Human Colon Carcinoma Caco-2 Cells Research Articles

Comparison of Drug Transporter Levels in Normal Colon, Colon Cancer, and Caco-2 Cells: Impact on Drug Disposition and Discovery

A critical step in early phase drug development is the determination of oral bioavailability. In part, the ability to predict whether a drug will be effectively transported across the gastrointestinal mucosa can be estimated from the physicochemical properties of the compound. Although advancements through rational drug design have more correctly predicted bioavailability, considerable variability remains to be explained. Transporter expression throughout the gastrointestinal tract may explain much of this variation. ATP-binding cassette (ABC) transporters were the first family of transporters identified to modify bioavailability. More recently, the solute carrier family has also been shown to alter the pharmacokinetic profile of drugs. Currently, the Caco-2 human colon carcinoma cell line is often used by the pharmaceutical industry to evaluate intestinal absorption of drugs; however, in vivo/in vitro permeabilities with carrier mediated drugs do not correlate well, suggesting that Caco-2 transporter expression varies from that of the small intestine. With this is mind, we integrated U133A GeneChip expression data from the NCBIs Gene Expression Omnibus (GEO) collection and then compared the expression pattern of Caco-2 cells to normal colon to determine if the Caco-2 cell line is a reliable model for colonic delivery. Furthermore, transporter expression of Caco-2 cells was compared to that of human colon tumors to assess whether this cell line could be useful to predict drug absorption for colon cancer. Our analysis shows that the expression pattern for Caco-2 cells closely resembles the gene expression profile of transporters within the normal colon, suggesting that this cell line may serve as an in vitro model of colonic drug adsorption. However, the molecular "fingerprint" of Caco-2 was distinctly different from tumor samples, indicating that the Caco-2 model would unlikely predict accurate drug absorption for colon cancer sites.

Stimulation of IL-8 Production byAralia cordateLectin in Human Colon Carcinoma Caco-2 Cells

Some lectins are known to stimulate interleukin-8 (IL-8) productions in human colon carcinoma Caco-2 cells. Since IL-8 may cause deleterious effects, we examined this stimulatory activity of Aralia cordate lectin (ACL) and Wasabia japonica lectin (WJL), both of which we isolated recently. The results indicate that ACL exhibited strong stimulatory activity for IL-8 protein production, while WJL showed marginal activity. The activity of ACL was associated with high enhancement of IL-8 gene expression. The effect of ACL was abolished almost completely in the presence of brefeldin A, indicating that internalization into cells is necessary for ACL to exert activity. The findings suggest that ingestion of a large amount of raw vegetable Aralia cordate might cause unfavorable effects on the colon.

Inhibitory effects of anti-CXCR4 antibodies on human colon cancer cells.

CXCR4, the chemokine receptor for CXCL12, has recently been involved in the metastatic process of several neoplasms. The expression of CXCR4 was evaluated by immunohistochemistry of colorectal tissue samples and by flow cytometry on Caco2, GEO, SW480, SW48, Lovo and SW620 human colon carcinoma cell lines. Correlations with pathological characteristics of the specimens were analysed with chi-square test. To verify the functional status of CXCR4, cell lines were tested in adhesion, migration, and proliferation assays. We studied the expression of CXCR4 in 88 human colorectal tissues and we found that CXCR4 was expressed in > 10% of epithelial cells in 50% of normal mucosae (7/14), in 55% of polyps (29/53), in all of carcinomas (16/16) and hepatic metastasis (5/5). Notably, CXCR4 was significantly over-expressed in cancerous lesions (carcinomas and metastasis) compared to non-cancerous lesions (normal mucosa and polyps) (P = 0.003) and in adenomatous polyps versus hyperplastic polyps (P = 0.009). The diameter of a polyp was also significantly associated with CXCR4 expression (P = 0.031). SW480, SW48 and SW620 cell lines showed the highest levels of CXCR4 (60-80% of positive cells). Adhesion, migration, and proliferation increased in response to the CXCL12 chemokine. These effects were abrogated by the addition of anti-CXCR4 antibodies. Further, CXCL12 activated ERK1/2 in SW480 cells. These data suggest that CXCR4 might play a role in colon cancer cell properties and that anti-CXCR4 antibodies could have therapeutic effects against colorectal cancer.

