sodium-taurodeoxycholate and Colonic-Neoplasms

Mammalian matrix metalloproteinases (MMPs) which degrade extracellular matrix facilitate colon cancer cell invasion into the bloodstream and extra-colonic tissues; in particular, MMP1 expression correlates strongly with advanced colon cancer stage, hematogenous metastasis and poor prognosis. Likewise, muscarinic receptor signaling plays an important role in colon cancer; muscarinic receptors are over-expressed in colon cancer compared to normal colon epithelial cells. Muscarinic receptor activation stimulates proliferation, migration and invasion of human colon cancer cells. In mouse intestinal neoplasia models genetic ablation of muscarinic receptors attenuates carcinogenesis. In the present work, we sought to link these observations by showing that MMP1 expression and activation plays a mechanistic role in muscarinic receptor agonist-induced colon cancer cell invasion. We show that acetylcholine, which robustly increases MMP1 expression, stimulates invasion of HT29 and H508 human colon cancer cells into human umbilical vein endothelial cell monolayers - this was abolished by pre-incubation with atropine, a non-selective muscarinic receptor inhibitor, and by pre-incubation with anti-MMP1 neutralizing antibody. Similar results were obtained using a Matrigel chamber assay and deoxycholyltaurine (DCT), an amidated dihydroxy bile acid associated with colon neoplasia in animal models and humans, and previously shown to interact functionally with muscarinic receptors. DCT treatment of human colon cancer cells resulted in time-dependent, 10-fold increased MMP1 expression, and DCT-induced cell invasion was also blocked by pre-treatment with anti-MMP1 antibody. This study contributes to understanding mechanisms underlying muscarinic receptor agonist-induced promotion of colon cancer and, more importantly, indicates that blocking MMP1 expression and activation has therapeutic promise to stop or retard colon cancer invasion and dissemination.

Topics: Acetylcholine; Cell Movement; Colonic Neoplasms; HT29 Cells; Humans; Matrix Metalloproteinase 1; Muscarinic Agonists; Neoplasm Invasiveness; Receptors, Muscarinic; Taurodeoxycholic Acid

Conjugated secondary bile acids promote human colon cancer cell proliferation by activating EGF receptors (EGFR). We hypothesized that bile acid-induced EGFR activation also mediates cell survival by downstream Akt-regulated activation of NF-kappaB. Deoxycholyltaurine (DCT) treatment attenuated TNF-alpha-induced colon cancer cell apoptosis, and stimulated rapid and sustained NF-kappaB nuclear translocation and transcriptional activity (detected by NF-kappaB binding to an oligonucleotide consensus sequence and by activation of luciferase reporter gene constructs). Both DCT-induced NF-kappaB nuclear translocation and attenuation of TNF-alpha-stimulated apoptosis were dependent on EGFR activation. Inhibitors of nuclear translocation, proteosome activity, and IkappaBalpha kinase attenuated NF-kappaB transcriptional activity. Cell transfection with adenoviral vectors encoding a non-degradable IkappaBalpha 'super-repressor' blocked the actions of DCT on both NF-kappaB activation and TNF-alpha-induced apoptosis. Likewise, transfection with mutant akt and treatment with a chemical inhibitor of Akt attenuated effects of DCT on NF-kappaB transcriptional activity and TNF-alpha-induced apoptosis. Chemical inhibitors of Akt and NF-kappaB activation also attenuated DCT-induced rescue of H508 cells from ultraviolet radiation-induced apoptosis. Collectively, these observations indicate that, downstream of EGFR, bile acid-induced colon cancer cell survival is mediated by Akt-dependent NF-kappaB activation. These findings provide a mechanism whereby bile acids increase resistance of colon cancer to chemotherapy and radiation.

