosmetics-related consumer products is of public concern because of the lack of information about its safety. In this study, two widely used in vitro cell culture models, human liver HepG2 and colon Caco2 cells, and the flow cytometric micronucleus (FCMN) assay were evaluated as tools for rapid predictive screening of the potential genotoxicity of nanosilver. Recently, we reported the genotoxicity of 20 nm nanosilver using these systems. In the current study presented here, we tested the hypothesis that the nanoparticle size and cell types were critical determinants of its genotoxicity. To test this hypothesis, we used the FCMN assay to evaluate the genotoxic potential of 50 nm nanosilver of the same shape, composition, surface charge and obtained from the same commercial source using the same experimental conditions and in vitro models (HepG2 and Caco2) as previously tested for the 20 nm silver. Results of our study show that up to the concentrations tested in these cultured cell test systems, the smaller (20 nm) nanoparticle is genotoxic to both the cell types by inducing micronucleus (MN). However, the larger (50 nm) nanosilver induces MN only in HepG2 cells, but not in Caco2 cells. Also in this study, we evaluated the contribution of ionic silver to the genotoxic potential of nanosilver using silver acetate as the representative ionic silver. The MN frequencies in HepG2 and Caco2 cells exposed to the ionic silver in the concentration range tested are not statistically significant from the control values except at the top concentrations for both the cell types. Therefore, our results indicate that the ionic silver may not contribute to the MN-forming ability of nanosilver in HepG2 and Caco2 cells. Also our results suggest that the HepG2 and Caco2 cell cultures and the FCMN assay are useful tools for rapid predictive screening of a genotoxic potential of food- and cosmetics-related chemicals including nanosilver.

  6. Human oral isolate Lactobacillus fermentum AGR1487 reduces intestinal barrier integrity by increasing the turnover of microtubules in Caco-2 cells.

    Directory of Open Access Journals (Sweden)

    Rachel C Anderson

    Full Text Available Lactobacillus fermentum is found in fermented foods and thought to be harmless. In vivo and clinical studies indicate that some L. fermentum strains have beneficial properties, particularly for gastrointestinal health. However, L. fermentum AGR1487 decreases trans-epithelial electrical resistance (TEER, a measure of intestinal barrier integrity. The hypothesis was that L. fermentum AGR1487 decreases the expression of intestinal cell tight junction genes and proteins, thereby reducing barrier integrity. Transcriptomic and proteomic analyses of Caco-2 cells (model of human intestinal epithelial cells treated with L. fermentum AGR1487 were used to obtain a global view of the effect of the bacterium on intestinal epithelial cells. Specific functional characteristics by which L. fermentum AGR1487 reduces intestinal barrier integrity were examined using confocal microscopy, cell cycle progression and adherence bioassays. The effects of TEER-enhancing L. fermentum AGR1485 were investigated for comparison. L. fermentum AGR1487 did not alter the expression of Caco-2 cell tight junction genes (compared to L. fermentum AGR1485 and tight junction proteins were not able to be detected. However, L. fermentum AGR1487 increased the expression levels of seven tubulin genes and the abundance of three microtubule-associated proteins, which have been linked to tight junction disassembly. Additionally, Caco-2 cells treated with L. fermentum AGR1487 did not have defined and uniform borders of zona occludens 2 around each cell, unlike control or AGR1485 treated cells. L. fermentum AGR1487 cells were required for the negative effect on barrier integrity (bacterial supernatant did not cause a decrease in TEER, suggesting that a physical interaction may be necessary. Increased adherence of L. fermentum AGR1487 to Caco-2 cells (compared to L. fermentum AGR1485 was likely to facilitate this cell-to-cell interaction. These findings illustrate that bacterial strains of the

  7. Comparison of the Caco-2, HT-29 and the mucus-secreting HT29-MTX intestinal cell models to investigate Salmonella adhesion and invasion.

    Science.gov (United States)

    Gagnon, Mélanie; Zihler Berner, Annina; Chervet, Noémie; Chassard, Christophe; Lacroix, Christophe

    2013-09-01

    Human intestinal cell models are widely used to study host-enteric pathogen interactions, with different cell lines exhibiting specific characteristics and functions in the gut epithelium. In particular, the presence of mucus may play an important role in adhesion and invasion of pathogens. The aim of this study was to evaluate the suitability of the mucus-secreting HT29-MTX intestinal epithelial cell model to test adhesion and invasion of Salmonella strains and compare with data obtained with the more commonly used Caco-2 and HT-29 models. Adhesion of Salmonella to HT29-MTX cell model was significantly higher, likely due to high adhesiveness to mucins present in the native human mucus layer covering the whole cell surface, compared to the non- and low-mucus producing Caco-2 and HT-29 cell models, respectively. In addition, invasion percentages of some clinical Salmonella strains to HT29-MTX cultures were remarkably higher than to Caco-2 and HT-29 cells suggesting that these Salmonellae have subverted the mucus to enhance pathogenicity. The transepithelial electrical resistances of the infected HT29-MTX cell model decreased broadly and were highly correlated with invasion ability of the strain. Staining of S. Typhimurium-infected cell epithelium confirmed the higher invasion by Salmonella and subsequent disruption of tight junctions of HT29-MTX cell model compared with the Caco-2 and HT-29 cell models. Data from this study suggest that the HT29-MTX cell model, with more physiologically relevant characteristics with the mucus layer formation, could be better suited for studying cells-pathogens interactions.

  8. Comparative detection of bacterial adhesion to Caco-2 cells with ELISA, radioactivity and plate count methods.

    Science.gov (United States)

    Le Blay, Gwenaëlle; Fliss, Ismaïl; Lacroix, Christophe

    2004-11-01

    Different methods are used to study bacterial adhesion to intestinal epithelial cells, which is an important step in pathogenic infection as well as in probiotic colonization of the intestinal tract. The aim of this study was to compare the ELISA-based method with more conventional plate count and radiolabeling methods for bacterial adhesion detection. An ELISA-based assay was optimized for the detection of Bifidobacterium longum and Escherichia coli O157:H7, which are low and highly adherent bacteria, respectively. In agreement with previous investigations, a percentage of adhesion below 1% was obtained for B. longum with ELISA. However, high nonspecific background and low positive signals were measured due to the use of polyclonal antibodies and the low adhesion capacity with this strain. In contrast, the ELISA-based method developed for E. coli adhesion detected a high adhesion percentage (15%). For this bacterium the three methods tested gave similar results for the highest bacterial concentrations (6.8 Log CFU added bacteria/well). However, differences among methods increased with the addition of decreased bacterial concentration due to different detection thresholds (5.9, 5.6 and 2.9 Log CFU adherent bacteria/well for radioactivity, ELISA and plate count methods, respectively). The ELISA-based method was shown to be a good predictor for bacterial adhesion compared to the radiolabeling method when good quality specific antibodies were used. This technique is convenient and allows handling of numerous samples.

  9. Gliadin peptides induce tissue transglutaminase activation and ER-stress through Ca2+ mobilization in Caco-2 cells.

    Directory of Open Access Journals (Sweden)

    Ivana Caputo

    Full Text Available BACKGROUND: Celiac disease (CD is an intestinal inflammatory condition that develops in genetically susceptible individuals after exposure to dietary wheat gliadin. The role of post-translational modifications of gliadin catalyzed by tissue transglutaminase (tTG seems to play a crucial role in CD. However, it remains to be established how and where tTG is activated in vivo. We have investigated whether gliadin peptides modulate intracellular Ca(2+ homeostasis and tTG activity. METHODS/PRINCIPAL FINDINGS: We studied Ca(2+ homeostasis in Caco-2 cells by single cell microfluorimetry. Under our conditions, A-gliadin peptides 31-43 and 57-68 rapidly mobilized Ca(2+ from intracellular stores. Specifically, peptide 31-43 mobilized Ca(2+ from the endoplasmic reticulum (ER and mitochondria, whereas peptide 57-68 mobilized Ca(2+ only from mitochondria. We also found that gliadin peptide-induced Ca(2+ mobilization activates the enzymatic function of intracellular tTG as revealed by in situ tTG activity using the tTG substrate pentylamine-biotin. Moreover, we demonstrate that peptide 31-43, but not peptide 57-68, induces an increase of tTG expression. Finally, we monitored the expression of glucose-regulated protein-78 and of CCAAT/enhancer binding protein-homologous protein, which are two biochemical markers of ER-stress, by real-time RT-PCR and western blot. We found that chronic administration of peptide 31-43, but not of peptide 57-68, induces the expression of both genes. CONCLUSIONS: By inducing Ca(2+ mobilization from the ER, peptide 31-43 could promote an ER-stress pathway that may be relevant in CD pathogenesis. Furthermore, peptides 31-43 and 57-68, by activating intracellular tTG, could alter inflammatory key regulators, and induce deamidation of immunogenic peptides and gliadin-tTG crosslinking in enterocytes and specialized antigen-presenting cells.

  10. Transport of Progesterone and Its Proliposome in Caco-2 Cells Monolayer%黄体酮及其前体脂质体制剂的Caco-2细胞转运研究

    Institute of Scientific and Technical Information of China (English)

    王梅; 高晓黎

    2012-01-01

    OBJECTIVE To study the absorption mechanism of progesterone and its proliposome in gastrointestinal (CI) tract METHODS Caco-2 cells monolayer was employed to investigate the transport of progesterone and its proliposome, and the effect of transport time, the drug concentration and inhibitors (cyclosporine and verapamil) on progesterone transport was further conducted. RESULTS The apparent permeability coefficient of progesterone changed with the variety of the drug concentration, and inhibitors could improve the accumulative transport flux and the apparent permeability coefficient of progesterone. Liposorae could also increase the transport of progesterone. But the transport of progesterone liposome was complied with a passive diffusion. CONCLUSION The absorption process of progesterone is affected by P-gp, which may lead to its low bioavailability. As progesterone belongs to the second kind drugs in biopharmaceutical classification system, its bioavailability can be improved as the solubility is increased. Liposome can also improve its absorption and bioavailability through changing its absorption mechanism, which means the transport could be a passive diffusion.%目的 研究黄体酮及其前体脂质体制剂的胃肠吸收转运机制.方法 采用Caco-2细胞模型考察黄体酮及其脂质体的转运,考察转运时间、药物浓度及抑制剂(环孢素和维拉帕米)对黄体酮转运的影响.结果 黄体酮表现渗透系数随浓度变化而变化,抑制剂的加入会提高黄体酮的表现渗透系数,黄体酮脂质体能使黄体酮累积转运量提高,表观渗透系数明显增加,黄体酮脂质体的转运符合被动扩散特征.结论 黄体酮的吸收受P-糖蛋白的介导,这可能是导致其生物利用度低的原因之一.黄体酮属生物药剂学分类系统中Ⅱ类药物,提高溶解度可提高其生物利用度,黄体酮脂质体能通过改变黄体酮的吸收机制,使黄体酮转运以被动扩散方式进行,从而明显促进黄体酮的吸收.

  11. A low-molecular-weight factor in human milk whey promotes iron uptake by Caco-2 cells.

    Science.gov (United States)

    Etcheverry, Paz; Miller, Dennis D; Glahn, Raymond P

    2004-01-01

    The iron bioavailability of human milk (HM) is substantially greater than that of cow's milk (CM), but the factor responsible for this high bioavailability is unknown. This study evaluated the effects of various HM and CM fractions on iron bioavailability. Milk was separated into fat, casein and whey fractions by ultracentrifugation. Whey was further fractionated by ultrafiltration with a 10-kDa membrane to produce a 10-kDa retentate (10kR) and a 10-kDa filtrate (10kF). Samples were prepared by mixing various combinations of the fractions, bringing the samples to prefractionation weight with minimum essential medium (MEM), and adding iron (10 micro mol/L) as ferrous sulfate. Samples were divided into two aliquots: one was subjected to in vitro digestion, the other was not. Bioavailability was assessed by applying the samples to Caco-2 cell monolayers and incubating for 24 h. Ferritin formation in the cells was used as an index of iron uptake. Removing the fat from undigested HM samples doubled the ferritin formation, but removing the whey or casein had no effect. Results with digested HM samples were similar, except that removing the whey decreased ferritin formation by 48%. Removing the fat from digested CM samples had no effect, but removing the casein doubled the ferritin formation. Removing the 10kF from HM reduced ferritin formation by 60%, but removing the 10kR had no effect. These data suggest that a low-molecular-weight factor (milk enhances iron absorption.

  12. Pectin from Prunus domestica L. induces proliferation of IEC-6 cells through the alteration of cell-surface heparan sulfate on differentiated Caco-2 cells in co-culture.

    Science.gov (United States)

    Nishida, Mitsutaka; Murata, Kazuma; Oshima, Kazuya; Itoh, Chihiro; Kitaguchi, Kohji; Kanamaru, Yoshihiro; Yabe, Tomio

    2015-05-01

    Dietary fiber intake provides various physiological and metabolic effects for human health. Pectin, a water-soluble dietary fiber, induces morphological changes of the small intestine in vivo. However, the molecular mechanisms underlying pectin-derived morphological alterations have not been elucidated. Previously, we found that pectin purified from Prunus domestica L. altered the sulfated structure of cell-surface heparan sulfate (HS) on differentiated Caco-2 cells via fibronectin and α5β1 integrin. In this study, we investigated the biological significance of the effect of pectin on HS in differentiated Caco-2 cells. An in vitro intestinal epithelium model was constructed by co-culture of differentiated Caco-2 cells and rat IEC-6 cells, which were used as models of intestinal epithelium and intestinal crypt cells, respectively. We found that pectin-treated differentiated Caco-2 cells promoted growth of IEC-6 cells. Real-time RT-PCR analysis and western blotting showed that relative mRNA and protein expression levels of Wnt3a were upregulated by pectin treatment in differentiated Caco-2 cells. Analysis by surface plasmon resonance spectroscopy demonstrated that pectin-induced structural alteration of HS markedly decreased the interaction with Wnt3a. However, depression in the secretion of Wnt3a from Caco-2 cells by anti-Wnt3a antibody did not affect the proliferation of IEC-6 cells in co-culture system. These observations indicated that pectin altered the sulfated structure of cell-surface HS to promote secretion of Wnt3a from differentiated Caco-2 cells and Wnt3a indirectly stimulated the proliferation of IEC-6 cells.

  13. Effect of different surfactants in biorelevant medium on the secretion of a lipophilic compound in lipoproteins using Caco-2 cell culture

    DEFF Research Database (Denmark)

    Karpf, Ditte M; Holm, René; Garafalo, Carole

    2006-01-01

    The impact of a pharmaceutical relevant metabolizable, ionic surfactant or two synthetic, nonionic surfactants on the absorption and lipoprotein incorporation of a lipophilic drug, retinol, was studied in the Caco-2 cell culture. Filter-grown monolayers of Caco-2 cells were incubated for 20 h...... was ultracentrifugated into different lipoprotein classes and their (3)H-retinol and (14)C-lipid concentrations were determined. The cells incubated with lyso-PC and Tween 80 increased the incorporation of (3)H-retinol and (14)C-lipid into chylomicrons and very low density lipoproteins (VLDL). The explored surfactants...... impacted the incorporation of (3)H-retinol and (14)C-lipid in chylomicrons and VLDL in a concentration-dependent manner. As these surfactants interfere with the intestinal lipoprotein secretion, inclusion of high concentrations of the surfactants in lipid-based formulations of poorly aqueous soluble drugs...

  14. The inhibitory and combinative mechanism of HZ08 with P-glycoprotein expressed on the membrane of Caco-2 cell line

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yanyan; Hu, Yahui; Feng, Yidong; Kodithuwakku, Nandani Darshika; Fang, Weirong [State Key Laboratory of Natural Medicines, Department of Physiology, China Pharmaceutical University, Nanjing 210009 (China); Li, Yunman, E-mail: yunmanlicpu@hotmail.com [State Key Laboratory of Natural Medicines, Department of Physiology, China Pharmaceutical University, Nanjing 210009 (China); Huang, Wenlong [Center of Drug Discovery, China Pharmaceutical University, Nanjing 210009 (China)

    2014-01-15

    Recently, the research and development of agents to reverse the phenomenon of multidrug resistance has been an attractive goal as well as a key approach to elevating the clinical survival of cancer patients. Although three generations of P-glycoprotein modulators have been identified, poor clearance and metabolism render these agents too toxic to be used in clinical application. HZ08, which has been under investigation for several years, shows a dramatic reversal effect with low cytotoxicity. For the first time, we aimed to describe the interaction between HZ08 and P-glycoprotein in Caco-2 cell line in which P-glycoprotein is overexpressed naturally. Cytotoxicity and multidrug resistance reversal assays, together with flow cytometry, fluorescence microscopy and siRNA interference as well as Caco-2 monolayer transport model were employed in this study to evaluate the interaction between HZ08 and P-glycoprotein. This study revealed that HZ08 was capable of reversing adriamycin resistance mediated by P-glycoprotein as a result of intracellular enhancement of adriamycin accumulation, which was found to be superior to verapamil. In addition, we confirmed that HZ08 suppressed the transport of Rhodamine123 in the Caco-2 monolayer model but had little effect on P-glycoprotein expression. The transport of HZ08 was diminished by P-glycoprotein inhibitors (verapamil and LY335979) and its accumulation was increased via siRNA targeting MDR1 in Caco-2 cells. Furthermore, considering the binding site of P-glycoprotein, verapamil performed as a competitive inhibitor with HZ08. In conclusion, as a P-glycoprotein substrate, HZ08 inhibited P-glycoprotein activity and may share the same binding site of verapamil to P-glycoprotein. - Highlights: • The cytotoxicity and reversing effect of HZ08 was measured in Caco-2 cell line. • HZ08 inhibited the transport of Rhodamine123 across Caco-2 cell monolayer. • The efflux ratio of HZ08 was dropped when combined with P

  15. Effect of nonfat dry milk and major whey components on interleukin-6 and interleukin-8 production in human intestinal epithelial-like Caco-2 cells.

    Science.gov (United States)

    Ustunol, Z; Wong, C

    2010-06-01

    Bovine nonfat dry milk (NDM) and major whey components (lactose, alpha-lactalbumin, and beta-lactoglobulin) were evaluated for their effects on IL-6 and IL-8 production in human intestinal-like Caco-2 cells unstimulated or stimulated with IL-1beta. All the whey components investigated and NDM induced IL-6 production by Caco-2 cells; the most significant increase was observed with beta-lactoglobulin. In the case of IL-1beta-stimulated cells, neither NDM nor the major whey components investigated contributed to the induction of IL-6 production after they were stimulated. Induction of IL-8 production by both alpha-lactalbumin and beta-lactoglobulin was higher than that by lactose and NDM; alpha-lactalbumin was a more potent inducer of IL-8 than beta-lactoglobulin and IL-1beta alone in both unstimulated and stimulated cells. In Caco-2 cells that were stimulated with IL1-beta, NDM and all the major whey components investigated had a synergistic effect on induction of IL-8 production, indicating that IL-8 induction was amplified by prior stimulation of cells by IL-1beta. This synergistic effect was not observed with IL-6. Our results suggest that immunomodulatory properties of milk components may be affected by other complex events in the gut.

  16. Adhesion, invasion, intracellular survival and cytotoxic activity of strains of Aeromonas spp. in HEp-2, Caco-2 and T-84 cell lines.

    Science.gov (United States)

    dos Santos, Paula Azevedo; Pereira, Ana Claudia Machado; Ferreira, Andréa Fonseca; de Mattos Alves, Maria Angélica; Rosa, Ana Cláudia Paula; Freitas-Almeida, Angela Corrêa

    2015-05-01

    The genus Aeromonas contains important pathogen for both humans and other animals, being responsible for the etiology of intestinal and extraintestinal diseases. The pathology caused by these bacteria involves several virulence factors, such as the ability to produce toxins, adhesion and invasion. The properties conferred by these factors have been extensively studied in experiments of interaction between bacterial strains and cell culture. We evaluate the interaction of eight Aeromonas spp. strains, previously isolated from human faeces, food and water with HEp-2, Caco-2 and T-84 cell lines. Cytotoxic effects, the pattern of adhesion, invasive capacity and intracellular survival were analyzed. The results showed that Aeromonas strains were adherent to three cells lines in 6 h of incubation, displaying the aggregative adherence pattern. Among eight strains studied, 50% produced cytotoxic effects on HEp-2 cells, while none of the strains produced cytotoxic effects on Caco-2 and T-84 cells at 48 h. This study demonstrated that subsets of Aeromonas isolated from different sources were able to invade intestinal (T-84, Caco-2) and epithelial (HEp-2) cell lines cultivated in vitro surviving in intracellular environments up to 72 h. Finally, our results support the pathogenic potential of Aeromonas, especially those of food and clinical sources.

  17. Candida albicans and Saccharomyces cerevisiae induce interleukin-8 production from intestinal epithelial-like Caco-2 cells in the presence of butyric acid.

    Science.gov (United States)

    Saegusa, Shizue; Totsuka, Mamoru; Kaminogawa, Shuichi; Hosoi, Tomohiro

    2004-07-01

    Intestinal epithelial cells (IEC) are important in initiation and regulation of immune responses against numerous foreign substances including food, microorganisms and their metabolites in the intestine. Since the responses of IEC against yeasts have not yet been well understood, we investigated the effects of Candida albicans, Saccharomyces cerevisiae, and their cell wall components on interleukin-8 (IL-8) secretion by the IEC-like Caco-2 cells. Live cells of both yeast species stimulated Caco-2 cells to produce IL-8 only in the presence of butyric acid, which is a metabolite produced by intestinal bacteria. S. cerevisiae zymosan and glucan also enhanced IL-8 secretion. Treatment of Caco-2 cells with butyric acid increased the expression of mRNAs coding for Toll-like receptor 1 (TLR1), TLR6 and dectin-1, which recognize zymosan. C. albicans induced more IL-8 secretion and also decreased transepithelial electrical resistance more rapidly than S. cerevisiae. These results suggest that both yeasts in the intestine stimulate the host's mucosal immune systems by interacting with IEC.

  18. LXR/RXR ligand activation enhances basolateral efflux of beta-sitosterol in CaCo-2 cells.

    Science.gov (United States)

    Field, F Jeffrey; Born, Ella; Mathur, Satya N

    2004-05-01

    To examine whether intestinal ABCA1 was responsible for the differences observed between cholesterol and beta-sitosterol absorption, ABCA1-facilitated beta-sitosterol efflux was investigated in CaCo-2 cells following liver X receptor/retinoid X receptor (LXR/RXR) activation. Both the LXR agonist T0901317 and the natural RXR/LXR agonists 22-hydroxycholesterol and 9-cis retinoic acid enhanced the basolateral efflux of beta-sitosterol without altering apical efflux. LXR-mediated enhanced beta-sitosterol efflux occurred between 6 h and 12 h after activation, suggesting that transcription, protein synthesis, and trafficking was likely necessary prior to facilitating efflux. The transcription inhibitor actinomycin D prevented the increase in beta-sitosterol efflux by T0901317. Glybenclamide, an inhibitor of ABCA1 activity, and arachidonic acid, a fatty acid that interferes with LXR activation, also prevented beta-sitosterol efflux in response to the LXR ligand activation. Influx of beta-sitosterol mass did not alter the basolateral or apical efflux of the plant sterol, nor did it alter ABCA1, ABCG1, ABCG5, or ABCG8 gene expression or ABCA1 mass. Similar to results observed with intestinal ABCA1-facilitated cholesterol efflux, LXR/RXR ligand activation enhanced the basolateral efflux of beta-sitosterol without affecting apical efflux. The results suggest that ABCA1 does not differentiate between cholesterol and beta-sitosterol and thus is not responsible for the selectivity of sterol absorption by the intestine. ABCA1, however, may play a role in beta-sitosterol absorption.

  19. Study on the Mechanism of Intestinal Absorption of Epimedins A, B and C in the Caco-2 Cell Model

    Directory of Open Access Journals (Sweden)

    Yan Chen

    2014-01-01

    Full Text Available Epimedium spp. is commonly used in Traditional Chinese Medicine. Epimedins A, B, and C are three major bioactive flavonoids found in Epimedium spp. that share similar chemical structures. In this study, the intestinal absorption mechanism of these three compounds was investigated using the Caco-2 cell monolayer model in both the apical-to-basolateral (A-B and the basolateral-to-apical (B-A direction. The absorption permeability (PAB of epimedins A, B, and C were extremely low and increased as the concentration of the epimedins increased from 5 to 20 μM, but, at 40 μM, the PAB values were reduced. Meanwhile, the amount of transported compounds increased in a time-dependent manner. The PAB of epimedins A and C were significantly increased and efflux ratios decreased in the presence of verapamil (an inhibitor of P-glycoprotein and dipyridamole (an inhibitor of breast cancer resistance protein while, in the presence of MK571 (an inhibitor of multidrug resistance proteins, the absorption of epimedins A and C did not change significantly, indicating that P-gp and BCRP might be involved in the transport of epimedins A and C. The PAB of epimedin B significantly increased while its secretory permeability (PBA significantly decreased in the presence of dipyridamole, indicating that BCRP might be involved in the transport of epimedin B. No obvious changes in the transport of epimedin B were observed in the presence of verapamil and MK571. In summary, our results clearly demonstrate, for the first time, that poor bioavailability of these three prenylated flavonoids is the result of poor intrinsic permeability and efflux by apical efflux transporters.

  20. Estrogen-related receptor ERRα regulation of human hydroxysteroid sulfotransferase (SULT2A1) gene expression in human Caco-2 cells.

    Science.gov (United States)

    Huang, Chaoqun; Zhou, Tianyan; Chen, Yue; Sun, Teng; Zhang, Shunfen; Chen, Guangping

    2014-01-01

    Human hydroxysteroid sulfotransferase, SULT2A1, is important for xenobiotic detoxification and the maintenance of hydroxysteroid homeostasis. Our published report suggested that estrogen-related receptor ERRα downregulates SULT2A1 in Hep G2 cells. The results shown in this study suggest that ERRα upregulates SULT2A1 transcription in Caco-2 cells. The deletion analysis suggested that SULT2A1 promoter region between -65 and -44 is important for this upregulation. Our further investigation suggested that ERRα binding element, ERRE51, mediates ERRα activation of SULT2A1 promoter transcription in Caco-2 cells. The interaction of ERRE51 with ERRα was confirmed by electrophoretic mobility shift assay and chromatin immunoprecipitation analysis. Results also suggest that the difference of constitutive androstane receptor transcription levels in Hep G2 and Caco-2 cells at least partially contribute to the cell type dependent ERRα modulation of SULT2A1 promoter transcription. ERRα regulates human SULT2A1 transcription by competing with other nuclear receptors binding to the DNA-promoter region.

  1. Poly-L-arginine-Induced internalization of tight junction proteins increases the paracellular permeability of the Caco-2 cell monolayer to hydrophilic macromolecules.

    Science.gov (United States)

    Yamaki, Tsutomu; Ohtake, Kazuo; Ichikawa, Keiko; Uchida, Masaki; Uchida, Hiroyuki; Oshima, Shinji; Ohshima, Shinji; Juni, Kazuhiko; Kobayashi, Jun; Morimoto, Yasunori; Natsume, Hideshi

    2013-01-01

    We investigated whether poly-L-arginine (PLA) enhances the paracellular permeability of the Caco-2 monolayer to hydrophilic macromolecules and clarified the disposition of tight junction (TJ) proteins. The transepithelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC)-dextran (FD-4) permeation were determined after treatment with PLA. TJ proteins were visualized using immunofluorescence microscopy after PLA exposure and depletion, and their expression levels were determined. The barrier function of TJs was also evaluated by measuring the alterations in the TEER and in the localization of TJ proteins. PLA induced an increase in hydrophilic macromolecule, FD-4, permeation through Caco-2 cell monolayers and a decrease in the TEER in a concentration-dependent manner, without any significant impact on the cell viability. This increased paracellular permeability induced by PLA was found to be internalized of claudin-4, ZO-1, tricellulin and mainly occludin from cell-cell junction to the subcellular space. ZO-1 appeared to play an important role in the reconstitution of TJ strand structures following PLA depletion. These results indicate that the PLA led to the internalization of TJ proteins to the subcellular space, subsequently increasing the permeability of the Caco-2 cell monolayer to FD-4 via a paracellular route.

  2. CFTR depletion results in changes in fatty acid composition and promotes lipogenesis in intestinal Caco 2/15 cells.

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    Geneviève Mailhot

    Full Text Available BACKGROUND: Abnormal fatty acid composition (FA in plasma and tissue lipids frequently occurs in homozygous and even in heterozygous carriers of cystic fibrosis transmembrane conductance regulator (CFTR mutations. The mechanism(s underlying these abnormalities remained, however, poorly understood despite the potentially CFTR contributing role. METHODOLOGY/PRINCIPAL FINDINGS: The aim of the present study was to investigate the impact of CFTR depletion on FA uptake, composition and metabolism using the intestinal Caco-2/15 cell line. shRNA-mediated cftr gene silencing induced qualitative and quantitative modifications in FA composition in differentiated enterocytes as determined by gas-liquid chromatography. With the cftr gene disruption, there was a 1,5 fold increase in the total FA amount, largely attributable to monounsaturated and saturated FA compared to controls. The activity of delta-7 desaturase, estimated by the 16:1(n-7/16:0, was significantly higher in knockdown cells and consistent with the striking elevation of the n-7 FA family. When incubated with [14C]-oleic acid, CFTR-depleted cells were capable of quick incorporation and export to the medium concomitantly with the high protein expression of L-FABP known to promote intracellular FA trafficking. Accordingly, lipoprotein vehicles (CM, VLDL, LDL and HDL, isolated from CFTR knockdown cells, exhibited higher levels of radiolabeled FA. Moreover, in the presence of [14C]-acetate, knockdown cells exhibited enhanced secretion of newly synthesized phospholipids, triglycerides, cholesteryl esters and free FA, thereby suggesting a stimulation of the lipogenic pathway. Conformably, gene expression of SREBP-1c, a key lipogenic transcription factor, was increased while protein expression of the phosphorylated and inactive form of acetylCoA carboxylase was reduced, confirming lipogenesis induction. Finally, CFTR-depleted cells exhibited lower gene expression of transcription factors (PPARalpha

  3. Procyanidin B2 and a cocoa polyphenolic extract inhibit acrylamide-induced apoptosis in human Caco-2 cells by preventing oxidative stress and activation of JNK pathway.

    Science.gov (United States)

    Rodríguez-Ramiro, Ildefonso; Ramos, Sonia; Bravo, Laura; Goya, Luis; Martín, María Ángeles

    2011-12-01

    Humans are exposed to dietary acrylamide (AA) during their lifetime; it is therefore necessary to investigate the mechanisms associated with AA induced toxic effects. Accumulating evidence indicates that oxidative stress may contribute to AA cytotoxicity, but the link between oxidative stress and AA cytotoxicity in the gastrointestinal tract, the primary organ in contact with dietary AA, has not been described. In this study, we evaluate the alterations of the redox balance induced by AA in Caco-2 intestinal cells as well as the potential protective role of natural antioxidants such as a well-standardized cocoa polyphenolic extract (CPE) and its main polyphenol components epicatechin (EC) and procyanidin B2 (PB2). We found that AA-induced oxidative stress in Caco-2 cells is evidenced by glutathione (GSH) depletion and reactive oxygen species (ROS) overproduction. AA also activated the extracellular-regulated kinases and the c-Jun N-amino terminal kinases (JNKs) leading to an increase in caspase-3 activity and cell death. Studies with appropriate inhibitors confirmed the implication of oxidative stress and JNKs activation in AA-induced apoptosis. Additionally, AA cytotoxicity was counteracted by CPE or PB2 by inhibiting GSH consumption and ROS generation, increasing the levels of gamma-glutamyl cysteine synthase and glutathione-S-transferase and blocking the apoptotic pathways activated by AA. Therefore, AA-induced cytotoxicity and apoptosis are closely related to oxidative stress in Caco-2 cells. Interestingly, natural dietary antioxidant such as PB2 and CPE were able to suppress AA toxicity by improving the redox status of Caco-2 cells and by blocking the apoptotic pathway activated by AA.

  4. The expression of apoB mRNA editing factors is not the sole determinant for the induction of editing in differentiating Caco-2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Galloway, Chad A. [Department of Biochemistry and Biophysics, University of Rochester School of Medicine, 601 Elmwood Ave., Rochester, NY 14642 (United States); Smith, Harold C., E-mail: harold.smith@rochester.edu [Department of Biochemistry and Biophysics, University of Rochester School of Medicine, 601 Elmwood Ave., Rochester, NY 14642 (United States)

    2010-01-01

    Apolipoprotein B mRNA is edited at cytidine 6666 in the enterocytes lining the small intestine of all mammals; converting a CAA codon to a UAA stop codon. The conversion is {approx}80% efficient in this tissue and leads to the expression of the truncated protein, ApoB48, essential for secretion of dietary lipid as chylomicrons. Caco-2 cell raft cultures have been used as an in vitro model for the induction of editing activity during human small intestinal cell differentiation. This induction of apoB mRNA editing has been ascribed to the expression of APOBEC-1. In agreement our data demonstrated differentiation-dependent induction of expression of the editing enzyme APOBEC-1 and in addition we show alternative splicing of the essential auxiliary factor ACF. However, transfection of these editing factors in undifferentiated proliferating Caco-2 cells was not sufficient to induce robust apoB mRNA editing activity. Only differentiation of Caco-2 cells could induce more physiological like levels of apoB mRNA editing. The data suggested that additional regulatory mechanism(s) were induced by differentiation that controlled the functional activity of editing factors.

  5. Avaliação da atividade antioxidante do extrato hidroalcoólico da romã (Punica granatum, L. sobre células da linhagem Caco-2 Antioxidant activity evaluation of the pomegranate (Punica granatum, L. hidroalcoholic extract at Caco-2 cell line

    Directory of Open Access Journals (Sweden)

    Fernanda Archilla Jardini

    2007-08-01

    Full Text Available A absorção dos compostos antioxidantes presentes no extrato hidroalcoólico da polpa da romã (Punica granatum, L. foi avaliada utilizando-se o modelo de cultura de células, cuja linhagem escolhida foi a Caco-2, provenientes de um adenocarcinoma do cólon. O extrato hidroalcoólico da polpa apresentou um conteúdo de 832 µg equivalente de ácido gálico.mL-1 de extrato e sua atividade antioxidante mostrou valores de 5,9% (0,1 ppm a 93,09% (8 ppm de capacidade de redução do radical DPPH•. As células tratadas com o extrato apresentaram uma inibição de crescimento celular de 4,16% (200 ppm, e a absorção dos compostos antioxidantes foi de 63,25%. Para o ácido gálico, os resultados mostraram uma inibição de 2,98% (400 ppm e uma absorção de 72,54% dos compostos antioxidantes. Os resultados mostraram que o método de avaliação de absorção através de cultura de células foi eficaz, e que os compostos antioxidantes foram absorvidos pelas células, que se apresentaram viáveis, após um período de exposição prolongado aos compostos.The absorption of antioxidant compounds present in hydroalcoholic extract of pomegranate pulp (Punica granatum, L. was evaluated by a cell culture model, using the Caco-2 cells (from an adenocarcinom of the colon. The hydroalcoholic extract of the pulp showed a content of reducing compounds of approximately 832 µg equivalent to gallic acid.mL-1 of the extract and its antioxidant activity was from 5.9% (0.1 ppm to 93.09% (8 ppm, measured by the capacity of reducing the DPPH• radical. The cells that were treated with the extract showed a 4.16% of growing inibition (at a concentration of 200 ppm, and an absorption of the antioxidants compounds of approximately 63.25%. The gallic acid showed, at 400 ppm of concentration, a growing inhibition of 2.98% and anabsorption of 72.54% of the antioxidant compounds. These results pointed out that the method of evaluating the absorption by using cell culture was

  6. Effects of Ursolic Acid Derivatives on Caco-2 Cells and Their Alleviating Role in Streptozocin-Induced Type 2 Diabetic Rats

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    Panpan Wu

    2014-08-01

    Full Text Available In this study, the effect and mechanism of a series of ursolic acid (UA derivatives on glucose uptake were investigated in a Caco-2 cells model. Their effect on hyperglycemia, hyperlipidemia and oxidative stress were also demonstrated in streptozocin (STZ-induced diabetic rats. 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-ylamino]-2-deoxy-glucose (2-NBDG was used as a fluorescein in Caco-2 cells model to screen UA derivatives by glucose uptake and expression of glucose transporter protein (SGLT-1, GLUT-2. Moreover, STZ-induced diabetic rats were administered with these derivatives for 4 weeks of treatment. The fasting blood glucose (FBG, insulin levels, biochemical parameters, lipid levels, and oxidative stress markers were finally evaluated. The results of this study indicated that compounds 10 and 11 significantly inhibited 2-NBDG uptake under both Na+-dependent and Na+-independent conditions by decreasing SGLT-1 and GLUT-2 expression in the Caco-2 cells model. Further in vivo studies revealed that compound 10 significantly reduced hyperglycemia by increasing levels of serum insulin, total protein, and albumin, while the fasting blood glucose, body weight and food intake were restored much closer to those of normal rats. Compounds 10 and 11 showed hypolipidemic activity by decreasing the total amounts of cholesterol (TC and triglycerides (TG. Furthermore, compound 10 showed antioxidant potential which was confirmed by elevation of glutathione (GSH and superoxide dismutase (SOD and reduction of malondialdehyde (MDA levels in the liver and kidney of diabetic rats. It was concluded that compound 10 caused an apparent inhibition of intestinal glucose uptake in Caco-2 cells and hypoglycemia, hypolipidemia and augmented oxidative stress in STZ-induced diabetic rats. Thus, compound 10 could be developed as a potentially complementary therapeutic or prophylactic agent for diabetics mellitus and its complications.

