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Article

Highly Unsaturated (n-3) Fatty Acids, but Not -Linolenic, Conjugated Linoleic or -Linolenic Acids, Reduce Tumorigenesis in Apc^Min/+ Mice¹

Melissa B. Hansen Petrik, Michael F. McEntee^*, Benjamin T. Johnson, Mark G. Obukowicz and Jay Whelan²

Departments of Nutrition and ^* Pathology, University of Tennessee, Knoxville, TN and Discovery Pharmacology, G. D. Searle, c/o Monsanto Company, St. Louis, MO

²To whom correspondence should be addressed.

	ABSTRACT

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We showed previously that dietary eicosapentaenoic acid [EPA, 20:5(n-3)] is antitumorigenic in the Apc^Min/+ mouse, a genetic model of intestinal tumorigenesis. Only a few studies have evaluated the effects of dietary fatty acids, including EPA and docosahexaenoic acid [DHA, 22:6(n-3)], in this animal model and none have evaluated the previously touted antitumorigenicity of -linolenic acid [ALA, 18:3(n-3)], conjugated linoleic acid [CLA, 77% 18:2(n-7)], or -linolenic acid [GLA, 18:3(n-6)]. Stearidonic acid [SDA, 18:4(n-3)], the 6-desaturase product of ALA, which is readily metabolized to EPA, has not been evaluated previously for antitumorigenic efficacy. This study was undertaken to evaluate the antitumorigenicity of these dietary fatty acids (ALA, SDA, EPA, DHA, CLA and GLA) compared with oleic acid [OA, 18:1(n-9)] at a level of 3 g/100 g in the diets of Apc^Min/+ mice and to determine whether any alterations in tumorigenesis correspond to alterations in prostaglandin biosynthesis. Tumor multiplicity was significantly lower by 50% in mice fed SDA or EPA compared with controls, whereas less pronounced effects were observed in mice fed DHA (P = 0.15). ALA, CLA and GLA were ineffective at the dose tested. Although lower tumor numbers coincided with significantly lower prostaglandin levels in SDA- and EPA-fed mice, ALA and DHA supplementation resulted in equally low prostaglandin levels, despite proving less efficacious with regard to tumor number. Prostaglandin levels did not differ significantly in the CLA and GLA groups compared with controls. These results suggest that SDA and EPA attenuate tumorigenesis in this model and that this effect may be related in part to alterations in prostaglandin biosynthesis.

KEY WORDS: • tumor • cancer • (n-3) fatty acids • intestine • Apc • mice

	INTRODUCTION

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Colorectal cancer is the second leading cause of cancer deaths in the United States with 56,503 deaths reported in 1997 (Hoyert et al. 1999 ). Several lines of evidence overwhelmingly implicate environmental factors, specifically dietary components, as major variables influencing colorectal cancer incidence. Western-style diets characterized by high intakes of energy, fat, meat, refined grains and sugar, combined with low intakes of fiber, calcium, and fruits and vegetables have been strongly linked to an increased risk of colorectal cancer (Giovannucci and Willett 1994 , Lipkin et al. 1999 , Slattery et al. 1999 ). Among these, the amount and type of dietary fat consumed is of particular importance (Caygill et al. 1996 , Hansen Petrik et al. 2000 , Reddy 1992 , Willett et al. 1990 ).

