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The nuclear factor-erythroid 2-related factor 2 (Nrf2) plays a critical role in protecting various tissues against inflammation, which is a potential risk factor for colorectal and other cancers. Our previously published mouse model work showed that Nrf2 helps protect against dextran sulfate sodium (DSS)–induced colitis/inflammation, and others have shown that Nrf2 helps protect against inflammation-associated colorectal carcinogenesis (aberrant crypt foci). The present study extended these important earlier findings by exploring the role of Nrf2 in colitis-associated colorectal cancer in a mouse model involving azoxymethane/DSS–induced colorectal carcinogenesis in Nrf2 knockout mice. Azoxymethane/DSS–treated Nrf2 knockout mice had increased incidence, multiplicity, and size of all colorectal tumors, including adenomas, versus treated wild-type (WT) mice, and the proportion of tumors that were adenocarcinoma was much higher in knockout (80%) versus WT (29%) mice. Compared with WT mice, knockout mice also had increased markers of inflammation in tumor tissue (cyclooxygenase-2 and 5-lipoxygenase expressions and prostaglandin E2 and leukotriene B4 levels) and in inflamed colonic mucosa (nitrotyrosine expression), supporting the association of knockout mouse tumor formation with inflammation. The phase II detoxifying/antioxidant enzymes NAD(P)H-quinone reductase 1 and UDP-glucurosyltransferase 1A1 were elevated in the normal mucosa of WT, but not Nrf 2 knockout, mice treated with azoxymethane/DSS. Our findings show that Nrf2 plays a critical role in protecting against inflammation-associated colorectal cancer.

Perspective from Increased Susceptibility of Nrf2 Knockout Mice to Colitis-Associated Colorectal Cancer

Cancer chemopreventive agent sulforaphane (SFN) and dibenzoylmethane (DBM) showed antitumorigenesis effects in several rodent carcinogenesis models. In this study, we investigated the cancer chemopreventive effects and the underlying molecular mechanisms of dietary administration of SFN and DBM alone or in combination in the ApcMin/+ mice model. Male ApcMin/+ mice (12 per group) at age of 5 weeks were given control AIN-76A diet, diets containing 600 ppm SFN and 1.0% DBM, or a combination of 300 ppm SFN and 0.5% DBM for 10 weeks. Mice were then sacrificed, and tumor numbers and size were examined. Microarray analysis, Western blotting, ELISA, and immunohistochemical staining were done to investigate the underlying molecular mechanisms of cancer chemopreventive effects of SFN and DBM. Dietary administrations of SFN and DBM alone or in combination significantly inhibited the development of intestinal adenomas by 48% (P = 0.002), 50% (P = 0.001), and 57% (P < 0.001), respectively. Dietary administration of 600 ppm SFN and 1.0% DBM also reduced colon tumor numbers by 80% (P = 0.016) and 60% (P = 0.103), respectively, whereas the combination of SFN and DBM treatment blocked the colon tumor development (P = 0.002). Both SFN and DBM treatments resulted in decreased levels of prostaglandin E2 or leukotriene B4 in intestinal polyps or apparently normal mucosa. Treatments also led to the inhibition of cell survival and growth-related signaling pathways (such as Akt and extracellular signal-regulated kinase) or biomarkers (such as cyclooxygenase-2, proliferating cell nuclear antigen, cleaved caspases, cyclin D1, and p21). In conclusion, our results showed that both SFN and DBM alone as well as their combination are potent natural dietary compounds for chemoprevention of gastrointestinal cancers. [Cancer Res 2007;67(20):9937–44]

Supplementary Tables 1-2 from Chemoprevention of Familial Adenomatous Polyposis by Natural Dietary Compounds Sulforaphane and Dibenzoylmethane Alone and in Combination in ApcMin/+ Mouse

This chapter examines scientific evidence for a dietary etiology of colon cancer and proposes dietary guidelines for the primary and secondary prevention of colon cancer. It also examines various diet-related initiators and promoters involved in colon carcinogenesis and the mechanism whereby dietary factors modulate colon carcinogenesis. The concept that dietary fat and certain fibers distinct from chemical contaminants of diet and from other environmental and genetic factors are important determinants of colon cancer risk is reinforced by biochemical epidemiologic studies in humans and laboratory animal studies. Cancer of the colon has been the subject of several epidemiologic reviews. In several populations consuming the diets high in total fat, dietary fiber acts as a protective factor for colon cancer risk. The biological plausibility of the nutritional factors in the etiology of colon cancer is reflected in a number of hypotheses for the mechanism of action.

Epidemiology studies suggest that statins and nonsteroidal anti-inflammatory drugs reduce the risk of prostate cancer. In the present study, LNCaP cells were cultured in regular medium containing fetal bovine serum or in medium supplemented with charcoal-stripped fetal bovine serum to mimic androgen deprivation treatment. We found that atorvastatin (Lipitor) or celecoxib (Celebrex) treatment of LNCaP cells cultured in regular or androgen-depleted medium inhibited growth and stimulated apoptosis. A combination of atorvastatin and celecoxib was more effective than either agent alone. In animal studies, severe combined immunodeficient mice were injected s.c. with LNCaP cells in Matrigel. After 4 to 6 weeks, mice with LNCaP tumors (about 0.6 cm wide and 0.6 cm long) were surgically castrated and received daily i.p. injections of vehicle, atorvastatin (10 μg/g body weight/d), celecoxib (10 μg/g/d), or a combination of atorvastatin (5 μg/g/d) and celecoxib (5 μg/g/d) for 42 days. In all groups, the androgen-dependent LNCaP tumors regressed initially in response to castration, but the tumors eventually progressed to androgen independence and started to grow. Treatment of the mice with atorvastatin or celecoxib alone suppressed the regrowth of LNCaP tumors after castration. A combination of low doses of atorvastatin and celecoxib had a more potent effect in inhibiting the growth and progression of LNCaP tumors to androgen independence than a higher dose of either agent alone. Our results indicate that administration of a combination of atorvastatin and celecoxib may be an effective strategy for the prevention of prostate cancer progression from androgen dependence to androgen independence. Cancer Prev Res; 3(1); 114–24

Gamma-tocopherol (gamma-T) alone or in combination with alpha-tocopherol has been shown to suppress biomarkers of oxidative stress in asthamatics and human subjects with metabolic syndrome. Oxidative stress has been implicated as a key event in prostate carcinogenesis. Hence, the purpose of this study was to examine the effects of gamma-tocopherol-enriched mixed tocopherol diet on prostate carcinogenesis in a murine prostate cancer model (TRAMP). 8 week old TRAMP males were fed 0.1% gamma-T-enriched mixed tocopherol diet that contained 20-fold higher levels of gamma-tocopherol, and roughly 3-fold higher levels of alpha-tocopherol. The effect of such diet on tumor and PIN development was observed. The expression of phase II detoxifying, antioxidant enzymes and Nrf2 mRNA and protein were determined by RT-PCR, immunohistochemistry and western blotting techniques. Treatment with gamma-T-enriched mixed tocopherols significantly suppressed the incidence of palpable tumor and Prostate Intraepithelial Neoplasia (PIN) development without affecting the expression of the transgene (SV-40). Tumor progression occurred with a significant suppression of antioxidant enzymes such as catalase, superoxide dismutase, glutathione peroxidase, heme-oxygenase-1 and phase II detoxifying enzymes. Treatment with gamma-T-enriched mixed tocopherol diet upregulated the expression of most detoxifying and antioxidant enzymes. Nrf2-a redox sensitive transcription factor known to mediate the expression of phase II detoxifying enzymes, was also significantly upregulated following treatment with gamma-T-enriched mixed tocopherol diet. Gamma-T-enriched mixed tocopherols significantly up-regulated the expression of Nrf2 and its related detoxifying and antioxidant enzymes thereby suppressing PIN and tumor development.