Topics: Active Transport, Cell Nucleus; Apoptosis; Bile Acids and Salts; Cell Line, Tumor; Cell Nucleus; Cell Survival; Chromones; Colonic Neoplasms; ErbB Receptors; Humans; I-kappa B Kinase; Leupeptins; Morpholines; Mutation; NF-kappa B; Nitriles; Peptides; Proto-Oncogene Proteins c-akt; Sulfones; Taurodeoxycholic Acid; Tumor Necrosis Factor-alpha; Ultraviolet Rays

Recent studies indicate that secondary bile acids promote colon cancer cell proliferation but their role in maintaining cell survival has not been explored. We found that deoxycholyltaurine (DCT) markedly attenuated both unstimulated and TNF-alpha-stimulated programmed cell death in colon cancer cells by a phosphatidylinositol 3-kinase (PI3K)-dependent mechanism. To examine the role of bile acids and PI3K signaling in maintaining colon cancer cell survival, we explored the role of signaling downstream of bile acid-induced activation of the epidermal growth factor receptor (EGFR) in regulating both apoptosis and proliferation of HT-29 and H508 human colon cancer cells. DCT caused dose- and time-dependent Akt (Ser(473)) phosphorylation, a commonly used marker of activated PI3K/Akt signaling. Both EGFR kinase and PI3K inhibitors attenuated DCT-induced Akt phosphorylation and Akt activation, as demonstrated by reduced phosphorylation of a GSK-3-paramyosin substrate. Transfection of HT-29 cells with kinase-dead EGFR (K721M) reduced DCT-induced Akt phosphorylation. In HT-29 cells, EGFR and PI3K inhibitors as well as transfection with dominant negative AKT attenuated DCT-induced cell proliferation. DCT-induced PI3K/Akt activation resulted in downstream phosphorylation of GSK-3 (Ser(21/9)) and BAD (Ser(136)), and nuclear translocation (activation) of NF-kappaB, thereby confirming that DCT-induced activation of PI3K/Akt signaling regulates both proproliferative and prosurvival signals. Collectively, these results indicate that DCT-induced activation of post-EGFR PI3K/Akt signaling stimulates both colon cancer cell survival and proliferation.

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colonic Neoplasms; Enzyme Activation; ErbB Receptors; Humans; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; Taurodeoxycholic Acid; Transfection; Tumor Necrosis Factor-alpha

Prior evidence indicates that bile acids stimulate colon cancer cell proliferation by muscarinic receptor-induced transactivation of epidermal growth factor receptors (EGFR). To explore further the mechanism underlying this action, we tested the hypothesis that bile acids activate a matrix metalloproteinase (MMP) that catalyzes release of an EGFR ligand. Initial studies showed that non-selective MMP inhibitors blocked the actions of deoxycholyltaurine (DCT), thereby indicating a role for MMP-catalyzed release of an EGFR ligand. DCT-induced cell proliferation was reduced by increasing concentrations of EGFR kinase inhibitors, by antibodies to the ligand binding domain of EGFR, by neutralizing antibodies to heparin binding-EGF-like growth factor (HB-EGF) and by CRM197, an inhibitor of HB-EGF release. These findings and our observations with more selective MMP inhibitors suggested that MMP-7, an enzyme known to release HB-EGF, plays a key role in mediating bile acid-induced H508 colon cancer cell proliferation. We observed that recombinant HB-EGF and MMP-7 mimicked both the signaling and proliferative actions of bile acids. Strikingly, reducing MMP-7 expression with either neutralizing antibody or small interfering RNA attenuated the actions of DCT. MMP-7 expression in H508 cells was confirmed using quantitative reverse transcription PCR. DCT stimulated a greater than 10-fold increase in MMP-7 gene transcription. Co-localization of pro-MMP-7 and pro-HB-EGF at the cell surface (immunofluorescence microscopy) was demonstrated, indicating proximity of the enzyme to its substrate. These findings provide strong evidence that in H508 human colon cancer cells, DCT-induced transactivation of EGFR is mediated by MMP-7-catalyzed release of the EGFR ligand HB-EGF.

