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Sample records for intestinal caco-2 cells

  1. Effect of neuronal PC12 cells on the functional properties of intestinal epithelial Caco-2 cells.

    Science.gov (United States)

    Satsu, Hideo; Yokoyama, Tatsuya; Ogawa, Nobumasa; Fujiwara-Hatano, Yoko; Shimizu, Makoto

    2003-06-01

    The effect of neuronal cells on the functional properties of intestinal epithelial cells was examined by using an in vitro coculture system. Two cell lines, Caco-2 and PC12, were respectively used as intestinal epithelial and enteric neuronal cell models. Coculture of differentiated Caco-2 cells with PC12 caused a significant decrease in the transepithelial electrical resistance (TER) value of the Caco-2 monolayer. The permeability to lucifer yellow (LY) was also significantly increased, suggesting that the tight junction (TJ) of the Caco-2 monolayers was modulated by coculturing with PC12. To identify the TJ-modulating factor presumably secreted from PC12, the effects of the major neurotransmitters on the TER value and LY transport were examined, but no influence was apparent. The TJ-modulating effect of PC12 was prevented by exposing PC12 to cycloheximide, suggesting that new protein synthesis in PC12 was necessary for this regulation.

  2. Determination of Autophagy in the Caco-2 Spontaneously Differentiating Model of Intestinal Epithelial Cells.

    Science.gov (United States)

    Tunçer, Sinem; Banerjee, Sreeparna

    2017-08-27

    The Caco-2 colorectal cancer cell line is widely used as a model for intestinal differentiation and barrier function. These cells, upon reaching confluency, spontaneously differentiate into enterocyte-like cells, synthesize intestinal enzymes, and form domes. Caco-2 cells also undergo autophagy in the course of differentiation. The criteria to establish the induction of autophagy in cells are already well established. Here, we describe the protocol for the spontaneous differentiation of Caco-2 cells and the detection of autophagy using Western blot, flow cytometry, and immunofluorescence.

  3. Bioavailability of genistein, daidzein, and their glycosides in intestinal epithelial Caco-2 cells

    NARCIS (Netherlands)

    Steensma, A.; Noteborn, H.P.J.M.; Jagt, van der R.C.M.; Polman, Th.H.G.; Mengelers, M.J.B.; Kuiper, H.A.

    1999-01-01

    In this study information was obtained on bioavailability of genistein, daidzein and their glycosides in human intestinal epithelial Caco-2 cells grown on semi-permeable filters. The integrity of Caco-2 monolayers was confirmed by transepithelial electrical resistance measurements and by

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

    Science.gov (United States)

    Milovic, Vladan; Turchanowa, Lyudmila; Stein, Jürgen; Caspary, Wolfgang F.

    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 S adenosylmethionine decarboxylase. However, EGF did not have any significant influence on putrescine flux across the Caco- 2 cell monolayers. Excretion of putrescine from Caco-2 cells 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. PMID:11819759

  5. 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....

  6. Substrate specificity and some properties of phenol sulfotransferase from human intestinal Caco-2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Baranczyk-Kuzma, A.; Garren, J.A.; Hidalgo, I.J.; Borchardt, R.T. (Univ. of Kansas, Lawrence (United States))

    1991-01-01

    The phase 2 metabolic reactions, sulfation and glucuronidation, were studied in a human colon carcinoma cell line (Caco-2), which has been developed as a model of intestinal enterocytes. Phenol sulfotransferase was isolated from Caco-2 cells cultured for 7, 14 and 21 days. The enzyme catalyzed the sulfation of both p-nitrophenol and catecholamines as well as most catecholamine metabolites. The affinity (K{sub m}) of PST for dopamine was much higher than for p-nitrophenol, and the specific activity of PST with both substrates increased with the age of the cells. The thermal stability of Caco-2 PST increased with cell age and was not dependent on the acceptor substrate used. The thermolabile PST from 7-day old cells was more sensitive to NEM than was the thermostable enzyme from 21-day old cells. No UDP-glucuronyltransferase activity was detected in 7-, 14- and 21-day old Caco-2 cells with any of the methods used.

  7. Vitamin A metabolism in the human intestinal Caco-2 cell line

    Energy Technology Data Exchange (ETDEWEB)

    Quick, T.C.; Ong, D.E. (Vanderbilt Univ. School of Medicine, Nashville, TN (USA))

    1990-12-01

    The human intestinal Caco-2 cell line, described as enterocyte-like in a number of studies, was examined for its ability to carry out the metabolism of vitamin A normally required in the absorptive process. Caco-2 cells contained cellular retinol-binding protein II, a protein which is abundant in human villus-associated enterocytes and may play an important role in the absorption of vitamin A. Microsomal preparations from Caco-2 cells contained retinal reductase, acyl-CoA-retinol acyltransferase (ARAT), and lecithin-retinol acyltransferase (LRAT) activites, which have previously been proposed to be involved in the metabolism of dietary vitamin A in the enterocyte. When intact Caco-2 cells were provided with {beta}-carotene, retinyl acetate, or retinyl acetate, or retinol, synthesis of retinyl palmitoleate, oleate, palmitate, and small amounts of stearate resulted. However, exogenous retinyl palmitate or stearate was not used by Caco-2 cells as a source of retinol for ester synthesis. While there was a disproportionate synthesis of monoenoic fatty acid esters of retinol in Caco-2 cells compared to the retinyl esters typically found in human chylomicrons or the esters normally synthesized in rat intestine, the pattern was consistent with the substantial amount of unsaturated fatty acids, particularly 18:1 and 16:1, found in the sn-1 position of Caco-2 microsomal phosphatidylcholine, the fatty acyl donor for LRAT. Both ARAT and LRAT have been proposed to be responsible for retinyl ester synthesis in the enterocyte. These data suggest the LRAT may be the physiologically important enzyme for the esterification of retinol in Caco-2 cells.

  8. Inhibitory Influence of Panax notoginseng Saponins on Aspirin Hydrolysis in Human Intestinal Caco-2 Cells.

    Science.gov (United States)

    Sun, Zongxi; Wu, Yali; Yang, Bing; Zhu, Baochen; Hu, Shaonan; Lu, Yang; Zhao, Bo; Du, Shouying

    2018-02-18

    Herb-drug interactions are important safety concerns in clinical practice. The interactions occur firstly in the intestinal absorption for orally administered drugs. Aspirin and Panax notoginseng saponins (PNS)-based drugs are often combined in China to prevent larger-artery atherosclerosis. Here, we aimed to characterize the aspirin transport across Caco-2 cell monolayers, a model of the intestinal absorption, and further to evaluate the influence of PNS on aspirin hydrolysis and the relating mechanisms. Transcellular transport of aspirin and the influence of PNS were explored using Caco-2 cell monolayers. The protein expression of human carboxylesterase 1 (hCE1) and hCE2 in Caco-2 cells after PNS treatment was analyzed by ELISA, and the mRNA level were determined by qRT-PCR. In the study, Caco-2 cells showed high level of hydrolase activity, and most aspirin was hydrolyzed inside the cells during the transport process. Interestingly, PNS were demonstrated to inhibit the esterase activities responsible for aspirin hydrolysis in Caco-2 cells. PNS could also decrease the protein expression of hCE1 and hCE2, whereas exhibited minor effect on the mRNA expression. These results indicated that oral administration of PNS-based drugs might inhibit the hydrolysis of aspirin during intestinal absorption thus promoting its bioavailability.

  9. Transport of Antihypertensive Peptide RVPSL, Ovotransferrin 328-332, in Human Intestinal Caco-2 Cell Monolayers.

    Science.gov (United States)

    Ding, Long; Wang, Liying; Zhang, Yan; Liu, Jingbo

    2015-09-23

    The objective of this study was to investigate the transepithelial transport of RVPSL (Arg-Val-Pro-Ser-Leu), an egg-white-derived peptide with angiotensin I-converting enzyme (ACE) inhibitory and antihypertensive activity, in human intestinal Caco-2 cell monolayers. Results revealed that RVPSL could be passively transported across Caco-2 cell monolayers. However, during the process of transport, 36.31% ± 1.22% of the initial RVPSL added to the apical side was degraded, but this degradation decreased to 23.49% ± 0.68% when the Caco-2 cell monolayers were preincubated with diprotin A (P transport from the apical side to the basolateral side was investigated, the apparent permeability coefficient (Papp) was (6.97 ± 1.11) × 10(-6) cm/s. The transport route of RVPSL appears to be the paracellular pathway via tight junctions, as only cytochalasin D, a disruptor of tight junctions (TJs), significantly increased the transport rate (P transport across Caco-2 cell monolayers was studied by mutation of RVPSL. It was found that N-terminal Pro residues were more beneficial for transport of pentapeptides across Caco-2 cell monolayers than Arg and Val. Furthermore, RVPSL could be more easily transported as smaller peptides, especially in the form of dipeptides and tripeptides.

  10. Astilbin from Engelhardtia chrysolepis enhances intestinal barrier functions in Caco-2 cell monolayers.

    Science.gov (United States)

    Nakahara, Tatsuo; Nishitani, Yosuke; Nishiumi, Shin; Yoshida, Masaru; Azuma, Takeshi

    2017-06-05

    Astilbin, which is one of polyphenolic compounds isolated from the leaves of Engelhardtia chrysolepis H ANCE (Chinese name, huang-qui), is available as the effective component in food and cosmetics because of its anti-oxidant and anti-inflammatory effects. The tight junction (TJ) proteins, which protect the body from foreign substances, are related to adhesion between a cell and a cell. Previously, the enhancement of TJ's functions induced by aglycones of flavonoids has been demonstrated, but the effects of the glycosides such as astilbin have not been observed yet. In this study, we investigated the effects of astilbin on the TJ's functions, and human colon carcinoma Caco-2 cell monolayers were used to evaluate the effects of astilbin on transepithelial electrical resistance (TER) value and the mRNA and proteins expressions of TJ-related molecules. Astilbin increased the TER value, mRNA expression levels of claudin-1 and ZO-2, and protein expression levels of occludin and ZO-2 in Caco-2 cells. Astilbin also increased the TER value in Caco-2 cells co-stimulated with TNF-α plus IFN-γ, and moreover upregulated the protein expression of TJ-related molecules in Caco-2 cells co-treated with TNF-α plus IFN-γ. These results suggest that astilbin can enhance the expressions of TJ-related molecules, leading to upregulation of the barrier functions in the intestinal cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Polarity of fatty acid uptake and metabolism in a human intestinal cell line (CACO-2)

    Energy Technology Data Exchange (ETDEWEB)

    Trotter, P.J.; Storch, J. (Harvard School of Public Health, Boston, MA (United States))

    1990-02-26

    Free fatty acids (ffa) can enter the intestinal cell via the apical (AP) or basolateral (BL) membrane. The authors are using the Caco-2 intestinal cell line to examine the polarity of ffa uptake and metabolism in the enterocyte. Cells are grown on permeable polycarbonate Transwell filters in order to obtain access to both AP and BL compartments. Differentiated Caco-2 cells form tight polarized monolayers which express small intestine-specific enzymes and are impermeable to the fluid phase marker Lucifer Yellow. Submicellar concentrations of {sup 3}H-palmitic acid (2uM) were added to AP or BL sides of Caco-2 monolayers at 37{degrees}C and cells were incubated for various times between 2 and 120 minutes. Total AP and BL uptake is similar; however, when relative membrane surface areas are accounted for, AP uptake is about 2-fold higher. The metabolism of AP and BL ffa is not significantly different: triacylglycerol and phosphatidylcholine account for most of the metabolites (32{plus minus}4 and 24{plus minus}2% respectively at 5 minutes). Little ffa oxidation is observed. Preincubation with albumin-bound 2-monoolein (100uM) and palmitate (50uM) increases the level of TG metabolites. The results suggest that in this cell line the uptake of AP ffa may be greater than BL ffa, but that AP (dietary) ffa and BL (plasma) ffa are metabolized similarly.

  12. Polarity of fatty acid uptake and metabolism in a human intestinal cell line (CACO-2)

    International Nuclear Information System (INIS)

    Trotter, P.J.; Storch, J.

    1990-01-01

    Free fatty acids (ffa) can enter the intestinal cell via the apical (AP) or basolateral (BL) membrane. The authors are using the Caco-2 intestinal cell line to examine the polarity of ffa uptake and metabolism in the enterocyte. Cells are grown on permeable polycarbonate Transwell filters in order to obtain access to both AP and BL compartments. Differentiated Caco-2 cells form tight polarized monolayers which express small intestine-specific enzymes and are impermeable to the fluid phase marker Lucifer Yellow. Submicellar concentrations of 3 H-palmitic acid (2uM) were added to AP or BL sides of Caco-2 monolayers at 37 degrees C and cells were incubated for various times between 2 and 120 minutes. Total AP and BL uptake is similar; however, when relative membrane surface areas are accounted for, AP uptake is about 2-fold higher. The metabolism of AP and BL ffa is not significantly different: triacylglycerol and phosphatidylcholine account for most of the metabolites (32±4 and 24±2% respectively at 5 minutes). Little ffa oxidation is observed. Preincubation with albumin-bound 2-monoolein (100uM) and palmitate (50uM) increases the level of TG metabolites. The results suggest that in this cell line the uptake of AP ffa may be greater than BL ffa, but that AP (dietary) ffa and BL (plasma) ffa are metabolized similarly

  13. 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.

  14. Dynamics of absorption, metabolism, and excretion of 5-aminolevulinic acid in human intestinal Caco-2 cells.

    Science.gov (United States)

    Saito, Kei; Fujiwara, Tohru; Ota, Urara; Hatta, Shunsuke; Ichikawa, Satoshi; Kobayashi, Masahiro; Okitsu, Yoko; Fukuhara, Noriko; Onishi, Yasushi; Ishizuka, Masahiro; Tanaka, Tohru; Harigae, Hideo

    2017-09-01

    5-Aminolevulinic acid (ALA) is a precursor for the biosynthesis of porphyrins and heme. Although the oral administration of ALA has been widely applied in clinical settings, the dynamics of its absorption, metabolism, and excretion within enterocytes remain unknown. In this study, after enterocytic differentiation, Caco-2 cells were incubated with 200 µM ALA and/or 100 µM sodium ferrous citrate (SFC) for up to 72 h. Both ALA and the combination of ALA and SFC promoted the synthesis of heme, without affecting the expression of genes involved in intestinal iron transport, such as DMT1 and FPN . The enhanced heme synthesis in Caco-2 cells was more pronounced under the effect of the combination of ALA and SFC than under the effect of ALA alone, as reflected by the induced expression of heme oxygenase 1 (HO-1), as well as a reduced protein level of the transcriptional corepressor Bach1. Chromatin immunoprecipitation analysis confirmed Bach1 chromatin occupancy at the enhancer regions of HO-1, which were significantly decreased by the addition of ALA and SFC. Finally, Transwell culture of Caco-2 cells suggested that the administered ALA to the intestinal lumen was partially transported into vasolateral space. These findings enhance our understanding of the absorption and metabolism of ALA in enterocytes, which could aid in the development of a treatment strategy for various conditions such as anemia.

  15. Dynamics of absorption, metabolism, and excretion of 5-aminolevulinic acid in human intestinal Caco-2 cells

    Directory of Open Access Journals (Sweden)

    Kei Saito

    2017-09-01

    Full Text Available 5-Aminolevulinic acid (ALA is a precursor for the biosynthesis of porphyrins and heme. Although the oral administration of ALA has been widely applied in clinical settings, the dynamics of its absorption, metabolism, and excretion within enterocytes remain unknown. In this study, after enterocytic differentiation, Caco-2 cells were incubated with 200 µM ALA and/or 100 µM sodium ferrous citrate (SFC for up to 72 h. Both ALA and the combination of ALA and SFC promoted the synthesis of heme, without affecting the expression of genes involved in intestinal iron transport, such as DMT1 and FPN. The enhanced heme synthesis in Caco-2 cells was more pronounced under the effect of the combination of ALA and SFC than under the effect of ALA alone, as reflected by the induced expression of heme oxygenase 1 (HO-1, as well as a reduced protein level of the transcriptional corepressor Bach1. Chromatin immunoprecipitation analysis confirmed Bach1 chromatin occupancy at the enhancer regions of HO-1, which were significantly decreased by the addition of ALA and SFC. Finally, Transwell culture of Caco-2 cells suggested that the administered ALA to the intestinal lumen was partially transported into vasolateral space. These findings enhance our understanding of the absorption and metabolism of ALA in enterocytes, which could aid in the development of a treatment strategy for various conditions such as anemia.

  16. 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.

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

    Science.gov (United States)

    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. PMID:26550018

  18. Pseudomonas fluorescens alters epithelial permeability and translocates across Caco-2/TC7 intestinal cells

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    Madi Amar

    2010-11-01

    Full Text Available Abstract Background Pseudomonas fluorescens has long been considered as a psychrotrophic microorganism. Recently, we have shown that clinical strains of P. fluorescens (biovar 1 are able to adapt at a growth temperature of 37°C or above and induce a specific inflammatory response. Interestingly, a highly specific antigen of P. fluorescens, I2, is detected in the serum of patients with Crohn's disease but the possible role of this bacterium in the disease has not yet been explored. In the present study, we examined the ability of a psychrotrophic and a clinical strain of P. fluorescens to modulate the permeability of a Caco-2/TC7 intestinal epithelial model, reorganize the actin cytoskeleton, invade the target cells and translocate across the epithelium. The behaviour of these two strains was compared to that of the well known opportunistic pathogen P. aeruginosa PAO1. Results Both strains of P. fluorescens were found to decrease the transepithelial resistance (TER of Caco-2/TC7 differentiated monolayers. This was associated with an increase in paracellular permeability and F-actin microfilaments rearrangements. Moreover, the invasion and translocation tests demonstrated that the two strains used in this study can invade and translocate across the differentiated Caco-2/TC7 cell monolayers. Conclusions The present work shows for the first time, that P. fluorescens is able to alter the intestinal epithelial barrier function by disorganizing the F-actin microfilament network. Moreover, we reveal that independently of their origins, the two P. fluorescens strains can translocate across differentiated Caco-2/TC7 cell monolayers by using the transcellular pathway. These findings could, at least in part, explain the presence of the P. fluorescens specific I2 antigen in the serum of patients with Crohn's disease.

  19. Toxicity of graphene oxide on intestinal bacteria and Caco-2 cells.

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

    2015-05-01

    In recent years, novel nanomaterials have received much attention due to their great potential for applications in agriculture, food safety, and food packaging. Among them, graphene and graphene oxide (GO) are emerging as promising nanomaterials that may have a profound impact on food packaging. However, there are some concerns from consumers and the scientific community about the potential toxicity and biocompatibility of nanomaterials. In this study, we investigated the antibacterial properties of GO against human intestinal bacteria. The cytotoxicity of GO was also studied in vitro using the Caco-2 cell line derived from a colon carcinoma. Electron microscopy was used to investigate the morphology of GO and the interaction between GO flakes and Caco-2 cells. GO at different concentrations (10 to 500 μg/ml) exhibited no toxicity against the selected bacteria and a mild cytotoxic action on Caco-2 cells after 24 h of exposure. The results show that weak adsorption of medium nutrients may contribute to GO's low toxicity. This study suggests that GO is biocompatible and has a potential to be used in agriculture and food science, indicating that more studies are needed to exploit its potential applications.

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

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    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.

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

    Science.gov (United States)

    Li, Na; Lin, Pei; Li, Yunfei; Mao, Xiaojian; Bao, Getuzhaori; Zhao, Ronghua

    2014-01-01

    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 (P app) 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 P app. PMID:24693324

  2. Yogurt inhibits intestinal barrier dysfunction in Caco-2 cells by increasing tight junctions.

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    Putt, Kelley K; Pei, Ruisong; White, Heather M; Bolling, Bradley W

    2017-01-25

    Chronic inflammation disrupts intestinal barrier function and may contribute to the pathology of obesity and other diseases. The goal of this study was to determine the mechanism by which yogurt improves intestinal barrier function. Caco-2 cells were differentiated on Transwell inserts and used as a model of intestinal barrier permeability. Transepithelial electrical resistance (TEER) and flux of 4 kDa fluorescein isothiocyanate-dextran (FD) and lucifer yellow (LY) were used as indicators of monolayer integrity and paracellular permeability. Immunofluorescence microscopy and real time quantitative polymerase chain were used to assess the localization and expression of tight junction proteins known to regulate intestinal permeability. Differentiated cells were treated with a vehicle control (C), inflammatory stimulus (I) (interleukin-1β, tumor necrosis factor-α, interferon-γ, and lipopolysaccharide), or I and 0.03 g mL -1 yogurt (IY). After 48 h, I reduced Caco-2 TEER by 46%, while IY reduced TEER by only 27% (P effect on barrier function was reduced at latter stages of digestion.

  3. Metabolism and transfer of the mycotoxin zearalenone in human intestinal Caco-2 cells.

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    Videmann, Bernadette; Mazallon, Michelle; Tep, Jonathan; Lecoeur, Sylvaine

    2008-10-01

    The mycotoxin zearalenone (ZEA) is found worldwide as contaminant in cereals and grains. It is implicated in reproductive disorders and hyperestrogenic syndromes in animals and humans exposed by food. We investigated metabolism and transfer of ZEA using the human Caco-2 cell line as a model of intestinal epithelial barrier. Cells exposed to 10-200 microM ZEA showed efficacious metabolism of the toxin. alpha-zearalenol and beta-zearalenol were the measured preponderant metabolites (respectively 40.7+/-3.1% and 31.9+/-4.9% of total metabolites, after a 3h exposure to 10 microM ZEA), whereas ZEA-glucuronide and alpha-zearalenol glucuronide were less produced (respectively 8.2+/-0.9% and 19.1+/-1.3% of total metabolites, after a 3h exposure to 10 microM ZEA). Cell production of reduced metabolites was strongly inhibited by alpha-and beta-hydroxysteroid dehydrogenase inhibitors, and Caco-2 cells exhibited alpha-hydroxysteroid dehydrogenase type II and beta-hydroxysteroid dehydrogenase type I mRNA. After cell apical exposure to ZEA, alpha-zearalenol was preponderantly found at the basal side, whereas beta-zearalenol and both glucuronides were preferentially excreted at the apical side. As alpha-zearalenol shows the strongest estrogenic activity, the preferential production and basal transfer of this metabolite suggests that intestinal cells may contribute to the manifestation of zearalenone adverse effects.

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

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    Determining the effect of selenium (Se) chemical form on uptake and transport in human intestinal cells is critical to assess Se bioavailability. In the present study, we measured the uptake and transport of various Se compounds in the human intestinal Caco-2 cell model. We found that two sources...

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

    Science.gov (United States)

    Determining the effect of selenium (Se) chemical form on uptake and transport in human intestinal cells is critical to assess Se bioavailability. In the present study, we measured the uptake and transport of various Se compounds in the human intestinal Caco-2 cell model. We found that two sources of...

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

    Directory of Open Access Journals (Sweden)

    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

  7. Enhanced intestinal absorption of curcumin in Caco-2 cell monolayer using mucoadhesive nanostructured lipid carriers.

    Science.gov (United States)

    Chanburee, Sanipon; Tiyaboonchai, Waree

    2018-02-01

    This study aimed to compare the intestinal permeation of curcumin-loaded polymer coated nanostructured lipid carriers (NLCs) and uncoated NLCs using the Caco-2 cell model. The uncoated NLCs were prepared using a warm microemulsion technique, while polymer-coated NLCs were prepared with the same method but were followed by coating particle surface with polyethylene glycol (PEG) 400 or polyvinyl alcohol (PVA). After lyophilization, all formulations possessed a mean size of  0.05) compared to those freshly prepared formulations. Considered overall, polymer coated NLCs are an important strategy to improve the oral bioavailability of curcumin. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 734-741, 2018. © 2017 Wiley Periodicals, Inc.

  8. Modulation of intestinal and liver fatty acid-binding proteins in Caco-2 cells by lipids, hormones and cytokines.

    NARCIS (Netherlands)

    Dube, N.; Delvin, E.; Yotov, W.; Garofalo, C.; Bendayan, M.; Veerkamp, J.H.; Levy, E.

    2001-01-01

    Intestinal and liver fatty acid binding proteins (I- and L-FABP) are thought to play a role in enterocyte fatty acid (FA) trafficking. Their modulation by cell differentiation and various potential effectors was investigated in the human Caco-2 cell line. With the acquisition of enterocytic

  9. Dephosphorylation of myo-inositol phosphates in the in vitro intestinal Caco-2 cell model.

    Science.gov (United States)

    Briviba, Karlis; Schollenberger, Margit; Rodehutscord, Markus; Greiner, Ralf

    2018-02-01

    Plant and microbial phytases present in raw materials can cause a dephosphorylation of phytate (myo-inositol hexakisphosphate) (InsP 6 )) during food processing resulting in a broad range of different myo-inositol phosphates such as pentakisphosphate (InsP 5 ) and tetrakisphosphate (InsP 4 ) in foods. Here, we investigated whether the human intestinal epithelium is able to dephosphorylate myo-inositol phosphates (InsP 6 , InsP 5 -, InsP 4 -, InsP 3 -isomers) using an in vitro model with differentiated human Caco-2 cells cultured on semipermeable inserts. Incubation of InsP 6 and an InsP 5 -isomer with cells for 3 h showed no dephosphorylation of both InsPs. Treatment of cells with a mixture of different InsP 4 -isomers, however, caused a formation of about 3.5% of an InsP 3 -isomer (Ins(1,5,6)P 3 ) and treatment with a mixture of different InsP 3 -isomers caused about 20% formation of InsP 2 -isomers, respectively. Thus, human intestinal cells can contribute to the dephosphorylation of myo-inositol phosphates of partly dephosphorylated forms such as InsP 3 and InsP 4 .

  10. 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. Copyright © 2015 John Wiley & Sons, Ltd.

  11. Carrier-mediated ¿-aminobutyric acid transport across the basolateral membrane of human intestinal Caco-2 cell monolayers

    DEFF Research Database (Denmark)

    Nielsen, Carsten Uhd; Carstensen, Mette; Brodin, Birger

    2012-01-01

    and the anticancer prodrug d-aminolevulinic acid across the apical membrane of small intestinal enterocytes. Little is however known about the basolateral transport of these substances. We investigated basolateral transport of GABA in mature Caco-2 cell monolayers using isotope studies. Here we report that, at least...

  12. 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. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

  13. Uptake of phenolic compounds from plant foods in human intestinal Caco-2 cells.

    Science.gov (United States)

    Hithamani, Gavirangappa; Kizhakayil, Dhanya; Srinivasan, Krishnapura

    2017-12-01

    In continuation of our studies on the bioaccessibility of phenolic compounds from food grains as influenced by domestic processing, we examined the uptake of phenolics from native/sprouted finger millet (Eleucine coracana) and green gram (Vigna radiata) and native/heat-processed onion (Allium cepa) in human Caco-2 cells. Absorption of pure phenolic compounds, as well as the uptake of phenolic compounds from finger millet, green gram, and onion, was investigated in Caco-2 monolayer model. Transport of individual phenolic compounds from apical compartment to the basolateral compartment across Caco-2 monolayer was also investigated. Sprouting enhanced the uptake of syringic acid from both these grains. Open-pan boiling reduced the uptake of quercetin from the onion. Among pure phenolic compounds, syringic acid was maximally absorbed, while the flavonoid isovitexin was least absorbed. Apparent permeability coefficient P (app) of phenolic compounds from their standard solutions was 2.02 x 10 -6 cm/s to 8.94 x 10 -6 cm/s. Sprouting of grains enhanced the uptake of syringic acid by the Caco-2 cells. Open-pan boiling drastically reduced the uptake of quercetin from the onion. The permeability of phenolic acids across Caco-2 monolayer was higher than those of flavonoids.

  14. Influence of Physicochemical Properties and PEG Modification of Magnetic Liposomes on Their Interaction with Intestinal Epithelial Caco-2 Cells.

    Science.gov (United States)

    Kono, Yusuke; Jinzai, Hitomi; Kotera, Yota; Fujita, Takuya

    2017-12-01

    The present study aimed to investigate the effect of particle size (100, 500 nm), surface charge (cationic, neutral and anionic) and polyethylene glycol (PEG) modification of magnetic liposomes on their interaction with the human intestinal epithelial cell line, Caco-2. The cellular associated amount of all the magnetic liposomes was significantly increased by the presence of a magnetic field. The highest association and internalization into Caco-2 cells was observed with magnetic cationic liposomes. Moreover, small magnetic liposomes were more efficiently associated and taken up into the cells, than large ones. In contrast, PEG modification significantly attenuated the enhancing effect of the magnetic field on the cellular association of magnetic liposomes. We also found that magnetic cationic liposomes had the highest retention properties to Caco-2 cells. Moreover, the retention of large magnetic liposomes to the cells was much longer than that of small ones. In addition, magnetic cationic and neutral liposomes had relatively high stability in Caco-2 cells, whereas magnetic anionic liposomes rapidly degraded. These results indicate that the physicochemical properties and PEG modification of magnetic liposomes greatly influences their intestinal epithelial transport.

  15. Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D.

    Science.gov (United States)

    Anabazhagan, Arivarasu N; Chatterjee, Ishita; Priyamvada, Shubha; Kumar, Anoop; Tyagi, Sangeeta; Saksena, Seema; Alrefai, Waddah A; Dudeja, Pradeep K; Gill, Ravinder K

    2017-03-16

    The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing a barrier to foreign particles. Impaired epithelial transport (ion, nutrient, or drugs) has been associated with many diseases and can have consequences that extend beyond the normal physiological functions of the transporters, such as by influencing epithelial integrity and the gut microbiome. Understanding the function and regulation of transport proteins is critical for the development of improved therapeutic interventions. The biggest challenge in the study of epithelial transport is developing a suitable model system that recapitulates important features of the native intestinal epithelial cells. Several in vitro cell culture models, such as Caco-2, T-84, and HT-29-Cl.19A cells are typically used in epithelial transport research. These cell lines represent a reductionist approach to modeling the epithelium and have been used in many mechanistic studies, including their examination of epithelial-microbial interactions. However, cell monolayers do not accurately reflect cell-cell interactions and the in vivo microenvironment. Cells grown in 3D have shown to be promising models for drug permeability studies. We show that Caco-2 cells in 3D can be used to study epithelial transporters. It is also important that studies in Caco-2 cells are complemented with other models to rule out cell specific effects and to take into account the complexity of the native intestine. Several methods have been previously used to assess the functionality of transporters, such as everted sac and uptake in isolated epithelial cells or in isolated plasma membrane vesicles. Taking into consideration the challenges in the field with respect to models and the measurement of transport function, we demonstrate here a protocol to grow Caco-2 cells in 3D and describe the use of an Ussing chamber as an effective approach to measure serotonin

  16. Evaluation of the Intestinal Absorption Mechanism of Casearin X in Caco-2 Cells with Modified Carboxylesterase Activity.

    Science.gov (United States)

    Moreira da Silva, Rodrigo; Verjee, Sheela; de Gaitani, Cristiane Masetto; Moraes de Oliveira, Anderson Rodrigo; Pires Bueno, Paula Carolina; Cavalheiro, Alberto José; Peporine Lopes, Norberto; Butterweck, Veronika

    2016-04-22

    The clerodane diterpene casearin X (1), isolated from the leaves of Casearia sylvestris, is a potential new drug candidate due to its potent in vitro cytotoxic activity. In this work, the intestinal absorption mechanism of 1 was evaluated using Caco-2 cells with and without active carboxylesterases (CES). An LC-MS method was developed and validated for the quantification of 1. The estimation of permeability coefficients was possible only under CES-inhibited conditions in which 1 is able to cross the Caco-2 cell monolayer. The mechanism is probably by active transport, with no significant efflux, but with a high retention of the compound inside the cells. The enzymatic hydrolysis assay demonstrates the susceptibility of 1 to first-pass metabolism as substrate for specific CES expressed in human intestine.

  17. Suppressive effect of nobiletin and epicatechin gallate on fructose uptake in human intestinal epithelial Caco-2 cells.

    Science.gov (United States)

    Satsu, Hideo; Awara, Sohei; Unno, Tomonori; Shimizu, Makoto

    2018-04-01

    Inhibition of excessive fructose intake in the small intestine could alleviate fructose-induced diseases such as hypertension and non-alcoholic fatty liver disease. We examined the effect of phytochemicals on fructose uptake using human intestinal epithelial-like Caco-2 cells which express the fructose transporter, GLUT5. Among 35 phytochemicals tested, five, including nobiletin and epicatechin gallate (ECg), markedly inhibited fructose uptake. Nobiletin and ECg also inhibited the uptake of glucose but not of L-leucine or Gly-Sar, suggesting an inhibitory effect specific to monosaccharide transporters. Kinetic analysis further suggested that this reduction in fructose uptake was associated with a decrease in the apparent number of cell-surface GLUT5 molecules, and not with a change in the affinity of GLUT5 for fructose. Lastly, nobiletin and ECg suppressed the permeation of fructose across Caco-2 cell monolayers. These findings suggest that nobiletin and ECg are good candidates for preventing diseases caused by excessive fructose intake.

  18. Mechanisms of Iron Uptake from Ferric Phosphate Nanoparticles in Human Intestinal Caco-2 Cells

    Directory of Open Access Journals (Sweden)

    Antonio Perfecto

    2017-04-01

    Full Text Available Food fortification programs to reduce iron deficiency anemia require bioavailable forms of iron that do not cause adverse organoleptic effects. Rodent studies show that nano-sized ferric phosphate (NP-FePO4 is as bioavailable as ferrous sulfate, but there is controversy over the mechanism of absorption. We undertook in vitro studies to examine this using a Caco-2 cell model and simulated gastrointestinal (GI digestion. Supernatant iron concentrations increased inversely with pH, and iron uptake into Caco-2 cells was 2–3 fold higher when NP-FePO4 was digested at pH 1 compared to pH 2. The size and distribution of NP-FePO4 particles during GI digestion was examined using transmission electron microscopy. The d50 of the particle distribution was 413 nm. Using disc centrifugal sedimentation, a high degree of agglomeration in NP-FePO4 following simulated GI digestion was observed, with only 20% of the particles ≤1000 nm. In Caco-2 cells, divalent metal transporter-1 (DMT1 and endocytosis inhibitors demonstrated that NP-FePO4 was mainly absorbed via DMT1. Small particles may be absorbed by clathrin-mediated endocytosis and micropinocytosis. These findings should be considered when assessing the potential of iron nanoparticles for food fortification.

  19. Chemical composition and effect on intestinal Caco-2 cell viability and lipid profile of fixed oil from Cynomorium coccineum L.

    Science.gov (United States)

    Rosa, Antonella; Rescigno, Antonio; Piras, Alessandra; Atzeri, Angela; Scano, Paola; Porcedda, Silvia; Zucca, Paolo; Assunta Dessì, M

    2012-10-01

    Cynomorium coccineum L. is a non-photosynthetic plant, spread over Mediterranean countries, amply used in traditional medicine. We investigated the composition and effect on intestinal Caco-2 cell viability and lipid profile of fixed oil obtained from dried stems of the plant. Oil isolation has been performed by supercritical fractioned extraction with CO2. 13C NMR spectroscopy has been used to study the molecular composition of oil lipids; fatty acid composition was identified using GC and HPLC techniques. The fixed oil was composed mainly by triacylglycerols and derivates. The main fatty acids were 18:1 n-9 (38%), 18:2 n-6 (20%), 16:0 (15%), and 18:3 n-3 (10.8%). The oil showed a significant in vitro inhibitory effect on the growth of colon cancer undifferentiated Caco-2 cells. Moreover, cell viability, lipid composition, and lipid peroxidation were measured in intestinal epithelial cells (differentiated Caco-2 cells) after 24 h incubation with fixed oil. The oil did not show a toxic effect on colon epithelial cell viability but induced a significant change in fatty acid composition, with a significant accumulation of the essential fatty acids 18:3 n-3 and 18:2 n-6. The results showed remarkable biological activity of Maltese mushroom oil, and qualify it as a potential resource for food/pharmaceutical applications. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells

    Energy Technology Data Exchange (ETDEWEB)

    Artursson, P.; Karlsson, J. (Uppsala Univ., (Sweden))

    1991-03-29

    Monolayers of a well differentiated human intestinal epithelial cell line, Caco-2, were used as a model to study passive drug absorption across the intestinal epithelium. Absorption rate constants (expressed as apparent permeability coefficients) were determined for 20 drugs and peptides with different structural properties. The permeability coefficients ranged from approximately 5 x 10{sup {minus} 8} to 5 x 10{sup {minus} 5} cm/s. A good correlation was obtained between data on oral absorption in humans and the results in the Caco-2 model. Drugs that are completely absorbed in humans had permeability coefficients greater than 1 x 10{sup {minus} 6} cm/s. Drugs that are absorbed to greater than 1% but less than 100% had permeability coefficients of 0.1-1.0 x 10{sup {minus} 6} cm/s while drugs and peptides that are absorbed to less than 1% had permeability coefficients of less than or equal to 1 x 10{sup {minus} 7} cm/s. The results indicate that Caco-2 monolayers can be used as a model for studies on intestinal drug absorption.

  1. Tick-Borne Encephalitis Virus Replication, Intracellular Trafficking, and Pathogenicity in Human Intestinal Caco-2 Cell Monolayers

    Science.gov (United States)

    Möller, Lars; Schulzke, Joerg D.; Niedrig, Matthias; Bücker, Roland

    2014-01-01

    Tick-borne encephalitis virus (TBEV) is one of the most important vector-borne viruses in Europe and Asia. Its transmission mainly occurs by the bite of an infected tick. However, consuming milk products from infected livestock animals caused TBEV cases. To better understand TBEV transmission via the alimentary route, we studied viral infection of human intestinal epithelial cells. Caco-2 cells were used to investigate pathological effects of TBEV infection. TBEV-infected Caco-2 monolayers showed morphological changes including cytoskeleton rearrangements and cytoplasmic vacuolization. Ultrastructural analysis revealed dilatation of the rough endoplasmic reticulum and further enlargement to TBEV containing caverns. Caco-2 monolayers maintained an intact epithelial barrier with stable transepithelial electrical resistance (TER) during early stage of infection. Concomitantly, viruses were detected in the basolateral medium, implying a transcytosis pathway. When Caco-2 cells were pre-treated with inhibitors of cellular pathways of endocytosis TBEV cell entry was efficiently blocked, suggesting that actin filaments (Cytochalasin) and microtubules (Nocodazole) are important for PI3K-dependent (LY294002) virus endocytosis. Moreover, experimental fluid uptake assay showed increased intracellular accumulation of FITC-dextran containing vesicles. Immunofluorescence microscopy revealed co-localization of TBEV with early endosome antigen-1 (EEA1) as well as with sorting nexin-5 (SNX5), pointing to macropinocytosis as trafficking mechanism. In the late phase of infection, further evidence was found for translocation of virus via the paracellular pathway. Five days after infection TER was slightly decreased. Epithelial barrier integrity was impaired due to increased epithelial apoptosis, leading to passive viral translocation. These findings illuminate pathomechanisms in TBEV infection of human intestinal epithelial cells and viral transmission via the alimentary route. PMID

  2. Tick-borne encephalitis virus replication, intracellular trafficking, and pathogenicity in human intestinal Caco-2 cell monolayers.

    Directory of Open Access Journals (Sweden)

    Chao Yu

    Full Text Available Tick-borne encephalitis virus (TBEV is one of the most important vector-borne viruses in Europe and Asia. Its transmission mainly occurs by the bite of an infected tick. However, consuming milk products from infected livestock animals caused TBEV cases. To better understand TBEV transmission via the alimentary route, we studied viral infection of human intestinal epithelial cells. Caco-2 cells were used to investigate pathological effects of TBEV infection. TBEV-infected Caco-2 monolayers showed morphological changes including cytoskeleton rearrangements and cytoplasmic vacuolization. Ultrastructural analysis revealed dilatation of the rough endoplasmic reticulum and further enlargement to TBEV containing caverns. Caco-2 monolayers maintained an intact epithelial barrier with stable transepithelial electrical resistance (TER during early stage of infection. Concomitantly, viruses were detected in the basolateral medium, implying a transcytosis pathway. When Caco-2 cells were pre-treated with inhibitors of cellular pathways of endocytosis TBEV cell entry was efficiently blocked, suggesting that actin filaments (Cytochalasin and microtubules (Nocodazole are important for PI3K-dependent (LY294002 virus endocytosis. Moreover, experimental fluid uptake assay showed increased intracellular accumulation of FITC-dextran containing vesicles. Immunofluorescence microscopy revealed co-localization of TBEV with early endosome antigen-1 (EEA1 as well as with sorting nexin-5 (SNX5, pointing to macropinocytosis as trafficking mechanism. In the late phase of infection, further evidence was found for translocation of virus via the paracellular pathway. Five days after infection TER was slightly decreased. Epithelial barrier integrity was impaired due to increased epithelial apoptosis, leading to passive viral translocation. These findings illuminate pathomechanisms in TBEV infection of human intestinal epithelial cells and viral transmission via the alimentary

  3. Tick-borne encephalitis virus replication, intracellular trafficking, and pathogenicity in human intestinal Caco-2 cell monolayers.

    Science.gov (United States)

    Yu, Chao; Achazi, Katharina; Möller, Lars; Schulzke, Joerg D; Niedrig, Matthias; Bücker, Roland

    2014-01-01

    Tick-borne encephalitis virus (TBEV) is one of the most important vector-borne viruses in Europe and Asia. Its transmission mainly occurs by the bite of an infected tick. However, consuming milk products from infected livestock animals caused TBEV cases. To better understand TBEV transmission via the alimentary route, we studied viral infection of human intestinal epithelial cells. Caco-2 cells were used to investigate pathological effects of TBEV infection. TBEV-infected Caco-2 monolayers showed morphological changes including cytoskeleton rearrangements and cytoplasmic vacuolization. Ultrastructural analysis revealed dilatation of the rough endoplasmic reticulum and further enlargement to TBEV containing caverns. Caco-2 monolayers maintained an intact epithelial barrier with stable transepithelial electrical resistance (TER) during early stage of infection. Concomitantly, viruses were detected in the basolateral medium, implying a transcytosis pathway. When Caco-2 cells were pre-treated with inhibitors of cellular pathways of endocytosis TBEV cell entry was efficiently blocked, suggesting that actin filaments (Cytochalasin) and microtubules (Nocodazole) are important for PI3K-dependent (LY294002) virus endocytosis. Moreover, experimental fluid uptake assay showed increased intracellular accumulation of FITC-dextran containing vesicles. Immunofluorescence microscopy revealed co-localization of TBEV with early endosome antigen-1 (EEA1) as well as with sorting nexin-5 (SNX5), pointing to macropinocytosis as trafficking mechanism. In the late phase of infection, further evidence was found for translocation of virus via the paracellular pathway. Five days after infection TER was slightly decreased. Epithelial barrier integrity was impaired due to increased epithelial apoptosis, leading to passive viral translocation. These findings illuminate pathomechanisms in TBEV infection of human intestinal epithelial cells and viral transmission via the alimentary route.

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

    Directory of Open Access Journals (Sweden)

    Rame Taha

    2010-07-01

    Full Text Available 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.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.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.

  5. Enhanced uptake and transport of (+)-catechin and (−)-epigallocatechin gallate in niosomal formulation by human intestinal Caco-2 cells

    Science.gov (United States)

    Song, Qinxin; Li, Danhui; Zhou, Yongzhi; Yang, Jie; Yang, Wanqi; Zhou, Guohua; Wen, Jingyuan

    2014-01-01

    The aim of this study was to evaluate (+)-catechin and (−)-epigallocatechin gallate (EGCG) cellular uptake and transport across human intestinal Caco-2 cell monolayer in both the absence and presence of niosomal carrier in variable conditions. The effect of free drugs and drug-loaded niosomes on the growth of Caco-2 cells was studied. The effects of time, temperature, and concentration on drug cellular uptake in the absence or presence of its niosomal delivery systems were investigated. The intestinal epithelial membrane transport of the drug-loaded niosomes was examined using the monolayer of the human Caco-2 cells. The kinetics of transport, and the effect of temperature, adenosine triphosphate inhibitor, permeability glycoprotein inhibitor, multidrug resistance-associated protein 2 inhibitor, and the absorption enhancer on transport mechanism were investigated. It was found that the uptake of catechin, EGCG, and their niosomes by Caco-2 cells was 1.22±0.16, 0.90±0.14, 3.25±0.37, and 1.92±0.22 μg/mg protein, respectively (n=3). The apparent permeability coefficient values of catechin, EGCG, and their niosomes were 1.68±0.16, 0.88±0.09, 2.39±0.31, and 1.42±0.24 cm/second (n=3) at 37°C, respectively. The transport was temperature- and energy-dependent. The inhibitors of permeability glycoprotein and multidrug resistance-associated protein 2 and the absorption enhancer significantly enhanced the uptake amount. Compared with the free drugs, niosomal formulation significantly enhanced drug absorption. Additionally, drug-loaded niosomes exhibited stronger stability and lower toxicity. These findings showed that the oral absorption of tea flavonoids could be improved by using the novel drug delivery systems. PMID:24855353

  6. Intestinal Absorption and First-Pass Metabolism of Polyphenol Compounds in Rat and Their Transport Dynamics in Caco-2 Cells

    Science.gov (United States)

    Zhang, Feng; Huan, Menglei; Cao, Weidong; Li, Kangchu; Yang, Jingyue; Cao, Dayong; Zhou, Siyuan; Mei, Qibing

    2012-01-01

    Background Polyphenols, a group of complex naturally occurring compounds, are widely distributed throughout the plant kingdom and are therefore readily consumed by humans. The relationship between their chemical structure and intestinal absorption, transport, and first-pass metabolism remains unresolved, however. Methods Here, we investigated the intestinal absorption and first-pass metabolism of four polyphenol compounds, apigenin, resveratrol, emodin and chrysophanol, using the in vitro Caco-2 cell monolayer model system and in situ intestinal perfusion and in vivo pharmacokinetic studies in rats, so as to better understand the relationship between the chemical structure and biological fate of the dietary polyphenols. Conclusion After oral administration, emodin and chrysophanol exhibited different absorptive and metabolic behaviours compared to apigenin and resveratrol. The differences in their chemical structures presumably resulted in differing affinities for drug-metabolizing enzymes, such as glucuronidase and sulphatase, and transporters, such as MRP2, SGLT1, and P-glycoprotein, which are found in intestinal epithelial cells. PMID:22253753

  7. 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.

  8. Different responses of Fe transporters in Caco2/HT29-MTX cocultures than in independent Caco-2 cell cultures

    Science.gov (United States)

    The human intestinal epithelium is composed of several cell types; mainly enterocytes and globet (mucin-secreting) cells. This study compares the cellular response for Fe transporters in Caco-2, HT29-MTX, and Caco-2/HT29-MTX coculture models for Fe bioavailability studies. Under culture, Caco-2 cell...

  9. Intestinal Absorption of Triterpenoids and Flavonoids from Glycyrrhizae radix et rhizoma in the Human Caco-2 Monolayer Cell Model.

    Science.gov (United States)

    Wang, Xiao-Xue; Liu, Gui-Yan; Yang, Yan-Fang; Wu, Xiu-Wen; Xu, Wei; Yang, Xiu-Wei

    2017-09-29

    Glycyrrhizae radix et rhizoma has been used as a traditional Chinese medicine for the treatment of various diseases. Triterpenoids and flavonoids from the plant have many beneficial effects and their chemical structures are modified in the gastrointestinal tract after oral administration. However, absorption of these triterpenoids and flavonoids still needs to be defined. Here, the uptake and transepithelial transport of the selected major triterpenoids, glycyrrhizin ( 1 ), glycyrrhetic acid-3- O -mono-β-d-glucuronide ( 2 ), and glycyrrhetinic acid ( 3 ); and the selected major flavonoids, licochalcone A ( 4 ), licochalcone B ( 5 ), licochalcone C ( 6 ), echinatin ( 7 ), isoliquiritin apioside ( 8 ), liquiritigenin ( 9 ), liquiritin apioside ( 10 ) isolated from Glycyrrhizae radix et rhizoma , were investigated in the human intestinal epithelium-like Caco-2 cell monolayer model. Compounds 3 , 5 - 7 , and 9 were designated as well-absorbed compounds, 2 and 4 were designated as moderately absorbed ones, and 1 , 8 , and 10 were assigned for the poorly absorbed ones. The absorption mechanism of well and moderately absorbed compound was mainly passive diffusion to pass through the human intestinal Caco-2 cell monolayer. These findings provided useful information for predicting their oral bioavailability and the clinical application.

  10. Bioaccessibility, Cellular Uptake, and Transport of Astaxanthin Isomers and their Antioxidative Effects in Human Intestinal Epithelial Caco-2 Cells.

    Science.gov (United States)

    Yang, Cheng; Zhang, Hua; Liu, Ronghua; Zhu, Honghui; Zhang, Lianfu; Tsao, Rong

    2017-11-29

    The bioaccessibility, bioavailability, and antioxidative activities of three astaxanthin geometric isomers were investigated using an in vitro digestion model and human intestinal Caco-2 cells. This study demonstrated that the trans-cis isomerization of all-E-astaxanthin and the cis-trans isomerization of Z-astaxanthins could happen both during in vitro gastrointestinal digestion and cellular uptake processes. 13Z-Astaxanthin showed higher bioaccessibility than 9Z- and all-E-astaxanthins during in vitro digestion, and 9Z-astaxanthin exhibited higher transport efficiency than all-E- and 13Z-astaxanthins. These might explain why 13Z- and 9Z-astaxanthins are found at higher concentrations in human plasma than all-E-astaxanthin in reported studies. All three astaxanthin isomers were effective in maintaining cellular redox homeostasis as seen in the antioxidant enzyme (CAT, SOD) activities ; 9Z- and 13Z- astaxanthins exhibited a higher protective effect than all-E-astaxanthin against oxidative stress as demonstrated by the lower cellular uptake of Z-astaxanthins and lower secretion and gene expression of the pro-inflammatory cytokine IL-8 in Caco-2 cells treated with H 2 O 2 . We conclude, for the first time, that Z-astaxanthin isomers may play a more important role in preventing oxidative stress induced intestinal diseases.

  11. Structure characteristics for intestinal uptake of flavonoids in Caco-2 cells.

    Science.gov (United States)

    Fang, Yajing; Liang, Fuqiang; Liu, Kunyuan; Qaiser, Shakeel; Pan, Siyi; Xu, Xiaoyun

    2018-03-01

    Flavonoids are a large group of polyphenols and widely distributed in plant foods. Flavonoids exhibit various biological activities, such as anti-cancer, antioxidant and anti-inflammatory while poor oral bioavailability has been considered as a major hurdle in their use as functional foods. Cellular uptake and efflux of flavonoid implicates their bioavailability. To investigate the cellular uptake and efflux of flavonoids, 27 flavonoids were measured for their cellular uptake in Caco-2 cells with (CUV) and without (CU) the inhibitor of P-glycoprotein (P-gp) verapamil. Then, a quantitative structure-absorption relationship (QSAR) model containing 21 compounds as training set was obtained from their corresponding CU. The model showed good robustness and predictivity with a high cross-validation coefficient (Q 2 ) value of 0.809 and Log of the octanol/water partition coefficient (SlogP) and atomic charge on carbon 5 (Q C5 ) were related to flavonoid uptake. The CUV of some flavonoids were significantly (ppumped out by P-gp. The structure-affinity relationship of flavonoids as substrates of P-gp was determined with the presence of 4'-OCH 3 , 3'-OCH 3 and the absence of 3'-OH, 3-OH and 4'-OH favorable for the affinity of flavonoids. These results provide valuable information for screening flavonoids with good absorption and low affinity with transporters. Copyright © 2017. Published by Elsevier Ltd.

  12. Characterization of Caco-2 cells stably expressing the protein-based zinc probe eCalwy-5 as a model system for investigating intestinal zinc transport.

    Science.gov (United States)

    Maares, Maria; Keil, Claudia; Thomsen, Susanne; Günzel, Dorothee; Wiesner, Burkhard; Haase, Hajo

    2018-01-29

    Intestinal zinc resorption, in particular its regulation and mechanisms, are not yet fully understood. Suitable intestinal cell models are needed to investigate zinc uptake kinetics and the role of labile zinc in enterocytes in vitro. Therefore, a Caco-2 cell clone was produced, stably expressing the genetically encoded zinc biosensor eCalwy-5. The aim of the present study was to reassure the presence of characteristic enterocyte-specific properties in the Caco-2-eCalwy clone. Comparison of Caco-2-WT and Caco-2-eCalwy cells revealed only slight differences regarding subcellular localization of the tight junction protein occludin and alkaline phosphatase activity, which did not affect basic integrity of the intestinal barrier or the characteristic brush border membrane morphology. Furthermore, introduction of the additional zinc-binding protein in Caco-2 cells did not alter mRNA expression of the major intestinal zinc transporters (zip4, zip5, znt-1 and znt-5), but increased metallothionein 1a-expression and cellular resistance to higher zinc concentrations. Moreover, this study examines the effect of sensor expression level on its saturation with zinc. Fluorescence cell imaging indicated considerable intercellular heterogeneity in biosensor-expression. However, FRET-measurements confirmed that these differences in expression levels have no effect on fractional zinc-saturation of the probe. Copyright © 2018 Elsevier GmbH. All rights reserved.

  13. The Role of Turmerones on Curcumin Transportation and P-Glycoprotein Activities in Intestinal Caco-2 Cells

    Science.gov (United States)

    Yue, Grace G.L.; Cheng, Sau-Wan; Yu, Hua; Xu, Zi-Sheng; Lee, Julia K.M.; Hon, Po-Ming; Lee, Mavis Y.H.; Kennelly, Edward J.; Deng, Gary; Yeung, Simon K.; Cassileth, Barrie R.; Fung, Kwok-Pui; Leung, Ping-Chung

    2012-01-01

    Abstract The rhizome of Curcuma longa (turmeric) is often used in Asia as a spice and as a medicine. Its most well-studied component, curcumin, has been shown to exhibit poor bioavailability in animal studies and clinical trials. We hypothesized that the presence of lipophilic components (e.g., turmerones) in turmeric extract would affect the absorption of curcumin. The effects of turmerones on curcumin transport were evaluated in human intestinal epithelial Caco-2 cells. The roles of turmerones on P-glycoprotein (P-gp) activities and mRNA expression were also evaluated. Results showed that in the presence of α- and aromatic turmerones, the amount of curcumin transported into the Caco-2 cells in 2 hours was significantly increased. α-Turmerone and verapamil (a P-gp inhibitor) significantly inhibited the efflux of rhodamine-123 and digoxin (i.e., inhibited the activity of P-gp). It is interesting that aromatic turmerone significantly increased the rhodamine-123 efflux and P-gp (MDR1 gene) mRNA expression levels. The effects of α- and aromatic turmerones on curcumin transport as well as P-gp activities were shown here for the first time. The presence of turmerones did affect the absorption of curcumin in vitro. These findings suggest the potential use of turmeric extract (including curcumin and turmerones), rather than curcumin alone, for treating diseases. PMID:22181075

  14. Development of a serum-free co-culture of human intestinal epithelium cell-lines (Caco-2/HT29-5M21

    Directory of Open Access Journals (Sweden)

    Schneider Yves-Jacques

    2006-05-01

    Full Text Available Abstract Background The absorptive and goblet cells are the main cellular types encountered in the intestine epithelium. The cell lineage Caco-2 is a model commonly used to reproduce the features of the bowel epithelium. However, there is a strong debate regarding the value of Caco-2 cell culture to mimick in vivo situation. Indeed, some authors report in Caco-2 a low paracellular permeability and an ease of access of highly diffusible small molecules to the microvilli, due to an almost complete lack of mucus. The HT29-5M21 intestinal cell lineage is a mucin-secreting cellular population. A co-culture system carried out in a serum-free medium and comprising both Caco-2 and HT29-5M21 cells was developed. The systematic use of a co-culture system requires the characterization of the monolayer under a given experimental procedure. Results In this study, we investigated the activity and localization of the alkaline phosphatase and the expression of IAP and MUC5AC genes to determine a correlation between these markers and the cellular composition of a differentiated monolayer obtained from a mixture of Caco-2 and HT29-5M21 cells. We observed that the culture conditions used (serum-free medium did not change the phenotype of each cell type, and produced a reproducible model. The alkaline phosphatase expression characterizing Caco-2 cells was influenced by the presence of HT29-5M21 cells. Conclusion The culture formed by 75% Caco-2 and 25% HT29-5M21 produce a monolayer containing the two main cell types of human intestinal epithelium and characterized by a reduced permeability to macromolecules.

