Permeation of hesperidin glycosides across Caco-2 cell monolayers via the paracellular pathway

The intestinal permeability to hesperidin glycosides was investigated by using a cultured monolayer of Caco-2 as a model for the small intestinal epithelium. Hesperidin glycosides were added to the apical side of the monolayer, and the substances that permeated to the basolateral side were determined by HPLC. Whereas hesperidin did not permeate across the Caco-2 monolayer, probably owing to its low solubility, the hesperidin glycosides did permeate. The transepithelial transport of hesperidin glycosides occurred in time- and dose-dependent manners. The transport was observed to be energy-independent, and was inversely correlated with the transepithelial electrical resistance (TEER) of the monolayer. These results suggest that hesperidin glycosides permeate across the Caco-2 cell monolayer via the paracellular pathway.     

The effect of various liposome formulations on insulin penetration across Caco-2 cell monolayer

The aim of the study was to determine the penetration properties of various insulin containing liposome formulations through Caco-2 cell monolayer and to compare the in vitro test results with in vivo tests. The effect of sodium taurocholate as a penetration enhancer when it was added to the liposome formulation was also investigated. In vitro permeation experiments were performed in diffusion cells with the Caco-2 cell monolayer used as the membrane. Permeability values of various insulin containing liposome formulations through Caco-2 cells were determined (log k(insulin-solution) = -2.217 +/- 0.0723 cm.h(-1), log k(insulin-liposome) = -2.141 +/- 0.0625 cm.h(-1), log k(insulin-sodium tauroholate liposome)= -1.952 +/- 0.0623 cm.h(-1)). In vivo tests were performed in mice. Formulations were administered orally and blood glucose levels were determined and penetrations were compared with the Caco-2 cell experiment results. In conclusion, the permeability of insulin was increased across Caco-2 cell monolayer when the liposome sodium taurocholate (NaTC) formulation was used. The oral administration of insulin and NaTC incorporated liposomes significantly decreased blood glucose levels. Furthermore, it was shown that a high in vitro/in vivo correlation was observed using the Caco-2 cell monolayer model.     

Monitoring Caco-2 to enterocyte-like cells differentiation by means of electric impedance analysis on printed sensors

BackgroundColorectal adenocarcinoma cells (Caco-2) are a widely used model of intestinal barrier to study cancer development, toxicological assessments, absorption and metabolism in food science or drug discovery. Caco-2 spontaneously differentiate into a monolayer expressing several specific characteristics, typically showed by mature enterocytes. For in vitro experiments, it is crucial to identify non-invasive and non-destructive techniques able to evaluate the integrity and differentiation of the cells monolayer. Thus, we aimed to assess these properties by analyzing electrical impedance measurements.MethodsCaco-2 cells were differentiated for 21 days. The monolayer integrity and differentiation were primarily evaluated by means of morphological, biochemical and molecular data. Impedance measurements in a range of frequencies from 400 Hz to 50 kHz were performed using a dedicated set up, including customized Aerosol Jet Printed carbon-based sensors.ResultsThe trends of RI observed at three different frequencies were able to describe cell growth and differentiation. In order to evaluate which frequencies better correlate with cell differentiation, Principal Component Analysis have been employed and the concordance analysis between RI magnitude and morphological, biochemical and molecular data, highlighted 40 kHz as the optimal frequency to assess Caco-2 cells differentiation process.ConclusionWe demonstrated the feasibility and reliability of applying impedance-based measurements not only to provide information about the monolayer status, but also for cell differentiation monitoring.General significanceThis study underlined the possibility to use a dedicated sensor to assess the integrity and differentiation of Caco-2 monolayer, as a reliable non-destructive alternative to conventional approaches.     