  7. Cytotoxic Effect and Permeability Activities of Curcumin Analogue; 2, 6-Bis (2, 5-dimethoxybenzy-lidene cyclohexanone (BDMC33 in Caco-2 Cell Model

    Directory of Open Access Journals (Sweden)

    N. Yakubu

    2015-11-01

    Full Text Available Previously, curcumin analogue, 2, 6-bis (2, 5-dimethoxybenzylidene cyclohexanone (BDMC33 with high anti-inflammatory activity was chemically synthesized in our laboratory to enhance the biological activity of curcumin. In this study, the toxicity and permeability activities of 2,6-bis(2,5-dimethoxybenzy-lidenecyclohexanone (BDMC33 in Caco-2 cells was investigated. Toxicity effects using MTT assay and apparent permeability coefficient (Papp, uptake (UR and efflux (ER ratios, and mass balance of BDMC33 after permeation in Caco-2 cells for 180 min were evaluated in apical (A to basolateral (B and basolateral (B to apical (A directions. The similar analyses on 3-(2-fluoro-benzylidene-5-(2-fluorocyclohexylmethylene-piperidin-4-one; (EF-24 (check control were also conducted. The 24 hr LC50 value for BDMC33 and EF-24 on Caco-2 cells were both 50 µM. The Papp value in A→B direction was 3.37 ± 0.47 cm/s (BDMC33 and 2.47 ± 0.15 cm/s (EF-24. Whereas in B→A direction, it was 1.9 ± 0.36 cm/s (BDMC33 and 1.8 ± 0.15 cm/s (EF-24 upon 120 min incubation. The UR and ER ratios calculated were 1.77% and 0.56%, respectively, and the mass balance calculated were 41-44% (BDMC33 and 31-34% (EF-24 in A→B and B→A direction. This study has suggested BDMC33 to be more absorbable than EF-24 in Caco-2 cells. Therefore, BDMC33 could be a leading feature, the anti-inflammatory agent, as it biological activities would be expected outside the intestine.

  8. Evaluation of the cytotoxicity and genotoxicity of extracts of mussels originating from Moroccan Atlantic coast, in human colonic epithelial cells Caco-2.

    Science.gov (United States)

    Nasser, Boubker; Moustaid, Khadija; Moukha, Serge; Mobio, Théophile A; Essamadi, Abdelkhalid; Creppy, Edmond E

    2008-08-01

    Industrial processing of phosphates generates chemical wastes which are, without any treatment, discharged directly into the Atlantic Ocean at Jorf Lasfar (JL), located 120 km south of Casablanca (Morocco) were shellfish are also collected by people without any control. Marine bivalves concentrate these pollutants by filtration and serve as vectors in human's exposure. The objective of this study was to test and compare in vitro on human intestinal cells (Caco-2) the cytotoxicity and genotoxicity of mussels (Mytilus galloprovincialis) extracts (either hydrophilic or lipophilic) collected at two coastal sites; JL (neighboring a phosphate processing plat-form) and Oualidia (OL) (a vegetable growing area) located 160 km south of Casablanca (i.e. 40 km south of JL). Using Caco-2 cells, the following end-points have been evaluated, cytotoxicity as measured by MTS test, inhibition of cellular macromolecules syntheses (DNA and protein) and genotoxicity evaluated by DNA fragmentation in agarose gel electrophoresis. The results indicated, that hydrophilic and lipophilic OL mussels extracts are cytotoxic and inhibit cellular macromolecules syntheses. Moreover these extracts damage the DNA in Caco-2 cells. The lipophilic JL mussels extract is cytotoxic, inhibits cellular macromolecules syntheses, and damages the DNA in Caco-2 cells whereas the hydrophilic extract of JL mussels fails to inhibit protein synthesis and does not damage the DNA. This extract rather enhances protein synthesis, suggesting possible metallothioneins induction by metal ions. Altogether these in vitro data indicate that mussels collected from OL could be more harmful than those from JL even though the later is closer to the pollution site than OL. Nevertheless consumption of mussels from all these areas may present a risk for humans. Epidemiological studies will be needed for global risk assessment in humans living in these areas especially those consuming see food regularly.

  9. Wild Raspberry Subjected to Simulated Gastrointestinal Digestion Improves the Protective Capacity against Ethyl Carbamate-Induced Oxidative Damage in Caco-2 Cells.

    Science.gov (United States)

    Chen, Wei; Xu, Yang; Zhang, Lingxia; Li, Ya; Zheng, Xiaodong

    2016-01-01

    Ethyl carbamate (EC), a probable human carcinogen, occurs widely in many fermented foods. Previous studies indicated that EC-induced cytotoxicity was associated with oxidative stress. Wild raspberries are rich in polyphenolic compounds, which possess potent antioxidant activity. This study was conducted to investigate the protective effect of wild raspberry extracts produced before (RE) and after in vitro simulated gastrointestinal digestion (RD) on EC-induced oxidative damage in Caco-2 cells. Our primary data showed that ethyl carbamate could result in cytotoxicity and genotoxicity in Caco-2 cells and raspberry extract after digestion (RD) may be more effective than that before digestion (RE) in attenuating toxicity caused by ethyl carbamate. Further investigation by fluorescence microscope revealed that RD may significantly ameliorate EC-induced oxidative damage by scavenging the overproduction of intracellular reactive oxygen species (ROS), maintaining mitochondrial function and preventing glutathione (GSH) depletion. In addition, HPLC-ESI-MS results showed that the contents of identified polyphenolic compounds (esculin, kaempferol O-hexoside, and pelargonidin O-hexoside) were remarkably increased after digestion, which might be related to the better protective effect of RD. Overall, our results demonstrated that raspberry extract undergoing simulated gastrointestinal digestion may improve the protective effect against EC-induced oxidative damage in Caco-2 cells.

  10. Insoluble fraction of buckwheat (Fagopyrum esculentum Moench) protein possessing cholesterol-binding properties that reduce micelle cholesterol solubility and uptake by Caco-2 cells.

    Science.gov (United States)

    Metzger, Brandon T; Barnes, David M; Reed, Jess D

    2007-07-25

    Buckwheat (Fagopyrum esculentum Moench) protein (BWP) exhibits hypocholesterolemic activity in several animal models by increasing fecal excretion of neutral and acidic sterols. In the current study, the ability of BWP to disrupt micelle cholesterol solubility by sequestration of cholesterol was investigated. When BWP (0.2%) was incubated with cholesterol and micelle lipid components prior to micelle formation, cholesterol solubility was reduced 40%. In contrast, cholesterol solubility was not decreased when BWP (0.2%) was incubated after micelle formation and incorporation of soluble cholesterol. Buckwheat flour, from which BWP was derived, had no significant effect on cholesterol solubility. Cholesterol uptake in Caco-2 cells from micelles made in the presence of BWP (0.2%) was reduced by 47, 36, 35, and 33% when compared with buckwheat flour, bovine serum albumin, casein, and gelatin, respectively. Reduction in cholesterol uptake in Caco-2 cells was dose-dependent, with maximum reductions at 0.1-0.4% BWP. In cholesterol-binding experiments, 83% of the cholesterol was associated with an insoluble BWP fraction, indicating strong cholesterol-binding capacity that disrupts solubility and uptake by Caco-2 cells.

  11. Glucose and calcium ions may modulate the efficiency of bovine β-casomorphin-7 permeability through a monolayer of Caco-2 cells.

    Science.gov (United States)

    Jarmołowska, Beata; Teodorowicz, Małgorzata; Fiedorowicz, Ewa; Sienkiewicz-Szłapka, Edyta; Matysiewicz, Michał; Kostyra, Elżbieta

    2013-11-01

    Milk and dairy products provide a lot of valuable nutritive elements. They are also sources of biologically active peptides, including β-casomorphins that manifest the properties of morphine. An activity of DPPIV seems to be most crucial factor decreasing the efficiency of the β-casomorphin-7 (BCM7) transport. The increase of BCM7 concentration in blood may intensify symptoms of apparent life threatening events (ALTE), autism, schizophrenia, and allergy. This study aimed at identifying the influence of several selected substances on a transport efficiency of bovine BCM7 through an intestinal monolayer in a Caco-2 cell model system. Applying the ELISA method, the permeability coefficient of BCM7 through the Caco-2 monolayer was calculated. TEER values were used to evaluate the integrity of Caco-2 cell monolayers. An increase of glucose and Ca(2+) concentrations in the culture medium was accompanied by an increase of the BCM7 transport efficiency. The lowest permeability coefficients of BCM7 were observed for the membranes with high electrical resistances. The transport was enhanced in the presence of milk infant formulas, whereas no changes were observed when using μ-opioid receptor antagonist (casoxin-6). The results may be useful in understanding the pathogenesis of inflammation and food allergy in infants.

  12. Mathematical modelling of the transport of hydroxypropyl-β-cyclodextrin inclusion complexes of ranitidine hydrochloride and furosemide loaded chitosan nanoparticles across a Caco-2 cell monolayer.

    Science.gov (United States)

    Sadighi, Armin; Ostad, S N; Rezayat, S M; Foroutan, M; Faramarzi, M A; Dorkoosh, F A

    2012-01-17

    Chitosan nanoparticles (CS-NPs) have been used to enhance the permeability of furosemide and ranitidine hydrochloride (ranitidine HCl) which were selected as candidates for two different biopharmaceutical drug classes having low permeability across Caco-2 cell monolayers. Drugs loaded CS-NPs were prepared by ionic gelation of CS and pentasodium tripolyphosphate (TPP) which added to the drugs inclusion complexes with hydroxypropyl-β-cyclodextrin (HP-βCD). The stability constants for furosemide/HP-βCD and ranitidine HCl/HP-βCD were calculated as 335 M(-1) and 410 M(-1), whereas the association efficiencies (AE%) of the drugs/HP-βCD inclusion complexes with CS-NPs were determined to be 23.0 and 19.5%, respectively. Zetasizer and scanning electron microscopy (SEM) were used to characterise drugs/HP-βCD-NPs size and morphology. Transport of both nano and non-nano formulations of drugs/HP-βCD complexes across a Caco-2 cell monolayer was assessed and fitted to mathematical models. Furosemide/HP-βCD-NPs demonstrated transport kinetics best suited for the Higuchi model, whereas other drug formulations demonstrated power law transportation behaviour. Permeability experiments revealed that furosemide/HP-βCD and ranitidine HCl/HP-βCD nano formulations greatly induce the opening of tight junctions and enhance drug transition through Caco-2 monolayers.

  13. Related research of building intestinal barrier modal with Caco-2 Cells%关于Caco-2细胞构建肠粘膜屏障模型的相关研究

    Institute of Scientific and Technical Information of China (English)

    吴茂军; 侯龙龙

    2016-01-01

    目的 通过用Caco-2细胞的体外培养,建立肠粘膜屏障的模型.方法 通过对Caco-2细胞在体外transwell小室中连续培养21 d,并对培养的细胞进行形态学观察、跨膜电阻测定、inulin-FITC通透率测定,评估肠屏障模型是否构建成功.结果 通过体外连续培养Caco-2细胞形成了致密单层,可见细胞层单层排列,相互融合,边界清晰,细胞间紧密连接清晰可以见,21 dTER值为496.32±6.02(Ω·cm2),inulin-FITC的通透率<0.5%.结论 通过细胞形态学观察,跨膜电阻测定及inulin-FITC的通透率显示Caco-2细胞体外构建肠屏障模型成功,此模型对于相关疾病的研究具有一定意义.

  14. Cobalt chloride decreases fibroblast growth factor-21 expression dependent on oxidative stress but not hypoxia-inducible factor in Caco-2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Yanlong [School of Pharmacy, Wenzhou Medical College, Wenzhou (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Wang, Chunhong [Second Hospital, Jilin University, Changchun (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Wang, Yuhua [College of Food Science and Engineering, Jilin Agricultural University, Changchun (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Ma, Zhenhua [First Hospital, Xi' an Jiaotong University, Xi' an (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Xiao, Jian [School of Pharmacy, Wenzhou Medical College, Wenzhou (China); McClain, Craig [Department of Medicine, University of Louisville, Louisville, KY (United States); Department of Pharmacology and Toxicology, University of Louisville, Louisville, KY (United States); Alcohol Research Center, University of Louisville, Louisville, KY (United States); Robley Rex Veterans Affairs Medical Center, Louisville, KY (United States); Li, Xiaokun [School of Pharmacy, Wenzhou Medical College, Wenzhou (China); Feng, Wenke, E-mail: wenke.feng@louisville.edu [School of Pharmacy, Wenzhou Medical College, Wenzhou (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Alcohol Research Center, University of Louisville, Louisville, KY (United States)

    2012-10-15

    Fibroblast growth factor-21 (FGF21) is a potential metabolic regulator with multiple beneficial effects on metabolic diseases. FGF21 is mainly expressed in the liver, but is also found in other tissues including the intestine, which expresses β-klotho abundantly. The intestine is a unique organ that operates in a physiologically hypoxic environment, and is responsible for the fat absorption processes including triglyceride breakdown, re-synthesis and absorption into the portal circulation. In the present study, we investigated the effects of hypoxia and the chemical hypoxia inducer, cobalt chloride (CoCl{sub 2}), on FGF21 expression in Caco-2 cells and the consequence of fat accumulation. Physical hypoxia (1% oxygen) and CoCl{sub 2} treatment decreased both FGF21 mRNA and secreted protein levels. Gene silence and inhibition of hypoxia-inducible factor-α (HIFα) did not affect the reduction of FGF21 mRNA and protein levels by hypoxia. However, CoCl{sub 2} administration caused a significant increase in oxidative stress. The addition of n-acetylcysteine (NAC) suppressed CoCl{sub 2}-induced reactive oxygen species (ROS) formation and completely negated CoCl{sub 2}-induced FGF21 loss. mRNA stability analysis demonstrated that the CoCl{sub 2} administration caused a remarkable reduction in FGF21 mRNA stability. Furthermore, CoCl{sub 2} increased intracellular triglyceride (TG) accumulation, along with a reduction in mRNA levels of lipid lipase, hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), and an increase of sterol regulatory element-binding protein-1c (SREBP1c) and stearoyl-coenzyme A (SCD1). Addition of both NAC and recombinant FGF21 significantly attenuated the CoCl{sub 2}-induced TG accumulation. In conclusion, the decrease of FGF21 in Caco-2 cells by chemical hypoxia is independent of HIFα, but dependent on an oxidative stress-mediated mechanism. The regulation of FGF21 by hypoxia may contribute to intestinal lipid metabolism and

  15. HYDROGEN-RICH MEDIUM AMELIORATES LIPOPOLYSACCHARIDE-INDUCED BARRIER DYSFUNCTION VIA RHOA-MDIA1 SIGNALING IN CACO-2 CELLS.

    Science.gov (United States)

    Yang, Tao; Wang, Lu; Sun, Ruiqiang; Chen, Hongguang; Zhang, Hongtao; Yu, Yang; Wang, Yanyan; Wang, Guolin; Yu, Yonghao; Xie, Keliang

    2016-02-01

    Gastrointestinal barrier dysfunction is associated with the severity and prognosis of sepsis. Hydrogen gas (H2) can ameliorate multiple organ damage in septic animals. Ras homolog gene family member A (RhoA) and mammalian diaphanous-related formin 1 (mDia1) are important to regulate tight junction (TJ) and adherens junction (AJ), both of which determine the integrity of the intestinal barrier. This study was aimed to investigate whether H2 could modulate lipopolysaccharide (LPS)-stimulated dysfunction of the intestinal barrier and whether RhoA-mDia1 signaling is involved. Caco-2 cells were exposed to different concentrations of LPS (1 μg/mL-1 mg/mL). The permeability of the intestinal barrier was evaluated by transepithelial resistance (TER) and fluorescein-isothiocyanate-dextran flux. Expression and distribution of occludin and E-cadherin were analyzed by Western blot and immunofluorescence. RhoA activity was measured by G-Lisa assay, and mDia1 expression was assessed by Western blot. LPS (100 μg/mL) decreased TER and increased fluorescein-isothiocyanate-dextran flux, which were alleviated by H2-rich medium. Also, H2 down-regulated LPS-induced oxidative stress. Moreover, H2 improved the down-regulated expression and redistribution of occludin and E-cadherin caused by LPS. Additionally, H2 alleviated LPS-caused RhoA activation, and the beneficial effects of H2 on barrier were counteracted by RhoA agonist CN03. Rho inhibitor C3 exoenzyme mitigated LPS-induced barrier breakdown. Furthermore, H2-rich medium increased mDia1 expression, and mDia1 knockdown abolished protections of H2 on barrier permeability. mDia1 knockdown eliminated H2-induced benefits for occludin and E-cadherin. These findings suggest that H2 improves LPS-induced hyperpermeability of the intestinal barrier and disruptions of TJ and AJ by moderating RhoA-mDia1 signaling.

  16. Permeability of ferulic acid in Caco-2 cell model and its absorption properties in rats in vivo%阿魏酸在Caco-2细胞模型的通透性及其在大鼠体内吸收特性研究

    Institute of Scientific and Technical Information of China (English)

    莫李立; 王素军; 杨本坤

    2012-01-01

    目的 研究阿魏酸在大鼠体内的绝对生物利用度(Fabs)及其吸收特点.方法 建立人源结肠腺癌细胞系Caeo-2单层细胞模型,LC-MS/MS法分析阿魏酸由绒毛面(AP侧)到基底面(BL侧)和由BL侧到AP侧两个方向的转运过程;用大鼠原位肠肝血管灌流模型研究阿魏酸在肠道的吸收率;通过测定大鼠ig和iv阿魏酸后的血药浓度计算其Fabs.结果 阿魏酸的表观渗透系数(Papp)分别为Papp AP→BL:(10.24±1.58)×10 6cm/s,Papp BL→AP:(11.25±1.45)×10-6 cm/s;在肠肝血管灌流模型中的吸收率为59.00%;在大鼠体内的Fabs为64.91%.结论 LC-MS/MS定量分析法灵敏、简单、专属性强;阿魏酸在Caco-2细胞具有良好的转运,在肠肝血管灌流模型中吸收速度快,主要吸收部位为小肠,阿魏酸ig给药后在大鼠体内达峰时间短,吸收、分布、消除较快.%Objective To study the absolute bioavailability (Fabs) of ferulic acid in rats and its absorption properties. Methods Based on the LC-MS/MS method, Caco-2 (the human colon adenocarcinoma cell lines) cell monolayer model was applied to studying the absorption and transport of ferulic acid from apical (AP) to basolateral (BL) side and from BLto AP side; the in situ vascularly perfused intestine-liver preparation was used to investigate the intestinal absorption rate; the Fabs was calculated by measuring the plasma concentration after iv or ig administration of ferulic acid in rats. Results The apparent permeability coefficients of ferulic acid from AP to BL side (Papp AP→BL) and from BL to AP side (Papp BL→AP) were (10.24 ± 1.58) × 10-6 and (11.25 ± 1.45) x 10-6cm/s, respectively; the intestinal absorption rate in the in situ vascularly perfused intestine-liver preparation was 59.00%; the Fababswas 64.91 % in rats. Conclusion LC-MS/MS method is a simple, sensitive, and specific approach for the quantitative analysis; ferulic acid shows a good permeability in Caco-2 cells; a fast

  17. Transport Characteristics of Ibuprofen in Caco-2 Cell Monolayers%布洛芬在Caco-2细胞模型中的转运行为考察

    Institute of Scientific and Technical Information of China (English)

    耿俊; 栾立标

    2010-01-01

    采用Caco-2细胞单层模型考察了布洛芬的吸收机制.用HPLC法测定了磷酸盐缓冲液中布洛芬的转运量.结果显示,布洛芬的转运量受培养时间、浓度和P-糖蛋白抑制剂维拉帕米的影响,其表观渗透系数为1×10-6~7×10-6cm/s,且双向转运的表观渗透性存在方向性差异.这提示布洛芬在Caco-2细胞模型中以主动转运和被动扩散两种机制吸收,且存在P-糖蛋白介导的外排机制.

  18. Hydrophobicity of Antifungal β-Peptides Is Associated with Their Cytotoxic Effect on In Vitro Human Colon Caco-2 and Liver HepG2 Cells

    Science.gov (United States)

    Mora-Navarro, Camilo; Méndez-Vega, Janet; Caraballo-León, Jean; Lee, Myung-ryul; Palecek, Sean; Torres-Lugo, Madeline; Ortiz-Bermúdez, Patricia

    2016-01-01

    The widespread distribution of fungal infections, with their high morbidity and mortality rate, is a global public health problem. The increase in the population of immunocompromised patients combined with the selectivity of currents treatments and the emergence of drug-resistant fungal strains are among the most imperative reasons to develop novel antifungal formulations. Antimicrobial β-peptides are peptidomimetics of natural antimicrobial peptides (AMPs), which have been proposed as developmental platforms to enhance the AMPs selectivity and biostability. Their tunability allows the design of sequences with remarkable activity against a wide spectrum of microorganisms such as the human pathogenic Candida spp., both in planktonic and biofilm morphology. However, the β-peptide’s effect on surrounding host cells remains greatly understudied. Assessments have mainly relied on the extent of hemolysis that a candidate peptide is able to cause. This work investigated the in vitro cytotoxicity of various β-peptides in the Caco-2 and HepG2 mammalian cell lines. Results indicated that the cytotoxic effect of the β-peptides was influenced by cell type and was also correlated to structural features of the peptide such as hydrophobicity. We found that the selectivity of the most hydrophobic β-peptide was 2–3 times higher than that of the least hydrophobic one, for both cell types according to the selectivity index parameter (IC50/MIC). The IC50 of Caco-2 and HepG2 increased with hydrophobicity, which indicates the importance of testing putative therapeutics on different cell types. We report evidence of peptide-cell membrane interactions in Caco-2 and HepG2 using a widely studied β-peptide against C. albicans. PMID:26992117

  19. Hydrophobicity of Antifungal β-Peptides Is Associated with Their Cytotoxic Effect on In Vitro Human Colon Caco-2 and Liver HepG2 Cells.

    Directory of Open Access Journals (Sweden)

    Camilo Mora-Navarro

    Full Text Available The widespread distribution of fungal infections, with their high morbidity and mortality rate, is a global public health problem. The increase in the population of immunocompromised patients combined with the selectivity of currents treatments and the emergence of drug-resistant fungal strains are among the most imperative reasons to develop novel antifungal formulations. Antimicrobial β-peptides are peptidomimetics of natural antimicrobial peptides (AMPs, which have been proposed as developmental platforms to enhance the AMPs selectivity and biostability. Their tunability allows the design of sequences with remarkable activity against a wide spectrum of microorganisms such as the human pathogenic Candida spp., both in planktonic and biofilm morphology. However, the β-peptide's effect on surrounding host cells remains greatly understudied. Assessments have mainly relied on the extent of hemolysis that a candidate peptide is able to cause. This work investigated the in vitro cytotoxicity of various β-peptides in the Caco-2 and HepG2 mammalian cell lines. Results indicated that the cytotoxic effect of the β-peptides was influenced by cell type and was also correlated to structural features of the peptide such as hydrophobicity. We found that the selectivity of the most hydrophobic β-peptide was 2-3 times higher than that of the least hydrophobic one, for both cell types according to the selectivity index parameter (IC50/MIC. The IC50 of Caco-2 and HepG2 increased with hydrophobicity, which indicates the importance of testing putative therapeutics on different cell types. We report evidence of peptide-cell membrane interactions in Caco-2 and HepG2 using a widely studied β-peptide against C. albicans.

  20. Multiorganelle Localization of Metallated Phthalocyanine Photosensitizer in Colorectal Cancer Cells (DLD-1 and CaCo-2 Enhances Efficacy of Photodynamic Therapy

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    Palesa Rose Sekhejane

    2014-01-01

    Full Text Available Colorectal cancer is the third most commonly diagnosed cancer. Amongst treatments that have been explored, photodynamic therapy (PDT is a treatment that is of interest as it poses ideal advantages such as affinity for cancer cells. This study aimed to determine the correlation between the localization site of a sulfonated zinc phthalocyanine (ZnPcSmix photosensitizer (PS and its associated cell death pathway in vitro in colorectal cancer cell lines (DLD-1 and CaCo-2. Visible morphological changes were observed in PDT treated cells after 24 h. Reactive oxygen species (ROS were detected and visualized 1 h after PDT. ZnPcSmix was predominantly localized in lysosomes and partially in the mitochondria. FITC Annexin V staining showed a significant decrease in the percentage of viable DLD-1 and CaCo-2 cells 24 h after PDT, with an increase in apoptotic cell population. Moreover, there was a significant increase in both cathepsin D and cytochrome C at 1 and 24 h. In conclusion, ZnPcSmix showed the ability of inducing apoptotic cell death features in PDT treated cells.

  1. Lactobacillus S-layer protein inhibition of Salmonella-induced reorganization of the cytoskeleton and activation of MAPK signalling pathways in Caco-2 cells.

    Science.gov (United States)

    Li, Pengcheng; Yu, Qinghua; Ye, Xiaolan; Wang, Zhisheng; Yang, Qian

    2011-09-01

    Surface layer (S-layer) proteins are crystalline arrays of proteinaceous subunits that are present as the outermost component of the cell wall in several Lactobacillus species. The S-layer proteins have been shown to play a role in the antimicrobial activity of certain lactobacilli. However, it is not fully understood how the S-layer proteins exert this biological function. The aim of this study was to test the hypothesis that Lactobacillus acidophilus S-layer proteins antagonize Salmonella Typhimurium (S. Typhimurium) infection by protecting against F-actin cytoskeleton rearrangements and the activation of mitogen-activated protein kinase (MAPK) signalling pathways. Monolayer transepithelial electrical resistance (TER) was measured after S. Typhimurium infection in Caco-2 cultured human intestinal cells with L. acidophilus S-layer proteins. F-actin rearrangement and MAPK activation were also assessed by immunofluorescence staining or Western blotting. The results showed that when S. Typhimurium was co-incubated with S-layer proteins, the S. Typhimurium-induced Caco-2 cell F-actin rearrangement was reduced, and the S. Typhimurium-induced TER decrease and interleukin 8 (IL-8) secretion were attenuated. Additionally, L. acidophilus S-layer proteins could inhibit S. Typhimurium-induced phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun amino-terminal kinase (JNK) and p38. This study indicates that L. acidophilus S-layer proteins are able to inhibit S. Typhimurium infection through blocking S. Typhimurium-induced F-actin rearrangements and S. Typhimurium-induced ERK1/2, JNK and p38 activation in Caco-2 cells. These data provide a rationale for the use of lactobacillus S-layer proteins as therapeutic and preventative agents, at least in infectious diarrhoea.

  2. The role of the intestinal microvasculature in inflammatory bowel disease: studies with a modified Caco-2 model including endothelial cells resembling the intestinal barrier in vitro

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    Kasper JY

    2016-12-01

    Full Text Available Jennifer Y Kasper,1 Maria Iris Hermanns,1 Christian Cavelius,2 Annette Kraegeloh,2 Thomas Jung,3 Rolf Danzebrink,3 Ronald E Unger,1 Charles James Kirkpatrick1 1Institute of Pathology, University Medical Center, Mainz, 2Leibniz Institute for New Materials, 3NanoGate AG, Goettelborn, Saarbrücken, Germany Abstract: The microvascular endothelium of the gut barrier plays a crucial role during inflammation in inflammatory bowel disease. We have modified a commonly used intestinal cell model based on the Caco-2 cells by adding microvascular endothelial cells (ISO-HAS-1. Transwell filters were used with intestinal barrier-forming Caco-2 cells on top and the ISO-HAS-1 on the bottom of the filter. The goal was to determine whether this coculture mimics the in vivo situation more closely, and whether the model is suitable to evaluate interactions of, for example, prospective nanosized drug vehicles or contrast agents with this coculture in a physiological and inflamed state as it would occur in inflammatory bowel disease. We monitored the inflammatory responsiveness of the cells (release of IL-8, soluble intercellular adhesion molecule 1, and soluble E-selectin after exposure to inflammatory stimuli (lipopolysaccharide, TNF-α, INF-γ, IL1-β and a nanoparticle (Ba/Gd: coprecipitated BaSO4 and Gd(OH3, generally used as contrast agents. The barrier integrity of the coculture was evaluated via the determination of transepithelial electrical resistance and the apparent permeability coefficient (Papp of NaFITC. The behavior of the coculture Caco-1/ISO-HAS-1 was compared to the respective monocultures Caco-2 and ISO-HAS-1. Based on transepithelial electrical resistance, the epithelial barrier integrity of the coculture remained stable during incubation with all stimuli, whereas the Papp decreased after exposure to the cytokine mixture (TNF-α, INF-γ, IL1-β, and Ba/Gd. Both the endothelial and epithelial monocultures showed a high inflammatory response in

  3. Coordinated induction of GST and MRP2 by cAMP in Caco-2 cells: Role of protein kinase A signaling pathway and toxicological relevance

    Energy Technology Data Exchange (ETDEWEB)

    Arana, Maite Rocío, E-mail: arana@ifise-conicet.gov.ar [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Tocchetti, Guillermo Nicolás, E-mail: gtocchetti@live.com.ar [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Domizi, Pablo, E-mail: domizi@ibr-conicet.gov.ar [Instituto de Biología Molecular y Celular de Rosario (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Arias, Agostina, E-mail: agoarias@yahoo.com.ar [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Rigalli, Juan Pablo, E-mail: jprigalli@gmail.com [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Ruiz, María Laura, E-mail: ruiz@ifise-conicet.gov.ar [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); and others

    2015-09-01

    The cAMP pathway is a universal signaling pathway regulating many cellular processes including metabolic routes, growth and differentiation. However, its effects on xenobiotic biotransformation and transport systems are poorly characterized. The effect of cAMP on expression and activity of GST and MRP2 was evaluated in Caco-2 cells, a model of intestinal epithelium. Cells incubated with the cAMP permeable analog dibutyryl cyclic AMP (db-cAMP: 1,10,100 μM) for 48 h exhibited a dose–response increase in GST class α and MRP2 protein expression. Incubation with forskolin, an activator of adenylyl cyclase, confirmed the association between intracellular cAMP and upregulation of MRP2. Consistent with increased expression of GSTα and MRP2, db-cAMP enhanced their activities, as well as cytoprotection against the common substrate 1-chloro-2,4-dinitrobenzene. Pretreatment with protein kinase A (PKA) inhibitors totally abolished upregulation of MRP2 and GSTα induced by db-cAMP. In silico analysis together with experiments consisting of treatment with db-cAMP of Caco-2 cells transfected with a reporter construct containing CRE and AP-1 sites evidenced participation of these sites in MRP2 upregulation. Further studies involving the transcription factors CREB and AP-1 (c-JUN, c-FOS and ATF2) demonstrated increased levels of total c-JUN and phosphorylation of c-JUN and ATF2 by db-cAMP, which were suppressed by a PKA inhibitor. Co-immunoprecipitation and ChIP assay studies demonstrated that db-cAMP increased c-JUN/ATF2 interaction, with further recruitment to the region of the MRP2 promoter containing CRE and AP-1 sites. We conclude that cAMP induces GSTα and MRP2 expression and activity in Caco-2 cells via the PKA pathway, thus regulating detoxification of specific xenobiotics. - Highlights: • cAMP positively modulates the expression and activity of GST and MRP2 in Caco-2 cells. • Such induction resulted in increased cytoprotection against chemical injury. • PKA

  4. Cocoa polyphenols prevent inflammation in the colon of azoxymethane-treated rats and in TNF-α-stimulated Caco-2 cells.

    Science.gov (United States)

    Rodríguez-Ramiro, Ildefonso; Ramos, Sonia; López-Oliva, Elvira; Agis-Torres, Angel; Bravo, Laura; Goya, Luis; Martín, Maria Angeles

    2013-07-28

    Numerous lines of evidence support a relationship between intestinal inflammation and cancer. Therefore, much attention has recently been focused on the identification of natural compounds with anti-inflammatory activities as a strategy to suppress the early stages of colorectal cancer. Because cocoa is a rich source of bioactive compounds, the present study investigated its anti-inflammatory properties in a rat model of azoxymethane (AOM)-induced colon carcinogenesis and in TNF-α-stimulated Caco-2 cells. A total of forty male rats were fed with control or cocoa-enriched diets (12 %) during 8 weeks and injected with saline or AOM (20 mg/kg body weight) during the third and fourth week (n 10 rats/group). At the end of the experiment, colon samples were evaluated for markers of inflammation. The anti-inflammatory activity of a cocoa polyphenolic extract (10 μg/ml) was examined in TNF-α-stimulated Caco-2 cells, an in vitro model of experimentally induced intestinal inflammation. The signalling pathways involved, including NF-κB and the mitogen-activated protein kinase family such as c-Jun NH₂-terminal kinases (JNK), extracellular signal-regulated kinases and p38, were also evaluated. The results show that the cocoa-rich diet decreases the nuclear levels of NF-κB and the expression of pro-inflammatory enzymes such as cyclo-oxygenase-2 and inducible NO synthase induced by AOM in the colon. Additionally, the experiments in Caco-2 cells confirm that cocoa polyphenols effectively down-regulate the levels of inflammatory markers induced by TNF-α by inhibiting NF-κB translocation and JNK phosphorylation. We conclude that cocoa polyphenols suppress inflammation-related colon carcinogenesis and could be promising in the dietary prevention of intestinal inflammation and related cancer development.

  5. Effect of apelin on mitosis, apoptosis and DNA repair enzyme OGG 1/2 expression in intestinal cell lines IEC-6 and Caco-2.