  15. Development of a serum-free co-culture of human intestinal epithelium cell-lines (Caco-2/HT29-5M21)

    Science.gov (United States)

    Nollevaux, Géraldine; Devillé, Christelle; El Moualij, Benaïssa; Zorzi, Willy; Deloyer, Patricia; Schneider, Yves-Jacques; Peulen, Olivier; Dandrifosse, Guy

    2006-01-01

    Background The absorptive and goblet cells are the main cellular types encountered in the intestine epithelium. The cell lineage Caco-2 is a model commonly used to reproduce the features of the bowel epithelium. However, there is a strong debate regarding the value of Caco-2 cell culture to mimick in vivo situation. Indeed, some authors report in Caco-2 a low paracellular permeability and an ease of access of highly diffusible small molecules to the microvilli, due to an almost complete lack of mucus. The HT29-5M21 intestinal cell lineage is a mucin-secreting cellular population. A co-culture system carried out in a serum-free medium and comprising both Caco-2 and HT29-5M21 cells was developed. The systematic use of a co-culture system requires the characterization of the monolayer under a given experimental procedure. Results In this study, we investigated the activity and localization of the alkaline phosphatase and the expression of IAP and MUC5AC genes to determine a correlation between these markers and the cellular composition of a differentiated monolayer obtained from a mixture of Caco-2 and HT29-5M21 cells. We observed that the culture conditions used (serum-free medium) did not change the phenotype of each cell type, and produced a reproducible model. The alkaline phosphatase expression characterizing Caco-2 cells was influenced by the presence of HT29-5M21 cells. Conclusion The culture formed by 75% Caco-2 and 25% HT29-5M21 produce a monolayer containing the two main cell types of human intestinal epithelium and characterized by a reduced permeability to macromolecules. PMID:16670004

  16. In vitro impact of five pesticides alone or in combination on human intestinal cell line Caco-2

    Directory of Open Access Journals (Sweden)

    Sylvain Ilboudo

    2014-01-01

    Full Text Available In Burkina Faso, as in most Sahelian countries, the failure to follow good agricultural practices coupled with poor soil and climate conditions in the locust control context lead to high environmental contaminations with pesticide residues. Thus, consumers being orally exposed to a combination of multiple pesticide residues through food and water intake, the digestive tract is a tissue susceptible to be directly exposed to these food contaminants. The aim of our work was to compare in vitro the impact of five desert locust control pesticides (Deltamethrin DTM, Fenitrothion FNT, Fipronil FPN, Lambda-cyalothrine LCT, and Teflubenzuron TBZ alone and in combination on the human intestinal Caco-2 cells viability and function. Cells were exposed to 0.1–100 μM pesticides for 10 days alone or in mixture (MIX. Our results showed a cytotoxic effect of DTM, FNT, FPN, LCT, and TBZ alone or in combination in human intestinal Caco-2 cells. The most efficient were shown to be FPN and FNT impacting the cell layer integrity and/or barrier function, ALP activity, antioxidant enzyme activity, lipid peroxidation, Akt activation, and apoptosis. The presence of antioxidant reduced lipid peroxidation level and attenuated the pesticides-induced cell toxicity, suggesting that key mechanism of pesticides cytotoxicity may be linked to their pro-oxidative potential. A comparative analysis with the predicted cytotoxic effect of pesticides mixture using mathematical modeling shown that the combination of these pesticides led to synergistic effects rather than to a simple independent or dose addition effect.

  17. Inhibition of p38 mitogen-activated protein kinase attenuates butyrate-induced intestinal barrier impairment in a Caco-2 cell monolayer model.

    Science.gov (United States)

    Huang, Xiao-Zhong; Li, Zhong-Rong; Zhu, Li-Bin; Huang, Hui-Ya; Hou, Long-Long; Lin, Jing

    2014-08-01

    Butyrate is well known to induce apoptosis in differentiating intestinal epithelial cells. The present study was designed to examine the role of p38 mitogen-activated protein kinase (MAPK) in butyrate-induced intestinal barrier impairment. The intestinal barrier was determined by measuring the transepithelial electrical resistance (TER) in a Caco-2 cell monolayer model. The permeability was determined by measuring transepithelial passage of fluorescein isothiocyanate-conjugated inulin (inulin-FITC). The morphology of the monolayers was examined with scanning electron microscopy. The apoptosis status was determined by annexin V-FITC labeling and flow cytometry. The activity of p38 MAPK was determined by the phosphorylation status of p38 with Western blotting. Butyrate at 5 mM increases the apoptosis rate of Caco-2 cells and induces impairment of intestinal barrier functions as determined by decreased TER and increased inulin-FITC permeability. Butyrate treatment activates p38 MAPK in a concentration- and time-dependent manner. SB203580, a specific p38 inhibitor, inhibits butyrate-induced Caco-2 cell apoptosis. Treatment of SB203580 significantly attenuates the butyrate-induced impairment of barrier functions in the Caco-2 cell monolayer model. p38 MAPK can be activated by butyrate and is involved in the butyrate-induced apoptosis and impairment of intestinal barrier function. Inhibition of p38 MAPK can significantly attenuate butyrate-induced intestinal barrier dysfunction.

  18. Anti-inflammatory properties of an extract of M. ilicifolia in the human intestinal epithelial Caco-2 cell line.

    Science.gov (United States)

    Wonfor, Ruth; Natoli, Manuela; Parveen, Ifat; Beckman, Manfred; Nash, Robert; Nash, Deborah

    2017-09-14

    Maytenus ilicifolia is a Celastracea plant used in traditional medicine to alleviate digestive tract inflammatory disorders. We investigated anti-inflammatory properties of M. ilicifolia crude extract towards Caco-2 cell line, as a model of Toll-like Receptor 2 (TLR-2) inflammatory pathway. Toxicity was assessed following culture of Caco-2 with M. ilicifolia, using apparent cell permeability and trans-epithelial electric resistance. Anti-inflammatory properties of M. ilicifolia were assessed through IL-8 secretion and TLR-2 associated gene expression of Caco-2 cells with or without an LTA challenge. M. ilicifolia was not toxic to Caco-2 cells. M. ilicifolia down-regulated TLR2 expression with and without LTA challenge but had no effect on other genes. Following LTA challenge of Caco-2 cells, 100 and 200µg/mL M. ilicifolia abrogated IL-8 secretion. We provide preliminary data for some M. ilicifolia anti-inflammatory properties. Further research must establish the full extent and mode of action on particular inflammatory pathways. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

  19. The transepithelial transport mechanism of polybrominated diphenyl ethers in human intestine determined using a Caco-2 cell monolayer.

    Science.gov (United States)

    Yu, Yingxin; Wang, Mengmeng; Zhang, Kaiqiong; Yang, Dan; Zhong, Yufang; An, Jing; Lei, Bingli; Zhang, Xinyu

    2017-04-01

    Oral ingestion plays an important role in human exposure to polybrominated diphenyl ethers (PBDEs). The uptake of PBDEs primarily occurs in the small intestine. The aim of the present study is to investigate the transepithelial transport characteristics and mechanisms of PBDEs in the small intestine using a Caco-2 cell monolayer model. The apparent permeability coefficients of PBDEs indicated that tri- to hepta-BDEs were poorly absorbed compounds. A linear increase in transepithelial transport was observed with various concentrations of PBDEs, which suggested that passive diffusion dominated their transport at the concentration range tested. In addition, the pseudo-first-order kinetics equation can be applied to the transepithelial transport of PBDEs. The rate-determining step in transepithelial transport of PBDEs was trans-cell transport including the trans-pore process. The significantly lower transepithelial transport rates at low temperature for bidirectional transepithelial transport suggested that an energy-dependent transport mechanism was involved. The efflux transporters (P-glycoprotein, multidrug resistance-associated protein, and breast cancer resistance protein) and influx transporters (organic cation transporters) participated in the transepithelial transport of PBDEs. In addition, the transepithelial transport of PBDEs was pH sensitive; however, more information is required to understand the influence of pH. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Development of Yam Dioscorin-Loaded Nanoparticles for Paracellular Transport Across Human Intestinal Caco-2 Cell Monolayers.

    Science.gov (United States)

    Hsieh, Hung-Ling; Lee, Chia-Hung; Lin, Kuo-Chih

    2018-02-07

    Dioscorins, the major storage proteins of yam tubers, exert immunomodulatory activities. To improve oral bioavailability of dioscorins in the intestine, recombinant dioscorin (rDioscorin) was coated with N,N,N-trimethyl chitosan (TMC) and tripolyphosphate (TPP), resulting in the formation of TMC-rDio-TPP nanoparticles (NPs). The loading capacity and entrapment efficiency of rDioscorin in the NPs were 26 ± 0.7% and 61 ± 1.4%, respectively. The NPs demonstrated a substantial release profile in the pH environment of the jejunum. The rDioscorin released from the NPs stimulated proliferation and phagocytosis of the macrophage RAW264.7 and activated the gene expression of IL-1β and IL-6. Incubation of the NPs in the Caco-2 cell monolayer led to a 5.2-fold increase of P app compared with rDioscorin alone, suggesting that rDioscorin, with the assistance of TMC, can be promptly transported across the intestinal epithelia. These results demonstrate that the TMC-rDio-TPP NPs can be utilized for elucidating the immunopharmacological effects of dioscorins through oral delivery.

  1. 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

    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...... 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...... 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....

  2. Human Oral Isolate Lactobacillus fermentum AGR1487 Reduces Intestinal Barrier Integrity by Increasing the Turnover of Microtubules in Caco-2 Cells

    Science.gov (United States)

    Anderson, Rachel C.; Young, Wayne; Clerens, Stefan; Cookson, Adrian L.; McCann, Mark J.; Armstrong, Kelly M.; Roy, Nicole C.

    2013-01-01

    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 same species can

  3. Genome-wide analysis of CDX2 binding in intestinal epithelial cells (Caco-2)

    DEFF Research Database (Denmark)

    Boyd, Mette; Hansen, Morten; Jensen, Tine G K

    2010-01-01

    , revealing a critical role of CDX2 in the formation of the normal intestinal identity. The identification of direct targets of transcription factors is a key problem in the study of gene regulatory networks. The ChIP-seq technique combines chromatin immunoprecipitation (ChIP) with next generation sequencing...

  4. The effect of onion exposure on gene expression profiles in intestinal Caco-2 cells

    NARCIS (Netherlands)

    Wit, de N.J.W.; Govers, C.C.F.M.; Boekschoten, M.V.; Mes, J.J.

    2016-01-01

    Background: Human intestinal tissue samples are barely accessible to study potential health benefits of nutritional compounds. Numbers of animals used in animal trials, however, need to be minimalized. Therefore, in this study we explored the applicability of an in vitro model, namely human

  5. Enhanced Intestinal Permeability of Bufalin by a Novel Bufalin-Peptide-Dendrimer Inclusion through Caco-2 Cell Monolayer.

    Science.gov (United States)

    Chan, Chi-On; Jing, Jing; Xiao, Wei; Tan, Zhexu; Lv, Qiuyue; Yang, Jingyu; Chen, Sibao

    2017-11-29

    Bufalin (BFL) has excellent physiological activities such as defending tumors, improving cardiac function, and so on. However, due to its poor water-solubility and bioavailability, the clinical application of BFL remains limited. In order to improve bioavailability of BFL, in our previous research, a novel peptide-dendrimer (PD) was synthesized and applied to encapsulate BFL. In the present study, we investigate the absorption property and mechanism of BFL in free form and BFL-peptide-dendrimer inclusion (BPDI) delivery system by using the Caco-2 cell monolayer model in vitro. The apparent permeability coefficient ( P app ) values of BFL in free or BPDI form were over 1.0 × 10 -6 cm/s. Meanwhile, their almost equal bi-directional transport and linear transport percentage with time and concentration course indicated that BFL in both forms was absorbed mainly through passive diffusion. The most important result is that the P app values of BFL increased about three-fold more BPDI than those of its free form, which indicated the intestinal permeability of BFL could be improved while BFL was encapsulated in BPDI form. Therefore, PD encapsulation may be a potential delivery system to increase the bioavailability of BFL.

  6. Adaptation in Caco-2 Human Intestinal Cell Differentiation and Phenolic Transport with Chronic Exposure to Blackberry (Rubus sp.) Extract.

    Science.gov (United States)

    Redan, Benjamin W; Albaugh, George P; Charron, Craig S; Novotny, Janet A; Ferruzzi, Mario G

    2017-04-05

    As evidence mounts for a health-protective role of dietary phenolics, the importance of understanding factors influencing bioavailability increases. Recent evidence has suggested chronic exposure to phenolics may impact their absorption and metabolism. To explore alterations occurring from chronic dietary exposure to phenolics, Caco-2 cell monolayers were differentiated on Transwell inserts with 0-10 μM blackberry (Rubus sp.) total phenolics extracts rich in anthocyanins, flavonols, and phenolic acids. Following differentiation, apical to basolateral transport of phenolics was assessed from an acute treatment of 100 μM blackberry phenolics from 0 to 4 h. Additionally, differences in gene expression of transport and phase II metabolizing systems including ABC transporters, organic anion transporters (OATs), and uridine 5'-diphospho (UDP) glucuronosyltransferases (UGTs) were probed. After 4 h, 1 μM pretreated monolayers showed a significant (P transport including less epicatechin (42.1 ± 0.53), kaempferol glucoside (23.5 ± 0.29), and dicaffeoylquinic acid (31.9 ± 0.20) compared to control. Finally, significant (P transport proteins were observed with treatment. Therefore, adaptation to blackberry extract exposure may impact intestinal transport and metabolism of phenolics.

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

    Directory of Open Access Journals (Sweden)

    Geneviève Mailhot

    2010-05-01

    Full Text Available 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.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, LXRalpha, LXRbeta and RXRalpha

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

    Directory of Open Access Journals (Sweden)

    Almudena Pérez-Sánchez

    Full Text Available 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.

  9. 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

  10. [Absorption and transport of isoflavonoid compounds from Tongmai formula across human intestinal epithelial (Caco-2) cells in vitro].

    Science.gov (United States)

    Wang, Fu-Rong; Yang, Xiu-Wei

    2017-08-01

    Tongmai formula (TMF) is a drug combination of three components including Puerariae Lobatae Radix [roots of Pueraria lobata], Salviae Miltiorrhizae Radix (roots of Salvia miltiorrhiza) and Chuanxiong Rhizoma (rhizomes of Ligusticum chuanxiong) in a weight ratio of 1∶1∶1. The absorption and transport of isoflavonoid compounds from Tongmai formula across human intestinal epithelial (Caco-2) cells in vitro were studied in this paper. The assay isoflavonoid compounds include daidzein, formononetin, 5-hydroxylononin, ononin, daidzin, 3'-methoxypuerarin, genistin, puerarin, formononetin-8-C-β-D-apiofuranosyl-(1→6)-O-β-D-glucopyranoside, formononetin-7-O-β-D-apiofuranosyl-(1→6)-O-β-D-glucopyranoside, lanceolarin, kakkanin, daidzein-7,4'-di-O-β-D-glucopyranoside, mirificin, 3'-hydroxypuerarin, 3'-methoxydaidzin, formononetin-8-C-β-D-xylopyranosyl-(1→6)-O-β-D-glucopyranoside, genistein-8-C-β-D-apiofuranosyl-(1→6)-O-β-D-glucopyranoside, genistein-7-O-β-D-apiofuranosyl-(1→6)-O-β-D-glucopyranoside (ambocin), 3'-hydroxymirificin, 6″-O-β-D-xylosylpuerarin, biochanin A-8-C-β-D-apiofuranosyl-(1→6)-O-β-D-glucopyranoside, 3'-methoxydaidzein-7,4'-di-O-β-D-glucopyranoside, daidzein-7-O-β-D-glucopyranosyl-(1→4)-O-β-D-glucopyranoside, and daidzein-7-O-α-D-glucopyranosyl-(1→4)-O-β-D-glucopyranoside. By using human Caco-2 monolayer as an intestinal epithelial cell model in vitro, the permeability of above-mentioned 25 isoflavonoids in TMF were studied from the apical (AP) side to basolateral (BL) side or from the BL side to AP side. The assay compounds were determined by reversed phased high-performance liquid chromatography (HPLC) coupled with UV detector. Transport parameters and apparent permeability coefficients (Papp) were then calculated and and compared with those of propranolol and atenolol, which are the transcellular transport marker and as a control substance for high and poor permeability, respectively. The Papp values of daidzein and

  11. 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

    2016-01-01

    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

  12. Administration of Protein kinase D1 induce an immunomodulatory effect on lipopolysaccharide-induced intestinal inflammation in a co-culture model of intestinal epithelial Caco-2 cells and RAW 264.7 macrophage cells

    DEFF Research Database (Denmark)

    Nielsen, Ditte Søvsø Gundelund; Fredborg, Marlene; Andersen, Vibeke

    2017-01-01

    the effects of human PKD1 in relation to intestinal inflammation, using a co-culture model of intestinal epithelial Caco-2 cells and RAW264.7 macrophages. An inflammatory response was induced in the macrophages by lipopolysaccharide (LPS), upregulating the expression of tumour necrosis factor alpha (TNF......-α), interleukin- (IL-) 1β, and IL-6 besides increasing the secretion of TNF-α protein. The effect of administering PKD1 to Caco-2 was evaluated in relation to both amelioration of inflammation and the ability to suppress inflammation initiation. Administration of PKD1 (10–100 ng/ml) following induction...

  13. A role for the non-receptor tyrosine kinase ACK1 in TNF-alpha-mediated apoptosis and proliferation in human intestinal epithelial caco-2 cells.

    Science.gov (United States)

    Zhao, Xinmei; Lv, Chaolan; Chen, Shengbo; Zhi, Fachao

    2017-09-16

    The roles of tumor necrosis factor alpha (TNF-alpha) and its mediators in cellular processes related to intestinal diseases remain elusive. In this study, we aimed to determine the biological role of activated Cdc42-associated kinase 1 (ACK1) in TNF-alpha-mediated apoptosis and proliferation in Caco-2 cells. ACK1 expression was knocked down using ACK1-specific siRNAs, and ACK1 activity was disrupted using a small molecule ACK1 inhibitor. The Terminal deoxynucleotidyl transferase biotin-dUTP Nick End Labeling (TUNEL) and the BrdU incorporation assays were used to measure apoptosis and cell proliferation, respectively. ACK1-specific siRNA and the pharmacological ACK1 inhibitor significantly abrogated the TNF-alpha-mediated anti-apoptotic effects and proliferation of Caco-2 cells. Interestingly, TNF-alpha activated ACK1 at tyrosine 284 (Tyr284), and the ErbB family of proteins was implicated in ACK1 activation in Caco-2 cells. ACK1-Tyr284 was required for protein kinase B (AKT) activation, and ACK1 signaling was mediated through recruiting and phosphorylating the down-stream adaptor protein AKT, which likely promoted cell proliferation in response to TNF-alpha. Moreover, ACK1 activated AKT and Src enhanced nuclear factor-кB (NF-кB) activity, suggesting a correlation between NF-кB signaling and TNF-alpha-mediated apoptosis in Caco-2 cells. Our results demonstrate that ACK1 plays an important role in modulating TNF-alpha-induced aberrant cell proliferation and apoptosis, mediated in part by ACK1 activation. ACK1 and its down-stream effectors may hold promise as therapeutic targets in the prevention and treatment of gastrointestinal cancers, in particular, those induced by chronic intestinal inflammation. © 2017 The Authors. Cell Biology International Published by John Wiley & Sons Ltd on behalf of International Federation of Cell Biology.

  14. Diamine Oxidase from White Pea (Lathyrus sativus) Combined with Catalase Protects the Human Intestinal Caco-2 Cell Line from Histamine Damage.

    Science.gov (United States)

    Jumarie, Catherine; Séïde, Marilyne; Marcocci, Lucia; Pietrangeli, Paola; Mateescu, Mircea Alexandru

    2017-07-01

    Diamine oxidase (DAO) administration has been proposed to treat certain gastrointestinal dysfunctions induced by histamine, an immunomodulator, signaling, and pro-inflammatory factor. However, H 2 O 2 resulting from the oxidative deamination of histamine by DAO may be toxic. The purpose of this study was to investigate to which extent DAO from white pea (Lathyrus sativus), alone or in combination with catalase, may modulate histamine toxicity in the human intestinal Caco-2 cell line. The results show that histamine at concentrations higher than 1 mM is toxic to the Caco-2 cells, independently of the cell differentiation status, with a LC 50 of ≅ 10 mM following a 24-h exposure. Depending on its concentration, DAO increased histamine toxicity to a greater extent in differentiated cells compared to undifferentiated cultures. In the presence of catalase, the DAO-induced increase in histamine toxicity was completely abolished in the undifferentiated cells and only partially decreased in differentiated cells, showing differences in the sensitivity of Caco-2 cells to the products resulting from histamine degradation by DAO (H 2 O 2 , NH 3 , or imidazole aldehyde). It appears that treatment of food histaminosis using a combination of vegetal DAO and catalase would protect against histamine toxicity and prevent H 2 O 2 -induced damage that may occur during histamine oxidative deamination.

  15. Transport of the Glucosamine-Derived Browning Product Fructosazine (Polyhydroxyalkylpyrazine) Across the Human Intestinal Caco-2 Cell Monolayer: Role of the Hexose Transporters.

    Science.gov (United States)

    Bhattacherjee, Abhishek; Hrynets, Yuliya; Betti, Mirko

    2017-06-14

    The transport mechanism of fructosazine, a glucosamine self-condensation product, was investigated using a Caco-2 cell model. Fructosazine transport was assessed by measuring the bidirectional permeability coefficient across Caco-2 cells. The mechanism of transport was evaluated using phlorizin, an inhibitor of sodium-dependent glucose cotransporters (SGLT) 1 and 2, phloretin and quercetin, inhibitors of glucose transporters (GLUT) 1 and 2, transcytosis inhibitor wortmannin, and gap junction disruptor cytochalasin D. The role of hexose transporters was further studied using downregulated or overexpressed cell lines. The apparent permeability (P a,b ) of fructosazine was 1.30 ± 0.02 × 10 -6 cm/s. No significant (p > 0.05) effect was observed in fructosazine transport by adding wortmannin and cytochalasin D. The presence of phlorizin, phloretin, and quercetin decreased fructosazine transport. The downregulated GLUT cells line was unable to transport fructosazine. In human intestinal epithelial Caco-2 cells, GLUT1 or GLUT2 and SGLT are mainly responsible for fructosazine transport.

  16. 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.

  17. Quantification of egg ovalbumin hydrolysate-derived anti-hypertensive peptides in an in vitro model combining luminal digestion with intestinal Caco-2 cell transport.

    Science.gov (United States)

    Grootaert, Charlotte; Jacobs, Griet; Matthijs, Bea; Pitart, Judit; Baggerman, Geert; Possemiers, Sam; Van der Saag, Hans; Smagghe, Guy; Van Camp, John; Voorspoels, Stefan

    2017-09-01

    Food-derived peptides can impact blood pressure through several mechanisms. However, their fate in the gastro-intestinal tract and bioavailability are difficult to assess because of their fast degradation and challenging analysis in physiologically relevant matrices. The aim of this study was to construct an in vitro bioavailability methodology in which luminal digestion is combined with Caco-2 cell transport. Egg ovalbumin hydrolysate, both in pure form and mixed with a food matrix, was used as a test case. Results indicate that a food matrix protected bioactive peptides from luminal digestion, especially in small intestine. Moreover, the Caco-2 absorption peak was extended over a longer time period (>60min) compared to the pure peptide solutions (~15min) which in total resulted in a 3-12 times higher absorption of the bioactive sequences after 60min compared to fasted conditions. These results suggest further investigation is warranted towards peptide-based functional foods with improved gastro-intestinal stability and longer-term release in the blood. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Dipeptide model prodrugs for the intestinal oligopeptide transporter. Affinity for and transport via hPepT1 in the human intestinal Caco-2 cell line

    DEFF Research Database (Denmark)

    Nielsen, C U; Andersen, R; Brodin, Birger

    2001-01-01

    -moieties for benzyl alcohol have been shown to maintain affinity for hPepT1. The primary aim of the present study was to investigate if modifications of the benzyl alcohol model drug influence the corresponding D-Glu-Ala and D-Asp-Ala model prodrugs' affinity for hPepT1 in Caco-2 cells. A second aim...... was to investigate the transepithelial transport and hydrolysis parameters for D-Asp(BnO)-Ala and D-Glu(BnO)-Ala across Caco-2 cell monolayers. In the present study, all investigated D-Asp-Ala and D-Glu-Ala model prodrugs retained various degrees of affinity for hPepT1 in Caco-2 cells. These affinities are used....... Transepithelial transport studies performed using Caco-2 cells of D-Asp(BnO)-Ala and D-Glu(BnO)-Ala showed that the K(m) for transepithelial transport was not significantly different for the two compounds. The maximal transport rate of the carrier-mediated flux component does not differ between the two model...

  19. Effects of Marine Oils, Digested with Human Fluids, on Cellular Viability and Stress Protein Expression in Human Intestinal Caco-2 Cells.

    Science.gov (United States)

    Tullberg, Cecilia; Vegarud, Gerd; Undeland, Ingrid; Scheers, Nathalie

    2017-11-04

    In vitro digestion of marine oils has been reported to promote lipid oxidation, including the formation of reactive aldehydes (e.g., malondialdehyde (MDA) and 4-hydroxy-2-hexenal (HHE)). We aimed to investigate if human in vitro digestion of supplemental levels of oils from algae, cod liver, and krill, in addition to pure MDA and HHE, affect intestinal Caco-2 cell survival and oxidative stress. Cell viability was not significantly affected by the digests of marine oils or by pure MDA and HHE (0-90 μM). Cellular levels of HSP-70, a chaperone involved in the prevention of stress-induced protein unfolding was significantly decreased (14%, 28%, and 14% of control for algae, cod and krill oil, respectively; p ≤ 0.05). The oxidoreductase thioredoxin-1 (Trx-1) involved in reducing oxidative stress was also lower after incubation with the digested oils (26%, 53%, and 22% of control for algae, cod, and krill oil, respectively; p ≤ 0.001). The aldehydes MDA and HHE did not affect HSP-70 or Trx-1 at low levels (8.3 and 1.4 μM, respectively), whilst a mixture of MDA and HHE lowered Trx-1 at high levels (45 μM), indicating less exposure to oxidative stress. We conclude that human digests of the investigated marine oils and their content of MDA and HHE did not cause a stress response in human intestinal Caco-2 cells.

  20. Effects of Marine Oils, Digested with Human Fluids, on Cellular Viability and Stress Protein Expression in Human Intestinal Caco-2 Cells

    Directory of Open Access Journals (Sweden)

    Cecilia Tullberg

    2017-11-01

    Full Text Available In vitro digestion of marine oils has been reported to promote lipid oxidation, including the formation of reactive aldehydes (e.g., malondialdehyde (MDA and 4-hydroxy-2-hexenal (HHE. We aimed to investigate if human in vitro digestion of supplemental levels of oils from algae, cod liver, and krill, in addition to pure MDA and HHE, affect intestinal Caco-2 cell survival and oxidative stress. Cell viability was not significantly affected by the digests of marine oils or by pure MDA and HHE (0–90 μM. Cellular levels of HSP-70, a chaperone involved in the prevention of stress-induced protein unfolding was significantly decreased (14%, 28%, and 14% of control for algae, cod and krill oil, respectively; p ≤ 0.05. The oxidoreductase thioredoxin-1 (Trx-1 involved in reducing oxidative stress was also lower after incubation with the digested oils (26%, 53%, and 22% of control for algae, cod, and krill oil, respectively; p ≤ 0.001. The aldehydes MDA and HHE did not affect HSP-70 or Trx-1 at low levels (8.3 and 1.4 μM, respectively, whilst a mixture of MDA and HHE lowered Trx-1 at high levels (45 μM, indicating less exposure to oxidative stress. We conclude that human digests of the investigated marine oils and their content of MDA and HHE did not cause a stress response in human intestinal Caco-2 cells.

  1. Factors Affecting the Bioaccessibility and Intestinal Transport of Difenoconazole, Hexaconazole, and Spirodiclofen in Human Caco-2 Cells Following in Vitro Digestion.

    Science.gov (United States)

    Shi, Yan-Hong; Xiao, Jin-Jing; Feng, Rong-Peng; Liu, Yu-Ying; Liao, Min; Wu, Xiang-Wei; Hua, Ri-Mao; Cao, Hai-Qun

    2017-10-18

    This study examined how gastrointestinal conditions affect pesticide bioaccessibility and intestinal transepithelial transport of pesticides (difenoconazole, hexaconazole, and spirodiclofen) in humans. We used an in vitro model combining human gastric and intestinal digestion, followed with Caco-2 cell model for human intestinal absorption. Bioaccessibility of three tested pesticides ranged from 25.2 to 76.3% and 10.6 to 79.63% in the gastric and intestinal phases, respectively. A marked trend similar to the normal distribution was observed between bioaccessibility and pH, with highest values observed at pH 2.12 in gastric juice. No significant differences were observed with increasing digestion time; however, a significant negative correlation was observed with the solid-liquid (S/L) ratio, following a logarithmic equation. R 2 ranged from 0.9198 to 0.9848 and 0.9526 to 0.9951 in the simulated gastric and intestinal juices, respectively, suggesting that the S/L ratio is also a major factor affecting bioaccessibility. Moreover, significant dose- and time-response effects were subsequently observed for intestinal membrane permeability of difenoconazole, but not for hexaconazole or spirodiclofen. This is the first study to demonstrate the uptake of pesticides by human intestinal cells, aiding quantification of the likely effects on human health and highlighting the importance of considering bioaccessibility in studies of dietary exposure to pesticide residues.

  2. Absorption, Conjugation and Efflux of the Flavonoids, Kaempferol and Galangin, Using the Intestinal CACO-2/TC7 Cell Model.

    Science.gov (United States)

    Barrington, Robert; Williamson, Gary; Bennett, Richard N; Davis, Barry D; Brodbelt, Jennifer S; Kroon, Paul A

    2009-01-01

    Flavonoids are biologically active compounds in food with potential health effects. We have used the Caco-2 cell monolayer model to study the absorption and metabolism of two flavonols, a class of flavonoids, specifically kaempferol and galangin. Metabolism experiments allowed identification of 5 kaempferol conjugates: 3-, 7- and 4'-glucuronide, a sulphate and a glucurono-sulphate; and 4 galangin conjugates: 3-, 5- and 7-glucuronides, and a sulphate, using specific enzyme hydrolysis, HPLC-MS, and HPLC with post column metal complexation/tandem MS. Transport studies showed that the flavonols were conjugated inside the cells then transported across the monolayer or effluxed back to the apical side. Sulphated conjugates were preferentially effluxed back to the apical side, whereas glucuronides were mostly transported to the basolateral side. For kaempferol, a small amount of the unconjugated aglycone permeated in both directions, indicating some passive diffusion. When kaempferol-3-glucuronide and quercetin7-sulphate were applied to either side of the cells, no permeation in either direction was observed, indicating that conjugates cannot re-cross the cell monolayer. Formation of apical kaempferol-7- and 4'-glucuronides was readily saturated, whereas formation of other conjugates at the apical side and all at the basolateral side increased with increasing concentration of kaempferol, implying different transporters are responsible at the apical and basolateral sides. The results highlight the important but complex metabolic changes occurring in flavonoids during absorption.

  3. Zinc Supplementation, via GPR39, Upregulates PKCζ to Protect Intestinal Barrier Integrity in Caco-2 Cells Challenged by Salmonella enterica Serovar Typhimurium.

    Science.gov (United States)

    Shao, Yu-Xin; Lei, Zhao; Wolf, Patricia G; Gao, Yan; Guo, Yu-Ming; Zhang, Bing-Kun

    2017-07-01

    Background: Zinc has been shown to improve intestinal barrier function against Salmonella enterica serovar Typhimurium ( S. typhimurium ) infection, but the mechanisms involved in this process remain undefined. Objective: We aimed to explore the roles of G protein-coupled receptor (GPR)39 and protein kinase Cζ (PKCζ) in the regulation by zinc of intestinal barrier function. Methods: A Transwell Caco-2 monolayer was pretreated with 0, 50, or 100 μM Zn and then incubated with S. typhimurium for 0-6 h. Afterward, cells silenced by the small interfering RNA for GPR39 or PKCζ were pretreated with 100 μM Zn and incubated with S. typhimurium for 3 h. Finally, transepithelial electrical resistance (TEER), permeability, tight junction (TJ) proteins, and signaling molecules GPR39 and PKCζ were measured. Results: Compared with controls, S. typhimurium decreased TEER by 62.3-96.2% at 4-6 h ( P 0.1). Silencing GPR39 decreased ( P zinc-activated PKCζ and blocked ( P zinc on epithelial integrity. Furthermore, silencing PKCζ counteracted the protective effect of zinc on epithelial integrity but did not inhibit GPR39 ( P = 0.138). Conclusion: We demonstrated that zinc upregulates PKCζ by activating GPR39 to enhance the abundance of ZO-1, thereby improving epithelial integrity in S. typhimurium- infected Caco-2 cells. © 2017 American Society for Nutrition.

  4. 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. PMID:26226276

  5. Impact of copper oxide nanomaterials on differentiated and undifferentiated Caco-2 intestinal epithelial cells; assessment of cytotoxicity, barrier integrity, cytokine production and nanomaterial penetration.

    Science.gov (United States)

    Ude, Victor C; Brown, David M; Viale, Luca; Kanase, Nilesh; Stone, Vicki; Johnston, Helinor J

    2017-08-23

    Copper oxide nanomaterials (CuO NMs) are exploited in a diverse array of products including antimicrobials, inks, cosmetics, textiles and food contact materials. There is therefore a need to assess the toxicity of CuO NMs to the gastrointestinal (GI) tract since exposure could occur via direct oral ingestion, mucocillary clearance (following inhalation) or hand to mouth contact. Undifferentiated Caco-2 intestinal cells were exposed to CuO NMs (10 nm) at concentrations ranging from 0.37 to 78.13 μg/cm 2 Cu (equivalent to 1.95 to 250 μg/ml) and cell viability assessed 24 h post exposure using the alamar blue assay. The benchmark dose (BMD 20), determined using PROAST software, was identified as 4.44 μg/cm 2 for CuO NMs, and 4.25 μg/cm 2 for copper sulphate (CuSO 4 ), which informed the selection of concentrations for further studies. The differentiation status of cells and the impact of CuO NMs and CuSO 4 on the integrity of the differentiated Caco-2 cell monolayer were assessed by measurement of trans-epithelial electrical resistance (TEER), staining for Zonula occludens-1 (ZO-1) and imaging of cell morphology using scanning electron microscopy (SEM). The impact of CuO NMs and CuSO 4 on the viability of differentiated cells was performed via assessment of cell number (DAPI staining), and visualisation of cell morphology (light microscopy). Interleukin-8 (IL-8) production by undifferentiated and differentiated Caco-2 cells following exposure to CuO NMs and CuSO 4 was determined using an ELISA. The copper concentration in the cell lysate, apical and basolateral compartments were measured with Inductive Coupled Plasma Optical Emission Spectrometry (ICP-OES) and used to calculate the apparent permeability coefficient (P app ); a measure of barrier permeability to CuO NMs. For all experiments, CuSO 4 was used as an ionic control. CuO NMs and CuSO 4 caused a concentration dependent decrease in cell viability in undifferentiated cells. CuO NMs and CuSO 4

  6. 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.

  7. 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 < 0.01), indicating that the established MRLs for AFM1 should be re-evaluated considering its frequent co-occurrence with other mycotoxins in baby food which contains milk and cereals. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Butyrate Enhances the Intestinal Barrier by Facilitating Tight Junction Assembly via Activation of AMP-Activated Protein Kinase in Caco-2 Cell Monolayers12

    Science.gov (United States)

    Peng, Luying; Li, Zhong-Rong; Green, Robert S.; Holzman, Ian R.; Lin, Jing

    2009-01-01

    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. PMID:19625695

  9. 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.

  10. In vitro study of soil arsenic release by human gut microbiota and its intestinal absorption by Caco-2 cells.

    Science.gov (United States)

    Yin, Naiyi; Cai, Xiaolin; Du, Huili; Zhang, Zhennan; Li, Zejiao; Chen, Xiaochen; Sun, Guoxin; Cui, Yanshan

    2017-02-01

    Arsenic (As) speciation is essential in assessing health risks from As-contaminated soil. Release of soil-bound arsenic, As transformation by human gut microbiota, and the subsequent intestinal absorption of soil As metabolites were evaluated. A colon microbial community in a dynamic human gut model and the intestinal epithelial cell line Caco-2 were cultured. Arsenic speciation analysis and absorption of different As species were undertaken. In this study, soil As release (3.7-581.2 mg kg -1 ) was observed in the colon. Arsenic in the colon digests was transformed more quickly than that in the soil solid phase. X-ray absorption near-edge spectroscopy (XANES) analysis showed that 44.2-97.6% of arsenite [As(III)] generated due to arsenate [As(V)] reduction was in the soil solid phase after the colon phase. We observed a high degree of cellular absorption of soil As metabolites, exhibiting that the intestinal absorption of monomethylarsonic acid and As(III) (33.6% and 30.2% resp.) was slightly higher than that of dimethylarsinic acid and As(V) (25.1% and 21.7% resp.). Our findings demonstrate that human gut microbiota can directly release soil-bound arsenic, particularly As-bearing amorphous Fe/Al-oxides. Determining As transformation and intestinal absorption simultaneously will result in an accurate risk assessment of human health with soil As exposures. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Intestinal toxicity evaluation of TiO2 degraded surface-treated nanoparticles: a combined physico-chemical and toxicogenomics approach in caco-2 cells

    Directory of Open Access Journals (Sweden)

    Fisichella Matthieu

    2012-05-01

    Full Text Available Abstract Background Titanium dioxide (TiO2 nanoparticles (NPs are widely used due to their specific properties, like UV filters in sunscreen. In that particular case TiO2 NPs are surface modified to avoid photocatalytic effects. These surface-treated nanoparticles (STNPs spread in the environment and might release NPs as degradation residues. Indeed, degradation by the environment (exposure to UV, water and air contact … will occur and could profoundly alter the physicochemical properties of STNPs such as chemistry, size, shape, surface structure and dispersion that are important parameters for toxicity. Although the toxicity of surface unmodified TiO2 NPs has been documented, nothing was done about degraded TiO2 STNPs which are the most likely to be encountered in environment. The superoxide production by aged STNPs suspensions was tested and compared to surface unmodified TiO2 NPs. We investigated the possible toxicity of commercialized STNPs, degraded by environmental conditions, on human intestinal epithelial cells. STNPs sizes and shape were characterized and viability tests were performed on Caco-2 cells exposed to STNPs. The exposed cells were imaged with SEM and STNPs internalization was researched by TEM. Gene expression microarray analyses were performed to look for potential changes in cellular functions. Results The production of reactive oxygen species was detected with surface unmodified TiO2 NPs but not with STNPs or their residues. Through three different toxicity assays, the STNPs tested, which have a strong tendency to aggregate in complex media, showed no toxic effect in Caco-2 cells after exposures to STNPs up to 100 μg/mL over 4 h, 24 h and 72 h. The cell morphology remained intact, attested by SEM, and internalization of STNPs was not seen by TEM. Moreover gene expression analysis using pangenomic oligomicroarrays (4x 44000 genes did not show any change versus unexposed cells after exposure to 10 μg/ mL, which

  12. Digestive stability, micellarization, and uptake by Caco-2 human intestinal cell of chlorophyll derivatives from different preparations of pea (Pisum sativum L.).

    Science.gov (United States)

    Gallardo-Guerrero, Lourdes; Gandul-Rojas, Beatriz; Mínguez-Mosquera, M Isabel

    2008-09-24

    The digestive stability, efficiency of micellarization, and cellular accumulation of the chlorophyll pigments of different preparations of pea were investigated, using an in vitro digestion procedure coupled with human intestinal Caco-2 cells. Fresh pea (FP), cooked fresh pea (CFP), frozen pea (FZP), cooked frozen pea (CFZP), and canned pea (CP) were subjected to simulated digestion. Although after digestion the pigment profile was modified for all samples, except CP, allomerization reactions and greater destruction of chlorophylls were observed in only FP, which should be due to enzymes in FP that were denaturalized in the rest of the test foods. A pigment extract of CFZP was also subjected to in vitro digestion, showing a positive effect of the food matrix on the pigment digestive stability. The transfer of the chlorophyll pigments from the digesta to the micellar fraction was significantly more efficient in CFZP (57%, p 0.05) different between CFP, FZP, and CP (28-35%), and lowest in FP (20%). Pheophorbide a stood out as the most-micellarized chlorophyll derivative in all of the samples, reaching levels of up to 98%. Incubation of Caco-2 cells with micellar fractions at the same concentration prepared from each test food showed that pigment absorption was considerably lower ( p < 0.006) in cells incubated with FP, whereas there were no differences among the rest of the preparations. Therefore, factors associated with the food matrix could inhibit or mediate the chlorophyll pigment absorption. These results demonstrated that the industrial preservation processes of peafreezing and canningas well as the cooking have a positive effect on the bioaccessibility and bioavailability of the chlorophyll pigments with respect to the FP sample, emphasizing CFZP with greater bioaccesibilty degree.

  13. Baicalin pharmacokinetic profile of absorption process using novel in-vitro model: cytochrome P450 3A4-induced Caco-2 cell monolayers combined with rat intestinal rinse fluids.

    Science.gov (United States)

    Morisaki, Tomoko; Hou, Xiao-Long; Takahashi, Kyoko; Takahashi, Koichi

    2013-10-01

    This study was designed to investigate baicalin (BG) pharmacokinetic profile in absorption process using a new model and evaluate the potentiality as a new model. The effects of BG on intestinal cytochrome P450 3A4 (CYP3A) protein/mRNA expression, activity and permeability glycoprotein (P-gp) were evaluated in CYP3A4-induced Caco-2 cell monolayers or rats. Intestinal rinse fluids (IF) were obtained from rat were added to modified Caco-2 monolayers. Orally administered BG (7 days pretreatment) inhibited intestinal CYP3A activity and protein expression. Baicalein (B) converted from BG by IF was detected in the upper jejunum in a portion-dependent manner. Subsequently, most BG were converted to B in the caecum. In modified Caco-2 monolayers, BG exhibited no effect on CYP3A4 activity or mRNA, whereas B and BG treated with IF inhibited CYP3A4 transcription and activity. Intestinal CYP3A was inhibited following oral administration of BG to rat. Correspondingly, BG-mediated CYP3A inhibition was shown in vitro using modified Caco-2 monolayers treated with IF. Hence, in-vivo intestinal absorption pharmacokinetic was reproduced in vitro. IF is a key determinant of intestinal absorption, and it facilitated inhibition of CYP3A by B, not BG. © 2013 Royal Pharmaceutical Society.

  14. 1-alpha,25-Dihydroxyvitamin D3up-regulates the expression of 2 types of human intestinal alkaline phosphatase alternative splicing variants in Caco-2 cells and may be an important regulator of their expression in gut homeostasis.

    Science.gov (United States)

    Noda, Seiko; Yamada, Asako; Nakaoka, Kanae; Goseki-Sone, Masae

    2017-10-01

    Vitamin D insufficiency is associated with a greater risk of osteoporosis and also influences skeletal muscle functions, differentiation, and development. The principal function of vitamin D in calcium homeostasis is to increase the absorption of calcium from the intestine, and the level of alkaline phosphatase (ALP) activity, a differentiation marker for intestinal epithelial cells, is regulated by vitamin D. Intestinal-type ALP is expressed at a high concentration in the brush border membrane of intestinal epithelial cells, and is known to be affected by several kinds of nutrients. Recent reviews have highlighted the importance of intestinal-type ALP in gut homeostasis. Intestinal-type ALP controls bacterial endotoxin-induced inflammation by dephosphorylating lipopolysaccharide and is a gut mucosal defense factor. In this study, we investigated the influence of vitamin D on the expression of 2 types of alternative mRNA variants encoding the human alkaline phosphatase, intestinal (ALPI) gene in human Caco-2 cells as an in vitro model of the small intestinal epithelium. After treatment with 1-alpha,25-dihydroxyvitamin D 3 , the biologically active form of vitamin D 3 , there were significant increases in the ALP activities of Caco-2 cells. Inhibitor and thermal inactivation experiments showed that the increased ALP had properties of intestinal-type ALP. Reverse transcription-polymerase chain reaction analysis revealed that expression of the 2 types of alternative mRNA variants from the ALPI gene was markedly enhanced by vitamin D in Caco-2 cells. In conclusion, these findings agree with the hypothesis: vitamin D up-regulated the expression of 2 types of human intestinal alkaline phosphatase alternative splicing variants in Caco-2 cells; vitamin D may be an important regulator of ALPI gene expression in gut homeostasis. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Zinc enhances intestinal epithelial barrier function through the PI3K/AKT/mTOR signaling pathway in Caco-2 cells.

    Science.gov (United States)

    Shao, Yuxin; Wolf, Patricia G; Guo, Shuangshuang; Guo, Yuming; Gaskins, H Rex; Zhang, Bingkun

    2017-05-01

    Zinc plays an important role in maintaining intestinal barrier function as well as modulating cellular signaling recognition and protein kinase activities. The phosphatidylinositol 3-kinase (PI3K) cascade has been demonstrated to affect intercellular integrity and tight junction (TJ) proteins. The current study investigated the hypothesis that zinc regulates intestinal intercellular junction integrity through the PI3K/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway. A transwell model of Caco-2 cell was incubated with 0, 50 and 100 μM of zinc at various time points. Transepithelial electrical resistance (TEER), paracellular permeability, TJ proteins, cell proliferation, differentiation and cell damage were measured. Compared with controls, 50 and 100 μM of zinc increased cell growth at 6, 12 and 24 h and the expression of proliferating cell nuclear antigen at 24 h. Zinc (100 μM) significantly elevated TEER at 6-24 h and reduced TJ permeability at 24 h, accompanied by the up-regulation of alkaline phosphatase (AP) activity and zonula occludens (ZO)-1 expression. In addition, zinc (100 μM) affected the PI3K/AKT/mTOR pathway by stimulating phosphorylation of AKT and the downstream target mTOR. Inhibition of PI3K signaling by LY294002 counteracted zinc promotion, as shown by a decrease in AP activity, TEER, the abundance of ZO-1 and phosphorylation of AKT and mTOR. Additionally, TJ permeability and the expression of caspase-3 and LC3II (markers of cell damage) were increased by addition of PI3K inhibitor. In conclusion, the activation of PI3K/AKT/mTOR signaling by zinc is involved in improving intestinal barrier function by enhancing cell differentiation and expression of TJ protein ZO-1. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Transport of chlorpromazine in the Caco-2 cell permeability assay: a kinetic study

    NARCIS (Netherlands)

    Broeders, J.J.W.; Eijkeren, J.C.H.; Blaauboer, B.J.; Hermens, J.L.M.

    2012-01-01

    The intestinal transport of compounds can be measured in vitro with Caco-2 cell monolayers. We took a closer look at the exposure and fate of a chemical in the Caco-2 cell assay, including the effect of protein binding. Transport of chlorpromazine (CPZ) was measured in the absorptive and secretory

  17. The proton-coupled amino acid transporter hPAT1 is the main transporter involved in vigabatrin uptake in intestinal Caco-2 cells

    DEFF Research Database (Denmark)

    Nøhr, Martha Kampp; Hansen, Steen Honore'; Brodin, Birger

    2012-01-01

    transporter hPAT1. The aim of the project was to identify if transporters are involved in cellular uptake of vigabatrin in Caco-2 cells. Methods: The uptake rate of vigabatrin was measured in Caco-2 cells at pH 6.0 or 7.4 for 15 min after application of 0.1 – 25.0 mM vigabatrin. The inhibitory effect...... of selected amino acids and -derivatives on the apical vigabatrin uptake in Caco-2 cells was investigated. Vigabatrin samples were analyzed using liquid chromatography (LC) coupled to a mass selective detector (MSD). Results: The uptake rate of vigabatrin in Caco-2 cells was pH-dependent. The uptake...... of vigabatrin was saturable at pH 6.0 with a Michaelis constant, Km of 12.7 ± 3.7 mM and a maximal flux, Jmax of 3.7 ± 0.5 nmol•min-1•cm-2. The presences of hPAT1 ligands significantly inhibited the uptake of vigabatrin in Caco-2 cells at pH 6.0, whereas hPAT1 non-ligands did not. Discussion: The saturability...

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

    Science.gov (United States)

    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.

  19. Transport of curcumin derivatives in Caco-2 cell monolayers.

    Science.gov (United States)

    Zeng, Zhen; Shen, Zhe L; Zhai, Shuo; Xu, Jia L; Liang, Hui; Shen, Qin; Li, Qing Y

    2017-08-01

    Curcumin (Cur) is a strong natural antioxidant, who can prevent multiple diseases such as anti-cancer, anti-inflammatory, have a resistance to alzheimer's disease and various malignant diseases. But it has poor oral bioavailability due to its poor aqueous solubility, as well as instability. While its novel derivatives (CB and FE), showed better anti-tumor activity, better anti-oxidant activity and better stability than the original drug (Cur). The aim of this study was to study the intestinal transport of Cur, CB and FE using an in vitro Caco-2 cell monolayer model. The results showed that Cur had a lower permeability coefficient (1.13×10 -6 ±0.11×10 -6 cm/s) for apical-to-basolated (AP-BL) transport at 25μM, while the transport rate for AP to BL flux of CB (3.18×10 -6 ±0.31×10 -6 cm/s) and FE (5.28×10 -6 ±0.83×10 -6 cm/s) were significantly greater than that of Cur. The efflux ratio (ER) value at the concentration of 25μM was 1.31 for Cur, 1.26 for CB and 1.33 for FE, suggesting there was no active efflux involved in the translocation across the Caco-2 cell monolayers for the three compounds. Furthermore, the transport flux of CB and FE was in a concentration dependent manner, suggesting the intestinal transport mechanism in them was passive transport. In summary, the results demonstrated that both the intestinal permeability of CB and FE across Caco-2 cell monolayers was significantly improved compare to Cur. Thus they might show a higher oral bioavailability in vivo, and show the potential application in clinic or nutraceutical. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. SGLT1-Mediated Transport in Caco-2 Cells Is Highly Dependent on Cell Bank Origin

    DEFF Research Database (Denmark)

    Steffansen, Bente; Pedersen, Maria D L; Laghmoch, Abdel M

    2017-01-01

    The human colon adenocarcinoma (Caco-2) cell line is a well-established in vitro model for studying transport phenomena for prediction of intestinal nutrient and drug absorption. However, substances depending on transporters such predictions are complicated due to variable transporter expression...... and limited knowledge about transporter function during multiple cell passaging and cell thawings. In the case of sodium glucose transporter 1 (SGLT1), a key transporter of oral absorption of d-glucose, one reason for compromised prediction could be inadequate expression of SGLT1 in Caco-2 cells and thereby...... limited sensitivity in the determination of SGLT1-mediated permeability (PSGLT1). Here, the objective is to characterize and compare SGLT1-mediated uptake in Caco-2 cells obtained from different cell banks. SGLT1-mediated uptake of the standard SGLT1 substrate, methyl-α-d-glucopyranoside, in Caco-2 cells...

  1. Glutathione S-transferase expression and isoenzyme composition during cell differentiation of Caco-2 cells

    International Nuclear Information System (INIS)

    Scharmach, E.; Hessel, S.; Niemann, B.; Lampen, A.

    2009-01-01

    The human colon adenocarcinoma cell line Caco-2 is frequently used to study human intestinal metabolism and transport of xenobiotica. Previous studies have shown that both Caco-2 cells and human colon cells constitutively express the multigene family of detoxifying enzymes glutathione S-transferases (GSTs), particularly GST alpha and GST pi. GSTs may play a fundamental role in the molecular interplay between phase I, II enzymes and ABC-transporters. The gut fermentation product, butyrate, can modulate the potential for detoxification. The aim of this study was to investigate the basal expression of further cytosolic GSTs in Caco-2 cells during cell differentiation. In addition, a comparison was made with expression levels in MCF-7 and HepG2, two other cell types with barrier functions. Finally, the butyrate-mediated modulation of gene and protein expression was determined by real time PCR and western blot analysis. In Caco-2, gene and protein expression levels of GST alpha increased during cell differentiation. High levels of GSTO1 and GSTP1 were constantly expressed. No expression of GSTM5 and GSTT1 was detected. HepG2 expressed GSTO1 and MCF-7 GSTZ1 most intensively. No expression of GSTA5, GSTM5, or GSTP1 was detected in either cell. Incubation of Caco-2 cells with butyrate (5 mM) significantly induced GSTA1 and GSTM2 in proliferating Caco-2 cells. In differentiated cells, butyrate tended to increase GSTO1 and GSTP1. The results of this study show that a differentiation-dependent expression of GSTs in Caco-2 cells may reflect the in vivo situation and indicate the potential of butyrate to modify intestinal metabolism. GSTA1-A4 have been identified as good markers for cell differentiation. The Caco-2 cell line is a useful model for assessing the potential of food-related substances to modulate the GST expression pattern.

  2. 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

    , and methylseleninic acid (MeSeA), were applied to Caco-2 cells in a concentration of 10 μM, and fluxes in both directions were studied for 2 h. Fluxes of selenite and MeSeA in the presence of excess reduced glutathione (selenite + GSH and MeSeA + GSH) and flux of MeSeA in the presence of excess cysteine (MeSeA + Cys...

  3. Interaction of cruciferin-based nanoparticles with Caco-2 cells and Caco-2/HT29-MTX co-cultures.

    Science.gov (United States)

    Akbari, Ali; Lavasanifar, Afsaneh; Wu, Jianping

    2017-12-01

    The objective of this work was to assess the potential of Cruciferin/Calcium (Cru/Ca) and Cruciferin/Chitosan (Cru/Cs) nanoparticles for oral drug delivery. For this purpose, Cru/Ca and Cru/Cs nanoparticles were developed through cold gelation of Cruciferin, a major canola protein, and in interaction with calcium and chitosan, respectively. The extent and rate of particle uptake in Caco-2 cells and Caco-2/HT29 co-culture was then evaluated by fluorescence spectroscopy as well as flow cytometry. Through pre-incubation of Caco-2 cell monolayer with specific endocytosis inhibitors, the mechanism of cell uptake was investigated. Our results showed that the uptake of negatively-charged Cru/Ca particles to be ∼3 times higher than positively-charged Cru/Cs ones by Caco-2 cells. Presence of mucus secreted by HT29 cells in their co-culture with Caco-2 had negligible influence on the uptake and transport of both particles. In contrast to Cru/Ca particles which were dissociated in the simulated gastrointestinal conditions, digestion of Cru/Cs particles resulted in 6- and 2-fold increase in the cellular uptake and transport of encapsulated coumarin in the latter particles, respectively. While the presence of mucus in Caco-2/HT29 co-culture caused 40-50% decrease of cellular uptake and transport for coumarin encapsulated in digested Cru/Cs particles, it had no significant effect on the cell uptake and transport of coumarin associated with Cru/Ca particles after digestion. Energy-dependent mechanisms were the dominant mechanism for uptake of both undigested and digested particles. Therefore, in Caco-2/HT29 co-culture which closely simulated intestinal epithelial cells, undigested Cru/Ca and Cru/Cs particles had the ability to penetrate mucus layers, while digested Cru/Cs particles showed mucoadhesive property, and digested Cru/Ca particles were dissociated. Our results points to a potential for cruciferin based nanoparticles for oral drug delivery. The long-term objective of

  4. Determination of the active transport of fucoidan derived from Okinawa Mozuku across the human intestinal Caco-2 cells as assessed by size-exclusion chromatography.

