Chemoprevention of colonic aberrant crypt foci by an inducible nitric oxide synthase-selective inhibitor

Inducible nitric oxide synthase (iNOS) is overexpressed in colonic tumors of humans and also in rats treated with a colon carcinogen. iNOS appear to regulate cyclooxygenase-2 (COX-2) expression and production of proinflammatory prostaglandins, which are known to play a key role in colon tumor development. Experiments were designed to study the inhibitory effects of S,S'-1,4-phenylene-bis(1,2-ethanediyl)bis-isothiourea (PBIT) a selective iNOS-specific inhibitor, measured against formation of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF). Beginning at 5 weeks of age, male F344 rats were fed experimental diets containing 0 or 50 p.p.m. of PBIT, or 2000 p.p.m. of curcumin (non-specific iNOS inhibitor). One week later, rats were injected s.c. with AOM (15 mg/kg body wt, once weekly for 2 weeks). At 17 weeks of age, all rats were killed, colons were evaluated for ACF formation and colonic mucosa was assayed for isoforms of COX and NOS activities. Both COX and iNOS activities in colonic mucosa of the AOM-treated rats were significantly induced. Importantly, 50 p.p.m. PBIT suppressed AOM-induced colonic ACF formation to 58% (P < 0.0001) and crypt multiplicity containing four or more crypts per focus to 78% (P < 0.0001); it also suppressed AOM-induced iNOS activity. Curcumin inhibited colonic ACF formation by 45% (P < 0.001). These observations suggest that iNOS may play a key regulatory role in colon carcinogenesis. Developing iNOS-specific inhibitors may provide a selective and safe chemopreventive strategy for colon cancer treatment.     

Suppression of occurrence and advancement of beta-catenin-accumulated crypts, possible premalignant lesions of colon cancer, by selective cyclooxygenase-2 inhibitor, celecoxib.

Suppression of occurrence and advancement of premalignant lesions is important for cancer prevention. Our previous studies clarified that beta-catenin-accumulated crypts, independent of aberrant crypt foci (ACF), are probably direct precursors of colon cancers in rats. Here we investigated the effects of a selective cyclooxygenase-2 inhibitor, celecoxib, on the development of beta-catenin-accumulated crypts in comparison with those on ACF. Male F344 rats were divided into 4 groups. Groups 1 - 3 were administered azoxymethane (AOM) s.c. at a dose of 15 mg / kg body weight, once weekly for 3 weeks to induce beta-catenin-accumulated crypts. Groups 2 and 3 also received experimental diet containing celecoxib (500 and 1500 ppm, respectively) for 8 weeks, starting a week before the first dosing of AOM. At termination, the frequency and crypt multiplicity (number of crypts / lesion) of beta-catenin-accumulated crypts of groups 2 and 3 were significantly less than that of group 1. Furthermore, numbers of silver-stained nucleolar organizer regions (AgNORs) / nucleus in beta-catenin-accumulated crypts were also decreased by exposure to celecoxib. In this study, celecoxib had greater effects on the frequency and growth of beta-catenin-accumulated crypts than on those of ACF. These findings represent additional evidence that beta-catenin-accumulated crypts are premalignant lesions of colon cancer. The results also suggest that beta-catenin-accumulated crypts could be a novel target for evaluation of possible chemopreventive agents against colon carcino-genesis, and indicate that possible chemopreventive effects of celecoxib on the initial stage of colon carcinogenesis may be related to modulation of cell proliferation activity in such early lesions.     

