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Chemopreventive Effects of Omega-3 Fatty Acids Joanne R. Lupton, PhD and Robert S. Chapkin, PhD CONTENTS INTRODUCTION THE ESSENTIAL FATTY ACIDS (18:2N−6) AND (18:3N−3) CHEMOPREVENTION BY OMEGA-3 FATTY ACIDS TREATMENT OF CANCER WITH OMEGA-3 FATTY ACIDS CONCLUSION REFERENCES 1. INTRODUCTION

Evidence increasingly suggests that 1ω−3 fatty acids, particularly eicosapentaenoic acid (20:5n−3) (EPA) and docosahexaenoic acid (22:6n−3) (DHA) are protective against cancer, and the data is strongest for breast and colon cancer. These protective effects are mediated by a variety of different mechanisms, including the incorporation of n−3 fatty acids into cell membranes, which changes membrane fluidity, may affect the association of proteins within cell membranes, and/or may initiate different signal-transduction processes. Effects of n−3 fatty acids on eicosanoid synthesis are well documented, although it is not yet determined which products of lipoxygenase (LOX) or cyclooxygenase (COX) are responsible for the observed effects and the specific role of EPA as compared to DHA on these pathways. Omega-3 fatty acids have also been shown to decrease cell proliferation and/or increase apoptosis during the tumorigenic process. Of interest is lipid peroxidation and the role it may play in initiating apoptosis. The role of n−3 fatty acids in the generation of new blood vessels is significant in both tumor growth and metastases, and these fatty acids now appear to play a role in modulating angiogenesis. With new methodology available to document changes in gene expression as a function of n−3 administration, it seems to be the right time to test hypotheses in humans that have previously been explored in vitro or in animals. It has not been determined whether the beneficial effects of n−3 fatty acids are dependent upon a specific amount of these fatty acids or on their ratio to n−6 fatty acids.

2. THE ESSENTIAL FATTY ACIDS (18:2N–6) AND (18:3N–3) 2.1. Biochemistry and Food Sources α-Linolenic acid (18:3n−3) and linoleic acid (18:2n−6) are considered to be the two essential fatty acids, which cannot be synthesized in the body and thus must be obtained from the diet. The major polyunsaturated fatty acid (PUFA) in most diets is linoleic acid (18:2n−6), found in vegetable seeds and oils such as those from corn, soybean, safflower, and sunflower. α-Linolenic acid (18:3n−3) is the primary n−3 fatty acid and, is found in soybean and canola oils (as well as in linseed, rapeseed, walnut, and blackcurrant oils) and in dark green leafy plants (1). Deep, cold-water fatty fish such as herring, sardines, salmon, and tuna are rich sources of EPA (20:5n−3) and DHA (22:6n−3) (2), which are incorporated into the fatty acids of these fish from the plankton and algae on which they feed. The relative amounts of EPA and DHA contained in fish oils are highly variable across species (3). Both α-linolenic acid and linoleic acid can be metabolized to longer-chain fatty acids. For example, α-linolenic acid can be elongated and desaturated to EPA (20:5n−3) and DHA (22:6n−3), and linoleic acid is the precursor of arachidonic acid (AA) (4). However, the efficiency of conversion of α-linolenic acid to the longer-chain fatty acids in the n−3 family is not optimal. A recent study addressing in vivo metabolism of n−3 fatty acids using isotope tracer methodology in healthy humans determined that only

From: Cancer Chemoprevention, Volume 1: Promising Cancer Chemoprevention Agents Edited by: G. J. Kelloff, E. T. Hawk, and C. C. Sigman © Humana Press Inc., Totowa, NJ

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about 0.2% of plasma 18:3n−3 was used for biosynthesis of 20:5n−3. In contrast, the synthesis of 22:5n−3 and 22:6n−3 from their immediate substrate precursors was highly effective. The authors concluded that dietary 18:3n−3 may be an inadequate substrate for 22:5n−3 synthesis, even under dietary conditions intended to enhance long-chain PUFA production (5). The inefficiency of the conversion of 18:3n−3 to 20 5n−3 indicates that the biosynthesis of long-chain n−3 PUFA from α-linolenic acid is limited in healthy individuals. This is significant because the primary source of 20:5n−3 and 22:6n−3 is marine fish (2), yet Americans typically do not eat high amounts of these rich sources of EPA and DHA (6). This means, as noted by Pawlosky et al., (5) that the major source of EPA and DHA for most Americans may be its production from α-linolenic acid (a highly ineffective process).

2.2.1. METABOLIC AND FUNCTIONAL DIFFERENCES BETWEEN SUBCLASSES OF N–3 FATTY ACIDS (18:3N–3) (20:5N–3) (22:6N–3) EPA and DHA are known to have different effects on plasma lipids (12). They also have different effects on eicosanoid production; EPA is a competitive inhibitor of both the COX and LOX pathways (13), and DHA only inhibits the COX pathway (14). Their roles in cell membranes also differ. DHA reduces membrane cholesterol content and increases membrane fluidity, an effect that is not observed with EPA (15). This may result in differential effects on tumorigenesis. For example, Kafrawy et al. (16) measured tumor cell death as a function of the incorporation of different fatty acids in different positions and in different phospholipids, and found that DHA in phosphatidylcholine is cytotoxic to T27A tumor cells, whereas EPA and α-linolenic acid did not have this effect (16).

2.2. Metabolic and Functional Differences Between n–6 and n–3 Fatty Acids

2.3. Ratio of n–6 to n–3 Fatty Acids

An increased intake of PUFA has been promoted as being “heart healthy,” as PUFA tend to lower blood cholesterol values compared to saturated fats (7), and high blood cholesterol values are considered a risk factor for coronary heart disease. However, the distinction between the major subclasses of PUFA (n−3 and n−6) is often lost in these recommendations. Clearly, with respect to their effects on cancer development (as described in Subheading 3), the two subclasses have very different effects. These two classes of PUFAs are biochemically and functionally distinct and have different physiological functions. Competition exists between the n−3 and n−6 fatty acids for the ∆ 5 and ∆ 6 desaturases, and the n−3 fatty acids have greater affinities for these enzymes. Thus, increasing the dietary intake of α-linolenic acid, EPA, or DHA may reduce the desaturation of linoleic acid and therefore the synthesis of AA (4,8,9). Also, n−3 fatty acids may inhibit the uptake of other fatty acids into cells. In a series of studies, Sauer et al. examined the effects of n−6 and n−3 fatty acids on the growth of a transplantable rat tumor, finding a direct relationship between the rate of linoleic acid uptake and tumor growth in vivo (10), which a later study from the same laboratory found could be inhibited by the addition of α-linolenic acid, EPA, or DHA to arterial blood during perfusion of the hepatoma, perfusion also inhibited tumor linoleic acid uptake (11).

