Reduction of hepatocarcinogenesis by ursodeoxycholic acid in rats

Kenji Oyama1, Goshi Shiota1,3, Hisao Ito2, Yoshikazu Murawaki1 and Hironaka Kawasaki1

1 Second Department of Internal Medicine, Faculty of Medicine, Tottori University, Yonago 683-8504, Japan and
2 First Department of Pathology, Faculty of Medicine, Tottori University, Yonago 683-8504, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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-dependant manner. The data of the present study suggest that UDCA treatment reduces hepatocarcinogenesis via inducing apoptosis of `initiated hepatocytes' as well as inhibiting proliferation.

Abbreviations: AOM, azoxymethane; CA, cholic acid; DCA, deoxycholic acid; DEN, diethylnitrosamine; GST-P, glutathione S transferase-P; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; IFN, interferon; MPT, mitochondrial permeability transition; PBC, primary biliary cirrhosis; PCNA, proliferating cell nuclear antigen; PMSF, phenylmethylsulfonyl fluoride; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick labeling; UDCA, ursodeoxycholic acid.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Hepatocellular carcinoma (HCC) is one of the most common cancers with a poor prognosis (1). There are about 500 000–1 000 000 new HCC cases per year (2). Major risk factors for HCC include cirrhosis, aflatoxin exposure, hepatitis B virus (HBV) and hepatitis C virus (HCV) infection (3). HCC can develop late in the course of viral infection, especially of chronic HCV infection, after the development of cirrhosis. HCV carriers demonstrate higher rates of developing HCC than HBV carriers; the incidence of HCC in HCV carriers is 4.2-fold higher than in HBV carriers, and it is 572-fold higher than in normal individuals (4,5). Therefore, prophylaxis of HCC occurrence from chronic viral hepatitis is likely as important as the progress of HCC therapy.

The efficacy of ursodeoxycholic acid (UDCA) in patients with primary biliary cirrhosis (PBC) has been demonstrated in several randomized clinical trials. UDCA improved biochemical and histological parameters, slowed the progression of PBC, and elongated survival (6–10). Since UDCA was reported to be effective in improving liver function of chronic hepatitis C (11), UDCA is now widely used for chronic viral hepatitis, especially type C. Although interferon (IFN) was first introduced as an agent for chronic viral hepatitis, it is now known that IFN inhibits occurrence of HCC as well as improving liver function (12). Surprisingly, it is shown that Sho-saiko-to and glycyrrhizin, which are cytoprotective but not inhibitory on viral replication, reduce HCC in human and animal models (13–16). In this context, it is interesting to clarify whether UDCA has inhibitory effects on hepatocarcinogenesis or not. Supplemental dietary UDCA was reported to be chemopreventive in the azoxymethane (AOM)-induced model of experimental colonic carcinogenesis (17). Recent reports suggested the strong relationship between arachidonic acid metabolism and colorectal cancer (18) and suppressive effect of UDCA on colon carcinogenesis was due to reduced expression of group II phospholipase A2, a rate-limiting enzyme of the arachidonic acid (19,20). UDCA also increases the activities of alkaline sphingomyelinase and caspase 3 in clonic mucosa (21). Interestingly, UDCA and its derivative induce apoptosis of HepG2 human hepatoblastoma cells (22,23). Taken together, UDCA may inhibit HCC development by inducing apoptosis of precancerous cells.

Experimental hepatocarcinogenesis in the rodent occurs in distinctly defined stages as follows: initiation, promotion, progression (24). Although apoptosis plays a role in each of these stages, it is especially important during the stage of promotion where the clonal expansion of initiated hepatocytes occurs. This expansion is caused by a selective increase in cell proliferation and by a selective decrease in apoptosis of preneoplastic hepatocytes. A number of studies have now demonstrated that several promoting agents which are effective in rat hepatocarcinogenesis selectively inhibit apoptosis of preneoplastic hepatocytes. These include 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), phenobarbital and cyproterone acetate (25,26). In addition, several other promoters have been shown to inhibit apoptosis. Indeed, withdrawal of a promoting agent during rat hepatocarcinogenesis may cause a dramatic increase in apoptosis of hepatocytes and preneoplastic foci (27). Thus, assessment of apoptosis is very important for chemically induced liver cancer experiments. Therefore, we examined the effect of UDCA on hepatocarcinogenesis by assessing cell growth and apoptosis.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
Diethylnitrosamine (DEN) was purchased from Sigma Chemical Co., MI. UDCA and cholic acid (CA) were kindly provided from Tokyo Mitsubishi Pharmaceuticals, Tokyo, Japan.

