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Carcinogenesis Advance Access originally published online on February 23, 2006
Carcinogenesis 2006 27(8):1607-1616; doi:10.1093/carcin/bgi365
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Hepatic oval cell response to the choline-deficient, ethionine supplemented model of murine liver injury is attenuated by the administration of a cyclo-oxygenase 2 inhibitor

Richard A. Davies1,2,3, Belinda Knight1,2, Yan Wu Tian1,2, George C.T. Yeoh1,3 and John K. Olynyk1,2,4,*

1 University of Western Australia Centre for Medical Research, Western Australian Institute for Medical Research Crawley, 6009
2 School of Medicine and Pharmacology Crawley, 6009
3 School of Biomedical and Chemical Sciences, University of Western Australia Crawley, 6009
4 Department of Gastroenterology, Fremantle Hospital Fremantle, 6160, Western Australia

*To whom correspondence should be addressed. Tel: +618 9431 3774; Fax: +618 9431 2977; E-mail: jolynyk{at}cyllene.uwa.edu.au


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Oval cell proliferation precedes neoplasia in many rodent models of hepatocellular carcinoma and prevention of this proliferative response can reduce the risk of subsequent carcinoma. This study aimed to determine whether a selective cyclo-oxygenase-2 (COX-2) inhibitor, SC-236, affects (i) the oval cell response to liver injury in a mouse model of hepatocarcinogenesis and (ii) an oval cell line. Four-week-old mice were fed either normal chow or a choline deficient, ethionine supplemented (CDE) diet in the presence or absence of SC-236. Liver histology and oval cell numbers were determined after 2, 4, 12 and 52 weeks of treatment. Oval cells were scored using morphological criteria and positive immuno-staining for the M2-isozyme of pyruvate kinase (M2PK) or A6. An immortalized oval cell line (PIL-2) was used to study the in vitro effects of SC-236 on oval cell proliferation, apoptosis and Akt phosphorylation. The percentage of M2PK-positive oval cells and COX-2-positive cells was reduced by 80% and 45%, respectively, in CDE-fed mice receiving SC-236 compared with CDE-fed animals not receiving SC-236. Some M2PK-positive oval cells were also COX-2 positive. The percentage of A6-positive cells was not affected by SC-236 administration to CDE-fed mice. Administration of SC-236 increased apoptosis as evidenced by a 73% increase in the number of TUNEL-positive cells at 2 weeks in CDE-fed mice. Primary oval cells and PIL-2 cells expressed COX-2. In vitro treatment of PIL-2 cells with SC-236 resulted in a dose-dependent preferential death of A6-negative cells. Administration of 25 and 50 µM Prostaglandin E2 partially attenuated SC-236 induced cell death by 25%. In vitro oval cell death was associated with apoptosis and a 70% reduction in Akt phosphorylation. These results suggest that the SC-236 induced reduction of M2PK-positive oval cell numbers may be due to COX-2 dependent inhibition of Akt phosphorylation and induction of apoptosis.

Abbreviations: AST, aspartate transaminase; CDE diet, choline deficient ethionine supplemented diet; CDE–D, CDE diet without SC-236; CDE+D, CDE diet with SC-236; COX-(1&2), cyclo-oxygenase-(1&2); H&E, hematoxylin and eosin; HCC, hepatocellular carcinoma; IFN{gamma}, interferon {gamma}; IHC, immunohistochemistry; M2PK, M2-pyruvate kinase; PGE2, prostaglandin E2; SC2I, selective COX-2 inhibitor; TNF, tumor necrosis factor.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Hepatocellular carcinoma (HCC) is the major malignancy complicating chronic liver disease (1). Preventative approaches to therapy are limited and treatment by resection, chemotherapy or transplantation is expensive and has limited success with high recurrence rates (2–4). Thus, new therapeutic approaches for the prevention of HCC are required. Emerging evidence suggests that HCC may arise by the transformation of adult hepatic progenitor cells, called ‘oval’ cells in mice and ‘intermediate hepatobiliary’ cells in humans (5–7). Oval cells have the capacity to differentiate into hepatocytes and biliary epithelial cells during liver regeneration (8–11). Oval cells proliferate in response to liver injury in the early stages of most rodent models of hepatocarcinogenesis and are strongly associated with the presence of inflammation (12–16). They share antigenic epitopes with liver tumor cells, including M2-pyruvate kinase (M2PK) and alpha-fetoprotein (AFP) (17–19). In humans, hepatic progenitor cells proliferate following chronic liver injury due to chronic viral hepatitis, alcoholic liver disease or metabolic liver conditions, and their numbers increase in direct proportion with the disease severity (20,21). We have shown that when mice have an attenuated M2PK-positive oval cell response they have a reduced incidence of HCC following prolonged liver injury via a tumor necrosis factor (TNF) dependent mechanism (22). As TNF is a pro-inflammatory cytokine, this suggests that anti-inflammatory agents may be effective in inhibiting the proliferation of oval cells and, therefore, HCC formation.