    Science.gov (United States)

    Nagamine, Takeaki; Hayakawa, Kou; Nakazato, Kyoumi; Iha, Masahiko

    2015-08-01

    In order to clarify the mechanism of fucoidan transport, we developed the chromatographic determination method. A size-exclusion chromatography (SEC) method for the determination of Okinawa-fucoidan using Develosil 300 Diol-5 (60×8.0mm I.D., 30nm pore-diameter) with the eluent containing 1% non-ionic detergent is developed. Determination range (UV at 210nm) is from 0 to 100ng of fucoidan with the linear calibration line inserting to zero. A transport activity of fucoidan is demonstrated by using Caco-2 cells (model of gut transport system); i.e., the initial transport velocity 12nmol/h/mg of protein (25-fold slower rate as compared to a bacterial l-alanine active-transport activity 300nmol/h/mg of protein) is found to occur. Since this fucoidan transport is inhibited by 10mM sodium azide (respiration inhibitor) and 0.05mM FCCP (uncoupler), this transport by Caco-2 cells is found to be an active one requiring energy-source. On the other hand, colchicine (inhibitor of phagocytosis/pinocytosis) and mannitol (putative competitive-inhibitor of tight-junction transport) cannot inhibit the fucoidan transport at all. We firstly report that the active transport occurs for such a high molecular-weight sulphated-polyfucose of fucoidan in vitro using Caco-2 cells. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Characterization of zinc (Zn) transport across Caco-2 cell monolayers

    Energy Technology Data Exchange (ETDEWEB)

    Wapnir, R.A.; Raffaniello, R.; Shihyu Lee (North Shore Univ. Hospital, Manhasset, NY (United States))

    1991-03-15

    Colonic carcinoma Caco-2 cells grown as a monolayer exhibit many of the characteristics of the small intestine including inducibility of metallothionein by Zn. The authors have now characterized the transport of Zn across confluent, 14 day in vitro Caco-2 monolayers grown on polycarbonate support membranes. Both uptake and transport across the cells were linear for at least 10 min. Zn cell uptake in the mucosa-to-serosa (m-s) direction was saturable, with a K{sub t} = 23.8 {plus minus} 7.8 {mu}M. J{sub max} was 0.46 {plus minus} 0.08 {mu}mol/cm{sup 2} {times} 10 min. Transcellular passage was concentration dependent. In the (s-m) direction, the K{sub t} of Zn cell uptake was 59 {plus minus} 13 {mu}M and that of the transcellular transport was 103 {plus minus} 53 {mu}M. Cell uptake J{sub max} was 0.98 {plus minus} 0.11 {mu}mol/cm{sup 2} {times} 10 min, and transcellular transport J{sub max} was 2.90 {plus minus} 0.47 {mu}mol/cm{sup 2} {times} 10 min. Low molecular weight ligands (histidine, cysteine, proline or glutathione at 1 mM each), added to the mucosal side, did not alter Zn cell uptake or transmembrane passage. Potential transport inhibitors such as ouabain or vanadate, or metabolic inhibitors also had no effect. Zn transport and cell uptake by Caco-2 cells are kinetically comparable to those of in vivo systems and could serve as a valid model for Zn absorption studies.

  6. Highly variable contents of phenolics in St. John's Wort products affect their transport in the human intestinal Caco-2 cell model: pharmaceutical and biopharmaceutical rationale for product standardization.

    Science.gov (United States)

    Gao, Song; Jiang, Wen; Yin, Taijun; Hu, Ming

    2010-06-09

    The purposes of this study were to determine content uniformity of phenolics in the St. John's wort (SJW) supplements and to demonstrate how variations in the product matrices affect their absorption and efflux. LC and LC-MS/MS methods were used to determine the phenolic contents of 12 different products purchased locally or from the Internet. Three representative extracts were further submitted to Caco-2 cell transport experiment, and transport of rutin, hyperoside, and isoquercitrin was evaluated. The results indicated that the 12 different products displayed 12 different HPLC fingerprints, but all products contained the following major compounds: rutin, hyperoside, isoquercitrin, quercitrin, quercetin, and amentoflavone. The content uniformity of these major compounds was poor across products, with the smallest difference in the amounts of amentoflavone (3.6-fold) and largest difference in that of isoquercitrin (28.8-fold). The Caco-2 experiments indicated transport of rutin in products was vectorial, with the permeabilities varied about 3.6-fold in both directions of transport. The vectorial permeabilities of hyperoside and isoquercitrin were similarly different. Use of efflux transporter inhibitor studies suggested that MRP2 was involved in isoquercitrin's efflux and the product matrix affected the extent of its efflux. In conclusion, different SJW supplements had highly variable contents of phenolics, and the variability in product matrix and phytochemical compositions affected the permeabilities of key phenolics across the Caco-2 monolayers, which may further affect their bioavailabilities. Therefore, standardization will be necessary to ensure safe and efficacious using of supplements such as SJW.

  7. Highly Variable Contents of Phenolics in St John's Wort Products Impact Their Transport in the Human Intestinal Caco-2 Cell Model: Pharmaceutical and Biopharmaceutical Rationale for Product Standardization

    Science.gov (United States)

    Gao, Song; Jiang, Wen; Yin, Taijun; Hu, Ming

    2010-01-01

    The purposes of this study were to determine content uniformity of phenolics in the St John's Wort (SJW) supplements, and to demonstrate how variations in the product matrices affect their absorption and efflux. LC and LC-MS/MS methods were used to determine the phenolic contents of twelve different products purchased locally or from the internet. Three representative extracts were further submitted to Caco-2 cell transport experiment and transport of rutin, hyperoside, and isoquercitrin were evaluated. The results indicated that twelve different products displayed twelve different HPLC fingerprints, but all products contained the following major compounds: rutin, hyperoside, isoquercitrin, quercitrin, quercetin, and amentoflavone. The content uniformity of these major compounds was poor across products, with the smallest difference in the amounts of amentoflavone (2.6 folds) and largest difference in that of isoquercitrin (28.8 folds). The Caco-2 experiments indicated transport of rutin was vectorial, with the permeabilities varied about 2 folds in both direction of transport. The vectorial permeabilities of hyperoside and isoquercitrin were similarly different. Use of efflux transporter inhibitors studies suggested that MRP2 was involved in isoquercitrin's efflux and the product matrix affected the extent of its efflux. In conclusion, different SJW supplements had highly variable contents of phenolics, and the variability in product matrix and phytochemical compositions affected the permeabilities of key phenolics across the Caco-2 monolayers, which may further impact their bioavailabilities. Therefore, standardization will be necessary to ensure safe and efficacious use of supplements such as SJW. PMID:20450158

  8. 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.

  9. 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

    2015-01-01

    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. PMID:26370977

  10. 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.

  11. Evaluation of a possible direct effect by casein phosphopeptides on paracellular and vitamin D controlled transcellular calcium transport mechanisms in intestinal human HT-29 and Caco2 cell lines.

    Science.gov (United States)

    Colombini, Alessandra; Perego, Silvia; Ardoino, Ilaria; Marasco, Emiliano; Lombardi, Giovanni; Fiorilli, Amelia; Biganzoli, Elia; Tettamanti, Guido; Ferraretto, Anita

    2013-08-01

    Intestinal cells are continuously exposed to food whose components are able to modulate some of their physiological functions. Among the bioactive food derivatives are casein phosphopeptides (CPPs), coming from the in vitro or in vivo casein digestion, which display the ability to form aggregates with calcium ions and to increase the uptake of the minerals in differentiated intestinal human HT-29 and Caco2 cells. Since extracellular calcium is a known inactivator of the TRPV6 channel, which is also involved in the colon cancer progression, the present study aims to determine a possible modulation by CPPs of the molecular structures responsible for paracellular and/or transcellular calcium absorption in these two cell lines. The paracellular calcium transport was determined by TEER measurements in Caco2 cells and by Lucifer Yellow flow in HT-29 cells. The possible modulation of transcellular calcium absorption machinery by CPPs was investigated by determining the mRNA expression for both the TRPV6 calcium channel and the VDR receptor in 1,25(OH)₂D₃ pre-treated undifferentiated/differentiated cells. The results obtained point out that: (i) CPPs do not affect paracellular calcium absorption; (ii) 1,25(OH)₂D₃ increases the TRPV6 mRNA expression in both types of cells. In the case of HT-29 cells this is the first determination of the presence of the TRPV6 channel; (iii) CPPs per se are not able to affect the VDR and TRPV6 mRNA expression; (iv) CPP administration does not affect the TRPV6 mRNA expression in 1,25(OH)₂D₃ pre-treated HT-29 cells and Caco2 cells. Unlike peptides coming from the digestion of cheese whey protein digest, the digestion of milk casein produces peptides with no effects on TRPV6 calcium channel expression, though the same peptides are able to determine a calcium uptake by the intestinal cells.

  12. Labile complexes facilitate cadmium uptake by Caco-2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Verheyen, L., E-mail: Liesbeth.Verheyen@ees.kuleuven.be [Division of Soil and Water Management, K.U.Leuven, Kasteelpark Arenberg 20, Box 2459, 3001 Heverlee (Belgium); Degryse, F. [Division of Soil and Water Management, K.U.Leuven, Kasteelpark Arenberg 20, Box 2459, 3001 Heverlee (Belgium); Niewold, T. [Division Animal-nutrition-quality, K.U.Leuven, Kasteelpark Arenberg 30, Box 2456, 3001 Heverlee (Belgium); Smolders, E. [Division of Soil and Water Management, K.U.Leuven, Kasteelpark Arenberg 20, Box 2459, 3001 Heverlee (Belgium)

    2012-06-01

    The Free Ion Activity Model (FIAM) predicts that metal uptake in biota is related to the free ion activity in the external solution and that metal complexes do not contribute. However, studies with plants have shown that labile metal complexes enhance metal bioavailability when the uptake is rate-limited by transport of the free ion in solution to the uptake site. Here, the role of labile complexes of Cd on metal bioavailability was assessed using Caco-2 cells, the cell model for intestinal absorption. At low Cd{sup 2+} concentration (1 nM), the CdCl{sub n}{sup 2-n} complexes contributed to the uptake almost to the same extent as the free ion. At large Cd{sup 2+} concentration (10 {mu}M), the contribution of the complexes was much smaller. At constant Cd{sup 2+} concentration, Cd intake in the cells from solutions containing synthetic ligands such as EDTA increased as the dissociation rate of the cadmium complexes increased, and correlated well with the Cd diffusion flux in solution measured with the Diffusive Gradient in Thin Films technique (DGT). The Cd intake fluxes in the cells were well predicted assuming that the specific uptake is limited by diffusion of the free Cd{sup 2+} ion to the cell surface. Our results underline that speciation of Cd has a major effect on its uptake by intestinal cells, but the availability is not simply related to the free ion concentration. Labile complexes of Cd enhance metal bioavailability in these cells, likely by alleviating diffusive limitations. - Highlights: Black-Right-Pointing-Pointer We examined the role of labile complexes on the uptake of Cd and Zn by Caco-2 cells. Black-Right-Pointing-Pointer The availability of Cd and Zn is not simply related to the free ion activity. Black-Right-Pointing-Pointer Labile complexes of Cd enhance metal bioavailability in the Caco-2 cells at low free ion activities. Black-Right-Pointing-Pointer The active uptake is limited by diffusion of the free Cd{sup 2+} ion to the cell surface.

  13. SGLT1-Mediated Transport in Caco-2 Cells Is Highly Dependent on Cell Bank Origin.

    Science.gov (United States)

    Steffansen, Bente; Pedersen, Maria D L; Laghmoch, Abdel M; Nielsen, Carsten U

    2017-09-01

    The human colon adenocarcinoma (Caco-2) cell line is a well-established in vitro model for studying transport phenomena for prediction of intestinal nutrient and drug absorption. However, substances depending on transporters such predictions are complicated due to variable transporter expression and limited knowledge about transporter function during multiple cell passaging and cell thawings. In the case of sodium glucose transporter 1 (SGLT1), a key transporter of oral absorption of d-glucose, one reason for compromised prediction could be inadequate expression of SGLT1 in Caco-2 cells and thereby limited sensitivity in the determination of SGLT1-mediated permeability (P SGLT1 ). Here, the objective is to characterize and compare SGLT1-mediated uptake in Caco-2 cells obtained from different cell banks. SGLT1-mediated uptake of the standard SGLT1 substrate, methyl-α-d-glucopyranoside, in Caco-2 cells was shown to be highly dependent on cell bank origin. The most robust and reliable SGLT1 functionality was identified in Caco-2 cells from Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ), whereas cells from the American Type Culture Collection and European Collection of Authenticated Cell Cultures have lower SGLT1 transport activity. Transepithelial P SGLT1 across Caco-2 cells from DSMZ showed that P SGLT1 likely accounts for approximately 97% of absorptive methyl-α-d-glucopyranoside P app(a-b) . In conclusion, Caco-2 cells from DSMZ provide a robust in vitro model for studying SGLT1-mediated uptake and transport-over multiple cell passages and independent cell stock thawings. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  14. The effect of hyperosmosis on paracellular permeability in Caco-2 cell monolayers.

    Science.gov (United States)

    Inokuchi, Hitoshi; Takei, Takuto; Aikawa, Katsuyoshi; Shimizu, Makoto

    2009-02-01

    The intestinal epithelium is a significant barrier to oral absorption of hydrophilic compounds, and their passage through the intercellular space is restricted by the tight junctions. In this study we found that hyperosmosis is a significant factor altering paracellular transport in Caco-2 cell monolayers. Osmotic regulators, such as sodium chloride, mannitol, and raffinose, decreased transepithelial electrical resistance and enhanced lucifer yellow permeability. The effect of these osmotic regulators on Caco-2 cell monolayers was not likely to be caused by gross cytotoxicity. Although certain amino acids and oligosaccharides have been reported to have specific tight junction-modulating activity, we found that the increased paracellular permeability of Caco-2 monolayers induced by these compounds was at least partly due to the increased osmotic pressure of the test solutions. These findings provide a new potential precaution in the evaluation of paracellular permeability-modulating substances using the Caco-2 cell monolayer system.

  15. Different sucrose-isomaltase response of Caco-2 cells to glucose and maltose suggests dietary maltose sensing

    Science.gov (United States)

    Using the small intestine enterocyte Caco-2 cell model, sucrase-isomaltase (SI, the mucosal alpha-glucosidase complex) expression and modification were examined relative to exposure to different mono- and disaccharide glycemic carbohydrates. Caco-2/TC7 cells were grown on porous supports to post-con...

  16. Shiga Toxin Glycosphingolipid Receptors in Human Caco-2 and HCT-8 Colon Epithelial Cell Lines.

    Science.gov (United States)

    Kouzel, Ivan U; Pohlentz, Gottfried; Schmitz, Julia S; Steil, Daniel; Humpf, Hans-Ulrich; Karch, Helge; Müthing, Johannes

    2017-10-25

    Shiga toxins (Stxs) released by enterohemorrhagic Escherichia coli (EHEC) into the human colon are the causative agents for fatal outcome of EHEC infections. Colon epithelial Caco-2 and HCT-8 cells are widely used for investigating Stx-mediated intestinal cytotoxicity. Only limited data are available regarding precise structures of their Stx receptor glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), and lipid raft association. In this study we identified Gb3Cer and Gb4Cer lipoforms of serum-free cultivated Caco-2 and HCT-8 cells, chiefly harboring ceramide moieties composed of sphingosine (d18:1) and C16:0, C22:0 or C24:0/C24:1 fatty acid. The most significant difference between the two cell lines was the prevalence of Gb3Cer with C16 fatty acid in HCT-8 and Gb4Cer with C22-C24 fatty acids in Caco-2 cells. Lipid compositional analysis of detergent-resistant membranes (DRMs), which were used as lipid raft -equivalents, indicated slightly higher relative content of Stx receptor Gb3Cer in DRMs of HCT-8 cells when compared to Caco-2 cells. Cytotoxicity assays revealed substantial sensitivity towards Stx2a for both cell lines, evidencing little higher susceptibility of Caco-2 cells versus HCT-8 cells. Collectively, Caco-2 and HCT-8 cells express a plethora of different receptor lipoforms and are susceptible towards Stx2a exhibiting somewhat lower sensitivity when compared to Vero cells.

  17. Experimental Evaluation of the Transport Mechanisms of PoIFN-α in Caco-2 Cells.

    Science.gov (United States)

    Liu, Xin; Zheng, Sidi; Qin, Yue; Ding, Wenya; Tu, Yabin; Chen, Xingru; Wu, Yunzhou; Yanhua, Li; Cai, Xuehui

    2017-01-01

    For the development of an efficient intestinal delivery system for Porcine interferon-α (PoIFN-α), the understanding of transport mechanisms of which in the intestinal cell is essential. In this study, we investigated the absorption mechanisms of PoIFN-α in intestine cells. Caco-2 cells and fluorescein isothiocyanate-labeled (FITC)-PoIFN-α were used to explore the whole transport process, including endocytosis, intracellular trafficking, exocytosis, and transcytosis. Via various techniques, the transport pathways of PoIFN-α in Caco-2 cells and the mechanisms were clarified. Firstly, the endocytosis of PoIFN-α by Caco-2 cells was time, concentration and temperature dependence. And the lipid raft/caveolae endocytosis was the most likely endocytic pathway for PoIFN-α. Secondly, both Golgi apparatus and lysosome were involved in the intracellular trafficking of PoIFN-α. Thirdly, the treatment of indomethacin resulted in a significant decrease of exocytosis of PoIFN-α, indicating the participation of cyclooxygenase. Finally, to evaluate the efficiency of PoIFN-α transport, the transepithelial electrical resistance (TEER) value was measured to investigate the tight junctional integrity of the cell monolayers. The fluorescence microscope results revealed that the transport of PoIFN-α across the Caco-2 cell monolayers was restricted. In conclusion, this study depicts a probable picture of PoIFN-α transport in Caco-2 cells characterized by non-specificity, partial energy-dependency and low transcytosis.

  18. Experimental Evaluation of the Transport Mechanisms of PoIFN-α in Caco-2 Cells

    Directory of Open Access Journals (Sweden)

    Xin Liu

    2017-11-01

    Full Text Available For the development of an efficient intestinal delivery system for Porcine interferon-α (PoIFN-α, the understanding of transport mechanisms of which in the intestinal cell is essential. In this study, we investigated the absorption mechanisms of PoIFN-α in intestine cells. Caco-2 cells and fluorescein isothiocyanate-labeled (FITC-PoIFN-α were used to explore the whole transport process, including endocytosis, intracellular trafficking, exocytosis, and transcytosis. Via various techniques, the transport pathways of PoIFN-α in Caco-2 cells and the mechanisms were clarified. Firstly, the endocytosis of PoIFN-α by Caco-2 cells was time, concentration and temperature dependence. And the lipid raft/caveolae endocytosis was the most likely endocytic pathway for PoIFN-α. Secondly, both Golgi apparatus and lysosome were involved in the intracellular trafficking of PoIFN-α. Thirdly, the treatment of indomethacin resulted in a significant decrease of exocytosis of PoIFN-α, indicating the participation of cyclooxygenase. Finally, to evaluate the efficiency of PoIFN-α transport, the transepithelial electrical resistance (TEER value was measured to investigate the tight junctional integrity of the cell monolayers. The fluorescence microscope results revealed that the transport of PoIFN-α across the Caco-2 cell monolayers was restricted. In conclusion, this study depicts a probable picture of PoIFN-α transport in Caco-2 cells characterized by non-specificity, partial energy-dependency and low transcytosis.

  19. 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.

  20. 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)

    O'Brien, Patrick; Corpe, Christopher Peter

    2016-01-01

    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. To study the direct model of intestinal sugar transport using CaCo-2 cells, a well-established in vitro model of the human enterocyte. 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. 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. 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 inhibitory effect on glucose transport

  1. 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.

    Directory of Open Access Journals (Sweden)

    Patrick O'Brien

    Full Text Available 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.To study the direct model of intestinal sugar transport using CaCo-2 cells, a well-established in vitro model of the human enterocyte.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.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.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 inhibitory effect on glucose

  2. Low uptake of silica nanoparticles in Caco-2 intestinal epithelial barriers.

    Science.gov (United States)

    Ye, Dong; Bramini, Mattia; Hristov, Delyan R; Wan, Sha; Salvati, Anna; Åberg, Christoffer; Dawson, Kenneth A

    2017-01-01

    Cellular barriers, such as the skin, the lung epithelium or the intestinal epithelium, constitute one of the first obstacles facing nanomedicines or other nanoparticles entering organisms. It is thus important to assess the capacity of nanoparticles to enter and transport across such barriers. In this work, Caco-2 intestinal epithelial cells were used as a well-established model for the intestinal barrier, and the uptake, trafficking and translocation of model silica nanoparticles of different sizes were investigated using a combination of imaging, flow cytometry and transport studies. Compared to typical observations in standard cell lines commonly used for in vitro studies, silica nanoparticle uptake into well-developed Caco-2 cellular barriers was found to be very low. Instead, nanoparticle association to the apical outer membrane was substantial and these particles could easily be misinterpreted as internalised in the absence of imaging. Passage of nanoparticles through the barrier was very limited, suggesting that the low amount of internalised nanoparticles was due to reduced uptake into cells, rather than a considerable transport through them.

  3. Alpha-Melanocyte Stimulating Hormone Protects against Cytokine-Induced Barrier Damage in Caco-2 Intestinal Epithelial Monolayers.

    Directory of Open Access Journals (Sweden)

    Judit Váradi

    Full Text Available Alpha-melanocyte-stimulating hormone (α-MSH is a potent anti-inflammatory peptide with cytoprotective effect in various tissues. The present investigation demonstrates the ability of α-MSH to interact with intestinal epithelial cell monolayers and mitigate inflammatory processes of the epithelial barrier. The protective effect of α-MSH was studied on Caco-2 human intestinal epithelial monolayers, which were disrupted by exposure to tumor necrosis factor-α and interleukin-1β. The barrier integrity was assessed by measuring transepithelial electric resistance (TEER and permeability for marker molecules. Caco-2 monolayers were evaluated by immunohistochemistry for expression of melanocortin-1 receptor and tight junction proteins ZO-1 and claudin-4. The activation of nuclear factor kappa beta (NF-κB was detected by fluorescence microscopy and inflammatory cytokine expression was assessed by flow cytometric bead array cytokine assay. Exposure of Caco-2 monolayers to proinflammatory cytokines lowered TEER and increased permeability for fluorescein and albumin, which was accompanied by changes in ZO-1 and claudin-4 immunostaining. α-MSH was able to prevent inflammation-associated decrease of TEER in a dose-dependent manner and reduce the increased permeability for paracellular marker fluorescein. Further immunohistochemistry analysis revealed proinflammatory cytokine induced translocation of the NF-κB p65 subunit into Caco-2 cell nuclei, which was inhibited by α-MSH. As a result the IL-6 and IL-8 production of Caco-2 monolayers were also decreased with different patterns by the addition of α-MSH to the culture medium. In conclusion, Caco-2 cells showed a positive immunostaining for melanocortin-1 receptor and α-MSH protected Caco-2 cells against inflammatory barrier dysfunction and inflammatory activation induced by tumor necrosis factor-α and interleukin-1β cytokines.

  4. Alpha-Melanocyte Stimulating Hormone Protects against Cytokine-Induced Barrier Damage in Caco-2 Intestinal Epithelial Monolayers

    Science.gov (United States)

    Váradi, Judit; Harazin, András; Fenyvesi, Ferenc; Réti-Nagy, Katalin; Gogolák, Péter; Vámosi, György; Bácskay, Ildikó; Fehér, Pálma; Ujhelyi, Zoltán; Vasvári, Gábor; Róka, Eszter; Haines, David; Deli, Mária A.; Vecsernyés, Miklós

    2017-01-01

    Alpha-melanocyte-stimulating hormone (α-MSH) is a potent anti-inflammatory peptide with cytoprotective effect in various tissues. The present investigation demonstrates the ability of α-MSH to interact with intestinal epithelial cell monolayers and mitigate inflammatory processes of the epithelial barrier. The protective effect of α-MSH was studied on Caco-2 human intestinal epithelial monolayers, which were disrupted by exposure to tumor necrosis factor-α and interleukin-1β. The barrier integrity was assessed by measuring transepithelial electric resistance (TEER) and permeability for marker molecules. Caco-2 monolayers were evaluated by immunohistochemistry for expression of melanocortin-1 receptor and tight junction proteins ZO-1 and claudin-4. The activation of nuclear factor kappa beta (NF-κB) was detected by fluorescence microscopy and inflammatory cytokine expression was assessed by flow cytometric bead array cytokine assay. Exposure of Caco-2 monolayers to proinflammatory cytokines lowered TEER and increased permeability for fluorescein and albumin, which was accompanied by changes in ZO-1 and claudin-4 immunostaining. α-MSH was able to prevent inflammation-associated decrease of TEER in a dose-dependent manner and reduce the increased permeability for paracellular marker fluorescein. Further immunohistochemistry analysis revealed proinflammatory cytokine induced translocation of the NF-κB p65 subunit into Caco-2 cell nuclei, which was inhibited by α-MSH. As a result the IL-6 and IL-8 production of Caco-2 monolayers were also decreased with different patterns by the addition of α-MSH to the culture medium. In conclusion, Caco-2 cells showed a positive immunostaining for melanocortin-1 receptor and α-MSH protected Caco-2 cells against inflammatory barrier dysfunction and inflammatory activation induced by tumor necrosis factor-α and interleukin-1β cytokines. PMID:28103316

  5. Evaluation of drug permeation under fed state conditions using mucus-covered Caco-2 cell epithelium

    DEFF Research Database (Denmark)

    Birch, Ditlev; Diedrichsen, Ragna G; Christophersen, Philip C

    2018-01-01

    The absence of a surface-lining mucus layer is a major pitfall for the Caco-2 epithelial model. However, this can be alleviated by applying biosimilar mucus (BM) to the apical surface of the cell monolayer, thereby constructing a mucosa mimicking in vivo conditions. This study aims to elucidate...... the influence of BM as a barrier towards exogenic compounds such as permeation enhancers, and components of fed state simulated intestinal fluid (FeSSIF). Caco-2 cell monolayers surface-lined with BM were exposed to several compounds with distinct physicochemical properties, and the cell viability...... and permeability of the cell monolayer was compared to that of cell monolayers without BM and well-established mucus-secreting epithelial models (HT29 monolayers and HT29/Caco-2 co-culture monolayers). Exposure of BM-covered cells to constituents from FeSSIF revealed that it comprised a strong, hydrophilic barrier...

  6. Interactions between organic anions on multiple transporters in Caco-2 cells

    DEFF Research Database (Denmark)

    Grandvuinet, Anne Sophie; Steffansen, Bente

    2011-01-01

    Caco-2 cell line may be used as an overall model to predict interactions on multiple membrane transporters in the intestine. Taurocholic acid (TCA) and estrone-3-sulfate (E1S) were used as model substrates. Possible inhibitors studied were TCA, E1S, taurolithocholic acid, fluvastatin, and glipizide...

  7. 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.

  8. Toxoplasma gondii Infection Promotes Epithelial Barrier Dysfunction of Caco-2 Cells

    Science.gov (United States)

    Briceño, Marisol Pallete; Nascimento, Layane Alencar Costa; Nogueira, Nathalia Pires; Barenco, Paulo Victor Czarnewski; Ferro, Eloisa Amália Vieira; Rezende-Oliveira, Karine; Goulart, Luiz Ricardo; Alves, Patrícia Terra; Barbosa, Bellisa de Freitas; Lima, Wânia Rezende; Silva, Neide Maria

    2016-01-01

    After oral infection, Toxoplasma gondii invades intestinal cells, induces breakdown of intestinal physiology and barrier functions, and causes intestinal pathology in some animal species. Although parasites’ invasion into host cells is a known phenomenon, the effects of T. gondii infection in the intestinal barrier are still not well established. To evaluate morphological and physiological modifications on the colorectal adenocarcinoma-derived Caco-2 cell line during T. gondii infection, microvilli, tight junction integrity, and transepithelial electrical resistance (TEER) were investigated under infection. It was observed that the dextran uptake (endocytosis) and distribution were smaller in infected than in noninfected Caco-2 cells. The infection leads to the partial loss of microvilli at the cell surface. Claudin-1, zonula occludens-1 (ZO-1), and occludin expressions were colocalized by immunofluorescence and presented discontinuous net patterns in infected cells. Immunoblotting analysis at 24 hr postinfection revealed decreasing expression of occludin and ZO-1 proteins, whereas claudin-1 presented similar expression level compared with noninfected cells. T. gondii decreased TEER in Caco-2 cells 24 hr after infection. Our results suggest that T. gondii infection may lead to the loss of integrity of intestinal mucosa, resulting in impaired barrier function. PMID:27370796

  9. Evaluation of drug permeation under fed state conditions using mucus-covered Caco-2 cell epithelium.

    Science.gov (United States)

    Birch, Ditlev; Diedrichsen, Ragna G; Christophersen, Philip C; Mu, Huiling; Nielsen, Hanne M

    2018-03-07

    The absence of a surface-lining mucus layer is a major pitfall for the Caco-2 epithelial model. However, this can be alleviated by applying biosimilar mucus (BM) to the apical surface of the cell monolayer, thereby constructing a mucosa mimicking in vivo conditions. This study aims to elucidate the influence of BM as a barrier towards exogenic compounds such as permeation enhancers, and components of fed state simulated intestinal fluid (FeSSIF). Caco-2 cell monolayers surface-lined with BM were exposed to several compounds with distinct physicochemical properties, and the cell viability and permeability of the cell monolayer was compared to that of cell monolayers without BM and well-established mucus-secreting epithelial models (HT29 monolayers and HT29/Caco-2 co-culture monolayers). Exposure of BM-covered cells to constituents from FeSSIF revealed that it comprised a strong, hydrophilic barrier effect as 90% of BM-covered cells remained viable for >4 h, and the permeation rate of hydrophobic drugs was reduced. In contrast, the permeation rate of hydrophilic drugs was largely unaffected. Control monolayers displayed a loss of barrier function and Caco-2 cell monolayers surface-lined with BM constitute a valuable in vitro model that makes it possible to mimic intestinal fed state conditions when studying drug permeation. Copyright © 2017. Published by Elsevier B.V.

  10. Human influenza A viruses are proteolytically activated and do not induce apoptosis in CACO-2 cells

    International Nuclear Information System (INIS)

    Zhirnov, Oleg; Klenk, Hans-Dieter

    2003-01-01

    Replication of human influenza A/H3N2 and A/H1N1 viruses was studied in human CACO-2 cells, a continuous line of intestinal epithelial differentiated cells. Hemagglutinin (HA) was cleaved in these cells by an endogenous protease. Thus, infectious virus was produced that underwent multiple cycle replication and plaque formation in the absence of trypsin added to the media. Cleavage of de novo-synthesized HA occurred at a late stage of the exocytic pathway as indicated by pulse-chase labeling and by experiments employing endoglycosidase H and brefeldin A treatment. However, surface-labeling experiments employing biotinylation suggested that there is no cleavage at the plasma membrane. Unlike HA of serotypes H5 and H7 cleaved at multibasic cleavage sites by furin, the HAs with monobasic cleavage sites analyzed here were not cleaved in CACO-2 cells in the presence of aprotinin, a natural inhibitor of trypsinlike proteases. Growing CACO-2 cells were able to cleave HA of incoming virus, although influenza virus activating protease was not detected in culture medium. These observations indicate that the activating enzyme of CACO-2 cells is a trypsinlike protease functioning in the trans-Golgi network and presumably endosomes. In support of this concept immune staining with antibodies specific to human and bovine trypsin revealed the presence of a trypsinlike protease in CACO-2 cells. Unlike MDCK and CV-1 cells undergoing rapid apoptosis after influenza virus infection, CACO-2 cells showed no apoptosis but displayed cytopathic effects with necrotic signs significantly later after infection. It follows from these data that, depending on the cell type, influenza virus may kill cells either by apoptosis or by necrosis

  11. 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,

  12. Tumor necrosis factor alpha increases epithelial barrier permeability by disrupting tight junctions in Caco-2 cells.

    Science.gov (United States)

    Cui, W; Li, L X; Sun, C M; Wen, Y; Zhou, Y; Dong, Y L; Liu, P

    2010-04-01

    The objectives of this study were to determine the effect of tumor necrosis factor alpha (TNF-alpha) on intestinal epithelial cell permeability and the expression of tight junction proteins. Caco-2 cells were plated onto Transwell microporous filters and treated with TNF-alpha (10 or 100 ng/mL) for 0, 4, 8, 16, or 24 h. The transepithelial electrical resistance and the mucosal-to-serosal flux rates of the established paracellular marker Lucifer yellow were measured in filter-grown monolayers of Caco-2 intestinal cells. The localization and expression of the tight junction protein occludin were detected by immunofluorescence and Western blot analysis, respectively. SYBR-Green-based real-time PCR was used to measure the expression of occludin mRNA. TNF-alpha treatment produced concentration- and time-dependent decreases in Caco-2 transepithelial resistance and increases in transepithelial permeability to the paracellular marker Lucifer yellow. Western blot results indicated that TNF-alpha decreased the expression of phosphorylated occludin in detergent-insoluble fractions but did not affect the expression of non-phosphorylated occludin protein. Real-time RT-PCR data showed that TNF-alpha did not affect the expression of occludin mRNA. Taken together, our data demonstrate that TNF-alpha increases Caco-2 monolayer permeability, decreases occludin protein expression and disturbs intercellular junctions.

  13. Tumor necrosis factor alpha increases epithelial barrier permeability by disrupting tight junctions in Caco-2 cells

    Directory of Open Access Journals (Sweden)

    W. Cui

    2010-04-01

    Full Text Available The objectives of this study were to determine the effect of tumor necrosis factor alpha (TNF-α on intestinal epithelial cell permeability and the expression of tight junction proteins. Caco-2 cells were plated onto Transwell® microporous filters and treated with TNF-α (10 or 100 ng/mL for 0, 4, 8, 16, or 24 h. The transepithelial electrical resistance and the mucosal-to-serosal flux rates of the established paracellular marker Lucifer yellow were measured in filter-grown monolayers of Caco-2 intestinal cells. The localization and expression of the tight junction protein occludin were detected by immunofluorescence and Western blot analysis, respectively. SYBR-Green-based real-time PCR was used to measure the expression of occludin mRNA. TNF-α treatment produced concentration- and time-dependent decreases in Caco-2 transepithelial resistance and increases in transepithelial permeability to the paracellular marker Lucifer yellow. Western blot results indicated that TNF-α decreased the expression of phosphorylated occludin in detergent-insoluble fractions but did not affect the expression of non-phosphorylated occludin protein. Real-time RT-PCR data showed that TNF-α did not affect the expression of occludin mRNA. Taken together, our data demonstrate that TNF-α increases Caco-2 monolayer permeability, decreases occludin protein expression and disturbs intercellular junctions.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  15. Effect of dodecylmaltoside (DDM) on uptake of BCS III compounds, tiludronate and cromolyn, in Caco-2 cells and rat intestine model.

    Science.gov (United States)

    Deshmukh, Deepali D; Nagilla, Rakesh; Ravis, William R; Betageri, Guru V

    2010-04-01

    The efficacy of n-lauryl-beta-D-maltopyranoside, (dodecylmaltoside, DDM) as a permeability-enhancer for tiludronate and cromolyn (BCS Class III, water-soluble compounds with oral bioavailability Lucifer Yellow) were collected over a 40 min period; accumulation in the serosal fluid and intestinal tissue was measured. At lower concentration DDM had no effect on cromolyn permeability and a marginal increase was observed at higher concentration. Tiludronate permeability in the presence of DDM showed greater enhancement as compared to cromolyn. At higher concentration DDM appeared to cause permanent damage to the cell monolayer (irreversible change in TEER). In the intestinal tissue, DDM caused increased tissue accumulation of test compounds. This indicated that transport was not restricted to the paracellular route and damage to the intestinal tissue could not be ruled out. Based on the results obtained in this study it can be concluded that at concentrations that are non-toxic DDM appears to have a limited use to improve the oral absorption of cromolyn and tiludronate.

  16. Formation of a Hydroxymethylfurfural-Cysteine Adduct and Its Absorption and Cytotoxicity in Caco-2 Cells.

    Science.gov (United States)

    Zhao, Qianzhu; Zou, Yueyu; Huang, Caihuan; Lan, Ping; Zheng, Jie; Ou, Shiyi

    2017-11-15

    Adducts of 5-hydroxymethylfurfural (HMF)-amino acids are formed during food processing and digestion; the elimination capacity of in vitro intestinal digests of biscuits, instant noodles, and potato crisps for HMF is 652, 727, and 540 μg/g, respectively. However, the safety of these adducts is unknown. In this study, an HMF-cysteine adduct named 1-dicysteinethioacetal-5-hydroxymehtylfurfural (DCH), which was found to be produced in the gastrointestinal tract after HMF intake, was prepared to test its effect toward Caco-2 cells. Compared with HMF, the adduct displayed lower cytotoxicity against Caco-2 cells with an IC 50 value of 31.26 mM versus 14.95 mM (HMF). The DCH did not induce cell apoptosis, whereas HMF significantly increased the apoptosis rate after incubation at concentrations of 16, 32, and 48 mM for 72 h. DCH showed an absorption rate considerably lower than that of HMF by Caco-2 cells. Lower absorption of DCH may result in lower toxicity compared with HMF against Caco-2 cells. Intracellular transformation of DCH has been observed.

  17. Transport in Caco-2 cell monolayers of antidiabetic cucurbitane triterpenoids from Momordica charantia fruits.

    Science.gov (United States)

    Wu, Shi-Biao; Yue, Grace G L; To, Ming-Ho; Keller, Amy C; Lau, Clara B S; Kennelly, Edward J

    2014-07-01

    Bitter melon, the fruit of Momordica charantia L. (Cucurbitaceae), is a widely-used treatment for diabetes in traditional medicine systems throughout the world. Various compounds have been shown to be responsible for this reputed activity, and, in particular, cucurbitane triterpenoids are thought to play a significant role. The objective of this study was to investigate the gastrointestinal transport of a triterpenoid-enriched n-butanol extract of M. charantia using a two-compartment transwell human intestinal epithelial cell Caco-2 monolayer system, simulating the intestinal barrier. Eleven triterpenoids in this extract were transported from the apical to basolateral direction across Caco-2 cell monolayers, and were identified or tentatively identified by HPLC-TOF-MS. Cucurbitane triterpenoids permeated to the basolateral side with apparent permeability coefficient (P app) values for 3-β-7-β,25-trihydroxycucurbita-5,23(E)-dien-19-al and momordicines I and II at 9.02 × 10(-6), 8.12 × 10(-6), and 1.68 × 10(-6)cm/s, respectively. Also, small amounts of these triterpenoids were absorbed inside the Caco-2 cells. This is the first report of the transport of the reputed antidiabetic cucurbitane triterpenoids in human intestinal epithelial cell monolayers. Our findings, therefore, further support the hypothesis that cucurbitane triterpenoids from bitter melon may explain, at least in part, the antidiabetic activity of this plant in vivo. Georg Thieme Verlag KG Stuttgart · New York.

  18. 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

  19. Co-incubation of PMN and CaCo-2 cells modulates inflammatory potential.

    Science.gov (United States)

    Schaefer, M B; Schaefer, C A; Hecker, M; Morty, R E; Witzenrath, M; Seeger, W; Mayer, K

    2017-05-20

    Polymorphonuclear granulocytes (PMN) are activated in inflammatory reactions. Intestinal epithelial cells are relevant for maintaining the intestinal barrier. We examined interactions of PMN and intestinal epithelial cell-like CaCo-2 cells to elucidate their regulation of inflammatory signalling and the impact of cyclooxygenase (COX), nitric oxide (NO) and platelet-activating factor (PAF). Human PMN and CaCo-2 cells, separately and in co-incubation, were stimulated with the calcium ionophore A23187 or with N-Formyl-methionyl-leucyl-phenylalanin (fMLP) that activates PMN only. Human neutrophil elastase (HNE) and respiratory Burst were measured. To evaluate the modulation of inflammatory crosstalk we applied inhibitors of COX (acetyl salicylic acid; ASA), NO-synthase (N-monomethyl-L-arginin; L-NMMA), and the PAF-receptor (WEB2086). Unstimulated, co-incubation of CaCo-2 cells and PMN led to significantly reduced Burst and elevated HNE as compared to PMN. After stimulation with A23187, co-incubation resulted in an inhibition of Burst and HNE. Using fMLP co-incubation failed to modulate Burst but increased HNE. Without stimulation, all three inhibitors abolished the effect of co-incubation on Burst but did not change HNE.  ASA partly prevented modulation of Burst L-NMMA and WEB2086 did not change Burst but abolished mitigation of HNE. Without stimulation, co-incubation reduced Burst and elevated HNE. Activation of PMN and CaCo-2 cells by fMLP as compared to A23187 resulted in a completely different pattern of Burst and HNE, possibly due to single vs. dual cell activation. Anti-inflammatory effect of co-incubation might in part be due to due to COX-signalling governing Burst whereas NO- and PAF-dependent signalling seemed to control HNE release.

  20. Caco-2 cell permeability modelling: a neural network coupled genetic algorithm approach

    Science.gov (United States)

    Di Fenza, Armida; Alagona, Giuliano; Ghio, Caterina; Leonardi, Riccardo; Giolitti, Alessandro; Madami, Andrea

    2007-04-01

    The ability to cross the intestinal cell membrane is a fundamental prerequisite of a drug compound. However, the experimental measurement of such an important property is a costly and highly time consuming step of the drug development process because it is necessary to synthesize the compound first. Therefore, in silico modelling of intestinal absorption, which can be carried out at very early stages of drug design, is an appealing alternative procedure which is based mainly on multivariate statistical analysis such as partial least squares (PLS) and neural networks (NN). Our implementation of neural network models for the prediction of intestinal absorption is based on the correlation of Caco-2 cell apparent permeability ( P app) values, as a measure of intestinal absorption, to the structures of two different data sets of drug candidates. Several molecular descriptors of the compounds were calculated and the optimal subsets were selected using a genetic algorithm; therefore, the method was indicated as Genetic Algorithm-Neural Network (GA-NN). A methodology combining a genetic algorithm search with neural network analysis applied to the modelling of Caco-2 P app has never been presented before, although the two procedures have been already employed separately. Moreover, we provide new Caco-2 cell permeability measurements for more than two hundred compounds. Interestingly, the selected descriptors show to possess physico-chemical connotations which are in excellent accordance with the well known relevant molecular properties involved in the cellular membrane permeation phenomenon: hydrophilicity, hydrogen bonding propensity, hydrophobicity and molecular size. The predictive ability of the models, although rather good for a preliminary study, is somewhat affected by the poor precision of the experimental Caco-2 measurements. Finally, the generalization ability of one model was checked on an external test set not derived from the data sets used to build the models

  1. Regional expression levels of drug transporters and metabolizing enzymes along the pig and human intestinal tract and comparison with Caco-2 cells

    NARCIS (Netherlands)

    Vaessen, S.F.C.; Lipzig, M.M.H. van; Pieters, R.H.H.; Krul, C.A.M.; Wortelboer, H.M.; Steeg, E. van de

    2017-01-01

    Intestinal transporter proteins and metabolizing enzymes play a crucial role in the oral absorption of a wide variety of drugs. The aim of the current study was to characterize better available intestinal in vitro models by comparing expression levels of these proteins and enzymes between porcine

  2. Regional expression levels of drug transporters and metabolizing enzymes along the pig and human intestinal tract and comparison with Caco-2 cells

    NARCIS (Netherlands)

    Vaessen, Stefan F C; van Lipzig, Marola M H; Pieters, Raymond H H; Krul, Cyrille A M; Wortelboer, Heleen M; van de Steeg, Evita

    2017-01-01

    Intestinal transporter proteins and metabolizing enzymes play a crucial role in the oral absorption of a wide variety of drugs. The aim of the current study was to better characterize available intestinal in vitro models by comparing expression levels of these proteins and enzymes between porcine

  3. Short-Chain Fatty Acids Activate AMP-Activated Protein Kinase and Ameliorate Ethanol-Induced Intestinal Barrier Dysfunction in Caco-2 Cell Monolayers

    NARCIS (Netherlands)

    Eamin, E.E.; Masclee, A.A.; Dekker, J.; Pieters, H.J.; Jonkers, D.M.

    2013-01-01

    Short-chain fatty acids (SCFAs) have been shown to promote intestinal barrier function, but their protective effects against ethanol-induced intestinal injury and underlying mechanisms remain essentially unknown. The aim of the study was to analyze the influence of SCFAs on ethanol-induced barrier

  4. Adhesion of some probiotic and dairy Lactobacillus strains to Caco-2 cell cultures.

    Science.gov (United States)

    Tuomola, E M; Salminen, S J

    1998-05-05

    The adhesion of 12 different Lactobacillus strains was studied using Caco-2 cell line as an in vitro model for intestinal epithelium. Some of the strains tested have been used as probiotics, and most of them are used in the dairy and food industry. Human and bovine enterotoxigenic Escherichia coli strains were used as positive and negative control, respectively. Bacterial adhesion to Caco-2 cell cultures was quantitated using radiolabelled bacteria. The adherence of bacteria was also observed microscopically after Gram staining. Viability of bacteria prior to adhesion was verified using flow cytometry. Among the tested strains, L. casei (Fyos) was the most adhesive strain and L. casei var. rhamnosus (Lactophilus) was the least adhesive strain, approximately 14 and 3% of the added bacteria adhered to Caco-2 cell cultures, respectively. The corresponding values for positive and negative control E. coli strains were 14 and 4%, respectively. The Lactobacillus strains tested could not be divided into distinctly adhesive or non-adhesive strains, since there was a continuation of adhesion rates. The four most adhesive strains were L. casei (Fyos), L. acidophilus 1 (LC1), L. rhamnosus LC-705 and Lactobacillus GG (ATCC 53103). No significant differences in the percentage adhesion were observed between these strains. Adhesion of all the strains was dependent on the number of bacteria used, since an approximately constant number of Caco-2 cells was used, indicating that the Caco-2 cell binding sites were not saturated. Viability of bacteria was high since approximately 90% of the bacteria were viable with the exception of L. acidophilus 1 which was 74% viable. Microscopic evaluations agreed with the radiolabelled binding as evidenced by observing more bacteria in Gram-stained preparations of good adhering strains compared to poorly adhering strains.

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

    Science.gov (United States)

    Bhattacharjee, Sourav; van Opstal, Edward J.; Alink, Gerrit M.; Marcelis, Antonius T. M.; Zuilhof, Han; Rietjens, Ivonne M. C. M.

    2013-06-01

    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/size 45 nm) and polystyrene nanoparticles (PSNPs/size 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).

  6. 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).

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

    International Nuclear Information System (INIS)

    Bhattacharjee, Sourav; Opstal, Edward J. van; Alink, Gerrit M.; Marcelis, Antonius T. M.; Zuilhof, Han; Rietjens, Ivonne 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/size ∼45 nm) and polystyrene nanoparticles (PSNPs/size ∼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).

  8. Uptake of label-free graphene oxide by Caco-2 cells is dependent on the cell differentiation status.

    Science.gov (United States)

    Kucki, Melanie; Diener, Liliane; Bohmer, Nils; Hirsch, Cordula; Krug, Harald F; Palermo, Vincenzo; Wick, Peter

    2017-06-21

    Understanding the interaction of graphene-related materials (GRM) with human cells is a key to the assessment of their potential risks for human health. There is a knowledge gap regarding the potential uptake of GRM by human intestinal cells after unintended ingestion. Therefore the aim of our study was to investigate the interaction of label-free graphene oxide (GO) with the intestinal cell line Caco-2 in vitro and to shed light on the influence of the cell phenotype given by the differentiation status on cellular uptake behaviour. Internalisation of two label-free GOs with different lateral size and thickness by undifferentiated and differentiated Caco-2 cells was analysed by scanning electron microscopy and transmission electron microscopy. Semi-quantification of cells associated with GRM was performed by flow cytometry. Undifferentiated Caco-2 cells showed significant amounts of cell-associated GRM, whereas differentiated Caco-2 cells exhibited low adhesion of GO sheets. Transmission electron microscopy analysis revealed internalisation of both applied GO (small and large) by undifferentiated Caco-2 cells. Even large GO sheets with lateral dimensions up to 10 µm, were found internalised by undifferentiated cells, presumably by macropinocytosis. In contrast, no GO uptake could be found for differentiated Caco-2 cells exhibiting an enterocyte-like morphology with apical brush border. Our results show that the internalisation of GO is highly dependent on the cell differentiation status of human intestinal cells. During differentiation Caco-2 cells undergo intense phenotypic changes which lead to a dramatic decrease in GRM internalisation. The results support the hypothesis that the cell surface topography of differentiated Caco-2 cells given by the brush border leads to low adhesion of GO sheets and sterical hindrance for material uptake. In addition, the mechanical properties of GRM, especially flexibility of the sheets, seem to be an important factor for

  9. Comparison of Caco-2, IEC-18 and HCEC cell lines as a model for intestinal absorption of genistein, daidzein and their glycodides

    NARCIS (Netherlands)

    Steensma, A.; Noteborn, H.P.J.M.; Kuiper, H.A.

    2004-01-01

    Genistein and daidzein receive much attention because of their potential to prevent hormone-related cancer and cardiovascular diseases. Limited information is available on the pharmacokinetics of these compounds like, for instance, intestinal uptake by humans and systematic bioavailability. In this

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

    Directory of Open Access Journals (Sweden)

    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.

  11. Bioavailability of iron from spinach using an in vitro/human Caco-2 cell bioassay model

    Science.gov (United States)

    Rutzke, Corinne J.; Glahn, Raymond P.; Rutzke, Michael A.; Welch, Ross M.; Langhans, Robert W.; Albright, Louis D.; Combs, Gerald F Jr; Wheeler, Raymond M.

    2004-01-01

    Spinach (Spinacia oleracea) cv Whitney was tested for iron bioavailabilty using an in vitro human intestinal cell culture ferritin bioassay technique previously developed. Spinach was cultured in a growth chamber for 33 days, harvested, and freeze-dried. Total iron in the samples was an average of 71 micrograms/g dry weight. Spinach was digested in vitro (pepsin and 0.1 M HCl followed by pancreatin and 0.1 M NaHCO3) with and without the addition of supplemental ascorbic acid. Caco-2 cell cultures were used to determine iron bioavailability from the spinach mixtures. Production of the iron-binding protein ferritin in the Caco-2 cells showed the supplemental ascorbic acid doubled bioavailability of iron from spinach. The data show fresh spinach is a poor source of iron, and emphasize the importance of evaluation of whole meals rather than single food items. The data support the usefulness of the in vitro/Caco-2 cell ferritin bioassay model for prescreening of space flight diets for bioavailable iron.

  12. pH-dependent and carrier-mediated transport of salicylic acid across Caco-2 cells.

    Science.gov (United States)

    Takanaga, H; Tamai, I; Tsuji, A

    1994-07-01

    The transport of monocarboxylic acid drugs such as salicylic acid was examined in the human colon adenocarcinoma cell line, Caco-2 cells that possess intestinal epithelia-like properties. [14C]Salicylic acid transport was pH-dependent and appeared to follow the pH-partition hypothesis. However, 10 mM unlabelled salicylic acid significantly reduced the permeability coefficient of [14C]salicylic acid. Kinetic analysis of the concentration dependence of the permeation rate of salicylic acid across Caco-2 cells showed both saturable (Kt = 5.28 +/- 0.72 mM Jmax = 36.6 +/- 3.54 nmol min-1 (mg protein)-1) and nonsaturable (kd = 0.37 +/- 0.08 microL min-1 (mg protein)-1) processes. The permeation rate of [14C]salicylic acid was competitively inhibited by both acetic acid and benzoic acid, which were demonstrated in our previous studies to be transported in the carrier-mediated-transport mechanism which is responsible for monocarboxylic acids. Furthermore, certain monocarboxylic acids significantly inhibited [14C]salicylic acid transport, whereas salicylamide and dicarboxylic acids such as succinic acid did not. From these results, it was concluded that the transcellular transport of [14C]salicylic acid across Caco-2 cells is by the pH-dependent and carrier-mediated transport mechanism specific for monocarboxylic acids.

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

    International Nuclear Information System (INIS)

    Song, Yijuan; Guan, Rongfa; Lyu, Fei; Kang, Tianshu; Wu, Yihang; Chen, Xiaoqiang

    2014-01-01

    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 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 than

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

    Energy Technology Data Exchange (ETDEWEB)

    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

  15. Defining conditions for the co-culture of Caco-2 and HT29-MTX cells using Taguchi design.

    Science.gov (United States)

    Chen, Xiu-Min; Elisia, Ingrid; Kitts, David D

    2010-01-01

    The co-culture of Caco-2 and HT29 cells for testing intestinal drug and nutrient transport and metabolism provides the presence of both absorptive and goblet cells, both of which have different culture requirements for optimal growth and function. The research on the co-culture of Caco-2 and HT29 cells is very limited in respect to refining specific conditions that reduce intra- and inter-laboratory variations. In the present study we reported conditions that enable reproducible results to be obtained for drug permeability using in vitro co-culture of Caco-2 and HT29-MTX based on Taguchi experimental design. The selection of four factors that specified cell culture conditions, namely culture medium, seeding time, seeding density, and Caco-2:HT29-MTX ratio on TEER value and individual permeability coefficients of propranolol, ketoprofen and furosemide was established. Based on the selected conditions for co-culture, we also confirmed the functionality of the final chosen culture condition using nitric oxide as an indicator of intestinal inflammation. Choice of cell culture time and culture medium represented two of the most important factors that affected TEER values and the permeability coefficients of the model drugs. On the other hand, the seeding density and the Caco-2:HT29-MTX ratio exerted no significant influence on TEER values and the drug permeability coefficients. No absolute optimal cell culture condition could be obtained for all drugs; however subsequent confirmation experiments concluded that excellent precision for TEER values and drug permeability coefficients was obtained from the two operators using the following combination of conditions, namely an initial seeding density of 1 x 10(5) Caco-2 and HT29-MTX cells/cm(2) at a ratio of 9:1, followed by a 21day culture time in MEM medium. Finally, functionality of the co-culture model system using the above selected in vitro conditions resulted in comparable nitric oxide synthesis to that of a Caco-2

  16. 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

    of vigabatrin in Caco-2 cells, a cell culture model of the small intestinal epithelium. The uptake and transepithelial flux of vigabatrin was measured using an LC-MS method for quantification. Transepithelial transport of vigabatrin was shown to be proton-dependent and polarized in the apical-to-basolateral (A...... 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....

  17. Impact of lipid-based drug delivery systems on the transport and uptake of insulin across Caco-2 Cell monolayers

    DEFF Research Database (Denmark)

    Li, Ping; Nielsen, Hanne Mørck; Müllertz, Anette

    2016-01-01

    on the transport and uptake mechanisms of insulin across the intestinal membrane. For this purpose, 3 SNEDDS were prepared, and Caco-2 cell monolayers were used to study transport and uptake. The prepared SNEDDSs were all in the range of 35-50 nm and had a negative zeta potential (between -8 and -25 m...... space of the Caco-2 cell monolayer, indicating transport by paracellular diffusion. In contrast, the fluorescein isothiocyanate-labeled insulin in LCT SNEDDS was taken up by the cells. In conclusion, the present study demonstrated that MCT1 and MCT2 SNEDDS, but not LCT SNEDDS increased...

  18. Normalization genes for quantitative RT-PCR in differentiated Caco-2 cells used for food exposure studies

    NARCIS (Netherlands)

    Vreeburg, R.A.M.; Bastiaan-Net, S.; Mes, J.J.

    2011-01-01

    Exposure of food products to small-intestinal-like Caco-2 cells, combined with a gene expression based response analysis can be a valuable tool to classify potential bioactive effects of food homogenates. In order to study changes in gene expression upon food exposure, a robust set of stably

  19. Simulating kinetic parameters in transporter mediated permeability across Caco-2 cells. A case study on estrange-3-sulphate

    DEFF Research Database (Denmark)

    Rolsted, Kamilla; Rapin, Nicolas; Steffansen, Bente

    2011-01-01

    Substances that compete for the same saturable intestinal transporters may when dosed together lead to altered permeability and hence influence bioavailability. The aim was to simulate kinetic parameters, i.e. K(m) and J(max), for transporter mediated E(1)S permeability across Caco-2 cells...

