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Pharmacology & Therapeutics 83 (1999) 217244

Associate editor: S.T. Mayne

Omega-3 fatty acids as cancer chemopreventive agents


David P. Rose*, Jeanne M. Connolly
Division of Nutrition and Endocrinology, American Health Foundation, One Dana Road, Valhalla, NY 10595, USA

Abstract
There is both epidemiologic and experimental evidence that the long-chain omega-3 fatty acids (FAs), which occur at high levels in some fish oils, exert protective effects against some common cancers, notably those of breast, colon, and, perhaps, prostate. Multiple mechanisms are involved in this chemopreventive activity, including suppression of neoplastic transformation, cell growth inhibition and enhanced apoptosis, and antiangiogenicity; however, a common feature of most of these biological effects is the inhibition of eicosanoid production from omega-6 FA precursors. Several of the known risk factors for breast, and colon, cancer may be favorably modified by dietary omega-3 FA supplementation, and the implementation of clinical chemoprevention trials is now feasible. 1999 Elsevier Science Inc. All rights reserved.
Keywords: Breast cancer; Colon cancer; Chemoprevention; Omega-3 fatty acids; Eiscosanoids

Abbreviations: AA, arachidonic acid; COX, cyclooxygenase; DHA, docosahexaenoic acid; DMBA, dimethylbenz[a]anthracene; DPA, docosapentaenoic acid; EGF, epidermal growth factor; EPA, eicosapentaenoic acid; FA, fatty acid; HETE, hydroxyeicosatetraenoic acid; HPETE, hydroperoxyeicosatetraenoic acid; 13-HODE, 13(S)-hydroxyoctadecadienoic acid; LA, linoleic acid; LNA, -linolenic acid; LOX, lipoxygenase; LT, leukotriene; ODC, ornithine decarboxylase; PG, prostaglandin; PGI2, prostacyclin; PKC, protein kinase C; TX, thromboxane.

Contents 1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Nomenclature, chemistry, and metabolic conversion of unsaturated fatty acids . . . . . . . . . 3. Dietary sources of omega-3 fatty acids, intestinal absorption, and tissue and subcellular distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Dietary sources. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Intestinal absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Tissue and subcellular distribution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. The eicosanoids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Arachidonic acid-derived eicosanoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Eicosanoids derived from eicosapentaenoic acid. . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Eicosanoid biosynthesis and n-3:n-6 fatty acid ratio . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. 13(S)-Hydroxyoctadecadienoic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Biomarkers of n-3 fatty acid nutritional status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Omega-3 fatty acids and cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Breast cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.1. Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.2. Experimental studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.3. The relative potency of eicosapentaenoic acid and docosahexaenoic acid in breast cancer animal models. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Colon cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.1. Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2.2. Experimental studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.1. Breast cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.2. Colon cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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* Corresponding author. Tel.: 914-789-7145; fax: 914-592-6317. E-mail address: david@westnet.com (D.P. Rose) 0163-7258/99/$ see front matter 1999 Elsevier Science Inc. All rights reserved. PII: S0163-7258(99)00 0 2 6 - 1

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7.

8.

Omega-3 fatty acids and cancer chemoprevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Breast cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1.1. Mammographic densities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1.2. Atypical hyperplasia and carcinoma in situ . . . . . . . . . . . . . . . . . . . . . . . . . 7.1.3. Chemoprevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1.4. Omega-3 fatty acid supplementation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1.5. Dietary modification targeting other fatty acids: The n-3:n-6 fatty acid ratio. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1.6. Intermediate response biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Commentary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction It is becoming increasingly recognized that, at least in the long term, future advances in clinical cancer research will come from an emphasis on prevention rather than the treatment of metastatic disease. This research effort encompasses epidemiological studies, such as those responsible for the recognition that high consumption of vegetables and fruits is associated with a reduced risk of some cancers, laboratory experiments to evaluate potential natural and synthetic products as chemopreventive agents, and the execution of clinical preventive trials (Greenwald et al., 1993). As always, these different approaches are complementary: for example, work with animal models showed that nonsteroidal anti-inflammatory drugs such as aspirin possess chemopreventive activity against experimental colonic carcinogenesis, epidemiological observations supported such a potential role in human populations, and several of this class of compounds are now undergoing evaluation in clinical trials for colon cancer prevention. The progression from the initial steps in the carcinogenic process to the establishment of clinically manifest, invasive disease occurs in a series of steps, each of which merges into the next (Fig. 1). Intervention strategies follow much the same path: primary chemoprevention merges with chemosuppression, which itself is indistinguishable from the chemotherapy of early disease. However, the distinction between chemoprevention and chemosuppression is useful because it recognizes that it is not necessary to prevent the development of premalignant, or even frankly cancerous, foci in order to suppress progression to symptomatic, lifethreatening cancer. Thus, while the antiestrogen tamoxifen may have a place as a truly chemopreventive agent, its action in preventing the development of a new primary cancer in the contralateral breast of a postmenopausal woman previously treated surgically for primary breast cancer is most likely one of chemosuppression (Jordan, 1991). Experimental studies performed in our laboratory suggest that the omega-3 fatty acids (FAs), which are the subject of this review, can have a similar chemosuppressive effect on the progression of microscopic metastatic foci (Rose et al., 1996). Some drugs that were introduced originally for the treatment of metastatic cancer are now having their role extended to include chemoprevention. This changing applica-

tion carries with it the need for increased vigilance in order to avoid the introduction of undesirable side effects; for example, the long-term risk of causing a tumor at another organ site, while acceptable when treating advanced cancer, will prohibit the use of the same drug as a chemopreventive agent in healthy individuals. As cancer chemoprevention research has progressed, interest has turned to the investigation of natural products, a number of which have been found to be active in animal models when administered at levels that appear to lack systemic toxicity. Prominent among these compounds are nutrients, including vitamins and the omega-3 FA; food additives such as curcumin; and food-associated natural products, such as indole 3-carbinol and lycopene. This review deals exclusively with the omega-3 FAs. These remarkable nutrients have attracted interest because of their importance in normal brain development (Neuringer et al., 1988), as dietary supplements for the prevention and treatment of chronic cardiovascular disease (Simopoulos, 1991), and the treatment of arthritic disorders (Kremer, 1991) and diabetes mellitus (Malasanos & Stacpoole, 1991). They have been, and are currently, the subject of various clinical trials in which they have shown a lack of complications or serious side effects. Epidemiological studies, investigations utilizing a range of animal models, and mechanistic experiments in vitro all support their potential as cancer chemopreventive and chemosuppressive agents, and as auxiliary agents for cancer therapy. The tumor targets include carcinomas of the breast and colon, which are two of the most common cancers in North America and Europe. Our objective is to provide a background general discussion of the omega-3 FAs, review the current status of research into their chemopreventive activities and the biological mechanisms involved, and to consider in some depth the development of a clinical trial in women at high breast cancer risk.

2. Nomenclature, chemistry, and metabolic conversion of unsaturated fatty acids The unsaturated FAs comprise monounsaturates and polyunsaturates. The conventional chemical nomenclature is to begin the systematic numbering of carbon atoms from

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Fig. 1. The progression of premalignant benign breast disease to metastatic carcinoma.

the carboxyl terminal group. The carbon atoms number 2 and 3 from the carboxyl group are referred to as the and carbons, respectively, the last carbon is the - or n-carbon, and the position of a double bond is indicated by the symbol , followed by a number: for example, 9 refers to a double bond between carbon atoms 9 and 10 from the carboxyl group. However, an accepted practice in describing the chemical structure of FA molecules is to start numbering the carbons at the methyl group ( - or n-). Oleic acid has its single double bond located between the 9th and 10th carbon atoms from the methyl end, and so it is designated an 9 (or n-9) monounsaturated FA (Fig. 2). It can be synthesized by all mammals, including humans. The omega-3 (n-3) and omega-6 (n-6) polyunsaturated FAs cannot be synthesized by mammals, and because they must be obtained from the diet, they are referred to as essential fatty acids. In Fig. 2, the n-3 FAs are represented by -linolenic acid (LNA) and the n-6 FAs by linoleic acid (LA). Both LNA and LA are metabolized to longer-chain FAs, largely in the liver; LNA is converted to eicosapentaenoic acid (EPA), and thence to docosahexaenoic acid (DHA), while LA is the metabolic precursor of arachidonic acid (AA). These events, which are summarized in Fig. 3, involve increases in chain length and degree of unsaturation that are achieved by adding extra double bonds between the existing double bond and the carboxyl group (de Gomez Dumm & Brenner, 1975). Competition exists between the n-3 and the n-6 FAs for the 4 and 6 desaturases, with the n-3 FAs having greater affinities for the enzymes. In consequence, increasing the dietary intake of n-3 FAs reduces the desaturation of LA and so, the production of AA (de Gomez Dumm & Brenner, 1975; Hague & Christoffersen, 1984), an inhibition that is achieved by EPA and DHA, as well as LNA (Christiansen et al., 1991).

Humans can produce EPA from LNA, but the extent of the capacity to perform this conversion has not been well defined (Hunter, 1990), and there is evidence that human 6 desaturase activity decreases with age (de Gomez Dumm & Brenner, 1975). In one study (Mantzioris et al., 1994), feeding a low LA-containing diet to human volunteers, thus reducing the enzymic competition referred to above, did allow effective conversion of LNA to EPA, with corresponding elevations in the tissue DHA levels. However, Grimsgaard et al. (1997) observed a 15% decrease in the serum phospholipid DHA level in a group of adult males whose diet was supplemented with 3.8 g EPA/day. In addition to any forward metabolic sequence, retroconversion of DHA to EPA does occur in humans (Von Schacky & Weber, 1985; Conquer & Holub, 1997; Nelson et al., 1997; Grimsgaard et al., 1997). Dietary LA generally is considered to be the major source of tissue AA, although lean meats and meat fat are direct sources in the human diet (Li et al., 1998). LA is the principal polyunsaturated FA in the American diet, amounting to 10 15 g/day (Jonnalagadda et al., 1995), and most of the AA in serum and platelets is derived from dietary LA (Grnn et al., 1991). However, in another human study, James et al. (1993) found no changes in the neutrophil phospholipid AA levels in response to LA intakes ranging from 2.5 to 17.5% of energy. Similarly, Whelan et al. (1993) showed that feeding AA to male Syrian hamsters increased the phospholipid AA levels in a variety of normal tissues, whereas when dietary LA was increased by as much as 50%, there was no such effect. They concluded that while dietary LA may influence eicosanoid formation by increasing the tissue AA pool, this contribution diminishes as dietary AA intake increases. In rats, incremental elevations in LA intake did produce corresponding increases in liver AA (Lands et al., 1990b;

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Fig. 2. Structures of oleic acid (an n-9 monounsaturated FA), LA (an n-6 polyunsaturated FA), and LNA (an n-3 polyunsaturated FA). The n-numbers are counted from the methyl terminal.

Marangoni et al., 1992), but while increasing dietary LA intake results in greater hepatic conversion of LA to AA, in extrahepatic tissues, the enhanced availability of LA can result in displacement of AA. For example, when LA in the human diet was increased to about 8% of total energy, the levels of AA in the platelet phospholipids, and especially the AA/LA ratio, were decreased (Tremoli et al., 1986). One consequence of the AA displacement is that feeding sufficiently high levels of LA can actually result in a tissue-specific reduction in the biosynthesis of AA-derived eicosanoids (Galli et al., 1981; Croft et al., 1984; Tremoli et al., 1986).

