Dietary sphingolipids in colorectal cancer prevention.

Sphingolipids are widespread membrane components that are found in all eukaryotic cells. They consist of a long chain sphingoid-base, usually sphingosine, which is acylated at the 2-amino position, forming a ceramide. All together, sphingolipids may represent the most structurally diverse category of lipids in nature. There is no known nutritional requirement for sphingolipids. Nonetheless, studies with experimental animals have shown that consumption of sphingolipids inhibits colon carcinogenesis, reduces serum low-density lipoprotein cholesterol and elevates high-density lipoproteins, which suggest that they are 'functional' components of food. In animal models (CF1 mice) sphingomyelin supplementation reduces the number of aberrant colonic crypt foci by approximately 70% and, with longer feeding, reduces the number of colonic adenocarcinomas. A possible mechanism of action of sphingolipids in suppressing colon carcinogenesis is that exogenously supplied sphingolipids bypass a sphingolipid signalling defect that is important in cancer (for example, a loss of cellular sphingomyelin turnover to produce ceramide and sphingosine). Indirect evidence suggests that sphingolipids can inhibit colon cancer in humans: sphingosine and ceramide induce apoptosis in a human adenocarcinoma cell line and feeding sphingolipids to Min mice reduces the number of colon tumours.     

Diet Induced Obesity Increases the Risk of Colonic Tumorigenesis in Mice

A large body of epidemiological data indicates that obesity increases the risk of colon cancer in humans. There are limited studies using rodent models where the relationship between obesity and colon cancer has been studied. In this study, wild-type diet-induced obese (DIO) mice and lean wild-type controls were used to investigate the influence of obesity on the risk of colon cancer. We hypothesized that the obese phenotype would exhibit increased colonic tumorigenesis. Colon cancer was chemically induced by injecting the mice with azoxymethane (AOM) at levels that we experimentally determined to result in equivalent AOM concentrations in circulating blood. Risk of colon cancer was assessed via microscopic examination of entire colons for aberrant crypts, aberrant crypt foci and proliferation levels. The DIO mice were found to have significantly more aberrant crypts and aberrant crypt foci as well as increased proliferation of colonocytes per mouse compared to wild-type control mice, supporting the epidemiological data that obesity increases the risk of colonic tumorigenesis.     

Silk protein, sericin, suppresses colon carcinogenesis induced by 1,2-dimethylhydrazine in mice

Male 5-week old ICR mice were examined for the effect of feeding silk protein, sericin on colon carcinogenesis. In experiment 1, mice were fed the diets supplemented with 1.5% or 3% sericin for five weeks, and given weekly injections of 1,2-dimethylhydrazine (DMH) for the initial three weeks. Supplemental sericin caused a dose-dependent decrease in the development of colonic aberrant crypt foci. In experiment 2, mice were fed the diet supplemented with 3% sericin for 115 days, and given weekly injections of DMH for the initial ten weeks. The incidence and number of colon tumors were suppressed by consumption of sericin. The results suggest a potential usefulness of sericin as a chemopreventive agent for colon carcinogenesis.     

Risk of Colonic Cancer is Not Higher in the Obese Lepob Mouse Model Compared to Lean Littermates

Epidemiological data suggest that obesity increases the risk of colorectal cancer in humans. Given that diet-induced obesity mouse models verified the epidemiological data, the present study aimed to determine whether obese C57BL/6J-Lep^ob male mice (a different obesity in vivo model) were at greater risk of colonic cancer than their lean male littermates. Risk of colonic tumorigenesis was assessed by numbers of aberrant crypts, aberrant crypt foci and colonic tumors. Proliferation of the colonic epithelia was assessed histochemically following administration of BrdU. Availability of the procarcinogen, azoxymethane (AOM) to target tissues was assessed by quantifying via HPLC plasma AOM concentrations during the 60 min period following AOM injection. When obese and lean mice were injected with azoxymethane (AOM) at doses calculated to provide equivalent AOM levels per kg lean body mass, obese animals had significantly fewer aberrant crypts/colon and fewer aberrant crypt foci/colon than the lean animals. Tumors were identified in the colonic mucosa of lean (4 tumors in 14 mice) but not obese (0 tumors in 15 mice) mice. Colonic cell proliferation was not significantly different for obese and lean mice. Because these results were unexpected, plasma AOM concentrations were measured and were found to be lower in the obese than lean mice. When plasma AOM levels were comparable for the lean and obese mice, the Lep^ob mice continued to have significantly fewer aberrant crypt foci/colon than the lean mice, but differences were not statistically different for aberrant crypts/colon. Interestingly, obese Lep^ob mice did not exhibit increased risk of colonic cancer as expected. Instead, Lep^ob mice exhibited equivalent or lower risk of colon cancer when compared to the lean group. These results taken together with in vivo results from diet-induced obesity studies, imply that leptin may be responsible for the increased risk of colon cancer associated with obesity.     

