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Objective: Bromotetrandrine (W198) was reported as a P-glycoprotein (P-gp) inhibitor. We aimed to prepare oral W198 micelles following by paclitaxel (PTX) micelles (W198/PTX micelles) to improve the clinical application of PTX., Significance: The poor water solubility, intestinal permeability, and multidrug resistance (MDR) of PTX can be improved in the multistage oral delivery system., Methods: The novel W198/PTX oral micelles were developed by water-bath ultrasound method and were evaluated in vivo and in vitro in 4T1 orthotopic tumor-bearing mice model., Results: PTX micelles and W198 micelles were prepared to be round and uniform. W198 micelles pre-administrated group showed higher cellular uptake efficiency of PTX on Caco-2 cells and more prominent cytotoxicity compared with W198-untreated group on 4T1 cells. The oral bioavailability of W198/PTX micelles group was nearly 5.7-folds higher than the PTX micelles only group. In addition, W198/PTX micelles showed enhanced anticancer efficacy., Conclusions: We established a multistage oral delivery system to improve oral bioavailability and anticancer efficacy of PTX.

Colorectal cancer is frequently diagnosed at an advanced stage due to the absence of early clinical indicators. Hence, the identification of new targeting molecules is crucial for an early detection and development of targeted therapies. This study aimed to identify and characterize novel peptides specific for the colorectal cancer cell line RKO using a phage-displayed peptide library. After four rounds of selection plus a negative step with normal colorectal cells, CCD-841-CoN, there was an obvious phage enrichment that specifically bound to RKO cells. Cell-based enzyme-linked immunosorbent assay (ELISA) was performed to assess the most specific peptides leading to the selection of the peptide sequence CPKSNNGVC. Through fluorescence microscopy and cytometry, the synthetic peptide RKOpep was shown to specifically bind to RKO cells, as well as to other human colorectal cancer cells including Caco-2, HCT 116 and HCT-15, but not to the normal non-cancer cells. Moreover, it was shown that RKOpep specifically targeted human colorectal cancer cell tissues. A bioinformatics analysis suggested that the RKOpep targets the monocarboxylate transporter 1, which has been implicated in colorectal cancer progression and prognosis, proven through gene knockdown approaches and shown by immunocytochemistry co-localization studies. The peptide herein identified can be a potential candidate for targeted therapies for colorectal cancer.

This study aimed to screen the stability, disintegration, and swelling behavior of chemically modified anionic polymers. Investigated polymers were well-known and widely used staples of the pharmaceutical and medical field, namely, alginate (AL), carboxymethyl cellulose (CMC), polycarbophil (PC), and hyaluronic acid (HA). On the basis of amide bond formation between the carboxylic acid moieties of anionic polymers and the primary amino group of the modification ligand cysteine (CYS), the modified polymers were obtained. Unmodified polymers served as controls throughout all studies. With the Ellman's assay, modification degrees were determined of synthesized polymeric excipients. Stability assay in terms of erosion study at physiological conditions were performed. Moreover, water uptake of compressed polymeric discs were evaluated and further disintegration studies according to the USP were carried out to define the potential ranking. Results ranking figured out PCCYS > CMCCYS > HACYS > ALCYS in terms of water uptake capacity compared to respective controls. Cell viability assays on Caco-2 cell line as well as on RPMI 2650 (ATTC CCL30) proved modification not being harmful to those. Due to the results of this study, an intense screening of prominent anionic polymer derivate was performed in order to help the pharmaceutical research for the best choice of polymeric excipients for developments of controlled drug release systems.

Context: Prediction of the in vivo absorption of poorly soluble drugs may require simultaneous dissolution/permeation experiments. In vivo predictive media have been modified for permeation experiments with Caco-2 cells, but not for excised rat intestinal segments., Objective: The present study aimed at improving the setup of dissolution/permeation experiments with excised rat intestinal segments by assessing suitable donor and receiver media., Methods: The regional compatibility of rat intestine in Ussing chambers with modified Fasted and Fed State Simulated Intestinal Fluids (Fa/FeSSIF mod ) as donor media was evaluated via several parameters that reflect the viability of the excised intestinal segments. Receiver media that establish sink conditions were investigated for their foaming potential and toxicity. Dissolution/permeation experiments with the optimized conditions were then tested for two particle sizes of the BCS class II drug aprepitant., Results: Fa/FeSSIF mod were toxic for excised rat ileal sheets but not duodenal sheets, the compatibility with jejunal segments depended on the bile salt concentration. A non-foaming receiver medium containing bovine serum albumin (BSA) and Antifoam B was nontoxic. With these conditions, the permeation of nanosized aprepitant was higher than of the unmilled drug formulations., Discussion: The compatibility of Fa/FeSSIF mod depends on the excised intestinal region. The chosen conditions enable dissolution/permeation experiments with excised rat duodenal segments. The experiments correctly predicted the superior permeation of nanosized over unmilled aprepitant that is observed in vivo., Conclusion: The optimized setup uses FaSSIF mod as donor medium, excised rat duodenal sheets as permeation membrane and a receiver medium containing BSA and Antifoam B.

Context: Transporting drugs through the lymphatic system has attracted increasing attention. Lipid-based formulations have been proved to be an effective way to improve systemic bioavailability of highly lipophilic drugs by increasing intestinal lymphatic transport., Objective: The formulation of polymer micelle was developed for probucol to improve its intestinal lymphatic transport., Materials and Methods: Methoxy-polyethylenelglycol-distearyl phosphatidyl-ethanolamine (mPEG-DSPE) polymer was chosen to develop the micelles for probucol. The physicochemical properties were characterized. Caco-2 cell model, unconscious and conscious lymph duct cannulated rat models were established for in vitro and in vivo evaluation of lymphatic transport., Results: In vitro evaluation in the Caco-2 cell model showed that the micellar formulation could significantly increase the uptake and transport of probucol. The study in unconscious and conscious lymph duct cannulated rat models further verified the significant enhancement of lymphatic transport of probucol by mPEG-DSPE micelles., Discussion and Conclusion: These results suggested that mPEG-DSPE micellar formulation could provide a useful alternative approach for improving the lymphatic transport of hydrophobic compounds.