3. Dietary sources of omega-3 fatty acids, intestinal absorption, and tissue and subcellular distribution 3.1. Dietary sources To a considerable extent, the FA content of fish oils is determined by the food that is available to the fish, and this is influenced strongly by the geographic area in which the fish live, the season of the year, and the fluctuations that occur from year to year. In consequence, published data on the classes and levels of FAs in various species of fish vary widely, and they still may not represent the true situation

Fig. 3. The metabolism of LNA to EPA and DHA and of LA to -linolenic acid (n-6; C20:3) and AA. Each metabolic sequence competes for the same enzyme systems, with the affinity being greater for n-3 FA than n-6 FA. -Linolenic acid, AA, and EPA are the substrates for the biosynthesis of 1-series PG and 2-series LT, 2 series-PG and 4-series LT, and 3-series PG and 5-series LT, respectively.

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(Stansby, 1986). Puustinen et al. (1985) analyzed the total lipid content and the FA composition of 12 commonly eaten northern European fish. They found that the range of total lipid amounts in different fish species was greater than the variation of the percentage amount of EPA and DHA, which led them to conclude that any beneficial dietary effects would be best achieved by the inclusion of the fattier fish. The United States Department of Agriculture published fat and FA values for 30 fish of different species or location (Simopoulos, 1991); 14 of these are listed in Table 1. The total amount of fat in these fish ranged from a low of 0.7 g/ 100 g in cod and haddock to highs of 13.8 and 13.9 g/100 g in Greenland halibut and Pacific herring, respectively. The relative amounts of EPA and DHA contained in fish oils vary considerably between species (Childs et al., 1990). Moreover, some freshwater fish oils contain relatively low levels of EPA, but substantial amounts of AA, compared with those present in marine fish oils and so, their consumption will have very different effects on the cell membrane FA profiles and prostanoid production (Innis et al., 1995). In addition to fish and fish oils, soybean and canola (low erucic acid rapeseed) oils may provide a significant source of dietary n-3 FA in the form of LNA (Hunter, 1990), the major FA in chloroplast lipids. In North American diets, the principal food sources of LNA are salad and cooking oils and salad dressing products. The per capita intake in the United States has been estimated to be 1620 g/day for men and 12 g/day for women (Kim et al., 1984). The changes in the FA content of the typical human diet that have occurred over time since the Paleolithic period were discussed by Simopoulos (1991). She pointed out that the early diet contained small, but approximately equal, amounts of n-6 and n-3 FAs, whereas the modern Western diet contains a rela-

tive excess of n-6 FA. It is this imbalance of n-6 FA to n-3 FA that some investigators have associated with increased risks for both cardiovascular disease and some cancers, including carcinoma of the breast. While this aspect of nutritional cancer epidemiology will be discussed in detail in Section 7.1.5, one instructive example may be mentioned at this point: the increased breast cancer risk in Japanese women, which has taken place over the past four decades and which correlates with a decrease in the dietary n-3:n-6 FA ratio (Rose, 1997a). 3.2. Intestinal absorption The form in which dietary n-3 FAs are ingested affects their absorption from the intestinal tract. In human subjects, EPA ethyl ester was not absorbed as efficiently as the free FA (El Boustani et al., 1987). Lawson & Hughes (1988) found the relative absorption capacities to be: docosapentaenoic acid (DPA; C22:5) EPA and DHA. Also, the relative absorption of the different forms of EPA, DHA, and C22:5, compared with that of an 18:3 n-3 control, was free FA triglyceride ethyl ester. Fish and fish oil products are mostly ingested as the triglycerides; much of the EPA and DHA in the Western diet comprises long-chain n-3 FAs that are absorbed as 2-monoglyceride (Nelson & Ackman, 1988). Hudson & Tisdale (1994) compared the effect of free EPA and EPA ethyl ester on cachectic weight loss and MAC16 adenocarcinoma growth in mice, and related this to their rates of intestinal absorption. Oral administration of the free EPA resulted in prevention of body weight loss and suppression of tumor growth, effects that were associated with elevations in plasma and tumor EPA concentrations. In contrast, the ethyl ester of EPA failed to exert a favorable effect on body weight, and the tumor volumes were not significantly different from those of control animals. This lack of therapeutic efficacy was reflected in the lower tumor EPA concentrations and plasma EPA responses to EPA ethyl ester. Nordy et al. (1991) assessed the intestinal absorption of both EPA and DHA as the triglycerides or ethyl esters in healthy adults, and in contrast to the reports by El Boustani et al. (1987) and Lawson & Hughes (1988), found them to be equally well absorbed. 3.3. Tissue and subcellular distribution In humans and other mammals, DHA occurs at relatively high levels in the cerebral cortex (OBrien & Sampson, 1965), the retina (Anderson, 1970), and testis (Poulos et al., 1975). Dietary deficiency of LNA in developing animals results in reduced CNS DHA levels, which have been associated with learning disabilities and impaired visual function (Innis, 1991). In response to a DHA-supplemented diet, the n-3 FA also concentrates in the heart muscle, as well as the liver and lung (Innis et al., 1995). Both n-3 and n-6 FAs are important constituents of the cell membranes, and cell membrane FA composition of both normal and neoplastic tissues undergoes modification in animals

Table 1 The total lipid and omega-3 fatty contenta of 14 edible fish that are widely available in the United States Fish species Herring, Pacific Halibut, Greenland Mackerel, king Salmon, chinook Bluefish Tuna, albacore Herring, round Salmon, pink Trout, rainbow Halibut, Pacific Swordfish Plaice, European Cod, Atlantic Haddock
a

Total lipid (g/100 g) 13.9 13.8 13.0 10.4 6.5 4.9 4.4 3.4 3.4 2.3 2.1 1.5 0.7 0.7

EPA (g/100 g) 1.0 0.5 1.0 0.8 0.4 0.3 0.4 0.4 0.1 0.1 0.1 0.1 0.1 0.1

DHA (g/100 g) 0.7 0.4 1.2 0.6 0.8 1.0 0.8 0.6 0.4 0.3 0.1 0.1 0.2 0.1

LNA (g/100 g) 0.1 Tr () 0.1 () 0.2 0.1 Tr 0.1 0.1 () Tr Tr Tr

Per 100-g raw, edible, portion. (), lack of reliable data; Tr, trace ( 0.05 g/100 g). Reproduced from Simopoulos (1991), with permission of the author and the copyright holder, Williams & Wilkins, Baltimore.

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fed different high-fat diets, reflecting the FA content of the consumed lipid. The n-3 FAs accumulate in phosphatidylcholine, phosphatidylethanolamine, and triglycerides (de Bravo et al., 1991). The phospholipid pool is the major site of FA incorporation in both cultured normal and transformed cells. In a human breast cancer cell line, LA was found to be preferentially esterified to phosphatidylcholine, whereas EPA was incorporated largely into phosphatidylethanolamine. EPA was also effective in channeling LA from phospholipid to the neutral lipid pool (Hatala et al., 1994).

4. The eicosanoids 4.1. Arachidonic acid-derived eicosanoids AA (5,8,11,14-eicosatetraenoic acid) is an omega-6 polyunsaturated FA metabolically derived from LA. It is the substrate for two classes of enzymes, the prostaglandin (PG) synthases (EC 1.14.99.1), which produce the prostanoidsPGs, prostacyclin (PGI2), and thromboxanes (TXs), and the lipoxygenases (LOXs) (EC 1.13.11.12), which catalyze the biosynthesis of the hydroxyeicosatetraenoic acids (HETEs) and leukotrienes (LTs). These eicosanoids, so called because of their common

origin from a 20-carbon (eicosa-) polyunsaturated FA, behave as local hormones (autacoids), and are chemical transmitters for a variety of intercellular and intracellular signals. The biosynthesis of the n-6 FA-derived eicosanoids is initiated by the release of AA from membrane stores, this cleavage from phospholipids being mediated by membrane-associated phospholipase A2 activity (Verheij et al., 1981). PG synthase catalyzes two sequential reactions: first, the cyclooxygenase (COX) activity of the enzyme converts AA to PGG2, and then the peroxidase activity reduces PGG2 to PGH2 (Oates et al., 1988) (the subscripts indicate the presence of double bonds). However, in spite of these two distinct reactions, it has become commonplace for the complete entity to be referred to as COX. The PGH2 formed from AA is then converted to the various prostanoids (Fig. 4). There are two forms of COX, of which COX-1 is constitutively expressed in most tissues and is considered to generate PG for normal physiological function (Herschman, 1994; Kargman et al., 1995a). COX-2 undergoes rapid induction in response to a variety of stimuli, including mitogens, cytokines, and hormones (Herschman, 1994). While COX-2 is undetectable in normal intestine (Kargman et al., 1996) and breast tissue (Hwang et al., 1998), it is expressed

Fig. 4. The metabolism of AA to the prostanoid subfamily of eicosanoids (2-series PG and TXA2).

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Fig. 5. The metabolism of LA to 13-HODE and of AA to 5-, 12-, and 15-HETE. Not shown are the intermediate compounds 5-, 12-, and 15-HPETE, which are converted to the corresponding HETE by peroxidase activity.

in the majority of colonic carcinomas (Kargman et al., 1995b) and in some primary breast cancers (Parrett et al., 1997; Hwang et al., 1998). The PGs possess a broad spectrum of biological activities, but once PGH2 has been produced, specific cell types are usually highly selective in their formation of bioactive prostanoids. For example, PGI2 is synthesized primarily by vascular endothelial cells and inhibits platelet activation and aggregation, while TXA2, a product of TX synthase-mediated metabolism of PGH2 (Hamberg et al., 1975), is the principal COX product in platelets, has opposing actions to PGI2, and promotes platelet aggregation. Both PGI2 and TXA2 have important roles in cancer biology because they are involved in tumor cell-vascular endothelial cell and tumor cell-platelet interactions, but whereas PGI2 inhibits metastasis, TXA2 exerts an enhancing effect (Honn, 1983; Chen et al., 1992). The 5-, 12-, and 15-LOXs insert molecular oxygen into polyunsaturated FA (Fig. 5). The incorporation of 1 mol of oxygen forms the corresponding 5-, 12-, or 15-hydroperoxyeicosatetraenoic acid (HPETE), which is reduced to the hydroxy analogue (5-, 12-, and 15-HETE), but in addition, 5-HPETE is converted to the bioactive LT (Malle et al., 1987; Spector et al., 1988) (Fig. 6). The HETEs are involved in the regulation of a wide range of biological activities, which are only now being understood, but include ion transport, hormone secretion, and the immune response. They have been associated with rheumatic and collagen diseases, atherosclerosis, and ischemic injury. Within the lung, LOX activity and 5-, 12-, and 15-HETE production occur in alveolar macrophages (Peters-Golden & Thebert,

1987), and LTs are believed to have a role in the pathogenesis of asthma, cystic fibrosis, and pulmonary hypertension. There may be a place for n-3 FAs in the management of these respiratory diseases: in one study, the levels of 12-HETE in lung homogenates were reduced by approximately 60% in mice fed fish oil compared with those in mice fed LA-rich safflower oil (Zhang & German, 1996), and AA, 5-HETE, and 12-HETE concentrations were all reduced in the lungs of rats fed a range of n-3 FA levels with a constant amount of n-6 FA (Hwang et al., 1988). Both LTs and HETEs influence various aspects of tumor cell biology, and there is considerable interest in the potential for pharmacological inhibitors of their biosynthesis as a new approach to cancer chemoprevention and therapy (Rose & Hatala, 1994; Rioux & Castonguay, 1998). 4.2. Eicosanoids derived from eicosapentaenoic acid EPA is a precursor of the prostanoids of the 3-series, with 3 double bonds (Needleman et al., 1979; Fischer & Weber, 1984), and LTs of the 5-series, with 5 double bonds (Prescott, 1984; Lee et al., 1984; Strasser et al., 1985). The n-3 FA is converted by COX to the 3-series endoperoxide PGH3, by way of PGG3, which is further metabolized to PGE3, PGI3, and TXA3, and thence to the inactive metabolites 17-6-keto-PGF1 and TXB3 (Fig. 7). These pathways correspond to those by which AA is converted to the 2-series prostanoids, and EPA competes effectively with the n-6 FA for available COX activity (Needleman et al., 1979). In humans, feeding an EPA-supplemented diet caused an in-

