PURPOSE: Cytotoxicity of the bile acids on colon cancer cell lines was studied to know which bile acid was most cytotoxic to colonic mucosal epithelium. We performed agarose gel electrophoresis whether this toxicity was caused by detergent effect of the bile acids or by apoptotic pathway. MATERIALS AND METHODS: HT29, LoVo, SW620 colon cancer cell lines were exposed to lithocholate, cholate, deoxycholate and chenodeoxycholate with 50, 100, 150, 200, 250, 300 pM as final concentration in DMEM culture media for short time (for 2 hours) and for long time (for 5 days). Agarose gel electrophoresis was performed on each colon cancer cell lines (HT29, LoVo, SW620, SW480) after 1, 2, 3, 4, 5 days exposure to deoxycholate with 150 pM concentration to detect intemucleosomal fragmentation. RESULTS: There was no toxicity after short time exposure in all bile acids concentration and in all colon cancer cell lines. Of the bile acids, deoxycholate was most toxic for all colon cancer cell lines. And DNA fragmentation was noticed after 2 days exposure with deoxycholate. Only LoVo cell line showed apoptotic DNA pattern after 4 days of exposure with deoxycholate. CONCLUSION: Bile acids (especially deoxycholate) are suggested to be possible agents to cause apoptosis in colonic mucosal epithelium.
Apoptosis ; Bile Acids and Salts* ; Bile* ; Cell Line* ; Chenodeoxycholic Acid ; Cholates ; Colon* ; Colonic Neoplasms* ; Culture Media ; Deoxycholic Acid ; Detergents ; DNA ; DNA Fragmentation ; Electrophoresis, Agar Gel ; Epithelium ; Lithocholic Acid

We studied the effects of bile acids on the induction of apoptosis in HepG2 human hepatocellular carcinoma cells. Treatment with either ursodeoxycholic acid (UDCA) or lithocholic acid (LCA) resulted in a dose- and time-dependent decrease in cell viability assessed by MTT assay. Both UDCA and LCA also induced genomic DNA fragmentation, a hallmark of apoptosis, indicating that the mechanism by which these bile acids induce cell death was through apoptosis. Cycloheximide, a protein synthesis inhibitor, blocked the apoptosis induced by these bile acids, implying that new protein synthesis may be required for the apoptosis. Intracellular Ca2+ release blockers (dantrolene and 3,4,5-trimethoxybenzoic acid-8-(diethylamino)octyl ester) inhibited decreased cell viability and DNA fragmentation induced by these bile acids. Treatment of HepG2 cells with calcium ionophore A23187 induced DNA fragmentation. These results suggest that UDCA and LCA induce apoptosis in the HepG2 cells and that the activation of intracellular Ca2+ signals may play an important role in the apoptosis induced by these bile acids.
Apoptosis* ; Bile Acids and Salts* ; Bile* ; Calcimycin ; Calcium ; Carcinoma, Hepatocellular* ; Cell Death ; Cell Survival ; Cycloheximide ; DNA Fragmentation ; Hep G2 Cells ; Humans* ; Lithocholic Acid ; Ursodeoxycholic Acid

BACKGROUND: Bacterial and Helicobacter gene were commonly detected in diseased human bile, although the meaning of the presence of Helicobacter in biliary tract is still unclear. The aim of this study was to evaluate the changes of bile acid composition in bacterial and Helicobacter infected bile, and to determine whether Helicobacter pylori might grow in human bile or not. METHODS: Thirty bile samples were obtained by percutaneous transhepatic biliary drainage or gallbladder puncture during cholecystectomy. According to the polymerase chain reaction analysis using bacterial 16S rRNA and Helicobacter genus specific 16S rRNA primers, 3 groups were divided; Group I; no presence of any bacterial DNA, Group II; positive bacterial DNA only, Group III; positive bacterial and Helicobacter DNA. Bile acid analysis for deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), lithocholic acid (LCA), and ursodeoxycholic acid (UDCA) was performed by high performance liquid chromatography. And then Helicobacter pylori was tried to culture in broth mixed with human bile at a final bile concentration of 50%. RESULTS: The concentrations of DCA in group II and III were very low and significantly reduced compared to group I (p<0.01, respectively). The concentrations of LCA or UDCA were not shown any relationships between groups. Helicobacter pylori has grown actively in the broth mixed with human bile containing both of less than 0.1 gm/L of DCA and CDCA, compared to no growth in media mixed with human bile containing more than 3.0 gm/L of DCA and/or CDCA. CONCLUSION: DCA seems to have the strongest antibacterial effect. Helicobacter pylori is likely to grow in human bile containg very low concentrations of CDCA and DCA.
Bile* ; Biliary Tract Diseases* ; Biliary Tract* ; Chenodeoxycholic Acid ; Cholecystectomy ; Chromatography, Liquid ; Deoxycholic Acid ; DNA ; DNA, Bacterial ; Drainage ; Gallbladder ; Helicobacter Infections* ; Helicobacter pylori ; Helicobacter* ; Humans ; Lithocholic Acid ; Polymerase Chain Reaction ; Punctures ; Ursodeoxycholic Acid