Suppression of Occurrence and Advancement of β-Catenin-accumulated Crypts, Possible Premalignant Lesions of Colon Cancer, by Selective Cyclooxygenase-2 Inhibitor, Celecoxib

Suppression of occurrence and advancement of premalignant lesions is important for cancer prevention. Our previous studies clarified that β-catenin-accumulated crypts, independent of aberrant crypt foci (ACF), are probably direct precursors of colon cancers in rats. Here we investigated the effects of a selective cyclooxygenase-2 inhibitor, celecoxib, on the development of β-catenin-accumulated crypts in comparison with those on ACF. Male F344 rats were divided into 4 groups. Groups 1-3 were administered azoxymethane (AOM) s.c. at a dose of 15 mg/kg body weight, once weekly for 3 weeks to induce β-catenin-accumulated crypts. Groups 2 and 3 also received experimental diet containing celecoxib (500 and 1500 ppm, respectively) for 8 weeks, starting a week before the first dosing of AOM. At termination, the frequency and crypt multiplicity (number of crypts/lesion) of β-catenin-accumulated crypts of groups 2 and 3 were significantly less than that of group 1. Furthermore, numbers of silver-stained nucleolar organizer regions (AgNORs)/nucleus in β-catenin-accumulated crypts were also decreased by exposure to celecoxib. In this study, celecoxib had greater effects on the frequency and growth of β-catenin-accumulated crypts than on those of ACF. These findings represent additional evidence that β-catenin-accumulated crypts are premalignant lesions of colon cancer. The results also suggest that β-catenin-accumulated crypts could be a novel target for evaluation of possible chemopreventive agents against colon carcino-genesis, and indicate that possible chemopreventive effects of celecoxib on the initial stage of colon carcinogenesis may be related to modulation of cell proliferation activity in such early lesions.     

Azoxymethane-induced beta-catenin-accumulated crypts in colonic mucosa of rodents as an intermediate biomarker for colon carcinogenesis

It is now well established that bile acids act as colon tumor promoters. However, a previous study provided conflicting data showing that dietary exposure of cholic acid (CHA), a primary bile acid, inhibits the carcinogen-induced formation of aberrant crypt foci (ACF), possible preneoplastic lesions, in colonic mucosa of rodents. Recently we found beta-catenin-accumulated crypts (BCAC) in colonic mucosa of rats initiated with azoxymethane (AOM) and provided evidence that BCAC might be preneoplastic lesions independent from ACF. In the present study, we investigated the modifying effects of dietary CHA on the formation of BCAC as well as ACF in male F344 rats after exposure to AOM to determine if the differences in the effect of CHA on these lesions could account for this discrepancy. The results indicate that administration of CHA (0.5%) in the diet during the post-initiation phase significantly reduced the total number, multiplicity and size of ACF (P < 0.00001) in AOM-exposed colonic mucosa as reported previously. The number of ACF even with >4 aberrant crypts/focus was also decreased significantly (P < 0.0002), suggesting that the large ACF are little resistant to continuous feeding of 0.5% CHA diet. Interestingly, the dietary CHA significantly enhanced both the multiplicity (P < 0.002) and size (P < 0.00001), but not the incidence, of AOM-induced BCAC when compared with the control diet group. Importantly, the number of large BCAC with >6 crypts/lesion was increased significantly by the dietary CHA (P < 0.003). Our results support the concept that BCAC are precursors of colon tumors and indicate the usefulness of BCAC as intermediate biomarkers for colon carcinogenesis, although the methodology for their detection requires further improvement.     

Suppression of azoxymethane-induced colon cancer development in rats by a cyclooxygenase-1 selective inhibitor, mofezolac

We demonstrated recently that mofezolac, a cyclooxygenase-1 (COX-1) selective inhibitor, suppresses the development of azoxymethane (AOM)-induced colonic aberrant crypt foci in F344 rats and intestinal polyps in APC1309 mice. In the present study, we therefore investigated the effects of mofezolac on colon cancer development. Male F344 rats were injected subcutaneously with 15 mg/kg body weight of AOM in the back twice at 7-day intervals from 5 weeks of age, and fed a diet containing 600 or 1200 ppm mofezolac for 32 weeks, starting 1 day before the first dosing of AOM. Treatment with 1200 ppm mofezolac significantly reduced the incidence, multiplicity and volume of colon carcinomas to 79%, 2.15 +/- 1.65 and 7.5 +/- 11.8 mm3, respectively, compared with 94%, 3.19 +/- 1.87 and 23.7 +/- 31.2 mm3 in the AOM treatment alone. Administration of 600 ppm mofezolac showed only a slight reduction. No side effects were observed in any of the groups. These results confirm that COX-1, as well as COX-2, contributes to colon carcinogenesis and that mofezolac may be a good chemopreventive agent for human colon cancer.     