P53 gene mutation increases progastrin dependent colonic proliferation and colon cancer formation in mice

Transgenic mice overexpressing human progastrin (hGAS) show colonic crypt hyper-proliferation and elevated susceptibility to colon carcinogenesis. We aimed to investigate effects of p53 mutation on colon carcinogenesis in hGAS mice. We show that introducing a p53 gene mutation further increases progastrin dependent BrdU labeling and results in markedly elevated number of aberrant crypt foci (ACF) and colonic tumors. We demonstrate that hGAS/Lgr5-GFP mice have higher number of Lgr5+ colonic stem cells per crypt when compared to Lgr5-GFP mice indicating that progastrin changes crypt biology through increased stem cell numbers and additional p53 mutation leads to more aggressive phenotype in this murine colon cancer model.     

Reduction in formation of 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP)-induced aberrant crypt foci in the rat colon by docosahexaenoic acid (DHA)

Docosahexaenoic acid (DHA), a major component of fish oil, suppresses the formation and growth of aberrant crypt foci induced by 1,2- dimethylhydrazine and azoxymethane. In the present study we examined the effects of intragastric gavage administration of DHA on the yield of rat colonic aberrant crypt foci due to treatment with a heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which induces colon cancer in male F344 rats and is considered to be a possible human colon carcinogen. Male F344 rats were given a standard diet (AIN-76A) and received 10 doses of PhIP (75 mg/kg body wt, by intragastric intubation, on days 1-5 and 8-12) with or without intragastric application of 1 ml DHA 4 h prior to each carcinogen treatment, followed by further DHA dosing. The numbers of PhIP-induced aberrant crypt foci per colon after 4 and 12 weeks DHA administration were significantly reduced to 47 and 38% respectively of the values obtained when PhIP alone was used. The mean number of aberrant crypts per focus was also decreased by DHA treatment. At week 4 the PhIP-DNA adduct levels in the colon of rats from the PhIP+DHA group were approximately two thirds of the PhIP group value. The results thus suggest that DHA exerts a preventive effect on PhIP-induced colon carcinogenesis.      

Induction of aberrant crypt foci in the large intestine of F344 rats by oral administration of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

Carcinogenicity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to rat colon was investigated using the appearance of colonic aberrant crypt (AC), a preneoplastic lesion, as a marker. The number of AC foci per colon at experimental week 4 was 1.3 +/- 0.8; almost half the level of AC foci induced by 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), which is a known colon carcinogen. No ACs were observed in rats of the control group. A repeat experiment showed that induction of AC foci by PhIP administration was reproducible and a significant increase in the number of AC foci, 3.0 +/- 0.0, was observed after 12 weeks of PhIP administration. The majority of ACs induced by PhIP were localized in the distal part of the colon. The distribution was similar to those induced by Glu-P-1 and 1,2-dimethylhydrazine. Those data suggested that PhIP is possibly carcinogenic to rat colon.     

Inhibition of aberrant crypt growth by non-steroidal anti-inflammatory agents and differentiation agents in the rat colon

Aberrant crypts are aggregates of single to multiple colonic crypts evidencing hallmarks of dysplasia and may be the earliest detectable pathological lesions for colon cancer. The aberrant crypt assay has been developed in 2 protocols. In one, putative chemoprevention agents are tested for inhibitory effects when administered concomitantly with a carcinogen. In the other, the objective of this study, aberrant crypts were induced in F344 rats by parenteral injection of the colon carcinogen azoxymeth-ane (AOM) and allowed to develop for 4 weeks, when an average of 90-100 aberrant crypt foci per colon were found in the methylene blue-stained colon. Then, during the second 4 weeks of the experiment, aberrant crypts were allowed to further develop to a frequency of > 150 foci per colon, a time when multi-crypt foci were observed. During this time we tested the inhibitory effects of 4 analgesic drugs and 2 differentiation agents for effects of aberrant crypt growth and development. We found the non-steroidal anti-inflammatory drugs piroxicam, aspirin and ibuprofen, but not acetaminophen, to be effective in suppressing aberrant crypt formation or the progression to foci of multiple aberrant crypts. Treatment with chemo-suppressing agents 13-cis-retinoic acid (13-cRA) and 4-hydroxy-phenretinamide (4-HPR), known differentiating agents, however, did suppress expansion of aberrant crypt foci, with 13-cRA being the much more potent agent.     

