Reactive oxygen species and NADPH oxidase 4 induced by transforming growth factor ß1 are the therapeutic targets of polyenylphosphatidylcholine in the suppression of human hepatic stellate cell activation.

OBJECTIVE AND DESIGN: To clarify the molecular mechanism of polyenylphosphatidylcholine (PPC), we examined the involvement of reactive oxygen species (ROS) and NADPH oxidase 4 (Nox4) in human hepatic stellate cells (HSCs). MATERIAL: Using human LX-2 HSC cells, we examined the effects of PPC on expression of α-smooth muscle actin (α-SMA) and collagen 1, generation of ROS, Nox4 expression, p38 activation and cell proliferation, induced by transforming growth factor ß1 (TGFß1). RESULTS: PPC suppressed ROS which are induced by TGFß1, phosphorylation of p38MAPK, and expression levels of α-SMA and collagen 1 in a dose-dependent manner. Higher concentrations of PPC also suppressed Nox4 levels. CONCLUSION: These results suggest that ROS and Nox4 induced by TGFß1 are the therapeutic targets of PPC in the suppression of human hepatic stellate cell activation.

A novel biomarker TERTmRNA is applicable for early detection of hepatoma.

BACKGROUNDS: We previously reported a highly sensitive method for serum human telomerase reverse transcriptase (hTERT) mRNA for hepatocellular carcinoma (HCC). alpha-fetoprotein (AFP) and des-gamma-carboxy prothrombin (DCP) are good markers for HCC. In this study, we verified the significance of hTERTmRNA in a large scale multi-centered trial, collating quantified values with clinical course. METHODS: In 638 subjects including 303 patients with HCC, 89 with chronic hepatitis (CH), 45 with liver cirrhosis (LC) and 201 healthy individuals, we quantified serum hTERTmRNA using the real-time RT-PCR. We examined its sensitivity and specificity in HCC diagnosis, clinical significance, ROC curve analysis in comparison with other tumor markers, and its correlations with the clinical parameters using Pearson relative test and multivariate analyses. Furthermore, we performed a prospective and comparative study to observe the change of biomarkers, including hTERTmRNA in HCC patients receiving anti-cancer therapies. RESULTS: hTERTmRNA was demonstrated to be independently correlated with clinical parameters; tumor size and tumor differentiation (P < 0.001, each). The sensitivity/specificity of hTERTmRNA in HCC diagnosis showed 90.2%/85.4% for hTERT. hTERTmRNA proved to be superior to AFP, AFP-L3, and DCP in the diagnosis and underwent an indisputable change in response to therapy. The detection rate of small HCC by hTERTmRNA was superior to the other markers. CONCLUSIONS: hTERTmRNA is superior to conventional tumor markers in the diagnosis and recurrence of HCC at an early stage.

Partial analysis of hepatitis B virus DNA from hepatocellular carcinoma showing negative hepatitis B virus surface antigen: an analysis of two Japanese cases.

It has been reported that hepatitis B virus (HBV) DNA is detected in serum and/or liver in patients with hepatocellular carcinoma (HCC) without HBsAg. To adress this issue, we analyzed HBV genome in 2 HCC cases without HBsAg. The DNA from serum from patients with HCC was amplified with a nested PCR, and 'a' determinant of S region, core promoter region and precore region were sequenced. The first case, a 50 years-old male, was negative for HBsAg and HBeAg, and positive for anti-HBs, anti-HBe and anti-HBc. Viral load of HBV in serum was 4.0 log genome equivalent/ml by TMA assay, and was 1.1 X 105 copy/ml by real-time PCR system. A nucleotide analysis of the common 'a' determinant of S gene showed that the 5 first amino acids of 'a' determinant, CTIPA, were changed to CKTCTTPA. The second case, a 76 years-old male, was positive for anti-HBe, but negative for HBsAg, anti-HBs, HBeAg and anti-HBc. No missense or nonsense mutations were seen in 'a' determinant of S region. Viral load of serum HBV was < 3.7 log genome equivalent/ml by TMA assay, but was 2.4X103 copy/ml by real-time PCR system. The results of the present study suggest that the mechanisms of HBsAg loss are diverse among HCC patients without HBsAg, and that an analysis of HBV genome is a useful tool to dissolve molecular mechanisms losing HBs antigenicity.

