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

Carcinogenesis vol.23 no.2 pp.245–256, 2002 Increased expression of cyclooxygenase-2 protein during rat hepatocarcinogenesis caused by a choline-deficient, L-amino aciddefined diet and chemopreventive efficacy of a specific inhibitor, nimesulide Ayumi Denda3, Wakashi Kitayama, Akiko Murata1,2, Hideki Kishida, Yasutaka Sasaki, Osamu Kusuoka, Toshifumi Tsujiuchi, Masahiro Tsutsumi, Dai Nakae, Hidetoshi Takagi1 and Yoichi Konishi 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. Department of Oncological Pathology, Cancer Center, Nara Medical Univesity, 840 Shijo-cho, Kashihara, Nara 634-8521 and 1Wyeth Lederle Japan Ltd, 1-6-34 Kashiwa-cho, Shiki, Saitama 353-8511, Japan Introduction 2Present address: Department of Customer Service, Ventana Japan K.K., 2-4-1 Shibakoen, Minato-ku, Tokyo 105-0011, Japan 3To whom correspondence should be addressed Email: Expression of cyclooxygenase (COX)-2 protein during rat hepatocarcinogenesis associated with fatty change, fibrosis, cirrhosis and oxidative DNA damage, caused by a cholinedeficient, 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 enzymealtered 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 Abbreviations: CDAA, choline-deficient, L-amino acid-defined; CDML, choline-deficient, methionine-low; COX, cyclooxygenase; CSAA, cholinesupplemented, 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, 8hydroxydeoxyguanosine; 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. © Oxford University Press 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 (1–4). We have found that a cholinedeficient, 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 (5–7). 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 (7,11–13), 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 β-catenin, however, have not been found (18–20). Recently, we established the preventive potential of nonsteroidal 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 (21–24). 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 (25–28). 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). 245 A.Denda et al. 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 PGF2α involvement in liver regeneration, although the responsible COX isozymes and their producing cells remain largely unknown (38–46). 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 (47–51). Moreover, elevated levels of COX-2 have been described in human hepatocellular carcinomas (HCCs) as well as under condi (...truncated)