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PubMed1995

Characterization of the effect of SR48692 on inositol monophosphate, cyclic GMP and cyclic AMP responses linked to neurotensin receptor activation in neuronal and non-neuronal cells.

1. Neurotensin stimulated inositol monophosphate (IP1) formation in both human colonic carcinoma HT29 cells and in mouse neuroblastoma N1E115 cells with EC50 values of 3.5 +/- 0.5 nM (n = 4) and 0.46 +/- 0.02 nM (n = 3), respectively. Neurotensin also stimulated cyclic GMP production with an EC50 of 0.47 +/- 1.2 nM and inhibited cyclic AMP accumulation induced by forskolin (0.5 microM) with an IC50 of 1.33 +/- 1.5 nM (n = 3) on the N1E115 cell line. 2. The competitive antagonism by the non-peptide neurotensin receptor antagonist, SR48692 of neurotensin-induced IP1 formation revealed pA2 values of 8.7 +/- 0.2 (n = 3) for HT29 and 10.1 +/- 0.2 (n = 3) for N1E115 cells. SR48692 also antagonized the cyclic GMP and cyclic AMP responses induced by neurotensin in the N1E115 cell line with pA2 values of 10.7 +/- 0.7 (n = 3) and 9.8 +/- 0.3 (n = 3), respectively. 3. In CHO cells transfected with the rat neurotensin receptor, neurotensin stimulated IP1 and cyclic AMP formation with EC50 values of 3.0 +/- 0.5 nM (n = 3) and 72.2 +/- 20.7 nM (n = 3), respectively. Both effects were antagonized by SR48692, giving pA2 values of 8.4 +/- 0.1 (n = 3) for IP1 and 7.2 +/- 0.4 (n = 3) for cyclic AMP responses. 4. Radioligand binding experiments, performed with [125I]-neurotensin (0.2 nM), yielded IC50 values of 15.3 nM (n = 2) and 20.4 nM (n = 2) for SR48692 versus neurotensin receptor binding sites labelled in HT29 and N1E115 cells, respectively. 5 In conclusion, SR48692 appears to be a potent, species-independent antagonist of the signal transduction events triggered by neurotensin receptor activation in both neuronal and non-neuronal cell systems.

Animals↗
PubMed2006

Ergosterol peroxide from an edible mushroom suppresses inflammatory responses in RAW264.7 macrophages and growth of HT29 colon adenocarcinoma cells.

BACKGROUND AND PURPOSE: 5alpha,8alpha-Epidioxy-22E-ergosta-6, 22-dien-3beta-ol (ergosterol peroxide) is a major antitumour sterol produced by edible or medicinal mushrooms. However, its molecular mechanism of action has yet to be determined. Here, we examine the anticancer and anti-inflammatory effects of ergosterol peroxide. EXPERIMENTAL APPROACH: After treating RAW264.7 macrophages with LPS and purified ergosterol peroxide or ergosterol, we determined LPS-induced inflammatory cytokines, nuclear DNA binding activity of transcription factors and phosphorylation of MAP kinases (MAPKs). HT29 colorectal adenocarcinoma cells were treated with ergosterol peroxide for 5 days. To investigate the antitumour properties of ergosterol peroxide, we performed DNA microarray and RT-PCR analyses and determined the reactive oxygen species (ROS) in HT29 cells. KEY RESULTS: Ergosterol peroxide suppressed LPS-induced TNF-alpha secretion and IL-1alpha/beta expression in RAW264.7 cells. Ergosterol peroxide and ergosterol suppressed LPS-induced DNA binding activity of NF-kappaB and C/EBPbeta, and inhibited the phosphorylation of p38, JNK and ERK MAPKs. Ergosterol peroxide down-regulated the expression of low-density lipoprotein receptor (LDLR) regulated by C/EBP, and HMG-CoA reductase (HMGCR) in RAW264.7 cells. In addition, ergosterol peroxide showed cytostatic effects on HT29 cells and increased intracellular ROS. Furthermore, ergosterol peroxide induced the expression of oxidative stress-inducible genes, and the cyclin-dependent kinase inhibitor CDKN1A, and suppressed STAT1 and interferon-inducible genes. CONCLUSION AND IMPLICATION: Our results suggest that ergosterol peroxide and ergosterol suppress LPS-induced inflammatory responses through inhibition of NF-kappaB and C/EBPbeta transcriptional activity, and phosphorylation of MAPKs. Moreover, ergosterol peroxide appears to suppress cell growth and STAT1 mediated inflammatory responses by altering the redox state in HT29 cells.