Topics: Bile Acids and Salts; Cell Proliferation; Colonic Neoplasms; Dose-Response Relationship, Drug; Epidermal Growth Factor; Genes, erbB-1; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Matrix Metalloproteinase 7; Mitogen-Activated Protein Kinase 3; Molecular Mimicry; Phosphorylation; Recombinant Proteins; Taurodeoxycholic Acid

LT97 human colonic adenoma cells reflecting early premalignant genotype and growth characteristics have been posed to tumor promoting bile acids in order to identify marker genes that permit identification of tumor promoters in vitro. Physiologically relevant concentrations of desoxycholate (DOC) and chenodesoxycholate (CDC) upregulated expression of c-fos and COX-2 in a concentration- and time-dependent manner. Transient induction of c-fos was seen with the non-promoting taurodesoxycholate (TDOC) as well as DOC, however extended induction at 3 h was only achieved by DOC and CDC reaching 3-6-fold as compared to the control. Stimulation of COX-2 expression was completely specific for the tumor promoting analogs DOC and CDC. It was about 4-fold in the 80 microM DOC and CDC groups after 3 h and increased to 12- and 7-fold respectively after 6 h. Expression of VEGF was stimulated 4-5-fold in the tumor promoter (DOC and CDC) groups and about 2-fold in the non-promoting controls TDOC and GCDC. At later times the tumor promoter specific difference was lost. Our results show that all three genes are modulated in a tumor promoter dependent way and that their upregulation in LT97 adenoma cells can be used for in vitro testing of colon tumor promoters and chemopreventive compounds.

Topics: Adenoma; Bile Acids and Salts; Biomarkers, Tumor; Cell Line, Tumor; Chenodeoxycholic Acid; Colonic Neoplasms; Cyclooxygenase 2; Deoxycholic Acid; Dose-Response Relationship, Drug; Gene Amplification; Gene Expression Regulation, Neoplastic; Glyceraldehyde-3-Phosphate Dehydrogenases; Humans; Isoenzymes; Membrane Proteins; Polymerase Chain Reaction; Prostaglandin-Endoperoxide Synthases; Proto-Oncogene Proteins c-fos; RNA; Taurodeoxycholic Acid; Time Factors; Tumor Necrosis Factor-alpha; Up-Regulation; Vascular Endothelial Growth Factor A

Previous studies showed that bile salts had a promoting effect on colon cancer development and this effect was inhibited by ursodeoxycholate (UDC). We recently found that both human colorectal adenomas and carcinomas were associated with a specific decrease in alkaline sphingomyelinase activity. In this work, we compared the effects of ursodeoxycholate and other bile salts on the levels of rat intestinal alkaline sphingomyelinase both in the intestinal loops and after oral administration. Bile salts at different concentrations were injected into intestinal loops and the dissociation of alkaline sphingomyelinase from the mucosa was assayed. We found that bile salts, including taurocholate, taurodeoxycholate, glycocholate, glycochenodeoxycholate, and 3-(3-cholamidopropyl dimethylammonio)-1-propanesulonate (CHAPS), dose dependently dissociated alkaline sphingomyelinase from the intestinal mucosa. UDC alone did not dissociate the enzyme but significantly inhibited the dissociation caused by other bile salts and CHAPS. Feeding rats with 0.3% (w/w) taurocholate for four days decreased peak activity of intestinal alkaline sphingomyelinase by 39% and total activity in the intestine by 20% and increased the output of the enzyme in the feces. In contrast, feeding 0.3% (w/w) UDC for four days increased the peak activity of alkaline sphingomyelinase in the small intestine by 87% and the activity in the colon by 187%. The total activity of alkaline sphingomyelinase was increased by 80% and the output of the enzyme in the feces was only slightly increased by UDC administration. The changes in alkaline phosphatase after feeding taurocholate and UDC were much smaller. Our results indicate that UDC and other bile salts have different effects on the levels of alkaline sphingomyelinase, which may be implicated in their different influences on cancer development reported previously.