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Fig. 6. The metabolism of AA to the 4-series LTs, with 4 double bonds, via 5-HPETE. The LTA synthase is a dehydrase.

crease in PGI3 production, as reflected in the urinary excretion of 17-2,3-dinor-6-keto-PGF1 (Fischer & Weber, 1984). Because the PGI3 derived from EPA is as antiaggregatory as PGI2 derived from AA, whereas EPA-derived TXA3 has only weak aggregatory activity, the net result is that the COX products of EPA metabolism are associated with diminished platelet aggregation (Fischer & Weber, 1984; Kramer et al., 1996). EPA can also be metabolized by way of the 5-LOX pathway, which parallels the metabolism of AA to the 4-series LT (Fig. 8). The immediate product of this 5-LOX activity, LTA5, is metabolized to LTB5, which possesses only 510% of the activity of LTB4 as a chemotactic and aggregating factor for polymorphonuclear leukocytes (Lee et al., 1984). Some tumor cell types have been shown to produce LTs of the 5-series from EPA (Jakschik et al., 1980; Murphy et al., 1981), and these biosynthetic pathways are subject to dietary manipulation (Murphy et al., 1981; Strasser et al., 1985). The synthesis of LTB5 and LTC5 by mastocytomas in fish oil-fed mice was accompanied by pronounced reductions in LTB4 and LTC4 (Murphy et al., 1981). However, we are not aware of any studies that have related these LTs to the cancer chemopreventive properties of EPA. Although the earlier studies largely attributed the inhibitory effects of dietary fish oil on eicosanoid biosynthesis to EPA, DHA was found to be a potent inhibitor of PG production in vitro (Corey et al., 1983) and of LTB4, LTC4, and 12-HETE, products of LOX-catalyzed AA metabolism (Lokesh et al., 1988). When DHA is incorporated into platelet and other cell membranes, it is not metabolized to a TX (Aveldao & Spre-

cher, 1983) or to LT (Corey et al., 1983). Even so, it can competitively inhibit eicosanoid biosynthesis from AA. 4.3. Eicosanoid biosynthesis and n-3:n-6 fatty acid ratio A number of investigators have stressed the importance of the dietary n-3:n-6 FA ratio, rather than the absolute levels of the two classes of unsaturated FAs, in modulating eicosanoid biosynthesis from AA. This arises because the competitive inhibition that exists between n-3 and n-6 FAs for the desaturases and elongases results in the capacity of dietary n-3 FA to suppress the conversion of LA to AA, being dependent not only on the amount of n-3 FA, but also on the amount of n-6 FA in the diet. In one study (Boudreau et al., 1991), it was found that there was no dose-dependent change in tissue AA or in the levels of TXB2, 6-keto-PGF1 , and 12-HETE when groups of rats were fed 10% total fatcontaining diets providing different amounts of fish oil, but with equivalent increases in safflower oil so as to maintain a constant n-3:n-6 FA ratio of 0.3. This competition between the two classes of polyunsaturated FAs was also demonstrated in human volunteers by feeding them a fixed level of dietary n-3 FA, but in combination with high or low intakes of n-6 FA. Here, the EPA content in serum, platelets, and neutrophil phospholipids was significantly higher in those fed the low LA diet (Grnn et al., 1991; Cleland et al., 1992). The importance of the dietary n-3:n-6 FA ratio has been re-emphasized by two cancer-related studies. In one, concerned with dietary n-3 FAs as potential modifiers of colon cancer risk, a relatively high n-3:n-6 FA ratio was required

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presence of EGF (Bandyopadhyay et al., 1987). On the other hand, in contrast to these stimulatory effects, 13HODE opposes the 15-HETE and 12-HETE stimulation of tumor cell adhesion, which is an integral part of the metastatic process (Bastida et al., 1990; Liu et al., 1991).

5. Biomarkers of n-3 fatty acid nutritional status The FA composition of the plasma lipids and the membranes of platelets and erythrocytes provides an assessment of the dietary intake of the long-chain n-3 FAs over the preceding 2 days to 18 weeks (Bjerve et al., 1993; Andersen et al., 1996; Grnn et al., 1991; Innis et al., 1988; Brown et al., 1991). However, for epidemiological studies aimed at determining the association of dietary FAs with disease risk or outcome, it is necessary to obtain an index of consumption over a relatively long period of time. For this purpose, most investigators have utilized adipose tissue obtained by percutaneous biopsy, for which the turnover time for FAs has been estimated to be 13 years (Field & Clandinin, 1984). Marckmann et al. (1995) found adipose tissue DHA and DPA levels to be much higher than that of EPA, although the food record estimates of dietary EPA and DHA intakes were both considerably higher than that of DPA. The apparent discrepancy may have resulted from the elongation of EPA to the less metabolically active DPA. In this study, the DHA content of adipose tissue provided a better marker of fish and marine n-3 FA intake than did EPA, a result that was also obtained by Tjnneland et al. (1993). Two other studies provided a comparison of data from food-frequency questionnaires, diet records, and subcutaneous adipose tissue FA analyses in American postmenopausal women and men, respectively (London et al., 1991; Hunter et al., 1992). Both showed significant correlations between the percentage of n-3 FAs in adipose tissue, and the percentage of n-3 FAs in the diet, as determined by a foodfrequency questionnaire. In the study of American males, dietary intakes were also assessed by 7-day diet records, which are generally regarded as more accurate. However, this method of evaluation gave a similar level of correlation to that obtained with the semi-quantitative food-frequency questionnaire, suggesting that the less complex method provides an equally reliable assessment. In neither study were separate data collected for EPA and DHA and so, there were no results upon which to judge the relative merits of the two n-3 FAs as markers of n-3 FA intake. One point worth making is that the instruments of n-3 FA intake used in this and other epidemiological studies do not distinguish between the types of fish consumed, which, given the wide variations in n-3 content, may reduce their sensitivity. Although the analysis of adipose tissues is necessary to evaluate dietary FA intake over extended periods of time and is unaffected by temporary variations in diet, their acquisition is not amenable to the large-scale sampling required for many epidemiological studies, and the presence

Fig. 7. The metabolism of EPA to 3-series prostanoids. The only structural difference between PGH3 and PGH2 is that PGH3 has an additional, 17, unsaturation.

to obtain suppression of PGE2 production in human rectal mucosa (Bartram et al., 1995a). In the other, a multinational epidemiological study, the ratio of n-3 FAs to n-6 FAs in adipose tissue biopsies, but not the absolute levels of the two classes of FAs per se, was inversely associated with breast cancer risk (Simonsen et al., 1998). These two studies are discussed in more detail in Section 6. 4.4. 13(S)-Hydroxyoctadecadienoic acid LA can form an oxidation product, 13(S)-hydroxyoctadecadienoic acid (13-HODE), both under the catalytic influence of 15-LOX (Fig. 5) and by nonenzyme autoxidation (Welsch, 1995). This metabolite is attracting the attention of tumor biologists because it may be involved in both carcinogenesis and cancer progression. In some cell types, 15-LOX metabolism of LA is stimulated by epidermal growth factor (EGF), and 13-HODE modulates the EGF mitogenic signal (Glasgow et al., 1992). Welsch (1995) has described the stimulation of mouse mammary epithelial cell proliferation by 13-HODE, and the LA-stimulated growth of these cells in vitro requires the

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Fig. 8. The metabolism of EPA and AA to their LT products of the 5- and 4-series, via 5-hydroperoxyeicosapentaenoic (5-HPEPE) and 5-HPETE, respectively.

of large amounts of nonessential FAs can obscure the relatively low levels of n-3 FAs. With this in mind, it is encouraging that a carefully executed comparative study by Godley et al. (1996) found that the EPA and DHA contents of adipose tissue aspirates and erythrocyte membranes had similar correlations with fish consumption, as estimated by a food-frequency questionnaire. Because the n-3 FAs are very susceptible to peroxidation, appropriate storage is a prime concern when accruing samples for later analysis. Two studies that have addressed this issue concluded that the DHA and EPA contents of adipose tissue were even stable at room temperature ( 20 ) over a 7-month period (Deslypere et al., 1993), and that, at least when stored at 70 , the n-3 FA in erythrocytes exhibited little loss over 12 months (Stanford et al., 1991). The authors suggested that the remarkable stability of DHA and EPA in adipose tissue may be due to the presence of high levels of vitamin E, which prevents deterioration due to lipid peroxidation because of its antioxidant properties.

6. Omega-3 fatty acids and cancer 6.1. Breast cancer 6.1.1. Epidemiology Breast cancer is the most common cancer among woman in the United States, and is second to lung cancer as the most frequent cause of cancer-related deaths. It was esti-

mated that 178,700 new cases would be diagnosed in 1998, and that 43,500 American women would die of the disease (American Cancer Society, 1998). The influence of dietary fat intake on breast cancer risk continues to be a topic of considerable interest, and controversy (Hunter et al., 1996; Wynder et al., 1997). While this important issue is unlikely to be resolved in the immediate future, ecologic studies do show a wide spread in breast cancer incidence and mortality rates between countries and correspondingly large variations in dietary practices. Moreover, migration studies consistently have shown that when women move from countries such as Japan, in which breast cancer risk and dietary fat intake are relatively low, to countries such as the United States, for which the reverse is true, their breast cancer incidence rates increase within one generation (Ziegler et al., 1993). Even within Japan, a correlation was found between dietary total fat intake and breast cancer mortality rates for different districts, with both being higher in urban locations (Hirayama, 1978). One possible explanation for the conflicting evidence concerning dietary fat and breast cancer risk is that the emphasis has been largely on the total amounts consumed rather than the nature of FA contained in those fats (Rose, 1997b). Support for this interpretation came when Kaizer et al. (1989) pointed out the inverse association between breast cancer incidence and mortality rates and dietary fish intake for the same 32 countries that previously had been shown to exhibit a direct relationship for total fat consumption (Arm-