Average linoleic acid intake in the United States has risen from approx 3% of energy in the 1950s to 6–7% in the 1990s (17). Simopoulos has noted that the 1950s diet contained small but approximately equal amounts of n−6 and n−3 fatty acids, in contrast to higher amounts of n−6 fatty acids that are presently consumed (3). The ratio of n−6 to n−3 fatty acids in most Western diets today is considered to be too high, with a ratio of 10–20× more n−6 than n−3 PUFAs (18,19). The rationale for lowering the ratio of n−6 to n−3 comes predominantly from studies on fatty acid intake and various cancers—e.g., which have found an increased incidence of breast cancer and colon cancer in the Japanese, which correlates with a decrease in the n− 3:n−6 fatty acid ratio. However, from a meta-analysis of linoleic acid intake and cancer risk (20), the authors concluded: “It seems unlikely that a high intake of linoleic acid substantially raises the risks of breast, colorectal, or prostate cancer in humans.” Others have also questioned whether or not n−6 fatty acids such as linoleic acid and AA are actually negative dietary factors (21). However, 18:2n−6 does promote colonic tumors in a rat multiorgan carcinogenesis model (22). A compromise in recommendations for fatty acid intake centers around monounsaturated fatty acids (n−9). The Mediterranean diet (high in olive oil; 18:1n−9) is believed to be both cardioprotective and chemoprotective. In the Lyon Diet Heart Study (23), investigators compared cancer rates among patients randomized to either a Mediterranean-type diet or a control diet close

Chapter 39 / Omega-3 Fatty Acids to the Step 1 American Heart Association Prudent Diet. The Mediterranean diet produced a healthier profile than the American Heart Association Prudent Diet; the reduction in risk for Mediterranean diet subjects compared to those on the American Heart Association diet was 56% for total deaths (p = 0.03) and 61% (p = 0.05) for cancers. However, this does not necessarily mean that n−9 fatty acids are more protective than n−6 fatty acids, as omega-3 fatty acids were higher (p < 0.001) and omega-6 fatty acids were lower (p < 0.001) in the Mediterranean diet compared to the American Heart Association Prudent Diet.

3. CHEMOPREVENTION BY OMEGA-3 FATTY ACIDS 3.1. General Epidemiological evidence suggests that individuals with diets rich in fish containing high levels of n−3 fatty acids have a low incidence of cancer in general, and of breast and colon cancer in particular (24–26). There are some excellent previously published reviews on the relationship of lipids to cancer (27–33).

3.2. Breast/Mammary Cancer 3.2.1. EPIDEMIOLOGICAL STUDIES Ecological studies comparing the incidence of breast cancer and mortality rates with fish consumption have generally found an inverse association between the percentage of calories from fish and breast cancer rates (24). Sasaki et al. (34) analyzed data from 30 countries and found a significant negative correlation between fish intake and the incidence of breast cancer. In this same analysis, using the variable of dietary animal fat minus fish fat intake, a highly significant positive relationship with breast cancer mortality rates for women over 50 yr of age was detected (34). It has long been known that native Greenland Eskimos, whose diet is very high in fish and aquatic mammals and thus very rich in n−3 fatty acids (35), have a very low risk for cancer in general and breast cancer in particular (2,36), despite a high total fat intake. Data from a number of case-control studies also suggest a protective effect of diets high in fish against the risk of breast cancer (37–40), but this is not true of all such studies (41–44). The importance of separating out n−3 fatty acids from n−6 fatty acids is illustrated by Boyd et al. (45), who evaluated the relationship between total PUFA intake and relative risk of breast

593 cancer in nine case-control studies published before 1993. They calculated a combined relative risk of 0.92 from the nine case-control studies; two showed a lower risk of breast cancer with higher PUFA intake, and the other seven studies showed no significant effect. As some have observed (33), the ratio of n−6 to n−3 fatty acids may be more important than the amount of n−3 fatty acids per se. In support of this hypothesis, a casecontrol study (46) and a cohort study (47) indicated that breast cancer patients have a lower proportion of n−6 PUFAs in their red blood cell (RBC) membranes (46) and serum phospholipids (47), but no association was found between levels of serum phospholipid n−3 PUFAs (47) and the risk of the disease. In contrast, Pala et al. (48) analyzed the association between the risk of breast cancer and fatty acid composition of erythrocyte membranes in 4,052 healthy postmenopausal women who resided in northern Italy and were enrolled in the Hormone and Diet Etiology Study of Breast Cancer (ORDET) (49). They found that DHA, inversely associated with the risk of breast cancer, was the only fatty acid associated with fish consumption. Although it has been not confirmed that the fatty acid composition of erythrocyte membranes is similar to that of breast tissue, it has been shown to be correlated in a number of animal studies (50,51). Further confirmation for the importance of the n−6 to n−3 fatty acid ratio comes from a study in which the fatty acid content of adipose tissue in postmenopausal breast cancer cases and controls from five European countries in the European Community Multicenter Study on Antioxidants, Myocardial Infarction, and Cancer (EURAMIC) was used to test the hypothesis that n−3 fatty acids inhibit breast cancer with the degree of inhibition depending on background levels of n−6 fatty acids (52). The level of n−3 fatty acids in adipose tissue was not consistently associated with breast cancer; however, the ratio of n−3 fatty acids to total n−6 showed an inverse association with breast cancer in four of five centers in this trial (52). More recently (53), the authors examined fatty acid composition in adipose tissue from 241 patients with invasive, nonmetastatic breast carcinoma and 88 patients with benign breast disease in Tours, France. They found inverse associations between the risk of breast cancer and n−3 fatty acid levels in breast adipose tissue. Women in the highest tertile of the long-chain n− 3/total n−6 ratio had an odds ratio of 0.33 compared to women in the lowest tertile (p = 0.0002). The authors concluded that their data suggest a protective effect of