  20. Selected Phytochemicals and Culinary Plant Extracts Inhibit Fructose Uptake in Caco-2 Cells.

    Science.gov (United States)

    Lee, Yurim; Lim, Yeni; Kwon, Oran

    2015-09-18

    This study compared the ability of nine culinary plant extracts containing a wide array of phytochemicals to inhibit fructose uptake and then explored the involvement of intestinal fructose transporters and phytochemicals for selected samples. The chemical signature was characterized by high performance liquid chromatography with mass spectrometry. Inhibition of [(14)C]-fructose uptake was tested by using human intestinal Caco-2 cells. Then, the relative contribution of the two apical-facing intestinal fructose transporters, GLUT2 and GLUT5, and the signature components for fructose uptake inhibition was confirmed in naive, phloretin-treated and forskolin-treated Caco-2 cells. HPLC/MS analysis of the chemical signature revealed that guava leaf contained quercetin and catechin, and turmeric contained curcumin, bisdemethoxycurcumin and dimethoxycurcumin. Similar inhibition of fructose uptake (by ~50%) was observed with guava leaf and turmeric in Caco-2 cells, but with a higher contribution of GLUT2 for turmeric and that of GLUT5 for guava leaf. The data suggested that, in turmeric, demethoxycurcumin specifically contributed to GLUT2-mediated fructose uptake inhibition, and curcumin did the same to GLUT5-mediated fructose uptake inhibition, but GLUT2 inhibition was more potent. By contrast, in guava leaf, catechin specifically contributed to GLUT5-mediated fructose uptake inhibition, and quercetin affected both GLUT5- and GLUT2-mediated fructose uptake inhibition, resulting in the higher contribution of GLUT5. These results suggest that demethoxycurcumin is an important contributor to GLUT2-mediated fructose uptake inhibition for turmeric extract, and catechin is the same to GLUT5-mediated fructose uptake inhibition for guava leaf extract. Quercetin, curcumin and bisdemethoxycurcumin contributed to both GLUT5- and GLUT2-mediated fructose uptake inhibition, but the contribution to GLUT5 inhibition was higher than the contribution to GLUT2 inhibition.

  1. Interactions between organic anions on multiple transporters in Caco-2 cells

    DEFF Research Database (Denmark)

    Grandvuinet, Anne Sophie; Steffansen, Bente

    2011-01-01

    Caco-2 cell line may be used as an overall model to predict interactions on multiple membrane transporters in the intestine. Taurocholic acid (TCA) and estrone-3-sulfate (E1S) were used as model substrates. Possible inhibitors studied were TCA, E1S, taurolithocholic acid, fluvastatin, and glipizide......-dependent bile acid transporter and the organic solute transporter α/β, and to less extent by the organic anion transporting polypeptide 2B1. However, interactions on efflux transporters were not detected, although they were expected from the literature on the investigated compounds. Biosimulation methods may...

  2. Comparison of the sensitivity of different toxicological endpoints in Caco-2 cells after cadmium chloride treatment

    Energy Technology Data Exchange (ETDEWEB)

    Boveri, M.; Pazos, P.; Gennari, A.; Casado, J.; Hartung, T.; Prieto, P. [ECVAM, Inst. for Health and Consumer Protection, Joint Research Centre, European Commission, Ispra (Italy)

    2004-04-01

    The human colorectal adenocarcinoma cell line Caco-2 is a widely used in vitro model of the intestinal barrier. Cadmium chloride (CdCl{sub 2}) is a highly toxic metal compound, ubiquitous in the biosphere, able to enter the food chain and to reach the intestinal epithelium, causing structural and functional damages. The aim of this work was to characterise cadmium toxicity in Caco-2 cells and, in particular, to compare the sensitivity of different endpoints revealing damage both on the epithelial barrier and at the cellular or molecular level. After 24-h exposure of the cells to CdCl{sub 2}, lactate dehydrogenase (LDH) leakage showed cadmium-induced cell toxicity, significant from 25 {mu}M CdCl{sub 2} and above, and analysis of different cell death pathways indicated the presence of necrosis after treatment with 50 {mu}M CdCl{sub 2}. At the molecular level, we observed an increase in the protective protein heat shock protein 70 (HSP70), starting at 10 {mu}M CdCl{sub 2}. At the barrier level, transepithelial electrical resistance (TEER) decreased while paracellular permeability (PCP) significantly increased after the treatment, showing an EC{sub 50} of 6 and 16 {mu}M CdCl{sub 2}, respectively, and indicating the loss of barrier integrity. In conclusion, our data reveal that CdCl{sub 2} toxicity in Caco-2 cells can be detected at the barrier level at very low concentrations; also, HSP70 was shown to be a sensitive marker for detecting in vitro cadmium-induced toxicity. (orig.)

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

    Science.gov (United States)

    Coyuco, Jurja C; Liu, Yuanjie; Tan, Bee-Jen; Chiu, Gigi NC

    2011-01-01

    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 (f SWCNT-COOH) and poly(ethylene glycol) functionalized single-walled carbon nanotubes (f SWCNT-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, f SWCNT-COOH significantly reduced transepithelial electrical resistance and enhanced transport of Lucifer Yellow across the Caco-2 monolayer. Confocal fluorescence microscopy showed that f SWCNT-COOH treated cells had the highest perturbation in the distribution of ZO-1, a protein marker of tight junction, suggesting that f SWCNT-COOH could enhance paracellular permeability via disruption of tight junctions. This modulating effect of f SWCNT-COOH can be reversed over time. Furthermore, cellular accumulation of the P-gp substrate, rhodamine-123, was significantly increased in cells treated with f SWCNT-COOH, suggestive of P-gp inhibition. Of note, f SWCNT-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 delivery, and the differential effects on the intestinal epithelium imparted by different types of CNMs would create unique opportunities for drug-specific oral

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

    Science.gov (United States)

    Coyuco, Jurja C; Liu, Yuanjie; Tan, Bee-Jen; Chiu, Gigi N C

    2011-01-01

    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 (f SWCNT-COOH) and poly(ethylene glycol) functionalized single-walled carbon nanotubes (f SWCNT-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, f SWCNT-COOH significantly reduced transepithelial electrical resistance and enhanced transport of Lucifer Yellow across the Caco-2 monolayer. Confocal fluorescence microscopy showed that f SWCNT-COOH treated cells had the highest perturbation in the distribution of ZO-1, a protein marker of tight junction, suggesting that f SWCNT-COOH could enhance paracellular permeability via disruption of tight junctions. This modulating effect of f SWCNT-COOH can be reversed over time. Furthermore, cellular accumulation of the P-gp substrate, rhodamine-123, was significantly increased in cells treated with f SWCNT-COOH, suggestive of P-gp inhibition. Of note, f SWCNT-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 delivery, and the differential effects on the intestinal epithelium imparted by different types of CNMs would create unique opportunities for drug-specific oral

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

    NARCIS (Netherlands)

    Dihal, A.A.; Woutersen, R.A.; Ommen, van B.; 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

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

    NARCIS (Netherlands)

    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

  7. [In vitro absorption mechanism of strychnine and the transport interaction with liquiritin in Caco-2 cell monolayer model].

    Science.gov (United States)

    Wang, Jun-jun; Liao, Xiao-huan; Ye, Min; Chen, Yong

    2010-09-01

    To study the effect of liquiritin (Liq) on the transport of strychnine (Str) in Caco-2 cell monolayer model, the transport parameters of Str, such as apparent permeability coefficient (P app (B-->A) and P app (A-->B)) and cumulative transport amount (TRcum), were determined and comparatively analyzed when Str was used solely and co-used with Liq. The effect of drug concentrations, conveying times, P-glycoprotein (P-gp) inhibitor verapamil and conveying liquor pH values on the transport of Str were also investigated. The results indicated that the absorption of Str in Caco-2 cell monolayer model was well and the passive transference was the main intestinal absorption mechanism of Str in the Caco-2 monolayer model, along with the excretion action mediated by P-gp. Liq enhanced the absorption of Str. Meanwhile, conveying liquor pH value had significant influence on the excretion transport of Str.

  8. Synergistic Effects of Zinc Oxide Nanoparticles and Fatty Acids on Toxicity to Caco-2 Cells

    DEFF Research Database (Denmark)

    Cao, Yi; Roursgaard, Martin; Kermanizadeh, Ali

    2015-01-01

    epithelial (Caco-2) cells. The ZnO NPs exposure concentration dependently induced cytotoxicity to Caco-2 cells showing as reduced proliferation and activity measured by 3 different assays. PA exposure induced cytotoxicity, and coexposure to ZnO NPs and PA showed the largest cytotoxic effects. The presence...

  9. 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.

  10. Human rotavirus strain Wa downregulates NHE1 and NHE6 expressions in rotavirus-infected Caco-2 cells.

    Science.gov (United States)

    Chen, Honglang; Song, Lijun; Li, Guixian; Chen, Wenfeng; Zhao, Shumin; Zhou, Ruoxia; Shi, Xiaoying; Peng, Zhenying; Zhao, Wenchang

    2017-06-01

    Rotavirus (RV) is the most common cause of severe gastroenteritis and fatal dehydration in human infants and neonates of different species. However, the pathogenesis of rotavirus-induced diarrhea is poorly understood. Secretory diarrhea caused by rotavirus may lead to a combination of excessive secretion of fluid and electrolytes into the intestinal lumen. Fluid absorption in the small intestine is driven by Na + -coupled transport mechanisms at the luminal membrane, including Na + /H + exchanger (NHE). Here, we performed qRT-PCR to detect the transcription of NHEs. Western blotting was employed for protein detection. Furthermore, immunocytochemistry was used to validate the NHE's protein expression. Finally, intracellular Ca 2+ concentration was detected by confocal laser scanning microscopy. The results demonstrated that the NHE6 mRNA and protein expressed in the human colon adenocarcinoma cell line (Caco-2). Furthermore, RV-Wa induced decreased expression of the NHE1 and NHE6 in Caco-2 cell in a time-dependent manner. In addition, intracellular Ca 2+ concentration in RV-Wa-infected Caco-2 cells was higher than that in the mock-infected cells. Furthermore, RV-Wa also can downregulate the expression of calmodulin (CaM) and calmodulin kinase II (CaMKII) in Caco-2 cells. These findings provides important insights into the mechanisms of rotavirus-induced diarrhea. Further studies on the underlying pathophysiological mechanisms that downregulate NHEs in RV-induced diarrhea are required.

  11. Digestive Stability and Transport of Norbixin, a 24-Carbon Carotenoid, across Monolayers of Caco-2 Cells

    Science.gov (United States)

    Polar-Cabrera, Karina; Huo, Tianyao; Schwartz, Steven J.; Failla, Mark L.

    2013-01-01

    Annatto is a natural pigment widely used in the food industry to add yellow to red colors to dairy and cereal products. Here, the in vitro bioaccessibility and potential bioavailability of norbixin, the abundant 24-carbon carotenoid in annatto, were investigated. Norbixin added to milk was highly stable during simulated digestion, and bile salts enhanced partitioning of this carotenoid in the aqueous fraction during the small intestinal phase of digestion. Apical uptake of norbixin by Caco-2 cells was proportional to the concentration in apical medium, but cellular content increased only slightly after 60 min. Transport of norbixin to the basolateral compartment was maximum at 120 min. Both all-trans and cis isomers of norbixin were present in cells and basolateral medium. The results suggest that ingested norbixin is stable during gastric and small intestinal phases of digestion and that both cis and all-trans isomers are bioavailable. PMID:20408560

  12. Blackcurrant anthocyanins stimulated cholesterol transport via post-transcriptional induction of LDL receptor in Caco-2 cells.

    Science.gov (United States)

    Kim, Bohkyung; Bae, Minkyung; Park, Young-Ki; Ma, Hang; Yuan, Tao; Seeram, Navindra P; Lee, Ji-Young

    2018-02-01

    We previously showed that polyphenol-rich blackcurrant extract (BCE) showed a hypocholesterolemic effect in mice fed a high fat diet. As direct cholesterol removal from the body via the intestine has been recently appreciated, we investigated the effect of BCE on the modulation of genes involved in intestinal cholesterol transport using Caco-2 cells as an in vitro model. Caco-2 cells were treated with BCE to determine its effects on mRNA and protein expression of genes important for intestinal cholesterol transport, low-density lipoprotein (LDL) uptake, cellular cholesterol content, and cholesterol transport from basolateral to apical membrane of Caco-2 cell monolayers. Cells were also treated with anthocyanin-rich or -poor fraction of BCE to determine the role of anthocyanin on BCE effects. BCE significantly increased protein levels of LDL receptor (LDLR) without altering its mRNA, which consequently increased LDL uptake into Caco-2 cells. This post-transcriptional induction of LDLR by BCE was markedly attenuated in the presence of rapamycin, an inhibitor of mechanistic target of rapamycin complex 1 (mTORC1). In addition, BCE altered genes involved in cholesterol transport in the enterocytes, including apical and basolateral cholesterol transporters, in such a way that could enhance cholesterol flux from the basolateral to apical side of the enterocytes. Indeed, BCE significantly increased the flux of LDL-derived cholesterol from the basolateral to the apical chamber of Caco-2 monolayer. LDLR protein levels were markedly increased by anthocyanin-rich fraction, but not by anthocyanin-free fraction. mTORC1-dependent post-transcriptional induction of LDLR by BCE anthocyanins drove the transport of LDL-derived cholesterol to the apical side of the enterocytes. This may represent a potential mechanism for the hypocholesterolemic effect of BCE.

  13. A Co-culture Method to Investigate the Crosstalk Between X-ray Irradiated Caco-2 Cells and PBMC.

    Science.gov (United States)

    Babini, Gabriele; Morini, Jacopo; Barbieri, Sofia; Baiocco, Giorgio; Ivaldi, Giovanni Battista; Liotta, Marco; Tabarelli de Fatis, Paola; Ottolenghi, Andrea

    2018-01-30

    The protocol adopted in this work aims at unraveling how X-rays perturb the functioning of the intestinal barrier, focusing on the interplay between colorectal tumor cells and the immune system. Colorectal carcinoma is among the most common type of cancer, typically treated by surgery, chemotherapy, and radiotherapy. Advantages of radiotherapy in targeting the tumor are well known. However, even limited exposures of healthy tissues are of great concern, particularly regarding the effects on the intestinal barrier and the immune system. The adopted setup allows to study the interplay between two cell populations in a condition more similar to the physiological one, when compared to normal cell cultures. For this purpose, we resort to different techniques and we used an in vitro co-culture model, based on Caco-2 cells differentiated as a monolayer and PBMC, sharing the same culture medium. This protocol has been developed to focus on both macroscopic effects, i.e. cell viability and Trans-Epithelial Electrical Resistance (TEER), and, through western blot, molecular alterations, i.e. the activation of inflammatory pathway in immune cells and the tight junction protein expression in Caco-2 cells. Initial evaluation of radiation effects on Caco-2 cell viability was assessed via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Trypan blue assays, while TEER was measured at fixed time intervals through an ohmmeter specifically designed for co-culture systems. In this way, the effects due to radiation, the presence of Peripheral Blood Mononuclear Cells (PBMC), and eventually their synergistic effect, can be demonstrated. Through these complementary techniques, we observed a high radio-resistance of Caco-2 within the range of 2 - 10 Gy of X-rays and an increased Caco-2 monolayer permeability when PBMCs were added. In particular, PBMC presence was found to be associated with the variation in the tight junction scaffold proteins expression.

  14. Extracellular Signals of a Human Epithelial Colorectal Adenocarcinoma (Caco-2) Cell Line Facilitate the Penetration of Pseudomonas aeruginosa PAO1 Strain through the Mucin Layer

    Science.gov (United States)

    Hayashi, Naoki; Yokotani, Atsushi; Yamamoto, Masami; Kososhi, Mariko; Morita, Mayu; Fukunishi, Chiaki; Nishizawa, Nagisa; Gotoh, Naomasa

    2017-01-01

    Pseudomonas aeruginosa can penetrate the layer of mucus formed by host intestinal epithelial cells, often resulting in sepsis in immunocompromised patients. We have previously demonstrated that P. aeruginosa can penetrate the mucin layer by flagellar motility and the degradation of the mucin layer. However, it remains unclear how P. aeruginosa initially recognizes epithelial cells. Using the human epithelial colorectal adenocarcinoma (Caco-2) cell line, we investigated extracellular signaling that could facilitate the penetration of P. aeruginosa through the mucin layer. The supernatant from Caco-2 cell cultures increased penetration of P. aeruginosa through an artificial mucin layer. The Caco-2 cell supernatant increased bacterial flagella-dependent swarming motility, but it did not influence P. aeruginosa growth or protease activity. Filtering of the Caco-2 cell supernatant indicated that proteins weighing Caco-2 cell supernatant attracted P. aeruginosa cells. Finally, we identified that growth-regulated oncogene-α (GRO-α) secreted by Caco-2 cells was a factor facilitating flagellar filament rotation and swarming motility, although it did not attract the bacteria. We conclude that penetration of the mucin layer by P. aeruginosa is facilitated by small proteins (Caco-2 cells, both by inducing acceleration of flagellar motility and increasing chemotaxis. PMID:28983473

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

    Science.gov (United States)

    Chen, Gao; Lianqin, Zhu; Fenghua, Zhu; Fang, Zheng; Mingming, Song; Kai, Huang

    2015-04-01

    Different concentrations of CuSO4, 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 app) of CuSO4 and nano-CuO increased with the Cu concentration in the culture medium ( p Caco-2 cells ( p > 0.05). When the Cu concentration in the culture medium was in the range 31.25-500 μM, the P app value of Caco-2 cells incubated with nano-CuO was significantly higher than that obtained with CuSO4. The latter was also significantly higher than that when cells were incubated with micro-CuO ( p 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.

  16. Uptake of perfluorooctanoic acid by Caco-2 cells: Involvement of organic anion transporting polypeptides.

    Science.gov (United States)

    Kimura, Osamu; Fujii, Yukiko; Haraguchi, Koichi; Kato, Yoshihisa; Ohta, Chiho; Koga, Nobuyuki; Endo, Tetsuya

    2017-08-05

    The mechanism underlying the intestinal absorption of perfluorooctanoic acid (PFOA) was investigated using Caco-2 cells. The uptake of PFOA from the apical membrane of Caco-2 cells was fast, and pH, temperature, and concentration dependent, but Na + independent. Coincubation with sulfobromophthalein (BSP), glibenclamide, estron-3-sulfate, cyclosporine A or rifamycin SV, which are typical substrates or inhibitors of organic anion transporting polypeptides (OATPs), significantly decreased the uptake of PFOA. However, coincubation with probenecid or p-aminohippuric acid, typical substrates of organic anion transporters, did not decrease the uptake of PFOA. Furthermore, coincubation with l-lactic acid or benzoic acid, substrates of monocarboxylic acid transporters, did not decrease PFOA uptake. The relationship between the initial uptake of PFOA and its concentration was saturable, suggesting the involvement of a carrier-mediated process. The calculated K m and uptake clearance (V max /K m ) values for PFOA were 8.3μM and 55.0μL/mg protein/min, respectively. This clearance value was about 3-fold greater than that of the non-saturable uptake clearance (K d : 18.1μL/mg protein/min). Lineweaver-Burk plots revealed that BSP competitively inhibits the uptake of PFOA, with a K i value of 23.1μM. These results suggest that the uptake of PFOA from the apical membranes of Caco-2 cells could be, at least in part, mediated by OATPs along with BSP. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Oleic Acid Uptake Reveals the Rescued Enterocyte Phenotype of Colon Cancer Caco-2 by HT29-MTX Cells in Co-Culture Mode

    Science.gov (United States)

    Berger, Emmanuelle; Nassra, Merian; Atgié, Claude; Plaisancié, Pascale; Géloën, Alain

    2017-01-01

    Gastrointestinal epithelium is the unique route for nutrients and for many pharmaceuticals to enter the body. The present study aimed to analyze precisely whether co-culture of two colon cancer cell lines, mucus-producing cells HT29-MTX and enterocyte-like Caco-2 cells, ameliorate differentiation into an in vitro intestinal barrier model and the signaling pathways involved. Differentiated Caco-2 cells gene datasets were compared first to intestinal or cancer phenotypes and second to signaling pathway gene datasets. Experimental validations were performed in real-time experiments, immunochemistry, and gene expression analyses on Caco-2 versus co-cultures of Caco-2 and HT29-MTX (10%) cells. Partial maintenance of cancer-cell phenotype in differentiated Caco-2 cells was confirmed and fatty acids merged as potential regulators of cancer signaling pathways. HT29-MTX cells induced morphological changes in Caco-2 cells, slightly increased their proliferation rate and profoundly modified gene transcription of phenotype markers, fatty acid receptors, intracellular transporters, and lipid droplet components as well as functional responses to oleic acid. In vitro, enterocyte phenotype was rescued partially by co-culture of cancer cells with goblet cells and completed through oleic acid interaction with signaling pathways dysregulated in cancer cells. PMID:28726765

  18. Effect of chitosan-coated alginate microspheres on the permeability of Caco-2 cell monolayers.

    Science.gov (United States)

    Silva, Catarina M; Veiga, Francisco; Ribeiro, António J; Zerrouk, Naïma; Arnaud, Philippe

    2006-10-01

    Alginate microspheres were prepared by emulsification/internal gelation and coated with chitosan. The ability of chitosan-coated alginate microspheres to increase the paracellular transport across Caco-2 cell monolayers was evaluated in comparison to uncoated microspheres and chitosan solutions. Transport studies were performed by using a permeability marker, Lucifer Yellow (LY), and by measuring the transepithelial electric resistance (TEER) variations. Furthermore, the occurrence of cytotoxic effects was assessed by evaluating neutral red uptake in viable cells and lactate dehydrogenase (LDH) release from damaged cells. A 3-fold increase on LY permeability was obtained for coated microspheres when compared to chitosan solutions. TEER variations were in agreement with permeability results. Chitosan solutions exhibited a dose-dependent toxicity, but coated microspheres did not decrease the viability of cells. Chitosan-coated alginate microspheres have potential to be used as carriers of poorly absorbable hydrophilic drugs to the intestinal epithelia and possibly increase their oral bioavailability.

  19. Hypoxia Decreases Invasin-Mediated Yersinia enterocolitica Internalization into Caco-2 Cells.

    Science.gov (United States)

    Zeitouni, Nathalie E; Dersch, Petra; Naim, Hassan Y; von Köckritz-Blickwede, Maren

    2016-01-01

    Yersinia enterocolitica is a major cause of human yersiniosis, with enterocolitis being a typical manifestation. These bacteria can cross the intestinal mucosa, and invade eukaryotic cells by binding to host β1 integrins, a process mediated by the bacterial effector protein invasin. This study examines the role of hypoxia on the internalization of Y. enterocolitica into intestinal epithelial cells, since the gastrointestinal tract has been shown to be physiologically deficient in oxygen levels (hypoxic), especially in cases of infection and inflammation. We show that hypoxic pre-incubation of Caco-2 cells resulted in significantly decreased bacterial internalization compared to cells grown under normoxia. This phenotype was absent after functionally blocking host β1 integrins as well as upon infection with an invasin-deficient Y. enterocolitica strain. Furthermore, downstream phosphorylation of the focal adhesion kinase was also reduced under hypoxia after infection. In good correlation to these data, cells grown under hypoxia showed decreased protein levels of β1 integrins at the apical cell surface whereas the total protein level of the hypoxia inducible factor (HIF-1) alpha was elevated. Furthermore, treatment of cells with the HIF-1 α stabilizer dimethyloxalylglycine (DMOG) also reduced invasion and decreased β1 integrin protein levels compared to control cells, indicating a potential role for HIF-1α in this process. These results suggest that hypoxia decreases invasin-integrin-mediated internalization of Y. enterocolitica into intestinal epithelial cells by reducing cell surface localization of host β1 integrins.

  20. Bioavailability and metabolism of rosemary infusion polyphenols using Caco-2 and HepG2 cell model systems.

    Science.gov (United States)

    Achour, Mariem; Saguem, Saad; Sarriá, Beatriz; Bravo, Laura; Mateos, Raquel

    2018-01-12

    Rosmarinus officinalis is an aromatic plant used in folk medicine as a result of the therapeutic properties associated with its phenolic composition, being rich in rosmarinic acid (RA) and caffeic acid (CA). To better understand the bioactivity of these compounds, their absorption and metabolism were assessed in human Caco-2 and HepG2 cells, as small intestine and liver models, respectively, using RA and CA standards, as well as a rosemary infusion and ferulic acid (FA). Test compounds were partially up-taken and metabolized by Caco-2 and HepG2 cells, although a higher metabolization rate was observed after hepatic incubation compared to intestinal incubation. CA was the compound best absorbed followed by RA and FA, showing metabolites percentages of 30.4%, 11.8% and 4.4% in Caco-2 and 34.3%, 10.3% and 3.2% in HepG2 cells, respectively. RA in the rosemary infusion showed improved bioavailability compared to pure RA. Methyl derivatives were the main metabolites detected for CA and RA after intestinal and hepatic metabolism, followed by methyl-glucuronidates and glucuronidates. RA was also minimally hydrolyzed into CA, whereas FA only was glucuronidated. Rosemary polyphenols followed the same biotransformation pathways as the standards. In addition, phase II derivatives of luteolin were observed. Rosemary polyphenols are partially metabolized in both the intestine and liver. © 2018 Society of Chemical Industry. © 2018 Society of Chemical Industry.

  1. Acamprosate permeability across Caco-2 cell monolayer is predominantly paracellular

    DEFF Research Database (Denmark)

    Antonescu, Irina-Elena; Steffansen, Bente

    Background. The human oral bioavailability (BA) of acamprosate is 11% and its oral absorption is permeability limited (BCS class III). Acamprosate is not metabolized, therefore it’s BA has the same nominal value as its fraction absorbed (fa). It is however controversial whether the intestinal...... in the different regions of the rodent small intestine and colon. Biopharm Drug Dispos. 2017;38(2):94-114. 2. Avdeef A. Leakiness and size exclusion of paracellular channels in cultured epithelial cell monolayers-interlaboratory comparison. Pharm Res. 2010;27(3):480-9. 3. Avdeef A. Absorption and Drug Development...

  2. Effect of Penicillium roqueforti mycotoxins on Caco-2 cells: Acute and chronic exposure.

    Science.gov (United States)

    Hymery, Nolwenn; Mounier, Jérome; Coton, Emmanuel

    2018-04-01

    Penicillium roqueforti is a common food and feed contaminant. However, it is also worldwide renowned for its use as a technological culture responsible for the typicity of blue-veined cheese. Members of the P. roqueforti species are also known to be able to produce secondary metabolites including mycophenolic acid (MPA) and roquefortine C (ROQ C) mycotoxins. In order to more closely simulate the reality of mycotoxin exposure through contaminated food consumption, this work investigated the toxicological effects of MPA and ROQ C not only in acute but also in chronic (i.e. 21-days continuous exposure) conditions on Caco-2 cells. Acute exposure to high MPA or ROQ C concentrations induced an increase of IL-8 secretion. Effects of 21-days continuous exposure on barrier integrity, based on concentrations found in blue-veined cheese and mean of blue cheese intake by French consumers, were monitored. Concerning exposure to ROQ C, no alteration of the intestinal barrier was observed. In contrast, the highest tested MPA concentration (780 μM) induced a decrease in the barrier function of Caco-2 cell monolayers, but no paracellular passage of bacteria was observed. This study highlighted that exposure to MPA and ROQ C average concentrations found in blue-veined cheese does not seem to induce significant toxicological effects in the tested conditions. Copyright © 2018 Elsevier Ltd. All rights reserved.

  3. Micellar lipid composition profoundly affects LXR-dependent cholesterol transport across CaCo2 cells

    NARCIS (Netherlands)

    Petruzzelli, Michele; Groen, Albert K.; van Erpecum, Karel J.; Vrins, Carlos; van der Velde, Astrid E.; Portincasa, Piero; Palasciano, Giuseppe; van Berge Henegouwen, Gerard P.; Sasso, Giuseppe Lo; Morgano, Annalisa; Moschetta, Antonio

    2009-01-01

    Intraluminal phospholipids affect micellar solubilization and absorption of cholesterol. We here study cholesterol transport from taurocholate-phospholipid-cholesterol micelles to CaCo2 cells, and associated effects on ABC-A1 mediated cholesterol efflux. Micellar incorporation of

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

    Directory of Open Access Journals (Sweden)

    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.

  5. Adsorption of hematite nanoparticles onto Caco-2 cells and the cellular impairments: effect of particle size

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Wen; Chen Yongsheng [School of Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta, GA 30332 (United States); Kalive, Madhavi; Capco, David G, E-mail: yongsheng.chen@ce.gatech.edu [School of Life Sciences, Arizona State University, Tempe, AZ 85287 (United States)

    2010-09-03

    The increasing applications of engineered nanomaterials nowadays have elevated the potential of human exposure through various routes including inhalation, skin penetration and digestion. To date there is scarce information on a quantitative description of the interactions between nanoparticles (NPs) and cell surfaces and the detrimental effects from the exposure. The purpose of this work was to study in vitro exposure of Caco-2 cells to hematite ({alpha}-Fe{sub 2}O{sub 3}) NPs and to determine the particle size effects on the adsorption behaviors. Cellular impairment was also investigated and compared. Hematite NPs were synthesized as part of this study with a discrete size distribution and uniform morphology examined by dynamic light scattering (DLS) and confirmed by transmission electron microscopy (TEM). Caco-2 cells were cultured as a model epithelium to mirror human intestinal cells and used to evaluate the impacts of the exposure to NPs by measuring transepithelial electrical resistance (TEER). Cell surface disruption, localization and translocation of NPs through the cells were analyzed with immunocytochemical staining and confocal microscopy. Results showed that hematite NPs had mean diameters of 26, 53, 76 and 98 nm and were positively charged with minor aggregation in the buffer solution. Adsorption of the four sizes of NPs on cells reached equilibrium within approximately 5 min but adsorption kinetics were found to be size-dependent. The adsorption rates expressed as mg m{sup -2} min{sup -1} were greater for large NPs (76 and 98 nm) than those for small NPs (26 and 53 nm). However, adsorption rates, expressed in units of m{sup -2} min{sup -1}, were much greater for small NPs than large ones. After the adsorption equilibrium was reached, the adsorbed mass of NPs on a unit area of cells was calculated and showed no significant size dependence. Longer exposure time (>3 h) induced adverse cellular effects as indicated by the drop in TEER compared to the

  6. Adsorption of hematite nanoparticles onto Caco-2 cells and the cellular impairments: effect of particle size

    Science.gov (United States)

    Zhang, Wen; Kalive, Madhavi; Capco, David G.; Chen, Yongsheng

    2010-09-01

    The increasing applications of engineered nanomaterials nowadays have elevated the potential of human exposure through various routes including inhalation, skin penetration and digestion. To date there is scarce information on a quantitative description of the interactions between nanoparticles (NPs) and cell surfaces and the detrimental effects from the exposure. The purpose of this work was to study in vitro exposure of Caco-2 cells to hematite (α-Fe2O3) NPs and to determine the particle size effects on the adsorption behaviors. Cellular impairment was also investigated and compared. Hematite NPs were synthesized as part of this study with a discrete size distribution and uniform morphology examined by dynamic light scattering (DLS) and confirmed by transmission electron microscopy (TEM). Caco-2 cells were cultured as a model epithelium to mirror human intestinal cells and used to evaluate the impacts of the exposure to NPs by measuring transepithelial electrical resistance (TEER). Cell surface disruption, localization and translocation of NPs through the cells were analyzed with immunocytochemical staining and confocal microscopy. Results showed that hematite NPs had mean diameters of 26, 53, 76 and 98 nm and were positively charged with minor aggregation in the buffer solution. Adsorption of the four sizes of NPs on cells reached equilibrium within approximately 5 min but adsorption kinetics were found to be size-dependent. The adsorption rates expressed as mg m - 2 min - 1 were greater for large NPs (76 and 98 nm) than those for small NPs (26 and 53 nm). However, adsorption rates, expressed in units of m - 2 min - 1, were much greater for small NPs than large ones. After the adsorption equilibrium was reached, the adsorbed mass of NPs on a unit area of cells was calculated and showed no significant size dependence. Longer exposure time (>3 h) induced adverse cellular effects as indicated by the drop in TEER compared to the control cells without the exposure

  7. 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...

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

    Directory of Open Access Journals (Sweden)

    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.

  9. 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

  10. Study of Structure and Permeability Relationship of Flavonoids in Caco-2 Cells.

    Science.gov (United States)

    Fang, Yajing; Cao, Weiwei; Xia, Mengmeng; Pan, Siyi; Xu, Xiaoyun

    2017-11-29

    Flavonoids exhibit a broad range of biological activities. However, poor absorption of some flavonoids is a major limitation for use of flavonoids as nutraceuticals. To investigate the structure requirements for flavonoids intestinal absorption, transepithelial transport and cellular accumulation (CA) of 30 flavonoids were determined using the Caco-2 cell monolayer. The bilateral permeation of five types of flavonoids followed the order: flavanones ≥ isoflavones > flavones ≥ chalcones > flavonols. The concentration of flavonoids accumulated in cells did not correlate with cell penetration since the correlation coefficient between the apparent permeability coefficient ( P app ) and their corresponding CA was poor ( R ² < 0.3). Most flavonoids exhibited a ratio of 0.8-1.5 for P app A to B / P app B to A , suggesting passive diffusion pathways. However, luteolin, morin and taxifolin may involve the efflux mechanisms. The quantitative structure-permeability relationship (QSPR) study demonstrated that the intestinal absorption of flavonoids can be related to atomic charges on carbon 3' ( Q C3' ), molecule surface area ( S log P_V 3), balance between the center of mass and position of hydrophobic region ( vsurf_ID 1) and solvation energy of flavonoids ( E_sol ). These results provide useful information for initially screening of flavonoids with high intestinal absorption.

  11. 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

  12. Insights into Caco-2 cell culture structure using coherent anti-Stokes Raman scattering (CARS) microscopy.

    Science.gov (United States)

    Saarinen, Jukka; Sözeri, Erkan; Fraser-Miller, Sara J; Peltonen, Leena; Santos, Hélder A; Isomäki, Antti; Strachan, Clare J

    2017-05-15

    We have used coherent anti-Stokes Raman scattering (CARS) microscopy as a novel and rapid, label-free and non-destructive imaging method to gain structural insights into live intestinal epithelial cell cultures used for drug permeability testing. Specifically we have imaged live Caco-2 cells in (bio)pharmaceutically relevant conditions grown on membrane inserts. Imaging conditions were optimized, including evaluation of suitable membrane materials and media solutions, as well as tolerable laser powers for non-destructive imaging of the live cells. Lipid structures, in particular lipid droplets, were imaged within the cells on the insert membranes. The size of the individual lipid droplets increased substantially over the 21-day culturing period up to approximately 10% of the volume of the cross section of individual cells. Variation in lipid content has important implications for intestinal drug permeation testing during drug development but has received limited attention to date due to a lack of suitable analytical techniques. CARS microscopy was shown to be well suited for such analysis with the potential for in situ imaging of the same individual cell-cultures that are used for permeation studies. Overall, the method may be used to provide important information about cell monolayer structure to better understand drug permeation results. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Adhesion of Two Lactobacillus gasseri Probiotic Strains on Caco-2 Cells

    Directory of Open Access Journals (Sweden)

    Mojca Narat

    2003-01-01

    Full Text Available Previous in vitro and in vivo studies showed that two human isolates of Lactobacillus gasseri, LF221 and K7 are able to survive the passage through the gastrointestinal tract and to colonise intestines of pigs at least temporarily. The aim of this study was to examine the adhesion ability of LF221 and K7 strains to Caco-2 cells. Adhesion of lactobacilli from early stationary growth phase was examined at two pH values of DMEM buffer (4.5 and 7. Lactobacillus rhamnosus GG, a widely used strain with clinical evidences of its efficiency, served as a positive control. The number of lactobacilli added to each well was found to be crucial in the adhesion assay. When added, lactobacilli were in range of 2.5 · 106 to 2.5 · 108 cfu/well, the linear correlation between the number of adhered cells (log cfu and the number of added cells (log cfu was found for all three strains (R2 > 0.99 at both pH values (4.5 and 7. At the highest concentration of added K7 and GG cells tested (app. 109 cfu/well, the efficiency of adhesion was reduced. pH value of the medium strongly affected the adhesion, which was promoted in acidic conditions (pH=4.5. The adhesion of K7 strain was slightly weaker compared to GG strain at both pH values, while at pH=4.5 the adhesion of LF221 strain was even better than GG adhesion, at least at lower concentration of lactobacilli. The direct comparison of these strains was possible by regression analysis. At lower concentration of lactobacilli (2.5 · 106, the best efficiency of adhesion (% of adhered bacteria was observed for the strain LF221, reaching the values of 7.8 and 1.9 % at pH=4.5 and 7, respectively, while at higher lactobacilli concentration the ration of adhesion was higher for GG strain (3.3 % at pH=4.5. In conclusion, strains LF221 and K7 were demonstrated to be adhesive, especially in acidic conditions. The level of adhesion of K7 and GG strains positively correlates with the number of added lactobacilli only up to the

  14. Secretagogue-induced protein phosphorylation and chloride transport in Caco-2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Burnham, D.B.; Fondacaro, J.D.

    1989-04-01

    The effects of vasoactive intestinal polypeptide (VIP), 16,16-dimethyl prostaglandin E2 (DMPGE2) and dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) on protein phosphorylation were studied in relation to stimulation of chloride transport in cell suspensions of the human colon epithelial cell line Caco-2. In /sup 36/Cl-loaded cells, VIP and DMPGE2 within 1 min decreased cellular chloride content 35-40%, with half-maximal effects being elicited at 1.0 and 85 nM concentration, respectively. A similar effect on chloride content occurred after 10 min of treatment with 0.5 mM DBcAMP. For all three secretagogues, decreases in cellular chloride content were associated with increases in membrane permeability to chloride. DMPGE2 and VIP within 1 min, and DBcAMP within 10 min, increased the phosphorylation of an unidentified soluble protein of Mr = 42,000 and pI = 6.1, and of a protein of Mr = 20,200 and pI = 4.9 identified as myosin regulatory light chain. Between 10 and 30 min of stimulation, however, phosphorylation of the Mr = 42,000 protein and chloride transport activity remained elevated in DMPGE2- and DBcAMP-treated cells, whereas light chain phosphorylation returned to control level. No effect of secretagogues on phosphorylation was detected in the total particulate fraction or an integral membrane protein fraction. It is concluded that increased membrane permeability to chloride induced by cAMP-mediated secretagogues in Caco-2 is temporally associated with the increased phosphorylation of a Mr = 42,000 soluble protein.

  15. Acamprosate permeability across Caco-2 cell monolayer is predominantly paracellular

    DEFF Research Database (Denmark)

    Antonescu, Irina-Elena; Steffansen, Bente

    was mathematically accounting for the unstirred boundary layer permeability (PUBL), the filter permeability (Pf), the intrinsic passive transcellular permeability (Ptrans,0) and Ppara (1-3). The mathematical model thereby accounted for (i) the physical-chemical properties of acamprosate and mannitol (molecular...... role in acamprosate permeability, as only a very low fraction of acamprosate is in the neutral form at pH 7.4. The estimated acamprosate Ppara accounts for nearly 100% of the mathematically determined acamprosate Papp, calc (0.20 ± 0.10 x 10-6 cm/s), which matches well with the experimentally...... in the different regions of the rodent small intestine and colon. Biopharm Drug Dispos. 2017;38(2):94-114. 2. Avdeef A. Leakiness and size exclusion of paracellular channels in cultured epithelial cell monolayers-interlaboratory comparison. Pharm Res. 2010;27(3):480-9. 3. Avdeef A. Absorption and Drug Development...

  16. Caco-2 Cell Conditions Enabling Studies of Drug Absorption from Digestible Lipid-Based Formulations.

    Science.gov (United States)

    Keemink, Janneke; Bergström, Christel A S

    2018-02-26

    To identify conditions allowing the use of cell-based models for studies of drug absorption during in vitro lipolysis of lipid-based formulations (LBFs). Caco-2 was selected as the cell-based model system. Monolayer integrity was evaluated by measuring mannitol permeability after incubating Caco-2 cells in the presence of components available during lipolysis. Pure excipients and formulations representing the lipid formulation classification system (LFCS) were evaluated before and after digestion. Porcine mucin was evaluated for its capacity to protect the cell monolayer. Most undigested formulations were compatible with the cells (II-LC, IIIB-LC, and IV) although some needed mucin to protect against damaging effects (II-MC, IIIB-MC, I-LC, and IIIA-LC). The pancreatic extract commonly used in digestion studies was incompatible with the cells but the Caco-2 monolayers could withstand immobilized recombinant lipase. Upon digestion, long chain formulations caused more damage to Caco-2 cells than their undigested counterparts whereas medium chain formulations showed better tolerability after digestion. Most LBFs and components thereof (undigested and digested) are compatible with Caco-2 cells. Pancreatic enzyme is not tolerated by the cells but immobilized lipase can be used in combination with the cell monolayer. Mucin is beneficial for critical formulations and digestion products.

  17. Four Cation-Selective Transporters Contribute to Apical Uptake and Accumulation of Metformin in Caco-2 Cell Monolayers

    Science.gov (United States)

    Han, Tianxiang (Kevin); Proctor, William R.; Costales, Chester L.; Cai, Hao; Everett, Ruth S.

    2015-01-01

    Metformin is the frontline therapy for type II diabetes mellitus. The oral bioavailability of metformin is unexpectedly high, between 40 and 60%, given its hydrophilicity and positive charge at all physiologic pH values. Previous studies in Caco-2 cell monolayers, a cellular model of the human intestinal epithelium, showed that during absorptive transport metformin is taken up into the cells via transporters in the apical (AP) membrane; however, predominant transport to the basolateral (BL) side occurs via the paracellular route because intracellular metformin cannot egress across the BL membrane. Furthermore, these studies have suggested that the AP transporters can contribute to intestinal accumulation and absorption of metformin. Transporter-specific inhibitors as well as a novel approach involving a cocktail of transporter inhibitors with overlapping selectivity were used to identify the AP transporters that mediate metformin uptake in Caco-2 cell monolayers; furthermore, the relative contributions of these transporters in metformin AP uptake were also determined. The organic cation transporter 1, plasma membrane monoamine transporter (PMAT), serotonin reuptake transporter, and choline high-affinity transporter contributed to approximately 25%, 20%, 20%, and 15%, respectively, of the AP uptake of metformin. PMAT-knockdown Caco-2 cells were constructed to confirm the contribution of PMAT in metformin AP uptake because a PMAT-selective inhibitor is not available. The identification of four intestinal transporters that contribute to AP uptake and potentially intestinal absorption of metformin is a significant novel finding that can influence our understanding of metformin pharmacology and intestinal drug-drug interactions involving this highly prescribed drug. PMID:25563903

  18. 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. © 2016 Institute of Food Technologists®

  19. Egg-Yolk Sphingomyelin and Phosphatidylcholine Attenuate Cholesterol Absorption in Caco-2 Cells.

    Science.gov (United States)

    Yang, Fang; Chen, Guoxun; Ma, Meihu; Qiu, Ning; Zhu, Lingjiao; Li, Jing

    2018-02-01

    Phospholipids have been shown to modulate intestinal cholesterol absorption in cells and animals, a process that is regulated by several transporter proteins. Of these proteins, Niemann-Pick C1-Like 1 (NPC1L1) is a major contributor to this process. The mechanism by which phospholipids modulate cholesterol absorption remains unknown. Here, we evaluate the effects of egg-yolk phospholipids on cholesterol absorption and transport in human colon carcinoma cell line (Caco-2 cells) and on the expression of NPC1L1 and others proteins associated with cholesterol absorption (ABCG5, ABCG8, ABCA1, ACAT2, MTP, CAV-1, ANX-2). The roles of SREBP-1 and SREBP-2 in this process were also investigated. The results show that egg-yolk sphingomyelin (CerPCho) and phosphatidylcholine (PtdCho) inhibit cholesterol transport in the Caco-2 monolayer in a dose-dependent manner. These might be due to the decrease of the cholesterol solubility in micelles as well as to the increases in the micellar sizes and the bile acid-binding capacity. Furthermore, the treatments with egg-yolk CerPCho or PtdCho at 1.2 mmol/L reduced the expression levels of NPC1L1 protein to 21 or 22%, respectively, and its mRNA to 9 or 31% of that in the control group (p egg-yolk PtdCho and CerPCho on the mRNA levels of SREBP-1, and SREBP-2. These results suggest that the inhibitory effect of egg-yolk CerPCho and PtdCho on cholesterol transport might be due to their interference with the physicochemical properties of micelles and their regulations on the expression of the NPC1L1 gene. © 2018 AOCS.

  20. Immunomodulatory activity of enzymatically synthesized glycogen and its digested metabolite in a co-culture system consisting of differentiated Caco-2 cells and RAW264.7 macrophages.

    Science.gov (United States)

    Yasuda, Michiko; Furuyashiki, Takashi; Nakamura, Toshiyuki; Kakutani, Ryo; Takata, Hiroki; Ashida, Hitoshi

    2013-09-01

    Previously, we developed enzymatically synthesized glycogen (ESG) from starch, and showed its immunomodulatory and dietary fiber-like activities. In this study, we investigated the metabolism of ESG and its immunomodulatory activity using differentiated Caco-2 cells as a model of the intestinal barrier. In a co-culture system consisting of differentiated Caco-2 cells and RAW264.7 macrophages, mRNA expression of IL-6, IL-8, IL-1β and BAFF cytokines was up-regulated in Caco-2 cells and IL-8 production in basolateral medium was induced after 24 h apical treatment with 5 mg ml(-1) of ESG. The mRNA level of iNOS was also up-regulated in RAW264.7 macrophages. After characterization of the binding of anti-glycogen monoclonal antibodies (IV58B6 and ESG1A9) to ESG and its digested metabolite resistant glycogen (RG), an enzyme-linked immunosorbent assay (ELISA) system was developed to quantify ESG and RG. Using this system, we investigated the metabolism of ESG in differentiated Caco-2 cells. When ESG (7000 kDa, 5 mg ml(-1)) was added to the apical side of Caco-2 monolayers, ESG disappeared and RG (about 3000 kDa, 3.5 mg ml(-1)) appeared in the apical solution during a 24 h incubation. Neither ESG nor RG was detected in the basolateral solution. In addition, both ESG and RG were bound to TLR2 in Caco-2 cells. In conclusion, we suggest that ESG is metabolized to a RG-like structure in the intestine, and this metabolite activates the immune system via stimulation of the intestinal epithelium, although neither ESG nor its metabolite could permeate the intestinal cells under our experimental conditions. These results provide evidence for the beneficial function of ESG as a food ingredient.

  1. 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.

  2. Organic acid bioavailability from banana and sweet potato using an in vitro digestion and Caco-2 cell model.

    Science.gov (United States)

    Sabboh-Jourdan, Houda; Valla, Fanny; Epriliati, Indah; Gidley, Michael J

    2011-02-01

    Organic acids from plant food have been shown to play an important role in the prevention of chronic diseases (osteoporosis, obesity), inherent to western diets, but little is known about their bioavailability in the small intestine, information that needs to be determined in order to quantify likely effects on human health. An in vitro model of human digestion was carried out, comprising simulated oral, gastric and pancreatic digestion followed by an in vitro model of small intestine absorption using Caco-2 cell monolayers. As models for fruits and vegetables, freeze-dried or raw samples of banana and sweet potato were used. Organic acids have been found to be slowly released from the food matrix during simulated digestion of both banana and sweet potato, either raw or after freeze-drying. In the Caco-2 cell assay, malic and oxalic acids were absorbed more than citric acid. Oxalic and citric acids, but not malic acid, were transported across the cell monolayer. The release and uptake of major organic acids from model fruits and vegetables using established in vitro simulation processes was not quantitative and varied with acid type. Partial uptake is consistent with a dual nutritional role for organic acids as alkalinising agents (fraction which is taken up) and as modulators of large intestinal function (fraction which is not taken up in the small intestine). Studies of in vivo digestive release and uptake are needed in order to identify the contribution of organic acids to the nutritional value of fruits and vegetables.

  3. Antioxidant potential of buffalo and cow milk Cheddar cheeses to tackle human colon adenocarcinoma (Caco-2) cells.

    Science.gov (United States)

    Huma, Nuzhat; Rafiq, Saima; Sameen, Aysha; Pasha, Imran; Khan, Muhammad Issa

    2018-02-01

    The aim of present study was to assess the anti-oxidant potential of water-soluble peptides (WSPs) extract derived from buffalo and cow milk Cheddar cheeses at different stages of ripening. The antioxidant potential of WSPs extract was assessed through 2,2'-azinobis-3-ethylbenzothiazoline-6sulfonic acid (ABTS)-radical scavenging activity. In addition, impact of WSPs extract on cell viability and production of reactive oxygen species (ROS) in human colon adenocarcinoma Caco-2 (tert-butylhydroperoxide-induced) cell lines was also evaluated. The ABTS-radical scavenging activity increased progressively with ripening period and dose-dependently in both cheeses. However, peptide extract from buffalo milk Cheddar cheese demonstrated relatively higher activity due to higher contents of water-soluble nitrogen. Intracellular ROS production in Caco-2 cells decreased significantly (pCaco-2 cell line. On the basis of current in vitro study, the Cheddar cheese WSPs extract can protect intestinal epithelium against oxidative stress due to their antioxidant activity.

  4. Antioxidant potential of buffalo and cow milk Cheddar cheeses to tackle human colon adenocarcinoma (Caco-2 cells

    Directory of Open Access Journals (Sweden)

    Nuzhat Huma

    2018-02-01

    Full Text Available Objective The aim of present study was to assess the anti-oxidant potential of water-soluble peptides (WSPs extract derived from buffalo and cow milk Cheddar cheeses at different stages of ripening. Methods The antioxidant potential of WSPs extract was assessed through 2,2’-azinobis-3-ethylbenzothiazoline-6sulfonic acid (ABTS-radical scavenging activity. In addition, impact of WSPs extract on cell viability and production of reactive oxygen species (ROS in human colon adenocarcinoma Caco-2 (tert-butylhydroperoxide-induced cell lines was also evaluated. Results The ABTS-radical scavenging activity increased progressively with ripening period and dose-dependently in both cheeses. However, peptide extract from buffalo milk Cheddar cheese demonstrated relatively higher activity due to higher contents of water-soluble nitrogen. Intracellular ROS production in Caco-2 cells decreased significantly (p<0.05 till 150th day of cheese ripening and remained constant thereafter. Additionally, dose-dependent response of WSPs extract on antioxidant activity was noticed in the Caco-2 cell line. Conclusion On the basis of current in vitro study, the Cheddar cheese WSPs extract can protect intestinal epithelium against oxidative stress due to their antioxidant activity.

  5. Cytotoxicity assessment of lipid-based self-emulsifying drug delivery system with Caco-2 cell model: Cremophor EL as the surfactant.

    Science.gov (United States)

    Bu, Pengli; Narayanan, Silpa; Dalrymple, Damon; Cheng, Xingguo; Serajuddin, Abu T M

    2016-08-25

    Caco-2 cells are used extensively for in vitro prediction of intestinal drug absorption. However, toxicity of excipients and formulations used can artificially increase drug permeation by damaging cell monolayers, thus providing misleading results. The present study aimed to investigate cytotoxicity of common lipid-based excipients and formulations on Caco-2 cells. Medium-chain monoglycerides alone or in mixture with the surfactant Cremophor EL, with and without a medium-chain triglyceride, were prepared and incubated with Caco-2 cells from a series of culture stages with varying maturity. Cell viability was evaluated and cell membrane integrity assessed. Cytotoxicity of lipid-based formulations was influenced by the maturity of Caco-2 cells and formulation composition. One-day culture was most sensitive to lipids. When cultured for 5days, viability of Caco-2 cells was significantly improved. The 21-day Caco-2 monolayers maintained the highest survival rate. Microemulsion formulations exhibited significantly less cytotoxicity than neat lipids or surfactant at all stages of cell maturity, and microemulsions containing 1:1 mixtures of monoglyceride and triglyceride appeared to be best tolerated among all the formulations tested. Mechanistically, the observed cytotoxicity was partially due to lipid-induced rupture of cell membrane. Microemulsions of lipid-surfactant mixtures have less cytotoxicity than lipid alone. Maturity of Caco-2 cells renders significant resistance to cytotoxicity, and monolayers with 21-day maturity are more relevant to in vivo conditions and appear to be a more accurate in vitro model for cytotoxicity assessment. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Barbara Lieder

    Full Text Available 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.

  7. 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. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. The cytotoxic effect of palytoxin on Caco-2 cells hinders their use for in vitro absorption studies.

    Science.gov (United States)

    Pelin, M; Sosa, S; Della Loggia, R; Poli, M; Tubaro, A; Decorti, G; Florio, C

    2012-02-01

    Palytoxin (PLTX), found in Palythoa zoanthids and Ostreopsis dinoflagellates, has also been detected in crabs and fish, through which it can enter into the food chain. Indeed, PLTX is considered the causative agent of several cases of human seafood poisoning resulting in systemic symptoms. Available epidemiological data on PLTX human toxicity suggest that the intestinal tract may be one of its in vivo targets and its potential site of access into the bloodstream. Hence, the purpose of this study was to investigate the suitability of the human intestinal Caco-2 cell line for evaluating PLTX oral absorption. A detailed analysis of PLTX cytotoxicity revealed a high sensitivity of Caco-2 cells: 4h toxin exposure reduced mitochondrial activity (MTT assay, EC(50) of 8.9±3.7×10(-12)M), cell density (SRB assay, EC(50) of 2.0±0.6×10(-11)M) and membrane integrity (LDH release, EC(50) of 4.5±1.4×10(-9)M and PI uptake, EC(50) of 1.0±0.8×10(-8)M). After low PLTX concentration (1.0×10(-11)M) exposure for 1-8h, followed by 24h recovery time in toxin-free medium, cell density reduction was only partially reversible. These results indicate that, due to the high susceptibility to PLTX cytotoxic effects, Caco-2 cells do not represent an appropriate and reliable model for investigating intestinal barrier permeation by this toxin. Copyright © 2011 Elsevier Ltd. All rights reserved.

  9. Effects of Tea Phenolics on Iron Uptake from Different Fortificants by Caco-2 cells

    Science.gov (United States)

    The in vitro effects of tea phenolics on Fe uptake from different fortificants (FeSO4, FeCl3, FeEDTA) by Caco-2 cells were compared. Cell cultures were exposed to catechin, tannic acid, green or black tea solutions added within Fe-containing solution, or used to pre-treat cell cultures before Fe-exp...

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

    Directory of Open Access Journals (Sweden)

    Saura C. Sahu

    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. Keywords: Nanosilver, Silver nanoparticles, Nanoparticles, Toxicogenomics, DNA microarray, Global gene expression profiles, Caco2 cells

  11. 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...

  12. Lactobacillus plantarum L9 but not Lactobacillus acidophilus LA reduces tumour necrosis factor induced bacterial translocation in Caco-2 cells.

    Science.gov (United States)

    Wang, B; Chen, J; Wang, S; Zhao, X; Lu, G; Tang, X

    2017-05-30

    Translocation of bacteria across the intestinal barrier is important in the pathogenesis of systemic sepsis and multiple organ dysfunction syndromes. Inflammatory cytokines increase paracellular permeability that allows increased luminal bacteria to translocate across mucosal epithelium and further deteriorate the gut barrier. In order to reduce this risk, the prophylactic use of probiotics has been recently addressed. In this paper, we investigate the protective role toward tumour necrosis factor (TNF)-α induced non-pathogenic Escherichia coli translocation across Caco-2 monolayers of Lactobacillus strains. According to our experimental data, Lactobacillus plantarum L9 and Lactobacillus acidophilus LA have good capacities to adhere to Caco-2 cells. Addition of L. plantarum L9 and L. acidophilus LA to the enterocyte monolayer surface result in significant inhibition of E. coli adhesion and cell internalisation. However, L. plantarum L9 and L. acidophilus LA did not inhibit the growth of the non-pathogenic E. coli B5 after 24 h incubation. Exposure to TNF-α for 6 h caused a dramatic increase in E. coli B5 translocation across Caco-2 cells, which was uncoupled from increases in paracellular permeability. Pretreatment with L. plantarum L9 prevent TNF-α induced transcellular bacterial translocation and IL-8 production in Caco-2 cells. L. plantarum L9 also did not affect the integrity of the monolayers, as indicated by lactate dehydrogenase release, horseradish peroxidase permeability, and transepithelial electrical resistance. L. plantarum L9 showed the potential to protect enterocytes from an acute inflammatory response and therefore could be good potential prophylactic agents in counteracting bacterial translocation.

