Omega-3 fatty acids in colorectal cancer prevention.

Abstract

Colon cancer, which is the fourth most common cancer in the world, is one of the leading causes of cancer death in both men and women in the United States, Canada, Northern and Western Europe, Australia and New Zealand.1, 2 It is markedly less frequent in Asia, Africa and South America.1, 2 Therefore, it is a major public health problem. Migrant and temporal trend studies suggest that colon cancer is determined largely by environmental exposures, especially nutritional habits.1 Marked international differences in the incidence and mortality of colon cancer and increase of risk in populations migrating from low- to high-risk areas such as from Japan, China and the Philippines to the United States within 1 or 2 generations suggest that environmental factors, specifically dietary habits, rather than the genetic factors play an important role in the etiology of this cancer. This upward trend in incidences of colon cancer among Japanese immigrants in Hawaii and California compared to Japanese in Japan stimulated epidemiologists to investigate the reasons for this increase. Although the relationship between nutrition and cancer is complex and sometimes perplexing to nutritionists and to those who visualize carcinogenesis in terms of a specific carcinogen, it should be recognized that nutritional factors and diet may relate to cancer risk in several ways; first, food additives, contaminants, a particular dietary component, or products formed during food preparation may act as carcinogens, cocarcinogens and/or promoters; second, nutrient deficiencies and excesses may lead to biochemical/molecular alterations that may promote neoplastic processes; third, changes in the intake of selected macronutrients may induce metabolic, biochemical and molecular abnormalities that enhance cancer risk; and fourth, certain dietary constituents act as anticarcinogens or chemopreventives. During the last 3 decades, substantial progress has been made in understanding the relationship between dietary constituents and colon cancer risk. Fish oils are unique because they contain high levels of polyunsaturated omega-3 fatty acids (n-3 PUFAs) that are not present in vegetable oils or in saturated fats. Omega-3 fatty acids that are present in fish oil include docosahexaenoic acid (DHA; C22:6), eicosapentaenoic acid (EPA; C20:5) and docosapentaenoic acid (DPA; C22:5). Vegetable oils including corn oil and safflower oil contain high levels of linoleic acid (LA; C18:2). LA has the terminal double-bond 6 carbon atoms from the terminal (omega) methyl group of fatty acid, whereas DHA has the terminal double-bond 3 carbon atoms from the terminal (omega) methyl group of fatty acid (Fig. 1). Chemical structures of omega-3 and omega-6 fatty acids. Nutritional epidemiologic studies have provided evidence that dietary factors are important determinants of colorectal cancer in different populations worldwide. Cancer statistics in Japan for 2001 published by the Foundation for Promotion of Cancer Research indicate that there is an upward trend in age-adjusted mortality rates for colon cancer from 1955 to 1999.3 According to this report, the death rates due to colon cancer in Japanese men and women in 1955 were 2.9 and 3.0, respectively, whereas they increased to 14.7 and 9.8 in 1999. This upward trend in death rates due to colon cancer is mainly attributable to Westernization of Japanese food habits.3, 4 In addition, the report by the Foundation for Promotion of Cancer Research provided the data on the time trends in food consumption, which show increased dietary intakes of animal fat and meat and decreased consumption of whole grains from 1960 to 1999. For example, animal fat consumption in 1960 was about 25 g/day (per capita), whereas in 1999 it increased to about 58 g/day. Meat intake was increased from 19 to 78 g/day (per capita), whereas grain consumption decreased from 453 to 245 g/day during these years.3 The importance of types of dietary fat differing in fatty acid composition rather than total fat cannot be discounted because several preclinical studies using well-established colon cancer models strongly supported the notion that the colon tumor-promoting effect of dietary fat or lack of such effect depends on its fatty acid composition.5 A recent report by an expert panel assembled by the American Institute for Cancer Research/World Cancer Research Fund came to the scientific consensus that evidence for an association between the intake of saturated fat and/or animal fat and colon cancer risk is very strong.6 Continuing population studies revealed that diets particularly high in total fat, especially animal fat, are generally associated with increased risk of developing colon cancer, whereas high dietary fish oil or fish