BACKGROUND Prostate tissue microenvironment is susceptible to several risk factors including carcinogens, dietary factors, hormones, cytokines and growth factors that could induce chronic inflammation. Because of the difference in the serum levels and the intrinsic ability of monocytes/macrophages to cause harm, the transcriptional responses triggered by inflammatory stimuli must be controlled. Unfortunately, an in-depth association between prostate cancer and potential mediators of inflammation has not been completely investigated. METHODS To determine whether activated macrophage (infiltrating monocytes), iNOS and NF-κB are primary mediators of inflammation, besides COX-2, in prostate carcinogenesis, we examined tissue sections of rat prostate tumor induced by N-methyl-N-nitrosourea (MNU) plus testosterone in a follow-up study. We performed H&E and immunohsitochemical staining of the prostate tissue to detect specific markers of inflammation. RESULTS We report an increase in infiltrating monocyte, iNOS, NF-κBp65, VEGF and TNF-α at the early and advanced stages of tumor growth in MNU plus testosterone treated rats. Monocyte infiltration was often found in the stromal and perivascular regions of the DL prostate. We conclude for the first time that prostate cancer induced by MNU plus testosterone partly involves mediators of inflammation which could trigger the process of carcinogenesis and cause loss of apoptosis. Selective COX-2 inhibitor celecoxib at a dose of 500 mg/kg/bw administered for 52 weeks reduced infiltrating monocytes, inhibited iNOS, NF-κB p65 expression, induced apoptosis and tumor growth inhibition. CONCLUSION Carcinogen plus testosterone induced prostate carcinogenesis showing activation of macrophage, iNOS and NF-κBp65 could be prevented by celecoxib or related anti-inflammatory agents. Prostate 69: 133–141, 2009. © 2008 Wiley–Liss, Inc.

Human intervention studies have suggested an exciting synergistic action between calcium supplementation and aspirin intake in reducing the risk of colorectal cancer. The aim of this study was to determine whether such a synergy can be demonstrated on azoxymethane (AOM)-induced colon aberrant crypt foci (ACF) formation in mice and rats. Female CF-1 mice and male F344 rats were injected subcutaneously with AOM and then received diet treatments for 8 wk. The basal control diet contained high fat (20% mixed lipids by weight) and low calcium (1.4 mg/g diet) to mimic the average Western diet. The treatment diets contained enriched calcium (5.2 mg calcium/g diet), aspirin (0.2 mg aspirin/g diet), or calcium plus aspirin (5.2 mg calcium plus 0.2 mg aspirin/g diet). Treatment with calcium, aspirin, or their combination significantly decreased the number of total ACF and aberrant crypt per mouse (by 43-59%) or rat (by 23-38%), but statistically significant differences among the 3 groups were not observed. A hint of additivity between calcium and aspirin was observed in mice but not in rats. These results indicate that the combination of calcium and aspirin did not produce a synergistic effect on the ACF formation in AOM-treated mice and rats.

Previous studies in animal models have shown enhanced efficacy of a combined treatment of statins and Nonsteroidal anti-inflammatory drugs against colorectal cancer development. In our study, we investigated the combinational effects of atorvastatin and celecoxib in 2 human colon cancer cell lines HCT116 and HT29. Celecoxib moderately inhibited the growth of both cell lines with a similar IC(50) of 40-50 microM, whereas atorvastatin showed stronger growth inhibitory effect in HCT116 cells than in HT29 cells (IC(50) of 5-8 microM vs. 30-35 microM) after treatment for 48-72 hr. The combination of these 2 agents produced strong synergistic actions, as determined by isobologram analysis. Flow cytometry analysis indicated that the combination treatment for 24 hr caused extensive cell cycle arrest in G0/G1 phase; whereas at 48 hr or longer, apoptosis was induced significantly. The effects produced by the combination were much stronger than that by atorvastatin or celecoxib alone. Our results further demonstrated that the combinational effects of atorvastatin/celecoxib were associated with increased levels of p21(Cip1/Waf1), p27(Kip1), and phospho-JNK; decreased levels of phospho-AKT and hyper-phosphorylated Rb; and activation of caspase cascade. Atorvastatin/celecoxib combination also selectively modified membrane localization of small G-proteins, such as RhoA, RhoB and RhoC, which may contribute to the anti-cancer effects. Taken together, the results demonstrated a strong synergy between the actions of atorvastatin and celecoxib in growth inhibition and killing of human colon cancer cells. The present work suggests the possible therapeutic application of this combination and provides leads for mechanistic and biomarker investigations in clinical trials.

Green tea and its constituents have shown cancer-preventive activities in many animal models. In order to prepare for a human trial on the inhibition of colon carcinogenesis, we conducted a study with green tea polyphenols as the preventive agent in an azoxymethane (AOM)-induced rat colon cancer model using aberrant crypt foci (ACF) as an end point. F344 rats were given two weekly injections of AOM (15 mg/kg), and then fed a 20% high-fat diet with or without 0.12 or 0.24% Polyphenon E (PPE, a standardized green tea preparation consisting 65% of (-)-epigallocatechin-3-gallate and 22% of other catechins) for 8 weeks. Colorectal ACF were analyzed under a microscope after methylene blue staining. Dietary PPE administration was found to significantly and dose dependently decrease the total number of ACF per rat and the total number of aberrant crypt per rat. Moreover, treatment with 0.24% PPE also significantly decreased the percentage of large ACF (four or more crypts) and the percentage of ACF with high-grade dysplasia in total ACF. The high-grade dysplastic ACF from 0.24% PPE-treated group had increased apoptosis and decreased nuclear expression levels of beta-catenin and cyclin D1. Retinoid X receptor (RXR)alpha expression was reduced in high-grade dysplastic ACF, adenoma and adenocarcinoma during AOM-induced colon carcinogenesis, and the PPE treatment partially prevented the loss of RXRalpha expression in high-grade dysplastic ACF. Taken together, our results strongly suggest the colon cancer-preventive activity of PPE and identified possible molecular markers for future colon cancer prevention studies.