Epidemiologic studies indicate that consumption of fish and fish oil correlates with a reduced risk of colorectal cancer (Caygill et al. 1996 , Fernandez et al. 1999 , Kato et al. 1997 ). Fish oil is rich in the (n-3) polyunsaturated fatty acids (PUFA)³ eicosapentaenoic acid [EPA, 20:5(n-3)] and docosahexaenoic acid [DHA, 22:6(n-3)]. Studies in humans and in chemically induced colonic tumor animal models overwhelmingly indicate a protective effect of (n-3) PUFA (in the form of fish, fish oils or EPA ethyl ester), and the mechanism is largely thought to be related to interference with biosynthesis of 2-series prostaglandins from arachidonic acid [AA, 20:4(n-6)] (Anti et al. 1992 , Chang et al. 1998 , Latham et al. 1999 , Minoura et al. 1988 , Reddy 1992 ). We and others demonstrated recently that diets containing EPA, and possibly DHA, are also antitumorigenic in a murine model of human colorectal cancer, and this effect is related at least in part to interference with AA metabolism (Hansen Petrik et al. 2000 , Oshima et al. 1995 , Paulsen et al. 1997 ). -Linolenic acid [ALA, 18:3(n-3)], the parent fatty acid of the (n-3) family, has been studied on a more limited basis, but with some promising results. Dietary perilla oil and flaxseed oil, both rich sources of ALA, have been shown to decrease chemically induced colonic tumors and aberrant crypt foci (ACF) in a rat model (Hirose et al. 1990 , Narisawa et al. 1994 , Onogi et al. 1996 , Serraino and Thompson 1992 ). These results have coincided with competitive exclusion of (n-6) PUFA from membrane phospholipids and associated reductions in prostaglandin E₂ (PGE₂) concentrations in colonic mucosa (Onogi et al. 1996 ). Stearidonic acid [SDA, 18:4(n-3)], the 6-desaturase product of ALA found naturally in blackcurrant (Ribes nigrum) seed oil and oils derived from some members of the Boraginaceae family (Phillips and Huang 1996 ), has not been studied previously as an antitumorigenic agent, although it has been shown to strongly inhibit growth of NIH-3T3 cells (Cantrill et al. 1993 ). The use of dietary SDA as an antitumorigenic agent is promising because it is not dependent on the 6-desaturase reaction, the rate-limiting step in de novo biosynthesis of long-chain PUFA. As such, SDA increases tissue levels of EPA more effectively than does ALA (Huang et al. 1991 , Yamazaki et al. 1992 ). Furthermore, other studies suggest that SDA may reduce formation of AA-derived eicosanoids in vitro (Guichardant et al. 1993 , Kockmann et al. 1989 ). This collective evidence, albeit limited, warrants investigation into the antitumorigenic potential of SDA.

Other fatty acids, including some (n-6) PUFA and their derivatives, have also been investigated for purported antitumorigenic effects. Conjugated linoleic acid (CLA) refers collectively to several positional and geometric isomers of linoleic acid [LA, 18:2(n-6)] in which the double bonds are in conjugation, typically at positions 9 and 11 or 10 and 12 (Ha et al. 1987 ). CLA, predominantly as 9(Z),11(E)-18:2(n-7), occurs naturally in small amounts in cooked meats, dairy products and ruminant meats (Chin et al. 1992 ), and is potently antitumorigenic in chemically induced rat mammary tumors and murine skin tumors (Belury et al. 1996 , Ip et al. 1991 ). It has recently been suggested that CLA exerts its antitumorigenic effect by inhibiting metabolism of LA to AA, thereby decreasing biosynthesis of AA-derived prostaglandins (Banni et al. 1999 , Belury and Kempa-Steczko 1997 , Kavanaugh et al. 1999 ). Compared with studies investigating the efficacy of CLA on mammary tumorigenesis, evidence for protection against colorectal cancer is less definitive. To date, gavage treatment with CLA has been shown to result in fewer chemically induced colonic ACF and gastric neoplasia in mice (Ha et al. 1990 , Liew et al. 1995 ), and CLA treatment reduced proliferation of human colon tumor cells in vitro (O’Shea et al. 1999 , Shultz et al. 1992 ).

