deoxycholic-acid and Cell-Transformation--Neoplastic

Obesity is a known risk factor for the development of gastroesophageal reflux disease (GERD), Barrett's Esophagus (BE) and the progression to esophageal adenocarcinoma. The mechanisms by which obesity contributes to GERD, BE and its progression are currently not well understood. Recently, changes in lipid metabolism especially in the context of a high fat diet have been linked to GERD and BE leading us to explore whether fatty acid oxidation plays a role in the disease progression from GERD to esophageal adenocarcinoma. To that end, we analyzed the expression of the rate-limiting enzyme, carnitine palmytoyltransferase 1A (CPT1A), in human tissues and cell lines representing different stages in the sequence from normal squamous esophagus to cancer. We determined uptake of palmitic acid, the most abundant fatty acid in human serum, with fluorescent dye-labeled lipids as well as functional consequences of stimulation with palmitic acid relevant to Barrett's tumorigenesis, e.g., proliferation, characteristics of stemness and IL8 mediated inflammatory signaling. We further employed different mouse models including a genetic model of Barrett's esophagus based on IL1β overexpression in the presence and absence of a high fat diet and deoxycholic acid to physiologically mimic gastrointestinal reflux in the mice. Together, our data demonstrate that CPT1A is upregulated in Barrett's tumorigenesis and that experimental palmitic acid is delivered to mitochondria and associated with increased cell proliferation and stem cell marker expression.

Topics: Adenocarcinoma; Animals; Barrett Esophagus; Carcinogenesis; Carnitine; Carnitine O-Palmitoyltransferase; Cell Proliferation; Cell Transformation, Neoplastic; Deoxycholic Acid; Esophageal Neoplasms; Fluorescent Dyes; Gastroesophageal Reflux; Humans; Interleukin-8; Mice; Obesity; Palmitic Acid

Although the unconjugated secondary bile acids, specifically deoxycholic acid (DCA) and lithocholic acid (LCA), are considered to be risk factors for colorectal cancer, the precise mechanism(s) by which they regulate carcinogenesis is poorly understood. We hypothesize that the cytotoxic bile acids may promote stemness in colonic epithelial cells leading to generation of cancer stem cells (CSCs) that play a role in the development and progression of colon cancer.. Normal human colonic epithelial cells (HCoEpiC) were used to study bile acid DCA/LCA-mediated induction of CSCs. The expression of CSC markers was measured by real-time qPCR. Flow cytometry was used to isolate CSCs. T-cell factor/lymphoid-enhancing factor (TCF/LEF) luciferase assay was employed to examine the transcriptional activity of β-catenin. Downregulation of muscarinic 3 receptor (M3R) was achieved through transfection of corresponding siRNA.. We found DCA/LCA to induce CSCs in normal human colonic epithelial cells, as evidenced by the increased proportion of CSCs, elevated levels of several CSC markers, as well as a number of epithelial-mesenchymal transition markers together with increased colonosphere formation, drug exclusion, ABCB1 and ABCG2 expression, and induction of M3R, p-EGFR, matrix metallopeptidases, and c-Myc. Inhibition of M3R signaling greatly suppressed DCA/LCA induction of the CSC marker ALDHA1 and also c-Myc mRNA expression as well as transcriptional activation of TCF/LEF.. Our results suggest that bile acids, specifically DCA and LCA, induce cancer stemness in colonic epithelial cells by modulating M3R and Wnt/β-catenin signaling and thus could be considered promoters of colon cancer.

Topics: ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily G, Member 2; beta Catenin; Biomarkers, Tumor; Cell Line; Cell Transformation, Neoplastic; Colon; Deoxycholic Acid; Epithelial Cells; Epithelial-Mesenchymal Transition; ErbB Receptors; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Lithocholic Acid; Luciferases; Neoplasm Proteins; Neoplastic Stem Cells; Proto-Oncogene Proteins c-myc; Receptor, Muscarinic M3; RNA, Small Interfering; Signal Transduction; T Cell Transcription Factor 1