Animal model
A total of 105 eight-week-old adult male Fisher 344 rats weighing about 150 g were obtained from Charles River Japan (Yokohama, Japan). Rats were maintained in a temperature-controlled room with a 12 h light/dark illumination cycle. The animal experiment protocol was approved by an institutional animal experimentation review board of our university. On arrival, the animals were quarantined for 1 week, and then divided into the following experimental groups (Figure 1Go). The rats of Group 1 (n = 30) were fed a standard diet (CE-2 diet, CLEA Japan Inc., Tokyo, Japan). The rats of Group 2 (n = 30) were fed a standard diet supplemented with 0.1% (w/w) UDCA. The rats of Groups 3 (n = 30) were fed a standard diet supplemented with 0.3% UDCA. Three rats were housed in one cage, and daily food intake was measured. The rats of Groups 1, 2 and 3 were fed their assigned diets for 2 weeks, and then given an intraperitoneal (i.p.) injection of 38 mg/kg body weight of DEN once a week for 3 weeks, followed by an i.p. injection of 50 mg/kg body weight of DEN (28). The rats of Group 4 were fed a diet supplemented with 0.3% UDCA without DEN treatment. The rats in each group were serially killed at 5, 10 and 18 weeks after the last injection of DEN. At sampling, rats were killed under ether anesthesia, and blood was taken from aorta by vascular canulation. The livers were weighed, and sectioned into 2–3 mm strips for examination of the presence of tumors. The tumors with diameters >3 mm were counted as tumors and histologically examined. Strips of liver were fixed in 4% paraformaldehyde for 24 h, and then embedded in paraffin. Sectioned paraffin blocks were stained with hematoxylin and eosin. In the histopathological examination, liver lesions were diagnosed according to the criteria described previously (29). Two independent investigators performed the histological assessment blinded to other information (KO and GS).



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Fig. 1. Experimental schedule. Experimental schedules of Groups 1, 2, 3 and 4 were shown. The rats in Group 1 to 3 were given an i.p. injection of DEN, however, the rats of Group 4 were not treated with DEN. The rats were killed at 5, 10 and 18 weeks after the last injection of DEN. The number of killed at each time point was 10 in Groups 1 to 3, and that in Group 4 was 5.

 
TUNEL staining
Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) was performed by using ApoTag Plus Peroxidase in situ Apoptosis Detection Kit (Intergen) according to the manufacturer's instruction. In brief, paraffin-sections were de-waxed, rehydrated through a graded alcohol series and washed in distilled water. After digestion in 20 µg/ml of proteinase K (Boehringer Mannheim/Yamaouchi, Tokyo, Japan) for 10–20 min at room temperature, the sections were washed in tap water. The sections were treated with 2% H2O2/methanol and washed in distilled water. Then, terminal deoxynucleotidyl transferase (TdT) buffer (100 mM potassium cacodylate, 2 mM cobalt chloride, 0.2 mM dithiothreitol, pH 7.2) containing 0.3 U/l TdT (Gibco BRL, MD, USA) and 0.04 nmol/l biotinylated dUTP (Boehringer Mannheim/Yamanouchi) were added to cover the sections, which were incubated in a humidified atmosphere for 90 min at 37°C. The sections were washed in TB buffer (300 mM sodium chloride, 300 mM sodium citrate) for 15 min at room temperature. After washing with phosphate buffer, they were subsequently incubated with peroxidase-labeled streptavidin–H2O2. The sections were counterstained with methyl green. The TUNEL-positive cells were determined by analyzing 1000 cells in the fields randomly selected.

