Dietary fish oil inhibits the expression of farnesyl protein transferase and colon tumor development in rodents

Although epidemiological and experimental studies indicate a strong relationship between different dietary fats and risk of colon cancer, the modulating effects of these nutritional factors at the molecular level are not fully elucidated. Activated ras genes have been implicated in the etiology of many human malignancies, including colon cancer. It is well established that the transforming ability of ras-p21 depends on its correct localization in plasma membrane. We have previously demonstrated that ingestion of a relatively higher amount of dietary fish oil leads to reduced plasma membrane levels of ras-p21 with concomitant increase in its cytoplasmic contents during the promotion and progression phases of chemically-induced colon tumorigenesis. In this follow-up experiment, we have found that intake of a high amount of corn oil, one of the most widely used fats in the American diet, enhances the expression of farnesyl protein transferase (FPTase). This enzyme catalyses farnesylation of ras precursors in a critical step during post-translational modification of ras oncoproteins, thereby enabling their anchorage to plasma membrane. In contrast, consumption of high amounts of fish oil, which is rich in omega-3 polyunsaturated fatty acids, reduces the levels of FPTase expression, thus inhibiting post-translational processing of ras precursors resulting in decreased ras function both in colonic mucosa as well as in colon tumors. These results correlate with increased incidence and multiplicity of grossly visibly colon tumors in carcinogen-treated animals fed a high corn oil diet versus decreased incidence and multiplicity of colon tumors in their counterparts fed the high fish oil diet. This dietary inhibition of FPTase may have a practical chemopreventive potential.      

Down-regulation of PLK3 gene expression by types and amount of dietary fat in rat colon tumors.

Epidemiological studies suggest that high intake of dietary fat rich in saturated fatty acids increases the colon cancer risk whereas dietary fish oil high in omega-3 fatty acids reduces the colon cancer risk. Previously, we reported that consumption of omega-6 fatty acid rich diets such as corn oil strongly promotes azoxymethane (AOM)-induced colon carcinogenesis in rats as compared to ingestion of a diet with equivalent amount of fat containing fish oil (HFFO) or low-fat diet (LFCO). Expression of PLK3 (Polo-like kinase-3, previously named Prk) is negatively correlated with the development of certain cancers. Ectopic expression of human PLK3 results in cell cycle arrest or induces apoptosis. To understand the role of PLK3 in colon carcinogenesis and to study the effect of types and amount of dietary fat on the expression levels of PLK3 in colon tumors, we analyzed the colon tumors and mucosa of rats administered the diets containing fish oil and corn oil for PLK mRNA expression. Here we report that expression of PLK3 was down-regulated in rat colon tumors. Quantitative polymerase chain reaction demonstrated that PLK3 mRNA levels were significantly lower in carcinogen (azoxymethane)-induced rat colon tumors than their uninvolved normal colonic mucosa. Among the normal mucosa isolated from rats fed on diets with various levels of fat (LFCO, or high fat diet with corn oil, HFCO, or supplemented with fish oil, HFFO), no significant changes in PLK3 mRNA expression was detected. Tumors isolated from rats fed with HFCO diet contained a very low level of PLK3 mRNA expression. Interestingly, tumors from rats fed the HFFO diet did not exhibit as dramatic down-regulation of PLK3 as the tumors of animals fed the HFCO diet. Furthermore, our results also indicate that the ectopic expression of a kinase active PLK3 construct induced apoptosis in HT-29 colon carcinoma cells. These observations suggest for the first time that a decreased activity of PLK3 may play a key role in colon tumor development as well as in HFCO-induced colon tumorigenesis.     

Modifying effect of tuna orbital oil rich in docosahexaenoic acid and vitamin D3 on azoxymethane-induced colonic aberrant crypt foci in rats

The modifying effect of dietary tuna (Thunnus thynnus orientalis) orbital oil rich in docosahexaenoic acid (DHA) and vitamin D3 (VD3) on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) was investigated in male F344 rats. Animals were given three weekly subcutaneous injections of AOM (15 mg/kg body weight) to induce ACF. The rats were fed the experimental diet containing 5% tuna orbital oil (low fish oil), 23.5% tuna orbital oil (high fish oil), 5% corn oil (low corn oil) or 23.5% corn oil (high corn oil) for 5 weeks, starting 1 week before the first dose of AOM. Animals were sacrificed 2 weeks after the last AOM injection to count colonic ACF and assay the expression of cyclooxygenase (COX)-1 and -2. High corn oil diet significantly increased the development of ACF, when compared with low corn oil diet (P<0.005). High fish oil diet also increased ACF formation compared with low fish oil diet (P<0.01), but the increase was smaller than high corn oil diet. The frequency of ACF was significantly lower in the rats fed high fish oil diet than high corn oil diet (P<0.02). Moreover, frequency of ACF consisted of 4 or more crypts in rats fed the high fish oil diet was significantly lower than that of rats given high corn oil diet. COX-1 and COX-2 expression did not significantly differ among the groups. These results suggest that fish oil derived from tuna, which contains high amounts of DHA and VD3, suppresses the formation and growth of ACF without affecting COX-1 and COX-2 expression, and may have a preventive effect on colon carcinogenesis.     

