Generation of Caco-2 cells stably expressing CYP3A4·POR·UGT1A1 and CYP3A4·POR·UGT1A1*6 using a PITCh system.

The small intestine plays a critical role in the absorption and metabolism of orally administered drugs. Therefore, a model capable of evaluating drug absorption and metabolism in the small intestine would be useful for drug discovery. Patients with genotype UGT1A1*6 (exon 1, 211G > A) treated with the antineoplastic drug SN-38 have been reported to exhibit decreased glucuronide conjugation and increased incidence of intestinal toxicity and its severe side effects, including severe diarrhea. To ensure the safety of drugs, we must develop a drug metabolism and toxicity evaluation model which considers UGT1A1*6. In this study, we generated CYP3A4·POR·UGT1A1 KI- and CYP3A4·POR·UGT1A1*6 KI-Caco-2 cells for pharmaceutical research using a PITCh system. The CYP3A4·POR·UGT1A1 KI-Caco-2 cells were shown to express functional CYP3A4 and UGT1A1. The CYP3A4·POR·UGT1A1*6 KI-Caco-2 cells were sensitive to SN-38-induced intestinal toxicity. We thus succeeded in generating CYP3A4·POR·UGT1A1 KI- and CYP3A4·POR·UGT1A1*6 KI-Caco-2 cells, which can be used in pharmaceutical research. We also developed an intestinal epithelial cell model of patients with UGT1A1*6 and showed that it was useful as a tool for drug discovery.

A new nuclear protease with cathepsin L properties is present in HeLa and Caco-2 cells.

Recently many authors have reported that cathepsin L can be found in the nucleus of mammalian cells with important functions in cell-cycle progression. In previous research, we have demonstrated that a cysteine protease (SpH-protease) participates in male chromatin remodeling and in cell-cycle progression in sea urchins embryos. The gene that encodes this protease was cloned. It presents a high identity sequence with cathepsin L family. The active form associated to chromatin has a molecular weight of 60 kDa, which is higher than the active form of cathepsin L described until now, which range between 25 and 35 kDa. Another difference is that the zymogen present in sea urchin has a molecular weight of 75 and 90 kDa whereas for human procathepsin L has a molecular weight of 38-42 kDa. Based on these results and using a polyclonal antibody available in our laboratory that recognizes the active form of the 60 kDa nuclear cysteine protease of sea urchin, ortholog to human cathepsin L, we investigated the presence of this enzyme in HeLa and Caco-2 cells. We have identified a new nuclear protease, type cathepsin L, with a molecular size of 60 kDa, whose cathepsin activity increases after a partial purification by FPLC and degrade in vitro histone H1. This protease associates to the mitotic spindle during mitosis, remains in the nuclei in binuclear cells and also translocates to the cytoplasm in non-proliferative cells.

Evaluation of in vitro models for screening alkaline phosphatase-mediated bioconversion of phosphate ester prodrugs.

Generating a phosphate prodrug is one of the common approaches for circumventing poor solubility issues of a parent drug. Alkaline phosphatase (ALP) level was determined in rat intestine mucosa scraps, human colon carcinoma (Caco-2) cells, and Madin-Darby canine kidney (MDCK) cells to characterize in vitro models for ALP-mediated phosphate prodrug conversion. In addition, fosphenytoin and fosfluconazole were used as probe prodrugs to evaluate the models. The highest amount of ALP was detected in rat intestinal mucosa scraps, whereas ALP in 5-day cultured MDCK cells was minimal. As anticipated, ALP levels correlated with the parent drug conversion; the shortest cleavage half-life (t(1/2)) was observed in rat mucosa scraps; and MDCK cells showed the slowest conversion. Furthermore, the polarized conversion for the prodrugs was observed in Caco-2 monolayer cells, suggesting the polarized localization of alkaline in differentiated Caco-2 cells. The rate of ALP-mediated conversion was prodrug concentration-dependent with Michaelis-Menten constants of 1160 and 351 microM for fosphenytoin and fosfluconazole, respectively, determined in Caco-2 cells. The results revealed that whereas the intestinal mucosa scraps reserved the highest ALP activities and were shown as a promising in vitro tool for screening the bioconversion of phosphate prodrug, Caco-2 monolayers could provide the predictive information of bioconversion and further offer the capability in characterizing the permeability of prodrug and parent drug.