Transferrin-Oligomers as Potential Carriers in Anticancer Drug Delivery

To investigate if the cross-linking of transferrin receptor (TfR) induced by Tf-oligomers alters the endocytosis of receptor-ligand complexes in cultured tumor cells and hence increases intracellular drug release. An average of 3.5 Tf molecules per aggregate were cross-linked either by using homobifunctional linker (1, 11-bis-maleimidotetraethyleneglycol) [Tf(3.5-BM(PEO)4)] or heterobifunction linker [succinimidyl 4-(-p-maleimidophenyl)-butyrate] (Tf(3.5-SMPB)). Cell surface binding and competition experiments with 125I-Tf for TfR binding were studied to demonstrate that Tf-oligomers maintain specificity of the TfR-binding. To determine the degradation of Tf-oligomers in TfR-mediated endocytosis, cultured tumor cells were pulsed for 15 min with 125I-Tf-oligomers and chased for 2 h at 37 degrees C in the presence of excess unlabeled Tf. The chase medium was subjected to TCA precipitation to separate the intact and degraded Tf. To investigate if the alteration of TfR-trafficking facilitates the intracellular release of the drug from the Tf-conjugated form, methotrexate (MTX) was conjugated to Tf-oligomer (Agg-Tf-MTX) and its antiproliferative activity was compared with monomeric-Tf-MTX (Mono-Tf-MTX) in human colon carcinoma (Caco-2) cells, human breast adenocarcinoma (MCF-7) cells, wild-type Chinese hamster ovary (CHO) cells, and MTX-resistant CHO (CHO-MTX-RII) cells. TfR-mediated degradation of Tf-oligomers was higher than that of monomeric Tf in both Caco-2 and MCF-7 cells. The IC50 of Agg-Tf-MTX was lower than that of Mono-Tf-MTX in both tumor cell lines. The IC50 of MTX and Mono-Tf-MTX in CHO-MTX-RII cells was higher than that in wild-type CHO cells, whereas the Agg-Tf-MTX was almost identical in both the resistant and wild-type cells. Cross-linking of TfR induced by oligomeric Tf binding alters the intracellular trafficking of Tf-TfR complexes, redirects them out of the recycling pathway, and targets them to intracellular degradation in cultured tumor cells. The alteration of TfR-trafficking facilitates the intracellular release of the drug from the Tf-conjugated form. Consequently, Agg-Tf-MTX is more effective than Mono-Tf-MTX as a TfR-mediated antiproliferative agent in tumor cells, as well as in MTX-resistant transport deficient cells. Therefore, Tf-oligomers are potentially effective TfR-targeting carriers for intracellular delivery of anticancer drugs.

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

A DNA fragment encoding the cholera toxin A2 subunit (CTA 2) 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 NSP4 22. Anti-CFA/I antibodies recognized a single band of approximately 72-kDa in transformed potato tuber tissue consistent with CFA/I–CTA 2 and CTB–NSP4 fusion protein assembly into a cholera holotoxin-like structure. Enzyme-linked immunosorbent assay (G M1 ELISA) indicated that the CFA/I–CTA 2 fusion protein bound specific G M1 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.

C-jun N-terminal kinase modulates 1,25-dihydroxyvitamin D3-induced cytochrome P450 3A4 gene expression.