Immunohistochemistry
De-waxed paraffin sections were immunostained using the avidin–biotin–peroxidase complex. The sections were incubated with 0.3% H2O2 in methanol for 30 min. They were heated in 10 mmol/l sodium citrate buffer (pH 6.0) at 600 W for 15 min. They were incubated with normal horse or goat serum. After washing, they were stained using the following primary antibodies: anti-proliferating cell nuclear antigen (PCNA) monoclonal antibody (Novocastra Laboratories, Newcastle upon Tyne) diluted 1:100 and rabbit anti-GST-P polyclonal antibody (MBL, Nagoya, Japan) diluted 1:1000 at 4°C overnight. Then, they were incubated with biotin-conjugated horse anti-mouse immunoglobulin G or biotin-conjugated goat anti-rabbit antibody (Vector Laboratories, CA) for 60 min. Immunoreactive cells were visualized using a Vectastain ABC-PO (mouse IgG) Kit or Vectastain ABC-PO (rabbit IgG) Kit (Vector Laboratories). For double immunostaining of GST-P with PCNA and GST-P with TUNEL, GST-P-positive areas were first stained with Vectastain ABC-AP (rabbit IgG) kit (Vector Laboratories), and the nuclei positive for PCNA and TUNEL were then stained with Vectastain-PO (mouse IgG) kit or ApoTag Plus Peroxidase in situ Apoptosis Detection Kit, respectively. The GST-P-positive cells and TUNEL-positive cells in the fields containing a total of at least 1000 hepatocyte nuclei were counted in each rat. The labeling index was determined by calculating the mean of the numbers of positive hepatocytes divided by the total number of counted hepatocytes in each field.

Preparation of mitochondria and cytosol fractions
Preparation of mitochondrial and cytosolic fractions was done previously described (30). For isolation of mitochondria, the liver tissue was minced on ice, resuspended in 10 ml of ice-cold Buffer A (200 mM mannitol, 50 mM sucrose, 10 mM KCl, 1 mM EDTA, 10 mM HEPES–KOH, pH 7.4, 0.1% bovine serum albumin, 10 µg/ml of aprotinin, and 1 mM phenylmethylsulfonyl fluoride), and homogenized with a glass Dounce homogenizer and a tight Teflon pestle. Homogenates were centrifuged at 600 g for 15 min at 4°C, and supernatants were then centrifuged at 3500 g for 15 min at 4°C. After floating lipid layers were aspirated, the mitochondrial pellets were resusupended in Buffer A. After centrifugation at 1500 g for 5 min at 4°C, the supernatants were recentrifuged at 5500 g for 10 min. The last two steps were repeated twice, and used for western blot analysis. For preparation of cytosolic extracts, the liver was homogenized in ice-cold buffer (20 mM Herpes–KOH, pH 7.0, 10 mM KCl, 1.5 mM MgCl2, 1 mM sodium EDTA, 1 mM sodium EGTA, 1 mM dithiothreitol, 250 mM sucrose, 10 µg/ml of aprotinin, and 1 mM phenylmethylsulfonyl fluoride) by Dounce homogenizer. Supernatants were centrifuged at 14 000 g for 15 min in a microcentrifuge. The resulting supernatants were used for western blot analysis.

Western blot analysis
The mitochondrial and cytosolic fractions were lysed in the modified RIRA buffer (50 mM Tris HCl, pH 7.4, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1% NP-40, 1 µg/ml of aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM Na3VO4, and 1 mM NaF). Fifty micrograms of protein of each fraction were boiled for 5 min and electrophoresed on 15% sodium dodecyl sulfate polyacrylamide gels. Then, the proteins were transferred onto Hybond ECL membranes (Amersham Pharmacia Biotech, IL) using a semi-dry blotting apparatus. The mitochondrial protein on the membranes was incubated with 1:500 dilution of rabbit anti-Bax polyclonal antibody (Santa Cruz Biotechnology, CA), anti-Bcl-2 antibody (Santa Cruz Biotechnology, CA), anti-Bcl-XL antibody (Santa Cruz Biotechnology, CA), anti-Bcl-Xs antibody (Santa Cruz Biotechnology, CA), respectively. The same filter was erased and reprobed with monoclonal antibody (Molecular Probes, WO, USA) against cytochrome c oxidase, the mitochondria membrane bound protein which was used as a marker of mitochondria (31). The cytosolic protein on the membrane was incubated with mouse anti-cytochrome c monoclonal antibody (Pharmingen International, CA) at 4°C overnight. As an internal control of cytosolic protein, anti-ß-actin monoclonal antibody (Sigma, MO) was used. After washing, the membranes were incubated with horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham Pharmacia Biotech) or horseradish peroxidase-conjugated sheep anti-mouse IgG (Amersham Pharmacia Biotech) at room temperature for 1 h. Immunoreactive bands were visualized with the ECL western blotting detection kit (Amersham Pharmacia Biotech) according to the manufacturer's protocol.