BACKGROUND/AIMS: Metabolomics is a powerful tool for measuring low-molecular-weight metabolites in an organism at a specified time under specific environmental conditions. The aim of this study was to determine the usefulness of metabolomics in identifying the metabolites in stool-fat-positive specimens, and to establish whether the results could be used to predict the long-term prognosis. METHODS: Fecal specimens were collected from 52 subjects with bowel habit change. The subjects were accessed using Rome III questionnaires and Bristol stool scale form, and followed after three years. The feces samples were centrifuged and the resulting extracts reconstituted for liquid chromatography/mass spectrometry analysis. The datasets were autoscaled, log-transformed, and mean-centered in a column-wise fashion prior to principal-components analysis and partial least-squares-discrimination analysis modeling. RESULTS: Fecal samples from 10 of the 52 patients gave a positive stool-fat result of 30-100 microm; those of the remaining 42 contained neither fatty acids nor neutral fats. The peak intensities of lithocholic acid (p=0.001), lysophosphatidyl ethanolamine (lysoPE) 16:0 (p=0.015), and lysoPE 18:1/0:0 (p=0.014) were correlated with the size of the fatty acid. Subjects with positive stool-fat result showed higher score in Bristol stool scale form than those with negative stool-fat result at initial (p=0.040) and after three years (p=0.012). CONCLUSIONS: The metabolomic assay of stool fatty acid revealed mainly lysoPEs and lithocholic acid. The size of the fatty acid was correlated with higher concentrations of lysoPEs and lithocholic acid in stool-fat-test-positive specimens and related to loose stool even after three years of follow-up period.
Adult ; Aged ; Chromatography, High Pressure Liquid ; Fatty Acids/*analysis ; Feces/*chemistry ; Female ; Follow-Up Studies ; Humans ; Least-Squares Analysis ; Lithocholic Acid/analysis ; Lysophospholipids/analysis ; Male ; *Metabolomics ; Middle Aged ; Principal Component Analysis ; Questionnaires ; Spectrometry, Mass, Electrospray Ionization

PURPOSE: Gastric stump cancer is defined as a cancer that develops in the stomach after a resection in cases of non-malignant or malignant gastric disease. The interval between the gastrectomy and the detection of gastric stump cancer must be over 5 years. Since duodenogastric reflux gastritis is a precancerous condition and one of the most important factors inducing gastric stump cancer, we compared the bile-acid content of gastric juice between gastric stump cancer patients and controls. MATENRIALS AND METHODS: To evaluate retrospectively the surgical treatment of patients with gastric stump cancer, we reviewed the cases histories of 1016 stomach cancer patients who had been operated on at the Department of General Surgery, Kosin University Gospel Hospital, between 1995 and 1998. The gastric juice was collected during the operations on the gastric stump cancer patients by using a needle puncture of the fundus of the stomach and during the endoscopic examinations of the control subjects. The samples were analyzed for various bile acids (gas chromatography/mass spectrometry). RESULTS: The 6 gastric stump cancer cases accounted for 0.6% of all gastric cancer patients; 5 patients were first operated on for a peptic ulcer and the remaining one for an adenocarcinoma of the stomach. All of the cases were men. The reconstruction method after the initial gastrectomy was a Billroth II in all cases. The sites of the gastric stump cancer were the anastomotic sitein 2 patients, the upper body in 2, the fundus in 1 and the cardia in 1. The operative methods were 3 total gastrectomies, 2 subtotal gastrectomies with Roux en Y anastomosis, and 1 partial gastrectomy with lymph node dissection and had a curative intention in all patients. All of the patients were still surviving at the time of this report. The gastric juices of 4 gastric stump patients showed significantly higher contents of cholic acid (36.42microgram/ ml) compared to the gastric juices of 35 control subjects (12.82microgram/ml)(p< or =0.0001). Chenodeoxycholic acid and lithocholic acid were not significantly different. CONCLUSION: The gastric juice of gastric stump cancer patients contained a significantly higher cholic acid content. At the time of the initial gastrectomy, an operative method that prevents duodenogastric reflux may prevent or minimize the development of gastric stump cancer, and more aggressive surgical treatment may improve survival.
Adenocarcinoma ; Anastomosis, Roux-en-Y ; Bile Acids and Salts ; Cardia ; Chenodeoxycholic Acid ; Cholic Acid ; Duodenogastric Reflux ; Gastrectomy ; Gastric Juice ; Gastric Stump* ; Gastritis ; Gastroenterostomy ; Humans ; Intention ; Lithocholic Acid ; Lymph Node Excision ; Male ; Needles ; Peptic Ulcer ; Precancerous Conditions ; Punctures ; Retrospective Studies ; Stomach ; Stomach Diseases ; Stomach Neoplasms

