Kengo Matsunaga

The inhibitory effects of mangiferin, a naturally occurring glucosylxanthone, in bowel carcinogenesis of male F344 rats

Mangiferin, 1,3,6,7-tetrahydroxyxanthone-C2-beta-D-glucoside, is one of xanthone derivatives and C-glucosylxanthones, is widely distributed in higher plants and is one of constituents of folk medicines. Recent studies showed that mangiferin has a potential as an anti-oxidant and an anti-viral agent. In this study, we examined the effects of mangiferin in rat colon carcinogenesis induced by chemical carcinogen, azoxymethane (AOM). We performed two experiments: a short-term assay to investigate the effects of mangiferin on the development of preneoplastic lesions by AOM, aberrant crypt foci (ACF), and the following long-term assay for the influence of mangiferin on tumorigenesis induced by AOM. In the short-term assay, 0.1% mangiferin in a diet significantly inhibited the ACF development in rats treated with AOM compared to rats treated with AOM alone (64.6+/-22.0 vs. 108.3+/-43.0). In the long-term assay, the group treated with 0.1% mangiferin in initiation phase of the experimental protocol had significantly lower incidence and multiplicity of intestinal neoplasms induced by AOM (47.3 and 41.8% reductions of the group treated with AOM alone for incidence and multiplicity, respectively). In addition, the cell proliferation in colonic mucosa was reduced in rats treated with mangiferin (65-85% reductions of the group treated with AOM alone). These results suggest that mangiferin has potential as a naturally-occurring chemopreventive agent.

Modifying effects of ferulic acid on azoxymethane-induced colon carcinogenesis in F344 rats

The modifying effects of dietary administration of ferulic acid (FA) on azoxymethane (AOM)-induced colon carcinogenesis were examined in three experiments with male 344 rats. In the first experiment, the modifying effect of FA on AOM (15 mg/kg body weight, once a week, for 3 weeks)-induced formation of aberrant crypt foci (ACF) was examined in five groups. Numbers of ACF/colon of groups 2 (AOM+250 ppm FA) and 3 (AOM+500 ppm FA) at the termination (5 weeks after the start) were smaller than of group 1 (AOM alone). Those of ACF/cm(2) of group 3 were also smaller than of group 1 (P<0.05). In the second experiment, a long-term assay for the effects of FA was conducted with seven groups. At the termination (35 weeks), groups 2 and 3 which were given FA during the initiation phase at doses of 250 and 500 ppm, respectively, had lower incidences of colonic carcinomas (23 and 27%, respectively) than group 1 which was given AOM alone (59%; P<0.05). In the third experiment, to determine whether FA could modify the activities of phase II detoxifying enzymes, glutathione S-transferase (GST) and quinone reductase (QR) in liver and colon, 60 rats were gavaged with FA at four doses (0, 25, 50, 100 mg/kg body weight). Dosing of 100 mg/kg significantly elevated GST activity in liver (P<0.03), and QR activities in liver and colonic mucosa (P<0.01 and P<0.02, respectively), suggesting that detoxifying enzymes are related to the blocking effect of FA on AOM-induced colon carcinogenesis.

Chemoprevention of Azoxymethane-induced Rat Colon Carcinogenesis by a Xanthine Oxidase Inhibitor, 1′-Acetoxychavicol Acetate