    Science.gov (United States)

    Antushevich, Hanna; Krawczynska, Agata; Kapica, Malgorzata; Herman, Andrzej Przemyslaw; Zabielski, Romuald

    2014-01-01

    Apelin is a regulatory peptide, identified as an endogenous ligand of the Apelin receptor (APJ). Both the apelin and the APJ were detected in brain, lung, heart, mammary gland, kidney, placenta, adipose tissues and the gastrointestinal tract. Apelin is considered an important regulatory gut peptide with a potential physiological role in gastrointestinal cytoprotection, regulation of food intake and drinking behaviour. The aim of the present study was to assess the effect of the apelin on mitosis, apoptosis and the expression of DNA repair enzyme (OGG 1/2), and APJ receptor in intestinal cell lines: rat crypt (IEC-6) and human enterocyte model (Caco-2). The cell cultures were incubated with the apelin-12 (10-8 M) for 4, 6, 12, 24 and 48 h and the apoptosis (caspase 3), mitosis (Ki-67) and DNA repair enzyme (OGG1/2) markers were studied by Real-Time qRT-PCR and immunofluorescent methods. The results of Real-Time qRT-PCR and immunocytochemical analysis showed that the levels of mRNAs were inversely related to the expression level of corresponding proteins. Immunofluorescent studies revealed inhibitory effect of apelin-12 on apoptosis, mitosis and the expression of OGG1/2 in the intestinal crypt cell line IEC-6. However, in the enterocyte model Caco-2 cells apelin stimulated apoptosis and mitosis, and reduced OGG1/2 expression. These findings suggest that apelin may be involved in the control of epithelial cell turnover in the gastrointestinal tract.

  6. Assessing the bioavailability of polyphenols and antioxidant properties of extra virgin argan oil by simulated digestion and Caco-2 cell assays. Comparative study with extra virgin olive oil.

    Science.gov (United States)

    Seiquer, Isabel; Rueda, Ascensión; Olalla, Manuel; Cabrera-Vique, Carmen

    2015-12-01

    Argan oil is becoming increasingly popular in the edible-oil market as a luxury food with healthy properties. This paper analyzes (i) the bioavailability of the polyphenol content and antioxidant properties of extra virgin argan oil (EVA) by the combination of in vitro digestion and absorption across Caco-2 cells and (ii) the protective role of the oil bioaccessible fraction (BF) against induced oxidative stress. Results were compared with those obtained with extra virgin olive oil (EVO). Higher values of polyphenols and antioxidant activity were observed in the BF obtained after the in vitro digestion of oils compared with the initial chemical extracts; the increase was higher for EVA but absolute BF values were lower than EVO. Bioaccessible polyphenols from EVA were absorbed by Caco-2 cells in higher proportions than from EVO, and minor differences were observed for antioxidant activity. Preincubation of cell cultures with BF from both oils significantly protected against oxidation, limiting cell damage and reducing reactive oxygen species generation.

  7. Absorption and metabolism of cis-9,trans-11-CLA and of its oxidation product 9,11-furan fatty acid by Caco-2 cells.

    Science.gov (United States)

    Buhrke, Thorsten; Merkel, Roswitha; Lengler, Imme; Lampen, Alfonso

    2012-04-01

    Furan fatty acids (furan-FA) can be formed by auto-oxidation of conjugated linoleic acids (CLA) and may therefore be ingested when CLA-containing foodstuff is consumed. Due to the presence of a furan ring structure, furan-FA may have toxic properties, however, these substances are toxicologically not well characterized so far. Here we show that 9,11-furan-FA, the oxidation product of the major CLA isomer cis-9,trans-11-CLA (c9,t11-CLA), is not toxic to human intestinal Caco-2 cells up to a level of 100 μM. Oil-Red-O staining indicated that 9,11-furan-FA as well as c9,t11-CLA and linoleic acid are taken up by the cells and stored in the form of triglycerides in lipid droplets. Chemical analysis of total cellular lipids revealed that 9,11-furan-FA is partially elongated probably by the enzymatic activity of cellular fatty acid elongases whereas c9,t11-CLA is partially converted to other isomers such as c9,c11-CLA or t9,t11-CLA. In the case of 9,11-furan-FA, there is no indication for any modification or activation of the furan ring system. From these results, we conclude that 9,11-furan-FA has no properties of toxicological relevance at least for Caco-2 cells which serve as a model for enterocytes of the human small intestine.

  8. Effect of soy and milk protein-related compounds on Listeria monocytogenes infection in human enterocyte Caco-2 cells and A/J mice.

    Science.gov (United States)

    Kuda, Takashi; Nakamura, Shinsuke; An, Choa; Takahashi, Hajime; Kimura, Bon

    2012-10-15

    Listeria monocytogenes causes listeriosis in humans, mainly through the consumption of ready-to-eat foods such as cheese. Immunocompromised persons, the elderly, and pregnant women and their fetuses or newborns are at the highest risk for the infection. We examined the effects of dietary milk-casein (MC) and soy-protein (SP), and their digested compounds tryptone (TP) and phytone peptone (PP), respectively, on L. monocytogenes invasion and infection in human enterocyte-like Caco-2 cells and A/J mice. Invasion into Caco-2 cells tended to be high with TP. In A/J mice orally infected with L. monocytogenes, viable numbers in the liver and spleen showed a tendency of decreasing with the 20% SP diet compared to the 20% MC diet. SP suppressed the inflammation marker tumour necrosis factor-α in spleen tissue. Furthermore, bacteria lipopolysaccharide (LPS)-stimulated nitric oxide (NO) secretion from murine macrophage RAW 264.7 cells was suppressed by PP more than TP. These results suggest that major dietary proteins might affect infection and inflammation by L. monocytogenes.

  9. Determination of S-methyl-L-methionine (SMM) from Brassicaceae Family Vegetables and Characterization of the Intestinal Transport of SMM by Caco-2 Cells.

    Science.gov (United States)

    Song, Ji-Hoon; Lee, Hae-Rim; Shim, Soon-Mi

    2017-01-01

    The objectives of the current study were to determine S-methyl-L-methionine (SMM) from various Brassicaceae family vegetables by using validated analytical method and to characterize the intestinal transport mechanism of SMM by the Caco-2 cells. The SMM is well known to provide therapeutic activity in peptic ulcers. The amount of SMM from various Brassicaceae family vegetables ranged from 89.08 ± 1.68 μg/g to 535.98 ± 4.85 μg/g of dry weight by using validated ultra-performance liquid chromatography-electrospray ionization-mass spectrometry method. For elucidating intestinal transport mechanism, the cells were incubated with or without transport inhibitors, energy source, or a metabolic inhibitor. Phloridzin and verapamil as inhibitors of sodium glucose transport protein (SGLT1) and P-glycoprotein, respectively, were not responsible for cellular uptake of SMM. Glucose and sodium azide were not affected by the cellular accumulation of SMM. The efflux ratio of SMM was 0.26, implying that it is not effluxed through Caco-2 cells. The apparent coefficient permeability (Papp ) of SMM was 4.69 × 10(-5) cm/s, indicating that it will show good oral absorption in in vivo.

  10. Antagonistics against pathogenic Bacillus cereus in milk fermentation by Lactobacillus plantarum ZDY2013 and its anti-adhesion effect on Caco-2 cells against pathogens.

    Science.gov (United States)

    Zhang, Zhihong; Tao, Xueying; Shah, Nagendra P; Wei, Hua

    2016-04-01

    Lactobacillus plantarum ZDY2013 is a potential probiotic isolated from fermented bean acid. In this study, we aimed to evaluate the in vitro antimicrobial activity of this organism against Bacillus cereus in milk fermentation, the antiadhesion ability on intestinal epithelial cells, as well as its ability to abrogate the cytotoxic effect and expression levels of genes. We found no antimicrobial activity produced by L. plantarum once the pH was adjusted to 6.0 and 7.0. The pH decreased continuously when L. plantarum and B. cereus were co-incubated during milk fermentation, which caused a decrease in the B. cereus counts. Antiadhesion assays showed that L. plantarum can significantly inhibit the adhesion of enterotoxin-producing B. cereus ATCC14579 and pathogenic B. cereus HN001 by inhibition, competition, and displacement. The supernatants of B. cereus, either alone or in conjunction with L. plantarum, caused damage to the membrane integrity of Caco-2 cells to release lactate dehydrogenase. In addition, L. plantarum tended to attenuate proinflammatory cytokine and oxidative stress gene expression on Caco-2 cells, inducing with B. cereus HN001 supernatants. This study provided systematic insights into the antagonistic effect of L. plantarum ZDY2013, and the information may be helpful to explore potential control measures for preventing food poisoning by lactic acid bacteria.

  11. Calcium Channels and Oxidative Stress Mediate a Synergistic Disruption of Tight Junctions by Ethanol and Acetaldehyde in Caco-2 Cell Monolayers.

    Science.gov (United States)

    Samak, Geetha; Gangwar, Ruchika; Meena, Avtar S; Rao, Roshan G; Shukla, Pradeep K; Manda, Bhargavi; Narayanan, Damodaran; Jaggar, Jonathan H; Rao, RadhaKrishna

    2016-12-13

    Ethanol is metabolized into acetaldehyde in most tissues. In this study, we investigated the synergistic effect of ethanol and acetaldehyde on the tight junction integrity in Caco-2 cell monolayers. Expression of alcohol dehydrogenase sensitized Caco-2 cells to ethanol-induced tight junction disruption and barrier dysfunction, whereas aldehyde dehydrogenase attenuated acetaldehyde-induced tight junction disruption. Ethanol up to 150 mM did not affect tight junction integrity or barrier function, but it dose-dependently increased acetaldehyde-mediated tight junction disruption and barrier dysfunction. Src kinase and MLCK inhibitors blocked this synergistic effect of ethanol and acetaldehyde on tight junction. Ethanol and acetaldehyde caused a rapid and synergistic elevation of intracellular calcium. Calcium depletion by BAPTA or Ca(2+)-free medium blocked ethanol and acetaldehyde-induced barrier dysfunction and tight junction disruption. Diltiazem and selective knockdown of TRPV6 or CaV1.3 channels, by shRNA blocked ethanol and acetaldehyde-induced tight junction disruption and barrier dysfunction. Ethanol and acetaldehyde induced a rapid and synergistic increase in reactive oxygen species by a calcium-dependent mechanism. N-acetyl-L-cysteine and cyclosporine A, blocked ethanol and acetaldehyde-induced barrier dysfunction and tight junction disruption. These results demonstrate that ethanol and acetaldehyde synergistically disrupt tight junctions by a mechanism involving calcium, oxidative stress, Src kinase and MLCK.

  12. Characterization of the intestinal absorption of seven flavonoids from the flowers of Trollius chinensis using the Caco-2 cell monolayer model.

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    Lijia Liu

    Full Text Available The human Caco-2 cell monolayer model was used to investigate the absorption property, mechanism, and structure-property relationship of seven representative flavonoids, namely, orientin, vitexin, 2"-O-β-L-galactopyranosylorientin, 2"-O-β-L-galactopyranosylvitexin, isoswertisin, isoswertiajaponin, and 2"-O-(2"'-methylbutanoylisoswertisin from the flowers of Trollius chinensis. The results showed that these flavonoids were hardly transported through the Caco-2 cell monolayer. The compounds with 7-OCH3 including isoswertisin, isoswertiajaponin and 2"-O-(2"'-methylbutanoylisoswertisin were absorbed in a passive diffusion manner, and their absorbability was increased in the same order as their polarity. The absorption of the remaining compounds with 7-OH including orientin, vitexin, 2"-O-β-L-galactopyranosylorientin, and 2"-O-β-L-galactopyranosylvitexin involved transporter mediated efflux in addition to passive diffusion. Among the four compounds with 7-OH, those with a free hydroxyl group at C-2" such as orientin and vitexin were the substrates of P-glycoprotein (P-gp and that with a free hydroxyl group at C-2' such as 2"-O-β-L-galactopyranosylorientin was the substrate of multidrug resistance protein 2 (MRP2. The results of this study also implied that the absorbability of the flavonoids should be taken into account when estimating the effective components of T. chinensis.

  13. Effects of Curcumin Analogue, 2, 6-Bis (2, 5-Dimethoxybenzylidene Cyclohexanone (BDMC33 on the Activities of Drug-Metabolizing Enzymes in Cultured Caco-2 Cell Model

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    Ndatsu Yakubu

    2016-01-01

    Full Text Available Poor systemic delivery of curcumin outside the gut due to its rapid metabolism has severely limited its application to many chronic diseases. Previously, our research group synthesized curcumin analogues 2, 6-bis (2, 5-dimethoxybenzylidene cyclohexanone (BDMC33 that has potent anti-inflammatory activities. Therefore, the aim of this study is to evaluate the effects of curcumin analog (BDMC33 on the activities of drug metabolizing enzymes in Caco-2 cells, which was compared with that of curcumin and 3-(2-Fluoro-benzylidene-5-(2-fluorocyclohexylmethylene-piperidin-4-one (EF-24. BDMC-33 was synthesized through the appropriate reaction of the aromatic aldehyde with cyclohexanone, under base catalyzed aldol condensation, at the ratio of ketone: aldehyde (1:2. Activity of drug metabolizing enzymes such as NADPH-cytochrome p450 reductase (CPR, UDP-glucuronosyltransferase (UGT, glutathione-S-transferase (GST and Sulfotransferase (SULT in Caco-2 cells were evaluated upon exposure to 50µM of BDMC33, curcumin, and EF-24, separately, for 4 hours. The BDMC33, EF-24, and curcumin treatments did not affect the activities of UGT, GST, SULT, and CPR in respect to their controls (29.45, 27.18, 23.64 and 2.08µmol/mg, respectively, at all periods of incubation. Hence, BDMC33 was able to maintain the activities of both phases I and II drug metabolizing enzymes, and therefore it could be a potential lead, anti-inflammatory agents.

  14. Capsaicin, nonivamide and trans-pellitorine decrease free fatty acid uptake without TRPV1 activation and increase acetyl-coenzyme A synthetase activity in Caco-2 cells.

    Science.gov (United States)

    Rohm, Barbara; Riedel, Annett; Ley, Jakob P; Widder, Sabine; Krammer, Gerhard E; Somoza, Veronika

    2015-01-01

    Red pepper and its major pungent component, capsaicin, have been associated with hypolipidemic effects in rats, although mechanistic studies on the effects of capsaicin and/or structurally related compounds on lipid metabolism are scarce. In this work, the effects of capsaicin and its structural analog nonivamide, the aliphatic alkamide trans-pellitorine and vanillin as the basic structural element of all vanilloids on the mechanisms of intestinal fatty acid uptake in differentiated intestinal Caco-2 cells were studied. Capsaicin and nonivamide were found to reduce fatty acid uptake, with IC₅₀ values of 0.49 μM and 1.08 μM, respectively. trans-Pellitorine was shown to reduce fatty acid uptake by 14.0±2.14% at 100 μM, whereas vanillin was not effective, indicating a pivotal role of the alkyl chain with the acid amide group in fatty acid uptake by Caco-2 cells. This effect was associated neither with the activation of the transient receptor potential cation channel subfamily V member 1 (TRPV1) or the epithelial sodium channel (ENaC) nor with effects on paracellular transport or glucose uptake. However, acetyl-coenzyme A synthetase activity increased (p<0.05) in the presence of 10 μM capsaicin, nonivamide or trans-pellitorine, pointing to an increased fatty acid biosynthesis that might counteract the decreased fatty acid uptake.

  15. Hypocholesterolaemic Activity of Lupin Peptides: Investigation on the Crosstalk between Human Enterocytes and Hepatocytes Using a Co-Culture System Including Caco-2 and HepG2 Cells

    Directory of Open Access Journals (Sweden)

    Carmen Lammi

    2016-07-01

    Full Text Available Literature indicates that peptic and tryptic peptides derived from the enzymatic hydrolysis of lupin protein are able to modulate cholesterol metabolism in human hepatic HepG2 cells and that part of these peptides are absorbed in a small intestine model based on differentiated human Caco-2 cells. In this paper, a co-culture system, including Caco-2 and HepG2 cells, was investigated with two objectives: (a to verify whether cholesterol metabolism in HepG2 cells was modified by the peptides absorption through Caco-2 cells; (b to investigate how lupin peptides influence cholesterol metabolism in Caco-2 cells. The experiments showed that the absorbed peptides, not only maintained their bioactivity on HepG2 cells, but that this activity was improved by the crosstalk of the two cells systems in co-culture. In addition, lupin peptides showed a positive influence on cholesterol metabolism in Caco-2 cells, decreasing the proprotein convertase subtilisin/kexin type 9 (PCSK9 secretion.

  16. Involvement of CDX2 in the cross talk between TNF-α and Wnt signaling pathway in the colon cancer cell line Caco-2

    DEFF Research Database (Denmark)

    Coskun, Mehmet; Olsen, Anders Krüger; Bzorek, Michael

    2014-01-01

    influence on the Wnt signaling-related genes and progression of colorectal cancer. Although several inflammatory signaling pathways, including TNF-α, have been reported to promote Wnt/β-catenin activity and development of cancer, the underlying molecular mechanisms remain unclear. The aim was to investigate...... the signaling pathways involved in the TNF-α-mediated downregulation of CDX2, and its influence on Wnt/β-catenin signaling components in colon cancer cells. The expression of TNF-α and CDX2 at the invasive front were evaluated by immunohistochemical staining and showed reduced CDX2-positive cells in tumor...... buddings in areas with TNF-α expression in the surrounding inflammatory cells. In vitro studies revealed that TNF-α treatment showed a dose-dependent decrease of CDX2 messenger RNA (mRNA) and protein expression in Caco-2 cells. Inhibition of nuclear factor-kappaB or p38 pathways showed...

  17. The role of the intestinal microvasculature in inflammatory bowel disease: studies with a modified Caco-2 model including endothelial cells resembling the intestinal barrier in vitro.

    Science.gov (United States)

    Kasper, Jennifer Y; Hermanns, Maria Iris; Cavelius, Christian; Kraegeloh, Annette; Jung, Thomas; Danzebrink, Rolf; Unger, Ronald E; Kirkpatrick, Charles James

    The microvascular endothelium of the gut barrier plays a crucial role during inflammation in inflammatory bowel disease. We have modified a commonly used intestinal cell model based on the Caco-2 cells by adding microvascular endothelial cells (ISO-HAS-1). Transwell filters were used with intestinal barrier-forming Caco-2 cells on top and the ISO-HAS-1 on the bottom of the filter. The goal was to determine whether this coculture mimics the in vivo situation more closely, and whether the model is suitable to evaluate interactions of, for example, prospective nanosized drug vehicles or contrast agents with this coculture in a physiological and inflamed state as it would occur in inflammatory bowel disease. We monitored the inflammatory responsiveness of the cells (release of IL-8, soluble intercellular adhesion molecule 1, and soluble E-selectin) after exposure to inflammatory stimuli (lipopolysaccharide, TNF-α, INF-γ, IL1-β) and a nanoparticle (Ba/Gd: coprecipitated BaSO4 and Gd(OH)3), generally used as contrast agents. The barrier integrity of the coculture was evaluated via the determination of transepithelial electrical resistance and the apparent permeability coefficient (Papp) of NaFITC. The behavior of the coculture Caco-1/ISO-HAS-1 was compared to the respective monocultures Caco-2 and ISO-HAS-1. Based on transepithelial electrical resistance, the epithelial barrier integrity of the coculture remained stable during incubation with all stimuli, whereas the Papp decreased after exposure to the cytokine mixture (TNF-α, INF-γ, IL1-β, and Ba/Gd). Both the endothelial and epithelial monocultures showed a high inflammatory response in both the upper and lower transwell-compartments. However, in the coculture, inflammatory mediators were only detected on the epithelial side and not on the endothelial side. Thus in the coculture, based on the Papp, the epithelial barrier appears to prevent a potential inflammatory overreaction in the underlying endothelial cells

  18. Degradation of the transcription factors NF-κB, STAT3, and STAT5 is involved in Entamoeba histolytica-induced cell death in Caco-2 colonic epithelial cells.

    Science.gov (United States)

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

    2014-10-01

    Entamoeba histolytica is a tissue-invasive protozoan parasite causing dysentery in humans. During infection of colonic tissues, amoebic trophozoites are able to kill host cells via apoptosis or necrosis, both of which trigger IL-8-mediated acute inflammatory responses. However, the signaling pathways involved in host cell death induced by E. histolytica have not yet been fully defined. In this study, we examined whether calpain plays a role in the cleavage of pro-survival transcription factors during cell death of colonic epithelial cells, induced by live E. histolytica trophozoites. Incubation with amoebic trophozoites induced activation of m-calpain in a time- and dose-dependent manner. Moreover, incubation with amoebae resulted in marked degradation of STAT proteins (STAT3 and STAT5) and NF-κB (p65) in Caco-2 cells. However, IκB, an inhibitor of NF-κB, was not cleaved in Caco-2 cells following adherence of E. histolytica. Entamoeba-induced cleavage of STAT proteins and NF-κB was partially inhibited by pretreatment of cells with a cell-permeable calpain inhibitor, calpeptin. In contrast, E. histolytica did not induce cleavage of caspase-3 in Caco-2 cells. Furthermore, pretreatment of Caco-2 cells with a calpain inhibitor, calpeptin (but not the pan-caspase inhibitor, z-VAD-fmk) or m-calpain siRNA partially reduced Entamoeba-induced DNA fragmentation in Caco-2 cells. These results suggest that calpain plays an important role in E. histolytica-induced degradation of NF-κB and STATs in colonic epithelial cells, which ultimately accelerates cell death.

  19. Oxygen restriction increases the infective potential of Listeria monocytogenes in vitro in Caco-2 cells and in vivo in guinea pigs

    DEFF Research Database (Denmark)

    Andersen, Jens Bo; Roldgaard, Bent; Christensen, Bjarke Bak

    2007-01-01

    : Infection of Caco-2 cells revealed that Listeria cultivated under oxygen-restricted conditions were approximately 100 fold more invasive than similar cultures grown without oxygen restriction. This was observed for exponentially growing bacteria, as well as for stationary-phase cultures. Oral dosage......Background: Listeria monocytogenes has been implicated in several food borne outbreaks as well as sporadic cases of disease. Increased understanding of the biology of this organism is important in the prevention of food borne listeriosis. The infectivity of Listeria monocytogenes ScottA, cultivated...... with and without oxygen restriction, was compared in vitro and in vivo. Fluorescent protein labels were applied to allow certain identification of Listeria cells from untagged bacteria in in vivo samples, and to distinguish between cells grown under different conditions in mixed infection experiments. Results...

  20. The importance of the stem cell marker prominin-1/CD133 in the uptake of transferrin and in iron metabolism in human colon cancer Caco-2 cells.

    Directory of Open Access Journals (Sweden)

    Erika Bourseau-Guilmain

    Full Text Available As the pentaspan stem cell marker CD133 was shown to bind cholesterol and to localize in plasma membrane protrusions, we investigated a possible function for CD133 in endocytosis. Using the CD133 siRNA knockdown strategy and non-differentiated human colon cancer Caco-2 cells that constitutively over-expressed CD133, we provide for the first time direct evidence for a role of CD133 in the intracellular accumulation of fluorescently labeled extracellular compounds. Assessed using AC133 monoclonal antibody, CD133 knockdown was shown to improve Alexa488-transferrin (Tf uptake in Caco-2 cells but had no impact on FITC-dextran or FITC-cholera-toxin. Absence of effect of the CD133 knockdown on Tf recycling established a role for CD133 in inhibiting Tf endocytosis rather than in stimulating Tf exocytosis. Use of previously identified inhibitors of known endocytic pathways and the positive impact of CD133 knockdown on cellular uptake of clathrin-endocytosed synthetic lipid nanocapsules supported that CD133 impact on endocytosis was primarily ascribed to the clathrin pathway. Also, cholesterol extraction with methyl-β-cyclodextrine up regulated Tf uptake at greater intensity in the CD133(high situation than in the CD133(low situation, thus suggesting a role for cholesterol in the inhibitory effect of CD133 on endocytosis. Interestingly, cell treatment with the AC133 antibody down regulated Tf uptake, thus demonstrating that direct extracellular binding to CD133 could affect endocytosis. Moreover, flow cytometry and confocal microscopy established that down regulation of CD133 improved the accessibility to the TfR from the extracellular space, providing a mechanism by which CD133 inhibited Tf uptake. As Tf is involved in supplying iron to the cell, effects of iron supplementation and deprivation on CD133/AC133 expression were investigated. Both demonstrated a dose-dependent down regulation here discussed to the light of transcriptional and post

  1. Estimating intestinal absorption of inorganic and organic selenium compounds by in vitro flux and biotransformation studies in Caco-2 cells and ICP-MS detection

    DEFF Research Database (Denmark)

    Gammelgaard, Bente; Rasmussen, Laura Hyrup; Gabel-Jensen, Charlotte

    2012-01-01

    selenite and MeSeA fluxes correlated to poor in vivo absorption. Speciation analysis of cell lysate and donor and receptor solutions by LC-ICP-MS showed limited transformation of all selenium compounds. Extensive transformation as well as significantly increased absorptive flux was observed when co......SeA, including volatile species, whereas no significant increases in fluxes were observed. In summary, the absorption of selenite selenate and the selenoamino acids is considered complete under physiological conditions, but the absorption mechanisms and metabolism of the compounds are different. © 2011 Springer......The aim of the present work was to compare and estimate absorption and biotransformation of selected selenium compounds by studying their fluxes across Caco-2 cells. Five different selenium compounds, selenomethionine (SeMet), Se-methylselenocysteine (MeSeCys), selenate, selenite...

  2. Hydroxytyrosol and tyrosol sulfate metabolites protect against the oxidized cholesterol pro-oxidant effect in Caco-2 human enterocyte-like cells.

    Science.gov (United States)

    Atzeri, Angela; Lucas, Ricardo; Incani, Alessandra; Peñalver, Pablo; Zafra-Gómez, Alberto; Melis, M Paola; Pizzala, Roberto; Morales, Juan C; Deiana, Monica

    2016-01-01

    The aim of this study was to investigate the ability of the sulfate metabolites of hydroxytyrosol (HT) and tyrosol (TYR) to act as antioxidants counteracting the pro-oxidant effect of oxidized cholesterol in intestinal cells. For this purpose, we synthesized sulfate metabolites of HT and TYR using a chemical methodology and examined their antioxidant activity in Caco-2 monolayers in comparison with the parent compounds. Exposure to oxidized cholesterol led to ROS production, oxidative damage, as indicated by the MDA increase, a decrease of reduced glutathione concentration and an enhancement of glutathione peroxidase activity. All the tested compounds were able to counteract the oxidizing action of oxidized cholesterol; HT and TYR sulfate metabolites showed an efficiency in protecting intestinal cells comparable to that of the parent compounds, strengthening the assumption that the potential beneficial effect of the parent compounds is retained, although extensive metabolisation occurs, the resulting metabolites being able to exert a biological action themselves.

  3. 灯盏细辛提取物中3种活性成分在Caco-2细胞模型吸收机制的研究%In vitro absorption mechanism of Erigeron breviscapus extract in Caco-2 cell monolayer model

    Institute of Scientific and Technical Information of China (English)

    胡杰; 侯佳; 李月婷; 王爱民; 黄勇

    2016-01-01

    Aim To study the absorption mechanism of apigenin-7-O-glucronide, 3,4-Dihydroxycinnamic acid and chlorogenic acid in Erigeron breviscapus extract ( Ebe) by Caco-2 cell monolayer model. Methods The three active ingredients were quantified by UPLC-MS/MS method, and the effect of Ebe concentrations, conveying times, pH values and P-glycoprotein inhibi-tor on the transport of three active ingredients were also investigated. Results Apigenin-7-O-glucronide and chlorogenic acid in Caco-2 cell monolayer model were time-dependent and concentration-dependent. The 3, 4-Dihydroxycinnamic acid in Caco-2 cell uptake was concentration-saturate and P-glycoprotein inhibitor was involved in the uptake process. Conclusion The mechanism of apigenin-7-O-glucronide and chlorogenic acid absorption in cells is mainly through passive trans-port, and the absorption of 3, 4-Dihydroxycinnamic acid is mainly realized by the carrier transport.%目的:研究灯盏细辛提取物( erigeron breviscapus ex-tract,Ebe)中3种活性成分灯盏甲素、咖啡酸和绿原酸的吸收机制。方法建立人结肠腺癌细胞系( the human colon carcinoma cell line,Caco-2细胞)模型;利用该模型研究Ebe的细胞摄取规律,通过UPLC-MS/MS法测定Caco-2细胞中灯盏甲素、咖啡酸和绿原酸的浓度,研究pH、时间以及药物浓度对3种活性成分的转运影响;考察在P-糖蛋白抑制剂(维拉帕米、环孢素A)存在与否时3种活性成分在 Caco-2细胞模型中的转运情况。结果受试物Ebe在所设置的浓度范围内(0.1~25.0 g·L-1)对 Caco-2细胞无毒副作用。灯盏甲素和绿原酸在Caco-2细胞摄取实验中具有浓度、时间依赖性,呈正相关,渗透系数维持在一个平衡状态。咖啡酸具有浓度饱和性,且加入P-糖蛋白抑制剂后咖啡酸的细胞摄取量明显增加。结论 Ebe中灯盏甲素和绿原酸主要表现为被动转运,P-糖蛋白参与了咖啡酸的摄取过程,咖啡酸的吸收主要由载体媒介转运实现。

  4. Effect of diffusely adherent Escherichia coli strains isolated from diarrhoeal patients and healthy carriers on IL-8 secretion and tight junction barrier integrity of Caco-2 cells.

    Science.gov (United States)

    Tanimoto, Yoshihiko; Arikawa, Kentaro; Nishikawa, Yoshikazu

    2013-03-15

    The pathogenesis of diffusely adherent Escherichia coli (DAEC) remains to be elucidated. Previously, we found that afimbrial adhesin gene (afa)-positive motile DAEC strains isolated from patients with diarrhoea induce high levels of IL-8 secretion in Caco-2 cells via toll-like receptor 5 (TLR-5), while non-motile strains did not. The aim of this study was to compare virulence properties, including the phylogenetic groups, afa subtypes, IL-8 secretion levels, and the effects on tight junctions, of DAEC strains isolated from healthy persons with those isolated from patients with diarrhoea. Induction of IL-8 secretion in Caco-2 cells was examined for a total of 36 afa-positive strains: 19 from diarrhoeal patients and 17 from healthy carriers. Irrespective of the source, all strains were classified into the phylogenetic group B2 or D, with the exception of two strains. All 7 motile strains isolated from diarrhoeal patients induced high levels of IL-8 secretion, while only 6 of 15 motile strains from healthy carriers induced IL-8 secretion to the same levels as the diarrhoeal strains. We speculated that additional virulence factors other than afa and motility cause the loosening of tight junctions that allows flagellin to reach TLR-5 located on the basolateral side of the epithelium. However, no differences in the TER and dextran permeability were observed between cells infected with diarrhoeal strains and those from healthy persons. Thus, diarrhoeagenic DAEC seems to possess additional factors, in addition to adhesin and flagellin, which can induce high IL-8 secretion.

  5. Effect of phytate reduction of sorghum, through genetic modification, on iron and zinc availability as assessed by an in vitro dialysability bioaccessibility assay, Caco-2 cell uptake assay, and suckling rat pup absorption model.

    Science.gov (United States)

    Kruger, Johanita; Taylor, John R N; Du, Xiaogu; De Moura, Fabiana F; Lönnerdal, Bo; Oelofse, André

    2013-11-15

    Improved iron and zinc availability from sorghum, a commonly consumed staple, will benefit many malnourished communities in rural Africa burdened with high prevalence of iron and zinc deficiency. This research compared the effect of genetic phytate reduction in sorghum on iron and zinc bioaccessibility and uptake measured by in vitro dialysability and Caco-2 cell uptake assays to that of iron and zinc absorption measured by a suckling rat pup model. The phytate reduction (80-86%) in these sorghums significantly increased zinc availability. The Caco-2 cell method, but not the dialysability assay, proved useful in estimating zinc absorption. The measured increase in iron availability differed between the methods, possibly due to the effect of varying mineral (Ca, Fe, Zn, P) contents of the sorghums. This effect was most prominent in the iron uptake results. More research is needed to determine the effect of naturally occurring variations in mineral contents of sorghum on the iron uptake by Caco-2 cells.

  6. Effect of molecular weight on the transepithelial transport and peptidase degradation of casein-derived peptides by using Caco-2 cell model.

    Science.gov (United States)

    Wang, Bo; Li, Bo

    2017-03-01

    The transepithelial transport routes of casein-derived peptides with different molecular weights (MWs) were investigated using a Caco-2 cell monolayer. The peptidase hydrolysis during transport was also studied. The results indicate that the paracellular route was the main pathway for F1 (1600-1300Da) and F2 (1000-500Da), and the bioavailabilities were 10.66% and 9.54%, respectively. Peptidase hydrolysis results reveal that brush-border peptidases (BBPs) as well as some other peptidases were responsible for peptide degradation in the paracellular route. The maximum hydrolysis rate of the former was 6.91 and 5.59μM Gly/min for the latter. However, PepT1 was involved in the transport of F3 (transport and the maximum hydrolysis rate was 11.4μM Gly/min. Furthermore, we found that the amino acid sequence of di- and tripeptides might affect their bioavailabilities significantly.

  7. HPLC-MS analysis of Schisandra lignans and their metabolites in Caco-2 cell monolayer and rat everted gut sac models and in rat plasma

    Directory of Open Access Journals (Sweden)

    Jia-ming Yang

    2011-06-01

    Full Text Available The absorption profiles of Schisandra chinensis were evaluated using the human Caco-2 cell monolayer and rat everted gut sac models, as well as in rat plasma. By analyzing the chromatographic and MSn characteristics of individual peak acquired by HPLC-DAD-APCI-MSn determination, thirteen lignans were identified as the major in vitro absorbable components of the Schisandra extract. Most of these compounds were also detected and identified in rat plasma after an oral administration of the Schisandra extract, except for angeloyl(tigloylgomisin H and angeloyl(tigloylgomisin Q, whose structures possess an ester group at the cyclooctadiene ring. In addition, four metabolites, corresponding to the hydroxylation and demethylation products of schisandrin and the hydrolysis derivative of angeloyl(tigloylgomisin Q, were tentatively identified. The results demonstrate that Schisandra lignans are the major absorbable components of this crude drug, and hydroxylation, demethylation and hydrolysis are important metabolic transformations of the absorbable lignans.

  8. Comparative QSAR studies on PAMPA/modified PAMPA for high throughput profiling of drug absorption potential with respect to Caco-2 cells and human intestinal absorption

    Science.gov (United States)

    Verma, Rajeshwar P.; Hansch, Corwin; Selassie, Cynthia D.

    2007-01-01

    Despite the dramatic increase in speed of synthesis and biological evaluation of new chemical entities, the number of compounds that survive the rigorous processes associated with drug development is low. Thus, an increased emphasis on thorough ADMET (absorption, distribution, metabolism, excretion and toxicity) studies based on in vitro and in silico approaches allows for early evaluation of new drugs in the development phase. Artificial membrane permeability measurements afford a high throughput, relatively low cost but labor intensive alternative for in vitro determination of drug absorption potential; parallel artificial membrane permeability assays have been extensively utilized to determine drug absorption potentials. The present study provides comparative QSAR analysis on PAMPA/modified PAMPA for high throughput profiling of drugs with respect to Caco-2 cells and human intestinal absorption.

  9. Characterization of Polyphenols from Lycium ruthenicum Fruit by UPLC-Q-TOF/MS(E) and Their Antioxidant Activity in Caco-2 Cells.

    Science.gov (United States)

    Wu, Tao; Lv, Haiyang; Wang, Fengzhong; Wang, Yi

    2016-03-23

    The fruit of Lycium ruthenicum Murr. (LRF) has long been used in folk medicine. Nevertheless, detailed information related to its polyphenol compositions remains scarce. In this study, we confirmed that the total phenolic and anthocyanin contents of LRF fruit extracts (LRFEs) were 4906.5 ± 60.6 mg of gallic acid equivalents/100 g DW and 787.6 ± 34.1 mg of cyanindin-3-glucoside equivalents/100 g DW, respectively. A characterization of LRFEs was performed by ultrahigh performance liquid chromatography/quadrupole time-of-flight mass spectrometry using an MS(E) data acquisition. A total of 26 polyphenols were tentatively identified, of which 19 represent the first reports of these polyphenols in LRFEs. Furthermore, the cellular antioxidant array showed that LRFEs could protect Caco-2 cells against H2O2-induced oxidative damage based on microscopic fluorometric imaging.

