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© 2004 The American Society for Nutritional Sciences J. Nutr. 134:1162-1166, May 2004

Nutrition and Cancer

Conjugated Eicosapentaenoic Acid (EPA) Inhibits Transplanted Tumor Growth via Membrane Lipid Peroxidation in Nude Mice

Tsuyoshi Tsuzuki, Miki Igarashi and Teruo Miyazawa1

Food and Biodynamic Chemistry Laboratory, Graduate School of Life Science and Agriculture, Tohoku University, Sendai, 981-8555, Japan

1To whom correspondence should be addressed. E-mail: miyazawa{at}biochem.tohoku.ac.jp.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Both conjugated linoleic acid (CLA) and eicosapentaenoic acid (EPA) have an antitumor effect. Hence, we hypothesized that a combination of conjugated double bonds and an (n-3) highly unsaturated fatty acid would produce stronger bioactivity. To verify the antitumor effect of conjugated EPA (CEPA), we transplanted DLD-1 human colon tumor cells into nude mice, and compared the tumor growth between CEPA-fed mice and CLA- and EPA-fed mice. After tumor cell inoculation, mice were assigned to 1 of 4 groups (control, CLA, EPA, and CEPA) consisting of 10 mice each. The control group received only safflower oil fatty acids, whereas the remaining groups received a mixture of safflower oil fatty acids and 20 g/100 g of total fatty acids as CLA, EPA, or CEPA. Mice were fed once every 2 d for 4 wk at a dose of 50 mg/mouse at each feeding. After 4 wk, tumor growth in CEPA-fed mice was significantly suppressed, compared with that in CLA- (P < 0.005) and EPA-fed mice (P < 0.001). DNA fragmentation in the tumor tissues of the CEPA-fed mice occurred more frequently than in the CLA- (P < 0.001) and EPA-fed mice (P < 0.001), suggesting that CEPA induced apoptosis in the tumor tissues. To further investigate the mechanism, the level of oxidative stress in the tumor tissues was determined. The CEPA-fed mice showed significant lipid peroxidation, compared with the CLA- (P < 0.001) and EPA-fed mice (P < 0.001). Therefore, we verified that CEPA has a stronger in vivo antitumor effect than EPA and CLA, and that CEPA acts through induction of apoptosis via lipid peroxidation.

KEY WORDS: • conjugated linoleic acid • conjugated eicosapentaenoic acid • nude mice • lipid peroxidation • conjugated fatty acid

Fatty acids with conjugated double bonds exist in nature, but occur only in small quantities. A conjugated linoleic acid (CLA; 18:2),2 a geometrical and positional isomer of linoleic acid (LA; 9Z12Z-18:2), is found in dairy products such as milk and cheese; its basic component is 9Z11E-CLA (1) (Fig. 1). The seeds of some plants include conjugated triene fatty acids such as {alpha}-eleostearic acid (9Z11E13E-18:3) and calendic acid (8E10E12Z-18:3), and tetraene fatty acids such as parinaric acid (9Z11E13E15Z-18:4) (2,3); seaweeds such as red and green algae contain more highly unsaturated conjugated fatty acids, i.e., conjugated EPA (CEPA; 5Z7E9E14Z17Z-20:5) (Fig. 1), bosseopentaenoic acid (5Z8Z10E12ZE14Z-20:5) and stellaheptaenoic acid (4Z7Z9E11E13Z16Z19Z-22:7) (4,5).



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  		FIGURE 1 Chemical structures of LA, CLA, EPA, and CEPA.

 
It was reported that CLA has very useful bioactive properties, including antitumor activity (1,6–8). Highly unsaturated (n-3) fatty acids such as eicosapentaenoic acid (EPA; 5Z8Z11Z14Z17Z-20:5) and docosahexaenoic acid (DHA; 4Z7Z10Z13Z16Z19Z-22:6) were also reported to have an antitumor effect (9) (Fig. 1). Therefore, we hypothesized that a combination of conjugated double bonds and an (n-3) highly unsaturated fatty acid would produce stronger bioactivity. In addition, we found previously that a mixture of conjugated fatty acids prepared by alkaline treatment of {alpha}-linolenic acid, EPA, and DHA induced a strong and selective apoptosis in cultured human tumor cells, with the mechanism proceeding via lipid peroxidation (10,11).

To verify the in vivo antitumor effect of CEPA, the fatty acid that was most effective in cultured cell lines, in this study, we transplanted DLD-1 human colon tumor cells into nude mice, and fed the mice CEPA, CLA, or EPA. We then compared the tumor cell growth among the CEPA-, CLA- and EPA-fed mice and investigated the mechanism of the antitumor activity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
    Materials. RPMI 1640 medium (containing 0.3 g/L L-glutamine and 2.0 g/L sodium bicarbonate) was obtained from Sigma Chemical. Fetal bovine serum (FBS) was purchased from Dainippon Pharmaceutical. Penicillin and streptomycin were products of Gibco BRL. EPA (98% purity) was a gift from Bizen Chemical. Safflower oil and CLA (80% purity) were kind gifts from Rinoru Oil Mills.