Cancer-related inflammation recently has been proposed as a major physiological hallmark of malignancy. Some genetic alterations known to promote cellular proliferation and induce malignant transformation also may participate in an intrinsic inflammatory pathway that produces a cancer-promoting inflammatory microenvironment. Little is known about this intrinsic inflammatory pathway in Barrett's esophagus. We have used a series of nontransformed and transformed human Barrett's epithelial cell lines developed in our laboratory to explore the potential contribution of interleukin (IL)-6 and signal transducer and activator of transcription (STAT3) (key molecules in the intrinsic inflammatory pathway) to Barrett's carcinogenesis. We determined IL-6 mRNA expression and protein secretion and protein expression of activated phospho-STAT3 and its downstream target myeloid cell leukemia (mcl)-1 (Mcl-1). We used an IL-6 blocking antibody and two JAK kinase inhibitors (AG490 and JAK inhibitor I) to assess whether STAT3 activation is IL-6 dependent. We also used small interfering RNAs (siRNAs) to STAT3 and Mcl-1 to assess effects of STAT3 pathway inhibition on apoptosis. Phospho-STAT3 was expressed only by transformed Barrett's cells, which also exhibited higher levels of IL-6 mRNA and of IL-6 and Mcl-1 proteins than nontransformed Barrett's cells. STAT3 phosphorylation could be blocked by IL-6 blocking antibody and by AG490 and JAK inhibitor I. In transformed Barrett's cells, rates of apoptosis following exposure to deoxycholic acid were significantly increased by transfection with siRNAs for STAT3 and Mcl-1. We conclude that activation of the IL-6/STAT3 pathway in transformed Barrett's epithelial cells enables them to resist apoptosis. These findings demonstrate a possible contribution of the intrinsic inflammatory pathway to carcinogenesis in Barrett's esophagus.

Topics: Apoptosis; Barrett Esophagus; Cell Line, Transformed; Cell Transformation, Neoplastic; Cyclin-Dependent Kinase Inhibitor p16; Deoxycholic Acid; Epithelial Cells; Esophageal Neoplasms; Esophagitis; Esophagus; Genes, ras; Humans; Interleukin-6; Janus Kinases; Myeloid Cell Leukemia Sequence 1 Protein; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-bcl-2; RNA Interference; RNA, Messenger; Signal Transduction; STAT3 Transcription Factor; Telomerase; Time Factors; Transfection; Tumor Microenvironment; Tumor Suppressor Protein p53; Tyrosine

Gastroesophageal reflux is associated with adenocarcinoma in Barrett's esophagus, but the incidence of this tumor is rising, despite widespread use of acid-suppressing medications. This suggests that refluxed material other than acid might contribute to carcinogenesis. We looked for potentially carcinogenetic effects of two bile acids, deoxycholic acid (DCA) and ursodeoxycholic acid (UDCA), on Barrett's epithelial cells in vitro and in vivo. We exposed Barrett's (BAR-T) cells to DCA or UDCA and studied the generation of reactive oxygen/nitrogen species (ROS/RNS); expression of phosphorylated H2AX (a marker of DNA damage), phosphorylated IkBα, and phosphorylated p65 (activated NF-κB pathway proteins); and apoptosis. During endoscopy in patients, we took biopsy specimens of Barrett's mucosa before and after esophageal perfusion with DCA or UDCA and assessed DNA damage and NF-κB activation. Exposure to DCA, but not UDCA, resulted in ROS/RNS production, DNA damage, and NF-κB activation but did not increase the rate of apoptosis in BAR-T cells. Pretreatment with N-acetyl-l-cysteine (a ROS scavenger) prevented DNA damage after DCA exposure, and DCA did induce apoptosis in cells treated with NF-κB inhibitors (BAY 11-7085 or AdIκB superrepressor). DNA damage and NF-κB activation were detected in biopsy specimens of Barrett's mucosa taken after esophageal perfusion with DCA, but not UDCA. These data show that, in Barrett's epithelial cells, DCA induces ROS/RNS production, which causes genotoxic injury, and simultaneously induces activation of the NF-κB pathway, which enables cells with DNA damage to resist apoptosis. We have demonstrated molecular mechanisms whereby bile reflux might contribute to carcinogenesis in Barrett's esophagus.

