Increased expression of cyclooxygenase-2 protein during rat hepatocarcinogenesis caused by a choline-deficient, L-amino acid-defined diet and chemopreventive efficacy of a specific inhibitor, nimesulide

Ayumi Denda,3, Wakashi Kitayama, Akiko Murata1,2, Hideki Kishida, Yasutaka Sasaki, Osamu Kusuoka, Toshifumi Tsujiuchi, Masahiro Tsutsumi, Dai Nakae, Hidetoshi Takagi1 and Yoichi Konishi

Department of Oncological Pathology, Cancer Center, Nara Medical Univesity, 840 Shijo-cho, Kashihara, Nara 634-8521 and
1 Wyeth Lederle Japan Ltd, 1-6-34 Kashiwa-cho, Shiki, Saitama 353-8511, Japan

Abstract

Expression of cyclooxygenase (COX)-2 protein during rat hepatocarcinogenesis associated with fatty change, fibrosis, cirrhosis and oxidative DNA damage, caused by a choline-deficient, L-amino acid-defined (CDAA) diet were investigated in F344 male rats, along with the chemopreventive efficacy of the specific COX-2 inhibitor, nimesulide (NIM). Nimesulide, which was administered in the diet at concentrations of 200, 400, 600 and 800 p.p.m. for 12 weeks, decreased the number and size of preneoplastic enzyme-altered liver foci, levels of oxidative DNA damage, and the grade and incidence of fibrosis in a dose-dependent manner. A preliminary long-term study of 65 weeks also revealed that 800 p.p.m. NIM decreased the multiplicity of neoplastic nodules and hepatocellular carcinomas and prevented the development of cirrhosis. Western blot analysis revealed that COX-2 protein was barely expressed in control livers and increased ~2.9-fold in the livers of rats fed on a CDAA diet for 12 weeks and ~4.5–5.4-fold in tumors, with a diameter larger than 5 mm, at 80 weeks. Immunohistochemically, COX-2 protein was positive in sinusoidal and stromal cells in fibrotic septa, which were identified by immunoelectron microscopy as Kupffer cells, macrophages, either activated Ito cells or fibroblasts, after exposure to the CDAA diet for 12 weeks, whereas it was only occasionally weakly positive in sinusoidal, probably Kupffer, cells in control livers. In neoplastic nodules in rats fed on a CDAA diet for 30 and 80 weeks, sinusoidal cells and cells with relatively large round nuclei and scanty cytoplasm were strongly positive for COX-2 protein, with the neoplastic hepatocytes in the minority of the nodules, but not the cancer cells, being moderately positive. These results clearly indicate that rat hepatocarcinogenesis, along with fatty change, fibrosis and cirrhosis, is associated with increased expression of COX-2 protein, and point to the chemopreventive efficacy of a selective COX-2 inhibitor against, at least, the early stages of hepatocarcinogenesis.

Abbreviations: CDAA, choline-deficient, L-amino acid-defined; CDML, choline-deficient, methionine-low; COX, cyclooxygenase; CSAA, choline-supplemented, L-amino acid-defined; DNase, deoxyribonuclease; GST-P, glutathione S-transferase placental form; HCC, hepatocellular carcinoma; HE, hematoxylin and eosin; HGF, hepatocyte growth factor; 8-OHdG, 8-hydroxydeoxyguanosine; LPS, lipopolysaccharide; MEM, minimum essential medium; NIM, nimesulide; NSAID, non-steroidal anti-inflammatory drug; PG, prostaglandin; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; TGF, transforming growth factor

Introduction

Prolonged feeding of a choline-deficient, methionine-low (CDML) diet, a lipotrope-deficient diet with deficiencies in choline, methionine, folic acid or vitamin B12 (all of which are involved in the generation of labile methyl groups) is known to cause hepatocellular carcinomas, associated with fatty change, hepatocyte injury, fibrosis, cirrhosis and generation of oxidative DNA damage via 8-hydroxydeoxyguanosine (8-OHdG) in rodents (14). We have found that a choline-deficient, L-amino acid-defined (CDAA) diet, which is a L-amino acid-defined and completely choline-devoid CDML diet, possesses a greater capacity to cause these lesions than semisynthetic diets (57). This provides us with a useful experimental model for hepatocarcinogenesis that is caused by endogenous factors, and which has similarities in its histopathological sequence to human hepatocellular carcinoma development with cirrhosis (8). So far, repeating cycles of hepatocyte injury and regeneration (9), inhibition of apoptosis (10), oxidative stress (7,1113), hypomethylation of DNA and RNA, including the 5'-upstream region of the c-myc gene (14,15), and chronic activation of protein kinase C (16,17) have all been postulated to be involved. Major roles for gene mutations in Ki-ras, p53, p16, p21 and ß-catenin, however, have not been found (1820).

Recently, we established the preventive potential of non-steroidal anti-inflammatory drugs (NSAID)s including aspirin and piroxicam, basically inhibitors of cyclooxygenases (COX)s, against the development of lesions, other than fatty change, caused by a CDAA diet in rats (2124). The two COX isozymes COX-1 and COX-2, both rate-limiting enzymes in the production of prostanoids, prostaglandins (PGs), thromboxanes and prostacyclins from arachidonic acid, have only ~60% homology, but their active site residues are almost entirely preserved. In contrast to COX-1, a constitutively expressed housekeeping gene contributing to normal physiological functions in the majority of tissues, COX-2 is an inducible immediate early gene which has recently been postulated to be involved not only in inflammation but also in carcinogenesis, impacting on cell proliferation, differentiation, apoptosis, angiogenesis, metastasis and immunological surveillance (2528). In fact, the hypothesis that COX-2 could be a chemopreventive target molecule is supported by evidence of up-regulated expression of COX-2 mRNA and protein in various human and animal tumors, such as colon, stomach, breast, skin, pancreas, lung, esophagus, head and neck and urinary bladder cancers, and the prevention of carcinogenesis by specific COX-2 inhibitors (25–37 and references therein), as well as by double knockout of the COX-2 gene in APC gene knockout mice (27).

It has been postulated that prostanoids play a role in the physiological function of the liver, including maintenance of the microcirculation, glucose metabolism, bile flow and lipoprotein secretion, especially with PGE2 and PGF2{alpha} involvement in liver regeneration, although the responsible COX isozymes and their producing cells remain largely unknown (3846). Recently, up-regulated expression of COX-2 mRNA and protein has been reported in the liver and liver cells associated with ethanol-induced liver injury and hyperosmolarity, and by treatments with tumor promoters (4751). Moreover, elevated levels of COX-2 have been described in human hepatocellular carcinomas (HCCs) as well as under conditions of chronic hepatitis and liver cirrhosis (52,53). Nevertheless, information on the involvement of COX-2 in hepatocarcinogenesis is limited. In the present study, expression of COX-2 protein during rat hepatocarcinogenesis caused by a CDAA diet, as well as the chemopreventative potential of specific COX-2 inhibitor nimesulide (NIM), were therefore investigated.

Materials and methods

Chemicals
Nimesulide (NIM) was obtained from Helsinn Healthcare SA (Pambio Noranco, Switzerland).

Anti-COX-2 antibodies, consisting of a mouse monoclonal antibody against rat COX-2 (C-terminal protein fragment corresponding to amino acids 368–604), were obtained from Transduction Laboratories (No. C22420; Lexington, KY), and rabbit and goat polyclonal antibodies against synthetic peptides corresponding to C-terminal sequences of murine and human COX-2 were obtained from Oxford Biomedical Research (No. PG26; Oxford MI) and Santa Cruz Biotechnology (No. sc-1747; Santa Cruz, CA), respectively. Anti-COX-1 antibody, a rabbit polyclonal antibody against rat COX-1 (C448 synthetic peptide), was obtained from IBL (No. 18521; Gunma, Japan). Rabbit polyclonal antibody against rat GST-P was purchased from Medical and Biological Laboratories (Nagoya, Japan) and 8-OHdG was purchased from Wako Pure Chemical Industries (Osaka, Japan). Collagenase (Type I), trypsin inhibitor (Type II) and deoxyribonuclease (DNase) I (DN-25) were purchased from Sigma (St Louis MO), and proteinase E was purchased from Merck (Darmstadt, Germany). Joklik-minimum essential medium (MEM) and Hank's solution were purchased from Gibco-BRL (Grand Island, NY).

