The fatty acid compositions of the microsomal and mitochondrial membrane fractions of liver, colon and MDA-MB 231 human breast tumor were analyzed by gas chromatography. The individual percent compositions for α-linolenic, eicosapentaenoic and docosahexaenoic acids were summed to estimate the fraction of n-3 fatty acids. The individual percent compositions for linoleic and arachidonic acids were summed to estimate the fraction of n-6 fatty acids. The ratios of the n-3 to n-6 fatty acids in each tissue and subcellular fraction were subjected to statistical analyses. A two way ANOVA revealed that the n-3 to n-6 ratio (Table 1) was significantly higher in mice that consumed FOC than in mice that consumed corn oil (CO) indicating that n-3 fatty acids were incorporated in cellular membranes when FOC was provided in the diet of the diet. Supplemental Vitamin E in the diet did not effect the ratio of n-3 to n-6 fatty acids.

To determine the effect of consumption of FOC on endogenous antioxidant enzyme activity, the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) were determined in the liver, colon and tumor of mice that had consumed the diet for two weeks. As summarized in Table 2, two way ANOVA revealed that the activities of SOD, CAT and GPX were not significantly altered in the liver or colon due to the consumption of FOC or of supplemental Vitamin E for two weeks. However, GPX activity was significantly less in the tumors of mice that consumed FOC than in the tumors of mice that consumed CO and supplemental Vitamin E did not significantly alter GPX activity. Thus, the effect of consumption of FOC on GPX activity was different in the tumor than in the normal host liver or colon.

Lipid peroxidation was assayed in the livers, colons and tumors of mice sacrificed after two weeks consumption of each diet and after two weeks consumption of the diets and a single injection of DOX 24 h before sacrifice (Table 3). Three way, two way and one way ANOVA revealed that in the:

Figure 1 illustrates the mean growth rates of the tumors of each dietary group following DOX treatment. ANOVA revealed that the growth rates of the tumors of both groups that consumed FOC were significantly less than the mean growth rates of both of the groups of mice that consumed either CO diet.

Values for the means of SOD, CAT, GPX activities and TBARS from the tumors of groups of mice killed after two weeks on each diet with or without DOX treatment and the mean tumor growth rate of mice fed each diet and treated with DOX for five weeks were entered into a forward multiple regression analyses. Multiple regression analyses can address the question: What variable(s) present in the tumor prior to or after DOX treatment best correlates with the response of the tumor to DOX treatment? The results of the analyses (Table 4) revealed that GPX activity in the tumor prior to DOX treatment explained 78% of the variation in tumor growth rate (the correlation coefficient (r) between GPX activity in the tumor before DOX treatment and the tumor growth rate following DOX treatment was 0.88). Increased TBARS following DOX treatment added an additional 19% explanation for the variation in tumor growth rate. Thus, 97% of the variation in tumor growth rate could be explained by the diet induced decrease in GPX activity and by increased lipid peroxidation in the tumor following DOX treatment.

The mean body weight change of each group of tumor bearing mice following consumption of each diet for two weeks or following DOX treatment for five weeks was determined. Two way ANOVA (Table 5) revealed that addition of Vitamin E to the diet did not significantly effect body weight but that mice that consumed FOC prior to DOX treatment gained significantly more weight than did mice that consumed CO. Neither fat type nor Vitamin E supplementation significantly altered the change in body weight during five weeks of DOX treatment.

It has been reported that omega 3 fatty acids can decrease tumor cachexia [19-21]. It is important to note that tumor bearing mice that received only 3% FOC in their diet gained more weight prior to initiation of DOX therapy than mice that did not receive FOC. At the initiation of DOX therapy, the tumors of FOC fed mice were slightly, (but not significantly) smaller than the tumors of the CO fed mice and at the end of the study the tumors of FOC fed mice were significantly smaller than the tumors of CO fed mice, thus the higher weight gain has to be body weight, not tumor weight. The significantly higher body weight gain is likely due to reduced tumor related cachexia.

CO and FOC fed groups did not lose different amounts of body weight during DOX treatment. This fact and the maintenance of antioxidant enzyme production in colon and liver are indications that the toxicity of DOX to normal host tissues was not increased by the FOC diet.