The effects of dietary selenomethionine on polyamines and azoxymethane-induced aberrant crypts

We evaluated the effects of dietary selenomethionine supplementation on colonic polyamine levels and the ability of L-selenomethionine supplementation to modulate the carcinogenic activity of azoxymethane (AOM) in the rat colon. Four-week-old male F344 rats were treated with 15 mg/kg body weight of AOM once a week for 2 weeks. Dietary selenomethionine at a concentration of either 1 or 2 ppm was administered in AIN-76A rodent diet to AOM-treated animals for 16 weeks. Aberrant crypt foci (ACF), precursor lesions of colon cancer, were investigated after the 16 week treatment course. Selenomethionine given in the diet at 2 ppm markedly reduced the number of aberrant crypt foci. The multiplicity of ACFs (i.e. the number of aberrant crypts/focus) and the percentage of microadenomas were also affected by selenomethionine in a dose dependent manner. However, evaluation of the colonic tissue polyamine levels between control and treated groups showed no significant difference. These results demonstrate that selenomethionine can modulate the development of AOM-induced premalignant lesions through a polyamine-independent mechanism.     

A xanthine oxidase inhibitor 1'-acetoxychavicol acetate inhibits azoxymethane-induced colonic aberrant crypt foci in rats

The modifying effect of dietary administration of a xanthine oxidase inhibitor 1'-acetoxychavicol acetate (ACA) present in an edible plant Languas galanga in Thailand on the development of azoxymethane (AOM)- induced colonic aberrant crypt foci (ACF) was investigated in rats. Male F344 rats were given s.c. injections of AOM (15 mg/kg body wt) once a week for 3 weeks to induce colonic ACF. They were fed the diets containing 100 or 200 ppm ACA for 5 weeks, starting 1 week before the first dosing of AOM. At the termination of the study (week 5), AOM induced 118 +/- 28 ACF/colon. Dietary administration of ACA caused significant reduction in the frequency of ACF (41% inhibition by 100 ppm ACA feeding and 37% inhibition by 200 ppm ACA feeding, P<0.01). Such inhibition might be associated with suppression of the proliferation biomarkers' expression such as ornithine decarboxylase activity in the colonic mucosa, number of silver-stained nucleolar organizer regions' protein in the colonic mucosal cell nuclei and blood polyamine content. These results indicate that ACA could inhibit the development of AOM-induced ACF through its suppression of cell proliferation in the colonic mucosa and ACA might be a possible chemopreventive agent against colon tumourigenesis.      