Tartary buckwheat is a gluten-free crop with great potential as a wheat substitute. Iron (Fe) is an important mineral element in staple foods which is required in sufficient bioaccessible quantities. The aim of the study was to investigate how processing of grains into groats (hydrothermal processing to remove the husk) and sprouts (7-day-old seedlings) affected Fe speciation (Fe(2+) or Fe(3+)), Fe ligand composition and Fe bioaccessibility to human Caco-2 cells. Groats contained the least Fe (23.8 ± 1.65 mg kg(-1)) and the lowest amounts of Fe(2+) (8%). Grains and sprouts had comparable Fe concentrations (78.2 ± 2.65 and 68.9 ± 2.73 mg kg(-1)) and similar proportions of Fe(2+) (15% and 18%). The main ligands for Fe in Tartary buckwheat material were phytate and citrate. Phytate was less abundant in sprouts, which did not correlate with greater Fe bioaccessibility. Iron bioaccessibility was 4.5-fold greater for grains than groats, suggesting that Fe is more bioaccessible in the husk than in the rest of the grain., (Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.)

This study evaluated the health-promoting properties of Pinot Noir juices (Vitis vinifera L.) obtained at different maceration times after pulsed electric fields (PEF) using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and human intestinal Caco-2 cells assays. Juice quality, anthocyanins, total phenolics and vitamin C were also determined. The evaluation of bioprotective capacity of the juice against H2O2-induced oxidative stress in Caco-2 cells was determined using biomarkers for cellular health and integrity: cell viability and lactate dehydrogenase (LDH) leakage. Compared to untreated grape juice, PEF pre-treatment on grapes enhanced the release of the major anthocyanin found in Pinot Noir, i.e. malvidin-3-O-glucoside (+224%). Increase in the content of total phenolic (+61%) and vitamin C (+19%) as well as improvement in the DPPH scavenging activity (+31%) and bioprotective capacity (+25% for cell viability and +30% for LDH leakage) were observed in grape juices following PEF treatment. Bioprotective capacity determined by the cellular biomarkers had significant linear correlations with malvidin-3-O-glucoside content (0.71⩽r⩽0.73) whereas DPPH scavenging activity was not well correlated with malvidin-3-O-glucoside (r=0.30) and total phenolics (r=0.30). Therefore, evaluation of the bioprotective capacities using Caco-2 cell assay performed in this study makes a novel contribution to the current knowledge that demonstrates the capability of PEF technology to produce plant-based foods with better phytochemical composition and exhibiting the capacity to protect cells from oxidative stress., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The aim of the study was to evaluate the ability of poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NP) to enhance lutein bioavailability. The bioavailability of free lutein and PLGA-NP lutein in rats was assessed by determining plasma pharmacokinetics and deposition in selected tissues. Lutein uptake and secretion was also assessed in Caco-2 cells. Compared to free lutein, PLGA-NP increased the maximal plasma concentration (Cmax) and area under the time-concentration curve in rats by 54.5- and 77.6-fold, respectively, while promoting tissue accumulation in the mesenteric fat and spleen. In comparison with micellized lutein, PLGA-NP lutein improved the Cmax in rat plasma by 15.6-fold and in selected tissues by ⩾ 3.8-fold. In contrast, PLGA-NP lutein had a lower uptake and secretion of lutein in Caco-2 cells by 10.0- and 50.5-fold, respectively, compared to micellized lutein. In conclusion, delivery of lutein with polymeric NP may be an approach to improve the bioavailability of lutein in vivo., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The transepithelial transport of an antioxidative and ACE inhibitory peptide, VLPVPQK (named peptide C) derived from casein hydrolysates was investigated along with extensively studied opioid peptide β-casomorphin using a human intestinal cell (Caco-2) monolayer. The susceptibility to the brush-border peptidases and route of transepithelial transport were observed to be the primary factors influencing the transport of these peptides. The apical to basal transport mechanism was studied using bradykinin as control as it shows resistance to cellular peptidases and its route of transepithelial transport had been established. VLPVPQK and BCM 5 were hydrolyzed by cellular peptidases while bradykinin was found intact. The transport of VLPVPQK (1.0%) was found to be relatively much higher than BCM 5 (0.03%) and bradykinin (0.1%). Interestingly the effect of some inhibitors on the transport of VLPVPQK suggested involvement of PepT1 like transporters/SOPT2 while BCM 5, its hydrolytic product and bradykinin were suggested to be transported mainly via the intracellular transcytosis pathway., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Curcumin (CCM) is a bioactive polyphenolic compound that suffers a low bioavailability because of its low water solubility. In this work β-lactoglobulin (β-Lg) and nanoemulsion were used as carriers to deliver curcumin. The pH stability of β-Lg-CCM was investigated. The digestion of β-Lg-CCM and the nanoemulsion was studied using an in vitro gastrointestinal model. The effect of different carriers on the permeability of curcumin was assessed using the Caco-2 cell monolayer model. The results revealed that the water solubility and the pH stability of curcumin significantly increased by binding with β-Lg. In SDS-PAGE experiments the β-Lg-CCM complex and nanoemulsion were found to be resistant to pepsin digestion but sensitive to trypsin. In the permeability experiment it was shown that the digested nanoemulsion and β-Lg-CCM improved significantly the permeation rate of curcumin., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