Transepithelial transport of the bioactive tripeptide, Val-Pro-Pro, in human intestinal Caco-2 cell monolayers

Some of the food-derived tripeptides with angiotensin converting enzyme (ACE)-inhibitory activity have been reported to be hypotensive after being orally administered. The mechanism for the intestinal transport of these tripeptides was studied by using monolayer-cultured human intestinal Caco-2 cells which express many enterocyte-like functions including the peptide transporter (PepT1)-mediated transport system. Val-Pro-Pro, an ACE-inhibitory peptide from fermented milk, was used as a model tripeptide. A significant amount of intact Val-Pro-Pro was transported across the Caco-2 cell monolayer. This transport was hardly inhibited by a competitive substrate for PepT1. Since no intact Val-Pro-Pro was detected in the cells, Val-Pro-Pro apically taken by Caco-2 cells via PepT1 was likely to have been quickly hydrolyzed by intracellular peptidases, producing free Val and Pro. These findings suggest that PepT1-mediated transport was not involved in the transepithelial transport of intact Val-Pro-Pro. Paracellular diffusion is suggested to have been the main mechanism for the transport of intact Val-Pro-Pro across the Caco-2 cell monolayer.     

Insulin-cell penetrating peptide hybrids with improved intestinal absorption efficiency

Cell-penetrating peptide (CPP) was linked to insulin to form insulin-CPP hybrids. The intestinal absorption efficiency of CPP hybridized insulin was 6-8 times increased compared to normal insulin as tested on Caco-2 cell monolayer, a widely used in vitro model for intestinal absorption. Insulin-CPP hybrid transportation seemed to be through an active and transcytosis-like mechanism. Importantly, insulin in hybrids kept intact after they passed through the Caco-2 cell monolayer. This study provides a new clue for oral insulin development.     

Epithelial properties of human intestinal Caco-2 cells cultured in a serum-free medium

Human intestinal Caco-2 cells were cultured under serum-free conditions on an insoluble collagen and FCS matrix (Caco-2-SF), and a comparison was made between several characteristics of Caco-2 and Caco-2-SF cells. Their morphological appearance was identical. Slight differences were found in cell growth and expression of brush border enzymes between Caco-2 and Caco-2-SF cells. Similar levels of activity of Gly-Gly transport were expressed in both types of cell. Caco-2 cells cultured on permeable filters showed high transepithelial electrical resistance (TEER), indicating the high monolayer integrity. The transepithelial transport activity for glucose, alanine and Gly-Gly was detected by measuring the change in short-circuit current (Isc) after adding each of these nutrients to the apical chamber. In Caco-2-SF cells, such parameters as TEER and Isc were reduced drastically, suggesting that the monolayer integrity and cell polarity that are important for transepithelial transport were not attained. These parameters, however, could be restored by adding FCS or by milk whey. The result suggested that FCS and milk whey contain factors which regulate the formation of the tight junctions and, consequently, the development of cell polarity. Thus the Caco-2-SF cell-culture system will provide a useful model for studying factors which regulate the intestinal transepithelial transport functions.Abbreviations BCECF 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein - TEER transepithelial electrical resistance - LY lucifer yellow CH lithium salt     

Correlation of in vitro and in vivo models for the oral absorption of peptide drugs

The aim of this study was to evaluate two in vitro models, Caco-2 monolayer and rat intestinal mucosa, regarding their linear correlation with in vivo bioavailability data of therapeutic peptide drugs after oral administration in rat and human. Furthermore the impact of molecular mass (Mm) of the according peptides on their permeability was evaluated. Transport experiments with commercially available water soluble peptide drugs were conducted using Caco-2 cell monolayer grown on transwell filter membranes and with freshly excised rat intestinal mucosa mounted in Using type chambers. Apparent permeability coefficients (P (app)) were calculated and compared with in vivo data derived from the literature. It was shown that, besides a few exceptions, the Mm of peptides linearly correlates with permeability across rat intestinal mucosa (R (2) = 0.86; y = -196.22x + 1354.24), with rat oral bioavailability (R (2) = 0.64; y = -401.90x + 1268.86) as well as with human oral bioavailability (R (2) = 0.91; y = -359.43x + 1103.83). Furthermore it was shown that P (app) values of investigated hydrophilic peptides across Caco-2 monolayer displayed lower permeability than across rat intestinal mucosa. A correlation between P (app) values across rat intestinal mucosa and in vivo oral bioavailability in human (R (2) = 0.98; y = 2.11x + 0.34) attests the rat in vitro model to be a very useful prediction model for human oral bioavailability of hydrophilic peptide drugs. Presented correlations encourage the use of the rat in vitro model for the prediction of human oral bioavailabilities of hydrophilic peptide drugs.     