594 n−3 fatty acids on the risk of breast cancer and support the hypothesis that the balance between n−3 and n−6 fatty acids plays a role in breast cancer (53). 3.2.2. CLINICAL STUDIES Although the studies by Zaridze et al. (46), Vatten et al. (47), Pala et al. (48), and Berrino et al. (49) tested the relationship between a biochemical parameter (fatty acid composition of erythrocyte membranes) and the incidence of breast cancer and may thus be considered epidemiological, we consider them clinical studies because of the biochemical assessment. Similarly, Simonsen et al. (52) and Maillard et al. (53) measured fatty acid composition of adipose tissue and related these data to the incidence of breast cancer. Again, these studies could be considered preclinical in nature. However, there are no clinical intervention trials in which omega-3 fatty acids are provided in the diet and measurements of the incidence of breast cancer are evaluated. 3.2.3. EXPERIMENTAL STUDIES Although at first all PUFAs were believed to promote experimentally induced mammary cancer, n−3 fatty acids have been shown to delay their development, whereas n−6 fatty acids have the opposite effect (54–58, reviewed in 59,60). For example, Ip et al. observed a direct positive relationship between the concentration of dietary linoleate (0.5–4.4%) and breast tumor proliferation (61). Karmali (58) was one of the first to show that n−3 fatty acids, but not n−6 fatty acids, were protective against rodent mammary tumors. Several studies after those by Karmali showed that diets high in n−6 fatty acids enhance breast and colon tumorigenesis in rodents, whereas fish oil that is high in n−3 PUFAs reduces carcinogenesis (29). With respect to individual fatty acids and mammary tumor development, both DHA and EPA have proven to be protective in a number of studies. For example, DHA was provided as a triglyceride to female nude mice, injected in their mammary fat pads with MDA-MB-231 cells (62). The addition of 4% DHA to a 4% linoleic acid-containing diet reduced the tumor growth rate. Tumor weights were also reduced in the 4% DHA-fed mice compared with the 4% linoleic acid control group (p = 0.02) (62). A separate study investigated the effects of linoleic acid, EPA, and DHA on the growth, metastasis, and cell proliferation of a murine mammary tumor transplanted into mice. Growth of the primary tumor, the number of metastatic tumors in the lung, and cell proliferation of

Lupton and Chapkin the tumor were significantly inhibited in EPA and DHA groups compared to control and/or linoleic acid groups (63). In contrast, an in vitro study by Abdidezfuli et al. that used EPA and DHA in the medium of MCF-7 human breast cancer cells found that only EPA inhibited their proliferation. In a number of instances, in vitro studies do not reflect the effects found in vivo. Although it did not assess the effect of DHA or EPA on mammary tumor incidence, one study (64) determined their effect on the recurrence and/or metastases of mammary tumors excised from female nude mice. Although there were no differences in the incidence of local recurrence between groups, EPA and DHA both inhibited the development of lung metastases. As pioneers in the area of experimental mammary cancer and fatty acids, Rose et al. (65) have compared the effects of diets containing linoleic acid, EPA, and DHA on the growth and metastasis of human breast cancer cells in the nude mouse model. They have also determined how such effects relate to observed changes in the chemical content of tumor fatty acids and eicosanoid production. Results showed that growth of primary tumors was retarded in mice fed diets supplemented with EPA and DHA compared with those fed linoleic acid (65). Fay et al. (66) conducted a metaanalysis on the incidence of mammary tumors and fat intake from 97 reports of experiments involving more than 12,800 mice and rats. Despite numerous positive effects of n−3 fatty acids against mammary cancer cited here, results of their analysis showed that n−6 PUFAs have a strong promoting effect on mammary cancer, whereas n−3 PUFAs have a small and statistically nonsignificant protective effect. 3.2.4. POTENTIAL MECHANISMS 3.2.4.1. Overview There is a growing body of literature on how n-3 fatty acids may ultimately decrease mammary carcinogenesis. These fatty acids may incorporate into cell membranes and differentially affect signaling pathways, which in turn can up- or downregulate oncogenes and tumor suppressor genes, activate cell death pathways, or inactivate events that lead to an increase in cell proliferation. In addition, these long-chain PUFAs undergo lipid peroxidation, resulting in oxidative products that may be cytotoxic to tumor cells. Omega-3 fatty acids may inhibit COX activity and depress synthesis of certain prostaglandins (PG), known stimulants to cell proliferation. Examples of each of these follow.

Chapter 39 / Omega-3 Fatty Acids 3.2.4.2. Alterations in Signaling Pathways Both the protein kinase C (PKC) and protein kinase A (PKA) signaling pathways have been shown to be affected by administration of n–3 fatty acids. For example, the human breast cancer cell line MDA-MB-231 exposed to physiological concentrations of EPA and DHA significantly decreased expression of the RIa regulatory subunit of PKA and PKCa isozyme (67), which are capable of modulating cell cytokinetics. n–3 Fatty acids may also mediate the development of mammary cancer through their effect on the epidermal growth-factor receptor (EGFR) mitogen-activated PK signal-transduction cascade (reviewed in 68). The effectiveness of n–3 fatty acids to inhibit growth of human breast cancer cells may depend upon expression of a mammary-derived growth inhibitor (MRG) (a DHA-selective fatty acid binding protein). In one study, MRG-transfected cells or MRG-protein treated cells were more sensitive to DHAinduced growth inhibition than MRG-negative or untreated control cells (69). Another pathway that may be affected by n–3 fatty acids is production of mevalonate, an intermediate in cholesterol biosynthesis. Mevalonate production was shown to be decreased in mammary glands of fish oil-fed rats (70). This may be significant because inhibitors of cholesterol synthesis (such as isoprenoids) are also inhibitors of tumor cell growth. The precise mechanism by which this inhibition of the key regulatory step in cholesterol biosynthesis (e.g., production of mevalonate) may be antitumorigenic is unknown, but could include such diverse outcomes as changes in membrane fluidity affecting signaling pathways to a downregulation of Ras farnesylation, which could result in a lower activity of the Ras protein that is often overexpressed during tumorigenesis. Omega-3 fatty acids may also have a direct effect on gene expression (71). Thoennes et al. (71) tested a variety of fatty acids for their effects on the transcriptional activity of peroxisome proliferator-activated receptor γ (PPARγ), in human breast cancer cell lines. Omega-3 fatty acids inhibited transactivation of PPARγ to levels below controls, and omega-6 fatty acids stimulated the activity of the transcriptional reporter (71). Although not specific to mammary tumorigenesis, Palakurthi et al. (72) have elucidated a relationship between EPA-induced depletion of intracellular calcium stores and the inhibition of cell proliferation and tumor growth. A series of experiments showed that EPA may release calcium from inositol-tris-1,4,5-phosphatesensitive calcium pools and prevent the refilling of these pools. This in turn triggers PKR-mediated