reduces this risk.7, 8 A recent ecologic study suggests that mortality data for colorectal cancer in 22 European countries, the United States and Canada correlate with the consumption of animal fat.7 That eating a diet rich in n-3 PUFAs may decrease the risk of colorectal cancer has been hypothesized in relation to fish and fish oil.7 Caygill and Hill et al.8 reported an inverse correlation between fish and fish oil consumption and colorectal cancer when expressed as a proportion of total or animal fat. This inverse relationship was significant for both male and female colorectal cancer, whether the intakes were in the current period or 10 years or 23 years before cancer mortality, It is noteworthy that these effects were only observed in countries with a high (> 85 g/caput/day) animal fat intake.8 Also, Mediterranean diet rich in olive oil and fish is associated with a low risk of colorectal cancer.9 On the basis of epidemiologic evidence, it is reasonable to suggest that diets high in saturated fats increase the risk of colorectal cancer, whereas diets high in fish and fish oil rich in n-3 PUFAs reduce the risk. Animal models have contributed significantly to understanding of the carcinogenesis process and to study the multiple environmental factors against the pathogenesis of colon cancer.10 Several studies have utilized these relevant animal models to investigate the modulation of colon carcinogenesis by nutritional and chemopreventive agents. These animal models include induction of colon tumors in rats by administration of aromatic amines such as 3,2′-dimethyl-4-aminobi-phenyl (DMBA); derivatives and analogs of cycacin, such as methylazoxymethanol (MAM), 1,2-dimethyl-hydrazine (DMH) and azoxymethane (AOM) in rats and mice of selected strains; direct-acting carcinogens of the type of alkylureas, such as methylnitrosourea (MNU) or N-methyl-N′-nitro-N-nitrosoguanidine (MNNG); and 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 agents is similar to various types of neoplastic lesions observed in the colorectum of humans. Azoxymethane (AOM), which is a potent inducer of carcinomas of the large intestine in various strains of male and female rats, has been used extensively by many investigators to induce colon tumors and to study the effects of nutritional factors and chemopreventive agents in colon carcinogenesis.10, 11, 12, 13, 14, 15, 16, 17 Colons of Fischer (F344) rats treated with AOM seem to have light and electron microscopic morphology as well as histochemical properties that are quite similar to that of humans and the biologic behavior of AOM-induced rat colon carcinomas is similar to that of human colon carcinomas.10, 18 Other characteristics of the human disease process reflected in the AOM rat model are the occurrence of both adenomas and adenocarcinomas.18 Also, aberrant crypt foci (ACF), which are recognized as early appearing preneoplastic lesions, develop in experimentally induced colon carcinogenesis in laboratory rodents as well as in the colonic mucosa of patients with colon cancer.19, 20 Recently, β-catenin-accumulated crypts were identified in the colonic mucosa at the early stages of AOM-induced colon carcinogenesis and are considered as early-appearing preneoplastic lesions.21 Therefore, ACF are now regarded as putative preneoplastic lesions for colon cancers and are used as biomarkers to evaluate potential chemopreventive agents against colon carcinogenesis.22 AOM treatment also induces oncogene mutations at codon 12 of K- and H-ras and increases in the expression of the ras family of protooncogenes have been causally associated with colon tumor development.23, 24 Enhanced ras oncogene expression has been observed in a variety of human colon tumors.25 AOM-induced colon tumors also demonstrate enhanced cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression similar to human colon tumors.13, 26 Mutations in the tumor suppressor gene, APC, are known to be early events in the colon cancer process in humans and have been identified in patients with familial adenomatous polyposis, who have germline mutation in one of the APC alleles, and in sporadic colorectal cancer.27, 28 Evidence in humans thus implicates the APC suppressor gene as causal in large bowel carcinogenesis. Recent studies indicating the presence of APC mutation in AOM-induced colon tumors in rats strengthens the concept that these models are appropriate for human colon cancer studies.29 It has been increasingly apparent that β-catenin signaling pathway is closely associated with the development of colon cancer.30 Also, frequent mutations in the β-catenin gene are confirmed in AOM-induced colon tumors in rodent models.30 Furthermore, it has recently been indicated that the expression of nuclear β-catenin is correlated with the size of colon neoplasms.31 