Effect of different levels of dietary trans fat or corn oil on azoxymethane-induced colon carcinogenesis in F344 rats

The effect of various levels of dietary corn oil or trans fat on azoxymethane (AOM; CAS: 25843-45-2)-induced carcinogenesis was investigated in female F344 rats fed the AIN-76 semipurified diets. Starting at 5 weeks of age, groups of rats were fed the low-fat diet containing 5% corn oil (designated as low-fat control diet). At 7 weeks of age, all animals except the vehicle-treated controls, were given sc injections of AOM (15 mg/kg body wt, once weekly) for 3 weeks. After 1 week, groups of animals were transferred to semipurified diets containing 13.6% corn oil and 23.5% corn oil or high-fat diets containing 5.9% corn oil plus 5.9% trans fat plus 11.8% Oleinate (low trans fat), 5.9% corn oil plus 11.8% trans fat plus 5.9% Oleinate (intermediate trans fat), and 5.9% corn oil plus 17.6% trans fat (high trans fat). Fecal bile acids were measured in vehicle-treated rats. All animals were necropsied 34 weeks after the last AOM injection. The animals fed the 23.5% corn oil diet had a higher incidence of colon tumors than did those in the groups fed the 5 and 13.6% corn oil diets. There was no difference in colon tumor incidence between the 5 and 13.6% corn oil diet groups. The animals fed the high-fat diets containing low trans fat, intermediate trans fat, and high trans fat developed significantly fewer liver and colon tumors and more small intestinal tumors than did the rats fed 23.5% corn oil diet. The excretion of fecal deoxycholic acid, lithocholic acid, and 12-ketolithocholic acid was higher in animals fed the 23.5% corn oil diet compared to the excretion in animals fed the other diets.     

Predictive value of proliferation, differentiation and apoptosis as intermediate markers for colon tumorigenesis

In order to determine the prognostic significance of proliferation, differentiation, and apoptosis as intermediate markers for colon tumor development, these indices were measured during the promotion phase of tumorigenesis. Two hundred and sixty male Sprague-Dawley rats were provided with one of two fats (corn oil and fish oil) and two fibers (pectin and cellulose) plus or minus the carcinogen azoxymethane (AOM) and killed at two time points (18 and 36 wk) in a 2x2x2x2 factorial design. In vivo cell proliferation was measured immunohistochemically using incorporation of bromodeoxyuridine into DNA. Differentiation was assessed by binding of Dolichos biflorus agglutinin (DBA) to colonocytes. Apoptosis was measured by immunoperoxidase detection of digoxigenin-labeled genomic DNA. Adenocarcinoma incidence results at week 36 were 70.3% for corn oil + AOM and 56.1% for fish oil + AOM treatment (P < 0.05); no main effect of fiber was observed. At week 18, AOM treatment increased the number of cells per crypt column in the proximal colon compared with saline controls (P = 0.0358) and increased the proliferative zone in the distal colon compared with controls (P = 0.0073). However, changes in cell proliferation at week 18 did not predict the beneficial effect of fish oil versus corn oil. In contrast, DBA binding (the marker for differentiation) was higher in fish oil versus corn oil fed animals in both the proximal and distal colon and in each portion of the crypt (P = 0.0001). There were a greater number of apoptotic cells/crypt column in the proximal colon (P = 0.0019) and distal colon (P = 0.0358) with fish oil compared with corn oil, and indices of apoptosis also predicted certain fat/fiber interactions. Measurements of differentiation and apoptosis had greater prognostic value to detect dietary effects on tumor incidence than did measurements of cell proliferation.      