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is known to induce the expression of cytochrome P450 3A4 (CYP3A4) in human colon carcinoma Caco-2 cells. Recently, it was demonstrated that the vitamin D receptor (VDR) regulates 1,25(OH)(2)D(3)-induced CYP3A4 gene expression through the xenobiotic-responsive element and the vitamin D-responsive element located on the 5'-flanking region of the CYP3A4 gene. On the other hand, we previously reported that protein kinases such as protein kinase C and tyrosine kinases contribute to the induction of CYP3A4 mRNA by 1,25(OH)(2)D(3). In the present study, we examined the involvement of mitogen-activated protein kinases (MAPKs) in the 1,25(OH)(2)D(3)-induced CYP3A4 gene expression using MAPK inhibitors. Curcumin, a c-Jun N-terminal kinase (JNK) pathway inhibitor, and anthra[1,9-cd]pyrazole-6(2H)-one (SP600125), a JNK inhibitor, suppressed the induction of CYP3A4 mRNA by 1,25(OH)(2)D(3), but not 2'-amino-3'-methoxyflavone (PD098059), a mitogen-activated protein kinase kinase-extracellular signal-regulated kinase (ERK) pathway inhibitor, or 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580), a p38 inhibitor. In addition, we demonstrated that SP600125 dose-dependently inhibited the CYP3A4 promoter activity induced by 1,25(OH)(2)D(3) using the reporter plasmid of the CYP3A4 promoter. However, SP600125 did not affect 1,25(OH)(2)D(3)-induced transactivation of the DR3 via VDR. These results indicate that JNK, but not ERK or p38, is required for the optimal activation of the CYP3A4 gene induced by 1,25(OH)(2)D(3).

Cold-Storage Affects Antioxidant Properties of Apples in Caco-2 Cells

Data on the composition of phenolic antioxidant compounds present in food plants and assessment of their activity are essential for epidemiological explanation of the health benefits of fruit and vegetables. Various factors such as cultivation methods, industrial processing, and storage may affect the final concentrations of phytochemicals in food plants and their eventual bioactivity. This study investigated the influence of commercial cold-storage periods on the antioxidant properties of apples grown either by organic or integrated systems. In both cases, total phenolics and total antioxidant activity decreased only in the first 3 mo and only in apples with skin (P < 0.05), suggesting that cold storage rapidly impoverishes these properties in skin but not in pulp. Assessment of antioxidant bioactivity in vitro, measured in terms of intracellular antioxidant, cytoprotective, and antiproliferative activity in human colon carcinoma (Caco-2) cells (differentiated to normal intestinal epithelia for intracellular antioxidant and cytoprotective effects), showed strong, time-related decreases over 6 mo of cold storage for all 3 parameters (P < 0.01), irrespective of the cultivation system. These findings with integrated and organic apples further support the concept that organic systems of cultivation do not generally provide real health benefits. Moreover, the data from the present study clearly show that factors such as cold storage may affect the antioxidant properties of apples. Epidemiological studies on the cancer-preventive benefits of fruits and vegetables should take into account the cold-storage bias for apples, and possibly for other products.

Uptake, Hydrolysis, and Metabolism of Pyridoxine-5′-β-D-Glucoside in Caco-2 Cells

An important dietary source of vitamin B-6, pyridoxine-5'-beta-D-glucoside (PNG), exhibits only partial bioavailability, which is limited by the extent of enzymatic cleavage of the beta-glucosidic bond to release metabolically available pyridoxine (PN). This laboratory showed that the intestinal hydrolysis of PNG is catalyzed by cytosolic PNG hydrolase (PNGH) and brush border lactase-phlorizin hydrolase (LPH). LPH-catalyzed PNG hydrolysis in vitro is competitively inhibited by lactose. In the present study, the uptake and hydrolysis of PNG were examined in Caco-2 human colon carcinoma cells, which express a functional LPH but exhibit no PNGH activity. PNG uptake at 37 degrees C was linear over 5-500 micromol/L PNG. Uptake was not significantly reduced when Na(+) was substituted with K(+), Li(+), or Tris in the medium. Increasing PNG concentration in the medium did not change intracellular concentrations of PN, pyridoxamine (PM), pyridoxamine 5'-phosphate (PMP), or pyridoxal 5'-phosphate (PLP); however, intracellular pyridoxal (PL) concentration increased. Intracellular PNG concentration was not significantly reduced in the presence of lactose, but the concentration of PL declined in proportion to extracellular lactose (P = 0.01). These results indicate that PNG can be absorbed intact in a Na(+)-independent process and is taken up by passive diffusion. The presence of lactose in this in vitro model of intestinal uptake reduced the enzymatic hydrolysis of PNG by lactase.

Effect of rabeprazole on MDR1-mediated transport of Rhodamine 123 in Caco-2 and Hvr100-6 cells.