Purpose: To investigate the effects and mechanisms of atorvastatin and celecoxib administered individually or in combination on human prostate cancer PC-3 cells cultured in vitro or grown as xenograft tumors in immunodeficient mice. Experimental Design: Human prostate cancer PC-3 cells in culture were treated with atorvastatin and celecoxib alone or in combination. Severe combined immunodeficient (SCID) mice were injected s.c. with PC-3 cells. The mice received daily i.p injections starting 2 days before tumor cell inoculation and continuing during the course of treatment with atorvastatin (10 μg/g body weight/d), celecoxib (10 μg/g/d), a combination of atorvastatin (10 μg/g/d) and celecoxib (10 μg/g/d), or a combination of atorvastatin (5 μg/g/d) and celecoxib (5 μg/g/d). Results: Atorvastatin in combination with celecoxib had stronger effects on growth inhibition and apoptosis of PC-3 cells than either agent used individually. Atorvastatin and celecoxib in combination also had a stronger inhibitory effect on activation of nuclear factor-κB and extracellular signal-regulated kinase 1/2 in PC-3 cells than either agent alone. Treatment of SCID mice with combinations of atorvastatin and celecoxib more effectively inhibited the formation and growth of PC-3 tumors in the mice than either agent administered alone. Conclusions: A combination of atorvastatin and celecoxib had a more potent inhibitory effect on the growth of PC-3 cells cultured in vitro or grown in SCID mice than either agent alone. A combination of atorvastatin and celecoxib may be an effective strategy for the prevention of prostate cancer.

Purpose: Epidemiologic studies have linked the consumption of fruits and vegetables to reduced risk of several types of cancer. Laboratory animal model studies have provided evidence that stilbenes, phenolic compounds present in grapes and blueberries, play a role in inhibiting the risk of certain cancers. Pterostilbene, a naturally occurring stilbene from blueberries, was tested for its preventive activity against colon carcinogenesis. Experimental Design: Experiments were designed to study the inhibitory effect of pterostilbene against the formation of azoxymethane-induced colonic aberrant crypt foci (ACF) preneoplastic lesions in male F344 rats. Beginning at 7 weeks of age, rats were treated with azoxymethane (15 mg/kg body weight s.c., once weekly for 2 weeks). One day after the second azoxymethane treatment, rats were fed experimental diets containing 0 or 40 ppm of pterostilbene. At 8 weeks after the second azoxymethane treatment, all rats were sacrificed, and colons were evaluated for ACF formation and for inhibition of inducible nitric oxide synthase (iNOS) and proliferating cell nuclear antigen. Effects on mucin MUC2 were also determined. Results: Administration of pterostilbene for 8 weeks significantly suppressed azoxymethane-induced formation of ACF (57% inhibition, P < 0.001) and multiple clusters of aberrant crypts (29% inhibition, P < 0.01). Importantly, dietary pterostilbene also suppressed azoxymethane-induced colonic cell proliferation and iNOS expression. Inhibition of iNOS expression by pterostilbene was confirmed in cultured human colon cancer cells. Conclusions: The results of the present study suggest that pterostilbene, a compound present in blueberries, is of great interest for the prevention of colon cancer.

This study was designed to investigate the cancer preventive activities of wheat bran (WB) oil. We studied the colon cancer preventive effects of WB oil and its subfractions in the Apc(min/+) mouse model, a recognized mouse model for human colorectal cancer, and used human colon cancer cell lines (HCT-116 and HT-29) to identify possible active fractions in WB oil. Our results showed that the oil fraction of WB was more active than the water fraction against the growth of human colon cancer cell lines and that 2% WB oil significantly inhibited the overall tumorigenesis by 35.7% (p < 0.0001) in the Apc(min/+) mouse model. The WB oil was further fractioned into nonpolar lipids and phytochemicals and the phytochemical fraction was fractionated into phytosterols and phytosterol ferulates, 5-alk(en)ylresorcinols, and unidentified constituents by normal phase silica gel column chromatography. Results on cell culture showed that the phytochemical fraction had a higher inhibitory effect on HCT-116 human colon cancer cells than that of WB oil, whereas the nonpolar lipid fraction had less growth inhibitory effectiveness. However, neither fractions showed a stronger inhibition than WB oil in the Apc(min/+) mouse model. The current results demonstrate, for the first time, the intestinal cancer preventive activity of WB oil. The active ingredients, however, remain to be identified.

Sulforaphane (SFN) is a potent and promising naturally occurring dietary cancer chemopreventive compound that exerts its cancer protective effects by the induction of genes including cellular defensive genes such as phase II detoxifying and antioxidant enzymes. This gene induction primarily occurs via the transcription factor Nrf2 acting on the antioxidant response element (ARE) located at the 5'-flanking region of these genes. In the present study, transcriptional expression profiles of the livers obtained from the nrf2 wild-type mice and the nrf2 knockout (-/-) mice after treatments with vehicle or SFN at 3 and 12h were generated using the Affymetrix 39K oligonucleotide microarray. The Nrf2-dependent, SFN-inducible genes were identified which include detoxification phase I, II drug metabolizing enzymes and phase III transporters. Unexpected clusters of genes include genes for heat shock proteins (HSP), ubiquitin/26S proteasome subunits, and lipid metabolism genes. Collectively, SFN increases the expression of genes through the Nrf2 signaling pathway that directly detoxify exogenous toxins/carcinogens or endogenous reactive oxygen species, and genes involved in the recognition and repair/removal of damaged proteins, which could provide secondary protection against DNA or protein damage that enhance cell survival.

Electrophiles generated during metabolic activation of carcinogens and reactive oxygen species formed from endogenous and exogenous sources might play a significant role in carcinogenesis. Cancer chemoprevention by induction of phase II detoxifying enzymes to counteract the insults of these reactive intermediates is under intensive investigation. Nrf2, a bZIP transcription factor, plays a central role in the regulation of phase II genes by binding to the antioxidant response element (ARE) in their promoters. Identification of novel Nrf2-regulated genes is likely to provide insight into cellular defense systems against the toxicities of electrophiles and oxidants and may define effective targets for achieving cancer chemoprevention. Phenethyl isothiocyanate (PEITC) is a promising chemopreventive agent that exerts its effects by induction of phase II enzymes via activation of Nrf2. In the present study, a transcriptional profile of liver of the wild-type (Nrf2+/+) and knock-out (Nrf2-/-) mice after treatments with vehicle or PEITC at 3 h and at 12 h was generated using the Affymetrix Mouse Genome 430 2.0 Array. Comparative analysis of gene expression changes between different treatment groups of wild-type and Nrf2-deficient mice facilitated identification of numerous genes regulated by Nrf2. These Nrf2-dependent and PEITC-inducible genes include known detoxication enzymes, as well as novel xenobiotic-metabolizing genes regulated by Nrf2 such as CYP 2c55, CYP 2u1 and aldehyde oxidase. Unexpected clusters included genes for heat shock proteins, ubiquitin/26 S proteasome subunits, and lipid metabolism molecules. Collectively, the identification of these genes not only provides novel insight into the effect of PEITC on global gene expression and chemoprevention, but also reveals the role of Nrf2 in those processes, which would confer cancer chemopreventive future.