  10. The Small Colony Variant Of Listeria Monocytogenes Is More Tolerant To Antibiotics And Grows Better Within Caco-2 Epithelial Cells Than The Wild Type

    DEFF Research Database (Denmark)

    Curtis, Thomas; Gram, Lone; Knudsen, Gitte Maegaard

    2015-01-01

    , the SCV E18 survived significantly better than the wild type N53-1 (one and three log10 higher CFU/ml) when exposed to super-MIC concentrations of most tested antibiotics, indicating a persister-like phenotype of the SCV. While SCV E18 displayed sensitivity towards oxygen, it was significantly more...... tolerant of 20mM H2O2 as compared to the wild type, with 6.3 log10 CFU/ml and 3.7 log10 CFU/ml, respectively. The SCV E18 had lower survival rate in unactivated macrophages, however, it was able to survive and multiply to almost 100-fold higher CFU/ml than the wild type in CaCo-2 epithelial cells...

  11. Assessment and modulation of acamprosate intestinal absorption: comparative studies using in situ, in vitro (CACO-2 cell monolayers) and in vivo models.

    Science.gov (United States)

    Zornoza, Teodoro; Cano-Cebrián, María José; Nalda-Molina, Ricardo; Guerri, Consuelo; Granero, Luis; Polache, Ana

    2004-08-01

    The purpose of this study was to explore the intestinal absorption mechanism of acamprosate and to attempt to improve the bioavailability (BA) of the drug through modulation of its intestinal absorption using two enhancers (polysorbate 80 and sodium caprate) based on in situ, in vitro and in vivo models and comparing the results obtained. Intestinal transport of the drug, in the absence and in presence of polysorbate 80 (0.06, 0.28 and 9.6 mM) or sodium caprate (13 and 16 mM) was measured by using an in situ rat gut technique and Caco-2 cell monolayers. Additionally, the effect of sodium caprate on drug oral bioavailability, measured as urinary recovery, was quantified by performing in vivo experiments with the rat as animal model. Only sodium caprate was able to increase the absorption rate constant (ka) of acamprosate in the mid-intestine of the rats from 0.29 +/- 0.07 h-1 in the absence of the promoter to 0.51 +/- 0.19 h-1 in the presence of C10 16 mM, along with the apparent permeability (Papp) obtained in Caco-2 cells (around two-fold). However, the drug bioavailability in rats (around 20%) did not improve in the presence of any of the concentrations tested (13, 16 and 50 mM). It is concluded that acamprosate absorption likely occurs via paracellular pathway and can be enhanced by sodium caprate in situ and in vitro but not in vivo-thus suggesting that although in situ and in vitro studies could be useful in early screening to select a potential promoter, in vivo studies in animal models are necessary to confirm the utility of the enhancer and to determine the influence of physiological variables.

  12. 治疗剂量下4种抗结核药物与Caco-2细胞上P-gp相互作用研究%Initial Study on Interaction between Therapeutic Doses of Four Anti-Tuberculosis Drugs and P-Glycoprotein in Caco-2 Cells

    Institute of Scientific and Technical Information of China (English)

    方平飞; 高维; 李焕德; 刘艺平

    2011-01-01

    目的 研究治疗剂量下4种抗结核药物(异烟肼、左氧氟沙星、乙胺丁醇、吡嗪酰胺)对Caco-2细胞上P-糖蛋白功能、表达及MDR1 mRNA表达的影响,从而解释联合抗痨治疗中不同组合的合理性.方法 采用流式细胞仪测定细胞内罗丹明-123的浓度,考察药物对P-糖蛋白功能的影响,流式细胞术分析药物对Caco-2细胞上P-糖蛋白表达的影响,实时荧光定量PCR技术分析药物对Caco-2细胞MDRI基因mRNA水平表达的影响.结果 含药培养20 d后,异烟肼和乙胺丁醇减少了罗丹明-123在Caco-2细胞内的蓄积(P<0.05),为诱导作用;异烟肼、乙胺丁醇均上调了Caco-2细胞上P-糖蛋白的表达(P<0.05),其P-糖蛋白表达量分别为阴性对照组的3.5和3.8倍;同时上调了Caco-2细胞上MDR1 mRNA的表达(P<0.05),其MDRImRNA的表达量分别为阴性对照组的11.5和11倍.左氧氟沙星增加了罗丹明-123在Caco-2细胞内的蓄积(P<0.05),为抑制作用;下调了Caco-2细胞上P-糖蛋白和MDR1 mRNA的表达(P<0.05),其P-糖蛋白和MDR1 mRNA表达量分别为阴性对照组的50%和32%.而吡嗪酰胺与P-糖蛋白无明显相互作用.结论 治疗剂量的异烟肼和乙胺丁醇为P-糖蛋白的诱导剂,左氧氟沙星为P-糖蛋白的抑制剂,而吡嗪酰胺对P-糖蛋白功能和表达无明显影响.%OBJECTIVE To study the effects of four anti-tuberculosis drugs on the function and expression of P-glycoprotein and the MDR1 mRNA expression, for explaining the rationality of different combination in anti-tuberculosis chemotherapy. METHODS The effect of the drugs on P-glycoprotein function was analyzed using Rh-123 assay. The flow cytometry was used to determine the intracellular Rh-123 concentration and the expression of P-glycoprotein in Caco-2 cells. Real-time fluorescent quantitative polymerase chain reaction was used to measure the expression of MDR1 gene mRNA in Caco-2 cells. RESULTS After the intervention for 20 d, the

  13. Interaction of GABA-mimetics with the taurine transporter (TauT, Slc6a6) in hyperosmotic treated caco-2, LLC-PK1 and rat renal SKPT cells

    DEFF Research Database (Denmark)

    Rasmussen, Rune Nørgaard; Lagunas, Candela; Plum, Jakob Munk;

    2016-01-01

    The aim of the present study was to investigate if basic GABA-mimetics interact with the taurine transporter (TauT, Slc6a6), and to find a suitable cell based model that is robust towards extracellular changes in osmolality during uptake studies. Taurine uptake was measured in human Caco-2 cells...

  14. Heme iron uptake by Caco-2 cells is a saturable, temperature sensitive and modulated by extracellular pH and potassium.

    Science.gov (United States)

    Arredondo, Miguel; Kloosterman, Janneke; Núñez, Sergio; Segovia, Fabián; Candia, Valeria; Flores, Sebastián; Le Blanc, Solange; Olivares, Manuel; Pizarro, Fernando

    2008-11-01

    It is known that heme iron and inorganic iron are absorbed differently. Heme iron is found in the diet mainly in the form of hemoglobin and myoglobin. The mechanism of iron absorption remains uncertain. This study focused on the heme iron uptake by Caco-2 cells from a hemoglobin digest and its response to different iron concentrations. We studied the intracellular Fe concentration and the effect of time, K+ depletion, and cytosol acidification on apical uptake and transepithelial transport in cells incubated with different heme Fe concentrations. Cells incubated with hemoglobin-digest showed a lower intracellular Fe concentration than cells grown with inorganic Fe. However, uptake and transepithelial transport of Fe was higher in cells incubated with heme Fe. Heme Fe uptake had a low Vmax and Km as compared to inorganic Fe uptake and did not compete with non-heme Fe uptake. Heme Fe uptake was inhibited in cells exposed to K+ depletion or cytosol acidification. Heme oxygenase 1 expression increased and DMT1 expression decreased with higher heme Fe concentrations in the media. The uptake of heme iron is a saturable and temperature-dependent process and, therefore, could occur through a mechanism involving both a receptor and the endocytic pathway.

  15. Supplemental inulin does not enhance iron bioavailability to Caco-2 cells from milk- or soy-based, probiotic-containing, yogurts but incubation at 37 oC does

    Science.gov (United States)

    The in vitro effects of supplemental inulin (4%) on iron (Fe) availability in two different probiotic-containing yogurts were examined. Milk or soy-based yogurts, with and without inulin, were incubated (37 deg C) or not for 48h before comparison by an in vitro gastrointestinal digestion/Caco-2 cell...

  16. Effects of prefermented cereals or the end products of fermentation on growth and metabolism of enterocyte-like Caco-2 cells and on intestinal health of restrictedly fed weanling pigs

    NARCIS (Netherlands)

    Bruininx, E.M.A.M.; Koninkx, J.F.J.G.; Binnendijk, G.P.; Zandstra, T.; Heetkamp, M.J.W.; Peet-Schwering, van der C.M.C.; Gerrits, W.J.J.

    2010-01-01

    To unravel the underlying mechanisms that explain the positive effects of prefermented cereals on in vivo gastrointestinal (GI) architecture and function, an in vitro experiment using a human small intestinal epithelial cell model (Caco-2) was performed. A range of dilutions (0% to 10%) of the super

  17. 慢病毒载体介导的RNAi双敲除ABCG2和UGT1A1的Caco-2细胞模型%Construction of Caco-2 cell line with double knockdown of ABCG2 and UGT1A1 using lentivirus-mediated RNA interference

    Institute of Scientific and Technical Information of China (English)

    龚淑敏; 李光明; 董莉真; 吴志敏; 程彬; 张斌; 朱泽

    2013-01-01

    Objective:To determine the contribution of ABCG2 and UGTIA1 to absorption and metabolism of drug in intestine and con struct a Caco-2 cell line with stable double knockdowns of ABCG2 and UGT 1A 1 by lentiviral vector-mediated RNAi.Methods:Lentiviral vectors targeting ABCG2 and UGT1A1 were constructed and subsequently used to transfect Caco-2 cells.Expression of ABCG2 and UGT1A1 mRNA were analysised by RT-PCR,real-time PCR.While protein levels were measured by Western blotting.High interference expression Caco-2 cell line was chosen to continuously extend monitoring interference ABCG2 and UGT1A1 gene expression modificatiens.Results:Successfully construct ABCG2 and UGT1A1 double knockdown Caco-2 cell model by lentivirus-mediated RNAi.RNAi ABCG2 and UGT1A1 gene interferes efficiency was 75% and 88% respectively.Expression of ABCG2 and UGT1A1 in mRNA levels was respectively down-regulated to 72.3%(after) with 85.7% (initial); and to 69.7%(after) from 88.7%(initial).No significant difference were found between ABCG2 and the RNAi UGT1A1 gene interference and final double interference effect (P>0.05).ABCG2 and UGT1A1 protein expression levels were also significantly reduced accordingly by western blotting methods.Conclusion:It is the first time the con-struction of ABCG2 and UGT1A1 simultaneous double knockdown Caco-2 cells by lentiviral vectors RNAi is applied to further study the coupling function of ABCG2 and UGT1A 1 in absorption and metabolism of orally administered drugs and exogenous compounds in intestine.%目的:构建以Caco-2细胞为研究对象,由慢病毒载体介导ABCG2和UGT1A1基因RNAi干扰的肠道药物吸收和代谢模型.方法:应用慢病毒载体介导的RNAi技术敲除Caco-2细胞的ABCG2和UGT1A1基因,构建双RNAi干扰ABCG2和UGT1A1的Caco-2细胞系.应用RT-PCR,Real-time PCR,Western blotting等方法检测ABCG2和UGT1A1在mRNA及蛋白水平的表达.然后选择干扰高表达细胞株,连续传代监测干扰相应干

  18. Bioaccessibility, biotransformation, and transport of alpha-mangostin from Garcinia mangostana (Mangosteen) using simulated digestion and Caco-2 human intestinal cells.

    Science.gov (United States)

    Bumrungpert, Akkarach; Kalpravidh, Ruchaneekorn W; Suksamrarn, Sunit; Chaivisuthangkura, Apinya; Chitchumroonchokchai, Chureeporn; Failla, Mark L

    2009-05-01

    alpha- and gamma-Mangostin are the most abundant prenylated xanthones present in the fruit of the mangosteen tree. These compounds have been reported to possess numerous bioactivities that have provided the impetus for use of mangosteen products as nutraceuticals and in functional foods and dietary supplements. The health-promoting benefits of mangosteen are dependent on delivery of the xanthones to target tissues. Here, we used simulated digestion and Caco-2 cells to investigate the digestive stability, bioaccessibility, and intestinal cell transport of alpha- and gamma- mangostin. Recovery of alpha- and gamma-mangostin after simulated digestion of pericarp and fruit pulp exceeded 90%. Transfer of alpha- and gamma-mangostin to the aqueous fraction during simulated digestion was efficient (65-74%) and dependent on bile salts suggesting that micellarization is required for optimal bioaccessibility of xanthones. Cell uptake of xanthones from micelles was dose dependent and intracellular concentrations were maximum by 1 h. Both free and phase II metabolites of alpha-mangostin were transported in the basolateral compartment and metabolites also effluxed into the apical chamber. Transepithelial transport of alpha-mangostin was increased during prandial-like compared to fasted conditions suggesting that absorption is enhanced by dietary fat.

  19. Biphasic effect of IL-1beta on the activity of argininosuccinate synthetase in Caco-2 cells. Involvement of nitric oxide production.

    Science.gov (United States)

    Brasse-Lagnel, Carole; Lavoinne, Alain; Fairand, Alain; Vavasseur, Karine; Deniel, Nicolas; Husson, Annie

    2006-06-01

    The expression of the argininosuccinate synthetase gene (ASS), the limiting enzyme of arginine synthesis, was previously shown to be rapidly induced by a short-term (4 h) exposure to IL-1beta in Caco-2 cells [Biochimie, 2005, 403-409]. The present report shows that, by contrast, a long-term (24 h) exposure to IL-1beta inhibited the ASS activity despite an increase in both specific mRNA level and protein amount, demonstrating a post-translational effect. Concerning the mechanism involved, we demonstrate that the inhibiting effect is linked to the production of nitric oxide (NO) induced by IL-1beta. Indeed, the inhibiting effect of IL-1beta was totally blocked in the presence of l-NMMA, an inhibitor of the inducible nitric oxide synthase, or by culturing the cells in an arginine-deprived medium. Moreover, a decrease in the ASS activity was induced by culturing the cells in the presence of SNAP, a NO donor. Conversely, blocking the action of NO by antioxidant agents, the stimulatory effect of IL-1beta on ASS activity was restored, as measured at 24 h. Finally, such an inhibiting effect of NO on ASS activity may be related, at least in part, to S-nitrosylation of the protein. The physiological relevance of the antagonistic effects of IL-1beta and NO on ASS is discussed.

  20. Oxygen restriction increases the infective potential of Listeria monocytogenes in vitro in Caco-2 cells and in vivo in guinea pigs

    Directory of Open Access Journals (Sweden)

    Licht Tine

    2007-06-01

    Full Text Available Abstract Background Listeria monocytogenes has been implicated in several food borne outbreaks as well as sporadic cases of disease. Increased understanding of the biology of this organism is important in the prevention of food borne listeriosis. The infectivity of Listeria monocytogenes ScottA, cultivated with and without oxygen restriction, was compared in vitro and in vivo. Fluorescent protein labels were applied to allow certain identification of Listeria cells from untagged bacteria in in vivo samples, and to distinguish between cells grown under different conditions in mixed infection experiments. Results Infection of Caco-2 cells revealed that Listeria cultivated under oxygen-restricted conditions were approximately 100 fold more invasive than similar cultures grown without oxygen restriction. This was observed for exponentially growing bacteria, as well as for stationary-phase cultures. Oral dosage of guinea pigs with Listeria resulted in a significantly higher prevalence (p Listeria in fecal samples was observed after dosage with oxygen-restricted bacteria. These differences were seen after challenge with single Listeria cultures, as well as with a mixture of two cultures grown with and without oxygen restriction. Conclusion Our results show for the first time that the environmental conditions to which L. monocytogenes is exposed prior to ingestion are decisive for its in vivo infective potential in the gastrointestinal tract after passage of the gastric barrier. This is highly relevant for safety assessment of this organism in food.

  1. Mycotoxins modify the barrier function of Caco-2 cells through differential gene expression of specific claudin isoforms: Protective effect of illite mineral clay.

    Science.gov (United States)

    Romero, Alejandro; Ares, Irma; Ramos, Eva; Castellano, Víctor; Martínez, Marta; Martínez-Larrañaga, María-Rosa; Anadón, Arturo; Martínez, María-Aránzazu

    2016-04-15

    Aflatoxin B1 (AFB1), fumonisin B1 (FB1), ochratoxin A (OTA) and T-2 toxin (T2) are mycotoxins that commonly contaminate the food chain and cause various toxicological effects. Their global occurrence is regarded as an important risk factor for human and animal health. In this study, the results demonstrate that, in human Caco-2 cells, AFB1, FB1, OTA and T2 origin cytotoxic effects, determining cell viability through MTT assay and LDH leakage, and decrease trans-epithelial electrical resistance (TEER). The decrease in barrier properties is concomitant with a reduction in the expression levels of the tight junction constituents claudin-3, claudin-4 and occludin. The protective effect of mineral clays (diosmectite, montmorillonite and illite) on alterations in cell viability and epithelial barrier function induced by the mycotoxins was also evaluated. Illite was the best clay to prevent the mycotoxin effects. Illite plus mycotoxin co-treatment completely abolished AFB1 and FB1-induced cytotoxicity. Also, the decreases in the gene expression of claudins and the reduction of TEER induced by mycotoxins were reversed by the illite plus mycotoxin co-treatment. In conclusion, these results demonstrated that mycotoxins AFB1, FB1, T2 and OTA disrupt the intestinal barrier permeability by a mechanism involving reduction of claudin isoform expressions, and illite counteracts this disruption.

  2. Low-molecular-weight fucoidan and high-stability fucoxanthin from brown seaweed exert prebiotics and anti-inflammatory activities in Caco-2 cells

    Directory of Open Access Journals (Sweden)

    Pai-An Hwang

    2016-08-01

    Full Text Available Background: The aim of this study is to investigate the anti-inflammatory effects of low-molecular-weight fucoidan (LMF and high-stability fucoxanthin (HS-Fucox in a lipopolysaccharide-induced inflammatory Caco-2 cell line co-culture with B. lactis. Methods: We used various methods such as transepithelial resistance (TER assay, cytokine secretion assay, and tight junction protein mRNA expression assay to examine LMF and HS-Fucox anti-inflammatory properties. Results: LMF and HS-Fucox activated probiotic growth and reduced the inflammation of the intestinal epithelial cells. Moreover, the combination of LMFHS-Fucox dramatically enhanced the intestinal epithelial barrier and immune function against the lipopolysaccharide effect by inhibiting IL-1β and TNF-α and promoting IL-10 and IFN-γ. Conclusion: These findings suggested that LMF and HS-Fucox, alone or in combination, could be the potential natural compounds to enhance the immune system and have an anti-inflammatory effect on the intestinal cells.

  3. In vitro potential modulation of baicalin and baicalein on P-glycoprotein activity and expression in Caco-2 cells and rat gut sacs.

    Science.gov (United States)

    Miao, Qing; Wang, Zhiyong; Zhang, Yuanyuan; Miao, Peipei; Zhao, Yuanyuan; Zhang, Yujie; Ma, Shuangcheng

    2016-09-01

    Context Previous studies have shown that Scutellariae Radix, the dried root of Scutellaria baicalensis Georgi (Labiatae), has a certain inhibitory effect on P-glycoprotein (P-gp), but the effects of its main active constituents on P-gp are still ambiguous. Objectives In vitro studies were performed to investigate the effects of its main active constituents (baicalin and its aglycone, baicalein) on the activity and expression of P-gp in intestine using Caco-2 cells and rat gut sacs. Materials and methods In Caco-2 cell experiments, the effects of baicalin and baicalein on P-gp activity were investigated using a P-gp substrate, rhodamine 123 and non-substrate fluorescein Na, by determining their intracellular fluorescence accumulation, and their effects on P-gp expression were determined using flow cytometry. In addition, rat gut sac model was selected to investigate the effects of baicalin and baicalein on the transport of verapamil, a classical P-gp substrate. The gut sacs of male Sprague-Dawley rats were filled with 0.4 mL the test solution contained verapamil (0.2575 mg/mL) and the drugs [baicalin and baicalein, at concentrations of 1/8 IC50 (59.875, 41.5 μg/mL), 1/4 IC50 (119.75, 83 μg/mL) and 1/2 IC50 (239.5, 166 μg/mL)], and then incubated in Tyrode's solution for a period of time. After termination of the incubation, the incubated solution was processed for the subsequent detection. Results According to the results of MTT assay, the IC50 values of verapamil, baicalin and baicalein were 104, 479, 332 μg/mL, respectively. The obtained results from the two models were confirmed mutually. As a result, baicalin exhibited no obvious effect on intracellular accumulation of Rh-123, and almost had no effect on P-gp expression and verapamil transportation, while baicalein significantly increased intracellular accumulation of Rh-123 (p P-gp expression (p p P-gp inhibitor, which presented obvious inhibitory effects on P-gp activity and expression

  4. Lutein transport by Caco-2 TC-7 cells occurs partly by a facilitated process involving the scavenger receptor class B type I (SR-BI).

    Science.gov (United States)

    Reboul, Emmanuelle; Abou, Lydia; Mikail, Céline; Ghiringhelli, Odette; André, Marc; Portugal, Henri; Jourdheuil-Rahmani, Dominique; Amiot, Marie-Josèphe; Lairon, Denis; Borel, Patrick

    2005-04-15

    The carotenoid lutein is thought to play a role in the human eye and to protect against age-related macular degeneration. Lutein transport in the human intestine has not been characterized. We examined lutein transport processes using Caco-2 TC-7 monolayers as a model for human intestinal epithelium. Purified lutein was mixed with phospholipids, lysophospholipids, cholesterol, mono-olein, oleic acid and taurocholate to obtain lutein-rich mixed micelles that mimicked those found under physiological conditions. The micelles were added to the apical side of Caco-2 TC-7 cell monolayers for 30 min or 3 h at 37 degrees C. Absorbed lutein, i.e. the sum of lutein recovered in the scraped cells and in the basolateral chamber, was quantified by HPLC. Transport rate was measured (i) as a function of time (from 15 to 60 min), (ii) as a function of micellar lutein concentration (from 1.5 to 15 microM), (iii) at 4 degrees C, (iv) in the basolateral to apical direction, (v) after trypsin pretreatment, (vi) in the presence of beta-carotene and/or lycopene, (vii) in the presence of increasing concentrations of antibody against SR-BI (scavenger receptor class B type 1) and (viii) in the presence of increasing concentrations of a chemical inhibitor of the selective transfer of lipids mediated by SR-BI, i.e. BLT1 (blocks lipid transport 1). The rate of transport of lutein as a function of time and as a function of concentration was saturable. It was significantly lower at 4 degrees C than at 37 degrees C (approx. 50%), in the basal to apical direction than in the opposite direction (approx. 85%), and after trypsin pretreatment (up to 45%). Co-incubation with beta-carotene, but not lycopene, decreased the lutein absorption rate (approx. 20%) significantly. Anti-SR-BI antibody and BLT1 significantly impaired the absorption rate (approx. 30% and 57% respectively). Overall, these results indicate that lutein absorption is, at least partly, protein-mediated and that some lutein is taken up

  5. Effect of ganoderma lucidum polysaccharides peptide on the expression and function of p-glycoprotein in Caco-2 cells%灵芝多糖肽对Caco-2细胞上P糖蛋白功能、表达的影响

    Institute of Scientific and Technical Information of China (English)

    吴芬; 成日华; 李焕德

    2012-01-01

    目的 研究灵芝多糖肽(ganoderma lucidum polysaccharides peptide,GLPP)对Caco-2细胞P糖蛋白(P-gp)功能和表达的影响.方法 用流式细胞仪测定Caco-2细胞中P-gp底物罗丹明123和抗体的荧光强度,分析药物对P-gp功能和表达的影响.双向跨膜转运实验考查GLPP对罗丹明123转运的影响.结果 10、20、50、100μg·mL-1的GLPP均抑制了Rh-123的外排,其外排分别降低了8%、16%、19%、33%,具有浓度依赖性.高、中浓度的GLPP(50、100 μg·mL-1)对Rh-123的双向转运有一定的抑制,外排率(ER)较阴性对照组降低了15%、18%.细胞与药物作用72 h后,细胞抗体荧光强度降低,P-gp的表达降低.结论 GLPP对P-gp的活性有一定的抑制作用,能在较短的时间内增加细胞内Rh-123的蓄积,抑制Rh-123的双向转运.长期使用GLPP可抑制P-gp的表达.%Objective To determine the effect of ganoderma lucidum polysaccharides peptide (GLPP) on the expression and function of p-glycoprotein in Caco-2 cells. Methods The effect of GLPP on P-glycoprotein function and expression in Caco-2 cells was analyzed by rhodanmine-123 and antibody assay. Flowcytometry was used to determine the intracel-lular Rhodanmine-123 concentration and antibody concentration. The effect of GLPP on the transportation of rhodanmine-123 was detected by Caco-2 cell monolayer model. Results Compared with that of the controls, the efflux quantity of rhodamnine-123 was reduced by 8%, 16%, 19% and 33%. When treated with GLPP at 10, 20, 50 and 100 μg · mL-1 . The inhibitive effect of GLPP was concentration-dependent. Rhodanmine-123 bidirectional transport showed that the efflux rate of rhodanmine-123 was reduced by 15% and 18% when treated with GLPP at 50 and 100 μg · mL-1. The P-gp protein level was decreased after 72 h incubation. Conclusion GLPP is an inhibitor of P-gp which can inhibit the function of P-gp in 1 h and decrease the expression of P-gp after 72 h incubation with Caco-2 cells

  6. Knockdown of copper-transporting ATPase 1 (Atp7a) impairs iron flux in fully-differentiated rat (IEC-6) and human (Caco-2) intestinal epithelial cells.

    Science.gov (United States)

    Ha, Jung-Heun; Doguer, Caglar; Collins, James F

    2016-09-01

    Intestinal iron absorption is highly regulated since no mechanism for iron excretion exists. We previously demonstrated that expression of an intestinal copper transporter (Atp7a) increases in parallel with genes encoding iron transporters in the rat duodenal epithelium during iron deprivation (Am. J. Physiol.: Gastrointest. Liver Physiol., 2005, 288, G964-G971). This led us to postulate that Atp7a may influence intestinal iron flux. Therefore, to test the hypothesis that Atp7a is required for optimal iron transport, we silenced Atp7a in rat IEC-6 and human Caco-2 cells. Iron transport was subsequently quantified in fully-differentiated cells plated on collagen-coated, transwell inserts. Interestingly, (59)Fe uptake and efflux were impaired in both cell lines by Atp7a silencing. Concurrent changes in the expression of key iron transport-related genes were also noted in IEC-6 cells. Expression of Dmt1 (the iron importer), Dcytb (an apical membrane ferrireductase) and Fpn1 (the iron exporter) was decreased in Atp7a knockdown (KD) cells. Paradoxically, cell-surface ferrireductase activity increased (>5-fold) in Atp7a KD cells despite decreased Dcytb mRNA expression. Moreover, increased expression (>10-fold) of hephaestin (an iron oxidase involved in iron efflux) was associated with increased ferroxidase activity in KD cells. Increases in ferrireductase and ferroxidase activity may be compensatory responses to increase iron flux. In summary, in these reductionist models of the mammalian intestinal epithelium, Atp7a KD altered expression of iron transporters and impaired iron flux. Since Atp7a is a copper transporter, it is a logical supposition that perturbations in intracellular copper homeostasis underlie the noted biologic changes in these cell lines.

  7. Study of the Biotransformation of Tongmai Formula by Human Intestinal Flora and Its Intestinal Permeability across the Caco-2 Cell Monolayer.

    Science.gov (United States)

    Wu, Shuai; Xu, Wei; Wang, Fu-Rong; Yang, Xiu-Wei

    2015-10-15

    Tongmai formula (TMF) is a well-known Chinese medicinal preparation that contains isoflavones as its major bioactive constituents. As traditional Chinese medicines (TCMs) are usually used by oral administration, their fate inside the intestinal lumen, including their biotransformation by human intestinal flora (HIF) and intestinal absorption deserves study. In this work TMF extract was incubated with human intestinal bacteria under anaerobic conditions and the changes in the twelve main constituents of TMF were then investigated. Their intestinal permeabilities, i.e., the transport capability across the intestinal brush border were investigated with a human colon carcinoma cell line (Caco-2) cell monolayer model to predict the absorption mechanism. Meanwhile, rapid HPLC-DAD methods were established for the assay. According to the biotransformation curves of the twelve constituents and the permeability coefficients, the intestinal absorption capacity of the typical compounds was elevated from the levels of 10(-7) cm/s to 10(-5) cm/s from those of the original compounds in TMF. Among them the main isoflavone glycosides puerarin (4), mirificin (6) and daidzin (7) were transformed into the same aglycone, daidzein (10). Therefore it was predicted that the aglycone compounds might be the real active ingredients in TMF. The models used can represent a novel path for the TCM studies.

  8. Effects of colored and noncolored phenolics of Echium plantagineum L. bee pollen in Caco-2 cells under oxidative stress induced by tert-butyl hydroperoxide.

    Science.gov (United States)

    Sousa, Carla; Moita, Eduarda; Valentão, Patrícia; Fernandes, Fátima; Monteiro, Pedro; Andrade, Paula B

    2015-02-25

    Bee pollen is used as a dietary supplement, being promoted as a health food. Echium plantagineum L. bee pollen fractions enriched in flavonols (fraction I) or anthocyanins (fraction II) and the whole extract were characterized by HPLC-DAD. Both in the whole extract and in fraction II seven flavonols and five anthocyanins were identified, while fraction I contained six flavonols (in higher levels than fraction II) and small amounts of petunidin-3-O-rutinoside. Antioxidant capacity was evaluated in Caco-2 cells under oxidative stress induced by tert-butyl hydroperoxide (t-BHP). Fraction I pre-exposure imparted a tendency to protect cells, while fraction II and the whole extract aggravated t-BHP toxicity at some concentrations. The protective effects seem to be correlated with the levels of total glutathione, while no correlation between cellular viability and reactive species was seen. The extracts displayed no significant effect on antioxidant enzymes activity. Overall, anthocyanins seem to abrogate the antioxidant potential of flavonoid-rich extracts.

  9. Effect of Pluronic on Cellular Accumulation of Rhodamine 123, a P-gp Substrate in Caco-2 Cells%普流罗尼克对P-gp底物罗丹明123在Caco-2细胞蓄积的影响

    Institute of Scientific and Technical Information of China (English)

    关延彬; 左岚; 饶子超; 斯陆勤; 李高

    2011-01-01

    OBJECTIVE To determine whether Pluronic, P123, F127, P85, F68 and L61 possess ability to modulate P-glycoprotein(P-gp) mediated efflux of drugs. METHODS Caco-2 cell viability was measured using MTT assay in the presence of Pluronic at different concentrations. To evaluate the effects of Pluronic and verapamil( inhibitor of P-gp)on cellular drug accumulation in caco-2 cells, rhodamine 123(R-123), a P-gp substrate, was selected as fluorescent probe. A HPLC-fluorescence method was used to determined the concentration of R-123 in Caco-2 cells. Caco-2 cell membranes were exposed to various Pluronic solutions( P123 or F127),and then the ATPase activity of P-gp was assayed by determining liberated inorganic phosphate. RESULTS The viability of Pluronictreated cells was above 80% except L61, which suggested that Caco-2 cells could maintain their viability under the experiment conditions. In the presence of verapamil and Pluronic at concentrations ranging from 0.00 1 -50 mg · mL-1, R-123 accumulation were increased significantly to differernt extent. R-123 accumulation reached maximum levels when Pluronic concentrations were close to or above the CMC, Higher Pluronic concentrations resulted in a drop in accumulation compared to that of R-123 control. Furthermore,P123 and F127 treatments decreased P-gP ATPase activity in Caco-2 cell membranes expressing P-gp. CONCLUSION Pluronic (F68, F127, P123 and P85) can modulate drug effiux by inhibiting P-gp activity in Caco-2 cell membranes. It may be possible to improve the oral biuavailability of P-gp substrates by co-administration of Pluronic.%目的 研究普流罗尼克(Pluronic F127,F68,P85,P123)对小肠P-糖蛋白(P-gP)的调控作用.方法 采用MTT法检测不同质量浓度的Pluronic 时Caco-2细胞生长抑制作用;以P-gp底物罗丹明123(R-123)为荧光探针,评价各种Pluronic辅料和P-gp抑制剂维拉帕米对R-123细胞蓄积的影响,细胞内的R-123浓度采用高效液相-

  10. Uptake of cadmium by rice grown on contaminated soils and its bioavailability/toxicity in human cell lines (Caco-2/HL-7702).

    Science.gov (United States)

    Aziz, Rukhsanda; Rafiq, Muhammad Tariq; Li, Tingqiang; Liu, Di; He, Zhenli; Stoffella, P J; Sun, Kewang; Xiaoe, Yang

    2015-04-01

    Cadmium (Cd) enters the food chain from polluted soils via contaminated cereals and vegetables; therefore, an understanding of Cd bioaccessibility, bioavailability, and toxicity in humans through rice grain is needed. This study assessed the Cd bioaccessibility, bioavailability, and toxicity to humans from rice grown on Cd-contaminated soils using an in vitro digestion method combined with a Caco-2/HL-7702 cell model. Cadmium bioaccessibility (18.45-30.41%) and bioavailability (4.04-8.62%) were found to be significantly higher in yellow soil (YS) rice than calcareous soil (CS) rice with the corresponding values of 6.89-11.43 and 1.77-2.25%, respectively. Toxicity assays showed an initial toxicity in YS rice at 6 mg kg(-1) Cd, whereas CS rice did not show any significant change due to low Cd concentrations. The acidic soils of Cd-contaminated areas can contribute to a higher dietary intake of Cd. Therefore, it is imperative to monitor Cd concentration in rice to minimize human health risk.

  11. Influence of surfactants in self-microemulsifying formulations on enhancing oral bioavailability of oxyresveratrol: Studies in Caco-2 cells and in vivo.

    Science.gov (United States)

    Sangsen, Yaowaporn; Wiwattanawongsa, Kamonthip; Likhitwitayawuid, Kittisak; Sritularak, Boonchoo; Graidist, Potchanapond; Wiwattanapatapee, Ruedeekorn

    2016-02-10

    Self-microemulsifying drug delivery systems (SMEDDS) containing two types (Tween80 and Labrasol) and two levels (low; 5% and high; 15%) of co-surfactants were formulated to evaluate the impact of surfactant phase on physical properties and oral absorption of oxyresveratrol (OXY). All formulations showed a very rapid release in the simulated gastric fluid (SGF) pH 1.2. After dilution with different media, the microemulsion droplet sizes of the Tween80-based (∼26 to 36 nm) were smaller than that of the Labrasol-based systems (∼34 to 45 nm). Both systems with high levels of surfactant increased the Caco-2 cells permeability of OXY compared to those with low levels of surfactant (1.4-1.7 folds) and the unformulated OXY (1.9-2.0 folds). It was of interest, that there was a reduction (4.4-5.3 folds) in the efflux transport of OXY from both systems compared to the unformulated OXY. The results were in good agreement with the in vivo absorption studies of such OXY-formulations in rats. Significantly greater values of Cmax and AUC(0-10h) (psurfactant in the SMEDDS to enhance oral drug bioavailability.

  12. Transport of Schisandra chinensis extract and its biologically-active constituents across Caco-2 cell monolayers - an in-vitro model of intestinal transport.