595 phosphorylation of eIF2α, with the ultimate effect of cell-cycle arrest in G1. 3.2.4.3. Alterations in Eicosanoid Synthesis The literature on breast carcinoma and n−3 fatty acids is reviewed in Noguchi et al. (73). A number of studies in both cell lines and rodents have shown a link between downregulation of eicosanoid synthesis and protection against mammary tumor growth. In one study, rats fed a menhaden oil diet and treated with 7,12-dimethylbenz[a]anthracene (DMBA) had both lower mammary tumor incidence and lower levels of PGE2, 6-ketoPGF1α, and LTB4 than their corn oil-fed counterparts (74). Which eicosanoids may be responsible for the enhanced tumorigenesis seen with corn oil-supplemented rats has not been determined, but LOX rather than COX inhibitors have proven to be effective in inhibiting human breast cancer cell growth both in vivo (75) and in vitro (76,77). For example, in one study, inhibition of DMBA-induced rat mammary tumorigenesis was associated with both reduction of tumor LTB4 production and inhibition of 5-LOX (56). Treatment of W256 cells with LOX, but not COX, inhibitors induced apoptosis that was partially reversed by exogenous 12-hydroxyeicosatetraenoic acid (HETE) or 15-HETE (78). In fact, it is hypothesized that promotive effects of linoleic acid (high in corn oil) are really the result of its conversion to 13-hydroxyoctadecadienoic acid (HODE) by LOX rather than to linoleic acid itself (79). This hypothesis is supported by findings that 14C linoleic acid added to arterial blood is recovered as 14C13-HODE in tumor venous blood (10); the addition of a LOX inhibitor to drinking water inhibited formation of 13-HODE in the tumor and caused regression of growth, but had no effect on linoleic acid uptake. However, other studies have shown that PGE2 concentrations are elevated in mammary tumors (80,81); fish oil has been shown to decrease PGE2 levels by 90% in human mammary tumor MX-1 in the athymic nude mouse (82). Similarly, inhibitors of COX (e.g., indomethacin) decrease the growth of tumors (83,84). Also unresolved is the mechanism by which downregulation of either COX-2 or 12-LOX expression results in a lower incidence of mammary tumors. One connection appears to be through downregulation of apoptosis, when 12-LOX or COX-2 is overexpressed. For example, treatment of nude mice with MCF-7 breast cancer cells that overexpress 12-LOX resulted in increased tumor cell growth and inhibition of apop-

596 tosis (85). Rose summarizes these data accordingly: “With this background, it seems reasonable to postulate that dietary n−3 fatty acids, which inhibit 12HETE, 15-HETE, and PGE2 production by human breast cancer cells growing as solid tumors in nude mice, may exert their suppressive effect on tumormass acquisition, at least in part, by activation of the apoptotic pathway” (86). 3.2.4.4. Alterations in Cell Kinetics Both EPA and DHA inhibit growth of human breast cancer cell lines in vitro, although DHA has been more effective than EPA in suppressing growth of certain estrogen-independent breast cancer cells (76). With respect to an estrogendependent breast cancer cell line, EPA, DHA, and alinolenic acid all inhibited growth, although a-linolenic acid did so to a lesser degree than the other two longerchain n–3 fatty acids (87). There appears to be a fine line between the inhibition of growth by non-cytotoxic means seen at low concentrations of n–3 fatty acids, and cytotoxic effects seen at higher levels (88). The growth of human breast cancer cell lines in vitro, or mammary tumors in vivo, is dependent upon an increase in cell proliferation and/or a decrease in apoptosis. One study addressed the kinetics of cell proliferation in vivo using a double label (BrdU labeling and DNA analysis by flow cytometry) (89). They found a longer S-phase duration (15.0 vs 9.1 h, p < 0.001) in cells from fish oil-fed rats than in those from safflower oil-fed rats, accounting for the difference in tumor growth rates. A study in nude mice with the MDA-MB-231 human breast cancer cell line growing as solid tumors showed reduced tumor growth, decreased cell proliferation, and increased apoptosis when 4% DHA was added to a 20% fat diet containing 4% linoleic acid (90). The relationship of n−3 fatty acids to cell kinetics is reviewed in (59,60). 3.2.4.5. Lipid Peroxidation and Cytostatic and/or Cytotoxic Effects Oxidation of n–3 fatty acids can produce a number of products that may have cytostatic or cytotoxic effects on tumor cells (reviewed in 91,92). In one study, female athymic nude mice were implanted with human breast carcinoma MDA-MB-231 (93) and provided with diets containing various amounts of corn oil and fish oil (plus or minus antioxidants). Tumor volume and thiobarbituric acid reactive substances (TBARS) were evaluated after 6–8 wk. Tumor growth was suppressed in mice that received fish oil diets without antioxidants in a dose-dependent manner. However, the addition of antioxidants to the fish oil reversed the benefits of fish oil feeding (93). The level

Lupton and Chapkin of increase in TBARS was directly related to the increase in dietary fish oil, suggesting that lipid peroxidation products were responsible for the depression in tumor growth. The effect of n–3 fatty acids on mammary tumorigenesis as a function of lipid peroxidation has been reviewed by Welsch (94). The mechanism(s) by which these lipid peroxidation products of n–3 fatty acids may inhibit breast cancer cell growth is unclear, and could be a direct or indirect cytotoxic effect that initiates an apoptotic cascade. 3.2.4.6. Effects on Hormones Surprisingly, there is little information on the effect of n−3 fatty acids on hormones associated with breast cancer, although steroid hormones are known to play a role in breast cancer development. In one study (95), women with either a predisposition to breast cancer or a carcinoma in situ were given fish oil, which reduced the 16αhydroxylation of estradiol. This is significant because studies in women have shown that elevated 16αhydroxylation of estradiol may be a biomarker for an increased risk of breast cancer (96). 3.2.4.7. Effects on Angiogenesis In order for a tumor to grow and for cancers to metastasize, the formation of new blood vessels is required. Thus, inhibition of neovascularization is considered to be an important target for chemoprevention and/or cancer therapy. High angiogenic activity was seen in athymic nude mice injected with MCF-7 human breast carcinoma cells that stably overexpressed 12-LOX (and secreting high levels of 12-HETE) (85). In contrast, a number of factors known to promote blood vessel growth are also known to be downregulated by n–3 fatty acids. Examples of these factors include COX and LOX products—e.g., PGs (97) and 12-HETE (98). This suggests a role for n–3 fatty acids as antiangiogenic agents (99).