Preclinical and clinical studies have established evidence that cyclooxygenase-2 (COX-2) inhibitors and statins [hydroxy-3-methylglutaryl CoA reductase (HMGR) inhibitors] inhibit colon carcinogenesis. Chronic use of high doses of COX-2 inhibitors may induce side effects, and combining the low doses of agents may be an effective way to increase their efficacy and minimize the side effects. We assessed the chemopreventive efficacy of atorvastatin (Lipitor) and celecoxib individually or in combination in an animal model of familial adenomatous polyposis. Six-week-old male C57BL/6J-APCmin/+ mice were either fed diets containing 0 or 100 ppm atorvastatin or 300 ppm celecoxib, or a combination of both for ∼80 days. Mice were sacrificed, and their intestines were scored for tumors. Normal-seeming mucosa and intestinal tumors were harvested and assayed for apoptosis (terminal deoxynucleotidyl transferase–mediated nick-end labeling) and HMGR and COX-2 protein expression and activity. We observed that 100 ppm atorvastatin significantly (P < 0.002) suppressed intestinal polyp formation. As anticipated, 300 ppm celecoxib decreased the rate of formation of intestinal polyps by ∼70% (P < 0.0001). Importantly, the combination of 100 ppm atorvastatin and 300 ppm celecoxib in the diet suppressed the colon polyps completely and small intestinal polyps by >86% (P < 0.0001) compared with the control group. The inhibition of tumor formation by the atorvastatin and celecoxib combination was significant (P < 0.005) when compared with tumor inhibition by celecoxib alone. In addition, increased rates of apoptosis in intestinal tumors (P < 0.01-0.0001) were observed in animals fed with atorvastatin and celecoxib and more so with the combinations. Tumors of animals fed atorvastatin showed a significant decrease in HMGR-R activity. Similarly, tumors of mice exposed to celecoxib showed significantly lower levels of COX-2 activity. These observations show that atorvastatin inhibits intestinal tumorigenesis and that, importantly, when given together with low doses of celecoxib, it significantly increases the chemopreventive efficacy in an APCmin mice. (Cancer Res 2006; 66(14): 7370-7)

Epidemiological data indicate that colon cancer incidence is associated mainly with high dietary fat intake. Studies in metabolic epidemiology have shown a strong association between dietary fat intake, level of fecal bacteria, fecal acid, and neutral sterols, and the risk of colon cancer among different populations. Current concepts visualize that colonic bile acids and cholesterol metabolites play a modifying role in large bowel carcinogenesis, and that these compounds are derived from dietary factors, directly or indirectly, and subsequently are modified by the intestinal bacteria. In animal models, lithocholic acid and deoxycholic acid, which are present in high concentration in the large bowel of man, acted as promoters of colon carcinogenesis. The carcinogenic effect of azoxymethane in rats was enhanced by the increase of bile salts in the colon induced by surgical means. Animals fed a high fat diet were more susceptable to colon tumor induction by dimethylhydrazine compared with rats fed a normal fat diet. Our data also demonstrate that the intestinal microflora played a modifying role in accelerating colon tumor production by dimethylhydrazine.

Nitric oxide–releasing nonsteroidal anti-inflammatory drugs (NO-NSAID) are promising chemoprevention agents; unlike conventional NSAIDs, they seem free of appreciable adverse effects, while they retain beneficial activities of their parent compounds. Their effect on colon carcinogenesis using carcinoma formation as an end point is unknown. We assessed the chemopreventive properties of NO-indomethacin (NCX 530) and NO-aspirin (NCX 4016) against azoxymethane-induced colon cancer. Seven-week-old male F344 rats were fed control diet, and 1 week later, rats received two weekly s.c. injections of azoxymethane (15 mg/kg body weight). Two weeks after azoxymethane treatment, rats (48 per group) were fed experimental diets containing NO-indomethacin (0, 40, or 80 ppm), or NO-aspirin (1,500 or 3,000 ppm), representing 40% and 80% of the maximum tolerated dose. All rats were killed 48 weeks after azoxymethane treatment and assessed for colon tumor efficacy and molecular changes in colonic tumors and normally appearing colonic mucosa of different dietary groups. Our results suggest that NO-indomethacin at 40 and 80 ppm and NO-aspirin at 3,000 ppm significantly suppressed both tumor incidence (P < 0.01) and multiplicity (P < 0.001). The degree of inhibition was more pronounced with NO-indomethacin at both dose levels (72% and 76% inhibition) than with NO-aspirin (43% and 67%). NO-indomethacin at 40 and 80 ppm and NO-aspirin at 3,000 ppm significantly inhibited the colon tumors' (P < 0.01 to P < 0.001) total cyclooxygenase (COX), including COX-2 activity (52–75% inhibition) and formation of prostaglandin E2 (PGE2), PGF2α, and 6-keto-PGF1α, and TxB2 from arachidonic acid (53–77% inhibition). Nitric oxide synthase 2 (NOS-2) activity and β-catenin expression were suppressed in animals given NO-NSAID. In colonic crypts and tumors of animals fed these two NO-NSAIDs, there was a significant decrease in proliferating cell nuclear antigen labeling when compared with animals fed the control diet. The results of this study provide strong evidence that NO-NSAIDs possess strong inhibitory effect against colon carcinogenesis; their effect is associated with suppression of COX and NOS-2 activities and β-catenin levels in colon tumors. These results pave the way for the rational design of human clinical trials. [Mol Cancer Ther 2006;5(6):1530–8]

Preclinical and clinical studies have provided evidence that aspirin, celecoxib, (cyclooxygenase-2 inhibitor), and statins (3-hydroxy-3-methylglutaryl CoA reductase inhibitors) inhibit colon carcinogenesis. Chronic use of high doses of these agents may induce side effects in ostensibly normal individuals. Combining low doses of agents may be an effective way to increase their efficacy and minimize toxicity. We assessed the efficacy of atorvastatin (lipitor), celecoxib, and aspirin, given individually at high dose levels and in combination at lower doses against azoxymethane-induced colon carcinogenesis, in male F 344 rats. One day after the last azoxymethane treatment (15 mg/kg body weight, s.c., once weekly for 2 weeks), groups of male F 344 rats were fed the AIN-76A diet or AIN-76A diet containing 150 ppm atorvastatin, 600 ppm celecoxib, and 400 ppm aspirin, 100 ppm atorvastatin + 300 ppm celecoxib, and 100 ppm atorvastatin + 200 ppm aspirin. Rats were killed 42 weeks later, and colon tumors were processed histopathologically and analyzed for cell proliferation and apoptosis immunohistochemically. Administration of these agents individually and in combination significantly suppressed the incidence and multiplicity of colon adenocarcinomas. Low doses of these agents in combination inhibited colon carcinogenesis more effectively than when they were given individually at higher doses. Inhibition of colon carcinogenesis by these agents is associated with the inhibition of cell proliferation and increase in apoptosis in colon tumors. These observations are of clinical significance because this can pave the way for the use of combinations of these agents in small doses against colon cancer. (Cancer Res 2006; 66(8): 4542-6)