Because of similarities of histopathology of adenomas and adenocarcinomas, ACF and several molecular parameters between human colon tumors and AOM-induced colon tumors, it is indicated that the AOM model system and other chemically induced colon models appear to be appropriate colon cancer models. Recent progress in the area of molecular carcinogenesis has identified multiple molecular targets for the chemoprevention and nutritional modulation of colon cancer. The multiple intestinal neoplasia (Min) mouse, which carries a fully penetrant dominant mutation converting codon 850 of the murine APC gene from a lucine to a stop codon, is markedly different from that of patients with familial adenomatous polyposis (FAP) in which adenomas are found exclusively in the colon and duodenum, whereas in Min mouse adenomas are detectable in the small intestine and rarely in the colon. APCΔ716 knockout mice also develop numerous intestinal polyps at an early age.32 These are potential limitations of APCMin mouse and knockout models for testing agents for their potential chemopreventive properties against colon carcinogenesis. The development of strategies for prevention of colorectal cancer by dietary modification has been markedly facilitated by the use of relevant laboratory animal models, including carcinogen-induced colon cancer mimicking the neoplastic process that occurs in humans. Ample and consistent experimental evidence from preclinical efficacy studies conducted earlier have provided convincing evidence that not only the amount but also types of dietary fat differing in fatty acid composition are important factors in determining modulating effect of this nutrient in colon tumor development.33, 34, 35, 36 Studies conducted in our laboratory and those of others have consistently demonstrated that diets high in beef tallow, lard and corn oil (20–23% in the diet) significantly increased chemically induced colon carcinogenesis in F344 and Sprague-Dawley rats as compared to diets low (5%) in these fats.11, 33, 34, 35, 36, 37, 38 Additional studies conducted in our laboratory also demonstrate that male F344 rats fed diets containing 20% lard or 20% corn oil rich in n-6 PUFAs were more susceptible to 1,2-dimethylhydrazine-induced colon carcinogenesis compared with those fed diets containing 5% lard or 5% corn oil.38 Deschner et al.39 demonstrated that dietary n-3 PUFAs (fish oil) inhibits methylazoxymethanol (metabolite of azoxymethane)-induced focal areas of dysplasia and colon tumors, whereas n-6 PUFAs (corn oil) enhance colon tumorigenesis in rats. In a recent study, Chang et al.11 reported a protective effect of dietary fish oil against AOM-induced colon carcinogenesis in male Sprague-Dawley rats. High dietary fish oil significantly inhibited colon tumors as compared to high corn oil diet. In addition, colon tumor inhibition by fish oil diet was associated with lower levels of DNA damage in the distal colon compared with corn oil diet.11, 40 These studies provided evidence in preclinical models that diets containing high amount of saturated fat of animal origin or n-6 PUFAs had a greater colon tumor-enhancing effect than diets low in such fatty acids, whereas diets high in n-3 PUFAs had no such enhancing effect Further studies in our laboratory have evaluated the modulating effects of high dietary corn oil and safflower oil rich in n-6 PUFAs, olive oil high in monounsaturated fatty acid oleic acid, coconut oil high in medium-chain fatty acids such as lauric acid and fish oil during the postinitiation stage of AOM-induced colon carcinogenesis in male F344 rats.14, 37, 38, 41 Animals fed diets containing high corn oil or safflower oil (23.5%) had a higher incidence of colon tumors than did those fed diets low in fat (5%). By contrast, diets high in coconut oil, olive oil or menhaden fish oil had no such colon tumor-enhancing effect. The varied effects of different types of fat on colon carcinogenesis during postinitiation stage suggest that fatty acid composition is one of the determining factors in colon tumor promotion by a dietary fat and that the influence of types and amount of dietary fat is exerted mostly during the postinitiation phase of carcinogenesis.34, 41, 42 In this connection, it is interesting to note that in a phase 2 clinical trial of patients with colonic polyps, dietary fish oil supplements have in fact inhibited cell proliferation in the colonic mucosa.43 Thus far, progress has been made with regard to the relationship between dietary fat intake and colon cancer risk in that we know of the tumor-promoting effects of diets rich in n-6 PUFAs and saturated fatty acids and lack of such effects by n-3 PUFAs. However, it should be recognized that among the sources of dietary fat, animal fat with its high-saturated fatty acid content is by far the