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strong & Doll, 1975). However, this analysis was heavily dependent on the inclusion of data from Japan, a country with an exceptionally high fish consumption and low breast cancer mortality rate. Sasaki et al. (1993) analyzed the data from 30 countries, including Japan, by multiple regression analyses using dietary animal fat minus fish fat intake. When this was done, there was a highly significant positive relationship with breast cancer mortality rates for women over 50 years of age; a separate analysis for fish intake showed the expected negative correlation. Despite these widely quoted results from ecologic studies, most case-control studies have not supported a protective effect of fish consumption (reviewed by Willett, 1997). A similar absence of benefit was found in prospective studies performed in the United States (Stampfer et al., 1987) and in Norway (Vatten et al., 1990), a country with a relatively high consumption of n-3 FA-rich-cold water fish. However, as noted in Sections 3.1 and 4.3, it may be the ratio of n-3 FA to n-6 FA in the diet, rather than the absolute levels of their intake, which is critical in influencing cancer promotion and progression. This distinction is likely to be particularly important in Western countries, where the levels of dietary n-6 FAs are generally high, whereas those of n-3 FAs are low. This interpretation is supported by the results of a multi-national case-control study of adipose tissue FA profiles that was performed in 5 European countries with an approximately 2-fold range in breast cancer incidence (Simonsen et al., 1998). Although the level of n-3 or n-6 FA showed no consistent association, the ratio of longchain n-3 FA to total n-6 FA was inversely correlated with breast cancer in four of the five centers. Interestingly, the relationship between breast cancer and the ratio of LNA to n-6 FA was inconsistent across centers. Although Japanese women continue to have a relatively low breast cancer risk, this has risen over the past 40 years, and an increasing breast cancer mortality rate since 1960 has been accompanied by dietary changes, prominent among which is an increase in the average fat consumption from 9% of total energy in 1955 to 25% in 1987 (Wynder et al., 1991). However, in addition to an increase in the consumption of red meat, there has been an increase in the use of LA-rich vegetable oils, a decrease in fish consumption, and hence, a reduction in the dietary n-3:n-6 ratio (Lands et al., 1990a). While ecologic studies have generally associated high breast cancer risk with high dietary fat intakes, the native Eskimos of Greenland and Alaska provide an instructive exception. Their diet is high in fat, but this comes largely from marine mammals (Bang et al., 1976) and fish (Nobmann et al., 1992). In consequence, they have exceptionally high intakes of n-3 FA, which are reflected in the plasma and erythrocyte membrane EPA and DHA levels, and the n-3:n-6 ratios (Innis et al., 1988; Parkinson et al., 1994). A survey conducted for the years 19691973 showed that although the total cancer incidence rates among Alaska Eskimos and Aleuts did not differ significantly from that of the United

States white population, there were significantly lower rates for cancers of the breast, endometrium, and prostate (Lanier et al., 1976). Although these observations were consistent with earlier reports, a further survey of cancer incidence for the years 19891993 found that there were significant increases in the incidence rates for cancers of the breast and endometrium in women, and for cancers of the prostate and colon in men (Lanier et al., 1996). A contributing factor to these changes may be urbanization of the Alaska native population, and altered dietary practices. Nielsen & Hansen (1980) had drawn a similar conclusion from an analysis of breast cancer incidence among indigenous Greenlandic women for the years 19501979. A recent study of Canadian Eskimos found that plasma DHA and, to a lesser extent, EPA levels were positively correlated with age, whereas age was negatively correlated with plasma n-6 FA (Rode et al., 1995). This age-related difference in plasma FA and the n-3:n-6 ratio was due to a high consumption of marine mammals and fish by the older Eskimos and replacement of this fat source by vegetable oils in the younger members of the community. Although these epidemiological studies of special populations support a protective role for dietary n-3 FA, Miller & Gaudette (1996), while remarking on a low breast cancer risk among Inuit populations in the circumpolar region, rightly point out that other known protective factors, early age at first pregnancy and prolonged lactation, are also prevalent among the Inuit. These also may be changing over time in response to Western influences. Indeed, it has to be recognized that all epidemiological evidence for an association between dietary n-3 FA intake and cancer risk is inferential and correlative, and liable to confounding by other factors. 6.1.2. Experimental studies Although the emphasis of research has been on the effects of n-3 FA on the later stages of carcinogenesis in vivo, there is some direct evidence that they also affect cell transformation. Takahashi et al. (1992) used ionizing radiation of mouse embryo fibroblasts, and, in separate experiments, the transfection of these and NIH 3T3 cells with the H-ras oncogene, as models to demonstrate suppression of transformation by EPA and DHA, and they associated this with the inhibition of eicosanoid production from AA. An analogous study was performed by Telang et al. (1988) using explant cultures of mammary glands from mice harboring the murine mammary tumor virus. In their experiment, it was shown that both LA and AA enhanced the formation of preneoplastic hyperplastic alveolar lesions in vitro, whereas EPA exerted inhibitory activity. In association with these opposing effects, the n-6 FAs increased the levels of ras p21 protein, whereas the n-3 FAs decreased this indicator of ras proto-oncogene expression. It is well established that chemically induced rat mammary carcinogenesis is promoted by dietary n-6 FA, but is inhibited by feeding n-3 FA-rich menhaden oil (Jurkowski &

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Cave, 1985; Braden & Carroll, 1986; Abou-El-Ela et al., 1988, 1989; Cohen et al., 1993). Abou-El-Ela et al. (1988) showed that high tumor production rates of PGE2, 6-keto-PGF1 , and LTB4 were associated with enhanced mammary carcinoma development in dimethylbenz[a]anthracene (DMBA) exposed rats fed a 20% corn oil, LA-rich diet, whereas the levels of these eicosanoids were reduced and tumorigenesis was partially suppressed, in animals fed a 20% menhaden oil diet. In their second study, Abou-El-Ela et al. (1989) obtained inhibition of mammary tumorigenesis and of PGE2 and LTB4 production by tumor cells when they fed a diet containing FAs with an n-3:n-6 FA ratio of 1.2 (15% menhaden oil and 5% corn oil) compared with a 20% corn oil diet. The optimal level of menhaden oil to be fed in order to suppress experimental mammary tumor development remains uncertain, but so also does the relevance of these feeding studies to the chemoprevention of human breast cancer by n-3 FA. There is evidence that when menhaden oil is fed as 18% of the diet, it may actually enhance mammary tumor development (Cohen et al., 1993), but it is questionable whether such an extreme situation has any implications for the design of human dietary intervention trials, where there is no possibility of attempting such high intakes. In addition to their effects on mammary carcinogenesis in animal models, FAs affect both the growth of transplanted tumors and the expression of the metastatic phenotype. Gabor et al. (1985) showed an enhancing effect of dietary LA on the growth of transplanted mammary carcinomas in BALB/c mice, and subsequently reported that feeding a diet containing 10% menhaden oil suppressed growth, or more specifically, enhanced tumor cell loss (Gabor & Abraham, 1986). Earlier, Karmali et al. (1984) had also reported inhibition of R3230AC mammary tumor growth in rats fed MaxEPA, a fish oil concentrate that contains approximately 18% EPA and 12% DHA. Human breast cancer cells growing as xenografts in athymic nude mice have proved invaluable for assessing the influence of dietary FAs on metastasis, despite the limitation that estrogen-dependent cell lines do not metastasize, or do so rarely, so that the model cannot be used to study estrogen-FA interactions on the metastatic process (Rose & Connolly, 1997). A number of studies have shown that diets rich in the long-chain n-3 FA inhibit the growth (Borgeson et al., 1989; Gonzalez et al., 1991; Rose & Connolly, 1993; Rose et al., 1995) and metastasis (Rose & Connolly, 1993; Rose et al., 1995) of human breast cancer cell lines growing as solid tumors in nude mice. While fish oil was used in the earlier studies, when pure EPA and DHA ethyl esters became available, these were shown to be effective in suppressing tumor progression when fed at levels of 8% and 4% (wt/wt) in a diet containing a total of 20% fat (Rose et al., 1995). As generally used, the nude mouse model is of limited clinical relevance because the tumor growing at the injection site is left in situ throughout the experiment. A more realistic approach was introduced when the ethyl es-

ters of EPA or DHA were used as nutritional adjuvant therapy after the surgical removal of the primary breast cancer (Rose et al., 1996). In this way, the focus was on the chemosuppression of residual disease and of microscopic metastases. 6.1.3. The relative potency of eicosapentaenoic acid and docosahexaenoic acid in breast cancer animal models The recent availability of EPA and DHA in bulk quantities and of unique DHA-rich oils derived from microalgae has offered a new approach to n-3 FA chemoprevention and the suppression of cancer progression. It is also now possible to compare the efficacy of the two long-chain n-3 FAs. Noguchi et al. (1997) used the DMBA-induced mammary tumor model to examine the effects of low doses of EPA and DHA ethyl esters on tumor incidence and growth in rats fed a high-fat, 20% corn oil, diet. In this experiment, 0.5 mL of the 98% pure n-3 FA was given by gastric gavage twice a week to provide a calculated dietary n-3:n-6 FA ratio of 1:1.8. The DHA-treated rats showed a 69% reduction and the EPA-treated rats a 47% reduction in tumor incidence compared with the incidence in the high-fat-fed controls. These relative degrees of efficacy of the two n-3 FAs were paralleled by a partial, but statistically insignificant, suppression of growth once the tumors had developed. A transplantable mouse mammary carcinoma was used by Kinoshita et al. (1994) to compare the effects of EPA and DHA, again given at low dose by intragastric intubation, on primary tumor growth and metastasis to the lungs. Some partial suppression was observed, with no significant differences between the two n-3 FAs. When we compared the two n-3 FAs in experiments using nude mice bearing human breast cancer xenografts, we found no difference between EPA and DHA ethyl esters in achieving partial suppression of tumor growth. There was, however, some indication that EPA was more effective in inhibiting metastasis (Rose et al., 1995). A second study in which the n-3 FAs were employed in an experimental protocol designed to mimic postsurgery, adjuvant chemotherapy produced opposite results. The extent of lung metastasis was reduced to a greater degree by feeding 2% or 4% DHA, compared with the same levels of EPA (Rose et al., 1996). More recently, we have had the opportunity to evaluate DHA triglyceride prepared from microalgae in the MDAMB-435 breast cancer cell/nude mouse model (Connolly & Rose, 1998a) The control of primary tumor growth and metastasis appeared to be superior to that obtained with EPA in our earlier experiment. 6.2. Colon cancer 6.2.1. Epidemiology Colon cancer is the fourth most common cancer in the United States and ranks second among cancer-related causes of death. It was estimated that 24,600 American women and 23,100 American men would die of the disease

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in 1998 (American Cancer Society, 1998). Some of the international comparisons of breast cancer mortality rates apply also to colon cancer (Rose et al., 1986), and a positive correlation was found between the geographic distribution of the breast and colorectal cancer mortality rates within the United States (Jansson et al., 1975). In Finland, an excess risk of colon cancer was observed in women who had a previous history of breast cancer (Teppo et al., 1985), and other studies have shown an increased incidence of precancerous colonic polyps in breast cancer patients (Bremond et al., 1984; Jouin et al., 1989). Rozen et al. (1990) reported that rectal mucosal cell proliferation, a presumptive marker of colon cancer risk, was elevated in treated breast cancer patients. These relationships suggest the presence of either a common risk factor(s) or the same relative lack of factor(s) that are protective against the two tumor types. Among these, it is tempting to focus on dietary FA, but recent epidemiological studies indicate that we cannot ignore shared endocrine mechanisms (Fernandez et al., 1996; HbertCroteau, 1998). In an analysis involving 24 European countries, an inverse correlation was found in males between colorectal cancer mortality and current fish intake. A similar trend for females did not achieve statistical significance, and there was no association for breast cancer (Caygill & Hill, 1995). However, the importance of the dietary n-3:n-6 FA ratio was stressed in Section 4.3, and so it is of particular interest that when those data were reanalyzed for breast and colorectal cancer, there was evidence of a protective effect of a high fish intake relative to that of dietary sources of n-6 FAs for both tumor types (Caygill et al., 1996). Familial adenomatous polyposis is an example of a genetically determined precancerous condition in which numerous polyps develop in the colon and rectum. These appear in young adulthood, and virtually all of these individuals develop colon adenocarcinoma by age 40 years (DeCosse et al., 1977). The standard therapy is total colectomy. In a controlled clinical trial, the nonsteroidal antiinflammatory drug and unselective COX inhibitor sulindac reduced the number and size of polyps, but its effect was incomplete (Giardiello et al., 1993). This precancerous condition is an obvious target for chemopreventive intervention, and while the present focus is on selective COX-2 inhibitors, the place for dietary n-3 FA supplementation merits careful evaluation. 6.2.2. Experimental studies A number of studies have shown that experimental rat colon carcinogenesis is inhibited by feeding a diet containing a high level of fish oil (Reddy & Maruyama, 1986; Nelson et al., 1988; Reddy & Sugie, 1988; Deschner et al., 1990; Reddy et al., 1991) or supplemented with n-3 FAs (Minoura et al., 1988). In the experiment performed by Minoura et al. (1988), a diet containing 5% total fat and supplemented with 4.7% EPA (91% pure; without DHA) together with 0.3% LA to prevent essential FA deficiency

