Transgenic expression of human MGMT protects against azoxymethane-induced aberrant crypt foci and G to A mutations in the K-ras oncogene of mouse colon

Transgenic mice over-expressing MGMT, which codes for the humanprotein O⁶-alkylguanine-DNA alkyltransferase, are protectedfrom methylating agent-induced thymic lymphomas. In this studywe evaluated the ability of transgenic overexpression of MGMTin the colon to protect mice from the development of azoxymethane(AOM)-inducedaberrant crypt foci (ACF) and mutations in K-ras. Colonic alkyltransferasein MGMT+ transgenic mice was > 5-fold higher than in nontransgenics:10.5 ± 1.1 vs 2.2 ± 1.1 fmol/µg DNA, P => 0.0001. Mice received 20 mg AOM/kg i.p. at 6 weeks or 15mg AOM/kg at 6 and 7 weeks of age, and 8 wks later colons wereexamined for ACF. A significant protective effect of MGMT wasseen in mice given single dose of 20 mg AOM/kg. The incidenceof ACF/colon was lower in MGMT+ mice (2.0 ± 1.2) thanin nontransgenic mice (3.9 ± 1.8, P = 0.02). G to A mutationsin codon 12 of K-ras were detected by PCR-RFLP in ACF and inrandom samples of normal appearing mucosa. The incidence ofACF with mutant K-ras in MGMT transgenic mice (0.6 ±0.7/colon) was significantly reduced compared to nontransgenicmice (2.3 ± 1.7/colon, P = 0.02). We propose that AOMinduces at least two overlapping but not identical premalignantlesions (aberrant crypt foci and K-ras mutations) which canbe prevented by over-expression of MGMT. Thus, MGMT may protectcolonic mucosa from carcinogenesis involving methylating agentssuch as AOM.     

Intake of beer inhibits azoxymethane-induced colonic carcinogenesis in male Fischer 344 rats

Modulatory effects of beer consumption on azoxymethane (AOM)-induced rat colonic carcinogenesis in male Fischer 344 rats were investigated. Single cell gel electrophoresis assay indicated that DNA damage of colonocytes, induced by a single AOM injection (15 mg/kg body weight), was significantly reduced in rats fed beer or malt extract for 2 weeks. Examination of aberrant crypt foci (ACF) formation in colonic mucosa, induced by AOM (15 mg/kg body weight; twice weekly), revealed that feeding of beer during the whole experimental period of 5 weeks significantly reduced the number of ACF by 35%. In the post-initiation protocol, a reduction in ACF formation by 26% was not significant. The efficacy in inhibition of ACF formation varied with the brand of beer. ACF formation was significantly reduced in rats treated with freeze-dried beer (FD Beer), but not with ethanol, suggesting that nonvolatile components of beer are responsible for the reduction. Significant suppression of ACF formation was observed in groups treated with hot water extract of malt, especially with extracts of colored malts, although no reduction was observed by feeding with hops extract. A long-term experiment of 42 weeks indicated that intake of beer decreased tumor incidence by 22% and decreased the number of neoplastic lesions, including adenocarcinomas and adenomas, by 44%. These results suggest that components of beer have chemopreventive effects on colonic carcinogenesis induced by AOM and that intake of beer may contribute to a reduction in the risk of cancer susceptibility.     

Metformin suppresses azoxymethane‐induced colorectal aberrant crypt foci by activating AMP‐activated protein kinase