New Model for Studying the Migration of Immune Cells into Intestinal Epithelial Cell Monolayers

A novel cell culture system was constructed to analyze the direct interaction between intestinal epithelial cells and immune cells. Human intestinal epithelial Caco-2 cells were monolayer-cultured on the under side of a permeable membrane (12 μm pore size) in a Millicell insert. Integrated monolayers of Caco-2 cells had formed after 12 days of culture. Human monocyte/macrophage-like THP-1 cells were then added to the upper chamber of the insert, and their migration into the Caco-2 cell monolayers was observed by confocal laser scanning microscopy, after staining the cells with specific antibodies. When MCP-1, a β-chemokine, was added to the apical side of the monolayer, a greater number of THP-1 cells migrated into the Caco-2 cell monolayers. This cell culture system will be useful for studying the behavior of macrophages in the intestinal epithelial cell monolayers at the initial stage of an intestinal immune reaction.     

Caco-2 cell permeability and stability of two d-glucopyranuronamide conjugates of thyrotropin-releasing hormone

Caco-2 cell permeability and stability assays were used as an in vitro model to study the intestinal epithelial transport and stability of two analogues of thyrotropin-releasing hormone (TRH; Pyr-His-Pro-NH2). Peptide 1 (Pyr-His-Pro-D-glucopyranuronamide) was more permeable across the Caco-2 cell monolayer compared with the permeability of the parent TRH peptide (Papp=5.10+/-1.89x10(-6) cm/s c.f. Papp=0.147+/-0.0474x10(-6) cm/s respectively). The permeability of peptide 1 was improved threefold by attaching a 2-aminooctanoic acid moiety to the N-terminus to form peptide 2 (2-aminooctanoic acid-Gln-His-Pro-D-glucopyranuronamide) (Papp=16.3+/-2.47x10(-6) cm/s). The half-life for both peptide 1 and peptide 2 was approximately 20 min in a homogenate of Caco-2 cells compared with the half-life of TRH which is approximately 3 min. It was concluded that the permeability of peptides 1 and 2 was enhanced because of their increased stability, while the higher permeability of peptide 2 compared with peptide 1 may be attributed to its increased lipophilicity which results in enhanced passive diffusion.     

Docosahexaenoic acid and eicosapentaenoic acid-enriched phosphatidylcholine liposomes enhance the permeability, transportation and uptake of phospholipids in Caco-2 cells

The influence of docosahexaenoic acid (DHA)- and eicosapentaenoic acid (EPA)-enriched phosphatidylcholine (PC) on the permeability, transport and uptake of phospholipids was evaluated in Caco-2 cells. The cells were grown on permeable polycarbonate transwell filters, thus allowing separate access to the apical and basolateral chambers. The monolayers of the cells were used to measure lucifer yellow permeability and transepithelial electrical resistance (TEER). Transcellular transportation of diphenylhexatriene (DPH) labeled-PC small unilamellar vesicles (SUV) from the apical to basolateral chamber, and uptake of the same SUV was monitored in the cell monolayers. Cell-membrane perturbation was evaluated to measure the release of lactate dehydrogenase and to determine the cell viability with sodium 2-(4-iodophenyl)-3-(4-nitrophenyl) -5-(2, 4-disulfophenyl)-2H-tetrazolium dye reduction assay. The lucifer yellow flux was 1.0 and 1.5 nmol/h/cm² with 50 μM PC, and 17.0 and 23.0 nmol/h/cm² with 100 μM PC when monolayers of Caco-2 cells were treated with DHA- and EPA-enriched PC, respectively. TEER decreased to 24 and 27% with 50 and 100 μM DHA-enriched PC, and to 25 and 30% with 50 and 100 μM EPA-enriched PC, respectively. Our results show that DHA- and EPA-enriched PC increases tight junction permeability across the Caco-2 cell monolayer whereas soy PC has no effect on tight junction permeability. Transportation and uptake of DHA- and EPA-enriched PC SUV differed significantly (P < 0.01) from those of soy PC SUV at all doses. We found that PC SUV transported across Caco-2 monolayer and was taken up by Caco-2 cells with very slight injury of the cell membrane up to 100 μM PC. Lactate dehydrogenase release and cell viability did not differ significantly between the treatment and control, emphasizing that injury was minimal. Our results suggest that DHA- and EPA-enriched PC enhance the permeability, transport and uptake of PC SUV across monolayers of Caco-2 cells. (Mol Cell Biochem xxx: 1–9, 2005)     

