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.

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.

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.

Modulation of CYP1A1 activity by a Ginkgo biloba extract in the human intestinal Caco-2 cells.

Ginkgo biloba is a widely consumed dietary supplement. Some dietary active compounds modulate the activity of biotransformation enzymes inside the enterocytes and more interestingly of cytochrome P-450 1A1 (CYP1A1). This enzyme is of a particular interest because of its implication in the metabolism of some exogenous pro-carcinogens or endogenous molecules. In the present work, we have used Caco-2 cells to study the effect of a standard reference material of a Ginkgo biloba extract (GBE) (10-400 µg/ml), as well as of its major individual active compounds (kaempferol, quercetin, isorhamnetin, ginkgolides and bilobalide), alone or in mixtures, at realistic intestinal concentrations, on the induction of CYP1A1 activity, in the presence or absence of benzo[a]pyrene (B[a]P) (0.1 µg/ml), a well-known CYP1A1 inducer. 3-O-rutinosides of kaempferol, quercetin and isorhamnetin were also tested. We have demonstrated a strong induction (p < 0.005) of CYP1A1 activity and a slight, but significant (p < 0.005), decrease of this activity in the presence of B[a]P by the GBE at the realistic exposure level of 100 µg/ml. The inductive effect was explained, in part, by quercetin and kaempferol after 24h exposure while unknown compounds seem to be responsible for the strong CYP1A1 induction observed after 6h exposure. The inhibitory potency of flavonols on CYP1A1 activity in presence of B[a]P was much stronger for the aglycones than for the 3-O-rutinosides, explaining the slight effect observed with the GBE, mainly composed of glycosylated flavonoids. These results indicate that GBEs may disturb intestinal CYP1A1 activity and, in turn, affect the metabolism of other compounds. The present paper thus highlights the necessity to take these side effects into account when administrating Ginkgo biloba herbal supplements.

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.

Xanthine oxidoreductase - clinical significance in colorectal cancer and in vitro expression of the protein in human colon cancer cells.

Xanthine oxidoreductase (XOR) is a key enzyme in degradation of DNA and RNA, and has previously been shown to be decreased in aggressive breast and gastric cancer. In this study, XOR expression was assessed in tissue microarray specimens of 478 patients with colorectal cancer and related to clinical parameters. In addition, we performed in vitro studies of XOR activity, protein and mRNA in colon cancer cells (Caco-2). Results from the tissue expression analyses show that XOR was decreased in 62% and undetectable in 22% of the tumours as compared to normal tissue. Loss of XOR was associated with poor grade of differentiation (p=0.006) and advanced Dukes stage (p=0.03). In multivariate survival analysis, XOR was a prognostic factor (p=0.008), independent of Dukes stage, histological grade, age and tumour location. The in vitro analyses show that XOR is not measurable in undifferentiated Caco-2 cells, but appears and increases with differentiation. We conclude that XOR expression is associated with histological grade of differentiation and extent of disease in colorectal cancer, and it provides significant prognostic information independently of established factors.

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.

Curcumin decreases acid sphingomyelinase activity in colon cancer Caco-2 cells.

Curcumin has been shown to inhibit cell growth and induce apoptosis in colon cancer cells. The metabolism of sphingomyelin has implications in the development of colon cancert. We examined whether curcumin affects the enzymes that hydrolyse sphingomyelin in Caco-2 cells. The cells were cultured in both monolayer and polarized conditions and stimulated with curcumin. The activities of sphingomyelinases were determined. Sphingomyelin and its hydrolytic products were analysed by thin layer chromatography. The changes of acid sphingomyelinase protein were examined by Western blotting. We found that curcumin reduced the hydrolytic capacity of the cells against choline-labelled sphingomyelin, associated with a mild increase of cellular sphingomyelin in the cells. Analysis of the hydrolytic products revealed that the activity was derived from acid sphingomyelinase not from phospholipase D. The curcumin-induced reduction of acid SMase required more than 8 h stimulation. Western blotting showed reduced acid sphingomyelinase protein after curcumin stimulation. The inhibitory effect was more potent in monolayer cells than in polarised cells. No changes of other sphingomyelinases were identified. In the concentrations inhibiting acid sphingomyelinase, curcumin inhibited DNA synthesis and induced cell death. In conclusion, curcumin inhibits acid sphingomyelinase and the effect might be involved in its antiproliferative property against colon cancer cells.

An NSP4-dependant mechanism by which rotavirus impairs lactase enzymatic activity in brush border of human enterocyte-like Caco-2 cells.