Metformin is widely used for the treatment of diabetes mellitus. Adenosine monophosphate‐activated protein kinase (AMPK) is known to be activated by metformin and to inhibit the mammalian target of rapamycin (mTOR) pathway. The mTOR pathway plays an important role in the protein translational machinery and cell proliferation. We examined the effect of metformin on the suppression of colorectal carcinogenesis in chemical carcinogen‐induced models. Seven‐wk‐old BALB/c mice were intraperitoneally (i.p.) injected with azoxymethane (AOM, 10 mg/kg) and then treated with or without metformin (250 mg/kg/d) for 6 wk (for the investigation of aberrant crypt foci [ACF] formation) or 32 wk (for polyp formation). We next investigated colonic epithelial proliferation using bromodeoxyuridine (BrdU) and the proliferating cell nuclear antigen (PCNA) labeling indices. Furthermore, to examine the indirect effect of metformin, the insulin resistance status and the serum lipid levels were assessed. Treatment with metformin significantly reduced ACF formation. The effect of metformin on colon polyp inhibition was relatively modest. No significant difference in body weight or glucose concentration was observed. The BrdU and PCNA indices decreased in mice treated with metformin. A Western blot analysis revealed that the phosphorylated mTOR, S6 kinase, and S6 protein levels in the colonic mucosa decreased significantly in mice treated with metformin. In conclusion, metformin suppresses colonic epithelial proliferation via the inhibition of the mTOR pathway through the activation of AMPK. As metformin is already used daily as an antidiabetic drug, it might be a safe and promising candidate for the chemoprevention of colorectal cancer. © 2010 Wiley‐Liss, Inc.     

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.      

Catalpa seed oil rich in 9t,11t,13c-conjugated linolenic acid suppresses the development of colonic aberrant crypt foci induced by azoxymethane in rats

Catalpa (Catalpa ovata) seed oil (CPO) is a unique oil that contains a high amount of 9trans,11trans,13cis-conjugated linolenic acid. In the present study, we investigated whether dietary administration with CPO affects the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) in male F344 rats to elucidate its possible cancer chemopreventive efficiency. Also, the effect of CPO on the fatty acid composition of liver tissue and colonic mucosa, the serum levels of total cholesterol and triglyceride, and the mRNA expression of cyclooxygenase (COX)-2 in the colonic mucosa were measured. In addition, the cell proliferation activity and apoptotic index in the colonic mucosa were estimated immunohistochemically. Animals were given two weekly subcutaneous injections of AOM (20 mg/kg body weight). They also received the experimental diet containing 0.01%, 0.1% or 1% CPO for 4 weeks, starting one week before the first dosing of AOM. AOM exposure produced a substantial number of ACF (99+/-28) at the end of the study (week 4). Dietary administration of CPO reduced the number of ACF (AOM + 0.01% CPO, 32+/-11, P<0.001; AOM + 0.1% CPO, 35+/-18, P<0.001; AOM + 1% CPO, 18+/-10, P<0.001). 9t,11t-conjugated linoleic acid was detected in the liver tissue and colonic mucosa of rats fed the CPO-containing diet. Additionally, dietary administration with CPO decreased the serum triglyceride level and the expression of COX-2 mRNA in the colonic mucosa. The indices of cell proliferation and apoptosis in the colonic mucosa of rats treated with AOM and 1% CPO have significant differences when compared with the AOM alone group. These findings suggest the possible chemopreventive activity of CPO in the early phase of colon carcinogenesis.     

Suppressing Effects of 6-(2,5-Dichlorophenyl)-2,4-diamino-1,3,5-triazine and Related Synthetic Compounds on Azoxymethane-induced Aberrant Crypt Foci in Rat Colon

The modifying effects of dietary administration of 6-(2,5-dichlorophenyl)-2,4-diamino-1,3,5-triazine and 5 related compounds on the occurrence of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) were investigated in rats. Male F344 rats were given s.c. injections of AOM (15 mg/kg body weight) once a week for 3 weeks to induce ACF. They also received the diet containing 200 ppm test compound for 5 weeks, starting one week before the first dosing of AOM. At the termination of experiment, all of the compounds had caused a significant reduction in ACF frequency, which might be associated with suppression of the expression of proliferation biomarkers. The apoptotic index in the colonic mucosal epithelium of rats killed at 6 h after the first AOM exposure revealed no blocking activity of the compounds.     