The cyclo-oxygenase (COX) isozymes COX-1 and COX-2 mediate the conversion of arachidonic acid into a range of prostaglandins that have effects on inflammation and carcinogenesis. Whilst COX-1 plays a constitutive homeostatic role, COX-2 is induced locally during inflammation and carcinogenesis (23–25). COX-2 expression is up-regulated in subjects with chronic hepatitis, cirrhosis and HCC, and is expressed in non-parenchymal cells during experimental hepatocarcinogenesis (26–29). Investigations now show that the administration of a selective COX-2 inhibitor (SC2I) may reduce the incidence of breast, colon, pancreatic, skin and stomach tumors (30,31). Recent studies have also shown that SC2Is can reduce COX-2 expression and the development of pre-neoplastic foci, fibrosis and HCC in rats fed a choline-deficient diet (28,32–35). The mechanism of this anti-tumorigenic effect of SC2Is was not explored in these studies, however other reports have shown that COX-2 inhibitors are able to induce apoptosis in liver tumor cells (36–39). We hypothesized that SC2Is may modulate hepatocarcinogenesis by selectively reducing the hepatic oval cell response during the early stages of liver disease. Thus, the aim of this study was to determine the effect of an SC2I, SC-236, on oval cell numbers during pre-neoplastic liver injury in mice fed a carcinogenic diet, and their ability to directly modulate oval cell behavior in vitro.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Experimental design
Four-week-old C57Bl/6J mice were housed under specific pathogen-free conditions, in accordance with the guidelines of the National Health and Medical Research Council of Australia and the Animal Ethics Committee of the University of Western Australia. Mice were fed either normal chow and drinking water (control diet) or choline-deficient chow (ICN, Irvine, CA, USA) and drinking water supplemented with 0.165% ethionine (Sigma-Aldrich, St. Louis, MO, USA) (Choline-deficient ethionine supplemented diet [CDE diet]). To study the effect of SC2I administration, mice were given either SC-236 (Pfizer, New York, NY, USA; 5 mg/kg/day) or vehicle alone (0.05% Tween 20 and 0.95% polyethylene glycol 200; Sigma-Aldrich, St. Louis, MO, USA) in drinking water, according to the method previously described (40). Mice were divided into four groups and fed: (i) normal chow + SC-236, (ii) CDE diet, (iii) CDE diet + vehicle or (iv) CDE diet + SC-236 (CDE+D). At 2, 4, 12 and 52 weeks, 5–7 mice from each group were killed and their livers and serum collected. Throughout the experiments there were no significant differences between (a) CDE and (b) CDE + vehicle so these groups were combined and named CDE–D.

Tissue and serum preparation
Mice were anaesthetized and whole bloods taken via cardiac puncture for serum collection. Whole body and liver weights were recorded at the time of killing. The liver was perfused with phosphate-buffered saline (PBS) and portions of liver tissue (~5 mm3) were either snap frozen in liquid nitrogen or fixed in neutral buffered formalin for 24 h and embedded in paraffin. Serum aspartate aminotransferase (AST) activity was measured using a kit, according to the manufacturer's instructions (Sigma-Aldrich, St. Louis, MO, USA).

