Chemoprevention of biliary carcinogenesis in Syrian hamsters by the novel carboxamide derivative IS-741 after initiation with N-nitrosobis(2-oxopropyl)amine (BOP)
Yoshifumi Ogura4,
Shinsuke Matsuda1,
Morihiro Ito2,
Rui Niimi3,
Masaki Sumitomo3 and
Yoshifumi Kawarada3
Department of Surgery, National Mie Chuo Hospital, 2158-5 Myojin, Hisai City, 514-1101,
1 Department of Surgery, Nagai General Hospital,
2 Department of Microbiology and
3 First Department of Surgery, Mie University School of Medicine Hisai and Tsu City, Japan
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Abstract
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To elucidate the possible inhibitory effect of a novel carboxamide derivative (IS-741) on biliary carcinogenesis, Syrian hamsters were subjected to cholecystoduodenostomy and ligation of the distal end of the common duct, and then given a regular diet (group I) or a diet containing 200 p.p.m. of IS-741 (group II). All hamsters were subcutaneously injected with N-nitrosobis(2-oxopropyl)amine until 10 weeks after surgery, and continued to feed on their respective dietary regimen until termination of the experiment at 16 weeks after surgery. Biliary adenocarcinomas were evaluated histologically. Non-cancerous and cancerous hepatobiliary tract tissues were analyzed for phospholipase A2 (PLA2) activity, myeloperoxidase (MPO) activity, and the concentrations of prostaglandin (PG), i.e., prostaglandin E2, 6-ketoprostaglandin F1
and thromboxane B2. IS-741 significantly inhibited the development and multiplicity of hepatobiliary adenocarcinomas and reduced the proliferating cell nuclear antigen labeling indices in non-cancerous hepatobiliary tissues, compared with group I. The anti-cancerous effect of IS-741 was associated with a significant inhibition of PLA2 and MPO levels in non-cancerous tissues of the extrahepatic biliary tract and the liver, and in cancerous tissue of the liver. Furthermore, IS-741 reduced the production of PGs in non-cancerous hepatobiliary tissues, compared with group I. Although the precise mechanism of action of IS-741 in preventing biliary tumorigenesis remains to be elucidated, it is likely to be related to modulation of arachidonic acid metabolism and/or suppression of neutrophil accumulation.
Abbreviations: 6-KF, 6-ketoprostaglandin F1
; AA, arachidonic acid; BOP, N-nitrosobis(2-oxopropyl)amine; COX, cyclooxygenase; IS-741, novel N-(2-sulfonylamino)-5-trifluoromethyl-3-pyridyl carboxamide derivative; LOX, lipoxygenase; MPO, myeloperoxidase; PCNA, proliferating cell nuclear antigen; PG, prostaglandin; PGE2, prostaglandin E2; PLA2, phospholipase A2; PLC, phospholipase C; TXB2, thromboxane B2.
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Introduction
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The biosynthesis of prostaglandins (PGs) through the cyclooxygenase (COX) system and of hydroxy fatty acids via the lipoxygenase (LOX) pathway from arachidonic acid (AA) has been well documented. These AA metabolites exert a variety of biological activities. Several studies have shown that COX metabolites, particularly PGs of the type-2 series, modulate cell proliferation, tumor growth and immune responses (1,2). LOX metabolites, on the other hand, influence various biological responses, including chemotoxic responses, hormone secretion, iron transport, tumor cell adhesion, stimulation of tumor cell spreading and regulation of tumor cell metastatic potential (35). Generation of AA for biosynthesis of COX and LOX metabolites involves both degradation of phosphatidylinositol via a sequence of reactions regulated by phospholipase C (PLC) and a direct action of phospholipase A2 (PLA2) on phospholipid (2,6,7). The rate-limiting step for PG synthesis is the release of AA from membrane phospholipids by the lipolytic enzyme PLA2, which catalyzes the specific hydrolysis of the fatty acyl ester bond at the sn-2 position of 3-glycerophospholipids (8). Membrane-associated (cellular or type 2) PLA2 exhibits selectivity towards AA in the sn-2 position and is involved in COX and LOX metabolite synthesis, whereas pancreatic (secretary group or type1) PLA2 has no preference for fatty acids in the sn-2 position and is involved in digestion (9,10).