-Linolenic acid [GLA, 18:3(n-6)], the 6-desaturase product of LA, is found predominantly in only a few dietary sources, including evening primrose (Oenothera biennis) oil, borage (Boraginaceae) oil, blackcurrant seed oil and spirulina (Phillips and Huang 1996 ). By bypassing the rate-limiting 6-desaturase reaction, dietary GLA is rapidly metabolized to dihomo--linolenic acid [DGLA, 20:3(n-6)]. As a 20-carbon PUFA, DGLA purportedly competes with AA for cyclooxygenase (COX) and 15-lipoxygenase activity to produce prostaglandin E₁ (PGE₁) and 15-hydroxyeicosatrienoic acid (15-HETrE), respectively, in some cell types, and may thereby attenuate formation of AA-derived metabolites (Borgeat et al. 1976 , Fan et al. 1997 , Johnson et al. 1997 ). A second proposed mechanism whereby GLA might elicit antitumorigenic effects is via increased lipid peroxidation (Devi and Das 1994 , Kokura et al. 1997 , Takeda et al. 1992 ). Nevertheless, the actual antitumorigenicity of GLA has been only narrowly investigated to date. GLA treatment has been shown to limit the metastatic potential of human colon cancer cell lines and block cell cycle progression in vitro (Jiang et al. 1995 and 1998 ). Accordingly, arterial GLA injections have been shown to inhibit growth of implanted hepatoma cells in rats (Kokura et al. 1997 ), and intratumoral injections of lithium-GLA resulted in smaller tumor volumes in mice implanted with pancreatic tumor cells (Ravichandran et al. 1998 ). However, dietary GLA has demonstrated potential efficacy as an antitumorigenic agent only in 7,12-dimethylbenz()anthracene (DMBA)-induced mammary tumors in rats, a study in which a diet containing evening primrose oil (20 g/100 g) resulted in a lower tumor incidence than did corn oil (Abou El-Ela et al. 1987 ), and in nude mice bearing breast carcinoma xenografts (Pritchard et al. 1989 ). A preventive role for dietary GLA in gastrointestinal tumorigenesis has yet to be established.

Over the last several years, the Apc^Min/+mouse model has been used to evaluate the effects of nutritional intervention on intestinal tumorigenesis because of its germline mutation in the murine adenomatous polyposis coli (Apc) gene (Hansen Petrik et al. 2000 , Oshima et al. 1995 , Paulsen et al. 1997 , Wasan et al. 1997 ). After somatic mutation of the wild-type allele, these mice spontaneously develop adenomas throughout the intestinal tract with preferred localization in the small intestine (Chiu et al. 1997 ). Development of colorectal cancer in humans from dysplastic crypts to metastatic carcinoma involves a series of genetic mutations, the earliest often involving APC (Kinzler and Vogelstein 1996 , Powell et al. 1992 ). Individuals with familial adenomatous polyposis (FAP), like Apc^Min/+ mice, possess a germline mutation in APC, and mutational damage or loss of the wild type allele initiates intestinal tumor formation (Levy et al. 1994 , Miyoshi et al. 1992 ). Although FAP accounts for <1% of all human colorectal cancer cases, somatic mutations resulting in loss of full length APC protein also occur early in spontaneous forms of the disease (Miyoshi et al. 1992 , Powell et al. 1992 ), indicating that an APC defect is associated with a majority of human colorectal cancers (Jen et al. 1994 , Smith et al. 1993 ). Therefore, in this study, we investigated effects of the dietary fatty acids ALA, SDA, EPA, DHA, CLA and GLA on intestinal tumorigenesis in Apc^Min/+ mice fed diets based on a typical Western diet.

	MATERIALS AND METHODS

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Animals.

Male C57BL/6J Apc^Min/+ mice (n = 77; 37 d old) (Jackson Laboratories, Bar Harbor, ME) were randomly assigned to eight dietary groups (n = 9–10 mice/group) on arrival. They were housed in a temperature-controlled room with a 12-h light:dark cycle and given free access to food and water. The health of the mice was checked daily. Food was withheld overnight before they were killed. All animal procedures were approved by the University of Tennessee Animal Care and Use Committee and were in accordance with the NIH Guidelines (NRC 1985 ).

Diets.

All diets were based on the composition of a typical Western diet with the following energy distribution: carbohydrate, 44.4%; fat, 35.4%; and protein, 21.2%. These diets were predicated on the "US17" diet formulated by Monsanto (St. Louis, MO) and Research Diets (New Brunswick, NJ) with input from the authors. The following fatty acid ethyl esters were added (31 g/kg) to the base diet at the expense of Trisun high oleic acid [OA, 18:1(n-9)] sunflower oil (Monsanto, St. Louis, MO): 1) conjugated linoleic acid [CLA, 9,11–18:2(n-7)] isomers, 77%; [10,12–18:2(n-6)] isomers, 13%; mixed isomers, 5%, 2) -linolenic acid [GLA, 18:3(n-6)], 95% pure, 3) -linolenic acid [ALA, 18:3(n-3)], 95% pure, 4) stearidonic acid [SDA, 18:4(n-3)], 85% pure, eicosapentaenoic acid [EPA, 20:5(n-3)], 95% pure, or docosahexaenoic acid [DHA, 22:6(n-3)], 90% pure. The diet containing the nonsteroidal anti-inflammatory drug (NSAID) sulindac (320 mg/kg) was used as a positive control (Table 1 ). Overall fatty acid compositions of the diets are shown in Table 2 . All diets were divided into daily aliquots and stored under an atmosphere of nitrogen at -80°C to prevent oxidation. All mice were provided fresh food daily. Body weights were recorded weekly.