inhibited azoxymethane-induced colon carcinogenesis in rats compared with tumorigenesis in rats fed a 5% LA control diet. It should be noted that the LA content of the control diet generally would be considered as providing a low intake of the n-6 FA. Therefore, in human terms, at least for Western countries, the EPA was exerting its protective effect in the presence of an already favorable low-fat, lowLA diet. The effects of different fish oil-containing diets, formulated to provide several n-3:n-6 FA ratios, on the early pathological stages of experimental colon carcinogenesis were studied by Deschner et al. (1990). They used an azoxymethane-induced rat model fed diets that all contained a total of 20.4% fat; the fish oil concentrate MaxEPA was the source of n-3 FA. Epithelial cell proliferation in the colonic crypts showed the expected elevation as a result of exposure to azoxymethane, and this increase was partially suppressed by feeding the highest level of MaxEPA (16% wt/wt) with an n-3:n-6 FA ratio of approximately 1.5. The 16% MaxEPA-containing diet also inhibited the increase in DNA-synthesizing, S-phase, cells in the middle and upper thirds of the crypts, which is a feature of a carcinogenic response. Similarly, the occurrence of early dysplastic foci was reduced significantly in carcinogen-exposed animals fed either 16% or 10.2%, but not 4.4%, MaxEPA. In a second part of this study, Deschner et al. (1990) examined the effect of the same levels of MaxEPA on the full development of azoxymethane-induced colonic carcinomas. The highest tumor incidence occurred in rats fed 20.4% corn oil (5055% LA) or 16% corn oil with the lowest (4.4%) level of MaxEPA. Diets containing 4.4% corn oil without any n-3 FA addition, or 10.2% or 16% MaxEPA, all produced similar reductions in colonic tumor incidence. We have described the study by Deschner et al. in some detail because it is one of the few in which different levels of n-3 FAs, with differing n-3:n-6 FA ratios, have been compared for their chemopreventive efficacy. One additional point that came out in analyzing the results of this experiment is that both tumor incidence and number of tumors per animal were actually higher in rats fed the 16% corn oil:4.4% MaxEPA diet than in those fed 20.4% corn oil. This could be interpreted as a direct adverse effect of diets containing supplemental n-3 FA, at relatively low levels, on colon carcinogenesis. However, as suggested by the authors, it could also be a reflection of the dietary LA level and the reduced production of AA-derived eicosanoids that can occur with very high LA intakes (Section 2), such as that provided by a diet containing 20.4% corn oil. The effect of n-3 FA on the early stages of colon carcinogenesis was also examined by Takahashi et al. (1993). However, the experimental design was somewhat unusual in that DHA ethyl ester (0.7 mL) was given by intragastric intubation in single doses 5 times a week. This regimen reduced the formation of 1,2-dimethylhydrazine-induced aberrant crypt foci, and when these colonic preneoplastic lesions did occur, there was retardation of their subsequent growth.

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Various germ line mutations of the murine Apc gene result in multiple intestinal polyposis, and provide a model for human familial adenomatous polyposis (Moser et al., 1990; Fodde et al., 1994). The inhibitory effects of DHA and an EPA/DHA-enriched fish oil concentrate on the development and progression of intestinal polyps have been demonstrated in two of these models. Oshima et al. (1995) fed a 3% DHA ethyl ester-containing diet, of unspecified total and n-6 FA content, to female and male Apc 716 heterozygotes. The females showed a 69% reduction in polyp formation and a significant suppression of polyp growth. In the males, there was no effect on polyp number and a lesser effect on polyp size. The mechanism for this sex difference is obscure and was not reproduced in a similar study performed by Paulsen et al. (1997). These investigators used the Min (multiple intestinal neoplasia) mouse, which is heterozygous for a nonsense mutation of the Apc gene at codon 850, and fed a 12% total fat diet and a fish oil concentrate containing 54.4% EPA, 30.3% DHA, and 8.3% other n-3 FAs. Both tumor numbers and size were reduced in male and female mice fed the n-3 FA-rich diet. As in the case of breast cancer, the administration of pure EPA or DHA has also been shown to inhibit colon cancer cell metastasis to the lungs in experimental animal models (Iigo et al., 1997; Suzuki et al., 1997). In both cases, these inhibitory effects on tumor progression were associated with a reduction in matrix metalloproteinase (EC 3.4.24.7) activity, which is a critical part of the invasive phenotype (Liu & Rose, 1995; Suzuki et al., 1997). 6.3. Mechanisms 6.3.1. Breast cancer 6.3.1.1. Mitogenesis and apoptosis Both EPA and DHA inhibit the growth of human breast cancer cell lines in vitro. In experiments performed by Rose & Connolly (1990), DHA was considerably more effective than EPA in suppressing the growth of estrogen-independent MDA-MB231 breast cancer cells. While there was a 50% reduction in cell number when 2 g/mL of DHA was added to the culture medium, trypan blue exclusion indicated that the cells present were viable, suggesting that the cell loss was due to inhibition of mitosis rather than cytotoxic cell death. The two long-chain n-3 FAs, and to a lesser degree LNA, also inhibited the growth of the estrogen-dependent MCF-7 breast cancer cell line (Grammatikos et al., 1994). Again, this inhibition was not due to cytotoxicity, which can occur due to the formation of peroxidation products from polyunsaturated FAs (Welsch, 1995). Although nonspecific cytotoxicity does not appear to be responsible for the growth inhibition induced by n-3 FA at low concentration, at high levels, both n-3 FA and n-6 FA caused lysis of human breast cancer cells in vitro (Begin et al., 1986, 1988; Begin & Ells, 1987). This cytotoxic activity was shown to correlate with the levels of intracellular thiobarbituric acid-reactive substances, the determination of which provides a crude index of lipid peroxidation. How-

ever, DHA produced the least thiobarbituric acid-reactive substances, and the highest levels were generated by -linolenic acid and AA (Begin et al., 1988), a result that is not consistent with the overall experience concerning the relative effects of n-3 FAs and n-6 FAs on breast cancer cell growth (Rose & Connolly, 1990; Buckman et al., 1991), and brings into question the physiological and clinical relevance of these cell culture experiments performed in vitro with very high FA concentrations. The acquisition of solid tumor mass and increasing cell number is the consequence of active mitogenesis, reduced programmed cell death (apoptosis), or a combination of these two cellular events (McDonnell, 1993; Kerr et al., 1994). In contrast to cytotoxin-induced necrosis, apoptosis is a physiological process important to tissue development and organ modeling, and is carefully regulated by the expression of specific genes. Although there is direct evidence that the long-chain n-3 FAs can induce apoptosis, this appears to be cell-type specific. Thus, while culture with EPA resulted in apoptosis in a human pancreatic cancer cell line (Lai et al., 1996), this n-3 FA produced an inhibition of Morris hepatocarcinoma 3924A growth in vivo that was primarily due to reduced proliferation. In contrast, DHA had no effect on mitosis, but it induced apoptosis (Calviello et al., 1998). We are not aware of any published studies that have investigated the effects of n-3 FAs on breast cancer cell apoptosis, but this may be inferred from a publication by Gabor & Abraham (1986), which predated the current intense interest in apoptosis and cancer. They reported that the inhibition of growth of a transplantable mouse mammary adenocarcinoma by dietary n-3 FA was a result of cell loss and suggested the involvement of PG in this process. As discussed in Section 6.1.2, the inhibition of DMBAinduced rat mammary carcinogenesis by feeding n-3 FArich menhaden oil is associated with a reduction in tumor PG and in LOX-mediated AA product levels. The relative importance of these eicosanoid families in mammary tumorigenesis and breast cancer progression is uncertain, although LOX rather than COX inhibitors were effective in inhibiting human breast cancer cell growth in vitro (Rose & Connolly, 1990; Buckman et al., 1991) and DMBA-induced tumor growth in vivo (Kitagawa & Noguchi, 1994). Similarly, Abou-El-Ela et al. (1989) concluded that a reduction in tumor LTB4 production due to 5-LOX inhibition was causally associated with n-3 FA inhibition of DMBAinduced rat mammary tumorigenesis. Both COX-2 (Tsujii & DuBois, 1995) and 12-LOX (Tang et al., 1996) expression have been shown to downregulate the apoptotic pathway, and overexpression of 12LOX in MCF-7 breast cancer cells resulted in increased growth in nude mice and suppressed apoptosis (Connolly & Rose, 1998b). Treatment of W256 cells, a monocytoid cell line that arose spontaneously in the mammary gland of a pregnant rat, with LOX, but not COX, inhibitors induced apoptosis that was partially reversed by exogenous 12-

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HETE or 15-HETE (Tang et al., 1996). With this background, it seems reasonable to postulate that dietary n-3 FAs, which inhibit 12-HETE, 15-HETE, and PGE2 production by human breast cancer cells growing as solid tumors in nude mice, may exert their suppressive effect on tumor mass acquisition, at least in part, by activation of the apoptotic pathway (Rose & Connolly, 1997). 6.3.1.2. Angiogenesis Neovascularization, or angiogenesis, is essential for solid tumor growth (Folkman, 1990), and also provides the tumor cells with access to the vascular circulatory system, thus establishing the potential for metastatic disease progression. Angiogenic activity in primary breast cancers has been associated with metastasis and a poor prognosis (Heimann et al., 1996; Fox et al., 1997) in benign breast lesions with a propensity for progression to malignant disease (Guinebretire et al., 1994; Fregene et al., 1994) and in breast ductal carcinoma in situ with later invasiveness (Heffelfinger et al., 1996). The process of neovascularization is promoted by a variety of angiogenic factors, which include both PGs (Form & Auerbach, 1983; Spisni et al., 1992) and 12-HETE (Tang et al., 1995). We have shown that transfection of the MCF-7 human breast cancer cell line so that it stably overexpresses 12-LOX and secretes high levels of 12-HETE, causes rapid growth with not only reduced apoptosis, but also high angiogenic activity (Connolly & Rose, 1998b). Again, dietary n-3 FAs may prove to be antiangiogenic because of their suppressive effects on eicosanoid biosynthesis (Connolly et al., 1997b). 6.3.1.3. Estrogen metabolism The natural estrogen 17 estradiol, in combination with other steroid and polypeptide hormones and growth factors, stimulates not only normal mammary development, but also neoplastic transformation. A series of studies has shown that a shift in estradiol metabolism in favor of 16 -hydroxylation and away from the formation of catechol derivatives produces aberrant hyperproliferation in mammary explant experiments, and clinical studies suggest that an elevated C16 -hydroxylation of estradiol may provide a biomarker for breast cancer risk (Telang et al., 1997). In a preliminary study, Osborne et al. (1988) found that feeding an n-3 FA-rich fish oil supplement to women with either a family history of breast cancer, breast atypical hyperplasia, or carcinoma in situ (Section 7.1.2) reduced the extent of 16 -hydroxylation. This potentially important observation should be pursued in any future trials of n-3 FAs as breast cancer chemopreventive agents. 6.3.1.4. Mevalonate synthesis 3-Hydroxy-3-methylglutaryl co-enzyme A reductase (EC 1.1.1.34) is the rate-limiting enzyme in the cholesterol biosynthetic pathway and catalyzes the synthesis of mevalonate. Mevalonate exerts a number of effects that signal increased cell proliferation, and compounds such as dietary isoprenoids, which inhibit mevalonate synthesis, are inhibitors of tumor cell growth (Elson, 1995).

