Kunihiro Kawabata

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.

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. β‐Glucuronidase activity, PGE2 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.

Chemopreventive effect of dietary flavonoid morin on chemically induced rat tongue carcinogenesis

The modifying effects of dietary exposure of the flavonoid morin on 4‐nitroquinoline 1‐oxide (4‐NQO)‐induced tongue tumorigenesis, the activities of phase II detoxifying enzymes glutathione S‐transferase (GST) and quinone reductase (QR) in liver and tongue, and cell proliferation activity in tongue were investigated in male F344 rats. At 7 weeks of age, all animals except those treated with morin alone and control group were given 4‐NQO (20 ppm) in drinking water for 8 weeks to induce oral neoplasms. Starting 7 days before 4‐NQO exposure, experimental groups were fed experimental diets containing morin (100 and 500 ppm) for 10 weeks (“initiation feeding”). Starting 1 week after the cessation of exposure to 4‐NQO, other experimental groups given 4‐NQO and a basal diet were given experimental diets for 22 weeks (“post‐initiation feeding”). At week 32 week, “initiation feeding” of morin caused a significant reduction in the incidence of tongue carcinoma (by 44–100%). “Post‐initiation feeding” with morin also significantly decreased the frequency of tongue carcinoma (by 44%). Morin feeding elevated liver GST and QR activities and GST activity in the anterior portion of tongue. Feeding with morin significantly lowered QR activity of the posterior part of the tongue. Dietary exposure to morin significantly decreased the proliferating cell nuclear antigen‐positive index in the posterior portion. Also, morin feeding lowered tongue polyamine levels, especially in the “post‐initiation feeding” group. Our results indicate that morin acts as a chemopreventive agent against tongue carcinogenesis induced by 4‐NQO through modification of detoxifying enzyme activities and/or cell proliferation activities. Int. J. Cancer 83:381–386, 1999.

Chemopreventive Effect of Curcumin on N‐Nitrosomethylbenzylamine‐induced Esophageal Carcinogenesis in Rats

Modifying effects of curcumin (derived from the rhizome of Curcuma longa L.) during the initiation or post‐initiation phase of N‐nitrosomethylbenzylamine (NMBA)‐induced esophageal carcinogenesis were investigated in male F344 rats. Five‐week‐old rats were divided into 5 groups, and groups 1, 2 and 3 were given intraperitoneal injections of NMBA (0.5 mg/kg body weight/injection 15 times) for 5 weeks from 7 weeks old to induce esophageal neoplasms. Groups 2 and 3 were fed the diet containing 500 ppm curcumin during the initiation and post‐initiation phases, respectively. Group 4 was given the diet containing curcumin throughout the experiment, and group 5 was kept on the basal diet alone and served as an untreated control. Incidence and multiplicity of esophageal neoplasms of group 1 (NMBA alone) were 66.7% and 0.83 ±0.70, respectively. Those of groups 2 and 3 were significantly less than those of group 1 (39.3%, 0.46±0.64, P < 0.05; 33.3%, 0.36±0.56, P < 0.05, respectively). Furthermore, the incidence and multiplicity of esophageal preneoplastic lesions (moderate or severe epithelial dysplasia) of group 2 (57.1%, 0.61±0.57; 40%, 0.29±0.46) or 3 (56.7%, 0.67±0.66; 23.3%, 0.23±0.43) were less than those of group 1 (100%, 1.67±0.70; 70.8%, 0.92±0.72) (P < 0.05). In this experiment, feeding of curcumin significantly decreased the expression of cell proliferation biomarkers (5‐bromo‐2′‐deoxyuridine labeling index) in the non‐lesional esophageal epithelium (P < 0.01). These findings indicate that curcumin inhibits NMBA‐induced esophageal carcinogenesis when given during the post initiation as well as initiation phase. This inhibition may be related to suppression of the increased cell proliferation induced by NMBA in the esophageal epithelium.

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.

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.

Dietary Crocin Inhibits Colitis and Colitis-Associated Colorectal Carcinogenesis in Male ICR Mice

A natural carotenoid crocin is contained in saffron and gardenia flowers (crocuses and gardenias) and is used as a food colorant. This study reports the potential inhibitory effects of crocin against inflammation-associated mouse colon carcinogenesis and chemically induced colitis in male ICR mice. In the first experiment, dietary crocin significantly inhibited the development of colonic adenocarcinomas induced by azoxymethane (AOM) and dextran sodium sulfate (DSS) in mice by week 18. Crocin feeding also suppressed the proliferation and immunohistochemical expression of nuclear factor- (NF-) κB but increased the NF-E2-related factor 2 (Nrf2) expression, in adenocarcinoma cells. In the second experiment, dietary feeding with crocin for 4 weeks was able to inhibit DSS-induced colitis and decrease the mRNA expression of tumor necrosis factor α, interleukin- (IL-) 1β, IL-6, interferon γ, NF-κB, cyclooxygenase-2, and inducible nitric oxide synthase in the colorectal mucosa and increased the Nrf2 mRNA expression. Our results suggest that dietary crocin suppresses chemically induced colitis and colitis-related colon carcinogenesis in mice, at least partly by inhibiting inflammation and the mRNA expression of certain proinflammatory cytokines and inducible inflammatory enzymes. Therefore, crocin is a candidate for the prevention of colitis and inflammation-associated colon carcinogenesis.

Suppression of N-nitrosomethylbenzylamine-induced rat esophageal tumorigenesis by dietary feeding of 1'-acetoxychavicol acetate.

The modifying effects of 1′‐acetoxychavicol acetate (ACA) on N‐nitrosomethylbenzylamine (NMBA)‐induced esophageal tumorigenesis were investigated in male F344 rats. At 5 weeks of age, all test animals, except those given the test chemical alone, and the control rats received s.c. injections of NMBA (0.5 mg/kg body weight/injection, three times per week) for 5 weeks. At the termination of the study (20 weeks), 75% of rats treated with NMBA alone had esophageal neoplasms (papillomas). However, the groups given a dose of 500 ppm ACA during the initiation phase developed a significantly reduced incidence of tumors (29%; P < 0.01). Exposure to ACA (500 ppm) during the post‐initiation phase also decreased the frequency of the tumors (38%; P < 0.05). A reduction of the incidence of preneoplastic lesions (hyperplasia or dysplasia) was obtained when ACA was administered in the initiation phase (P < 0.01). Cell proliferation in the esophageal epithelium, determined by assay of proliferating cell nuclear antigen (PCNA), was lowered by ACA (P < 0.05). Blood polyamine contents in rats given NMBA and the test compound were also smaller than those of rats given the carcinogen (P < 0.05). These findings suggest that dietary ACA is effective in inhibiting the development of esophageal tumors by NMBA when given during the initiation or post‐initiation phase, and such inhibition is related to suppression of cell proliferation in the esophageal epithelium.

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.