Topics: Administration, Oral; Animals; Bile Acids and Salts; Cholic Acids; Colonic Neoplasms; Glycochenodeoxycholic Acid; Glycocholic Acid; Intestinal Mucosa; Rats; Rats, Sprague-Dawley; Sphingomyelin Phosphodiesterase; Taurocholic Acid; Taurodeoxycholic Acid; Ursodeoxycholic Acid

Bile acids have been implicated as tumor promoters that enhance epithelial proliferation and the development of colonic tumors. This study investigated the effects of bile acids on the growth of in vitro models of human colonic epithelial cells. Cell lines with varying degrees of differentiation (Caco2, HT29, LS174T, and Lovo) were studied. Cell viability and number were measured by a tetrazolium (MTT) spectrophotometric assay. Enhanced cell growth was not observed with any bile acid over the range 10 nmol/L to 2.5 mmol/L. Cytotoxicity was consistently observed at concentrations of unconjugated bile acids greater than 0.1 mmol/L. The bile acid concentration at which 50% growth inhibition occurred was similar for all cell lines and increased in the following order: deoxycholic acid = chenodeoxycholic acid < taurodeoxycholic acid < ursodeoxycholic acid < taurochenodeoxycholic acid < cholic acid < tauroursodeoxycholic acid. Coincubation of tauroursodeoxycholic acid (TUDC) with taurodeoxycholic acid (TDC) or taurochenodeoxycholic acid (TDCD) reversed the short-term (30-minute) cytotoxicity and release of glycoprotein induced by TDC or TCDC regardless of differentiation status. In contrast, TUDC did not reverse the cytotoxicity of deoxycholic acid. Unconjugated ursodeoxycholic acid did not alter short-term cytotoxicity of any bile acid. These data indicate that bile acids do not stimulate cell growth in undifferentiated or differentiated colon cancer cell lines, in contrast to normal colonic epithelium in vivo. Bile acid cytotoxicity correlates with the relative hydrophobicity of the bile acid. Because tauroursodeoxycholic acid alters the cytotoxicity of hydrophobic bile acids in vitro, further understanding of bile acid interactions in the colon may have important implications in altering tumor promotion.

Topics: Bile Acids and Salts; Carcinoma; Cell Division; Colonic Neoplasms; Cytotoxins; Humans; Taurodeoxycholic Acid; Tumor Cells, Cultured; Ursodeoxycholic Acid

Whole-cell patch-clamp techniques and fluorescence measurements of intracellular Ca2+ concentration, (Ca2+)i, were used to investigate the mechanism of taurodeoxycholate (TDC) stimulation of Cl- secretion in the T84 colonic cell line. During perforated whole-cell recordings, the cell membrane voltage was alternately clamped to EK and ECl. Initially, TDC (0.75 mM) stimulated inward nonselective cation currents that were composed of discrete large conductance single-channel events. This initial response was followed by activation of K+ and Cl- currents with peak values of 385 +/- 41 pA and 98 +/- 28 pA, respectively (n = 12). The K+ and Cl- currents oscillated while TDC was present and returned to baseline levels upon its removal. The threshold for activation of the oscillatory currents was 0.1 mM TDC. Taurocholate, a bile acid that does not stimulate colonic Cl- secretion, induced no current response. The TDC-induced currents could be activated in Ca(2+)-free bathing solutions. Preincubation of cells with the Ca2+ chelator, bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethy)-ester (20 microM), (BAPTA-AM), eliminated the K+ and Cl- current responses, although the nonselective cation channel events were still present. Replacement of bath Na+ with NMDG+ inhibited the TDC-induced nonselective cation current but did not affect the K+ or Cl- currents. TDC induced a transient (Ca2+)i rise of 575 +/- 70 nM from a baseline of 71 +/- 5 nM (n = 15); thereafter, (Ca2+)i either plateaued or oscillated. TDC-induced (Ca2+)i oscillations were observed in the absence of bath Ca2+; however, removal of bath Ca2+ during the TDC response caused (Ca2+)i to return to near baseline values. Simultaneous K+ current and (Ca2+)i measurements confirmed that the initial nonselective cation current was independent of (Ca2+)i, while K+ current oscillations were in phase with the (Ca2+)i oscillations. TDC induced inositol monophosphate (IP) accumulation, reflecting production of inositol 1,4,5-trisphosphate (IP3) during TDC stimulation. The response to TDC during standard whole-cell patch-clamp was similar to that observed with perforated whole-cell recordings, except the nonselective cation current was prolonged. When heparin (1 mg/ml) was added to the pipette under these conditions, the Ca(2+)-activated currents were inhibited, but the nonselective cation currents were unaffected. These data suggest that TDC induces a Ca(2+)-independent nonselective cation conduc

