HT-29 Colon Adenocarcinoma Cells Research Articles

Polyunsaturated fatty acids sensitize human colon adenocarcinoma HT-29 cells to death receptor-mediated apoptosis

The proliferative and apoptotic response to TNF-α and anti-Fas antibody (CH-11) in human colon adenocarcinoma HT-29 cells was modulated by pretreatment with arachidonic (AA, 20:4, n−6) or docosahexaenoic (DHA, 22:6, n−3) fatty acids, which alone increased reactive oxygen species production and lipid peroxidation, and decreased the S-phase of the cell cycle. The higher amount of floating cells, subG 0/G 1 population and apoptotic cells detected in pre-treated cells was potentiated by cycloheximide. The effects of CH-11 were associated with activation of caspase-8, -9, and -3, cleavage of poly(ADP-ribose)polymerase-PARP, and decreased mitochondrial membrane potential (MMP), but these parameters were not significantly changed after PUFA pretreatment.

Piperine Enhances the Bioavailability of the Tea Polyphenol (−)-Epigallocatechin-3-gallate in Mice

(-)-Epigallocatechin-3-gallate (EGCG), from green tea (Camellia sinensis), has demonstrated chemopreventive activity in animal models of carcinogenesis. Previously, we reported the bioavailability of EGCG in rats (1.6%) and mice (26.5%). Here, we report that cotreatment with a second dietary component, piperine (from black pepper), enhanced the bioavailability of EGCG in mice. Intragastric coadministration of 163.8 micromol/kg EGCG and 70.2 micromol/kg piperine to male CF-1 mice increased the plasma C(max) and area under the curve (AUC) by 1.3-fold compared to mice treated with EGCG only. Piperine appeared to increase EGCG bioavailability by inhibiting glucuronidation and gastrointestinal transit. Piperine (100 micromol/L) inhibited EGCG glucuronidation in mouse small intestine (by 40%) but not in hepatic microsomes. Piperine (20 micromol/L) also inhibited production of EGCG-3"-glucuronide in human HT-29 colon adenocarcinoma cells. Small intestinal EGCG levels in CF-1 mice following treatment with EGCG alone had a C(max) = 37.50 +/- 22.50 nmol/g at 60 min that then decreased to 5.14 +/- 1.65 nmol/g at 90 min; however, cotreatment with piperine resulted in a C(max) = 31.60 +/- 15.08 nmol/g at 90 min, and levels were maintained above 20 nmol/g until 180 min. This resulted in a significant increase in the small intestine EGCG AUC (4621.80 +/- 1958.72 vs. 1686.50 +/- 757.07 (nmol/g.min)). EGCG appearance in the colon and the feces of piperine-cotreated mice was slower than in mice treated with EGCG alone. The present study demonstrates the modulation of the EGCG bioavailablity by a second dietary component and illustrates a mechanism for interactions between dietary chemicals.

Early enterocytic differentiation of HT-29 cells: biochemical changes and strength increases of adherens junctions

We have characterized the modulation of cell–cell adhesion and the structure of adherens junctions in the human colon adenocarcinoma HT-29 cell line that differentiates into enterocytes after glucose substitution for galactose in the medium. We demonstrate that differentiated cells (HT-29 Gal) rapidly established E-cadherin-mediated interactions in aggregation assays. This effect is not due to an increase in E-cadherin expression during this early stage of cell differentiation, but rather results from the maturation of preexisting adherens junctions. These junctions are characterized by the redistribution of E-cadherin to the basolateral membrane and its co-localization with the actin cytoskeleton. Subcellular fractionation studies indicate that actin-associated E-cadherins bind β-catenin and p120 ctn. Furthermore, the p120 ctn/E-cadherin association is upregulated. These data reveal a cooperative interaction between p120 ctn and E-cadherin that corresponds to mature functional adherens junctions able to initiate tight cell–cell adhesion required for epithelium architecture and further affirm the gatekeeper role of p120 ctn.