    Science.gov (United States)

    Madgula, Vamsi L M; Avula, Bharathi; Choi, Young W; Pullela, Srinivas V; Khan, Ikhlas A; Walker, Larry A; Khan, Shabana I

    2008-03-01

    We have determined the intestinal transport of Schisandra chinensis extract and its lignans (gomisin A, gomisin N and schisandrin C) in the Caco-2 cell monolayer model. The transport across monolayers was examined for 2 h in absorptive and secretory directions. Quantitation of lignans was performed by HPLC. Out of the three lignans, gomisin A exhibited bi-directional transport, with P(app) values in the range of 25-29 x 10(-6) cm s(-1), indicating a passive diffusion. Gomisin N, mixture and Schisandra extract displayed a higher transport in the secretory direction with efflux ratios in the range of 2.2-5.2. The efflux was decreased in the presence of inhibitors of multidrug resistance protein (MRP) transporter (MK-571) and P-glycoprotein (verapamil) indicating a possible involvement of an efflux pump and MRP in the transport of Schisandra lignans. Poor transport of schisandrin C was observed which could not be quantitated. The permeability of gomisin A in the isolated form was significantly different compared with the mixture or extract.

  13. Evaluation of the intestinal transport of a phenylethanoid glycoside-rich extract from Cistanche deserticola across the Caco-2 cell monolayer model.

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    Yuan Gao

    Full Text Available Phenylethanoid glycosides (PhGs, a class of polyphenolic compounds, are considered one of major bioactive constituents of Cistanche deserticola Y.C. Ma (CD, whose extract is orally used in traditional Chinese medicine. Although previous pharmacological studies have reported that PhGs exert many activities, their intestinal transport profiles have not been clarified. In this study, we investigated the intestinal permeability of a PhG-rich extract (PRE from CD as an integrated system in the Caco-2 cell monolayer model using a bioassay system. The results showed that PRE is primarily transported via poorly absorbed passive diffusion down a concentration gradient without efflux, which provides the pharmacokinetic basis for the clinical application of PhGs in CD. We also determined the intestinal permeability of three major PhGs [acteoside (AC, isoacteoside (IS and echinacoside (EC] by HLPC. Furthermore, we developed a novel HPLC-fluorescence detection method to accurately determine the flux amount of AC and IS. As expected, the transport characteristics of the three PhGs are consistent with those of PRE, indicating that the present bioassay system is appropriate and reliable for the evaluation of the transport characteristics of active ingredient groups (AIG in PRE. Moreover, this system may also be suitable for other plant extracts given appropriate bioactivity.

  14. In vitro digestion/Caco-2 cell model to estimate cadmium and lead bioaccessibility/bioavailability in two vegetables: the influence of cooking and additives.

    Science.gov (United States)

    Fu, Jin; Cui, Yanshan

    2013-09-01

    The estimation of heavy metal bioaccessibility and bioavailability in vegetables is helpful for human health risk assessment. Using an in vitro digestion/Caco-2 cell model, the bioaccessibility and bioavailability of cadmium (Cd) and lead (Pb) in raw/cooked pakchoi (Brassica rapa L., Chinensis Group) and Malabar spinach (Basella rubra L.) were studied. The effect of the addition of iron, calcium and acetic acid to the samples was also determined. The results indicated that Cd bioaccessibility was higher in the gastric phase and Pb bioaccessibility was higher in the small intestinal phase. Cadmium and Pb bioavailability were 11.2% and 9.4% in the raw vegetables, respectively, and found to be higher significantly than the cooked vegetables with 6.1% for Cd and 3.2% for Pb. The results showed that it will be overestimating the risk of Pb and Cd based on the data of raw vegetables ingestion. Using bioavailability values, average Cd and Pb daily intake by adult were 23% and 28% respectively, of the base bioaccessibility values. Our study will be better understanding the possible health risks of some vegetables base on the bioaccessibility or bioavailability.

  15. Butyrate enhances the intestinal barrier by facilitating tight junction assembly via activation of AMP-activated protein kinase in Caco-2 cell monolayers.

    Science.gov (United States)

    Peng, Luying; Li, Zhong-Rong; Green, Robert S; Holzman, Ian R; Lin, Jing

    2009-09-01

    Butyrate, one of the SCFA, promotes the development of the intestinal barrier. However, the molecular mechanisms underlying the butyrate regulation of the intestinal barrier are unknown. To test the hypothesis that the effect of butyrate on the intestinal barrier is mediated by the regulation of the assembly of tight junctions involving the activation of the AMP-activated protein kinase (AMPK), we determined the effect of butyrate on the intestinal barrier by measuring the transepithelial electrical resistance (TER) and inulin permeability in a Caco-2 cell monolayer model. We further used a calcium switch assay to study the assembly of epithelial tight junctions and determined the effect of butyrate on the assembly of epithelial tight junctions and AMPK activity. We demonstrated that the butyrate treatment increased AMPK activity and accelerated the assembly of tight junctions as shown by the reorganization of tight junction proteins, as well as the development of TER. AMPK activity was also upregulated by butyrate during calcium switch-induced tight junction assembly. Compound C, a specific AMPK inhibitor, inhibited the butyrate-induced activation of AMPK. The facilitating effect of butyrate on the increases in TER in standard culture media, as well as after calcium switch, was abolished by compound C. We conclude that butyrate enhances the intestinal barrier by regulating the assembly of tight junctions. This dynamic process is mediated by the activation of AMPK. These results suggest an intriguing link between SCFA and the intracellular energy sensor for the development of the intestinal barrier.

  16. The relationship between the anthocyanin and vitamin C contents of red-fleshed sweet cherries and the ability of fruit digests to reduce hydrogen peroxide-induced oxidative stress in Caco-2 cells.

    Science.gov (United States)

    Leong, Sze Ying; Burritt, David J; Hocquel, Alice; Penberthy, Ari; Oey, Indrawati

    2017-07-15

    The present work investigated the bioprotective capacities of red-fleshed sweet cherry cultivars (Prunus avium; Lapins, Stella, Sweetheart and Staccato), with distinct differences in anthocyanins and vitamin C contents, on human intestinal Caco-2 cells exposed to hydrogen peroxide (H2O2)-induced oxidative stress. Three assays of cell health, the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl-tetrazolium-bromide cell viability assay, the lactate dehydrogenase membrane leakage assay and nitric oxide production, were used to determine if these cherry digests differed in their ability to protect Caco-2 cells from H2O2. Cells treated with digests from cherries identified as containing high anthocyanins provided the greatest protection against H2O2. A strong linear correlation (-0.82stress than vitamin C.

  17. Upregulation of Salmonella-Induced IL-6 Production in Caco-2 Cells by PJ-34, PARP-1 Inhibitor: Involvement of PI3K, p38 MAPK, ERK, JNK, and NF-κB

    Directory of Open Access Journals (Sweden)

    Fu-Chen Huang

    2009-01-01

    Full Text Available Following Salmonella invasion, intestinal epithelial cells release a distinct array of proinflammatory cytokines. Interleukin (IL-6 produced by enterocytes may have anti-inflammatory and cell-protective effects, and may counteract some of the injurious effects of sepsis and endotoxemia. Recent studies in a variety of rodent models of experimental colitis by using PJ-34, a potent poly (ADP-ribose polymerase-1 (PARP-1 inhibitor, support the concept that the marked beneficial effect of PJ-34 can be exploited to treat human inflammatory diseases. The present study was to investigate the effect of PJ-34 on Salmonella-induced enterocyte IL-6 production and its mechanisms. We found that PJ-34 enhanced Salmonella-induced IL-6 production in Caco-2 cells, either secreted protein or mRNA expression. PJ-34 treatment enhanced the activity of NF-κB in Salmonella-infected Caco-2 cells. Besides, the involvement of PJ-34 in up-regulating IL-6 production in S. typhimurium-infected Caco-2 cells might be also through the ERK but not p38 MAPK, JNK or PI3K/Akt pathways, as demonstrated by Western blot of phosphorylated ERK, p38, JNK and Akt proteins. It suggests that PJ-34 may exert its protective effect on intestinal epithelial cells against invasive Salmonella infection by up-regulating IL-6 production through ERK and NF-κB but not P38 MAPK, JNK or PI3K/Akt signal pathways.

  18. Protamine coated proliposomes of recombinant human insulin encased in Eudragit S100 coated capsule offered improved peptide delivery and permeation across Caco-2 cells.

    Science.gov (United States)

    Sharma, Shiva; Jyoti, Kiran; Sinha, Richa; Katyal, Anju; Jain, Upendra Kumar; Madan, Jitender

    2016-10-01

    In present investigation, recombinant human insulin loaded proliposomes and protamine sulphate coated proliposomes (rh insulin-proliposomes and Pt-rh insulin proliposomes) were encased in Eudragit S100 coated capsule to offer peptide release in simulated intestinal conditions. The particle size and zeta potential of Pt-rh insulin proliposomes were measured to be 583.2±10.2nm/+28.3±3.7mV significantly (P<0.05) higher than 569.7±14.9nm/-37.9±4.3mV and 534.6±24.6nm/-42.7±2.8mV of rh insulin proliposomes and proliposomes, respectively. Next, shape and surface morphology analysis pointed out the successful transformation of proliposomes in to spherical shaped liposomes. Furthermore, in vitro release study specified that free rh insulin solution encapsulated in uncoated gelatine capsule released 97.8% of peptide within 1h in SGF (pH~1.2). On other hand, rh insulin-proliposomes and Pt-rh insulin proliposomes encased in Eudragit S100 coated capsule released 93.2% and 81.6% of peptide, up to 24 h in SIF (pH~7.2). SDS-PAGE and circular dichroism (CD) ascertained the stability and intactness of isolated rh insulin from tailored dosage forms. In last, cellular uptake in Caco-2 cells indicating the superiority of Pt-rh insulin proliposomes in comparison to rh-insulin proliposomes and free rh insulin solution, respectively. In conclusion, Pt-rh insulin proliposomes displayed promising results and may be considered for further investigations.

  19. EPS-SJ exopolisaccharide produced by the strain Lactobacillus paracasei subsp. paracasei BGSJ2-8 is involved in adhesion to epithelial intestinal cells and decrease on E. coli association to Caco-2 cells

    Directory of Open Access Journals (Sweden)

    Milica eZivkovic

    2016-03-01

    Full Text Available The aim of this study was to determine the role of an exopolysaccharide produced by natural dairy isolate Lactobacillus paracasei subsp. paracasei BGSJ2-8, in the adhesion to intestinal epithelial cells and a decrease in E. coli’s association with Caco-2 cells. Annotation of the BGSJ2-8 genome showed the presence of a gene cluster, epsSJ, which encodes the biosynthesis of the strain-specific exopolysaccharide EPS-SJ, detected as two fractions (P1 and P2 by size exclusion chromatography (SEC coupled with multi-angle laser light scattering (MALLS detection. SEC-MALLS analysis revealed that an EPS-SJ‒ mutant (EPS7, obtained by insertion mutagenesis of the glps_2198 gene encoding primary glycosyltransferase does not produce the P2 fraction of EPS-SJ. Transmission electron microscopy showed that EPS7 mutant has a thinner cell wall compared to the EPS-SJ+ strain BGSJ2-83 (a plasmid free-derivative of BGSJ2-8. Interestingly, strain BGSJ2-83 showed higher adhesion to Caco-2 epithelial intestinal cell line than the EPS7 mutant. Accordingly, BGSJ2-83 effectively reduced E. coli ATCC25922’s association with Caco-2 cells, while EPS7 did not show statistically significant differences. In addition, the effect of EPS-SJ on the proliferation of lymphocytes in gastrointestinal associated lymphoid tissue (GALT was tested and the results showed that the reduction of GALT lymphocyte proliferation was higher by BGSJ2-83 than by the mutant. To the best of our knowledge this is the first report indicating that the presence of EPS (EPS-SJ on the surface of lactobacilli can improve communication between bacteria and intestinal epithelium, implying its possible role in gut colonization.

  20. Co-culture of Caco-2 Intestinal Cell and Intestinal Floras to Build an In Vitro Intestinal Model%Caco-2细胞与肠道菌共培养初建体外肠道共生模型

    Institute of Scientific and Technical Information of China (English)

    陈晓清; 焦红; 程树军; 易敏英; 刘静宇; 许龙岩; 刘超

    2012-01-01

    [Objective] To build an in vitro intestinal model which will be used to assess the harm risk caused by foodborne antimicrobial and the resistance bacteria to human health. [Method] The Caco-2 intestinal epithelial models after 21 days culture was built, 4 groups were divided; negative control group, mix floras group, Salmonella group, mix floras & Salmonella group, the time for co-culture was 4h. The 16S rDNA fragment of each flora before and after co-culture by quantitative fluorescence PCR was quantified respectively, and the changes of Caco-2 cells intestinal integrity were evaluated by detecting Trans-epithelial electrical resistance (TEER) and the immunofluorescence staining image of tight junction protein ZO-1.[ Results] From the 16S r DNA fragment of four intestinal floras quantified by qPCR, there were no significant changes between alone cultured and the mixed culture (P > 0.05), the 2 aerobes grown significantly faster than the growth of the 2 anaerobes (P < 0.01). B/E was near 100:1, showed that the aerobes were dominant bacteria. Compared with the negative control, TEER and ZO-1 distribution in mix floras group had no significant change and revealed that there were no significant damages to the integrity of Caco-2 intestinal epithelial cells, while in Salmonella group, TEER reduced significantly, and the destruction of ZO-1 structure was observed. But the damages in mix floras & Salmonella group (mixed floras pre-planted) could be some extent reduced. [Conclusion] This research built an primary in vitro co-culture intestinal epithelial model with Caco-2 cell and 4 intestinal Horas-Bifidobacterium lactis, Lactobacillus plantum, Escherichia coli and Enterococcus faecalis. The model showed the certain stability and biological activity. Normal intestinal flora had inhibitory effect to invasive salmonella. Furthermore, we will refine the model to lay the foundation to study the interaction of foodborne antimicrobial drug, drug resistant bacteria and

  1. Bioavailability of iron in geophagic earths and clay minerals, and their effect on dietary iron absorption using an in vitro digestion/Caco-2 cell model.

    Science.gov (United States)

    Seim, Gretchen L; Ahn, Cedric I; Bodis, Mary S; Luwedde, Flavia; Miller, Dennis D; Hillier, Stephen; Tako, Elad; Glahn, Raymond P; Young, Sera L

    2013-08-01

    Geophagy, the deliberate consumption of earth, is strongly associated with iron (Fe) deficiency. It has been proposed that geophagy may be practiced as a means to improve Fe status by increasing Fe intakes and, conversely, that geophagy may cause Fe deficiency by inhibiting Fe absorption. We tested these hypotheses by measuring Fe concentration and relative bioavailable Fe content of 12 samples of geophagic earth and 4 samples of pure clay minerals. Further, we assessed the impact of these samples on the bioavailability of Fe from an Fe-rich test meal (cooked white beans, WB). Fe concentrations were measured with inductively coupled plasma atomic emission spectroscopy. Fe bioavailability was determined using an in vitro digestion/Caco-2 cell model in which ferritin formation was used as an index of Fe bioavailability. Geophagic earth and clay mineral samples were evaluated with this model, both alone and in combination with WB (1 : 16 ratio, sample : WB). Median Fe concentration of the geophagic earth was 3485 (IQR 2462, 14 ,571) μg g⁻¹ and mean Fe concentration in the clay minerals was 2791 (±1782) μg g⁻¹. All specimens had Fe concentrations significantly higher (p ≤ 0.005) than the Fe concentration of WB (77 μg g⁻¹). Ferritin formation (i.e. Fe uptake) in cells exposed to geophagic earths and clay minerals was significantly lower than in cells exposed to WB (p ≤ 0.05) and Fe uptake responses of 11 of the 16 samples were not significantly different from the blank, indicating no bioavailable Fe. When samples were combined with WB, 5 of 16 had mean ferritin levels that were significantly lower (p ≤ 0.05, one tail) than the WB alone, indicating that the samples inhibited Fe uptake from the WB. None of the ferritin responses of cells exposed to both WB and earth/clay were significantly higher than WB alone. Thus, although geophagic earths and mineral clays are high in total Fe, very little of this Fe is bioavailable. Further, some

  2. Lactobacillus johnsonii N6.2 stimulates the innate immune response through Toll-like receptor 9 in Caco-2 cells and increases intestinal crypt Paneth cell number in biobreeding diabetes-prone rats.

    Science.gov (United States)

    Kingma, Sandra D K; Li, Nan; Sun, Frank; Valladares, Ricardo B; Neu, Joe; Lorca, Graciela L

    2011-06-01

    Lactobacillus johnsonii (Ljo) N6.2 has been shown to mitigate the development of type 1 diabetes when administered to diabetes-prone rats. The specific mechanisms underlying this observed response remain under investigation. The objective of this study was to assess the effect of Ljo N6.2 on mucosal inflammatory response using differentiated Caco-2 monolayers. The mRNA expression levels of CCL20, CXCL8, and CXCL10 chemokines were determined by qRT-PCR. Ljo at 10(11) CFU/L induced a strong response in all chemokines examined. To assess the specific host-signaling pathways involved, we performed RT-PCR amplification of Toll-like receptors (TLR) and nucleotide-binding oligomerization domain-like receptors. TLR7 and TLR9 expression levels were induced 4.2- and 9-fold, respectively, whereas other TLR and nucleotide-binding oligomerization domain receptors were not modified. A similar effect was observed in Caco-2 monolayers treated with Ljo cell-free extract or purified nucleic acids (NA). Increased levels of IFN type 1 and IFN regulators Stat1 and IRF7 followed the upregulation of TLR9. Activation of TLR9 was also evidenced by increased Frizzled 5 expression in Ljo-treated Caco-2 cells and an increase in the number of Paneth cells in Ljo-fed, diabetes-prone rats. These results are in agreement with the polarizing-tolerizing mechanism recently described in which the apical stimulation of TLR9 in intestinal epithelial cells leads to a higher state of immunologic alertness. Furthermore, these results suggest that live probiotics could be, in the future, replaced with select cellular components.

  3. Transport of trans-tiliroside (kaempferol-3-β-D-(6"-p-coumaroyl-glucopyranoside) and related flavonoids across Caco-2 cells, as a model of absorption and metabolism in the small intestine.

    Science.gov (United States)

    Luo, Zijun; Morgan, Michael R A; Day, Andrea J

    2015-01-01

    1. Absorption and metabolism of tiliroside (kaempferol 3-β-D-(6"-p-coumaroyl)-glucopyranoside) and its related compounds kaempferol, kaempferol-3-glucoside and p-coumaric acid were investigated in the small intestinal Caco-2 cell model. Apparent permeation (Papp) was determined as 0.62 × 10(-6) cm/s, 3.1 × 10(-6) cm/s, 0 and 22.8 × 10(-6) cm/s, respectively. 2. Mechanistic study showed that the transportation of tiliroside, kaempferol-3-glucoside and p-coumaric acid in Caco-2 model were transporter(s) involved, while transportation of kaempferol was solely by passive diffusion mechanism. 3. Efflux transporters, multi-drug-resistance-associated protein-2 (MRP2), were shown to play a role in limiting the uptake of tiliroside. Inhibitors of MRP2, (MK571 and rifampicin) and co-incubation with kaempferol (10 μM), increased transfer from the apical to the basolateral side by three to five fold. 4. Metabolites of kaempferol-3-glucoside and p-coumaric acid were not detected in the current Caco-2 model, while tiliroside was metabolised to a limited extent, with two tiliroside mono-glucuronides identified; and kaempferol was metabolised to a higher extent, with three mono-glucuronides and two mono-sulfates identified. 5. In conclusion, tiliroside was metabolised and transported across Caco-2 cell membrane to a limited extent. Transportation could be increased by applying MRP2 inhibitors or co-incubation with kaempferol. It is proposed that tiliroside can be absorbed by human; future pharmacokinetics studies are warranted in order to determine the usefulness of tiliroside as a bioactive agent.

  4. 一种新的小檗碱衍生物CPU-86017在人小肠上皮细胞(Caco-2细胞)中的转运与摄取特性%Transport and uptake characteristics of a new derivative of berberine (CPU-86017) by human intestinal epithelial cell line: Caco-2

    Institute of Scientific and Technical Information of China (English)

    杨海涛; 王广基

    2003-01-01

    AIM: The characteristics of transepithelial transport and uptake of CPU-86017 {[7-(4-chlorbenzyl)-7,8,13,13α tetrahydroberberine chloride, CTHB] }, a new antiarrhythmia agent and a new derivative of berberine, were investi gated on epithelial cell line (Caco-2) to further understand the absorption mechanism of berberine and its derivatives. METHODS: Caco-2 cell was used. RESULTS: 1) The permeability coefficient from the apical (AP) to basolateral (BL) of CPU-86017 was approximately 5 times higher than that from BL-to-AP transport. The effects of a P-glycoprotein (P-gp) inhibitor-cyclosporin A, some surfactants, and lower pH on the transepithelial transport of CPU-86017 were also observed. Cyclosporine A at 7.5 mg/L had no effect on the transepithelial electrical resistance (TEER); an about 4-fold enhancement on the transepithlial transport of CPU-86017 was observed. Some surfac tants (sodium citrate, sodium deoxycholate, and sodium dodecyl sulfate) at 100 μmol/L and low pH (pH=6.0) induced a reversible decrease of TEER; enhancements of the transepithelial transport of CPU-86017 were also observed with some surfactants; 2) In the process of uptake of CPU-86017, the initial uptake rates of CPU-86017 were saturable with a Vmax of (250+39) μg.min-1.g-1 (protein) and Km of (0.90+0.12) mmol/L. This process was enhanced by cyclosporine A (7.5 mg/L) with a Vmax of (588+49) μg.min-1.g-1 (protein) and Km (0.42+0.08) mmol/L. CONCLUSION: Some surfactants and P-gp inhibitors can be considered as enhancers of its transepithelial trans port and uptake.

  5. PLC-γ directly binds activated c-Src, which is necessary for carbachol-mediated inhibition of NHE3 activity in Caco-2/BBe cells.

    Science.gov (United States)

    Zachos, Nicholas C; Lee, Luke J; Kovbasnjuk, Olga; Li, Xuhang; Donowitz, Mark

    2013-08-01

    Elevated levels of intracellular Ca(2+) ([Ca(2+)]i) inhibit Na(+)/H(+) exchanger 3 (NHE3) activity in the intact intestine. We previously demonstrated that PLC-γ directly binds NHE3, an interaction that is necessary for [Ca(2+)]i inhibition of NHE3 activity, and that PLC-γ Src homology 2 (SH2) domains may scaffold Ca(2+) signaling proteins necessary for regulation of NHE3 activity. [Ca(2+)]i regulation of NHE3 activity is also c-Src dependent; however, the mechanism by which c-Src is involved is undetermined. We hypothesized that the SH2 domains of PLC-γ might link c-Src to NHE3-containing complexes to mediate [Ca(2+)]i inhibition of NHE3 activity. In Caco-2/BBe cells, carbachol (CCh) decreased NHE3 activity by ∼40%, an effect abolished with the c-Src inhibitor PP2. CCh treatment increased the amount of active c-Src as early as 1 min through increased Y(416) phosphorylation. Coimmunoprecipitation demonstrated that c-Src associated with PLC-γ, but not NHE3, under basal conditions, an interaction that increased rapidly after CCh treatment and occurred before the dissociation of PLC-γ and NHE3 that occurred 10 min after CCh treatment. Finally, direct binding to c-Src only occurred through the PLC-γ SH2 domains, an interaction that was prevented by blocking the PLC-γ SH2 domain. This study demonstrated that c-Src 1) activity is necessary for [Ca(2+)]i inhibition of NHE3 activity, 2) activation occurs rapidly (∼1 min) after CCh treatment, 3) directly binds PLC-γ SH2 domains and associates dynamically with PLC-γ under elevated [Ca(2+)]i conditions, and 4) does not directly bind NHE3. Under elevated [Ca(2+)]i conditions, PLC-γ scaffolds c-Src into NHE3-containing multiprotein complexes before dissociation of PLC-γ from NHE3 and subsequent endocytosis of NHE3.

  6. Investigation of anti-oxidative, cytotoxic, DNA-damaging and DNA-protective effects of plant volatiles eugenol and borneol in human-derived HepG2, Caco-2 and VH10 cell lines.

    Science.gov (United States)

    Slamenová, Darina; Horváthová, Eva; Wsólová, Ladislava; Sramková, Monika; Navarová, Jana

    2009-01-01

    Plant volatiles, which can get into the human organism in food, medicines, or cosmetic preparations, frequently manifest antibacterial, antifungal, antiviral and other effects. We studied anti-oxidative, cytotoxic, genotoxic and possible DNA-protective effects of eugenol and borneol. Anti-oxidative activities of aqueous and ethanolic solutions of these two volatile compounds of plants were determined by a spectrophotometric method by the use of the stable DPPH radical. Borneol did not show any anti-oxidative activity even at the highest concentrations soluble in water or ethanol (eugenol did manifest anti-oxidative activity, and at much lower concentrations (5-100 microM). The cytotoxicity of eugenol and borneol as well as their DNA-damaging effects and their influence on sensitivity of cells against the DNA-damaging effects of H(2)O(2) were investigated in three different cell lines, i.e. malignant HepG2 hepatoma cells, malignant Caco-2 colon cells, and nonmalignant human VH10 fibroblasts. The trypan-blue exclusion assay showed that in the three cell lines the cytotoxicity of eugenol was significantly higher than that of borneol. Single-cell gel electrophoresis revealed that borneol did not cause any DNA strand-breaks at the concentrations studied, but showed that all concentrations of eugenol (eugenol were not observed in metabolically active HepG2 hepatoma cells. Borneol and eugenol differed also with respect to their DNA-protective effects. While borneol protected HepG2 and, to a lesser extent, VH10 cells (but not Caco-2) against H(2)O(2)-induced DNA damage, eugenol either did not change the cellular sensitivity to H(2)O(2) (HepG2 cells) or it even increased the sensitivity (Caco-2 and VH10 cells). These results do not indicate any correlation between the DNA-protective and the anti-oxidative capacities of eugenol and borneol.

  7. Indicaxanthin inhibits NADPH oxidase (NOX)-1 activation and NF-κB-dependent release of inflammatory mediators and prevents the increase of epithelial permeability in IL-1β-exposed Caco-2 cells.

    Science.gov (United States)

    Tesoriere, L; Attanzio, A; Allegra, M; Gentile, C; Livrea, M A

    2014-02-01

    Dietary redox-active/antioxidant phytochemicals may help control or mitigate the inflammatory response in chronic inflammatory bowel disease (IBD). In the present study, the anti-inflammatory activity of indicaxanthin (Ind), a pigment from the edible fruit of cactus pear (Opuntia ficus-indica, L.), was shown in an IBD model consisting of a human intestinal epithelial cell line (Caco-2 cells) stimulated by IL-1β, a cytokine known to play a major role in the initiation and amplification of inflammatory activity in IBD. The exposure of Caco-2 cells to IL-1β brought about the activation of NADPH oxidase (NOX-1) and the generation of reactive oxygen species (ROS) to activate intracellular signalling leading to the activation of NF-κB, with the over-expression of inflammatory enzymes and release of pro-inflammatory mediators. The co-incubation of the cells with Ind, at a nutritionally relevant concentration (5-25 μM), and IL-1β prevented the release of the pro-inflammatory cytokines IL-6 and IL-8, PGE2 and NO, the formation of ROS and the loss of thiols in a dose-dependent manner. The co-incubation of the cells with Ind and IL-1β also prevented the IL-1β-induced increase of epithelial permeability. It was also shown that the activation of NOX-1 and NF-κB was prevented by Ind and the expression of COX-2 and inducible NO synthase was reduced. The uptake of Ind in Caco-2 cell monolayers appeared to be unaffected by the inflamed state of the cells. In conclusion, our findings suggest that the dietary pigment Ind may have the potential to modulate inflammatory processes at the intestinal level.

  8. Absorption characteristic of total alkaloids from Sophora flavescens and their monomers in Caco-2 cell model%苦参总生物碱及其单体在Caco-2细胞模型的吸收特征研究

    Institute of Scientific and Technical Information of China (English)

    张蕾; 冯志强; 陈孝健; 谢智勇

    2012-01-01

    目的 建立同时测定Caco-2细胞模型中苦参碱、氧化槐果碱和氧化苦参碱的HPLC方法,探讨苦参总生物碱在Caco-2细胞模型的吸收机制.方法 利用人源结肠腺癌细胞系Caco-2细胞单层模型,研究苦参碱、氧化槐果碱和氧化苦参碱由细胞绒毛膜面供给侧(AP)→基底外侧(BL)和BL→AP侧两个方向的转运过程;HPLC-UV法测定上述3个生物碱的量;计算转运参数和表观渗透系数(Papp).结果 苦参总碱给药后,苦参碱、氧化槐果碱和氧化苦参碱由AP→BL侧的Papp分别为(1.098±0.092)×10-5、(1.434±0.098)×10-5、(3.87±0.64)×10-6cm/s,由BL→AP侧的Papp分别为(1.104±0.098)×10-5、(1.034±0.079)×10-5、(2.75±0.33)×10-6 cm/s,与文献报道的单体化合物给药相比,氧化槐果碱和氧化苦参碱双向转运的Papp明显增大.苦参碱、氧化槐果碱和氧化苦参碱的表观渗透率值分别为1.01、0.72、0.71.结论 苦参总生物碱中苦参碱、氧化槐果碱和氧化苦参碱仍主要以被动吸收方式进入体内,但比各单体给药吸收更好.%Objective To establish an HPLC method for the simultaneous quantification of matrine (M), oxysophocarpine (OSP), and oxymatrine (OM), and to investigate the absorption mechanism of the total alkaloids from Sophora flavescens (TASF) in Caco-2 cell model. Methods By using Caco-2 cell monolayer model as an intestinal epithelial cell model, the transport processes of M, OSP, and OM from the apical (AP) side to the basolateral (BL) side or from BL side to AP side were studied. The contents of the three alkaloids were measured by HPLC-UV method. Transport parameters and apparent permeability coefficients (Papp) were then calculated. Results After TASF treatment, the Rvalues of M, OSP, and OM were (1.098 ±0.092) * 10-5, (1.434 ±0.098) x 10-5, and (3.87 ± 0.64) x 10-6cm/sfrom APside to BL side, and (1.104 ± 0.098) x 10~5, (1.034 ± 0. 079) x l(T5, and (2.75 ± 0.33) x 10~6cm/sfrom BLside to AP

  9. Acute Effects of Sugars and Artificial Sweeteners on Small Intestinal Sugar Transport: A Study Using CaCo-2 Cells As an In Vitro Model of the Human Enterocyte

    Science.gov (United States)

    2016-01-01

    Background The gastrointestinal tract is responsible for the assimilation of nutrients and plays a key role in the regulation of nutrient metabolism and energy balance. The molecular mechanisms by which intestinal sugar transport are regulated are controversial. Based on rodent studies, two models currently exist that involve activation of the sweet-taste receptor, T1R2/3: an indirect model, whereby luminal carbohydrates activate T1R2/3 expressed on enteroendocrine cells, resulting in the release of gut peptides which in turn regulate enterocyte sugar transport capacity; and a direct model, whereby T1R2/3 expressed on the enterocyte regulates enterocyte function. Aims To study the direct model of intestinal sugar transport using CaCo-2 cells, a well-established in vitro model of the human enterocyte. Methods Uptake of 10mM 14C D-Glucose and D-Fructose into confluent CaCo-2/TC7 cells was assessed following 3hr preincubation with sugars and artificial sweeteners in the presence and absence of the sweet taste receptor inhibitor, lactisole. Expression of the intestinal sugar transporters and sweet-taste receptors were also determined by RT-PCR. Results In response to short term changes in extracellular glucose and glucose/fructose concentrations (2.5mM to 75mM) carrier-mediated sugar uptake mediated by SGLT1 and/or the facilitative hexose transporters (GLUT1,2,3 and 5) was increased. Lactisole and artificial sweeteners had no effect on sugar transport regulated by glucose alone; however, lactisole increased glucose transport in cells exposed to glucose/fructose. RT-PCR revealed Tas1r3 and SGLT3 gene expression in CaCo-2/TC7 cells, but not Tas1r2. Conclusions In the short term, enterocyte sugar transport activities respond directly to extracellular glucose levels, but not fructose or artificial sweeteners. We found no evidence of a functional heterodimeric sweet taste receptor, T1R2/3 in CaCo-2 cells. However, when glucose/fructose is administered together there is an

  10. Molecular analysis and anticancer properties of two identified isolates, Fusarium solani and Emericella nidulans isolated from Wady El-Natron soil in Egypt against Caco-2 (ATCC) cell line

    Institute of Scientific and Technical Information of China (English)

    Hala F Mohamed

    2012-01-01

    Objective: To characterize, identify and investigate the anticancer properties of two new soil fungal isolates, Emericella nidulans and Fusarium solani isolated from Wady El-Natron in Egypt against colon cancer Caco-2 (ATCC) cell line. Methods: Soil sample was cultured and two strains were chosen for morphological and phenotypical characterization. Partial sequences of the 18s rRNA gene and the internal transcribed spacer region ITS of the two isolates were amplified by PCR. Phylogenetic tree construction and analysis of the resulted multiple sequences from the two fugal isolates were also carried out. In vitro anticancer activity of the two strains was done against colon Caco-2 cancer cell line. Reverse transcription – PCR was carried out to detect level of expression of p53 in Caco-2 cell line. Results: HF.1 displayed morphological and genotypic characteristics most similar to that of Fusarium solani while HF.2 was most similar to Emericella nidulans with high similarity of 99% and 97% respectively. The multiple sequence alignment of the two fungal isolates showed that, the maximum identical conserved domains in the 18s rRNA genes were identified with the nucleotide regions of 51st to 399th base pairs, 88th to 525th base pairs respectively. While those in the ITS genes were identified with the nucleotide regions of 88th to 463rdand 51st to 274th. The two isolates showed IC50 value with (6.24±5.21) and (9.84±0.36) μg/mL) concentrations respectively at 28h. Reverse transcription – PCR indicated that these cells showed high level of expression for p53 mRNA. Conclusions: The morphology and molecular analysis identified HF.1 and HF.2 to be Fusarium solani and Emericella nidulans; new isolates of anticancer producing fungi from Wady El-Natroon city in Egypt. Treatment with the two isolates caused P53 expression in Caco-2 cell line. These two isolates can be used as an anticancer agents.

  11. Sijunzi Decoction attenuates 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats and ameliorates TNBS-induced claudin-2 damage via NF-κB pathway in Caco2 cells

    OpenAIRE

    Lu, Yue; Lin, HanJie; Zhang, Jinwei; Wei, JianAn; Jing SUN; Han, Ling

    2017-01-01

    Background SijunziDecoction (SJZD) is a traditional Chinese medicine prescription used to treat the diseases of gastrointestinal tract since ancient times. The objective of this study was to investigate the protective effects of SJZD on TNBS-induced colitis in rats and TNBS-damaged Caco2 cells. Methods The rat colitis model was induced by 2, 4, 6-trinitrobenzene sulfonic acid (TNBS). SJZD (2.8 5.6, 11.2 g/kg) or salazosulfapyridine (SASP) (0.4 g/kg) was administrated orally in rats for 7 days...