Usefulness of a novel Caco-2 cell perfusion system II. Characterization of monolayer properties and peptidase activity.

In this study, the enzymatic activity and the influence of support filters and extracellular matrix proteins on the differentiation of Caco-2 cells grown in a perfusion system (Minucells and MinutissueTM) were examined and compared to traditional culturing approaches. Differences were observed regarding the differentiation of Caco-2 cells using the traditional approach and perfusion system such that the cell monolayers grown in a perfusion system showed a significant increase in dipeptidase activities (18.20 +/- 0.43nmol x min(-1) x cm(-2)) compared to the cells cultivated using the 21-day protocol (9.45 +/- 0.50 nmol x min(-1) x cm(-2)). The peptidase activity of Caco-2 cells was strikingly inhibited when Matrigel extracellular protein was used for coating polycarbonate support filters. While the enzymatic activities of the cell monolayers differentiated in the perfusion system were up-regulated, the transepithelial electrical resistance values of the cell monolayers (171 +/- 52 and 251 +/- 62 omega x cm2 for polycarbonate and polyester, respectively) decreased compared to the traditional Snapwell inserts (644 +/- 119 omega x cm2). The results suggested that the perfusion systems were useful permeability models which reduce workload, resources and manpower needed to obtain useful Caco-2 monolayers. In addition, the approach offers an efficient tool for long-term culturing of highly differentiated Caco-2 cell monolayers.

Suppression of pyrimidine biosynthesis by targeting DHODH enzyme robustly inhibits rotavirus replication.

Rotavirus infection remains a great health burden worldwide especially in some developing countries. It causes severe dehydrating diarrhea in infants, young children, as well as immunocompromised and organ transplanted patients. Viral replication heavily relies on the host to supply nucleosides. Thus, host enzymes involved in nucleotide biosynthesis represent potential targets for antiviral development. Dihydroorotate dehydrogenase (DHODH) is the rate-limiting enzyme in the de novo biosynthesis pathway of pyrimidines. In this study, we demonstrated that two specific DHODH enzyme inhibitors, brequinar (BQR) and leflunomide (LFM) robustly inhibited rotavirus replication in conventional human intestinal Caco2 cell line as well as in human primary intestinal organoids. The antiviral effect is conserved in both laboratory strain SA11 and rotavirus strain 2011K isolated from clinical sample. Mechanistic study indicated that BQR and LFM exerted their anti-rotavirus effect through targeting DHODH to deplete pyrimidine nucleotide pool. Therefore, targeting pyrimidine biosynthesis represents a potential approach for developing antiviral strategies against rotavirus.

Proanthocyanidin from grape seeds inactivates the PI3-kinase/PKB pathway and induces apoptosis in a colon cancer cell line.

The aim of this investigation was to evaluate the chemopreventative/antiproliferative potential of a grape seed proanthocyanidin extract (GSPE) against colon cancer cells (CaCo2 cells) and to investigate its mechanism of action. GSPE (10-100 microg/ml) significantly inhibited cell viability and increased apoptosis in CaCo2 cells, but did not alter viability in the normal colon cell line (NCM460). The increased apoptosis observed in GSPE-treated CaCo2 cells correlated with an attenuation of PI3-kinase (p110 and p85 subunits) and decreased PKB Ser(473) phosphorylation. GSPE might thus exert its beneficial effects by means of increased apoptosis and suppression of the important PI3-kinase survival-related pathway.

New methodological approach to induce a differentiation phenotype in Caco-2 cells prior to post-confluence stage.