Development of a novel assay to quantify serum human telomerase reverse transcriptase messenger RNA and its significance as a tumor marker for hepatocellular carcinoma.

Currently available tumor markers for hepatocellular carcinoma (HCC) are alpha-fetoprotein (AFP), lens culinaris agglutinin-reactive AFP (AFP-L3), and Des-gamma-carboxy prothrombin (DCP). However, their positive rate can not surpass abdominal ultrasonography (US) as modalities to detect small HCC at early stage, resulting in a possible delay of its diagnosis. There is a need to develop an additional sensitive marker to improve the early detection of HCC. We here introduced a newly developed quantitative detection method for serum hTERT mRNA, which has a clinical significance in HCC diagnosis. Briefly, we examined its sensitivity and specificity in HCC diagnosis, clinical significance in comparison with other tumor markers, and its correlations with the clinical parameters. Serum hTERT mRNA showed higher values in patients with HCC than those with chronic liver diseases. hTERT mRNA expression independently correlated with clinical parameters such as differentiation degree (p < 0.001). The sensitivity/specificity of hTERT mRNA in HCC diagnosis showed 88.2/70.0%. hTERT mRNA proved to be expectedly superior to AFP mRNA , AFP and DCP in HCC diagnosis. Importantly, hTERT mRNA in serum correlated with that in HCC tissue. Thus, we report that serum hTERT mRNA is a novel and available marker for HCC diagnosis.

Significance of urinary excretion of 8-hydroxy-2'-deoxyguanosine in healthy subjects and liver disease patients.

BACKGROUND/AIMS: Although the importance of reactive oxygen species (ROS) in the pathogenesis of various diseases is stressed, clinical significance of the markers reflecting DNA oxidation such as 8-hydroxy-2'-deoxyguanosine (8-OHdG) remains to be clarified. METHODOLOGY: To examine clinical usefulness of 8-OHdG in healthy individuals in comparison with liver disease patients, urinary excretion of 8-OHdG was measured in 336 healthy individuals and 110 patients with liver disease. RESULTS: In healthy persons, the 8-OHdG excretion was increased in an age-dependent manner. It was positively correlated with cigarettes smoked a day and negatively correlated with body mass index (BMI) (P < 0.05, each). Age, smoking and BMI were independent predictors of urinary 8-OHdG excretion (P < 0.01, P < 0.01 and P < 0.05, respectively). In liver disease, the excretion of 8-OHdG was not changed, as compared with healthy individuals. However, the liver disease patients under the age of 40 had higher values of 8-OHdG than healthy persons. In addition, the urinary excretion of 8-OHdG was higher in patients with hepatitis C virus (HCV) infection than those with hepatitis B virus (HBV) infection. CONCLUSIONS: The results of the present study suggest that measurement of urinary 8-OHdG excretion is useful in assessing DNA oxidation caused by aging, smoking, body composition and liver disease.

Naked gene therapy of hepatocyte growth factor for dextran sulfate sodium-induced colitis in mice.

Ulcerative colitis (UC) is progressive and relapsing disease. To explore the therapeutic effects of naked gene therapy of hepatocyte growth factor (HGF) on UC, the SRalpha promoter driving HGF gene was intrarectally administered to the mice in which colitis was induced by dextran sulfate sodium (DSS). Expression of the transgene was seen in surface epithelium, lamina propria, and muscularis mucosae. The HGF-treated mice showed reduced colonic mucosal damage and increased body weights, compared with control mice (P < 0.01 and P < 0.05, respectively). The HGF-treated mice displayed increased number of PCNA-positive cells and decreased number of apoptotic cells than in control mice (P < 0.01, each). Phosphorylated AKT was dramatically increased after HGF gene administration, however, phosphorylated ERK1/2 was not altered. Microarray analysis revealed that HGF induced expression of proliferation- and apoptosis-associated genes. These data suggest that naked HGF gene delivery causes therapeutic effects through regulation of many downstream genes.

Expression of oxidative stress-related molecules in circulating leukocytes and urine in patients with chronic viral hepatitis.