Histopathology and immunohistochemistry
For histology, 4-µm sections were cut from paraffin embedded liver and stained with hematoxylin and eosin (H&E). Sections from 52 week samples were stained with H&E and Masson's trichrome (41) and examined blindly by a histopathologist (Dr. Ross Glancy, Fremantle Hospital, Perth, Australia) for morphology, dysplasia and fibrosis. Five micrometer frozen sections were stained using A6 antibody (1:30, gift from Dr. V.Factor) and detected using a two-step indirect method (42). Immunohistochemistry (IHC) on formalin-fixed liver was performed using antibodies directed against M2PK (1:50, Schebo Tech, Germany), COX-2 (1:500, Santa Cruz, Hercules, CA, USA) and AFP (1:2000, ICN, Irvine, CA, USA). Before IHC, sections to be stained with M2PK or COX-2 antibodies underwent antigen retrieval by boiling tissues in EDTA buffer (1 mM, pH 8.0) or citrate buffer (10 mM, pH 6.0) for 15 min. Immunodetection was performed using a three-step indirect method (20). Oval cells were identified and scored using a combination of morphological and immunohistochemical criteria as previously described (42). The number of oval cells and COX-2-positive cells was determined by counting 10 peri-portal non-overlapping fields at 40x magnification. Cell numbers were expressed as the average percentage of total cell number per field of view. Double IHC was performed as described in Tian et al. (14), using antibodies directed against COX-2 (1:500, Santa Cruz, CA, USA), M2PK (1:200, Rockland Immunochemicals, Gilbertsville, PA, USA) or pan-cytokeratin (1:200, Dako, Carpinteria, CA, USA).

In vivo apoptosis studies
Apoptosis was detected in 4-µm paraffin embedded sections using the DeadEnd Colorimetric TUNEL System (Promega Corporation, Madison, WI, USA) according to manufacturer's protocol. The number of TUNEL-positive cells was determined by counting 10 peri-portal non-overlapping fields at 40x magnification. Oval-like cells were identified as previously described (42) using morphological criteria; care was taken to exclude small hepatocytes and endothelial cells. Apoptotic cells were expressed as the average percentage of TUNEL-positive cells relative to total cell number per field of view.

Prostaglandin E2 (PGE2) measurement
PGE2 concentration in liver tissue and culture media was determined using an immunoassay kit following the manufacturer's instructions (Cayman Chemicals, Ann Arbor, USA). For liver tissue, 50 mg of frozen tissue was homogenized in 0.5 ml of homogenization buffer (0.1 M Tris–HCl, 10 µM indomethacin) on ice. The homogenate was centrifuged for 10 min at 16 000 g and the supernatant was removed to measure the PGE2 content using a PGE2 purification kit (Cayman Chemicals, USA). Culture media (50 µl) was used to measure PGE2 concentration.

In vitro oval cell studies
A well characterized immortalized murine oval cell line (termed PIL-2), which exhibits phenotypic similarities with oval cells in vivo, was used for the in vitro work (19). PIL-2 cells are immuno-reactive for the hepatocyte markers albumin and transferrin and the oval cell markers AFP, A6 and M2PK. They are capable of differentiating into both hepatic and biliary lineages (unpublished results) and exhibit increased proliferation in response to increasing concentrations of serum (43). PIL-2 cells were maintained in culture as previously described (19). For analysis of COX-2 expression, primary oval cells were isolated from the livers of mice fed the CDE diet for 2 weeks as previously described (44). For growth assays, PIL-2 cells were seeded in 96-well microtiter plates at a medium density of 3000 cells/well. Following adherence, cells were exposed to media containing either DMSO alone (vehicle), LY-294002 (an inhibitor of phosphatidylinositol-3 kinase and Akt phosphorylation, Sigma-Aldrich, St. Louis, MO, USA) or SC-236 dissolved in DMSO at various concentrations (0.5–100 µM) for 24 h. To determine whether exogenous PGE2 could prevent the effects of SC-236, separate experiments were also conducted with the addition of PGE2 (MO Biomedicals, Kansas City, MO, USA) at various concentrations (0–50 µM). Numbers of viable cells present at the end of the experiment were determined by MTT-based assay (43). A6 immunocytochemistry was performed as previously described (19).