Animals and diets
Fischer 344 male rats were obtained from Japan SLC (Hamamatsu, Japan) and were 8 weeks of age, weighing 180–200 g, at the commencement of all experiments. CDAA diet (No. 518753) and the control choline-supplemented, L-amino acid-defined (CSAA) diet (No. 518754) were obtained from Dyets (Bethlehem, PA). Details of the composition and the lipotrope contents of the CDAA and CSAA diets have been previously described (22). CDAA and CSAA diets supplemented with 200, 400, 600 and 800 p.p.m. NIM were stored at 4°C in the dark, and given to the animals by freshly replenishing the feed trays twice a week. Diet was available ad libitum and body weights and food consumption were measured weekly.

Experimental protocol for the chemopreventive potential of NIM
Animals were divided into seven groups consisting of 9–10 rats each except 20, 15 and 12 rats for groups 1, 5 and 6, respectively. Group 1 was fed the CDAA diet alone and groups 2, 3, 4 and 5 were given the same diet supplemented with 200, 400, 600 and 800 p.p.m. NIM, respectively. Groups 6 and 7 received the CSAA diet alone and the same diet supplemented with 800 p.p.m. NIM, respectively. All animals were continuously fed the respective diets and killed at 12 weeks, except that 10, five and four rats from groups 1, 5 and 6, respectively, were killed at 65 weeks after the commencement of the experiment. For the 12 week point analysis, the livers, single tissue slices from each lobe, were fixed in either acetone under dehydration as previously described (22), or 10% buffered neutral formalin for 24 h, routinely processed for paraffin embedding, sectioned and subjected to immunohistochemical and histological analyses for glutathione S-transferase placental form (GST-P)-positive foci which are preneoplastic liver lesions, expression of COX-2 and COX-1 proteins and fibrosis. Portions of the livers were frozen in liquid nitrogen and subjected to biochemical analysis for 8-OHdG, a marker of oxidative DNA damage, and western blot analysis for the expression of COX-2 and COX-1 proteins. For the 65 week point analysis, the entire livers were step-sliced at 3 mm thickness and every other slice was fixed and processed for paraffin embedding as mentioned above, submitting to the histological diagnosis for neoplastic nodules and HCCs as previously described (22).

Analysis of GST-P-positive foci and fibrosis
Three serial liver sections were made from each acetone-fixed paraffin block and subjected to GST-P, hematoxylin and eosin (HE) and azan-Mallory staining. GST-P protein expression was demonstrated immunohistochemically and the number and area of GST-P-positive foci were analyzed using a color image processor SPICCA II (Olympus Optical, Tokyo, Japan) as previously described (22). Fibrosis was assessed by the degree of azan-Mallory staining.

Measurement of 8-OHdG levels
From samples of frozen liver tissue, DNA was extracted using a DNA Extractor WB kit (Wako Pure Chemical Industries), denatured, and hydrolyzed into nucleosides as described previously (22). 8-OHdG was determined, basically according to the method of Kasai, using a HPLC system connected to an electrochemical detector Coulochem II (ESA, Bedford, MA) eluting with 10 mM NaH2PO4 solution containing 5% methanol, as detailed previously (22).

Specimens used for further western blot, immunohistochemical and immunoelectron microscopic analyses
Forty-five animals were given either a CDAA or CSAA diet and killed after 12, 20, 30 or 80 weeks (5–10 rats each for the CDAA diet and 3–5 for the CSAA diet). Liver tissue and various sizes of excised nodules and isolated hepatocytes and nonparenchymal cells from three rats each were subjected to western blotting. The livers were also subjected to immunohistochemical analysis for COX-2 and COX-1 proteins, and immunoelectron microscopic analysis for COX-2 protein.

Separation of hepatocytes and nonparenchymal cells
Hepatocytes and nonparenchymal cells were isolated basically according to the methods of Yaswen et al. (54) and Sirica and Cihla (55). In brief, the livers, after intravenous injection with 100 U heparin, were perfused through the portal vein with O2-saturated Ca2+-free Hank's solution containing 20 mM HEPES (pH 7.4) for 10 min followed by O2-saturated 0.1% collagenase, 0.01% trypsin inhibitor in 67 mM NaCl, 6.7 mM KCl, 4.8 mM CaCl2, 100.7 mM HEPES (pH 7.6) for 15 min. The excised livers, after mincing only for the CDAA-treated livers, were gently rotated in 0.1% collagenase, 0.004% DNase I in 20 mM HEPES, Joklik-MEM (pH 7.4) at room temperature for 10–30 min. Hepatocytes were obtained from the filtrate through a 253 µm nylon mesh by centrifugation at 50 g for 1.5 min. The debris left was minced, incubated in 0.1% collagenase, 0.1% pronase E, 0.006% DNase I in 20 mM HEPES, Joklik-MEM (pH 7.4) at 37°C for 20 min, filtered through a 253 µm nylon mesh and centrifuged at 500 g for 10 min. Nonparenchymal cells were obtained from the sediment after removal of the hepatocytes at 50 g for 1.5 min and filtering through a 60 µm nylon mesh in 0.006% DNase I, 5% calf serum in 20 mM HEPES, Joklik-MEM (pH 7.4), by centrifugation at 500 g for 10 min. Hepatocytes and nonparenchymal cells were also isolated from nodules which were excised after perfusing with collagenase.

Western blot analysis of COX-2 and COX-1 proteins
Particulate fractions were obtained from the frozen liver samples basically according to the method of Liu and Rose, (56) as previously described (32,34, 35) and subjected to western blotting. Protein concentrations were determined using Coomassie brilliant blue G-250 solution (Nacalai Tsque, Kyoto, Japan). Protein (180 µg) was mixed with sample buffer, boiled for 5 min, electrophoresed and electrophoretically transferred to polyvinylidene difluoride membranes (No. IPVH000 10; Millipore, Bedford, MA). The membranes were blocked with 5% non-fat dried milk in 0.05 M Tris-buffered saline (pH 7.6) (TBS) containing 0.1% Tween-20 and incubated with primary antibodies to COX-2 and COX-1 for 1 h at a dilution of 1:1000 (No. C22420), 1:200 (No. sc-1747) and 1:50 (No. 18521), respectively. Secondary horseradish peroxidase-linked sheep anti-mouse, donkey anti-rabbit (Amersham Pharmacia Biotech, Piscataway, NJ) and donkey anti-goat (Santa Cruz Biotechnology) IgG antibodies were then employed and the membranes were analyzed using an enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia Biotech). The antibody to COX-2 (No. PG26), according to the method of Fukuda et al. (57), was pre-incubated with the secondary antibody Envision+ (K4003; Dako, Carpinteria, CA) in TBS, 0.1% Tween 20, 1% non-fat dried milk for 1 h, followed by normal rabbit serum for 1 h at room temperature with gentle rotation, then applied to the blocked membrane at a dilution of 1:600, followed by analysis by ECL. Mouse macrophage lysate, prepared from the RAW 264.7 cell line activated by interferon-{gamma} and lipopolysaccharide (LPS) (Transduction Laboratories), and COX-2 and COX-1 proteins from sheep placenta and ram seminal vesicles (Nos 360120 and 360100; Cayman, Ann Arbor, MI), respectively, were used as positive controls.

Immunohistochemical analysis for expression of COX-2 and COX-1 protein
Sections were made from formalin-fixed paraffin blocks and subjected to immunohistochemical analyses for COX-2 and COX-1 proteins as previously described (32,3435). In brief, antigens were retrieved for COX-2 analysis by treating with 0.05% protease XXVII (Sigma) in TBS at 37°C for 5 min, and for COX-1 analysis by microwaving for 50 min in 0.01 M citrate buffer (pH 6.0). Sections were blocked with 0.3% H2O2 in methanol for 45 min and incubated with a primary antibody (No. C22420, 1:100 dilution for COX-2; No. 18521, 1:60 dilution for COX-1) for 2 h. Immunoreactivity was detected using a Dako LSAB 2 kit for use with rat specimens (K609; Dako) for COX-2, and a Vectastain Elite ABC kit (PK-6101; Vector Laboratories, Burlingame, CA) for COX-1, and 3,3'-diaminobenzidine hydrochloride (Sigma) followed by counterstaining with Mayer's hematoxylin. Non-immune serum, mouse IgG1 (Dako, Japan, Kyoto) or rabbit IgG (Dako), as well as antibodies preabsorbed with the antigens were used as controls for the primary antibody binding. In the preabsorption experiment, COX-2 antibody was preabsorbed by incubating with 3-fold higher molar ratios of COX-2 and COX-1 proteins from sheep placenta and ram seminal vesicles (Nos 360120 and 360100; Cayman), respectively, and COX-1 antibody with 4-fold higher molar ratios of the antigen peptide (No. 18522; IBL) at 4°C overnight.