The aim of our study was to investigate the effects of rabeprazole, a proton pump inhibitor, on MDR1 expressed on human colon carcinoma cell line, Caco-2, and MDR1-overexpressing human cervical carcinoma cell line, HeLa cells selected by exposure to 100 nM vinblastine (Hvr100-6 cells). Inhibitory effects of rabeprazole on MDR1-mediated transport of Rhodamine123 were examined in these cells. A thousand micro molar rabeprazole increased Rhodamine 123 uptakes in Caco-2 and Hvr100-6 cells by 68% and 185%, respectively. No significant effects of rabeprazole were observed at the concentration of 1-100 microM. Since rabeprazole did not show any effects on Rhodamine 123 transport via MDR1 at the plasma levels (approximately 1 microM), it was considered that the drug interaction with MDR1 substrates would be minimal even though the interaction occurred in the patients with rabeprazole treatment.

Molecular cloning of the human beta1,4 N-acetylgalactosaminyltransferase responsible for the biosynthesis of the Sd(a) histo-blood group antigen: the sequence predicts a very long cytoplasmic domain.

The Sd(a) antigen is a carbohydrate determinant expressed on erythrocytes, the colonic mucosa and other tissues. This epitope, whose structure is Siaalpha2,3[GalNAcbeta1,4]Gal beta1,4GlcNAc, is synthesized by a beta1,4 N-acetylgalactosaminyltransferase (beta4GalNAc-T) that transfers a beta1,4-linked GalNAc to the galactose residue of an alpha2,3-sialylated chain. We have cloned from human colon carcinoma Caco2 cells a cDNA whose transfection in COS cells induces a GalNAc-T active on sialylated but not on asialylated fetuin and putatively represents the human Sd(a) beta4GalNAc-T. The cDNA predicts a 566 aa protein showing 66.6% and 39% identity with mouse CT beta4GalNAc-T and human GM2/GD2 synthase, respectively, with a typical type II glycosyltransferase organization, no potential N-glycosylation sites and a 67 aa cytoplasmic tail, which is probably the longest among the glycosyltransferases cloned to date. The gene maps in chromosome 17q23, and is composed of at least 11 exons. Exons 2-11 are homologous to exons 2-11 of the previously cloned CT beta4GalNAc-T from murine cytotoxic T lymphocytes while exons 1 of the two enzymes are totally different. The mRNA is expressed at a high level in differentiated Caco2 cells and in colonic mucosa and at a much lower level in lymphocytes and other colon cancer cell lines.

Muc4/Sialomucin Complex, the Intramembrane ErbB2 Ligand, Translocates ErbB2 to the Apical Surface in Polarized Epithelial Cells

Muc4/Sialomucin complex (SMC) acts as an intramembrane ligand for the receptor tyrosine kinase ErbB2, inducing a limited phosphorylation of the receptor. Because Muc4/SMC is found at the apical surfaces of polarized epithelial cells and ErbB2 is often basolateral, the question arises as to whether these components become associated in polarized cells. To address this question, we examined the localization of these proteins in polarized human colon carcinoma CACO-2 cells. Dual color immunofluorescence analysis by confocal microscopy demonstrated the basolateral localization of the ErbB2 in these cells; it is primarily co-localized with E-cadherin at adherens junctions. Expression of apical Muc4/SMC in these cells by transient transfection results in the localization of the ErbB2 at the apical surface. Two-color confocal microscopy indicated that ErbB2 is colocalized with Muc4/SMC in the transfected cells but not in untransfected cells in the same culture. The change of localization of ErbB2 was confirmed by cell surface biotinylation of apical and basolateral proteins, followed by streptavidin precipitation and the subsequent detection of ErbB2 by immunoblotting. In contrast, Na+/K+-ATPase maintains its basolateral localization in Muc4/SMC-transfected cells, indicating that the translocation of ErbB2 is not the result of depolarization of the cells. A potential physiological role for the apical localization of ErbB2 is indicated by the fact that ErbB2 phosphorylated at tyrosine 1248 is found predominantly in Muc4/SMC-transfected cells, but not in untransfected cells, and is co-localized with the apical Muc4/SMC. The ability of Muc4/SMC to alter the localization of ErbB2, particularly a phosphorylated form of it, in epithelial cells, suggests that it has an important role in regulating ErbB2 signaling.