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

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.     

Study on the intestinal permeability of lamivudine using Caco-2 cells monolayer and Single-pass intestinal perfusion

BackgroundThe aim of this work is to investigate the intestinal permeability of lamivudine and explore its absorption mechanism.MethodCaco-2 cells monolayer and single-pass intestinal perfusion (SPIP) were selected for the investigation of lamivudine under different conditions, such as different concentration, absorption time, bidirectional transportation, and transportation with efflux transporters inhibitor. The concentration of lamivudine both in Caco-2 cells monolayer samples and SPIP samples was detected by HPLC-UV. Then the permeability parameters were calculated.ResultsThe established HPLC-UV method reach the requirements for detection. There is no statistically difference between absorption parameters of lamivudine both in Caco-2 cells monolayer and SPIP (P > 0.05) under different dose groups. After transportation with efflux transporters inhibitor, the efflux rate of lamivudine in three dose groups was significantly decreased from 2.67, 2.59 and 2.59 to 1.78, 1.61, and 1.81 respectively. Lamivudine exhibits an absorption mechanism of passive diffusion.ConclusionThe absorption of lamivudine may be related to efflux transporters. In addition, lamivudine is a moderate-permeability drug in Biopharmaceutics Classification System.     

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

Milk and dairy products provide a lot of valuable nutritive elements. They are also sources of biologically active peptides, including β-casomorphins that manifest the properties of morphine. An activity of DPPIV seems to be most crucial factor decreasing the efficiency of the β-casomorphin-7 (BCM7) transport. The increase of BCM7 concentration in blood may intensify symptoms of apparent life threatening events (ALTE), autism, schizophrenia, and allergy. This study aimed at identifying the influence of several selected substances on a transport efficiency of bovine BCM7 through an intestinal monolayer in a Caco-2 cell model system. Applying the ELISA method, the permeability coefficient of BCM7 through the Caco-2 monolayer was calculated. TEER values were used to evaluate the integrity of Caco-2 cell monolayers. An increase of glucose and Ca²⁺ concentrations in the culture medium was accompanied by an increase of the BCM7 transport efficiency. The lowest permeability coefficients of BCM7 were observed for the membranes with high electrical resistances. The transport was enhanced in the presence of milk infant formulas, whereas no changes were observed when using μ-opioid receptor antagonist (casoxin-6). The results may be useful in understanding the pathogenesis of inflammation and food allergy in infants.     

Insulin and IGF-1 Receptors in A Human Intestinal Adenocarcinoma Cell Line (Caco-2): Regulation of Na+ Glucose Transport Across the Brush Border

Abstract

Both insulin and IGF–1 receptors are present in intestinal mucosal cells, although their role in this tissue is unclear. We have characterized these receptors in a human adenocarcinoma cell line, Caco-2, and examined their role in the regulation of glucose transport and absorption in these cells. The Caco-2 cells demonstrated specific insulin and IGF-1 receptors. They also bound cytochalasin B, suggesting the presence of a glucose transporter-like protein. When grown on membranes, the Caco-2 cells formed columnar, bipolar cells with tight junctions. The monolayer selectively transported D-glucose, and methyl-D-glucose, with complete exclusion of L-glucose, D-mannitol and inulin. The absorption of glucose across the monolayer occurred via a Na⁺/glucose cotransporter, as indicated by a change in short circuit current after addition of glucose to the apical membrane. When examined under several conditions, neither insulin nor IGF-1 had an affect on the transport of glucose across the Caco-2 monolayer, nor the production of lactate by the cells. It is concluded that the insulin and IGF-1 receptors of Caco-2 cells do not regulate glucose transport.     