Several studies have detected increased levels of PLA2 activity in human colon tumors, breast tumors and melanomas, compared with normal tissues (1113). In this regard, it is noteworthy that the COX metabolite, PGE2, which significantly modulates tumor growth and immune responses, has been found at high levels in tumors induced experimentally in animal tumors as well as in human tumors (1,14,15). Other studies have shown that COX inhibitors such as aspirin, indomethacin, piroxicam, sulindac and ibuprofen suppress colon carcinogenesis in laboratory animal models (1,1517). Thus, it is possible that changes both in PLA2 activity, which is involved in signal transudation and AA release, and in the COX pathway of AA metabolism induced by exogenous agents, may alter tumorigenesis.
Clinically, there is a very high incidence of extrahepatic biliary adenocarcinoma in choledochal cysts, are reflux of pancreatic juice into the biliary tract in association with anomalous arrangement of the pancreaticobiliary junction is thought to promote the development of biliary adenocarcinoma. In 1994, Tajima et al. (18) described a new model with a high incidence of adenocarcinoma development in the extrahepatic bile ducts and gallbladder induced by N-nitrosobis(2-oxopropyl)amine (BOP) in hamsters. We have also reported enhancement of the carcinogenic effects of this agent on biliary epithelium that had proliferated in response to and/or been injured by activated pancreatic enzymes. In the hamster model, activated pancreatic enzymes reflux into the biliary tract and lead to increased free-radical activity, resulting in a high frequency of adenocarcinoma in the biliary tract (19). It was therefore of interest to study the efficacy of a novel N-(2-sulfonylamino-5-trifluoromethyl-3-pyridyl) carboxamide derivative, IS-741, which has been synthesized and evaluated as a PLA2 inhibitor (20), as a chemopreventive agent in the above hamster model.
In the present study, we investigated the chemopreventive efficacy of IS-741 on BOP-induced biliary carcinogenesis in Syrian golden hamsters. In addition, we analyzed the effects of IS-741 on PLA2 activity, myeloperoxidase (MPO) activity and PG levels in the liver, extrahepatic biliary tract and adenocarcinoma to understand the possible modulating role of this agent in biliary carcinogenesis.
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Materials and methods
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Animals
A total of 104 5-week-old female Syrian golden hamsters (Shizuoka Laboratory Animal Center Co., Shizuoka, Japan) were housed three per cage and maintained under standard laboratory conditions in the Laboratory Animal Center for Biochemical Research, Mie University School of Medicine. The animals were provided with a standard pellet diet and water ad libitum throughout the study. All experiments were conducted in accordance with the Guidelines for Animal Experimentation of Mie University.
Surgical techniques
The extrahepatic biliary tract of the hamster is composed of the gallbladder and extrahepatic bile ducts, i.e. common bile duct and common duct. The surgical procedure consisted of cholecystoduodenostomy with double ligation of the extrahepatic bile ducts at the distal end of the common duct to allow backward flow of the pancreatic juice into the biliary tract. Following intraperitoneal anesthesia with Nembutal (50 mg/kg of body weight), a midline upper abdominal incision was made, and the distal end of the common duct was doubly ligated with 9-0 nylon sutures. A 1 mm long incision was made in both the gallbladder fundus and duodenal wall, approximately 10 mm distal to the pyloric ring, and cholecystoduodenostomy was performed by a continuous suture with 9-0 nylon under a surgical microscope. Sham-operated hamsters were subjected to a simple laparotomy.
Carcinogenesis study
Chemicals.
The carcinogen used was BOP (Nakarai Chemical Co., Kyoto, Japan) dissolved in physiological saline. IS-741, a novel N-(2-sulfonylamino-5-trifluoromethyl-3-pyridyl) carboxamide derivative, was a generous gift from Ishihara Sangyo Kaisya (Kusatsu, Japan). IS-741 mixed with the diet at a concentration of 1000 p.p.m. was found to have no significant toxic effects in rats (20). Our study was designed to use low levels of IS-741 in the diet (200 p.p.m., equivalent to five times the dose used as an anti-pancreatitis agent).