选择性环氧合酶2抑制剂对人膀胱癌细胞T24增殖和凋亡的作用

背景与目的:环氧合酶2(cyclooxygenase-2,COX-2)与膀胱癌的发生发展密切相关,因此COX-2抑制剂对于膀胱癌可能具有潜在的抗肿瘤效应.本实验旨在探讨选择性COX-2抑制剂对人膀胱癌细胞T24增殖和凋亡的抑制作用.方法:用四甲基偶氮唑蓝还原法(MTT)研究选择性COX-2抑制剂SC-58125、塞来昔布(celecoxib)和非选择性COX抑制剂吲哚美辛(indomethacin)对T24细胞增殖的影响;用流式细胞术、DNA电泳和Hoechst33258荧光染色3种方法检测SC-58125作用下T24细胞的凋亡,同时逆转录聚合酶链反应(RT-PCR)检测细胞凋亡相关基因Bcl-2和Bax基因的变化.结果:在12.5 μmol/L至200 μmol/L的剂量范围内,SC-58125、塞来昔布及吲哚美辛均能不同程度地抑制T24细胞的增殖,以SC-58125的抑制作用最强,IC50在25 μmol/L～50 μmol/L之间;3种凋亡检测方法均观察到SC-58125作用后凋亡细胞的比例增加,其中100 μmol/L的SC-58125作用于T24细胞6 h和12 h后,流式细胞仪测得凋亡细胞的比例分别为(7.95±1.88)%和(12.5±2.42)%,较对照组增加(P＜0.05),但细胞内与凋亡相关的Bcl-2和Bax基因表达没有变化.结论:选择性COX-2抑制剂体外可抑制T24细胞的增殖,并诱导细胞凋亡.     

Chemoprevention of colon cancer by specific cyclooxygenase-2 inhibitor, celecoxib, administered during different stages of carcinogenesis

Epidemiological observations and laboratory research have suggested that nonsteroidal anti-inflammatory drugs (NSAIDs) reduce the risk of colon cancer and that the inhibition of colon carcinogenesis by NSAIDs is mediated through the modulation of prostaglandin production by rate-limiting enzymes known as cyclooxygenases (COXs). Because traditional NSAIDs inhibit both COX-1 and COX-2, these drugs induce side effects, such as gastrointestinal ulceration and renal toxicity, through the inhibition of the constitutive COX-1. Overexpression of COX-2 has been observed in colon tumors; therefore, specific inhibitors of COX-2 could serve as chemopreventive agents. Our previous study has shown that celecoxib, an inhibitor of COX-2, while sparing COX-1, inhibited azoxymethane (AOM)-induced colon tumorigenesis when administered during both initiation and postinitiation stages, ie., celecoxib administered continuously before, during, and after carcinogen treatment. This study examined the dose-response effect of celecoxib when administered during the initiation and postinitiation stages. In addition, the chemopreventive effects of high-dose celecoxib administered during the promotion/progression stage of colon carcinogenesis, ie., continuous celecoxib administration beginning 14 weeks after the carcinogen treatment, was determined in male F344 rats. We also measured the steady-state levels of celecoxib in the plasma of animals given this inhibitor. Groups of 5-week-old male F344 rats were fed either a control diet or experimental diets containing 500, 1000, or 1500 ppm celecoxib. At 7 and 8 weeks of age, rats scheduled for carcinogen treatment were injected s.c. with AOM at a dose rate of 15 mg/kg body weight/week. Groups of animals destined for the promotion/ progression study and initially receiving the control diet were switched to a diet containing 1500 ppm celecoxib beginning 14 weeks after the second AOM treatment. All rats remained on their respective dietary regimens until the termination of the study, ie., 52 weeks, and were then sacrificed. Colon tumors were evaluated histopathologically. Administration of 500, 1000, or 1500 ppm celecoxib during the initiation and postinitiation stages significantly inhibited the incidence (P < 0.01 to P < 0.0001) as well as the multiplicity (P < 0.01 to P < 0.0001) of adenocarcinomas of the colon in a dose-dependent manner. Importantly, administration of 1500 ppm celecoxib during the promotion/progression stage also significantly suppressed the incidence and multiplicity of adenocarcinomas of the colon (P < 0.01). Also, administration of celecoxib to the rats during the initiation and postinitiation periods and throughout the promotion/progression stage strongly suppressed colon tumor volume (P < 0.0002 to P < 0.001). The steady-state plasma concentration of celecoxib increases somewhat with the dose. Thus, in this model system, the chemopreventive efficacy of celecoxib is dose-dependent when this COX-2 inhibitor is administered during the initiation and postinitiation periods. This study provides the first evidence that celecoxib is also very effective when it is given during the promotion/progression stage of colon carcinogenesis, indicating that the chemopreventive efficacy is achieved during the later stages of colon tumor development. This suggests that celecoxib may potentially be an effective chemopreventive agent for the secondary prevention of colon cancer in patients with familial adenomatous polyposis and sporadic polyps.     