In our studies to find natural compounds with chemopreventive efficacy in foods, using azoxymethane (AOM)‐induced colonic aberrant crypt foci and colonic mucosal cell proliferation as biomarkers, a x an thine oxidase inhibitor, 1′‐acetoxychavicol acetate (ACA), present in the edible plant Languas galanga from Thailand was found to be effective. This study was conducted to test the ability of ACA to inhibit AOM‐induced colon tumorigenesis when it was fed to rats during the initiation or post‐initiation phase. Male F344 rats were given three weekly s.c. injections of AOM (15mg/kg body weight) to induce colonic neoplasms. They were fed diet containing 100 or 500 ppm ACA for 4 weeks, starting one week before the first dosing of AOM (the initiation feeding). The other groups were fed the ACA diet for 34 weeks, starting one week after the last AOM injection (the post‐initiation feeding). At the termination of the study (week 38), AOM had induced 71% incidence of colonic adenocarcinoma (12/17 rats). The initiation feeding with ACA caused significant reduction in the incidence of colon carcinoma (54% inhibition by 100 ppm ACA feeding and 77% inhibition by 500 ppm ACA feeding, P=0.03 and P=0.001, respectively). The post‐initiation feeding with ACA also suppressed the incidence of colonic carcinoma (45% inhibition by 100 ppm ACA feeding and 93% inhibition by 500 ppm ACA feeding, P=0.06 and P=0.00003, respectively). Such inhibition was dose‐dependent and was associated with suppression of proliferation biomarkers, such as ornithine decarboxylase activity in the colonic mucosa, and blood and colonic mucosal polyamine contents. ACA also elevated the activities of phase II enzymes, glutathione S‐transferase (GST) and quinone reductase (QR), in the liver and colon. These results indicate that ACA could inhibit the development of AOM‐induced colon tumorigenesis through its suppression of cell proliferation in the colonic mucosa and its induction of GST and QR. The results confirm our previous finding that ACA feeding effectively suppressed the development of colonic aberrant crypt foci. These findings suggest possible chemopreventive ability of ACA against colon tumorigenesis.

Inhibitory effect of NS-398, a selective cyclooxygenase-2 inhibitor, on azoxymethane-induced aberrant crypt foci in colon carcinogenesis of F344 rats

Prostaglandin E2, which is produced by cyclooxygenase (COX) during arachidonic acid metabolism, is considered to be related to colon Carcinogenesis. Therefore, the effect of NS‐398 (N‐(2‐cyclohexyl‐oxy‐4‐nitrophenyl)methanesulfonamide), a COX‐2 inhibitor, was examined in azoxymethane (AOM)‐induced colon Carcinogenesis in rats in this study. In the first experiment, groups 1–3 were treated with AOM (15 mg/kg, s.c.) 3 times at intervals of a week from 5 weeks of age. Groups 2 and 3 were respectively given 1 mg/kg and 10 mg/kg of NS‐398 in 5% gum arable aqueous solution 3 times per week by oral garage during the experiment. Six weeks after the first exposure to AOM, aberrant crypt foci (ACF) were counted in the colonic mucosa of all rats. The mean occurrence of ACF per length in rats given 1 mg/kg b.w. or 10 mg/kg b.w. of NS‐398 was reduced to 65.7% or 52.8%, respectively, of that in rats treated with only AOM. Levels of COX‐2 mRNA expression in groups treated with AOM, regardless of NS‐398, were slightly higher than that in the group treated with NS‐398 alone as judged from reverse transcription‐polymerase chain reaction analysis. In the second experiment, the effect of NS‐398 at different times, i.e., during initiation and post‐initiation, was examined. Treatment with NS‐398 in both phases significantly inhibited the appearance of ACF. The results imply that NS‐398 might have a chemopreventive potential.

Chemopreventive Effect of N‐(2‐Cyclohexyloxy‐4‐nitrophenyl)methane Sulfonamide (NS‐398), a Selective Cyclooxygenase‐2 Inhibitor, in Rat Colon Carcinogenesis Induced by Azoxymethane

Non‐steroidal anti‐inflammatory drugs (NSAIDs) such as sulindac and indomethacin inhibit colon carcinogenesis, and selective cyclooxygenase (COX)‐2 inhibitors are considered to be potential chemopreventive agents without the side effects of usual NSAIDs. We reported that NS‐398, N‐(2‐cyclohexyloxy‐4‐nitrophenyl)methane sulfonamide, suppressed the formation of preneoplastic lesions, aberrant crypt foci (ACF), induced by azoxymethane (AOM) in a short‐term assay of rat colon carcinogenesis. In this study, we examined the effects of long‐term NS‐398 administration on rat colon carcinogenesis. After three AOM treatments at weekly intervals, a dose of 10 mg/kg of NS‐398 in 5% Arabic gum solution was administered by gavage three times per week in group 2 until the termination of the experiment. Rats in group 1 were fed in a basal diet and given 5% Arabic gum solution alone after AOM treatment. At 40 weeks after the first AOM treatment, all rats were killed and the whole intestines including colon were examined. While the incidences of whole intestinal and colon neoplasms in group 1 were 84.6% and 80.8%, respectively, those in group 2 (given NS‐398) were 51.9% and 44.4% respectively (P=0.0177 and P=0.0103 by Fishers exact test, respectively). The multiplicities in group 2 (0.67 ± 0.78 and 0.48 ± 0.58) were also decreased significantly compared with those (1.39 ± 1.10 and 1.08 ± 0.74) in group 1 (P < 0.01 by Welchs method and P < 0.002 by Students t test, respectively). In immunohistochemistry for proliferative cell nuclear antigen (PCNA), the PCNA‐stained cell index (7.40 ± 0.5) in group 2 was significantly decreased from that in group 1 (14.03 ± 0.82) (P < 0.001 by Welchs method). The results suggest that NS‐398, a selective COX inhibitor, has a chemopreventive activity against colon carcinogenesis without side‐effects such as gastric ulceration.