Prevention of colonic preneoplastic lesions by the probiotic Lactobacillus acidophilus NCFMTM in F344 rats

The experiments described here were aimed at developing novel probiotic strains that may aid in the reduction of colon cancer risk. We assessed the potential anticancer properties of Lactobacillus acidophilus NCFMTM in male F344 rats using inhibition of the formation of azoxymethane (AOM)-induced aberrant crypt foci (ACF) in the colon as the measure of preventive efficacy. At 6 weeks of age, groups of rats were fed the experimental diets containing 0, 2% or 4% lyophilized cultures of L. acidophilus NCFMTM. At 7 weeks of age, all animals in each dietary group, except the vehicle-treated rats, were s.c. injected with AOM (15 mg/kg body weight) once weekly for two weeks. The vehicle-treated groups were given s.c. injections of normal saline. All rats were necropsied 10 weeks after the last AOM injection and ACF in formalin-fixed, methylene blue-stained colonic tissues were counted under the light microscope. The contents of the cecum were analyzed for bacterial beta-glucuronidase activity. Diet supplementation with the probiotic strain NCFMTM significantly suppressed AOM-induction of colonic ACF, in terms of total number, as well as crypt multiplicity and number of ACF/cm2 colon (P<0.01 - 0.001). NCFMTM inhibited AOM-induced colonic ACF formation in a dose-dependent manner (P<0.01). A significant dose-dependent reduction of cecal beta-glucuronidase activities was observed in the rats fed 2% (P<0.04) and 4% (P<0.0001) NCFMTM. These results suggest that Lactobacillus acidophilus NCFMTM may potentially prevent colon cancer development. Further studies are warranted to determine the full potential of this probiotic strain in preclinical efficacy studies.     

Chemoprevention for colorectal tumorigenesis associated with chronic colitis in mice via apoptosis

The mechanism of the suppressive effect of nonsteroidal anti-inflammatory drugs in azoxymethan and dextran sulfate sodium-induced colonic aberrant crypt foci/tumors associated with chronic colitis in mice was studied. With administration of sulindac, a cyclooxygenase-1 and -2 inhibitor, the mean number of colonic aberrant crypt foci/tumors was significantly smaller than that of controls. There was no significant difference in prostaglandin E2 content in the colonic mucosa between the groups. Furthermore, nimesulid, a cyclooxygenase-2 selective inhibitor, also suppressed colonic aberrant crypt foci/tumors as well as sulindac. Administration of nimesulid caused apoptosis indices to be significantly higher along with cyclooxygenase-2 expression being significantly lower than in controls. Apoptosis indices of 400 ppm group of nimesulid were significantly higher than that of 200 ppm group. Nonsteroidal anti-inflammatory drugs distinctly suppress the occurrence of aberrant crypt foci/tumors in this murine colitis-associated neoplasia model. Induction of apoptosis is a more important factor for chemoprevention than this reduction of prostaglandin E2.     

Induction of Aberrant Crypt Foci in the Large Intestine of F344 Rats by Oral Administration of 2-Amino-l-methyl-6-phenylimidazo[4,5-b]pyridine

Carcinogenicity of 2-amino-l-methyl-6-phenylimidazo[4,5- b ]pyridine (PhIP) to rat colon was investigated using the appearance of colonic aberrant crypt (AC), a preneoplastic lesion, as a marker. The number of AC foci per colon at experimental week 4 was 1.3 ± 0.8; almost half the level of AC foci induced by 2-amino-6-methyIdipyrido[l,2- a :3',2'- d ]imidazole (Glu-P-1), which is a known colon carcinogen. No ACs were observed in rats of the control group. A repeat experiment showed that induction of AC foci by PhIP administration was reproducible and a significant increase in the number of AC foci, 3.0 ±0.0, was observed after 12 weeks of PhIP administration. The majority of ACs induced by PhIP were localized in the distal part of the colon. The distribution was similar to those induced by Glu-P-1 and 1,2-dimethylhydrazine. Those data suggested that PhIP is possibly carcinogenic to rat colon.     