Adenocarcinoma↗
PubMed1994

Quercetin exerts a preferential cytotoxic effect on active dividing colon carcinoma HT29 and Caco-2 cells.

The effect of the naturally occurring flavonol, quercetin, was investigated on cell growth and metabolism of two human carcinoma cell lines, HT29 and Caco-2 cells, both during the exponentially growing phase and after confluence. Our results show clearly that, after a 48-h period of treatment, quercetin (in the range of concentration from 15 microM to 120 microM) exerted a preferential cytotoxic effect on active proliferating cells. This effect was dose dependent and was accompanied by a simultaneous inhibition of lactate release and a dramatic decrease of total cellular ATP content. In contrast, in confluent cells, quercetin failed to affect cell viability or lactate release, but led nevertheless to a depletion of cellular ATP level. In conclusion, the cytotoxicity of quercetin is markedly higher in actively growing cells in comparison with confluent cells.

Adenosine Triphosphate↗
PubMed1998

Regulation of tumour cell fatty acid oxidation by n-6 polyunsaturated fatty acids.

Fatty acids have been shown to regulate the expression of mRNA for both lipogenic and glycolytic enzymes in rat liver. The role of fatty acids in the regulation of carnitine palmitoyltransferase (CPT) I and II activity in tumour cells was investigated. The polyunsaturated fatty acids, gamma-linolenic and arachidonic acid, caused 60-70% inhibition of tumour cell CPT I activity and 45-50% inhibition of [14C]-palmitic acid oxidation to 14CO2. These effects were blocked by the cyclooxygenase inhibitor, indomethacin. Prostaglandins E1 and E2 caused marked inhibition of both CPT I and CPT II activity and inhibition of cell proliferation. Prostaglandin E2 production by tumour cells was increased in the presence of arachidonic acid and inhibited when indomethacin was present. The proliferation of the HT29 cell line was unaffected as was its CPT I and II activity by both fatty acids and prostaglandins. CPT I mRNA expression was not inhibited by fatty acids, indeed it increased-in the presence of arachidonic acid and prostaglandin E1. These results strongly suggest that polyunsaturated n-6 fatty acids are able, via prostaglandin products, to regulate the CPT activity of certain tumour cells. This may have a considerable impact on mitochondrial beta-oxidation and cellular metabolism of fatty acids, reflected in the marked inhibition of cell proliferation by these fatty acids.

Alprostadil↗
PubMed2004

MRP1 and glucosylceramide are coordinately over expressed and enriched in rafts during multidrug resistance acquisition in colon cancer cells.