Peroxisome Proliferator-activated Receptor α Induces Hepatic Expression of the Human Bile Acid Glucuronidating UDP-glucuronosyltransferase 2B4 Enzyme

Glucuronidation, a major metabolic pathway for a large variety of endobiotics and xenobiotics, is catalyzed by enzymes belonging to the UDP-glucuronosyltransferase (UGT) family. Among UGT enzymes, UGT2B4 conjugates a large variety of endogenous and exogenous molecules and is considered to be the major bile acid conjugating UGT enzyme in human liver. In the present study, we identify UGT2B4 as a novel target gene of the nuclear receptor peroxisome proliferator-activated receptor alpha (PPAR alpha), which mediates the hypolipidemic action of fibrates. Incubation of human hepatocytes or hepatoblastoma HepG2 and Huh7 cells with synthetic PPAR alpha agonists, fenofibric acid, or Wy 14643 resulted in an increase of UGT2B4 mRNA levels. Furthermore, treatment of HepG2 cells with Wy 14643 induced the glucuronidation of hyodeoxycholic acid, a specific bile acid UGT2B4 substrate. Analysis of UGT2B mRNA and protein levels in PPAR alpha wild type and null mice revealed that PPAR alpha regulates both basal and fibrate-induced expression of these enzymes in rodents also. Finally, a PPAR response element was identified in the UGT2B4 promoter by site-directed mutagenesis and electromobility shift assays. These results demonstrate that PPAR alpha agonists may control the catabolism of cytotoxic bile acids and reinforce recent data indicating that PPAR alpha, which has been largely implicated in the control of lipid and cholesterol metabolism, is also an important modulator of the metabolism of endobiotics and xenobiotics in human hepatocytes.

Induction of cytotoxicity and ssDNA breaks by 9-bromo-5-morpholino-tetrazolo[1,5-c]quinazoline in tumor cells cultured in vitro

9-Bromo-5-morpholino-tetrazolo[1,5-c]quinazoline (BMTQ) acted cytotoxically on murine leukemia cell line L1210 and human colon carcinoma cells Caco-2. We found the two highest concentrations of BMTQ (149.2 and 74.6 μM) induced an acute cytotoxic effect, however other tested concentrations (<74.6 μM) manifested a concentration/dependent and time/dependent cytotoxic effect. The sensitivity of murine leukemia cells L1210 and human colon carcinoma cells Caco-2 was expressed in the same order. The cytotoxicity of BMTQ was not accompanied by changes of the cell cycle profile. Following the cytotoxicity-related effects of BMTQ we observed the induction of ssDNA breaks after BMTQ treatment. All the concentrations of BMTQ increased the level of ssDNA breaks 1.3–2.9 times (after 2 h of treatment) and 1.6–2.8 times (after 4 h of treatment) in Caco-2 cells compared to the control. No apoptotic DNA fragmentation induced by BMTQ in Caco-2 cells was recorded.

Gene transfer by adenovirus-mimetic peptides in the presence of a cationic lipid and/or adenovirus. Analysis of the contribution of the viral and nonviral components.

Peptide and cationic lipid-based gene transfer vectors have shown promise for gene therapy but are still less efficient than viral gene transfer vectors. We have examined the mechanism of gene transfer of different adenovirus-mimetic peptides in the presence and absence of a cationic lipid, lipofectamine and/or adenovirus with the aim of improving the design of nonviral vectors for efficient gene transfer. Three polylysine-adenovirus-mimetic peptides were synthesised and examined for their efficacy for gene transfer. Transfection levels in four cell lines: adenovirus permissive human tracheal epithelial (56FHTE8o(-)), human lung carcinoma (A549), human colon carcinoma (Caco-2) cells, and adenovirus low-permissive Chinese hamster ovary (CHO) cells, were examined. The polylysine-adenovirus-mimetic peptides increased the level of transfection of a reporter transgene in all cell lines. Transfection was substantially increased when an adenovirus was added to cells after pre-incubation with the vector complexes. Formulation of the peptide vector complexes with lipofectamine increased their transfection efficacy and the subsequent addition of an adenovirus increased transfection levels even further but only in permissive cells. Pre-incubation of cells with lipofectamine-peptide vector complexes increased cell binding of the adenovirus but uptake was only increased in intermediate- or non-permissive cells. The addition of lipofectamine increased transgene expression of a recombinant adenovirus in non-permissive cells but not in permissive cells. Enhancement with an adenovirus of peptide vector gene transfer is probably due to more efficient endosome escape while enhancement of gene transfer by peptide vectors complexed to lipofectamine is due to an increase in cellular binding and/or internalisation of the adenovirus.