Experimental protocol.
Cholecystoduodenostomy was performed with double ligation of the distal end of the common duct. One week after surgery, the hamsters were subcutaneously injected with BOP (10 mg/kg body wt, once per week for 10 consecutive weeks), and then provided with either regular diet (group I; 26 animals) or a diet containing 200 p.p.m. IS-741 (group II; 15 animals) for 16 weeks after surgery. The sham-operated group (n = 18) was subjected to simple laparotomy and injected with the same doses of BOP. This group was provided with the regular diet and observed for the same periods as the animals in the two experimental groups. The 12 hamsters in the normal control group were not subjected to any surgery and did not receive BOP injections, but were observed for the same period as the animals in the other three groups. At 16 weeks after surgery or commencement of the experiment, the surviving animals were killed with an overdose of Nembutal. Animals that died during the period of the experiment were excluded from the analysis. Immediately after termination, the gallbladder and extrahepatic bile ducts were removed en bloc together with the liver, pancreas and part of the duodenum, and fixed in 10% neutral formalin.
Histological examination.
Formalin-fixed specimens were cut into 1013 sections and embedded in paraffin, then processed using standard techniques for staining with hematoxylin and eosin (H&E). The number of hamsters with histologically verified adenocarcinomas of the hepatobiliary tract, i.e. of the gallbladder, extra- and intrahepatic bile ducts, was noted, with particular attention to the location and growth pattern of any induced tumors. Adenocarcinoma was diagnosed on the basis of disruption of the polarity of the epithelial cells, evidence of an invasion, and cytoplasmic or nuclear atypia.
Cell kinetic studies.
Immunohistochemical staining for proliferating cell nuclear antigen (PCNA) was performed by the streptavidinbiotin-complex method (sABC kit, Nichirei, Tokyo). Tissue was deparaffinized with xylene and dehydrated with a graded alcohol series. The sections were immersed in 0.3% hydrogen peroxide in absolute methanol for 30 min at room temperature and washed in phosphate-buffered saline (PBS; pH 7.2). Following incubation with normal rabbit serum (Nichirei, Tokyo) for 10 min, the sections were incubated with primary antibody for 12 h in a humidified chamber at room temperature. The primary antibody used was a 1:100 dilution of anti-PCNA (PC-10, Dako, Glostrup, Denmark). After incubation, the sections were washed with PBS three times and treated with biotinylated goat anti-mouse IgG and peroxidase-conjugated streptavidin for 30 min. They were then reacted with 0.02% diaminobezidine tetrahydrochloride containing 0.005% hydrogen peroxide for 3 min, counter-stained with H&E, dehydrated, cleared and mounted. The PCNA labeling index (LI) was determined by selecting the most PCNA-positive area from several high-power microscopic fields (x400) and counting labeled and unlabeled cells until reaching a total of 500 cells in the area selected. The LI was then calculated as the ratio of PCNA-labeled cells to the total number of cells counted, and expressed as a percentage.
Preparation of tissue sections for determination of PLA2 activity, MPO activity and prostaglandin production.
Specimens of the liver, extrahepatic biliary tract consisting of gallbladder and extrahepatic bile duct, and portions of tumors to be stored at 80°C until extraction of PLA2 and MPO, and other specimens of these tissues to be used for PG analysis, were rinsed in 10 mM EDTA2Na with 104 M indomethacin for 5 min, and then frozen in liquid nitrogen and stored at 80°C. The non-cancerous and cancerous regions of the extrahepatic biliary tract were extracted under a stereoscopic microscope.
Assay of PLA2 activity.