This study examined the antibacterial and anti-adhesive properties of pure plant extracts (PPEs) of green tea (GT), black tea (BT) and soybean individually or in combination with milk. Fermented phenolic enriched-milk (fPEM) was prepared by combining PPEs with milk and fermented with lactic acid bacteria. Antimicrobial activity of extracts was evaluated by broth-dilution and agar diffusion assay. Anti-adhesive property of extracts was evaluated in Caco-2 cell model. Results from antibacterial tests showed that PPEs exhibited a dose-dependent growth inhibitory effect. Tea extracts were more effective in inhibiting Gram-positive bacteria while soybean extract exhibited similar effects against all pathogens tested. For fPEM, although total phenolic contents decreased compared with those in PPEs, growth inhibitory effect of fPEM containing tea extracts was greatly enhanced. All extracts showed significant inhibition against pathogen adhesion to Caco-2 cells. In particular, adhesion inhibition against Staphylococcus aureus and Listeria monocytogenes was >89% when fPEM extracts were applied., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Strawberry fruits are highly valued for their taste and nutritional value. However, results describing the bioaccessibility and intestinal absorption of phenolic compounds from strawberries are still scarce. In our study, a combined in vitro digestion/Caco-2 absorption model was used to mimic physiological conditions in the gastrointestinal track and identify compounds transported across intestinal epithelium. In the course of digestion, the loss of anthocyanins was noted whilst pelargonidin-3-glucoside remained the most abundant compound, amounting to nearly 12 mg per 100 g of digested strawberries. Digestion increased the amount of ellagic acid available by nearly 50%, probably due to decomposition of ellagitannins. Only trace amounts of pelargonidin-3-glucoside were found to be absorbed in the intestine model. Dihydrocoumaric acid sulphate and p-coumaric acid were identified as metabolites formed in enterocytes and released at the serosal side of the model., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Collagen XVII has a well-established role as an adhesion molecule and a cell surface receptor located in the type I hemidesmosome of stratified epithelia. Its ectodomain is constitutively shed from the cell surface and suggested to regulate the adhesion, migration, and signaling of cutaneous epithelial cells. Collagen XVII was not previously thought to be expressed by colon epithelial cells. Immunohistochemical analysis of tissue microarray samples of 141 cases of colorectal carcinoma showed that collagen XVII is expressed in normal human colonic mucosa and colorectal carcinoma. In colorectal carcinoma, increased collagen XVII expression was significantly associated with higher TNM stage. It also correlated with infiltrative growth pattern and tumor budding as well as lymph node and distant metastasis. Increased collagen XVII expression was associated with decreased disease-free and cancer-specific survival. Immunofluorescence staining of collagen XVII and its well-known binding partner laminin γ2 chain demonstrated a partial colocalization in normal and tumor tissue. In vitro, the overexpression of murine collagen XVII promoted the invasion of CaCo-2 colon carcinoma cells through Matrigel (BD Biosciences; Bedford, MA). To conclude, this study reports for the first time the expression of collagen XVII in colon epithelium and the association of increased collagen XVII immunoexpression with poor outcome in colorectal carcinoma., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Resveratrol oligomers are biologically active polyphenols found in wine. No information about the bioavailability of these polyphenols is available. In order to discover if the resveratrol oligomers can pass the intestinal barrier, transport of the dimer ε-viniferin and the tetramer hopeaphenol was studied in the Caco-2 transwell system. A flux through the cell monolayer could neither be observed for ε-viniferin nor for hopeaphenol (apparent permeability coefficient (Papp)<1×10(-6)cms(-1)). In contrast, resveratrol showed a Papp of 11.9×10(-6)cms(-1). Nevertheless, about 16-30% of the oligomers were found in the lysed cellular fraction. This leads to the conclusion that the intestinal absorption rate of the two resveratrol oligomers, ε-viniferin and hopeaphenol, is low and negligible when compared to resveratrol. Therefore, it is unlikely that the oligomers could elicit a systemic biological effect after dietary intake. However, the compounds may act locally on the intestinal epithelium., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background and Objective: Early detection of colon adenomas at high risk of progression and early-stage colorectal cancer (CRC) is an effective approach to reduce CRC death rates. Current screening methods lack specificity as they detect many adenomas that will never progress to CRC. The authors aimed to identify cell surface protein biomarkers with extracellular domains that could be targeted for molecular imaging and discriminate low-risk adenomas and normal colon from high-risk adenomas and CRC., Design: Cell surface proteins of five CRC cell lines were biotinylated, isolated and analysed by in-depth proteomics using gel electrophoresis and nanoliquid chromatography coupled to tandem mass spectrometry. Differential expression in adenomas and CRCs was based on mRNA expression and verified by immunohistochemical staining of tissue microarrays., Results: In total, 2609 proteins were identified in the cell surface fractions. Of these, 44 proteins were selected as promising cell surface candidate biomarkers for adenoma-to-carcinoma progression based on the following criteria: protein identification in at least four out of five cell lines, a predicted (trans)membrane location and increased mRNA expression in CRCs compared to adenomas. Increased protein expression in high-risk adenomas and CRCs compared to low-risk adenomas was confirmed by immunohistochemistry for glucose transporter type 1 (gene symbol SLC2A1; p<0.00001) and prion protein (gene symbol PRNP; p<0.005)., Conclusion: This study revealed glucose transporter type 1, prion protein and 42 other cell surface candidate biomarkers for adenoma-to-carcinoma progression that could potentially serve as targets for emerging molecular imaging modalities like optical imaging, ¹⁹F-MRI and positron emission tomography.

Aim: To characterise differences between three widely used colorectal cancer cell lines using ultrastructural selective staining for glycogen to determine variation in metastatic properties., Methods: Transmission electron microscopy was used in this investigation to help identify intracellular structures and morphological features which are precursors of tumor invasion. In addition to morphological markers, we used selective staining of glycogen as a marker for neoplastic cellular proliferation and determined whether levels of glycogen change between the three different cell lines., Results: Ultrastructural analysis revealed morphological differences between the cell lines, as well as differentiation into two sub-populations within each cell line. Caco-2 cells contained large glycogen deposits as well as showing the most obvious morphological changes between the two sub-populations. SW480 cells also contained large glycogen stores as well as deep cellular protrusions when grown on porous filter membranes. HT-29 cells had trace amounts of glycogen stores with few cellular projections into the filter pores and no tight junction formation., Conclusion: Morphology indicative of metastatic properties coincided with larger glycogen deposits, providing strong evidence for the use of selective staining to determine the neoplastic properties of cells.