  12. 肠上皮细胞模型不同培养条件的优化及适应性研究%Comparative Study on Different Culture Conditions for Caco-2 Cell Model

    Institute of Scientific and Technical Information of China (English)

    马博; 孙桂波; 杨志宏; 李明; 孙晓波

    2011-01-01

    目的:建立肠上皮(Caeo-2)细胞单层模型,探讨不同培养条件对细胞单层模型的影响,优化适宜的条件,提供稳定、重复性好的Caco-2细胞模型.方法:将细胞接种Transwell培养板上进行体外培养,并比较3种Transwell培养板及包被鼠尾胶原蛋白对细胞单层的影响,通过透射电镜和倒置显微镜观察细胞单层的形态学特征,通过测定细胞单层的电阻值、细胞两侧碱性磷酸晦含量、荧光素钠的透过量验证细胞单层的完整性、极性、通透性.结果:经过21 d培养,24孔Transwell培养的细胞单层的完整性、极性高于12孔Transweil培养的细胞单层,而透过性低于12孔Transwell培养的细胞单层;包被鼠尾胶原蛋白的Transwell于细胞单层生长前期跨上皮细胞电阻值(transepithelium electrical resistance,TEER)迅速升高,对细胞单层生长的中后期无显著性影响.结论:3种Transwell均可成功培养Caco-2细胞单层,24孔站立式Transweli适用于样品需求量少、检测灵敏度高的物质,可应用于大量样品的高通量筛选;12孔悬挂式Transwell提供检测的样品量和透过率较大,适于实验室的常规操作和常用仪器的样品需求.%Objective:To study the impacts of culture conditions on the Caco-2 cell model,to optimize the appropriate conditions for a stable, reproducible Caco-2 model. Mehtod:The environment of small intestine was in vitro simulated,and rat tail collagen was coated on three types of Transwell monolayerly and compared. Morphological features were observed by microscope, the integrity, polarity and permeability of monolayer were measured by the resistance,alkaline phosphatase, the amount of sodium fluorescein. Result: After culture of 21 days, the integrity,polarity of Caco-2 monolayer cultured on 24-well Transwell were higher than that on 12-well Transwell, but the permeability were the opposite. The TEER of the monolayer coated with rat tail collagen was increased rapidly

  13. Effect of salt stress on morphology and membrane composition of Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium bifidum, and their adhesion to human intestinal epithelial-like Caco-2 cells.

    Science.gov (United States)

    Gandhi, Akanksha; Shah, Nagendra P

    2016-04-01

    The effects of NaCl reduction (10.0, 7.5, 5.0, 2.5, and 0% NaCl) and its substitution with KCl (50% substitution at each given concentration) on morphology of Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium longum was investigated using transmission electron microscopy. Changes in membrane composition, including fatty acids and phospholipids, were investigated using gas chromatography and thin layer chromatography. Adhesion ability of these bacteria to human intestinal epithelial-like Caco-2 cells, as affected by NaCl and its substitution with KCl, was also evaluated. Bacteria appeared elongated and the intracellular content appeared contracted when subjected to salt stress, as observed by transmission electron microscopy. Fatty acid content was altered with an increase in the ratio of unsaturated to saturated fatty acid content on increasing the NaCl-induced stress. Among the phospholipids, phosphatidylglycerol was reduced, whereas phosphatidylinositol and cardioplipin were increased when the bacteria were subjected to salt stress. There was a significant reduction in adhesion ability of the bacteria to Caco-2 cells when cultured in media supplemented with NaCl; however, the adhesion ability was improved on substitution with KCl at a given total salt concentration. The findings provide insights into bacterial membrane damage caused by NaCl.

  14. In vitro Evaluation of Cytotoxic Activities of Essential Oil from Moringa oleifera Seeds on HeLa, HepG2, MCF-7, CACO-2 and L929 Cell Lines.

    Science.gov (United States)

    Elsayed, Elsayed Ahmed; Sharaf-Eldin, Mahmoud A; Wadaan, Mohammad

    2015-01-01

    Moringa oleifera Lam. (Moringaceae) is widely consumed in tropical and subtropical regions for their valuable nutritional and medicinal characteristics. Recently, extensive research has been conducted on leaf extracts of M. oleifera to evaluate their potential cytotoxic effects. However, with the exception of antimicrobial and antioxidant activities, little information is present on the cytotoxic activity of the essential oil obtained from M. oleifera seeds. Therefore, the present investigation was designed to investigate the potential cytotoxic activity of seed essential oil obtained from M. oleifera on HeLa, HepG2, MCF-7, CACO-2 and L929 cell lines. The different cell lines were subjected to increasing oil concentrations ranging from 0.15 to 1 mg/mL for 24h, and the cytotoxicity was assessed using MTT assay. All treated cell lines showed a significant reduction in cell viability in response to the increasing oil concentration. Moreover, the reduction depended on the cell line as well as the oil concentration applied. Additionally, HeLa cells were the most affected cells followed by HepG2, MCF-7, L929 and CACO-2, where the percentages of cell toxicity recorded were 76.1, 65.1, 59.5, 57.0 and 49.7%, respectively. Furthermore, the IC50 values obtained for MCF-7, HeLa and HepG2 cells were 226.1, 422.8 and 751.9 μg/mL, respectively. Conclusively, the present investigation provides preliminary results which suggest that seed essential oil from M. oleifera has potent cytotoxic activities against cancer cell lines.

  15. Regulation of cholesterol synthesis in four colonic adenocarcinoma cell lines.

    Science.gov (United States)

    Cerda, S R; Wilkinson, J; Broitman, S A

    1995-12-01

    Colon tumor cells, unlike normal human fibroblasts, exhibited an uncoupling of low density lipoprotein (LDL)-derived cholesterol from cellular growth, when endogenous cholesterol synthesis was inhibited by mevinolin, a hydroxymethylglutaryl-CoA reductase (HMG-CoAR) competitive inhibitor [Fabricant, M., and Broitman, S.A. (1990) Cancer Res. 50, 632-636]. Further evaluation of cholesterol metabolism was conducted in two undifferentiated (SW480, SW1417) and two differentiated (HT29, CACO2) colonic adenocarcinoma (adeno-CA) cell lines and an untransformed human fibroblast, AG1519A. Cells grown in monolayer culture to near subconfluency were used to assess endogenous cholesterol synthesis by 14C-acetate incorporation, in response to the following treatments in lipoprotein-deficient serum (LPDS)-supplemented minimum essential medium (MEM): LPDS alone, LDL, mevinolin, mevinolin with LDL, and 25-hydroxy-cholesterol (25-OH-CH). Complete fetal bovine serum (FBS)-supplemented MEM was used as control. All colon tumor lines exhibited similarly high endogenous cholesterol synthesis in both FBS and LPDS relative to the fibroblasts which demonstrated low basal levels in FBS and maximal synthesis in LPDS. LDL treatment did not inhibit cholesterol synthesis in colon tumor cells, but suppressed that in the fibroblast by 70%. Sterol repression of cholesterol synthesis mediated by 25-OH-CH occurred in all cells. Mevinolin caused a reduction in cholesterol synthesis in the colonic cancer cell lines, which was not further decreased by concurrent addition of LDL. In contrast, in mevinolin-treated fibroblasts, LDL further inhibited cholesterol synthesis. When the effect of cell density on cholesterol synthesis regulation was evaluated under conditions of sparse density in SW480 and SW147, results indicated that (i) basal rates of cholesterol synthesis were higher, (ii) LDL inhibited cholesterol synthesis more effectively, and (iii) mevinolin or 25-OH-CH had a more pronounced effect than in

  16. 顺式-和反式-阿霍烯在Caco-2细胞模型中的体外摄取、转运和外排特性%Characteristics of uptake, transport and efflux of Z- and E-ajoenes in Caco-2 cell monolayers in vitro

    Institute of Scientific and Technical Information of China (English)

    田莉; 杨秀伟; 王莹; 徐嵬

    2007-01-01

    研究顺式-阿霍烯(Z-Ajo)和反式-阿霍烯(E-Ajo)的肠细胞摄取、转运和外排特性.采用体外培养的人结肠Caco-2细胞单层模型评价,应用高效液相色谱法测定Z-Ajo和E-Ajo的含量.结果表明,仅能在Caco-2细胞单层的顶侧检测到Z-Ajo或E-Ajo;阿霍烯在Caco-2细胞中的代谢可被抗氧化剂维生素C、细胞色素P450药物代谢酶3A亚型抑制剂甲吡酮和ATP抑制剂叠氮化钠所抑制.Z-Ajo和E-Ajo皆不能透过Caco-2单层细胞而被迅速代谢,其代谢与CYP450药物代谢酶有关.

  17. Rifaximin improves Clostridium difficile toxin A-induced toxicity in Caco-2 cells by the PXR-dependent TLR4/MyD88/NF-κB pathway

    Directory of Open Access Journals (Sweden)

    Giuseppe eEsposito

    2016-05-01

    Full Text Available Background: Clostridium difficile infections (CDIs caused by Clostridium difficile toxin A (TcdA lead to severe ulceration, inflammation and bleeding of the colon, and are difficult to treat.Aim: The study aimed to evaluate the effect of rifaximin on TcdA-induced apoptosis in intestinal epithelial cells and investigate the role of PXR in its mechanism of action.Methods: Caco-2 cells were incubated with TcdA and treated with rifaximin (0.1−10 µM with or without ketoconazole (10 µM. The transepithelial electrical resistance (TEER and viability of the treated cells was determined. Also, the expression of zona occludens-1 (ZO-1, toll-like receptor 4 (TLR4, Bcl-2-associated X protein (Bax, transforming growth factor-β-activated kinase-1 (TAK1, myeloid differentiation factor 88 (MyD88 and nuclear factor-kappaB (NF-κB was determined.Results Rifaximin treatment (0.1, 1.0 and 10 µM caused a significant and concentration-dependent increase in the TEER of Caco-2 cells (360%, 480% and 680% vs TcdA treatment 24 hours after the treatment and improved their viability (61%, 79% and 105%. Treatment also concentration-dependently decreased the expression of Bax protein (–29%, –65% and –77% and increased the expression of ZO-1 (25%, 54% and 87% and occludin (71%, 114% and 262% versus TcdA treatment. The expression of TLR4 (–33%, –50% and –75%, MyD88 (–29%, –60% and –81% and TAK1 (–37%, –63% and –79% were also reduced with rifaximin versus TcdA treatment. Ketoconazole treatment inhibited these effects.Conclusions: Rifaximin improved TcdA-induced toxicity in Caco-2 cells by the PXR-dependent TLR4/MyD88/NF-κB pathway mechanism, and may be useful in the treatment of CDIs.

  18. Study on Transport Mechanism of Amiodarone and Effect of Atorvastatin on Transport of Amiodarone by Caco-2 Cell Monolayer Model%采用Caco-2细胞转运模型研究胺碘酮转运机制及阿托伐他汀对其转运的影响

    Institute of Scientific and Technical Information of China (English)

    孔令提; 刘冬; 宋春丽; 朱裕林

    2016-01-01

    OBJECTIVE:To probe into the intestinal absorption characteristics of amiodarone , and to evaluate the effects of combination of atorvastatin on intestinal absorption of amiodarone .METHODS: The apparent permeability coefficients(Papp) of amiodarone in both the apical-to-basolateral(AP→BL) and the basolateral-to-apical(BL→AP) direction were studied by using the Caco-2 cell monolayer model , the transport mechanism of membrane-disrupting were also evaluated .And the effects of verapamil and atorvastatin on the Papp value was determined .RESULTS:The bilateral transfer capacity of amiodarone increased with the ascent of concentration (10μmol/L, 20μmol/L, 40μmol/L) . There was no significant difference in Papp values of different concentrations ( P>0.05) , and the outside ratio were all less than 1.5.There was no obvious effects on Papp values with verapamil or atorvastatin (P>0.05), which indicated that the transport mechanism of intestinal absorption of amiodarone was passive diffusion .CONCLUSIONS:The passive diffusion is the main transport mechanism of intestinal absorption of amiodarone , and combination of atorvastatin does not affect the intestinal absorption of amiodarone .%目的:探讨胺碘酮的肠吸收特性,评价联合应用阿托伐他汀是否影响胺碘酮的肠吸收。方法:采用Caco-2细胞转运模型,研究胺碘酮在不同浓度下从绒毛面(apical,AP)侧到基底面(basolateral,BL)侧,以及从BL侧到AP侧2个方向的转运过程,计算表观渗透系数(apparent permeability coefficient ,Papp),考察其跨膜转运机制,同时考察维拉帕米、阿托伐他汀对其Papp值的影响。结果:不同浓度(10、20、40μmol/L)胺碘酮的双侧转运量均随浓度增高而相应增大;不同浓度胺碘酮双向转运Papp值的差异无统计学意义(P>0.05),且外排比率均<1.5;加入维拉帕米或阿托伐他汀对胺碘酮的 Papp值无显著影响(P>0.05);

  19. Contribution of ionic silver to genotoxic potential of nanosilver in human liver HepG2 and colon Caco2 cells evaluated by the cytokinesis-block micronucleus assay.

    Science.gov (United States)

    Sahu, Saura C; Roy, Shambhu; Zheng, Jiwen; Ihrie, John

    2016-04-01

    Extensive human exposure to food- and cosmetics-related consumer products containing nanosilver is of public concern because of the lack of information about their safety. Genotoxicity is an important endpoint for the safety and health hazard assessment of regulated products including nanomaterials. The in vitro cytokinesis-block micronucleus (CBMN) assay is a very useful test for predictive genotoxicity testing. Recently, we have reported the genotoxicity of 20 nm nanosilver in human liver HepG2 and colon Caco2 cells evaluated using the CBMN assay. The objective of our present study was three-fold: (i) to evaluate if HepG2 and Caco2 cells are valuable in vitro models for rapid genotoxicity screening of nanosilver; (ii) to test the hypothesis that the nanoparticle size and cell types are critical determinants of its genotoxicity; and (iii) to determine if ionic silver contributes to the nanosilver genotoxicity. With these objectives in mind, we evaluated the genotoxic potential of 50 nm nanosilver of the same shape, composition, surface charge, obtained from the same commercial source, under the same experimental conditions and the same genotoxic CBMN endpoint used for the previously tested 20 nm silver. The ionic silver (silver acetate) was also evaluated under the same conditions. Results of our study show that up to the concentrations tested in these cell types, the smaller (20 nm) nanosilver induces micronucleus formation in both the cell types but the larger (50 nm) nanosilver and the ionic silver provide a much weaker response compared with controls under the same conditions.

  20. Lactobacillus amylovorus Inhibits the TLR4 Inflammatory Signaling Triggered by Enterotoxigenic Escherichia coli via Modulation of the Negative Regulators and Involvement of TLR2 in Intestinal Caco-2 Cells and Pig Explants

    Science.gov (United States)

    Finamore, Alberto; Roselli, Marianna; Imbinto, Ambra; Seeboth, Julie; Oswald, Isabelle P.; Mengheri, Elena

    2014-01-01

    Inflammation derived from pathogen infection involves the activation of toll-like receptor (TLR) signaling. Despite the established immunomodulatory activities of probiotics, studies relating the ability of such bacteria to inhibit the TLR signaling pathways are limited or controversial. In a previous study we showed that Lactobacillus amylovorus DSM 16698T, a novel lactobacillus isolated from unweaned pigs, protects the intestinal cells from enterotoxigenic Escherichia coli (ETEC) K88 infection through cytokine regulation. In the present study we investigated whether the ability of L. amylovorus to counteract the inflammatory status triggered by ETEC in intestine is elicited through inhibition of the TLR4 signaling pathway. We used the human intestinal Caco-2/TC7 cells and intestinal explants isolated from 5 week-old crossbreed Pietrain/Duroc/Large-White piglets, treated with ETEC, L. amylovorus or L. amylovorus cell free supernatant, either alone or simultaneously with ETEC. Western blot analysis showed that L. amylovorus and its cell free supernatant suppress the activation of the different steps of TLR4 signaling in Caco-2/TC7 cells and pig explants, by inhibiting the ETEC induced increase in the level of TLR4 and MyD88, the phosphorylation of the IKKα, IKKβ, IκBα and NF-κB subunit p65, as well as the over-production of inflammatory cytokines IL-8 and IL-1β. The immunofluorescence analysis confirms the lack of phospho-p65 translocation into the nucleus. These anti-inflammatory effects are achieved through modulation of the negative regulators Tollip and IRAK-M. We also found that L. amylovorus blocks the up-regulation of the extracellular heat shock protein (Hsp)72 and Hsp90, that are critical for TLR4 function. By using anti-TLR2 antibody, we demonstrate that TLR2 is required for the suppression of TLR4 signaling activation. These results may contribute to develop therapeutic interventions using L. amylovorus in intestinal disorders of piglets and humans

  1. Cell line-dependent cytotoxicity of poly(isobutylcyanoacrylate) nanoparticles coated with chitosan and thiolated chitosan: Insights from cultured human epithelial HeLa, Caco2/TC7 and HT-29/MTX cells.

    Science.gov (United States)

    Pradines, Bénédicte; Lievin-Le Moal, Vanessa; Vauthier, Christine; Ponchel, Gilles; Loiseau, Philippe M; Bouchemal, Kawthar

    2015-08-01

    Nanoparticles composed of poly(isobutylcyanoacrylate) core coated with a mixture of chitosan and thiolated chitosan have already shown promising results in terms of mucoadhesion and permeation enhancement properties of pharmaceutical active drugs delivered via mucosal routes. In the present work, the cytotoxicity of these nanoparticles was first investigated using direct contact assay on undifferentiated human cervix epithelial HeLa cells. The results showed strong toxicity in HeLa cells for the two investigated concentrations 25 and 50 μg/mL. The cytotoxic effect was mainly attributed to the poly(isobutylcyanoacrylate) core since no significant differences in nanoparticle cytotoxicity were reported when nanoparticle shell composition was modified by adding chitosan or thiolated chitosan. In contrast, lower nanoparticle toxicity was reported using human fully-differentiated enterocyte-like Caco-2/TC7, and fully-differentiated mucus-secreting HT-29/MTX cells forming monolayer in culture mimicking an intestinal epithelial barrier. This study demonstrated that the toxicity of poly(isobutylcyanoacrylate) nanoparticles is highly cell line-dependent.

  2. 乳酸杆菌对肿瘤坏死因子α诱导Caco-2细胞合成IL-8的影响%Effect of alive lactobacillus rhamnosus GG (LGG) on IL-8 production in Caco-2 cells induced by TNF-α

    Institute of Scientific and Technical Information of China (English)

    章丽燕; 陈涵强; 王玮; Li Nan; Josef Neu

    2006-01-01

    目的探讨乳酸杆菌(lactobacillus rhamnosus GG,LGG)对肿瘤坏死因子(tumor necrosis factor-α,TNF-α)诱导肠道上皮Caco-2细胞合成白细胞介素8(IL-8)的影响.方法培养2周的第15~16代Caco-2细胞分别用LGG和TNF-α处理,采用酶联免疫吸附法(ELISA)测定培养上清液中IL-8含量.结果TNF-α刺激Caco-2细胞合成IL-8的表达在12 h开始增加,24 h及48 h明显增加(分别为5.33 ng/mg与7.36 ng/mg,15.69 ng/mg与32.29 ng/mg,P<0.01),合成IL-8的量与TNF-α的量呈正相关(P<0.01).与对照组比较,LGG组合成IL-8量无差别;与TNF-α组比较,预先给予LGG处理36 h,再给予TNF-α组IL-8量明显减少(26 ng/mg与37 ng/mg,P<0.05).同时在培养液中加入LGG和TNF-α 24 h,LGG未减少TNF-α诱导合成IL-8的量(P>0.05).结论在肠道上皮Caco-2细胞中预先给予LGG能减少TNF-α诱导合成促炎症细胞因子IL-8的表达.

  3. Differential transcytosis and toxicity of the hNGAL receptor ligands cadmium-metallothionein and cadmium-phytochelatin in colon-like Caco-2 cells: implications for in vivo cadmium toxicity.

    Science.gov (United States)

    Langelueddecke, Christian; Lee, Wing-Kee; Thévenod, Frank

    2014-04-21

    The environmental toxicant cadmium (Cd) enters the food chain. A substantial proportion of Cd in nutrients of plant origin is present as Cd-metallothionein (CdMT) and Cd-phytochelatin (CdPC) complexes, which may be absorbed and transcytosed intact by colonic enterocytes following bacterial fermentation and contribute to systemic Cd toxicity, e.g. in liver and kidneys. We have recently demonstrated that the receptor for human neutrophil gelatinase-associated lipocalin (hNGAL) is expressed in human colon and colon-like Caco-2 BBE cells where it mediates transcytosis of MT and PC. Here we show in colon-like Caco-2 BBE cells that hNGAL receptor (hNGAL-R) dependent toxicity is significantly higher with CdMT than with CdPC3 (2.5-50μM Cd(2+) complexed to MT or PC3 for ≤24h), using MTT assay. Fluorescence-labelled A546-MT, but not A488-PC3 (both 700nM), co-localizes with the lysosomal marker cathepsin-B, as determined by confocal microscopy. In transwell experiments with confluent monolayers, transcytosis efficiency (i.e. the ratio of basal delivery to apical decrease) of A546-MT is decreased compared to A488-PC3 (both 700nM). The tubulin polymerization disruptor nocodazole (16.7μM) almost abolished CdMT and CdPC3 toxicity, reduced apical uptake of both A546-MT and A488-PC3, but increased transcytosis efficiency of A546-MT compared to that of A488-PC3 by preventing trafficking of A546-MT to lysosomes. Hence, following hNGAL-R dependent endocytosis of CdMT/CdPC3 in colonic epithelia, a nocodazole-sensitive trafficking pathway may preferentially target CdMT, but not CdPC3, to lysosomes, causing increased colonic epithelial toxicity but reduced systemic toxicity.

  4. A Fluorescence Labeling Method for the Study on Mechanism of Adhesion of Lactobacillus plantarum ST-Ⅲ on Caco-2 Cells%荧光标记法初探植物乳杆菌ST-Ⅲ对Caco-2细胞的粘附机理

    Institute of Scientific and Technical Information of China (English)

    陈臣; 周方方; 吴正钧; 王荫榆; 陈卫; 张灏; 郭本恒

    2010-01-01

    利用荧光探针CFDA-SE标记植物乳杆菌ST-Ⅲ,测定其对Caco-2细胞粘附能力的变化,提取相关物质,探讨其粘附机理.化学和酶处理ST-Ⅲ菌悬液发现,经胃蛋白酶、胰蛋白酶、氯化锂、苯酚、盐酸胍和热处理能显著降低ST-Ⅲ的粘附性,表明表面蛋白或脂磷壁酸(Lipoteichoic acid)可能参与了ST-Ⅲ对Caco-2细胞的粘附.粘附抑制试验和可逆性结合实验证实表面蛋白而非脂磷壁酸参与了ST-Ⅲ对Caco-2细胞的粘附.结果表明ST-Ⅲ的表面蛋白粗提物可能含有粘附素类的S-层蛋白(S-layer protein),经SDS-PAGE电泳分析,此粗提物主要成分的分子量分别为72.7、34.1和24.3 kD.

  5. Proinflammatory signal transduction pathway induced by Shigella flexneri porins in caco-2 cells Via de transdução de sinal pró-inflamatória induzida por porinas de Shigella flexneri em células caco-2

    Directory of Open Access Journals (Sweden)

    Grimaldi Elena

    2009-09-01

    Full Text Available The recognition of bacterial components on the intestinal epithelial cells occurs through the toll-like receptors and is followed by the induction of an effective innate immune response. We analyzed receptor expression and signaling pathways involved in activation of human colon adenocarcinoma cells after stimulation with porins and LPS of Shigella flexneri. We also analyzed the expression and production of some cytokines, of intercellular adhesion molecule-1, of antimicrobial peptides human ²-defensins, and of the inducible form of nitric oxide synthase. Our data demonstrate that TLR2 is involved in porin recognition, whereas TLR4 with MD2, is required for LPS recognition.O reconhecimento de componentes bacterianos nas células epiteliais intestinais ocorre através de receptores toll-like e é seguido de indução de uma resposta imune inata efetiva. Neste estudo foram analisadas as vias de expressão do receptor e sinalização envolvidas na ativação de células humanas de adenocarcinoma do colon após a estimulação com porinas e LPS de Shigella flexneri. Foram também analisadas a expressão e produção de algumas citoquinas, da molécula -1 de adesão intercelular, de ²-defensinas humanas a peptídios antimicrobianos e da forma indutível de oxido nítrico sintase. Os resultados demonstraram que TLR-2 está envolvido no reconhecimento de porinas, enquanto TLR4 com MD2 é necessário para o reconhecimento de LPS.

  6. Anti-proliferative and pro-apoptotic activity of whole extract and isolated indicaxanthin from Opuntia ficus-indica associated with re-activation of the onco-suppressor p16{sup INK4a} gene in human colorectal carcinoma (Caco-2) cells

    Energy Technology Data Exchange (ETDEWEB)

    Naselli, Flores; Tesoriere, Luisa; Caradonna, Fabio; Bellavia, Daniele; Attanzio, Alessandro; Gentile, Carla; Livrea, Maria A., E-mail: maria.livrea@unipa.it

    2014-07-18

    Highlights: • Cactus pear fruit extract and indicaxanthin cause apoptosis of colon cancer cells. • Indicaxanthin does not cause ROS formation, but affects epigenoma in Caco-2 cells. • Indicaxanthin reverses methylation of oncosuppressor p16{sup INK4a} gene in Caco-2 cells. • Indicaxanthin reactivates retinoblastoma in Caco-2 cells. • Bioavailable indicaxanthin may have chemopreventive activity in colon cancer. - Abstract: Phytochemicals may exert chemo-preventive effects on cells of the gastro-intestinal tract by modulating epigenome-regulated gene expression. The effect of the aqueous extract from the edible fruit of Opuntia ficus-indica (OFI extract), and of its betalain pigment indicaxanthin (Ind), on proliferation of human colon cancer Caco-2 cells has been investigated. Whole extract and Ind caused a dose-dependent apoptosis of proliferating cells at nutritionally relevant amounts, with IC{sub 50} 400 ± 25 mg fresh pulp equivalents/mL, and 115 ± 15 μM (n = 9), respectively, without toxicity for post-confluent differentiated cells. Ind accounted for ∼80% of the effect of the whole extract. Ind did not cause oxidative stress in proliferating Caco-2 cells. Epigenomic activity of Ind was evident as de-methylation of the tumor suppressor p16{sup INK4a} gene promoter, reactivation of the silenced mRNA expression and accumulation of p16{sup INK4a}, a major controller of cell cycle. As a consequence, decrease of hyper-phosphorylated, in favor of the hypo-phosphorylated retinoblastoma was observed, with unaltered level of the cycline-dependent kinase CDK4. Cell cycle showed arrest in the G2/M-phase. Dietary cactus pear fruit and Ind may have chemo-preventive potential in intestinal cells.

  7. The effect of calcium salts, ascorbic acid and peptic pH on calcium, zinc and iron bioavailabilities from fortified human milk using an in vitro digestion/Caco-2 cell model.

    Science.gov (United States)

    Etcheverry, Paz; Wallingford, John Charles; Miller, Dennis Dean; Glahn, Raymond Philip

    2005-05-01

    The calcium, zinc, and iron bioavailabilities of human milk with commercial and noncommercial human milk fortifiers (HMFs) were evaluated under a variety of conditions: peptic digestion at pH 2 and pH 4, supplementation of ascorbic acid, and addition of three calcium salts. The noncommercial HMFs consisted of casein phosphopeptides (CPPs), alpha-lactalbumin, colostrum, and hydrolyzed whey protein concentrate (WPC). They were mixed with human milk (HM) and calcium, zinc, and iron were added. Ascorbic acid (AA) was added in certain studies. The commercial HMFs were Nestlé FM-85, Similac HMF (SHMF), and Enfamil HMF (EHMF). All HMFs were compared to S-26/SMA HMF. Results showed that the peptic pH (2 vs. 4) had no effect on mineral bioavailability. Addition of different calcium salts had no effect on calcium cell uptake and cell ferritin levels (an indicator of iron uptake), however, the addition of calcium glycerophosphate/gluconate increased zinc uptake by Caco-2 cells. Addition of AA significantly increased ferritin levels, with no effect on calcium or zinc uptake. Among the commercial HMFs, FM-85 was significantly lower in zinc uptake than S-26/SMA, and HM+EHMF was significantly higher than HM+S-26/SMA. Cell ferritin levels were significantly higher for HM+S-26/SMA than for all other commercial fortifiers. None of the commercial HMFs were different from HM+S-26/SMA in calcium uptake.

  8. Mineral and/or milk supplementation of fruit beverages helps in the prevention of H2O2-induced oxidative stress in Caco-2 cells La adición de minerales y/o leche a bebidas a base de zumo de frutas ayuda en la prevención del estrés oxidativo inducido por H2O2 en celulas Caco-2

    Directory of Open Access Journals (Sweden)

    A. Cilla

    2011-06-01

    Full Text Available Introduction: Fruit beverages are commonly supplemented with milk, vitamins and/or minerals in order to improve their healthy effects by providing some bioactive components that can act additively or synergistically against oxidative stress. Aims: To test whether iron, zinc, and milk added to fruit beverages do not affect the cytoprotective effect against oxidative damage to Caco-2 cells through GSH-related enzymes induction and cell cycle progression preservation, in comparison with non-supplemented fruit beverage. Methods: Caco-2 cells were incubated 24 h with the bioaccessible fraction (BF of eight fruit beverages with/without iron and/or zinc, and/or milk, and then challenged with H2O2 (5 mmol L-1 -2 h. Mitochondrial enzyme activities (MTT test, GSH-Rd and GSH-Px enzyme activities, cell cycle progression and caspase-3 activity were measured. Results and discussion: Fruit beverages prevented the deleterious effect of H2O2 on cell viability, with almost all samples reaching control basal levels. Only independent iron or zinc supplementation with/without milk exerted positive effects upon GSH-Rd activity. Both minerals with milk, afforded improved preservation of GSH-Px activity. All samples prevented the decrease in the G1 phase of cell cycle induced by H2O2, except iron supplemented samples with/without milk, but none of them avoided the increase in sub-G1 phase. However, this fact was not associated to caspase-3 activity, with a probable positive effect of zinc upon this parameter. Conclusion: Mineral and/or milk supplementation of fruit beverages helps in the prevention of oxidative stress in Caco-2 cells based on cell viability maintenance, GSH-related enzymes activation, cell cycle distribution preservation and inhibition of caspase-3 activation.Introducción: En la actualidad las bebidas a base de zumo de frutas llevan adicionadas leche, vitaminas y/o minerales con objeto de mejorar sus efectos beneficiosos para la salud mediante el

  9. Quorum sensing in sourdough Lactobacillus plantarum DC400: induction of plantaricin A (PlnA) under co-cultivation with other lactic acid bacteria and effect of PlnA on bacterial and Caco-2 cells.

    Science.gov (United States)

    Di Cagno, Raffaella; De Angelis, Maria; Calasso, Maria; Vincentini, Olimpia; Vernocchi, Pamela; Ndagijimana, Maurice; De Vincenzi, Massimo; Dessì, Maria Rita; Guerzoni, Maria Elisabetta; Gobbetti, Marco

    2010-06-01

    This work aimed at showing the effect of pheromone plantaricin A (PlnA) by Lactobacillus plantarum DC400 towards other sourdough lactic acid bacteria and the potential of PlnA to protect the function of the human intestinal barrier. Growth and survival of sourdough lactic acid bacteria were differently affected by co-cultivation with L. plantarum DC400. Compared to mono-cultures, Lactobacillus sanfranciscensis DPPMA174 and Pediococcus pentosaceus 2XA3 showed growth inhibition and decreased viability when co-cultured with L. plantarum DC400. L. sanfranciscensis DPPMA174 induced the highest synthesis of PlnA. Survival of strain DPPMA174 only slightly varied by comparing the addition of PlnA to the culture medium and the co-cultivation with L. plantarum DC400. Compared to mono-culture, the proteome of L. sanfranciscensis DPPMA174 grown in co-culture with L. plantarum DC400 showed the variation of expression of 58 proteins (47 over expressed and 11 repressed). Thirty-four of them were also over expressed or repressed during growth of DPPMA174 with PlnA. Fifty-one of the above 58 proteins were identified. They had a central role in stress response, amino acid, energy and nucleotide metabolisms, membrane transport, regulation of transcription, and cell redox homeostasis. PlnA markedly increased the viability of human Caco-2/TC7 cells and the transepithelial electrical resistance.

  10. Effect of anthralin on cell viability in human prostate adenocarcinoma.

    Science.gov (United States)

    Raevskaya, A A; Gorbunova, S L; Savvateeva, M V; Severin, S E; Kirpichnikov, M P

    2012-07-01

    The study revealed the key role of serine protease hepsin activity in transition of in situ prostate adenocarcinoma into the metastasizing form. Inhibition of hepsin activity suppresses the invasive growth of the tumor. Hepsin is an convenient target for pharmacological agents, so the study of its inhibitory mechanisms is a promising avenue in drug development. Assay of proteolytic activity in various tumor cell lines in vitro showed that this activity in prostate adenocarcinoma cells significantly surpasses proteolytic activity in other examined tumor cell lines. Selective cytotoxic action of anthralin, an inhibitor of hepsin activity, on human adenocarcinoma cells was demonstrated in comparison with other tumor cell lines.

  11. Interaction of GABA-mimetics with the taurine transporter (TauT, Slc6a6) in hyperosmotic treated Caco-2, LLC-PK1 and rat renal SKPT cells.

    Science.gov (United States)

    Rasmussen, Rune Nørgaard; Lagunas, Candela; Plum, Jakob; Holm, René; Nielsen, Carsten Uhd

    2016-01-20

    The aim of the present study was to investigate if basic GABA-mimetics interact with the taurine transporter (TauT, Slc6a6), and to find a suitable cell based model that is robust towards extracellular changes in osmolality during uptake studies. Taurine uptake was measured in human Caco-2 cells, porcine LLC-PK1 cells, and rat SKPT cells using radiolabelled taurine. Hyperosmotic conditions were obtained by incubation with raffinose (final osmolality of 500mOsm) for 24h prior to the uptake experiments. Expression of the taurine transporter, TauT, was investigated at the mRNA level by real-time PCR. Uptake of the GABA-mimetics gaboxadol and vigabatrin was investigated in SKPT cells, and quantified by liquid scintillation or HPLC-MS/MS analysis, respectively. The uptake rate of [(3)H]-taurine was Na(+) and Cl(-) and concentration dependent with taurine with an apparent Vmax of 6.3±1.6pmolcm(-2)min(-1) and a Km of 24.9±15.0μM. β-alanine, nipecotic acid, gaboxadol, GABA, vigabatrin, δ-ALA and guvacine inhibited the taurine uptake rate in a concentration dependent manner. The order of affinity for TauT was β-alanine>GABA>nipecotic acid>guvacine>δ-ALA>vigabatrin>gaboxadol with IC50-values of 0.04, 1.07, 2.02, 4.19, 4.94, 31.4 and 39.9mM, respectively. In conclusion, GABA mimetics inhibited taurine uptake in hyperosmotic rat renal SKPT cells. SKPT cells, which seem to be a useful model for investigating taurine transport in the short-term presence of high concentrations of osmolytes. Furthermore, analogues of β-alanine appear to have higher affinities for TauT than GABA-analogues.

  12. Mixture Genotoxicity of 2,4-Dichlorophenoxyacetic Acid, Acrylamide, and Maleic Hydrazide on Human Caco-2 Cells Assessed with Comet Assay

    DEFF Research Database (Denmark)

    Syberg, Kristian; Binderup, Mona-Lise; Cedergreen, Nina;

    2015-01-01

    ), and maleic hydrazide (MH), in an experiment with a fixed ratio design setup. The genotoxic effects were assessed with the single-cell gel electrophoresis assay (comet assay) for both single chemicals and the ternary mixture. The concentration ranges used were 0-1.4, 0-20, and 0-37.7 mM for 2,4-D, AA, and MH...

  13. Comparison of isorhamnetin absorption properties in total flavones of Hippophae rhamnoides L. with its pure form in a Caco-2 cell model mediated by multidrug resistance-associated protein.