A new antigenic epitope appears in the catalytic subunit of viscumin during intracellular transport

The plant toxin viscumin (60 kD) consists of B- (binding) and A- (active) subunits joined by a disulfide bond. The B-subunit is a lectin interacting with galactose-containing glycolipids and glycoproteins of the cell surface. The A-subunit possesses N-glycosidase activity which modifies 28S ribosomal RNA. This results in irreversible inhibition of protein synthesis. After binding and receptor-mediated endocytosis viscumin-containing vesicles are transported to endoplasmic reticulum where the A- (catalytic) subunit is subsequently translocated to cytosol. It is possible that translocation of A-subunit requires its unfolding. For identification of epitopes which might appear during such unfolding, we developed hybridomas producing monoclonal antibodies against denatured viscumin A-chain. Resistance of hybridoma cells to cytotoxic action of viscumin suggests antibody–toxin interaction inside these cells. TA7 hybridoma cells against an epitope which appears only in denatured viscumin are insensitive to the toxin. This suggests that antibody–toxin interaction occurs before transmembrane translocation of the catalytic A-chain into the cytoplasm. Consequently, toxin resistance of TA7 hybridoma cells implies the appearance of a new epitope in viscumin during its intracellular transportation inside of vesicles. Sixty five octapeptides have been synthesized and epitopes have been identified for monoclonal TA7 antibody and immune mouse serum by means of ELISA. Based on the epitopic mapping the peptide A96-ETHLFTGT-T105 was chemically synthesized and binding of this peptide to the monoclonal antibody TA7 and conformation of antigenic determinant (L100-FTGT-T105) was investigated by means of ¹H-NMR spectroscopy.     

Expression of SLC30A10 and SLC23A3 Transporter mRNAs in Caco-2 Cells Correlates with an Increase in the Area of the Apical Membrane

Drug bioavailability studies commonly employ in vitro barrier tissue models consisting of epithelial and endothelial cells. These experiments require that the cell barrier quality be assessed regularly, which is usually performed using various labeled substrates and/or evaluation of transepithelial (transendothelial) electrical resistance (TEER). This technique provides information on the integrity of the monolayer, but not on differentiation-induced changes in the cell morphology. The present work shows that impedance spectroscopy can be applied to monitor both the integrity of the monolayer and the morphological changes of Caco-2 cells. The growth kinetics of the apical membrane was determined by calculating the electrical capacitance of the cell monolayer. In the course of differentiation, the most pronounced changes in the expression levels were observed for the mRNAs that encode SLC30A10 and SLC23A3 transporters. Their increase correlated with an increase in the apical membrane area, indicating that SLC30A10 and SLC23A3 mRNA levels assessed by qRT-PCR may be employed as cell differentiation biomarkers in Caco-2 models.     

Transepithelial permeability of myricitrin and its degradation by simulated digestion in human intestinal Caco-2 cell monolayer

Myricitrin permeated the human intestinal Caco-2 cell monolayer via the paracellular pathway in a time- and concentration-dependent manner. Myricitrin was not conjugated by Caco-2 cells. Myricitrin was degraded by simulated intestinal digestion, but permeability did not change significantly.     