Inhibitory effects of nabumetone, a cyclooxygenase-2 inhibitor, and esculetin, a lipoxygenase inhibitor, on N-methyl-N-nitrosourea-induced mammary carcinogenesis in rats.

We investigated the modifying effects of nabumetone, a relatively selective cyclooxygenase‐2 inhibitor, and esculetin, a lipoxygenase inhibitor, on N‐methyl‐N‐nitrosourea(MNU)‐induced mammary carcinogenesis in female Sprague‐Dawley rats. A total of 124 rats, 6 weeks old, were divided into 6 groups. At 50 days of age, groups 1, 2, and 3 were treated with MNU (50 mg/kg body weight) by subcutaneous injection. From the age of 8 weeks, groups 2 and 4 were given 0.03% nabumetone in the diet and groups 3 and 5 were given 0.03% esculetin in the diet. All rats were necropsied at the termination (25 weeks after the start of experiment). The incidence and multiplicity of neoplasms in group 2 were significantly smaller than those in group 1 (P<0.005 and P<0.001, respectively). The incidence of neoplasms in group 3 was also significantly smaller than that in group 1 (P<0.05). These results indicate that the intake of nabumetone or esculetin during the time corresponding to the post initiation phase has a chemopreventive effect on MNU‐induced mammary carcinogenesis in rats.

Regressive Effects of Various Chemopreventive Agents on Azoxymethane-induced Aberrant Crypt Foci in the Rat Colon

Regressive effects of four chemopreventive agents [5‐hydroxy‐4‐(2‐phenyl‐(E)‐ethenyl)‐2(5Hfura‐none (KYN‐54), S‐methyl metbanethiosulfonate (MMTS), chlorogenic acid (CA), and piroxicam] on azoxymethane (AOM)‐induced aberrant crypt foci (ACF) in the colon of male F344 rats were examined by dietary exposure. At six weeks of age, 60 rats of groups 1 through 5 received subcutaneous injections of AOM (15 mg/kg body weight) once a week for three weeks. Twelve weeks after the first carcinogen injection, wben the occurrence of ACF was maximal, the rats in groups 2 through 5 were started on diet containing the test chemicals as follows: group 2, KYN‐54 (0.02%); group 3, MMTS (0.01%); group 4, CA (0.025%); and group 5, piroxicam (0.0125%). Group 1 (20 rats) was kept on the basal diet alone, and group 6 (12 rate) served as an untreated control. Rats in each group were killed at 6, 12, 18, or 24 weeks after the start of the experiment, and the yield of ACF in the colon of each group at 18 or 24 weeks was compared with that at 12 weeks. The number of ACF per rat colon of each group at 18 or 24 weeks was smaller than that at 12 weeks. The reduction rates at 18 weeks were 7% in group 1 (AOM alone), 11% in group 2 (AOM+KYN‐54), 10% in group 3 (AOM+MMTS), 51% in group 4 (AOM + CA) (P 0.01), and 33% in group 5 (AOM+piroxicam) (P<0.02), while at 24 weeks they were 12%, 26%, 51% (P<0.002), 43% (P <0.05), and 70% (P <0.001), respectively. These results indicate that chemopreventive agents for large bowel carcinogenesis, i.e., KYN‐54, MMTS, CA, and piroxicam, are not only able to prevent the development of ACF, but also can regress ACF, which are regarded as precursor lesions of colorectal cancer.