Caspase 3 activity
Caspase 3 activity was measured by ApoAlert Caspase 3 Colorimetric Assay Kit (Clontech Laboratories, CA). A portion of liver tissue was homogenized in 50 µl of chilled cell lysis buffer, incubated on ice for 10 min, and centrifuged in a microcentrifuge at 12 000 rpm for 3 min. The supernatant was transferred to the new microcentrifuge tube. Fifty µl of 2x reaction buffer was added to 50 µl of sample solution (about 4 mg/ml protein concentration). Five µl of 1 mM conjugated substrate (50 µM final concentration) was added to the tube and incubated at 37°C for 1 h. The samples were read in a spectrometer at 400 nm. The reaction in each sample was performed in duplicate for the following samples: induced, uninduced (negative control), induced plus inhibitor (0.5 µl of DEVD-fmk inhibitor), and induced minus substrate. The caspase 3 activity was expressed as units/mg protein, where unit caspase 3 activity is equal to ({Delta}ODU/h) x / curver slope and {Delta}ODU/h is equal to the difference in ODU between T0 and T1.

Cell viability assay
Human HCC HUH7 cells and rat HCC Fao cells at 2 x 104/ml cell density were incubated with DMEM (Cosmo Bio Co., Tokyo, Japan) supplemented with 10% FBS in 100 µl of 96-well plate (Sumitomo Bakelite, Tokyo, Japan) at 37°C for 4 h under 5% CO2 in air. Then, the medium was changed to serum-free medium and incubated overnight. After washing in PBS, the medium was changed to various concentrations of UDCA or CA contained medium. The cells were incubated at 37°C for 72 h in a humidified, 5% CO2 atmosphere. At 4 h after addition of 20 µl/well of Cell Titer 96 Aqueous One Solution Reagent (Promega, WI), the absorbance at 490 nm was recorded using a 96-well plate reader. The cell viability was calculated as the absorbance at each concentration divided by the absorbance at 0 µM.

Agarose gel analysis of DNA fragmentation
For the qualitative analysis, the cells were harvested, washed with cold PBS, and digested with 200 µg/ml proteinase K in 10 mM Tris, pH 7.6, 100 mM NaCl, 10 mM EDTA and 0.5% SDS at 55°C for 4 h. After extraction with phenol/chloroform, the pellets were digested with 300 µg/ml DNase-free RNase A (Nippon gene Corp., Toyama, Japan). The electrophoresis of DNA was performed on a 1.5% agarose gel in TBE buffer (0.09 M Tris–HCl, 0.002 M EDTA).

Determination of bile acids
Bile acids in serum and liver were determined by high performance liquid chromatography according to the procedures previously described (32,33).

Statistics
Values are expressed as mean ± SEM. Means were compared using the Mann–Whitney U-test. A P-value of <0.5 was considered to be significant.