    Science.gov (United States)

    Xie, Yan; Duan, Jingze; Fu, Qingxue; Xia, Mengxin; Zhang, Lei; Li, Guowen; Wu, Tao; Ji, Guang

    2015-06-20

    Total flavones of Hippophae rhamnoides L. (TFH) are extracted from the widely distributed thorny bush Sea buckthorn (Hippophae rhamnoides L.). Isorhamnetin (IS) is one of the representative ingredients in TFH. In this study, the absorption properties of IS in TFH and its pure form were compared through transepithelial transport and cellular uptake experiments in a Caco-2 cell model. Our results show that the absorption properties of IS in TFH and its pure form were remarkably different: (1) Both PappAB and PappBA of IS in TFH were dramatically increased compared with those of IS pure form; consequently, its Pratio was 2.3-fold higher than that of IS; (2) Both the accumulation and efflux of IS in TFH were significantly enhanced compared with the single compound. One likely reason for these differences is that the multiple components in TFH significantly down regulated the mRNA expression level of MRP2, which lead to a decrease in the protein level of MRP2, based on western blotting and RT-PCR assays. This study highlights the significant differences in the absorption properties of flavonoid components in different forms and the potential multi-component interactions in TFH.

  14. Study of the Biotransformation of Tongmai Formula by Human Intestinal Flora and Its Intestinal Permeability across the Caco-2 Cell Monolayer

    Directory of Open Access Journals (Sweden)

    Shuai Wu

    2015-10-01

    Full Text Available Tongmai formula (TMF is a well-known Chinese medicinal preparation that contains isoflavones as its major bioactive constituents. As traditional Chinese medicines (TCMs are usually used by oral administration, their fate inside the intestinal lumen, including their biotransformation by human intestinal flora (HIF and intestinal absorption deserves study. In this work TMF extract was incubated with human intestinal bacteria under anaerobic conditions and the changes in the twelve main constituents of TMF were then investigated. Their intestinal permeabilities, i.e., the transport capability across the intestinal brush border were investigated with a human colon carcinoma cell line (Caco­2 cell monolayer model to predict the absorption mechanism. Meanwhile, rapid HPLC-DAD methods were established for the assay. According to the biotransformation curves of the twelve constituents and the permeability coefficients, the intestinal absorption capacity of the typical compounds was elevated from the levels of 10−7 cm/s to 10−5 cm/s from those of the original compounds in TMF. Among them the main isoflavone glycosides puerarin (4, mirificin (6 and daidzin (7 were transformed into the same aglycone, daidzein (10. Therefore it was predicted that the aglycone compounds might be the real active ingredients in TMF. The models used can represent a novel path for the TCM studies.

  15. Optimization on condition of epigallocatechin-3-gallate (EGCG) nanoliposomes by response surface methodology and cellular uptake studies in Caco-2 cells

    Science.gov (United States)

    Luo, Xiaobo; Guan, Rongfa; Chen, Xiaoqiang; Tao, Miao; Ma, Jieqing; Zhao, Jin

    2014-06-01

    The major component in green tea polyphenols, epigallocatechin-3-gallate (EGCG), has been demonstrated to prevent carcinogenesis. To improve the effectiveness of EGCG, liposomes were used as a carrier in this study. Reverse-phase evaporation method besides response surface methodology is a simple, rapid, and beneficial approach for liposome preparation and optimization. The optimal preparation conditions were as follows: phosphatidylcholine-to-cholesterol ratio of 4.00, EGCG concentration of 4.88 mg/mL, Tween 80 concentration of 1.08 mg/mL, and rotary evaporation temperature of 34.51°C. Under these conditions, the experimental encapsulation efficiency and size of EGCG nanoliposomes were 85.79% ± 1.65% and 180 nm ± 4 nm, which were close with the predicted value. The malondialdehyde value and the release test in vitro indicated that the prepared EGCG nanoliposomes were stable and suitable for more widespread application. Furthermore, compared with free EGCG, encapsulation of EGCG enhanced its inhibitory effect on tumor cell viability at higher concentrations.

  16. [Gastric signet ring cell adenocarcinoma: A distinct entity].

    Science.gov (United States)

    Tabouret, Tessa; Dhooge, Marion; Rouquette, Alexandre; Brezault, Catherine; Beuvon, Frédéric; Chaussade, Stanislas; Coriat, Romain

    2014-04-01

    Gastric signet ring cell carcinoma (GSRC) is a distinct entity. Their incidence is increasing. The pathologist plays a central role in the identification of this entity. Diagnosis is based on an adenocarcinoma containing a majority of signet ring cells (above 50 %). The prognosis of GSRC is the same as gastric adenocarcinoma while GSRC appeared more aggressive. Signet ring cells present a low sensitivity to chemotherapy. This review aimed to discuss the histological, the prognostic and the therapeutic aspect of this entity.

  17. Molecular mechanisms of bortezomib resistant adenocarcinoma cells.

    Directory of Open Access Journals (Sweden)

    Erika Suzuki

    Full Text Available Bortezomib (Velcade™ is a reversible proteasome inhibitor that is approved for the treatment of multiple myeloma (MM. Despite its demonstrated clinical success, some patients are deprived of treatment due to primary refractoriness or development of resistance during therapy. To investigate the role of the duration of proteasome inhibition in the anti-tumor response of bortezomib, we established clonal isolates of HT-29 adenocarcinoma cells adapted to continuous exposure of bortezomib. These cells were ~30-fold resistant to bortezomib. Two novel and distinct mutations in the β5 subunit, Cys63Phe, located distal to the binding site in a helix critical for drug binding, and Arg24Cys, found in the propeptide region were found in all resistant clones. The latter mutation is a natural variant found to be elevated in frequency in patients with MM. Proteasome activity and levels of both the constitutive and immunoproteasome were increased in resistant cells, which correlated to an increase in subunit gene expression. These changes correlated with a more rapid recovery of proteasome activity following brief exposure to bortezomib. Increased recovery rate was not due to increased proteasome turnover as similar findings were seen in cells co-treated with cycloheximide. When we exposed resistant cells to the irreversible proteasome inhibitor carfilzomib we noted a slower rate of recovery of proteasome activity as compared to bortezomib in both parental and resistant cells. Importantly, carfilzomib maintained its cytotoxic potential in the bortezomib resistant cell lines. Therefore, resistance to bortezomib, can be overcome with irreversible inhibitors, suggesting prolonged proteasome inhibition induces a more potent anti-tumor response.

  18. CD36 and SR-BI are involved in cellular uptake of provitamin A carotenoids by Caco-2 and HEK cells, and some of their genetic variants are associated with plasma concentrations of these micronutrients in humans.

    Science.gov (United States)

    Borel, Patrick; Lietz, Georg; Goncalves, Aurélie; Szabo de Edelenyi, Fabien; Lecompte, Sophie; Curtis, Peter; Goumidi, Louisa; Caslake, Muriel J; Miles, Elizabeth A; Packard, Christopher; Calder, Philip C; Mathers, John C; Minihane, Anne M; Tourniaire, Franck; Kesse-Guyot, Emmanuelle; Galan, Pilar; Hercberg, Serge; Breidenassel, Christina; González Gross, Marcela; Moussa, Myriam; Meirhaeghe, Aline; Reboul, Emmanuelle

    2013-04-01

    Scavenger receptor class B type I (SR-BI) and cluster determinant 36 (CD36) have been involved in cellular uptake of some provitamin A carotenoids. However, data are incomplete (e.g., there are no data on α-carotene), and it is not known whether genetic variants in their encoding genes can affect provitamin A carotenoid status. The objectives were 1) to assess the involvement of these scavenger receptors in cellular uptake of the main provitamin A carotenoids (i.e., β-carotene, α-carotene, and β-cryptoxanthin) as well as that of preformed vitamin A (i.e., retinol) and 2) to investigate the contribution of genetic variations in genes encoding these proteins to interindividual variations in plasma concentrations of provitamin A carotenoids. The involvement of SR-BI and CD36 in carotenoids and retinol cellular uptake was investigated in Caco-2 and human embryonic kidney (HEK) cell lines. The involvement of scavenger receptor class B type I (SCARB1) and CD36 genetic variants on plasma concentrations of provitamin A carotenoids was assessed by association studies in 3 independent populations. Cell experiments suggested the involvement of both proteins in cellular uptake of provitamin A carotenoids but not in that of retinol. Association studies showed that several plasma provitamin A carotenoid concentrations were significantly different (P < 0.0083) between participants who bore different genotypes at single nucleotide polymorphisms and haplotypes in CD36 and SCARB1. In conclusion, SR-BI and CD36 are involved in cellular uptake of provitamin A carotenoids, and genetic variations in their encoding genes may modulate plasma concentrations of provitamin A carotenoids at a population level.

  19. Piperine counteracts the inhibitory effect of lipopolysaccharide and staphylococcus aureus cells on the expression of defensins by human colon carcinoma HT-29 and Caco-2 cells%胡椒碱对脂多糖和金黄色葡萄球菌抑制人结肠腺癌细胞系表达防御素的拮抗作用

    Institute of Scientific and Technical Information of China (English)

    侯小凤; 潘浩; 徐丽慧; 何贤辉; 欧阳东云

    2015-01-01

    目的:研究胡椒碱(piperine,PIP)对脂多糖(LPS)和灭活金黄色葡萄球菌(SAC)菌体抑制人结肠腺癌HT-29和 Caco-2细胞表达防御素(Defensin)的影响.方法:WST-1法检测 PIP 对 HT-29和 Caco-2细胞增殖的作用;定量 PCR(qRT-PCR)检测人α防御素 DEFA5(HD5)和 DEFA6(HD6),人β防御素 HBD-1以及胰岛再生源蛋白(Reg)Reg IIIγ等 mRNA 的表达水平.结果:PIP 处理可剂量依赖性地抑制 HT-29和 Caco-2细胞的增殖,半数抑制浓度 IC50分别为328.5μmol/L 和155.1μmol/L,表明 PIP 对细胞的毒性较小;定量 PCR 检测显示,PIP 处理对 LPS或 SAC 下调 HT-29和 Caco-2细胞 DEFA5和 DEFA6 mRNA 表达水平具有显著的拮抗作用,对 DEFB1和 Reg IIIγmRNA 的表达没有明显影响.结论:胡椒碱可能通过拮抗病原体诱导细胞表达防御素的抑制作用,进而发挥其抗肠炎作用.%Aim:To explore the effect of piperine on the down-regulation of human defensins caused by lipopolysaccharide (LPS)and Staphylococcus aureus cells (SAC)stimulation in HT-29 and Caco-2 cells.Methods:The effect of piperine on cell proliferation was measured by WST-1 assay.The mRNA levels of human defensins (DEFA5,DEFA6 and DEFB1)and regenerating islet-derived protein (Reg) were detected by quantitative real-time RT-PCR.Results:Piperine could inhibit the proliferation of HT-29 and Caco-2 cells in a dose-dependent manner with their IC50 values of 328.5 μmol /L and 155.1μmol /L,respectively,demonstrating that the cytotoxicity of piperine was relatively low.RT-PCR showed that LPS and SAC greatly suppressed the expression of human defensins DEFA5 and DEFA6 in HT-29 cells and Caco-2 cells while piperine significantly counteracted the inhibitory effect of LPS and SAC on the expression of DEFA5 and DEFA6 mRNAs,instead of DEFB1 and Reg IIIγmRNA.Conclusion:Pip-erine might exert its anti-colitis effect through antagonizing the inhibitory effect of

  20. Effects of S-layer proteins from lactobacillus against Salmonella typhimurium adhesion and invasion on Caco-2 cells%乳酸杆菌S-层蛋白对鼠伤寒沙门氏菌黏附及入侵Caco-2细胞的拮抗作用

    Institute of Scientific and Technical Information of China (English)

    李鹏成; 叶小兰; 王志胜; 庾庆华; 杨倩

    2010-01-01

    [目的]对嗜酸乳杆菌的S-层蛋白(S-layer protein)进行提纯,研究嗜酸乳酸杆菌和S-层蛋白对鼠伤寒沙门氏菌黏附和入侵的拮抗作用.[方法]应用阴离子交换柱(DE52)对嗜酸乳酸杆菌的S-层蛋白进行提纯,然后分别研究了嗜酸乳酸杆菌和S-层蛋白对鼠伤寒沙门氏菌黏附及入侵Caco-2细胞的作用.[结果]S-层蛋白能显著地抑制鼠伤寒沙门氏菌的黏附及入侵;在竞争、排斥、置换3种黏附试验中,S-层蛋白可显著降低鼠伤寒沙门氏菌的黏附,其相对黏附力分别为1.17%±5.97%、8.71%±1.36%、10.56%±0.92%,差异极显著(P<0.01),其中竞争试验效果最好;并且S-层蛋白对鼠伤寒沙门氏菌黏附抑制作用极显著高于嗜酸乳酸杆菌(P<0.01);此外,S-层蛋白也能显著抑制鼠伤寒沙门氏菌入侵.[结论]乳酸杆菌S-层蛋白对鼠伤寒沙门氏菌可产生显著的拮抗作用,这可能与S-层蛋白和鼠伤寒沙门氏菌的宿主黏附受体存在竞争作用有关;提示乳酸杆菌S-层蛋白可用于预防和治疗鼠伤寒沙门氏菌感染,并有望成为抗生素的替代品.

  1. Transport of hop aroma compounds across Caco-2 monolayers.

    Science.gov (United States)

    Heinlein, A; Metzger, M; Walles, H; Buettner, A

    2014-11-01

    ,β-unsaturated alcohols 1-octen-3-ol and 3-methyl-2-buten-1-ol in Caco-2 cells.

  2. Involvement of ERK1/2, p38 MAPK, and PI3K/Akt signaling pathways in the regulation of cell cycle progression by PTHrP in colon adenocarcinoma cells.

    Science.gov (United States)

    Calvo, Natalia; Martín, María Julia; de Boland, Ana Russo; Gentili, Claudia

    2014-08-01

    Parathyroid hormone-related peptide (PTHrP) is distributed in most fetal and adult tissues, and its expression correlates with the severity of colon carcinoma. Recently we obtained evidence that in Caco-2 cells, a cell line from human colorectal adenocarcinoma, exogenous PTHrP increases the number of live cells, via ERK1/2, p38 MAPK, and PI3-kinase and induces the expression of cyclin D1, a cell cycle regulatory protein. In this study, we further investigated the role of PTHrP in the regulation of the cell cycle progression in these intestinal cells. Flow cytometry analysis revealed that PTHrP treatment diminishes the number of cells in the G0/G1 phase and increases the number in both S and G2/M phases. The hormone increases the expression of CDK6 and diminishes the amount of negative cell cycle regulators p27Kip1, p15INK4B, and p53. However, PTHrP does not modify the expression of cyclin D3, CDK4, and p16INK4A. In addition, inhibitors of ERK1/2 (PD98059), p38 MAPK (SB203580), and PI3Kinase (LY294002) reversed PTHrP response in Caco-2 cells. Taken together, our results suggest that PTHrP positively modulates cell cycle progression and changes the expression of proteins involved in cell cycle regulation via ERK1/2, p38 MAPK, and PI3K signaling pathways in Caco-2 cells.

  3. Prediction of dissolution-absorption relationships from a continuous dissolution/Caco-2 system.

    Science.gov (United States)

    Ginski, M J; Taneja, R; Polli, J E

    1999-01-01

    The objectives were 1) to design a continuous dissolution/Caco-2 system to predict the dissolution-absorption relationships for fast and slow dissolving formulations of piroxicam, metoprolol tartrate, and ranitidine HCl, and compare the predicted relationships with observed relationships from clinical studies; 2) to estimate the effect of croscarmellose sodium on ranitidine dissolution-absorption relationships; and 3) to estimate the effect of solubilizing agents on piroxicam dissolution-absorption relationships. A continuous dissolution/Caco-2 system was constructed from a dissolution apparatus and a diffusion cell, such that drug dissolution and permeation across a Caco-2 monolayer would occur sequentially and simultaneously. The continuous system generally matched observed dissolution-absorption relationships from clinical studies. For example, the system successfully predicted the slow metoprolol and slow ranitidiine formulations to be permeation-rate-limited. The system predicted the slow piroxicam formulation to be dissolution-rate-limited, and the fast piroxicam formulation to be permeation-rate-limited, in spite of piroxicam's high permeability and low solubility. Additionally, the system indicated croscarmellose sodium enhanced ranitidine permeability and predicted solubilizing agents to not modulate permeability. These results suggest a dissolution/Caco-2 system to be an experimentally based tool that may predict dissolution-absorption relationships from oral solid dosage forms, and hence the relative contributions of dissolution and permeation to oral drug absorption kinetics.

  4. Anti-proliferative and pro-apoptotic activity of whole extract and isolated indicaxanthin from Opuntia ficus-indica associated with re-activation of the onco-suppressor p16(INK4a) gene in human colorectal carcinoma (Caco-2) cells.

    Science.gov (United States)

    Naselli, Flores; Tesoriere, Luisa; Caradonna, Fabio; Bellavia, Daniele; Attanzio, Alessandro; Gentile, Carla; Livrea, Maria A

    2014-07-18

    Phytochemicals may exert chemo-preventive effects on cells of the gastro-intestinal tract by modulating epigenome-regulated gene expression. The effect of the aqueous extract from the edible fruit of Opuntia ficus-indica (OFI extract), and of its betalain pigment indicaxanthin (Ind), on proliferation of human colon cancer Caco-2 cells has been investigated. Whole extract and Ind caused a dose-dependent apoptosis of proliferating cells at nutritionally relevant amounts, with IC50 400±25 mg fresh pulp equivalents/mL, and 115±15 μM (n=9), respectively, without toxicity for post-confluent differentiated cells. Ind accounted for ∼80% of the effect of the whole extract. Ind did not cause oxidative stress in proliferating Caco-2 cells. Epigenomic activity of Ind was evident as de-methylation of the tumor suppressor p16(INK4a) gene promoter, reactivation of the silenced mRNA expression and accumulation of p16(INK4a), a major controller of cell cycle. As a consequence, decrease of hyper-phosphorylated, in favor of the hypo-phosphorylated retinoblastoma was observed, with unaltered level of the cycline-dependent kinase CDK4. Cell cycle showed arrest in the G2/M-phase. Dietary cactus pear fruit and Ind may have chemo-preventive potential in intestinal cells.

  5. 马钱子碱体外吸收机制及其与甘草苷的转运相互作用研究%Study on the absorption mechanism of brucine in vitro and its transport interaction with liquiritin in Caco-2 cell monolayer model

    Institute of Scientific and Technical Information of China (English)

    叶敏; 王俊俊; 简静; 陈勇

    2013-01-01

    Objective: To study the absorption mechanism of brucine in vitro and its transport interaction with liquiritin in Caco-2 cell monolayer model. Methods: The cumulative transport concentration (TRcum), apparent permeability coefficient (PappB→A and /PappA→B) of brucine were determined after Caco-2 cell monolayer model were treated by brucine and combined with liquiritin. The effects of drug concentration, conveying time, P-glycoprotein inhibitor verapamil and medium pH value on the transport of brucine in Caco-2 cell monolayer model were also investigated. Results: The Papp value of brucine was larger than 10-6cm/s, and the ratio of PAPPB→A versus PAPPA→B was less than 1.5. The co-treatment of brucine combined with liquiritin increase the ratio of PAPPB→A versus PAPPA→B whereas the co-treatment of brucine combined with verapamil decreased the ratio of PAPPB→A versus PAPPA→B Moreover, the PAPPA→B IIN medium pH6.0 was less than that in medium pH7.4. Conclusion: The absorption of brucine in Caco-2 cell monolayer model was well and the passive transference was its main intestinal absorption mechanism. P-glycoprotein and liquiritin inhibited the absorption of brucine, and medium pH had important effect on the transport of brucine.%目的:研究马钱子碱在Caco-2单层细胞模型中的吸收与转运情况,以及与甘草苷合用对马钱子碱吸收与转运的影响.方法:以Caco-2单层细胞为体外研究模型,考察转运时间、药物作用浓度、P-糖蛋白抑制剂维拉帕米、介质pH值对马钱子碱单用及与甘草苷合用时,马钱子碱的累积吸收浓度(TRcum)和表观渗透系数(Papp)的影响.结果:马钱子碱单用时,其Papp值均>10-6cm/s,PappB→A/PappA→B均<1.5.马钱子碱与甘草苷合用,明显升高了PappB→A/PappA→B值;马钱子碱与维拉帕米合用,显著降低了PappB →A/P appA→B值.介质pH6.0时的PappA→B值显著<介质pH7.4时PappA→B值.结论:马钱子碱以被动转运为

  6. Omeprazole decreases magnesium transport across Caco-2 monolayers

    Institute of Scientific and Technical Information of China (English)

    Narongrit Thongon; Nateetip Krishnamra

    2011-01-01

    AIM: To elucidate the effect and underlying mechanisms of omeprazole action on Mg2+ transport across the intestinal epithelium. METHODS: Caco-2 monolayers were cultured in various dose omeprazole-containing media for 14 or 21 d before being inserted into a modified Ussing chamber apparatus to investigate the bi-directional Mg2+ transport and electrical parameters. Paracellular permeability of the monolayer was also observed by the dilution potential technique and a cation permeability study. An Arrhenius plot was performed to elucidate the activation energy of passive Mg2+ transport across the Caco-2 monolayers. RESULTS: Both apical to basolateral and basolateral to apical passive Mg2+ fluxes of omeprazole-treated epithelium were decreased in a dose- and time-dependent manner. Omeprazole also decreased the paracellular cation selectivity and changed the paracellular selective permeability profile of Caco-2 epithelium to Li+, Na+, K+, Rb+, and Cs+ from series Ⅶ to series Ⅵ of the Eisenman sequence. The Arrhenius plot revealed the higher activation energy for passive Mg2+ transport in omeprazoletreated epithelium than that of control epithelium, indicating that omeprazole affected the paracellular channel of Caco-2 epithelium in such a way that Mg2+ movement was impeded. CONCLUSION: Omeprazole decreased paracellular cation permeability and increased the activation energy for passive Mg2+ transport of Caco-2 monolayers that led to the suppression of passive Mg2+ absorption.

  7. Anthocyanin Absorption and Metabolism by Human Intestinal Caco-2 Cells—A Review

    Directory of Open Access Journals (Sweden)

    Senem Kamiloglu

    2015-09-01

    Full Text Available Anthocyanins from different plant sources have been shown to possess health beneficial effects against a number of chronic diseases. To obtain any influence in a specific tissue or organ, these bioactive compounds must be bioavailable, i.e., effectively absorbed from the gut into the circulation and transferred to the appropriate location within the body while still maintaining their bioactivity. One of the key factors affecting the bioavailability of anthocyanins is their transport through the gut epithelium. The Caco-2 cell line, a human intestinal epithelial cell model derived from a colon carcinoma, has been proven to be a good alternative to animal studies for predicting intestinal absorption of anthocyanins. Studies investigating anthocyanin absorption by Caco-2 cells report very low absorption of these compounds. However, the bioavailability of anthocyanins may be underestimated since the metabolites formed in the course of digestion could be responsible for the health benefits associated with anthocyanins. In this review, we critically discuss recent findings reported on the anthocyanin absorption and metabolism by human intestinal Caco-2 cells.

  8. Collision tumor of kidney: A case of renal cell carcinoma with metastases of prostatic adenocarcinoma

    Directory of Open Access Journals (Sweden)

    Monika Vyas

    2013-01-01

    Full Text Available Simultaneous occurrence of prostatic adenocarcinoma and renal cell carcinoma is well documented in the literature. However, metastatic prostatic adenocarcinoma in a kidney harboring a renal cell carcinoma (RCC is quite rare. Although renal cell carcinoma is the most common tumor that can harbor metastasis, metastatic prostatic adenocarcinoma in a kidney harboring a RCC is quite rare. There are four cases in the literature showing metastasis of prostatic adenocarcinoma to RCC. However, as per our knowledge, this is the first case of a collision between RCC and metastatic prostatic adenocarcinoma.

  9. Morphological and release characterization of nanoparticles formulated with poly (dl-lactide-co-glycolide) (PLGA) and lupeol: In vitro permeability and modulator effect on NF-κB in Caco-2 cell system stimulated with TNF-α.

    Science.gov (United States)

    Cháirez-Ramírez, M H; Sánchez-Burgos, J A; Gomes, C; Moreno-Jiménez, M R; González-Laredo, R F; Bernad-Bernad, M J; Medina-Torres, L; Ramírez-Mares, M V; Gallegos-Infante, J A; Rocha-Guzmán, N E

    2015-11-01

    Lupeol exhibits anti-inflammatory effects; unfortunately it shows low water solubility. An alternative to overcome this is the development of nanomaterials. Several methods for nanomaterial production are available. One of them is emulsification/solvent-evaporation. The objective of the present work was to evaluate physical properties, transport and in vitro modulator effects on NF-κB of poly (lactide-co-glycolide) (PLGA) nanoparticles loaded with lupeol. Nanonutraceuticals were prepared with 16% (w/v) of lupeol. Size distribution and morphology were measured by particle size analyzer and TEM. In vitro release of lupeol was studied by three different models: Higuchi, Siepmann & Peppas, and Power law. Transport of nanonutraceutical was studied in a Caco-2 cell model and by GC-MS. Modulator effect on NK-κB was studied by western blot analysis. Nanonutraceuticals were 10% larger than the nanoparticles without lupeol (372 vs 337 nm) and presented a broader size distribution (0.28 vs 0.22). TEM results displayed spherical structures with a broader size distribution. Entrapment efficiency of lupeol was 64.54% and it in vitro release data fitted well to the Power law and Higuchi equation (R > 0.84-0.84). Strong regulation of NF-κB of nanonutraceutical was observed. It was not observed any transport across the Caco-2 cell model at the different experimental conditions.

  10. Reversing multidrug resistance in Caco-2 by silencing MDR1, MRP1, MRP2, and BCL-2/BCL-xL using liposomal antisense oligonucleotides.

    Directory of Open Access Journals (Sweden)

    Yu-Li Lo

    Full Text Available Multidrug resistance (MDR is a major impediment to chemotherapy. In the present study, we designed antisense oligonucleotides (ASOs against MDR1, MDR-associated protein (MRP1, MRP2, and/or BCL-2/BCL-xL to reverse MDR transporters and induce apoptosis, respectively. The cationic liposomes (100 nm composed of N-[1-(2,3-dioleyloxypropyl]-n,n,n-trimethylammonium chloride and dioleoyl phosphotidylethanolamine core surrounded by a polyethylene glycol (PEG shell were prepared to carry ASOs and/or epirubicin, an antineoplastic agent. We aimed to simultaneously suppress efflux pumps, provoke apoptosis, and enhance the chemosensitivity of human colon adenocarcinoma Caco-2 cells to epirubicin. We evaluated encapsulation efficiency, particle size, cytotoxicity, intracellular accumulation, mRNA levels, cell cycle distribution, and caspase activity of these formulations. We found that PEGylated liposomal ASOs significantly reduced Caco-2 cell viability and thus intensified epirubicin-mediated apoptosis. These formulations also decreased the MDR1 promoter activity levels and enhanced the intracellular retention of epirubicin in Caco-2 cells. Epirubicin and ASOs in PEGylated liposomes remarkably decreased mRNA expression levels of human MDR1, MRP1, MRP2, and BCL-2. The combined treatments all significantly increased the mRNA expressions of p53 and BAX, and activity levels of caspase-3, -8, and -9. The formulation of epirubicin and ASOs targeting both pump resistance of MDR1, MRP1, and MRP2 and nonpump resistance of BCL-2/BCL-xL demonstrated more superior effect to all the other formulations used in this study. Our results provide a novel insight into the mechanisms by which PEGylated liposomal ASOs against both resistance types act as activators to epirubicin-induced apoptosis through suppressing MDR1, MRP1, and MRP2, as well as triggering intrinsic mitochondrial and extrinsic death receptor pathways. The complicated regulation of MDR highlights the necessity

  11. Development of On-chip Coculture System for Cytotoxicity Test Using Caco-2 and Hep G2

    Science.gov (United States)

    Kimura, Hiroshi; Nakayama, Hidenari; Yamamoto, Takatoki; Sakai, Yasuyuki; Fujii, Teruo

    We developed a chip-based coculture system for cytotoxicity test, as our continuous effort to develop a multi-functional micro culture device realized by integration of fluidic control. The culture zone in the device was divided into two compartments separated by a microporous membrane through which substances in culture medium can freely come-and-go to induce the mutual interactions between the cells cultured at each compartment. In this work, it was examined that 1) coculture and 2) cytotoxicity model through oral intake, using Caco-2 and Hep G2 cell as a model cell of small intestine and liver respectively. As a result of test 1), Hep G2 cells cocultured with Caco-2 show same albumin secretion activity as the one not cocultured with Caco-2 cells. As a result of test 2), The cytotoxicity of caffeine and paraquat on Hep G2 cells was successfully measured with and without association of a selective chemical barrier function of Caco-2 cells.

  12. Procyanidin induces apoptosis of esophageal adenocarcinoma cells via JNK activation of c-Jun. : Procyanidin induces apoptosis of esophageal adenocarcinoma cells via JNK activation of c-Jun.

    NARCIS (Netherlands)

    Connor, Carol A; Adriaens, Michiel; Pierini, Roberto; Johnson, Ian T; Belshaw, Nigel J

    2014-01-01

    Procyanidins are polymeric flavanols found in fruits and vegetables and have shown anticarcinogenic/chemopreventive properties. We previously showed that oligomeric procyanidin extracted from apples induced cell cycle arrest and apoptosis in esophageal adenocarcinoma (OA) cells. To understand the me

  13. Simultaneous large cell neuroendocrine carcinoma and adenocarcinoma of the stomach

    Institute of Scientific and Technical Information of China (English)

    Tadashi Terada; Hirotoshi Maruo

    2011-01-01

    A large cell neuroendocrine carcinoma (LCNEC) of the stomach is very rare. A 76-year-old Japanese man was admitted to our hospital because of epigastralgia and nausea. Endoscopy revealed 2 large tumors in the stomach. He did not have multiple endocrine neoplasia type Ⅰ or Zollinger-Ellison syndrome. Imaging modali-ties, including computed tomography and magnetic resonance imaging, revealed no other tumors. Gas-trectomy, cholecystectomy, and lymph node dissection were performed. The resected stomach had 2 tumors: one was an antral ulcerated type 3 tumor measuring 5 cm x 5 cm, and the other was a polypoid type 1 tumor measuring 6 cm x 6 cm x 3 cm in the fundus. Micro-scopically, the antral ulcerated tumor was a well differ-entiated adenocarcinoma with deep invasion. The fun-dus polypoid tumor was a LCNEC, being composed of malignant large cells arranged in trabecular and nested patterns. The tumor cells were large and the nuclei were vesicular. Nucleoli were frequently present, and there were many mitotic figures, apoptotic bodies, and necrotic areas. Much lymphovascular permeation was seen. Seven out of 29 dissected lymph nodes showed metastatic foci; 6 were from the LCNEC and 1 from the adenocarcinoma. Many intravascular tumor emboli of LCNEC were seen in the peritoneum around the lymph nodes. Mucins were present in the adenocarcinoma but not in the LCNEC. Immunohistochemically, the LCNEC tumor cells were positive for pancytokeratins, synaptophysin (50% positive), chromogranin A (10% positive), Ki-67 (90% labeled), and platelet-derived growth factor-α (80% positive). They were negative for KIT, p53, CD56, and neuron-specific enolase. The non-cancerous stomach showed a normal number of endocrine cells. The patient is now treated with adju-vant chemotherapy.

  14. A case with primary signet ring cell adenocarcinoma of the prostate and review of the literature

    Directory of Open Access Journals (Sweden)

    Orcun Celik

    2014-06-01

    Full Text Available Primary signet cell carcinoma of the prostate is a rare histological variant of prostate malignancies. It is commonly originated from the stomach, colon, pancreas, and less commonly in the bladder. Prognosis of the classical type is worse than the adenocarcinoma of the prostate. Primary signet cell adenocarcinoma is diagnosed by eliminating the adenocarcinomas of other organs such as gastrointestinal tract organs. In this case report, we present a case with primary signet cell adenocarcinoma of the prostate who received docetaxel chemotherapy because of short prostate specific antigen doubling time.

  15. Clear cell adenocarcinoma of a female urethra: A case report and review of the literature

    Directory of Open Access Journals (Sweden)

    Amel Trabelsi

    2009-01-01

    Full Text Available Context : Clear cell adenocarcinoma of the urethra is an extremely rare tumour. Its histogenetic derivation remains controversial. Case report : We report a new case of clear cell adenocarcinoma of the proximal urethra in a 56-year-old woman who presented with grossly hematuria. Urethral cystoscopy revealed a tumour protruding from the posterior urethral wall at the bladder neck. Treatment consisted of urethrocystectomy with pelvic lymph node dissection. Histologically, the neoplasm consisted of clear cell adenocarcinoma of the urethra. Conclusion : It appears that female urethral adenocarcinoma has more than one tissue of origin.

  16. 黑酵母β-1,3/1,6-葡聚糖对感染肠炎沙门氏菌Caco-2细胞促炎症和抗炎症细胞因子基因mRNA表达水平的影响%Sophy Beta-1, 3/1,6-Glucan Affects mRNA Expression Levels of Pro-Inflammatory and Anti-Inflammatory Cytokine Genes in Caco-2 Cells Challenged with Salmonella enteritidis

    Institute of Scientific and Technical Information of China (English)

    李晓敏; 刘红宾; 呙于明; 王忠

    2013-01-01

    本试验采用实时荧光定量PCR分析黑酵母β-1,3/1,6-葡聚糖对肠炎沙门氏菌(Salmonella enteritidis)诱导Caco-2细胞表达细胞因子基因mRNA表达水平的影响.试验选用Caco-2细胞,分成4个组:对照组(A组)、肠炎沙门氏菌感染组(B组)、β-1,3/1,6-葡聚糖组(C组)和β-1,3/1,6-葡聚糖+肠炎沙门氏菌共培养组(D组).C、D组用黑酵母β-1,3/1,6-葡聚糖(50 μg/mL)预处理24 h,然后B、D组各孔接种1×108 CFU/mL肠炎沙门氏菌处理3h.实时荧光定量PCR方法分析细胞肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-8(IL-8)、白细胞介素-10(IL-10)和转化生长因子-β(TGF-β)基因mRNA表达水平.结果表明:肠炎沙门氏菌和β-1,3/1,6-葡聚糖均极显著地上调了IL-1β、TNF-α、IL-8和IL-10基因mRNA表达水平(P<0.01),但对TGF-β基因mRNA表达水平无显著影响(P>0.05).与肠炎沙门氏菌感染组相比,添加β-1,3/1,6-葡聚糖可显著地下调肠炎沙门氏菌诱导Caco-2细胞的细胞因子IL-1β、IL-8和TNF-α基因mRNA表达水平(P<0.05),并且显著上调IL-10、TGF-β基因mRNA表达水平(P<0.05).由此可知,黑酵母β-1,3/1,6-葡聚糖能抑制肠炎沙门氏菌感染引起的肠道上皮细胞免疫炎症反应.