Previously we have described a novel multidrug-resistant cell line, HT29(col), which displayed over expression of the multidrug-resistance protein 1 (MRP1) and an altered sphingolipid composition, including enhanced levels of glucosylceramide (GlcCer; Kok JW, Veldman RJ, Klappe K, Koning H, Filipeanu C, Muller M. Int J Cancer 2000;87:172-8). In our study, long-term screening revealed that, during colchicine-induced acquisition of multidrug resistance in a new HT29(col) cell line, increases in GlcCer occurred concomitantly with upregulation of MRP1 expression. Both MRP1 and GlcCer were found enriched in Lubrol-insoluble membrane domains. The expression of MRP1 and GlcCer were tightly correlated, as indicated also by a reversal of both at the later stage of colchicine consolidation. Resistance to colchicine was determined by MRP1, while glucosylceramide synthase (GCS) did not contribute: 1). Resistance was fully inhibited by MK571. 2). GCS expression and activity were not upregulated in HT29(col) cells. 3). Inhibition of GCS did not affect MRP1-mediated efflux function or sensitivity to colchicine. Instead, overall sphingolipid metabolism was upregulated through an increased rate of ceramide biosynthesis. In conclusion, upregulation of MRP1 occurs in concert with upregulation of GlcCer during multidrug-resistance acquisition, and both are enriched in rafts. The increased GlcCer pool does not directly modulate MRP1 function and cell survival.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Inhibition of Gal beta1, 4GlcNAc alpha2,6 sialyltransferase expression by antisense-oligodeoxynucleotides.

Treatment of human colorectal carcinoma cells HT29 with specific antisense oligodeoxynucleotides led to a decreased Gal beta1,4GlcNAc alpha2,6 sialyltransferase activity on the level of protein expression as well as on the mRNA level. Antisense treatment did not effect cell viability or cell growth. Oligodeoxynucleotides which were complementary to the region upstream of the initiation codon were particularly effective in inhibition of enzyme expression. No such inhibition was found by treatment of cells with oligodeoxynucleotides complementary to the region downstream of the initiation codon or by treatment of cells with scrambled controls or sense oligodeoxynucleotides.

Cell Division↗
PubMed2001

[Influence of HLA class I molecules expression on tumor cell resistance to NK lysis and the IFN-gamma regulatory effect].

OBJECTIVE: To investigate the relation between the NK lysis and HLA molecules expressed on the target cells as well as the regulatory effect of IFN-gamma. METHODS: The level of HLA-ABC molecules on seven human tumor cell lines were detected through the indirect immune fluorescence stain. NK lysis changes were observed after the blocking of HLA molecules on the target cells with the anti-HLA monoclonal antibodies or treating target cells with IFN-gamma. RESULTS: 1. Most of the tumor cell lines showed a complete or partial loss of HLA-ABC molecules, 2. After the HLA molecules had been marked on the target cells with the anti-HLA-ABC antibodies, the tumor cell susceptibility to the lysis of NK cells attack increased significantly and 3. After having being treated with IFN-gamma 500 U/ml for more than 48 hours, the HLA-ABC molecule levels on K562, M21 and PG cells went up. At the same time, their susceptibility to NK lysis was reduced. However, the resistance to Karpas, HL60 and HT29 NK lysis demonstrated a noticeable increase. The IFN-gamma promoted the apoptosis of HL60 and HT29 cells. CONCLUSION: The NK cells are capable of recognizing the HLA molecules on the target cells and show no lysis in providing a negative signal with the KIRs, an effect which the anti-HLA monoclonal antibodies are able to eliminate. IFN-gamma can be applied to make up for the loss of HLA molecules on some of the tumor cells, it can also facilitate some of the tumor cells' apoptosis.

Animals↗
PubMed1990

The concentration of glucose in the culture medium determines the effect of suramin on the growth and differentiation of the human colonic adenocarcinoma cell clone HT29-D4.

Suramin, a drug currently used for advanced malignancy, induces the differentiation of the human colonic adenocarcinoma cell clone HT29-D4 and this process is correlated with a decreased glycolytic activity. We investigated the effects of suramin on HT29-D4 cells in the presence of various glucose concentrations. The main result of this study is that suramin has only an effect on HT29-D4 cell growth and differentiation when the concentration of glucose is above 10 mM. Therefore the efficiency of suramin as an anticancer drug may be greater on poorly differentiated tumoral cells with a high proliferative capacity.

Adenocarcinoma↗
PubMedDate not supplied

Monitoring of the intracellular activation of 5-fluorouracil to deoxyribonucleotides in HT29 human colon cell line: application to modulation of metabolism and cytotoxicity study.