    Results
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 Materials and methods
 Results
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 References
 
Effects of UDCA on tumor development and GST-P-positive lesion
To examine the effect of UDCA on tumor development, tumor formation was assessed at 5, 10 and 18 weeks. Liver tumors were histologically composed of neoplastic nodules and HCC (29). Neither tumors nor GST-positive cells were found in rats of Group 4 throughout the experiment. At 5 weeks, no tumors were observed in these rats. At 10 weeks, tumors developed in Groups 1, 2 and 3, however, there were no differences in tumor number among the groups. At 18 weeks, the numbers of tumor in Group 2 and Group 3 were significantly lower than those in Group 1 (P < 0.05, each) (Figure 2AGo). At 5 weeks the GST-P-positive hepatocytes were observed, but no differences in number were found among three groups. The percentages of the GST-P-positive cells in Group 2 and Group 3 were significantly lower than those in Group 1 at 10 weeks (P < 0.05, each, Figures 2B, 3AGoGo) and at 18 weeks (P < 0.01, each), respectively. The tumor was yellowish and was sharp with well delineated (data not shown). HCC was discriminated from the surrounding tissue and had a trabecular structure, which is a typical morphological feature of HCC. At the periphery they compress or extend into the surrounding parenchyma. Cystic dilatation of bile ducts and mild fatty changes were features of the surrounding tissues of tumor.



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Fig. 2. Effect of UDCA on tumor development and GST-P-positive lesion. (A) Tumor development at 5, 10 and 18 weeks were expressed as tumor number per liver. {circ}: Group 1, {square}: Group 2, •: Group 3, *P < 0.05. (B) The percentage of GST-P-positive hepatocytes was counted in 1000 hepatocytes in Groups 1–3 at 5, 10 and 18 weeks. {circ}: Group 1, {square}: Group 2, •: Group 3, **P < 0.01.

 


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Fig. 3. Immunohistochemical analysis of liver tissues. (A) The GST-P-positive hepatocytes in Group 3 at 10 weeks were indicated by the arrows (magnification x100). (B) The TUNEL-positive hepatocyte in GST-P-negative area was shown. The arrow indicates the apoptotic cell from a rat liver in Group 1 at 5 weeks (magnification x400). (C) The PCNA-positive hepatocytes in GST-P-positive area were shown in a rat liver of Group 1 at 18 weeks. The nuclear staining was indicated by the arrow (magnification x200). (D) The TUNEL-positive hepatocytes in GST-P-positive area were shown in a rat liver in Group 3 at 18 weeks. The nuclear staining of the apoptotic hepatocytes was indicated by the arrow (magnification x200).

 
Bile acid concentrations
Hepatic UDCA concentrations in Group 1 were 11.2 nmol/g liver at 5 weeks, 11.9 nmol/g liver at 10 weeks and 19.4 nmol/g liver at 18 weeks, respectively. On the other hand, those in 0.1% UDCA group and 0.3% UDCA group were 67.1 nmol/g liver and 164.0 nmol/g liver at 5 weeks, 53.4 nmol/g liver and 135.8 nmol/g liver at 10 weeks, and 84.9 nmol/g liver and 188.6 nmol/g liver at 18 weeks, respectively. Serum UDCA concentrations were similarly increased in Groups 2 and 3, compared with Group 1, however, their values were much lower than those in liver (data not shown).

Effect of UDCA on cell proliferation and apoptosis
To examine the effect of UDCA on cell proliferation and apoptosis in cancerous and non-cancerous tissues, the PCNA-positive hepatocytes and the TUNEL-positive hepatocytes were counted in both the GST-P-positive and GST-P-negative areas (Figure 3B –DGo). At 5 and 10 weeks, the number of PCNA-positive cells was comparable among three groups (Figure 4Go). However, in the GST-P-negative area, the PCNA-positive cells in Group 3 were significantly decreased compared with those in Group 1 at 18 weeks (P < 0.05, Figure 4AGo). In the GST-P-positive area, the PCNA-positive cells in Group 2 and Group 3 were significantly decreased compared with those in Group 1 (P < 0.05, each, Figure 4BGo). Thus, UDCA treatment did not affect cell proliferation at 5 and 10 weeks, however, decreased cell proliferation at 18 weeks. On the other hand, in the GST-P-negative area, the numbers of the TUNEL-positive cells in Group 2 and Group 3 at 5 weeks were significantly higher than in Group 1 (P < 0.01, each, Figure 5AGo). Although the TUNEL-positive cells were gradually decreased at 10 and 18 weeks, those in Group 2 and Group 3 were still higher in Group 1 at 10 and 18 weeks (P < 0.01, each). In the GST-P-positive area, there were no differences in TUNEL-positive cells among three groups at 5 and 10 weeks. However, at 18 weeks, they were significantly increased in Group 3 compared with in Group 1 (P < 0.05, Figure 5BGo). Thus, UDCA administration stimulated apoptosis of hepatocytes in DEN-induced hepatocarcinogenesis model.