Troglitazone, a Ligand for Peroxisome Proliferator-activated Receptor γ Inhibits Chemically-induced Aberrant Crypt Foci in Rats

The biological roles of peroxisome proliferator-activated receptors (PPARs) in various diseases, including inflammation and cancer, have been highlighted recently. Although PPARγ ligand is suspected to play an important role in carcinogenesis, its effects on colon tumorigenesis remain undetermined. The present tune-course study was conducted to investigate possible modifying effects of a PPARγ ligand, troglitazone, on the development and growth of aberrant crypt foci (ACF), putative precursor lesions for colon carcinoma, induced by azoxymethane (AOM) or dextran sodium sulfate (DSS) in male F344 rats. Oral troglitazone (10 or 30 mg/kg body weight (b.w.)) significantly reduced AOM (two weekly subcutaneous injections, 20 mg/kg b.w.)-induced ACF. Treatment with troglitazone increased apoptosis and decreased polyamine content and ornithine decarboxylase (ODC) activity in the colonic mucosa of rats treated with AOM. Gastric gavage of troglitazone also inhibited colitis and ACF induced by DSS (1% in drinking water), in conjunction with increased apoptosis and reduced colonic mucosal polyamine level and ODC activity. Our results suggest that troglitazone, a synthetic PPARγ ligand, can inhibit the early stage of colon tumorigenesis with or without colitis.     

Chemopreventive effect of squalene on colon cancer

Epidemiologic and laboratory studies suggest a cancer protective effect and/or lack of a tumor promoting effect by dietary olive oil as compared with other types of non-marine oils. Squalene, a constituent of olive oil, and a key intermediate in cholesterol synthesis may be regarded as partially responsible for the beneficial effects of olive oil, which include decreased mortality rates among populations with high olive oil consumption. Thus, in this study we have assessed the chemopreventive efficacy of squalene on azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF). In addition, we measured the effect of squalene on serum cholesterol levels in the rats. Male F34 rats (5 weeks old) were fed the control diet (modified AIN-76A) or experimental diets containing 1% squalene or 320 p.p.m. sulindac. Two weeks later, all animals except those in vehicle (normal saline)-treated groups were s.c. injected with AOM (15 mg/kg body wt, once weekly for 2 weeks). At 16 weeks of age, all rats were killed, colons were evaluated for ACF and serum was assayed for the cholesterol levels. As expected, dietary administration of sulindac suppressed ACF development and reduced crypt multiplicity, i.e. number of aberrant crypts/focus. Administration of dietary squalene inhibited total ACF induction and crypt multiplicity by approximately >46% (P < 0.001). Further, squalene at a level of 1% did not show any significant effect on serum cholesterol levels. Our finding that squalene significantly suppresses colonic ACF formation and crypt multiplicity strengthens the hypothesis that squalene possesses chemopreventive activity against colon carcinogenesis.      

Citrus auraptene inhibits chemically induced colonic aberrant crypt foci in male F344 rats

The modifying effect of dietary administration of auraptene isolated from the peel of citrus fruit (Citrus natsudaidai Hayata) 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 ACF. They also received diets containing 100 or 500 p.p.m. auraptene for 5 weeks, starting 1 week before the first dose of AOM. At termination of the study (week 5) dietary administration of auraptene caused a significant reduction in the frequency of ACF in a dose- dependent manner (P < 0.05). Feeding of auraptene suppressed expression of cell proliferation biomarkers (5-bromo-2'-deoxyuridine labeling- index, ornithine decarboxylase activity, polyamine content and number of silver stained nucleolar organizer region protein particles) in the colonic mucosa and the occurrence of micronuclei caused by AOM. Also, auraptene increased the activities of phase II enzymes (glutathione S- transferase and quinone reductase) in the liver and colon. These findings might suggest that inhibition of AOM-induced ACF may be associated, in part, with increased activity of phase II enzymes in the liver and colon and suppression of cell proliferation in the colonic mucosa.      

Tumor formation is correlated with expression of beta-catenin-accumulated crypts in azoxymethane-induced colon carcinogenesis in mice