BACKGROUND: Various differentiation-inducing agents or harvesting of spontaneously late post-confluence cultures have been used to differentiate the human colon carcinoma Caco-2 cell line. We report a new procedure to generate pre-confluent subcultures of Caco-2 population at various stages of differentiation without altering culture conditions. MATERIALS AND METHODS: Ultrastructural analysis, cell proliferation activity and biochemical markers of differentiation were evaluated at different passages. RESULTS: Subcultures of Caco-2 cells at pre-confluence, exhibiting progressive acquisition of a more benign differentiation phenotype, were generated. Early passages of Caco-2 cells showed a well-developed brush border and incomplete junctional apparatus; subsequent subcultures yielded cell populations with well-developed junctions similar to those of small intestinal cells. CONCLUSION: These culture conditions represent a new versatile model not only to progressively induce the differentiation program in Caco-2 cells at pre-confluence without changes of culture media, but also to explore mechanistic modes of drug transport and tumor development.

Comparison of dietary triacylglycerol oil and diacylglycerol oil in protein kinase C activation.

The objective of the present study was to compare the effects of dietary diacylglycerol (DAG) oil with triacylglycerol (TAG) oil with a similar fatty acid composition (fatty acid chain range: C14-C22, C18 fatty acid chain: >90%) on protein kinase C (PKC) activation and on 1,2-DAG levels. Using male Wistar rats, no differences in cytosolic and membrane PKC activities in the lingual, esophageal, gastric, small intestinal, cecal, proximal colonic, and distal colonic mucosa were found between the 5% DAG and TAG oil groups, or between the 23% DAG and TAG oil groups after 1 month of feeding. The 1,2-DAG levels in the cecum and colon contents and in the feces and serum in male Wistar rats after a diet containing either 10% DAG or TAG oil feeding were similar between the groups. Moreover, exposure of Caco-2 cells to DAG and TAG oils had no effect on PKC activity in the membrane fraction, but 1,2-dioctanoyl glycerol composed of short-chain fatty acids (C8) did, suggesting the absence of an influence on PKC activity in DAG and TAG oils composed of long-chain fatty acids. In summary, the effects of DAG oil ingestion on PKC activity in the digestive tract and lingual mucosa, and on 1,2-DAG levels in the cecum and colon contents and in the feces and serum were similar to those observed for TAG oil ingestion.

The critical role of 15-lipoxygenase-1 in colorectal epithelial cell terminal differentiation and tumorigenesis.

Terminal differentiation is an important event for maintaining normal homeostasis in the colorectal epithelium, and the loss of apoptosis is an important mechanism underlying colorectal tumorigenesis. The very limited current data on the role of lipoxygenase (LOX) metabolism in tumorigenesis suggests that the oxidative metabolism of linoleic and arachidonic acid possibly shifts from producing antitumorigenic 15-LOX-1 and 15-LOX-2 products to producing protumorigenic 5-LOX and 12-LOX products. We examined whether this shift occurs in vitro in the human colon cancer cell line Caco-2 in association with the loss of terminal differentiation and apoptosis, or in vivo during the formation of colorectal adenomas in patients with familial adenomatous polyposis (FAP). Restoring terminal differentiation and apoptosis of Caco-2 cells increased the mRNA levels of 5-LOX, 15-LOX-2, and 15-LOX-1, but the only significant increases in protein expression and enzymatic activity were of 15-LOX-1. In FAP patients, 15-LOX-1 expression and activity were significantly down-regulated in adenomas (compared with paired nonneoplastic epithelial mucosa), whereas 5-LOX and 15-LOX-2 protein expressions and enzymatic activities were not. We conducted a validation study with immunohistochemical testing in a second group of FAP patients; 15-LOX-1 expression was down-regulated in colorectal adenomas (compared with nonneoplastic epithelial mucosa) in 87% (13 of 15) of this group. We confirmed the mechanistic relevance of these findings by demonstrating that ectopically restoring 15-LOX-1 expression reestablished apoptosis in Caco-2 cells. Therefore, 15-LOX-1 down-regulation rather than a shift in the balance of LOXs is likely the dominant alteration in LOX metabolism which contributes to colorectal tumorigenesis by repressing apoptosis.

Essential amino acid mixtures drive cancer cells to apoptosis through proteasome inhibition and autophagy activation.