The cyclooxygenase-2 inhibitor celecoxib is a potent preventive and therapeutic agent in the min mouse model of adenomatous polyposis

Epidemiological and animal studies suggest that nonsteroidal anti-inflammatory drugs (NSAIDs) may reduce colon cancer risk. NSAIDs nonselectively inhibit both the constitutive cyclooxygenase (COX) 1 associated with side effects and the desired therapeutic target COX-2, which is induced in inflammation and neoplasia. We used the adenomatous polyposis coli (Apc) mutant Min mouse model to determine whether the selective COX-2 inhibitor celecoxib is effective for adenoma prevention and/or regression, and whether it might be safer than the nonselective NSAID previously shown to be most effective in this model, piroxicam. Min mice (n = 120) were randomized to treatment with celecoxib (0, 150, 500, or 1500 ppm celecoxib mixed in the diet) or piroxicam. To distinguish prevention from regression effects, groups were treated either "early" (before adenomas develop) or "late" (after most adenomas are established). Celecoxib caused dramatic reductions in both the multiplicity and size of tumors in a dose-dependent manner (P < 0.01). Early treatment with 1500 ppm of celecoxib was effective for prevention, decreasing tumor multiplicity to 29% and tumor size to only 17% of controls (P < 0.01). Late treatment demonstrated regression effects, reducing tumor multiplicity and size by about half. In contrast to the significant toxicity of piroxicam, which caused ulcers complicated by perforation and bleeding, celecoxib caused no gastrointestinal side effects and did not inhibit platelet thromboxane B2 at plasma drug levels similar to those obtained in early clinical trials in humans. These results provide the first evidence that selective inhibitors of COX-2 are safe and effective for the prevention and regression of adenomas in a mouse model of adenomatous polyposis and strongly support ongoing clinical trials in humans with the same syndrome. The broader population of patients with common sporadic adenomas that have somatic mutations of the same gene (APC) may also benefit from this treatment approach.     

Modifying effect of dietary sesaminol glucosides on the formation of azoxymethane-induced premalignant lesions of rat colon

Sesame, which has been reported to have preventive effects against various disordered conditions, contains small quantities of lignans and several precursors to them such as sesaminol glucosides (SG). The lignans have the potent antioxidative activity and are suggested to have chemopreventive property. In the present study, we evaluated the modulating effect of SG on the development of colon precancerous lesions, aberrant crypt foci (ACF) and beta-catenin-accumulated crypts (BCAC), in the azoxymethane (AOM)-induced short-term model using male F344 rats. Dietary SG (500 ppm) significantly decreased the incidence of AOM-induced ACF when compared to the control (P<0.01). The incidences of AOM-induced BCAC in the SG-treated groups (250 or 500 ppm) were also significantly lower than that of the control group (P<0.01). Interestingly, administration of 500 ppm SG clearly decreased serum triglyceride level and mRNA expression of intestinal fatty acid-binding protein in the colonic mucosa, as compared to the control. These findings indicate that dietary SG inhibits AOM-induced carcinogenesis and suggest SG as a possible chemopreventive agent.     

Pterostilbene, an active constituent of blueberries, suppresses aberrant crypt foci formation in the azoxymethane-induced colon carcinogenesis model in rats.