  13. 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

  14. Conditioned medium from LS 174T goblet cells treated with oxyresveratrol strengthens tight junctions in Caco-2 cells.

    Science.gov (United States)

    Hwang, Dahyun; Jo, HyunA; Hwang, Seonwook; Kim, Jeong-Keun; Kim, In-Ho; Lim, Young-Hee

    2017-01-01

    Strengthening of intestinal tight junctions provides an effective barrier from the external environment. Goblet cell-derived trefoil factor 3 (TFF3) increases transepithelial resistance by upregulating the expression of tight junction proteins. Oxyresveratrol (OXY) is a hydroxyl-substituted stilbene found in the roots, leaves, stems, and fruit of many plants and known to have various biological activities. In this study, we investigated the strengthening effect of OXY on intestinal tight junctions through stimulation of TFF production in goblet cells. We prepared conditioned medium from LS 174T goblet cells treated with OXY (GCO-CM) and investigated the effect of GCO-CM on strengthening tight junctions of Caco-2 cells. The mRNA and protein expression levels of major tight junction components (claudin-1, occludin, and ZO-1) were measured by quantitative real-time PCR and western blotting, respectively. Transepithelial electric resistance (TEER) was measured using an ohm/V meter. Monolayer permeability was evaluated by paracellular transport of fluorescein isothiocyanate-dextran. OXY showed a strong antioxidant activity. It significantly increased the expression level of TFF3 in LS 174T goblet cells. GCO-CM prepared by treatment with 2.5, 5, and 10μg/ml OXY did not show cytotoxicity in Caco-2 cells. GCO-CM increased the mRNA and protein expression levels of claudin-1, occludin, and ZO-1. It also significantly increased tight junction integrity and reduced permeability in a dose-dependent manner. OXY stimulates the expression of TFF3 in goblet cells, which might increase the integrity of the intestinal tight junction barrier. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  15. Titanium dioxide nanoparticles activate IL8-related inflammatory pathways in human colonic epithelial Caco-2 cells

    Science.gov (United States)

    Krüger, Kristin; Cossais, François; Neve, Horst; Klempt, Martin

    2014-05-01

    Nanosized titanium dioxide (TiO2) particles are widely used as food additive or coating material in products of the food and pharmaceutical industry. Studies on various cell lines have shown that TiO2 nanoparticles (NPs) induced the inflammatory response and cytotoxicity. However, the influences of TiO2 NPs' exposure on inflammatory pathways in intestinal epithelial cells and their differentiation have not been investigated so far. This study demonstrates that TiO2 NPs with particle sizes ranging between 5 and 10 nm do not affect enterocyte differentiation but cause an activation of inflammatory pathways in the human colon adenocarcinoma cell line Caco-2. 5 and 10 nm NPs' exposures transiently induce the expression of ICAM1, CCL20, COX2 and IL8, as determined by quantitative PCR, whereas larger particles (490 nm) do not. Further, using nuclear factor (NF)-κB reporter gene assays, we show that NP-induced IL8 mRNA expression occurs, in part, through activation of NF-κB and p38 mitogen-activated protein kinase pathways.

  16. Impacts of Salmonella enterica Serovar Typhimurium and Its speG Gene on the Transcriptomes of In Vitro M Cells and Caco-2 Cells

    Science.gov (United States)

    Wang, Ke-Chuan; Huang, Chih-Hung; Huang, Ching-Jou

    2016-01-01

    Microfold or membranous (M) cells are specialized intestinal epithelial cells responsible for host immunity. The speG mutant of Salmonella Typhimurium (S. Typhimurium) is a nonreplicating strain within human cells to be a candidate vaccine vector for interacting with M cells. We conducted this study to identify the genes are differently expressed between in vitro M cells and Caco-2 cells, and to determine whether S. Typhimurium and speG affect the transcriptomes of both cell types. In vitro M cells and Caco-2 cells were infected with wild-type (WT) S. Typhimurium, its ΔspeG mutant, or none for 1 h for RNA microarrays; the transcriptomes among the 6 pools were pairwisely compared. Genetic loci encoding scaffold (e.g., HSCHR7_CTG4_4, HSCHR9_CTG9_35), long noncoding RNA, membrane-associated protein (PITPNB), neuron-related proteins (OR8D1, OR10G9, and NTNG2), and transporter proteins (MICU2 and SLC28A1) were significantly upregulated in uninfected M cells compared with uninfected Caco-2 cells; and their encoding proteins are promising M-cell markers. Significantly upregulated HSCHR7_CTG4_4 of uninfected in vitro M cells were speG-independently downregulated by S. Typhimurium infection that is a remarkable change representing an important but unreported characteristic of M cells. The immune responses of in vitro M cells and Caco-2 cells can differ and reply on speG or not, with speG-dependent regulation of KYL4, SCTR, IL6, TNF, and CELF4 in Caco-2 cells, JUN, KLF6, and KCTD11 in M cells, or speG-independent modulation of ZFP36 in both cells. This study facilitates understanding of the immune responses of in vitro M cells after administering the S. Typhimurium ΔspeG mutant as a future vaccine vector. PMID:27064787

  17. Transcriptome changes during intestinal cell differentiation

    DEFF Research Database (Denmark)

    Tadjali, Mehrdad; Seidelin, Jakob B; Olsen, Jørgen

    2002-01-01

    The expression of 18149 genes have been analysed during the differentiation of the human intestinal cell line Caco-2. cDNA probes from undifferentiated and differentiated Caco-2 cells were separately hybridised to EST DNAs spotted in an array on a nylon membrane. A remarkable change in the transc......The expression of 18149 genes have been analysed during the differentiation of the human intestinal cell line Caco-2. cDNA probes from undifferentiated and differentiated Caco-2 cells were separately hybridised to EST DNAs spotted in an array on a nylon membrane. A remarkable change...

  18. Low molecular weight heparin nanoparticles: mucoadhesion and behaviour in Caco-2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Lamprecht, Alf [Laboratory of Pharmaceutical Engineering, University of Besancon (France); Koenig, Petra [Center for Bioinformatics, Saarland University, Saarbruecken (Germany); Ubrich, Nathalie [Laboratory of Pharmaceutical Technology, University of Nancy (France); Maincent, Philippe [Laboratory of Pharmaceutical Technology, University of Nancy (France); Neumann, Dirk [Center for Bioinformatics, Saarland University, Saarbruecken (Germany)

    2006-08-14

    Nanoparticles (NPs) have shown their efficiency in increasing the oral bioavailability of macromolecular drugs, among them heparin. However, mechanisms of absorption are still unclear. Here, heparin-loaded NPs were prepared from different polymers (Eudragit[reg] RS, poly(lactic-co-glycolic acid) (PLGA), and their respective mixtures) and analysed for their mucoadhesive properties using a resonant mirror system. Subsequent binding and drug transport studies of the free heparin and heparin-loaded NPs were carried out on Caco-2 cells. Cationic NPs were found to be mucoadhesive, while pure drug and polyester NPs were not. The adsorption of anionic heparin masked the positive surface charge of the particles, thus partially diminishing the adhesiveness to mucin. Increased binding to Caco-2 cells was found for all NP formulation, with RS/PLGA NPs showing maximum binding. However, the transport of heparin was the same for the RS/PLGA NPs and the PLGA NPs and slightly higher than for the free drug. In all cases, no NP transport across the cell layer was observed. When Caco-2 cells were coated with an additional mucin layer, cell binding of RS NPs and RS/PLGA NPs was further increased. Transport across Caco-2 cells demonstrated similar tendencies to results obtained without mucin. In contrast, cationic NPs led to higher heparin transport in the presence of mucin. The mechanism of drug absorption associated with RS NPs was concluded to be independent of typical transcellular NP transport.

  19. Low molecular weight heparin nanoparticles: mucoadhesion and behaviour in Caco-2 cells

    Science.gov (United States)

    Lamprecht, Alf; Koenig, Petra; Ubrich, Nathalie; Maincent, Philippe; Neumann, Dirk

    2006-08-01

    Nanoparticles (NPs) have shown their efficiency in increasing the oral bioavailability of macromolecular drugs, among them heparin. However, mechanisms of absorption are still unclear. Here, heparin-loaded NPs were prepared from different polymers (Eudragit® RS, poly(lactic-co-glycolic acid) (PLGA), and their respective mixtures) and analysed for their mucoadhesive properties using a resonant mirror system. Subsequent binding and drug transport studies of the free heparin and heparin-loaded NPs were carried out on Caco-2 cells. Cationic NPs were found to be mucoadhesive, while pure drug and polyester NPs were not. The adsorption of anionic heparin masked the positive surface charge of the particles, thus partially diminishing the adhesiveness to mucin. Increased binding to Caco-2 cells was found for all NP formulation, with RS/PLGA NPs showing maximum binding. However, the transport of heparin was the same for the RS/PLGA NPs and the PLGA NPs and slightly higher than for the free drug. In all cases, no NP transport across the cell layer was observed. When Caco-2 cells were coated with an additional mucin layer, cell binding of RS NPs and RS/PLGA NPs was further increased. Transport across Caco-2 cells demonstrated similar tendencies to results obtained without mucin. In contrast, cationic NPs led to higher heparin transport in the presence of mucin. The mechanism of drug absorption associated with RS NPs was concluded to be independent of typical transcellular NP transport.

  20. Cytotoxicity and morphological effects induced by carvacrol and thymol on the human cell line Caco-2.

    Science.gov (United States)

    Llana-Ruiz-Cabello, María; Gutiérrez-Praena, Daniel; Pichardo, Silvia; Moreno, F Javier; Bermúdez, José María; Aucejo, Susana; Cameán, Ana María

    2014-02-01

    Essential oils used as additives in the food industry due to its flavour, antimicrobial and antioxidant properties. Therefore, human can be exposed orally to these compounds through the ingestion of foods. In this sense, the present work aims to assess toxicological effects of oregano essential oil on the digestive tract. In concrete, the cytotoxic effects of two components of the oregano essential oils, carvacrol and thymol, and their mixture, on the intestinal cells line Caco-2 after 24 and 48 h of exposure are studied. The basal cytotoxicity endpoints assayed (total protein content, neutral red uptake and the tetrazolium salt reduction) and the annexin/propidium iodide staining indicated that carvacrol and the mixture carvacrol/thymol induced toxic effects. Moreover, a morphological study was performed in order to determine the ultrastructural cellular damages caused by these substances. The main morphological alterations were vacuolated cytoplasm, altered organelles and finally cell death. In addition, although no cytotoxic effects were recorded for thymol at any concentration and time of exposure, ultrastructural changes evidenced cellular damage such as lipid degeneration, mitochondrial damage, nucleolar segregation and apoptosis. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. 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.

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

    International Nuclear Information System (INIS)

    Chen, Gao; Lianqin, Zhu; Fenghua, Zhu; Fang, Zheng; Mingming, Song; Kai, Huang

    2015-01-01

    Different concentrations of CuSO 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 app ) of CuSO 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 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 app value of Caco-2 cells incubated with nano-CuO was significantly higher than that obtained with CuSO 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 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 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

  3. Low Levels of GSTA1 Expression Are Required for Caco-2 Cell Proliferation

    Science.gov (United States)

    Adnan, Humaira; Quach, Holly; MacIntosh, Kimberley; Antenos, Monica; Kirby, Gordon M.

    2012-01-01

    The colonic epithelium continuously regenerates with transitions through various cellular phases including proliferation, differentiation and cell death via apoptosis. Human colonic adenocarcinoma (Caco-2) cells in culture undergo spontaneous differentiation into mature enterocytes in association with progressive increases in expression of glutathione S-transferase alpha-1 (GSTA1). We hypothesize that GSTA1 plays a functional role in controlling proliferation, differentiation and apoptosis in Caco-2 cells. We demonstrate increased GSTA1 levels associated with decreased proliferation and increased expression of differentiation markers alkaline phosphatase, villin, dipeptidyl peptidase-4 and E-cadherin in postconfluent Caco-2 cells. Results of MTS assays, BrdU incorporation and flow cytometry indicate that forced expression of GSTA1 significantly reduces cellular proliferation and siRNA-mediated down-regulation of GSTA1 significantly increases cells in S-phase and associated cell proliferation. Sodium butyrate (NaB) at a concentration of 1 mM reduces Caco-2 cell proliferation, increases differentiation and increases GSTA1 activity 4-fold by 72 hours. In contrast, 10 mM NaB causes significant toxicity in preconfluent cells via apoptosis through caspase-3 activation with reduced GSTA1 activity. However, GSTA1 down-regulation by siRNA does not alter NaB-induced differentiation or apoptosis in Caco-2 cells. While 10 mM NaB causes GSTA1-JNK complex dissociation, phosphorylation of JNK is not altered. These findings suggest that GSTA1 levels may play a role in modulating enterocyte proliferation but do not influence differentiation or apoptosis. PMID:23251616

  4. Caco-2 cell monolayer integrity and effect of probiotic Escherichia coli Nissle 1917 components

    Czech Academy of Sciences Publication Activity Database

    Štětinová, V.; Smetanová, L.; Květina, J.; Svoboda, Z.; Zídek, Zdeněk; Tlaskalová-Hogenová, Helena

    2010-01-01

    Roč. 31, - (2010), s. 51-56 ISSN 0172-780X R&D Projects: GA ČR GA305/08/0535; GA MZd NS9775 Institutional support: RVO:68378041 ; RVO:61388971 Keywords : probiotics * lipopolysaccharide (LPS) * Caco -2 cells Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 1.621, year: 2010

  5. Approaching the cellular processes involved in the positive effect of glycosaminoglycans on Fe uptake to Caco-2 cells

    Science.gov (United States)

    This study constitutes an approach to understand the enhancing effect of glycosaminoglycans (GAGs) on Fe uptake to Caco-2 cells. The high-sulfated GAGs fraction was isolated and purified from cooked haddock. An in vitro digestion/Caco-2 cell culture model was used to monitor Fe uptake (cell ferritin...

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

    Science.gov (United States)

    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.

  7. Isolation of Rice Bran Lectins and Characterization of Their Unique Behavior in Caco-2 Cells.

    Science.gov (United States)

    Nakata, Hajime; Lin, Ching Yu; Abolhassani, Maryam; Ogawa, Tomohisa; Tateno, Hiroaki; Hirabayashi, Jun; Muramoto, Koji

    2017-05-13

    Rice bran lectins, named as RBA1 and RBA2, were isolated from Oryza sativa in two chromatography steps: affinity chromatography and cation-exchange chromatography. RBA1 was found to be composed of a covalently linked heterodimer of 20- and 12-kDa subunits, and RBA2 was a noncovalently linked dimer of 12-kDa subunits. Both RBA1 and RBA2 bound to desialylated complex glycoproteins such as fetuin, α1-acid glycoprotein, and transferrin, and agalactosylated complex glycoproteins such as agalacto fetuin, agalacto-α1-acid glycoprotein, and agalacto-transferrin, in addition to chitooligosacchrides. RBAs were heat stable up to 80 °C and stable at pH 4-10. RBA1 increased the transport of the fluorescent marker, rhodamine 123, which is known to be transported via the P-glycoprotein-mediated efflux pathway across human intestinal Caco-2 cell monolayers. Furthermore, RBA1 itself was transported to the basolateral side of the monolayers via an endocytotic pathway.

  8. The effect of plant sterol-enriched turkey meat on cholesterol bio-accessibility during in vitro digestion and Caco-2 cell uptake.

    Science.gov (United States)

    Grasso, S; Harrison, S M; Monahan, F J; Brayden, D; Brunton, N P

    2018-03-01

    This study evaluated the effect of a plant sterol-enriched turkey product on cholesterol bio-accessibility during in vitro digestion and cholesterol uptake by Caco-2 monolayers. Turkey products, one plant sterol-enriched (PS) and one plant sterol-free (C), were produced in an industrial pilot plant. Before simulated digestion, matrices were spiked with cholesterol (1:5 weight ratio of cholesterol to plant sterol). Plant sterols were included at a concentration equivalent to the minimum daily intake recommended by the European Food Safety Authority (EFSA) for cholesterol lowering. After simulated digestion, the percentage of cholesterol micellarization and uptake by Caco-2 cells in the presence of PS meat were measured. Compared to C meat, PS meat significantly inhibited cholesterol micellarization on average by 24% and Caco-2 cell accumulation by 10%. This study suggests that plant sterols in meat can reduce cholesterol uptake by intestinal epithelia and it encourages efforts to make new PS-based functional foods.

  9. Inhibition of Glucose Transport by Tomatoside A, a Tomato Seed Steroidal Saponin, through the Suppression of GLUT2 Expression in Caco-2 Cells.

    Science.gov (United States)

    Li, Baorui; Terazono, Yusuke; Hirasaki, Naoto; Tatemichi, Yuki; Kinoshita, Emiko; Obata, Akio; Matsui, Toshiro

    2018-02-14

    We investigated whether tomatoside A (5α-furostane-3β,22,26-triol-3-[O-β-d-glucopyranosyl (1→2)-β-d-glucopyranosyl (1→4)-β-d-galactopyranoside] 26-O-β-d-glucopyranoside), a tomato seed saponin, may play a role in the regulation of intestinal glucose transport in human intestinal Caco-2 cells. Tomatoside A could not penetrate through Caco-2 cell monolayers, as observed in the transport experiments using liquid chromatography-mass spectrometry. The treatment of cells with 10 μM tomatoside A for 3 h resulted in a 46.0% reduction in glucose transport as compared to untreated cells. Western blotting analyses revealed that tomatoside A significantly (p transporter 2 (GLUT2) in Caco-2 cells, while no change in the expression of sodium-dependent glucose transporter 1 was observed. In glucose transport experiments, the reduced glucose transport by tomatoside A was ameliorated by a protein kinase C (PKC) inhibitor and a multidrug resistance-associated protein 2 (MRP2) inhibitor. The tomatoside A-induced reduction in glucose transport was restored in cells treated with apical sodium-dependent bile acid transporter (ASBT) siRNA or an ASBT antagonist. These findings demonstrated for the first time that the nontransportable tomato seed steroidal saponin, tomatoside A, suppressed GLUT2 expression via PKC signaling pathway during the ASBT-influx/MRP2-efflux process in Caco-2 cells.

  10. 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

  11. 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

  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. 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...

  14. The Potential Therapeutic Agent Mepacrine Protects Caco-2 Cells against Clostridium perfringens Enterotoxin Action

    Science.gov (United States)

    Freedman, John C.; Hendricks, Matthew R.

    2017-01-01

    ABSTRACT Clostridium perfringens enterotoxin (CPE) causes the diarrhea associated with a common bacterial food poisoning and many antibiotic-associated diarrhea cases. The severity of some CPE-mediated disease cases warrants the development of potential therapeutics. A previous study showed that the presence of mepacrine inhibited CPE-induced electrophysiology effects in artificial lipid bilayers lacking CPE receptors. However, that study did not assess whether mepacrine inactivates CPE or, instead, inhibits a step in CPE action. Furthermore, CPE action in host cells is complex, involving the toxin binding to receptors, receptor-bound CPE oligomerizing into a prepore on the membrane surface, and β-hairpins in the CPE prepore inserting into the membrane to form a pore that induces cell death. Therefore, the current study evaluated the ability of mepacrine to protect cells from CPE. This drug was found to reduce CPE-induced cytotoxicity in Caco-2 cells. This protection did not involve mepacrine inactivation of CPE, indicating that mepacrine affects one or more steps in CPE action. Western blotting then demonstrated that mepacrine decreases CPE pore levels in Caco-2 cells. This mepacrine-induced reduction in CPE pore levels did not involve CPE binding inhibition but rather an increase in CPE monomer dissociation due to mepacrine interactions with Caco-2 membranes. In addition, mepacrine was also shown to inhibit CPE pores when already present in Caco-2 cells. These in vitro studies, which identified two mepacrine-sensitive steps in CPE-induced cytotoxicity, add support to further testing of the therapeutic potential of mepacrine against CPE-mediated disease. IMPORTANCE Clostridium perfringens enterotoxin (CPE) causes the gastrointestinal (GI) symptoms of a common bacterial food poisoning and several nonfoodborne human GI diseases. A previous study showed that, via an undetermined mechanism, the presence of mepacrine blocks CPE-induced electrophysiologic activity in

  15. In vitro toxicity of different-sized ZnO nanoparticles in Caco-2 cells

    Science.gov (United States)

    Kang, Tianshu; Guan, Rongfa; Chen, Xiaoqiang; Song, Yijuan; Jiang, Han; Zhao, Jin

    2013-11-01

    There has been rapid growth in nanotechnology in both the public and private sectors worldwide, but concern about nanosafety exists. To assess size-dependent cytotoxicity on human cancer cells, we studied the cytotoxic effect of three kinds of zinc oxide nanoparticles (ZnO NPs) on human epithelial colorectal adenocarcinoma (Caco-2) cells. Nanoparticles were first characterized by size, distribution, and intensity. Multiple assays have been adopted to measure the cell activity and oxidative stress. The cytotoxicity of ZnO NPs was time dependent and dose dependent. The 24-h exposure was chosen to confirm the viability and accessibility of the cells and taken as the appropriate time for the following test system. The IC50 value was found at a low concentration. The oxidative stress elicited a significant reduction in glutathione with increase in reactive oxygen species and lactate dehydrogenase. The toxicity resulted in a deletion of cells in the G1 phase and an accumulation of cells in the S and G2/M phases. One type of metallic oxide (ZnO) exerted different cytotoxic effects according to different particle sizes. Data from the previous experiments showed that 26-nm ZnO NPs appeared to have the highest toxicity to Caco-2 cells. The study demonstrated the toxicity of ZnO NPs to Caco-2 cells and the impact of particle size, which could be useful in the medical applications.

  16. Organic Cation Transporter 1 (OCT1/mOct1) Is Localized in the Apical Membrane of Caco-2 Cell Monolayers and Enterocytes

    Science.gov (United States)

    Han, Tianxiang (Kevin); Everett, Ruth S.; Proctor, William R.; Ng, Chee M.; Costales, Chester L.; Brouwer, Kim L. R.

    2013-01-01

    Organic cation transporters (OCTs) are members of the solute carrier 22 family of transporter proteins that are involved in absorption, distribution, and excretion of organic cations. OCT3 is localized in the apical (AP) membrane of enterocytes, but the literature is ambiguous about OCT1 (mOct1) localization, with some evidence suggesting a basolateral (BL) localization in human and mouse enterocytes. This is contrary to our preliminary findings showing AP localization of OCT1 in Caco-2 cell monolayers, an established model of human intestinal epithelium. Therefore, this study aims at determining the localization of OCT1 (mOct1) in Caco-2 cells, and human and mouse enterocytes. Functional studies using OCT1-specific substrate pentamidine showed transporter-mediated AP but not BL uptake in Caco-2 cells and human and mouse intestinal tissues. OCT1 inhibition decreased AP uptake of pentamidine by ∼50% in all three systems with no effect on BL uptake. A short hairpin RNA-mediated OCT1 knockdown in Caco-2 cells decreased AP uptake of pentamidine by ∼50% but did not alter BL uptake. Immunostaining and confocal microscopy in all three systems confirmed AP localization of OCT1 (mOct1). Our studies unequivocally show AP membrane localization of OCT1 (mOct1) in Caco-2 cells and human and mouse intestine. These results are highly significant as they will require reinterpretation of previous drug disposition and drug-drug interaction studies where conclusions were drawn assuming BL localization of OCT1 in enterocytes. Most importantly, these results will require revision of the regulatory guidance for industry in the United States and elsewhere because it has stated that OCT1 is basolaterally localized in enterocytes. PMID:23680637

  17. 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

    Tumor necrosis factor-α (TNF-α) is highly upregulated in inflammation and reduces the expression of the intestinal transcription factor, Caudal-related homeobox transcription factor 2 (CDX2). Wnt/β-catenin signaling is critical for intestinal cell proliferation, but a decreased CDX2 expression ha...... 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...... targets were significantly elevated in TNF-α-treated Caco-2 cells. These findings were associated with reduced binding of CDX2 to promoter or enhancer regions of APC, AXIN2 and GSK3β. In conclusion, it was found that TNF-α induces the expression of Wnt signaling components through a downregulation...

  18. Concord and Niagara Grape Juice and Their Phenolics Modify Intestinal Glucose Transport in a Coupled in Vitro Digestion/Caco-2 Human Intestinal Model

    Directory of Open Access Journals (Sweden)

    Sydney Moser

    2016-07-01

    Full Text Available While the potential of dietary phenolics to mitigate glycemic response has been proposed, the translation of these effects to phenolic rich foods such as 100% grape juice (GJ remains unclear. Initial in vitro screening of GJ phenolic extracts from American grape varieties (V. labrusca; Niagara and Concord suggested limited inhibitory capacity for amylase and α-glucosidase (6.2%–11.5% inhibition; p < 0.05. Separately, all GJ extracts (10–100 µM total phenolics did reduce intestinal trans-epithelial transport of deuterated glucose (d7-glu and fructose (d7-fru by Caco-2 monolayers in a dose-dependent fashion, with 60 min d7-glu/d7-fru transport reduced 10%–38% by GJ extracts compared to control. To expand on these findings by assessing the ability of 100% GJ to modify starch digestion and glucose transport from a model starch-rich meal, 100% Niagara and Concord GJ samples were combined with a starch rich model meal (1:1 and 1:2 wt:wt and glucose release and transport were assessed in a coupled in vitro digestion/Caco-2 cell model. Digestive release of glucose from the starch model meal was decreased when digested in the presence of GJs (5.9%–15% relative to sugar matched control. Furthermore, transport of d7-glu was reduced 10%–38% by digesta containing bioaccessible phenolics from Concord and Niagara GJ compared to control. These data suggest that phenolics present in 100% GJ may alter absorption of monosaccharides naturally present in 100% GJ and may potentially alter glycemic response if consumed with a starch rich meal.

  19. 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. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Plant-synthesized E. coli CFA/I fimbrial protein protects Caco-2 cells from bacterial attachment.

    Science.gov (United States)

    Lee, Jin-Yong; Yu, Jie; Henderson, David; Langridge, William H R

    2004-11-25

    A DNA fragment encoding the cholera toxin A2 subunit (CTA2) linked to the enterotoxigenic Escherichia coli (ETEC) colony forming fimbrial antigen CFA/I was inserted into a plant expression vector containing the cholera toxin B subunit (CTB) fused to the rotavirus enterotoxin 22 amino acid epitope NSP422. Anti-CFA/I antibodies recognized a single band of approximately 72-kDa in transformed potato tuber tissue consistent with CFA/I-CTA2 and CTB-NSP4 fusion protein assembly into a cholera holotoxin-like structure. Enzyme-linked immunosorbent assay (GM1 ELISA) indicated that the CFA/I-CTA2 fusion protein bound specific GM1 ganglioside membrane receptors and made up approximately 0.002% of the total soluble tuber protein. Oral immunization of BALB/c mice with transformed tuber tissues generated anti-CFA/I serum and intestinal IgG and IgA secretory antibodies. Attachment of ETEC H10407 to enterocyte-like Caco-2 human colon carcinoma cells incubated with antiserum from immunized mice was reduced by 15% in comparison with Caco-2 cells incubated with serum from unimmunized mice. Immunogold staining of bacterial preparations revealed deposition of gold particles on E. coli H10407 fimbria incubated with immune serum but not on fimbria treated with sera from unimmunized mice demonstrating the specificity of antibodies in the immune serum for binding to CFA/I protein containing fimbria. The protection against toxic E. coli binding to Caco-2 cells generated by antisera from mice immunized with plant-synthesized CFA/I antigen demonstrates the feasibility of plant-based multi-component vaccine protection against enterotoxigenic E. coli, rotavirus and cholera, three enteric diseases that together exert the highest levels of child morbidity and mortality in economically emerging countries.

  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. Acamprosate has no impact on the permeability of paracellular markers across Caco-2 cells

    DEFF Research Database (Denmark)

    Antonescu, Irina; Steffansen, Bente; Neuhoff, Sibylle

    of the paracellular markers, mannitol and Lucifer Yellow (LY), was investigated. Methods: Ppara of LY and [14C]-mannitol was investigated across filter grown human epithelial colorectal adenocarcinoma (Caco-2) cell monolayers. Changes in the transepithelial electrical resistance (TEER) across the monolayers were...... the [14C]-mannitol, Papp values of 0.71±0.2x10-6 and 0.51±0.17x10-6 cm/s were obtained. TEER values at the end of all experiments were in the range of 426-444 ohm*cm2. Summary/Conclusion: Acamprosate has no impact on the paracellular pathway across Caco-2 cell monolayers of LY and mannitol, or on the TEER...

  3. Evaluation of the inflammatory response to Kudoa septempunctata genotype ST3 isolated from olive flounder (Paralichthys olivaceus in Caco-2 cells

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    Ahn Meejung

    2018-01-01

    Full Text Available Kudoa septempunctata (Myxosporea, Multivalvulida is a parasite of the trunk muscle of cultured olive flounder (Paralichthys olivaceus. We investigated whether K. septempunctata genotype ST3 spores induce cell damage and the secretion of inflammatory mediators in Caco-2cells, which exhibit characteristics similar to human intestinal epithelial cells. Purified K. septempunctata spores were heated at 95 °C for 5 min. Lactate dehydrogenase (LDH release was measured to determine the efficacy of denaturation. Naïve and heated spores, lipopolysaccharide (positive control and vehicle (negative control were added to Caco-2cells. Cells were subjected to the cytotoxic LDH assay and western blot analysis to examine the expression of inducible nitric oxide synthase (iNOS and cyclooxygenase (COX-2. Supernatants were collected to measure nitric oxide (NO and prostaglandin E2 (PGE2. Most spores were denaturated by heating, and the spore morphology was found to be wrinkled with shell valves and polar capsules. In addition, cytotoxicity and inflammatory mediators, such as NO, PGE2, iNOS, and COX-2, remained unchanged in Caco-2cells following exposure to naïve and heated spores compared with the positive controls. Collectively, the findings of this study imply that spores of K. septempunctata genotype ST3 do not cause inflammation in Caco-2cells.

  4. Transepithelial transport efficiency of bovine collagen hydrolysates in a human Caco-2 cell line model.

    Science.gov (United States)

    Feng, Mengmeng; Betti, Mirko

    2017-06-01

    Collagen was extracted from raw bovine hide and hydrolyzed by one of three enzymes - Alcalase, Flavourzyme, or trypsin - or by using a combination of two or three of these enzymes. The Alcalase-containing enzymatic hydrolysis treatments generated a greater proportion of hydrolysates with molecular weight (MW) Alcalase/Flavourzyme combination - generated the greatest transport efficiency across Caco-2 cell monolayers (21.4%), two-fold more than that of the collagen control. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Unsaturated fatty acids promote bioaccessibility and basolateral secretion of carotenoids and α-tocopherol by Caco-2 cells.

    Science.gov (United States)

    Failla, Mark L; Chitchumronchokchai, Chureeporn; Ferruzzi, Mario G; Goltz, Shellen R; Campbell, Wayne W

    2014-06-01

    Bioavailability of carotenoids and tocopherols from foods is determined by the efficiency of transfer from food/meal to mixed micelles during digestion, incorporation into chylomicrons for trans-epithelial transport to lymphatic/blood system, and distribution to target tissues. Fats and oils are important factors for facilitating the absorption of lipophilic compounds. However, dietary fats and oils are composed of various types of saturated and unsaturated fatty acids which may differentially impact the bioavailability of carotenoids and tocopherols from foods. We have investigated the effects of several common commercial lipids on bioavailability using an in vitro digestion model and Caco-2 human intestinal cells. Meals consisted of mixed salad vegetables containing a single test lipid. Micellarization and cellular uptake of β-carotene (βC) and lycopene (LYC) during small intestinal digestion was increased by lipids rich in unsaturated fatty acids: soybean oil > olive > canola > butter. In contrast, type of lipid minimally affected the bioaccessibility of lutein (LUT) and zeaxanthin (ZEA). To examine the influence of type of dietary triglyceride on uptake and basolateral secretion of carotenoids, Caco-2 cells grown on Transwell membranes were incubated with micellar mixtures of fatty acids (1.0 mM) mimicking the types and ratio of saturated to unsaturated (mono- + poly-unsaturated) fatty acids (FA) present in butter (70 : 30), olive oil (7 : 93) and soybean oil (11 : 89). Cells were exposed to micelles containing βC, LUT, α-tocopherol (α-TC) and a mixture of test fatty acids. Uptake and basolateral secretion of βC, LUT and α-TC were greater in cells pre-treated with mixtures enriched in unsaturated compared to saturated FA and these effects were mediated by increased assembly and secretion of chylomicrons. These results suggest that dietary fats/oils rich in unsaturated fatty acids promote carotenoid and α-TC bioavailability by enhancing their

  6. Proteomic analysis and bioluminescent reporter gene assays to investigate effects of simulated microgravity on Caco-2 cells.

    Science.gov (United States)

    La Barbera, Giorgia; Capriotti, Anna Laura; Michelini, Elisa; Piovesana, Susy; Calabretta, Maria Maddalena; Zenezini Chiozzi, Riccardo; Roda, Aldo; Laganà, Aldo

    2017-08-01

    Microgravity is one of the most important features in spaceflight. Previous evidence from in-vitro studies has shown that significant changes occur under simulated microgravity. For this reason, human colon adenocarcinoma Caco-2 cells were selected as cell model of intestinal epithelial barrier and their response to altered gravity conditions was investigated, especially on the protein level. In this study, we combined label-free shotgun proteomics and bioluminescent reporter gene assays to identify key proteins and pathways involved in the response of Caco-2 cells under reference and microgravity conditions. A two-dimensional clinostat was modified with 3D-printed adaptors to hold conventional T25 culture flasks. The comparative proteome analysis led to identify 38 and 26 proteins differently regulated by simulated microgravity after 48 and 72 h, respectively. Substantial fractions of these proteins are involved in regulation, cellular and metabolic processes and localization. Bioluminescent reporter gene assays were carried out to investigate microgavity-induced alterations on the transcriptional regulation of key targets, such as NF-kB pathway and CYP27A1. While no significant difference was found in the basal transcription, a lower NF-kB basal activation in simulated microgravity conditions was reported, corroborating the hypothesis of reduced immunity in microgravity conditions. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Effect of chum salmon egg lectin on tight junctions in Caco-2 cell monolayers.

    Science.gov (United States)

    Nemoto, Ryo; Yamamoto, Shintaro; Ogawa, Tomohisa; Naude, Ryno; Muramoto, Koji

    2015-05-05

    The effect of a chum salmon egg lectin (CSL3) on tight junction (TJ) of Caco-2 cell monolayers was investigated. The lectin opened TJ as indicated by the decrease of the transepithelial electrical resistance (TER) value and the increase of the permeation of lucifer yellow, which is transported via the TJ-mediated paracellular pathway. The effects of CSL3 were inhibited by the addition of 10 mM L-rhamnose or D-galactose which were specific sugars for CSL3. The lectin increased the intracellular Ca2+ of Caco-2 cell monolayers, that could be inhibited by the addition of L-rhamnose. The fluorescence immunostaining of β-actin in Caco-2 cell monolayers revealed that the cytoskeleton was changed by the CSL3 treatment, suggesting that CSL3 depolymerized β-actin to cause reversible TJ structural and functional disruption. Although Japanese jack bean lectin and wheat germ lectin showed similar effects in the decrease of the TER values and the increase of the intracellular Ca2+, they could not be inhibited by the same concentrations of simple sugars, such as D-glucose and N-acetyl-D-glucosamine.

  8. Effect of Chum Salmon Egg Lectin on Tight Junctions in Caco-2 Cell Monolayers

    Directory of Open Access Journals (Sweden)

    Ryo Nemoto

    2015-05-01

    Full Text Available The effect of a chum salmon egg lectin (CSL3 on tight junction (TJ of Caco-2 cell monolayers was investigated. The lectin opened TJ as indicated by the decrease of the transepithelial electrical resistance (TER value and the increase of the permeation of lucifer yellow, which is transported via the TJ-mediated paracellular pathway. The effects of CSL3 were inhibited by the addition of 10 mM L-rhamnose or D-galactose which were specific sugars for CSL3. The lectin increased the intracellular Ca2+ of Caco-2 cell monolayers, that could be inhibited by the addition of L-rhamnose. The fluorescence immunostaining of β-actin in Caco-2 cell monolayers revealed that the cytoskeleton was changed by the CSL3 treatment, suggesting that CSL3 depolymerized β-actin to cause reversible TJ structural and functional disruption. Although Japanese jack bean lectin and wheat germ lectin showed similar effects in the decrease of the TER values and the increase of the intracellular Ca2+, they could not be inhibited by the same concentrations of simple sugars, such as D-glucose and N-acetyl-D-glucosamine.

  9. 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 H 2 O 2 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Live and heat-killed Lactobacillus rhamnosus GG upregulate gene expression of pro-inflammatory cytokines in 5-fluorouracil-pretreated Caco-2 cells.

    Science.gov (United States)

    Fang, Shiuh-Bin; Shih, Hsin-Yu; Huang, Chih-Hung; Li, Li-Ting; Chen, Chia-Chun; Fang, Hsu-Wei

    2014-06-01

    This study investigates whether post-chemotherapeutic use of live and heat-killed Lactobacillus rhamnosus GG can modulate the expression of three pro-inflammatory cytokines in 5-fluorouracil (5-FU)-induced intestinal mucositis in vitro. Live L. rhamnosus GG and heat-killed L. rhamnosus GG were observed using scanning electron microscopy. To establish the duration required for optimal expression of tumor necrosis factor-α (TNF-α), monocyte chemotactic protein-1 (MCP-1), and interleukin-12 (IL-12), 5 μM of 5-FU was selected to treat 10-day-old Caco-2 cells for 4, 6, 8, and 24 h. Caco-2 cells were treated with 5-FU (5 μM) for 4 h, followed by the administration of live L. rhamnosus GG (multiplicity of infection = 25), and heat-killed L. rhamnosus GG for 2 and 4 h. Finally, total cellular RNA was isolated to quantify mRNA expression of TNF-α, MCP-1, and IL-12 using real-time PCR. The results demonstrated that heat-killed L. rhamnosus GG remained structurally intact with elongation. A biphasic upregulated expression of TNF-α, MCP-1, and IL-12 was observed in 5-FU-treated Caco-2 cells at 4 and 24 h. Compared to non-L. rhamnosus GG controls in 5-FU-pretreated Caco-2 cells, a 2-h treatment of heat-killed L. rhamnosus GG significantly upregulated the MCP-1 expression (p GG treatments lasting 4 h upregulated the TNF-α and MCP-1 expression (p GG upregulated the IL-12 expression (p GG can upregulate the gene expression of 5-FU-induced pro-inflammatory cytokines in Caco-2 cells. Human intestinal epithelium may be vulnerable to the post-chemotherapeutic use of L. rhamnosus GG in 5-FU-induced mucositis that requires further in vivo studies for clarification.

  11. 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

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

    Science.gov (United States)

    Feng, Dan; Ohlsson, Lena; Duan, Rui-Dong

    2010-04-19

    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. 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. 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 muM curcumin for 24 h and found that such a treatment dose-dependently inhibited cholesterol uptake with 40% inhibition obtained by 100 muM 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. Curcumin inhibits cholesterol uptake through suppression of NPC1L1

  13. The influence of breast milk and infant formulae hydrolysates on bacterial adhesion and Caco-2 cells functioning.

    Science.gov (United States)

    Fiedorowicz, Ewa; Markiewicz, Lidia Hanna; Sidor, Katarzyna; Świątecka, Dominika; Cieślińska, Anna; Matysiewicz, Michał; Piskorz-Ogórek, Krystyna; Sienkiewicz-Szłapka, Edyta; Teodorowicz, Małgorzata; Świątecki, Aleksander; Kostyra, Elżbieta

    2016-11-01

    The aim of the study was to determine the concentration of BCM7 in human milk and infant formulae (IF) before and after eznymatic hydrolysis, and to evaluate the effect of obtained hydrolysates on interleukin-8 (IL-8) secretion and on proliferation of enterocytes in the in vitro model (Caco-2 cells). This study evaluates also the effect of hydrolysates on the adhesion of intestinal microbiota isolated from faeces of both healthy (H) and allergic (A) infants. In the study we investigated breast milk delivered by mothers of healthy ('healthy milk'; HM) and allergic ('allergic milk'; AM) infants. Three infant formulae were investigated: from hydrolysed cow casein (IF1), from hydrolysed cow whey (IF2) and from whole cow milk (IF3). Intestinal bacteria: Bifidobacterium, lactic acid bacteria, Enterobacteriaceae, Clostridium and Enterococcus were isolated from faeces of five healthy and five allergic infants. Mixtures of bacterial isolates and bacteria adhering to Caco-2 cells were characterised qualitatively with PCR-DGGE, and quantitavely with FISH. Concentration of BCM7 in breast milk and infant formulae was 1.6 to 8.9 times higher after enzymatic hydrolysis in comparison to undigested samples. The presence of this peptide resulted in alteration of intestinal epithelial proliferation and increase in secretion of IL-8. The quantitative profile of adherred bacteria applied as a mix of all isolates from healthy infants (H-MIX) was unchanged in the presence of HM hydrolysate and was modulated (increased number of beneficial Bifidobacterium and reduced commensal Enterobacteriaceae) in the presence of all IF hydrolysates. The presence of IF hydrolysates affected the profile of adhering isolates obtained from allergic infants (A-MIX) and reduced the adhesion of Enterobacteriaceae; the IF2 and IF3 hydrolysates decreased also the total number of adhering bacteria (TBN). However, a stimulating effect of AM hydrolysate on A-MIX adhesion (increased TBN) was observed. Copyright

  14. Simultaneous assessment of absorption characteristics of coumarins from Angelicae Pubescentis Radix: In vitro transport across Caco-2 cell and in vivo pharmacokinetics in rats after oral administration.

    Science.gov (United States)

    Yang, Yan-Fang; Zhang, Lei; Zhang, You-Bo; Yang, Xiu-Wei

    2017-08-15

    Angelicae Pubescentis Radix (APR), a well-known traditional Chinese medicine, is widely used for the treatments of rheumatism and headache for centuries. To assess the absorption characteristics of coumarins from APR, a sensitive and reliable UPLC-MS/MS method was established for the simultaneous determination of sixteen coumarins from APR, including psoralen, xanthotoxin, bergapten, bergaptol, isoimperatorin, imperatorin, columbianetin, columbianetin acetate, columbianadin, oxypeucedanin hydrate, angelol B, umbelliferone, scopoletin, osthole, meranzin hydrate and nodakenetin. The specificity, linearity, sensitivity, precision, accuracy, recovery, matrix effect and stability of the method were all validated to be satisfactory. The method was then applied to the in vitro transport of APR extract (APRE) across human intestinal epithelial Caco-2 cell and in vivo pharmacokinetics in rats after oral administration of APRE. All of the tested coumarins were well or moderately absorbed across Caco-2 monolayers, and could be quickly absorbed into rat blood circulation after oral administration. Columbianetin was the most easily absorbed compound across Caco-2 cell, and also had extremely highest plasma concentration in vivo. Excellent correlation between in vitro absorption across Caco-2 cell monolayers and in vivo pharmacokinetics of coumarins from APRE was well verified. The results provided valuable information for the overall absorption characteristics of the coumarins from APR, as well as for its further studies of in vivo active substances in the further. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Influence Of Magnetic Iron Oxide Nanoparticles And Perfluoroalkylated Amphiphilic Phosphates On Red Blood Cells And Caco-2 Cells

    Science.gov (United States)

    Moersdorf, Daniel; Hugounenq, Pierre; Phuoc, Lai Truonc; Mamlouk-Chaouachi, Hind; Nikolova, Gergana; Krafft, Marie Pierre; Waton, Gilles; Felder-Flesch, Delphine; Begin-Colin, Sylvie; Pourroy, Geneviève; Bernhardt, Ingolf

    2010-10-01

    The interactions of two types of cells (red blood cells (RBCs), Caco-2 cells) with magnetic iron oxide nanoparticles (non-grafted, citrate-grafted, dendrimer-grafted) of 11 nm in size, and with three perfluoroalkylated amphiphilic phosphates varying in chain length and number of fluorine atoms have been investigated. We focused on two important physiological parameters of the cells, the intracellular pH and the intracellular Ca2+ content. The results show that the nanoparticles do not have a significant influence on the pH and Ca2+ content of Caco-2 cells. The Ca2+ content of RBCs is also not affected but the intracellular pH is slightly reduced. In addition, the perfluoroalkylated phosphates do not affect the Caco-2 cells. However, these molecules have a small effect on the intracellular pH of RBCs. Furthermore, they influence significantly the Ca2+ content of a subpopulation of the RBCs.

  16. A cellular uptake and cytotoxicity properties study of gallic acid-loaded mesoporous silica nanoparticles on Caco-2 cells

    Science.gov (United States)

    Rashidi, Ladan; Vasheghani-Farahani, Ebrahim; Soleimani, Masoud; Atashi, Amir; Rostami, Khosrow; Gangi, Fariba; Fallahpour, Masoud; Tahouri, Mohammad Taher

    2014-03-01

    In this study, the effects of intracellular delivery of various concentrations of gallic acid (GA) as a semistable antioxidant, gallic acid-loaded mesoporous silica nanoparticles (MSNs-GA), and cellular uptake of nanoparticles into Caco-2 cells were investigated. MSNs were synthesized and loaded with GA, then characterized using transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy, N2 adsorption isotherms, X-ray diffraction, and thermal gravimetric analysis. The cytotoxicity of MSNs and MSNs-GA at low and high concentrations were studied by means of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) test and flow cytometry. MSNs did not show significant toxicity in various concentrations (0-500 μg/ml) on Caco-2 cells. For MSNs-GA, cell viability was reduced as a function of incubation time and different concentrations of nanoparticles. The in vitro GA release from MSNs-GA exhibited the same antitumor properties as free GA on Caco-2 cells. Flow cytometry results confirmed those obtained using MTT assay. TEM and fluorescent microscopy confirmed the internalization of MSNs by Caco-2 cells through nonspecific cellular uptake. MSNs can easily internalize into Caco-2 cells without deleterious effects on cell viability. The cell viability of Caco-2 cells was affected during MSNs-GA uptake. MSNs could be designed as suitable nanocarriers for antioxidants delivery.

  17. 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

  18. Fusobacterium nucleatum-Induced Impairment of Autophagic Flux Enhances the Expression of Proinflammatory Cytokines via ROS in Caco-2 Cells.

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    Bin Tang

    Full Text Available Fusobacterium nucleatum (F. nucleatum plays a critical role in gastrointestinal inflammation. However, the exact mechanism by which F. nucleatum contributes to inflammation is unclear. In the present study, it was revealed that F. nucleatum could induce the production of proinflammatory cytokines (IL-8, IL-1β and TNF-α and reactive oxygen species (ROS in Caco-2 colorectal adenocarcinoma cells. Furthermore, ROS scavengers (NAC or Tiron could decrease the production of proinflammatory cytokines during F. nucleatum infection. In addition, we observed that autophagy is impaired in Caco-2 cells after F. nucleatum infection. The production of proinflammatory cytokines and ROS induced by F. nucleatum was enhanced with either autophagy pharmacologic inhibitors (3-methyladenine, bafilomycin A1 or RNA interference in essential autophagy genes (ATG5 or ATG12 in Caco-2 cells. Taken together, these results indicate that F. nucleatum-induced impairment of autophagic flux enhances the expression of proinflammatory cytokines via ROS in Caco-2 Cells.

  19. Fusobacterium nucleatum-Induced Impairment of Autophagic Flux Enhances the Expression of Proinflammatory Cytokines via ROS in Caco-2 Cells.

    Science.gov (United States)

    Tang, Bin; Wang, Kun; Jia, Yin-Ping; Zhu, Pan; Fang, Yao; Zhang, Zhu-Jun; Mao, Xu-Hu; Li, Qian; Zeng, Dong-Zhu

    2016-01-01

    Fusobacterium nucleatum (F. nucleatum) plays a critical role in gastrointestinal inflammation. However, the exact mechanism by which F. nucleatum contributes to inflammation is unclear. In the present study, it was revealed that F. nucleatum could induce the production of proinflammatory cytokines (IL-8, IL-1β and TNF-α) and reactive oxygen species (ROS) in Caco-2 colorectal) adenocarcinoma cells. Furthermore, ROS scavengers (NAC or Tiron) could decrease the production of proinflammatory cytokines during F. nucleatum infection. In addition, we observed that autophagy is impaired in Caco-2 cells after F. nucleatum infection. The production of proinflammatory cytokines and ROS induced by F. nucleatum was enhanced with either autophagy pharmacologic inhibitors (3-methyladenine, bafilomycin A1) or RNA interference in essential autophagy genes (ATG5 or ATG12) in Caco-2 cells. Taken together, these results indicate that F. nucleatum-induced impairment of autophagic flux enhances the expression of proinflammatory cytokines via ROS in Caco-2 Cells.

  20. 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.

  1. Galacto-oligosaccharides Protect the Intestinal Barrier by Maintaining the Tight Junction Network and Modulating the Inflammatory Responses after a Challenge with the Mycotoxin Deoxynivalenol in Human Caco-2 Cell Monolayers and B6C3F1 Mice

    NARCIS (Netherlands)

    Akbari, Peyman; Braber, Saskia; Alizadeh, Arash; Verheijden, Kim; Schoterman, Margriet Hc; Kraneveld, Aletta D; Garssen, Johan; Fink-Gremmels, Johanna

    BACKGROUND: The integrity of the epithelial layer in the gastrointestinal tract protects organisms from exposure to luminal antigens, which are considered the primary cause of chronic intestinal inflammation and allergic responses. The common wheat-associated fungal toxin deoxynivalenol acts as a

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

    Science.gov (United States)

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

    2010-01-01

    Abstract 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 3H-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 μg/ml) had no significant cytotoxic effect on proliferating and differentiated Caco-2 cells. Rhein (0.1 and 1 μg/ml) significantly reduced cell proliferation as well as mitogen-activated protein (MAP) kinase activation; by contrast, at high concentration (10 μg/ml) rhein significantly increased cell proliferation and extracellular-signal-related kinase (ERK) phosphorylation. Moreover, rhein (0.1–10 μg/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 H2O2-induced DNA damage and (iii) significantly inhibited the increase in malondialdehyde and reactive oxygen species (ROS) levels induced by H2O2/Fe2+. 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. PMID:19538468

  3. The Interplay between Radioresistant Caco-2 Cells and the Immune System Increases Epithelial Layer Permeability and Alters Signaling Protein Spectrum

    Science.gov (United States)

    Morini, Jacopo; Babini, Gabriele; Barbieri, Sofia; Baiocco, Giorgio; Ottolenghi, Andrea

    2017-01-01

    Colorectal cancer is one of the most frequent type of cancer, with a higher incidence in the developed countries. Colorectal cancer is usually managed with both surgeries, chemotherapy and radiotherapy. Radiotherapy has the well-known advantage of targeting the tumor, minimizing normal tissue exposure. Nevertheless, during radiation treatment, exposure of healthy tissues is of great concern, in particular because of the effects on the intestinal barrier functions and on cells belonging to the immune system. The functional role of intestinal barrier in avoiding paracellular trafficking and controlling bacterial spread from gut it is well known and it is due to the presence of tight junction complexes. However, intestinal barrier is fundamental in participating to the interplay with immune system, especially considering the gut-associated lymphoid tissue. Until few years ago, radiotherapy was considered to bear only a depressive action on the immune system. However, it is now recognized that the release of pro-inflammatory signals and phenotypic changes in tumoral cells due to ionizing radiation could trigger the immune system against the tumor. In this work, we address how intestinal barrier functions are perturbed by X-ray doses in the range 0–10 Gy, focusing on the interplay between tumoral cells and the immune system. To this aim, we adopted a coculture model in which Caco-2 cells can be grown in presence/absence of peripheral blood mononuclear cells (PBMC). We focused our attention on changes in the proliferation, trans-epithelial electrical resistance (TEER), cytokine release, and proteins of the junctional complexes. Our results indicate a high radioresistance of Caco-2 in the investigated dose range, and an increased permeability of the tumoral cell layer due to the presence of PBMC. This is found to be correlated with activation of PBMC, inhibiting the apoptotic pathway, with the enhancement of cytokine release and with variation of tight junction

  4. Alisertib Induces Cell Cycle Arrest, Apoptosis, Autophagy and Suppresses EMT in HT29 and Caco-2 Cells

    Science.gov (United States)

    Ren, Bao-Jun; Zhou, Zhi-Wei; Zhu, Da-Jian; Ju, Yong-Le; Wu, Jin-Hao; Ouyang, Man-Zhao; Chen, Xiao-Wu; Zhou, Shu-Feng

    2015-01-01

    Colorectal cancer (CRC) is one of the most common malignancies worldwide with substantial mortality and morbidity. Alisertib (ALS) is a selective Aurora kinase A (AURKA) inhibitor with unclear effect and molecular interactome on CRC. This study aimed to evaluate the molecular interactome and anticancer effect of ALS and explore the underlying mechanisms in HT29 and Caco-2 cells. ALS markedly arrested cells in G2/M phase in both cell lines, accompanied by remarkable alterations in the expression level of key cell cycle regulators. ALS induced apoptosis in HT29 and Caco-2 cells through mitochondrial and death receptor pathways. ALS also induced autophagy in HT29 and Caco-2 cells, with the suppression of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR), but activation of 5′ AMP-activated protein kinase (AMPK) signaling pathways. There was a differential modulating effect of ALS on p38 MAPK signaling pathway in both cell lines. Moreover, induction or inhibition of autophagy modulated basal and ALS-induced apoptosis in both cell lines. ALS potently suppressed epithelial to mesenchymal transition (EMT) in HT29 and Caco-2 cells. Collectively, it suggests that induction of cell cycle arrest, promotion of apoptosis and autophagy, and suppression of EMT involving mitochondrial, death receptor, PI3K/Akt/mTOR, p38 MAPK, and AMPK signaling pathways contribute to the cancer cell killing effect of ALS on CRC cells. PMID:26729093

  5. Alisertib Induces Cell Cycle Arrest, Apoptosis, Autophagy and Suppresses EMT in HT29 and Caco-2 Cells

    Directory of Open Access Journals (Sweden)

    Bao-Jun Ren

    2015-12-01

    Full Text Available Colorectal cancer (CRC is one of the most common malignancies worldwide with substantial mortality and morbidity. Alisertib (ALS is a selective Aurora kinase A (AURKA inhibitor with unclear effect and molecular interactome on CRC. This study aimed to evaluate the molecular interactome and anticancer effect of ALS and explore the underlying mechanisms in HT29 and Caco-2 cells. ALS markedly arrested cells in G2/M phase in both cell lines, accompanied by remarkable alterations in the expression level of key cell cycle regulators. ALS induced apoptosis in HT29 and Caco-2 cells through mitochondrial and death receptor pathways. ALS also induced autophagy in HT29 and Caco-2 cells, with the suppression of phosphoinositide 3-kinase (PI3K/protein kinase B (Akt/mammalian target of rapamycin (mTOR, but activation of 5′ AMP-activated protein kinase (AMPK signaling pathways. There was a differential modulating effect of ALS on p38 MAPK signaling pathway in both cell lines. Moreover, induction or inhibition of autophagy modulated basal and ALS-induced apoptosis in both cell lines. ALS potently suppressed epithelial to mesenchymal transition (EMT in HT29 and Caco-2 cells. Collectively, it suggests that induction of cell cycle arrest, promotion of apoptosis and autophagy, and suppression of EMT involving mitochondrial, death receptor, PI3K/Akt/mTOR, p38 MAPK, and AMPK signaling pathways contribute to the cancer cell killing effect of ALS on CRC cells.

  6. 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.

  7. 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...

  8. Evaluation of cellular purine transport and metabolism in the Caco-2 cell using comprehensive high-performance liquid chromatography method for analysis of purines.