Topics: Aged; Analysis of Variance; Animals; Apoptosis; Barrett Esophagus; Cell Line; Cell Transformation, Neoplastic; Deoxycholic Acid; DNA Damage; Epithelial Cells; Histones; Humans; I-kappa B Proteins; Male; Middle Aged; NF-kappa B; NF-KappaB Inhibitor alpha; Rats; Reactive Nitrogen Species; Reactive Oxygen Species; Signal Transduction; Ursodeoxycholic Acid

High-mobility group box 1 (HMGB1) is a nuclear protein that acts as a ligand of the receptor for advanced glycation end products (RAGE) and its expression enhances progression of cancer. However, the mechanism underlying HMGB1 secretion is still unclear. In this study, we examined the effect of deoxycholic acid (DCA), a promoter of colon carcinogenesis, on HMGB1 secretion.. We used an in vitro transformation model comprised of IEC6 intestinal epithelial cells treated with azoxymethane (AOM) and/or DCA. HMGB1 expression and secretion were examined by Western and Northern blot analyses, and ELISA. Intracellular translocation of HMGB1 was examined by protein fractionation.. AOM + DCA-treated IEC6 cells showed upregulation of HMGB1 mRNA expression and increased level of HMGB1 protein in culture medium, but decreased level of HMGB1 protein in the nucleus. AOM + DCA treatment increased level of histone H4 acetylation, which induced translocation of HMGB1 from the nucleus to the cytoplasm and increased HMGB1 secretion. Leptomycin B inhibited extranuclear translocation and secretion of the HMGB1 protein.. These findings suggest that DCA affects intracellular localization and secretion of HMGB1.

Topics: Acetylation; Animals; Antibiotics, Antineoplastic; Azoxymethane; Carcinogens; Cell Line; Cell Transformation, Neoplastic; Cholagogues and Choleretics; Colon; Deoxycholic Acid; Enzyme Inhibitors; Epithelial Cells; Fatty Acids, Unsaturated; Histones; HMGB1 Protein; Hydroxamic Acids; Intestinal Mucosa; Male; Rats; Rats, Inbred F344; RNA, Messenger; Up-Regulation

Deoxycholic acid (DCA) is a secondary bile acid implicated in various cancers of the gastrointestinal (GI) tract. In oesophageal adenocarcinoma, DCA is believed to contribute to carcinogenesis during reflux where stomach contents enter the lower oesophagus. It is imperative that we understand the mechanisms whereby oesophageal carcinogens function in order that therapeutic options may be developed. We have previously shown that DCA can damage chromosomes and does so through its generation of reactive oxygen species (ROS). We show here, after detailed experiments, that DCA appears to have a non-linear dose response for DNA damage. DCA induces DNA damage (as measured by the micronucleus assay) at doses of 100 microM and higher in oesophageal OE33 cells, but fails to induce such DNA damage below this cut-off dose. We also show that in terms of NF-kappaB activation (as measured by up-regulation of two NF-kappaB target genes) by DCA, a similar dose response is observed. This dose-response data may be important clinically as DCA exposure to the oesophagus may be used as a way to identify the 10% of Barrett's oesophagus patients currently progressing to cancer from the 90% of patients who do not progress. Only quantitative studies measuring DCA concentrations in refluxates correlated with histological progression will answer this question. We further show here that ROS are behind DCAs ability to activate NF-kappaB as antioxidants (epigallocatechin gallate, resveratrol and vitamin C) abrogate DCAs ability to up-regulate NF-kappaB-controlled genes. In conclusion, low doses of DCA appear to be less biologically significant in vitro. If this were to be confirmed in vivo, it might suggest that reflux patients with low DCA concentrations may be at a lower risk of cancer progression compared to patients with high levels of DCA in their refluxate. Either way, antioxidant supplementation may possibly help prevent the deleterious effects of DCA in the whole GI tract.

