Role of the prostaglandin E receptor subtype EP1 in colon carcinogenesis.

Although the cyclooxygenase pathway of the arachidonic acid cascade has been suggested to play an important role in colon carcinogenesis, the molecular species of prostanoids and receptors involved have not been fully elucidated yet. We examined the development of aberrant crypt foci (ACFs), putative preneoplastic lesions of the colon, in two lines of knockout mice, each deficient in prostaglandin E receptors, EP1 and EP3, by treatment with the colon carcinogen, azoxymethane. Formation of ACFs was decreased only in the EP1-knockout mice to approximately 60% of the level in wild-type mice. Administration of 250, 500, or 1000 ppm of a novel selective EP1 antagonist, ONO-8711, in the diet to azoxymethane-treated C57BL/6J mice also resulted in a dose-dependent reduction of ACF formation. Moreover, when Min mice, having a nonsense mutation in the adenomatous polyposis coli gene, were given 500 ppm ONO-8711 in the diet, the number of intestinal polyps was significantly reduced to 57% of that in the basal diet group. These results strongly suggest that prostaglandin E2 contributes to colon carcinogenesis to some extent through its action at the EP1 receptor. Thus, EP1 antagonists may be good candidates as chemopreventive agents for colon cancer.

Suppression of azoxymethane-induced colonic aberrant crypt foci by a nitric oxide synthase inhibitor.

Nitric oxide synthase (NOS), an important bioregulator of a variety of biological processes, is overexpressed in colonic tumors of humans and rodents. In this study, effects of L-N(G)-nitroarginine methyl ester (L-NAME), a NOS inhibitor, on development of aberrant crypt foci (ACF) induced by azoxymethane (AOM) in F344 male rats were investigated. Six-week-old male F344 rats were fed diets containing 0 or 100 ppm L-NAME, and given s.c. injections of AOM at 15 mg/kg body wt, once a week for 2 weeks. At 17 weeks of age, all animals were sacrificed and their colons were evaluated for numbers of ACF. Feeding of 100 ppm L-NAME inhibited the development of ACF in different sizes by 24-39%, those containing four or more crypts being most markedly affected. Assessment of silver-stained nucleolar organizer regions protein (AgNORs)/nucleus further revealed a 44% reduction by administration of L-NAME. These results suggest that the NOS inhibitor, L-NAME, may be an effective chemopreventive agent against colon carcinogenesis due to depression of cell proliferation.

Inhibitory effect of a prostaglandin E receptor subtype EP(1) selective antagonist, ONO-8713, on development of azoxymethane-induced aberrant crypt foci in mice.

We previously reported that prostaglandin E(2) contributes to colon carcinogenesis through its binding to the prostaglandin E receptor subtype EP(1) using a genetic approach in EP(1)-knockout mice and a pharmacological approach with the EP(1) selective antagonist, ONO-8711. In the present study, we examined the effects of another more selective EP(1) receptor antagonist, ONO-8713, on development of azoxymethane (AOM)-induced aberrant crypt foci (ACFs) in male C57BL/6J mice treated i.p. with 10mg/kg body weight AOM once a week for 3weeks. Administration of ONO-8713 at doses of 250, 500 and 1000ppm in diet during and post-AOM treatment for 5weeks resulted in a dose-dependent reduction of ACF formation, being 15, 30 and 36% inhibition of the control value, respectively. The level of inhibition was similar to that with ONO-8711. Moreover, ONO-8713 suppressed the development of ACF when administered at post-AOM, as in the case of ONO-8711. The data confirm EP(1) receptor involvement in colon carcinogenesis.

Effects of non-steroidal anti-inflammatory drugs on prostaglandin E2 production by cyclooxygenase-2 from endogenous and exogenous arachidonic acid in rat peritoneal macrophages stimulated with lipopolysaccharide.