In vitro apoptosis study
PIL-2 cells were cultured in the presence of either vehicle alone or 25 µM of SC-236 for 6 h. Cells undergoing apoptosis were identified using Annexin V-FITC (Molecular Probes, Eugene, OR, USA) according to the manufacturer's protocol.

RNA isolation and RT-PCR
Total RNA was isolated from whole liver or cultured cells using TRIzol (Invitrogen, Mount Waverley VIC, Australia) according to the manufacturer's instructions. RNA was converted to cDNA using the Thermoscript Reverse Transcription System (Invitrogen, USA) according to the instructions of the manufacturer. PCR was performed using specific primers directed against murine COX-2 (forward primer: 5'-AAAACCGTGGGGAATGTATGAGCAC-3', reverse primer: 5'-AAACTTCGCAGGAGGGGGATGTTG-3') or ß-Actin (forward primer: 5'-CTGGCACCACACCTTCTA-3'reverse primer: 5'-GGGCACAGTGTGGGTGAC-3'). For analysis of whole liver COX-2 levels, quantitative PCR was performed in real time using a RotorGene 2000 (Corbett, Australia) incorporating SYBR green to measure the formation of double stranded amplicons. Data were normalized against a plasmid construct containing the COX-2 fragment, generated exactly as previously described (44). Quantitation data for COX-2 PCR was normalized against that of ß-Actin. PCR products were separated using agarose gel electrophoresis and stained using ethidium bromide. Resultant bands were compared with a molecular weight marker to confirm the products were of the expected size.

Western immunoblotting
Cells were washed in ice cold PBS and harvested on ice using lysis buffer (20 mM HEPES [pH 7.7], 2.5 mM MgCl2, 0.1 mM EDTA, 20 mM ß-glycerophosphate, 100 mM NaCl, 0.05% Triton X-100, 500 µM DTT, 100 µM Na3VO4, 20 µg/ml leupeptin, 100 µg/ml PMSF and 20 µg/ml aprotonin transylol). The insoluble material was excluded by centrifugation at 16 000 g at 4°C for 20 min and the resultant supernatant aspirated. Protein (25 µg) was separated on an 8% SDS-polyacrylamide gel on a Minigel apparatus (Hoefer, San Francisco, CA, USA) and transferred onto a PVDF membrane (Immobilon, Millipore, USA) with a semi-dry transfer cell (Owl Separation Systems, Portsmouth, NH, USA). After transblotting, membranes were dried and then incubated overnight at 4°C with primary antibody diluted 1:1000 for COX-2, ß-Actin (both from Santa Cruz, CA, USA), Akt, p-Akt Thr308, p-Akt Ser473 (all three from Cell Signalling Technologies, Danvers, MA, USA). Following overnight incubation, membranes were washed three times in PBS. Primary antibodies were detected using either goat anti-rabbit HRP (1:2000, Santa Cruz, CA, USA) or donkey anti-goat HRP (1:2000, Santa Cruz, CA, USA) at room temperature for 1 h, and washed with PBS three times. Bound antibodies were detected using ECL Plus (Amersham, UK) and visualized using the VersadocTM Imager (Biorad, Hercules, CA, USA). Band densities were quantified using Quantity One software, version 4.5.0 (Biorad, USA).

Statistical methods
Each in vitro experiment was performed three times and in duplicate. Data are reported as the mean ± SEM. Comparisons between groups were performed using unpaired t-test or Fisher's exact test (GraphPad Prism version 4.00 for Windows, GraphPad Software, San Diego, CA, USA). Data were deemed to be significantly different when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Early changes following administration of the CDE diet
At 2 and 4 weeks, mice fed the CDE diet exhibited mild macrovesicular steatosis and inflammatory infiltration consistent with previously published observations (42). There were no discernible differences in these parameters between CDE-fed mice treated with or without SC-236. Serum AST levels were elevated at 2 weeks in mice fed the CDE diet but were not different from control diet animals thereafter (Figure 1). Administration of SC-236 did not affect the total body or liver weights (data not shown), or serum AST levels of the CDE-fed mice (Figure 1). The hepatic PGE2 content of mice fed the control diet was 481 ± 18 pg/mg protein. Administration of the CDE diet for 2 weeks increased the PGE2 content to 863 ± 35 pg/mg protein (P < 0.05). Treatment with SC-236 did not affect hepatic PGE2 content in mice fed the CDE diet (800 ± 30 pg/mg protein).