PLA2 levels were assayed according to the modified method described by Grass et al. (21). Tissue samples to be assayed were suspended (1:4) in 100 mM Tris pH 9.0, 4 mM CaCl2. Then, 50 µl of each sample was added, on ice, to a 100 µl reaction mixture containing 100 mM Tris pH 9.0, 4 mM CaCl2 and 50 000 c.p.m. [3H]arachidonic acid-labeled E.coli membranes (Dupont/NEN, Wilmington, DE), and the mixture was incubated at 37°C for 30 min. After stopping the reaction with 50 µl of 2 N HCl, 100 µl of fatty acid-free bovine serum albumin (50 mg/ml in PBS) was added, and the mixture was briefly vortexed. After centrifugation in an Eppendorf microfuge at 14 000 g for 5 min, the radioactivity released into the 200 µl supernatant fluid was determined. The protein content of the tissue sample was measured using a protein assay (Bio-Rad Laboratories, Hercules, CA). PLA2 levels are expressed as the amount of AA (in c.p.m.) per milligram of protein.
Assay of tissue prostaglandin.
Liver, extrahepatic biliary tract consisting of gallbladder and extrahepatic bile ducts, and portions of the tumor from some of the animals were homogenized in 2 ml cold ethanol with a motor-driven Teflon homogenizer. The samples were then centrifuged at 3000 r.p.m. at 4°C for 15 min, and the supernatant fractions were stored at 20°C. Tissue concentrations of prostaglandin E2 (PGE2), 6-ketoprostaglandin-F1
(6-KF) and thromboxane B2 (TXB2) were determined at Special Reference Laboratories (Tokyo, Japan) as described previously (22,23).
A 1 ml volume of each supernatant was mixed and stirred for 10 min with 1 ml octadecylsilyl suspension and 0.1 ml of 1 N HCl. After centrifugation (3000 r.p.m.), the precipitate was washed twice with 1 ml of ethanol/0.05 N HCl and once with 1 ml of petroleum ether. The precipitate was then extracted twice with 1 ml of ethyl acetate, and the extracts were combined and evaporated under N2 gas. The residue was applied to a silica minicolumn (Bond Elut Si, Analytichem International, Harbor City, CA) and, after eluting the fractions with a solvent (acetonitrilecholoroformacetic acid, 50:50:0.5), they were assayed for PGE2, 6-KF and TXB2 with RIA kits (New England Nuclear, Boston, MA). The protein content of the tissue sample was measured by the method of Lowry et al. (24). PG content is expressed as amount per milligram of protein.
Assay of myeloperoxidase activity.
Tissue MPO levels were measured by the method described previously (25,26). Briefly, tissue samples were homogenized in 100 mg/ml of 50 mM potassium phosphate buffer (pH 6.0) containing 0.01% soybean trypsin inhibitor and 0.1 mM phenylmethylsulfonyl fluoride and centrifuged at 12 000 g for 10 min at 4°C. The supernatants were discarded and the insoluble pellets were re-homogenized in potassium phosphate buffer (pH 6.0) containing 0.5% hexa-decyltrimethylammonium bromide. The homogenates were then sonicated with three bursts of 10 s and centrifuged at 12 000 g for 10 min. A 100 µl volume of the supernatant was added to a 1 cm light path cuvette containing 2.9 ml of the assay solution. The assay solution was prepared fresh daily from the following ingredients: 26.9 ml of H2O, 3.0 ml of 0.1 M sodium phosphate buffer (pH 7.0), 0.1 ml of 0.1 M H2O2 and 48 µl guaiacol, and it was stored in a capped opaque-glass flask at room temperature. The cuvette components were quickly mixed, and the absorbance was determined at 470 nm for 1 min with a spectrophotometer (Shimadzu UV-2100, Japan). One unit of enzyme was defined as the amount that consumes 1 µmol H2O2/min at 25°C. Four moles of H2O2 are required to produce 1 mol of the terraguaiacol product which had an extinction coefficient (E) of 26.6 mM1 cm1 at 470 nm. Units were calculated as follows:
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where Vt = total volume (ml); Vs = sample volume (ml),
OD = density change and
t = duration of measurement (min).