Immunoelectron microscopic analysis for expression of COX-2 proteins
The livers were fixed in periodate–lysine–paraformaldehyde (PLP) for 4 h, and after washing in 0.01 M phosphate buffered saline (pH 7.2) (PBS) and 10, 15, 20, 25% sucrose in PBS for 4 h to overnight, were frozen in OCT compound (Tissue-Tek; Miles, Elkhart, IN). The frozen sections at 6 µm thickness, after being dried and treated with acetone for 1–2 s, were rinsed with PBS for 15 min, blocked with 10% fetal calf serum for 10 min and treated with a primary antibody to COX-2 (No. C22420, 1:40 dilution), followed by secondary horseradish peroxidase-linked goat anti-mouse Fab fragment (1:75 dilution; MBL, Nagoya, Japan) at 4°C overnight. After post-fixing with 1% glutaraldehyde in PBS for 5 min, immunoreactivity was detected by reacting with 3,3'-diaminobenzidine hydrochloride followed by 1% osmium tetroxide for 2 h, and the sections were processed for embedding in Epon 812 (Oken, Tokyo).

Statistical analysis
Quantitative differences between group values were statistically analyzed using ANOVA (analysis of variance) with multiple comparison post-hoc testing by Dunnett, Student's t-test, {chi}2 or Fisher's exact tests.

Results

Chemopreventive potential of NIM
No rats died in any of the groups during the 12 week experimental period. As is evident from the data summarized in Table IGo, NIM did not significantly affect final body weights or food intake during the experimental period. However, liver weight of rats given 600 or 800 p.p.m. NIM fed on either the CDAA or CSAA diet were significantly increased in terms of both absolute values and ratio to body weight as compared with CDAA or CSAA diet alone groups, and their increasing rates 1.14 to 1.17 were similar between CDAA and CSAA diets. However, no explaination for the increments were found on histological analysis.


View this table:
[in this window]
[in a new window]
 
Table I. Body and liver weights and intake data for rats given the CDAA or CSAA diet containing nimesulide for 12 weeksa
 
The livers of rats given the CDAA diet alone for 12 weeks macroscopically exhibited fibrotic features with small and brownish nodules (Figure 1AGo), while those given the diet supplemented with 600 or 800 p.p.m. NIM were much less fibrotic and nodulous, particularly with 800 p.p.m. NIM where surfaces were smooth (Figure 1BGo). As summarized in Table IIGo, NIM exerted an inhibitory effect on the development of GST-P-positive foci with foci number/cm2 liver, percent liver area occupied, average size and fractions of >1 mm2 in size all being decreased in a dose-dependent manner as compared with the CDAA alone group values. No GST-P-positive foci were observed in the livers of rats given CSAA diet alone or the CSAA plus 800 p.p.m. NIM diet. In accordance with the macroscopic findings, NIM also exerted an inhibitory effect on the development of fibrosis, decreasing the grade and incidence of fibrosis in a dose-dependent manner (Table IIGo, Figure 1C and DGo).



View larger version (103K):
[in this window]
[in a new window]
 
Fig. 1. Representative macroscopic appearance (A, B, E and F) and findings of azan-Mallory staining (magnifications: C and D, x6.6; G, x5.0; H, x6.6) for livers of rats given a CDAA diet alone for 12 (A and C) and 65 (E and G) weeks, or the CDAA plus 800 p.p.m. NIM diet for 12 (B and D) and 65 (F and H) weeks. Histological findings of well (I) and moderately (J) differentiated hepatocellular carcinomas developed in rat livers given a CDAA diet for 65 weeks (hematoxylin and eosin staining, x20)

 

View this table:
[in this window]
[in a new window]
 
Table II. Effects of nimesulide on the induction of GST-P-positive foci in the liver of rats fed a CDAA diet for 12 weeksa
 
Results for the preliminary long-term studies of 65 weeks are summarized in Table IIIGo. One rat each from groups 1, 5 and 6 died of pneumonia. Nimesulide at a dose of 800 p.p.m. did not significantly affect food intake during the experimental period (10.2 ± 0.9, 11.2 ± 1.1 and 10.9 ± 1.8 g/day/rat in groups 1, 5 and 6, respectively) or final body weight, but tended to decrease (which was not significant) the liver weight in terms of both absolute weight and ratio to body weight, reflecting a decrease in the number of liver tumors compared with the control CDAA diet alone group. The livers of rats given the CDAA diet alone for 65 weeks macroscopically exhibited cirrhotic features with several tumorous nodules (Figure 1EGo), while those given the CDAA plus 800 p.p.m. NIM diet, exhibited a smooth surface with a single tumorous nodule (Figure 1FGo). Nimesulide at a dose of 800 p.p.m. inhibited the development of neoplastic nodules and HCCs caused by a CDAA diet, significantly decreasing the multiplicity despite the small numbers of animals, but not the incidence of nodules and HCCs compared with the CDAA diet alone group. Nevertheless, these results were clearly not conclusive because of the small number of animals involved. The majority of HCCs were well differentiated (Figure 1IGo) but 1/2 (50%) and 2/17 HCCs (11.8%) in the CDAA plus 800 p.p.m. NIM diet and CDAA diet alone groups, respectively, were moderately differentiated (Figure 1JGo) with lung metastasis. As is evident from Figure 1E, F, G and H,Go NIM further decreased the grade of fibrosis and inhibited the development of cirrhosis caused by a CDAA diet.


View this table:
[in this window]
[in a new window]
 
Table III. Effects of nimesulide on the development of neoplastic nodules and hepatocellular carcinomas in the liver of rats fed a CDAA diet for 65 weeksa
 
Levels of 8-OHdG in liver DNA
Nimesulide at 200 p.p.m. or above significantly decreased the 8-OHdG levels in liver DNA caused by the CDAA diet feeding for 12 weeks (Table IVGo). NIM at a dose of 800 p.p.m. also exhibited a significant decrease in 8-OHdG levels of rat livers given the control CSAA diet.


View this table:
[in this window]
[in a new window]
 
Table IV. Effects of nimesulide on the generation of 8-OHdG in the liver of rats fed a CDAA diet for 12 weeksa
 
Western blot analysis for COX-2 and COX-1 proteins
Representative western blots for COX-2 and COX-1 proteins and quantified density of each band using NIH imaging are shown in Figure 2A and BGo. The density of each band from different groups exhibited a reproducibility, although there was no internal control as no appropriate control protein for the particulate fractions could be found. COX-2 protein was slightly expressed while COX-1 protein was substantially expressed in the liver of rats given the control CSAA diet for 12 weeks. Feeding a CDAA diet for 12 weeks caused a significant increase in the expression of COX-2 protein in the liver, to a level ~2.9-fold that of the CSAA diet alone, along with a ~1.6-fold significant increase in that of COX-1 protein. Nimesulide, particularly at a dose of 800 p.p.m., significantly inhibited the up-regulated expression of COX-2, but not COX-1, caused by a CDAA diet.



View larger version (56K):
[in this window]
[in a new window]
 
Fig. 2. (A) A representative western blot for expression of COX-2 (top) and COX-1 (bottom) proteins in the liver of rats given, from left to right, the CSAA diet plus 800 p.p.m. NIM (two rats), a CSAA diet alone (three rats), a CDAA diet alone (three rats), the CDAA diet plus 800 p.p.m. NIM (three rats) and the CDAA diet plus 600 p.p.m. NIM (two rats) for 12 weeks. Note the COX-2 antibody (No. C22420), positive control proteins for COX-2 (prepared from the RAW 264.7 mouse macrophage cell line stimulated with interferon-{gamma} and lipopolysaccharide) and COX-1 (derived from sheep seminal vesicles). Similar blots were obtained for other COX-2 antibodies (Nos sc-1747 and PG26). (B) Quantified densities of the bands in (A) expressed as ratios to the density of the CSAA diet alone group. Significant difference between groups for *, COX-2 expression and #, COX-1 expression.