High fat diet elevates the activity of inducible nitric oxide synthase and 1,2-dimethylhydrazine-induced aberrant crypt foci in colon of rats

Rats were examined for the effect of dietary level of fat (beef tallow or safflower oil) on colonic activity of inducible nitric oxide synthase (iNOS) which has been suggested to be associated with colon carcinogenesis. In experiment 1, feeding high fat diets (20%) for 3 weeks caused higher activity of the iNOS compared to feeding low fat diets (5%). In experiment 2, rats were fed 20% or 5% beef tallow diet for 11 or 32 days, and given an injection of a carcinogen, 1,2-dimethylhydrazine on day 4. The activity of colonic iNOS and the development of colonic aberrant crypt foci were enhanced by high fat diet.     

Promotion of aberrant crypt foci and cancer in rat colon by thermolyzed protein.

BACKGROUND: We have previously shown that thermolyzed protein (casein) cooked with fat in the diet of the rat promotes the growth of aberrant crypt foci (putative precursors of colon cancer) assessed at 100 days. PURPOSE: To determine how thermolysis affects this promotion, we examined thermolysis conditions, quantity of thermolyzed protein in the diet, and duration of thermolysis. To determine whether the previous finding of promotion of aberrant crypt foci corresponds to promotion of cancers assessed much later, we carried out promotion studies until colon cancers appeared. METHODS: F344 rats were given an initiating dose of azoxymethane and were then randomly allocated to groups receiving diets differing in their quantity and quality of casein. The groups were examined for aberrant crypt foci and tumors in the colon. RESULTS: Aberrant crypt foci were promoted by diets containing thermolyzed casein (180 degrees C, 2 hours). Promotion increased with increasing level of thermolyzed casein in the diet (to 20%) and with increasing thermolysis time (to 4 hours). The number of animals with polyps and cancers was higher in the animals receiving thermolyzed protein (2 hours), 16/23 versus 9/26 (P less than .05) and 10/26 versus 3/27 (P less than .05), respectively. The number of aberrant crypts per focus and the number of large aberrant crypt foci were higher in the tumor-bearing animals. CONCLUSIONS: Thermolyzed casein promotes early colonic precursor lesions in a dose-dependent and thermolysis time-dependent manner; thermolyzed casein also promotes colon cancer. IMPLICATIONS: The promoter formed on thermolysis could be involved in colon cancers associated with diets cooked at elevated temperatures, such as can occur with high-fat diets.     

Dietary cholesterol enhances the induction and development of colonic preneoplastic lesions in C57BL/6J and BALB/cJ mice treated with azoxymethane.

The effect of dietary cholesterol on the induction and development of colonic precursor lesions was determined in two mouse strains, C57BL/6J and BALB/cJ, which differ in their metabolism of cholesterol. Mice were randomized into four groups and fed a cholesterol-free or a 1.25% cholesterol diet during and/or subsequent to four weekly injections of azoxymethane (5 mg/kg body wt.). Dietary cholesterol significantly increased the number of aberrant crypt foci (P less than 0.0001), enhanced cell proliferation (P less than 0.0001) and induced alterations in the proliferative pattern and crypt morphometrics in the colonic epithelium of both mouse strains. While C57BL/6J mice developed a greater number of aberrant crypt foci than BALB/cJ mice (p less than 0.0001), a significant diet-strain interaction effect was not observed. The present results indicate that dietary cholesterol enhances the induction and development of chemically-induced colonic precancerous lesions but this process is not affected by genetic differences in cholesterol metabolism, as represented by the two strains of mice studied.     

Pluronic F68 block polymer, a very potent suppressor of carcinogenesis in the colon of rats and mice

Polyethylene-glycol (PEG) is a strong inhibitor of colon cancer in rats, and the most potent suppressor of aberrant crypt foci. 9 PEG-like block copolymers were tested in rodents, after an azoxymethane injection. Dietary pluronic F68 led to a 98.6% reduction in the number of aberrant crypt foci in a first rat study (P< 0.0001). Next 3 studies confirmed this pluronic efficacy in rats and mice. This non-toxic laxative seems roughly 5 times more potent than PEG for chemoprevention.     