We have reported that β-catenin-accumulated crypts (BCAC) are independent of aberrant crypt foci (ACF) in the colonic mucosa of rats exposed to colorectal carcinogens, and we suggested that they may be premalignant lesions. In the present study, we performed a comparative study on the formation of the two types of early-appearing lesions (BCAC and ACF), and tumors of the colon in two mouse strains with different susceptibility to azoxymethane (AOM). SWR/J mice are known to be relatively susceptible to AOM, whereas AKR/J mice are reported to be virtually resistant. Both AKR/J and SWR/J mice, 6 weeks old, received subcutaneous injections of AOM (15 mg/kg body weight) once a week for 3 weeks, and were sacrificed at 16 and 41 weeks of age. Colons of the animals sacrificed at 16 and 41 weeks of age were processed to examine expression of the early-appearing lesions and neoplasms. Although AKR/J mice had a lower incidence of colonic tumors than SWR/J mice did, AKR/J mice showed a similar frequency of ACF to that in SWR/J mice. In both strains, ACF were detected at high frequency in the proximal colon, whereas tumors developed mainly in the distal colon. Importantly, the incidence of BCAC in SWR/J mice was significantly higher than that in AKR/J mice, and the highest frequency was observed in the distal segments of the colon. These results support the idea that BCAC are a reliable surrogate endpoint for colon carcinogenesis in mice.     

Suppression of Azoxymethane-induced Rat Colon Aberrant Crypt Foci by Dietary Protocatechuic Acid

The modifying effect of dietary exposure to protocatechuic acid (PCA) on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) was investigated in male F344 rats. The effects of PCA feeding on the silver-stained nucleolar organizer regions protein (AgNORs) count in the colonic epithelial cells and on the ornithine decarboxylase (ODC) activity in the colonic mucosa were also estimated. Animals were given weekly s.c. injections of AOM (15 mg/kg body weight) for 3 weeks to induce ACF. These rats were fed diet containing 1000 or 2000 ppm PCA for 5 weeks, starting one week before the first dosing of AOM. All rats were killed 2 weeks after the last AOM injection, to measure the number of ACF, ODC activity, and AgNORs count per nucleus in the colon. In rats given AOM and PCA, the frequency of ACF/colon was significantly decreased compared with that in rats given AOM alone ( P < 0.005 at 1000 and P < 0.05 at 2000 ppm). ODC activity in the colon of rats given AOM and PCA at both doses was also significantly lower than that of rats treated with AOM alone ( P < 0.05). Similarly, the mean AgNORs count in rats fed PCA was significantly smaller than that of rats treated with AOM alone ( P < 0.0001). Treatment with PCA alone did not affect these three biomarkers. These results provide further evidence that PCA could be a chemopreventive agent against rat colon carcinogenesis.     

Development of aberrant crypt foci in the colons of ob/ob and db/db mice: evidence that leptin is not a promoter

Leptin is elevated in obesity and has been suggested to increase the risk of colorectal cancer (CRC), although the evidence is conflicting. The objective of this study was to compare the susceptibility to colon carcinogenesis of db/db mice that have highly elevated circulating leptin and leptin-deficient ob/ob mice, both of which are obese. Seven-week-old male ob/ob, db/db, and WT mice received 4 weekly i.p. injections of 5 mg/kg azoxymethane (AOM) and were killed 14 wk later for the analysis of putative preneoplastic aberrant crypt foci (ACF). There were no differences in ACF number or multiplicity between ob/ob and db/db mice. Leptin has been shown to induce CYP2E1, the main enzyme that activates AOM, but we observed no differences in hepatic CYP2E1 activity or colonic CYP2E1 protein levels between ob/ob and db/db mice. We also induced ACF with 2 oral doses 3 d apart of 30 mg/kg methylnitrosourea (MNU), a direct-acting carcinogen. There were no differences in ACF number or multiplicity between the two groups of obese animals 5 wk following the last dose of MNU. The colonic mucosa of db/db mice expressed significantly lower mRNA levels of ObRa, the predominant short form of the leptin receptor, compared to ob/ob mice, and following i.p. injection with 1 mg/kg recombinant mouse leptin, exhibited significantly reduced p44/42 pMAPK compared to saline-treated controls. These results show that ObRa is functionally active in the colons of db/db mice. We conclude that leptin does not play a significant role in ACF development.     