 
In the liver of rats given a CDAA diet for 30 and 80 weeks (Figure 3A and BGo), approximately 3.1- and 3.8-fold increases in COX-2 protein were observed, respectively, compared with the CSAA diet alone group. Tumors >5 mm in diameter exhibited ~4.5–5.4-fold increases. Expression of COX-1 protein was also elevated ~2.5- and 3.0-fold, and tumors >10 mm in diameter exhibited ~4.2–5.0-fold increases (Figure 3A and BGo).



View larger version (67K):
[in this window]
[in a new window]
 
Fig. 3. (A) A representative western blot showing the expression of COX-2 (top) and COX-1 (bottom) proteins in the liver of rats given, from left to right, the CSAA diet alone for 12 (one rat) or 30 (one rat) weeks, the CDAA diet alone for 12 (two rats), 30 (three rats) or 80 (two rats) weeks, as well as pooled liver tumors of 2 and 5 mm and individual tumors of 10 mm, #1 and #2, in diameter developing with the CDAA diet for 80 weeks. Note the COX-2 antibody (No. PG26), positive control proteins for COX-2 (derived from sheep placenta) and COX-1 (derived from ram seminal vesicles). Similar blots were obtained for other COX-2 antibodies (Nos C22420 and sc-1747). (B) Quantified densities of the bands in (A) expressed as ratios to the average density of the CSAA diet alone group.

 
Immunohistochemical analysis of COX-2 and COX-1 proteins
In the liver of rats given the CSAA diet for 12 weeks, COX-2 protein was occasionally and weakly to moderately positive in sinusoidal cells of the periportal area (Figure 4AGo), whereas after the rats had received the CDAA diet for 12 weeks, it was moderately to strongly positive in the sinusoidal and stromal cells of the fibrotic septa (Figure 4BGo). In the liver of rats given the CDAA diet containing 800 p.p.m. NIM for 12 weeks, in accordance with the reduced expression of the protein in western blot analysis (Figure 2A and BGo) and the prevention of fibrosis, only sinusoidal cells were stained positive for COX-2 protein (Figure 4CGo). In all cases, the nuclear membrane and cytoplasm were positive. Non-immune serum gave negative staining (Figure 4DGo). Preabsorption of the COX-2 antibody with COX-2 but not COX-1 proteins reduced or abrogated staining (data not shown). COX-1 protein was moderately positive in sinusoidal cells, especially in the periportal to midzones, and weakly in endothelial and bile duct cells in the CSAA diet alone group (Figure 4EGo), whereas in the CDAA diet alone group, it was moderately to strongly positive in sinusoidal cells and occasionally moderately positive in stromal cells in fibrotic septa (Figure 4FGo). Non-immune serum gave negative staining, and preabsorption of the COX-1 antibody with the antigen peptide abrogated staining (data not shown).



View larger version (121K):
[in this window]
[in a new window]
 
Fig. 4. Immunohistochemical findings for COX-2 (A, B, C, G, I and K) and COX-1 (E, F, M and N) proteins, those stained with non-immune mouse IgG1 (D), and histological findings (hematoxylin and eosin staining, H, J and L) in livers of rats given a CSAA diet for 12 weeks (A and E), a CDAA diet for 12 (B, D and F), 30 (G, H and M) or 80 (I, J, K, L and N) weeks, and the CDAA plus 800 p.p.m. NIM diet for 12 weeks (C). Magnification: A, E and M, x25; G–H, K–L and N, x33; B–D and F, x40; I–J, x50.

 
In some neoplastic nodules developing in the liver of rats given a CDAA diet for 30 weeks, and in the majority at 80 weeks, COX-2 protein was strongly positive in sinusoidal cells and cells with relatively large round nuclei and scanty cytoplasm (Figure 4G and HGo). Small numbers of neoplastic hepatocytes with weakly COX-2-positive cytoplasm were occasionally scattered, especially at 80 weeks. In a minority of neoplastic nodules at 80 weeks, which totalled 37 (4.11 ± 2.26 per rat) out of nine rats examined, composed either of relatively small eosinophilic cells with occasional fatty change (18 nodules, 48.6%) (Figure 4I and JGo), or middle to large-sized eosinophilic cells (13 nodules, 35.1%), cytoplasm of 20–100% of the neoplastic hepatocytes was also stained moderately positive for COX-2 protein. Moreover, in all of the cancer lesions present in the nodules as shown in Figure 4K and LGo, which totalled six in nine rats examined (incidence 4/9, 44.4%, multiplicity 0.67 ± 0.87), well-differentiated hepatocellular carcinoma cells were negative while the surrounding relatively small eosinophilic neoplastic hepatocytes with occasional fatty change were moderately positive for COX-2 protein. On the other hand, COX-1 protein, in majority of nodules at 30 and 80 weeks, was diffusely and moderately to strongly positive in sinusoidal cells and cells with relatively large round nuclei and scanty cytoplasm but not neoplastic hepatocytes (Figure 4MGo). Hepatocellular carcinomas, in accordance with their reduction or absence of Kupffer cells, were negative for COX-1 (Figure 4NGo).

Immunoelectron microscopic analysis of COX-2 protein expression
Immunoelectron microscopically, nuclear membranes and rough endoplasmic reticulum of Kupffer cells in sinusoid (Figure 5AGo), and in macrophages (Figure 5BGo) and either activated Ito cells (stellate cells or fat-storing cells) (Figure 5CGo) or fibroblasts in the fibrotic septa were positive for COX-2 protein in the liver of rats given the CDAA diet for 12 weeks, whereas positive cells were hardly observed with the CSAA diet.



View larger version (117K):
[in this window]
[in a new window]
 
Fig. 5. Immunoelectron microscopic findings for COX-2 protein expression in a sinusoid (A) and fibrotic septa (B and C) of livers after the CDAA diet for 12 weeks. Note positive staining in nuclear membranes and rough endoplasmic reticulum of Kupffer cells (A), macrophages (B) and activated Ito cells (C). K, Kupffer cell; M, macrophage; I, Ito cell; H, hepatocytes; E, endothelial cell; R, rough endoplasmic reticulum; Y, lysosomes; L, lipid droplet. Magnification: A, x11 400; B, x9300; C, x12 900. Microscopic findings for isolated hepatocytes (D, F and H) and nonparenchymal cells (E and G) from livers of rats given the CSAA diet for 12 weeks (D and E) and the CDAA diet for 12 (F and G) and 20 (H) weeks. Papanicolaou staining, x60 (D, E and G) and x120 (F and H).

 
Western blot analysis of COX-2 and COX-1 proteins in isolated liver cells
Localization of COX-2 and COX-1 proteins was further examined by western blotting in isolated hepatocyte and nonparenchymal cell fractions from livers and pooled nodules of rats given a CDAA diet for 12 and 20 weeks. A representative western blot is shown in Figure 6A and BGo. In accordance with the immunohistochemical results, COX-2 protein was hardly expressed in hepatocytes but substantially in nonparenchymal cell fractions with a CSAA diet for 12 weeks, and increased approximately 3.7-, 4.4- and 4.9-fold in nonparenchymal cell fractions from the livers of rats given a CDAA diet for 12 and 20 weeks and from nodules at 20 weeks, respectively. The purity of these nonparenchymal cell fractions as well as that of parenchymal ones from the CSAA diet group were >98% (Figure 5D, E and GGo). However, discrepant results with hepatocyte fractions (respectively, approximately 5.8-, 8.4- and 4.0-fold increases), might have been ascribable to contamination with approximately 30, 35 and 15% of nonparenchymal cells (Figure 5F and HGo) because of rupture of the fat storing hepatocytes at severer conditions in which the nonparenchymal cells could be separated. On the other hand, in accordance with the immunohistochemical results, COX-1 protein was found to be expressed scarcely in hepatocytes but strongly in nonparenchymal cell fractions from the CSAA diet alone group, with increases of ~1.2–1.5-fold in the nonparenchymal cell fractions, and ~9.1-, 7.7- and 9.8-fold in the hepatocyte fractions (although still very low densities), from the livers of rats given the CDAA diet for 12 and 20 weeks and from nodules at 20 weeks, respectively.