most important contributor, amounting to about 60%, to the Western diet. Importantly, dietary fat intake in the United States and Canada and other Western countries, where colon cancer rates are high, consists predominantly of a mixture of saturated, monounsaturated and polyunsaturated fats.6, 44 A recent assay in mice demonstrated that high dietary fat simulating mixed lipid composition of the average Western-style diet produced dysplastic lesions in the colon indicative of tumorigenesis.44, 45 In view of the significance of mixed lipids in colon cancer and because of potential tumor-inhibitory properties of n-3 PUFAs, we have conducted a study to examine the effects of high-fat diets that contain mixed lipids rich in saturated fatty acids and to compare them with the effects of fish oil during the different stages of colon carcinogenesis in male F344 rats.13 Colonic preneoplastic lesions, ACF, were assessed in animals fed the experimental diets for 8, 23 and 38 weeks. ACF, which are putative preneoplastic lesions in the colon, were observed at high frequency in the colonic mucosa of patients with colon cancer46 and of rats and mice treated with colon carcinogenesis.19, 47 ACF and their growth are thought to be useful biomarkers of the effects of agents preventing carcinogenesis in the colon. ACF were predominately observed in the distal colons of carcinogen-treated rats. Rats fed the high-fat mixed lipids (HFML) diet showed a significantly greater number of ACF/colon compared with those fed the low-fat corn oil (LFCO) or high-fat fish oil (HFFO) diet at all time points. The incidence of multicrypt aberrant foci was also higher in the HFML diet group than in the HFFO or LFCO diet groups, suggesting that administration of the HFFO diet significantly inhibits the formation and growth of preneoplastic lesions in the colon, whereas the HFML diet promotes the growth of such lesions. Also, dietary HFML significantly increased colon tumor incidence and multiplicity when compared with the HFFO or LFCO diets. Importantly, rats fed the HFML diet showed 100% incidence of colonic adenocarcinomas compared with incidences of 63% and 69% in rats fed the LFCO and HFFO diets, respectively. Also, the multiplicity of adenocarcinomas was significantly higher in animals fed the HFML diet (about 4-fold increase) as compared to those fed the LFCO diet. Equally important, the HFFO diet containing 20% fat (mostly in the form of fish oil) induced fewer tumors than HFML diet containing the same amount of total fat (mostly from mixed lipids). This reinforces that both the type and the amount of fatty acids in the diet play a critical role in colon carcinogenesis. In general, overall evidence from preclinical studies is consistent with the epidemiologic data. The efficacy of dietary n-3 PUFAs including DHA and EPA against colon carcinogenesis has also been investigated in rodent models. Takahashi et al.48 reported that intragastric administration of 0.7 ml of DHA twice a week for 4 and 12 weeks significantly suppressed AOM-induced ACF in the colon. Takahashi et al.49 also reported that intragastric administration of 1 ml of DHA 5 times a week for 36 weeks significantly suppressed AOM-induced colon tumor multiplicity, specifically moderately differentiated adenocarcinomas in the middle and distal colon in male F344 rats. Minoura et al.12 compared dietary EPA at 4.7% plus LA at 0.3% or LA alone at 5% against AOM-induced colon carcinogenesis in male Donryu rats. The results indicate that the rats fed EPA had a significantly lower colon tumor incidence and multiplicity than those fed the LA diet. Histologic examination of tumors showed that the rats on EPA diet had fewer well-differentiated adenocarcinomas and more mucinous adenocarcinomas than those on the LA diet. Also, the content of PGE2 in the colon tumors of LA diet group was higher than that in the colon tumors of the EPA diet group. These results suggest that EPA exerts its inhibitory effect through the modulation of PGE2 synthesis in colon tumors. Several potential mechanisms have been proposed for colon cancer preventive activity of types of dietary fat. Several studies indicate that diets high in saturated fatty acids (beef tallow and lard) and n-6 PUFAs (corn oil or safflower oil) increase the concentration of colonic luminal secondary bile acids, including deoxycholic acid and lithocholic acid, whereas dietary fish oil high in n-3 PUFAs had no such enhancing effect.37 Metabolic epidemiologic studies demonstrated that populations who are on typical Western diet and at high risk for colon cancer excrete high levels of secondary bile acids.50, 51 Secondary bile acids have been shown to stimulate protein kinase C (PKC) in a manner similar to phorbol esters, induce cell proliferation and ornithine decarboxylase activity, a