Chemopreventive effect of 2-(allylthio)pyrazine (2-AP) on rat colon carcinogenesis induced by azoxymethane (AOM)

An investigation was conducted to assess the chemopreventive effects of 2-(allylthio)pyrazine (2-AP), synthesized for potential use as a chemopreventive agent, after administration during the pre-initiation and post-initiation stages in a rat colon carcinogenesis model with azoxymethane (AOM). One hundred, 5-week-old, male F344 rats were randomly divided into two experiments (n = 50 each). Experiment 1 rats were randomly divided into three groups: Group 1 rats were pre-treated with 2-AP (25 or 50 mg/kg body weight, 3 consecutive days through the route of intragastric intubations) before AOM (20 mg/kg body weight, single subcutaneous (s.c.) injection) initiation. Group 2 rats were treated with AOM alone. Group 3 rats were given 2-AP alone without AOM initiation. The animals were killed at the end of each experiment (week 5) and the aberrant crypt foci (ACF) of the colonic mucosa were assessed after staining with methylene blue. Experiment 2 rats were randomly divided into three groups: Group 1 rats were given 2-AP (10, 25 or 50 mg/kg body weight, five-times intragastric intubations per week for 5 weeks from week 3) after AOM (15 mg/kg body weight, three s.c. injections) initiation for 2 weeks. Group 2 rats were treated with AOM alone. Group 3 rats were given 2-AP alone without AOM initiation. The animals were killed at the end of the experiment (week 8) and the ACF of the colonic mucosa were quantified. Total numbers of ACF/colon in Group 1 rats (pre-treated with 2-AP) tended to decrease (2-AP, 50 mg/kg body weight) or increase (2-AP, 100 mg/kg body weight) depending on the dose level. Total numbers of ACF/colon in Group 1 rats (treated with AOM followed by 2-AP, all subgroups; 160.8 +/- 38.0; 161.8 +/- 38.1; 137.1 +/- 48.4) were decreased significantly compared with the values in Group 2 rats (AOM alone; 214.8 +/- 48.1) (P < 0.05 or 0.01). The highest dose group (2-AP, 50 mg/kg body weight) had the lowest levels of total numbers of ACF/colon among the three subgroups. Total numbers of aberrant crypts (AC)/colon of the highest dose group (340.1+/- 117.9) decreased significantly compared with the value for Group 2 rats (AOM alone; 545.1 +/- 38.3). These results thus suggest that 2-AP may have potential as a chemopreventive agent against rat colon carcinogenesis after administration of AOM during the post-initiation stage.     

Enhanced growth of colorectal aberrant crypt foci in fasted/refed rats involves changes in TGFbeta1 and p21CIP expressions

We previously demonstrated that fasting/refeeding enhances the initiation phase of liver and colorectal carcinogenesis in rats. The present study was undertaken to establish whether cycles of fasting/refeeding carried out during the promotion phase of carcinogenesis may also affect the formation of aberrant crypt foci (ACF), preneoplastic lesions induced in the colon by azoxymethane (AOM). We were also interested in studying whether this effect might be mediated by changes in the proliferation, apoptosis or expression of TGFbeta1 and p21CIP genes in the colon. 44 male Fisher 344 rats were given a single dose of AOM (20 mg/kg s.c.) and one week later, they were exposed to 5 cycles of 4 days fasting followed by 7-10 days of refeeding (refed rats); controls were regularly fed; the rats were killed 2, 8 or 30 days after the last cycle of fasting. Fasting/refeeding caused a dramatic increase in crypt multiplicity when compared with regularly fed rats (AC/ACF was 4.30 +/- 1.3 in refed and 2.38 +/- 0.4 in regularly fed rats, P < 0.005 means +/- SD), while no significant changes were observed in the number of ACF/colon. In the two experimental groups, cell proliferation was higher in ACF than in the surrounding mucosa, but proliferative indexes were higher and the apoptotic index lower in ACF of refed rats compared with regularly fed rats. TGFbeta1 expression was higher in the ACF of refed rats than in those of fully fed controls while p21CIP was less expressed in refed rats than in controls. These results suggest that fasting/refeeding is a risk factor for colon cancer and must be taken into account for cancer prevention in humans.     