View larger version (66K):
[in this window]
[in a new window]
 
Fig. 6. (A) A representative western blot showing the expression of COX-2 (top) and COX-1 (bottom) proteins in isolated hepatocyte and nonparenchymal cell fractions, from left to right, from livers of rats given the CSAA diet for 12 weeks, and the CDAA diet for 12 and 20 weeks, as well as from pooled liver nodules with the CDAA diet for 20 weeks. Note COX-2 antibody (No. PG26) and positive control proteins in legend of Figure 3Go. Similar blots were obtained for other COX-2 antibodies (Nos C22420 and sc-1747). (B) Quantified densities of the bands in (A) expressed as ratios to the densities of hepatocyte fractions from the CSAA diet alone group.

 
Discussion

The present study demonstrated an increase in expression of COX-2 protein with the CDAA diet in nonparenchymal, sinusoidal and stromal cells in fibrotic septa, immunoelectron microscopically identified as Kupffer cells, macrophages, activated Ito cells or fibroblasts, in the early stage, and neoplastic hepatocytes but not cancer cells in the later stage. In addition, clear preventive effects of a specific COX-2 inhibitor, NIM, on the development of preneoplastic lesions, fibrosis and induction of oxidative DNA damage, were evident. This preliminary long-term study also suggested prevention by NIM of the development of cirrhosis, neoplastic nodules and HCCs, which might partly be ascribable to the prevention of the earlier lesions. Nimesulide, a sulfonanilide class COX-2 inhibitor which can bind to the large catalytic moiety of COX-2 but not COX-1, preferentially inhibits sheep placenta COX-2 activity in vitro, with an IC50 of 0.07 µM (as compared with >100 or 300 µM for ram seminal vesicle COX-1) and appears to possess much less adverse effects on the gastrointestinal tract than non-specific NSAIDs (26,58,59). The average daily intake at 800 p.p.m. NIM, 35.5 mg/kg body weight, is ~10 times the maximum tolerated dose in man of 200 mg/person/day (58). The present results are the first, to the authors' knowledge, to provide direct evidence of an involvement of COX-2 at least in the early stage of hepatocarcinogenesis associated with fatty change, fibrosis and cirrhosis. Nevertheless, the biological significance of the present absence of COX-2 expression in cancer cells, which is inconsistent with its increases in most cancer cells of humans and rodents (2537), remains to be elucidated. Moreover, present inconsistent dose–response effects of NIM on the formation of preneoplastic lesions and 8-OHdG, a possible carcinogenic cause in this model, would be worth further studying.

In contrast to the present results in rats, in humans, hepatocytes have been demonstrated immunohistochemically to be the major site of COX-2 protein production, even in normal liver (although with faint to weak stainability), as well as in chronic hepatitis and cirrhosis, although the reported positive staining in dysplasia and well but not poorly differentiated hepatocellular carcinomas is in line with the present results (52,53). These discrepancies might be due partly to differences in the characteristics of the antibodies used, or in the causative mechanisms of liver disease, involving different cytokine and growth factor networks. For the present immunohistochemistry, anti-COX-2 and -COX-1 antibodies that gave a single band on western blotting were used, and in accordance with the immunohistochemical findings, western blots of separated liver cell fractions revealed COX-2 protein to be present primarily in nonparenchymal cells from the livers of rats given the CSAA diet. Whether the relatively strong expression of COX-2 protein in the hepatocyte fractions after the CDAA diet was administered for 12 and 20 weeks, could be ascribed to contaminating nonparenchymal cells, especially those expressing COX-2 in fibrotic septa, is unclear and requires further study. It also might be possible that diffusely and weakly expressed COX-2 protein in hepatocytes was unable to be immunohistochemically detected since the detection sensitivity of antibodies between western blotting and immunohistochemistry could be different. In this context, it is worth noting that nonparenchymal cells, especially Kupffer cells, have been postulated to be the major producers of prostanoids, affecting hepatocytes in a paracrine fashion (38). In fact, primary adult hepatocytes express no COX-2 protein (60), and Kupffer cells isolated from partial hepatectomized rats, overproduce PGE2 in response to small amounts of LPS (45). Further, COX-2 is up-regulated in Kupffer cells but not hepatocytes in the livers associated with ethanol-induced rat hepatocyte injury with fibrosis (47) as well as after administration of the liver tumor promoters phenobarbital and lindane (49). Moreover, the ability of cultured primary fetal hepatocytes to express COX-2 in response to LPS and pro-inflammatory cytokines such as tumor necrosis factor-{alpha} and interleukin-1ß1, and their combination, is abrogated in adult hepatocytes from a few days after birth (60), and only immortalized liver cells can express COX-2 in response to various tumor promoters, including peroxisome proliferators (50). The present finding of increased expression of COX-2 in relatively small eosinophilic neoplastic hepatocytes, suggests an altered regulation in early neoplasia. However, proliferation of cultured primary hepatocytes on stimulation with hepatocyte growth factor (HGF) and epidermal growth factor is accompanied by the production of PGE2 and PGF2{alpha}, with NSAIDs inhibiting the cell proliferation (44,61), so that further studies are warranted.

The significance of COX-2 for the development of pre- and neoplastic lesions and fibrosis and the production of reactive oxygen species largely remains to be elucidated. Mitogenic and anti-apoptotic activities of PGs acting in a paracrine fashion on altered hepatocytes (although autocrine roles can not be excluded) (38,4346,6264), might be important for preneoplastic lesion development. Autocrine antiapoptotic activity of COX-2, through induction of Bcl-2 or Bcl-XL and inhibition of caspase-3 activity (38,63,64), might be of advantage to the COX-2-positive neoplastic nodules, while cancer cells acquiring independence to COX-2. Reactive oxygen species produced through either COX-2 activity per se (65), or activation of enzymes such as NADP(H) oxidase by PGs (66), could cause oxidative DNA damage and together with the resultant lipid peroxidation products 4-hydroxynonenal or transforming growth factor (TGF)-ß1, stimulate Ito cells to produce extracellular matrix (67,68). TGF-ß1 and HGF, which have been reported to be overexpressed in rat livers given a CDML diet (69), are known in themselves to regulate COX-2 gene expression (64,70). Further, COX-2 and H2O2 are reportedly involved in expression of the procollagen {alpha}2 (I) gene (71). Paradoxically, in general, PGs, PGE2, PGI2 and HGF are cytoprotective in the animal and human liver injury by chemicals and viruses (38, 64), and are antifibrogenic, especially with PGEs inhibiting Ito cells from proliferating and producing collagen I, and up-regulating collagenase gene expression (72,73). Moreover, COX-2 has been postulated to be involved in the antiproliferative effects of endothelin-1 and tumor necrosis factor-{alpha} on Ito cells (74).

It should be noted that the present results show an increase in the expression of the COX-1 protein, a constitutively expressed housekeeping gene, with the CDAA diet. The anti-rat COX-1 antibody used in the present study reacted with the authentic sheep COX-1 but not COX-2 protein, and earlier exhibited no significant elevation in rat urinary bladder tumors (35) but a slight elevation in rat tongue tumors (34). Increased expression of COX-1 along with COX-2 has also been reported in mouse lung tumors (75). Reportedly, COX-1 can be up-regulated during differentiation of a human monocytic cell line by phorbol ester, a murine macrophage cell line by activin A or murine immature mast cells by c-kit (7678), and in endothelial cells co-cultured with cancer cells (79), although the regulating mechanisms are largely unknown. Moreover, immortalized endothelial cells which were forced to express COX-1 become tumorigenic (80). Thus, our present results, although there is a necessity for them to be confirmed by the use of a suitable internal control, appear to be sound, and the significance of the elevated COX-1 for the hepatocarcinogenesis, as well as the enhancing mechanisms, require further study.

In conclusion, in spite of the well-established cytoprotective and antifibrogenic roles of PGs, the present results, using a rat hepatocarcinogenesis model, indicate that hepatocarcinogenesis with fatty change, fibrosis and cirrhosis is associated with an increase in the expression of COX-2, and that a selective COX-2 inhibitor has preventive potential at least against the early stages of tumor development and fibrosis, suggesting a new approach for the prevention of liver neoplasia.