AIMS: Oxidative stress plays a role in pathogenesis of chronic viral hepatitis. Expression of oxidative stress-related molecules remains to be clarified. METHODS: 4-hydroxy-2-nonenal (4-HNE), 4-hydroxy-2-hexenal (4-HHE), catalase, superoxide dismutase-1 (SOD-1), glutathione peroxidase-1, thioredoxin (TRX) in leukocytes and urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) were examined in 164 persons, including 130 chronic viral hepatitis patients and 34 normal individuals, by Western blot analysis and enzyme-linked immunosorbent assay, respectively. Hepatic expression of these proteins was immunohistochemically examined in 12 patients with chronic viral hepatitis, compared with three persons without liver damage. RESULTS: The 4-HNE/beta-actin ratios in chronic viral hepatitis were significantly higher than those in normal individuals (P<0.01), and were significantly correlated with asparate aminotransferase (AST) and alanine aminotransferase (ALT) (P<0.01, each). The catalase/beta-actin and SOD-1/beta-actin ratios in chronic viral hepatitis were higher than those in normal individuals, and were significantly correlated with 4-HNE/beta-actin ratios (P<0.01, each). Hepatic expression of 4-HNE, 4-HHE, catalase, SOD-1 and TRX in chronic viral hepatitis was higher than that without liver damage. Urinary excretion of 8-OHdG was not changed in chronic viral hepatitis. CONCLUSIONS: The results of the present study suggest that expression of oxidative stress-related molecules in leukocytes is upregulated in relation to serum aminotransferase levels.

Postmarketing surveillance of rabeprazole in upper gastrointestinal peptic lesions in Japanese patients with coexisting hepatic disorders.

BACKGROUND: Many Japanese patients with hepatic disorders confirmed on diagnostic imaging and coexisting upper gastrointestinal (GI) peptic lesions receive treatment with proton pump inhibitors. Some pharmacotherapies used to treat peptic ulcers have been associated with adverse drug reactions (ADRs), including elevated liver enzyme levels. OBJECTIVE: The aim of this study was to determine the tolerability and effectiveness of rabeprazole sodium in treating peptic lesions in patients with coexisting hepatic disorders. METHODS: This open-label, practice-based, postmarketing surveillance investigation was conducted at 15 centers across Japan. Male and female patients aged ≥18 years with peptic lesions confirmed on upper GI endoscopy and with underlying hepatic disease were enrolled. Patients were randomly assigned to receive rabeprazole 10 or 20 mg PO (tablet) QD after a meal for up to 8 weeks. Tolerability was assessed using monitoring of the incidence of ADRs determined by direct patient questioning, spontaneous reporting, and laboratory assessment. All patients who received at least 1 dose of study drug were included in the tolerability assessment. Effectiveness was assessed at baseline and study end using the rates of achievement of improvement on endoscopy, relief of subjective/objective symptoms (rates of improvement in epigastric pain and heartburn), and global improvement. The effectiveness analysis included all patients with complete data before and after treatment. Subanalyses were conducted to determine the effectiveness of drug by identification of the proportion of patients with coexisting hepatic disorders (cirrhosis, chronic hepatitis, and other hepatic diseases [eg, alcoholic hepatitis, fatty liver]) and by peptic lesion (gastric ulcer, duodenal ulcer, stomal ulcer, and reflux esophagitis) who achieved improvement. RESULTS: A total of 114 patients were enrolled; 108 patients were included in the tolerability analysis (81 men, 27 women; mean age, 59.9 years; 10-mg dose, 90 patients; 20-mg dose, 18 patients) and 98 patients were included in the analysis of effectiveness. Twenty-one ADRs occurred in 11 (10.2%) patients. Serious ADRs occurred in 2 patients (elevated bilirubin level and hepatic encephalopathy, 1 patient each). Administration of rabeprazole was discontinued in 5 patients due to the occurrence of the following ADRs: constipation (1 patient); epigastric pain (1); dyslalia, disorientation, tremor, sleep disorder, and hepatic encephalopathy (1); diarrhea (1); and elevated alkaline phosphatase and y-glutamyl transpeptidase levels (1). On endoscopy, the proportion of patients achieving improvement with either dose was 30/33 (90.9%). The relief rates assessed using subjective symptoms were 47/55 (85.5%) and 47/56 (83.9%) for epigastric pain and heartburn, respectively. The proportion of patients achieving global improvement with either dose was 80/98 (81.6%) patients (49/62 [79.0%] for cirrhosis, 11/16 [68.8%] for chronic hepatitis, and 20/20 [100.0%] for other hepatic diseases [alcoholic hepatitis, fatty liver]). CONCLUSION: In this study in Japanese patients with hepatic disorders, rabeprazole was well tolerated and appeared effective for the treatment of upper GI peptic lesions.