PURPOSE: Epidemiologic studies have linked the consumption of fruits and vegetables to reduced risk of several types of cancer. Laboratory animal model studies have provided evidence that stilbenes, phenolic compounds present in grapes and blueberries, play a role in inhibiting the risk of certain cancers. Pterostilbene, a naturally occurring stilbene from blueberries, was tested for its preventive activity against colon carcinogenesis. EXPERIMENTAL DESIGN: Experiments were designed to study the inhibitory effect of pterostilbene against the formation of azoxymethane-induced colonic aberrant crypt foci (ACF) preneoplastic lesions in male F344 rats. Beginning at 7 weeks of age, rats were treated with azoxymethane (15 mg/kg body weight s.c., once weekly for 2 weeks). One day after the second azoxymethane treatment, rats were fed experimental diets containing 0 or 40 ppm of pterostilbene. At 8 weeks after the second azoxymethane treatment, all rats were sacrificed, and colons were evaluated for ACF formation and for inhibition of inducible nitric oxide synthase (iNOS) and proliferating cell nuclear antigen. Effects on mucin MUC2 were also determined. RESULTS: Administration of pterostilbene for 8 weeks significantly suppressed azoxymethane-induced formation of ACF (57% inhibition, P < 0.001) and multiple clusters of aberrant crypts (29% inhibition, P < 0.01). Importantly, dietary pterostilbene also suppressed azoxymethane-induced colonic cell proliferation and iNOS expression. Inhibition of iNOS expression by pterostilbene was confirmed in cultured human colon cancer cells. CONCLUSIONS: The results of the present study suggest that pterostilbene, a compound present in blueberries, is of great interest for the prevention of colon cancer.     

Silymarin,a naturally occurring polyphenolic antioxidant flavonoid,inhibits azoxymethane-induced colon carcinogenesis in male F344 rats

The modifying effect of dietary administration of the polyphenolic antioxidant flavonoid silymarin, isolated from milk thistle [Silybum marianum (L.) Gaertneri], on AOM-induced colon carcinogenesis was investigated in male F344 rats. In the short-term study, the effects of silymarin on the development of AOM-induced colonic ACF, being putative precursor lesions for colonic adenocarcinoma, were assayed to predict the modifying effects of dietary silymarin on colon tumorigenesis. Also, the activity of detoxifying enzymes (GST and QR) in liver and colonic mucosa was determined in rats gavaged with silymarin. Subsequently, the possible inhibitory effects of dietary feeding of silymarin on AOM-induced colon carcinogenesis were evaluated using a long-term animal experiment. In the short-term study, dietary administration of silymarin (100, 500 and 1,000 ppm in diet), either during or after carcinogen exposure, for 4 weeks caused significant reduction in the frequency of colonic ACF in a dose-dependent manner. Silymarin given by gavage elevated the activity of detoxifying enzymes in both organs. In the long-term experiment, dietary feeding of silymarin (100 and 500 ppm) during the initiation or postinitiation phase of AOM-induced colon carcinogenesis reduced the incidence and multiplicity of colonic adenocarcinoma. The inhibition by feeding with 500 ppm silymarin was significant (p < 0.05 by initiation feeding and p < 0.01 by postinitiation feeding). Also, silymarin administration in the diet lowered the PCNA labeling index and increased the number of apoptotic cells in adenocarcinoma. beta-Glucuronidase activity, PGE(2) level and polyamine content were decreased in colonic mucosa. These results clearly indicate a chemopreventive ability of dietary silymarin against chemically induced colon tumorigenesis and will provide a scientific basis for progression to clinical trials of the chemoprevention of human colon cancer.     

Reduction of UV-induced skin tumors in hairless mice by selective COX-2 inhibition.

UV light is a complete carcinogen, inducing both basal and squamous cell skin cancers. The work described uses the selective COX-2 inhibitor celecoxib to examine the efficacy of COX-2 inhibition in the reduction of UV light-induced skin tumor formation in hairless mice. UVA-340 sun lamps were chosen as a light source that effectively mimics the solar UVA and UVB spectrum. Hairless mice were irradiated for 5 days a week for a total dose of 2.62 J/cm(2). When 90% of the animals had at least one tumor, the mice were divided into two groups so that the tumor number and multiplicity were the same (P < 0.31). Half of the mice were then fed a diet containing 1500 p.p.m. celecoxib. Tumor number, multiplicity and size were then observed for the next 10 weeks. Ninety-five percent of the tumors formed were histopathologically evaluated as squamous cell carcinoma. COX-2 expression and activity were increased in tumors. After 10 weeks, the difference in tumor number and multiplicity in the drug-treated group was 56% of UV controls (P < 0.001). The results show that the orally administered selective COX-2 inhibitor celecoxib prevents new tumor formation after the onset of photocarcinogenesis and suggest that treatment with celecoxib may be very useful in preventing UV-induced skin tumors in humans.     