Lipopolysaccharide (LPS) stimulated prostaglandin E2 (PGE2) formation and induction of cyclooxygenase-2 (COX-2) expression without changing the levels of COX-1 protein in rat peritoneal macrophages. Non-steroidal anti-inflammatory drugs (NSAIDs) (nimesulide, indomethacin and ibuprofen) strongly inhibited LPS-stimulated PGE2 production without any effect on COX-2 protein expression, suggesting that NSAIDs are active in inhibiting the ability of COX-2 to convert arachidonic acid (AA) endogenously released in response to LPS stimulation. Exogenous AA can be converted to PGE2 by both COX isoforms even in LPS-stimulated macrophages. NSAIDs inhibited PGE2 production from exogenous AA mediated by both COX-1 and COX-2. However, the two isoforms interacted differentially with different NSAIDs. Furthermore, NSAIDs were distinctly more active in inhibiting PGE2 production from endogenous AA than that from exogenous AA. These data suggest that PGE2 production through COX-2 from exogenous AA may not be subject to the same regulatory processes as that from endogenous AA and the two metabolic processes may be differentially sensitive to different NSAIDs.

Effects of nimesulide, a preferential cyclooxygenase-2 inhibitor, on carrageenan-induced pleurisy and stress-induced gastric lesions in rats.

Intrapleural injection of carrageenan in rats increased prostaglandin E2 (PGE2) production and induced newly synthesized cyclooxygenase-2 (COX-2) in pleural exudate cells without affecting COX-1 levels. Nimesulide, a preferential inhibitor of COX-2, reduced pleural PGE2 production and was almost as active as indomethacin and 10 times more active than ibuprofen. Only COX-1, and nc COX-2, was detected in gastric mucosal cells, and PGE2 concentration of gastric mucosa was significantly decreased by indomethacin and ibuprofen. The decrease in gastric PGE2 production induced by indomethacin and ibuprofen was enhanced in stressed rats, resulting in aggravation of stress-induced gastric lesions at anti-inflammatory doses. However, nimesulide did not produce stress-induced gastric lesions even at 30 times the anti-inflammatory dose. This supports the hypothesis that inhibition of COX-1 causes unwanted side effects and inhibition of COX-2 produces anti-inflammatory effects.

Effect of caffeine on ibuprofen-induced gastric mucosal damage in rats.

During investigations on the effect of caffeine on ibuprofen-induced gastric mucosal lesions in rats, we have found that caffeine (p.o.) inhibits the development of ibuprofen-induced gastric lesions in a dose-dependent manner (ED50 18.4 mg kg(-1)). To investigate this protective effect of caffeine, we have studied the effect of caffeine on HCl-ethanol-induced gastric mucosal lesions with or without indomethacin pretreatment. Caffeine inhibited the development of HCl-ethanol-induced gastric lesions with and without indomethacin pretreatment. These results indicate that caffeine did not act as a mild irritant but, on the contrary, had protective effects. We measured the gastric mucosal prostaglandin E2 (PGE2) concentrations and gastric mucosal blood flow, as representative protective factors for gastric mucosa. Caffeine did not affect the gastric mucosal PGE2 concentrations 4h after administration of ibuprofen. However, topical administration of caffeine resulted in an increase in gastric mucosal blood flow, as measured by laser Doppler flowmetry. We investigated the gastric acid secretion and gastric mucosal myeloperoxidase activity as representative aggressive factors for gastric mucosa. When caffeine was administered intraduodenally in pylorus-ligated rats, gastric acid secretion decreased in a dose-dependent manner, with an ED50 of 44.9 mg kg(-1). Caffeine decreased ibuprofen-induced gastric myeloperoxidase activity in a dose-dependent manner, with an ED50 of 9.1 mg kg(-1). These findings indicate that caffeine, at least in rats, may inhibit the development of acute gastric mucosal injury. The mechanisms underlying the protective actions of caffeine are unclear, but may be related in part to an increase in gastric mucosal blood flow and suppression of neutrophil activation.

COX-2 and iNOS, good targets for chemoprevention of colon cancer.