PCLCA1 becomes a cAMP-dependent chloride conductance mediator in Caco-2 cells

Members of the CLCA protein family are expressed in airway and intestinal epithelium, where they may participate in secretory activity as mediators of chloride conductance. A calcium-dependent chloride conductance has been observed upon expression of CLCA proteins in non-epithelial cell lines. The pCLCA1 gene, cloned in our laboratory, codes for a product containing a unique A-kinase consensus acceptor site not found in other CLCA proteins. Calcium-dependent, but not cAMP-dependent, chloride conductance increased when pCLCA1 was expressed in NIH/3T3 fibroblasts. We transfected the Caco-2 human colon carcinoma cell line with pCLCA1 to investigate the regulation of CLCA-associated chloride conductance in this differentiated epithelial cell line. Expression of pCLCA1 in the Caco-2 cell line enhanced cAMP-responsive 36 Cl efflux, short circuit current, and whole cell chloride current in these cells. This cAMP-dependent chloride conductance was localized to the apical membrane of polarized Caco-2 cells.

Extracellular thiol/disulfide redox state affects proliferation rate in a human colon carcinoma (Caco2) cell line.

Redox mechanisms function in regulation of cell growth, and variation in redox state of plasma thiol/disulfide couples occurs in various physiologic conditions, including diabetes, chemotherapy, and aging. The present study was designed to determine whether a systematic variation in extracellular thiol/disulfide redox state (E(h)) over a range (0 mV to -150 mV) that occurs in human plasma altered proliferation of cultured cells. Experiments were performed with a human colon carcinoma cell line (Caco2), which grows slowly in the absence of serum and responds to peptide growth factors with increased rate of cell division. The extracellular redox states were established by varying concentrations of cysteine and cystine, maintaining constant pool size in terms of cysteine equivalents. Incorporation of 5-bromo-2-deoxyuridine (BrdU) was used to measure DNA synthesis and was lowest at the most oxidized extracellular E(h) (0 mV). Incorporation increased as a function of redox state, attaining a 100% higher value at the most reduced condition (-150 mV). Addition of insulin-like growth factor-1 (IGF-1) or epidermal growth factor (EGF) increased the rate of BrdU incorporation at more oxidizing redox conditions (0 to -80 mV) but had no effect at -150 mV. Cellular GSH was not significantly affected by variation in extracellular E(h). In the absence of growth factors, extracellular E(h) values were largely maintained for 24 h. However, IGF-1 or EGF stimulated a change in extracellular redox to values similar to that for cysteine/cystine redox in plasma of young, healthy individuals. The results show that extracellular thiol/disulfide redox state modulates cell proliferation rate and that this control interacts with growth factor signaling apparently independently of cellular glutathione.

Cytokine responses of human intestinal epithelial-like Caco-2 cells to the nonpathogenic bacterium Bacillus subtilis ( natto)