Combination of enprostil and cimetidine is more effective than cimetidine alone in treating gastric ulcer: prospective multicenter randomized controlled trial.

BACKGROUND/AIMS: Little is known about the clinical efficacy of co-therapy of enprostil, a prostaglandin E2 analogue, with a histamine H2-receptor antagonist. We aimed to assess the additive benefit of enprostil in combination with cimetidine for treating gastric ulcer in a prospective multicenter randomized controlled trial. METHODOLOGY: In 43 hospitals 171 intention-to-treat (ITT) patients, diagnosed as having gastric ulcer by endoscopy, were randomly allocated to receive either enprostil 25microg b.i.d. and cimetidine 400mg b.i.d. (Group E=85), or cimetidine 400mg b.i.d. alone (Group C=86) for 8 weeks. Healing was examined by endoscopy at 4 and 8 weeks. RESULTS: Per protocol (PP) analysis comprised 166 patients (E=82, C=84). Despite no significant advantage at 4 weeks (E=55.3%, C=42.2%), the combination yielded higher healing rates at 8 weeks by ITT (E=89.4%, C=68.6%; p<0.001) and PP analysis (E=92.7%, C=70.2%; p<0.001). Symptom relief rates [E, C] at 2, 4, and 8 weeks were [80.2%, 68.3%] (not significant), [97.4%, 88.3%] (p<0.05), and [95.6%, 87.0%] (p<0.05), respectively. Significant advantage was observed in the patients aged 40 or older, with solitary ulcer (>5mm in diameter), and without smoking or drinking habits. No adverse effects were critical. CONCLUSIONS: Enprostil safely and significantly augmented gastric ulcer healing and symptom relief by cimetidine.

Development of quantitative neuropsychological tests for diagnosis of subclinical hepatic encephalopathy in liver cirrhosis patients and establishment of diagnostic criteria-multicenter collaborative study in Japanese.

At present, there are no generally accepted diagnostic criteria or methods for subclinical hepatic encephalopathy (SHE) associated with liver cirrhosis. We therefore developed an easily conducted computer-aided quantitative neuropsychiatric function test system for use in routine medical practice. We established normal values in healthy Japanese subjects and determined differences between healthy persons and liver cirrhosis patients without clinical encephalopathy in a multi-center clinical trial. The test system consists of eight tests: number connection tests A and B, a figure position test, a digit symbol test, a block design test, and reaction time tests A, B and C. The test results were affected by age, but not by gender or facility. No learning effect was noted. The results were therefore reported by 5-year quartile ranges and differences were evaluated between 542 healthy subjects and 292 cirrhotic patients. When the cut-off value was set at the 10th/90th percentile of the results in healthy subjects, the results of each of the 8 tests were abnormal in about 25% of cirrhotic patients, and at least 1 of the 8 tests gave values greater than the 10th/90th percentile cut-off value in 58.2% of the 292 liver cirrhosis patients. SHE patients were thought to be included in these 58.2% of patients. The developed test makes it possible to quantitatively assess neuropsychiatric function, and the results obtained can be used as a basis for the diagnosis of SHE.

Serum levels of interleukin-10, interleukin-12 and soluble interleukin-2 receptor in chronic liver disease type C.