MEK Is a Key Modulator for TLR5-induced Interleukin-8 and MIP3α Gene Expression in Non-transformed Human Colonic Epithelial Cells

Flagellin, a specific ligand for Toll-like receptor 5 (TLR5), is a molecular pattern associated with several bacterial species. Recently, TLR signaling has been intensively studied. However, TLR5-associated signaling in non-transformed colonocytes has not been investigated. Here we studied the expression of cytokines induced by flagellin in non-transformed human colonic NCM460 cells and the signaling mechanisms mediating these responses. Cytokine expression array experiments showed that exposure of the cells to flagellin (100 ng/ml) for 12 h increased the expression of interleukin (IL)-8 and macrophage-inflammatory protein 3alpha (MIP3alpha) in a TLR5-specific manner. Flagellin also activated MAP kinases (ERK1/2, JNK, and p38) and degraded IkappaBalpha. Dominant negative MEK1 (a kinase that activates ERK1/2) blocked flagellin-stimulated IL-8 and MIP3alpha transcriptional activity, while the MEK-specific inhibitors PD98059 and U0126 reduced protein production of these cytokines. Conversely, transfection with a constitutively active MEK1 increased IL-8 and MIP3alpha transcriptional activity in a NFkappaB-independent manner. Furthermore, overexpression of the constitutively active MEK1 induced IL-8 and MIP3alpha protein production. We also demonstrated that C-terminal coiled-coil and TRAF-C domains of TRAF6, unable to mediate NFkappaB activation, are involved in MEK-mediated IL-8 and MIP3alpha expression. Thus, in non-transformed human colonocytes, MEK activation following flagellin/TLR5 engagement is a key modulator for NFkappaB-independent, IL-8 and MIP3alpha expression.

The effects of TNF-α and inhibitors of arachidonic acid metabolism on human colon HT-29 cells depend on differentiation status

The level of differentiation could influence sensitivity of colonic epithelial cells to various stimuli. In our study, the effects of TNF-α, inhibitors of arachidonic acid (AA) metabolism (baicalein, BA; indomethacin, INDO; niflumic acid, NA; nordihydroguaiaretic acid, NDGA), and/or their combinations on undifferentiated or sodium butyrate (NaBt)-differentiated human colon adenocarcinoma HT-29 cells were compared. NaBt-treated cells became growth arrested (blocked in G 0/G 1 phase of the cell cycle), and showed down-regulated Bcl-x L and up-regulated Bak proteins and increased expression of cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX). These cells were more perceptive to anti-proliferative and apoptotic effects of TNF-α. Both inhibitors of LOX (BA and NDGA) and COX (INDO and NA) in higher concentrations modulated cell cycle changes accompanying NaBt-induced differentiation and induced various level of cell death in undifferentiated and differentiated cells. Most important is our finding that TNF-α action on proliferation and cell death can be potentiated by co-treatment of cells with AA metabolism inhibitors, and that these effects were more significant in undifferentiated cells. TNF-α and INDO co-treatment was associated with accumulation of cells in G 0/G 1 cell cycle phase, increased reactive oxygen species production, and elevated caspase-3 activity. These results indicate the role of differentiation status in the sensitivity of HT-29 cells to the anti-proliferative and proapoptotic effects of TNF-α, AA metabolism inhibitors, and their combinations, and imply promising possibility for novel anti-cancer strategies.

The roles of JNK and apoptotic signaling pathways in PEITC-mediated responses in human HT-29 colon adenocarcinoma cells.

Phenethyl isothiocyanate (PEITC) is a potential chemopreventive agent that is present naturally in widely consumed vegetables, especially in watercress. It has been extensively investigated for its anticancer activities against lung, forestomach and esophageal tumorigenesis. Here we investigated the pro-apoptotic effect of PEITC in HT-29 human colorectal carcinoma cell line, and the mechanism of apoptosis induced by PEITC. PEITC-induced apoptosis was determined by DNA fragmentation assay and diamidino-2-phenylindole (DAPI) staining technique. To understand the mechanisms of apoptosis induced by PEITC, we studied the role of caspases, mitochondria-cytochrome c release, and mitogen-activated protein kinase (MAPK) signaling pathways involved in PEITC-induced apoptosis in HT-29 cells. Both the caspase-3 and -9 activities were stimulated by PEITC. The release of cytochrome c from the mitochondrial inter-space was time- and dose-dependent, with a maximal release at 50 micro M after 10 h treatment. Three MAPKs [JNK (c-Jun N-terminal kinase), extracellular signal-regulated protein kinase (ERK) and p38 kinase] were activated shortly after PEITC treatment in HT-29 cells. Importantly, the SP600125 compound, an anthrapyrazolone inhibitor of JNK, but not the ERK and p38 inhibitor, suppressed apoptosis induced by PEITC. Similarly, this JNK inhibitor attenuated both cytochrome c release and caspase-3 activation induced by PEITC. In summary, this study shows that PEITC can induce apoptosis in HT-29 cells in a time- and dose-dependent manner via the mitochondria caspase cascade, and the activation of JNK is critical for the initiation of the apoptotic processes. This mechanism of PEITC may play an important role in the killing of cancerous cells and offer a potential mechanism for its anticancer action in vivo.