After randomization into dietary groups, all mice were fed the assigned diets for 7 wk. At 87–89 d of age, mice were killed by cervical dislocation, and tumor number, size and location were determined as previously described (Chiu et al. 1997 ). Portions of normal-appearing intestine and several of the largest tumors were harvested for histologic examination based on previously reported features of intestinal development and dysplasia in this strain of mice. Phospholipid fatty acid composition and levels of PGE₂ and 6-keto-prostaglandin F₁(6-keto-PGF₁) were determined for each mouse from normal-appearing jejunal sections of the small intestine.

Fatty acid analysis.

Fatty acid methyl esters (FAME) of the tissue phospholipids were prepared and analyzed as described previously (Whelan et al. 1992 ). Briefly, tissues were homogenized in ice-cold saline and lipids were extracted with chloroform/methanol (1:2, v/v), followed by extraction (x2) with chloroform. The pooled chloroform extracts were evaporated and resuspended in a small volume of chloroform, and the phospholipids were separated by TLC on silica gel 60 HP-TLC plates (Merck, Darmstadt, Germany) with chloroform/methanol (8:1, v/v) as the solvent system. Bands corresponding to the phospholipids were scraped, dissolved in toluene and saponified with KOH (0.5 mol/L) in methanol for 8 min at 86°C. After acidification with HCl in methanol (0.7 mol/L), the fatty acids were extracted twice with hexane, evaporated and methylated with ethereal diazomethane. The FAME were resuspended in hexane and analyzed using a Hewlett-Packard model 5890 series II gas chromatograph (Palo Alto, CA) equipped with flame ionization detector and a DB23 fused silica capillary column (0.25 mm i.d. x 30 m x 0.25-µm film; J&W Scientific, Folsom, CA). Separation was achieved by temperature programming from 160 to 250°C at 3.5°C/min with hydrogen as the carrier gas. The internal standard, pentadecaenoic acid (15:0) methyl ester (Avanti Polar Lipids, Alabaster, AL) was added to each sample before the saponification step. The FAME were identified by comparison of retention times with those of known standards (Nu-Chek-Prep, Elysian, MN). Various CLA isomers and elongation products of GLA and SDA were identified by comparison of retention times to purified FAME standards (Matreya, Pleasant Gap, PA).

Measurement of prostaglandins.

After cervical dislocation, normal-appearing intestinal sections were quickly perfused with ice-cold saline (NaCl, 154 mmol/L), immediately snap-frozen in liquid nitrogen and stored at -80°C. For analysis, the tissues were homogenized using a Tenbroeck tissue grinder (Pyrex, England) in ice-cold Tris-HCl buffer (0.1 mol/L, pH 7.4) containing indomethacin (final concentration 1 mmol/L) to prevent ex vivo prostaglandin biosynthesis (basal levels of prostaglandins associated with the tissues). This was followed by methanolic acidification with 8.8% formic acid in 90% methanol (final pH 3.5). The resulting homogenate was vigorously mixed and centrifuged at 5000 x g for 5 min after which the supernatant was removed. The pellet was resuspended in a 20% solution of methanol in water (pH 3.5), vigorously mixed and centrifuged at 5000 x g for 5 min after which the supernatant was collected and pooled. Prostaglandins were isolated from pooled supernatants by solid-phase extraction using an octadecyl (C18) cartridge (Burdick & Jackson, Muskegon, MI) and eluted with 100% methanol. The recovery of prostaglandins was determined by adding 0.37 nBq [³H]-prostaglandin D₂ (PGD₂) to the sample as an internal standard before processing. The methanol was evaporated under a stream of nitrogen gas and the extracts were resuspended in PBS (pH 7.4) containing gelatin (1.0 g/L). PGE₂ and 6-keto-PGF₁were measured by RIA as described previously using antiserum obtained from PerSeptive Diagnostics, (Cambridge, MA) (Whelan et al. 1993 ). Cross-reactivities at half maximal binding of various prostanoids with PGE₂ antiserum are as follows: PGE₂ (100%), PGE₃ (26%), 6-keto-PGF₁ (< 1%), PGF₁ (1%), thromboxane B₂ (TXB₂) (<1%), PGD₂ (< 1%) and PGF₂ (1%). Cross-reactivities with various prostanoids with 6-keto-PGF₁ antiserum are as follows: 6-keto-PGF₁ (100%), -17, 6-keto-PGF₁ (14%), PGE₂ (<1%), PGF₂ (2%), PGF₁ (8%), PGD₂ (< 1%) and TXB₂ (<1%). All standards were purchased from Cayman Chemical (Ann Arbor, MI); [³H]-PGE₂, [³H]-PGD₂ and [³H]-6-keto-PGF₁ were obtained from New England Nuclear (Boston, MA). An aliquot of the homogenate from each sample was used to determine protein concentration by the modified Lowry protocol (Markwell et al. 1981 ).