Sulforaphane (SFN) is an isothiocyanate that is present in widely consumed vegetables. Previous studies have shown that SFN is effective in preventing carcinogenesis induced by carcinogens in rodents. Recently it was found that SFN could also suppress the growth of intestinal polyps in the ApcMin/+ mouse. In the present study, the acute effect of SFN on the gene expression profile in small intestinal polyps of ApcMin/+ mice using Affymetrix microarray was performed. SFN is a strong inducer for phase II drug metabolizing enzymes, which is believed to contribute to its chemopreventive properties. However, the results show that genes involved in apoptosis, cell growth and maintenance rather than the predicted phase II genes were modulated. The proapoptotic genes including MBD4, TNFR-7 and TNF (ligand)-11 were up-regulated while pro-survival genes including cyclin-D2, integrin-beta1 and Wnt-9A were down-regulated. Interestingly, two genes potentially involved in colorectal carcinogenesis, 15-LOX and COX-2 were found to be increased and decreased, respectively. In conclusion, the results show, for the first time, that chemopreventive agents such as SFN regulate different set of genes involving apoptosis, cell growth/maintenance and inflammation in the small intestinal polyps of ApcMin/+ mice, which could contribute to the overall chemopreventive pharmacological effects.

Many naturally occurring compounds, including beta-phenylethyl isothiocyanate (PEITC) and curcumin, exhibit significant anti-cancer chemopreventive effects. In this study, we investigated the combined effects of PEITC and curcumin in PC-3 human prostate cancer cells and in PC-3 cells that were stably transfected with an NF-kappaB luciferase plasmid (PC-3 C4). We found an additive effect of PEITC and curcumin for the induction of apoptosis. To elucidate the potential mechanisms of this effect, we studied several critical cellular signaling pathways, including the critical NF-kappaB cell survival signal that is hyper-activated in PC-3 cells and many other cancers. PEITC and curcumin additively inhibited NF-kappaB luciferase activity. Furthermore, the combined treatment significantly increased the activity of poly(ADP-Ribose) polymerase and cleavage of caspase-3 in correlation with apoptotic cell death. Studying upstream signaling events, we found that the phosphorylations of IkappaBalpha and Akt (Ser473, Thr308) were significantly attenuated by the combination of PEITC and curcumin. As these events can be downstream of the activation of epidermal growth factor receptor (EGFR), we pretreated PC-3 cells with PEITC and curcumin and then stimulated them with EGF. EGFR phosphorylations (Y845 and Y1068) were dramatically suppressed by PEITC or curcumin, and more so by the combination. Importantly, the degree of Akt and PI3K phosphorylations induced by EGF were also significantly suppressed. We conclude that the simultaneous targeting of EGFR, Akt and NF-kappaB signaling pathways by PEITC and curcumin could be the molecular targets by which PEITC and curcumin exert their additive inhibitory effects on cell proliferation and ultimately lead to programmed cell death of tumor cells.

Epidemiological and preclinical studies suggest that diets that are rich in n-3 polyunsaturated fatty acids (PUFAs) and selenium (Se) reduce the risk of colon cancer. Studies conducted in our laboratory have indicated that synthetic organoselenium 1,4-phenylene bis(methylene) selenocyanate (p-XSC) is less toxic and more effective than inorganic Se and selenomethionine, the major Se compound in natural selenium yeast. Through cDNA microarray analysis, we have demonstrated earlier that the n-3 PUFA docosahexaenoic acid (DHA), modulated more than one signaling pathway by altering several genes involved in colon cancer growth. There is increasing interest in the use of combinations of low doses of chemopreventive agents that differ in their specific modes of action as this approach can minimize toxicity and increase efficacy in model assays. In the present study we assessed the efficacy of DHA and p-XSC individually and in combination at low doses in CaCo-2 colon cancer cells, using cell growth inhibition and apoptosis as measures of chemopreventive efficacy. On the basis of western blot and RT-PCR analysis, we also determined the effects of DHA and p-XSC on the levels of expression of cyclooxygenase-2, inducible nitric oxide synthase, cyclin D1, beta-catenin and nuclear factor kappaB, all of which presumably participate in colon carcinogenesis. A 48 h incubation of CaCo-2 cells with 5 microM each DHA or p-XSC induced cell growth inhibition and apoptosis and altered the expression of the above molecular parameters. Interestingly, the modulation of these cellular and molecular parameters was more pronounced in cells treated with low doses of DHA and p-XSC (2.5 microM each) in combination than in cells treated with these agents individually at higher concentrations (5.0 microM each). These findings are viewed as highly significant since they will provide the basis for the development of combinations of low dose regimens of DHA and p-XSC in preclinical models against colon carcinogenesis and, ultimately, in human clinical trials.

Purpose: Epidemiologic studies have revealed a decreased risk of colon cancer among people who have regularly taken cyclooxygenase (COX)-2 inhibitors such as aspirin or other nonsteroidal anti-inflammatory drugs (NSAIDs). Whereas the selective COX-2 inhibitor celecoxib and exisulind, a metabolic product of sulindac, have gained increasing attention as efficacious chemopreventive agents against colon and prostate cancer, not much is known about the underlying molecular targets and mechanisms. Moreover, the side effects of NSAIDs are a major obstacle for large-scale application to the prevention of cancer in humans; for example, in the United States in 1998, there were 16,550 deaths from NSAID-induced gastrointestinal complications. The toxicity associated with these compounds is raising concerns, and more needs to be known about their mode of action and molecular targets. Experimental Design: We used the transgenic mouse prostate (TRAMP) model, which exhibits similarities with human prostate cancer, including epithelial origin, progression from the PIN stage to adenocarcinoma, and metastasis by a transgene that is hormonally regulated by androgens. In addition to histologically analyzing the PIN lesions of the dorsolateral prostate from TRAMP mice, we delineated the molecular targets and mechanisms of celecoxib and exisulind against mouse PIN lesions. We performed Western blot analysis of the total protein lysate from the tissues of mouse PIN lesions to measure the level of expression of androgen receptor, vascular endothelial growth factor, nuclear factor-κB p65, BclII, AKT (total and phosphorylated Ser473), p53, cyclin-dependent kinase inhibitor p21WAF1/CIP1, p27, BAX, and caspase-3 to demonstrate the COX-2–independent mechanism involved in the inhibition of PIN lesions of the dorsolateral prostate by both celecoxib and exisulind. Results: We found for the first time that (a) both celecoxib and exisulind as dietary supplements induce strong inhibitory effects against prostate cancer at doses of 800 and 500 ppm, respectively, after 16 weeks; (b) the histologic analysis of the dorsolateral prostate after 2 weeks of treatment indicated a reduction of PIN lesions from 75% to 19% with celecoxib and to 16% with exisulind; (c) more importantly, those few PINs and adenocarcinomas in the groups treated with celecoxib or exisulind showed more apoptotic cells, lower levels of proliferating cell nuclear antigen, and a lower number of mitotic cells. To understand the molecular mechanisms involved in the inhibition of PIN lesions, first, we examined the expression of molecular targets involved in angiogenesis and inflammatory processes. It was clearly evident from Western blot analysis of the total protein lysate derived from the dorsolateral prostate tissues with PIN lesions that expression of androgen receptor, vascular endothelial growth factor, nuclear factor-κB p65, and BclII is down-regulated more effectively by celecoxib. Down-regulation of AKT protein (total and phosphorylated at Ser473) signaling by celecoxib clearly indicates an inhibition of the survival gene and the pathological process that could otherwise lead to adenocarcinoma. Conclusions: Overall, the findings from this study clearly show the effectiveness of celecoxib and exisulind in reducing the PIN lesions by modulating a cascade of molecular targets involved in COX-2–dependent and –independent mechanisms. Whereas these agents are already in clinical trial or in use as chemopreventive agents, findings from this study demonstrate the difference in their mode of action, thus helping us to understand the side effects.