Topics: Adenocarcinoma; Barrett Esophagus; Cell Line, Tumor; Cell Transformation, Neoplastic; Deoxycholic Acid; DNA; DNA Damage; Esophageal Neoplasms; Esophagus; Gene Expression; Gene Expression Regulation; Humans; Micronucleus Tests; Mutagens; NF-kappa B; Reactive Oxygen Species

Although progression to adenocarcinoma at the gastroesophageal junction reflects exposure to acid and bile acids associated with reflux, mechanisms mediating this transformation remain undefined. Guanylyl cyclase C (GC-C), an intestine-specific tumor suppressor, may represent a mechanism-based marker and target of transformation at the gastroesophageal junction. The present studies examine the expression of GC-C in normal tissues and tumors from esophagus and stomach and mechanisms regulating its expression by acid and bile acids.. Gene expression was examined by reverse-transcription polymerase chain reaction, promoter analysis, immunohistochemistry, immunoblotting, and functional analysis. Promoter transactivation was quantified by using luciferase constructs and mutational analysis. DNA binding of transcription factors was examined by electromobility shift analysis.. GC-C mRNA and protein were ectopically expressed in approximately 80% of adenocarcinomas arising in, but not in normal, esophagus and stomach. Similarly, in OE19 human esophageal cancer cells, deoxycholate and acid induced expression of GC-C. This was associated with the induction of expression of Cdx2, a transcription factor required for GC-C expression. In turn, induction of Cdx2 expression by deoxycholate was mediated by binding sites in the proximal promoter for nuclear factor kappaB (NF-kappaB). Furthermore, deoxycholate increased NF-kappaB activity, associated with nuclear translocation and Cdx2 promoter binding of the NF-kappaB subunit p50. Moreover, a dominant negative construct for NF-kappaB prevented deoxycholate-induced p50 nuclear translocation and activation of the Cdx2 promoter.. Transformation associated with reflux at the gastroesophageal junction reflects activation by bile acid and acid of a transcriptional program involving NF-kappaB and Cdx2, which mediate intestinal metaplasia and ectopic expression of GC-C.

Topics: Adenocarcinoma; Bile Acids and Salts; CDX2 Transcription Factor; Cell Line, Tumor; Cell Transformation, Neoplastic; Deoxycholic Acid; Esophageal Neoplasms; Esophagogastric Junction; Esophagus; Gene Expression; Guanylate Cyclase; Homeodomain Proteins; Humans; Intestines; NF-kappa B; Promoter Regions, Genetic; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; RNA, Messenger; Tissue Distribution; Transcription, Genetic

In this study, we designed an in vitro azoxymethane (AOM)-induced carcinogenesis model and analyzed the effect of deoxycholic acid (DCA) on growth, apoptosis, genotoxicity, and transformation of IEC6 intestinal cells. CYP2E1 production was confirmed in IEC6 cells. The growth of IEC6 cells was enhanced by DCA (100 microg/ml). However, IEC6 cells treated with DCA (200 microg/ml) were inhibited and disappeared at 48 hrs after treatment. Apoptotic cells increased 11.2 times by treatment with DCA (200 microg/ml) as compared to cells with no treatment. DNA injury detected by comet assay was found in cells treated with AOM, but not in cells treated with DCA (100 microg/ml) and AOM. The number of colony formation in soft agar increased by AOM treatment. However, the number of foci treated with DCA (100 microg/ml) plus AOM was 69% that of cells treated with AOM alone. Two out of the 6 mice subcutaneously injected with AOM-treated IEC6 cells showed tumorigenesis, whereas IEC6 cells treated with DCA (100 microg/ml) plus AOM or DCA (100 microg/ml) alone did not form any tumor. Reduced protein expression of MLH1, Bcl-2 was detected in IEC6 cells treated with DCA (100 microg/ml). Production of Bax, pJNK, TGF-beta, TGFBRI, TGFBRII, and beta-catenin were higher in IEC6 cells treated with DCA (100 microg/ml) than that in cells with no treatment. These results suggest that high-dose DCA induced apoptosis and inhibited AOM-induced in vitro transformation of IEC6 cells.