Chemoprevention of colon and small intestinal tumorigenesis in APC(Min/+) mice by licofelone, a novel dual 5-LOX/COX inhibitor: potential implications for human colon cancer prevention

Preclinical and clinical studies suggest that 5-lipoxygenase (5-LOX), such as COX-2, is a potential target for colon cancer inhibition and, in part, contributes to cardiovascular side effects associated with COX-2 inhibitors. Experiments were designed to assess the chemopreventive effects of a novel dual 5-LOX/COX inhibitor, licofelone {[6-(4-chlorophenyl)-2,2-dimethyl-7-phenyl-2,3-dihydro-1H-pyrrolizin-5-yl] acetic acid}, in APC(Min/+) mouse intestinal tumorigenesis. Six-week-old male and female APC(Min/+) mice (n = 10 per group) were fed with control American Institute of Nutrition-76A diet or diets containing 150 or 300 ppm licofelone for 14 weeks (～100 days), and intestinal tumors were evaluated for tumor multiplicity and size. Licofelone significantly inhibited total intestinal tumor multiplicity and size in a dose-dependent manner (P < 0.0001; mean tumors for 0, 150, and 300 ppm: 48.8, 17, and 8, respectively, in male mice; and 34.3, 8.8, and 5.5, respectively, in female mice). Licofelone at high dose showed more than 83% (P < 0.0001) tumor inhibition in both genders of mice. One hundred and fifty and 300 ppm licofelone resulted in 86% to 97% inhibition of polyps having size greater than 2 mm. One hundred and fifty and 300 ppm licofelone caused more than 72% and 100% inhibition of colonic tumors, respectively. Importantly, in mice fed with licofelone, tumors showed significantly reduced proliferating cell nuclear antigen expression (70%, P < 0.0001), increased terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cells (75%, P < 0.0001), and there was dose-dependent suppression of serum triglycerides (71%-83%, P < 0.0001), decreased inflammatory cytokines; and decreased COX and 5-LOX activities (57%-64%, P < 0.0001). Also, compared with 300 ppm celecoxib, 300 ppm licofelone provided better efficacy in suppressing tumor growth. These observations show that a novel dual 5-LOX/COX inhibitor dramatically suppresses small intestinal and colonic tumor formation in APC(Min/+) mice.     

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.     

Chemopreventive effects of a selective nitric oxide synthase inhibitor on carcinogen-induced rat esophageal tumorigenesis

The inducible nitric oxide synthase (iNOS) generates a high concentration of nitric oxide (NO) in tissues. Increased NO production is associated with many disorders including esophageal cancer. Previous studies in our laboratory demonstrated an association between increased iNOS expression and the development of N-nitrosomethylbenzylamine (NMBA)-induced tumors in the rat esophagus. On the basis of these observations, we initiated a bioassay to evaluate the ability of S,S'-1,4-phenylene-bis(1,2-ethanediyl)bis-isothiourea (PBIT), a selective iNOS inhibitor, to prevent the progression of esophageal tumors in rats preinitiated with NMBA. Rats were given s.c. injections of NMBA (0.25 mg/kg body weight) three times per week for 5 weeks. One week later, they were fed a synthetic diet containing either 50 or 100 ppm PBIT until the end of the bioassay (25 weeks). PBIT reduced the incidence of esophageal cancer from 96% in NMBA-treated rats to 83% and 77% (P < 0.05) in rats treated with 50 and 100 ppm PBIT, respectively. Tumor multiplicity was reduced from 3.64 +/- 0.42 tumors per esophagus in NMBA-treated rats to 1.79 +/- 0.25 (P < 0.001) and 1.50 +/- 0.24 (P < 0.0001) in rats treated with 50 and 100 ppm PBIT, respectively. PBIT reduced the production of NO in NMBA-induced preneoplastic and papillomatous esophageal lesions when compared with comparable lesions in rats treated with NMBA only. iNOS mRNA expression was not modulated by PBIT. These observations suggest that iNOS plays a role in tumor development and that its selective inhibitor, PBIT, significantly inhibits esophageal tumor progression presumably through reducing the production of NO.     