Animals ; Bridged Bicyclo Compounds ; metabolism ; Cytochrome P-450 CYP3A ; biosynthesis ; genetics ; Drug Design ; Drug Interactions ; Enzyme Induction ; Humans ; Lithocholic Acid ; metabolism ; Phloroglucinol ; analogs & derivatives ; metabolism ; Receptors, Cytoplasmic and Nuclear ; genetics ; metabolism ; physiology ; Receptors, Steroid ; genetics ; physiology ; Signal Transduction ; Terpenes ; metabolism ; Transcription Factors ; genetics ; metabolism

BACKGROUND/AIMS: Chronic liver disease leads to liver fibrosis, and although the liver does have a certain regenerative capacity, this disease is associated with dysfunction of the liver vessels. C-reactive protein (CRP) is produced in the liver and circulated from there for metabolism. CRP was recently shown to inhibit angiogenesis by inducing endothelial cell dysfunction. The objective of this study was to determine the effect of CRP levels on angiogenesis in a rat model of liver dysfunction induced by bile duct ligation (BDL). METHODS: The diameter of the hepatic vein was analyzed in rat liver tissues using hematoxylin and eosin (H&E) staining. The expression levels of angiogenic factors, albumin, and CRP were analyzed by real-time PCR and Western blotting. A tube formation assay was performed to confirm the effect of CRP on angiogenesis in human umbilical vein endothelial cells (HUVECs) treated with lithocholic acid (LCA) and siRNA-CRP. RESULTS: The diameter of the hepatic portal vein increased significantly with the progression of cirrhosis. The expression levels of angiogenic factors were increased in the cirrhotic liver. In contrast, the expression levels of albumin and CRP were significantly lower in the liver tissue obtained from the BDL rat model than in the normal liver. The CRP level was correlated with the expression of albumin in hepatocytes treated with LCA and siRNA-CRP. Tube formation was significantly decreased in HUVECs when they were treated with LCA or a combination of LCA and siRNA-CRP. CONCLUSION: CRP seems to be involved in the abnormal formation of vessels in hepatic disease, and so it could be a useful diagnostic marker for hepatic disease.
Angiogenic Proteins/genetics/metabolism ; Animals ; Bile Ducts/surgery ; C-Reactive Protein/*analysis/genetics/metabolism ; Cells, Cultured ; Disease Models, Animal ; Hepatic Veins/abnormalities ; Hepatocytes/cytology/metabolism ; Human Umbilical Vein Endothelial Cells ; Humans ; Lithocholic Acid/pharmacology ; Liver/metabolism/pathology ; Liver Cirrhosis/etiology ; Liver Diseases/metabolism/*pathology ; Male ; Microscopy, Fluorescence ; Mitochondria/drug effects/metabolism ; RNA Interference ; RNA, Small Interfering/metabolism ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Serum Albumin/genetics/metabolism