Cancer cells require both energy and material to survive and duplicate in a competitive environment. Nutrients, such as amino acids (AAs), are not only a caloric source, but can also modulate cell metabolism and modify hormone homeostasis. Our hypothesis is that the environmental messages provided by AAs rule the dynamics of cancer cell life or death, and the alteration of the balance between essential amino acids (EAAs) and non-essential amino acids (NEAAs) (lower and higher than 50%, respectively) present in nutrients may represent a key instrument to alter environment-dependent messages, thus mastering cancer cells destiny. In this study, two AA mixtures, one exclusively consisting of EAAs and the other consisting of 85% EAAs and 15% NEAAs, were tested to explore their effects on the viability of both normal and cancer cell lines and to clarify the molecular mechanisms involved. Both mixtures exerted a cell-dependent anti-proliferative, cytotoxic effect involving the inhibition of proteasome activity and the consequent activation of autophagy and apoptosis. These results, besides further validating the notion of the peculiar interdependence and extensive crosstalk between the ubiquitin-proteasome system (UPS) and autophagy, indicate that variation in the ratio of EAAs and NEAAs can deeply influence cancer cell survival. Consequently, customization of dietary ratios among EAAs and NEAAs by specific AA mixtures may represent a promising anticancer strategy able to selectively induce death of cancer cells through the induction of apoptosis via both UPS inhibition and autophagy activation.

Ursodeoxycholic acid differentially affects three types of sphingomyelinase in human colon cancer Caco 2 cells.

We investigated the effects of UDCA on sphingomyelinase (SMase) in Caco 2 cells cultured in monolayer and polarized conditions. Alkaline SMase activity was high in polarized cells whereas, acid and neutral SMase activities were high in monolayer cells. In polarized cells, UDCA increased alkaline SMase expression and caspase 3 activity but had no effect on acid and neutral SMases. In monolayer cells, UDCA reduced both acid and neutral SMase activities, inhibited cell proliferation, but had little effect on alkaline SMase and caspase 3 activities. In conclusion, UDCA differentially affects SMase activity, cell proliferation, and apoptosis in colonic cells depending on the cell conditions.

Decreased PKC-alpha expression increases cellular proliferation, decreases differentiation, and enhances the transformed phenotype of CaCo-2 cells.

Previous studies have shown that PKC-alpha protein expression is decreased in sporadic human colon cancers, as well as in colonic tumors of rats induced by chemical carcinogens. To elucidate the potential role of PKC-alpha on several phenotypic characteristics of colon cancer cells, we have transfected cDNAs for PKC-alpha in sense or antisense orientations into CaCo-2 cells, a human colonic adenocarcinoma cell line. Transfected clones were isolated that demonstrated approximately 3-fold increases (sense transfectants) and approximately 95% decreases (antisense transfectants) in PKC-alpha expression with no significant alterations in other PKC isoforms. Transfection of CaCo-2 cells with PKC-alpha in the antisense orientation resulted in enhanced proliferation and decreased differentiation, as well as in a more aggressive transformed phenotype compared with empty vector-transfected control cells. In contrast, cells transfected with PKC-alpha cDNA in the sense orientation demonstrated decreased proliferation, enhanced differentiation, and an attenuated tumor phenotype compared with these control cells. These data show that alterations in the expression of PKC-alpha induce changes in the proliferation, differentiation, and tumorigenicity of CaCo-2 cells. Furthermore, these findings indicate that loss of PKC-alpha expression in sporadic human and chemically induced colonic cancers may confer a relative growth advantage during colonic malignant transformation.

Differential effects of theaflavin monogallates on cell growth, apoptosis, and Cox-2 gene expression in cancerous versus normal cells.