To develop efficient synergistic or additive combinations of chemopreventive and nutritional agents to reduce the risk of colon cancer, experiments were designed to test the application of a selective cyclooxygenase-2 (COX-2) inhibitor together with dietary ω-3 polyunsaturated fatty acids (PUFAs), such as decosahexaenoic acid (DHA). Thus, individual application of celecoxib, a COX-2 inhibitor, DHA, a ω-3 PUFA, and combinations of both were tested for their effectiveness using cell proliferation, apoptosis, and COX-2 expression as markers in the human colon cancer HCA-7 cell line. HCA-7 cells exposed to various subtoxic doses of celecoxib, DHA, or combinations of both were analyzed for inhibition of cell proliferation by trypan blue exclusion and proliferating cell nuclear antigen methods, induction of apoptosis by 4′,6-diamidino-2-phenylindole method, and COX-2 by reverse transcription-PCR and Western blot analysis. In addition, we examined the inhibitory potential of celecoxib and DHA on 14C-arachidonic acid metabolism mediated by COX-2 in the HCA-7 cell line. We found that treatment with celecoxib (50–150 μm) or DHA (150–225 μm) individually induces apoptosis and inhibits cell proliferation only at high concentrations in HCA-7 cell lines. A synergistic effect was observed on induction of apoptosis and inhibition of proliferation when cells were exposed to low doses of celecoxib (50–100 μm) together with DHA (75 μm). At high concentrations, celecoxib and DHA blocked the increase in COX-2 protein and mRNA expression in HCA-7 cells. Importantly, the inhibition of COX-2 expression was more pronounced in cells treated with low-dose combinations than with individual agents at high concentrations. In addition, celecoxib and DHA at low-dose levels inhibited 14C-arachidonic acid metabolism (50–85%, P < 0.0001) leading to very low levels of type 2 series prostaglandin formation. These findings provide the basis for the development of combinations of low-dose regimens of a COX-2 inhibitor and ω-3 PUFAs such as DHA for the prevention and treatment of colon cancer. We are currently testing this concept in preclinical models.

Epidemiological studies suggest that carotenoids such as beta-carotene and lutein play an important role in reducing the risk for several cancers. However, in colon cancer there is ambiguity with regard to the role of these compounds in that both preventive effects and tumor promotion have been observed. In the present study we observed that male F344 rats were able to tolerate up to 2,500 ppm of beta-carotene as well as of lutein. We have then assessed the chemopreventive efficacy of beta-carotene and lutein at dose levels of approximately 4 and 8% of the 2,500 ppm tolerated dose (TD) and also approximately 40 and 80% of the TD on azoxymethane (AOM)-induced colon carcinogenesis, using aberrant crypt foci (ACF) as a surrogate biomarker for colon cancer. Throughout the experiments, 5-week-old male F344 rats were fed the control diet (modified AIN-76A) or experimental diets containing 100 or 200 ppm (approximately 4 or 8% of the 2,500 ppm TD), or 1,000 or 2,000 ppm ( approximately 40 or 80% of the 2,500 ppm TD) of beta-carotene and lutein (n=10 rats/group). After 2 weeks on the experimental or control diets, all animals were injected with AOM (15 mg/kg body wt., once weekly for 2 weeks). At 14 weeks of age, all rats were killed, and their colons were evaluated for ACF. Administration of 100 or 200 ppm of beta-carotene inhibited AOM-induced total colonic ACF formation by 24% (p

During recent years, multidisciplinary studies in epidemiology and molecular biology have contributed to our understanding of the etiology of colorectal cancer; more importantly they have enabled us to approach its prevention. An impressive body of epidemiological data suggests an inverse relationship between colorectal cancer risk and consumption of diets rich in omega (ω)-3 fatty acids (n-3 PUFAs) or the regular use of nonsteroidal antiinflammatory drugs (NSAIDs), including aspirin. The development of strategies for the chemoprevention of colorectal cancer have been facilitated by the use of relevant animal models mimicking the neoplastic processes that occur in humans, including similarities in histopathology and molecular and genetic lesions during both the early and promotion/progression stages of carcinogenesis. Studies with the azoxymethane-F344 rat model indicate that diets rich in n-3 PUFAs, NSAIDs, selective cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) inhibitors, and curcumin can reduce the incidence of colon cancer. Advances in the knowledge of the mechanisms by which chemopreventive agents act offer opportunities to use combinations of specific chemopreventive agents, having clinically beneficial aggregate activity with minimal toxicity. This approach is extremely important when a promising chemopreventive agent demonstrates apparent efficacy but may produce toxic effects at high doses. Our studies show that a combination of very low doses of piroxicam (NSAID) and difluoromethylornithine, a specific inhibitor of ornithine decarboxylase, or very low doses of COX-2 and HMG-CoA reductase inhibitors are more effective in inhibiting colon carcinogenesis than administration of these compounds as single agents even at higher levels. The natural history of colorectal cancer, from dysplastic aberrant crypts to adenomas and adenocarcinomas, offers multiple opportunities for assessment and intervention. Of further importance is to identify whether the molecular targets that are critical in the growth and survival of the malignant colorectal cell are modulated by n-3 PUFAs, NSAIDs, or COX-2 and iNOS inhibitors. Environ. Mol. Mutagen. 44:26–35, 2004. © 2004 Wiley-Liss, Inc.

16S ribosomal DNA terminal restriction fragment patterns from rat fecal samples were analyzed to track the dynamics of Lactobacillus acidophilus NCFM and discern bacterial populations that changed during feeding with NCFM. Lactobacillus johnsonii and Ruminococcus flavefaciens were tentatively identified as such bacterial populations. The presence of L. johnsonii was confirmed by isolation from feces.