Intestinal epithelial cells produce cytokines in response to pathogenic bacteria. However, cellular responses of these cells to nonpathogenic strains, such as Bacillus subtilis, are yet to be determined. In this study, we investigate whether epithelial-like human colon carcinoma Caco-2 cells produce cytokines in response to B. subtilis or B. subtilis ( natto). The latter strain is utilized for manufacturing the fermented soy food “natto”. Live cells of nonpathogenic B. subtilis JCM 1465 T, B. subtilis ( natto) and E. coli JCM 1649 T, as well as pathogenic S. enteritidis JCM 1652 and P. aeruginosa JCM 5516 strains, induced secretion of interleukin-6 (IL-6) and/or IL-8, but not IL-7, IL-15 or tumor necrosis factor α (TNF-α). Transepithelial electrical resistance (TER) of Caco-2 cell monolayers cultured with E. coli, S. enteritidis or P. aeruginosa decreased more rapidly than that of cells cultured with B. subtilis or B. subtilis ( natto). The amounts of cytokine induced by B. subtilis ( natto) cells were strain-dependent. Moreover, B. subtilis ( natto) cells subjected to hydrochloric acid treatment, but not autoclaving, induced a higher secretion of IL-6 and IL-8 than intact cells. Tyrosine kinase inhibitors, including AG126 and genistein, suppressed cytokine secretion. Our results suggest that the nonpathogenic B. subtilis ( natto) bacterium induces cytokine responses in intestinal epithelial cells via activation of an intracellular signaling pathway, such as that of nuclear factor-kappa B (NF-κB).

Transepithelial transport of HIV-1 by M cells is receptor-mediated.

Human colon carcinoma Caco-2 cell monolayers undergo conversion into cells that share morphological and functional features of M cells when allowed to interact with B lymphocytes. A lymphotropic (X4) HIV-1 strain crosses M cell monolayers and infects underlying CD4(+) target cells. Transport requires both lactosyl cerebroside and CXCR4 receptors, which are expressed on the apical surface of Caco-2 and M cells. Antibodies specific for each receptor block transport. In contrast, a monotropic (R5) HIV-1 strain is unable to cross M cell monolayers and infect underlying monocytes, despite efficient transport of latex beads. Caco-2 and M cells do not express CCR5, but transfection of these cells with CCR5 cDNA restores transport of R5 virus, which demonstrates that HIV-1 transport across M cells is receptor-mediated. The follicle-associated epithelium covering human gut lymphoid follicles expresses CCR5, but not CXCR4, and lactosyl cerebroside, suggesting that HIV-1 infection may occur through M cells and enterocytes at these sites.

Effects of vitamins C and E on cytotoxicity induced by N-nitroso compounds, N-nitrosomorpholine and N-methyl- N′-nitro- N-nitrosoguanidine in Caco-2 and V79 cell lines.

Since N-nitroso compounds as strong carcinogens are closely related to food and nutrition, the cytotoxic effects of N-nitrosomorpholine (NMOR) and N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) and their reduction by vitamins C and E were investigated in hamster V79 cells and human colon carcinoma Caco-2 cells. Cytotoxicity was evaluated by the trypan blue exclusion technique in Caco-2 cells and by the plating efficiency assay in V79 cells. NMOR caused a dose-dependent decline of viable cells in both cell lines; MNNG induced a dose-dependent cytotoxic effect only in V79 cells. Pretreatment of cells with vitamin C and vitamin E significantly reduced the cytotoxicity of NMOR, however, both vitamins had not effect on cytotoxicity induced by MNNG. These results suggest that different N-nitroso compounds react differently with cellular macromolecules. Measurement of the level of NMOR-induced DNA strand breaks and alkali-labile sites in both cell types using the alkaline comet assay also indicates a protective effect of both vitamins against the genotoxic effects of NMOR.

Digoxin Up-Regulates MDR1 in Human Colon Carcinoma Caco-2 Cells

Because MDR1 (P-glycoprotein) plays an important role in pharmacokinetics such as absorption and excretion of xenobiotics and multidrug resistance, an understanding of the factors regulating its function and expression is important. Here, the effects of digoxin on cell sensitivity to an anticancer drug, MDR1 function, and expression were examined by assessing the growth inhibition by paclitaxel, the transport characteristics of the MDR1 substrate Rhodamine123, and the level of MDR1 mRNA, respectively, using human colon carcinoma Caco-2 cells, which are widely used as a model of intestinal epithelial cells. The sensitivity to paclitaxel, an MDR1 substrate, in Caco-2 cells pretreated with digoxin was lower than that in non-treated cells. The accumulation of Rhodamine123 was reduced by pretreatment with digoxin and its efflux was enhanced. The level of MDR1 mRNA in Caco-2 cells was increased in a digoxin concentration-dependent manner. These results taken together suggested that digoxin up-regulates MDR1 in Caco-2 cells.