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Fig. 4. Changes of the PCNA-positive hepatocytes in GST-P-negative and GST-P-positive areas. (A) The percentages of the PCNA-positive cells in GST-P-negative area were shown. The PCNA-positive cells were counted in 1000 hepatocytes in each group. {circ}: Group 1, {square}: Group 2, •: Group 3, *P < 0.05. (B) The percentages of the PCNA-positive cells in GST-P-positive area were shown at 5, 10 and 18 weeks. The PCNA-positive cells were counted in 1000 hepatocytes in each group. {circ}: Group 1, {square}: Group 2, •: Group 3, *P < 0.05.

 


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Fig. 5. Changes of apoptotic hepatocytes in GST-P-negative and GST-P-positive areas. (A) The percentages of the apoptotic cells in GST-P-negative area were shown. The apoptotic cells were counted in 1000 hepatocytes in each group. They were decreased with time. {circ}: Group 1, {square}: Group 2, •: Group 3,**P < 0.01. (B) The percentages of the apoptotic cells in GST-P-positive area were shown. Apoptotic cells were counted in 1000 hepatocytes in each group. {circ}: Group 1, {square}: Group 2, •: Group 3, *P < 0.05.

 
Effect of UDCA on Bax protein, cytochrome c release and caspase 3 activity
To clarify the mechanism of induction of apoptosis by UDCA, Bcl-2 family proteins, Bax, Bcl-Xs, Bcl-2 and Bcl-XL were examined on their expression in mitochondria fraction. Expression of Bcl-2, Bcl-Xs and Bcl-XL was not changed significantly among three groups (data not shown). Expression of Bax in Group 2 and Group 3 was higher than that in Group 1 and it was over 3-fold higher in Group 3 than that in Group 1 (Group 1 in lanes 1 and 2, Group 2 in lanes 3–5, and Group 3 in lanes 6–8, Figure 6Go). The cytochrome c oxidase, the mitochondria membrane bound protein which was used as a marker of mitochondria (31), was used to show equal amount of protein loading. To examine the amount of cytochrome c release from mitochondria to cytosol, cytochrome c in the cytosol fraction was examined. The amount of cytochrome c in Group 2 and Group 3 was greater in Group 2 and Group 3 than in Group 1. Therefore, release of cytochrome c from mitochondria to cytosol was increased. We next examined the activity of caspase 3, the most important enzyme responsible for apoptosis (34). Caspase 3 activity in Group 1 was 75 ± 14 units/mg protein, which was significantly increased to 110 ± 11 units/mg protein in Group 2, and 140 ± 21 units/mg protein in Group 3 (P < 0.05 and P < 0.01, respectively). These data suggest that an increased expression of Bax protein stimulates cytochrome c release from mitochondria to cytosol, and activates caspase 3, resulting in induction of apoptosis.



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Fig. 6. Western blot analysis of Bax and cytochrome c. Rat liver mitochondria and cytosol fractions were prepared as described in Materials and methods. The mitochondria fractions were separated and 50 µg of the resulting supernatant were subjected to SDS/PAGE immunoblot analysis by using an anti-Bax antibody. Complete pelleting of mitochondria and input of equivalent amount of mitochondria was verified by reprobing with the antibody against cytochrome c oxidase, a mitochondria membrane-bound protein (31). The cytosol fractions were also prepared and 50 µg of the supernatants were subjected to SDS/PAGE immunoblot analysis by using an anti-cytochrome c antibody, and reprobed with anti-ß-actin antibody as an internal control. Rat livers were used from Group 1 (lanes 1, 2), Group 2 (lanes 3–5) and Group 3 (lanes 6–8) at 5 weeks.