Modulation of alterations in p53 tumor suppressor gene and its association with activation of ras proto-oncogenes during chemoprevention of colon cancer

Previously, we reported (Carcinogenesis 15: 1317-1323, 1994) a high rate of activating point mutations in I ns proto-oncogenes in azoxymethane (AOM)-induced colon tumors, and a significant suppression of these mutations by dietary administration of chemopreventive agents, D,L-alpha-difluoromethylornithine (DFMO) and piroxicam. To understand the role of p53 tumor suppressor gene in chemoprevention of colon cancer and to study the association of p53 gene alterations with activation of ras genes, we determined point mutations in conserved regions (exons 5-9) of p53 gene and analyzed the occurrence of double event of ms activation acid p53 mutation. Groups of male F344 rats were fed the modified AIN-76A diet containing 0, 4000 ppm DFMO, or 150 ppm piroxicam and administered s.c. AOM at a dose rate of 15 mg/kg body wt, once weekly, for 4 weeks. Vehicle controls received s.c. equal volume of normal saline. Animals were sacrificed 32 weeks after the last AOM or saline injection and their grossly visible colon tumors were analyzed to determine p53 mutations by PCR amplification based single strand conformation polymorphism (SSCP) and direct DNA sequencing. Our results demonstrate that about 57% tumors from animals fed the control diet contained predominantly missense but also nonsense mutations, whereas only 30% tumors from animals on piroxicam diet, and none (0%) from animals fed the DFMO diet had similar mutations. Analysis of data revealed that about half of the tumors from animals on control diet possessed both ms and p53 mutations together, only 27% of colon tumors from animals on piroxicam diet and none of the tumors from animals on DFMO diet exhibited both ms and p53 mutations. These results indicate that the administration of piroxicam, a non-steroidal anti-inflammatory drug, and DFMO, a irreversible inhibitor of ornithine decarboxylase, may inhibit selective proliferation of initiated cells containing activated las and/or mutant p53. Dietary DFMO exerted more pronounced inhibition of selective amplification of initiated cells containing mutated ras and/or p53.     

Effect of different levels of dietary corn oil and lard during the initiation phase of colon carcinogenesis in F344 rats

The effect of various levels of polyunsaturated fat (corn oil) and saturated fat (lard) fed during the initiation stage of colon carcinogenesis was studied in male F344 rats. The animals were fed the diets containing 5, 13.6, and 23.5% corn oil or lard 2 weeks before, during, and until 1 week after sc injection of 15 mg azoxymethane [(AOM) CAS: 25843-45-2]/kg body weight, once weekly for 2 weeks (designated as initiation). One week after AOM treatment, groups of animals were transferred to their respective 5% corn oil or lard diets. Additional groups consuming 5% corn oil or lard were transferred to 23.5% corn oil or lard, respectively (post-initiation stage). All animals were fed these diets until the termination of the experiment. Fecal bile acids and colonic mucosal ornithine decarboxylase activity were measured in vehicle-treated animals fed the experimental diets for 14 weeks. Body weights and intakes of total calories, protein, nonnutritive fiber, and micronutrients were comparable among the various dietary groups. The animals fed the 23.5% corn oil diet during the postinitiation stage had a higher incidence of colon tumors than did those fed the 5% corn oil diet, whereas feeding of 23.5 and 13.6% corn oil diets during the initiation stage had no effect. In contrast, animals fed the 23.5 and 13.6% lard diet during the initiation stage and 23.5% lard diet during the postinitiation stage developed more colon adenocarcinomas than did those fed the 5% lard diet. The excretion of fecal deoxycholic acid, lithocholic acid, and 12-ketolithocholic acid and the activity of colonic mucosal ornithine decarboxylase activity were higher in animals fed the 23.5% corn oil or lard diet during the postinitiation compared to the levels in animals fed the 5% corn oil or lard diet.     

Chemopreventive effects of dietary mustard oil on colon tumor development

Fatty acid composition of dietary fat is one of the detrimental factors in colon cancer development. Fats containing omega 6-polyunsaturated fatty acids (e.g. corn oil) enhance and omega 3-polyunsaturated fatty acids (e.g. fish oil) reduce chemically-induced colon cancer in animal studies. The purpose of this study is to investigate the effects of dietary mustard oil (containing omega 3-polyunsaturated fatty acid) on azoxymethane-induced colon cancer in rats and compare with corn and fish oil treated groups. Colon tumor incidence and multiplicity were found to be 90, 75, and 50% and 1.7, 0.8, and 0.4 tumors/rat in corn, fish and mustard oil treated groups respectively. Omega-3 polyunsaturated fatty acid levels were highest in serum and colon microsomal fractions of the fish oil group followed by the mustard oil group. Corn oil group had the highest level of omega 6-polyunsaturated fatty acid levels in serum and colon microsomal fractions. The results indicate that dietary mustard oil is more effective in preventing colon cancer in rats than dietary fish oil.     

The effect of high-risk and low-risk diets on aberrant crypt and colonic tumor formation in Fischer-344 rats.