Notes

2 Present address: Department of Customer Service, Ventana Japan K.K., 2-4-1 Shibakoen, Minato-ku, Tokyo 105-0011, Japan Back

3 To whom correspondence should be addressed Email: adenda{at}nmu-gw.cc.naramed-u.ac.jp Back

Acknowledgments

This work was supported in part by a Grant-in-Aid (09253104) for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture of Japan, Grants-in-Aid (10-36 and S10-1) for Cancer Research from the Ministry of Health, Labor and Welfare of Japan, and the 2nd-Term Comprehensive 10-Year Strategy for Cancer Control, Cancer Prevention from the Ministry of Health, Labor and Welfare of Japan.

References

  1. Shinozuka,H. and Katyal,S.L. (1985) Pathology of choline deficiency. In Sidransky,H. (ed.) Nutritional Pathology. Marcel Dekker, New York, pp. 279–320.
  2. Newberne,P.M. (1986) Lipotropic factors and oncogenesis. Adv. Exp. Med. Biol., 206, 223–251.[Medline]
  3. Ghoshal,A.K. and Farber,E. (1993) Choline-deficiency and the development of liver disease including liver cancer: a new perspective. Lab. Invest., 68, 255–260.[ISI][Medline]
  4. Zeisel,S.H. (1990) Choline deficiency. J. Nutr. Biochem., 1, 332–349.[ISI]
  5. Mikol,Y.B., Hoover,K.L., Creasia,D. and Poirier,L.A. (1983) Hepatocarcinogenesis in rats fed methyl-deficient, amino acid-defined diets. Carcinogenesis, 4, 1619–1629.[ISI][Medline]
  6. Nakae,D., Yoshiji,H., Mizumoto,Y., Horiguchi,K., Shiraiwa,K., Tamura,K., Denda,A. and Konishi,Y. (1992) High incidence of hepatocellular carcinomas induced by a choline-deficient, L-amino acid-defined diet in rats. Cancer Res., 52, 5042–5045.[Abstract]
  7. Nakae,D., Yoshiji,H., Kinugasa,T., Denda,A. and Konishi,Y. (1990) Production of both 8-hydroxydeoxyguanosine in liver DNA and {gamma}-glutamyltransferase-positive hepatocellular lesions in rats given a choline-deficient, L-amino acid-defined diet. Jpn. J. Cancer Res., 81, 1081–1085.[ISI][Medline]
  8. Flier,J.S. and Underhill,L.H. (1993) The cellular basis of hepatic fibrosis. Mechanisms and treatment strategies. New Engl. J. Med., 328, 1828–1835.[Free Full Text]
  9. Chander,N., Amenta,J., Kandala,J.C. and Lombardi,B. (1987) Liver cell turnover in rats fed a choline-devoid diet. Carcinogenesis, 8, 669–673.[Abstract]
  10. Zeisel,S.H., Albright,C.D., Shin,O.H., Mar,M.H., Salganik,R.I. and da Costa,K.A. (1997) Choline deficiency selects for resistance to p53-independent apoptosis and causes tumorigenic transformation of rat hepatocytes. Carcinogenesis, 18, 731–738.[Abstract]
  11. Rushmore,T.H., Ghazarian,D.M., Subrahmanyan,V., Farber,E. and Ghoshal,A.K. (1987) Probable free radical effects on rat liver nuclei during early hepatocarcinogenesis with a choline-devoid low-methionine diet. Cancer Res., 47, 6731–6740.[Abstract]
  12. Hinrichsen,L.I., Floyd,R.A. and Sudilovsky,O. (1990) Is 8-hydroxydeoxyguanosine a mediator of carcinogenesis by a choline-devoid diet in the rat liver? Carcinogenesis, 11, 1879–1881.[Abstract]
  13. Nakae,D., Kotake,Y., Kishida,H. et al. (1998) Inhibition by phenyl N-tert-butyl nitrone of early phase carcinogenesis in the livers of rats fed a choline-deficient, L-amino acid-defined diet. Cancer Res., 58, 4548–4551.[Abstract]
  14. Christman,J.K., Sheikhnejad,G., Dizik,M., Abileah,S. and Wainfan,E. (1993) Reversibility of changes in nucleic acid methylation and gene expression induced in rat liver by severe dietary methyl deficiency. Carcinogenesis, 14, 551–557.[Abstract]
  15. Tsujiuchi,T., Tsutsumi,M., Sasaki,Y., Takahama,M. and Konishi,Y. (1999) Hypomethylation of CpG sites and c-myc gene overexpression in hepatocellular carcinomas, but not hyperplastic nodules, induced by a choline-deficient, L-amino acid-defined diet in rats. Jpn. J. Cancer Res., 90, 909–913.[ISI][Medline]
  16. da Costa,K.A., Cochary,E.F., Blusztajn,J.K., Garner,S.C. and Zeisel,S.H. (1993) Accumulation of 1,2-sn-diradylglycerol with increased membrane-associated protein kinase C may be the mechanism for spontaneous hepatocarcinogenesis in choline-deficient rats. J. Biol. Chem., 268, 2100–2105.[Abstract/Free Full Text]
  17. da Costa,K.A., Garner,S.C., Chang,J. and Zeisel,S.H. (1995) Effects of prolonged (1 year) choline deficiency and subsequent re-feeding of choline on 1,2-sn-diradylglycerol, fatty acids and protein kinase C in rat liver. Carcinogenesis, 16, 327–334.[Abstract]
  18. Tsujiuchi,T., Kido,A., Nakae,D., Takahama,M., Majima,T., Kobitsu,K., Okajima,E., Tsutsumi,M., Denda,A. and Konishi,Y. (1996) Infrequent Ki-ras and absence of p53 mutations in hepatocellular carcinomas induced by a choline deficient L-amino acid defined diet in rats. Cancer Lett., 108, 137–141.[ISI][Medline]
  19. Tsujiuchi,T., Tsutsumi,M., Sasaki,Y., Takahama,M. and Konishi,Y. (1999) Different frequencies and patterns of ß-catenin mutations in hepatocellular carcinomas induced by N-nitrosodiethylamine and a choline-deficient L-amino acid-defined diet in rats. Cancer Res., 59, 3904–3907.[Abstract/Free Full Text]
  20. Sasaki,Y., Tsujiuchi,T., Murata,N., Kubozoe,T., Tsutsumi,M. and Konishi,Y. (2000) Absence of p16, p21 and p53 gene alterations in hepatocellular carcinomas induced by N-nitrosodiethylamine or a choline-deficient L-amino acid-defined diet in rats. Cancer Lett., 152, 71–77.[ISI][Medline]
  21. Denda,A., Tang,Q., Endoh,T., Tsujiuchi,T., Horiguchi,K., Noguchi,O., Mizumoto,Y., Nakae,D. and Konishi,Y. (1994) Prevention by acetylsalicylic acid of liver cirrhosis and carcinogenesis as well as generation of 8-hydroxydeoxyguanosine and thiobarbituric acid-reactive substances caused by a choline-deficient, L-amino acid-defined diet in rats. Carcinogenesis, 15, 1279–1283.[Abstract]
  22. Endoh,T., Tang,Q., Denda,A. et al. (1996) Inhibition by acetylsalicylic acid, a cyclo-oxygenase inhibitor, and p-bromophenacylbromide, a phospholipase A2 inhibitor, of both cirrhosis and enzyme-altered nodules caused by a choline-deficient, L-amino acid-defined diet in rats. Carcinogenesis, 17, 467–475.[Abstract]
  23. Denda,A., Endoh,T., Kitayama,W. et al. (1997) Inhibition by piroxicam of oxidative DNA damage, liver cirrhosis and development of enzyme-altered nodules caused by a choline-deficient, L-amino acid-defined diet in rats. Carcinogenesis, 18, 1921–1930.[Abstract]
  24. Denda,A., Endoh,T., Tang,Q., Tsujiuchi,T., Nakae,D. and Konishi,Y. (1998) Prevention by inhibitors of arachidonic acid cascade of liver carcinogenesis, cirrhosis and oxidative DNA damage caused by a choline-deficient, L-amino acid-defined diet in rats. Mutat. Res., 402, 279–288.[ISI][Medline]
  25. Pairet,M. and Engelhardt,G. (1996) Distinct isoforms (COX-1 and COX-2) of cyclooxygenase: possible physiological and therapeutic implications. Fundam. Clin. Pharmacol., 10, 1–15.[ISI][Medline]