An HPLC method was developed for in vitro detection and monitoring of intracellular metabolites of [3H]-5-fluorouracil (FUra). Results showed a preferential activation of FUra to ribonucleoside and ribonucleotide derivatives (FURd, FUMP, FUDP and FUTP) in the human colorectal HT29 cell line. We screened various agents so as to determine if they could act as modulators of metabolism and/or toxicity of FUra by reversing the activation pathway of FUra from ribo- to deoxyribonucleotides, thus enhancing FdUMP formation. Different drugs (efflux inhibitors, catabolism inhibitors and enzymatic cofactors) were tested for enhancement of cytotoxicity when associated with FUra. The most promising agents were further studied by assessment of their ability to modulate intracellular activation of FUra to enhance thymidylate synthase (TS) inhibition by FUra and to increase the subsequent induction of apoptosis. 2'-Deoxyinosine (d-Ino), a deoxyribose 1-phosphate donor increasing thymidine phosphorylase activity, stood out as the best modulating agent we screened. Results showed an up to 30-fold increase of cytotoxicity along with a stronger inhibition of TS when FUra was associated with d-Ino, while FUra alone exhibited a lesser effect on TS activity. Besides, HPLC analysis revealed a complete reversal of the activation pathway of FUra, thus leading to an intracellular accumulation of deoxyribonucleotides. Assessment of cell cycle distribution showed a marked increase (+480%) of apoptosis in cells exposed to FUra/d-Ino compared to FUra alone. The HPLC method we developed is a convenient tool for assessing to what extent modulators will actually act on the intracellular activation of FUra. This study confirms the potentiality of d-Ino to modulate FUra metabolism in vitro. It proved to be an agent able to orientate the mechanism of action of FUra towards the inhibition of TS in cells where the normal activation pathway of the drug does not result in the intracellular accumulation of the active metabolite FdUMP.

Cell Cycle↗
PubMed1994

Involvement of calmodulin in Ca(2+)-activated K+ efflux in human colonic cell line, HT29-19A.

The receptor-mediated agonist, neurotensin (NT) stimulated Ba(2+)- and charybdotoxin-sensitive 86Rb (K+) efflux in the HT29-19A colonic cell line. Efflux was also stimulated by ionomycin and thapsigargin and could be abolished by incubation with the intracellular Ca2+ chelator, BAPTA. Together, these data suggest a rise in [Ca2+]i is prerequisite for activation of K+ efflux in these cells. Comparison of the temporal profiles for NT-induced increases in [Ca2+]i and 86Rb efflux, however, failed to show a direct relationship between these parameters. The NT-stimulated increase in [Ca2+]i was transient, returning to baseline within 4-5 min, while efflux was sustained over a much longer period (> 12 min). Ca(2+)-activated 86Rb efflux was inhibited by pretreatment with calmodulin (CaM) antagonist, W7. W7 had no effect on basal efflux, but reduced both NT- and IM-activated efflux up to 80%, with a Ki of 38 microM. Other CaM antagonist inhibited efflux with an order of potency (TFP approximately W8 > W7 >> W5) consistent with inhibition of a CaM-dependent process. Inhibition by W7 was not abolished by ouabain or bumetanide, indicating its effects are not mediated by action upon K+ uptake processes. W7 did not inhibit NT-stimulated 125I efflux but significantly reduced efflux stimulated by the Ca2+ ionophore, ionomycin. NT-stimulated 86Rb+ efflux was localized to the basolateral membrane of HT29-19A monolayers grown on permeable supports. These data are consistent with the involvement of CaM in mediating Ca(2+)-dependent activation of K+ conductance in HT29-19A colonocytes.

Calcium↗
PubMed2001

[Effects of calcium channel antagonist on gastrin-induced proliferation of HT29 colon carcinoma cells].