Statistical analysis
All data are expressed as means ± SD. Differences between groups were examined for statistical significance by the Student's t-test. Differences in the incidences of adenocarcinoma production were analyzed by the
2 test. A P-value <0.05 was considered to indicate the presence of a statistically significant difference.
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Results
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Tumor incidence
The incidences of carcinoma of the gallbladder, extrahepatic bile duct and intrahepatic bile duct induced in each experimental group are shown in Table I
. There was no evidence of adenocarcinoma in the normal-control group, which was not treated with BOP. Surgery combined with BOP injections, however, resulted in the development of adenocarcinoma of the hepatobiliary system. The differences in the incidences of adenocarcinomas in the three anatomical locations between the animal fed the IS-741 diet (group II) and the regular diet (group I) were statistically significant (adenocarcinoma of: gallbladder, P < 0.01; extrahepatic bile duct, P < 0.05; intrahepatic bile duct, P < 0.05). The IS-741 diet (group II) also significantly inhibited adenocarcinoma multiplicity in all three regions (adenocarcinoma of: gallbladder, P < 0.01; extrahepatic bile duct, P < 0.01; intrahepatic bile duct, P < 0.05), when compared with the regular diet (group I) (Table I
).
PCNA labeling index
As summarized in Table II
, the PCNA LIs in the non-cancerous regions of the extrahepatic biliary tract (P < 0.05) and liver tissue (P < 0.05) of animals fed the IS-741 diet (group II) were significantly reduced, compared with the animals fed the regular diet (group I). Markedly higher percentages of PCNA LIs, 69.1 ± 19.5 and 47.4 ± 18.5%, were measured in the adenocarcinomas in group I and group II, when compared with the non-cancerous regions of the hepatobiliary tract tissues in all experimental groups, but there was no significant difference between the adenocarcinomas in groups I and II (Table II
).
Histological neutrophil infiltration
Figure 3
shows that neutrophil infiltration was confirmed histologically by H&E staining. Neutrophil infiltration was significantly suppressed in Glisson's sheath, that is, in the periportal and/or peribiliary areas of the liver tissue with adenocarcinoma (Figure 3C
) in group II, compared with the liver tissue with adenocarcinoma in group I (Figure 3A
). Neutrophil infiltration in Glisson's sheath was also significantly suppressed in the liver tissue without adenocarcinoma in group II (Figure 3D
), compared with the liver tissue without adenocarcinoma in group I (Figure 3B
).

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Fig. 3. Histological sections of liver tissue from hamsters in groups I and II. Magnification, x100. Neutrophil infiltration was significantly suppressed in Glisson's sheath, i.e. in the periportal and/or peribiliary areas of the liver tissue with adenocarcinoma (C) in group II, compared with the liver tissue with adenocarcinoma in group I (A). Neutrophil infiltration in Glisson's sheath was also significantly suppressed in the liver tissue without adenocarcinoma in group II (D), compared with the liver tissue without adenocarcinoma in group I (B).
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Biochemical studies
The PLA2 levels measured in the non-cancerous regions of the extrahepatic biliary tract, liver tissues and hepatobiliary adenocarcinomas are summarized in Figure 1
.

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Fig. 1. Effect of dietary IS-741 on the PLA2 activity of hepatobiliary non-cancerous regions and tumors. Group I, cholecystoduodenostomy and ligation of the distal end of the common duct, followed by administration of a regular diet. Group II, same surgical procedure and then given a diet containing 200 p.p.m. of IS-741. Sham-operated, simple laparotomy and then provided with regular diet. All animals in the above three groups were injected with BOP (10 mg/kg/body wt, once a week for 10 consecutive weeks). Normal control, no surgery and no BOP. Significantly different from group I by Student's t-test: *P < 0.05, **P < 0.01. Significantly different from non-cancerous liver tissues in group II by Student's t-test: #P < 0.05.