β‐Catenin (Ctnnb1) Gene Mutations in Diethylnitrosamine (DEN)‐induced Liver Tumors in Male F344 Rats

Alterations in multiple phosphorylation sites on exon 3 of the β‐catenin gene have recently been implicated in hepatocarcinogenesis in humans as well as mice. To identify genetic alterations which could be involved in the chemical‐induced hepatocarcinogenesis of rats, we analyzed the status of the sites in the β‐catenin gene (Ctnnb1) of liver neoplasms induced by diethylnitrosamine (DEN) in male F344 rats, using the polymerase chain reaction‐single strand conformation polymorphism method. In the present investigation, we examined 35 hepatocellular neoplasms (28 adenomas and 7 carcinomas) for the expression of mutations in the region of the β‐catenin gene. Point mutation at codon 32, 35, 37 or 41, which has been reported in human and mouse liver cell carcinomas and/or other cancers, was recognized in eleven (31%) out of 35 lesions (8 adenomas and 3 carcinomas). Our results indicate that Ctnnb1 mutations may contribute to hepatocarcinogenesis in rats. Our finding that Ctnnb1 mutation was present in adenomas as well as carcinomas also suggests that the mutation is a relatively early event in DEN‐induced hepatocarcinogenesis in rats.

Effects of protocatechuic acid, S-methylmethanethiosulfonate or 5-hydroxy-4-(2-phenyl-(E)ethenyl)-2(5H)-furanone(KYN-54) on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced pulmonary carcinogenesis in mice

Modifying effects of dietary exposure of protocatechuic acid (PCA), a natural monophenolic compound, S-methylmethanethiosulfonate (MMTS), an organosulfur compound newly isolated from cauliflower, and 5-hydroxy-4-(2-phenyl-(E)ethenyl)-2(5H)-furanone (KYN-54), a novel retinoidal butenolide compound, on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (10 micromol, [corrected] single i.p. injection)-induced pulmonary carcinogenesis were examined in female A/J mice. Each of the test chemicals was given in diets during initiation or post-initiation phases (PCA, 1000 ppm; MMTS, 100 ppm; KYN-54, 200 ppm). All of these which had been proved to be chemopreventive mainly in digestive-organs carcinogenesis did not exert any preventive effect in this model when the incidence or multiplicity of pulmonary tumors (adenomas) of mice given NNK and the test chemical at the termination of the experiment (4 months) was compared to that of mice exposed to the carcinogen alone. In contrast, the multiplicity of lung tumors of mice receiving KYN-54 during the post-initiation phase was significantly larger than of the animals with NNK alone (P < 0.05), showing that KYN-54 has a promoting effect on pulmonary carcinogenesis in mice. These data indicate an organotropic activity of these compounds and suggest that candidate compounds for cancer chemoprevention need to be carefully examined for effectiveness in multiple organs by different models.

Induction of apoptosis in colonic epithelium treated with 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and its modulation by a P4501A2 inducer, β-naphthoflavone, in male F344 rats

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is one of the mutagenic heterocyclic amines derived from cooked meat. In long-term experiments using rodents, carcinogenicity of PhIP in colon, mammary gland and prostate has been demonstrated. In this study, an experiment was designed to determine the apoptosis-inducing capacity of PhIP in colonic epithelium, a target organ for PhIP carcinogenicity, and possible modulating effects of beta-naphthoflavone (beta-NF), a P4501A2 inducer, on the apoptosis in rats. Out of eight groups of male F344 rats, four were given beta-NF in diet (1000 ppm) for a week beginning at 5 weeks of age. Four groups were given PhIP (100 mg/kg body weight) by gavage at 6 weeks of age. Twenty-four hours after the dosing of PhIP, cell death with typical morphology of apoptosis was apparent in the colon and the apoptotic index was significantly greater (P < 0.01) than of the control rats without exposure to PhIP. Prior administration of beta-NF caused significant acceleration of the induction of apoptosis by PhIP. Since PhIP requires metabolic activation by P4501A2 to exert genotoxic activities, the modulating effect of beta-NF on the PhIP-induced apoptosis will be through a P4501A2-dependent mechanism. Such assay of apoptotic indices in the colon may be useful not only for the evaluation of genotoxicity and/or the initiating capability of chemical agents with potentials for colorectal cancer, but also for the analysis of modifying agents on the carcinogenesis in the large bowel.