Role of the AP-1 element and redox factor-1 (Ref-1) in mediating transcriptional induction of DT-diaphorase gene expression by oltipraz: a target for chemoprevention

The dithiolethione oltipraz is a potent chemopreventive agent in preclinical models, and induces the expression of protective enzymes in the colon mucosa and peripheral mononuclear cells of treated human subjects. We investigated the effects of oltipraz on DT-diaphorase expression in HT29 colon adenocarcinoma cells. Following a 24-hr exposure to 100 μM oltipraz, elevated steady-state levels of mRNA for Jun and Fos family members were observed. A nuclear run-on assay showed induction of c-fos and c-jun transcripts at the end of the exposure, peaking at 12 hr after resuspension of cells in drug-free medium. Gel mobility shift analysis revealed a similar time-course of induced nuclear factor binding to an AP-1 probe. Supershift analysis verified the participation of Jun and Fos in the complexes. The redox coactivator Ref-1, a function of which is to enhance AP-1 binding, was induced 5-fold by oltipraz. Immunodepletion of Ref-1 partially inhibited factor binding to the AP-1 probe. Deletion analysis of the DT-diaphorase promoter in a CAT reporter construct revealed that loss of the AP-1 site accounted for approximately 65% of the induction by oltipraz. Mutation of the AP-1 element in a full-length promoter construct yielded similar results. These data suggest the importance of transcriptional activation mediated by AP-1 in the chemopreventive activity of oltipraz, and indicate that novel chemoprevention structures may be selected based upon agonist activity at this locus.

Benzo (a)pyrene Activates Extracellular Signal-Related and p38 Mitogen-Activated Protein Kinases in HT29 Colon Adenocarcinoma Cells: Involvement in NAD(P)H:quinone Reductase Activity and Cell Proliferation

Benzo (a)pyrene (BP) is a polyaromatic hydrocarbon generated from the combustion of fossil fuel. Human exposure results primarily through dietary sources and smoking. Little is known about the effect of BP on mitogen-activated protein (MAP) kinases, which include extracellular signal-related kinase (ERK), Jun N-terminal kinase (JNK), and p38. We investigated the participation of BP-induced MAP kinase activation in cell growth and increases in activity of the detoxification enzyme NAD(P)H:quinone reductase (QR). In HT29 human colon carcinoma cells, 10 nM BP activated ERK and p38 but not JNK after 24 h. Treatment with 10 nM BP increased QR activity within 24 h and tritiated thymidine ([ 3H]thyd) incorporation after 48 h and reduced cell viability after 72 h. Using the MAP kinase inhibitors PD98059 and SB203580, we investigated the relative contributions of ERK and p38 to QR activity and [ 3H]thyd cell incorporation. Inhibition of ERK eliminated BP-induced QR activity, whereas inhibition of p38 had no effect on QR activity. Treatment of cells with 10 nM BP increased [ 3H]thyd incorporation by 50% after 48 h. This increase was eliminated by ERK but not p38 inhibition. In conclusion, 10 nM BP activates ERK and p38, but only ERK contributes to BP-induced QR activity. ERK, but not p38 activation participated in [ 3H]thyd incorporation. In summary, BP influences cellular signaling pathways at concentrations present in routine human exposures.

Cancer chemoprevention by hydroxytyrosol isolated from virgin olive oil through G1 cell cycle arrest and apoptosis.