Topics: Animals; Antineoplastic Agents; Apoptosis; Azoxymethane; Carcinogens; Cell Line; Cell Proliferation; Cell Transformation, Neoplastic; Comet Assay; Cytochrome P-450 CYP2E1; Deoxycholic Acid; In Vitro Techniques; Mice; Mice, Nude; Neoplasms, Experimental; Rats

To examine the expression of the transcription factor nuclear factor kappa B (NF-kappaB) in Barrett's epithelium and adenocarcinoma and the impact of NF-kappaB expression on tumor stage and response to neoadjuvant chemotherapy and radiation therapy.. Progression of Barrett's esophagus to adenocarcinoma is associated with a wide range of cellular and molecular abnormalities. Nuclear factor-kappa B (NF-kappaB) regulates several genes involved in inflammatory, immune and apoptotic responses, but its role in esophageal inflammation and tumorigenesis has not been reported.. Mobility shift assay was used to measure NF-kappaB activity in nuclear extracts of fresh-frozen biopsies from tumor and uninvolved tissues (n = 30) and esophageal cell lines OE33, SKGT-4, and OE21. RelA expression was assessed by immunohistochemical staining (n = 97). The NF-kappaB/RelA and IkappaB protein expressions were also examined by Western blotting.. NF-kappaB was not expressed in normal esophageal squamous epithelium, in contrast to increased expression in 40% of patients with Barrett's epithelium. Sixty-one percent of resected tumors (n = 97) displayed NF-kappaB immunoreactivity, and 87.5% of the NF-kappaB-positive tumors were Stage IIb and III compared with only 12.5% of patients with Stage I and IIa disease (P < 0.05). The expression of NF-kappaB inversely correlated with major or complete pathologic responses to neoadjuvant chemotherapy and radiation therapy, with 15/20 (75%) responders in the NF-kappaB-negative group compared with 7/38 (18%) in the NF-kappaB-positive group (P < 0.00001). Moreover, incubation of esophageal cell lines OE33, SKGT-4, and OE21 with deoxycholic acid or low pH induced NF-kappaB expression.. Bile acids and low pH induce NF-kappaB expression in esophageal cell lines. NF-kappaB activation is common in esophageal adenocarcinoma. In patients with Barrett's epithelium and an associated esophageal adenocarcinoma, there is a progressive expression of NF-kappaB through Barrett's tumorigenesis. The absence of NF-kappaB expression in esophageal adenocarcinoma correlates with response to neoadjuvant chemoradiotherapy and may be of value in predicting response to neoadjuvant therapy.

Topics: Adenocarcinoma; Antineoplastic Agents; Barrett Esophagus; Bile Acids and Salts; Cell Line, Tumor; Cell Transformation, Neoplastic; Chemotherapy, Adjuvant; Deoxycholic Acid; Esophageal Neoplasms; Esophagectomy; Humans; Hydrogen-Ion Concentration; Neoadjuvant Therapy; Neoplasm Staging; NF-kappa B; Predictive Value of Tests; Radiotherapy, Adjuvant; Survival Analysis; Treatment Outcome

The possibility to use the rat visceral yolk sac as a model for the study of processes of cell transformation was studied. Yolk sac teratocarcinomas could be induced using the method of in vitro culture of yolk sacs in a medium containing a direct carcinogen and a tumor promoter with subsequent transplantation under the renal or testicular capsule of syngeneic rats. Biochemical, electron-microscopic and immunohistochemical methods were used to study the characteristic changes that accompanied cellular transformation. It was shown that even a short-term (3 h) exposure of the yolk sac cells to N-methyl-N'-nitro-N-nitrosoguanidine with or without deoxycholic acid in vitro decreased significantly the rate of yolk sac transport and changed their developmental potential with manifestation of carcinogenic antigens (polyclonal keratins, monoclonal vimentin and smooth muscle actin). This cancerous transformation was promoted following their in vivo transplantation into special anatomic sites.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Deoxycholic Acid; Endoderm; Female; Immunohistochemistry; Male; Mesonephroma; Methylnitronitrosoguanidine; Neoplasm Transplantation; Pregnancy; Rats; Rats, Inbred Strains; Teratoma; Testis; Tumor Cells, Cultured; Vimentin; Yolk Sac

Topics: Amphotericin B; Animals; Cell Division; Cell Transformation, Neoplastic; Deoxycholic Acid; Dose-Response Relationship, Drug; Melanoma, Experimental; Mice; Monophenol Monooxygenase; Tumor Cells, Cultured