Cyclooxygenase (COX)-2 has been suggested to play an important role in colon carcinogenesis. We found that the COX-2 selective inhibitor, nimesulide, reduces azoxymethane (AOM)-induced aberrant crypt foci (ACF) in rats and colon carcinogenesis in mice, as well as formation of intestinal polyps in Min mice. Thus, selective inhibitors of COX-2, which catalyzes the synthesis of prostanoids, could be good candidates as chemopreventive agents against colon cancer. Examination of the effect of prostanoid receptor deficiency and a selective antagonist of prostanoid receptor on the development of AOM-induced ACF in mice revealed the involvement of the EP1 receptor. Moreover, a selective EP1 antagonist reduced the number of intestinal polyps in Min mice. These results suggest that PGE2 contributes to colon carcinogenesis through binding to the EP1 receptor. Nitric oxide synthase (NOS) is known to be overexpressed in colon cancers of humans and rats, and a NOS inhibitor, L-NG-nitroarginine methyl ester, was found to inhibit the development of AOM-induced ACF in rats. Thus, NOS including iNOS could also be a good target for chemoprevention of colon cancer, as in the COX-2 case.

Frequent mutations of the beta-catenin gene in mouse colon tumors induced by azoxymethane.

The beta-catenin gene is frequently mutated at codons 33, 41 and 45 of the glycogen synthase kinase-3beta phosphorylation motif in human colon cancers in patients without APC mutations. Frequent mutations at codons 32 and 34, as well as 33 and 41, have been detected in rat colon tumors induced by azoxymethane (AOM), with the second G of CTGGA sequences being considered as a mutational hot-spot. In the present study, exon 3 of the beta-catenin gene in mouse colon tumors induced by AOM was amplified by PCR and mutations were detected by the single strand conformation polymorphism method, restriction enzyme fragment length polymorphism and direct sequencing. All 10 colon tumors tested were found to have beta-catenin mutations, four in codon 34, three in codon 33, two in codon 41 and one in codon 37, nine being G:C-->A:T transitions. However, no mutations were found in codon 32 of the mouse beta-catenin gene. On immmunostaining, beta-catenin was observed in the cytoplasm and nucleus of the tumor cells. The cytoplasmic staining was homogeneous, while both homogeneous and heterogeneous patterns were noted for the nuclei. Highly frequent mutations of the beta-catenin gene in AOM-induced mouse colon tumors suggest that consequent alterations in the stability and localization of the protein may play an important role in this colon carcinogenesis model.

Interaction between NSAIDs and steroid in rat stomach: safety of nimesulide as a preferential COX-2 inhibitor in the stomach.

The relative risk of development of peptic ulcer with the use of nonsteroidal antiinflammatory drugs (NSAIDs) has been reported to increase when these drugs are administered in combination with steroids. We investigated the ulcerogenic potential of a combination of NSAIDs and steroids in rats and the underlying pathogenic mechanisms. Indomethacin alone produced gastric lesions, and the severity of the lesions markedly increased with concomitant administration of prednisolone. However, nimesulide, even in excessive doses, did not produce any gastric lesions, regardless of concomitant administration with prednisolone. Furthermore, we showed that the ulcerogenic potential of indomethacin administered in combination with prednisolone may be related to the induction of physiological changes, such as endogenous prostaglandin deficiency, an increase in neutrophil activation, and gastric hypermotility, by indomethacin and alteration of normal epithelial renewal by the steroid. These results suggest that the ulcerogenic potential of preferential a COX-1 inhibitor increases following concomitant administration with a steroid, whereas, nimesulide, a preferential COX-2 inhibitor, is nonulcerogenic, even when administered concomitantly with a steroid, and is therefore a clinically useful antiinflammatory agent.

Suppression of intestinal polyp development by nimesulide, a selective cyclooxygenase-2 inhibitor, in Min mice.

Nonsteroidal anti-inflammatory drugs (NSAIDs) suppress colon carcinogenesis in man and experimental animals. However, conventional NSAIDs inhibit both cyclooxygenase (COX) isoforms, COX-1 and COX-2, and cause gastrointestinal side-effects. Nimesulide, a selective inhibitor of COX-2, is much less ulcerogenic. We, therefore, examined its influence on the development of intestinal polyps in Min mice. Female Min mice at 4 weeks old were given 400 ppm nimesulide in their diet for 11 weeks. This treatment resulted in a significant reduction of the numbers of both small and large intestinal polyps, the total being 52% of that in untreated control Min mice. The size of the polyps in the nimesulide-treated group was also significantly decreased. The results suggest that nimesulide is a good candidate as a chemopreventive agent for human colon cancer with low toxicity.