Eighty Fischer-344 male rats were divided into eight groups. Half the groups were given a high-risk diet (HRD) that was high in fat, low in fiber and low in calcium, while the remaining groups were given a low-risk diet (LRD) that was low in fat, high in fiber and high in calcium. After 4 weeks, four groups were then given two weekly s.c. injections of azoxymethane (AOM) (15 mg/kg body wt), and four groups were given saline injections. At the end of 6 weeks the rats were either continued on HRD, LRD, or crossed over from HRD to LRD, or LRD to HRD for an additional 6 weeks. The rats were then killed and the influence of different dietary regimens on the induction of foci of aberrant crypts (AC) in their colons was compared. The number of foci of AC was highest in the AOM-injected HRD/HRD dietary group (8.83 +/- 0.98), followed by the AOM-injected LRD/HRD group (5.37 +/- 0.75), the HRD/LRD group (3.32 +/- 0.36) and the LRD/LRD group (2.93 +/- 0.32). Except for the HRD/HRD control group, which developed a small but significant number of foci of AC (1.24 +/- 0.19), the other saline-injected dietary groups showed no significant numbers of foci of AC. Two groups of rats on HRD/HRD (AOM and saline-injected), and two groups of rats on HRD/LRD (AOM and saline-injected) were continued on HRD and LRD respectively for an additional 14 weeks. A continuous HRD in AOM-injected rats induced colon tumors in 92% of rats, while the crossover from HRD to LRD suppressed the tumor incidence to 33%. Neither diet induced any tumors in saline-injected rats. The results clearly show that the influence of HRD and LRD on the induction of foci of AC and their distribution in the colon, correlated with the induction and distribution of tumors in the colon.     

Morphodensitometric analysis of protein kinase C beta(II) expression in rat colon: modulation by diet and relation to in situ cell proliferation and apoptosis

We have recently demonstrated that overexpression of PKC beta(II) renders transgenic mice more susceptible to carcinogen-induced colonic hyperproliferation and aberrant crypt foci formation. In order to further investigate the ability of PKC beta(II) to modulate colonocyte cytokinetics, we determined the localization of PKC beta(II) with respect to cell proliferation and apoptosis along the entire colonic crypt axis following carcinogen and diet manipulation. Rats were provided diets containing either corn oil [containing n-6 polyunsaturated fatty acids (PUFA)] or fish oil (containing n-3 PUFA), cellulose (non-fermentable fiber) or pectin (fermentable fiber) and injected with azoxymethane (AOM) or saline. After 16 weeks, an intermediate time point when no macroscopic tumors are detected, colonic sections were utilized for immunohistochemical image analysis and immunoblotting. Cell proliferation was measured by incorporation of bromodeoxyuridine into DNA and apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling. In the distal colon, PKC beta(II) staining was localized to the upper portion of the crypt. In comparison, proximal crypts had more (P < 0.05) staining in the lower tertile. AOM enhanced (P < 0.05) PKC beta(II) expression in all regions of the distal colonic crypt (upper, middle and lower tertiles). There was also an interaction (P < 0.05) between dietary fat and fiber on PKC beta(II) expression (corn/pectin > fish/cellulose, fish/pectin > corn/cellulose) in all regions of the distal colonic crypt. With respect to colonic cell kinetics, proliferation paralleled the increase in PKC beta(II) expression in carcinogen-treated animals. In contrast, apoptosis at the lumenal surface was inversely proportional to PKC beta(II) expression in the upper tertile. These results suggest that an elevation in PKC beta(II) expression along the crypt axis in the distal colon is linked to enhancement of cell proliferation and suppression of apoptosis, predictive intermediate biomarkers of tumor development. Therefore, select dietary factors may confer protection against colon carcinogenesis in part by blocking carcinogen-induced PKC beta(II) expression.     

Effect of diets high in omega-3 and omega-6 fatty acids on initiation and postinitiation stages of colon carcinogenesis.