Caco-2 cells were used as a model for investigating and comparing the absorption of alpha-tocopherol (Tol) and alpha-tocopheryl acetate (Tac) solubilized in micelles based on a mixture of sodium taurocholate (NaTC) and oleic acid. Surprisingly, the uptake of Tac was found to be similar to that of Tol, and in both cases, the dose-response plots suggest that protein-mediated transport processes were involved. Moreover Tol or Tac were also secreted into the basolateral medium of Caco-2 cells but Tac was mainly hydrolyzed either prior to absorption or intracellularly. The solubilization of Tol or Tac by NaTC on the apical side of the cell monolayer is a prerequisite for the uptake process, although larger amounts of the bile salt are necessary to solubilize Tac than Tol. Caco-2 cells showed hydrolytic activity on Tac, and additional cholesterol esterase may be taken up by the cells, thus increasing the rates of intracellular hydrolysis of Tac. Based on our findings, a scheme is suggested accounting for the absorption of alpha-tocopheryl acetate by enterocytes.

Repeated mild heat shock treatment has been shown to have anti-aging effects on cellular mechanisms in vitro. Among these, the age-associated accumulation of advanced glycation end products (AGEs), such as N(epsilon)-(carboxymethyl)lysine (CML), has been demonstrated to be effectively prevented in glyoxal-exposed human skin fibroblasts following mild heat shock treatment. The biochemical mechanism responsible for this inhibition is not yet known. However, the involvement of heat shock proteins (HSPs) and the misfolded proteins degrading the ubiquitin-proteasome system have been hypothesized. As AGE-modified proteins are likely to be conformationally modified, we investigated whether treatment of human intestinal cells with casein-linked CML or nonprotein-linked CML affects the expression of HSPs and the ubiquitin-proteasome system by using matrix-assisted laser desorption/ionization-time-of-flight tandem mass spectroscopy (after protein separation by two-dimensional gel electrophoresis) and by Western blotting. Compared to nontreated control cells, expression of HSP90, HSP60, HSP70 chaperones, and the proteasome S26 ATPase subunit 2 were significantly upregulated in casein-CML and in CML-treated cells. Exposure of Caco-2 cells to beta-amyloid, a nonglycation product, revealed similar results. In conclusion, the results indicate that CML and casein-linked CML activate the expression of HSPs as well as the proteasome system, which are involved in the degradation of misfolded and possibly glycated proteins. Whether this mechanism is based on binding to cell surface receptors, such as the receptor for AGE, has to be clarified in future studies.

A chemical probe was developed for the detection of the emerging cancer marker galectin-3. The probe contains a benzophenone moiety which covalently attaches itself to the protein upon binding and irradiation. Introduction of a fluorescent label via'click' chemistry allows the labelled proteins to be visualized in a gel. With the probe, selective visualization of galectin-3 in protein mixtures was shown and remarkably even in cell lysates.

To explore the role of transcriptome polymorphism in adaptation of organisms to their environment, we evaluated this parameter for the Escherichia coli/Shigella bacterial species, which is composed of well-characterized phylogenetic groups that exhibit characteristic life styles ranging from commensalism to intracellular pathogenicity. Both the genomic content and the transcriptome of 10 strains representative of the major E. coli/Shigella phylogenetic groups were evaluated using macroarrays displaying the 4290 K12-MG1655 open reading frames (ORFs). Although Shigella and enteroinvasive E. coli (EIEC) are not monophyletic, phylogenetic analysis of the binary coded (presence/absence) gene content data showed that these organisms group together due to similar patterns of undetectable K12-MG1655 genes. The variation in transcript abundance was then analyzed using a core genome of 2880 genes present in all strains, after adjusting RNA hybridization signals for DNA hybridization signals. Nonrandom changes in gene expression during the evolution of the E. coli/Shigella species were evidenced. Phylogenetic analysis of transcriptome data again showed that Shigella and EIEC strains group together in terms of gene expression, and this convergence involved groups of genes displaying biologically coherent patterns of functional divergence. Unlike the other E. coli strains evaluated, Shigella and EIEC are intracellular pathogens, and therefore face similar selective pressures. Thus, within the E. coli/Shigella species, strains exhibiting a particular life style have converged toward a specific gene expression pattern in a subset of genes common to the species, revealing the role of selection in shaping transcriptome polymorphism.

We recently established the critical role of the lipid phosphatase activity of the PTEN tumor suppressor in stabilizing cell-cell contacts and suppressing invasiveness. To delineate the effector systems involved, we investigated the interaction of PTEN with E-cadherin junctional complexes in kidney and colonic epithelial cell lines. PTEN and the p85 regulatory subunit of phosphatidylinositol 3-OH kinase (PI3K) co-immunoprecipitated with E-cadherin and catenins. By using a yeast two-hybrid assay, we demonstrated that PTEN interacted indirectly with beta-catenin by binding the scaffolding protein MAGI-1b. This model was corroborated in various ways in mammalian cells. Ectopic expression of MAGI-1b potentiated the interaction of PTEN with junctional complexes, promoted E-cadherin-dependent cell-cell aggregation, and reverted the Src-induced invasiveness of kidney MDCKts-src cells. In this model, MAGI-1b slightly decreased the activity of AKT, a downstream effector of PI3K. By using dominant-negative and constitutively active AKT expression vectors, we demonstrated that this kinase was included in the pathways involved in Src-induced destabilization of junctional complexes and was necessary and sufficient to trigger invasiveness. We propose that the recruitment of PTEN at adherens junctions by MAGI-1b and the local down-regulation of phosphatidylinositol-3,4,5-trisphosphate pools and downstream effector systems at the site of cell-cell contacts are focal points for restraining both disruption of junctional complexes and induction of tumor cell invasion.