OBJECTIVE: This study aimed to assess the effects of nifedipine on gastrin-induced proliferation of HT29 colon carcinoma cells and inquire into the possible mechanisms. METHODS: Flow cytometry was used to monitor the cytoplasmic free calcium; MTT colorimetry was used to determine the proliferation of HT29 cells. RESULTS: The results showed that 2.5 x 10(-6) mol/L pentagastrin (PG) induced a quick rise of intracellular free calcium ([Ca2+]i) (P < 0.01). 10(-5) mol/L nifedipine can significantly inhibited the rise of [Ca2+]i induced by pentagastrin (P < 0.01), in parallel, at growth assay we demonstrated that 10(-5)-10(-6) mol/L nifedipine could obviously block the increase in cell number elicited by 2.5 x 10(-6) mol/L PG. CONCLUSION: These data indicate that nifedipine can stop the influx of Ca2+ and hence inhibit the pentagastrin-induced proliferation.

Biological Transport, Active↗
PubMed2002

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.

Adenocarcinoma↗
PubMed1991

Selected human immunodeficiency virus replicates preferentially through the basolateral surface of differentiated human colon epithelial cells.

We have used HIV1-NDK-infected HT29 cells grown on permeable substratum to study the polarity of virus maturation in human intestinal cells. When cultured in glucose-containing medium, these cells are mostly undifferentiated. The removal of glucose from the medium allowed the emergence of a selected differentiated subpopulation which continued to produce viral particles in the culture supernatant. The polarity of viral production was evaluated by harvesting virus from the two sides of the monolayer. Seventy-five percent of released HIV was found on the basolateral side of the monolayer. Mature viral particles were observed by electron microscopy near the apical (luminal) and the basolateral (serosal) membrane. These data suggest that epithelial cells of the colon productively infected by a selected strain of HIV are able to produce the virus through both sides of the epithelium but mainly through the serosal side.

Cell Differentiation↗
PubMed2003

Glutaminase isoform expression in cell lines derived from human colorectal adenomas and carcinomas.

This paper describes some properties of glutamine oxidation and glutaminase isoform expression in cell lines derived from human colorectal adenomas and carcinomas. The slow-growing adenoma-derived cell line AA/C1, and the rapidly proliferating carcinoma cell line HT29, both required glutamine for growth. The rate of (14)CO(2) production from [U-(14)C]glutamine was faster in AA/C1 cells than in HT29 cells. Conversely HT29 cells showed faster rates of glucose oxidation and lactate production. The activity of glutaminase was 3 times higher in AA/C1 cell extracts than in extracts of HT29 cells. Glutaminase activity in the two cell lines had similar K(m) values for glutamine, but the activity in AA/C1 cells had a higher K(0.5) for activation by phosphate. Glutaminase activity in extracts of both cells was inhibited by glutamate. Western blotting showed the presence, in both cell lines, of isoform(s) of glutaminase with an molecular mass of 63 kDa, intermediate between that of kidney glutaminase and liver glutaminase. PCR-based analysis showed that an mRNA species identical to the kidney-type isoform glutaminase C was present in both cell types as was an additional mRNA species identical to the liver-type glutaminase isoform from human breast tumour cells. Northern blotting using isoform-specific cDNA probes demonstrated that mRNA for both glutaminase isoforms was expressed at significant levels in both cell types. Similar results to those in AA/C1 cells and HT29 cells were obtained in two further adenoma and carcinoma cell lines respectively. These results contrast with those reported previously in hepatocyte/hepatoma model systems with respect to fuel selection, glutaminase activity and isoform expression. They also constitute the first demonstration of simultaneous expression of two glutaminase isoforms in a single cell type.

Cell Division↗
PubMed2003

NAD(P)H oxidase 1, a product of differentiated colon epithelial cells, can partially replace glycoprotein 91phox in the regulated production of superoxide by phagocytes.