3.3. Colon Cancer 3.3.1. EPIDEMIOLOGICAL STUDIES As noted in Subheading 3.2.1., it has long been known that native Alaskan and Greenland Eskimos, whose consumption of 20:5n−3 and 22:6n−3 fatty acids is higher than that of other North Americans (2,35,100), have a very low risk for cancer in general and breast and colon cancer in particular (2,36,35), despite a high total fat intake. Also, fish consumption in 24 European countries was found to be inversely related to colorectal cancer (CRC) in males, although a similar trend for females was not significant (25). Interestingly, when the data from these 24 countries were reanalyzed to consider both n−3 and n−6 fatty acid consumption into

Chapter 39 / Omega-3 Fatty Acids account, high fish intake had a protective effect relative to sources of n−6 fatty acids for both colon and breast cancer (26). This suggests, as noted in Subheading 2.3., that the ratio of n−6 to n−3 fatty acids rather than the absolute amounts of either of these classes of PUFAs may determine their protective effect. An epidemiological study on diet and colon cancer from South Africa (101) is illuminating for a variety of reasons. This study compared 101 men and women with a low incidence of colon cancer, who fished for their livelihood on the west coast of South Africa, to 99 age- and sex-matched urban Cape Town inhabitants with a higher rate of this disease. The daily intake of n−3 fatty acids was more than 5 × higher in the fishing population than in the urban Cape Town inhabitants (p < 0.0001). Surprisingly, despite the lower incidence of colon cancer, a higher proportion of those who fished were smokers and had hypertension, and they consumed fewer fruits and vegetables and fiber than did the urban population. Of particular interest is the finding that none of those who fished took vitamin supplements, compared to one-third of the urban Cape Town population who took supplements. This may be significant because of the ability of peroxidation products of n−3 fatty acids to either initiate apoptotic removal of DNA damaged cells and/or to delete DNA damaged cells by cytotoxic mechanisms (91,92) as noted in Subheading 3.2.4.5. Support for the potentially negative effect of antioxidant supplementation on tumor development was from the previously discussed study in female athymic nude mice implanted with human breast carcinoma MDAMB-231 and fed diets containing various amounts of corn oil and fish oil (plus or minus antioxidants). Adding antioxidants to the fish oil reversed the benefits of fish-oil feeding (93). This is consistent with the South African fishing community that did not consume vitamin supplements and yet had a low incidence of colon cancer, in contrast to the urban Cape Town inhabitants with a higher rate of this disease (one-third of these did consume such supplements). Similar to the effects observed in breast cancer, a stepwise increase in EPA concentrations in plasma phospholipids and in colonic mucosal phospholipids was associated with a stepwise reduction in colon tumorigenesis assessed in patients as CRC, sporadic adenoma, or a normal colon. A stepwise increase in EPA concentrations was seen from the most advanced colon cancer to the most benign adenoma (p = 0.009) (102).

597 3.3.2. CLINICAL STUDIES Clinical intervention trials on n−3 fatty acid consumption and intermediate markers for colon cancer are limited. Ulcerative colitis (UC) is considered to be a risk factor for colon cancer, and thus interventions that reduce the severity of this disease may be considered protective against later colon cancer development. In one such study (103), patients were randomized into two groups (either fish oil or sunflower oil supplementation) for 6 mo. At the end of 6 mo, the fish oil-supplemented group had a significant reduction in sigmoidoscopic and histological scores for UC compared to controls (103). Individuals with sporadic adenomatous colorectal polyps are also considered at increased risk to develop colon cancer. Anti et al. found that humans with sporadic adenomatous colorectal polyps who took fish oil in capsule form for 12 wk had lower indices of cell proliferation in the upper colonic crypt compared to placebo controls (104). In later work, the same group studied patients with sporadic adenomatous colorectal polyps to establish an optimal dose of fish oil for achieving a chemopreventive effect (105). After 30 d, the n−3 fatty acid-supplemented group showed doserelated increases in rectal mucosal EPA and DHA levels. These changes in rectal mucosal n−3 fatty acids were accompanied by decreased cell proliferation, but this effect was not dose-related. This study also compared n−3 fatty acid supplementation with a placebo over a 6-mo period. As in the first study, rectal cellproliferative indices were reduced by n−3 fatty acid supplementation in those with elevated cell proliferation prior to intervention. Unlike breast cancer, colon cancer is affected by the constituents passing through the gastrointestinal (GI) tract. Data are unclear on which fecal bile acids (if any) promote colon cancer, and what the appropriate fecal bacterial content may be. In one study (106), 24 healthy volunteers were supplemented for 4 wk with either fish oil or corn oil. Effects of these diet interventions were investigated with respect to fecal excretion of secondary bile acids, certain neutral sterols, and bacterial enzyme activities. No significant differences were noted for fecal microbial enzyme activities measured, and fecal bile acid excretion was not affected by treatment (107). Changes in rectal cell proliferation have been used as intermediate markers for later tumor development to evaluate whether or not a diet intervention is protective against colon cancer. Diets that decrease rectal cell proliferation are considered to be protective of colon cancer development. Two human studies were performed to

598 determine the effect of fish oil supplementation on rectal cell proliferation and PGE2 biosynthesis (107,108). A decrease in rectal cell proliferation was observed 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 fatty acid ratio of 0.25. This further supports the previously discussed concept, that the ratio of n−6 to n−3 fatty acids is more important in terms of chemoprevention than is the absolute amount of either of these PUFA categories. In a separate study, Bartoli et al. (109) investigated the effect of fish oil supplements in a 30-d clinical trial. They found that rectal cell proliferation was lower in the fish oil group than in the placebo group. 3.3.3. EXPERIMENTAL STUDIES Fish oil high in n−3 fatty acids has been shown to be protective against experimentally induced colon cancer in a large number of studies (110–117). This protective effect of fish oil has been shown to occur at initiation (118) and during promotion (111,112), and with fish oil and n−3 fatty acid supplementation (119). Athymic mice fed high-fat diets rich in coconut oil, olive oil, safflower oil, or fish oil were inoculated with HT29 cells to initiate colon tumor growth (120) and compared to mice fed a low-fat diet. There was no difference between resulting tumor sizes of mice fed fish oil compared to the low-fat diet, yet all other diets resulted in an increase in tumor size. Similarly, other investigators (115) found that a diet high in fish oil (23% by weight) did not enhance colon tumor development in rats injected with azoxymethane (AOM) compared with a diet low in corn oil (5%), whereas a diet high in corn oil did increase tumor incidence. We have found that fish oil-fed rats had higher levels of colonic epithelial cell differentiation and increased apoptosis, with no difference in proliferation compared to corn oil-fed rats (121). Fish oil-fed rats also had a lower incidence of tumors than their corn oil-fed counterparts (112). In one of the few studies that tested the effects of various ratios of n−3 to n−6 fatty acids, Deschner et al. (110) looked at both early and late stages of tumor development in rats injected with AOM. Providing 16% of the total 20.4% fat as fish oil (n−3:n−6 ratio of 1.5) partially suppressed the increase in cell proliferation, which occurred as a function of the carcinogen injection at the early stages of tumorigenesis. Similarly, the occurrence of early dysplastic foci was reduced significantly in AOM-injected animals fed either 16% or 10.2%, but not 4.4%, fish oil. The highest tumor incidence occurred in rats fed 20.4% corn