Fluorescent labels are commonly used to investigate the mechanisms of cellular uptake and intracellular distribution of cell-penetrating peptides. However, labels such as fluorescein and rhodamine are relatively large and very lipophilic and may significantly alter physicochemical properties of small peptides. To minimize the impact of the fluorescent probe on a tetrapeptide, we substituted one of the amino acids (Lys4) in a tetrapeptide ([Dmt1]DALDA, Dmt-D-Arg-Phe-Lys-NH2 where Dmt = 2',6'-dimethyltyrosine) with two different fluorescent amino acids (beta-dansyl-L-alpha,beta-diaminopropionic acid (dnsDap4) or beta-anthraniloyl-L-alpha,beta-diaminopropionic acid (atnDap4)). Initial studies with confocal laser scanning microscopy (CLSM) showed very different localization patterns for the two fluorescent analogs, with [Dmt1,atnDap4]DALDA showing mitochondrial localization and [Dmt1,dnsDap4]DALDA showing diffuse cytoplasmic localization. Studies with isolated mouse liver mitochondria suggested that [Dmt1,dnsDap4]DALDA targeted the mitochondrial matrix resulting in mitochondrial depolarization, opening of the permeability transition pore, mitochondrial swelling, and rapid release of the peptide into the cytoplasm. In contrast, [Dmt1,atnDap4]DALDA was retained in the inner mitochondrial membrane and did not induce mitochondrial swelling. Furthermore, [Dmt1,atnDap4]DALDA protected mitochondria against Ca2+-induced swelling. Importantly, the unlabeled parent peptide [Dmt1]DALDA behaved like [Dmt1,atnDap4]DALDA and was mitoprotective. These findings suggest that experimental results obtained with fluorescent labels must be interpreted with caution, and the use of multiple fluorophores, together with confirmation using the original or radiolabeled molecule, is recommended.

Loss of heterozygosity (LOH) on 8p occurs at high frequencies in many tumor types, including colorectal carcinoma (CRC). We previously used microcell-mediated chromosome transfer (MMCT) into the CRC cell line SW620 to map a approximately 7.7-Mb colorectal cancer-suppressor region (CRCSR) at 8p22-23.1. In the current study, we transferred small fragments of this CRCSR into SW620 to refine the region further. Two microcell hybrids containing a 321- to 898-kb region around the D8S552 marker at 8p23.1 showed suppression of soft agar clonicity and tumorigenicity in athymic mice when compared to control cell lines. These data suggest that the putative colorectal tumor-suppressor gene is within this small region. We analyzed two candidate genes within this region: FLJ23749 and KIAA1456. Expression of both genes was detected in normal colonic crypt cells and in mucosa. Quantitative reverse transcriptase polymerase chain reaction showed downregulation of KIAA1456 in 9 of 12 primary colorectal tumors compared to matching normal mucosa, but normal or increased expression of FLJ23749. FLJ23749 was expressed in all CRC cell lines tested; however, KIAA1456 was downregulated in three cell lines, including SW620, and was restored in the suppressed microcell hybrids. 5'aza-2'Deoxycytidine treatment of the downregulated cell lines restored expression of KIAA1456, but bisulfite genomic sequencing did not show a correlation between promoter methylation and expression. Forty percent of the primary tumors showed LOH at this CRCSR locus, and mutation analysis revealed somatic mutations in 1 of 88 primary colorectal tumors for both KIAA1456 and FLJ23749. Despite the rarity of somatic mutations, the expression data suggest that KIAA1456 is still a candidate for the putative 8p colorectal cancer tumor-suppressor gene., (Copyright 2004 Wiley-Liss, Inc.)

The effect of fatty acids on LXR (liver X receptors)-mediated enhancement of ABCA1 (ATP-binding cassette transporter A1) expression and cholesterol efflux was investigated in human intestinal cells CaCo-2. LXR activation by T0901317 increased basolateral cholesterol efflux to lipoprotein particles isolated at a density of 1.21 g/ml or higher. Oleic and arachidonic acids attenuated the amount of cholesterol isolated from these particles. Stearic, linoleic and docosahexaenoic acids also decreased cholesterol efflux from basolateral membranes, with the polyunsaturated fatty acids being the most potent. Although oleic, arachidonic and docosahexaenoic acids modestly decreased ABCA1 mRNA levels in response to LXR activation, stearic and linoleic acids did not. Except for oleic acid, all fatty acids substantially attenuated an increase in ABCA1 mass secondary to LXR activation. Inhibiting acyl-CoA:cholesterol acyltransferase activity prevented the decrease in cholesterol efflux caused by oleic acid. Thus, in response to LXR activation, all fatty acids decreased the efflux of cholesterol from the basolateral membrane of CaCo-2 cells. Although modest suppression of ABCA1 gene expression by oleic, arachidonic and docosahexaenoic acids cannot be completely excluded as a mechanism, the predominant effect of fatty acids on ABCA1 expression and cholesterol efflux is at a post-transcriptional level.

The intestinal Fc receptor, FcRn, functions in the maternofetal transfer of gamma globulin (IgG) in the neonatal rodent. In humans, most of this transfer is presumed to occur in utero via the placenta. Although the fetus swallows amniotic fluid that contains immunoglobulin, it is unknown whether this transfer also occurs via the fetal intestine. A human FcRn has been identified in the syncytiotrophoblast that mediates the maternofetal transfer of antibody. It has also been identified in human fetal intestine and is postulated to function in IgG transport. We hypothesize that the human fetal intestinal FcRn may play a role in IgG transport from the amniotic fluid into the fetal circulation. The aim of this study was to characterize the distribution of the FcRn along the human fetal intestine. Lysates prepared from human fetal intestine and from a nonmalignant human fetal intestinal epithelial cell line (H4) were subjected to Western blot analysis and probed using anti-FcRn antibodies. A 42-kD band, consistent with the known molecular weight of the FcRn, was detected along the human fetal intestine and in H4 cells. Expression of the human FcRn was confirmed with immunohistochemistry. Our study demonstrates the expression of FcRn along the human fetal intestine and in a human nonmalignant fetal intestinal epithelial cell line (H4), which by location indicates that FcRn could play a role in the uptake and transport of IgG in the human fetus.