Figure 1
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  		Fig. 1 SC-236 does not affect serum AST activity in mice fed the CDE diet. AST activity was measured at 2, 4, 12 and 52 weeks in mice fed control diet (white bars), CDE diet without SC-236 (CDE–D, dark gray bars) or CDE diet with SC-236 (CDE+D, black bars). Results are expressed as group mean ± SEM.

 
SC-236 administration decreases CDE-mediated pathologies
Mice fed the CDE diet for 52 weeks exhibited marked hepatocyte nuclear atypia and dysplasia compared with age-matched mice receiving a normal diet (Figure 2A and B). Mice fed the CDE diet developed AFP-positive foci (Figure 2C). Some also developed large white nodular lesions on their livers (Figure 2D) which stained positively for A6. Sixty percent (8 of 14) of CDE-fed mice exhibited peri-cellular fibrosis (Figure 2E) while none of the CDE-fed mice also receiving SC-236 had fibrosis (Figure 2F, P < 0.05). Overall SC-236 administration reduces the incidence of a range of pathologies which develop during long-term CDE diet administration (Table I).


Figure 2
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  		Fig. 2 Liver pathologies in mice fed the CDE diet after 52 weeks. H&E stained liver sections show hepatocyte nuclear atypia (broad arrow) and typia (small arrow) (A) and atypical hepatocyte foci (B). An AFP-positive focus in a CDE–D liver (C). Gross morphology of an A6-positive nodule in a CDE–D animal (D). Masson's trichrome staining illustrates mild peri-cellular fibrosis (white arrows) in CDE–D animals (E) and no fibrosis with administration of SC-236 (F). Each black bar represents 100 µm and the white bar represents 1 cm.

 

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  		Table I SC-236 reduces the incidence of pathologies induced in mice fed the CDE diet for 12 months

 
SC-236 reduces the number of M2PK-positive oval cells
The CDE diet induced the appearance of M2PK (Figure 3A and B) and A6-positive oval cells (Figure 3C and D). SC-236 administration significantly reduced the number of CDE-induced M2PK-positive oval cells by ~60% at 2 weeks (P < 0.01), 70% at 4 weeks (P < 0.01), 80% at 12 weeks and 50% at 52 weeks (P < 0.01) (Figure 3E). In contrast, the administration of SC-236 did not affect the numbers or distribution of A6-positive cells in mice fed the CDE diet (Figure 3F).


Figure 3
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  		Fig. 3 SC-236 reduces M2PK-positive oval cells and does not affect A6-positive cells in mice fed the CDE diet. IHC illustrates M2PK (A, B) or A6 (C, D) -positive oval cells (arrows) in CDE–D (A, C) and CDE+D (B, D) mice fed the CDE for 2 weeks. Each bar represents 100 µm. Quantitation of M2PK-positive oval cells (E) and A6-positive cells (F) demonstrates a significant reduction in the numbers of M2PK-positive cells in animals receiving SC-236. Data expressed as mean ± SEM. **P < 0.01.

 
SC-236 reduces the number of COX-2-positive cells
Administration of the CDE diet induced a 44-fold increase in hepatic COX-2 expression (Figure 4A). SC-236 administration to animals fed the CDE diet significantly reduced the number of COX-2-positive cells by 45% at 2 weeks (P < 0.05), 20% at 4 weeks (P < 0.05) and 30% at 52 weeks (P < 0.05) (Figure 4B). COX-2 IHC shows that Kupffer, sinusoidal and endothelial cells were immuno-reactive. Hepatocytes and cholangiocytes were not immuno-reactive for COX-2. Double IHC showed COX-2-positive cells also positive for M2PK, but not for cytokeratin (an alternative marker to A6 for staining biliary oval cells) (Figure 4C and D).