Statistical analyses.

Values are expressed as means ± SEM Levene’s test was used to determine homogeneity of variance among groups. Differences in tumor number, tumor size and biochemical parameters were analyzed statistically by one-way ANOVA followed by Fisher’s least significant difference multiple comparison method to determine differences among groups. For statistical analysis of tumor number, one extreme outlier (defined as a value > 3 interquartile ranges above the 75th percentile for each of the respective groups) was removed in each of the DHA and sulindac-supplemented groups. This did not affect the statistical results. By the same definition, one extreme outlier was removed from the EPA group for analysis of PGE₂ levels. This altered the statistical significance of PGE₂ levels in the EPA group compared with controls. Regression analysis was used to determine relationships among variables in the fatty acid–supplemented groups. The Statistical Analysis System (SAS Version 6.12, SAS Institute, Cary, NC) was used to evaluate the data. Repeated measures ANOVA was used to determine differences in body weight gain and dietary intake among groups (SPSS, Chicago, IL). Differences were considered significant at P < 0.05.

	RESULTS

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Food intake and body weights.

Repeated measures ANOVA showed no significant difference in weekly body weights among the dietary groups throughout the study. Food intake was not significantly different among the groups at each time point by one-way ANOVA. However, repeated measures ANOVA revealed a significant difference in intake over time. This was most likely due to the decline in food intake in the GLA group during the final 2 wk, probably related to the increasing tumor burden toward the end of the study.

Effect of diet treatment on tumor frequency and size.

As expected, the mice treated with sulindac (positive control) had 93% fewer overall tumors and significantly smaller tumors relative to the controls (Table 3 ). Supplementation with CLA and GLA had no effect on tumor number or size. Of the (n-3) PUFA, dietary SDA and EPA supplementation resulted in the smallest number and size of tumors. ALA, the parent fatty acid in the (n-3) family, had no effect, whereas DHA-supplemented mice had 30% fewer tumors (P = 0.15). Interestingly, only SDA-supplemented mice had significantly fewer colonic tumors, which are typically resistant to chemotherapeutic intervention. In this regard, SDA was as effective as sulindac. There were no histologic distinctions between tumors harvested from any of the groups.

Phospholipid fatty acid composition of normal-appearing small intestinal tissue appropriately reflected dietary fatty acid supplementation (Table 4 ). Conjugated linoleic acid supplementation resulted in detection of CLA isomers in phospholipids at a level of 2 mol/100 mol. Additionally, 11(Z),13(E)-20:2(n-7), the immediate elongation product of 9(Z),11(E)-18:2(n-7), was detected at low levels (0.02 mol/100 mol). However, we did not detect a conjugated AA derivative of CLA. Feeding CLA had little effect on long-chain PUFA composition of the phospholipids, whereas dietary GLA supplementation significantly enriched phospholipids with GLA, DGLA and AA at the expense of OA and LA. Inclusion of (n-3) PUFA in the diets progressively elevated concentrations of EPA and docosapentaenoic acid [22:5(n-3)] in phospholipids. In contrast, dietary SDA and EPA did not significantly alter phospholipid DHA levels, whereas ALA-supplemented mice had significantly higher levels of DHA than controls (by 0.6 mol/100 mol), and dietary DHA supplementation more than doubled the DHA content of phospholipids compared with controls. Elevations in levels of (n-3) PUFA were primarily at the expense of AA and, in most cases, coincided with elevations in LA content. SDA, the 6-desaturase product of ALA, was the fatty acid associated with the lowest AA content, and with the exception of dietary EPA, was associated with the highest tissue EPA content. Only minimal amounts of SDA were detected in intestinal phospholipids.