Caco-2 cells offer a means to rapidly screen permeability of drug candidates, allowing pharmaceutical companies to eliminate candidates unable to cross the intestinal barrier early in the discovery process. This screening process is typically performed by conventional liquid chromatography/tandem mass spectrometry (LC/MS/MS), which can require time-consuming method development. An alternative to LC/MS/MS, automated nanoelectrospray tandem mass spectrometry (nanoESI-MS/MS), is introduced. This novel approach requires an off-line ZipTip desalting step followed by automated nanoESI-MS/MS, using the NanoMate 100 and ESI Chip. In addition to reduced method development time, automated nanoESI-MS/MS also offers no carry-over between samples, low sample consumption, and ease-of-use as compared with conventional pulled-capillary nanoelectrospray. Furthermore, the infusion system described has the potential to be high-throughput. A comparison of Caco-2 samples analyzed both by LC/MS/MS and by automated nanoESI-MS/MS is presented. The permeability and recovery data of the two compounds analyzed in this study obtained from conventional LC/MS/MS and by automated nanoESI-MS/MS were in excellent agreement., (Copyright 2003 John Wiley & Sons, Ltd.)

Rapid renewing epithelia such as the epidermis and the intestinal epithelium are maintained by proliferation of undifferentiated stem cells located at specific locations. Recent experiments indicate that stem cells from adult organs might be able to populate tissues other than their tissue of origin. Such findings open the possibility that adult stem cells from different tissues might share common markers. We investigated this by two different approaches. In a first approach we compared the expression profiles from epidermal and intestinal epithelial cells at various stages of differentiation. We found that 108 of 1,176 genes analyzed were expressed above background in either keratinocytes or enterocytes and, among these, only 16 genes were expressed in both cell types. Of these 16 genes expressed in both cell types, only five displayed the same shift in expression level during cellular differentiation. Interestingly, all five genes were downregulated during cellular differentiation and represented ubiquitously expressed genes. In the second approach we analyzed the expression of the CC chemokine receptor 6 (CCR6), which we have recently identified as an early differentiation marker of epidermal cells, in the intestine. This analysis demonstrates that the CCR6 protein is found in enterocytes at later stages of differentiation.

Soy isoflavonoids have attracted much attention because of their estrogenic activity. To study the intestinal absorption of the isoflavonoids, we investigated the cellular uptake and metabolism of genistein and daidzein and their glucosides, genistin and daidzin, by Caco-2 cell monolayers as a model of the human intestinal epithelium. When Caco-2 monolayers were incubated with genistein or daidzein at 10 micromol/L from the apical (mucosal) side, aglycone was lost from the apical solution for 2.0 h (P < 0.05) and the glucuronide/sulfates appeared at the level of 1-2 micromol/L. In the basolateral (serosal) solution, both intact aglycones and their glucuronide/sulfates increased (P < 0.05) with time and reached approximately 20 and 15% of the initial dose, respectively. The transport of their glucosides, genistin and daidzin, through Caco-2 monolayers was less than one tenth that of the aglycones. The cellular metabolism of genistein was compared with quercetin, kaempferol, luteolin and apigenin. Only genistein aglycone was transported intact to the basolateral solution. Transport was greater (P < 0.05) than that of flavonoid aglycones and was without an appreciable decrease of transepithelial resistance. Radical scavenging activity was not related to the susceptibility to conjugation of flavonoids/isoflavonoids. Affinity to the liposomal membrane was increased in the order of genistin = daidzin < daidzein < genistein << flavonoid aglycones. These results strongly suggest that isoflavone aglycones are taken up into enterocytes more efficiently than their glucosides because of their moderate lipophilicity. Furthermore, they are generally transported to the basolateral side in intact form in contrast to flavonoids, probably due to their unique isoflavonoid structure.

The 5' boundary of the chromosomal domain of the human apolipoprotein B (apoB) gene in intestinal cells has been localized and characterized. It is composed of two kinds of boundary elements; the first, functional boundary is an insulator activity exhibited by a 1.8 kb DNA fragment located between -58 and -56 kb upstream of the human apoB promoter. In this region, an enhancer-blocking activity has been mapped to a CTCF binding site that is located upstream of two apoB intestinal enhancers (IEs), the 315 IE and the 485 IE. The CTCF site represents a boundary between two types of chromatin structure: an open, DNaseI-sensitive region 3' of the CTCF site containing the intestinal regulatory elements and a closed, DNaseI-resistant region 5' of the CTCF site. The 1.8 kb fragment harboring the CTCF site also insulated mini-white transgenes against position effects in Drosophila melanogaster. The second, structural boundary is represented by a nuclear matrix attachment region (MAR), situated about 3 kb 5' of the CTCF site. This MAR may represent the 5' anchorage site for a chromosomal loop that functions to bring the intestinal regulatory elements closer to the apoB promoter.

Reactive oxygen species are implicated in the development of gastrointestinal pathologies. Caco-2 monolayers are routinely used to study intestinal oxidative stress and its potential amelioration by pharmacological agents or dietary micronutrients. Little is known of the plasticity of Caco-2 antioxidant defenses with changes in culture conditions. We examined whether the frequency of culture media renewal alters the antioxidant-prooxidant status and integrity of Caco-2 monolayers. In comparison to monolayers subject to daily media renewal, increasing periods between media exchange resulted in varying degrees of suppression of catalase, glutathione reductase, and glutathione-S-transferase activity. No significant changes to superoxide dismutase activity, total glutathione, or intracellular ROS profiles were observed. Alkaline phosphatase activity, as a marker of differentiation, and mean monolayer cell population size were also unaffected. We suggest that Caco-2 antioxidant enzyme activities are differentially sensitive to changes in culture conditions. Studies employing this cell line for antioxidant-oxidative stress interactions will need to evaluate responses with respect to culture regime.