The effect of dietary menhaden oil containing omega-3 fatty acids and corn oil rich in omega-6 fatty acids fed during the initiation and/or postinitiation stages of colon carcinogenesis was investigated in male F344 rats. At 5 weeks of age, all animals were divided into seven groups (39 rats/group) and fed the semipurified diets containing 5% corn oil (LCO), 23.5% corn oil (HCO), or 18.5% menhaden oil plus 5% corn oil (HFO). At 7 weeks of age, all animals except the vehicle (normal saline)-treated groups were given two weekly s.c. injection of azoxymethane (AOM) at a dose rate of 15 mg/kg body weight, once weekly. Three days after the second injection of AOM, groups of animals fed LCO, LCO, HCO, HCO, HCO, HFO, or HFO diets were transferred, respectively, to LCO, HCO, LCO, HCO, HFO, HCO, or HFO and continued on these diets until termination of the experiment. All animals were necropsied 42 weeks after carcinogen treatment. Body weights of animals fed various experimental diets during the initiation and postinitiation periods were comparable. As expected, the HCO diet fed during the postinitiation period significantly increased the AOM-induced incidence and multiplicity of colon adenocarcinomas, whereas the HCO diet fed during the initiation phase of carcinogenesis had no effect. Colon tumor incidence and multiplicity were significantly reduced in groups fed the HFO diet at either initiation and/or postinitiation phases of carcinogenesis as compared with those fed the HCO diet. Whereas the precise mechanisms producing the difference between the high menhaden oil (HFO) diet as compared with high corn oil (HCO) diet remain to be elucidated, it is likely that the effect during the initiation and postinitiation phases may be due to alteration in carcinogen metabolism and to modulation of prostaglandin synthesis, respectively.     

Molecular markers as intermediate end-points in chemoprevention of colon-cancer - modulation of ras activation by sulindac and phenylhexylisothiocyanate during colon carcinogenesis

Recent evidence suggests that activation of ras proto-oncogenes and inactivation of suppressor genes induce malignant phenotype in colonic cells. Thus the identification of clonal population of cells expressing activated ras may lead to a valuable intermediate biomarker to detect premalignant lesions amenable to chemoprevention. Previously, we demonstrated that sulindac inhibited the carcinogen-induced colon tumor development whereas phenylhexylisothiocyanate (PHITC) promoted the tumor outcome. The present study was conducted to investigate the effect of sulindac and PHITC on azoxymethane (AOM)induced activation of ras proto-oncogenes in order to explore the plausibility of using ras as an intermediate biomarker in chemoprevention of colon cancer. Male F344 rats were fed the AIN-76A diet containing 0, 320 ppm sulindac or 640 ppm PHITC and administered s.c. AOM dissolved in normal saline at a dose rate of 15 mg/kg body wt/week for 2 weeks. Vehicle control groups received s.c. equal volume of normal saline. Animals were sacrificed 52 weeks after AOM or saline treatment and their colonic mucosa and tumors were analyzed for mutations in codon 12 and 13 of K-ras and the expression of ras p21. As an alternative non-invasive approach, we developed a simple and sensitive one-step mutant-enriched PCR method to detect these genetic lesions in stools collected at 16, and again at 24 weeks after AOM treatment. AOM-induced G to A transitions were observed at the second nucleotide of 12th codon of K-ras substituting amino acid asp with wild-type gly. Sulindac not only suppressed the selective amplification of initiated cells possessing AOM-induced mutated K-ras codon 12, but significantly inhibited the AOM-induced expression of total and mutant ras-p21. PHITC did not exert any inhibitory effect on AOM-induced ras activation. Results indicated a strong correlation between ras activation and tumor outcome. Data suggest that ras activation may be a useful intermediate molecular marker in chemoprevention of colon cancer.     

A high-fat diet enhances the inhibitory effect of dietary vitamin B6 on colon cell proliferation in mice

Previously we reported that dietary supplemental vitamin B6 (B6) reduced colon tumorigenesis and cell proliferation in mice receiving azoxymethane (AOM) for 22 weeks. This study was conducted to examine the influence of short-term consumption (5 weeks) of diets containing graded levels of B6 and fat on colonic cell proliferation in mice with or without receiving AOM. In experiment 1, mice were fed the 10% corn oil diet containing 1, 7, 14, 35 or 70 mg pyridoxine HCl/kg, and received weekly injections of AOM for the initial 3 weeks. In experiment 2, mice were fed 5 or 20% corn oil diet containing 1, 7, 14 or 35 mg pyridoxine HCl/kg, and received weekly injections of AOM or saline for the initial 3 weeks. In experiment 1, supplemental B6 caused a dose-dependent reduction of colon aberrant crypt foci and cell proliferation (BrdU-labeling index) among the 1-14 mg pyridoxine HCl/kg. There was no influence of B6 on these parameters among 14-70 mg pyridoxine HCl/kg. Immunohistochemical analysis of apoptosis labeling by TUNEL method indicated no influence of dietary B6 on colon apoptosis. In experiment 2, supplemental B6 significantly reduced colon cell proliferation regardless of AOM injection. This inhibitory effect on cell proliferation was markedly enhanced by a high-fat diet, but slightly affected by AOM treatment. The results suggest that dietary supplemental B6 inhibits colon cell proliferation from the early stage of colon carcinogenesis, and a high-fat diet markedly enhances the inhibitory effect.     