Epidemiological studies have linked consumption of broccoli to a reduced risk of colon cancer in individuals with the glutathione S-transferase M1 (GSTM1) null genotype. GSTs are involved in excretion and elimination of isothiocyanates (ITCs), which are major constituents of broccoli and other cruciferous vegetables and have cancer chemopreventive potential, so it is speculated that ITCs may play a role in protection against human colon cancer. However, there is a lack of data from animal studies to support this. We carried out a bioassay to examine whether sulforaphane (SFN) and phenethyl isothiocyanate (PEITC), major ITCs in broccoli and watercress, respectively, and their corresponding N:-acetylcysteine (NAC) conjugates, show any chemopreventive activity towards azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) in F344 rats. Groups of six male F344 rats were treated with AOM subcutaneously (15 mg/kg body wt) once weekly for 2 weeks. SFN and PEITC and their NAC conjugates were administered by gavage either three times weekly for 8 weeks (5 and 20 micromol, respectively) after AOM dosing (post-initiation stage) or once daily for 3 days (20 and 50 micromol, respectively) before AOM treatment (initiation stage). The bioassay was terminated on week 10 after the second AOM dosing and ACF were quantified. SFN, SFN-NAC, PEITC and PEITC-NAC all significantly reduced the formation of total ACF from 153 to 100-116 (P < 0.01) and multicrypt foci from 52 to 27-38 (more than four crypts/focus; P < 0.05) during the post-initiation treatment. However, only SFN and PEITC were effective during the initiation phase, reducing the total ACF from 153 to 109-115 (P < 0.01) and multicrypt foci from 52 to 35 (more than four crypts/focus; P < 0.05). The NAC conjugates were inactive as anti-initiators against AOM-induced ACF. These findings provide important laboratory evidence for a potential role of SFN and PEITC in the protection against colon cancer.

Large bowel cancer is not only the third most frequent cancer in the world but is one of the most common human malignancies in Western countries, including North America. In recent years, multidisciplinary research in epidemiology, molecular biology, and laboratory animal model studies have contributed much to our understanding of the aetiology of this cancer; more importantly, it has enabled us to devise preventive strategies. Several epidemiological studies have detected a 40 to 50% decrease in risk of colorectal cancer in individuals who regularly use aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs). Clinical trials with NSAIDs in patients with familial adenomatous polyposis have demonstrated that treatment with NSAIDs caused regression of pre-existing adenomas. Preclinical efficacy studies have provided scientifically sound evidence as to how NSAIDs act to retard, block, or reverse colonic carcinogenesis. Equally exciting are opportunities for effective chemoprevention with selective cyclo-oxygenase-2 inhibitors in a variety of animal models of colon cancer. Selective cyclo-oxygenase-2 inhibitors such as celecoxib have been proven to be effective chemopreventive agents against colonic carcinogenesis with minimal gastrointestinal toxicity. Our exploration of the multistep process of carcinogenesis has provided substantial insights into the mechanisms by which anti-inflammatory agents modulate these events. There is growing optimism for the view that realisation of preventive concepts in large bowel cancer will also serve as a model for preventing malignancies of the prostate and breast.

▪ Abstract Colorectal cancer is a significant cause of mortality in Western societies. The progression of the disease from normal colonic epithelium to the acquisition of the malignant phenotype is accompanied by numerous genetic and epigenetic alterations. Compelling experimental and epidemiological evidence indicates that diet and nutrition are key factors in the modulation of colorectal cancer. A salient case in point is the recent observation that a dietary regimen based on a Western-style diet provokes in the rodent colon the appearance of preneoplastic lesions in the absence of any genotoxic insult. This review mainly describes dietary factors that inhibit the development and progression of colorectal cancer. Much is unknown about the precise mechanisms of action of chemically disparate nutrients and how they interfere with the development and progression of this disease. Current knowledge about this important issue is summarized. We believe that continuing scrutiny and precise assessment of the benefits (and potential risks) of nutrients in the treatment and prevention of colorectal cancer will prove significant to controlling this devastating disease.

Oligofructose and inulin, selective fermentable chicory fructans, have been shown to stimulate the growth of bifidobacteria, which are regarded as beneficial strains in the colon. Studies were designed to evaluate inulin (Raftiline) and oligofructose (Raftilose) for their potential inhibitory properties against the development of colonic aberrant crypt foci (ACF) in rats. ACF are putative preneoplastic lesions from which adenomas and carcinomas may develop in the colon. The results of this study indicate that dietary administration of oligofructose and inulin inhibits the development of ACF in the colon, suggesting the potential colon tumor inhibitory properties of chicory fructans. The degree of ACF inhibition was more pronounced in animals given inulin than in those fed oligofructose. Because these prebiotics selectively stimulate the growth of bifidobacteria, ornithine decarboxylase (ODC) activities, ras-p21 ontoprotein expressions and tumor inhibitory activity of lyophilized cultures of Bifidobacterium longum against chemically induced colon and mammary carcinogenesis and against colonic tumor cell proliferation were examined. Dietary administration of lyophilized cultures of B. longum strongly suppressed colon and mammary tumor development and tumor burden. Inhibition of colon carcinogenesis was associated with a decrease in colonic mucosal cell proliferation and activities of colonic mucosal and tumor ornithine decarboxylase and ras-p21. Human clinical trials are likely to broaden our insight into the importance of the pre- and probiotics in health and disease.

Studies have demonstrated a reduced risk of colon cancer when populations with diets high in total fat switched to a diet high in total fiber and certain whole-grain foods. Case-control studies have shown convincingly the relationship between dietary fiber and colon cancer. Studies in animal models have demonstrated that the inhibitory effects of dietary fiber on the development of colonic neoplasms depend on the nature and source of the fiber; that wheat bran appears to inhibit colon tumor development more consistently than do other dietary sources of fiber, such as oat and corn bran; and that dietary administration of phytic acid, high levels of which are present in wheat bran, inhibits colon carcinogenesis. Human dietary intervention studies have also indicated that the modifying effect of dietary fiber on bacterial enzymes involved in the production of putative colon tumor promoters depends on the type of fiber consumed. Dietary wheat bran, but not oat or corn bran, significantly decreased the levels of several tumor promoters in the colon, independent of stool bulk.

In studies of various human diseases, it is critical that reliable animal models are developed that demonstrate similarity to the human disease. The relative rarity of spontaneous epithelial tumors of the colon in experimental animals and the absence of evidence of virally induced large bowel tumors provided rationale to develop chemically induced experimental models for colon carcinogenesis. Such animal models have been developed to study the multiple environmental factors involved in the pathogenesis of cancer of the colon. These animal models are the following: (1) induction of colon tumors in rats through aromatic amines such as 3,2′-dimethyl-4-aminobiphenyl (DMAB); (2) derivatives and analogues of cycacin such as methylazoxymethanol (MAM), 1,2-dimethylhydrazine (DMH), and azoxymethane (AOM) in rats and mice of selected strains; (3) direct-acting carcinogens of the type of alkylureas, such as methylnitrosourea (MNU) or N -methyl- N ′-nitro- N -nitrosoguanidine (MNNG); and (4) heterocyclic amines such as 2-amino-3-methylimidazo[4,5- f ]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5- b ]pyridine (PhIP). The spectrum of epithelial lesions induced in the colon by these chemical carcinogens is similar to various types of neoplastic lesions observed in the colorectum of humans. Some of the earlier studies tested the carcinogenicity of the agents. Subsequent studies were of histogenesis, cell proliferation kinetics, genetic susceptibilities, effects of dietary components, effect of fecal stream, bile acids, and bacterial flora, to cite a few. Some recent studies were directed toward inhibition of colon carcinogenesis by nutritional factors and chemopreventive agents. This brief review discusses the strengths and weaknesses of various animal models to study the relationship between the nutritional factors and chemopreventive agents and colon carcinogenesis. Animal models are extremely valuable in understanding human disease, but one must use extreme care in selecting realistic models. Indeed, one must be familiar with the limitations of the model system that is being used to study the human disease. Based on the analysis of weaknesses and strengths of several models, we found that the AOM model system appears to be appropriate, because of the similarities in histopathology of adenomas and adenocarcinomas induced in the colon and regional distribution of colon tumors with human large bowel tumors. Also, the ongoing nutritional and chemoprevention trials in humans are, in part, based on the results generated using this model system.