Lupton and Chapkin oil or 16% corn oil with the lowest (4.4%) level of fish oil. Diets containing 4.4% corn oil without any n−3 fatty acids, or 10.2% or 16% fish oil, resulted in a similarly low incidence of colon tumors. The specific effect of DHA was tested in a study by Takahashi et al. (119). DHA was provided as the ethyl ester by intragastric intubation in single doses 5 × weekly in rats injected with 1,2-dimethylhydrazine, and the development of aberrant crypt foci (ACF) was assessed. DHA treatment reduced the formation of ACF using this experimental paradigm. We have also provided purified n–3 fatty acid ethyl esters to rats in ratios that mimic those found in fish oil (122) in an attempt to determine whether purified fatty acids produce the same physiological effects as intact fish oil. We found that that these fatty acids incorporated similarly into mitochondrial phospholipids, and also had similar effects on mitochondrial membrane potential and caspase 3 activation (an early event in the apoptotic cascade) (122). This suggests that the benefits of fish oil seen in experimental studies are indeed the result of fatty acids, rather than some contaminant of fish oil itself. 3.3.4. POTENTIAL MECHANISMS 3.3.4.1. Overview Most potential mechanisms by which n–3 fatty acids may protect against colon cancer are common to breast cancer—alterations in signaling pathways, eicosanoid synthesis, cell kinetics, lipid peroxidation, effect on hormones, and effect on angiogenesis. In addition, n–3 fatty acids may have a direct effect on lumenal constituents within the GI tract and on colonic microflora. This class of PUFA also uniquely influences the immune system (123–125). As noted previously, n–3 fatty acids accumulate in biological membranes and may initiate signal-transduction processes or modify membrane structure (123), which may also be linked to changes in gene expression. 3.3.4.2. Alterations in Signaling Pathways One of the most important signaling pathways that may be modified during tumorigenesis is ras-mediated. Ras genes code for 21-Kd guanine nucleotide-binding proteins that play an important role in colonic epithelialcell growth, differentiation, and tumor formation (126,127). Singh et al. (128) investigated the effect of various types and amounts of dietary fat on Ras-p21 expression during AOM-induced colon carcinogenesis in rats. They found higher levels of Ras-p21 expression with advancing stages of colon tumorigenesis. However, feeding fish oil inhibited Ras-p21 expression,

Chapter 39 / Omega-3 Fatty Acids and decreased both the incidence and multiplicity of colon tumors. Ras protein is only active in the plasma membrane, and Singh et al. also found that fish oil feeding resulted in lower levels of membrane-bound Ras. Davidson et al. (129), using the rat AOM tumor model, demonstrated that colonic membrane Ras levels were decreased in fish oil- compared to corn oil-fed rats. This is important because prolonged Ras activation could result in a stimulation of cell proliferation (130). Another documented effect of prolonged Ras activation is a reduced susceptibility to apoptosis (131) and another signaling pathway shown to be affected by fish oil feeding is PKC (132). Specific PKC isoforms have different effects in colonocytes (133), and altered PKC isoform signaling appears to be involved in colon cancer development (134–136). A necessary prerequisite to cell proliferation and thus to tumor growth is polyamine synthesis. Ornithine decarboxylase (ODC) is considered to be the rate-limiting enzyme for polyamine biosynthesis, and this inducible enzyme is elevated during colon carcinogenesis (137). Craven and DeRubertis (138) reported an increase in ODC activity and PKC activation in colonic epithelial cells exposed to n−6 fatty acids. In an AOM rat colon cancer study, Reddy and Sugie (117) detected an inverse relationship between ODC activity and the level of menhaden oil in the diet. Investigators from the same laboratory (139) showed that an n−6 fatty acid-rich diet upregulated ODC activity in the colonic mucosa of rats exposed to AOM, whereas an n−3 fatty acid-rich diet resulted in relative suppression of the enzyme. 3.3.4.3. Alterations in Eicosanoid Synthesis Although it is not known how fish oil decreases cell division and enhances apoptosis (Subheading 3.3.4.4.), one hypothesis is that n–3 fatty acids inhibit PG production, which in turn decreases colonic cell proliferation and tumor formation (107,139). COX catalyzes the conversion of AA to PGs, and COX-2 expression is increased in colorectal adenomas and carcinomas compared to normal mucosa (140). Elevated levels of PGs have been observed in human colon carcinomas compared to normal mucosa (141), and in the colonic mucosa of rats injected with AOM during the initiation and postinitiation stages of carcinogenesis (142). Similarly, elevated levels of AA (the precursor for PG synthesis) have been reported in colon tumors from rats injected with the colon carcinogen (143). We have reported that fish oil diets result in lower levels of AA in colonic mucosal phospholipids of rats

599 than corn oil diets (144), and significantly lower levels of colonic mucosal PGE2 with fish oil-supplemented diets compared with those supplemented with corn oil or beef tallow (144). Similarly, Minoura et al. (145) found that partial suppression of rat colon carcinogenesis by EPA was associated with a reduction in PGE2 content in tumors of EPA-supplemented rats. PGE2 levels also have been shown to be lower in colonic biopsies from humans after fish oil consumption compared to corn oil consumption (107). Singh et al. (146) showed that COX-2 expression was positively correlated with the incidence and multiplicity of colon tumors in the rat AOM model, and that enzyme expression and the incidence of tumors were enhanced by feeding a high-fat, n−6 PUFA-rich diet, but suppressed by a high-fat, 20% menhaden oil diet. Corey et al. have reported that DHA is a strong inhibitor of PG but not leukotriene synthesis (147). In an in vitro study, Hussey and Tisdale (148) showed that growth of two murine colon adenocarcinoma cell lines was enhanced by linoleic acid and AA but blocked by a selective LOX inhibitor. Subsequent in vivo studies using a 12-LOX inhibitor provided similar results (149). The protective effect of nonsteroidal antiinflammatory drugs (NSAIDs) on CRC is believed to be mediated in part by inhibition of COX-2 (141,150,151). Epidemiological studies show a protective effect of aspirin ingestion on colon cancer incidence (152). Selective COX-2 inhibitors are also effective in experimental colon cancer chemoprevention (153,154). In a recent study, both EPA and DHA were shown to be potent inhibitors of COX-2-catalyzed PG biosynthesis (155). Rao et al. tracked the effects of diets high in n−3 and n−6 fatty acids on PG and thromboxane formation during different stages of AOM-induced colon tumorigenesis (156). They found that compared to saline injection, AOM treatment increased the formation of PG and thromboxane during the tumorigenic process. In contrast, both a high-fat n− 3 fatty acid-enriched diet and a low-fat corn oil diet resulted in lower amounts of PG and thromboxane B2 than a high-fat corn oil diet (156). The connection between PG synthesis and apoptosis was explored by Tsujii and DuBois (157), who overexpressed COX-2 in epithelial cells, and found that cells that overexpressed COX-2 were resistant to apoptosis induction by butyrate. In a separate study, Shiff et al. (158) reported that the PG synthesis inhibitor sulindac sulfide inhibits proliferation and induces apoptosis in HT-29 colon adenocarcinoma cells.