Growth inhibition of tumour cells by a liposome-encapsulated, mycolic acid-containing glycolipid, trehalose 2,3,6'-trimycolate.

In vivo growth of syngeneic tumour cells in the peritoneal cavity was strongly inhibited by intraperitoneal injection of a liposome-encapsulated, mycolic acid-containing glycolipid, trehalose 2,3,6'-trimycolate (TTM), derived from a non-pathogenic, acid-fast bacterium. Gordona aurantiaca. Peritoneal macrophages from mice after this treatment lysed tumour cells in vitro at a low effector/target ratio, and their culture supernatant inhibited tumour cell growth. The supernatant inhibited growth of not only tumour necrosis factor (TNF)-sensitive tumour cells, but also TNF-insensitive tumour cells. This inhibitory activity was enhanced by addition of lipopolysaccharide (LPS) to the culture medium of the macrophages. The macrophages released more superoxide (O2-), TNF and interleukin-1 (IL-1) on LPS triggering, and the releases of these compounds were further increased by addition of recombinant interferon-gamma (IFN-gamma) to the medium. Moreover, splenic T cells of TTM liposome-primed mice were found to produce eight times more IFN-gamma upon stimulation with LPS. These results indicated that priming with TTM liposomes resulted in strong activation of macrophages, which lysed tumour cells directly and also inhibited tumour cell growth by released factors.

Chemoprevention by nimesulide, a selective cyclooxygenase-2 inhibitor, of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced mammary gland carcinogenesis in rats.

Breast cancer is common in women all over the world, and exploration of chemopreventive approaches to this cancer is very important. Nimesulide, a selective inhibitor of cyclooxygenase-2 (COX-2), is a good candidate as a chemopreventive agent with low toxicity. We examined its effects on mammary tumor development in female Sprague-Dawley rats induced with the environmental carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Rats at 7 weeks of age received intragastric intubations of PhIP (85 mg / kg body weight) 4 times weekly for 2 weeks and were maintained on control diet (high fat diet) or experimental diet (high fat diet supplemented with 400 ppm nimesulide) throughout the experiment. COX-2 protein was over-expressed in epithelial cancer cells and stromal cells of the PhIP-induced mammary carcinomas, but was weak or not apparent in normal mammary gland cells. The development of mammary carcinomas was clearly suppressed by administration of nimesulide. The carcinoma incidence was 51% as compared to 71% for the control diet group. The average multiplicity of carcinomas in the experimental diet group was 1.2 +/- 0.2 (P < 0.05), significantly smaller than the control diet group value (2.6 +/- 0. 5). The size of carcinomas was also clearly decreased; 1.1 +/- 0.4 cm(3) / rat in experimental diet group (P < 0.05), 4.1 +/- 1.3 cm(3) / rat in the control diet group. The results therefore provide evidence that the selective COX-2 inhibitor, nimesulide, possesses chemopreventive activity against PhIP-induced mammary carcinogenesis in rats.

Suppressive effects of nimesulide, a selective inhibitor of cyclooxygenase-2, on azoxymethane-induced colon carcinogenesis in mice.

The effects of nimesulide, a selective inhibitor of cyclooxygenase-2 (COX-2) on azoxymethane (AOM)-induced colon carcinogenesis were investigated in mice. AOM at a dose of 10 mg/kg body wt was administered to male ICR mice once a week for 6 weeks. The animals were fed on AIN-76A powder diet containing nimesulide at doses of 200 or 400 p.p.m., starting the day before the first carcinogen treatment until the end of the experiment, at week 30. Administration of nimesulide reduced the incidence of colon carcinomas to 32 and 25% for the AOM + 200 and 400 p.p.m. nimesulide groups, respectively, compared with the AOM + basal diet group (50%). Multiplicities of colon carcinomas in the 200 and 400 p.p.m. nimesulide-treated groups were 0.70 +/- 0.28 and 0.35 +/- 0.11, respectively, being significantly smaller than the AOM alone value (1.79 +/- 0.47). The sizes of the colon carcinomas in the nimesulide-treated groups were also decreased. No significant influence on liver and lung tumor development was apparent. Thus, nimesulide exerted a suppressive effect on AOM-induced colon carcinogenesis in mice.