The tumor growth curves (Figure 1) illustrate the significant enhancement of the efficacy of DOX against MDA-MB 231 human breast cancer xenografts when FOC was incorporated in the diet. The tumor growth rate of mice consuming either FOC diet was significantly less than the tumor growth rate of mice consuming either CO diet. It is interesting that adding vitamin E to the CO diet slightly increased the mean growth rate of the tumors. Since vitamin E quenches oxidation and oxidative damage is one of the mechanisms of action of DOX [13], it would be expected that addition of vitamin E to the diet of mice bearing tumors with adequate GPX activity, might reduce the efficacy of DOX against the tumor.

Cultured MDA-MB 231 cells (American Type Culture Collection, Rockville, MD) were harvested, rinsed then suspended in serum-free M3D base culture medium (INCELL Corporation, LLC, San Antonio, TX). Cells in suspension were counted using a hemocytometer and the cell count was adjusted to 20 × 10⁶/ml. The suspension was kept well mixed during the time of injection. MDA-MB 231 cells (1 × 10⁶ cells in 0.05 ml of serum free media) were injected sc between the scapulae of each mouse.

Corn oil (CO) contains about 50% linoleic acid, 23% oleic acid, 10% C16 fatty acids and < 1% n-3 PUFAs. The n-3 ethyl ester concentrate of fish oil (FOC) containing > 65% n-3 ethyl esters, (> 33% EPA, > 22% DHA, 10% other n-3 fatty acids), was obtained from the Lipro AS, Norway and was supplied antioxidant-free. This oil is made in accordance with Good Manufacturing Practice and is approved as a food additive for humans. The oil is saturated with nitrogen to prevent oxidation during shipping and storage.

This experiment was conducted at the University of Texas Health Science Center. All animal use and handling was approved by the UTHSCSA Institutional Animal Care and Use Committee. Eighty female athymic nu/nu mice (Harlan Sprague Dawley Inc. Madison, WI), 3 months old received tumor cells. The mice were housed under aseptic conditions in a temperature (24°C) and light-controlled (12 h/day) room.

The tumor cell bearing mice were fed the AIN-76 semipurified diet from receipt until three weeks after injection of cells to allow the tumor to become established. Nude mice bearing growing MDA-MB-231 human breast carcinoma xenografts were then divided into four dietary groups (20 mice per group) such that the mean tumor size was not different between groups. One group received the standard AIN-76 diet containing 5% CO (the CO diet), one group received the 5% CO diet supplemented with 2000 IU/kg Vitamin E (the CO+E diet), one group, received the AIN-76 diet modified to contain 3% FOC and 2% CO (the FOC diet) and the final group received the 3% FOC and 2% CO supplemented with 2000 IU/kg Vitamin E (the FOC+E diet). Supplemental vitamin E was added to quench in vivo lipid peroxidation due to the diet. The diet composition is given in Table 6. Diets were prepared weekly, daily portions were individually packaged and stored at -20°C in sealed containers with nitrogen gas atmosphere to prevent lipid peroxidation. Mice were fed fresh food each day and food remaining in the cage was discarded.

Lengths and widths of tumors and body weights were measured three times weekly. Tumor sizes were calculated using the formula for the volume of a prolate spheroid: V = 4/3 * 3.14 * L/2 * W/2 * D/2. The width measurement was used as the depth of the tumor. The body weight change before DOX treatment was determined by subtracting the mouse body weight on the day of the diet change from the body weight on the day of the first DOX treatment, thus this represents body weight change due to effects of the growing tumor and of the diet. The body weight change after DOX treatment was determined by subtracting the mouse body weight on the day of the first DOX injection from the mouse body weight on the day of killing and represents body weight change due to diet, to the growing tumor and to DOX therapy.