Inhibition of the promotional phase of azoxymethane-induced colon carcinogenesis in the F344 rat by calcium lactate: effect of simulating two human nutrient density levels

The effects of 2 levels of dietary calcium and 2 types of dietary fat on the promotional phase phase of azoxymethane-induced colon cancer in the F344 rat were investigated. During the initiation phase of carcinogenesis all animals were fed a 5% corn oil AIN-76A diet containing 0.32% Ca in the form of calcium lactate. Rats were then injected with azoxymethane (AOM) weekly for 8 weeks. Thereafter, the rats were fed 1 of 3 diet formulations: a 5% corn oil diet or a 20% corn oil or 20% American Blend oil fat diet, with the level of Ca set at either 0.32% of the diet, a nutrient density simulating a daily human intake of approximately 1700 mg Ca/day, or at 0.04% of the diet, reflecting a human daily intake of approximately 200-250 mg of Ca/day, thus modeling 2 human nutrient density levels for calcium. Measurements of fecal pH during the experiment indicated an acidic adaptation of the large bowel to the lactate anion. Analysis of collected fecal samples showed more total fatty acids to be present in the colon when higher amounts of calcium were consumed. However, results of the tumorigenesis study indicated that calcium lactate fed at the 0.32% level significantly inhibited the development of colonic adenocarcinoma in all dietary groups. Taken together, this investigation supports the hypothesis that calcium supplementation can inhibit colon neoplasia in rats fed a high fat diet; however, under the conditions of this study, the 20% fat level did not significantly promote colon cancer as compared to a 5% fat level.     

4-(Methylnitrosamino)-I-(3-pyridyl)-1-butanone enhances the expression of apolipoprotein A-I and Clara cell 17-kDa protein in the lung proteomes of rats fed a corn oil diet but not a fish oil diet.

The nicotine-derived nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the most potent lung carcinogens in rodents. Several epidemiologic studies indicated that the development of lung cancer in smokers is influenced by the type and amount of dietary polyunsaturated fatty acids. A high corn oil diet has been shown to increase lung tumor volume and to decrease tumor latency in rats treated with NNK. In this study, we investigated the effects of dietary polyunsaturated fatty acids in the form of corn oil or fish oil on lung proteomes in F344 rats treated with or without NNK. The fish oil diet contained 17% fish oil and 3% corn oil, and the corn oil diet contained 20% corn oil. Rats were sacrificed after 3 months, and lungs were excised. Whole lung tissue proteins were separated by two-dimensional liquid chromatography, and differentially expressed proteins were identified by trypsin digestion and tandem mass spectrometry. Apolipoprotein A-I and Clara cell 17-kDa protein were overexpressed in the lungs of rats fed corn oil diet, compared with fish oil diet. NNK further enhanced their expression in rats fed corn oil diet; this effect was not observed in animals fed fish oil diet. The results suggest that the elevated levels of apolipoprotein A-I and Clara cell 17-kDa protein may be involved in the development of NNK-induced lung cancer in rats fed a high corn oil diet. Therefore, we propose that both proteins may serve as potential biomarkers in future molecular epidemiologic and clinical chemoprevention intervention studies.     

Effect of different levels of omega-3 and omega-6 fatty acids on azoxymethane-induced colon carcinogenesis in F344 rats