BACKGROUND/AIMS: It is still unclear whether and how Th1/Th2 type cytokines are involved in the progression of chronic liver disease type C. We therefore examined serum levels of IL-10, IL-12 and sIL-2R (soluble IL-2 receptor) in association with clinical parameters in chronic liver disease type C, whereas IL-12 and sIL-2R represent Th1 cytokine and IL-10 does Th2 cytokine, respectively. METHODOLOGY: Serum levels of IL-10, IL-12 and sIL-2R were measured in 110 patients, including 36 with chronic hepatitis, 24 with liver cirrhosis and 50 with hepatocellular carcinoma in comparison with 19 normal individuals, by an enzyme-linked immunosorbent assay. In 9 chronic hepatitis patients, serum levels of these cytokines were measured before and after interferon therapy. In 28 with hepatocellular carcinoma, they were also measured before and after transcatheter arterial embolization. RESULTS: Serum levels of IL-10 in chronic hepatitis, liver cirrhosis and hepatocellular carcinoma were 3.9 +/- 1.8 pg/mL, 5.7 +/- 6.4 pg/mL and 5.6 +/- 8.9 pg/mL, respectively. IL-10 level was significantly correlated with level of y-globulin. Serum levels of IL-12 in chronic hepatitis, liver cirrhosis and hepatocellular carcinoma were 347.4 +/- 150.3 pg/mL, 365.2 +/- 130.7 pg/mL and 399.4 +/- 258.2 pg/mL. sIL-2R levels in chronic hepatitis, liver cirrhosis and hepatocellular carcinoma were 614.6 +/- 223.5 U/mL, 878.7 +/- 330.5 U/mL and 1037.9 +/- 412.0 U/mL. Serum levels of IL-12 and sIL-2R were significantly elevated on day 7 after interferon therapy compared to day 0 (p < 0.05 and p < 0.001, respectively), while no significant difference was seen in IL-10. Serum level of IL-10 was significantly elevated on day 3, and that of sIL-2R was elevated on day 3 and 7 after transcatheter arterial embolization, while that of IL-12 was decreased on day 3 and 7. CONCLUSIONS: The results of the present study suggest that Th1/Th2 type cytokines are changed in association with progression of chronic liver disease type C and in response to therapy.

Combination therapy of ecabet sodium and cimetidine compared with cimetidine alone for gastric ulcer: prospective randomized multicenter study.

BACKGROUND AND AIM: Little is known about the clinical efficacy of co-therapy of ecabet sodium, a mucoprotective agent, and a histamine H2-receptor antagonist. The aim of the present study was to assess its additive benefit in combination with cimetidine for gastric ulcer. METHODS: In this prospective randomized study, after gastric ulcer was confirmed by endoscopy, 200 patients in 47 hospitals received either ecabet sodium 1 g b.i.d and cimetidine 400 mg b.i.d. (EC), or cimetidine 400 mg b.i.d. alone (C) for 8 weeks. Healing was examined by endoscopy at 4 and 8 weeks. RESULTS: Of the intention-to-treat (ITT) population (EC, 103; C, 97), 181 patients comprised the per protocol (PP) analysis (EC, 93; C, 88). At 4 weeks, healing rates were significantly higher in the EC group (60%) than in the C group (36%) ( p < 0.01). At 8 weeks, those by the ITT and PP analyses were 82% (EC) versus 58% (C), and 90% (EC) versus 64% (C), respectively ( p < 0.01 and p < 0.001). Symptom relief rates (EC vs C) at 2, 4 and 8 weeks were 73%versus 47% ( p < 0.01), 89%versus 66% ( p < 0.001), and 97%versus 73% ( p < 0.001), respectively. Significant additive effects of ecabet sodium were observed in patients aged 60 years or older, with solitary and medium to large ulcer, and without smoking or drinking habits. No adverse effects were critical. CONCLUSION: Ecabet sodium significantly augmented gastric ulcer healing and symptom relief by cimetidine, especially in the elderly.

Expression of transforming growth factor-beta 1 in hepatocellular carcinoma in comparison with the non-tumor tissue.

BACKGROUND/AIMS: TGF-beta 1 (transforming growth factor-beta 1) has been shown to be overexpressed in various cancer cells including hepatocellular carcinoma cells. In this study, we examined the association of the immunohistochemical expression of TGF-beta 1 in hepatocellular carcinoma tissues with the clinicopathological findings. METHODOLOGY: Thirty liver tumor biopsy specimens obtained with ultrasound guidance and in which tumor-tissue and non-tumor tissue existed were examined. TGF-beta 1 was stained using rabbit anti-human TGF-beta 1 antibody. RESULTS: TGF-beta 1 was expressed mainly in the cytoplasm and plasma membrane of the hepatocellular carcinoma cells, while it was stained in the fibrous septa and sinusoidal cells in the non-tumor tissue. When comparing the intensity of TGF-beta 1 staining in the hepatocellular carcinoma tissue with that in the non-tumor tissue, the former was more intense than the latter in 21 (70%) of the 30 cases. Although the expression of TGF-beta 1 in the hepatocellular carcinoma tissue was not related to the tumor size of hepatocellular carcinoma, it was correlated with the histological differentiation: the lower the histological differentiation grade of hepatocellular carcinoma was, the more intense was the TGF-beta 1 staining in hepatocellular carcinoma tissue. In addition, TGF-beta 1 tended to be overexpressed in the trabecular type of hepatocellular carcinoma. To elucidate the relationship of TGF-beta 1 staining with apoptosis in hepatocellular carcinoma cells, we performed terminal deoxynucleotidyl transferase-mediated DUTP biotinnick end labeling (TUNEL) in 11 of the 30 specimens. No positive cells for TUNEL were detected in the hepatocellular carcinoma tissues. CONCLUSIONS: These results indicated that the expression of TGF-beta 1 was associated with the histological differentiation of hepatocellular carcinoma cells.