Recent epidemiological evidence and animal studies suggest a relationship between the intake of olive oil and a reduced risk of several malignancies. The present study assesses the effect of hydroxytyrosol, a major antioxidant compound of virgin olive oil, on proliferation, apoptosis and cell cycle of tumour cells. Hydroxytyrosol inhibited proliferation of both human promyelocytic leukaemia cells HL60 and colon adenocarcinoma cells HT29 and HT29 clone 19A. The con-centrations of hydroxytyrosol which inhibited 50% of cell proliferation were approximately 50 and approximately 750 micromol/l for HL60 and both HT29 and HT29 clone 19A cells, respectively. At concentrations ranging from 50 to 100 micromol/l, hydroxytyrosol induced an appreciable apoptosis in HL60 cells after 24 h of incubation as evidenced by flow cytometry, fluorescence microscopy and internucleosomal DNA fragmentation. Interestingly, no effect on apoptosis was observed after similar treatment of freshly isolated human lymphocytes and polymorphonuclear cells. The DNA cell cycle analysis, quantified by flow cytometry, showed that the treatment of HL60 cells with hydroxytyrosol 50-100 micromol/l arrested the cells in the G0/G1 phase with a concomitant decrease in the cell percentage in the S and G2/M phases. These results support the hypothesis that hydroxytyrosol may exert a protective activity against cancer by arresting the cell cycle and inducing apoptosis in tumour cells, and suggest that hydroxytyrosol, an important component of virgin olive oil, may be responsible for its anticancer activity.

Homocamptothecin-daunorubicin association overcomes multidrug-resistance in breast cancer MCF7 cells.

The multidrug-resistance (MDR) status of a novel camptothecin analogue, homocamptothecin (hCPT), was investigated in human colon adenocarcinoma HT29 cells, myelogenous leukemia K562 cells and breast carcinoma MCF7 cells. The cytotoxicity of hCPT was not sensitive to the MDR status in K562 cell lines. However, its cytotoxicity was altered by MRP1, but not Pgp, in naturally MRP1-expressing HT29 cells, and etoposide- and doxorubicin-resistant MCF7/VP and MCF7/DOX cells, respectively. These cells were sensitized to hCPT in presence of MK571, probenecid but not verapamil. These results led to consider hCPT as a substrate for MRP1 and a potential modulator of MRP1 activity. The relationship between the cytotoxic effect of anthracyclines and their nuclear localization had been previously demonstrated. We show that MRPI mediated the daunorubicin (DNR) efflux in MCF7/VP and MCF7/DOX cells. The combination of sub-toxic doses of hCPT with DNR resulted in the potentiation of DNR activity, well-correlated with an increase in its nuclear accumulation in MCF7/VP cells. Simultaneous pattern was shown to provide higher cytotoxic response than sequential one. In agreement, hCPT increased also the DNR nuclear accumulation in low MRP1-expressing MCF7/DOX cells. However, the enhancement of cytotoxicity in the DNR-hCPT combination was poorly correlated with the nuclear concentration of DNR in MCF7/DOX cells. In addition to the increase in DNR accumulation, the potentiation of DNR activity by hCPT in MCF7/DOX cells implied a synergistic mechanism between both drugs. These data suggest that the present topoisomerase I/II inhibitors combination may be of clinical interest to overcome MDR phenotype in DNR-treated breast cancer patients.

Cytotoxicity and cellular differentiation activity of methylenebis(phosphonate) analogs of tiazofurin and mycophenolic acid adenine dinucleotide in human cancer cell lines

Mycophenolic acid (MPA) is a fungally-derived inhibitor of inosine 5′-monophosphate dehydrogenase (IMPDH). MPA binds IMPDH at the nicotinamide sub-site of the NAD cofactor binding domain leaving the adenosine sub-site empty. In order to improve the binding affinity we synthesized MPA analogs by linking adenosine 5′-methylenebis(phosphonate) with mycophenolic alcohols containing 2-, 4-, and 6-carbon atoms in their aliphatic side chain. Adenine dinucleotide analogs of tiazofurin, selenazofurin and benzamide riboside were synthesized as P 1, P 2-disubstituted pyrophosphates. Cytotoxicity of each analog was examined in human colon adenocarcinoma HT-29 and erythroleukemia K562 cells, and induction of differentiation in K562 cells by these agents was determined. Mycophenolic acid is currently used as an immunosuppressant but its anticancer action is limited by inactivation due to rapid glucuronidation. The new analogs show resistance to metabolism to inactive species and exhibit enhanced cytotoxicity in tumor cell lines, and therefore could be useful as anticancer agents.