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Cholecystoduodenostomy and ligation of the distal end of the common duct, so that pancreatic juice flowed backward into the biliary tract, elevated PLA2 activity in the non-cancerous regions of the extrahepatic biliary tract and liver, irrespective of dietary regimen. Although long-term IS-741 feeding did not suppress PLA2 levels in non-cancerous regions of the extrahepatic biliary tract, it resulted in a significant suppression of PLA2 activity in the non-cancerous regions of the liver (P < 0.05). Interestingly, significant increase in the PLA2 activity was noted in hepatobiliary tumors compared with the non-cancerous regions of the extrahepatic biliary tract and the livers of the sham-operated and normal-control groups, and there was a significant difference between the cancerous and non-cancerous regions of the liver tissue in the IS-741-treated group (P < 0.05).
We also measured tissue MPO activity to evaluate the degree of hepatobiliary tissue infiltration by neutrophil (Figure 2
). Treatment with IS-741 significantly reduced the levels of MPO in non-cancerous regions of the extrahepatic biliary tract and liver in group II compared with hamsters of group I fed regular diet (extrahepatic biliary tract, P < 0.05; liver, P < 0.01). The levels of MPO in the cancerous tissues were higher than in the non-cancerous tissues of the two groups, and there was significant difference between cancerous and non-cancerous regions of the liver tissue in group II (P < 0.05).

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Fig. 2. Tissue MPO activity of hepatobiliary non-cancerous regions and tumors. Significantly different from group I by Student's t-test: *P < 0.05, **P < 0.01. Significantly different from non-cancerous liver tissue in group II by Student's t-test: #P < 0.05.
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Table III
shows the effect of treatment on PG production in non-cancerous regions of the extrahepatic biliary tract and liver tissues. Significantly high concentrations of PGE2, 6-KF and TXB2 were measured in the extrahepatic biliary tract compared with the liver tissue, even in the sham-operated and normal-control groups. Markedly reduced levels of PGE2, 6-KF and TXB2 were observed in the non-cancerous regions of the extrahepatic biliary tract (PGE2, P < 0.05; 6-KF, P < 0.05; TXB2, P < 0.01) and liver tissues (PGE2, P < 0.01; 6-KF, P < 0.01; TXB2, P < 0.01) of animals fed the IS-741 diet (group II) compared with those fed regular diet (group I). Significantly high levels of PGE2 [388 ± 192 pg/mg protein (n = 8)], 6-KF [683 ± 282 pg/mg protein (n = 8)] and TXB2 [347 ± 164 pg/mg protein (n = 8)] were measured in the intrahepatic biliary adenocarcinomas, when compared with non-cancerous regions of the liver tissues in all experimental groups. However, there were no significant differences in PG levels in cancerous tissues in comparison with non-cancerous tissue in group II. The PG levels in the extrahepatic biliary adenocarcinomas could not be examined because their tissue specimens were too small in quantity.
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Table III. Effect of dietary IS-741 on the PG levels of non-cancerous regions of the extrahepatic biliary tract and liver
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Discussion
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The main aim of this study was to elucidate the possible inhibitory effect on biliary carcinogenesis of IS-741, which has been synthesized and evaluated as a PLA2 inhibitor. Previous studies have demonstrated that IS-741 had the highest and most stable therapeutic effect of the substances tested on acute hemorrhagic pancreatitis experimentally induced in rats by the closed duodenal loop method (20). In addition, Yamaguchi et al. (27) recently demonstrated that IS-741 significantly reduced pulmonary neutrophil infiltration, cytokine-induced neutrophil chemoattraction and accumulation of Mac-1 cells in the lungs of rats and, thus, prevents lung injury in cerulein-induced necrotizing pancreatitis associated with septicemia. These new findings prompted us to investigate the effect of this agent on cancer in a well-established biliary cancer model. To our knowledge, this is the first report that provides evidence of a significant inhibitory effect of IS-741 on the development and multiplicity of adenocarcinoma of the hepatobiliary tract in this established hamster model. Cells are most susceptible to the tumorigenic effects of chemical carcinogens in the DNA synthesis phase of the cell cycle (28), and DNA-synthesizing cells can be identified by immunohistochemical staining methods using anti-PCNA monoclonal antibody (29,30). In this study, IS-741 significantly reduced the PCNA labeling index in non-cancerous hepatobiliary tissues, compared with untreated group I. The results of the present study are of major interest because long-term treatment with IS-741 not only significantly inhibited PLA2 levels in non-cancerous hepatobiliary tract tissues, including gallbladder, extrahepatic bile duct and liver, but also reduced MPO levels in the same tissues. Although the precise mechanism by which IS-741 inhibits BOP-induced biliary carcinogenesis has not been established, it is likely that it involves the anti-inflammatory action of IS-741.