rate-limiting enzyme in polyamine biosynthesis, and act as promoters in colon carcinogenesis.52, 53, 54, 55, 56, 57 Collectively, these observations suggest that secondary bile acids that are modulated by types of dietary fat may be important for inducing cellular response in relation to colon tumor promotion. As discussed in detail by Hong et al.,58 select dietary PUFAs may modulate carcinogen activation, methylation-induced DNA damage, repair and deletion.58, 59, 60 Dietary n-3 PUFAs may protect against colon carcinogenesis by either decreasing DNA adduct formation and/or enhancing DNA repair.58 Lower levels of AOM-induced DNA adducts were detected in fish oil-fed rats as compared to those fed corn oil rich in n-6 PUFAs. Hong et al.58 and Chang et al.59 have also demonstrated that fish oil supplementation caused an increase in apoptosis in the colon compared with corn oil-fed rats. It is therefore reasonable to conclude that one of the mechanisms by which n-3 PUFAs protect against colon carcinogenesis is in part by reducing the level of DNA adducts and by enhancing the deletion of colonic cells through apoptosis.58 Also, feeding of fish oil or purified n-3 fatty acid ethyl esters selectively increased EPA and DHA levels in the mitochondrial phosplolipids of colonocytes at the expense of n-6 fatty acids.60 It has been shown that inhibition of colon carcinogenesis by DHA is mediated through the activation of retinoid X receptors (RXRs), a obligatory component of a large number of nuclear receptors,61 suggesting n-3 PUFAs mediate growth inhibitory effects in the colon through the RXR subunit of nuclear receptor heterodimers. Members of the nuclear receptor superfamily are transcription factors that selectively regulate cell differentiation and proliferation.62 There are studies to indicate that inducible nitric oxide synthase (iNOS), which is regulated primarily at the transcriptional levels, is overexpressed in human colon adenomas63 and in chemically induced colon tumors of laboratory animal models.64 Accumulating data also indicate that the overproduction of NO by iNOS is critical to carcinogenesis process and induces deaminated DNA lesions, thus resulting in DNA damage.65 Both NO and peroxinitrate produced in the tissues by family of NOS also activate cyclooxygenase (COX)-2. These data clearly suggest a key role for NO in tumor initiation, promotion and progression. Studies conducted in our laboratory indicate that deoxycholic acid induces iNOS activity in intestinal cells.66 Treatment with specific PKC inhibitors suppressed iNOS expression suggesting that one of the mechanisms by which tumor promoters including secondary bile acids may involve an increase in expression of iNOS through activation pathway that enhances colon carcinogenesis.66 It is known that the fatty acid composition of cells is sensitive to diet. Studies conducted in our laboratory indicate that increasing levels of dietary fish oil in rats increased the omega-3 fatty acids, namely, DHA and EPA in the colonic mucosal membrane phospholipid fractions at the expense of omega-6 PUFAs such as linoleic acid and arachidonic acid, suggesting the possibility that the DHA and EPA of fish oil can modulate the activity of membrane-bound enzymes by partially replacing arachidonic acid and linoleic acid in the phospholipid pool.67 Therefore, the types of dietary fat determine the fatty acid composition of colonic mucosal membrane phospholipids. It is well established that arachidonic acid and some of its metabolites, including prostaglandins (PGs), play an important role in the intracellular signaling pathway associated with cell proliferation and gene expression. PGs increase cell proliferation, promote angiogenesis and inhibit immune surveillance, all of which are involved in tumor growth. The mechanisms by which n-3 PUFAs inhibit colon carcinogenesis are summarized in Figure 2. Overexpression of COX-2 plays an important role in colon carcinogenesis.68 Tsujii and DuBois,69 who have implicated COX-2 activity in the regulation of apoptosis of rat intestinal epithelial cells, have shown that overexpression of COX-2 can lead to the suppression of apoptosis. Additionally, high intake of saturated fat and omega-6 PUFAs alters membrane phospholipid turnover, releasing membrane arachidonic acid from phospholipids and affecting prostaglandin synthesis via COX enzyme.34, 41 Elevated levels of COX-2 have been observed in human colon tumors and chemically induced colon tumors in rodents.70, 71 Normal adult rodent and human colon tissues express levels of COX-2 that are undetectable by Northern analysis. Recent reports have shown a link between the tumorigenic potential of APC mutations and arachidonic metabolism by observation that deletion of the COX-2 gene reduces the number of tumors in mice heterozygous