GLA and CLA did not significantly alter basal prostaglandin levels (PGE₂ and 6-keto-PGF₁) in normal-appearing small intestinal tissue (Table 5 ). In contrast, dietary (n-3) PUFA supplementation resulted in basal prostaglandin levels significantly lower than controls by 50%, which coincided with similarly low levels of tissue AA. SDA and EPA were the dietary fatty acids associated with the lowest prostaglandin levels. Furthermore, treatment with sulindac, an inhibitor of cyclooxygenase, the committed step in prostaglandin biosynthesis, resulted in prostaglandin levels 56% lower than those observed in control mice.

	DISCUSSION

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ALA is the parent fatty acid of the (n-3) family of fatty acids and is metabolized to SDA via 6-desaturase, the rate-limiting step in this biosynthetic pathway (Guichardant et al. 1993 ). Our results suggest that desaturation of ALA to SDA via the 6-desaturase may be an important step involved in the antitumorigenic effect of (n-3) fatty acids. However, it is unclear whether these differences are related to the formation of down-stream metabolites, the ability to antagonize AA and its metabolism [highly unsaturated (n-3) PUFA are 2.5 to 5 times more effective than ALA] (Whelan et al. 1991 ) and/or whether the degree of unsaturation of the (n-3) fatty acids has an independent effect on cell signaling/function. Feeding SDA did not dramatically enrich tissue phospholipids with SDA; its ability to alter intestinal tumor load appears to be related more to its conversion to EPA than DHA given the fact that SDA had no effect on DHA levels and DHA-supplemented mice appeared to have only modestly fewer tumors. With the exception of EPA, dietary SDA had the greatest effect on elevating tissue EPA content, but was the most potent fatty acid in its ability to lower tissue AA levels. Similarly, supplementing rats with dietary SDA (1 g/100 g) resulted in significantly elevated levels of EPA, but not SDA, in hepatic phospholipid, triglyceride and free fatty acid fractions, whereas phospholipid AA levels were significantly lower compared with controls (Yamazaki et al. 1992 ). Rescuing tissue AA levels by supplementing AA to diets containing EPA effectively normalizes tumor load in Apc^Min/+ mice, highlighting the importance of AA in maintaining tumor integrity (Hansen Petrik et al. 2000 ).

The ability of dietary DHA to reduce intestinal tumorigenesis effectively is more equivocal than that of SDA or EPA. DHA-supplemented mice appeared to have fewer tumors, but these differences were not significant (P = 0.15). However, partial retroconversion of DHA to EPA and its subsequent effect on AA may have accounted for its less-pronounced efficacy compared with EPA itself. Although we cannot rule out any independent effects of DHA, there was no significant correlation between DHA levels in the tissue phospholipids and tumor number (P = 0.19). In the only other study examining the efficacy of dietary DHA in a similar animal model, DHA resulted in fewer tumors in female knockout mice with Apc truncation at codon 716 (Apc⁷¹⁶), but not in their male counterparts (Oshima et al. 1995 ). Overall, these data suggest that the ability of SDA and EPA to lessen tumor multiplicity is not dependent upon their conversion to DHA.