    Science.gov (United States)

    Fukuuchi, T; Kobayashi, M; Yamaoka, N; Kaneko, K

    2016-12-01

    Using Caco-2 cells and our previously developed high-performance liquid chromatography method for quantification of purine bases, nucleosides, and nucleotides, we evaluated cellular purine transport and uptake. The analytes were separated using YMC-Triart C18 column with gradient elution. We used Caco-2 cells as intestinal model cells and monitored purine transport across a monolayer for 2 h. The degree of change of purine concentrations in the permeate was very slight; however, it was possible to simultaneously determine these parameters for all purines because of our method's high sensitivity. In the present study, the purine bases (adenine, guanine, hypoxanthine, and xanthine) showed a relatively high permeability as compared with the nucleosides (adenosine, guanosine, inosine, and xanthosine). Increased concentration of metabolites in the permeate was also observed following the addition of purines. In a cell uptake assay, both the cell culture medium (extracellular) and the cells extracted from Caco-2 with acetonitrile:water (7:3) (intracellular) were measured. The additional nucleoside did not increase significantly within the cells. On the other hand, we observed that nucleotide, such as ATP, increased in the cell in a time-dependent manner following the addition of nucleoside. The additional nucleosides were considered to be rather recycled via the salvage pathway than metabolized to purine bases and/or uric acid in the cell. Such differences might have affected the increase in the serum uric acid levels depending on purine form.

  9. Gastrointestinal Hormone Cholecystokinin Increases P-Glycoprotein Membrane Localization and Transport Activity in Caco-2 Cells.

    Science.gov (United States)

    Yano, Kentaro; Shimizu, Saori; Tomono, Takumi; Ogihara, Takuo

    2017-09-01

    It was reported that stimulation of taste receptor type 2 member 38 by a bitter substance, phenylthiocarbamide (PTC), increased P-glycoprotein (P-gp) mRNA level and transport activity via release of the gastrointestinal hormone cholecystokinin-8 (CCK-8) at 9 h. Therefore, we hypothesized that CCK-8 and PTC might also regulate P-gp activity more rapidly via a different mechanism. As a result, we found that the pretreatment of human colon adenocarcinoma (Caco-2) cells with 10-mM PTC significantly decreased the intracellular accumulation of P-gp substrate rhodamine 123 (Rho123) compared with the control after 90-min incubation. Moreover, CCK-8 treatments significantly reduced the accumulation of Rho123 within 30 min, compared with the control. On the other hand, when Caco-2 cells were pretreated with PTC, the efflux ratio of Rho123 was significantly increased compared with control. The efflux ratio of Rho123 in CCK-8 treatment cells was also significantly increased compared with control. Furthermore, CCK-8 increased the phosphorylation of the scaffold proteins ezrin, radixin, and moesin, which regulate translocation of P-gp to the plasma membrane. Therefore, our results indicate that PTC induced release of CCK-8, which in turn induced the phosphorylation of ezrin, radixin, and moesin proteins, leading to upregulation of P-gp transport activity via increased membrane localization of P-gp. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  10. para-Sulphonato-calix[n]arenes as selective activators for the passage of molecules across the Caco-2 model intestinal membrane.

    Science.gov (United States)

    Roka, Eszter; Vecsernyes, Miklos; Bacskay, Ildiko; Félix, Caroline; Rhimi, Moez; Coleman, Anthony W; Perret, Florent

    2015-06-07

    The passage of Lucifer Yellow across the Caco-2 intestinal model membrane has been studied for the para-sulphonato-calix[n]arenes, the results show that para-sulphonato-calix[4]arene and para-sulphonato-calix[8]arene activate membrane passage when used simultaneously with a transport probe, Lucifer Yellow, whereas para-sulphonato-calix[6]arene has no effect.

  11. Effect of food models and low-temperature storage on the adhesion of Lactobacillus rhamnosus GG to Caco-2 cells.

    Science.gov (United States)

    Deepika, G; Rastall, R A; Charalampopoulos, D

    2011-08-24

    This study evaluated the effects of fat and sugar levels on the surface properties of Lactobacillus rhamnosus GG during storage in food model systems, simulating yogurt and ice cream, and related them with the ability of the bacterial cells to adhere to Caco-2 cells. Freeze-dried L. rhamnosus GG cells were added to the model food systems and stored for 7 days. The bacterial cells were analyzed for cell viability, hydrophobicity, ζ potential, and their ability to adhere to Caco-2 cells. The results indicated that the food type and its composition affected the surface and adhesion properties of the bacterial cells during storage, with yogurt being a better delivery vehicle than ice cream in terms of bacterial adhesion to Caco-2 cells. The most important factor influencing bacterial adhesion was the storage time rather than the levels of fats and sugars, indicating that conformational changes were taking place on the surface of the bacterial cells during storage.

  12. Effect of dietary protein on heme iron uptake by Caco-2 cells.

    Science.gov (United States)

    Villarroel, Pía; Flores, Sebastián; Pizarro, Fernando; de Romaña, Daniel López; Arredondo, Miguel

    2011-12-01

    To study heme iron bioavailability and the role of dietary protein (animal and vegetable) on iron uptake using an in vitro model (Caco-2 cell line). Caco-2 cells were seeded in bicameral chambers with different animal (beef, chicken or fish) or vegetable (peas, lentils, and soybeans) proteins or with pure animal (collagen and casein) or vegetable (gliadin, zein, and glutein) protein extracts. The effect of each protein over heme iron absorption was assessed. Intact heme uptake was higher than either heme plus albumin or digested heme plus albumin, but lower than digested heme. White meal exerted the highest inhibitory effect on hemin uptake. Heme iron uptake decreased in the presence of all legume extracts, but was not significantly different among them (one-way ANOVA, NS). Pure animal (collagen and casein) and vegetable (zein and glutelin) proteins increased heme iron uptake, except for gliadin. Animal and vegetable protein in general decreased heme iron uptake. However, purified animal and vegetable protein induce an increase in heme iron uptake.

  13. Low uptake of silica nanoparticles in Caco-2 intestinal epithelial barriers

    NARCIS (Netherlands)

    Ye, Dong; Bramini, Mattia; Hristov, Delyan R.; Wan, Sha; Salvati, Anna; Åberg, Christoffer; Dawson, Kenneth A.

    2017-01-01

    Cellular barriers, such as the skin, the lung epithelium or the intestinal epithelium, constitute one of the first obstacles facing nanomedicines or other nanoparticles entering organisms. It is thus important to assess the capacity of nanoparticles to enter and transport across such barriers. In

  14. Bioactive-Chylomicrons for Oral Lymphatic Targeting of Berberine Chloride: Novel Flow-Blockage Assay in Tissue-Based and Caco-2 Cell Line Models.

    Science.gov (United States)

    Elsheikh, Manal A; Elnaggar, Yosra S R; Otify, Dina Y; Abdallah, Ossama Y

    2018-01-05

    To develop novel bioactive-chylomicrons to solve oral delivery obstacles of Berberine chloride and target the lymphatic system. Berberine-loaded bioactive-chylomicrons were prepared and underwent full in vitro characterization. Intestinal permeability was appraised via both non-everted gut sac model and Caco-2 cell model. Furthermore, Bioactive-chylomicrons' cellular uptake and distribution were examined by laser scanning confocal microscopy. Finally, a novel chylomicron flow-blockage assay on tissue and cellular levels were elaborated to assess the lymphatic targeting ability. Berberine-loaded chylomicrons showed spherical vesicles of size (175.6 nm), PDI (0.229), zeta potential (-16 .6 mV) and entrapment efficiency (95.5%). Ex-vivo intestinal permeability studies demonstrated 10.5 fold enhancement in permeability of Berberine-loaded chylomicrons over free Berberine. Moreover, Caco-2 studies revealed significant improvement in chylomicrons' permeability and cellular uptake. Furthermore, confocal microscopy analyses revealed 2 fold increase in berberine-loaded chylomicrons' intracellular fluorescence. Lymphatic targeting models were successfully elaborated using cycloheximide protein synthesis inhibitor. Such models demonstrated 47 and 27.5% reduction in ex-vivo and Caco-2 permeability respectively. Finally, a good rank order correlation was established between different permeability assessment techniques. The findings shed the light on the underlying mechanisms of Berberine bioavailability improvement. Consequently, berberine-loaded chylomicrons could be considered as promising bioactive-nanocarriers for Berberine lymphatic targeting and bioavailability improvement.

  15. Modified Dietary Fiber from Cassava Pulp and Assessment of Mercury Bioaccessibility and Intestinal Uptake Using an In Vitro Digestion/Caco-2 Model System.

    Science.gov (United States)

    Kachenpukdee, Natta; Santerre, Charles R; Ferruzzi, Mario G; Oonsivilai, Ratchadaporn

    2016-07-01

    The ability of modified dietary fiber (MDF) generated from cassava pulp to modulate the bioaccessibility and intestinal absorption of heavy metals may be helpful to mitigate health risk associated with select foods including select fish high in methyl mercury. Using a coupled in vitro digestion/Caco-2 human intestinal cell model, the reduction of fish mercury bioaccessibility and intestinal uptake by MDF was investiaged. MDF was prepared from cassava pulp, a byproduct of tapioca production. The highest yield (79.68%) of MDF was obtained by enzymatic digestion with 0.1% α-amylase (w/v), 0.1% amyloglucosidase (v/v) and 1% neutrase (v/v). MDF and fish tissue were subjected to in vitro digestion and results suggest that MDF may reduce mercury bioaccessibility from fish to 34% to 85% compared to control in a dose-dependent manner. Additionally, accumulation of mercury from digesta containing fish and MDF was only modestly impacted by the presence of MDF. In conclusion, MDF prepared from cassava pulp may be useful as an ingredient to reduce mercury bioavailability from food such as fish specifically by inhibiting mercury transfer to the bioaccessibile fraction during digestion. © 2016 Institute of Food Technologists®

  16. Are additive effects of dietary surfactants on intestinal tight junction integrity an overlooked human health risk? - A mixture study on Caco-2 monolayers.

    Science.gov (United States)

    Glynn, Anders; Igra, Annachiara Malin; Sand, Salomon; Ilbäck, Nils Gunnar; Hellenäs, Karl Erik; Rosén, Johan; Aspenström-Fagerlund, Bitte

    2017-08-01

    Surfactants may cause dysfunction of intestinal tight junctions (TJs), which is a common feature of intestinal autoimmune diseases. Effects of dietary surfactants on TJ integrity, measured as trans-epithelial resistance (TEER), were studied in Caco-2 cell monolayers. Cytotoxicity was assessed as apical LDH leakage. Monolayers were apically exposed for 60 min to the dietary surfactants solanine and chaconine (SC, potato glycoalkaloids, 0-0.25 mM), perfluorooctane sulfonic acid (PFOS, industrial contaminant, 0-0.8 mM), and sucrose monolaurate (SML, food emulsifier E 473, 0-2.0 mM) separately and as a mixture. Dose-response modelling of TEER EC 50 showed that SC were 2.7- and 12-fold more potent than PFOS and SML, respectively. The mixture was composed of 1 molar unit SC, 2.7 units PFOS and 12 units SML ("SC TEER equivalent" proportions 1:1:1). Mixture exposure (0-0.05 mM SC equivalents) dose-response modelling suggested additive action on TJ integrity. Increasing SC and SML concentrations caused increased LDH leakage, but PFOS decreased LDH leakage at intermediate exposure concentrations. In the mixture PFOS appeared to protect from extensive SC- and SML-induced LDH leakage. Complex mixtures of surfactants in food may act additively on intestinal TJ integrity, which should be considered in risk assessment of emulsifier authorisation for use in food production. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. 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...... was investigated. Nonpathogenic Escherichia coli were added either to the apical or the basolateral compartment of this transwell cell culture system, the latter of which contained human leucocytes. The synthesis of tumour necrosis factor (TNF-alpha) and interleukin (IL)-12 was significantly suppressed by CACO-2...... 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...

  18. 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

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

    International Nuclear Information System (INIS)

    Liu, Yanlong; Wang, Chunhong; Wang, Yuhua; Ma, Zhenhua; Xiao, Jian; McClain, Craig; Li, Xiaokun; Feng, Wenke

    2012-01-01

    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 2 ), on FGF21 expression in Caco-2 cells and the consequence of fat accumulation. Physical hypoxia (1% oxygen) and CoCl 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 2 administration caused a significant increase in oxidative stress. The addition of n-acetylcysteine (NAC) suppressed CoCl 2 -induced reactive oxygen species (ROS) formation and completely negated CoCl 2 -induced FGF21 loss. mRNA stability analysis demonstrated that the CoCl 2 administration caused a remarkable reduction in FGF21 mRNA stability. Furthermore, CoCl 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 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 absorption. -- Graphical abstract: Physical

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    International Nuclear Information System (INIS)

    Martien, Ronny; Loretz, Brigitta; Sandbichler, Adolf Michael; Schnuerch, Andreas Bernkop

    2008-01-01

    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

  2. Comparison of brain capillary endothelial cell-based and epithelial (MDCK-MDR1, Caco-2, and VB-Caco-2) cell-based surrogate blood-brain barrier penetration models.

    Science.gov (United States)

    Hellinger, Eva; Veszelka, Szilvia; Tóth, Andrea E; Walter, Fruzsina; Kittel, Agnes; Bakk, Mónika Laura; Tihanyi, Károly; Háda, Viktor; Nakagawa, Shinsuke; Duy, Thuy Dinh Ha; Niwa, Masami; Deli, Mária A; Vastag, Monika

    2012-10-01

    An accurate means of predicting blood-brain barrier (BBB) penetration and blood-brain partitioning of NCEs (new chemical entities) would fulfill a major need in pharmaceutical research. Currently, an industry-standard BBB drug penetration model is not available. Primary brain capillary endothelial cells, optionally co-cultured with astrocytes and/or pericytes, are the most valued models of BBB. For routine use, establishing and maintaining a co-culture system is too costly and labor intensive. Alternatively, non-cerebral cell lines such as MDCK-MDR1 are used, and most recently, the suitability of native and modified Caco-2 for predicting brain penetration has also come under investigation. This study provides comparative data on the morphology and functionality of the high integrity brain capillary endothelial BBB model (EPA: triple culture of brain capillary endothelial cells with pericytes and astrocytes) and the epithelial cell-based (native Caco-2, high P-glycoprotein expressing vinblastine-treated VB-Caco-2 and MDCK-MDR1) surrogate BBB models. Using a panel of 10 compounds VB-Caco-2 and MDCK-MDR1 cell lines show restrictive paracellular pathway and BBB-like selective passive permeability that makes them comparable to the rat brain BBB model, which gave correlation with the highest r(2) value with in vivo permeability data. In bidirectional assay, the VB-Caco-2 and the MDCK-MDR1 models identified more P-glycoprotein drug substrates than the rat brain BBB model. While the complexity and predictive value of the BBB model is the highest, for the screening of NCEs to determine whether they are efflux substrates or not, the VB-Caco-2 and the MDCK-MDR1 models may provide a simple and inexpensive tool. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. Ginger Extract Suppresses Inflammatory Response and Maintains Barrier Function in Human Colonic Epithelial Caco-2 Cells Exposed to Inflammatory Mediators.

    Science.gov (United States)

    Kim, Yunyoung; Kim, Dong-Min; Kim, Ji Yeon

    2017-05-01

    The beneficial effects of ginger in the management of gastrointestinal disturbances have been reported. In this study, the anti-inflammatory potential of ginger extract was assessed in a cellular model of gut inflammation. In addition, the effects of ginger extract and its major active compounds on intestinal barrier function were evaluated. The response of Caco-2 cells following exposure to a mixture of inflammatory mediators [interleukin [IL]-1β, 25 ng/mL; lipopolysaccharides [LPS], 10 ng/mL; tumor necrosis factor [TNF]-α, 50 ng/mL; and interferon [INF]-γ, 50 ng/mL] were assessed by measuring the levels of secreted IL-6 and IL-8. In addition, the mRNA levels of cyclooxygenase-2 and inducible nitric oxide synthase were measured. Moreover, the degree of nuclear factor (NF)-κB inhibition was examined, and the intestinal barrier function was determined by measuring the transepithelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC)-dextran transfer. It was observed that ginger extract and its constituents improved inflammatory responses by decreasing the levels of nitrite, PGE2, IL-6, and IL-8 via NF-κB inhibition. The ginger extract also increased the TEER and decreased the transfer of FITC-dextran from the apical side of the epithelium to the basolateral side. Taken together, these results show that ginger extract may be developed as a functional food for the maintenance of gastrointestinal health. © 2017 Institute of Food Technologists®.

  4. Transcytosis of Aminopeptidase N in caco-2 cells is mediated by a Non-cytoplasmic Signal

    DEFF Research Database (Denmark)

    Vogel, L K; Norén, Ove; Sjöström, H

    1995-01-01

    of the transmembrane or cytoplasmic domain of aminopeptidase N for transport of aminopeptidase N by the indirect pathway by analysis of mutated forms of aminopeptidase N recombinantly expressed in Caco-2 cells. A tail-less and two secretory forms of aminopeptidase N, all deprived of the cytoplasmic tail, were...... transported to the basolateral plasma membrane in proportions equivalent to the wild type enzyme. This shows that no cytoplasmic basolateral sorting signal is involved in directing aminopeptidase N to the basolateral plasma membrane. Both the wild type and the tail-less aminopeptidase N were transcytosed from...... the basolateral to the apical plasma membrane, whereas no transcytosis of two secretory forms could be detected, showing that the transmembrane domain is important for efficient transcytosis to take place. A significant difference in transcytosis kinetics of the human and the porcine wild type aminopeptidase N...

  5. Milk modulates campylobacter invasion into caco-2 intestinal epithelial cells

    NARCIS (Netherlands)

    Louwen, R.; Neerven, van R.J.J.

    2015-01-01

    Raw milk is a recognized source of Campylobacter outbreaks, but pasteurization is an effective way to eliminate the causative agent of Campylobacteriosis. Whereas breastfeeding is protective against infectious diseases, consumption of formula milk is thought to be not. However, in relation to

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

    Directory of Open Access Journals (Sweden)

    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. SULT1A3-Mediated Regiospecific 7-O-Sulfation of Flavonoids in Caco-2 Cells Can Be Explained by the Relevant Molecular Docking Studies

    Science.gov (United States)

    Meng, Shengnan; Wu, Baojian; Singh, Rashim; Yin, Taijun; Morrow, John Kenneth; Zhang, Shuxing; Hu, Ming

    2012-01-01

    Flavonoids are the polyphenolic compounds with various claimed health benefits, but the extensive metabolism by uridine-5'-diphospho-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) in liver and intestine led to poor oral bioavailabilities. The effects of structural changes on the sulfonation of flavonoids have not been systemically determined, although relevant effects of structural changes on the glucuronidation of flavonoids had. We performed the regiospecific sulfonation of sixteen flavonoids from five different subclasses of flavonoids, which are represented by apigenin (flavone), genistein (isoflavone), naringenin (flavanone), kaempherol (flavonol), and phloretin (chalcone). Additional studies were performed using 4 mono-hydroxyl flavonoids with –OH group at 3, 4’, 5 or 7 position, followed by 5 di-hydroxyl-flavonoids, and 2 tri-hydroxyl flavonoids by using expressed human SULT1A3 and Caco-2 cell lysates. We found that these compounds were exclusively sulfated at the 7-OH position by SULT1A3 and primarily sulfated at 7-OH position in Caco-2 cell lysates with minor amounts of 4’-O-sulfates formed as well. Sulfonation rates measured using SULT1A3 and Caco-2 cell lysates were highly correlated at substrate concentrations of 2.5 and 10 µM. Molecular docking studies provided structural explanations as to why sulfonation only occurred at the 7-OH position of flavones, flavonols and flavanones. In conclusion, molecular docking studies explain why SULT1A3 exclusively mediates sulfonation at the 7-OH position of flavones/flavonols, and correlation studies indicate that SULT1A3 is the main isoform responsible for flavonoid sulfonation in the Caco-2 cells. PMID:22352375

  8. Short communication: A study of Lactobacillus isolates' adherence to and influence on membrane integrity of human Caco-2 cells.

    Science.gov (United States)

    Jose, Neethu M; Bunt, Craig R; McDowell, Arlene; Chiu, Jasper Z S; Hussain, Malik A

    2017-10-01

    The selection criteria of ideal probiotic bacteria are complex and involve many factors. One key criterion is based on the ability of the probiotic bacteria to adhere to the epithelial lining of the gastrointestinal tract. The objective of this study was to evaluate and compare the adherence and influence on membrane integrity of 2 selected lactobacilli isolates-Lactobacillus rhamnosus MI13 (dairy food origin) and L. plantarum RC2 (bovine rumen origin)-to Caco-2 cells in the presence and absence of Escherichia coli. The adhesion and influence on membrane integrity properties of the 2 Lactobacillus isolates were compared with Escherichia coli, a human commensal bacterium. From the adhesion studies, we concluded that the bovine rumen isolate exhibited better adherence to Caco-2 cells than the dairy food isolate. In contrast, the dairy food isolate better protected the Caco-2 monolayer from damage induced by ethanol. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  9. Different responses of Caco-2 and MCF-7 cells to silver nanoparticles are based on highly similar mechanisms of action

    NARCIS (Netherlands)

    Zande, van der M.; Undas, A.K.; Kramer, E.H.M.; Monopoli, Marco P.; Peters, R.J.B.; Garry, David; Antunes Fernandes, E.C.; Hendriksen, P.J.M.; Marvin, H.J.P.; Peijnenburg, A.A.C.M.; Bouwmeester, H.

    2016-01-01

    The mode of action of silver nanoparticles (AgNPs) is suggested to be exerted through both Ag+ and AgNP dependent mechanisms. Ingestion is one of the major NP exposure routes, and potential effects are often studied using Caco-2 cells, a well-established model for the gut epithelium. MCF-7 cells are

  10. In vitro co-culture models to evaluate acute cytotoxicity of individual and combined mycotoxin exposures on Caco-2, THP-1 and HepaRG human cell lines.

    Science.gov (United States)

    Smith, Marie-Caroline; Gheux, Alexiane; Coton, Monika; Madec, Stéphanie; Hymery, Nolwenn; Coton, Emmanuel

    2018-02-01

    Deoxynivalenol (DON) and zearalenone (ZEA) are mycotoxins primarily produced by Fusarium species and commonly co-occur in European grains. Some in vitro studies reported synergistic combined effects on cell viability reduction for these two natural food contaminants. However, most of these studies were carried out on conventional cell culture systems involving only one cell type and thus did not include cell-cell communication that is closer to in vivo conditions. In this context, we developed easy bi- and tri-culture systems using the Caco-2 (intestinal epithelial cells), THP-1 (monocytes) and HepaRG (hepatic cells) human cell lines in a proliferating state. Individual and combined cytotoxic effects of DON and ZEA were then assessed using co-cultures during 48 h. In bi-culture systems, results showed that only the highest tested dose of ZEA (IC 30 ) induced a significant reduction in THP-1 viability with both Caco-2 and HepaRG cells cultured in transwells above. On the contrary, only the highest tested dose of DON (IC 30 ) significantly affected HepaRG cell viability located under the Caco-2 cell monolayer. In addition, the DON + ZEA combination seemed to induce higher cytotoxicity than each toxin alone. Mycotoxin quantification in the abluminal compartment by Q-TOF LC-MS suggested uptake of both mycotoxins by the different cell lines. According to the co-culturing cell type, possible cell-cell interactions were also observed. Finally, in the tri-culture system, no cytotoxic effects were observed, regardless of the treatment. These findings highlighted the importance of the proposed models to better decipher toxicological impacts of mycotoxins on more complex cellular systems. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. The effect of five artificial sweeteners on Caco-2, HT-29 and HEK-293 cells.

    Science.gov (United States)

    van Eyk, Armorel Diane

    2015-01-01

    Artificial sweeteners (AS) have been associated with tumor development (including colon cancer) in both animals and humans although evidence has been conflicting. Additional research was thus conducted by studying the effects of 5 AS on the morphology, cell proliferation and DNA in cells by utilizing Caco-2, HT-29 (colon) and HEK-293 (kidney) cell lines. Cells were exposed to sodium cyclamate, sodium saccharin, sucralose and acesulfame-K (0-50 mM) and aspartame (0-35 mM) over 24, 48 and 72 hours. Morphological changes were presented photographically and % cell viability was determined by using the MTT cell viability assay. Possible DNA damage (comet assay) induced by the AS (0.1, 1 and 10 mM, treated for 24, 48 and 72 hours) was studied. The appearance of "comets" was scored from no damage to severe damage (0-4). Cells became flatter and less well defined at higher AS concentrations (>10 mM). At concentrations >10 mM, decreased cell viability was noted with both increasing concentration and increasing incubation time for all cell lines tested. In general, HEK-293 cells seemed to be less affected then the colon cancer cells. Sucralose and sodium saccharin seemed to elicit the greatest degree of DNA fragmentation of all the sweeteners tested in all the cell lines used. Morphological cell alterations, cell viability and DNA fragmentation seemed to be more in the colon cancer cells. Further studies have to be performed to clarify mechanisms involved causing these alterations in mammalian cells.

  12. Closed-Loop Doluisio (Colon, Small Intestine) and Single-Pass Intestinal Perfusion (Colon, Jejunum) in Rat-Biophysical Model and Predictions Based on Caco-2.

    Science.gov (United States)

    Lozoya-Agullo, Isabel; Gonzalez-Alvarez, Isabel; Zur, Moran; Fine-Shamir, Noa; Cohen, Yael; Markovic, Milica; Garrigues, Teresa M; Dahan, Arik; Gonzalez-Alvarez, Marta; Merino-Sanjuán, Matilde; Bermejo, Marival; Avdeef, Alex

    2017-12-29

    The effective rat intestinal permeability (P eff ) was deconvolved using a biophysical model based on parameterized paracellular, aqueous boundary layer, transcellular permeabilities, and the villus-fold surface area expansion factor. Four types of rat intestinal perfusion data were considered: single-pass intestinal perfusion (SPIP) in the jejunum (n = 40), and colon (n = 15), closed-loop (Doluisio type) in the small intestine (n = 78), and colon (n = 74). Moreover, in vitro Caco-2 permeability values were used to predict rat in vivo values in the rat data studied. Comparable number of molecules permeate via paracellular water channels as by the lipoidal transcellular route in the SPIP method, although in the closed-loop method, the paracellular route appears dominant in the colon. The aqueous boundary layer thickness in the small intestine is comparable to that found in unstirred in vitro monolayer assays; it is thinner in the colon. The mucosal surface area in anaesthetized rats is 0.96-1.4 times the smooth cylinder calculated value in the colon, and it is 3.1-3.6 times in the small intestine. The paracellular permeability of the intestine appeared to be greater in rat than human, with the colon showing more leakiness (higher P para ) than the small intestine. Based on log intrinsic permeability values, the correlations between the in vitro and in vivo models ranged from r 2 0.82 to 0.92. The SPIP-Doluisio method comparison indicated identical log permeability selectivity trend with negligible bias.

  13. 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

  14. 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.

  15. Role of germinant receptors in Caco-2 cell-initiated germination of Bacillus cereus ATCC 14579 endospores

    NARCIS (Netherlands)

    Hornstra, L.M.; Voort, van der M.; Wijnands, L.M.; Roubos-van den Hil, P.J.; Abee, T.

    2009-01-01

    Spores obtained from Bacillus cereus ATCC 14579 and mutant strains lacking each of seven germinant receptor operons were exposed to differentiated Caco-2 cells and monitored for germination. Spores of the gerI and gerL mutants showed a reduced germination response, pointing to a role for these

  16. 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...

  17. Inhibition by pectic oligosaccharides of the invasion of undifferentiated and differentiated Caco-2 cells by Campylobacter jejuni

    Science.gov (United States)

    The ability of pectic oligosaccharides (POS) to inhibit adherence to and invasion of undifferentiated (UC) and differentiated (DC) Caco-2 cells by Campylobacter jejuni (C. jejuni) was investigated. It was observed that both adherence and invasion were significantly higher in UC than in DC. POS (2.5 ...

  18. 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.|info:eu-repo/dai/nl/068570368; 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

  19. Effect of neutrase, alcalase, and papain hydrolysis of whey protein concentrates on iron uptake by Caco-2 cells

    NARCIS (Netherlands)

    Ou, K.Q.; Liu, Y.Z.; Zhang, L.B.; Yang, X.G.; Huang, Z.W.; Nout, M.J.R.; Liang, J.

    2010-01-01

    Effects of enzymatic hydrolysates of whey protein concentrates (WPC) on iron absorption were studied using in vitro digestion combined with Caco-2 cell models for improved iron absorption. Neutrase- and papain-treated WPC could improve iron absorption; especially hydrolysates by Neutrase could

  20. Different responses of Caco-2 and MCF-7 cells to silver nanoparticles are based on highly similar mechanisms of action

    NARCIS (Netherlands)

    Zande, van der Meike; Undas, Anna K.; Kramer, Evelien; Monopoli, Marco P.; Peters, Ruud J.; Garry, David; Antunes Fernandes, Elsa C.; Hendriksen, Peter J.; Marvin, Hans J.P.; Peijnenburg, Ad A.; Bouwmeester, Hans

    2016-01-01

    The mode of action of silver nanoparticles (AgNPs) is suggested to be exerted through both Ag+ and AgNP dependent mechanisms. Ingestion is one of the major NP exposure routes, and potential effects are often studied using Caco-2 cells, a well-established model for the gut epithelium.

  1. Pharmacokinetics and Bioavailability of the Isoflavones Formononetin and Ononin and Their in Vitro Absorption in Ussing Chamber and Caco-2 Cell Models.

    Science.gov (United States)

    Luo, Li-Yu; Fan, Miao-Xuan; Zhao, Hai-Yu; Li, Ming-Xing; Wu, Xu; Gao, Wen-Yuan

    2018-03-21

    Formononetin and its glycoside ononin are bioactive isoflavones widely present in legumes. The present study investigated the pharmacokinetics, bioavailability, and in vitro absorption of formononetin and ononin. After an oral administration to rats, formononetin showed a higher systemic exposure over ononin. The oral bioavailability of formononetin and ononin were 21.8% and 7.3%, respectively. Ononin was more bioavailable than perceived, and its bioavailability reached 21.7% when its metabolite formononetin was taken into account. Both formononetin and ononin exhibited better absorption in large intestine segments than that in small intestine segments. Formononetin displayed a better permeability in all intestinal segments over ononin. Transport of formononetin across Caco-2 cell monolayer was mainly through passive diffusion, while ononin was actively pumped out by MRP2 but not P-gp. The results provide evidence for better understanding of the pharmacological actions of formononetin and ononin, which advocates more in vivo evaluations or human trials.

  2. Biosynthesis and transport of lysosomal alpha-glucosidase in the human colon carcinoma cell-line Caco-2: secretion from the apical surface

    NARCIS (Netherlands)

    Klumperman, J.; Fransen, J.A.; Boekestijn, J.C.; Oude Elferink, R.P.; Matter, K.; Hauri, H.P.; Tager, J.M.; Ginsel, L.A.

    1991-01-01

    The human adenocarcinoma cell line Caco-2 was used for studies on the biosynthesis and transport of lysosomal acid alpha-glucosidase in polarized epithelial cells. Metabolic labelling revealed that in Caco-2 cells alpha-glucosidase is synthesized as a precursor form of 110 x 10(3) Mr. This form is

  3. Long-term effects of silver nanoparticles in caco-2 cells.

    Science.gov (United States)

    Vila, Laura; Marcos, Ricard; Hernández, Alba

    2017-08-01

    The high success of silver nanoparticles (AgNPs), mainly associated with their proved antimicrobial properties, has led to an increasing spread in our close environment. Although many studies have been carried out to detect potential toxicity of AgNPs, most of them have been developed under unrealistic exposure conditions. In terms of human risk, the evaluation of long-term exposures to subtoxic doses of NPs remains a challenge. Here, we have determined different transformation-related end points under a scenario of 6 weeks long-term exposure to low noncytotoxic AgNPs concentrations (0.5 and 1 μg/mL) in Caco-2 cells. A significant uptake of AgNPs was demonstrated by using confocal microscopy showing a high presence of AgNPs in both the cytoplasm and the nucleus. As for the assayed parameters of cell transformation such as ability to growth without requiring adherence to a surface (soft-agar assay), the secretion of extracellular matrix metalloproteinase to the medium (zymography), migration capacity and ability of the secretome of exposed cells to promote tumor growth, significant effects were detected in all cases, with the exception of the extracellular matrix metalloproteinases (MMP2 and MMP9) secretion. Our results point out the potential carcinogenic risk associated with AgNPs exposure under long-term exposure conditions, as well as the importance of using realistic exposure scenarios to test nanomaterials.

  4. Nanoemulsion-based delivery system for enhanced oral bioavailability and caco-2 cell monolayers permeability of berberine hydrochloride.

    Science.gov (United States)

    Li, Yong-Jiang; Hu, Xiong-Bin; Lu, Xiu-Ling; Liao, De-Hua; Tang, Tian-Tian; Wu, Jun-Yong; Xiang, Da-Xiong

    2017-11-01

    Berberine hydrochloride (BBH) has a variety of pharmacological activities such as antitumor, antimicrobial, anti-inflammation, and reduce irritable bowel syndrome. However, poor stability and low oral bioavailability limited its usage. Herein, an oil-in-water nanoemulsion system of BBH was developed to improve its stability and oral bioavailability. The pseudoternary phase diagrams were constructed for the determination of composition of various nanoemulsions. The nanoemulsions of BBH composed of Labrafil M 1944 CS (oil phase), RH-40 (surfactant), glycerin (co-surfactant), and water (aqueous phase). The O/W nanoemulsion of BBH showed a relative bioavailability of 440.40% compared with unencapsulated BBH and was stable in our 6-month stability study. Further, there was a significant increase in intestinal permeability of BBH as assessed by Caco-2 cell monolayers and a significant reduction in efflux of BBH by the multidrug efflux pump P-glycoprotein. This study confirmed that the nanoemulsion formulation could be used as an alternative oral formulation of BBH to improve its stability, oral bioavailability and permeability.

  5. 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.

  6. The advanced glycation end product Nϵ-carboxymethyllysine and its precursor glyoxal increase serotonin release from Caco-2 cells.

    Science.gov (United States)

    Holik, Ann-Katrin; Lieder, Barbara; Kretschy, Nicole; Somoza, Mark M; Ley, Jakob P; Hans, Joachim; Somoza, Veronika

    2018-03-01

    Advanced glycation end products (AGEs), comprising a highly diverse class of Maillard reaction compounds formed in vivo and during heating processes of foods, have been described in the progression of several degenerative conditions such as Alzheimer's disease and diabetes mellitus. N ϵ -Carboxymethyllysine (CML) represents a well-characterized AGE, which is frequently encountered in a Western diet and is known to mediate its cellular effects through binding to the receptor for AGEs (RAGE). As very little is known about the impact of exogenous CML and its precursor, glyoxal, on intestinal cells, a genome-wide screening using a customized microarray was conducted in fully differentiated Caco-2 cells. After verification of gene regulation by qPCR, functional assays on fatty acid uptake, glucose uptake, and serotonin release were performed. While only treatment with glyoxal showed a slight impact on fatty acid uptake (P < 0.05), both compounds reduced glucose uptake significantly, leading to values of 81.3% ± 22.8% (500 μM CML, control set to 100%) and 68.3% ± 20.9% (0.3 μM glyoxal). Treatment with 500 μM CML or 0.3 μM glyoxal increased serotonin release (P < 0.05) to 236% ± 111% and 264% ± 66%, respectively. Co-incubation with the RAGE antagonist FPS-ZM1 reduced CML-induced serotonin release by 34%, suggesting a RAGE-mediated mechanism. Similarly, co-incubation with the SGLT-1 inhibitor phloridzin attenuated serotonin release after CML treatment by 32%, hinting at a connection between CML-stimulated serotonin release and glucose uptake. Future studies need to elucidate whether the CML/glyoxal-induced serotonin release in enterocytes might stimulate serotonin-mediated intestinal motility. © 2017 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.

  7. Adhesion of staphylococcal and Caco-2 cells on diamond-like carbon polymer hybrid coating.

    Science.gov (United States)

    Kinnari, Teemu J; Soininen, Antti; Esteban, Jaime; Zamora, Nieves; Alakoski, Esa; Kouri, Vesa-Petteri; Lappalainen, Reijo; Konttinen, Yrjö T; Gomez-Barrena, Enrique; Tiainen, Veli-Matti

    2008-09-01

    Staphylococci cause the majority of the nosocomial implant-related infections initiated by adhesion of planktonic bacteria to the implant surface. It was hypothesized that plasma accelerating filtered pulsed arc discharge method enables combination of the advantageous properties of diamond with the antisoiling properties of polymers. Diamond-like carbon polytetrafluoroethylene hybrid (DLC-PTFE-h) coating was produced. The adhesion of S. aureus ATCC 25923 (10(8) colony-forming units/mL) to surfaces diminished from 2.32%, 2.35%, and 2.57% of high quality DLC, titanium, and oxidized silicon, respectively, to 1.93% of DLC-PTFE-h. For S. epidermidis ATCC 35984 the corresponding figures were 3.90%, 3.32%, 3.47%, and 2.57%. Differences in bacterial adhesion between recombinant DLC-PTFE-h and other materials were statistically significant (p DLC-PTFE-h as to DLC, titanium, or silicon, which were all in the MTT test found to be cytocompatible. DLC-PTFE-h coating can be used to modify the surface properties of any surgical implants and is an unfavorable substrate for staphylococcal cells, but compatible with human Caco-2 cells. DLC-PTFE-h coating may help in the combat against Staphylococcus-related implant infections which usually require both antibiotics and surgical removal of the implant for cure.

  8. Investigation of Enantioselective Membrane Permeability of α-Lipoic Acid in Caco-2 and MDCKII Cell

    Directory of Open Access Journals (Sweden)

    Ryota Uchida

    2016-01-01

    Full Text Available α-Lipoic acid (LA contains a chiral carbon and exists as two enantiomers (R-α-lipoic acid (RLA and S-α-lipoic acid (SLA. We previously demonstrated that oral bioavailability of RLA is better than that of SLA. This difference arose from the fraction absorbed multiplied by gastrointestinal availability (Fa × Fg and hepatic availability (Fh in the absorption phase. However, it remains unclear whether Fa and/or Fg are involved in enantioselectivity. In this study, Caco-2 cells and Madin–Darby canine kidney strain II cells were used to assess the enantioselectivity of membrane permeability. LA was actively transported from the apical side to basal side, regardless of the differences in its steric structure. Permeability rates were proportionally increased in the range of 10–250 µg LA/mL, and the permeability coefficient did not differ significantly between enantiomers. Hence, we conclude that enantioselective pharmacokinetics arose from the metabolism (Fh or Fg × Fh, and definitely not from the membrane permeation (Fa in the absorption phase.

  9. Uteroglobin, an apically secreted protein of the uterine epithelium, is secreted non-polarized form MDCK cells and mainly basolaterally from Caco-2 cells

    DEFF Research Database (Denmark)

    Vogel, L K; Suske, G; Beato, M

    1993-01-01

    A complete cDNA encoding rabbit uteroglobin was constructed and expressed in MDCK and Caco-2 cells. The MDCK cells secrete uteroglobin in approximately equal amounts to the apical and the basolateral side, whereas the Caco-2 cells secrete uteroglobin mainly to the basolateral side. Both MDCK...... and Caco-2 cells thus secrete uteroglobin in a non-sorted manner. It has, however, previously been shown that uteroglobin is secreted exclusively at the apical membrane in primary cell culture of endometrial epithelial cells [S.K. Mani et al. (1991) Endocrinology 128, 1563-1573]. This suggests that either...... the endometrial epithelium has an apical default pathway or recognises a sorting signal not recognised by MDCK cells and Caco-2 cells. Our data thus show that a soluble molecule can be secreted at the apical, the basolateral or both membranes depending on the cell type....

  10. Effect of proinflammatory cytokine IL-6 on efflux transport of rebamipide in Caco-2 cells.

    Science.gov (United States)

    Miyake, Masateru; Nakai, Daisuke

    2017-09-01

    1. Effect of IL-6, a pro-inflammatory cytokine, on efflux transport of rebamipide, an antiulcer drug, was investigated in Caco-2 cells. 2. Rebamipide had a greater basal-to-apical than apical-to-basal transport rate. Efflux transport of rebamipide was inhibited by cyclosporine A, a P-gp inhibitor, and probenecid, which is a general MRP inhibitor, but not by Ko143, a BCRP inhibitor. 3. By the addition of IL-6, mannitol transport was slightly increased in a concentration-dependent manner in both directions of absorption and efflux. The addition of IL-6 did not change efflux transport of rebamipide even though efflux transport of digoxin, a typical substrate of P-gp, was significantly decreased by the addition of IL-6, indicating decrease of the function of P-gp. 4. Therefore, it was suggested that increase of MRP(s)-mediated transport compensates for the decrease of P-gp mediated transport of rebamipide. These findings suggested that rebamipide absorption is unlikely to be changed in IBD patients.

  11. Effect of taurine on mRNA expression of thioredoxin interacting protein in Caco-2 cells.

    Science.gov (United States)

    Gondo, Yusuke; Satsu, Hideo; Ishimoto, Yoko; Iwamoto, Taku; Shimizu, Makoto

    2012-09-28

    Taurine (2-aminoethanesulfonic acid), a sulfur-containing β-amino acid, plays an important role in several essential biological processes; although, the underlying mechanisms for these regulatory functions remain to be elucidated, especially at the genetic level. We investigated the effects of taurine on the gene expression profile in Caco-2 cells using DNA microarray. Taurine increased the mRNA expression of thioredoxin interacting protein (TXNIP), which is involved in various metabolisms and diseases. β-Alanine or γ-aminobutyric acid (GABA), which are structurally or functionally related to taurine, did not increase TXNIP mRNA expression. These suggest the expression of TXNIP mRNA is induced specifically by taurine. β-Alanine is also known to be a substrate of taurine transporter (TAUT) and competitively inhibits taurine uptake. Inhibition of taurine uptake by β-alanine eliminated the up-regulation of TXNIP, which suggests TAUT is involved in inducing TXNIP mRNA expression. The up-regulation of TXNIP mRNA expression by taurine was also observed at the protein level. Furthermore, taurine significantly increased TXNIP promoter activity. Our present study demonstrated the taurine-specific phenomenon of TXNIP up-regulation, which sheds light on the physiological function of taurine. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. Effects of acylcarnitines on efflux transporting system in Caco-2 cell monolayers.

    Science.gov (United States)

    Tomita, Mikio; Doi, Nobuyuki; Hayashi, Masahiro

    2010-09-01

    This study examined the effects of the absorption enhancers, acylcarnitines, on efflux transporting systems, including P-glycoprotein (P-gp) and other efflux transporters, and elucidated the importance of acyl chain length and the concentration of acylcarnitine on the activity of efflux transport. The effects of two acyl (lauroyl and palmitoyl) carnitines on the influx and efflux of lucifer yellow and fluorescein isothiocyanate dextran 4,000, which have characteristic vectorial transport, were examined in Caco-2 cell monolayers. Lauroylcarnitine and palmitoylcarnitine increased influx and decreased efflux of these substrates, in a manner dependent on their concentration and acyl chain lengths by increasing influx and inhibiting efflux of the substrates. The results indicated that both the acyl moiety and long acyl chains play important roles in the modification of influx and efflux transport. Because no marked changes in the levels of P-gp protein or the leakage of LDH were observed at 1 h after the application of acylcarnitines, it was concluded that these acylcarnitines had an effect on modulation of the function of P-gp or other efflux transporters without cytotoxicity.

  13. In-depth evaluation of Gly-Sar transport parameters as a function of culture time in the Caco-2 cell model

    DEFF Research Database (Denmark)

    Bravo, Silvina A.; Nielsen, Carsten Uhd; Amstrup, Jan

    2004-01-01

    The aim of the present study was to investigate the influence of culture time on hPEPT1-mediated transport in Caco-2 cell monolayers. Peptide transport activity in Caco-2 cells grown in standard media and in a "rapid" 4-day model was first compared. The rapid 4-day Caco-2 cell model, cultured using...... a cocktail of growth factors and agonists, displayed lower peptide uptake capacity than Caco-2 cells grown for 4 days in conventional media, and was judged to be unsuitable for peptide transport studies. Peptide transport activity as well as monolayer integrity and tissue morphology were evaluated...... expression were studied using confocal laser scanning microscopy (CLSM) and conventional staining/ immunostaining. Caco-2 cells grown in conventional media became confluent after 3-4 days. Mannitol permeability decreased from day 5 to 21 and TEER increased steadily until ~day 21. Apical hPEPT1 uptake...

  14. Protective Effects of Bifidobacterium on Intestinal Barrier Function in LPS-Induced Enterocyte Barrier Injury of Caco-2 Monolayers and in a Rat NEC Model.

    Science.gov (United States)

    Ling, Xiang; Linglong, Peng; Weixia, Du; Hong, Wei

    2016-01-01

    Zonulin protein is a newly discovered modulator which modulates the permeability of the intestinal epithelial barrier by disassembling intercellular tight junctions (TJ). Disruption of TJ is associated with neonatal necrotizing enterocolitis (NEC). It has been shown bifidobacterium could protect the intestinal barrier function and prophylactical administration of bifidobacterium has beneficial effects in NEC patients and animals. However, it is still unknown whether the zonulin is involved in the gut barrier dysfunction of NEC, and the protective mechanisms of bifidobacterium on intestinal barrier function are also not well understood. The present study aims to investigate the effects of bifidobacterium on intestinal barrier function, zonulin regulation, and TJ integrity both in LPS-induced enterocyte barrier injury of Caco-2 monolayers and in a rat NEC model. Our results showed bifidobacterium markedly attenuated the decrease in transepithelial electrical resistance and the increase in paracellular permeability in the Caco-2 monolayers treated with LPS (P zonulin release (P zonulin (P zonulin protein release and improvement of intestinal TJ integrity.

  15. Effects of nonsteroidal anti-inflammatory drugs on the expression and function of P-glycoprotein/MDR1 in Caco-2 cells.

    Science.gov (United States)

    Takara, Kohji; Hayashi, Ryuhei; Kokufu, Misato; Yamamoto, Kazuhiro; Kitada, Noriaki; Ohnishi, Noriaki; Yokoyama, Teruyoshi

    2009-01-01

    The aim of this study was to examine the effects of 16 kinds of nonsteroidal anti-inflammatory drugs (NSAIDs) on P-glycoprotein/MDR1 in Caco-2 cells as an intestinal epithelial cell model. Cells were treated with NSAIDs for 24 hours, and then, the expression of MDR1 mRNA was evaluated by reverse-transcriptase polymerase chain reaction. The function of MDR1 in cells pretreated with NSAIDs for 48 hours was evaluated by measuring the cellular amount of rhodamine123, which is a substrate of MDR1. The expression of MDR1 mRNA was increased by diclofenac, fenbufen, indomethacin, and nimesulide and the tended to be increased by meloxicam, mepirizole, and sulindac. However, pretreatment for 48 hours with diclofenac, indomethacin, or nimesulide, but not fenbufen, resulted in a significant increase in the amount of rhodamine123 accumulated. Although NSAIDs without effects on the expression of MDR1 mRNA altered the accumulation of rhodamine123 significantly, the efflux of rhodamine123 from cells was unchanged. In conclusion, the expression of MDR1 mRNA in Caco-2 cells was demonstrated to be increased by treatment with some NSAIDs, although the transport function of MDR1 was unchanged. These findings imply that the NSAIDs did not cause the drug interaction via MDR1 induction.

  16. TNF-alpha induced junctional modulation enhances response to radiation in Caco-2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Moon, Yeonjoo; Ahn, Hyein; Park, Jina; Lee, Byungryong; Chung, Eunkyung [Hallym University, Seoul (Korea, Republic of); Yi, Jaeoun [KIRAMS, Seoul (Korea, Republic of)

    2007-10-15

    The Adhesion molecules mediated cell-cell and cell matrix interactions are essential for variety of physiological and pathological processes including maintenance of normal tissues integrity as well as tumor development and progression. Cell-cell interaction is initiated by interactions of tight junctional proteins with neighboring cells. Tight junctions govern the paracellular permeability of endothelial and epithelial cells. Aberrations of tight junction formation are an early and key event during vascular spread cancer and inflammation. TNF-alpha plays an important role in the intestinal inflammation by increase of intestinal epithelial tight junction permeability. It has been reported that TNF alpha- modulated intestinal epithelial tight junction barrier is mediated by myosin light-chain kinase protein expression through NFk-B activation. However, the alterations of tight junctional proteins involved in the TNF-alpha-induced increase of intestinal TJ permeability remain unclear. Claudin is essential to the formation and maintenance of tight junction (TJ) and has been identified 24 members so far. Claudin-1, 3, 4, 6, 10 and 16 have been shown altered in various cancers and they may have important roles in cell survival, motility, and invasion of cancer cells. However, the functions of these proteins in tumorigenesis and inflammation are still being elucidated.

  17. 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.

  18. Differential effect of ethanol and hydrogen peroxide on barrier function and prostaglandin E2 release in differentiated Caco-2 cells: selective prevention by growth factors.

    Science.gov (United States)

    Catalioto, Rose-Marie; Festa, Carla; Triolo, Antonio; Altamura, Maria; Maggi, Carlo Alberto; Giuliani, Sandro

    2009-02-01

    The present study investigates the effects of ethanol and hydrogen peroxide (H(2)O(2)) on the barrier function and prostaglandin E(2) (PGE(2)) release in differentiated Caco-2 cells. Epithelial barrier integrity was estimated by measuring transepithelial electrical resistance (TEER), the transport of reference compounds and lactate dehydrogenase leakage, the PGE(2) release by enzyme immunoassay. Ethanol and H(2)O(2) decreased TEER and increased the transport of lucifer yellow without affecting that of propranolol and phenylalanine. Only the effects of ethanol were accompanied by PGE(2) production and were reversible without causing long-term cytotoxicity. The cyclooxygenase-2 inhibitor, NS-398, prevented the effect of ethanol on both PGE(2) release and TEER, while inhibition of both cyclooxygenase-2 and tyrosine kinase drastically compromised cell viability and TEER recovery. Hepatocyte growth factor, keratinocyte growth factor or insulin prevented the effect of ethanol on cell permeability, but not on PGE(2) release. Their combination prevented the effect of H(2)O(2). In conclusion, ethanol and H(2)O(2) increased paracellular permeability in differentiated Caco-2 cells without affecting transcellular and active transport. Cyclooxygenase-2 stimulated PGE(2) release mediated the reversible effect of ethanol on tight junctions and, meanwhile, contributed to cell survival. Growth factors, normally present in the intestine, exerted a selective protective effect toward paracellular permeability increase induced by irritants.

  19. Identification of New Anti-inflammatory Peptides from Zein Hydrolysate after Simulated Gastrointestinal Digestion and Transport in Caco-2 Cells.

    Science.gov (United States)

    Liang, Qiufang; Chalamaiah, Meram; Ren, Xiaofeng; Ma, Haile; Wu, Jianping

    2018-02-07

    Chronic inflammation is an underlying contributor to various chronic diseases. The objectives of this study were to investigate the anti-inflammatory activity of zein hydrolysate after simulated gastrointestinal digestion and Caco-2 cell absorption and to identify novel anti-inflammatory peptides after transport across Caco-2 cells. Three zein hydrolysates were prepared and further digested using gastrointestinal proteases; their transports were studied in Caco-2 cells. Anti-inflammatory activity was studied in endothelial EA.hy926 cells. Three zein hydrolysates and their digests significantly decreased the expression of tumor necrosis factor-α (TNF-α) induced pro-inflammatory vascular cell adhesion molecule-1 (VCAM-1) by 37.3-66.0%. Eleven novel peptides with 5-9 amino acid residues were sequenced; three peptides showed strong anti-inflammatory activity by inhibiting the VCAM-1 by 54-38.9% and intercellular cell adhesion molecule-1 (ICAM-1) by 36.5-28.6% at 0.2 mM. A new approach to identify novel anti-inflammatory peptides that could survive gastrointestinal digestion and absorption was developed.

  20. Resistant Maltodextrin Decreases Micellar Solubility of Lipids and Diffusion of Bile Salt Micelles and Suppresses Incorporation of Micellar Fatty Acids into Caco-2 Cells.

    Science.gov (United States)

    Ikeda, Ikuo; Tamakuni, Kyouhei; Sakuma, Tomomi; Ozawa, Ran; Inoue, Nao; Kishimoto, Yuka

    2016-01-01

    Several studies have suggested that resistant maltodextrin (RMD) suppresses intestinal lipid absorption in experimental animals and humans. However, possible mechanisms underlying this effect are not known. In this study, effects of RMD on processes of the absorption of various lipids were investigated in vitro. RMD dose-dependently suppressed the solubility of various lipid components, including 1-mono-oleoylglycerol, oleic acid, and phosphatidylcholine in bile salt micelles in vitro. When the diffusion rate of bile salt micelles through a filter membrane was investigated in vitro, bile salt micelles containing RMD diffused more slowly than those without RMD. Incorporation of [1- 14 C] oleic acid into Caco-2 cells from the RMD-containing bile salt micelles was significantly smaller than that from the control micelles (without RMD). These results show that RMD suppresses intestinal absorption of lipids by decreasing their micellar solubility and the diffusion rate of bile salt micelles.

  1. 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.

  2. Tc-99m Radiolabeled Alendronate Sodium Microemulsion: Characterization and Permeability Studies across Caco-2 Cells.

    Science.gov (United States)

    Elitez, Yetkin; Ekinci, Meliha; Ilem-Ozdemir, Derya; Gundogdu, Evren; Asikoglu, Makbule

    2017-06-12

    Alendronate sodium (ALD) is used orally but it is poorly absorbed from the gastrointestinal (GI) tract. For this reason, microemulsion system was chosen to evaluate ALD from the GI tract after oral delivery. This study was aimed to prepare water-in-oil (w/o) microemulsion formulation of ALD and evaluate the permeability of ALD microemulsion from Caco-2 cell lines with radioactive and non-radioactive studies. The ALD microemulsion was developed by using pseudo-ternary phase diagram and composed of Soybean oil, Colliphor EL, Tween 80, Transcutol and distilled water. The prepared ALD microemulsion was characterized by physical appearance, droplet size, viscosity, pH, electrical conductivity and refractive index. The stability of the formulation was investigated for 6 months at 25±2°C/60±5% of relative humidity (RH) as well as at 40±2°C/75±5% RH. After that 1 mg of ALD was radiolabeled with 99mTc and added to microemulsion. The permeability studies were performed with both 99mTc-ALD microemulsion and ALD microemulsion. The experimental results suggested that ALD microemulsion presented adequate stability with droplet size varying from 37.8±0.9 to 39.9±1.2 nm during incubation time. In addition, ALD microemulsion was radiolabeled with high labeling efficiency (>95%). In non-radioactive study, ALD permeability was found to be 45 μg.mL-1 and microemulsion has high permeability percentage when compared to another study. The novel w/o microemulsion formulation has been developed for oral delivery of ALD. Based on the results, permeability of ALD could be significantly improved by the microemulsion formulation. In addition, 99mTc-ALD microemulsion in capsule can be used for bone disease treatment and diagnosis. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  3. In vitro assessment of bio-augmented minerals from peanut oil cakes fermented by Aspergillus oryzae through Caco-2 cells.

    Science.gov (United States)

    Sadh, Pardeep Kumar; Chawla, Prince; Bhandari, Latika; Kaushik, Ravinder; Duhan, Joginder Singh

    2017-10-01

    Present study was carried out to assess the significances of solid state fermentation of peanut oil cakes (POC) by Aspergillus oryzae on in vitro bioavailability of minerals (iron, zinc and calcium) and cellular transport, retention and uptake from POC through Caco-2 cells. Bioavailability of iron, zinc and calcium of POC was examined by means of a combined simulated gastrointestinal digestion/Caco-2 cell system. Bio-augmentation of minerals of fermented POC attributed a positive, statistically significant increased influence on minerals retention, transport and uptake values when compared with that of respective inorganic salts as reference. Results revealed increased cellular ferritin content from fermented POC digests than the digests of free form of respective inorganic salt. In prospect of the present investigation the fermented POC samples showed significantly higher iron, zinc and calcium bioavailability and enormous possible health benefits.