Figure 4
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  		Fig. 4 SC-236 reduces COX-2-positive cells in mice fed the CDE diet. The CDE diet increased hepatic COX-2 mRNA levels (A) and the number of COX-2-positive cells in CDE livers at each time point (B). The number of COX-2-positive cells was reduced by administration of SC-236. Double IHC for COX-2 and either M2PK (C) or cytokeratin (D) shows cells positive for COX-2 (brown stain; long arrows) in CDE-D mice after 2 weeks; some oval-shaped COX-2-positive cells were found to co-express M2PK (purple-black stain; thick arrows; C). Cells single-positive for either M2PK (blue stain; arrowhead; C) or cytokeratin (blue stain; arrowhead; D) were also observed. *P < 0.05 and **P < 0.01.

 
SC-236 increases the number of TUNEL-positive cells
Only hepatocytes, sinusoidal and oval-like cells were TUNEL-positive. Administration of the CDE diet increased the number of hepatic TUNEL-positive cells from 0.33% in control mice to 0.62% at 2 weeks (Figure 5, P < 0.05). Treatment of animals fed the CDE diet with SC-236 (CDE+D) caused a further 2-fold increase in the number of TUNEL-positive cells at 2 weeks (P < 0.05). SC-236 increased the percentage of TUNEL-positive oval-like cells from 0.1% in CDE–D to 0.4% in CDE+D mice at 2 weeks (Figure 5, P > 0.05).


Figure 5
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  		Fig. 5 SC-236 administration increases TUNEL-positive oval cells in mice fed the CDE diet for 2 weeks. Quantitation of all TUNEL-positive cells and TUNEL-positive oval-like cells in 2 week CDE animals. The number of TUNEL-positive cells increased following commencement of the CDE diet and was further increased by administration of SC-236. Data expressed as mean ± SEM, *P < 0.05 and **P < 0.01.

 
SC-236 induces cell death by apoptosis in an oval cell line
Primary and immortalized oval cell cultures expressed COX-2 mRNA (Figure 6A) and protein (Figure 6B). Following SC-236 treatment, cell viability decreased in a dose-dependent manner, with an estimated LD50 of 1 µM (Figure 6C). Administration of 25 µM SC-236 to PIL-2 cells resulted in a 60% reduction in PGE2 levels (P < 0.05). The addition of 25 or 50 µM PGE2 to PIL-2 cultures treated with SC-236 attenuated cell death by ~25% (P < 0.05). PIL-2 cultures exhibited heterogeneous A6 immuno-reactivity with ~10% of cells being A6-negative (Figure 7A and D). Following administration of SC-236, there was a preferential depletion of A6-negative cells, whereby almost all cells remaining following SC-236 treatment were A6-positive (Figure 7B and C). The proportion of A6-negative cells remaining following 24 h treatment was 1.5% and 0.8% when treated with 25 and 50 µM SC-236, respectively (Figure 7D, P < 0.001).


Figure 6
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  		Fig. 6 The mouse oval cell line PIL-2 expresses COX-2 mRNA and protein and undergoes dose-dependent cell death following exposure to SC-236. COX-2 transcript (A) was detected in PIL-2 cultures (lane 1) and primary oval cell cultures (lane 2). COX-2 protein was detected in PIL-2 cultures (B). Following SC-236 treatment, PIL-2 cell viability decreased in a dose-dependent manner, with an estimated LD50 of 1 µM (C).

 

Figure 7
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  		Fig. 7 SC-236 preferentially induces death of A6-negative PIL-2 cells. PIL-2 cells treated with vehicle (A), 25 µM (B) or 50 µM (C) of SC-236 were stained for A6. Small arrowheads indicate cells that are A6-negative. Scale bar represents 100 µm. Quantitation of A6-positive and A6-negative cells in PIL-2 cultures treated with SC-236 (D). Numbers of A6-positive cells were not reduced in the presence of SC-236 whilst A6-negative cells demonstrated markedly reduced in the presence of SC-236. Data expressed as group mean ± SEM for A6-positive and A6-negative PIL-2 cells. **P < 0.01; ***P < 0.001.