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.     

Progastrin Peptides Increase the Risk of Developing Colonic Tumors: Impact on Colonic Stem Cells

Preneoplastic lesions (aberrant crypt foci, hyperplastic/dysplastic polyps) are believed to be precursors of sporadic colorectal tumors (adenomas, adenocarcinomas). Aberrant crypt foci and hyperplastic/dysplastic polyps likely originate from abnormal growth of colonic crypts in response to aberrant queues in the microenvironment of colonic crypts. Thus, identifying factors which regulate homeostatic versus aberrant proliferation/apoptosis of colonocytes, especially stem/progenitor cells, may lead to effective preventative/treatment strategies. On the basis of this philosophy, the role of growth factors/peptide hormones potentially available in the circulation/microenvironment of colonic crypts is being examined extensively. Since the time gastrins were discovered as trophic (growth) factors for gastrointestinal cells, the effect of gastrins on the growth of normal/cancer cells has been investigated, leading to many discoveries. Seminal discoveries in the area of gastrins and colon cancer as it relates to molecular pathways associated with formation of colonic tumors are reviewed and the possible impact on diagnostic/preventative/treatment strategies is discussed.     

Aberrant crypt foci and colon tumors in F344 rats have similar increases in proliferative activity

Increased proliferative activity has been described frequently in the colons of animals treated with colon carcinogens and of patients at increased risk of colon cancer; it has been proposed as an intermediate biomarker of colon cancer. Aberrant crypt foci, microscopic lesions identified in whole-mount preparations of colons, are thought to be putative pre-neoplastic lesions. The present studies were carried out to evaluate the proliferative activity of aberrant crypt foci at several different time periods, and of tumors after a single dose of azoxymethane (AOM) in F344 rats. Rats were injected with 5-bromo-2′-deoxyuridine (BUdR) 1 hr before killing. Aberrant crypt foci and tumors were identified and marked in the whole-mount specimens, embedded in glycol methacrylate, and evaluated for histochemically demonstrable hexosaminidase activity. Hexosaminidase is known to be altered in over 95% of aberrant crypt foci. Serial sections were evaluated for BUdR incorporation immunohistochemically with a monoclonal antibody. The mean proliferative activity of aberrant crypt foci in the distal colons was found to be 'increased 3-to 4-fold over that of the adjacent normal crypts at every time period analyzed (4 to 36 weeks) and was comparable to that seen in benign and malignant colon tumors in the same animals. The observed increase in proliferative activity further supports the hypothesis that aberrant crypt foci are putative pre-neoplastic lesions. Similar aberrant crypt foci, identified in human colons at increased risk of colon cancer, may provide important biomarkers for this common human cancer.     

The suppression of aberrant crypt multiplicity in colonic tissue of 1,2-dimethylhydrazine-treated C57BL/6J mice by dietary flavone is associated with an increased expression of Krebs cycle enzymes

Colorectal cancer is the second leading cause of cancer deaths worldwide with diet playing a prominent role in disease initiation and progression. Flavonoids are secondary plant compounds that are suggested as protective ingredients of a diet rich in fruits and vegetables. We here tested whether flavone, a flavonoid that proved to be an effective apoptosis inducer in colon cancer cells in culture, can affect the development of aberrant crypt foci (ACFs) in C57BL/6J mice in vivo when preneoplastic lesions were induced by the carcinogen 1,2-dimethylhydrazine (DMH). Flavone applied at either a low dose (15 mg/kg body wt per day) or a high dose (400 mg/kg body wt per day) reduced the numbers of ACFs significantly, independent of whether it was supplied simultaneously with the carcinogen (blocking group) or subsequent to the tumor induction phase (suppressing group). Proteome analysis performed in colonic tissue samples revealed that flavone treatment increased the expression of a number of Krebs cycle enzymes in the suppressing group and this was associated with reduced crypt multiplicity. It suggests that mitochondrial substrate oxidation is increased by flavone in colonic cells in vivo as already observed in HT-29 cells in vitro as the prime mechanism underlying tumor cell apoptosis induction by flavone. In conclusion, flavone reduces the number of ACFs in DMH-treated mice at doses that can be achieved for flavonoids by a diet rich in fruits and vegetables. Moreover, reduction in crypt multiplicity by flavone is most probably due to the preservation of a normal oxidative metabolism.