The expression of microRNA-19b (miR-19b) in acute necrotizing pancreatitis (ANP) and its functional role in acinar cell necrosis of SD rats were investigated. Twelve SD rats were divided into two groups randomly, including control group and ANP group. The rat ANP models were established by intraperitoneal injection of L-arginine (2400 mg/kg body weight), and equal volume of 0.9% NaCl was injected in the control group. MiRNA chip assay was performed to examine the expression of miRNAs in the pancreas in two different groups. Besides, to further explore the role of miR-19b in ANP in vitro, taurolithocholic acid 3-sulfate disodium salt (TLC-S) (200 μmol/L) was administrated to treat the rat pancreatic acinar cell line, AR42J, for establishing the ANP cells model. The quantitative real-time PCR (qRT-PCR) was adopted to measure the miR-19b expression. Moreover, the mimic miRNA, miRNA antisense oligonucleotide (AMO) and control vector were used to transfect AR42J cells, the expression of miR-19b was confirmed by qRT-PCR and the necrotizing rate of AR42J cells was detected with AO/EB method. The expression of miR-19b was significantly higher in ANP group than in control group as displayed by the miRNA chip assay. Furthermore, after inducing necrosis of AR42J cells in vitro, the expression of miR-19b was significantly increased by 2.51±0.14 times in comparison with the control group. As revealed by qRT-PCR assay, the expression of miR-19b was 5.94±0.95 times higher in the mimic miRNA group than in the control vector group, companied with an obviously increased acinar cell necrotizing rate (50.3%±1.5% vs. 39.6%±2.3%, P<0.05). Moreover, the expression of miR-19b in the miRNA AMO group was 0.38±0.15 times lower than in the control vector group, and the cell necrosis rate was much lower accordingly (23.1%±3.3% vs. 39.6%±2.3%, P<0.05). Besides, there was no significant difference between the control vector cells and the cells without treatment (P>0.05). The expression of miR-19b was significantly induced in ANP. In addition, up-regulation of miR-19b could promote the necrosis of pancreatic acinar cells and miR-19b deficiency could decrease the rate of pancreatic acinar cell necrosis.
Acinar Cells ; metabolism ; pathology ; Animals ; Arginine ; toxicity ; Cell Line ; MicroRNAs ; genetics ; metabolism ; Necrosis ; Pancreatitis, Acute Necrotizing ; etiology ; metabolism ; Rats ; Rats, Sprague-Dawley ; Taurolithocholic Acid ; analogs & derivatives ; toxicity ; Up-Regulation

OBJECTIVE: To explore the effects of taurolithocholic acid (tLCA) and chenodeoxycholic acid (CDCA) on the expression of aorexigenic neuropeptide in mouse hypothalamus GT1-7 cells. METHODS: Mouse hypothalamic GT1-7 cells were treated with culture medium containing 10% FBS (control group, =3) or with 10 nmol/L, 100 nmol/L, 1 μmol/L and 10 μmol/L tLCA (tLCA group, =3) or CDCA (CDCA group, =3) for 12, 24 or 48 h. Real-time PCR was performed to determine the expression levels of proopiomelanocortin (POMC) mRNA in the cells, and the production levels of α-melanocyte-stimulating hormone (α-MSH) were assessed using an ELISA kit. Signal transduction and activator of transcription 3 phosphorylation (p-STAT3), threonine kinase phosphorylation (p-AKT), suppressor of cytokine signaling 3 (SOCS3), G protein-coupled bile acid receptor-1 (TGR5) and farnesoid X receptor (FXR) protein were detected by Western blotting. RESULTS: Western blotting results showed that mouse hypothalamic GT1-7 cells expressed two bile acid receptors, TGR5 and FXR, whose expressions were regulated by bile acids. Real-time PCR showed that the expression of POMC mRNA was significantly increased in the cells after treatment with 10 μmol/L tLCA or CDCA for 24 h. POMC-derived anorexigenic peptide α-MSH increased significantly in GT1-7 cells after treatment with 10 μmol/L tLCA or CDCA for 24 h. Treatment of the cells with tLCA or CDCA significantly increased the expressions of intracellular signaling proteins including p-STAT3, p-AKT and SOCS3. CONCLUSIONS Mouse hypothalamic GT1-7 cells express bile acid receptors TGR5 and FXR. Bile acids tLCA or CDCA can promote the expression of POMC mRNA and increase the production of the anorexigenic peptide α-MSH. The intracellular signaling proteins p-AKT, p-STAT3 and SOCS3 are likely involved in bile acid-induced anorexigenic peptide production.
Animals ; Cell Line ; Chenodeoxycholic Acid ; pharmacology ; Gene Expression Regulation ; drug effects ; Hypothalamus ; cytology ; Mice ; Neuropeptides ; genetics ; metabolism ; Pro-Opiomelanocortin ; genetics ; RNA, Messenger ; genetics ; STAT3 Transcription Factor ; metabolism ; Signal Transduction ; drug effects ; Suppressor of Cytokine Signaling 3 Protein ; metabolism ; Taurolithocholic Acid ; pharmacology ; alpha-MSH ; genetics