for an APC716 by more than 6-fold.32 Additional evidence supporting a role for COX-2 comes from our studies, which show a marked reduction in colon tumors in rodents with highly selective COX-2 inhibitor, celecoxib.26 Recent studies conducted in our laboratory have provided convincing evidence that an HFML diet enhances AOM-induced expression of COX-2 and eicosanoid formation from arachidonic acid in colon tumors of rats, whereas the HFFO diet inhibits the levels of COX-2.13 In this study, administration of the HFML diet produced 472 ± 33 pmol/min (mean ± SE) of eicosanoids in the colon tumors, significantly higher levels than the low-fat corn oil diet (380 ± 29 pmol/min) or the HFFO diet (348 ± 28 pmol/min), indicating higher COX activity. This suggests that inhibition of eicosanoid production through the modulation of COX-2 activity may be important for the ability of n-3 PUFAs to inhibit colon tumorigenesis. Also, colon tumors of animals fed the HFML diet showed a nearly 50% lower apoptotic index than was observed in the colon tumors of rats fed the HFFO diet. The results of these studies indicating that overexpression of COX-2 in the tumors of animals fed the HFML diet in contrast to the HFFO diet inhibits apoptosis and the consequent tumor burden support the contention that overexpression of COX-2 can lead to the suppression of apoptosis. In colon tumors, lowering the levels of PGs may be enough to slow growth by inhibiting proliferation and induction of apoptosis and thus tumor inhibition. A major question that remains to be answered is which signaling pathways are involved in downstream of the COX-2 enzyme. These could provide not only a key link between dietary fatty acids, eicosanoids, COX-2 and transcriptional regulation of colon carcinogenesis, but also additional molecular targets for colon cancer prevention strategies. Mechanisms by which n-3 PUFAs inhibit colon carcinogenesis. The schematic diagram presented here illustrated potential cellular and molecular events mediated by n-3 PUFAs against colon carcinogenesis. The cascade of molecular events modulated by n-3 PUFAs include proinflammatory genes including COX-2 and iNOS, activated ras, specific PKC isoforms and differentiating factors including RXRs that modulate cell differentiation and apoptosis. High dietary n-3 PUFAs exert antitumor activity by interfering with the posttranslational modification and membrane localization of ras-p21 through modulation of farnesyl protein transferase, thus inhibiting ras-p21 function. Recent studies from our laboratory have shown that high dietary n-6 PUFAs enhance activities of diverse enzymes, including PKC, that have been implicated directly or indirectly in colon tumor promotion, whereas high-fat diet containing n-3 PUFAs appears to suppress the activities of these enzymes.72 PKC constitutes a gene family of serine/threonine protein kinases that play central roles in transmembrane signaling events and are involved in cellular proliferation and differentiation. Members include several isoenzymes with unique functions. Many PKC isoenzymes translocate to the membrane from the cytosol upon activation.73, 74 Jiang et al.75 reported that chemopreventive efficacy of dietary fish oil is associated with the alterations in colonic PKC expression, a signal-dependent kinase that is activated upon stimulation by growth factors. PUFAs may influence the activity of the EGF receptor/mitogen-activated protein kinase pathway, which could activate a number of oncogenes. It is interesting that several kinases have been shown to participate in ras-mediated growth-promoting signal transduction pathways.76 The ras-p21, a guanine nucleotide-binding 21 kDa protein product of ras genes that is anchored to the cytoplasmic face of plasma membrane, functions in the regulation of cell proliferation. Mutational versions of ras-p21 are implicated in the etiology of human colon cancer.77 It is also known that trafficking of pro-ras from cytosol to plasma membrane is facilitated by a series of closely linked posttranslational modifications, including farnesylation, which is catalyzed by farnesyl protein transferase (FPTase). It appears that inhibition of ras farnesylation blocks membrane association of ras-p21 and prevents neoplastic transformation of cells. Studies conducted in our laboratory have provided data to indicate that high dietary n-6 PUFAs increases ras-p21 expression in colonic tumors, whereas high dietary n-3 PUFAs appear to exert antitumor activity by interfering with posttranslational modification and membrane localization of ras-p21 through the modulation of FPTase activity, thus inhibiting ras-p21 function.78 Several FPTase inhibitors have demonstrated selective antiproliferative activity against ras-transformed cells in both cell culture and laboratory