  26. Taketo,M.M. (1998) Cyclooxygenase-2 inhibitors in tumorigenesis (Part I). J. Natl Cancer Inst., 90, 1529–1536.[Abstract/Free Full Text]
  27. Taketo,M.M. (1998) Cyclooxygenase-2 inhibitors in tumorigenesis (Part II). J. Natl Cancer Inst., 90, 1609–1620.[Abstract/Free Full Text]
  28. Williams,C.S., Mann,M. and DuBois,R.N. (1999) The role of cyclooxygenases in inflammation, cancer, and development. Oncogene, 18, 7908–7916.[ISI][Medline]
  29. Fukutake,M., Nakatsugi,S., Isoi,T. et al. (1998) Suppressive effects of nimesulide, a selective inhibitor of cyclooxygenase-2, on azoxymethane-induced colon carcinogenesis in mice. Carcinogenesis, 19, 1939–1942.[Abstract]
  30. Müller-Decker,K., Kopp-Schneider,A., Marks,F., Seibert,K. and Fürstenberger,G. (1998) Localization of prostaglandin H synthase isozymes in murine epidermal tumors: suppression of skin tumor promotion by inhibition of prostaglandin H synthase-2. Mol. Carcinog., 23, 36–44.[ISI][Medline]
  31. Zimmermann,K.C., Sarbia,M., Weber,A.A., Borchard,F., Gabbert,H.E. and Schrör,K. (1999) Cyclooxygenase-2 expression in human esophageal carcinoma. Cancer Res., 59, 198–204.[Abstract/Free Full Text]
  32. Kitayama,W., Denda,A., Yoshida,J., Sasaki,Y., Takahama,M., Murakawa,K., Tsujiuchi,T., Tsutsumi,M. and Konishi,Y. (2000) Increased expression of cyclooxygenase-2 protein in rat lung tumors induced by N-nitrosobis(2-hydroxypropyl)amine. Cancer Lett., 148, 145–152.[ISI][Medline]
  33. Chan,G., Boyle,J.O., Yang,E.K. et al. (1999) Cyclooxygenase-2 expression is up-regulated in squamous cell carcinoma of the head and neck. Cancer Res., 59, 991–994.[Abstract/Free Full Text]
  34. Shiotani,H., Denda,A., Yamamoto,K., Kitayama,W., Endoh,T., Sasaki,Y., Tsutsumi,M., Sugimura,M. and Konishi,Y. (2001) Increased expression of cyclooxygenase-2 protein in 4-nitroquinoline-1-oxide-induced rat tongue carcinomas and chemopreventive efficacy of a specific inhibitor, nimesulide. Cancer Res., 61, 1451–1456.[Abstract/Free Full Text]
  35. Kitayama,W., Denda,A., Okajima,E., Tsujiuchi,T. and Konishi,Y. (1999) Increased expression of cyclooxygenase-2 protein in rat urinary bladder tumors induced by N-butyl-N-(4-hydroxybutyl)nitrosamine. Carcinogenesis, 20, 2305–2310.[Abstract/Free Full Text]
  36. Mohammed,S.I., Knapp,D.W., Bostwick,D.G., Foster,R.S., Khan,K.N.M., Masferrer,J.L., Woerner,B.M., Snyder,P.W. and Koki,A.T. (1999) Expression of cyclooxygenase-2 (COX-2) in human invasive transitional cell carcinoma (TCC) of the urinary bladder. Cancer Res., 59, 5647–5650.[Abstract/Free Full Text]
  37. Okajima,E., Denda,A., Ozono,S., Takahama,M., Akai,H., Sasaki,Y., Kitayama,W., Wakabayashi,K. and Konishi,Y. (1998) Chemopreventive effects of nimesulide, a selective cyclooxygenase-2 inhibitor, on the development of rat urinary bladder carcinomas initiated by N-butyl-N-(4-hydroxybutyl)nitrosamine. Cancer Res., 58, 3028–3031.[Abstract]
  38. Peltekian,K.M., Makowka,L., Williams,R., Blendis,L.M., Levy,G.A. and the Prostaglandins in Liver Transplantation Research Group. (1996) Prostaglandins in liver failure and transplantation: regeneration, immunomodulation, and cytoprotection. Liver Transplant Surg., 2, 171–184.[Medline]
  39. G'mez-Foix,A.M., Rodríguez-Gil,J.E., Guinovart,J.J. and Bosch,F. (1989) Prostaglandins E2 and F2{alpha} affect glycogen synthase and phosphorylase in isolated hepatocytes. Biochem. J., 261, 93–97.[ISI][Medline]
  40. G'mez-Foix,A.M., Rodríguez-Gil,J.E., Guinovart,J.J. and Bosch,F. (1991) Prostaglandins E2 and F2{alpha} increase fructose 2,6-bisphosphate levels in isolated hepatocytes. Biochem. J., 274, 309–312.[ISI][Medline]
  41. Beckh,K., Kneip,S. and Arnold,R. (1994) Direct regulation of bile secretion by prostaglandins in perfused rat liver. Hepatology, 19, 1208–1213.[ISI][Medline]
  42. Björnsson,Ó.G., Sparks,J.D., Sparks,C.E. and Gibbons,G.F. (1992) Prostaglandins suppress VLDL secretion in primary rat hepatocyte cultures: relationships to hepatic calcium metabolism. J. Lipid Res., 33, 1017–1027.[Abstract]
  43. Miura,Y. and Fukui,N. (1979) Prostaglandins as possible triggers for liver regeneration after partial hepatectomy. A review. Cell Mol. Biol., 25, 179–184.[ISI][Medline]
  44. Skouteris,G.G., Ord,M.G. and Stocken,L.A. (1988) Regulation of the proliferation of primary rat hepatocytes by eicosanoids. J. Cell. Physiol., 135, 516–520.[ISI][Medline]
  45. Callery,M.P., Mangino,M.J. and Flye,W. (1991) Kupffer cell prostaglandin-E2 production is amplified during hepatic regeneration. Hepatology, 14, 368–372.[ISI][Medline]
  46. Hashimoto,N., Watanabe,T., Ikeda,Y., Yamada,H., Taniguchi,S., Mitsui,H. and Kurokawa,K. (1997) Prostaglandins induce proliferation of rat hepatocytes through a prostaglandin E2 receptor EP3 subtype. Am. J. Physiol., 272, G597–G604.[Abstract/Free Full Text]
  47. Nanji,A.A., Miao,L., Thomas,P., Rahemtulla,A., Khwaja,S., Zhao,S., Peters,D., Tahan,S.R. and Dannenberg,A.J. (1997) Enhanced cyclooxygenase-2 gene expression in alcoholic liver disease in the rat. Gastroenterology, 112, 943–951.[ISI][Medline]
  48. Zhang,F., Warskulat,U., Wettstein,M., Schreiber,R., Henninger,H.P., Decker,K. and Häussinger,D. (1995) Hyperosmolarity stimulates prostaglandin synthesis and cyclooxygenase-2 expression in activated rat liver macrophages. Biochem. J., 312, 135–143.[ISI][Medline]
  49. Kroll,B., Kunz,S., Klein,T. and Schwarz,L.R. (1999) Effects of lindane and phenobarbital on cyclooxygenase-2 expression and prostanoid synthesis by Kupffer cells. Carcinogenesis, 20, 1411–1416.[Abstract/Free Full Text]
  50. Ledwith,B.J., Pauley,C.J., Wagner,L.K., Rokos,C.L., Alberts,D.W. and Manam,S. (1997) Induction of cyclooxygenase-2 expression by peroxisome proliferators and non-tetradecanoylphorbol 12,13-myristate-type tumor promoters in immortalized mouse liver cells. J. Biol. Chem., 272, 3707–3714.[Abstract/Free Full Text]
  51. Leung,L.K. and Glauert,H.P. (1998) Effect of the peroxisome proliferator ciprofibrate on hepatic cyclooxygenase and phospholipase A2 in rats. Toxicology, 126, 65–73.[ISI][Medline]
  52. Koga,H., Sakisaka,S., Ohishi,M. et al. (1999) Expression of cyclooxygenase-2 in human hepatocellular carcinoma: relevance to tumor dedifferentiation. Hepatology, 29, 688–696.[ISI][Medline]
  53. Kondo,M., Yamamoto,H., Nagano,H. et al. (1999) Increased expression of COX-2 in nontumor liver tissue is associated with shorter disease-free survival in patients with hepatocellular carcinoma. Clin. Cancer Res., 5, 4005–4012.[Abstract/Free Full Text]