Effect of posture on echographic examination for dialysate in patients treated with chronic ambulatory peritoneal dialysis as a model of ascites.

We evaluated the effect of posture on echographic examination for dialysate in 15 patients treated with chronic ambulatory peritoneal dialysis as a model of ascites. After gradually infusing 500 1000 and 1500 ml dialysate into their abdominal cavity, we compared echographic findings of infused fluids in dorsal, knee-elbow, and standing positions. The height of fluid in the standing position was largest and the width of fluid in Morison's pouch in the dorsal position was smallest. When calculating the volume of the infused fluid by modeling as an elliptic cone in the standing position or as a segment of a sphere in the knee-elbow position, the correlation coefficients between the calculated volumes and the infused volumes were 0.979 (P<0.0001) in the knee-elbow position and 0.920 (P<0.0001) in the standing position, respectively. Patients and examiners were required to endure an unnatural and awkward posture in the knee-elbow position. These findings suggest that echographic examination in the standing position is an easy and accurate method for the diagnostic paracentesis and the measurement of the volume of ascites in this model.

Increase in serum 7S domain of type IV collagen and N-terminal propeptide of type III procollagen levels with normal serum transaminase levels after long-term oral administration of Tegaful-Uracil.

To evaluate whether oral administration of Tegaful-Uracil (UFT(R)), a biochemical modulator of 5-fluorouracil that contains tegafur and uracil, can induce hepatic fibrosis, serum 7S domain of type IV collagen and N-terminal propeptide of type III procollagen levels were measured in 63 UFT(R)-treated, 38 tegafur-treated and 40 untreated patients. Serum transaminase and bilirubin levels were normal in almost all patients. Serum levels of 7S collagen and type III propeptide increased in 25 and 17% of the UFT(R)-treated patients, respectively, although a majority of tegafur-treated and untreated patients showed no increase in these markers. The patients with the elevated levels demonstrated mild or moderate hepatic fibrosis without necroinflammation in the liver. Both serum levels decreased markedly after the discontinuation of UFT(R). These findings suggest that long-term oral administration of UFT(R) can induce hepatic fibrosis without the elevation of serum transaminase levels and necroinflammation in the liver, and serum 7S collagen and type III procollagen are of diagnostic value for UFT(R)-induced hepatotoxicity.

Reduction of hepatocarcinogenesis by ursodeoxycholic acid in rats.

Ursodeoxycholic acid (UDCA) is used worldwide for treatment of primary biliary cirrhosis and chronic liver diseases. However, its action on hepatocarcinogenesis remains to be explored. To clarify its effect, in vivo and in vitro experiments were performed. Ninety Fisher 344 rats were fed a standard diet (Group 1, n = 30), a standard diet supplemented with 0.1% UDCA (Group 2, n = 30) and 0.3% UDCA (Group 3, n = 30). The rats were given an i.p. injection of diethylnitrosamine (DEN) weekly for 6 weeks. Fifteen additional rats were fed 0.3% UDCA supplemented diet without DEN treatment (Group 4). The rats were killed at 5, 10 and 18 weeks after the last injection of DEN. The number of liver tumor and percentage of the GST-P-positive hepatocytes were significantly reduced by UDCA treatment. The PCNA-positive cells were decreased by administration of UDCA at 18 weeks. The increased number of apoptotic cells was observed in the GST-P-negative area at 5, 10 and 18 weeks and in the GST-P-positive area at 18 weeks in the UDCA group. Expression of Bax in mitochondria and cytochrome c in cytosol was increased by UDCA treatment. Caspase 3 activity was also increased in the UDCA groups. The addition of UDCA into the culture of Huh7 and Fao hepatocellular carcinoma (HCC) cells induced apoptosis in a dose-dependent manner. The data of the present study suggest that UDCA treatment reduces hepatocarcinogenesis via inducing apoptosis of 'initiated hepatocytes' as well as inhibiting proliferation.