animal studies. Advances in recombinant DNA technology have greatly facilitated the identification of candidate genes involved in initiation, promotion and progression of colon cancer. Expression of PLK3 (Polo-like kinase-3) is negatively correlated with the development of certain tumors.79 Overexpression of PLK3 has also been shown to induce apoptosis, an effect that correlates with incomplete cytokinesis and inhibit cell proliferation. We have shown recently that expression of PLK3 was downregulated in colon tumors of rats than in uninvolved colon mucosa.80 Colon tumors isolated from rats fed the diet containing high levels of n-6 PUFAs contained very low levels of PLK3 mRNA expression, whereas the tumors from animals fed the diet containing high amount of n-3 PUFAs did not exhibit any downregulation of PLK3. These results correlate with colon tumor incidences by dietary n-3 and n-6 PUFAs. It would appear that modulation of apoptosis through PLK3 plays a significant role in n-3 PUFA-induced colon tumor inhibition and promotion. Additional studies conducted in our laboratory have demonstrated that DHA inhibits growth of CaCo-2 colon cancer cells in vitro and induces apoptosis.81 Using CaCo-2 cells, we also examined the effects of DHA on the genetic precursors of human colon cancer at the transcription level using DNA oligonucleotide arrays.82 Alterations in gene expression due to DHA treatment was observed to be in the multiple signaling pathways involved in the regulation of cell cycle regulatory genes, COX-2 target genes, lipoxygenases and peroxisome proliferators. Effects of DHA on cell cycle progression and induction of apoptosis were directly paralleled by an increase in the activation of several proapoptotic capases, inactivation of antiapoptotic Bcl-2 family of genes and activation of cyclin-dependent kinase inhibitors such as p21waf1/cip1 and p27. Comprehensive evaluation of several of these precursor genes and transcription factors will facilitate the determination of the chemopreventive efficacy of DHA and other important n-3 PUFAs present in fish oil and thus prevent colon cancer. Also, comprehensive evaluation of these precursor genes and transcription factors provided several simultaneously expressed biologic activities, many of which suggest themselves as molecular targets for effective intervention by selective chemopreventive agents, including nutritional factors. Weight loss is a major cause of morbidity and mortality in patients with advanced cancer, including colorectal cancer. Several clinical studies have provided evidence for beneficial effects of fish oil administration in cancer cachexia during therapy.81 Omega-3 fatty acids have been shown to have beneficial effects on skeletal muscle protein catabolism in cancer cachexia.83 Weight loss in patients with gastrointestinal cancer is refractory to therapeutic intervention and is associated with a shorter survival time and reduced quality of life.84 A fish oil-enriched nutritional supplement has the potential to be a safe and effective anticachectic agent. Administration of a mixed fish oil preparation (2.2 g of EPA and 1.4 g DHA) and a pure (6 g DHA) preparation daily stabilized weight in patients with unresectable pancreatic cancer.84, 85 The loss of muscle protein forms part of the syndrome of cachexia, which results in the loss of function of cancer patients and eventually death from hypostatic pneumonia, attributable to loss of respiratory function.83 EPA antagonizes loss of skeletal muscle proteins in cancer cachexia by downregulating proteasome expression, and this may be one of the mechanisms for inhibition of tumor growth.83 Also, fish oil at a daily dosage of 1.8 g during radiochemotherapy improved natural killer cells.86 In conclusion, on the basis of epidemiologic evidence from ecologic and case-control studies, it is reasonable to suggest that diets high in saturated fats increase the risk of colorectal cancer, whereas diets high in n-3 PUFAs does not increase its risk. The studies described here, both epidemiologic and preclinical, provide evidence for the beneficial effects of diets rich in n-3 PUFA in the prevention of colorectal cancer. Also, recent clinical trials clearly demonstrate beneficial effects of fish oil administration in cancer cachexia and during radio- and chemotherapy. Preclinical studies have provided convincing evidence that colon tumor-promoting effect of dietary fat depends on its fatty acid composition, suggesting that the composition of ingested dietary fatty acids is more critical to colon cancer risk than is the total amount of fat. Preclinical studies also demonstrate that a Western-style diet high in mixed lipids including saturated fats of animal origin as well as high dietary n-6 PUFAs had a higher potential to promote colon tumorigenesis