Theaflavin (TF-1), theaflavin-3-monogallate and theaflavin-3'-monogallate mixture (TF-2), and theaflavin-3,3'-digallate (TF-3) are the major black tea polyphenols. Here we compared the effects of these polyphenols on cell growth, apoptosis, and gene expression in normal and cancerous cells. We showed that TF-2 (10-50 microM) inhibited the growth of SV40 transformed WI38 human cells (WI38VA) and Caco-2 colon cancer cells but had little effect on the growth of their normal counterparts. The IC50s of TF-2 for the growth inhibition of WI38 and WI38VA cells were, respectively, 300 and 3 microM. The other two black tea polyphenols, TF-1 and TF-3, did not exhibit such differential growth-inhibitory effect. TF-2, but not TF-1 or TF-3, induced apoptosis in transformed WI38VA cells but not in normal WI38 cells, suggesting that apoptosis was responsible, at least in part, for the differential growth-inhibitory effect of TF-2. Cox-2 has been implicated in intestinal carcinogenesis. Among the tea polyphenols tested, TF-2 and, to a lesser degree, (-)-epigallocatechin gallate inhibited cyclooxygenase (Cox)-2 gene expression. TF-2 at 50 microM completely blocked the serum-induced Cox-2 gene expression at both mRNA and protein level. Other genes, including c-fos, c-myc, thymidine kinase, proliferating cell nuclear antigen, BRCA1, BRCA2, and Cox-1, were not significantly affected by TF-2. These findings suggest that TF-2 may be responsible, at least in part, for the chemopreventive activity in black tea extracts.

Dietary indoles and isothiocyanates that are generated from cruciferous vegetables can both stimulate apoptosis and confer protection against DNA damage in human colon cell lines.

The natural indoles 3,3'-diindolylmethane (DIM), ascorbigen (ASG), indole-3-carbinol (I3C), and indolo[3,2-b]carbazole (ICZ), as well as the natural isothiocyanates sulforaphane (SUL), benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC), all possess cancer chemopreventive properties. It is now shown that DIM, ICZ, SUL, and BITC can each stimulate apoptosis in human colon adenocarcinoma LS-174 and Caco-2 cells. Treatment of LS-174 cells with nontoxic doses of DIM, ASG, I3C, or ICZ affected an increase of up to 21-fold in cytochrome P450 1A1 (CYP1A1). None of these indoles caused an elevation in either aldo-keto reductase 1C1 (AKR1C1) or the gamma-glutamylcysteine synthetase heavy subunit (GCS(h)), but DIM, I3C, and ICZ produced a very modest increase in NAD(P)H:quinone oxidoreductase 1 (NQO1). By contrast, nontoxic doses of SUL, BITC, or PEITC failed to induce expression of CYP1A1 in LS-174 cells, but caused an increase of between 11- and 17-fold in the protein levels of AKR1C1, NQO1, and GCS(h). Treatment of the colon cell line with ICZ or SUL caused increases in the levels of mRNA for CYP1A1, AKR1C1, and NQO1 that were consistent with the enzyme data. Exposure of Caco-2 cells to media containing indoles or isothiocyanates gave similar results to those obtained using LS-174 cells. Evidence is presented that the ability of indoles and isothiocyanates to stimulate either xenobiotic response element- or antioxidant response element-driven gene expression accounts for the two groups of phytochemicals inducing different gene batteries. Pretreatment of LS-174 cells for 24 h with ICZ and SUL before exposure for 24 h to benzo(a)pyrene (BaP) reduced to <20% the number of single-strand DNA breaks produced by the carcinogen. Neither ICZ alone nor SUL alone were able to confer the same degree of protection against DNA damage produced by BaP as they achieved in combination. Similar results were obtained with H(2)O(2) as the genotoxic agent. Together, these phytochemicals may prevent colon tumorigenesis by both stimulating apoptosis and enhancing intracellular defenses against genotoxic agents.

IGF-II/IGF-I receptor pathway up-regulates COX-2 mRNA expression and PGE2 synthesis in Caco-2 human colon carcinoma cells.