Insulin induces PFKFB3 gene expression in HT29 human colon adenocarcinoma cells

Fructose 2,6-bisphosphate is present at high concentrations in many established lines of transformed cells. It plays a key role in the maintenance of a high glycolytic rate by coupling hormonal and growth factor signals with metabolic demand. The concentration of fructose 2,6-bisphosphate is controlled by the activity of the homodimeric bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2). We report here the PFKFB-3 gene expression control by insulin in the human colon adenocarcinoma HT29 cell line. The incubation of these cells with 1 μM insulin resulted in an increase in the PFK-2 mRNA level after 6 h of treatment, this effect being blocked by actinomycin D. Furthermore, insulin induced ubiquitous PFK-2 protein levels, that were evident after a lag of 3 h and could be inhibited by incubation with cycloheximide.

Production of Recombinant Large Proneurotensin/Neuromedin N-Derived Peptides and Characterization of Their Binding and Biological Activity

Proneurotensin/neuromedin N (pro-NT/NN) is the common precursor of two biologically active related peptides, neuromedin N (NN) and neurotensin (NT). It undergoes a tissue-specific processing leading to the formation in some tissues and cancer cell lines of large peptides ending with the NT (large NT) or NN (large NN) sequence. In this study, we prepared and purified high amounts of recombinant large NT and large NN using the Drosophila S2 cell expression system. The binding and pharmacological properties of recombinant large peptides were characterized and compared to those of NT and NN using either COS cells transfected with the human subtype-1 NT receptor (hNTS1) or the human colon adenocarcinoma HT29 cell line that endogenously expresses hNTS1. Furthermore, the metabolic stability of the large peptides, when exposed to HT29 cells, was compared to that of NT and NN. Both large NT and large NN were able to bind to and activate hNTS1 with potencies that were approximately 10 times lower than that of their small counterpart. In addition, the large forms proved to be far less sensitive to degradation than the small peptides. Taken together, these data suggest that the large forms might represent endogenous, long-lasting activators of hNTS1 in a number of physiopathological situations.

Immunolocalization of a new intestinal antiproliferative factor in human intestinal epithelial cells.

A new intestinal antiproliferative factor (IAF) with an approximate molecular weight of 120 kDa has been purified from the human small intestine. This factor blocks the progression of human colon adenocarcinoma cells HT-29 from the G1 to the S phase. IAF, specific of the lower part of the digestive tract, was detected rather late in mouse embryonic development. For determination of the specific intestinal cell producing IAF, long-term differentiated mucus-secreting HT-29 Cl 16E and enterocytic HT-29 Cl 19A cell lines were used. IAF is synthesized exclusively in the intestinal goblet cells; it is processed in the RER and Golgi complex before being excreted in secretory vesicles independently of mucin secretion. IAF can be considered a growth inhibitor of intestinal proliferation for the same reason as TGF-beta. However, two features differentiate it from TGF-beta: (1) the intestinal cell type synthesizing it, and (2) the delay in its expression in embryonic development. Particular interest was paid to IAF expression in pathological conditions using human colon biopsies. IAF was consistently recovered in biopsies from patients with inflammatory bowel diseases and benign tumors, but it was never detected in malignant tumors. IAF could represent a marker of colon cancer owing to its absence from malignant tumors.

Inhibition of proliferation of HT-29 colon adenocarcinoma cells by carboxylate NSAIDs and their acyl glucuronides

Many nonsteroidal anti-inflammatory drugs (NSAIDs) which have antiproliferative activity in colon cancer cells are carboxylate compounds forming acyl glucuronide metabolites. Acyl glucuronides are potentially reactive, able to hydrolyse, rearrange into isomers, and covalently modify proteins under physiological conditions. This study investigated whether the acyl glucuronides (and isomers) of the carboxylate NSAIDs diflunisal, zomepirac and diclofenac had antiproliferative activity on human adenocarcinoma HT-29 cells in culture. Included as controls were the carboxylate NSAIDs themselves, the non-carboxylate NSAID piroxicam, and the carboxylate non-NSAID valproate, as well as its acyl glucuronide and isomers. The compounds were incubated at 1–3000 μM with HT-29 cells for 24 hr, with [ 3H]-thymidine added for an additional 2 hr incubation. IC 50 values were calculated from the concentration-inhibition response curves for thymidine uptake. The four NSAIDs inhibited thymidine uptake, with IC 50 values about 200–500 μM. All of the NSAID acyl glucuronides (and isomers, tested in the case of diflunisal) showed antiproliferative activity broadly comparable to the parent drugs. This activity may stem from direct uptake of intact glucuronide/isomers followed by covalent modification of proteins critical in the cell replication process. However, hydrolysis during incubation and cellular uptake of liberated parent NSAID will play a role. In HT-29 cells incubated with zomepirac, covalently modified proteins in cytosol were detected by immunoblotting with a zomepirac antibody, suggesting that HT-29 cells do have the capacity to glucuronidate zomepirac. The anti-epileptic drug valproate had no effect on inhibition of thymidine uptake, though, surprisingly, its acyl glucuronide and isomers were active. The reasons for this are unclear at present.