The ability of dietary fatty acids to modify prostaglandin levels most likely contributes to their influence on tumorigenesis. Prostaglandin involvement in intestinal tumorigenesis was recently demonstrated in a study using prostaglandin E receptor (EP) knockout mice and an EP receptor antagonist (Watanabe et al. 1999 ). Currently, four EP receptors have been identified (EP1–EP4). Watanabe et al. (1999) treated C57BL/6J mice with the colon carcinogen azoxymethane (AOM) and observed significantly fewer early neoplastic lesions (ACF) in EP1 receptor knockout mice compared with wild-type controls. Similarly, AOM-treated wild-type mice developed fewer ACF in a dose-dependent manner after administration of ONO-8711, an EP1 receptor antagonist. They also showed that ONO-8711 treatment resulted in 44% fewer tumors in Apc^Min/+ mice (Watanabe et al. 1999 ), confirming the importance of EP1 and PGE to Apc-mediated tumorigenesis. The EP1 receptor acts through a phospholipase C-mediated signaling pathway resulting in the potential activation of protein kinase C (PKC) after the release of diacylglycerol. Overexpression of PKC ß_II results in downregulation of glycogen synthase kinase 3ß (GSK-3ß), an elevation in cellular ß-catenin levels and proliferation of colonic epithelium (Murray et al. 1999 ). The Apc gene product acts in concert with GSK-3ß to regulate the wnt/ß-catenin signaling pathway (Polakis 1999 ). Loss of full-length Apc protein, as occurs in the Apc^Min/+ mouse model, disables the cell’s ability to downregulate ß-catenin; as a result, free (not bound to E-cadherin) ß-catenin increases in the cytoplasm and moves into the nucleus where it acts in conjunction with the nuclear transcription factors LEF/TCF to induce expression of target genes (Polakis 1999 ). Treatment with NSAID, inhibitors of prostaglandin biosynthesis, may reduce tumor loads in this model, at least in part, by attenuating ß-catenin levels (Mahmoud et al. 1998 , McEntee et al. 1999 ), and (n-3) PUFA may also modulate this signaling pathway via reductions in AA and PKC activation (Jiang et al. 1997 ).

In our study, prostaglandin levels were significantly lower in all (n-3) fatty acid–supplemented groups, with dietary EPA having the greatest effect. However, lower prostaglandin levels did not always equate to a lower tumor number. For example, ALA significantly attenuated prostaglandin levels, but did not alter tumor multiplicity, suggesting that prostaglandins play a partial role in tumorigenesis. Furthermore, regression analysis revealed a small, but significant relationship between prostaglandin levels and tumor number, with prostaglandins accounting for 7–17% of the variability in tumor number (Fig. 1A, B ). It is possible that the degree of unsaturation associated with the (n-3) fatty acids could differentially affect the expression or, more importantly, the activity of COX (Laneuville et al. 1995 ), the committed step in prostaglandin biosynthesis (Hamid et al. 1999 , Singh et al. 1997 ). Intestinal tumors in Apc^Min/+ mice express both isozymes of COX, i.e., COX-1, which is constitutively expressed, and COX-2, the inducible isoform (Boolbool et al. 1996 , Hull et al. 1999 , Williams et al. 1996 ). Dietary fish oils (rich in EPA and DHA) reduce COX-2 expression in an AOM-induced rat colonic tumor model (Singh et al. 1997 ) and immunoreactive COX-1 and -2 protein levels in DMBA-induced mammary tumors (Hamid et al. 1999 ), whereas (n-6) fatty acids increase COX-2 expression (Singh et al. 1997 ). The involvement of COX in this animal model is clearly established: 1) inhibition of the COX isozymes by nonselective inhibitors results in 85–96% fewer tumors (Chiu et al. 1997 and 2000 , Jacoby et al. 1996 , Ritland and Gendler 1999 ); 2) selective inhibition of COX-2 results in 52% fewer tumors (Nakatsugi et al. 1997 ); 3) crossing COX-2 knockout mice with Apc⁷¹⁶ knockout mice results in fewer tumors in a gene-dose–responsive manner (Oshima et al. 1996 ); and 4) crossing COX-1 knockout mice with Apc^Min/+ mice results in 80–90% fewer tumors (Langenbach et al. 1999 ). Nevertheless, the precise mechanisms underlying the antitumorigenic effects of SDA and EPA remain unclear.

View larger version (19K): [in this window] [in a new window]   Figure 1. The relationship between total tumor numbers of male ApcMin/+ mice fed various fatty acid ethyl esters for 7 wk and prostaglandin (PG) levels in samples of normal-appearing small intestinal tissue was determined by regression analysis. There was a significant correlation between PGE2 and tumor number (A) and 6-keto-PGF1 and tumor number (B).