Nonsteroidal anti-inflammatory drugs reduce the risk of colon cancer and this effect is mediated in part through inhibition of type 2 prostaglandin endoperoxide synthase/ cyclo-oxygenase (COX-2). In the present study, we demonstrate that COX-2 expression and PGE2 synthesis are up-regulated by an IGF-II/IGF-I receptor autocrine pathway in Caco-2 colon carcinoma cells. COX-2 mRNA and PGE2 levels are higher in proliferating cells compared with post-confluent differentiated cells and in cells that constitutively overexpress IGF-II. Up-regulation of COX-2 expression by IGF-II is mediated through activation of IGF-I receptor because: (i) treatment of Caco-2 cells with a blocking antibody to the IGF-I receptor inhibits COX-2 mRNA expression; (ii) transfection of Caco-2 cells with a dominant negative IGF-I receptor reduces COX-2 expression and activity. Also, the blockade of the PI3-kinase, that mediates the proliferative effect of IGF-I receptor in Caco-2 cells, inhibits IGF-II-dependent COX-2 up-regulation and PGE2 synthesis. Moreover, COX-2 expression and activity inversely correlate with the increase of apoptosis in parental, IGF-II and dominant-negative IGF-I receptor transfected cells. This study suggests that induction of proliferation and tumor progression of colon cancer cells by the IGF-II/IGF-I receptor pathway may depend on the activation of COX-2-related events.

Endogenous lipase activity in Caco-2 cells.

Dietary triglycerides, the major precursors of long chain fatty acids (FA), require hydrolysis by pancreatic enzymes prior to their absorption by the small intestine. Although Caco-2 cells are frequently employed for the study of enterocyte lipid metabolism, the presence of an endogenous lipase activity has never been previously reported. The major goal of this investigation was to determine the presence of endogenous Caco-2 cell lipase activity, to examine its capacity to hydrolyze triglycerides, and to define its intracellular location. Caco-2 cells were found to have an endogenous lipase activity, capable of hydrolyzing [1-14C]triolein from the apical cell compartment. A time and concentration dependence of lipase activity was observed, with hydrolysis of triolein into free fatty acids and monoglyceride. The majority of the lipase activity was found in the cytosolic cell fraction and, to a lesser extent, in the apical brush border membrane and other organelles. Protamine sulfate markedly reduced the Caco-2 cell lipase activity, yet it remained relatively insensitive to high concentrations of NaCl, taurocholate, calcium, heparin and chloroquine. The addition of exogenous human gastric lipase to the medium of the apical compartment resulted in a significant increased rate of hydrolysis of triolein, followed by enhanced Caco-2 cell fatty acid uptake and basolateral lipid secretion. The major esterified intracellular lipids were triglycerides and phospholipids. We conclude that Caco-2 cells possess an endogenous lipase capable of hydrolyzing cytosolic triglycerides. Furthermore, activity present on the apical membrane and secreted into the apical medium, though quantitatively less important than the cytosolic lipase, may permit an additional route for energy uptake. The addition of gastric lipase to the Caco-2 cell cultures greatly enhanced FA uptake above that seen with the endogenous lipase alone.

Induction of gene expression of xenobiotic metabolism enzymes and ABC-transport proteins by PAH and a reconstituted PAH mixture in human Caco-2 cells.

It was shown recently that in epithelial Caco-2 cells the food contaminant benzo[a]pyrene (B[a]P) is metabolized and B[a]P-sulfate metabolites were transported out of the cells. The aim of this study was to investigate whether B[a]P and other polycyclic aromatic hydrocarbons (PAH) such as chrysene, phenanthrene, benzo[k]fluoranthene (B[k]F), dibenzo[a,l]pyrene (DB[a,l]P), and pyrene alone or in a mixture in a ratio as they occur in tobacco smoke have effects on gene expression of intestinal cytochrome P450 enzymes (CYP), Phase II enzymes and ATP-binding cassette (ABC)-transport proteins in the human Caco-2 cells. B[a]P induced its own metabolism. Treatment of the Caco-2 cells with B[a]P, chrysene, B[k]F, or DB[a,l]P induced mRNA expression of CYP1A1 and CYP1B1 specifically as measured by RT-PCR. In contrast, the mRNA expression of the microsomal epoxide hydrolase (mEH) was not affected by PAH. The gene expression of the Phase II enzymes UDP-glucuronosyltransferase 1A6 (UGT1A6) and UGT1A7 was also induced by these PAH but treatment with them had no effect on gene expression of sulfotransferases (SULT) at all. Of the ABC-transport proteins, MDR1 mRNA expression was induced by treatment with carcinogenic PAH, whereas MRP2 mRNA expression was not changed. The mixture of PAH also induced CYP1A1, CYP1B1, UGT1A6, and UGT1A7 mRNA expression. We conclude that B[a]P, chrysene, B[k]F, and DB[a,l]P have specific effects on intestinal CYP1A1, CYP1B1, UGT1A6, and UDP1A7 mRNA expression but no effects on the expression of SULT.