  4. Evaluation of some medicinal plants effect on Rhodamine 123 accumulation and efflux in Caco-2 cell line by flowcytometry

    Directory of Open Access Journals (Sweden)

    S.N. Sadati Lamardi*

    2017-11-01

    Full Text Available Background and objectives: In review of traditional Persian medicine (TPM literature concerning multi drug therapy, a group of medicinal plants that are called "convoy drugs", agents which penetrate fast into whole or specific part of the body and accelerate delivery of drugs into specific target has been mentioned. In this study, the inhibitory effect of the aqueous extracts of some selected medicinal plants on P-glycoprotein (P-gp was assessed in order to determine the possibility of herb-drug interactions. Methods: P-gp inhibitory effect of aqueous extracts (250 µg/mL from some medicinal plants and verapamile (5 µg/mL was measured using flow cytometry by Rhodamine 123 (Rh123 in Caco2 cell line. Inhibition percent of each sample (% was compared with a control group (Caco-2 cell with Rh 123. Results: According to the results pennyroyal, aniseed (p

  5. 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.

  6. Hydrolysis and transepithelial transport of two corn gluten derived bioactive peptides in human Caco-2 cell monolayers.

    Science.gov (United States)

    Ding, Long; Wang, Liying; Zhang, Ting; Yu, Zhipeng; Liu, Jingbo

    2018-04-01

    The objective of this paper was to investigate the transepithelial transport of two novel corn gluten-derived antioxidant peptides, YFCLT and GLLLPH, using Caco-2 cell monolayers. Results showed that both of YFCLT and GLLLPH could transport in intact form across Caco-2 cell monolayers with apparent permeability coefficient (P app ) values of (1.10±0.16)×10 -7 cm/s and (1.98±0.23)×10 -7 cm/s, respectively. However, it was found that the two peptides were susceptible and easily hydrolyzed by brush border membrane peptidases. In the presence of diprotin A, an inhibitor of dipeptidyl peptidase IV (DPPIV), the hydrolysis of YFCLT and GLLLPH decreased and their permeabilities increased significantly compared to control group (P0.05), suggesting that the transport of YFCLT and GLLLPH across Caco-2 cell monolayers was not mediated by PepT1. However, it was found that cytochalasin d, a tight junctions (TJs) disruptor, increased the permeability significantly (PCaco-2 cell monolayers. Copyright © 2018 Elsevier Ltd. All rights reserved.

  7. Comparison of the permeability of metoprolol and labetalol in rat, mouse, and Caco-2 cells: use as a reference standard for BCS classification.

    Science.gov (United States)

    Incecayir, Tuba; Tsume, Yasuhiro; Amidon, Gordon L

    2013-03-04

    The purpose of this study was to investigate labetalol as a potential high permeability reference standard for the application of Biopharmaceutics Classification Systems (BCS). Permeabilities of labetalol and metoprolol were investigated in animal intestinal perfusion models and Caco-2 cell monolayers. After isolating specific intestinal segments, in situ single-pass intestinal perfusions (SPIP) were performed in rats and mice. The effective permeabilities (Peff) of labetalol and metoprolol, an FDA standard for the low/high Peff class boundary, were investigated in two different segments of rat intestine (proximal jejunum and distal ileum) and in the proximal jejunum of mouse. No significant difference was found between Peff of metoprolol and labetalol in the jejunum and ileum of rat (0.33 ± 0.11 × 10(-4) vs 0.38 ± 0.06 × 10(-4) and 0.57 ± 0.17 × 10(-4) vs 0.64 ± 0.30 × 10(-4) cm/s, respectively) and in the jejunum of mouse (0.55 ± 0.05 × 10(-4) vs 0.59 ± 0.13 × 10(-4) cm/s). However, Peff of metoprolol and labetalol were 1.7 and 1.6 times higher in the jejunum of mouse, compared to the jejunum of rat, respectively. Metoprolol and labetalol showed segmental-dependent permeability through the rat intestine, with increased Peff in the distal ileum in comparison to the proximal jejunum. Most significantly, Peff of labetalol was found to be concentration-dependent. Decreasing concentrations of labetalol in the perfusate resulted in decreased Peff compared to Peff of metoprolol. The intestinal epithelial permeability of labetalol was lower than that of metoprolol in Caco-2 cells at both apical pH 6.5 and 7.5 (5.96 ± 1.96 × 10(-6) vs 9.44 ± 3.44 × 10(-6) and 15.9 ± 2.2 × 10(-6) vs 23.2 ± 7.1 × 10(-6) cm/s, respectively). Labetalol exhibited higher permeability in basolateral to apical (BL-AP) compared to AP-BL direction in Caco-2 cells at 0.1 times the highest dose strength (HDS) (46.7 ± 6.5 × 10(-6) vs 14.2 ± 1.5 × 10(-6) cm/s). The P

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

    OpenAIRE

    Boyer, Jeanelle; Brown, Dan; Liu, Rui Hai

    2005-01-01

    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 diges...

  9. Importance of Terminal Amino Acid Residues to the Transport of Oligopeptides across the Caco-2 Cell Monolayer.

    Science.gov (United States)

    Ding, Long; Wang, Liying; Yu, Zhipeng; Ma, Sitong; Du, Zhiyang; Zhang, Ting; Liu, Jingbo

    2017-09-06

    The objective of this paper was to investigate the effects of terminal amino acids on the transport of oligopeptides across the Caco-2 cell monolayer. Ala-based tetra- and pentapeptides were designed, and the N- or C-terminal amino acid residues were replaced by different amino acids. The results showed that the oligopeptides had a wide range of transport permeability across the Caco-2 cell monolayer and could be divided into four categories: non-/poor permeability, low permeability, intermediate permeability, and good permeability. Tetrapeptides with N-terminal Leu, Pro, Ile, Cys, Met, and Val or C-terminal Val showed the highest permeability, with apparent permeability coefficient (P app ) values over 10 × 10 -6 cm/s (p transport of tetrapeptides. Pentapeptides with N- or C-terminal Tyr also showed high permeability levels, with P app values of about 10 × 10 -6 cm/s. The amino acids Glu, Asn, and Thr at the N terminus or Lys, Asp, and Arg at the C terminus were also beneficial for the transport of tetra- and pentapeptides, with P app values ranging from 1 × 10 -6 to 10 × 10 -6 cm/s. In addition, peptides with amino acids replaced at the N terminus generally showed higher permeability than those with amino acids replaced at the C terminus (p transport of oligopeptides across the Caco-2 cell monolayer.

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

    International Nuclear Information System (INIS)

    Arana, Maite Rocío; Tocchetti, Guillermo Nicolás; Domizi, Pablo; Arias, Agostina; Rigalli, Juan Pablo; Ruiz, María Laura

    2015-01-01

    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

  11. 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

  12. Myofibroblast keratinocyte growth factor reduces tight junctional integrity and increases claudin-2 levels in polarized Caco-2 cells

    Science.gov (United States)

    Kim, Tae Il; Poulin, Emily J.; Blask, Elliot; Bukhalid, Raghida; Whitehead, Robert H.; Franklin, Jeffrey L.; Coffey, Robert J.

    2013-01-01

    The colonic epithelium is composed of a polarized monolayer sheathed by a layer of pericryptal myofibroblasts (PCMFs). We mimicked these cellular compartments in vitro to assess the effects of paracrine-acting PCMF-derived factors on tight junction (TJ) integrity, as measured by transepithelial electrical resistance (TER). Co-culture with 18Co PCMFs, or basolateral administration of 18Co conditioned medium (CM), significantly reduced TER of polarized Caco-2 cells. Amongst candidate paracrine factors, only keratinocyte growth factor (KGF) reduced Caco-2 TER; basolateral KGF treatment led to time- and concentration-dependent increases in claudin-2 levels. We also demonstrate amphiregulin (AREG), produced largely by Caco-2 cells, increased claudin-2 levels, leading to epidermal growth factor receptor-mediated TER reduction. We propose that colonic epithelial TJ integrity can be modulated by paracrine KGF and autocrine AREG through increased claudin-2 levels. KGF-regulated claudin-2 induction may have implications for inflammatory bowel disease, where both KGF and claudin-2 are upregulated. PMID:22946653

  13. Drug-transporter mediated interactions between anthelminthic and antiretroviral drugs across the Caco-2 cell monolayers.

    Science.gov (United States)

    Kigen, Gabriel; Edwards, Geoffrey

    2017-05-04

    Drug interactions between antiretroviral drugs (ARVs) and anthelminthic drugs, ivermectin (IVM) and praziquantel (PZQ) were assessed by investigating their permeation through the Caco-2 cell monolayers in a transwell. The impact of anthelminthics on the transport of ARVs was determined by assessing the apical to basolateral (AP → BL) [passive] and basolateral to apical (BL → AP) [efflux] directions alone, and in presence of an anthelminthic. The reverse was conducted for the assessment of the influence of ARVs on anthelminthics. Samples from the AP and BL compartments were taken at 60, 120, 180 and 240 min and quantified either by HPLC or radiolabeled assay using a liquid scintillating counter for the respective drugs. Transepithelial resistance (TEER) was used to assess the integrity of the monolayers. The amount of compound transported per second (apparent permeability, Papp) was calculated for both AP to BL (Papp AtoB ), and BL to AP (Papp BtoA ) movements. Samples collected after 60 min were used to determine the efflux ratio (ER), quotient of secretory permeability and absorptive permeability (PappBL-AP/PappAP-BL). The reverse, (PappAP-BL/PappBL-AP) constituted the uptake ratio. The impact of SQV, EFV and NVP on the transport of both IVM and PZQ were investigated. The effect of LPV on the transport of IVM was also determined. The influence of IVM on the transport of SQV, NVP, LPV and EFV; as well as the effect PZQ on the transport of SQV of was also investigated, and a two-tailed p value of <0.05 was considered significant. IVM significantly inhibited the efflux transport (BL → AP movement) of LPV (ER; 6.7 vs. 0.8, p = 0.0038) and SQV (ER; 3.1 vs. 1.2 p = 0.00328); and increased the efflux transport of EFV (ER; 0.7 vs. 0.9, p = 0.031) suggesting the possibility of drug transporter mediated interactions between the two drugs. NVP increased the efflux transport of IVM (ER; 0.8 vs. 1.8, p = 0.0094). The study provides in vitro

  14. ECHINACEA SANGUINEA AND ECHINACEA PALLIDA EXTRACTS STIMULATE GLUCURONIDATION AND BASOLATERAL TRANSFER OF BAUER ALKAMIDES 8 AND 10 AND KETONE 24 AND INHIBIT P-GLYCOPROTEIN TRANSPORTER IN CACO-2 CELLS

    OpenAIRE

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

    2013-01-01

    The use of Echinacea as a medicinal herb is prominent in the United States, and many studies have assessed the effectiveness of Echinacea as an immunomodulator. We hypothesized that Bauer alkamides 8, 10 and 11 and ketone 24 were absorbed similarly either as pure compounds or from Echinacea sanguinea and Echinacea pallida ethanol extracts, and that these Echinacea extracts could inhibit P-glycoprotein transporter (P-gp) in Caco-2 human intestinal epithelial cells. Using HPLC analysis, the per...

  15. Polydatin, a natural precursor of resveratrol, induces cell cycle arrest and differentiation of human colorectal Caco-2 cell

    Science.gov (United States)

    2013-01-01

    Background Human colon adenocarcinoma cells are resistant to chemotherapeutic agents, such as anthracyclines, that induce death by increasing the reactive oxygen species. A number of studies have been focused on chemo-preventive use of resveratrol as antioxidant against cardiovascular diseases, aging and cancer. While resveratrol cytotoxic action was due to its pro-oxidant properties. In this study, we investigate whether the Resveratrol (trans-3,5,49-trihydroxystilbene) and its natural precursor Polydatin (resveratrol-3-O-b-mono- D-glucoside, the glycoside form of resveratrol) combination, might have a cooperative antitumor effect on either growing or differentiated human adenocarcinoma colon cancer cells. Methods The polydatin and resveratrol pharmacological interaction was evaluated in vitro on growing and differentiated Caco-2 cell lines by median drug effect analysis calculating a combination index with CalcuSyn software. We have selected a synergistic combination and we have evaluated its effect on the biological and molecular mechanisms of cell death. Results Simultaneous exposure to polydatin and resveratrol produced synergistic antiproliferative effects compared with single compound treatment. We demonstrated that polydatin alone or in combination with resveratrol at 3:1 molar ratio synergistically modulated oxidative stress, cell cycle, differentiation and apoptosis. Worthy of note treatment with polydatin induced a nuclear localization and decreased expression of heat shock protein 27, and vimentin redistributed within the cell. Conclusions From morphological, and biochemical outcome we obtained evidences that polydatin induced a transition from a proliferative morphology to cell-specific differentiated structures and caused human CaCo-2 cell death by induction of apoptosis. Our data suggest the potential use of polydatin in combination chemotherapy for human colon cancer. PMID:24138806

  16. A dynamic ratio of the alpha+ and alpha- isoforms of the tight junction protein ZO-1 is characteristic of Caco-2 cells and correlates with their degree of differentiation.

    Science.gov (United States)

    Ciana, Annarita; Meier, Katharina; Daum, Nicole; Gerbes, Stefan; Veith, Michael; Lehr, Claus-Michael; Minetti, Giampaolo

    2010-05-07

    ZO-1 is a peripheral protein that plays a central role in the macromolecular assembly of tight junctions by interacting with integral proteins (occludin, claudins, JAMs) of the membrane of adjoining cells, with the actin cytoskeleton, and with nuclear factors. Human ZO-1 is expressed in all epithelia and some specialized endothelia as variable amounts of two related isoforms, which originate from the alternatively spliced mRNA transcripts alpha(+) and alpha(-) and whose specific differential role is still unknown. Moreover, little is known about the timing of expression of ZO-1 isoforms at the protein and mRNA level. This study shows that during growth of freshly plated Caco-2 cells, the alpha(+)/alpha(-) ratio increased as a result of simultaneous increase of alpha(+) and decrease of alpha(-). Differences in the isoform ratio also correlated with differences in epithelium differentiation. This was determined by aminopeptidase N measurements of cells grown on conventional substrates and on modified, micro/nano-patterned surfaces. A comparable shift of ZO-1 isoforms was not observed in other tumour cell lines of non-intestinal origin (A549, Calu-3). Pancreatic stem cells, propagated without exogenous differentiation stimuli, displayed a slight, stable prevalence of the alpha(-) isoform. Of the intestinal cell lines examined (Caco-2 and T84), only Caco-2 cells displayed a dramatic shift in isoform expression. This suggests that this tumour cell line retains to a higher degree a developmental programme related to the dynamic of enterocytic differentiation in vivo.

  17. Comparative cytotoxicity of nanosilver in human liver HepG2 and colon Caco2 cells in culture.

    Science.gov (United States)

    Sahu, Saura C; Zheng, Jiwen; Graham, Lesley; Chen, Lynn; Ihrie, John; Yourick, Jeffrey J; Sprando, Robert L

    2014-11-01

    The use of silver nanoparticles in food, food contact materials, dietary supplements and cosmetics has increased significantly owing to their antibacterial and antifungal properties. As a consequence, the need for validated rapid screening methods to assess their toxicity is necessary to ensure consumer safety. This study evaluated two widely used in vitro cell culture models, human liver HepG2 cells and human colon Caco2 cells, as tools for assessing the potential cytotoxicity of food- and cosmetic-related nanoparticles. The two cell culture models were utilized to compare the potential cytotoxicity of 20-nm silver. The average size of the silver nanoparticle determined by our transmission electron microscopy (TEM) analysis was 20.4 nm. The dynamic light scattering (DLS) analysis showed no large agglomeration of the silver nanoparticles. The concentration of the 20-nm silver solution determined by our inductively coupled plasma-mass spectrometry (ICP-MS) analysis was 0.962 mg ml(-1) . Our ICP-MS and TEM analysis demonstrated the uptake of 20-nm silver by both HepG2 and Caco2 cells. Cytotoxicity, determined by the Alamar Blue reduction assay, was evaluated in the nanosilver concentration range of 0.1 to 20 µg ml(-1) . Significant concentration-dependent cytotoxicity of the nanosilver in HepG2 cells was observed in the concentration range of 1 to 20 µg ml(-1) and at a higher concentration range of 10 to 20 µg ml(-1) in Caco2 cells compared with the vehicle control. A concentration-dependent decrease in dsDNA content was observed in both cell types exposed to nanosilver but not controls, suggesting an increase in DNA damage. The DNA damage was observed in the concentration range of 1 to 20 µg ml(-1) . Nanosilver-exposed HepG2 and Caco2 cells showed no cellular oxidative stress, determined by the dichlorofluorescein assay, compared with the vehicle control in the concentration range used in this study. A concentration-dependent decrease in

  18. Brush border membrane vesicle and Caco-2 cell line: Two experimental models for evaluation of absorption enhancing effects of saponins, bile salts, and some synthetic surfactants.

    Science.gov (United States)

    Moghimipour, Eskandar; Tabassi, Sayyed Abolghassem Sajadi; Ramezani, Mohammad; Handali, Somayeh; Löbenberg, Raimar

    2016-01-01

    The aim of this study was to investigate the influence of absorption enhancers in the uptake of hydrophilic compounds. The permeation of the two hydrophilic drug models gentamicin and 5 (6)-carboxyfluorescein (CF) across the brush border membrane vesicles and Caco-2 cell lines were evaluated using total saponins of Acanthophyllum squarrosum, Quillaja saponaria, sodium lauryl sulfate, sodium glycocholate, sodium taurodeoxycholate, and Tween 20 as absorption enhancers. Transepithelial electrical resistance (TEER) measurement was utilized to assess the paracellular permeability of cell lines. Confocal laser scanning microscopy (CLSM) was performed to obtain images of the distribution of CF in Caco-2 cells. These compounds were able to loosen tight junctions, thus increasing paracellular permeability. CLSM confirmed the effect of these absorption enhancers on CF transport across Caco-2 lines and increased the Caco-2 permeability via transcellular route. It was also confirmed that the decrease in TEER was transient and reversible after removal of permeation enhancers.

  19. Brush border membrane vesicle and Caco-2 cell line: Two experimental models for evaluation of absorption enhancing effects of saponins, bile salts, and some synthetic surfactants

    Directory of Open Access Journals (Sweden)

    Eskandar Moghimipour

    2016-01-01

    Full Text Available The aim of this study was to investigate the influence of absorption enhancers in the uptake of hydrophilic compounds. The permeation of the two hydrophilic drug models gentamicin and 5 (6-carboxyfluorescein (CF across the brush border membrane vesicles and Caco-2 cell lines were evaluated using total saponins of Acanthophyllum squarrosum, Quillaja saponaria, sodium lauryl sulfate, sodium glycocholate, sodium taurodeoxycholate , and Tween 20 as absorption enhancers. Transepithelial electrical resistance (TEER measurement was utilized to assess the paracellular permeability of cell lines. Confocal laser scanning microscopy (CLSM was performed to obtain images of the distribution of CF in Caco-2 cells. These compounds were able to loosen tight junctions, thus increasing paracellular permeability. CLSM confirmed the effect of these absorption enhancers on CF transport across Caco-2 lines and increased the Caco-2 permeability via transcellular route. It was also confirmed that the decrease in TEER was transient and reversible after removal of permeation enhancers.

  20. Brush border membrane vesicle and Caco-2 cell line: Two experimental models for evaluation of absorption enhancing effects of saponins, bile salts, and some synthetic surfactants

    Science.gov (United States)

    Moghimipour, Eskandar; Tabassi, Sayyed Abolghassem Sajadi; Ramezani, Mohammad; Handali, Somayeh; Löbenberg, Raimar

    2016-01-01

    The aim of this study was to investigate the influence of absorption enhancers in the uptake of hydrophilic compounds. The permeation of the two hydrophilic drug models gentamicin and 5 (6)-carboxyfluorescein (CF) across the brush border membrane vesicles and Caco-2 cell lines were evaluated using total saponins of Acanthophyllum squarrosum, Quillaja saponaria, sodium lauryl sulfate, sodium glycocholate, sodium taurodeoxycholate, and Tween 20 as absorption enhancers. Transepithelial electrical resistance (TEER) measurement was utilized to assess the paracellular permeability of cell lines. Confocal laser scanning microscopy (CLSM) was performed to obtain images of the distribution of CF in Caco-2 cells. These compounds were able to loosen tight junctions, thus increasing paracellular permeability. CLSM confirmed the effect of these absorption enhancers on CF transport across Caco-2 lines and increased the Caco-2 permeability via transcellular route. It was also confirmed that the decrease in TEER was transient and reversible after removal of permeation enhancers. PMID:27429925

  1. Cobalt chloride compromises transepithelial barrier properties of CaCo-2 BBe human gastrointestinal epithelial cell layers.

    Science.gov (United States)

    DiGuilio, K M; Valenzano, M C; Rybakovsky, E; Mullin, J M

    2018-01-05

    Elevation of the transcription factor HIF-1 is a prominent mediator of not only processes that accompany hypoxia, but also the tumor microenvironment and tissue regeneration. This study uses mediators of "chemical hypoxia" to ask the question whether HIF-1α elevation in a healthy epithelial cell layer leads to leakiness in its tight junctional seals. Transepithelial electrical resistance and transepithelial diffusion of 14 C-D-mannitol and other radiolabeled probes are used as indicators of transepithelial barrier function of CaCo-2 BBe human gastrointestinal epithelial cell layers cultured on permeable supports. Western immunoblot analyses of integral tight junctional proteins (occludin and claudins) are used as further indicators of barrier function change. Cobalt, an inhibitor of the prolyl hydroxylase enzymes governing HIF-1α breakdown in the cell, induces transepithelial leakiness in CaCo-2 BBe cell layers in a time and concentration-dependent manner. This increased leakiness is accompanied by significant changes in certain specific integral tight junctional (TJ) proteins such as a decreased level of occludin and increased level of claudin-5. Similar results regarding barrier function compromise also occur with other chemical inhibitors of HIF-1α breakdown, namely ciclopiroxolamine (CPX) and dimethyloxalylglycine (DMOG). The increased leak is manifested by both decreased transepithelial electrical resistance (R t ) and increased paracellular diffusion of D-mannitol (J m ). The induced transepithelial leak shows significant size selectivity, consistent with induced effects on TJ permeability. Less-differentiated cell layers were significantly more affected than well-differentiated cell layers regarding induced transepithelial leak. A genetically modified CaCo-2 variant with reduced levels of HIF-1β, showed reduced transepithelial leak in response to cobalt exposure, further indicating that elevation of HIF-1α levels induced by agents of "chemical hypoxia

  2. Modification of the cellular antioxidant activity (CAA) assay to study phenolic antioxidants in a Caco-2 cell line.

    Science.gov (United States)

    Kellett, Mary E; Greenspan, Phillip; Pegg, Ronald B

    2018-04-01

    In vitro assays are widely used to analyze the antioxidant potential of compounds, but they cannot accurately predict antioxidant behavior in living systems. Cell-based assays, like the cellular antioxidant activity (CAA) assay, are gaining importance as they provide a biological perspective. When the CAA assay was employed to study phenolic antioxidants using hepatocarcinoma (HepG2) cells, quercetin showed antioxidant activity in HepG2 cells; 25 and 250μM quercetin reduced fluorescence by 17.1±0.9% and 58.6±2.4%, respectively. (+)-Catechin, a phenolic antioxidant present in many foods, bestowed virtually no CAA in HepG2 cells. When Caco-2 cells were employed, more robust antioxidant activity was observed; 50μM (+)-catechin and quercetin reduced fluorescence by 54.1±1.4% and 63.6±0.9%, respectively. Based on these results, likely due to differences in active membrane transport between the cell types, the Caco-2-based CAA assay appears to be a more appropriate method for the study of certain dietary phenolics. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Improvement of paracellular transport in the Caco-2 drug screening model using protein-engineered substrates.

    Science.gov (United States)

    DiMarco, Rebecca L; Hunt, Daniel R; Dewi, Ruby E; Heilshorn, Sarah C

    2017-06-01

    The Caco-2 assay has achieved wide popularity among pharmaceutical companies in the past two decades as an in vitro method for estimation of in vivo oral bioavailability of pharmaceutical compounds during preclinical characterization. Despite its popularity, this assay suffers from a severe underprediction of the transport of drugs which are absorbed paracellularly, that is, which pass through the cell-cell tight junctions of the absorptive cells of the small intestine. Here, we propose that simply replacing the collagen I matrix employed in the standard Caco-2 assay with an engineered matrix, we can control cell morphology and hence regulate the cell-cell junctions that dictate paracellular transport. Specifically, we use a biomimetic engineered extracellular matrix (eECM) that contains modular protein domains derived from two ECM proteins found in the small intestine, fibronectin and elastin. This eECM allows us to independently tune the density of cell-adhesive RGD ligands presented to Caco-2 cells as well as the mechanical stiffness of the eECM. We observe that lower amounts of RGD ligand presentation as well as decreased matrix stiffness results in Caco-2 morphologies that more closely resemble primary small intestinal epithelial cells than Caco-2 cells cultured on collagen. Additionally, these matrices result in Caco-2 monolayers with decreased recruitment of actin to the apical junctional complex and increased expression of claudin-2, a tight junction protein associated with higher paracellular permeability that is highly expressed throughout the small intestine. Consistent with these morphological differences, drugs known to be paracellularly transported in vivo exhibited significantly improved transport rates in this modified Caco-2 model. As expected, permeability of transcellularly transported drugs remained unaffected. Thus, we have demonstrated a method of improving the physiological accuracy of the Caco-2 assay that could be readily adopted by

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

    Directory of Open Access Journals (Sweden)

    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.

  5. Motility, biofilm formation, apoptotic effect and virulence gene expression of atypical Salmonella Typhimurium outside and inside Caco-2 cells.

    Science.gov (United States)

    Chakroun, Ibtissem; Mahdhi, Abdelkarim; Morcillo, Patricia; Cordero, Hector; Cuesta, Alberto; Bakhrouf, Amina; Mahdouani, Kacem; Esteban, Maria Ángeles

    2018-01-01

    Disease outbreaks related to waterborne pathogen contamination throughout the world as well as challenges that lie ahead for addressing persistent infection are of renewed interest. In this research, we studied the effects of prolonged exposure of Salmonella enterica serovar Typhimurium to the cues encountered in the extracellular environment particularly in seawater microcosm on bacterial virulence and subsequent infection in Caco-2 cells. Our data show a significant difference in biofilm formation, swimming and swarming motilities between normal and stressed cells of S. Typhimurium under differing NaCl conditions (P Caco-2 epithelial cells were determined during infection with normal and stressed Salmonella. Furthermore, we compared the expression of SPI-1 virulence genes (sopA, sopB, sopD, sopE2 and hilA) of normal and stressed S. Typhimurium in response to salt conditions encountered in the extracellular environment in LB broth and after epithelial cell exposure. The interest of the present study is due to the fact that to investigate the bacterial survival strategies during its movement from the natural surroundings to the host cell is fundamental to our understanding of the infection process during the host-pathogen interactions. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Protective Effects of Bifidobacterium on Intestinal Barrier Function in LPS-Induced Enterocyte Barrier Injury of Caco-2 Monolayers and in a Rat NEC Model.

    Directory of Open Access Journals (Sweden)

    Xiang Ling

    Full Text Available Zonulin protein is a newly discovered modulator which modulates the permeability of the intestinal epithelial barrier by disassembling intercellular tight junctions (TJ. Disruption of TJ is associated with neonatal necrotizing enterocolitis (NEC. It has been shown bifidobacterium could protect the intestinal barrier function and prophylactical administration of bifidobacterium has beneficial effects in NEC patients and animals. However, it is still unknown whether the zonulin is involved in the gut barrier dysfunction of NEC, and the protective mechanisms of bifidobacterium on intestinal barrier function are also not well understood. The present study aims to investigate the effects of bifidobacterium on intestinal barrier function, zonulin regulation, and TJ integrity both in LPS-induced enterocyte barrier injury of Caco-2 monolayers and in a rat NEC model. Our results showed bifidobacterium markedly attenuated the decrease in transepithelial electrical resistance and the increase in paracellular permeability in the Caco-2 monolayers treated with LPS (P < 0.01. Compared with the LPS group, bifidobacterium significantly decreased the production of IL-6 and TNF-α (P < 0.01 and suppressed zonulin release (P < 0.05. In addition, bifidobacterium pretreatment up-regulated occludin, claudin-3 and ZO-1 expression (P < 0.01 and also preserved these proteins localization at TJ compared with the LPS group. In the in vivo study, bifidobacterium decreased the incidence of NEC from 88 to 47% (P < 0.05 and reduced the severity in the NEC model. Increased levels of IL-6 and TNF-α in the ileum of NEC rats were normalized in bifidobacterium treated rats (P < 0.05. Moreover, administration of bifidobacterium attenuated the increase in intestinal permeability (P < 0.01, decreased the levels of serum zonulin (P < 0.05, normalized the expression and localization of TJ proteins in the ileum compared with animals with NEC. We concluded that bifidobacterium may

  7. Coffee reduces KRAS expression in Caco-2 human colon carcinoma cells via regulation of miRNAs.

    Science.gov (United States)

    Nakayama, Takuya; Funakoshi-Tago, Megumi; Tamura, Hiroomi

    2017-07-01

    Previous epidemiological studies have demonstrated that moderate coffee consumption is associated with a lower risk of certain types of cancer, particularly colon cancer. To elucidate the molecular basis for this protective action, the effect of coffee on Caco-2 human colon carcinoma cells was investigated. Low concentrations of coffee (Caco-2 cells without affecting cell viability. Coffee also reduced KRAS proto-oncogene, GTPase (KRAS) gene expression in a dose-dependent manner; however, caffeine, caffeic acid and chlorogenic acid, three major constituents of coffee, did not exhibit this effect. Increasing the duration of coffee bean roasting increased the reduction in KRAS expression, suggesting that the active constituents responsible for this effect emerged during the roasting process. MicroRNA (miR) analysis revealed that coffee induced the expression of miR-30c and miR-96, both of which target the KRAS gene. The results of the present study suggested that daily coffee consumption may reduce KRAS activity, thereby preventing the malignant growth of colon carcinoma cells.

  8. Molecular characterization of gluten hydrolysing Bacillus sp. and their efficacy and biotherapeutic potential as probiotics using Caco-2 cell line.

    Science.gov (United States)

    Rashmi, B S; Gayathri, D

    2017-09-01

    To isolate and characterize indigenous gluten hydrolysing bacteria from wheat sourdough and curd samples and further evaluation of their probiotic potentiality. Indigenous gluten hydrolysing isolates GS 33, GS 143, GS 181 and GS 188 were identified as Bacillus sp. by molecular-typing methods and studied extensively for their functional and probiotic attributes. All the tested isolates could survive at pH 2 and toxicity of 0·3% bile and also exhibited cell surface hydrophobicity and autoaggregation phenotype. The isolates were adhered strongly to Caco-2 cells and coaggregated with Escherichia coli MTCC 433 and Listeria monocytogenes MTCC 1143 preventing pathogen invasion into Caco-2 cells in vitro. In addition, the minimum inhibitory concentration of selected antibiotics for all the investigated gluten hydrolysing isolates was within the breakpoint values as recommended by the European Food Safety Authority. The indigenous high intensity gluten hydrolysing bacteria exhibited high resistance to gastric and pancreatic stress and possessed antibacterial, aggregation, adhesion and pathogen exclusion properties, and as a potential probiotics, either alone or in consortium would be useful in the development of gluten-free wheat foods. Exploring new indigenous gluten hydrolysing bacteria from wheat sourdough and curd samples would be beneficial in developing gluten-free wheat foods using potential indigenous probiotics. © 2017 The Society for Applied Microbiology.

  9. In Vitro Intrinsic Permeability: A Transporter-Independent Measure of Caco-2 Cell Permeability in Drug Design and Development.

    Science.gov (United States)

    Fredlund, Linda; Winiwarter, Susanne; Hilgendorf, Constanze

    2017-05-01

    In vitro permeability data have a central place in absorption risk assessments in drug discovery and development. For compounds where active efflux impacts permeability in vitro, the inherent passive membrane permeability ("intrinsic permeability") gives a concentration-independent measure of the compound's permeability. This work describes the validation of an in vitro intrinsic permeability assay and application of the data in a predictive in silico model. Apparent intrinsic permeability (P app ) across Caco-2 cell monolayers is determined in the presence of an optimized cocktail of chemical inhibitors toward the three major efflux transporters ABCB1, ABCC2, and ABCG2. The intrinsic P app value gives an estimate of passive permeability, which is independent of transporter expression levels and not limited by solubility or cell toxicity. An in silico model has been established to predict the Caco-2 intrinsic permeability and shown to consistently identify highly permeable compounds. The new intrinsic permeability assay is useful for early absorption estimates and suitable for absorption risk assessment in DMPK and pharmaceutical development.

  10. 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.

  11. EPA blocks TNF-α-induced inhibition of sugar uptake in Caco-2 cells via GPR120 and AMPK.

    Science.gov (United States)

    Castilla-Madrigal, Rosa; Barrenetxe, Jaione; Moreno-Aliaga, María J; Lostao, María Pilar

    2018-03-01

    The aim of the present work was to investigate in Caco-2 cells whether eicosapentaenoic acid (EPA), an omega-3 polyunsaturated fatty acid, could block the inhibitory effect of tumor necrosis factor-α (TNF-α) on sugar transport, and identify the intracellular signaling pathways involved. After pre-incubation of the Caco-2 cells with TNF-α and EPA for 1 hr, EPA prevented the inhibitory effect of the cytokine on α-methyl-d-glucose (αMG) uptake (15 min) and on SGLT1 expression at the brush border membrane, measured by Western blot. The ERK1/2 inhibitor PD98059 and the AMPK activator AICAR also prevented the inhibitory effect of TNF-α on both αMG uptake and SGLT1 expression. Interestingly, the AMPK inhibitor, Compound C, abolished the ability of EPA to prevent TNF-α-induced reduction of sugar uptake and transporter expression. The GPR120 antagonist, AH7614, also blocked the preventive effect of EPA on TNF-α-induced decrease of αMG uptake and AMPK phosphorylation. In summary, TNF-α inhibits αMG uptake by decreasing SGLT1 expression in the brush border membrane through the activation of ERK1/2 pathway. EPA prevents the inhibitory effect of TNF-α through the involvement of GPR120 and AMPK activation. © 2017 Wiley Periodicals, Inc.

  12. The effects of probiotic Bacillus subtilis on the cytotoxicity of Clostridium perfringens type a in Caco-2 cell culture.

    Science.gov (United States)

    Poormontaseri, Maryam; Hosseinzadeh, Saeid; Shekarforoush, Seyed Shahram; Kalantari, Tahereh

    2017-07-04

    Some Bacillus strains have recently been identified for potential use as probiotics and food additives. The present study evaluated the antimicrobial effects of Bacillus subtilis ATCC 6633 and its metabolite on the enterotoxin and vegetative cells, spore and germinated spore of Clostridium perfringens type A in Caco-2 cells. We used flow cytometry and MTT assays to evaluate the cytotoxicity effect of treatments. According to the results, the most cell survival was found in the 4% crude antimicrobial substance (CAS) with the vegetative form of C. perfringens among co-cultured groups. Furthermore, the apoptosis and necrosis in co-cultured groups were significantly decreased (P < 0.05). The present results suggested the crucial role of the current probiotic in the control of various forms of C. perfringens type A which was investigated for the first time. Also, the majority of treatments showed higher cell viability in flow cytometry compared to the MTT assay.

  13. Pectin of Prunus domestica L. alters sulfated structure of cell-surface heparan sulfate in differentiated Caco-2 cells through stimulation of heparan sulfate 6-O-endosulfatase-2.

    Science.gov (United States)

    Nishida, Mitsutaka; Murata, Kazuma; Kanamaru, Yoshihiro; Yabe, Tomio

    2014-01-01

    Although previous reports have suggested that pectin induces morphological changes of the small intestine in vivo, the molecular mechanisms have not been elucidated. As heparan sulfate plays important roles in development of the small intestine, to verify the involvement of heparan sulfate (HS) in the pectin-induced morphological changes of the small intestine, the effects of pectin from Prunus domestica L. on cell-surface HS were investigated using differentiated Caco-2 cells. Disaccharide compositional analysis revealed that sulfated structures of HS were markedly changed by pectin administration. Real-time RT-PCR showed that pectin upregulated human HS 6-O-endosulfatase-2 (HSulf-2) expression and markedly inhibited HSulf-1 expression. Furthermore, inhibition analysis suggested that pretreatment with fibronectin III1C fragment, RGD peptide, and ERK1/2 inhibitor suppressed pectin-induced HSulf-2 expression. These observations indicate that pectin induced the expression of HSulf-2 through the interaction with fibronectin, α5β1 integrin, and ERK1/2, thereby regulating the sulfated structure of HS on differentiated Caco-2 cells.

  14. Apple extract induces increased epithelial resistance and claudin 4 expression in Caco-2 cells

    NARCIS (Netherlands)

    Vreeburg, R.A.M.; Wezel, van E.E.; Ocana-Calahorro, F.; Mes, J.J.

    2012-01-01

    BACKGROUND: The small intestinal epithelium functions both to absorb nutrients, and to provide a barrier between the outside, luminal, world and the human body. One of the passageways across the intestinal epithelium is paracellular diffusion, which is controlled by the properties of tight junction

  15. Chicken thigh, chicken liver, and iron-fortified wheat flour increase iron uptake in an in vitro digestion/Caco-2 cell model.

    Science.gov (United States)

    Pachón, Helena; Stoltzfus, Rebecca J; Glahn, Raymond P

    2008-12-01

    The objective of this study was to test meat and fortified-food combinations to identify those that optimize iron uptake in an in vitro digestion/Caco-2 cell model, a proxy for iron bioavailability. Four experiments tested combinations of meats such as chicken (blood, spleen, liver, thigh), beef (cube steak), and fish (whole-fish meal) with iron-fortified foods (rice cereal, maize-soy flour, wheat flour). Chicken liver, thigh, spleen, blood, or fish meal increased the Caco-2 cell iron uptake from these combined with rice cereal (Pflour (Pfortified foods to these meats did not increase the Caco-2 cell iron uptake (P >or= .05). Adding either meat to the 3 fortified foods increased the Caco-2 cell iron uptake of the fortified foods (Pflour were selected for an infant porridge because the combinations with the highest Caco-2 cell iron uptake were chicken thigh + wheat flour, chicken liver + wheat flour, and chicken liver + maize-soy flour, and wheat flour was the least expensive fortified food sold in the target population. Per unit of iron, the chicken thigh + wheat flour and chicken liver + wheat flour combinations resulted in the highest bioavailable iron. In the proportion of 3:1 fortified food:meat examined, meat increases the bioavailability of iron-fortified foods, but iron-fortified foods do not enhance total iron bioavailability when added to meat.

  16. Permeability through the Caco-2 cell monolayer of 42 bioactive compounds in the TCM formula Gegen-Qinlian Decoction by liquid chromatography tandem mass spectrometry analysis.

    Science.gov (United States)

    Wang, Qi; Kuang, Yi; Song, Wei; Qian, Yi; Qiao, Xue; Guo, De-An; Ye, Min

    2017-11-30

    Caco-2 cell monolayer model was used to evaluate the intestinal permeability of 42 bioactive compounds in the famous traditional Chinese medicine (TCM) formula Gegen-Qinlian Decoction (GQD). These compounds include alkaloids, flavonoids and glycosides, triterpenoid saponins, and coumarins. Their transportations across the cell monolayers in the forms of herb extract and formula extract were monitored by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) analysis. Most alkaloids from Huang-Lian; flavonoid C-glycosides from Ge-Gen and Huang-Qin; O-glycosides from Ge-Gen, Huang-Qin and Gan-Cao; O-glucuronides from Huang-Qin; and coumarins from Gan-Cao exhibited favorable permeability. Their P AB values were >1.05×10 -5 cm/s, and efflux ratios (ER, P BA /P AB ) were≤1.0. In contrast, triterpenoid saponins showed poor permeability (P AB ≤1.50×10 -6 cm/s, ER≤1.5), indicating a paracellular diffusion mechanism. Furthermore, GQD could remarkably improve the intestinal transport of alkaloids in Huang-Lian, flavonoid C-glycosides in Ge-Gen, as well as coumarins and flavonoid O-glycosides in Gan-Cao. These results indicate herb-herb interactions in GQD. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Effect of Passage Number and Culture Time on the Expression and Activity of Insulin-Degrading Enzyme in Caco-2 Cells

    Science.gov (United States)

    Mohammadi Farsani, Taiebeh; Motevaseli, Elahe; Neyazi, Nadia; Khorramizadeh, Mohammad Reza; Zafarvahedian, Elaheh; Ghahremani, Mohammad Hossein

    2018-01-01

    Insulin-degrading enzyme (IDE) is a conserved zinc metallopeptidase. Here, we have evaluated the effect of passage number and culture time on IDE expression and activity in colorectal adenocarcinoma cell line (Caco-2). Caco-2 cells were cultured with different passage ranges of 5-15, 25-35, 52-63 for 48, 72, and 120 hours. Subsequently, IDE expression and enzyme activity were assessed by Western blot analysis and fluorescent assay, respectively. Our results confirmed that the amount of IDE was higher in cell extract compared to supernatant, and different passage numbers and culture times had small effect on IDE expression. However, when cells were cultured in the passage number range of 5-15 for 72 hours, the IDE activity was 35% higher compared to other passage numbers (p Caco-2 cells at passage number range of 5-15 and culture time of 72 hours provides proper conditions for IDE-related studies.

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

    Directory of Open Access Journals (Sweden)

    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.

  19. Inhibitory effect of phospholipids on P-glycoprotein: cellular studies in Caco-2, MDCKII mdr1 and MDCKII wildtype cells and P-gp ATPase activity measurements.

    Science.gov (United States)

    Simon, S; Schubert, R

    2012-09-01

    Phospholipids are widely used excipients for pharmaceutical formulations, such as for preparing biphasic systems or to solubilize or encapsulate poorly soluble drugs. The present study investigates a new property of this class of substance: its ability to inhibit the efflux transporter Pglycoprotein (P-gp). P-gp is expressed in the intestinal epithelium, thereby significantly impairing the systemic absorption of various pharmaceutically active substances. The phospholipid screening performed in this study involved derivatives with different headgroups and fatty acid residues and a number of experimental parameters. For in vitro studies we carried out transport experiments and calcein accumulation assays in Caco-2- and MDCKII mdr1 and wildtype cell lines. The three compounds which displayed significant P-gp inhibition in both assays and in Caco-2 as well as in MDCKII mdr1, consisted of phosphatidylcholine (PC) and either two saturated fatty acid residues of eight (8:0 PC) or ten carbon atoms (10:0 PC), or of two unsaturated docosahexaeonic acid residues (cis-22:6 PC).Supported by P-gp ATPase activity measurements, 8:0 and 10:0 PC were assumed to function as direct P-gp inhibitors interacting with the transporter probably in their monomeric state, whereas a different, as yet unknown mechanism of action applied for cis-22:6 PC.Because of their proven ability to significantly inhibit P-gp in vitro, these phospholipids shall further be elucidated in vivo, whether they may truly serve to increase the bioavailability of orally applied drugs with a P-gp substrate character. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. Alginate inhibits iron absorption from ferrous gluconate in a randomized controlled trial and reduces iron uptake into Caco-2 cells.

    Science.gov (United States)

    Wawer, Anna A; Harvey, Linda J; Dainty, Jack R; Perez-Moral, Natalia; Sharp, Paul; Fairweather-Tait, Susan J

    2014-01-01

    Previous in vitro results indicated that alginate beads might be a useful vehicle for food iron fortification. A human study was undertaken to test the hypothesis that alginate enhances iron absorption. A randomised, single blinded, cross-over trial was carried out in which iron absorption was measured from serum iron appearance after a test meal. Overnight-fasted volunteers (n = 15) were given a test meal of 200 g cola-flavoured jelly plus 21 mg iron as ferrous gluconate, either in alginate beads mixed into the jelly or in a capsule. Iron absorption was lower from the alginate beads than from ferrous gluconate (8.5% and 12.6% respectively, p = 0.003). Sub-group B (n = 9) consumed the test meals together with 600 mg calcium to determine whether alginate modified the inhibitory effect of calcium. Calcium reduced iron absorption from ferrous gluconate by 51%, from 11.5% to 5.6% (p = 0.014), and from alginate beads by 37%, from 8.3% to 5.2% (p = 0.009). In vitro studies using Caco-2 cells were designed to explore the reasons for the difference between the previous in vitro findings and the human study; confirmed the inhibitory effect of alginate. Beads similar to those used in the human study were subjected to simulated gastrointestinal digestion, with and without cola jelly, and the digestate applied to Caco-2 cells. Both alginate and cola jelly significantly reduced iron uptake into the cells, by 34% (p = 0.009) and 35% (p = 0.003) respectively. The combination of cola jelly and calcium produced a very low ferritin response, 16.5% (p iron for the purpose of food fortification. ClinicalTrials.gov NCT01528644.

  1. Alginate inhibits iron absorption from ferrous gluconate in a randomized controlled trial and reduces iron uptake into Caco-2 cells.

    Directory of Open Access Journals (Sweden)

    Anna A Wawer

    Full Text Available Previous in vitro results indicated that alginate beads might be a useful vehicle for food iron fortification. A human study was undertaken to test the hypothesis that alginate enhances iron absorption. A randomised, single blinded, cross-over trial was carried out in which iron absorption was measured from serum iron appearance after a test meal. Overnight-fasted volunteers (n = 15 were given a test meal of 200 g cola-flavoured jelly plus 21 mg iron as ferrous gluconate, either in alginate beads mixed into the jelly or in a capsule. Iron absorption was lower from the alginate beads than from ferrous gluconate (8.5% and 12.6% respectively, p = 0.003. Sub-group B (n = 9 consumed the test meals together with 600 mg calcium to determine whether alginate modified the inhibitory effect of calcium. Calcium reduced iron absorption from ferrous gluconate by 51%, from 11.5% to 5.6% (p = 0.014, and from alginate beads by 37%, from 8.3% to 5.2% (p = 0.009. In vitro studies using Caco-2 cells were designed to explore the reasons for the difference between the previous in vitro findings and the human study; confirmed the inhibitory effect of alginate. Beads similar to those used in the human study were subjected to simulated gastrointestinal digestion, with and without cola jelly, and the digestate applied to Caco-2 cells. Both alginate and cola jelly significantly reduced iron uptake into the cells, by 34% (p = 0.009 and 35% (p = 0.003 respectively. The combination of cola jelly and calcium produced a very low ferritin response, 16.5% (p < 0.001 of that observed with ferrous gluconate alone. The results of these studies demonstrate that alginate beads are not a useful delivery system for soluble salts of iron for the purpose of food fortification.ClinicalTrials.gov NCT01528644.

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

    Directory of Open Access Journals (Sweden)

    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.

  3. 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.

  4. Estrone-1-sulphate (E1S) has impact on the kinetics parameters of transporter mediated taurine and glutamate influx in Caco-2 cells

    DEFF Research Database (Denmark)

    Steffansen, Bente; El-Sayed, F

    Previously, we have suggested estrone-1-sulfate (E1S) to be intercalated into the phospholipid membrane 1,2-dipalmitoyl-sn-glycero-3-phospho-choline (DPPC). The overall hypothesis of the present study was that E1S intercalation in the cell membrane of Caco-2 cells may changes the functionality...... of membrane transporters. The aim was therefore to investigate if addition of E1S to the growth medium of Caco-2 cells before but not during the influx study, change the kinetic parameters of transporter-mediated influx of taurine and glutamate by respective TAUT and EAAT transporters. The results show that 4...

  5. Physicochemical characterization of mineral (iron/zinc) bound caseinate and their mineral uptake in Caco-2 cells.

    Science.gov (United States)

    Shilpashree, B G; Arora, Sumit; Kapila, Suman; Sharma, Vivek

    2018-08-15

    Milk proteins (especially caseins) are widely accepted as good vehicle for the delivery of various bioactive compounds including minerals. Succinylation is one of the most acceptable chemical modification techniques to enhance the mineral binding ability of caseins. Addition of minerals to succinylated proteins may alter their physicochemical and biochemical properties. Physicochemical characteristics of succinylated sodium caseinate (S.NaCN)-mineral (iron/zinc) complexes were elucidated. Chromatographic behaviour and fluorescence intensity confirmed the structural modification of S.NaCN upon binding with minerals. The bound mineral from protein complexes showed significantly higher (P < 0.05) in vitro bioavailability (mineral uptake) than mineral salts in Caco-2 cells. Also, iron bound S.NaCN showed higher cellular ferritin formation than iron in its free form. These mineral bound protein complexes with improved bioavailability could safely replace inorganic fortificants in various functional food formulations. Copyright © 2018 Elsevier Ltd. All rights reserved.

  6. Evasin-displaying lactic acid bacteria bind different chemokines and neutralize CXCL8 production in Caco-2 cells.

    Science.gov (United States)

    Škrlec, Katja; Pucer Janež, Anja; Rogelj, Boris; Štrukelj, Borut; Berlec, Aleš

    2017-11-01

    Chemokines are key signals in the immune system and play an important role as proinflammatory mediators in the pathology of inflammatory bowel disease and colorectal cancer, making them an important target for therapy. Recombinant lactic acid bacteria (LAB) were engineered to bind CC and CXC chemokines by displaying chemokine-binding proteins evasin-1, evasin-3 and evasin-4 on their surface. Evasin genes were cloned into lactococcal surface display vector and overexpressed in L. lactis NZ9000 and NZ9000ΔhtrA in fusion with secretion signal and surface anchor. Evasin-displaying bacteria removed from 15% to 90% of 11 different chemokines from the solution as determined with ELISA and Luminex multiplexing assays, whereby L. lactis NZ9000ΔhtrA proved more efficient. Lactobacillus salivarius ATCC 11741 was coated with L. . lactis-expressed evasin fusion protein, and its ability to bind chemokines was also confirmed. Evasin-3-displaying L. lactis removed 76.0% of IL-1β-induced CXCL8 from the supernatant of Caco-2 epithelial cells. It also prevented secretion of CXCL8 from Caco-2 cells in a time-dependent manner when added before induction with IL-1β. Evasin-displaying LAB have the ability to bind multiple chemokines simultaneously and exert synergistic activity. This innovative treatment approach therefore has the potential for mucosal therapy of inflammatory bowel disease or colorectal cancer. © 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  7. 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.

  8. Molecular Characterization, Antimicrobial Resistance and Caco-2 Cell Invasion Potential of Campylobacter jejuni/coli from Young Children with Diarrhea.

    Science.gov (United States)

    Pan, Haijian; Ge, Yanling; Xu, Hao; Zhang, Jianmin; Kuang, Dai; Yang, Xiaowei; Su, Xudong; Huang, Zheng; Shi, Xianming; Xu, Xuebin; Meng, Jianghong

    2016-03-01

    Campylobacter is a major cause of bacterial gastroenteritis worldwide. Young children represent a particular age group affected by Campylobacter infection because of their limited diets and weak immune systems. In this study, a total of 110 Campylobacter (80 Campylobacter jejuni and 30 Campylobacter coli) isolated from children younger than 5 years of age with diarrhea in Shanghai, China in 2011 were examined for their genetic relationship and antimicrobial susceptibility. The presence of virulence genes and its association with invasion potential in Caco-2 cell were also determined. Multilocus sequence typing revealed 62 sequence types (STs) under 14 clonal complexes from C. jejuni and 15 STs under 2 clonal complexes from C. coli. High resistance rates among the 110 isolates were observed to nalidixic acid (88.2%), ciprofloxacin (87.3%) and tetracycline (87.3%), followed by ampicillin (30.9%), gentamicin (28.2%), clindamycin (21.8%), erythromycin (21.8%) and chloramphenicol (8.2%). Compared with that of C. jejuni (32.5%), a larger proportion of C. coli (83.3%) were resistant to multiple antimicrobials, including 16 isolates of ST-828 complex resistant to 6 antimicrobials: ciprofloxacin, clindamycin, erythromycin, gentamicin, nalidixic acid and tetracycline. Furthermore, 57 Campylobacter isolates were selected based on their distinct STs and the presence of virulence genes to determine their abilities to adhere to and invade Caco-2 cells. The level of invasion varied widely among isolates and had relatively weak correlation with the genotype data. Our findings provided baseline data on Campylobacter among young children. Active surveillance of Campylobacter is needed to better understand the epidemiology and antimicrobial resistance trends of this significant pathogen to help control and protect young children from such infections.

  9. Investigation of the effects of soluble fibers on the absorption of resveratrol and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PHIP) in the Caco-2 cellular model of intestinal absorption.

    Science.gov (United States)

    Willenberg, Ina; Wonik, Jasmin; Schebb, Nils Helge

    2015-01-01

    Soluble fibers are known to modulate intestinal absorption of non-polar compounds in the small intestine. Little is known about the modulation of absorption of more polar compounds. In the present study, we applied the Caco-2-transwell-system in order to investigate the modulation of intestinal bioavailability by soluble fibers. The system was tested using pectin and carrageenan as model soluble fibers at a concentration of 0.1% (w/v), which did not compromise the integrity of the cell monolayer. Modulation of absorption was evaluated for the heterocyclic amine aromatic 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PHIP) and the polyphenol resveratrol. Neither pectin nor carrageenan reduced the high flux of PHIP, apparent permeability coefficient (Papp) of 16 × 10(-6) cm s(-1). The low Papp of resveratrol was reduced by both soluble fibers, particularly by pectin. These results suggest that the low bioavailability of polyphenols could be further reduced by soluble fibers. Because of their co-occurrence in several fruits, these findings warrant further research.

  10. 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)...

  11. The von Hippel-Lindau (VHL) tumor-suppressor gene is down-regulated by selenium deficiency in Caco-2 cells and rat colon mucosa

    Science.gov (United States)

    To test the hypothesis that selenium affects DNA methylation and hence gene regulation we employed a methylation array (Panomics) in the human colonic epithelial Caco-2 cell model. The array profiles DNA methylation from promoter regions of 82 human genes. After conditioning cells to repeatedly redu...

  12. Dietary Factors Modulate Iron Uptake in Caco-2 Cells from an Iron Ingot Used as a Home Fortificant to Prevent Iron Deficiency

    Directory of Open Access Journals (Sweden)

    Ildefonso Rodriguez-Ramiro

    2017-09-01

    Full Text Available Iron deficiency is a major public health concern and nutritional approaches are required to reduce its prevalence. The aim of this study was to examine the iron bioavailability of a novel home fortificant, the “Lucky Iron Fish™” (LIF (www.luckyironfish.com/shop, Guelph, Canada and the impact of dietary factors and a food matrix on iron uptake from LIF in Caco-2 cells. LIF released a substantial quantity of iron (about 1.2 mM at pH 2 but this iron was only slightly soluble at pH 7 and not taken up by cells. The addition of ascorbic acid (AA maintained the solubility of iron released from LIF (LIF-iron at pH 7 and facilitated iron uptake by the cells in a concentration-dependent manner. In vitro digestion of LIF-iron in the presence of peas increased iron uptake 10-fold. However, the addition of tannic acid to the digestion reduced the cellular iron uptake 7.5-fold. Additionally, LIF-iron induced an overproduction of reactive oxygen species (ROS, similar to ferrous sulfate, but this effect was counteracted by the addition of AA. Overall, our data illustrate the major influence of dietary factors on iron solubility and bioavailability from LIF, and demonstrate that the addition of AA enhances iron uptake and reduces ROS in the intestinal lumen.

  13. 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.

  14. 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.

    2015-04-01

    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.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

  15. In Vitro Influence of Extracts from Snail Helix aspersa Müller on the Colon Cancer Cell Line Caco-2.

    Science.gov (United States)

    Matusiewicz, Magdalena; Kosieradzka, Iwona; Niemiec, Tomasz; Grodzik, Marta; Antushevich, Hanna; Strojny, Barbara; Gołębiewska, Małgorzata

    2018-04-03

    Colorectal cancer is the third most widely diagnosed cancer. Extracts from snails may modulate growth and development of colorectal cancer cells. The objective of this study was to determine the chemical composition of tissues derived from Helix aspersa Müller and red-ox properties of tissue extracts. Then, the influence of extracts and their fractions of different molecular weights on viability of Caco-2 cells was examined. Tissue lyophilisates contained antioxidants that could be important in the prevention of colorectal cancer. Moreover, we confirmed the presence of a wide array of compounds that might be used in treatment of this disease. The decrease of cell viability after the application of extracts from lyophilized mucus and foot tissues was affirmed. The effect of extract from mucus could be related to the content of some proteins and peptides, proper essential amino acids (EAA)/non-essential amino acids (NEAA) ratio, Met restriction and the presence of Cu, Ca, Zn, Se. The influence of the extract from foot tissues could be assigned additionally to the presence of eicosapentaenoic, α-linolenic, linoleic and γ-linolenic acids. The opposite effect was demonstrated by extract from lyophilized shells which increased cell viability. Further studies are needed to know whether dietary supplying of H. aspersa Müller tissues can be used as an approach in colorectal cancer management.