 
SC-236 induces apoptosis and reduces Akt phosphorylation in an oval cell line
PIL-2 cells became rounded or began to shrink or detach from the culture dish after SC-236 treatment (data not shown). SC-236 induced apoptosis of PIL-2 cells, as determined by Annexin V staining (Figure 8A–D). Phosphorylation of Akt in PIL-2 cells was decreased following treatment with SC-236 (Figure 8E). Levels of both phosphorylated forms of Akt (Thr308 and Ser473) were reduced by ~70% following treatment. Inhibition of phosphatidylinositol-3 kinase (an upstream inducer of Akt) using LY-294002 completely prevented Akt phosphorylation in PIL-2 cells (not shown).


Figure 8
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  		Fig. 8 PIL-2 cells undergo apoptosis and reduced Akt phosphorylation following SC-236 treatment. Annexin V staining shows that SC-236 treated cells (A) exhibit significantly more apoptosis than untreated PIL-2 cells (B). Phase-contrast photographs of the same fields indicate similar density of cell cultures (C, D). SC-236 reduced the amount of the phosphorylated forms of Akt; Thr308 and Ser473 in PIL-2 cells (E). ß-Actin was detected concurrently to ensure consistent sample quality and concentration for each RT-PCR and western blot.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
We tested the effect of a SC2I, SC-236, on (1) mice fed a CDE diet, for up to 52 weeks and (2) a well characterized oval cell line, PIL-2. SC-236 reduced a variety of CDE-induced pathologies such as atypical hepatocytes, AFP-positive foci and peri-cellular fibrosis. Administration of SC-236 in conjunction with the CDE diet resulted in a 60–80% reduction in the number of M2PK-positive oval cells but did not affect the number of A6-positive cells at each of the experimental time points. Some M2PK-positive oval cells co-expressed COX-2 while cytokeratin-positive cells did not. Primary oval cells and PIL-2 cells expressed COX-2 mRNA and protein. PIL-2 cultures underwent dose-dependent cell death following treatment with SC-236. PIL-2 cells that were immuno-negative for A6 were more sensitive to SC-236-induced toxicity than A6-positive cells. The induction of apoptosis was accompanied by a reduction in Akt phosphorylation. These results demonstrate that the administration of an SC2I attenuates the oval cell response induced by a CDE diet and its associated pathologies and suggest that this may occur through direct effects of SC-236 on oval cell apoptosis, via a pathway mediated by Akt activity.

There have been several studies reporting a reduction in the grade and incidence of fibrosis, pre-neoplastic foci and hepatocarcinogenesis following administration of various COX-2 inhibitors in rats fed a choline-deficient diet (28,32–35). However, these studies did not investigate the specific effects of an SC2I on oval cells, even though it is well established that oval cell proliferation is a key feature of liver injury in rodents fed a choline-deficient diet (45–47). Our study extends existing data by demonstrating that SC-236 reduces the number of oval cells and the severity of pre-neoplastic liver pathologies in mice fed the CDE diet. Interestingly, this attenuation in oval cell numbers occurred despite a down regulation in the number of hepatic COX-2 expressing cells following SC-236 treatment, suggesting that the effects may be mediated in part via COX-2 independent pathway(s). In accordance, we did not observe a reduction by SC-236 in hepatic PGE2 levels in CDE-fed animals. Analysis of in vivo programmed cell death by TUNEL staining showed significantly increased numbers of both total and oval-like cells undergoing apoptosis in SC-236 administered animals, suggesting that the attenuation in oval cell numbers may be mediated by pro-apoptotic effects of SC-236 on oval and other hepatic cell types.