IL-1beta stimulates argininosuccinate synthetase gene expression through NF-kappaB in Caco-2 cells.

Argininosuccinate synthetase (ASS) is limiting the arginine synthesis and can be stimulated by immunostimulants. We previously identified a putative NF-kappaB element in the human ASS gene promoter but its functionality was unknown (Husson et al., Eur. J. Biochem. 270 (2003) 1887). In the present study, using Caco-2 cells, a human enterocyte line, we demonstrate that IL-1beta rapidly induces the expression of the ASS gene at a transcriptional level through NF-kappaB activation. Using gel shift assay and double-strand oligonucleotide sequence of the identified putative NF-kappaB binding site of the ASS promoter, we provide evidence that NF-kappaB may functionally interact with this element.

Induction of phase-1 metabolizing enzymes by oltipraz, flavone and indole-3-carbinol enhance the formation and transport of benzo[a]pyrene sulfate conjugates in intestinal Caco-2 cells.

The small intestine is well equipped with various phase-1 and phase-2 xenobiotic metabolizing enzymes (XME), which contribute to the detoxification process of the body. Many XME are regulated via aryl hydrocarbon receptor (AhR)-dependent pathways, and numerous naturally occurring AhR agonists (e.g. flavonoids, dietary indoles) have been identified to date. In the present study we show that pretreatment of Caco-2 cells with food-associated compounds (flavone and indole-3-carbinol) and with the anticancer chemopreventive agent oltipraz enhances the formation of the major metabolites of the procarcinogen benzo[a]pyrene (BP) formed by intestinal Caco-2 cells, namely BP-1-sulfate and BP-3-sulfate, and their transport to the apical compartment of a Transwell chamber. Oltipraz treatment was most effective in this regard followed by flavone and indole-3-carbinol. The effect observed here after pretreatment with oltipraz, flavone and I3C was the result of the induction of both CYP1A1 and CYP1B1, as was confirmed by analysis of CYP1A1 (protein and mRNA) and CYP1B1 (mRNA) expression. In summary, our study shows that the induction of both CYP1A1 and CYP1B1 resulted in an accelerated metabolism and an enhanced clearance of the potent procarcinogen BP, indicating that flavone, indole-3-carbinol and oltipraz have an impact on the biochemical barrier against BP in intestinal cells.

An inter-laboratory study to evaluate the effects of medium composition on the differentiation and barrier function of Caco-2 cell lines.

Differentiated human intestinal Caco-2 cells are frequently used in toxicology and pharmacology as in vitro models for studies on intestinal barrier functions. Since several discrepancies exist among the different lines and clones of Caco-2 cells, comparison of the results obtained and optimisation of models for use for regulatory purposes are particularly difficult, especially with respect to culture conditions and morphological and biochemical parameters. An inter-laboratory study has been performed on the parental cell line and on three clonal Caco-2 cell lines, with the aim of standardising the culture conditions and identifying the best cell line with respect to parameters relevant to barrier integrity, namely, trans-epithelial electrical resistance (TEER) and mannitol passage, and of epithelial differentiation (alkaline phosphatase activity). Comparison of the cell lines maintained in traditional serum-supplemented culture medium or in defined medium, containing insulin, transferrin, selenium and lipids, showed that parameter performance was better and more reproducible with the traditional medium. The maintenance of the cell lines for 15 days in culture was found to be sufficient for the development of barrier properties, but not for full epithelial differentiation. Caco-2/TC7 cells performed better than the other three cell lines, both in terms of reproducibility and performance, exhibiting low TEER and mannitol passage, and high alkaline phosphatase activity.