IRF-1-Mediated CAS Expression Enhances Interferon-γ-Induced Apoptosis of HT-29 Colon Adenocarcinoma Cells

The expression of CAS is reported to be upregulated in a variety of human tumor cells, and such expression correlates with the development of tumors. CAS also plays a role in apoptosis. We investigated whether CAS expression affects the susceptibility of tumor cells to IFN-γ-induced apoptosis. Our data show that IFN-γ treatment induces CAS expression in HT-29 tumor cells. IFN-γ-induced gene expression is primarily mediated by the transcriptional factor, IRF-1. Our data show that IRF-1 mediates IFN-γ-induced CAS expression. Transfection of HT-29 cells with CAS expression vector did not induce apoptosis of cells; nevertheless, CAS overexpression greatly enhanced IFN-γ-induced apoptosis of cells. CPP32 is regarded as one of the central apoptosis executioner molecules. CAS overexpression enhances IFN-γ-induced CPP32 expression. These results indicate that tumor cells highly expressing CAS may be more susceptible to apoptosis induced by reagents that are capable of inducing CAS expression. Thus, CAS may be a target for the elimination of tumors.

Effects of Cyclooxygenase and Lipoxygenase Inhibitors on the Proliferation of Colon Cancer Cells and Their Production of Eicosanoids.

Epidemiological and laboratory studies suggest that nonsteroidal antiinflammatory drugs (NSAIDs) reduce the risk of colon cancer and that the inhibition of colon cancer is mediated through modulation of eicosanoid production. The present study examined the effect of cyclooxygenase (COX) and lipoxygenase (LOX) inhibitors on colon cancer cell growth and prostaglandin E(2) (PGE(2)) or leukotriene B(4) (LTB(4)) secretion by these cells. The human colon adenocarcinoma cell lines, Caco-2 and HT-29 cells, were cultured in serum-free medium with various concentrations of indomethacin, piroxicam or esculetin in the presence of 0.15nM or 10nM linoleic acid. Cell number was estimated by MTT assay and PGE(2) and LTB(4) were analyzed by enzyme immunoassay. The NSAIDs inhibited cell proliferation in a concentration-dependent manner. However, the potency and efficacy of each drug varied in the two cell lines. In Caco-2 cells, the effect of esculetin was higher than that of indomethacin, and piroxicam had no effect. In HT-29 cells, only indomethacin significantly inhibited cell proliferation. All three agents inhibited PGE(2) secretion in a dose-dependent manner; the effect of indomethacin was highest and that of esculetin lowest. The secretion of LTB4 was increased by indomethacin and piroxicam but decreased by esculetin. The effects of these drugs on cell proliferation and eicosanoid secretion were not influenced by linoleic acid concentrations in the culture media. Neither exogenous PGE2 nor LTB4 affected cell proliferation. The results of Pearson correlation analyses revealed that changes in cell proliferation were somewhat related to both concentrations of NSAIDs in the culture medium and production of PGE(2) and LTB(4). The present data suggests that the anti-proliferative effect of NSAIDs may not be entirely attributed to changes in the production of PGE2 and/or LTB4 in the two colon cancer cell lines. These NSAIDs may inhibit cell proliferation largely independent of their ability to modulate eicosanoid synthesis.

RhoA-dependent switch between alpha2beta1 and alpha3beta1 integrins is induced by laminin-5 during early stage of HT-29 cell differentiation.