Expression levels of heat shock proteins in enterocyte-like Caco-2 cells after exposure to Salmonella enteritidis

The enterocytes of the small intestine are occasionally exposed to pathogenic bacteria, such as Salmonella enteritidis 857, an etiologic agent of intestinal infections in humans. The expression of the heat shock response by enterocytes may be part of a protective mechanism developed against pathogenic bacteria in the intestinal lumen. We aimed at investigating whether S. enteritidis 857 is able to induce a heat shock response in crypt- and villus-like Caco-2 cells and at establishing the extent of the induction. To establish whether S. enteritidis 857 interfered with the integrity of the cell monolayer, the transepithelial electrical resistance (TEER) of filter-grown, differentiated (villus-like) Caco-2 cells was measured. We clearly observed damage to the integrity of the cell monolayer by measuring the TEER. The stress response was screened in both crypt- and villus-like Caco-2 cells exposed to heat (40-43 degrees C) or to graded numbers (10(1)-10(8)) of bacteria and in villus-like cells exposed to S. enteritidis 857 endotoxin. Expression of the heat shock proteins Hsp70 and Hsp90 was analyzed by polyacrylamide gel electrophoresis and immunoblotting with monoclonal antibodies. Exposure to heat or Salmonella resulted in increased levels of Hsp70 and Hsp90 in a temperature-effect or Salmonella-dose relationship, respectively. Incubation of Caco-2 cells with S. enteritidis 857 endotoxin did not induce heat shock gene expression. We conclude that S. enteritidis 857 significantly increases the levels of stress proteins in enterocyte-like Caco-2 cells. However, our data on TEER clearly indicate that this increase is insufficient to protect the cells.     

Regulation of protein phosphatase 2A by hydrogen peroxide and glutathionylation

The regulation of protein phosphatase 2A (PP2A) and protein threonine phosphorylation by H(2)O(2) was determined in Caco-2 cell monolayer. Incubation with H(2)O(2) (20 microM) resulted in threonine phosphorylation of a cluster of proteins at the molecular mass range of 170-250 kDa. PKC activity and plasma membrane localization of several isoforms of PKC were not affected by H(2)O(2). However, H(2)O(2) reduced 80-85% of okadaic acid-sensitive protein phosphatase activity. Immunocomplex protein phosphatase assay demonstrated that H(2)O(2) reduced the activity of PP2A, but not that of PP2C or PP1. Oxidized glutathione inhibited PP2A activity in plasma membranes prepared from Caco-2 cells and the phosphatase activity of an isolated PP2A. PP2A activity was also inhibited by N-ethylmaleimide, iodoacetamide, and p-chloromercuribenzoate. Inhibition of PP2A by oxidized glutathione was reversed by reduced glutathione. Glutathione also restored the PP2A activity in plasma membranes isolated from H(2)O(2)-treated Caco-2 cell monolayer. These results indicate that PP2A activity can be regulated by glutathionylation, and that H(2)O(2) inhibits PP2A in Caco-2 cells, which may involve glutathionylation of PP2A.     

Concurrent determination of topotecan and model permeability markers (atenolol, antipyrine, propranolol and furosemide) by reversed phase liquid chromatography: utility in Caco-2 intestinal absorption studies

A simple, sensitive, specific and high-resolution reversed-phase liquid chromatographic method utilizing ultraviolet detection has been developed and validated for simultaneous determination of topotecan and four intestinal permeability markers (atenolol, antipyrine, propranolol and furosemide) as suggested by US-FDA. Chromatography was carried out on C-18 column with mobile phase comprising water (pH 3.0) and acetonitrile gradient pumped at a flow rate of 1 ml min(-1). The validation parameters included specificity, accuracy, precision, sensitivity and stability studies. Topotecan, an anti-cancer drug widely used in metastatic carcinoma, is a P-glycoprotein substrate having oral bioavailability of 30% with large inter-patient variability. The present method was successfully applied for demonstrating P-gp mediated transport of topotecan and its inhibition using verapamil in Caco-2 cell monolayer. The method can be used in identification of novel P-gp inhibitors for topotecan and estimating the contribution of P-gp in affecting oral bioavailability of topotecan. The other applications of method include its use in validation of Caco-2 monolayer assay for getting biowaiver based on Biopharmaceutic Classification System and its extrapolation to in situ and/or in vivo studies.