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.     

Ursodeoxycholic acid and F(6)-D(3) inhibit aberrant crypt proliferation in the rat azoxymethane model of colon cancer: roles of cyclin D1 and E-cadherin.

We have previously demonstrated that ursodeoxycholic acid(UDCA) and a fluorinated analogue of vitamin D(3), F(6)-D(3),inhibited colonic carcinogenesis in the azoxymethane (AOM) model. Generalized colonic mucosal hyperproliferation and aberrant crypt foci (ACF) are intermediate biomarkers of colon cancer. Using these biomarkers, in this study we examined the anticarcinogenic mechanisms of these chemopreventive agents. Rats were maintained on AIN-76A chow or supplemented with 0.4% UDCA or F(6)-D(3) (2.5 nmol/kg chow) and treated weekly with AOM 20 mg i.p./kg wt or saline x 2 weeks. F(6)-D(3) was continued for an additional 2 weeks and UDCA for the duration of the study. At 40 weeks, animals received bromodeoxyuridine (BrdUrd) i.p. 2 h before sacrifice. A portion of each tumor was fixed in formalin and the remainder flash frozen. Colons were divided longitudinally and half-fixed in formalin and half in ethanol. The size and location of methylene blue-stained ACF were recorded. Cell proliferation (BrdUrd labeling) and apoptosis (terminal deoxynucleotidyl transferase-mediated nick end labeling assay) were measured in colonic crypts and tumors. Protein expression levels of several regulators of cell proliferation were analyzed by immunostaining and Western blotting. Colonic crypt cyclin D1 and E-cadherin mRNA levels were measured by real-time PCR. In saline injected controls, neither UDCA nor F(6)-D(3) alone had any effect on cytokinetic parameters or on the expression of mitogenic regulators. AOM significantly increased the proliferation (percentage of BrdUrd-positive cells) of both ACF (23.1 +/- 1.7%) and non-ACF crypts (17.6 +/- 1.6%), compared with normal colonic crypts (4.5 +/- 0.8%; P < 0.05). This hyperproliferation was accompanied by a 5-fold increase in cyclin D1 and >50% decrease in E-cadherin protein (P < 0.05) in ACF, both of which are predicted to be growth-enhancing alterations. UDCA and F(6)-D(3) significantly (P < 0.05) inhibited AOM-induced crypt cell hyperproliferation, ACF development, and tumor burden. These chemopreventive agents also significantly blocked AOM-induced alterations in cyclin D1 and E-cadherin protein in ACF and tumors. In ACF, changes in mRNA levels of cyclin D1, but not E-cadherin, paralleled alterations in protein expression. Cyclooxygenase-2 and inducible nitric oxide synthase were increased in AOM tumors but not in ACF, and these changes were blocked by UDCA and F(6)-D(3). UDCA and F(6)-D(3) significantly inhibited ACF development and hyperproliferation, in part, by preventing carcinogen-induced alterations in cyclin D1 and E-cadherin. In established tumors, UDCA and F(6)-D(3) also limited inductions of cyclooxygenase-2 and inducible nitric oxide synthase, which together with their effects on cyclin D1 and E-cadherin, contribute to their chemopreventive actions.