than ingestion of a diet on equivalent amount of fat containing n-3 PUFAs. Although the mechanisms by which diets high in saturated fats (such as those in Western diets) and n-6 PUFAs promote colon carcinogenesis are not fully known, the studies conducted thus far indicate that increased levels of colonic luminal secondary bile acids, modulation of ras-p21 activity, eicosanoid production via the influence on COX activity and the expression of apoptosis by the types of dietary fat, especially n-6 PUFAs, may play a key role in colon carcinogenesis. Further studies are deemed necessary to determine the role of n-3 PUFAs on the modulation of critical genes that are involved in colon and other types of cancer. The goal of prevention is to decrease the morbidity and mortality from colorectal cancer. Our results and those of others suggest that nutritional prevention has the potential to be a major component of colorectal cancer control, especially primary prevention in general population. Now the questions are how long and in what dosages n-3 PUFAs must be administered before they are effective for cancer prevention. Although there are no clear-cut data to indicate how much and how long n-3 PUFAs should be consumed, for primary prevention of colorectal cancer, levels of dietary n-3 PUFAs should be consistent with the recommendations based on epidemiologic studies of coronary heart disease as discussed by several nutritionists.87, 88, 89, 90, 91 These studies suggest that 1—2 fish meals per week or as little as 30–35 g/day of fish throughout life decreases the risk of coronary heart disease. Although a recommended dietary allowance (RDA) for n-3 PUFAs does not exist, there is a possibility that consumption of adequate amount of fish in our daily meals based on the above epidemiologic studies may also reduce the risk of colorectal cancer. Lands et al.88 has suggested that the ratio of n-6 PUFAs to n-3 PUFAs may be important for health. The varied risk for coronary heart diseases and several types of cancer among Japanese, Mediterranean and Western European populations may at least in part be explained on the basis of n-6 to n-3 PUFA ratios of 4, 6–8 and 10 in their diets, respectively.89 Based on preclinical and epidemiologic studies on arteriosclerosis and coronary heart disease, Okuyama et al.91 recommended for a reduction in the intake of linoleic acid and increase in the intake of n-3 PUFAs so that a n-6/n-3 ratio of 2 could be achieved for effective prevention of atherosclerosis and related diseases. They further recommended that this is necessary in Western countries for effective prevention of atherosclerosis and related diseases and probably diet-related cancers.90 This recommendation may well be applied for the effective primary prevention of colorectal cancer in the general population. Importantly, consumption of fibrous foods, fruits and vegetables is also necessary for those in Western countries to reduce the risk of colorectal cancer. In the secondary prevention of colorectal cancer in patients, if omega-3 fatty acids are taken in sufficient amounts, cachexia and reduced quality of life can be reversed. It should be recognized that intervention with nutritional supplements and/or diet modification alone may not be sufficient for secondary prevention of colorectal cancer in high–risk patients such as those with hereditary polyposis and sporadic colon polyps. This approach to colon cancer control is of growing importance as therapy modalities alone have not been fully effective in countering either the high incidence or low survival rate of colorectal cancer. However, intervention by diet modification as recommended to the general population, along with chemopreventive agents that either abolish or delay the development of those events, which begin with normal-appearing tissues and progress to invasion and metastases, is an ideal strategy for secondary prevention of colon cancer in these high-risk individuals. This approach is extremely important when promising chemopreventive agents demonstrate significant efficacy but may produce toxic effects at higher doses. It is certain that colon cancer prevention will be a significant focus of research and intervention in high-risk individual by identifying molecular targets that can alter or stop the process of carcinogenesis. Clearly, there is a need to carry out randomized double-blind controlled clinical trials in patients with sporadic colon polyps using the n-3 PUFA-rich diets in combination with a chemopreventive agent to prevent the progression of events leading to malignant neoplasms. The author thanks Ms. Laura McDermott for preparation of the manuscript. Current and past studies on n-3 PUFAs in colon cancer prevention are supported by the National Cancer Institute grants (CA-17613 and CA-37663).