  54. Yaswen,P., Hayner,N.T. and Fausto,N. (1984) Isolation of oval cells by centrifugal elutriation and comparison with other cell types purified from normal and neoplastic livers. Cancer Res., 44, 324–331.[Abstract]
  55. Sirica,A.E. and Cihla,H.P. (1984) Isolation and partial characterizations of oval and hyperplastic bile ductular cell-enriched populations from the livers of carcinogen and noncarcinogen-treated rats. Cancer Res., 44, 3454–3466.[Abstract]
  56. Liu,X.H. and Rose,D.P. (1996) Differential expression and regulation of cyclooxygenase-1 and -2 in two human breast cancer cell lines. Cancer Res., 56, 5125–5127.[Abstract]
  57. Fukuda,T., Tani,Y., Kobayashi,T., Hirayama,Y. and Hino,O. (2000) A new western blotting method using polymer immuno-complexes: detection of Tsc1 and Tsc2 expression in various cultured cell lines. Anal. Biochem., 285, 274–276.[ISI][Medline]
  58. Tavares,I.A., Bishai,P.M. and Bennett,A. (1995) Activity of nimesulide on constitutive and inducible cyclooxygenases. Arzneim. Forsch. Drug Res., 45, 1093–1095.
  59. Davis,R. and Brogden,R.N. (1994) Nimesulide. An update of its pharmacodynamic and pharmacokinetic properties, and therapeutic efficacy. Drugs, 48, 431–454.[ISI][Medline]
  60. Martín-Santz,P., Callejas,N.A., Casado,M., Díaz-Guerra,M.J.M. and Boscá,L. (1998) Expression of cyclooxygenase-2 in foetal rat hepatocytes stimulated with lipopolysaccharide and pro-inflammatory cytokines. Br. J. Pharmacol., 125, 1313–1319.[Abstract]
  61. Adachi,T., Nakashima,S., Saji,S., Nakamura,T. and Nozawa,Y. (1995) Roles of prostaglandin production and mitogen-activated protein kinase activation in hepatocyte growth factor-mediated rat hepatocyte proliferation. Hepatology, 21, 1668–1674.[ISI][Medline]
  62. Kroll,B., Kunz,S., Tu,N. and Schwarz,L. (1998) Inhibition of transforming growth factor-ß1 and UV light-induced apoptosis by prostanoids in primary cultures of rat hepatocytes. Toxicol. Appl. Pharmacol., 152, 240–250.[ISI][Medline]
  63. McGinty,A., Chang,Y.E., Sorokin,A., Bokemeyer,D. and Dunn,M.J. (2000) Cyclooxygenase-2 expression inhibits trophic withdrawal apoptosis in nerve growth factor-differentiated PC12 cells. J. Biol. Chem., 275, 12095–12101.[Abstract/Free Full Text]
  64. Nomi,T., Shiota,G., Isono,M., Sato,K. and Kawasaki,H. (2000) Adenovirus-mediated hepatocyte growth factor gene transfer prevents lethal liver failure in rats. Biochem. Biophys. Res. Commun., 278, 338–343.[ISI][Medline]
  65. Gunasekar,P.G., Borowitz,J.L. and Isom,G.E. (1998) Cyanide-induced generation of oxidative species: involvement of nitric oxide synthase and cyclooxygenase-2. J. Pharmacol. Exp. Therap., 285, 236–241.[Abstract/Free Full Text]
  66. Dana,R., Malech,H.L. and Levy,R. (1994) The requirement for phospholipase A2 for activation of the assembled NADPH oxidase in human neutrophils. Biochem. J., 287, 217–223.
  67. Parola,M., Pinzani,M., Casini,A., Albano,E., Poli,G., Gentilini,P. and Dianzani,M.U. (1993) Stimulation of lipid peroxidation or 4-hydroxynonenal treatment increases procollagen {alpha}1(I) gene expression in human liver fat-storing cells. Biochem. Biophys. Res. Commun., 194, 1044–1050.[ISI][Medline]
  68. Laskin,D.L. (1990) Nonparenchymal cells and hepatotoxicity. Semin. Liver Dis., 10, 293–304.[ISI][Medline]
  69. Masuhara,M., Katyal,S., Nakamura,T. and Shinozuka,H. (1992) Differential expression of hepatocyte growth factor, transforming growth factor-{alpha} and transforming growth factor-ß1 messenger RNAs in two experimental models of liver cell proliferation. Hepatology, 16, 1241–1249.[ISI][Medline]
  70. Gilbert,R.S., Reddy,S.T., Kujubu,D.A., Xie,W., Luner,S. and Herschman,H.R. (1994) Transforming growth factor ß1 augments mitogen-induced prostaglandin synthesis and expression of the TIS10/prostaglandin synthase 2 gene both in Swiss 3T3 cells and murine embryo fibroblasts. J. Cell. Physiol., 159, 67–75.[ISI][Medline]
  71. Nieto,N., Greenwel,P., Friedman,S.L., Zhang,F., Dannenberg,A.J. and Cederbaum,A.I. (2000) Ethanol and arachidonic acid increase {alpha}2(I) collagen expression in rat hepatic stellate cells overexpressing cytochrome P450 2E1. Role of H2O2 and cyclooxygenase-2. J. Biol. Chem., 275, 20136–20145.[Abstract/Free Full Text]
  72. Beno,D.W.A., Rapp,U.R. and Davis,B.H. (1994) Prostaglandin E suppression of platelet-derived-growth-factor-induced Ito cell mitogenesis occurs independent of rat perinuclear translocation and nuclear proto-oncogene expression. Biochim. Biophys. Acta, 1222, 292–300.[ISI][Medline]
  73. Bhatnagar,R., Schade,U., Rietschel,E.T. and Decker,K. (1982) Involvement of prostaglandin E and adenosine 3',5'-monophosphate in lipopolysaccharide-stimulated collagenase release by rat Kupffer cells. Eur. J. Biochem., 125, 125–130.[Abstract]
  74. Gallois,C., Habib,A., Tao,J., Moulin,S., Maclouf,J., Mallat,A. and Lotersztajn,S. (1998) Role of NF-{kappa}B in the antiproliferative effect of endothelin-1 and tumor necrosis factor-{alpha} in human hepatic stellate cells. J. Biol. Chem., 273, 23183–23190.[Abstract/Free Full Text]
  75. Bauer,A.K., Dwyer-Nield,L.D. and Malkinson,A.M. (2000) High cyclooxygenase 1 (COX-1) and cyclooxygenase 2 (COX-2) contents in mouse lung tumors. Carcinogenesis, 21, 543–550.[Abstract/Free Full Text]
  76. Smith,C.J., Morrow,J.D., Roberts,L.J. and Marnett,L.J. (1993) Differentiation of monocytoid THP-1 cells with phorbol ester induces expression of prostaglandin endoperoxide synthase-1 (COX)-1. Biochem. Biophys. Res. Commun., 192, 787–793.[ISI][Medline]
  77. Nüsing,R.M., Mohr,S. and Ullrich,V. (1995) Activin A and retinoic acid synergize in cyclooxygenase-1 and thromboxane synthase induction during differentiation of J774.1 macrophages. Eur. J. Biochem., 227, 130–136.[Abstract]
  78. Murakami,M., Matsumoto,R., Urade,Y., Austen,K.F. and Arm,J.P. (1995) c-Kit ligand mediates increased expression of cytosolic phospholipase A2, prostaglandin endoperoxide synthase-1, and hematopoietic prostaglandin D2 synthase and increased IgE-dependent prostaglandin D2 generation in immature mouse mast cells. J. Biol. Chem., 270, 3239–3246.[Abstract/Free Full Text]
  79. Tsujii,M., Kawano,S., Tsuji,S., Sawaokam,H., Hori,M. and DuBois,R.N. (1998) Cyclooxygenase regulates angiogenesis induced by colon cancer cells. Cell, 93, 705–716.[ISI][Medline]
  80. Narko,K., Ristimäki,A., MacPhee,M., Smith,E., Haudenschild,C.C. and Hla,T. (1997) Tumorigenic transformation of immortalized ECV endothelial cells by cyclooxygenase-1 overproduction. J. Biol. Chem., 272, 21455–21460.[Abstract/Free Full Text]
Received April 17, 2001; revised September 7, 2001; accepted September 13, 2001.