El-Sohemy & Archer (1997) extended these observations to n-3 FAs, which were known to reduce hepatic 3-hydroxy3-methylglutaryl co-enzyme A reductase activity, and found that both immunoreactive enzyme expression and mevalonate production were suppressed in the mammary glands of menhaden oil-fed female rats. Thus, the down-regulation of mevalonate synthesis in preneoplastic mammary epithelial cells provides another plausible mechanism that may contribute to the chemopreventive activity of n-3 FAs. 6.3.1.5. Lipid peroxidation The role of lipid peroxidation in the suppressive effects of n-3 FA on mammary tumorigenesis was reviewed by Welsch (1995), and in this section, we have already noted that the lytic effects of polyunsaturated FAs on cultured tumor cells correlated with the degree of lipid peroxidation product formation. Gonzalez et al. (1991) found that when diets containing 19% menhaden oil were fed to mice, there was both an inhibition of growth of human breast cancer cell xenografts and an accumulation of secondary products of lipid peroxidation in these tumors. Moreover, the inhibitory effects of the fish oil was reversed when antioxidants were added to the diet in sufficient amounts to reduce lipid peroxidation. The mechanism(s) by which these degradative products of n-3 FAs inhibit breast cancer cell growth is uncertain, as is the relevance of this effect of very high fish oil intakes to the clinical application of n-3 FA for breast cancer chemoprevention. We found that feeding DHA as the triglyceride at a level of 2% of diet retarded the growth of MDA-MB435 human breast cancer cell solid tumors in nude mice, and did so without increasing the tumor levels of secondary lipid peroxidation products to those that were necessary to inhibit growth in experiments reported by Gonzalez et al. (1993) (J. M. Connolly, R. Mongru, & D. P. Rose, unpublished data). 6.3.2. Colon cancer 6.3.2.1. Cyclooxygenase-2 inhibition Sakaguchi et al. (1984) showed that a high-fat, LA-rich diet enhanced colon carcinogenesis in rats treated with the chemical colonic carcinogen azoxymethane, and that the tumors contained elevated levels of AA. The colonic mucosal levels of PGE2 and 6keto-PGF1 are elevated during the initiation and postinitiation stages of carcinogenesis in this model (Kulkarni et al., 1992), and in human surgically excised colon cancers, the PGE2 concentrations are increased compared with the corresponding normal colonic mucosa (Rigas et al., 1993). In Section 6.2.2, we referred to a study by Minoura et al. (1988), and here, partial suppression of rat colon carcinogenesis by EPA was associated with a reduction in the PGE2 content of the tumors that did appear compared with that present in normal mucosa. Increased production of PG in colon cancers is associated with the up-regulation of COX-2 (Eberhart et al., 1994; Kargman et al., 1995b), and selective COX-2 inhibitors are effective in experimental colon cancer chemoprevention (Reddy et al., 1996a; Yoshimi et al., 1997). Singh et al.

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(1997) showed that there was a time-related increase in COX-2 expression in rat colonic mucosa after azoxymethane administration, and particularly high levels were present in the tumors that subsequently developed. This enzyme induction was enhanced further by feeding a high-fat, n-6 FArich diet, but was suppressed by a high-fat, 20% menhaden oil diet. These elevations in COX-2 expression correlated with the incidence and multiplicity of colon tumors, while the expression of COX-1 was unchanged. Thus, it appears that the suppressive effects of n-3 FAs on colon carcinogenesis, including the reduced formation and progression of aberrant crypts described by Takahashi et al. (1993), are intimately related to the down-regulation of COX-2. In this context, it should be noted that a selective inhibitor of COX-2 suppressed intestinal polyposis in the Apc 716 knockout mouse model for familial adenomatous polyposis (Oshima et al., 1996). However, while the current emphasis is on COX-2 inhibitors as potential colon cancer chemopreventive agents, the involvement of LOX products, the synthesis of which is also blocked by n-3 FA, deserves attention. Hussey & Tisdale (1994) showed that the growth in two murine colon adenocarcinoma cell lines was stimulated by both LA and AA in vitro, and that this effect could be blocked by a selective LOX inhibitor. In later experiments, these investigators obtained similar results in vivo and demonstrated the particular importance of 12-LOX inhibition in achieving growth suppression (Hussey & Tisdale, 1996). 6.3.2.2. Apoptosis Chang et al. (1997) assessed both cell proliferation and apoptosis in the colonic crypts of rats exposed to azoxymethane and fed either a high-corn oil or a high-fish oil diet, and found that the tumor-inhibitory effect of dietary n-3 FA was associated with increased apoptotic activity, but not with reduced cell proliferation. The apoptotic pathway in colon cancer cells is regulated by COX-2 expression. In one study, treatment of a human colon cancer cell line that possessed a high constitutive level of COX-2 expression and PGE2 production with a selective COX-2 inhibitor increased apoptotic activity, which was reversed by exogenous PGE2 (Sheng et al., 1998). 6.3.2.3. Angiogenesis As in the case of breast cancer and some other solid tumors, a high degree of angiogenic activity in colonic carcinomas has been associated with a poor prognosis (Takebayashi et al., 1996). Consistent with this relationship is the finding that microvessel density is higher in aggressively invasive colon cancers (Saeki et al., 1997). The calcium-independent and -inducible nitric oxide synthase-2 was found to be expressed at high level in approximately 60% of human colon adenomas and 2025% of colon carcinomas (Ambs et al., 1998), and both the inducible and endothelial constitutive nitric oxide synthases were elevated in rat colon tumors induced by azoxymethane (Takahashi et al., 1997). While the role for nitric oxide in tumor angiogenesis is currently under investigation (Garca-

Cardea & Folkman, 1998), it is noteworthy in the present context that nitric oxide is known to activate COX-2 and regulate PG biosynthesis (Salvemini et al., 1993; Sautebin et al., 1995). Given the relationships between both COX and LOX and angiogenesis referred to in Section 6.3.1.2, it seems very likely that the inhibition of PG and HETE production in colon cancer cells by n-3 FAs provides an antiangiogenic mechanism for their chemopreventive activity. 6.3.2.4. Protein kinase C (EC 2.7.10) Under physiological circumstances, protein kinase C (PKC) is activated by the combined effects of phospholipids and diacylglycerol (Hannun et al., 1986). The activation of PKC has been implicated in the stimulation of colonic epithelial cell growth by unsaturated FAs. Both LA and AA, as well as the monounsaturated oleic acid, stimulated [3H]thymidine incorporation, with concomitant PKC translocation from the soluble to the particulate fraction of colonic cells, the hallmark of PKC activation. When these FAs were instilled into the colon in vivo; in both cases, AA was the most effective of the three FAs (Craven & DeRubertis, 1988). Total PKC was found to be reduced in human colon adenocarcinomas compared with the uninvolved mucosa (Guillem et al., 1987) and in the histologically normal mucosa from colon cancer patients compared with colonic mucosa from patients without cancer (Sakanoue et al., 1991). Moreover, the proportion of the total PKC activity associated with the particulate, membrane, fraction was greater in mucosa from the colon cancer patients (Sakanoue et al., 1991). Baum et al. (1990) found that colonic mucosal PKC activity exhibited translocation to the membranes in fractions prepared from the colons of rats treated with the procarcinogen 1,2-dimethylhydrazine and examined prior to the appearance of microscopic neoplastic change. Over time, the extent of this translocation increased and there was a concomitant reduction in total PKC activity. In a second study, this same group of investigators showed that these changes in PKC were also present in the tumors that subsequently developed when they were accompanied by increases in 1,2-diacylglycerol (Wali et al., 1991). Reddy et al. (1996b) found that the exposure of rats to the direct-acting chemical colonic carcinogen azoxymethane increased diacylglycerol kinase activity in the colonic mucosa. This effect was enhanced by feeding a highfat, n-6 FA-rich diet, but not by a high-fat, n-3 FA-rich diet. In contrast to the experience of others, total PKC activity was higher in the colon tumors that developed compared with the corresponding mucosa. The reason for this contradictory result is unclear. Certainly, the reported down-regulation of total PKC activity in human and rat colon tumors contrasts with the elevated total activity and changes in PKC isoform expression seen in breast cancer (MorseGaudio et al., 1998). Chapkin et al. (1993) also examined the effect of dietary fish oil on PKC activity and intracellular distribution, but

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they found that the ratio of membrane to cytosolic PKC activity in normal colonic mucosa increased in rats fed a diet containing 11.5% menhaden oil compared with the activity ratio in animals fed a 15% corn oil, n-6 FA-rich diet. Also, there was a positive correlation between the activity ratio, the total membrane PKC activity. and colon cell proliferation. Thus, in spite of the report by Craven & DeRubertis (1988) that LA and AA increased the membrane PKC activity in colon cells in vivo and in vitro, n-3 FA appeared to have the potential for reversing the PKC changes associated with colon carcinogenesis. Aside from the total PKC activity, the colonic mucosa exhibits a distinct pattern of isoform expression (Doi et al., 1994; Davidson et al., 1994), and altered PKC isoform signaling appears to be involved in colon cancer development. In the present context, the report by Jiang et al. (1997) is of particular interest. These investigators found that dietary fish oil blocked the decrease in the steady-state levels of the atypical PKC isoforms, which is otherwise induced in the rat colonic mucosa by azoxymethane. There is clearly a need for further research on PKC activity, expression of the various isoforms in colon cancer, and the biological and molecular significance of the changes resulting from dietary FA modification. 6.3.2.5. Ornithine decarboxylase (EC 4.1.1.17) The polyamines that are produced from the metabolism of ornithine have a role in cell proliferation, and the intracellular content of these compounds is highly regulated. The first and generally the rate-limiting step in polyamine biosynthesis is catalyzed by ornithine decarboxylase (ODC), an inducible enzyme that is elevated during the promotion phase of multistage carcinogenesis, such as that which occurs in the colon. Increased ODC activity is present in premalignant adenomas, including adenomatous polyps, and in colorectal carcinoma tissues, compared with the corresponding uninvolved colonic mucosa (Narisawa et al., 1989; Hixson et al., 1993). Craven & DeRubertis (1988) found that in addition to PKC activation, there was an increase in ODC activity when colonic epithelial cells were exposed to n-6 FA. Reddy & Sugie (1988) assayed ODC activity in their study of the influence of dietary FA on azoxymethane-induced rat colon carcinogenesis and observed an inverse relationship between the enzyme and the level of menhaden oil in the diet. In a second study, Rao & Reddy (1993) demonstrated a stimulatory effect of an n-6 FA-rich diet on ODC activity and a relative suppression of the enzyme by dietary n-3 FA in colonic mucosa exposed to azoxymethane for 2 weeks. 6.3.2.6. Fecal bile acid and neutral sterol excretion Both epidemiologic and animal studies have provided evidence that colon cancer risk and the adverse influence of dietary fat on risk are associated with increased levels of secondary bile acids within the colonic lumen (Reddy & Wynder, 1973; Reddy et al., 1977). Bartram et al. (1995b) performed a short-term experi-