600 3.3.4.4. Alterations in Cell Kinetics We have shown (121) that fish oil has a greater effect on increasing apoptosis than on decreasing cell proliferation, and that this may be an important mechanism by which fish oil supplementation results in fewer colon tumors than corn oil (112). Inhibition of apoptosis now is believed to be an integral component of the genesis of colorectal adenomas and carcinomas (159). Of course, different signal transduction processes (see subheading 3.3.4.2.) may affect changes in both cell proliferation and apoptosis. In addition, COX-2 expression has been shown to downregulate apoptosis (160) (Subheading 3.3.4.3.). In an attempt to determine changes in gene expression related to cell kinetics as a function of n−3 fatty acids (161), CaCo-2 cells (a colon cancer cell line) were incubated with DHA for 48 h; gene-expression profiles were determined using DNA oligonucleotide arrays. The investigators reported elevated levels of a number of genes associated with apoptosis (e.g., cytochrome C and caspases), and also elevation of a number of growth arrest-specific proteins. Finally, consistent with studies reported in Subheading 3.3.4.3., there was an inactivation of the PG family of genes by DHA treatment. 3.3.4.5. Effect on Angiogenesis As shown previously for breast cancer, a high degree of angiogenic activity in colonic carcinomas has also been associated with a poor prognosis (162). Similarly, microvessel density is higher in aggressively invasive colon cancers than in less invasive cases (163). Nitric oxide (NO) is known to activate COX-2 and regulate PG biosynthesis (164), which in turn affects angiogenesis. One study (114) reports that both inducible and constitutive endothelial nitric oxide synthases (NOS) were elevated in rat colon tumors induced by AOM. Thus, it appears that the downregulation of COX by n−3 fatty acids, in addition to upregulating apoptosis (see Subheading 3.3.4.3.) may also lead to antiangiogenic effects. 3.3.4.6. Effects Specific to the Colon Fish oil feeding in rats has resulted in a different mixed population of colonic microflora relative to corn oil feeding (165). This in turn could result in differential bacterial metabolism in the colon. In one study, for example, a diet high in corn oil compared to one high in fish oil increased activity of the bacterial enzyme 7α-dehydroxylase, which converts primary bile acids to secondary bile acids. Lower levels of secondary bile acids were found with fish oil vs corn oil feeding (166), which may be significant because secondary bile acids are considered to be colon tumor promoters (167). Bartram et al. (168)

Lupton and Chapkin conducted a clinical study in which healthy volunteers were supplemented with either 11 g of fish oil or corn oil, and fecal bile acid excretion was determined. After 4 wk, excretion of lithocholic acid (a secondary bile acid) was lower after n−3 fatty acid than n−6 fatty acid administration, although the difference was not statistically significant.

3.4. Other Cancers 3.4.1. PROSTATE CANCER There is some epidemiological support for a protective effect of n−3 fatty acids on prostate cancer. Clinical trials, generally of short duration, with small numbers of participants, suggest that consumption of diets that are high in omega-3 fatty acids reduce the risk of prostate cancer. For example, in one study, 25 patients with prostate cancer (169) supplemented with 30 g per d of flaxseed (rich in n−3 fatty acids) who consumed 20% or less kilocalories from fat were compared to historic cases. Low fat combined with flaxseed supplementation resulted in lower levels of total testosterone, free androgen, and cell proliferation indices than historic controls. In addition, apoptotic indices were higher in this n−3 supplemented group. In an outpatient clinic-based study of 89 cases of prostate cancer and 38 controls (170), fatty acids in erythrocyte membranes and adipose tissue fatty acids from subcutaneous fat samples were analyzed and used to calculate odds ratios for the association of each fatty acid with prostate cancer. Linoleic acid in erythrocyte membranes was positively associated with prostate cancer (p < 0.04). n−3 Fatty acids had no significant protective effect, yet their sample size was quite small. One study that reported on the effects of diets containing different unsaturated fatty acids on human prostate cancer cell growth in nude mice (171) that consumed menhaden oil found a 30% reduction in tumor growth compared to mice that received corn oil or linseed oil diets (p < 0.001). Of the long chain n−3 fatty acids, it appears that both DHA and EPA inhibited androgen-stimulated cell growth, and prostate-specific antigen (PSA) was reduced by DHA and EPA in a dose-dependent manner (172). The amount of fatty acid administered also appears to be important. For example, in one study (173), EPA at lower concentrations had a promotive effect on human prostate cancer cell line growth. In contrast, EPA at higher concentrations inhibited prostate cell growth.

Chapter 39 / Omega-3 Fatty Acids 3.4.2. LUNG CANCER A study conducted in 36 countries found a significant inverse correlation between fish consumption and lung cancer mortality rates in nine of the 10 time periods studied, but only for men (174). Interestingly, this association of fish consumption with a reduced risk of lung cancer mortality was only observed in countries with high levels of cigarette smoking. Although the reason for this finding is not clear, it is interesting to note that smoking increases reactive oxygen species (ROS), which supports a protective effect of n−3 fatty acids by raising ROS production and perhaps initiating apoptosis through this process. In a separate epidemiological study in Norway (175), the relationship between the incidence of lung cancer and intake of fish and fish products was studied in 25,956 men and 25,496 women. A significantly lower risk for lung cancer was found for cod liver oil supplementation (RR = 0.5). 3.4.3. CERVICAL AND PANCREATIC CANCER These cancers have not been addressed in a concerted way with respect to the effect of fatty acid intake on their occurrence. With cervical cancer, one study examined the effect of linoleic acid, EPA, or DHA on human precancerous cervical keratinocytes immortalized with the oncogenic human papillomavirus (HPV) (176). Results showed that DHA inhibited growth of these cells in a dose-dependent manner to a greater extent than EPA; linoleic acid had no effect. Studies in human pancreatic cancer cell lines showed that EPA arrests cell growth and upregulates apoptosis (177,178).