Oligofructose and inulin, selective fermentable chicory fructans, have been shown to stimulate the growth of bifidobacteria which are regarded as beneficial strains in the colon. Studies were designed to evaluate inulin (Raftiline) and oligofructose (Raftilose), for their potential inhibitory properties against aberrant crypt foci (ACF) formation in the colon of rats. ACF are putative preneoplastic lesions from which adenomas and carcinomas may develop. The results of this study demonstrate that dietary administration of oligofructose and inulin inhibits the formation of preneoplastic lesions in the colon suggesting the potential colon tumour inhibitory properties of chicory fructans. Since these prebiotics selectively stimulate the growth of bifidobacteria, tumour inhibitory activity of lyophilized cultures of Bifidobacterium longum (BL) against azoxymethane (AOM)-induced colon carcinogenesis in rats and modulating effect of these cultures on colonic tumour cell proliferation, ornithine decarboxylase (ODC) activity, and ras-p21 oncoprotein expression were investigated. Dietary administration of lyophilized cultures of BL strongly suppressed AOM-induced colon tumour development. Inhibition of colon carcinogenesis was associated with a decrease in colonic mucosal cell proliferation and colonic mucosal and tumour ODC and ras-p21 activities.

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.

Many laboratory studies and human epidemiological data suggest that most cancer deaths are attributable to lifestyle, including nutritional factors and tobacco and alcohol consumption. Tobacco consumption is causally related to cancer of the lung, mouth, larynx, esophagus, bladder, kidney, and pancreas. Nutrients and non-nutrient dietary components probably account for cancer of the colon, breast, prostate, and stomach. This report is based on literature and our own data pertaining to the role of dietary fat, calories, and fiber in the development of colon and breast cancer. We also discuss the evidence from epidemiological, mechanistic, and preclinical efficacy studies indicating a protective effect of micronutrients, non-nutrients, and certain antioxidants in food against oral and lung cancers. Given the continuing cancer burden and the relatively slow impact of proven cancer treatment strategies in reducing cancer mortality, it is essential to evaluate promising nutrients and non-nutrients in foods as chemopreventive agents in persons at increased risk for cancer. Development of reliable intermediate biomarkers is valuable for clinical chemoprevention intervention trials. The purpose of this report is to provide the reader with plausible approaches to cancer control.

BACKGROUND & AIMS: Multiple studies show that continuous use of nonsteroidal anti-inflammatory drugs (NSAIDs) lowers the risk of colon cancer in humans and carcinogen-treated rodents. One target for NSAIDs is cyclooxygenase (COX), and two isoforms of this enzyme have been identified: COX-1 and COX-2. The present study was undertaken to determine if there is differential expression of COX in colonic tumors in azoxymethane-treated rats. METHODS: COX-1 and COX-2 messenger RNA levels were determined by Northern blot analysis of total RNA isolated from colonic tumors and normal adjacent mucosa. COX-2 protein levels were determined by Western blotting analysis. Quantitation of relative band densities was performed using standard densitometry scanning techniques. RESULTS: There was a marked increase in COX-2 RNA levels in six of six colonic tumors compared with paired normal mucosa. In contrast, there was equivalent intensity of the COX-1 RNA transcript between the normal mucosa and tumor in all of the specimens examined. Western blotting analysis showed an increase in the level of the COX-2 protein in four of five of the colonic tumor samples. CONCLUSIONS: COX- 2 but not COX-1 gene expression is markedly elevated in most colonic tumors examined in azoxymethane-treated rodents. COX-2 may provide a target for chemopreventive strategies for colorectal cancer. (Gastroenterology 1996 Apr;110(4):1259-62)

The available data support the concept that high-fat diets increase cytochrome P-450 activities in the liver, leading to increased rates of carcinogen metabolism and, in some instances, DNA adduct formation. Therefore we investigated whether a high-fat diet can also influence DNA methylation by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in the lungs of rats. Male F344 rats were fed a regular AIN-76A low-fat (5% corn oil) or AIN-76A high-fat (23.5% corn oil) diet. After three weeks on this dietary regimen, the animals were injected subcutaneously once daily for four days with NNK at 0.39 mmol/kg body wt. Groups of rats were sacrificed 4 and 24 hours after the last NNK administration; livers and lungs were excised for DNA isolation. We found that the high-fat diet significantly enhanced the formation of O6-methylguanine (O6-mGua) in the rat lung four hours (p < 0.01) after the last carcinogen administration. This may, in part, account for our previous finding in regard to the enhancing effect of the high-fat diet on NNK-induced lung carcinogenesis. There was no effect on O6-mGua or 7-mGua in the rat liver at either time point. To further elucidate the enhancing effect of the high-fat diet on DNA methylation by NNK in the lung, we determined its effect on the in vitro and in vivo metabolism of NNK. The in vitro data indicated that dietary fat has no measurable effect on liver and lung microsomal mixed-function oxidase in catalyzing the metabolic activation of NNK. The results of the metabolism study of NNK in vivo appear to be consistent with the in vitro finding, in that fat had no effect on the excretion pattern of NNK or on the distribution pattern of its urinary metabolites. It is apparent that the enhancing effect of the high-fat diet on O6-mGua in the lung of rats that was measured four hours after NNK injection requires future investigations.

Carcinogen induction of neoplasms in rodent colon has been used as a model for human colon cancer development and for evaluating chemopreventive regimens. We studied the regional distribution of small and large intestinal tumors in 229 rats given azoxymethane (AOM) once weekly for two weeks (15 mg/kg sc). The AOM regimen induced 63% more tumors in distal (DC) than in proximal colon (PC), although tumor volume was greater in PC. A high‐fat (23% com oil) diet increased tumors in PC and DC (p < 0.01). Caloric restriction of 10–30% of the ad libitum diet progressively reduced DC tumor formation but did not alter PC tumors. Tumor volume was unaffected by either regimen. Small intestinal tumors were concentrated in the proximal 15 cm of the intestine and were unaffected by dietary manipulation. This AOM model of colon tumor formation approximates human colon cancer distribution and is an appropriate model for rodent chemopreventive studies.