Initiation and promotion of colonic aberrant crypt foci in rats by 5-hydroxymethy1-2-furaldehyde in thermolyzed sucrose

We have previously shown that thermolyzed sucrose in the dietpromotes the growth of aberrant crypt foci (ACF) in the rat.HPLC analysis of the light caramel colored product showed thatit contained 1% 5-hydroxymethy1-2-furaldehyde (HMF), confirmedby mass and NMR spectroscopy. To determine whether HMF was responsiblefor the promotion of ACF by thermolyzed sucrose, 45 F344 femalerats were initiated with the colon carcinogen azoxymethane (AOM),and a week later were randomized to four groups receiving AIN-76diets containing untreated sucrose, 20% thermolyzed sucrose,20% butanol extracted thermolyzed sucrose (HMF free) or 1% HMF.Thermolyzed sucrose in the diet led to larger ACF as previouslyobserved. Thermolyzed sucrose extracted to remove HMF, did notaffect ACF size, but 1% HMF added to the diet led to a largerACF both with relation to average size and number of ACF oflarger sizes (P < 0.05). To determine whether HMF had initiatingeffects, 172 female F344 rats were given water, HMF (at dosesto 300 mg/kg) or AOM (5 mg/kg) by gavage twice and the totalnumber of ACF was scored 30 days later. The results demonstratedthat HMF induces ACF in a dose-dependent manner (P < 0.02),though the effect was much weaker than that of AOM. We concludethat sugar heated under household cooking conditions may actas both an initiator and a promoter of colon cancer becauseof the presence of HMF.     

Troglitazone, a ligand for peroxisome proliferator-activated receptor gamma, inhibits chemically-induced aberrant crypt foci in rats.

The biological roles of peroxisome proliferator-activated receptors (PPARs) in various diseases, including inflammation and cancer, have been highlighted recently. Although PPARgamma ligand is suspected to play an important role in carcinogenesis, its effects on colon tumorigenesis remain undetermined. The present time-course study was conducted to investigate possible modifying effects of a PPARgamma ligand, troglitazone, on the development and growth of aberrant crypt foci (ACF), putative precursor lesions for colon carcinoma, induced by azoxymethane (AOM) or dextran sodium sulfate (DSS) in male F344 rats. Oral troglitazone (10 or 30 mg / kg body weight (b.w.)) significantly reduced AOM (two weekly subcutaneous injections, 20 mg / kg b.w.)-induced ACF. Treatment with troglitazone increased apoptosis and decreased polyamine content and ornithine decarboxylase (ODC) activity in the colonic mucosa of rats treated with AOM. Gastric gavage of troglitazone also inhibited colitis and ACF induced by DSS (1% in drinking water), in conjunction with increased apoptosis and reduced colonic mucosal polyamine level and ODC activity. Our results suggest that troglitazone, a synthetic PPARgamma ligand, can inhibit the early stage of colon tumorigenesis with or without colitis.     

Resveratrol depresses the growth of colorectal aberrant crypt foci by affecting bax and p21(CIP) expression

We investigated whether resveratrol (RV) affects azoxymethane (AOM)-induced colon carcinogenesis, by administering RV (200 microg/kg/day in drinking water) to male F344 rats for 100 days, beginning 10 days before carcinogen treatment (two weekly doses of 15 mg/kg AOM). Aberrant crypt foci (ACF) were isolated and proliferation, apoptosis and expression of the cell cycle genes bax and p21 were determined. RV significantly reduced the number of ACF/colon [25.7 +/- 3.6 (mean +/- SEM) versus 39.4 +/- 3.3 in controls; P < 0.01] and their multiplicity (2.7 +/- 0.3 versus 4.9 +/- 0.6 in controls; P < 0.01), and also abolished large ACF. In RV-treated rats, bax expression was enhanced in ACF but not in the surrounding mucosa. In both controls and RV-treated rats, proliferation was higher in ACF than in normal mucosa. p21 was expressed in ACF of controls and of RV-treated rats and in normal mucosa of controls, but was lost in normal mucosa of RV-treated animals. In conclusion, the results suggest a protective role of RV in colon carcinogenesis with a mechanism involving changes in bax and p21 expression.     