  17. Trefoil factor 3 as a novel biomarker to distinguish between adenocarcinoma and squamous cell carcinoma.

    Science.gov (United States)

    Wang, Xiao-Nan; Wang, Shu-Jing; Pandey, Vijay; Chen, Ping; Li, Qing; Wu, Zheng-Sheng; Wu, Qiang; Lobie, Peter E

    2015-05-01

    In carcinoma, such as of the lung, the histological subtype is important to select an appropriate therapeutic strategy for patients. However, carcinomas with poor differentiation cannot always be distinguished on the basis of morphology alone nor on clinical findings. Hence, delineation of poorly differentiated adenocarcinoma and squamous cell carcinoma, the 2 most common epithelial-origin carcinomas, is pivotal for selection of optimum therapy. Herein, we explored the potential utility of trefoil factor 3 (TFF3) as a biomarker for primary lung adenocarcinoma and extrapulmonary adenocarcinomas derived from different organs. We observed that 90.9% of lung adenocarcinomas were TFF3-positive, whereas no expression of TFF3 was observed in squamous cell carcinomas. The subtype of lung carcinoma was confirmed by four established biomarkers, cytokeratin 7 and thyroid transcription factor 1 for adenocarcinoma and P63 and cytokeratin 5/6 for squamous cell carcinoma. Furthermore, expression of TFF3 mRNA was observed by quantitative PCR in all of 11 human lung adenocarcinoma cell lines and highly correlated with markers of the adenocarcinomatous lineage. In contrast, little or no expression of TFF3 was observed in 4 lung squamous cell carcinoma cell lines. By use of forced expression, or siRNA-mediated depletion of TFF3, we determined that TFF3 appeared to maintain rather than promote glandular differentiation of lung carcinoma cells. In addition, TFF3 expression was also determined in adenocarcinomas from colorectum, stomach, cervix, esophagus, and larynx. Among all these extrapulmonary carcinomas, 93.7% of adenocarcinomas exhibited TFF3 positivity, whereas only 2.9% of squamous cell carcinomas were TFF3-positive. Totally, 92.9% of both pulmonary and extrapulmonary adenocarcinomas exhibited TFF3 positivity, whereas only 1.5% of squamous cell carcinomas were TFF3-positive. In conclusion, TFF3 is preferentially expressed in adenocarcinoma and may function as an additional

  18. Monolayers of IEC-18 cells as an in vitro model for screening the passive transcellular and paracellular transport across the intestinal barrier: Comparison of active and passive transport with the human colon carcinoma Caco-2 cell line

    NARCIS (Netherlands)

    Versantvoort, C.H.M.; Ondrewater, R.C.A.; Duizer, E.; Sandt, J.J.M. van de; Gilde, A.J.; Groten, J.P.

    2002-01-01

    Purpose: previous studies have shown that the rat small intestinal cell line IEC-18 provides a size-selective barrier for paracellularly transported hydrophilic macromolecules. In order to determine the utility of IEC-18 cells as an in vitro model to screen the passive paracellular and transcellular

  19. Clear Cell Adenocarcinoma of the Urethra: Review of the Literature

    Directory of Open Access Journals (Sweden)

    Anthony Kodzo-Grey Venyo

    2015-01-01

    Full Text Available Background. Clear cell adenocarcinoma of the urethra (CCAU is extremely rare and a number of clinicians may be unfamiliar with its diagnosis and biological behaviour. Aims. To review the literature on CCAU. Methods. Various internet databases were used. Results/Literature Review. (i CCAU occurs in adults and in women in the great majority of cases. (ii It has a particular association with urethral diverticulum, which has been present in 56% of the patients; is indistinguishable from clear cell adenocarcinoma of the female genital tract but is not associated with endometriosis; and probably does not arise by malignant transformation of nephrogenic adenoma. (iii It is usually, readily distinguished from nephrogenic adenoma because of greater cytological a-typicality and mitotic activity and does not stain for prostate-specific antigen or prostatic acid phosphatase. (iv It has been treated by anterior exenteration in women and cystoprostatectomy in men and at times by radiotherapy; chemotherapy has rarely been given. (v CCAU is aggressive with low 5-year survival rates. (vi There is no consensus opinion of treatment options that would improve the prognosis. Conclusions. Few cases of CCAU have been reported. Urologists, gynaecologists, pathologists, and oncologists should report cases of CCAU they encounter and enter them into a multicentric trial to determine the best treatment options that would improve the prognosis.

  20. Clear-Cell Adenocarcinoma of Vesical Origin: A Case Study of Metastatic Disease Treated with Chemotherapy

    Directory of Open Access Journals (Sweden)

    Carolina Pena Álvarez

    2010-01-01

    Full Text Available Vesical clear cell adenocarcinoma is an uncommon tumour. The description of nearly all published cases focuses on histological issues, providing few clinical particulars and limited followup. The treatment choice is resection. No publications have been found regarding systemic treatments for advanced disease. We present a case of metastatic clear cell adenocarcinoma of the bladder treated with chemotherapy.

  1. Prostate Mucinous Adenocarcinoma with Signet Ring Cells: a Case Report and Literature Review

    Institute of Scientific and Technical Information of China (English)

    Yi Wang; Guang Sun; Jiangang Pan; Jiwu Chang; Shumin Zhang; Tao Li; Binghuang Ren

    2006-01-01

    @@ Prostate mucinous adenocarcinoma with signet ring cells(MCSRC)is a rare morphologic variant of prostate cancer,with only 12 cases reported to date.[1] Diagnosis of this carcinoma requires that at least 25% of the tumor tissue should consist of an extracellular mucin pool.[2] In this report, we present a case of prostate prostate mucinous adenocarcinoma with signet ring cells.

  2. Resveratrol engages selective apoptotic signals in gastric adenocarcinoma cells

    Institute of Scientific and Technical Information of China (English)

    William L Riles; Jason Erickson; Sanjay Nayyar; Mary Jo Atten; Bashar M Attar; Oksana Holian

    2006-01-01

    AIM: To investigate the intracellular apoptotic signals engaged by resveratrol in three gastric adenocarcinoma cancer cell lines, two of which (AGS and SNU-1) express p53 and one (KATO-Ⅲ) with deleted p53.METHODS: Nuclear fragmentation was used to quantitate apoptotic cells; caspase activity was determined by photometric detection of cleaved substrates; formation of oxidized cytochrome C was used to measure cytochrome C activity, and Western blot analysis was used to determine protein expression.RESULTS: Gastric cancer cells, irrespective of their p53 status, responded to resveratrol with fragmentation of DNA and cleavage of nuclear lamins A and B and PARP, Resveratrol, however, has no effect on mitochondria-associated apoptotic proteins Bcl-2, Bclxl, Bax, Bid or Smac/Diablo, and did not promote subcellular redistribution of cytochrome C, indicating that resveratrol-induced apoptosis of gastric carcinoma cells does not require breakdown of mitochondrial membrane integrity. Resveratrol up-regulated p53 protein in SNU-1 and AGS cells but there was a difference in response of intracellular apoptotic signals between these cell lines.SNU-1 cells responded to resveratrol treatment with down-regulation of survivin, whereas in AGS and KATO-Ⅲ cells resveratrol stimulated caspase 3 and cytochrome C oxidase activities.CONCLUSION: These findings indicate that even within a specific cancer the intracellular apoptotic signals engaged by resveratrol are cell type dependent and suggest that such differences may be related to differentiation or lack of differentiation of these cells.

  3. The effect of low concentrations of ethanol on gastric adenocarcinoma cell lines

    Directory of Open Access Journals (Sweden)

    Wu Lingjiao

    2013-01-01

    Full Text Available Chronic alcohol consumption was identified as a significant risk factor for cancer in humans. The aim of the study was to analyze the influence of low concentrations of ethanol on gastric adenocarcinoma cell viability, apoptosis, and changes in the expression of alcohol dehydrogenase with ethanol treatment. Gastric adenocarcinoma cell lines (MGC803, MGC823 and SGC7901 were treated with different concentrations of ethanol (0.03125%, 0.0625%, 0.125%, 0.25%, 0.5%, 1%, 2%, and 4%. The MTT (3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay and flow cytometry were used to analyze the effect of ethanol treatment on cell viability and apoptosis. Western blotting was used to analyze the expression of alcohol dehydrogenase in gastric carcinoma cells. Ethanol treatment inhibited cell proliferation in gastric adenocarcinoma cell lines in a significant dose-dependent manner. Ethanol induced apoptosis of gastric adenocarcinoma cells in a dose-dependent manner. The alcohol dehydrogenase activity of gastric adenocarcinoma cells increased with the increase in the concentration of ethanol. Ethanol inhibited cell viability and the growth of gastric adenocarcinoma cell lines. Low concentrations of ethanol also induced apoptosis and increased the expression of alcohol dehydrogenase of the gastric adenocarcinoma cell lines.

  4. The effect of low concentrations of ethanol on gastric adenocarcinoma cell lines

    Directory of Open Access Journals (Sweden)

    Wu Lingjiao

    2014-01-01

    Full Text Available Chronic alcohol consumption has been identified as a significant risk factor for cancer in humans. The aim of the study was to analyze the influence of low concentrations of ethanol on gastric adenocarcinoma cell viability, apoptosis, and changes in the expression of alcohol dehydrogenase with ethanol treatment. Gastric adenocarcinoma cell lines (MGC803, MGC823 and SGC7901 were treated with different concentrations of ethanol (0.03125%, 0.0625%, 0.125%, 0.25%, 0.5%, 1%, 2%, and 4%. An MTT (3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay and flow cytometry were used to analyze the effect of ethanol treatment on cell viability and apoptosis. Western blotting was used to analyze the expression of alcohol dehydrogenase in gastric carcinoma cells. Ethanol treatment inhibited cell proliferation in gastric adenocarcinoma cell lines in a significant dose-dependent manner. Ethanol was also able to induce the apoptosis of gastric adenocarcinoma cells in a dose-dependent manner. Alcohol dehydrogenase activity of gastric adenocarcinoma cells increased with the increase in the concentration of ethanol. Ethanol inhibited cell viability and growth of gastric adenocarcinoma cell lines. Low concentrations of ethanol also induced apoptosis and increased the expression of alcohol dehydrogenase of the gastric adenocarcinoma cell lines.

  5. File list: InP.Lng.10.AllAg.Lung_adenocarcinoma_cell_lines [Chip-atlas[Archive

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  14. File list: ALL.Lng.20.AllAg.Lung_adenocarcinoma_cell_lines [Chip-atlas[Archive

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  18. File list: InP.Lng.50.AllAg.Lung_adenocarcinoma_cell_lines [Chip-atlas[Archive

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  20. File list: ALL.Lng.50.AllAg.Lung_adenocarcinoma_cell_lines [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  1. File list: InP.Lng.20.AllAg.Lung_adenocarcinoma_cell_lines [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  2. File list: DNS.Lng.10.AllAg.Lung_adenocarcinoma_cell_lines [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  3. File list: Unc.Lng.50.AllAg.Lung_adenocarcinoma_cell_lines [Chip-atlas[Archive

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  4. File list: Unc.Lng.10.AllAg.Lung_adenocarcinoma_cell_lines [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  5. File list: ALL.Lng.10.AllAg.Lung_adenocarcinoma_cell_lines [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  6. File list: NoD.Lng.05.AllAg.Lung_adenocarcinoma_cell_lines [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  7. File list: Pol.Lng.10.AllAg.Lung_adenocarcinoma_cell_lines [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  8. File list: His.Lng.05.AllAg.Lung_adenocarcinoma_cell_lines [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  9. File list: DNS.Lng.50.AllAg.Lung_adenocarcinoma_cell_lines [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  10. File list: DNS.Lng.05.AllAg.Lung_adenocarcinoma_cell_lines [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  11. File list: Unc.Lng.20.AllAg.Lung_adenocarcinoma_cell_lines [Chip-atlas[Archive

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  12. File list: InP.Lng.05.AllAg.Lung_adenocarcinoma_cell_lines [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  13. File list: Oth.Lng.50.AllAg.Lung_adenocarcinoma_cell_lines [Chip-atlas[Archive

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  14. File list: His.Lng.20.AllAg.Lung_adenocarcinoma_cell_lines [Chip-atlas[Archive

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  15. File list: His.Lng.50.AllAg.Lung_adenocarcinoma_cell_lines [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  16. Stem cells as the root of pancreatic ductal adenocarcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Balic, Anamaria; Dorado, Jorge; Alonso-Gomez, Mercedes; Heeschen, Christopher, E-mail: christopher.heeschen@cnio.es

    2012-04-01

    Emerging evidence suggests that stem cells play a crucial role not only in the generation and maintenance of different tissues, but also in the development and progression of malignancies. For the many solid cancers, it has now been shown that they harbor a distinct subpopulation of cancer cells that bear stem cell features and therefore, these cells are termed cancer stem cells (CSC) or tumor-propagating cells. CSC are exclusively tumorigenic and essential drivers for tumor progression and metastasis. Moreover, it has been shown that pancreatic ductal adenocarcinoma does not only contain one homogeneous population of CSC rather than diverse subpopulations that may have evolved during tumor progression. One of these populations is called migrating CSC and can be characterized by CXCR4 co-expression. Only these cells are capable of evading the primary tumor and traveling to distant sites such as the liver as the preferred site of metastatic spread. Clinically even more important, however, is the observation that CSC are highly resistant to chemo- and radiotherapy resulting in their relative enrichment during treatment and rapid relapse of disease. Many laboratories are now working on the further in-depth characterization of these cells, which may eventually allow for the identification of their Achilles heal and lead to novel treatment modalities for fighting this deadly disease.

  17. Suppression of c-Myc is involved in multi-walled carbon nanotubes' down-regulation of ATP-binding cassette transporters in human colon adenocarcinoma cells.

    Science.gov (United States)

    Wang, Zhaojing; Xu, Yonghong; Meng, Xiangning; Watari, Fumio; Liu, Hudan; Chen, Xiao

    2015-01-01

    Over-expression of ATP-binding cassette (ABC) transporters, a large family of integral membrane proteins that decrease cellular drug uptake and accumulation by active extrusion, is one of the major causes of cancer multi-drug resistance (MDR) that frequently leads to failure of chemotherapy. Carbon nanotubes (CNTs)-based drug delivery devices hold great promise in enhancing the efficacy of cancer chemotherapy. However, CNTs' effects on the ABC transporters remain under-investigated. In this study, we found that multiwalled carbon nanotubes (MWCNTs) reduced transport activity and expression of ABC transporters including ABCB1/Pgp and ABCC4/MRP4 in human colon adenocarcinoma Caco-2 cells. Proto-oncogene c-Myc, which directly regulates ABC gene expression, was concurrently decreased in MWCNT-treated cells and forced over-expression of c-Myc reversed MWCNTs' inhibitory effects on ABCB1 and ABCC4 expression. MWCNT-cell membrane interaction and cell membrane oxidative damage were observed. However, antioxidants such as vitamin C, β-mecaptoethanol and dimethylthiourea failed to antagonize MWCNTs' down-regulation of ABC transporters. These data suggest that MWCNTs may act on c-Myc, but not through oxidative stress, to down-regulate ABC transporter expression. Our findings thus shed light on CNTs' novel cellular effects that may be utilized to develop CNTs-based drug delivery devices to overcome ABC transporter-mediated cancer chemoresistance.

  18. Nuclear distribution of claudin-2 increases cell proliferation in human lung adenocarcinoma cells.

    Science.gov (United States)

    Ikari, Akira; Watanabe, Ryo; Sato, Tomonari; Taga, Saeko; Shimobaba, Shun; Yamaguchi, Masahiko; Yamazaki, Yasuhiro; Endo, Satoshi; Matsunaga, Toshiyuki; Sugatani, Junko

    2014-09-01

    Claudin-2 is expressed in human lung adenocarcinoma tissue and cell lines, although it is absent in normal lung tissue. However, the role of claudin-2 in cell proliferation and the regulatory mechanism of intracellular distribution remain undefined. Proliferation of human adenocarcinoma A549 cells was decreased by claudin-2 knockdown together with a decrease in the percentage of S phase cells. This knockdown decreased the expression levels of ZONAB and cell cycle regulators. Claudin-2 was distributed in the nucleus in human adenocarcinoma tissues and proliferating A549 cells. The nuclear distribution of ZONAB and percentage of S phase cells were higher in cells exogenously expressing claudin-2 with a nuclear localization signal than in cells expressing claudin-2 with a nuclear export signal. Nuclear claudin-2 formed a complex with ZO-1, ZONAB, and cyclin D1. Nuclear distribution of S208A mutant, a dephosphorylated form of claudin-2, was higher than that of wild type. We suggest that nuclear distribution of claudin-2 is up-regulated by dephosphorylation and claudin-2 serves to retain ZONAB and cyclin D1 in the nucleus, resulting in the enhancement of cell proliferation in lung adenocarcinoma cells.

  19. NFAT5 promotes proliferation and migration of lung adenocarcinoma cells in part through regulating AQP5 expression

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Kai, E-mail: gk161@163.com [Department of Respiration, Tangdu Hospital, Fourth Military Medical University, Xi' an 710038 (China); Department of Respiration, 161th Hospital, PLA, Wuhan 430015 (China); Jin, Faguang, E-mail: jinfag@fmmu.edu.cn [Department of Respiration, Tangdu Hospital, Fourth Military Medical University, Xi' an 710038 (China)

    2015-09-25

    The osmoregulated transcription factor nuclear factor of activated T-cells 5(NFAT5), has been found to play important roles in the development of many kinds of human cancers, including breast cancer, colon carcinoma, renal cell carcinoma and melanoma. The aim of the present study was to determine whether NFAT5 is involved in the proliferation and migration of lung adenocarcinoma cells. We found that NFAT5 was upregulated in lung adenocarcinoma cells and knockdown of NFAT5 decreased proliferation and migration of the cells, accompanied by a significant reduction in the expression of AQP5. AQP5 was upregulated in lung adenocarcinoma cells and knockdown of AQP5 also inhibited proliferation and migration of the cells as knockdown of NFAT5 did. Moreover, overexpression of NFAT5 promoted proliferation and migration of lung adenocarcinoma cells, accompanied by a significant increase in the expression of AQP5. These results indicate that NFAT5 plays important roles in proliferation and migration of human lung adenocarcinoma cells through regulating AQP5 expression, providing a new therapeutic option for lung adenocarcinoma therapy. - Highlights: • NFAT5 expression is higher in lung adenocarcinoma cells compared with normal cells. • NFAT5 knockdown decreases proliferation and migration of lung adenocarcinoma cells. • Knockdown of NFAT5 reduces AQP5 expression in human lung adenocarcinoma cells. • Overexpression of NFAT5 promotes proliferation and migration of lung adenocarcinoma cells. • Overexpression of NFAT5 increases AQP5 expression in human lung adenocarcinoma cells.

  20. Identification and characterization of SP cells in human lung adenocarcinoma SPC-A1 cells

    Directory of Open Access Journals (Sweden)

    Yanliang ZHU

    2008-10-01

    Full Text Available Background and objective Recently, eloquent studies from some solid tumors have provided proofs that cancers originate from cancer stem cells (CSC. The discovery of CSC has changed our view of carcinogenesis and chemotherapy. The aim of this study is to identify and characterize the CSC population that drives and maintains lung adenocarcinoma growth and metastasis. Methods Side population (SP cell analysis combined with serum-free media (SFM were applied to established human lung adenocarcinoma cell lines. Properties of SP cells were evaluated by their proliferative index, colony-forming efficiency and tumorigenic potential. Results Characteristic SP cells could be detected by FACS in lung adenocarcinoma cell lines. And the proportion of SP cells is greatly increased after serum-free culture.SP cells have a greater proliferative index, a higher colony-forming efficiency and a greater ability to form tumor in vivo .Conclusion SP cells exist in human lung adenocarcinoma cell lines and they could be further enriched by preliminary serum-free culture before FACS sorting.

  1. Clear Cell Adenocarcinoma Arising from Abdominal Wall Endometriosis

    Directory of Open Access Journals (Sweden)

    Thouraya Achach

    2008-01-01

    Full Text Available Endometriosis is a frequent benign disorder. Malignancy arising in extraovarian endometriosis is a rare event. A 49-year-old woman is presented with a large painful abdominal wall mass. She underwent a myomectomy, 20 years before, for uterus leiomyoma. Computed tomography suggested that this was a desmoid tumor and she underwent surgery. Histological examination showed a clear cell adenocarcinoma associated with endometriosis foci. Pelvic ultrasound, computed tomography, and endometrial curettage did not show any malignancy or endometriosis in the uterus and ovaries. Adjuvant chemotherapy was recommended, but the patient was lost to follow up. Six months later, she returned with a recurrence of the abdominal wall mass. She was given chemotherapy and then she was reoperated.

  2. ADENOVIRUS-MEDIATED WILD-TYPE P53 EXPRESSION SUPPRESSES GROWTH OF LUNG ADENOCARCINOMA CELLS

    Institute of Scientific and Technical Information of China (English)

    Li Jian; Xia Yongjing; Jiang Lei; Li Hongxia; Hu Yajun; Yi Lin; Hu Shixue; Xu Hongji

    1998-01-01

    Objective: To study the growth suppression of lung adenocarcinoma cell by the introduction of wild-type P53gene and explore a gene therapy approach for lung adenocarcinoma. Methods: A replication-deficient adenovirus vector encoding a wild-type P53 was constructed and transfected into the cultured human lung adenocarcinoma cell line GLC-82. The efficiency of gene transfection and expression was detected by immunochemical staining and polymerase chain reaction. The cell growth rate and cell cycle were analysed by cell-counting and flow cytometry. Results: Wild-type P53 gene could be quickly and effectively transfected into the cells by adenovirus vector. Wild-type P53 expression could inhibit GLC-82 cell proliferation and induce apoptosis.Conclusion: The results indicated that recombinant adenovirus expressing wild-type P53 might be useful vector for gene therapy of human lung adenocarcinoma.

  3. Lactobacillus plantarum CUL66 can impact cholesterol homeostasis in Caco-2 enterocytes.

    Science.gov (United States)

    Michael, D R; Moss, J W E; Calvente, D Lama; Garaiova, I; Plummer, S F; Ramji, D P

    2016-06-01

    Hypercholesterolemia drives the development of cardiovascular disease, the leading cause of mortality in western society. Supplementation with probiotics that interfere with cholesterol metabolism may provide a contribution to disease prevention. Lactobacillus plantarum CUL66 (NCIMB 30280) has been assessed in vitro for its ability to impact cholesterol absorption. L. plantarum CUL66 tested positive for bile salt hydrolase activity and the ability to assimilate cholesterol from culture media. RT-qPCR analysis showed that the bacterium significantly decreased the expression of Niemann-Pick C1-like 1 and ATP-binding cassette transporter-1 in polarised Caco-2 cells after 6 h exposure. Conversely, the expression of ATP-binding cassette sub-family G member (ABCG)-5 and ABCG-8, and 3-hydroxy-3-methylglutaryl-CoA reductase were significantly increased. Using a radiolabelled assay, we also observed significant reductions in the uptake and basolateral efflux of cholesterol by Caco-2 cells exposed to L. plantarum CUL66. This in vitro study identified L. plantarum CUL66 as a cholesterol lowering bacteria by highlighting its ability to beneficially regulate multiple in vitro events associated with intestinal cholesterol metabolism and provides evidence of efficacy for its inclusion in future in vivo studies.

  4. 25-Hydroxycholesterol promotes migration and invasion of lung adenocarcinoma cells.

    Science.gov (United States)

    Chen, Li; Zhang, Lishan; Xian, Guozhe; Lv, Yinping; Lin, Yanliang; Wang, Yibing

    2017-03-18

    25-hydroxycholesterol (25-HC) is enzymatically produced by cholesterol 25-hydorxylase in various organs and is involved in many processes, including lipid metabolism, inflammation and the immune response. However, the role of 25-HC in the migration and invasion of lung adenocarcinoma (ADC) cells remains largely unknown. In this study, we demonstrated that 0.1 μM 25-HC promoted ADC cell migration and invasion without affecting cell proliferation, especially after coculture with THP1-derived macrophages. Further investigation showed that 0.1 μM 25-HC significantly stimulated interleukin-1β (IL-1β) secretion in a coculture system and increased the expression of LXR and Snail. IL-1β also mimicked the effect of 25-HC. LXR knockdown notably blocked the 25-HC-induced Snail expression, migration and invasion in both the monoculture system and the coculture system, but it did not impact the effect of IL-1β, which suggested that IL-1β functioned in an LXR-independent manner. These results suggested that 25-HC promoted ADC cell migration and invasion in an LXR-dependent manner in the monoculture system but that in the coculture system, the 25-HC-induced IL-1β secretion enhanced the effect of 25-HC in an LXR-independent manner.

  5. The HSP90 Inhibitor Ganetespib Radiosensitizes Human Lung Adenocarcinoma Cells

    Energy Technology Data Exchange (ETDEWEB)

    Gomez-Casal, Roberto; Bhattacharya, Chitralekha [The University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213 (United States); Department of Medicine, The University of Pittsburgh, Pittsburgh, PA 15213 (United States); Epperly, Michael W. [The University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213 (United States); Department of Radiation Oncology, The University of Pittsburgh, Pittsburgh, PA 15213 (United States); Basse, Per H. [The University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213 (United States); Department of Immunology, The University of Pittsburgh, Pittsburgh, PA 15213 (United States); Wang, Hong [The University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213 (United States); Department of Biostatistics, The University of Pittsburgh, Pittsburgh, PA 15213 (United States); Wang, Xinhui [Harvard Medical School, Harvard University, 25 Shattuck Street, Boston, MA 02115 (United States); Proia, David A. [Synta Pharmaceuticals Corp., 45 Hartwell Avenue, Lexington, MA 02421 (United States); Greenberger, Joel S. [The University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213 (United States); Department of Radiation Oncology, The University of Pittsburgh, Pittsburgh, PA 15213 (United States); Socinski, Mark A.; Levina, Vera, E-mail: levinav@upmc.edu [The University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213 (United States); Department of Medicine, The University of Pittsburgh, Pittsburgh, PA 15213 (United States)

    2015-05-22

    The molecular chaperone HSP90 is involved in stabilization and function of multiple client proteins, many of which represent important oncogenic drivers in NSCLC. Utilization of HSP90 inhibitors as radiosensitizing agents is a promising approach. The antitumor activity of ganetespib, HSP90 inhibitor, was evaluated in human lung adenocarcinoma (AC) cells for its ability to potentiate the effects of IR treatment in both in vitro and in vivo. The cytotoxic effects of ganetespib included; G2/M cell cycle arrest, inhibition of DNA repair, apoptosis induction, and promotion of senescence. All of these antitumor effects were both concentration- and time-dependent. Both pretreatment and post-radiation treatment with ganetespib at low nanomolar concentrations induced radiosensitization in lung AC cells in vitro. Ganetespib may impart radiosensitization through multiple mechanisms: such as down regulation of the PI3K/Akt pathway; diminished DNA repair capacity and promotion of cellular senescence. In vivo, ganetespib reduced growth of T2821 tumor xenografts in mice and sensitized tumors to IR. Tumor irradiation led to dramatic upregulation of β-catenin expression in tumor tissues, an effect that was mitigated in T2821 xenografts when ganetespib was combined with IR treatments. These data highlight the promise of combining ganetespib with IR therapies in the treatment of AC lung tumors.

  6. [A Case of Synchronous Multiple Esophageal Cancers Composed of Squamous Cell Carcinoma and Barrett's Adenocarcinoma].

    Science.gov (United States)

    Kobayashi, Toshiyuki; Shiozaki, Atsushi; Fujiwara, Hitoshi; Konishi, Hirotaka; Arita, Tomohiro; Kosuga, Toshiyuki; Morimura, Ryo; Murayama, Yasutoshi; Komatsu, Shuhei; Kuriu, Yoshiaki; Ikoma, Hisashi; Nakanishi, Masayoshi; Ichikawa, Daisuke; Okamoto, Kazuma; Otsuji, Eigo

    2015-11-01

    A 67-year-old man was admitted to our hospital for treatment for multiple superficial esophageal cancers. Screening upper gastrointestinal endoscopy examination revealed a superficial squamous cell carcinoma (SCC) at the middle thoracic esophagus and Barrett's epithelium and a superficial adenocarcinoma at the abdominal esophagus. We performed a subtotal esophagectomy with gastric tube reconstruction via the retrosternal route. Pathological examination revealed a Barrett's adenocarcinoma at the abdominal esophagus. Esophageal cancer is thought to be a multicentric disease, and we sometimes find multiple esophageal cancers. In Japan, most cases of multiple esophageal cancers are composed of SCCs, and the occurrence of multiple esophageal cancers composed of SCC and Barrett's adenocarcinoma is rare. In contrast, the number of the patients with Barrett's esophagus is increasing, and the number of the patients with Barrett's adenocarcinoma also seems to be on the rise. Therefore, it is important be aware of the possibility of multiple esophageal cancers composed of SCC and Barrett's adenocarcinoma while making diagnoses.

  7. Effects of the Spider Venom on proliferation of Human Lung Adenocarcinoma Cell A549

    Directory of Open Access Journals (Sweden)

    Zengxiang HU

    2010-10-01

    Full Text Available Background and objective The spider venom may inspire new drugs to treat cancer. The aim of this study is to investigate the effects and mechanisms of spider venom on lung adenocarcinoma cell A549. Methods The proliferation of lung adenocarcinoma A549 cells was detected by MTT. The apoptosis rate was observed with MTT assay and flow cytometer. The activity of catalase was detected by colorimetry. The malondialdehyde (MDA content was determined by improved thiobarbituric acid fluorometric method. The expression of P38MAPK protein was analyzed with Western blot. Results Spider venom can remarkably inhibite the proliferation of lung adenocarcinoma A549 cells, increased activity of catalase and MDA content, down-regulated expression of P38MAPK compared with the control group. Conclusion The reduced proliferation of lung adenocarcinoma A549 cells by spider venom is may be associated with the increased of activity of catalase and MDA content and decreased expression of P38MAPK.

  8. Acute Effects of Sugars and Artificial Sweeteners on Small Intestinal Sugar Transport:A Study Using CaCo-2 Cells As an In Vitro Model of the Human Enterocyte

    OpenAIRE

    O'Brien, Patrick; Corpe, Christopher Peter

    2016-01-01

    BACKGROUND: The gastrointestinal tract is responsible for the assimilation of nutrients and plays a key role in the regulation of nutrient metabolism and energy balance. The molecular mechanisms by which intestinal sugar transport are regulated are controversial. Based on rodent studies, two models currently exist that involve activation of the sweet-taste receptor, T1R2/3: an indirect model, whereby luminal carbohydrates activate T1R2/3 expressed on enteroendocrine cells, resulting in the re...

  9. Acute Effects of Sugars and Artificial Sweeteners on Small Intestinal Sugar Transport: A Study Using CaCo-2 Cells As an In Vitro Model of the Human Enterocyte

    OpenAIRE

    O’Brien, Patrick; Corpe, Christopher Peter

    2016-01-01

    Background The gastrointestinal tract is responsible for the assimilation of nutrients and plays a key role in the regulation of nutrient metabolism and energy balance. The molecular mechanisms by which intestinal sugar transport are regulated are controversial. Based on rodent studies, two models currently exist that involve activation of the sweet-taste receptor, T1R2/3: an indirect model, whereby luminal carbohydrates activate T1R2/3 expressed on enteroendocrine cells, resulting in the rel...

  10. The effect of low concentrations of ethanol on gastric adenocarcinoma cell lines

    OpenAIRE

    Wu Lingjiao; Chen Shaohua; Zhang Yu; Pan Hongming

    2014-01-01

    Chronic alcohol consumption has been identified as a significant risk factor for cancer in humans. The aim of the study was to analyze the influence of low concentrations of ethanol on gastric adenocarcinoma cell viability, apoptosis, and changes in the expression of alcohol dehydrogenase with ethanol treatment. Gastric adenocarcinoma cell lines (MGC803, MGC823 and SGC7901) were treated with different concentrations of ethanol (0.03125%, 0.0625%, 0.125%, 0....

  11. Circulating Tumor Cells in the Adenocarcinoma of the Esophagus

    Directory of Open Access Journals (Sweden)

    Giulia Gallerani

    2016-08-01

    Full Text Available Circulating tumor cells (CTCs are elements of indisputable significance as they seem to be responsible for the onset of metastasis. Despite this, research into CTCs and their clinical application have been hindered by their rarity and heterogeneity at the molecular and cellular level, and also by a lack of technical standardization. Esophageal adenocarcinoma (EAC is a highly aggressive cancer that is often diagnosed at an advanced stage. Its incidence has increased so much in recent years that new diagnostic, prognostic and predictive biomarkers are urgently needed. Preliminary findings suggest that CTCs could represent an effective, non-invasive, real-time assessable biomarker in all stages of EAC. This review provides an overview of EAC and CTC characteristics and reports the main research results obtained on CTCs in this setting. The need to carry out further basic and translational research in this area to confirm the clinical usefulness of CTCs and to provide oncologists with a tool to improve therapeutic strategies for EAC patients was herein highlighted.

  12. Suitability of the in vitro Caco-2 assay to predict the oral absorption of aromatic amine hair dyes.

    Science.gov (United States)

    Obringer, Cindy; Manwaring, John; Goebel, Carsten; Hewitt, Nicola J; Rothe, Helga

    2016-04-01

    Oral absorption is a key element for safety assessments of cosmetic ingredients, including hair dye molecules. Reliable in vitro methods are needed since the European Union has banned the use of animals for the testing of cosmetic ingredients. Caco-2 cells were used to measure the intestinal permeability characteristics (Papp) of 14 aromatic amine hair dye molecules with varying chemical structures, and the data were compared with historical in vivo oral absorption rat data. The majority of the hair dyes exhibited Papp values that indicated good in vivo absorption. The moderate to high oral absorption findings, i.e. ≥60%, were confirmed in in vivo rat studies. Moreover, the compound with a very low Papp value (APB: 3-((9,10-dihydro-9,10-dioxo-4-(methylamino)-1-anthracenyl)amino)-N,N-dimethyl-N-propyl-1-propanaminium) was poorly absorbed in vivo as well (5% of the dose). This data set suggests that the Caco-2 cell model is a reliable in vitro tool for the determination of the intestinal absorption of aromatic amines with diverse chemical structures. When used in combination with other in vitro assays for metabolism and skin penetration, the Caco-2 model can contribute to the prediction and mechanistic interpretation of the absorption, metabolism and elimination properties of cosmetic ingredients without the use of animals.

  13. Primary mucinous adenocarcinoma of the bladder with signet-ring cells: case report

    Directory of Open Access Journals (Sweden)

    Marcelo Lorenzi Marques

    Full Text Available CONTEXT: Primary adenocarcinomas of the bladder are uncommon and usually occur by contiguity with or hematogenic dissemination of other adenocarcinomas such as colorectal, prostate and gynecological tract carcinomas. Mucinous and signet-ring cell histological patterns are even rarer and it is often difficult to morphologically distinguish them from metastatic colorectal adenocarcinoma. CASE REPORT: We present and discuss a rare case of primary mucinous adenocarcinoma of the bladder with signet-ring cells in a 57-year-old male patient. Other primary sites for the tumor had been excluded and, in the absence of digestive tract tumor and for confirmation that it was a primary bladder tumor, an immunohistochemistry study was performed.

  14. Ipsilateral synchronous renal pelvic transitional cell carcinoma, squamous cell carcinoma and adenocarcinoma

    Institute of Scientific and Technical Information of China (English)

    韩平; 魏强; 石明; 杨宇如

    2004-01-01

    @@ Reports of multiple synchronous primary renal neoplasms in the literature are rare. Although primary renal tumors of 2 distinctively dissimilar origins have been sporadically described,1-6 to our knowledge there have been no reported cases of triple primary renal neoplasms in the same kidney. Here we report a very rare case of ipsilateral synchronous renal pelvic transitional cell carcinoma, squamous cell carcinoma and adenocarcinoma with marked hydronephrosis and multiple stones in the same kidney.

  15. Fluorouracil selectively enriches stem-like cells in the lung adenocarcinoma cell line SPC.

    Science.gov (United States)

    Shi, Mu-mu; Xiong, Yan-lei; Jia, Xin-shan; Li, Xin; Zhang, Li; Li, Xiao-lei; Wang, En-Hua

    2013-06-01

    Most adult stem cells are in the G0 or quiescent phase of the cell cycle and account for only a small percentage of the cells in the tissue. Thus, isolation of stem cells from tissues for further study represents a major challenge. This study sought to enrich cancer stem cells and explore cancer stem-like cell clones using 5-fluorouracil (5-FU) in the lung adenocarcinoma cell line, SPC. Proliferation inhibition was analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, according to which half maximal inhibitory concentration values were calculated. Expression levels of stem cell markers after treatment with 5-FU were examined using immunofluorescence and Western blotting. Additionally, side population (SP) cells were sorted using FACS. Properties of SP cells were evaluated by using Transwell, colony-forming assays, and tumor formation experiments. 5-FU greatly inhibits proliferation, especially of cells in S phase. SP cells possess greater invasive potential, higher clone-forming potential, and greater tumor-forming ability than non-SP