Simian virus 40-transformed 3T3 (SV-3T3) cells have been shown to possess on their surfaces binding sites for hyaluronate which mediate the divalent cation independent aggregation of these cells. To further characterize these binding sites, membranes were prepared from SV-3T3 cells and solubilized with the detergent, sodium deoxycholate. The binding activity present in the detergent solution was measured by the addition of [3H]hyaluronate followed by separation of free and bound ligand by (NH4)2SO4 precipitation. Using this assay, the soluble hyaluronate-binding protein was compared with the membrane-associated protein in situ. In both cases, binding was found to be saturable, linear with protein content, competitively inhibited by unlabeled hyaluronate and dependent on a minimum of 6 sugar residues of hyaluronate for recognition. However, the solubilized binding protein was found to differ from the membrane-associated protein in the following characteristics: a) the affinity of the interaction with hyaluronate was reduced (the Kd was higher), while the amount of ligand bound at saturation (Bmax) was increased; b) in competition experiments with unlabeled preparations of hyaluronate, the effect that the MWv of the hyaluronate had on its inhibitory potency was greatly reduced; and c) the binding was inhibited to a greater extent by chondroitin sulfate and dermatan sulfate. All of these differences can be accounted for by assuming that the detergent solubilization changes the nature of the hyaluronate-binding site interaction from one that is multivalent (i.e. one molecule of hyaluronate is attached to several sites) to one that is monovalent.

Topics: Animals; Carrier Proteins; Cell Membrane; Cell Transformation, Neoplastic; Cells, Cultured; Deoxycholic Acid; Hyaluronan Receptors; Hyaluronic Acid; Kinetics; Mice; Oligosaccharides; Simian virus 40; Solubility

The effects of two agents, 12-O-tetradecanoylphorbol-13-acetate (TPA) and deoxycholic acid (DOC), which act as tumor promoters in the gastrointestinal epithelium of experimental animals, were compared using primary cultures of human premalignant colonic epithelial cells at different stages in tumor progression. Both DOC and TPA enhanced the size of the proliferative fraction in colonies of early-stage premalignant cells, with DOC providing more stimulation. TPA-treated intermediate- and late-stage premalignant cells elongated and then disrupted the monolayer by forming rills several cells in thickness and then multicellular clusters. This multilayering was reminiscent of the areas of carcinoma found within adenomas. DOC had no such effects on morphology. Cell clustering was concomitant with secretion of a protease with characteristics of a plasminogen activator. Premalignant cells secreted severalfold higher levels of protease in response to TPA than did either TPA-treated primary cultures of colonic adenocarcinomas or established colon carcinoma cell lines. These results suggest that (a) DOC and TPA act sequentially during tumor promotion and (b) cell clustering and protease release may be associated with the transition of premalignant epithelial cells to colonic carcinoma.

Topics: Cell Division; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Colonic Neoplasms; Deoxycholic Acid; DNA Replication; Epithelium; Humans; Peptide Hydrolases; Phorbols; Plasminogen Activators; Precancerous Conditions; Protease Inhibitors; Tetradecanoylphorbol Acetate

Topics: Antigens, Neoplasm; Cell Line; Cell Transformation, Neoplastic; Deoxycholic Acid; gamma-Globulins; Molecular Weight; Neoplasm Proteins; Nucleoproteins; Polyribosomes; Protein Binding; Puromycin; Radioimmunoassay; Ribosomes; RNA, Messenger; Simian virus 40; Species Specificity

Topics: Ammonium Sulfate; Animals; Antibodies, Neoplasm; Antigens, Neoplasm; Binding Sites, Antibody; Buffers; Cell Membrane; Cell Transformation, Neoplastic; Cells, Cultured; Chromatography, DEAE-Cellulose; Chromatography, Gel; Cricetinae; Deoxycholic Acid; Dialysis; Electrophoresis, Disc; Fetus; Fluorescent Antibody Technique; Haplorhini; Iodine Isotopes; Kidney; Methods; Mice; Simian virus 40; Solubility; Vibration