Purpose: The purpose of this study was to elucidate the mechanisms by which an HMG-CoA reductase inhibitor, atorvastatin (an organic acid with a pKa of 4.46), was transported in the secretory and absorptive directions across Caco-2 cell monolayers., Methods: Caco-2 cells were grown on polycarbonate membrane inserts in 6-well Snapwell plates (Costar). The permeability of radiolabeled compounds across Caco-2 cell monolayers was determined using a side-by-side diffusion apparatus (NaviCyte) and an automated liquid handler (Hamilton Microlab 2200). The apical uptake of 14C-atorvastatin was also determined in Caco-2 cells. Cyclosporin A (20 microM) was present in the uptake media to block potential P-glycoprotein-mediated atorvastatin efflux., Results: Polarized permeation of atorvastatin was observed with the basolateral-to-apical (B-to-A) permeability being 7-fold greater than the A-to-B permeability (35.6 x 10(-6) and 4.9 x 10(-6) cm/s, respectively). The secretion of atorvastatin was a saturable process with an apparent Km of 115 microM. The B-to-A permeability of atorvastatin was significantly reduced by cyclosporin A (10 microM), verapamil (100 microM), and a P-glycoprotein specific monoclonal antibody, UIC2(10 microg/ml) (43%, 25%, and 13%, respectively). Furthermore, both CsA and verapamil significantly increased the A-to-B permeability of atorvastatin by 60%; however, UIC2 did not affect the A-to-B permeability of atorvastatin. CsA uncompetitively inhibited the B-to-A flux of atorvastatin with a Ki of 5 microM. In addition, atorvastatin (100 microM) significantly inhibited the B-to-A permeability of vinblastine by 61%. The apical uptake of atorvastatin increased 10.5-fold when the apical pH decreased from pH 7.4 to pH 5.5 while the pH in the basolateral side was fixed at pH 7.4. A proton ionophore, carbonylcyanide p-trifluoro-methoxyphenylhydrazone (FCCP) significantly decreased atorvastatin uptake. In addition, atorvastatin uptake was significantly inhibited by benzoic acid, nicotinic acid, and acetic acid each at 20 mM (65%, 14%, and 40%, respectively). Benzoic acid competitively inhibited atorvastatin uptake with a Ki of 14 mM. Similarly, benzoic acid, nicotinic acid, and acetic acid significantly, inhibited the A-to-B permeability of atorvastatin by 71%, 21%, and 66%, respectively., Conclusion: This study demonstrated that atorvastatin was secreted across the apical surface of Caco-2 cell monolayers via P-glycoprotein-mediated efflux and transported across the apical membrane in the absorptive direction via a H(+)-monocarboxylic acid cotransporter (MCT). In addition, this study provided the first evidence that negatively charged compounds, such as atorvastatin, can be a substrate for P-glycoprotein.

The specific mechanisms controlling intestinal cell differentiation remain largely undefined. The retinoblastoma (Rb) proteins (pRb, p130, and p107) appear crucial to the terminal differentiation process of certain cells through their association and repression of E2F transcription factors. We have examined the expression of pRb-related proteins p130 and p107 as well as the regulation of E2F during spontaneous differentiation of the Caco-2 intestinal cell line. Nuclear protein levels of p130 and p107 were increased with Caco-2 differentiation. Induction of a slower-migrating E2F complex was noted in postconfluent (i.e., differentiated) Caco-2 cells; p130 protein was the predominant component of this E2F complex with a minor contribution from cyclin-dependent kinase-2. A small component of p107 binding was identified by deoxycholate release gel shift assays. In contrast, no pRb binding to E2F was noted in Caco-2 cells. In addition to increased association with p130, E2F-4 phosphorylation was markedly decreased in differentiated Caco-2 cells, whereas E2F protein levels remained unchanged. Taken together, our findings suggest that the regulation of E2F function may be an important contributing factor in the cell cycle block and spontaneous differentiation of Caco-2 cells. This regulation of E2F occurs most likely through its increased association with p130 as well as decreased phosphorylation.

The mechanisms which regulate cell turnover in the intestinal epithelium are incompletely understood. The present study was performed to characterize the role of autocrine IGF system components in intestine epithelial cell proliferation and differentiation comparing rapidly growing crypt cells (IEC-6) with differentiating enterocytes (CaCo-2). The autocrine release of IGF-I, IGF-II and IGFBP-1 through -3 was determined by specific RIAs and western ligand blotting. In addition, binding and growth-promoting activity of insulin, IGF-I and IGF-II was investigated. Enterocytic differentiation was assessed by measuring the brush-border enzymes alkaline phosphatase and sucrase. During IEC-6 growth, the autocrine release of IGF-I and -II increased, whereas IGFBP-2 levels decreased. Specific receptors for IGF-I and IGF-II but not insulin could be detected. IGF-I was 100-fold more potent than insulin to stimulate IEC-6 cell proliferation. In contrast, CaCo-2 cells revealed higher binding of insulin than IGF-I/-II and no release of IGF-I. At switch from CaCo-2 cell proliferation to differentiation a marked increase in the secretion of IGF-II (10-fold), IGFBP-1 (2.5-fold), IGFBP-2 (3-fold), and IGFBP-3 (6-fold) was measured. Our data indicate that IGF system components differentially modulate enterocytic cell proliferation and differentiation.