Topics: Biological Transport; Calcium; Carrier Proteins; Cations; Cell Membrane; Chloride Channels; Choline; Colonic Neoplasms; Electric Conductivity; Fluorometry; Fura-2; Heparin; Humans; Inositol 1,4,5-Trisphosphate; Ion Channels; Microelectrodes; Organic Anion Transporters, Sodium-Dependent; Phosphatidylinositols; Potassium Channels; Signal Transduction; Sodium; Symporters; Taurodeoxycholic Acid; Tumor Cells, Cultured

Multiple alpha 1-->3fucosyltransferases occur in the human genome of which the Lewis (FucT-III), myeloid (FucT-IV), and plasma (FucT-V) enzyme forms have received the greatest attention. In this paper, the acceptor properties of the myeloid alpha 1-->3fucosyltransferase from human promyelocytic leukemia HL-60 cells have been studied. Fucose transfer into glycolipid acceptors was activated by detergents G-3634-A or taurodeoxycholate resulting in efficient transfer to neutral acceptors but poor transfer to sialylated acceptors, characteristic of the myeloid-type enzyme. Fucose transfer to nLc6 yielded both mono- and difucosyl derivatives under both detergent conditions. The nLc6 monofucosyl products were isolated and analyzed by TLC immunostaining and fast atom bombardment-mass spectroscopy analysis. The G-3634-A monofucosyl product was found to be composed solely of V3FucnLc6, whereas that with taurodeoxycholate was determined to be a mixture of III3FucnLc6 and V3FucnLc6 in near equal amounts. Analysis of reaction products using enzyme activation by phospholipids indicated that phosphatidylethanolamine and phosphatidylinositol behaved similarly to G-3634-A. In contrast, phosphatidylglycerol yielded results similar those of taurodeoxycholate. Fucose transfer to VI3NeuAcnLc6 yielded approximately 75% of the product as the III-GlcNAc monofucosyl derivative regardless of the detergent or phospholipid condition used. Analysis of fucose transfer to nLc6 catalyzed by the Lewis enzyme from Colo 205 cells using taurodeoxycholate yielded solely the III-GlcNAc monofucosyl derivative, whereas the predominant product with G-3634-A was V-GlcNAc fucosylated. In contrast, under both conditions only the V-GlcNAc monofucosylation product was found with the enzyme from NCI-H69 cells. The results indicate that subtle intrinsic differences exist between human alpha 1-->3fucosyltransferases. Modulation of enzyme specificity via the nature of the membrane environment could participate in regulation of expression of distinct cell surface antigens.

Topics: Acetylglucosamine; Carbohydrate Sequence; Colonic Neoplasms; Detergents; Fucose; Fucosyltransferases; Glycolipids; Granulocytes; Humans; Leukemia, Promyelocytic, Acute; Lung Neoplasms; Molecular Sequence Data; Phosphatidylethanolamines; Phosphatidylglycerols; Phosphatidylinositols; Phospholipids; Spectrometry, Mass, Fast Atom Bombardment; Substrate Specificity; Taurodeoxycholic Acid; Tumor Cells, Cultured