 
Effect of UDCA and CA on HCC cells in vitro
To clarify the direct effect of UDCA on cell viability of HCC cells, cell viability was assessed after incubation with various concentrations of UDCA (Figure 7Go). In Huh7 human HCC cells, cell viability was not affected up to 100 µM, however, it was greatly impaired in concentrations over 250 µM (Figure 7AGo). In Fao rat HCC cells, the similar tendency was observed in concentrations over 250 µM (Figure 7BGo). However, CA did not inhibit cell viability up to 500 µM and inhibited the cell viability at 750 µM in Huh7 cells (Figure 8AGo). In Fao cells, CA did not affect cell viability up to 500 µM, either. At 750 µM, CA reduced cell viability to ~40% (Figure 8BGo). The agarose gel electrophoresis showed that DNA fragmentation was clearly observed in Huh7 cells treated at 500 µM of UDCA for 72 h, suggesting that UDCA induced apoptosis of Huh7 HCC cells (Figure 9Go).



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Fig. 7. Effect of UDCA on cell viability. (A) Cell viability of Huh7 human HCC cells in various concentrations of UDCA was assessed after incubation for 72 h. The experiments were performed in triplicate. The data were expressed as means ± SEM. *P < 0.05, compared with 0. (B) Cell viability of Fao rat HCC cells in various concentrations of UDCA was examined. The experiments were performed in triplicate. The data were expressed as means ± SEM. *P < 0.05, compared with 0.

 


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Fig. 8. Effect of CA on cell viability. (A) Cell viability of Huh7 human HCC cells in various concentrations of CA was examined after incubation for 72 h. The experiments were performed in triplicate. The data were expressed as means ± SEM. *P < 0.05, compared with 0. (B) Cell viability of Fao rat HCC cells in various concentrations of CA was shown. The experiments were performed in triplicate. The data were expressed as means ± SEM. *P < 0.05, compared with 0.

 


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Fig. 9. Agarose gel electrophoresis of DNA. Huh7 cells were incubated with various concentrations of UDCA for 72 h. The DNA of the cells was electrophoresed on 1.5% agarose gel. At 250 µM, a small portion of the DNA seems like smear. At 500 µM of UDCA, the ladder formation was visualized.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the present study, UDCA administration reduced the number of liver tumor and the GST-P-positive hepatocytes in DEN induced liver cancer model. The number of GST-P-positive hepatocytes in the DEN groups was one third of the controls, suggesting that UDCA is actually capable of reducing tumor formation to one third. This is the first report that demonstrates that UDCA reduces liver tumor development in animal model. Our findings are very important, since the recurrence rate of HCC after treatment is so high that chemopreventive agents for HCC are currently required. Although interferon (IFN) reduces the risk of HCC in chronic hepatitis C (1235,36), the effect of IFN was less evident and severe side effects occurred more frequently in advanced liver diseases (37). Glycyrrhizin, widely used for chronic liver disease to improve liver function, has been reported to decrease the incidence of HCC in human and an animal model (13,14). In addition, acyclic retinoid was effective to prevent relapse of HCC after the first treatment (38). A Japanese herbal medicine, TJ-9, was also reported to prevent the development of HCC with cirrhosis, possibly with HCV (15). Our findings suggest that UDCA has an inhibitory effect on liver tumor and UDCA is one of a few drugs which are currently available and have a potency to inhibit malignant transformation in chronic liver disease.

UDCA, an endogenous tertiary bile acid in man, has been found to have several biologic effects, such as inhibition of cholesterol absorption in the intestine (39), and of secretion in bile (40), stabilization of cell membrane (41) and modulation of immunologic reactions (42). Recently, Earnest et al. demonstrated the chemopreventive effect of UDCA on colonic carcinogenesis in rats (17). One possible explanation for this effect is the decreased percentage of deoxycholic acid (DCA), which is cytotoxic to colonic mucosal cells, in the colonic luminal bile acids following UDCA administration (43). Decreased DCA may reduce the risk of transformation of colonic cells. The second explanation may include that UDCA induces alkaline sphingomyelinase, a physiologic inhibitor of colonic mucosal proliferation, and caspase 3, the most important enzyme responsible for apoptosis (30). The third explanation may be that UDCA suppresses group II phospholipase A2, leading to inhibition of arachidonic acid metabolism (20). Recently, UDCA and its derivative have been reported to induce apoptosis of HCC and breast cancer cells (22,23,44). Thus, UDCA and its derivative may inhibit carcinogenesis via different mechanisms in a variety of cell types.