The effect of various levels of dietary Menhaden fish oil containing omega-3 fatty acids plus corn oil containing omega-6 fatty acids fed during the postinitiation phase of colon carcinogenesis was studied in male F344 rats. Starting at 5 weeks of age, groups of animals were fed the 5% corn oil (5% CO) diet. At 7 weeks of age, all animals except the vehicle-treated controls were administered s.c. injections of azoxymethane (15 mg/kg body wt/week for 2 weeks). 4 days after carcinogen or vehicle treatment, groups of animals were transferred to experimental diets containing 4% Menhaden oil + 1% corn oil (4% MO + 1% CO), 23.5% corn oil (23.5% CO), 17.6% corn oil + 5.9% Menhaden oil (17.6% CO + 5.9% MO), 11.8% corn oil + 11.8% Menhaden oil (11.8% CO + 11.8% MO), or 5.9% corn oil + 17.6% Menhaden oil (5.9% CO + 17.6% MO) and fed these diets until termination of the experiment at Week 38 after carcinogen treatment. An additional group consuming a 5% CO diet was continued on these diets. Colon mucosal ornithine decarboxylase activity and microsomal fatty acid composition of colon mucosa were measured in vehicle-treated animals fed experimental diets for 14 weeks. Fatty acids were also analyzed in the microsomal fraction of colon tumors at termination of the experiment. The body weights of animals fed various experimental diets were comparable. Feeding of high fat diets containing 17.6% CO + 5.9% MO, 11.8% CO + 11.8% MO, or 5.9% CO + 17.6% MO significantly inhibited the incidence (percentage of animals with tumors) of colon adenocarcinomas compared to that of 23.5% CO diet. However, the multiplicity (number of tumors/rat) of colon adenocarcinomas was significantly inhibited only in groups fed the 5.9% CO + 17.6% MO compared to those fed the 23.5% CO diet. The incidence and multiplicity of adenocarcinomas were greater in animals fed the 23.5% CO diet compared to those fed the 5% CO diet. Colonic mucosal ornithine decarboxylase activity was lower in animals fed the 11.8% CO + 11.8% MO, 5.9% CO + 17.6% MO, 5% CO, and 4% MO + 1% CO diets compared to the levels in animals fed the 23.5% CO diet. The increasing levels of Menhaden oil in the diet significantly increased the omega-3 fatty acids such as eicosapentaenoic acid and docosahexaenoic acid and decreased the omega-6 fatty acids such as linoleic acid, linolenic acid, and arachidonic acid in microsomal fractions from colonic mucosa and tumors.     

Effect of chemopreventive agents on posttranslational plasmamembrane association of ras-p21 during chemoprevention of azoxymethane-induced colon carcinogenesis

Accumulating data suggest that activation of ms proto-oncogenes and inactivation of tumor suppressor genes induce malignant phenotype in colonic cells. However, the transforming ability of ras oncogenes critically depends on correct localization of ras-p21 in plasma membrane. In our previous studies, we demonstrated a strong correlation between the modulation of ras activation (both in terms of mutational activation and over-expression of ras genes) by chemopreventive agents and colon tumor outcome during different stages of azoxymethane (AOM)-induced colon carcinogenesis. In the present study, which is a part of our ongoing investigations on the role of ras in chemoprevention of colon cancer, we studied the effect of D,L-alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, and piroxicam, a non-steroidal antiinflammatory drug (NSAID), on the post-translational membrane association of ras-p21 during AOM-induced colon carcinogenesis. Groups of male F344 rats were fed the modified AIN-76A diets containing 0, 150 ppm piroxicam or 4000 ppm DFMO, and administered s.c. AOM dissolved in normal saline at a dose rate of 15 mg/kg body weight/week for 4 weeks. Vehicle control groups received equal volume of normal saline. Groups of animals were then sacrificed at 4, 16, 24, and 32 weeks after last AOM or saline injection and their colonic mucosa and tumors were analyzed for cytoplasmic as well as membrane bound ras-p21 levels. AOM-treatment resulted in increasingly higher levels of membrane-bound ras-p21 with advancing stages of colon tumorigenesis without any significant changes in cytoplasmic ras-p21. Dietary DFMO significantly suppressed AOM-induced membrane-bound ras-p21 in a time-dependent manner. Administration of piroxicam though resulted in significant inhibition of membrane-bound ras-p21, but concomitantly increased the cytosolic levels of ras-p21. Inhibition of membrane-bound ras-p21 levels by DFMO and piroxicam strongly correlated with the suppression of AOM-induced colon tumorigenesis by these agents. Data from the present and earlier studies suggest that DFMO may afford chemoprevention by suppressing DNA and protein biosynthesis by depleting intracellular polyamines, whereas piroxicam may exert its antitumor activity by interfering with post-translational membrane localization of ras-p21, in addition to modulating arachidonic acid metabolism.     

Mechanism of benzylselenocyanate inhibition of azoxymethane-induced colon carcinogenesis in F344 rats