Both the Na+-dependent glucose cotransporter (SGLT1) and the cystic fibrosis transmembrane conductance regulator (CFTR) modulate Na+ and fluid movement, although in opposite directions. Yet few studies have investigated a possible interrelationship between these two transporters. By using the Caco-2 human colon carcinoma cell line, we confirmed that the activities of these transporters increased with spontaneous differentiation to the enterocytic phenotype. We showed that SGLT1 was positively regulated by Cl- and that optimal activity of CFTR was dependent on the presence of glucose. We also demonstrated that inhibition of CFTR by glibenclamide or diphenylamine-2-carboxylate did not modify the activity of SGLT1 and inhibition of SGLT1 by phlorizin did not modify the activity of CFTR, although it resulted in inhibition of glycoconjugate synthesis. These results point to positive substrate-cross regulation of SGLT1 and CFTR and suggest that NaCl and glucose are important for not only Na+ absorption and fluid movement, but also for cAMP-dependent Cl- efflux, and glycoconjugate synthesis, functions that are known to be anomalous in cystic fibrosis.

To understand the molecular mechanisms underlying sorting of apical and basolateral membrane components in human intestinal epithelial cells, we have cloned the human homolog of rat syntaxin 3 and looked for its subcellular localization. Endogenous human syntaxin 3 was found to be localized at the apical membrane of colon epithelial and Caco-2 cells. This apical localization was confirmed by confocal microscopy after transfection of the cDNA coding for either full length or N-terminally truncated human syntaxin 3 in Caco-2 cells. Furthermore the signal(s) and machinery targeting human syntaxin 3 to the apical membrane of epithelial cells are conserved between species since human syntaxin 3 was also localized at the apical membrane of canine MDCK cells and of epithelial cells in transgenic Drosophila melanogaster.

Occludin is an integral membrane protein specifically associated with tight junctions. Previous studies suggest it is likely to function in forming the intercellular seal. In the present study, we expressed occludin under an inducible promotor in occludin-null fibroblasts to determine whether this protein confers intercellular adhesion. When human occludin is stably expressed in NRK and Rat-1 fibroblasts, which lack endogenous occludin and tight junctions but do have well developed ZO-1-containing adherens-like junctions, occludin colocalizes with ZO-1 to points of cell-cell contact. In contrast, L-cell fibroblasts which lack cadherin-based adherens junctions, target neither ZO-1 nor occludin to sites of cell contact. Occludin-induced adhesion was next quantified using a suspended cell assay. In NRK and Rat-1 cells, occludin expression induces adhesion in the absence of calcium, thus independent of cadherin-cadherin contacts. In contrast, L-cells are nonadhesive in this assay and show no increase in adhesion after induction of occludin expression. Binding of an antibody to the first of the putative extracellular loops of occludin confirmed that this sequence was exposed on the cell surface, and synthetic peptides containing the amino acid sequence of this loop inhibit adhesion induced by occludin expression. These results suggest that the extracellular surface of occludin is directly involved in cell-cell adhesion and the ability to confer adhesiveness correlates with the ability to colocalize with its cytoplasmic binding protein, ZO-1.

Using antibodies and recombinant DNA techniques, we have identified plakophilin 2, a novel desmosomal plaque protein of M(r) 100,000 (estimated from SDS-PAGE), which is a member of the arm-repeat family of proteins and can occur in two splice forms (2a and 2b) because of the insertion of a 44 amino acid (aa)-encoding exon. In its aa sequence (837 and 881 aa, calculated pIs: 9.33 and 9.38, mol wts 92,750 and 97,410 kD), it is conspicuously related to the 80-kD plakophilin 1, with which it shares a central region of 9 repeats of the arm-motif, preceeded by a long head region and followed by a very short (11 aa) carboxy-terminal sequence. Plakophilin 2 and its mRNA have been detected in a wide range of tissues and cell types, including cells devoid of desmosomes. By light and electron microscopical immunolocalization, plakophilin 2 has been localized to plaques of desmosomes of one-layered ("simple") and complex epithelia, carcinomas, diverse epithelium-derived cell culture lines, as well as cardiac tissue and the dendritic reticulum cells of lymphatic germinal centers, i.e., desmosomes in which plakophilin 1 is not detected. However, plakophilin 2 has also been localized in the desmosomes of certain but not all stratified epithelia where it coexists with plakophilin 1. Remarkably, plakophilin 2 is also enriched in the karyoplasm of a wide range of cell types, including many that lack desmosomes and in which, therefore, the nuclear state is the only locally enriched form of plakophilin 2 present. We conclude that plakophilins 2a and 2b are basic nuclear proteins that in certain cell types additionally assemble with other proteins to form the desmosomal plaque and serve general nuclear functions as well as a function specific to many but not all desmosomes.

Microtubules oriented in the apicobasal axis of columnar epithelial cells are arranged with a uniform polarity with minus ends toward the apical surface, suggesting that these cytoskeletal filaments might serve as a substrate for polarized movement of membrane vesicles within the cell. It is not known whether hepatocytes, a cuboidal epithelium in which transcellular transport is a requisite step in normal apical membrane biogenesis, contain microtubules arranged with a similar polarity. In the present study, we explore the question of microtubule polarity and possible mechanisms for nucleation in the epithelial cell lines WIF-B (hepatocyte), Caco-2 (intestine), and Madin-Darby canine kidney (MDCK). Caco-2 microtubules in the apicobasal axis had uniform polarity with minus ends nearest the apical surface. After cold and nocodazole-induced depolymerization, microtubule regrowth initiated in the apical region in all three cell types. The apex of WIF-B and Caco-2 cells contained two pools of gamma-tubulin: one associated with centrosomes and the other delocalized under the apical membrane. Non-centrosomal gamma-tubulin was present in complexes that sedimented between 10S and 29S; both forms could bind microtubules. The presence of both centrosomal and noncentrosomal gamma-tubulin in apical cytoplasm suggest multiple mechanisms by which microtubule nucleation might occur in epithelial cells.