Integrin-mediated interactions between the basement membrane and epithelial cells control the differentiation of epithelia. We characterized the modulation of adhesive behaviors to basement membrane proteins and of integrin function in the human colon adenocarcinoma HT-29 cell line, which differentiates into enterocytes after the substitution of galactose for glucose in the medium. We demonstrate an increased capability of these cells to adhere to collagen type IV during the early stage of differentiation. This effect occurs without any changes in integrin cell surface expression but rather results from an alpha2beta1/alpha3beta1 integrin switch, alpha3beta1 integrin becoming the major collagen receptor. The increase in laminin-5 secretion and deposit on the matrix is a key factor in the mechanism regulating cell adhesion, because it is responsible for the activation of alpha3beta1 integrin. Furthermore, down-regulation of RhoA GTPase activity occurs during HT-29 cell differentiation and correlates with the activation of the integrin alpha3beta1. Indeed, C3 transferase, a RhoA GTPase inhibitor, induces a similar alpha2beta1/alpha3beta1 switch in undifferentiated HT-29 cells. These results indicate that the decrease in RhoA activation is the biochemical mechanism underlying this integrin switch observed during cell differentiation. The physiological relevance of such modulation of integrin activity in the functioning of the crypt-villus axis is discussed.

Interaction between bacterial peptides, neutrophils and goblet cells: a possible mechanism for neutrophil recruitment and goblet cell depletion in colitis

Goblet cell depletion occurs in various forms of colitis, but its mechanism is unknown. We have investigated two linked hypotheses: (i) that bacterial peptides, such as formyl-methionyl-leucyl-phenylalanine (fMLP), interact with epithelial cells inducing the release of chemokines, including interleukin-8 (IL-8), which in turn leads to the recruitment of neutrophils which release mucin secretagogues; (ii) that fMLP acts directly on epithelial cells to cause mucus secretion. Studies were performed to measure the effects of fMLP on the synthesis and secretion of IL-8 and mucus by the goblet cell differentiated colon cancer cell lines HT29-MTX (methotrexate-conditioned HT29 colonic adenocarcinoma cell line) and LS174T, and to assess the effects of neutrophil-derived secretagogues on goblet cell secretion in these cell lines. fMLP (0.1 microM) increased the secretion of IL-8 by 105% (P<0.0001) in HT29-MTX cells and by 401% (P<0.0001) in LS174T cells. fMLP also increased the synthesis and secretion of mucins by these cell lines, with maximal effects of 65% above control values for synthesis (P<0.01) and 73% for secretion (P<0.01). A dose-related increase (up to 67%; P<0.01) in mucin secretion was demonstrated in HT29-MTX cells in response to incubation with supernatant from activated neutrophils. This effect was largely (83%; P<0.02) inhibited by ICI 200,355, a specific inhibitor of neutrophil elastase. In conclusion, the bacterial peptide fMLP and neutrophil elastase are both potent mucus secretagogues for colon epithelial cells. fMLP also elicits release of the potent neutrophil chemoattractant IL-8 from colon epithelial cells. These findings support the hypothesis that the mucosal inflammation and mucus depletion seen in ulcerative colitis could result from interaction between bacterial peptides and the mucosa.

Contribution of apoptosis to responses in the comet assay

Apoptosis, a physiological process of selected cell deletion, leads to DNA fragmentation in typical segments of 180 base pairs. DNA strand breaks are also an effect induced by genotoxic compounds. The aim of this study was to compare these two types of damaging potentials by a known genotoxic substance and an apoptosis-inducing agent in HT-29 colon adenocarcinoma cells. The cells were incubated for 24 h with N-methyl- N′-nitro- N-nitrosoguanidin (MNNG), a potent DNA damage-inducing agent, staurosporine, an inhibitor of proteine kinase C and apoptosis-inducing agent, and hydrogen peroxide, a source of reactive oxygen species. Apoptosis was measured with the Annexin V affinity assay which detects the translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the cytoplasmic membrane, an early event in the apoptotic process. DNA damage as an end point of genotoxicity was detected by single cell microgel electrophoresis, also called “comet assay”. The results show that apoptosis does not necessarily need to correlate or coincide with DNA damage observed with genotoxic substances in the comet assay. The representative apoptosis-inducing agent (staurosporine) did not induce strand breaks in the tested concentrations (0.5 and 1.0 μM); genotoxic doses of the strand break inducing agent MNNG did not induce apoptosis. Therefore, the comet assay can be used as a specific test for detecting genotoxicity, and the results are not necessarily confounded by concomittant processes leading to apoptosis.