To date, the efficacy of dietary GLA on intestinal tumorigenesis has not been determined. Therapeutic studies involving dietary GLA have largely utilized the DMBA-induced rat mammary tumor model or nude mouse mammary carcinoma xenograft model with encouraging results (Abou El-Ela et al. 1987 , Pritchard et al. 1989 ). The use of GLA in this study, however, did not alter intestinal tumor load in Apc^Min/+ mice, although tissue DGLA levels were significantly higher in the phospholipid fraction of the intestines. DGLA can be metabolized to 15-HETrE and PGE₁. Both of these compounds reportedly have antiproliferative properties [as reviewed by Fan and Chapkin (1998) ]. Like PGE₂, the actions of PGE₁ are mediated via the G-protein–coupled EP receptors (Boie et al. 1997 , Fan and Chapkin. 1998 , Funk et al. 1993 ). However, recent evidence indicates that antagonism, not agonism, of the EP1 receptor results in significantly fewer tumors in this mouse model (Watanabe et al. 1999 ). Furthermore, we found that feeding GLA resulted in 28% higher AA concentrations in intestinal phospholipids, although levels of PGE₂ and 6-keto-PGF₁ were not significantly different from control values, possibly accounting for the lack of efficacy of GLA. We determined previously that elevating tissue AA levels and prostaglandin levels, for that matter, above control values had no effect on tumor number or size in this animal model (Chiu et al. 1997 , Hansen Petrik et al. 2000 ).

In summary, dietary SDA and EPA supplementation resulted in 50% fewer intestinal tumors in Apc^Min/+ mice, a genetic murine model of colorectal cancer based on a mutation of the Apc tumor suppressor gene. SDA was the most efficacious (n-3) fatty acid, resulting in significantly fewer tumors in both the colon and small intestine plus significantly smaller tumors in the small intestine. The antitumorigenic effects of both SDA and EPA are likely related to alterations in tissue AA content and prostaglandin levels (Hansen Petrik et al. 2000 ). The fact that dietary ALA had no effect on tumor multiplicity suggests the importance of its conversion to SDA via the 6-desaturase, whereas the ability of DHA to affect tumorigenesis is less clear. Like ALA, CLA and GLA had no effect on intestinal tumorigenesis in this model.

	ACKNOWLEDGMENTS

 
We appreciate the valuable contributions of Ed Ulman, Research Diets, New Brunswick, NJ, and Julie Lindemann and Lisa Olsen, Nutrition and Consumer Sector, Monsanto Company, St. Louis, MO, in design of the experimental diets.

	FOOTNOTES

 
¹ Supported by a grant from Monsanto Company, St. Louis, MO.

³ Abbreviations used: AA, arachidonic acid; ACF, aberrant crypt foci; ALA, -linolenic acid; AOM, azoxymethane; Apc, murine adenomatous polyposis coli; APC, human adenomatous polyposis coli; Apc⁷¹⁶, Apc knockout mouse with Apc truncation at codon 716; Apc^Min/+, multiple intestinal neoplasia mouse; CLA, conjugated linoleic acid; COX, cyclooxygenase; DGLA, dihomo--linolenic acid; DHA, docosahexaenoic acid; DMBA, 7,12-dimethylbenz()anthracene; EP, prostaglandin E receptor; EPA, eicosapentaenoic acid; FAME, fatty acid methyl esters; FAP, familial adenomatous polyposis; GLA, -linolenic acid; GSK-3ß, glycogen synthase kinase-3ß; 15-HETrE, 15-hydroxyeicosatrienoic acid; 6-keto-PGF₁, 6-keto-prostaglandin F₁; LA, linoleic acid; OA, oleic acid; NSAID, nonsteroidal anti-inflammatory drug; PGD₂, prostaglandin D₂; PGE₁, prostaglandin E₁; PGE₂, prostaglandin E₂; PKC, protein kinase C; PUFA, polyunsaturated fatty acids; SDA, stearidonic acid; TXB₂, thromboxane B₂.

Manuscript received February 16, 2000. Initial review completed March 27, 2000. Revision accepted June 9, 2000.

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