Fasting/re-feeding before initiation enhances the growth of aberrant crypt foci induced by azoxymethane in rat colon and rectum

In contrast to the protective effect of chronic caloric restriction on tumor development, we have shown that fasting sustained tumor initiation in rat liver by a non-initiating dose of diethylnitrosamine. Here we investigated whether fasting had a similar favorable effect on initiation in the colorectal mucosa in 80 male F344 rats. Animals fasted for 4 days were given a single s.c. dose of azoxymethane (AOM) (20 mg/kg) on the first day of re-feeding, and rates of kinetic proliferative parameters, and development of the pre-neoplastic lesions such as aberrant crypt foci (ACF), were evaluated. Starvation before AOM treatment enhanced the growth of ACF, as shown by the significantly higher crypt multiplicity of fasted/re-fed rats as compared with fully fed rats (3.97 ± 0.50 vs. 2.64 ± 0.20, p ≤ 0.025). This difference was associated with perturbations in cell death and cell proliferation. Fasting induced apoptosis and depressed cell division, while re-feeding had opposite effects, resulting in a higher percentage of S-phase cells at the time of AOM injection and 2 days thereafter. Starvation-induced apoptosis may represent the mitogenic stimulus to an increase in the number of cells susceptible to AOM damage, and may favor its fixation, leading to enhanced growth of ACF. Our data therefore suggest that fasting/re-feeding enhances colon cancer. Int. J. Cancer 77:286–294, 1998.© 1998 Wiley-Liss, Inc.     

Inhibition of Azoxymethane-induced Colorectal Aberrant Crypt Foci in Mice Fed a High-fat Diet by Pleurotus eryngii (Eringi) and Hypsizygus marmoreus (Bunashimeji)

Obesity markedly increases the risk of colorectal cancer. Recently, the preventive effects of edible mushroomson triglyceride elevation and visceral fat accumulation have been reported. Here, the effects of Pleurotus eryngii(Eringi) and Hypsizygus marmoreus (Bunashimeji) on azoxymethane (AOM)-induced aberrant crypt foci (ACF;precancerous lesions) in the colorectums of mice fed a high-fat diet were examined. Eringi (ER) and Bunashimeji(BU) mushroom powder samples were used. Six-week-old male C57BL/6J mice received an intraperitonealinjection of AOM (10 mg/kg) once a week for two weeks, and were sacrificed and dissected at 6 weeks after thestart of the experiment. After the initiation of the experiment, they received a normal diet (ND), high-fat diet(HFD), HFD + ER (1 or 5% of diet), or HFD + BU (1 or 5% of diet). As a result, body and fat weights weresignificantly lower in the 5% ER and BU groups than in the HFD group. Liver triglyceride levels were alsosignificantly lower in the 5% ER and BU groups. Total liver cholesterol levels were significantly lower in the5% ER group. The numbers of ACF (especially large ACF) showed strong inhibitory effects in both ER and BUgroups. Measurement of the cell proliferation marker Ki-67 labeling index in the colonic mucosa demonstratedmore significant suppression in both ER and BU groups than in the HFD group. These results suggest that thesimultaneous intake of ER and BU may inhibit colorectal tumorigenesis in HFD-fed mice.     

Inhibition of azoxymethane-induced aberrant crypt foci in rats by natural compounds, caffeine, quercetin and morin.

The modifying effects of dietary administration of natural compounds, caffeine, quercetin and morin, which are present in our daily food, on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) were investigated in rats and compared to that of a metabolic inhibitor of AOM, disulfiram. Male F344 rats were given s. c. injections of AOM (15 mg/kg body weight) once a week for 3 weeks to induce ACF. They also received the experimental diets containing one of test compounds (500 ppm) for 5 weeks, starting one week before the first dosing of AOM. At the termination of the study (week 5), AOM exposure produced 101.0+/-10.2 ACF/rat. Disulfiram almost completely inhibited ACF development (0.60+/-0.90, 99% reduction). Dietary administration of test compounds caused significant reduction in the frequency of ACF: caffeine (70.4+/-16.6, 30% reduction), quercetin (53.0+/-8.4, 48% reduction) and morin (37. 6+/-18.1, 63% reduction). Numbers of cells positive for proliferative cell nuclear antigen in ACF and surrounding crypts were lowered by feeding of test compounds. Feeding of these test compounds also suppressed polyamine content in the colonic mucosa and blood as did disulfiram. These findings might indicate possible chemopreventive effects of caffeine, quercetin and morin, through their modulation of cell proliferation activity in crypt cells, on colon tumorigenesis.