ment to examine the effects of dietary fish oil on fecal bile acid excretion in which the daily diet of healthy volunteers was supplemented with either 11 g of fish oil (4.4 g n-3 FA) or 11 g of corn oil. After 4 weeks, there was no change in the total bile acid excretion. The excretion of the secondary bile acid lithocholic acid was lower after n-3 FA administration compared with that after dietary supplementation with the n-6 FA-rich corn oil, although the difference was not statistically significant. There was, however, a significant reduction in the excretion of the putative colon carcinogen 4-cholesten-3-one (Bartram et al., 1998). While these preliminary data need to be confirmed, they are supported by animal experimentation. Reddy et al. (1996b) determined fecal bile acid excretion in their studies of dietary fats and azoxymethane-induced rat colon carcinogenesis. They found that although the total bile acid excretion was unaffected by the type of FA in the diet, the fecal secondary bile acid concentrations, including lithocholic acid, were reduced in rats fed a high-fat, n-3 FA-rich diet compared with those in rats fed a high-fat, n-6 FA-rich diet. Moreover, the activity of cecal bacterial 7 -dehydroxylase, which is involved in the transformation of primary to secondary bile acids, was higher in the rats fed a high, 23.5%, corn oil-containing diet than that in animals fed a 20.5% menhaden oil and 3.0 % corn oil diet. Various studies have related these effects of bile acids and the associated fecal neutral sterols to colonic cell PKC and ODC activities, and to LOX products of AA metabolism. Bile salts stimulate colonic epithelial cell growth, and this potentially tumor-promoting effect is preceded by both the translocation of PKC from the cytosolic to the membrane fraction and increased ODC activity (Craven et al., 1987). Fitzer et al. (1987) showed that the unconjugated bile acids chenodeoxycholate and deoxycholate stimulate conventional (calcium and phospholipid-dependent) PKC activity in vitro when assayed in the presence of a high calcium concentration. Without this ionic excess, inhibition occurs because the bile acids interact with the Ca2 and the phosphatidylserine. The investigators postulated that the tumorpromoting activity of bile acids in colon carcinogenesis may be modified by the relative absorption of Ca2 and bile acids by the colonic mucosa, with calcium exerting a chemopreventive function and bile acids a promoting one. The expression of several biological characteristics of tumor cells has been shown to involve the regulation of PKC activity by the LOX-mediated product of AA metabolism 12-HETE. These include tumor cell adhesion to endothelial cells (Liu et al., 1991), cytoskeletal rearrangement (Timar et al., 1993), and invasive capacity (Liu et al., 1994). In the present context, it is noteworthy that tumor cell 12-HETE production is inhibited by dietary n-3 FA (Rose et al., 1995), and that EPA and DHA (Connolly & Rose, 1993) and a pharmacological inhibitor of PKC activity (Connolly et al., 1994) inhibited the invasive capacity of a human breast cancer cell line in an in vitro assay.

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This association of dietary FA, ODC activity, 12-HETE, bile salts, and colonic epithelial cell proliferation was brought together by DeRubertis et al. (1984). They showed that when deoxycholate stimulated the release of AA from rat colon in vivo, there was an accumulation of 12-HETE, which was followed by an elevation in colonic mucosal ODC activity and increased [3H]thymidine incorporation into DNA. Interestingly, treatment with indomethacin, a COX inhibitor, increased 12-HETE production, presumably by shifting AA metabolism into the LOX-mediated pathway, and this resulted in a further increase in ODC activity and thymidine incorporation. 7. Omega-3 fatty acids and cancer chemoprevention 7.1. Breast cancer A reduction in the breast cancer mortality rate is now evident in the United States, which is most likely attributable to the beneficial effect of mammographic screening on the clinical stage at diagnosis, together with the success of adjuvant chemohormonal therapy in the management of the postsurgically resected breast cancer patient (Chu et al., 1996). One consequence of routine breast cancer screening by mammography is that abnormalities that are known to constitute risk factors for invasive carcinoma of the breast are being recognized with increasing frequency. Some of these are direct radiological abnormalities, while others constitute histopathological features that are observed in breast tissue obtained by biopsy, itself performed in response to a mammographic abnormality. 7.1.1. Mammographic densities Wolfe (1976) originally defined four categories of mammographic parenchymal patterns and breast cancer risk. Risk is lowest in the N1 category, the parenchyma being composed largely of fat, and highest in the DY category, which is characterized by extensive nodular densities, socalled mammographic dysplasia. Later, attention focused on mammographic density per se, which, while approximating to the Wolfe classification in significance, is a distinct entity. Here, the proportion of the breast volume occupied by radiological densities is determined by digital planimetry (Lee-Han et al., 1995). Boyd et al. (1992) reported a study of women aged 40 49 years that showed that extensive mammographic densities predict a more than 9-fold increase in the likelihood of developing carcinoma in situ or atypical hyperplasia, two histopathological abnormalities that are the themselves risk factors for invasive breast cancer (see below). Of particular interest in the context of a chemoprevention trial, is the report by Lee et al. (1994) that cytological abnormalities present in exfoliated breast epithelial cells collected by nipple aspiration correlate with the presence of mammographic densities. Their elimination would provide an appropriate intermediate response end-point as part of a clinical trial of n-3 FA for women with mammographic densities.

Epidemiological studies have shown a positive correlation between dietary fat, and by implication LA, intake and mammographic dysplasia (Brisson et al., 1989; Nordevang et al., 1993), or benign breast disease with atypia (Lubin et al., 1989). Japanese women, with a traditional diet low in total fat and n-6 FAs, but high in n-3 FAs, more often have favorable mammographic patterns relative to breast cancer risk than do British women whose diet is high in fat, but low in n-3 FAs (Gravelle et al., 1991). 7.1.2. Atypical hyperplasia and carcinoma in situ Benign breast disease with hyperproliferation of the ductal epithelium, particularly if the epithelial cells exhibit morphological abnormalities, is associated with increased risk of invasive breast cancer (Dupont & Page, 1985; Page et al., 1988). In its severest form, this atypical hyperplasia merges into ductal carcinoma in situ, and the distinction between the two conditions may be difficult on histological examination. Ductal carcinoma in situ is the common form of noninvasive breast cancer. It is characterized by the proliferation of cancerous epithelial cells, but without invasion through the basement membrane into the surrounding stroma. Several studies assessed the risk of developing invasive breast cancer from a pre-existing in situ lesion and estimated that this occurs in 1030% of cases, with a latency of 1020 years (Page et al., 1995; Hetelekidis et al., 1995). There is a significant interaction between the presence of atypical hyperplasia and a family history of breast cancer involving first degree relatives (mother, sister, daughters). In the study by Dupont & Page (1985), the risk of developing breast cancer in patients with atypical hyperplasia was 5.3fold that of women with nonproliferative benign breast disease. When a first degree breast cancer family history was also present, this risk increased to approximately 11-fold. One consequence of mammographic screening is that ductal breast carcinoma in situ, which in the past was a relatively uncommon diagnosis, is being recognized with increasing frequency. This has created a new clinical problem because whereas the standard treatment for this condition was radical mastectomy, this is now difficult to justify, given that only a maximum of about one-third of carcinomas in situ will progress to invasive disease and that invasive breast cancer is now generally treated by local resection. There is no reliable means of predicting which cases of breast carcinoma in situ will progress to invasive, potentially life-threatening breast cancer. However, the presence of angiogenesis may be such a predictive marker in both carcinoma in situ and atypical hyperplasia (Heffelfinger et al., 1996; Fregene et al., 1994). 7.1.3. Chemoprevention The risk of invasive breast cancer in patients with carcinoma in situ is sufficiently high to merit the application of some form of chemoprevention. Its presence provided one criterion for entry into the National Surgical Adjuvant Breast and Bowel Projects Breast Cancer Prevention Trial,

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which demonstrated a 49% reduction in breast cancer incidence among women designated as being at high breast cancer risk and randomized to receive tamoxifen compared with those in the placebo-assigned group (Fisher et al., 1998). However, while this trial was discontinued on ethical grounds without assessing the long-term effects on breast cancer mortality, it did show a disturbing increase in the occurrence of endometrial cancer, pulmonary embolism, and deep vein thrombosis (Bruzzi, 1998). Another concern which needs emphasis is that ductal breast carcinomas in situ may be estrogen-independent and so, unresponsive to antiestrogen therapy. Holland et al. (1997) pointed out that more than 50% of ductal carcinomas in situ may be estrogen receptor negative, and showed that estrogen independence was particularly likely in the case of the comedo histological subtype, which, because of the presence of microcalcifications, are identified by mammography. Moreover, it is possible that the suppression of estrogen-dependent cell growth by tamoxifen would select for estrogen-independent cells, and, later, for invasive cancers that are not responsive to antiestrogen therapy. Alternative approaches to chemoprevention are likely to be needed for women who are not considered suitable for, or who reject, antiestrogen administration. At present, we believe that women with mammographic densities predictive of enhanced breast cancer risk and, perhaps, younger women with atypical hyperplasia or carcinoma in situ fall into this category. Omega-3 FA supplementation for breast cancer chemoprevention is strongly supported by the epidemiologic and experimental studies described in this review. Also, as discussed in Section 1, the n-3 FAs have attracted attention as potential interventions for a number of non-neoplastic diseases, and in all of these diverse situations, a notable feature was a lack of serious side effects. 7.1.4. Omega-3 fatty acid supplementation There are no published studies in which n-3FA has been fed to women with the intention of achieving a reduction in breast cancer risk. There are, however, many reports of cardiovascular disease clinical trials in which n-3 FA supplementation, usually as a fish oil preparation, has been evaluated, albeit largely in male subjects. Knapp (1997) has discussed the variation in FA composition and other components of fish oils and how this complicates the interpretation of clinical trial outcomes. He points out, however, that the few studies that have been performed using oils of defined composition, or pure n-3 FA ethyl esters, confirm much of the data acquired using cruder preparations. In an earlier commentary, Leaf (1992) concluded that the potential benefits of n-3 FA in the prevention of cardiovascular disease were evident, and that there was no cause for concern about postulated adverse side effects such as bleeding due to the suppression of TXA2 production and of platelet aggregation. He did, however, note that the co-administration of vitamin E should be considered in order to prevent the formation of lipid peroxidation products.

Knapp (1997) agreed that although hematological testing may show the bleeding time to be increased, this effect is usually of modest degree, and he noted that in no case has serious bleeding occurred. The possibility that an n-3 FAinduced bleeding tendency might become clinically significant during a surgical procedure has been considered. However, this was not a problem in patients undergoing cardiac surgical procedures, even with n-3 FA doses as high as 8 g/ day (Knapp, 1997). A trial reported by Saynor & Gillott (1992) merits special attention because of its size and duration. It was a 7-year dietary n-3 FA intervention in which the changes in blood lipids and fibrinogen were assessed in 304 male and 61 female patients at risk of fatal myocardial infarction. The fish oil preparation MaxEPA was administered to provide a daily supplement of 3.6 g of EPA for the first year and 1.8 g/day for the duration of the trial. The desired reductions in blood lipid profiles and fibrinogen concentrations were obtained without any detectable adverse effects. A suppressive effect of dietary n-3 FA supplementation on wound healing needs consideration in the context of breast cancer chemoprevention because the possible need for surgical intervention is always present. Leaf et al. (1994) reported that this did not appear to be a problem in the trial of fish oil, 8 g/day n-3 FA, to prevent restenosis after angioplasty, although here the presurgical n-3 FA supplementation lasted only 1214 days. Experiments with a standardized animal model did indicate that dietary n-3 FA administration may result in some impairment in wound healing (Albina et al., 1993), but the clinical relevance of this observation is uncertain.