After two weeks of consumption of the diets, five tumor bearing mice from each dietary group were killed for assay of the effects of the diet on the tumor and normal tissues of the mice. DOX therapy (5 mg/kg body weight each 4 days, i.v. in a lateral tail vein) was initiated on the remaining mice of each group. Five tumor bearing mice from each group were killed 24 h after one dose of DOX for determination of the acute effects of DOX on the sensitized tumors and normal tissues. After two weeks of DOX treatment, mice were losing excess weight at the given schedule of DOX treatment so the spacing of injections was increased to each seven days for the next three weeks and mouse weight stabilized. The remainder of the mice (10 per diet group) received DOX treatment for a total of five weeks to allow time to generate tumor growth curves and to determine the long term effects of FOC consumption and/or DOX treatment. Thus if day 0 was the day of the first injection, mice received DOX injections on days 0, 4, 8, 12, 19, 26, and 33 and were sacrificed on day 1 or on day 34.

Mice were deeply anesthetized using a ketamine/rompun solution prepared by the UTHSCSA veterinarian. The tumor, liver and large intestines were removed at necropsy. Portions of each tissue were placed individually in a labeled vial and flash frozen in liquid nitrogen. At a later date, frozen livers, colon or tumor were thawed and homogenized individually at 4°C in 280 mM mannitol with 10 mM HEPES buffer with 0.01% BHT using a Polytron homogenizer. The homogenate was divided into aliquots and frozen at -70°C until subsequent analyses.

The total protein content of an aliquot of each tissue homogenate was analyzed by the method of Bradford [22] using the Bio-Rad protein assay (micro-method). The thiobarbituric acid reactive substances (TBARS) assay was used to estimate lipid peroxidation in the homogenate. Malondialdehyde and other products of lipid peroxidation can be estimated spectrophotometrically at 535 nm after reaction with thiobarbituric acid to obtain an index for lipid peroxidation [23]. The absorbance values obtained were compared against a standard curve of known concentrations of malondialdehyde and normalized to the protein content of the specimen. The results were reported as nmol of TBARS per mg of protein.

The incorporation of fatty acids into the mitochondria and microsomes of colon, liver and tumor was determined in mice which consumed each diet. An aliquot of each whole tissue homogenate was fractioned into mitochondrial and microsomal fractions by successive centrifugation [600 × g for 10 min (remove large debris), 15,000 × g for 5 min (mitochondrial fraction), then 100,000 × g for 1 h (microsomal fraction)] for assay of lipid composition. Chloroform:methanol was used for lipid extraction of each fraction; lipids were esterified in acetyl chloride-methanol as described [24]. Gas chromatography was done using a Hewlett Packard 5890 Series 11 Gas Chromatograph (Palo Alto, CA) and conditions of: oven @ 170°C initial, 5°/min gradient to 220°C final temperature, injector temperature @ 225°C, flame-ionization detector @ 250°C, helium carrier gas @ 400 Kpa. Fatty acid methyl ester standards (Nu-Chek-Prep, Elysian, MN) were used for peak identification. The fatty acid methyl esters were reported as the percent of the total methylated fatty acids (area under the curve).

Catalase (CAT) activity in the tissue homogenates was assayed using a microplate adaptation of the method of Aebi [25]. In this method, the decomposition of H₂O₂ due to CAT activity was assayed by the decrease in the absorbance of H₂O₂ at 240 nm.

Superoxide dismutase (SOD) activity of tissue homogenates was determined using a microplate adaptation of the ferricytochrome C reduction assay of Flohé and Ötting [26]. In this method, the reduction of cytochrome C by superoxide radicals is monitored at 550 nm utilizing the xanthine-xanthine oxidase system as the source for superoxide.

Glutathione peroxidase (GPX) activity of tissue homogenates was determined using a microplate adaptation of the glutathione peroxidase assay of Paglia and Valentine [27]. This method follows the decrease in absorbance at 340 nm as NADPH is converted to NADP.

SAS computer software was used for statistical analyses. Tests for normality (basic statistics) were used on each data set. Two-way and one-way analyses of variance (ANOVA) followed by Student-Newman-Keuls (SNK) multiple range tests, as appropriate, were used to determine statistically significant (p < 0.05) differences in measured parameters due to the diet or to the chemotherapy. PRISM™ (GraphPad Software, San Diego, CA) was used to generate an ANOVA to test for differences between linear regression curves of tumor growth. Multiple linear regression analysis with forward selection was used to test for correlations in the data between the independent variables: SOD, CAT and GPX activity in the tumor prior to DOX therapy, and TBARS prior to or after DOX therapy and the dependent variable, the tumor growth rate following DOX treatment.