4. TREATMENT OF CANCER WITH OMEGA-3 FATTY ACIDS 4.1. Inhibition of Metastases and Tumor Growth The growth of a tumor at the original site or at a new site depends on a number of factors, including changes in cell kinetics, angiogenesis, and adhesion properties of tumor cells. n−3 Fatty acids can affect all these factors, as described in detail in the sections on cell kinetics and angiogenesis. The growth of a transplanted Morris hepatocarcinoma was reduced by providing rats with low doses of either EPA or DHA, but these reductions were apparently achieved by different mechanisms (179). EPA reduced cell proliferation of the tumor cells, and DHA upregulated apoptosis. In a separate study (180), DHA-containing liposomes were injected into tumor-bearing mice, and mouse survival was

601 charted. DHA-containing liposomes caused a statistically significant increase in the survival of tumor-bearing mice compared with those containing oleic acid. Treatment with n−3 fatty acids may also alter tumorcell sensitivity to immune cytolysis (181). An in vivo mouse study (182) correlated the incorporation of DHA into T27A leukemia cells grown as an ascites tumor in mice with an increased susceptibility to tumor cytolysis by T-lymphocytes. The adhesion of colon cancer cells to endothelial cells was tested in vitro by growing cells in an n−3 fatty acid-enriched medium. Binding of colon cancer cells to endothelial cells was reduced after incubation (183). Similarly, Connolly and Rose (184) used an in vitro invasion assay system to test the effect of linoleic acid, EPA, and DHA on the invasive capacity of MDA-MB-435 human breast cancer cells. Both n−3 fatty acids inhibited tumor cell invasion, whereas invasion was stimulated by linoleic acid. McCarty (185) suggests that one of the mechanisms by which n−3 fatty acids may exert their antitumor growth and antimetastatic effects is through the downregulation of PKC. According to McCarty, PKC can induce collagenase that aids angiogenesis; n−3 fatty acids reduce PKC. With respect to metastasis, angiogenesis plays an important role (see Subheadings 3.2.4.7. and 3.3.4.5.). Some researchers suggest that COX-2 upregulates the expression of vascular endothelial growth factor (VEGF), which is required for angiogenesis. As described previously, n−3 fatty acids downregulate COX-2 expression. In a recent study (186), COX-2, VEGF, PGE2, and microvessel density were immunohistochemically measured in tumors and adjacent normal mucosa from 31 surgical specimens. Both COX-2 and VEGF were significantly correlated with microvessel density, and COX-2 and VEGF genes were overexpressed in tumor specimens as compared to normal mucosa. Also, PGE2 levels were significantly higher in metastatic tumors than in nonmetastatic ones.

4.2. Immunonutrition A number of papers have appeared in the literature on recovery from surgery (for cancer and other surgical interventions) using normal enteral feeding, standard total parenteral nutrition, and what is called “immunonutrition” with an enteral formula enriched with arginine, omega-3 fatty acids, and RNA (187). In one such study (188), patients who had undergone surgery for GI cancers were provided with a standard diet or the same diet supplemented with glutamine,

602 arginine, and omega-3 fatty acids, within 38 h after surgery. The supplement had a positive effect on postsurgical immunosuppressive and inflammatory responses. Supplementation with n−3 fatty acids has also been shown to improve patient histological scores in UC (considered a risk factor for later colon cancer development) by suppressing immune reactivity (189). In addition, fish oil supplementation has reduced the rate of relapse in patients with Crohn’s disease (190). Diet interventions have differed in their timing and length of intervention. In most instances, immunonutrition has resulted in lower postoperative complications and less severe infectious complications (187,191). However, it has not shown an effect on overall mortality. Supplements of EPA have also been used to reduce the weight loss that is often associated with cancer (192). A recent review (193) summarized whether or not immunonutrition translates into an improvement in clinical outcomes, concluding that immunonutrition may decrease infectious complication rates, but is not associated with an overall mortality advantage. A similar conclusion was reached in a previous meta-analysis (194). A small intervention study (195) investigated the effect of dietary n−3 PUFA on T-cell subsets and natural killer (NK) cells of patients with solid tumors. Twenty (20) patients with solid tumors received 18 g fish oil/d for 40 d. At the end of 40 d, a significant increase in Thelper/T-suppressor cell ratio was seen, because of mainly a decrease in the number of suppressor T cells. The authors concluded that n−3 fatty acids may have a beneficial effect on the already compromised immune systems of patients with solid tumors. Also of interest is reversal of tumor cell drug resistance, as drug resistance has a profoundly negative impact on effective cancer chemotherapy. For example, in one study (196), EPA and DHA were shown to kill tumor cells in vitro and also to increase anticancer drug influx and decrease efflux in tumor cells. Similarly, a separate study (197) found that addition of DHA to cultured lymphoma cells enhanced the toxicity of chemotherapeutic agents. Bougnoux et al. (198) tested the association between levels of fatty acids in breast adipose tissue and tumor response to chemotherapy in 56 patients with an initially localized breast carcinoma. Levels of n−3 PUFA in adipose tissue were higher in the group of patients with complete or partial response to chemotherapy than in patients with no response or with tumor progression (p < 0.004). Specifically, DHA (22:6n−3) was an independent predictor for chemosensitivity. In a double-blind, randomized

Lupton and Chapkin study in dogs treated for lymphoblastic lymphoma with doxorubicin chemotherapy, increasing DHA blood levels were associated with a longer disease-free interval and survival time (199).

5. CONCLUSION Cogent evidence indicates that dietary n−3 PUFAs found in fish oil—e.g., EPA and DHA—confer protection against several forms of cancer, in part by activating apoptosis to enhance deletion of cells, and by suppressing the cell proliferation and gene functions associated with angiogenesis. These data support the contention that dietary n−3 PUFAs have chemoprotective value and should be adopted by health professionals who strive for cancer prevention. Therefore, it is essential to understand precisely how an important dietary constituent modulates cell phenotypes, so that recommendations regarding the health benefits derived from nutritional manipulation can be based on a firm scientific foundation.

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