7.1.5. Dietary modification targeting other fatty acids: The n-3:n-6 fatty acid ratio Most of the cardiovascular disease n-3 FA supplementation trials did not attempt to modify the consumption of other fat components, and specifically did not seek to reduce the intake of n-6 FA. However, there is convincing support for such a combined approach. A study performed by Grnn et al. (1991) showed that while a high intake of n-6 FA reduces the incorporation of dietary n-3 FA into the serum FA, and the platelet phospholipids (Section 2), the amounts of saturated and monounsaturated FAs consumed have no significant effect on n-3 FA incorporation. The comparison here was between diets containing 36 g of n-6 FA per day and diets with only 6 or 4 g of n-6 FA per day. The n-3 FA supplement provided 6 g per day from 10 capsules of Eicomarine 60 (Nycomed, Norway), each of which contained 280 mg of EPA, 260 mg of DHA, and 60 mg of DPA. The n-3:n-6 FA ratios with n-3 FA supplementation were 0.25, 1.23, and l.82. An important point that emerged from this study relates to the effects on LA and AA. In the serum, the accumulation of n-3 FA was at the expense of LA, but not AA. However, in platelets, cells in which there is active and physiologically

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important synthesis of eicosanoids from AA, both LA and AA levels were reduced in the membrane phospholipids. While there do not appear to be any published data on the effect of dietary n-3 FA on tumor phospholipid FA in breast cancer patients, in the nude mouse model, we have demonstrated a reduction in human breast cancer cell phospholipid AA, but not LA, with concomitant reductions in both AAderived COX and LOX products (Rose et al., 1995; J. M. Connolly, R. Mongru, & D. P. Rose, unpublished data.) Bartram et al. (1993, 1995a) performed two human studies in which fish oil supplementation was given in order to suppress rectal epithelial cell proliferation and PGE2 biosynthesis. This was achieved when the dietary n-3:n-6 ratio was 0.4, but not with the same absolute level of fish oil intake and an n-3:n-6 FA ratio of 0.25. Anti et al. (1994) studied patients with sporadic adenomatous colorectal polyps in order to establish an optimal dose of fish oil for achieving a chemopreventive effect. The levels of daily n-3 FA supplementation were 2.5, 5.1, and 7.7 g per day. Comparison was with a placebo-administered group consuming an estimated 0.55 g of n-3 FA per day, 0.4 g of which was LNA. After 30 days, the n-3 FA-supplemented groups showed dose-related increases in their rectal mucosa EPA and DHA levels and corresponding decreases in AA. There were no significant changes in LA content. These changes in rectal mucosal n-3 FA were accompanied by decreased cell proliferation, but this did not differ significantly between the three dose levels. In a second experiment, Anti et al. (1994) compared the low dose, 2.5 g/day, n-3 FA supplementation with a placebo over a 6-month period. As in the first study, all of the patients had elevated mucosal cell proliferative activity before the dietary intervention, and this was reduced by the n-3 FA administration. It is of interest that in the studies reported by Anti et al. (1994), the patients were maintained on their typical Italian diet, which 3-day food intake records showed provided a daily intake of 70 g of fat (29% of the total calories) with 47 g (67%) n-9 FA, essentially all oleic acid (C18:1), 17.5 g (25%) saturated FA, 4.9 g (7%) LA, and 0.55 g (0.6%) n-3 FA. This high level of n-9 FA, derived from olive oil, may be significant because there is evidence that the incorporation of n-3 FA into cell membranes is enhanced by feeding an olive oil-rich diet (Cleland et al., 1992). From this background, we suggest that in North American and Northern European patients, n-3 FA supplementation to favorably impact on potentially precancerous breast lesions be combined with a dietary modification in which the total fat intake is reduced to 2530% of energy intake, and the LA component reduced from the typical Western level of 1520 g/day to 5 g/day, with the difference being substituted by olive oil derived n-9 FA. This recommendation is supported by the results of both epidemiologic and experimental studies that suggest that a high olive oil consumption is associated with reduced breast cancer risk (reviewed by Rose, 1997b).

7.1.6. Intermediate response biomarkers 7.1.6.1. Mammographic densities Ursin et al. (1996) discussed the application of mammographic density as a potentially reversible intermediate marker of breast cancer risk in cancer prevention trials. From an examination of mammograms of the contralateral breast of premenopausal women diagnosed with unilateral breast cancer, and treated postsurgically with radiation, tamoxifen, or chemotherapy, they showed that regression of the putative risk marker could be detected in the tamoxifen-treated group. 7.1.6.2. Cytology and biochemistry of nipple aspirates The cytological examination of exfoliated breast epithelial cells in fluid collected by nipple aspiration provides a noninvasive procedure for acquiring material for pathological evaluation, including the detection of atypical hyperplasia and neoplasia (King et al., 1975, 1983; Sauter et al., 1997). King et al. (1983) reported an encouraging agreement between the cytological findings in nipple aspirates and the histopathological features observed in biopsy material, and a subsequent prospective study by the same group of investigators provided support for the hypothesis that as evidenced in these aspirated cells, hyperplasia and atypia is associated with an increased breast cancer risk (Wrensch et al., 1992). In a single report, it was also suggested that cytological atypia in specimens of breast fluid correlates with the presence of mammographic densities (Lee et al., 1994). While this preliminary study needs to be confirmed, the longitudinal evaluation of nipple aspirates appears to offer a promising intermediate response biomarker during dietary n-3 FA supplementation in women at increased breast cancer risk. Breast fluid obtained by nipple aspiration also contains steroid and protein hormones (Petrakis, 1986; Rose, 1986; Rose et al., 1988) and polypeptide growth factors (Connolly & Rose, 1988; Rose, 1992; Gann et al., 1997), which are considered to reflect the hormonal microenvironment of the ductal epithelium and may prove to be significant biological response markers. High levels of transforming growth factor- have been reported in fluids from patients with proliferative benign breast disease (Rose, 1992). Also, FA concentrations are high in nipple aspirates and are influenced by the dietary lipid composition (Petrakis et al., 1977; Bagga et al., 1994). 7.1.6.3. Urinary estrogen metabolites Given the important role of endogenously produced estrogens in breast cancer etiology, an endocrine-based intermediate response biomarker is particularly attractive. Unfortunately, assays of the principal serum estrogens, estradiol, estrone, and estrone sulfate, or of the biologically active unbound estradiol fraction, lack the precision and specificity and are too variable to provide a marker of risk in the individual woman. In Section 6.3.1.3, we referred to the hypothesis that increased metabolism of estrogen to 16 -hydroxyestrone, and away from the production of the catechol derivative

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2-hydroxyestrone, constitutes a breast cancer risk factor. Enzyme immunoassays have been developed for the two key estrogen metabolites 16 -hydroxyestrone and 2-hydroxyestrone, and the validity of using single (spot) urine samples has been established (Bradlow et al., 1997; Kabat et al., 1997). These methods were used for a case-control study, which demonstrated a strong positive association between the urinary 16 -hydroxyestrone excretion and breast cancer and an inverse one for the ratio of 2-hydroxyestrone to 16 hydroxyestrone (Kabat et al., 1997). However, the relationships were only evident in postmenopausal women. A shift in estrogen metabolism to obtain an increase in 2-hydroxyestrone would be expected to have a favorable influence on breast cancer risk, and in a combination of clinical and experimental studies, it has been shown that this effect may be achieved by inducing cytochrome P450 1A1dependent C2-hydroxylation of estradiol with indole-3-carbinol, a constituent of cruciferous vegetables (Telang et al., 1997). The very preliminary data published by Osborne et al. (1988) provide the only indication that a similar potentially chemopreventive effect can be obtained by dietary n-3 FA supplementation, but this observation certainly merits further examination.

8. Commentary In this review, we chose to focus our attention on the potential that long-chain n-3 FAs exhibit as breast and colon cancer chemopreventive agents. This seemed appropriate because in our view, there is sufficient support for their entry into clinical chemopreventive trials, relevant high-risk groups have been identified, and suitable intermediate response markers exist. Less well developed, but in the forefront of current interest, is the recognition that dietary fat and FAs also influence the biological behavior of prostate cancer cells (Rose & Connolly, 1992; Rose, 1997c). Once again, the mechanisms involve the biosynthesis of eicosanoids by prostate cancer cells (Rose & Connolly, 1991; Ghosh & Myers, 1998). Some epidemiological studies have suggested that n-3 FAs exert a protective effort (Mishina et al., 1985; Ewings & Bowie, 1996; Hebert et al., 1998), and feeding diets containing high levels of n-3 FA suppressed human prostate cancer cell growth in nude mice (Karmali et al., 1987; Rose & Cohen, 1988; Connolly et al., 1997a). One aspect of cell biology that we have scarcely touched upon is the role of the cell membrane. The long-chain n-3 FA can cause increased cell permeability (Jenski et al., 1991), and they may have critical effects on hormone and growth factor function (Cave & Jurkowski, 1984; Jurkowski & Cave, 1985) by altering the composition of lipid bilayers in cellular membranes. However, even here there is evidence for the involvement of the eicosanoids (Dave & Witorsch, 1983; Reichel et al., 1985). Cell membrane fluidity is also associated with the metastatic capacity of tumor cells (Taraboletti et al., 1989), and it has been proposed that the antimetastatic prop-

erties of DHA are ascribable, in part, to its effect on the tumor cell plasma membrane (Zerouga et al., 1997). An immediate decision to be made when designing a dietary n-3 FA cancer chemoprevention clinical trial is the source of the supplement and the specific n-3 FA to be used in order to achieve maximal biological activity. The problems of variation in n-3 FA composition, quality control, stability, and patient acceptance of fish oil preparations were recurring issues, but they have been resolved now that pure n-3 FA preparations and unique sources of high potency are available. This does, however, raise an additional question: Should the n-3 FA dietary supplement consist solely of DHA or EPA, or the two in combination, as exists in the commercially available fish oil concentrates? The two n-3 FAs do have different effects on the plasma lipids (Subbaiah et al., 1993) and lipoproteins (Childs et al., 1990), and in one study, the membrane fluidity of skin fibroblasts was increased when the cellular phospholipids were enriched with DHA, but not with EPA (Brown & Subbaiah, 1994). There is also evidence that DHA may depress phospholipid AA levels and so, indirectly reduce eicosanoid synthesis by both decreasing 6 and 5 desaturation (Section 2) and displacing the AA, whereas EPA only displaces the n-6 FA substrate (Grimsgaard et al., 1997). Nevertheless, as discussed in Section 6.1.3, the limited amount of experimental data that are available does not suggest that DHA and EPA differ significantly in their ability to suppress mammary carcinogenesis and tumor progression, and there is an urgent need to resolve this issue. We are now at a stage in our knowledge of n-3 FAs and cancer when we can consider the translation of our efforts from the laboratory to the clinic. Clinical trials should focus on women at increased breast cancer risk, those who have received surgical treatment for their breast cancer (secondary prevention), and, perhaps, patients with advanced disease. Because the basic mechanisms for therapeutic efficacy do not involve the reproductive endocrine system, an n-3 FA intervention would complement rather than compete with antiestrogen-based chemoprevention and adjuvant therapy. Moreover, the experimental evidence that n-3 FA may enhance the antitumor activity of cytotoxic drugs (Burns et al., 1988; Tsai et al., 1997) suggests that they merit a place in future trials of combination therapy. In colon cancer, there is currently a high level of interest in selective COX-2 inhibitors as chemopreventive agents. However, the likely involvement of products of LOX-mediated AA metabolism in colon carcinogenesis, tumor-associated angiogenesis, and cancer progression suggests that again there is a place for combined treatment with n-3 FA.

Acknowledgments We thank Ms. Roz Alexander for her assistance in preparing the manuscript. Our own work has been supported by NIH grant CA-53124, grants CN-100 and RPG-93-003-04-

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