Reactive oxygen species (ROS) serve several physiological functions; in some settings they act in host defense, while in others they function in cellular signaling or in biosynthetic reactions. We studied the expression and function of a recently described source of ROS, NAD(P)H oxidase 1 or Nox1, which has been associated with cell proliferation. In situ hybridization in mouse colon revealed high Nox1 expression within the lower two-thirds of colon crypts, where epithelial cells undergo proliferation and differentiation. Human multitumor tissue array analysis confirmed colon-specific Nox1 expression, predominantly in differentiated epithelial tumors. Differentiation of Caco2 and HT29 cells with 1alpha,25-dihydroxyvitamin D(3) or IFN-gamma enhances Nox1 expression and decreases cell proliferation, suggesting that Nox1 does not function as a mitogenic oxidase in colon epithelial cells. Transduction with retrovirus encoding Nox1 restored activation and differentiation-dependent superoxide production in gp91(phox)-deficient PLB-985 cells, indicating close functional similarities to the phagocyte oxidase (phox). Furthermore, coexpression of cytosolic components, p47(phox) and p67(phox), augments Nox1 activity in reconstituted K562 cells. Finally, Nox1 partially restores superoxide production in neutrophils differentiating ex vivo from gp91(phox)-deficient CD34(+) peripheral blood-derived stem cells derived from patients with X-linked chronic granulomatous disease. These studies demonstrate a significant functional homology (cofactor-dependent and activation-regulated superoxide production) between Nox1 and its closest homologue, gp91(phox), suggesting that targeted up-regulation of Nox1 expression in phagocytic cells could provide a novel approach in the molecular treatment of chronic granulomatous disease.

Animals↗
PubMed1992

des-(1-3)-IGF-I, an insulin-like growth factor analog used to mimic a potential IGF-II autocrine loop, promotes the differentiation of human colon-carcinoma cells.

HT29-D4 human colon-carcinoma cells have been shown to secrete insulin-like growth factor (IGF)-II and to simultaneously express type-I IGF receptors. However, the sequestration of IGF-II by several molecular forms of IGF-binding proteins (IGFBP) in the culture medium prevents the establishment of an operative IGF-II autocrine loop. IGFBPs secreted by HT29-D4 cells (HT29-D4 IGFBP) comprise isoforms of IGFBP-4 (25, 27 and 30 kDa) and 2 unidentified forms (34.5 and 32-34 kDa). This latter does not bind 125I-IGF-I. The net affinity of HT29-D4 IGFBP is about 12 times stronger for IGF-II (KD approx. 10(-10) M) than for IGF-I. All the HT29-D4 IGFBP molecular forms are unable to bind the N-terminally truncated IGF-I analog, des-(1-3)-IGF-I. In contrast, HT29-D4 cell-surface type-I IGF receptors bind IGF-I and des-(1-3)-IGF-I identically (KD approx. 5 x 10(-10) M). We have taken advantage of these particular binding properties to use des-(1-3)-IGF-I to mimic a potential IGF autocrine loop and to observe its biological consequences. Nanomolar concentrations of des-(1-3)-IGF-I induce HT29-D4 cells to develop into a differentiated phenotype, as judged by a substantial carcinoembryonic antigen release and the induction of numerous intercellular cysts with well-organized microvilli. In the same way, des-(1-3)-IGF-I early induces a slight inhibition of HT29-D4 cell proliferation. Based on these findings, we conclude that the type-I IGF receptor primarily controls the differentiation of these colonic cells, and that HT29-D4 cancer cells remain in an undifferentiated state because of their inability to use endogenous IGF-II as an autocrine regulatory factor.

Carcinoembryonic Antigen↗
PubMed2004

Upregulation of platelet derived endothelial cell growth factor/thymidine phosphorylase by interferon alpha.

Thymidine phosphorylase (TP) catalyzes the phosphorolytic cleavage of thymidine to thymine and deoxyribose-1-phosphate. TP, which is overexpressed in a wide variety of solid tumors, is involved in the activation and inactivation of fluoropyrimidines. TP is known to be regulated by several cytokines and interferons. In our HT29 cell line the TP mRNA and activity expression increased 2-3 fold after treatment with interferon alpha.

Catalysis↗