  16. In Vitro Influence of Extracts from Snail Helix aspersa Müller on the Colon Cancer Cell Line Caco-2

    Directory of Open Access Journals (Sweden)

    Magdalena Matusiewicz

    2018-04-01

    Full Text Available Colorectal cancer is the third most widely diagnosed cancer. Extracts from snails may modulate growth and development of colorectal cancer cells. The objective of this study was to determine the chemical composition of tissues derived from Helix aspersa Müller and red-ox properties of tissue extracts. Then, the influence of extracts and their fractions of different molecular weights on viability of Caco-2 cells was examined. Tissue lyophilisates contained antioxidants that could be important in the prevention of colorectal cancer. Moreover, we confirmed the presence of a wide array of compounds that might be used in treatment of this disease. The decrease of cell viability after the application of extracts from lyophilized mucus and foot tissues was affirmed. The effect of extract from mucus could be related to the content of some proteins and peptides, proper essential amino acids (EAA/non-essential amino acids (NEAA ratio, Met restriction and the presence of Cu, Ca, Zn, Se. The influence of the extract from foot tissues could be assigned additionally to the presence of eicosapentaenoic, α-linolenic, linoleic and γ-linolenic acids. The opposite effect was demonstrated by extract from lyophilized shells which increased cell viability. Further studies are needed to know whether dietary supplying of H. aspersa Müller tissues can be used as an approach in colorectal cancer management.

  17. 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...

  18. Iron uptake by Caco-2 cells following in vitro digestion: effects of heat treatments of pork meat and pH of the digests.

    Science.gov (United States)

    Sørensen, Anne D; Bukhave, Klaus

    2010-10-01

    The present in vitro studies report on iron uptake by Caco-2 cells from pepsin and pepsin+pancreatin-digested pork meat proteins at pH values between 4.6 and 7 mimicking conditions in the duodenum and the proximal jejunum, respectively. Heat treatment of the pork meat resulted in increased iron uptake from pepsin-digested samples to Caco-2 cells at pH 4.6. The major enhancing effects on iron uptake by Caco-2 cells were observed after pepsin digestion in the pH range 4.6-6.0, whereas the pepsin+pancreatin-digested samples resulted in negligible iron uptake in Caco-2 cells at pH 7. Thus, the results emphasize the importance of separating pepsin-digested and pepsin+pancreatin-digested proteins during in vitro studies on iron availability. Furthermore, the present results showed the pH dependency of iron uptake anticipated. The enhancing effect of ascorbic acid was verified by increased iron uptake from pepsin-digested pork meat samples at pH 4.6, while no effect of ascorbic acid was observed at pH 7 in pepsin+pancreatin-digested samples. Copyright © 2010 Elsevier GmbH. All rights reserved.

  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...

  20. CFTR-mediated anion secretion across intestinal epithelium-like Caco-2 monolayer under PTH stimulation is dependent on intermediate conductance K+ channels.

    Science.gov (United States)

    Jantarajit, Walailak; Lertsuwan, Kornkamon; Teerapornpuntakit, Jarinthorn; Krishnamra, Nateetip; Charoenphandhu, Narattaphol

    2017-07-01

    Parathyroid hormone (PTH), a pleiotropic hormone that maintains mineral homeostasis, is also essential for controlling pH balance and ion transport across renal and intestinal epithelia. Optimization of luminal pH is important for absorption of trace elements, e.g., calcium and phosphorus. We have previously demonstrated that PTH rapidly stimulated electrogenic [Formula: see text] secretion in intestinal epithelial-like Caco-2 monolayers, but the underlying cellular mechanism, contributions of other ions, particularly Cl - and K + , and long-lasting responses are not completely understood. Herein, PTH and forskolin were confirmed to induce anion secretion, which peaked within 1-3 min (early phase), followed by an abrupt decay and plateau that lasted for 60 min (late phase). In both early and late phases, apical membrane capacitance was increased with a decrease in basolateral capacitance after PTH or forskolin exposure. PTH also induced a transient increase in apical conductance with a long-lasting decrease in basolateral conductance. Anion secretion in both phases was reduced under [Formula: see text]-free and/or Cl - -free conditions or after exposure to carbonic anhydrase inhibitor (acetazolamide), CFTR inhibitor (CFTRinh-172), Na + /H + exchanger (NHE)-3 inhibitor (tenapanor), or K + channel inhibitors (BaCl 2 , clotrimazole, and TRAM-34; basolateral side), the latter of which suggested that PTH action was dependent on basolateral K + recycling. Furthermore, early- and late-phase responses to PTH were diminished by inhibitors of PI3K (wortmannin and LY-294002) and PKA (PKI 14-22). In conclusion, PTH requires NHE3 and basolateral K + channels to induce [Formula: see text] and Cl - secretion, thus explaining how PTH regulated luminal pH balance and pH-dependent absorption of trace minerals. Copyright © 2017 the American Physiological Society.

  1. 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

    2016-01-01

    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. PMID:27827957

  2. Trans-epithelial transport of the betalain pigments indicaxanthin and betanin across Caco-2 cell monolayers and influence of food matrix.

    Science.gov (United States)

    Tesoriere, L; Gentile, C; Angileri, F; Attanzio, A; Tutone, M; Allegra, M; Livrea, M A

    2013-04-01

    This study investigated the absorption mechanism of the phytochemicals indicaxanthin and betanin and the influence of their food matrix (cactus pear and red beet) on the intestinal transport. Trans-epithelial transport of dietary-consistent amounts of indicaxanthin and betanin in Caco-2 cell monolayers seeded on Transwell(R) inserts was measured in apical to basolateral (AP-BL) and basolateral to apical (BL-AP) direction, under an inwardly directed pH gradient (pH 6.0/7.4, AP/BL) mimicking luminal and serosal sides of human intestinal epithelium. The effect of inhibitors of membrane transporters on the absorption was also evaluated. Contribution of the paracellular route was investigated after EDTA treatment of the cell monolayer. In vitro digestion of betalainic food was performed to provide a post-intestinal fraction containing bioaccessible pigments. Apparent permeability coefficients (P(app)) in the absorptive direction were (4.4 ± 0.4) × 10⁻⁶ and (3.2 ± 0.3) × 10⁻⁶ cm s⁻¹ for indicaxanthin and betanin, respectively. Transport of indicaxanthin was non-polarized, linear as a function of time and concentration, and unaffected by inhibitors of membrane transporters. Betanin exhibited significantly different bidirectional P(app) values and non-linear efflux kinetics. The concentration-dependent betanin efflux was described by a kinetic model including one non-saturable (K(d) = 0.042 μL cm⁻² min⁻¹) and one saturable component identified as the apical multidrug resistance-associated protein 2 (MRP2; K(m) = 275 μM; J(max) = 42 pmol min⁻¹ cm⁻²). Permeation of both betalains increased remarkably after EDTA treatment of the cell monolayer. Neither indicaxanthin nor betanin underwent metabolic transformation. Food matrix did not affect trans-epithelial transfer of indicaxanthin, but reduced the absorption rate of betanin, red beet more than cactus pear. Dietary indicaxanthin and betanin can substantially be absorbed through paracellular

  3. Antimicrobial activities of therapeutic herbal plants against Listeria monocytogenes and the herbal plant cytotoxicity on Caco-2 cell.

    Science.gov (United States)

    Yoon, Y; Choi, K-H

    2012-07-01

    This study investigated the antimicrobial effect of various therapeutic herbal plants on Listeria monocytogenes, and their cytotoxicity effect on mammalian cells. The extracts from 69 therapeutic herbal plants were used to investigate the effect on the growth inhibition of L. monocytogenes, and their minimal inhibition concentrations and minimal bactericidal concentrations were determined. Among the plants, Psoraleae semen L. (Bogolji) and Sophorae radix L. (Gosam) extracts, which showed obvious antilisterial activity, were examined for the stability to heat, NaCl and acidic condition. Moreover, cytotoxicities of Bogolji and Gosam were tested, using Caco-2 cells. L. monocytogenes growth was completely inhibited by Bogolji and Gosam extracts at 3.2-6.3 and 50-100 AU ml(-1), respectively, and heat, NaCl and acidic condition did not affect the antilisterial activity of Bogolji and Gosam. Cytotoxic activities were observed only at high concentration (50 AU ml(-1)) of Bogolji extract. Bogolji and Gosam could be considered as potential phytochemicals to control L. monocytogenes. Use of therapeutic herbal plants should be useful in controlling L. monocytogenes, because most consumers have better acceptance for phytochemicals than synthetic chemicals. © 2012 The Authors. Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.

  4. MPA Modulates Tight Junctions' Permeability via Midkine/PI3K Pathway in Caco-2 Cells: A Possible Mechanism of Leak-Flux Diarrhea in Organ Transplanted Patients

    Science.gov (United States)

    Khan, Niamat; Binder, Lutz; Pantakani, D. V. Krishna; Asif, Abdul R.

    2017-01-01

    Mycophenolic acid (MPA) is prescribed to prevent allograft rejection in organ transplanted patients. However, its use is sporadically linked to leak flux diarrhea and other gastrointestinal (GI) disturbances in around 75% of patients through yet unknown mechanisms. Recently, we identified Midkine as a modulator of tight junctions (TJs) permeability in MPA treated Caco-2 monolayer. In the present study, we investigated the possible involvement of Midkine dependent PI3K pathway in alteration of TJs under MPA treatment. Caco-2 cells were grown as monolayer to develop TJs and were treated for 72 h with DMSO (control) or MPA in presence and absence of Midkine inhibitor (iMDK) or PI3K inhibitors (LY/AMG). Caco-2 monolayer integrity was assessed by transepithelial electrical resistance (TEER) and FITC-dextran assays. Our functional assays showed that PI3K inhibitors (LY/AMG) can significantly inhibit the compromised TJs integrity of MPA-treated Caco-2 cells monolayer. Chromatin immunoprecipitation analyses showed a significant epigenetic activation of Midkine, PI3K, Cdx-2, and Cldn-2 genes and epigenetic repression of Cldn-1 gene after MPA treatment. The MPA-induced epigenetic alterations were further confirmed by mRNA and protein expression analysis. Collectively, our data shows that PI3K pathway as the downstream target of Midkine which in turn modulates p38MAPK and pAKT signaling to alter TJs permeability in Caco-2 cell monolayers treated with MPA. These results highlight the possible use of either Midkine or PI3K inhibitors as therapeutic agents to prevent MPA induced GI disturbances. PMID:28694783

  5. Differential protein expression of Caco-2 cells treated with selenium nanoparticles compared with sodium selenite and selenomethionine

    Science.gov (United States)

    Fu, Linglin; Yan, Xuxia; Ruan, Xinming; Lin, Junda; Wang, Yanbo

    2014-10-01

    The study was designed to determine the differential protein expression of Caco-2 cells treated with different forms of selenium including sodium selenite, selenomethionine (Se-Met), and selenium nanoparticles (nano-Se). Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS) were used to identify the differentially expressed proteins. The results indicated that seven protein spots, ubiquitin-conjugating enzyme E2 (E2), glutathione synthetases (GS), triosephosphate isomerase (TSP), T-complex protein 1 subunit zeta (TCPZ), lamin-B1, heterogeneous nuclear ribonucleoprotein F (hnRNP F), and superoxide dismutase [Cu-Zn] (Cu, Zn-SOD) were significantly different among all the groups. According to the order of control, sodium selenite, Se-Met, and Nano-Se, the expression levels of two proteins (E2 and GS) increased and the other differential proteins were reverse. Except for E2, there were no significant differences in other protein expressions between the groups treated with nano-Se and Se-Met.

  6. Phosphorylation of Ser-525 in βPix impairs Nox1-activating ability in Caco-2 cells.

    Science.gov (United States)

    Kaito, Yuuki; Kataoka, Ryosuke; Mihara, Tatsuya; Takechi, Kento; Takahira, Akira; Watanabe, Shuhei; Han, Fei; Tamura, Minoru

    2018-01-15

    βPix activates Nox1, an O 2 - -generating NADPH oxidase, through Rac activation. In this study, we found that S525E mutation of βPix eliminated its Nox1-activating ability in transfected Caco-2 cells. Unexpectedly, affinity for Rac was not diminished but rather enhanced by S525E mutation, and guanine nucleotide exchange factor (GEF) activity was not altered. The N-terminal fragment (amino acids 1-400) showed similar Rac-binding and GEF activity to wild-type βPix. In contrast, the C-terminal fragment (amino acids 408-646) had higher Rac-binding activity, particularly for Rac-GTP, than wild-type βPix, and showed no GEF activity. These data suggest that a second Rac-binding site within the C-terminal region is opened by phosphorylation of Ser-525. The site may bind not only Rac-GDP but also Rac-GTP released from the N-terminal catalytic region, which interrupts Rac-GTP translocation to the membrane where Nox1 resides. If one considers that S340E mutation enhances Nox1 activation (Kaito et al., 2014), the present study suggests that βPix can also play an inhibitory role in O 2 - production, depending on the sites of phosphorylation. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Effect of neutrase, alcalase, and papain hydrolysis of whey protein concentrates on iron uptake by Caco-2 cells.

    Science.gov (United States)

    Ou, Keqin; Liu, Yunzhu; Zhang, Liebing; Yang, Xiaoguang; Huang, Zhenwu; Nout, M J Robert; Liang, Jianfen

    2010-04-28

    Effects of enzymatic hydrolysates of whey protein concentrates (WPC) on iron absorption were studied using in vitro digestion combined with Caco-2 cell models for improved iron absorption. Neutrase- and papain-treated WPC could improve iron absorption; especially hydrolysates by Neutrase could significantly increase iron absorption to 12.8% compared to 3.8% in the control. Hydrolysates by alcalase had negative effects to the lowest at 0.57%. Two new bands at molecular weights (MW) around and below 10 kDa occurred at tricine-SDS-PAGE of hydrolysates by Neutrase, and one new band at MW below 10 kDa occurred in hydrolysates by papain. No new band was observed in hydrolysates by alcalase. Concentration of free amino acids indicated that, except for tyrosine and phenylalanine, amino acids in papain-treated hydrolysates were higher than that of alcalase, and no cysteine and proline were found in hydrolysates by alcalase. The results suggested that hydrolysate by Neutrase-treated WPC is a promising facilitator for iron absorption. Peptides of MW around and lower than 10 kDa and aspartic acid, serine, glutamic acid, glycin, cysteine, histidine, and proline may be contributors to enhancement.

  8. Effects of Catechins and Their Related Compounds on Cellular Accumulation and Efflux Transport of Mitoxantrone in Caco-2 Cell Monolayers.

    Science.gov (United States)

    Sugihara, Narumi; Kuroda, Norihiko; Watanabe, Fumiya; Choshi, Tominari; Kamishikiryo, Jun; Seo, Makoto

    2017-05-01

    The ability of catechins and their related compounds to inhibit breast cancer resistance protein (BCRP) function in Caco-2 cell monolayers was investigated with mitoxantrone as a BCRP substrate. The gallate or pyrogallol moiety on the catechin structure seemed to promote increased cellular accumulation and inhibit efflux transport of mitoxantrone. The ability of gallate catechins such as (-)-epigallocatechin gallate (EGCG) and (-)-epicatechin gallate (ECG) to increase cellular accumulation and inhibit efflux transport of mitoxantrone was greater than that of nongallate catechins. Gallic acid octyl ester (GAO) also increased intracellular mitoxantrone accumulation. Experiments using GAO derivatives indicated that the gallate moiety required the presence of a long carbon chain for BCRP inhibition. Cellular accumulation and reduced efflux transport of mitoxantrone were greater with epigallocatechin 3-(3″-O-butyl) gallate than with EGCG. EGCG inhibition of BCRP seemed to be restricted by hydrophobicity. The co-administration of catechins, particularly EGCG and related compounds, with greater hydrophobicity may increase the therapeutic activities of BCRP substrates such as mitoxantrone. © 2017 Institute of Food Technologists®.

  9. 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

  10. Change in Caco-2 cells following treatment with various lavender essential oils.

    Science.gov (United States)

    Donadu, M G; Usai, D; Mazzarello, V; Molicotti, P; Cannas, S; Bellardi, M G; Zanetti, S

    2017-09-01

    Lavender is an aromatic evergreen shrub diffused in the Mediterranean basin appreciated since antiquity. The genus Lavandula is part of Lamiaceae family and includes more than 20 species, among which true lavender (L. vera D.C. or L. angustifolia Miller.) and spike lavender (L. latifolia Medikus); there are also numerous hybrids known as lavandins (L. hybrida Rev.). L. vera, spike lavender and several hybrids are the most intensely used breeding species for the production of essential oils. Lavender and lavandin essential oils have been applied in food, pharmaceutical and other agro industries as biological products. In their chemical composition, terpenes linalool and linalyl acetate along with terpenoids such as 1,8-cineole are mostly responsible for biological and therapeutic activities. This study evaluates cytotoxic activity of essential oils derived from four lavender species on human epithelial colorectal adenocarcinoma cells. Analysis of pre- and post-treatment cell morphology has been performed using scanning electron microscope.

  11. 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.

  12. The effect of milk components and storage conditions on the virulence of Listeria monocytogenes as determined by a Caco-2 cell assay.

    Science.gov (United States)

    Pricope-Ciolacu, Luminita; Nicolau, Anca Ioana; Wagner, Martin; Rychli, Kathrin

    2013-08-16

    Nearly all cases of human listeriosis have been associated with consumption of contaminated food, therefore the investigation of the virulence of Listeria (L.) monocytogenes after exposure to environmental conditions in food matrices is critical in order to understand and control its impact on public health. As milk and dairy products have been implicated in more than half of the listeriosis outbreaks, we investigated the in vitro virulence of L. monocytogenes incubated in different milk types at various storage conditions. Incubation in pasteurized milk at refrigeration conditions (4°C) revealed a higher invasion and intracellular proliferation of four different L. monocytogenes strains compared to raw milk using human intestinal epithelial Caco-2 cells. Furthermore the period of storage, which increased L. monocytogenes cell numbers, decreased in vitro virulence. However, L. monocytogenes stored for 3weeks at 4°C in milk are still able to invade and proliferate into the host cell. Interestingly abused storage temperatures (25°C and 30°C) for a short time period (2h) revealed an attenuated impact on the in vitro virulence of L. monocytogenes compared to the storage temperature of 4°C. Regarding the major milk compounds, the level of milk fat significantly affected the in vitro virulence of L. monocytogenes. Pre-incubation in milk with high fat content (3.6%) resulted in a lower invasion capability compared to milk with low fat content. In contrast casein and lactose did not influence the invasiveness of L. monocytogenes into the host cell. In conclusion our study shows that the milk environment and different storage conditions influence the in vitro virulence of L. monocytogenes, both of which have to be considered in the risk assessment of contaminated food. Copyright © 2013 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    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

  14. A Critical Evaluation of In Vitro Hesperidin 2S Bioavailability in a Model Combining Luminal (Microbial) Digestion and Caco-2 Cell Absorption in Comparison to a Randomized Controlled Human Trial.

    Science.gov (United States)

    Van Rymenant, Evelien; Salden, Bouke; Voorspoels, Stefan; Jacobs, Griet; Noten, Bart; Pitart, Judit; Possemiers, Sam; Smagghe, Guy; Grootaert, Charlotte; Van Camp, John

    2018-02-16

    Bioavailability strongly determines polyphenol bioactivity, and is strongly influenced by food matrix, enzymatic and microbial degradation, and gastrointestinal absorption. To avoid human trials for pre-screening of polyphenol bioavailability, studies have focused on in vitro model development. Nevertheless, their predictive value for bioavailability can be questioned. We used the orange flavonoid hesperidin 2S to validate a model combining digestion in the simulator of the human intestinal microbial ecosystem (SHIME) and Caco-2 cell transport, with a human intervention study. In vitro, hesperidin was resistant to degradation in the stomach and small intestine, but was rapidly deconjugated on reaching the proximal colon. Extensive and colon-region-specific degradation to smaller phenolics was observed. Hydrocaffeic and dihydroisoferulic acid accumulated in proximal, and hydroferulic acid in distal colon. Caco-2 transport was the highest for dihydroisoferulic acid. In humans, plasma and urine hesperetin-glucuronide levels increased significantly, whereas the impact on small phenolics was limited. In the combined in vitro model, smaller phenolics strongly accumulated, whereas in humans, hesperetin conjugates were the main bioavailable compounds. Future in vitro model development should focus on simulating faster polyphenol absorption and elimination of smaller phenolics to improve their predictive value of in vivo polyphenol bioavailability. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Homogenization, lyophilization or acid-extraction of meat products improves iron uptake from cereal-meat product combinations in an in vitro digestion/Caco-2 cell model.

    Science.gov (United States)

    Pachón, Helena; Stoltzfus, Rebecca J; Glahn, Raymond P

    2009-03-01

    The effect of processing (homogenization, lyophilization, acid-extraction) meat products on iron uptake from meat combined with uncooked iron-fortified cereal was evaluated using an in vitro digestion/Caco-2 cell model. Beef was cooked, blended to create smaller meat particles, and combined with electrolytic iron-fortified infant rice cereal. Chicken liver was cooked and blended, lyophilized, or acid-extracted, and combined with FeSO4-fortified wheat flour. In the beef-cereal combination, Caco-2 cell iron uptake, assessed by measuring the ferritin formed by cells, was greater when the beef was blended for the greatest amount of time (360 s) compared with 30 s (P meat products on iron absorption in iron-fortified cereals.

  16. DNA damage and apoptosis in blood neutrophils of inflammatory bowel disease patients and in Caco-2 cells in vitro exposed to betanin.

    Science.gov (United States)

    Zielińska-Przyjemska, Małgorzata; Olejnik, Anna; Dobrowolska-Zachwieja, Agnieszka; Łuczak, Michał; Baer-Dubowska, Wanda

    2016-04-06

    Inflammatory bowel diseases (IBD) are chronic, relapsing, inflammatory disorders of the gastrointestinal tract, and continuing colonic inflammation is considered an important risk factor in the development of colorectal cancer. Our previous studies showed that beetroot (Beta vulgaris var. rubra) products and their major component betanin modulate the reactive oxygen species (ROS) production and DNA damage in 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulated human polymorphonuclear neutrophils of healthy volunteers. The aim of the present study was to evaluate the effects of betanin on the oxidative DNA damage and apoptosis in neutrophils isolated from blood of patients with inflammatory bowel disease--ulcerative colitis (UC) and Crohn's disease (CD). The results were compared with those obtained in colon carcinoma-derived Caco-2 cells. Betanin treatment at the concentration of 100 μM for 24 h increased DNA damage assessed by comet assay in IBD patients' neutrophils. A similar effect although less pronounced was observed in Caco-2 cells. Treatment of Caco-2 cells with H2O2 caused a 4-fold increase of DNA strand breaks in comparison to untreated cells, but pre-treatment with betanin reduced DNA damage in these cells. Betanin also induced procaspase-3 cleavage and caspase-3 activity accompanied by the loss of mitochondrial transmembrane potential, indicating its pro-apoptotic activity. These results suggest that betanin may support mechanisms that lead to the release of ROS and apoptotic cell death. In this way betanin may exert anti-inflammatory and potentially cancer preventive activity.

  17. DNA damage and apoptosis in blood neutrophils of inflammatory bowel disease patients and in Caco-2 cells in vitro exposed to betanin

    Directory of Open Access Journals (Sweden)

    Małgorzata Zielińska-Przyjemska

    2016-04-01

    Full Text Available Inflammatory bowel diseases (IBD are chronic, relapsing, inflammatory disorders of the gastrointestinal tract, and continuing colonic inflammation is considered an important risk factor in the development of colorectal cancer. Our previous studies showed that beetroot (Beta vulgaris var. rubra products and their major component betanin modulate the reactive oxygen species (ROS production and DNA damage in 12-O-tetradecanoylphorbol 13-acetate (TPA stimulated human polymorphonuclear neutrophils of healthy volunteers. The aim of the present study was to evaluate the effects of betanin on the oxidative DNA damage and apoptosis in neutrophils isolated from blood of patients with inflammatory bowel disease– ulcerative colitis (UC and Crohn’s disease (CD. The results were compared with those obtained in colon carcinoma-derived Caco-2 cells. Betanin treatment at the concentration of 100 μM for 24 h increased DNA damage assessed by comet assay in IBD patients’ neutrophils. A similar effect although less pronounced was observed in Caco-2 cells. Treatment of Caco-2 cells with H2O2 caused a 4-fold increase of DNA strand breaks in comparison to untreated cells, but pre-treatment with betanin reduced DNA damage in these cells. Betanin also induced procaspase-3 cleavage and caspase-3 activity accompanied by the loss of mitochondrial transmembrane potential, indicating its pro-apoptoticactivity. These results suggest that betanin may support mechanisms that lead to the release of ROS and apoptotic cell death. In this way betanin may exert anti-inflammatory and potentially cancer preventive activity.

  18. Tannase enhances the anti-inflammatory effect of grape pomace in Caco-2 cells treated with IL-1beta

    Science.gov (United States)

    Grape pomace (GP) is rich in polymeric polyphenolics and glycosides which have lower bioefficacy than monomeric and aglycone counterparts. The aim of this study was to determine whether tannin acyl hydrolase [tannase (TNS)] can improve the antioxidant and anti-inflammatory actions of GP in Caco-2 ce...

  19. 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

  20. Decreased camptothecin sensitivity of the stem-cell-like fraction of Caco2 cells correlates with an altered phosphorylation pattern of topoisomerase I.

    Science.gov (United States)

    Roy, Amit; Tesauro, Cinzia; Frøhlich, Rikke; Hede, Marianne S; Nielsen, Maria J; Kjeldsen, Eigil; Bonven, Bjarne; Stougaard, Magnus; Gromova, Irina; Knudsen, Birgitta R

    2014-01-01

    The CD44+ and CD44- subpopulations of the colorectal cancer cell line Caco2 were analyzed separately for their sensitivities to the antitumor drug camptothecin. CD44+ cells were less sensitive to camptothecin than CD44- cells. The relative resistance of CD44+ cells was correlated with (i) reduced activity of the nuclear enzyme topoisomerase I and (ii) insensitivity of this enzyme to camptothecin when analyzed in extracts. In contrast, topoisomerase I activity was higher in extracts from CD44- cells and the enzyme was camptothecin sensitive. Topoisomerase I from the two subpopulations were differentially phosphorylated in a manner that appeared to determine the drug sensitivity and activity of the enzyme. This finding was further supported by the fact that phosphorylation of topoisomerase I in CD44+ cell extract by protein kinase CK2 converted the enzyme to a camptothecin sensitive, more active form mimicking topoisomerase I in extracts from CD44- cells. Conversely, dephosphorylation of topoisomerase I in extracts from CD44- cells rendered the enzyme less active and camptothecin resistant. These findings add to our understanding of chemotherapy resistance in the Caco2 CD44+ cancer stem cell model.

  1. Decreased camptothecin sensitivity of the stem-cell-like fraction of Caco2 cells correlates with an altered phosphorylation pattern of topoisomerase I.

    Directory of Open Access Journals (Sweden)

    Amit Roy

    Full Text Available The CD44+ and CD44- subpopulations of the colorectal cancer cell line Caco2 were analyzed separately for their sensitivities to the antitumor drug camptothecin. CD44+ cells were less sensitive to camptothecin than CD44- cells. The relative resistance of CD44+ cells was correlated with (i reduced activity of the nuclear enzyme topoisomerase I and (ii insensitivity of this enzyme to camptothecin when analyzed in extracts. In contrast, topoisomerase I activity was higher in extracts from CD44- cells and the enzyme was camptothecin sensitive. Topoisomerase I from the two subpopulations were differentially phosphorylated in a manner that appeared to determine the drug sensitivity and activity of the enzyme. This finding was further supported by the fact that phosphorylation of topoisomerase I in CD44+ cell extract by protein kinase CK2 converted the enzyme to a camptothecin sensitive, more active form mimicking topoisomerase I in extracts from CD44- cells. Conversely, dephosphorylation of topoisomerase I in extracts from CD44- cells rendered the enzyme less active and camptothecin resistant. These findings add to our understanding of chemotherapy resistance in the Caco2 CD44+ cancer stem cell model.

  2. Different transport mechanisms for cadmium and mercury in Caco-2 cells: inhibition of Cd uptake by Hg without evidence for reciprocal effects

    International Nuclear Information System (INIS)

    Aduayom, Ismaeel; Campbell, Peter G.C.; Denizeau, Francine; Jumarie, Catherine

    2003-01-01

    Cadmium/Hg interactions have been studied in the TC7 clone of the enterocytic-like Caco-2 cells to test the hypothesis that these metals may compete for intestinal transport. Comparison of the kinetic parameter values for 203 Hg(II) and 109 Cd(II) uptake in a serum-free medium revealed that Hg is accumulated much more rapidly and to higher concentrations. The very rapid uptake/binding step and the initial uptake rate of 109 Cd were both significantly inhibited by an excess of unlabeled Cd or Hg (apparent K i for Hg of 9.3 ± 1.2 μM) without reciprocal effects. 109 Cadmium uptake was highly sensitive to temperature and a significant fraction of accumulation (12%) was EDTA extractable. 203 Hg uptake remained insensitive to temperature or the EDTA washing procedure. However, the uptake of both tracers was half-decreased when an excess of the respective unlabeled metal was added in the stop solution, suggesting an exchange mechanism for adsorption. Cell pretreatment with N-ethylmaleimide (NEM) led to a 30% decrease or a 73% increase in the 3-min specific transport of 109 Cd when NEM was still present in or removed from the uptake medium, respectively. NEM had no effect on 203 Hg uptake. Overall our results suggest the involvement of a saturable specific mechanism for Cd, which is highly sensitive to inhibition by Hg and NEM under some conditions, and a nonspecific passive diffusion for Hg. The Hg- or NEM-induced inhibition of Cd uptake likely involves a thiol-mediated reaction, but our results suggest that NEM pretreatment may activate other cellular mechanisms leading to a stimulatory effect

  3. Echinacea sanguinea and Echinacea pallida extracts stimulate glucuronidation and basolateral transfer of Bauer alkamides 8 and 10 and ketone 24 and inhibit P-glycoprotein transporter in Caco-2 cells.

    Science.gov (United States)

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

    2013-03-01

    The use of Echinacea as a medicinal herb is prominent in the United States, and many studies have assessed the effectiveness of Echinacea as an immunomodulator. We hypothesized that Bauer alkamides 8, 10, and 11 and ketone 24 were absorbed similarly either as pure compounds or from Echinacea sanguinea and Echinacea pallida ethanol extracts, and that these Echinacea extracts could inhibit the P-glycoprotein transporter in Caco-2 human intestinal epithelial cells. Using HPLC analysis, the permeation rate of Bauer alkamides by passive diffusion across Caco-2 cells corresponded with compound hydrophilicity (alkamide 8 > 10 > 11), independent of the plant extract matrix. Both Echinacea ethanol extracts stimulated apparent glucuronidation and basolateral efflux of glucuronides of alkamides 8 and 10 but not alkamide 11. Bauer ketone 24 was totally metabolized to more hydrophilic metabolites when administered as a single compound, but was also glucuronidated when present in Echinacea extracts. Bauer alkamides 8, 10, and 11 (175-230 µM) and ethanol extracts of E. sanguinea (1 mg/mL, containing ~ 90 µM total alkamides) and E. pallida (5 mg/mL, containing 285 µM total alkamides) decreased the efflux of the P-glycoprotein transporter probe calcein-AM from Caco-2 cells. These results suggest that other constituents in these Echinacea extracts facilitated the metabolism and efflux of alkamides and ketones, which might improve therapeutic benefits. Alkamides and Echinacea extracts might be useful in potentiating some chemotherapeutics, which are substrates for the P-glycoprotein transporter. Georg Thieme Verlag KG Stuttgart · New York.

  4. The effects of baicalein or baicalin on the colloidal stability of ZnO nanoparticles (NPs) and toxicity of NPs to Caco-2 cells.

    Science.gov (United States)

    Li, Yining; Zhang, Cao; Liu, Liangliang; Gong, Yu; Xie, Yixi; Cao, Yi

    2018-03-01

    Recent study suggested that the presence of phytochemicals in food could interact with nanoparticles (NPs) and consequently reduce the toxicity of NPs, which has been attributed to the antioxidant properties of phytochemicals. In this study, we investigated the interactions between ZnO NPs and two flavonoids baicalein (Ba) or baicalin (Bn) as well as the influence of the interactions on the toxicity of ZnO NPs to Caco-2 cells. The antioxidant properties of Ba and Bn were confirmed by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assays, with Ba being stronger. However, the presence of Ba or Bn did not significantly affect cytotoxicity, intracellular superoxide or release of inflammatory cytokines of Caco-2 cells after ZnO NP exposure. When Ba was present, the cellular viability of Caco-2 cells after exposure to ZnO NPs was slightly increased, associated with a modest decrease of intracellular Zn ions, but these effects were not statistically different. Ba was more effective than Bn at changing the hydrodynamic sizes, Zeta potential and UV-Vis spectra of ZnO NPs, which indicated that Ba might increase the colloidal stability of NPs. Taken together, the results of the present study indicated that the anti-oxidative phytochemical Ba might only modestly protected Caco-2 cells from the exposure to ZnO NPs associated with an insignificant reduction of the accumulation of intracellular Zn ions. These results also indicated that when assessing the combined effects of NPs and phytochemicals to cells lining gastrointestinal tract, it might be necessary to evaluate the changes of colloidal stability of NPs altered by phytochemicals.

  5. 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

    Directory of Open Access Journals (Sweden)

    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.

  6. Inhibitory effects of a soluble dietary fiber from Amorphophallus konjac on cytotoxicity and DNA damage induced by fecal water in Caco-2 cells.

    Science.gov (United States)

    Yeh, Shu-Lan; Lin, Meng-Sjen; Chen, Hsiao-Ling

    2007-10-01

    The aims of this study were to determine the effects of konjac glucomannan (KGM), a water-soluble dietary fiber from Amorphophallu konjac C. Koch, on the cytotoxicity and DNA damage of fecal water-treated Caco-2 cells, a human colon adenocarcinoma cell line, and to compare these effects with those of inulin, oligofructose (FO), cellulose and no fiber diet. In addition, the possible mechanisms by which dietary fibers modulated the toxicity of feces were investigated. Seven-week-old BALB/c mice were randomly allocated to consume an AIN-93 diet that contained no dietary fiber (FF) or 5 % (w/w) KGM, inulin, FO, or cellulose for 3 weeks. Fresh feces were collected during days 18 - 21. Our results indicated that the survival rate (%) of fecal water-treated Caco-2 cells was similarly enhanced by each dietary fiber as compared with that of FF group, respectively. The inhibition of fecal water-induced DNA damage of Caco-2 cells was in the descending order of inulin> FO> cellulose, KGM group. Cellulose significantly exerted a bulk effect, while KGM, inulin and FO significantly increased the ratio of lactobacilli and bifidobacteria in feces, respectively, as well as the ferrous ion-chelating ability of fecal water, respectively. Therefore, this study suggests that KGM, inulin and FO may reduce the toxicity of fecal water mainly by increasing the fecal probiotics and ferrous ion-chelating activities while cellulose may act mainly by increasing the fecal bulk. FF: fiber free FO: oligofructose KGM: konjac glucomannan.

  7. 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

    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...

  8. 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

  9. The presence of oleate stabilized ZnO nanoparticles (NPs) and reduced the toxicity of aged NPs to Caco-2 and HepG2 cells.

    Science.gov (United States)

    Fang, Xin; Jiang, Leying; Gong, Yu; Li, Juan; Liu, Liangliang; Cao, Yi

    2017-12-25

    The presence of food components may alter the colloidal aspects and toxicity of nanoparticles (NPs). In this study, the toxicity of ZnO NPs to Caco-2 and HepG2 cells was assessed, with the emphasis on the interactions between ZnO NPs and oleate (OA). The presence of OA increased UV-Vis spectra and hydrodynamic sizes, decreased Zeta potential, and markedly reduced the release of Zn ions from the dissolution of ZnO NPs, which combined indicated that OA could coat ZnO NPs and stabilize ZnO NPs. Exposure to ZnO NPs significantly induced cytotoxicity to Caco-2 and HepG2 cells, associated with increased intracellular Zn ions but not superoxide. When OA was added to the freshly prepared ZnO NP suspensions, the cytotoxicity, intracellular Zn ions and superoxide induced by ZnO NPs were not significantly affected. However, when ZnO NPs were aged for 24 h with the presence of OA, the cytotoxicity of ZnO NPs to Caco-2 and HepG2 cells was significantly reduced, associated with a reduction of intracellular Zn ions. The results from this study suggested that the presence of OA could increase colloidal stability of ZnO NPs and consequently reduce the toxicity of ZnO NPs after aging associated with reduced accumulation of intracellular Zn ions. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Sulphonated modification of polysaccharides from Pleurotus eryngii and Streptococcus thermophilus ASCC 1275 and antioxidant activities investigation using CCD and Caco-2 cell line models.

    Science.gov (United States)

    Li, Siqian; Shah, Nagendra P

    2017-06-15

    Polysaccharides extracted from Pleurotus eryngii (PEPS) and Streptococcus thermophilus ASCC 1275 (EPS) were sulphonated and their composition determined. Antioxidant activities of crude and sulphonated polysaccharides (S.PEPS and S.EPS) were investigated using the ABTS radical scavenging test and CCD and Caco-2 cell models. Degrees of sulphonation for S.PEPS and S.EPS were 0.73 and 0.37, respectively. Results showed that S.PEPS and S.EPS had significantly higher scavenging capacities than PEPS and EPS in the ABTS radical scavenging test. Reactive oxygen species and malondialdehyde in H 2 O 2 treated CCD 841 CoN (CCD) and Caco-2 cells were significantly inhibited by PEPS, EPS, S.PEPS and S.EPS compared to the control group. Additionally, S.PEPS and S.EPS significantly improved superoxide dismutase, catalase and glutathione peroxidase activities in H 2 O 2 treated CCD and Caco-2 cells compared to PEPS, EPS and control groups. Results indicated that sulphonation was effective in improving antioxidant activities of both PEPS and EPS. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Ferric reductase activity of low molecular weight human milk fraction is associated with enhanced iron solubility and uptake in Caco-2 cells.

    Science.gov (United States)

    Pullakhandam, Raghu; Nair, Madhavan Krishnapillai; Kasula, Sunanda; Kilari, Sreenivasulu; Thippande, Tippeswamy Gowda

    2008-09-19

    It is known that the fractional absorption of extrinsic iron from human milk is higher in infants and adults. A low molecular weight milk fraction has been proposed to increase the bioavailability of iron from human milk. Nevertheless, the mechanisms remained elusive. Here in we demonstrate ferric reductase activity (Km7.73x10(-6)M) in low molecular weight human milk fraction (10kF, filtrate derived from ultra filtration of milk whey through 10kDa cutoff membrane), which increased ferric iron solubility and iron uptake in Caco-2 cells. The 10kF fraction was as effective as ascorbic acid (1:20 iron to ascorbic acid) in increasing the ferric iron solubility and uptake in Caco-2 cells. Further, gel filtration chromatography on peptide column led to co-elution of ferric reductase and iron solubilization activities at an apparent molecular mass of iron in Caco-2 cells. Thus, it is concluded that human milk possesses ferric reductase activity and is associated with ferric iron solubilization and enhanced absorption.

  12. 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.

  13. Anti-Inflammatory Phenolic Acid Esters from the Roots and Rhizomes of Notopterygium incisium and Their Permeability in the Human Caco-2 Monolayer Cell Model.

    Science.gov (United States)

    Wu, Xiu-Wen; Wei, Wei; Yang, Xiu-Wei; Zhang, You-Bo; Xu, Wei; Yang, Yan-Fang; Zhong, Guo-Yue; Liu, Hong-Ning; Yang, Shi-Lin

    2017-06-04

    A new ferulic acid ester named 4-methyl-3- trans -hexenylferulate ( 1 ), together with eight known phenolic acid esters ( 2 - 9 ), was isolated from the methanolic extract of the roots and rhizomes of Notopterygium incisium . Their structures were elucidated by extensive spectroscopic techniques, including 2D NMR spectroscopy and mass spectrometry. 4-Methoxyphenethyl ferulate ( 8 ) NMR data is reported here for the first time. The uptake and transepithelial transport of the isolated compounds 1 - 9 were investigated in the human intestinal Caco-2 cell monolayer model. Compounds 2 and 6 were assigned for the well-absorbed compounds, compound 8 was assigned for the moderately absorbed compound, and compounds 1 , 3 , 4 , 5 , 7 , and 9 were assigned for the poorly absorbed compounds. Moreover, all of the isolated compounds were assayed for the inhibitory effects against nitric oxide (NO) production in the lipopolysaccharide-activated RAW264.7 macrophages model and L-N ⁶-(1-iminoethyl)-lysine (L-NIL) was used as a positive control. Compounds 1 , 5 , 8 , and 9 exhibited potent inhibitory activity on NO production with the half maximal inhibitory concentration (IC 50 ) values of 1.01, 4.63, 2.47, and 2.73 μM, respectively, which were more effective than L-NIL with IC 50 values of 9.37 μM. These findings not only enriched the types of anti-inflammatory compounds in N. incisum but also provided some useful information for predicting their oral bioavailability and their suitability as drug leads or promising anti-inflammatory agents.

  14. Cytotoxic activity of extracts and crude saponins from Zanthoxylum armatum DC. against human breast (MCF-7, MDA-MB-468) and colorectal (Caco-2) cancer cell lines.

    Science.gov (United States)

    Alam, Fiaz; Najum Us Saqib, Qazi; Waheed, Abdul

    2017-07-17

    Zanthoxylum armatum DC has been an important traditional plant known for its medicinal properties. It is well known for its antimicrobial, larvicidal and cytotoxic activities. The potential anticancer effects of the methanol extract and the crude saponins from fruit, bark and leaves of Z. armatum on breast (MDA-MB-468 and MCF-7) and colorectal (Caco-2) cancer cell lines using MTT, neutral red uptake(NRU) and DAPI stain assays were evaluated. In MTT assay the methanol extract of fruit (Zf), bark (Zb) and leaves (Zl) of Zanthoxylum armatum, showed significant and dose dependent growth inhibition of MCF-7, MDA MB-468 and Caco-2 cancer cell lines in a dose of 200 μg/ml and above. The saponins (Zf.Sa, Zb.Sa and Zl.Sa) showed significant activity against MDA MB-468 (95, 94.5 and 85.3%) as compared to MCF-7 (79.8, 9.43, 49.08%) and Caco-2 (75.8, 61.8, 68.62%) respectively. The extracts were further tested in more sensitive NRU assay and its was found that Zf extract showed higher cytotoxic activity as compared to Zb and Zl extracts with 100 μg/ml concentration. The breast cancer cell lines showed more sensitivity toward the crude saponins from fruit and bark with maximum inhibition of up to 93.81(±2.32) % with respect to 71.19(± 2.76) of Actinomycin-D. DAPI staining experiment showed that saponins from fruit induced apoptosis mode of cell death in all three types of cell lines while saponins form leaves and bark showed similar results against MDA MB-468 indicated by nuclear fragmentation and chromatin condensation. The effect of saponins from fruit, bark and leaves (Zf.Sa, Zb.Sa and Zl.Sa) against Caco-2 cell lines inhibited the growth of Caco-2 by 53.16 (±3.31) %, 66.43 (± 3.24) and 45.96 (± 10.67) respectively with respect to Actinomycin-D (4 μM) which showed the growth inhibition of 65.40(±4.29) %. The current study clearly demonstrates that the extract and crude saponins from fruit, bark and leaves of traditional medicinal plant Zanthoxyllum armatum DC

  15. Epinephrine modulates Na+/K+ ATPase activity in Caco-2 cells via Src, p38MAPK, ERK and PGE2.

    Directory of Open Access Journals (Sweden)

    Layla El Moussawi

    Full Text Available Epinephrine, a key stress hormone, is known to affect ion transport in the colon. Stress has been associated with alterations in colonic functions leading to changes in water movements manifested as diarrhea or constipation. Colonic water movement is driven by the Na+-gradient created by the Na+/K+-ATPase. Whether epinephrine acts via an effect on the Na+/K+-ATPase hasn't been studied before. The aim of this work was to investigate the effect of epinephrine on the Na+/K+-ATPase and to elucidate the signaling pathway involved using CaCo-2 cells as a model. The activity of the Na+/K+-ATPase was assayed by measuring the amount of inorganic phosphate released in presence and absence of ouabain, a specific inhibitor of the enzyme. Epinephrine, added for 20 minutes, decreased the activity of the Na+/K+-ATPase by around 50%. This effect was found to be mediated by α2 adrenergic receptors as it was fully abolished in the presence of yohimbine an α2-blocker, but persisted in presence of other adrenergic antagonists. Furthermore, treatment with Rp-cAMP, a PKA inhibitor, mimicked epinephrine's negative effect and didn't result in any additional inhibition when both were added simultaneously. Treatment with indomethacin, PP2, SB202190, and PD98059, respective inhibitors of COX enzymes, Src, p38MAPK, and ERK completely abrogated the effect of epinephrine. The effect of epinephrine did not appear also in presence of inhibitors of all four different types of PGE2 receptors. Western blot analysis revealed an epinephrine-induced increase in the phosphorylation of p38 MAPK and ERK that disappeared in presence of respectively PP2 and SB2020190. In addition, an inhibitory effect, similar to that of epinephrine's, was observed upon incubation with PGE2. It was concluded that epinephrine inhibits the Na+/K+-ATPase by the sequential activation of α2 adrenergic receptors, Src, p38MAPK, and ERK leading to PGE2 release.

  16. Differential Effects of Falcarinol and Related Aliphatic C17-Polyacetylenes on Intestinal Cell Proliferation

    OpenAIRE

    Purup, Stig; Larsen, Eric; Christensen, Lars P.

    2009-01-01

    Quantitative major polyacetylenes of carrots (falcarinol and falcarindiol) and American ginseng roots (falcarinol and panaxydol) were isolated and tested in human intestinal epithelial cells of normal (FHs 74 Int.) and cancer (Caco-2) origin. A hormesis effect was seen for all isolated polyacetylenes when added to Caco-2 cells in concentrations ranging from 1 ng/mL to 20 ?g/mL. The relative inhibitory potency was falcarinol > panaxydol > falcarindiol. No hormesis effect was observed when addi...

  17. Caco-2 cell-based electrochemical biosensor for evaluating the antioxidant capacity of Asp-Leu-Glu-Glu isolated from dry-cured Xuanwei ham.

    Science.gov (United States)

    Xing, Lujuan; Ge, Qingfeng; Jiang, Donglei; Gao, Xiaoge; Liu, Rui; Cao, Songmin; Zhuang, Xinbo; Zhou, Guanghong; Zhang, Wangang

    2018-05-15

    A cell-based electrochemical biosensor was developed to determine the antioxidant activity of Asp-Leu-Glu-Glu (DLEE) isolated from dry-cured Chinese Xuanwei ham. A platinized gold electrode (Pt NPs/GE) covered with silver nanowires (Ag NWs) was fabricated to detect H 2 O 2 using redox signaling via cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS). Under optimal condition, the detection limit of the modified electrode was 0.12μM with a linear relationship from 0.2 to 2μM, which showed relatively outstanding catalytic effects towards the reduction of H 2 O 2 . Furthermore, the generation of reactive oxygen species (ROS) in the cell can be used to indirectly assess changes in intercellular oxidative stress by detecting variations in electrochemical signals. A 3D cell culture of alginate/graphene oxide (NaAlg/GO) was used to encapsulate and immobilize Caco-2 cells. Based on ROS generation and electrochemical results, we found that DLEE could effectively reduce oxidative stress level in Caco-2 cells under external stimulation. DLEE showed high antioxidant activity with a relative antioxidant capacity (RAC) rate of 88.17% at 1.5mg/mL. Finally, an efficient electrochemical biosensor was established using the active 3D Caco-2 cell platform. This system is sensitive and simple to operate with the property to evaluate the antioxidant activity of peptides by the detection of H 2 O 2 in cell membrane. In summary, this work describes a new method for assessing antioxidant capacity of peptide DLEE using cell-based electrochemical signaling with a rapid screening pattern. Copyright © 2018 Elsevier B.V. All rights reserved.

  18. Fermented soya bean (tempe) extracts reduce adhesion of enterotoxigenic Escherichia coli to intestinal epithelial cells

    NARCIS (Netherlands)

    Roubos-van den Hil, P.J.; Nout, M.J.R.; Beumer, R.R.; Meulen, van der J.; Zwietering, M.H.

    2009-01-01

    Aims: This study aimed to investigate the effect of processed soya bean, during the successive stages of tempe fermentation and different fermentation times, on adhesion of enterotoxigenic Escherichia coli (ETEC) K88 to intestinal brush border cells as well as Caco-2 intestinal epithelial cells; and

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

    Directory of Open Access Journals (Sweden)

    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.

  20. Optimization of process variables of zanamivir-loaded solid lipid nanoparticles and the prediction of their cellular transport in Caco-2 cell model.

    Science.gov (United States)

    Shi, Li-Li; Cao, Yue; Zhu, Xiao-Yin; Cui, Jing-Hao; Cao, Qing-Ri

    2015-01-15

    The aim of this study was to optimize the process variables of zanamivir-loaded solid lipid nanoparticles (SLNs) and to predict their cellular transport across Caco-2 cell monolayers. Zanamivir-loaded SLNs were prepared by a double emulsion solvent evaporation method. The effects of process variables on the mean particle size and zeta potential of SLNs were investigated and the physicochemical properties of SLNs were characterized. In addition, the cytotoxicity and transport ability of SLNs were also studied in Caco-2 cell model. The mean particle size of drug-loaded SLNs was significantly affected by the process variables. The SLNs were prepared using glyceryl monosterate (1% polyvinyl alcohol (PVA) or 1.5% poloxamer 188 as surfactant) had a regular shape with a relatively lower mean particle size (324.2 and 224.9 nm, respectively), higher encapsulation efficacy (55.7 and 42.4%, respectively) and sustained drug release over 12h. The crystalline form of drug had been partly changed and no physicochemical interaction had occurred between drug and carriers in SLNs. The cell viability was approximately 100% even at a high concentration of blank SLNs. There was no remarkable difference in drug permeation between drug-loaded SLNs and drug solution at 2h, while drug-loaded SLNs showed a significant decrease in drug permeation compared with the drug solution at 4h. The process variables of zanamivir-loaded SLNs were successfully optimized in this study. However, these SLNs significantly reduced the transport ability of zanamivir across Caco-2 cell monolayers. Further studies are needed to fully understand the real absorption mechanism of developed SLNs. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. The normal chain length distribution of the O antigen is required for the interaction of Shigella flexneri 2a with polarized Caco-2 cells

    Directory of Open Access Journals (Sweden)

    Anilei Hoare

    2012-01-01

    Full Text Available Shigella flexneri causes bacillary dysentery in humans. Essential to the establishment of the disease is the invasion of the colonic epithelial cells. Here we investigated the role of the lipopolysaccharide (LPS O antigen in the ability of S. flexneri to adhere to and invade polarized Caco-2 cells. The S. flexneri 2a O antigen has two preferred chain lengths: a short O antigen (S-OAg regulated by the WzzB protein and a very long O antigen (VL-OAg regulated by Wzz pHS2. Mutants with defined deletions of the genes required for O-antigen assembly and polymerization were constructed and assayed for their abilities to adhere to and enter cultured epithelial cells. The results show that both VL- and S-OAg are required for invasion through the basolateral cell membrane. In contrast, the absence of O antigen does not impair adhesion. Purified LPS does not act as a competitor for the invasion of Caco-2 cells by the wild-type strain, suggesting that LPS is not directly involved in the internalization process by epithelial cells.

  2. Antioxidant properties of chemical extracts and bioaccessible fractions obtained from six Spanish monovarietal extra virgin olive oils: assays in Caco-2 cells.

    Science.gov (United States)

    Borges, Thays H; Cabrera-Vique, Carmen; Seiquer, Isabel

    2015-07-01

    The antioxidant activity and the total phenolic content (TPC) of six Spanish commercial monovarietal extra virgin olive oils (Arbequina, Cornicabra, Hojiblanca, Manzanilla, Picual and Picudo) were evaluated in chemical extracts and in bioaccessible fractions (BF) obtained after in vitro digestion. Moreover, the effects of the BF on cell viability and the generation of reactive oxygen species (ROS) were investigated in Caco-2 cell cultures. The in vitro digestion process increased the TPC and antioxidant activity evaluated by different methods (ABTS, DPPH and FRAP) compared with chemical extracts. After digestion, the Picual variety showed better beneficial effects in preserving cell integrity than the other varieties studied. Significant reductions of ROS production were observed after incubation of Caco-2 cells with the BF of all the varieties and, moreover, a protective effect against the oxidative stress induced by t-BOOH was shown for Arbequina, Cornicabra, Hojiblanca, Manzanilla and Picual. These findings seem to be an additional reason supporting the health benefits of Spanish extra virgin olive oil varieties. Multivariate factor analysis and principal component analysis were applied to assess the contribution of antioxidant activity and TPC, before and after digestion, to the characterization of the different varieties.

  3. Ketogenesis contributes to intestinal cell differentiation.

    Science.gov (United States)

    Wang, Qingding; Zhou, Yuning; Rychahou, Piotr; Fan, Teresa W-M; Lane, Andrew N; Weiss, Heidi L; Evers, B Mark

    2017-03-01

    The intestinal epithelium undergoes a continual process of proliferation, differentiation and apoptosis. Previously, we have shown that the PI3K/Akt/mTOR pathway has a critical role in intestinal homeostasis. However, the downstream targets mediating the effects of mTOR in intestinal cells are not known. Here, we show that the ketone body β-hydroxybutyrate (βHB), an endogenous inhibitor of histone deacetylases (HDACs) induces intestinal cell differentiation as noted by the increased expression of differentiation markers (Mucin2 (MUC2), lysozyme, IAP, sucrase-isomaltase, KRT20, villin, Caudal-related homeobox transcription factor 2 (CDX2) and p21 Waf1 ). Conversely, knockdown of the ketogenic mitochondrial enzyme hydroxymethylglutaryl CoA synthase 2 (HMGCS2) attenuated spontaneous differentiation in the human colon cancer cell line Caco-2. Overexpression of HMGCS2, which we found is localized specifically in the more differentiated portions of the intestinal mucosa, increased the expression of CDX2, thus further suggesting the contributory role of HMGCS2 in intestinal differentiation. In addition, mice fed a ketogenic diet demonstrated increased differentiation of intestinal cells as noted by an increase in the enterocyte, goblet and Paneth cell lineages. Moreover, we showed that either knockdown of mTOR or inhibition of mTORC1 with rapamycin increases the expression of HMGCS2 in intestinal cells in vitro and in vivo, suggesting a possible cross-talk between mTOR and HMGCS2/βHB signaling in intestinal cells. In contrast, treatment of intestinal cells with βHB or feeding mice with a ketogenic diet inhibits mTOR signaling in intestinal cells. Together, we provide evidence showing that HMGCS2/βHB contributes to intestinal cell differentiation. Our results suggest that mTOR acts cooperatively with HMGCS2/βHB to maintain intestinal homeostasis.

  4. 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. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. Permeation of Insulin, Calcitonin and Exenatide across Caco-2 Monolayers: Measurement Using a Rapid, 3-Day System

    Science.gov (United States)

    Gupta, Vivek; Doshi, Nishit; Mitragotri, Samir

    2013-01-01

    Objectives Caco-2 monolayers are one of the most widely used in vitro models for prediction of intestinal permeability of therapeutic molecules. However, the conventional Caco-2 monolayer model has several drawbacks including labor-intensive culture process, unphysiological growth conditions, lack of reproducibility and limited throughput. Here, we report on the use of 3-day Caco-