Lactase-phlorizin hydrolase (LPH, EC 3.2.1.23-62) is a brush border membrane (BBM)-associated enzyme in intestinal cells that hydrolyse lactose, the most important sugar in milk. Impairing in lactase activity during rotavirus infection has been described in diseased infants but the mechanism by which the functional lesion occurs remains unknown. We undertook a study to elucidate whether rotavirus impairs the lactase enzymatic activity in BBM of human enterocyte cells. In this study we use cultured human intestinal fully differentiated enterocyte-like Caco-2 cells to demonstrate how the lactase enzymatic activity at BBM is significantly decreased in rhesus monkey rotavirus (RRV)-infected cells. We found that the decrease in enzyme activity is not dependent of the Ca(2+)- and cAMP-dependent signalling events triggered by the virus. The LPH biosynthesis, stability, and expression of the protein at the BBM of infected cells were not modified. We provide evidence that in RRV-infected cells the kinetic of lactase enzymatic activity present at the BBM was modified. Both BBM(control) and BBM(RRV) have identical K(m) values, but hydrolyse the substrate at different rates. Thus, the BBM(RRV) exhibits almost a 1.5-fold decreased V(max) than that of BBM(control) and is therefore enzymatically less active than the latter. Our study demonstrate conclusively that the impairment of lactase enzymatic activity at the BBM of the enterocyte-like Caco-2 cells observed during rotavirus infection results from an inhibitory action of the secreted non-structural rotavirus protein NSP4.

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.

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.

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.

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.

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.

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.

Identification of esterases expressed in Caco-2 cells and effects of their hydrolyzing activity in predicting human intestinal absorption.

The absorption characteristics of temocapril were investigated using Caco-2 cells, and the esterases expressed in Caco-2 cells were identified. Temocapril was almost completely hydrolyzed to temocaprilat during transport across Caco-2 cells. Hydrolysis experiments of temocapril in Caco-2 cell 9000g supernatant (S9) and brush-border membrane vesicles showed that temocapril was mainly hydrolyzed within the cells after uptake, after which the temocaprilat formed was transported to both the apical and basolateral surfaces. In native polyacrylamide gel electrophoresis by detection of hydrolase activity for 1-naphthylbutyrate, Caco-2 cell S9 showed a band with high esterase activity and another band with extremely low activity. The proteins in the major and minor bands were identified as carboxylesterase-1 (hCE-1) and carboxylesterase-2 (hCE-2). The abundant expression of hCE-1 in Caco-2 cells was supported by reverse transcription-polymerase chain reaction. In the normal human small intestine, hCE-2 is abundantly present, although the human liver expresses much higher levels of hCE-1 and lower levels of hCE-2. The expression pattern of carboxylesterases in Caco-2 cells is completely different from that in human small intestine but very similar to that in human liver. Since the substrate specificity of hCE-1 differs from that of hCE-2, it is suggested that the prediction of human intestinal absorption using Caco-2 cell monolayers should be performed carefully in the case of ester- and amide-containing drugs such as prodrugs.

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.

Butyrate-induced differentiation of colon cancer cells is PKC and JNK dependent.

Butyric acid, a short-chain fatty acid physiologically present in human large gut, is derived from bacterial fermentation of complex carbohydrates. It has been shown to reduce the growth and motility of colon cancer cell lines and to induce cell differentiation and apoptosis. Apoptosis is considered a result of normal colonocyte terminal differentiation in vivo. The aim of this study was to characterize the cellular mechanisms regulating differentiation of colon cancer cells stimulated with sodium butyrate (NaB). The two human colon cancer cell lines Caco-2 and HT-29 were treated with NaB at physiologically relevant concentrations. Alkaline phosphatase (ALP) activity, a marker of colonocyte differentiation, was increased 48 hr after treatment with 1 mM NaB. Higher doses of NaB (5 and 10 mM) induced apoptosis of the cells and failed to stimulate the colonocyte differentiation. Therefore, we assumed that butyrate augments cell differentiation and induces apoptosis, acting via various intracellular mechanisms, and butyrate-mediated programmed cell death cannot be considered a consequence of colonocyte terminal differentiation. The effect of NaB on ALP activity was significantly attenuated in the presence of inhibitors of protein kinase C and JNK. Inhibition of MEK-ERK signal transduction pathways augmented the impact of butyrate on colonocyte differentiation. These results suggest that butyrate could influence the colonocyte differentiation via modulation of the activity of cellular protein kinases and signal transduction.