Several studies have suggested a modulatory role for AA metabolites in various immunological and inflammatory diseases (7). The results of the present study demonstrated that the production of PGs in intrahepatic biliary tumors is higher than in non-cancerous regions of the liver, suggesting an increased synthesis of COX metabolites in such tumors. The role of COX metabolites, particularly PGE2, in colon tumor promotion has already been established (1,16,17). Earlier studies have shown that PG inhibitors, such as piroxicam, indomethacin, sulindac and aspirin, cause regression of colon tumors in rodents (1,14). The increased PLA2 activity and enhanced PG synthesis relative to the unaffected mucosa (16) may be attributable to macrophage infiltration of the tumors (17), and neutrophil accumulation and activation may be an important mechanism in the pathogenesis of BOP-induced biliary carcinogenesis.
In the present study, we determined the PLA2 levels in non-cancerous and cancerous hepatobiliary tissues. PLA2 is one of the enzymes involved in the generation of AA, and it acts directly on phospholipids, including 1,2-diacyl- or 1-O-alkyl-2-acyl-phophatidylinositol, phospatidylthylamine and phosphatidylcholine. Our results demonstrated that administration of IS-741 significantly inhibited PLA2 levels in cancerous and non-cancerous hepatobiliary tissues. Activated neutrophils release various substances, including products of AA metabolism, granular elements and oxygen radicals that cause tissue damage (31,32). In the present study, neutrophils were noted in the Glisson's sheath, i.e. periportal and/or peribiliary areas of the liver tissue in our model of BOP-induced biliary cancer, but the role infiltrating inflammatory cells in biliary carcinogenesis is not understood. In the present study, we measured tissue MPO levels, which are an unequivocal index of neutrophil accumulation in tissues (26), to evaluate the degree of inflammatory cell infiltration in BOP-induced biliary carcinogenesis. As shown in Figure 2
, the MPO level was significantly lower in non-cancerous regions of the hepatobiliary tract in hamsters treated with IS-741, compared with untreated hamsters, and these changes were confirmed histologically by examination of H&E-stained sections (Figure 3
). Thus, the results indicate that long-term treatment with IS-741 causes significant suppression of neutrophil infiltration. However, the exact mechanism by which IS-741 inhibits MPO levels is unclear.
It is possible that IS-741 exerts its inhibitory effect by acting directly on PLA2, or that, by acting on the regulators of PLA2, it reduces levels of AA and metabolite formation. Based on these findings, we speculate that IS-741 not only modulates PLA2, which alters endogenous AA available as a substrate for production of COX metabolites, but also the COX pathway itself. The second pathway involved in the generation of AA mediated by PLC
1 includes the degradation of phosphatidylinositol 4,5-biphosphate via a sequence of reactions that begins with PLC
1, which is followed by diglyceride lipase and monoglyceride lipase (33). These changes remain to be elucidated.
In conclusion, our study demonstrated that IS-741 significantly inhibits BOP-induced biliary carcinogenesis in Syrian hamsters. In addition, IS-741 was found to significantly suppress the PLA2 activity, MPO activity and PG levels, which are related to biliary carcinogenesis in non-cancerous hepatobiliary tract tissues. Although the exact mechanism of action of IS-741 remains to be elucidated, it is possible that the compound modulates AA metabolism and/or suppresses neutrophil infiltration of local tissues.
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Notes
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4 To whom correspondence should be addressed Email: yogura{at}alles.or.jp 
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Received July 28, 1999;
revised March 29, 2000;
accepted April 3, 2000.