Our findings are consistent with a previous work demonstrating that a reduction in the number of oval cells is associated with a reduced incidence of hepatocarcinogenesis in mice fed the CDE diet (22). In the context of human chronic liver disease, these results are of considerable relevance, since the risk of HCC is paralleled by changes in the number of hepatic progenitor cells (20). Whether hepatic progenitor cells directly give rise to HCC remains unresolved, however, they are at least closely associated with diverse liver diseases and pathologies that generally occur in association with HCC (48,49) including small cell dysplasia (50), parenchymal inflammation (15) and fibrosis (20,21). Therefore, hepatic progenitor cells represent a population that reflects the extent of liver damage and the likelihood for hepatocarcinogenesis. The administration of pharmaceutical agents such as SC2I may ultimately prove useful in the reduction of HCC risk associated with liver progenitor cell proliferation.

We recently established a transformed murine oval cell line (PIL-2) that exhibits morphologic and functional characteristics consistent with oval cells in vitro and in vivo (19). The current study demonstrated that PIL-2 cells express COX-2 mRNA and protein and upon SC-236 administration undergo a decrease in cell growth and induction of apoptosis, consistent with our in vivo observations. The partial protection provided by the addition of exogenous PGE2 to PIL-2 cells cultured in the presence of SC-236 again suggests that SC-236 exerts its effects through mechanisms both dependent and independent of COX-2 activity. Although the pathways by which SC2Is induce apoptosis are poorly understood, the Bcl-2 family and Akt have been implicated as mediators of these events in liver tumor cells (39,51–54). Our results show that a SC2I inhibits Akt phosphorylation in a mouse oval cell line, suggesting that similar pathways are invoked following SC2I treatment in mouse and human transformed liver cell lines. Thus, administration of an SC2I may reduce the risk of hepatocarcinogenesis by directly affecting the survival of oval cells, possibly via an Akt-dependent mechanism.

Hepatic oval cells are a heterogeneous population by virtue of their adaptive ability to differentiate into hepatocytes or cholangiocytes. During oval cell-mediated regeneration, the characteristics of the oval cell compartment will change, according to the relative demand for hepatocytes versus cholangiocytes. The exact relationship between the oval cell markers M2PK and A6 remains unresolved, however, previous work suggests they may mark distinct sub-populations of cells, possibly differing in regard to their lineage selectivity. Tian et al. showed that M2PK-positive oval cells co-express mature hepatocytic markers with some small hepatocytes being M2PK-positive during the early stages of the CDE diet (14). Conversely, oval cells expressing A6 are spatially associated with expanding bile ducts during oval cell proliferation in several mouse models (42,55). Our in vivo results suggest that SC-236 affects different oval cell sub-populations, as evidenced by a reduction in the numbers of M2PK-positive oval cells but not A6-positive oval cells. In accordance, our in vitro data also suggests that A6-positive and A6-negative PIL-2 sub-populations have different sensitivities to SC-236 administration. Double-staining of tissue sections showed some oval-shaped cells co-expressing COX-2 and M2PK, but none co-positive for COX-2 and cytokeratin, a marker shown recently to stain cells also positive for A6 (56). This suggests that the selective reduction in M2PK-positive oval cells in SC-236 treated liver may be related to differential COX-2 expression between the M2PK and A6/cytokeratin-positive oval cell subsets. The relevance of these different oval cell sub-populations to liver regeneration and hepatocarcinogenesis is unknown and clearly requires further evaluation.

In summary, our data confirm the inhibitory effects of SC2Is on hepatocarcinogenesis and suggest that this may be mediated by induction of oval cell apoptosis during the early stages of disease progression. We suggest that pharmaceutical agents with properties exhibited by SC2Is may be useful as preventative treatment strategies for HCC in patients with chronic liver disease.


   Acknowledgments
 
The authors would like to thank Dr. Ross Glancy for his assistance with the liver pathology. This study was supported by grants from Pfizer Inc (USA), the Cancer Council of Western Australia and The National Health and Medical Research Council of Australia. Richard Davies is supported by a scholarship from the Gastroenterological Society of Australia. Dr. Belinda Knight is the recipient of a Richard Walter Gibbon fellowship of the University of Western Australia.

Conflict of Interest Statement. None declared.


   References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received June 19, 2005; revised November 9, 2005; accepted February 11, 2006.


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