Hepatocarcinogenesis includes multistep processes. Of many processes, the stage where the clonal expansion of initiated hepatocytes occurs is very important (24). This expansion is caused by a selective increase in cell proliferation and by a selective decrease in apoptosis of preneoplastic hepatocytes. A number of studies have now demonstrated that several promoting agents which are effective in rat hepatocarcinogenesis selectively inhibit apoptosis in preneoplastic hepatocytes, suggesting that it is important to examine effect of chemopreventive drugs in terms of apoptosis (24) The above findings led us to investigate effect of UDCA on hepatocarcinogenesis in terms of apoptosis. In the present study, UDCA inhibited proliferation of hepatocytes in the GST-P-positive and -negative areas. However, this effect does not seem to be the main cause of cancer inhibition due to the following reasons; UDCA reduced the number of the GST-P-positive hepatocytes at 10 weeks, however, anti-proliferative effect by UDCA was found at 18 weeks, but not at 5 and 10 weeks. Taken together, the anti-tumor effect of UDCA seems to be attributable to both inhibition of proliferation and acceleration of apoptosis, however, the stimulation of apoptosis especially at early stage of hepatocarcinogenesis may be an important mechanism of the anti-tumor effect of UDCA.

In the present study, Bax protein was up-regulated in the mitochondria fraction. Bcl-2 family proteins have been reported to be involved in the regulation of apoptosis during cholestasis and liver regeneration (45,46). Indeed, Bax interacts directly with the mitochondrial permeability transition (MPT) pores in isolated mitochondria to induce permeability transition and cytochrome c release (47). In the present study, cytochrome c protein was increased in cytosol fraction and caspase 3 activity was up-regulated by UDCA treatment. Therefore, it was likely that increased bax protein in mitochondria by UDCA treatment induced an increased mitochondrial permeability, cytochrome c release into cytosol, and activation of caspase 3, leading to apoptosis of `initiated hepatocytes' and finally inhibiting liver tumor. Considering the inhibitory effect on tumor development in the present study, the apoptotic cells seem to be `initiated hepatocytes'. However, induction of apoptosis was observed in the GST-P-negative cells at the early stage. Therefore, we need to clarify whether these apoptotic cells induced by UDCA are really `initiated hepatocytes' in the future.

Recently, it has been reported that UDCA is anti-apoptotic for HCC cells (48). Hence, the pro-apoptotic action of UDCA observed in the present study seems contradictory. In the report, the authors studied the effect of UDCA in the `pro-apoptotic state' where apoptosis had been already induced by DCA, ethanol, TGF-ß1, Fas ligand and okdaic acid (48). The experimental condition in the present study is supposed to be `anti-apoptotic state', since the PCNA-positive cells in GST-P-negative areas outnumbered the apoptotic cells in the same area. Therefore, the experimental conditions seem to be attributable to the difference in the results.

The anti-tumor effect by 0.1% UDCA was comparable to that by 0.3% UDCA. Similarly, 0.1% UDCA had almost the same potential of inducing apoptosis and inhibiting proliferation as 0.3% UDCA. Therefore, 0.1% UDCA may be enough to act as `tumor inhibitor'. Considering hepatic UDCA concentrations, 50 nmol/g liver of UDCA may be required to exert an anti-tumor effect. In human, the UDCA concentration in liver tissues of patients who are administered 600 mg/day of UDCA was 40.1 ± 9.0 nmol/g liver (49). Taken together, our findings suggest that higher dose of UDCA can be applied for chronic liver disease to prevent HCC in human.

In conclusion, oral administration of UDCA is effective for reducing liver tumor development in rats. This effect may be exerted by inducing apoptosis as well as inhibiting proliferation of `initiated hepatocytes'.


    Notes
 
3 To whom correspondence should be addressed Email:gshiota{at}grape.med.tottori-u.ac.jp Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received July 30, 2001; revised February 18, 2002; accepted February 20, 2002.