Inhibitory effects of Bifidobacterium-fermented soy milk on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced rat mammary carcinogenesis, with a partial contribution of its component isoflavones.

High consumption of soybean and soybean-related products is hypothesized to contribute to protection against breast cancer. Soybean is a rich source of genistein, a putative cancer chemopreventive agent. Fermented soy milk (FSM), which is made of soy milk fermented with the Bifidobacterium breve strain Yakult, contains larger amounts of the isoflavone aglycones genistein and daidzein than unfermented soy milk. In the present study, we examined the effects of FSM and its component isoflavone mixture (genistein:daidzein 4:1) on 2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (PhIP)-induced mammary carcinogenesis in rats. Starting at 7 weeks of age, female Sprague-Dawley rats were given PhIP at a dose of 85 mg/kg body wt by intragastric administration four times a week for 2 weeks. They were fed control high fat basal diet or experimental high fat diet containing 10% FSM or 0.02 or 0.04% isoflavone mixture during and after carcinogen exposure. The incidences (percentage of rats with tumors) of mammary gland tumors were 71% in the control diet group, 51% in the FSM group and 68 and 61% in the groups treated with isoflavone mixture at 0.02 and 0.04%, respectively. Mammary tumor multiplicities (number of tumors per rat) were 1.2 +/- 0.2 for 10% FSM, 2.2 +/- 0.4 for 0.02% isoflavone mixture and 1.5 +/- 0.3 for 0.04% isoflavone mixture, being clearly smaller than the control diet value (2.6 +/- 0.5). Furthermore, feeding of FSM and the isoflavone mixture at both doses reduced the sizes of mammary tumors. Since the amounts of isoflavones in 10% FSM are approximately equivalent to those in the 0.02% isoflavone mixture, the chemopreventive activity of FSM could be partly attributable to the presence of isoflavones such as genistein and daidzein.

Chemopreventive effects of ONO-8711, a selective prostaglandin E receptor EP(1) antagonist, on breast cancer development.

Levels of prostaglandin E(2) (PGE(2)) in human and rodent breast cancers are higher than surrounding normal tissues. PGE(2) exhibits biological activity through binding to membrane receptors, EP(1-4). The present study was designed to investigate the effects of ONO-8711, a newly synthesized selective PGE receptor EP(1) antagonist, on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced breast cancer development. Starting at 7 weeks of age, female Sprague-Dawley (SD) rats were given PhIP (85 mg/kg body weight) by gavage four times weekly for two weeks. Dietary administration of ONO-8711 at 400 or 800 p.p.m. delayed occurrence of breast tumors for 2 or 4 weeks, respectively. At 20 weeks after the last dosing of PhIP, all animals were killed and complete autopsy was made. All breast tumors were diagnosed as invasive ductal adenocarcinomas histopathologically. Administration of ONO-8711 at 800 p.p.m. significantly decreased PhIP-induced breast cancer incidence, multiplicity and volume compared with those of rats fed the control diet (56% versus 79%, P < 0.05, 1.2 versus 2.5, P < 0.05, 0.7 versus 1.4 cm(3), P < 0.01, respectively). Apoptosis was significantly increased in breast cancer cells by feeding of ONO-8711 at 800 p.p.m. of 158% (P < 0.05). EP(1) receptor was detected by reverse transcription-polymerase chain reaction (RT-PCR) in breast cancers, not in normal tissues. These results suggest that EP(1) receptor is associated with breast cancer development and selective PGE receptor EP(1) antagonists may possess chemopreventive effects through the induction of apoptosis without any side effects.