Benzylselenocyanate (BSC), a novel organoselenium compound, has been found to inhibit azoxymethane (AOM)-induced colon carcinogenesis in rats during initiation. To investigate its mechanism of action, we examined the effects of BSC feeding on the following parameters: (a) metabolism of [14C]AOM to 14CO2 in vivo; (b) metabolic activation of AOM to MAM and of MAM to formic acid and methanol by rat liver microsomes in vitro; and (c) AOM-induced DNA methylation in rat livers and colons. Five-week-old male F344 rats were fed modified (23% corn oil) AIN-76A diets containing 0 (control), 25, or 50 ppm of BSC or benzylthiocyanate (BTC), a sulfur analogue of BSC which does not inhibit the colon carcinogenicity of AOM. After 3 weeks, rats were either sacrificed for the isolation of liver microsomes or were given 15 mg/kg of [14C]AOM s.c. to determine the rate of carcinogen metabolism in vivo. No difference in [14C]AOM metabolism was found between rats fed the BTC diets and those fed the control diet. In contrast, the rate of [14C]AOM metabolism, as determined by exhaled radioactivity, was 2-3 times higher in rats fed the BSC diets. While liver microsomes from rats fed the BTC diets metabolized AOM and MAM at rates not significantly different from those obtained with control liver microsomes, the metabolic activation of AOM as well as of MAM was stimulated severalfold when assayed with liver microsomes from rats fed the BSC diets. An increase in total liver cytochrome P-450 was also observed in the BSC-fed rats. Following the administration of 15 mg/kg AOM, significantly less O6-methylguanine and 7-methylguanine was present in the colon DNA from rats consuming the BSC diets than in rats fed the BTC or control diets. The body weight gains of rats fed the 25- and 50-ppm BSC-containing diets for 3 weeks were less (27 and 43%, respectively) than those of rats fed either the control or BTC-containing diets. These results indicate that dietary BSC significantly induces the hydroxylation of AOM and the oxidation of MAM in rat liver. An increase in the rates of AOM and MAM metabolism in the liver due to enzyme induction by BSC will result in decreased delivery of MAM to the colon via the bloodstream. This will be reflected in decreased DNA alkylation, as observed, and is likely to be a major factor in the inhibition of AOM-induced colon carcinogenesis by BSC.     

Effect of dietary fish oil on azoxymethane-induced colon carcinogenesis in male F344 rats

The effect of dietary intake of different levels of Menhaden fish oil on azoxymethane-induced carcinogenesis was examined in male F344 rats fed the semipurified diets. Starting at 5 weeks of age, groups of animals were fed the 5% corn oil (low corn oil) diet. At 7 weeks of age, all animals except the vehicle-treated controls were given s.c. injections of azoxymethane (15 mg/kg body weight/week for 2 weeks). After 4 days, groups of animals were fed the diets containing 4% Menhaden oil + 1% corn oil (low fish oil), 22.5% Menhaden oil + 1% corn oil (high fish oil), 5% corn oil, and 23.5% corn oil (high corn oil). Thirty-four weeks after azoxymethane injections, all animals were necropsied. High fish oil diet had no tumor promoting effect in the large intestine when compared to the high corn oil diet. There was no difference in large intestinal tumor incidence among the other dietary groups. The results of this study indicate that fish oils rich in highly polyunsaturated n-3 fatty acids do not enhance large bowel carcinogenesis and that the fatty acid composition of the dietary fat is one of the determining factors in large bowel carcinogenesis.     

Mutational and nonmutational activation of p21ras in rat colonic azoxymethane-induced tumors: effects on mitogen-activated protein kinase, cyclooxygenase-2, and cyclin D1

Azoxymethane (AOM)-induced colonic carcinogenesis involves a number of mutations, including those in the K-ras gene and CTNNB1, that codes for beta-catenin. Prior in vitro studies have also demonstrated that wild type p21(K-ras) can be activated by epigenetic events. We identified 15 K-ras mutations in 14 of 84 AOM-induced colonic tumors by three independent methods. By single strand conformational polymorphism, we also observed mutations in 22 of 68 tumors in exon 3 of CTNNB1. A highly sensitive method was then used to measure p21ras activation levels. All tumors assayed possessing K-ras mutations had significantly higher p21ras activation levels (8.8 +/- 1.5%; n = 13) compared with that of control colon (3.7 +/- 0.4; n = 6; P < 0.05) or tumors without such mutations (4.2 +/- 0.4%; n = 70; P < 0.05). Among tumors with wild-type K-ras, there was a subset of tumors (18 of 70) that had significantly higher p21ras activation levels (8.0 +/- 0.9%; n = 18) compared with control colons. In three of four tumors examined with activated wild-type p21ras, we observed increased c-erbB-2 receptor expression and decreased Ras-GAP expression. In contrast, only one of eight tumors examined with wild-type ras and nonactivated p21ras demonstrated these alterations. Mitogen-activated protein kinase (MAPK) activation and cyclooxygenase-2 (COX-2) expression were increased in tumors with mutated or activated wild-type p21ras, compared with their nonactivated counterparts. Although beta-catenin mutations did not alter COX-2 expression or MAPK activity, mutations in either K-ras or beta-catenin significantly increased cyclin D1 expression. In contrast, in tumors with wild-type but activated p21-ras, cyclin D1 expression was not enhanced. Thus, the spectrum of changes in MAPK, COX-2, and cyclin D1 is distinct among tumors with ras or beta-catenin mutations or nonmutational activation of p21ras.