    Preparation of safflower oil fatty acids and CEPA. Safflower oil fatty acid was prepared from safflower oil by saponification using a previously reported method (11). In our previous study, CEPA, which has a conjugated trienoic structure, had the strongest cytotoxic effect (10); in the current study, CEPA was prepared from EPA by alkaline isomerization using the same method as that previously reported (11,12). Potassium hydroxide at a concentration of 21 g/100 g in ethylene glycol was prepared, and nitrogen gas was bubbled through the solution for 10 min. EPA (100 mg) was added to 10 mL of the 4 mol/L KOH solution in a test tube (100 mL volume). Nitrogen gas was again bubbled through the mixture, and then the tube was screw-capped and allowed to stand for 10 min at 180°C. The reaction mixture was cooled, 10 mL of methanol was added, and then the mixture was acidified to below pH 2.0 with 20 mL of 6 mol/L HCl. After dilution with 2 mL of distilled water, the conjugated fatty acids were extracted with 5 mL of hexane. The hexane extract was then washed with 3 mL of 30% methanol and 3 mL of distilled water before evaporation under a nitrogen gas stream. The CEPA concentrate was stored at –20°C after being purged with nitrogen gas. UV/VIS spectrophotometric analysis of the conjugated fatty acid was performed with a Shimadzu UV-2400PC, and confirmed the presence of conjugated fatty acids containing dienes (absorption at 235 nm), trienes (268 nm), tetraenes (315 nm), pentaenes (345 nm), and hexaenes (375 nm) (13). The CEPA oil used in the subsequent experiments consisted of 57.6% conjugated dienes, 34.5% conjugated trienes, 7.7% conjugated tetraenes, and 1.2% conjugated pentaenes. The CEPA diet contained 20 g/100 g of total fatty acids as CEPA, and was fed to the mice for 4 wk.

    Cells and cell cultures. The human colon tumor cell line (DLD-1) was obtained from the Cell Resource Center for Biochemical Research at Tohoku University. The cell line was cultured in RPMI 1640 medium (containing 0.3 g/L L-glutamine and 2.0 g/L sodium bicarbonate) supplemented with 10% FBS, 100 kU/L penicillin, and 100 mg/L streptomycin. The DLD-1 cells were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO2.

    Animals and treatments. Male athymic nude mice (BALB/cA Jcl-nu nu/nu, 4 wk old) were obtained from Japan Clea and maintained in a clean environment. A commercial diet (CL-2)3 for the mice was purchased from Japan Clea. This is a vitamin-enriched feed that is used in the breeding of germ-free animals and immunodeficient animal models such as nude mice. After acclimatization for 1 wk, tumor cells (DLD-1) were subcutaneously inoculated into the nude mice. DLD-1 cells in culture were detached by trypsinization and washed with PBS. Cell suspensions of 5 x 106 cells in 100 mL of PBS were injected into the back region of each nude mouse using an 18-gauge needle. Mice were randomly divided into 4 groups 2 d after tumor cell inoculation; this day was considered to be the initial test day. The 4 groups were defined by the dietary test oils fed to the mice, as follows: the control group (n = 10), the CLA group (n = 10), the EPA group (n = 10), and the CEPA group (n = 10). The CLA oil, EPA oil, and CEPA oil contained CLA, EPA, and CEPA, respectively, as 20 g/100 g of the total fatty acid concentration. The test oil (50 mg) was administered orally to each mouse once every 2 d for 4 wk. Body weight and tumor size were measured once every 2 d after tumor cell inoculation. The tumor weight was estimated by the following formula (14):

The body weight of the host alone was estimated by the following formula (14):

The mice were caged individually, given free access to food and distilled water, and housed in a temperature- and humidity-controlled room with a 12-h light:dark cycle. All procedures were performed in accordance with the Animal Experiment Regulations of Tohoku University. After the 4-wk experimental feeding period, the mice were killed by decapitation and blood was collected into a EDTA-treated blood collection tubes. Plasma was prepared from the blood by centrifugation at 1000 x g for 15 min at 4°C, and was then stored at –80°C until analysis. Immediately after blood collection, all tumor and liver tissues were perfused in situ with ice-cold saline, removed, and stored at –80°C until assayed. After defrosting, DNA fragmentation in the tumor tissue was evaluated to confirm apoptosis in the tumor cells, and a DNA ladder assay was also performed (see below). Membrane phospholipid hydroperoxides and TBARS were also measured in the defrosted plasma, liver, and tumor tissues to confirm the occurrence of lipid peroxidation in vivo.

    GC analysis. Test oils with a known amount of heptadecanoic acid (17:0; Sigma) as an internal standard were treated with 4% HCl:methanol (v:v) for 20 min at 60°C to prepare the FAME. These were then subjected to GC (GC 353B, GL Sciences) with a flame-ionization detector and a Supelcowax-10 fused silica capillary column (60 m x 0.32 mm i.d., Supelco). The GC conditions were programmed as described previously (15). The respective test oils contained CLA, EPA, and CEPA as 20 g/100 g of the total fatty acids (Table 1). The test oils were prepared as follows: the control oil was prepared with safflower oil fatty acids (safflower oil fatty acids = 100%), the CLA oil with safflower oil fatty acids and CLA (safflower oil fatty acids:CLA = 75:25, v:v), the EPA oil with safflower oil fatty acids and EPA (safflower oil fatty acids:EPA = 78:22, v:v), and the CEPA oil with safflower oil fatty acids and CEPA (safflower oil fatty acids:CEPA = 78:22, v:v).


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  		TABLE 1 Fatty acid composition of test oil diet mixtures1

 
    DNA ladder assay. The outer part of the solid tumor was transferred into a glass tube, suspended in lysis buffer (5 mmol/L Tris, 20 mmol/L EDTA, 0.5% Triton X-100, pH 8.0), and incubated for 30 min at 4°C. After incubation, the tube was centrifuged at 15000 x g for 20 min to separate the intact chromatin from the DNA fragments. After centrifugation, 2 µL of RNase A (1 g/L) was added to the supernatant, and the mixture was incubated at 37°C for 1 h. Proteinase K (2 µL; 1 g/L) was then added and incubation was continued for an additional 1 h. DNA was precipitated with a mixture of 20 µL of 5 mol/L NaCl and 120 µL of 2-propanol overnight at –20°C. After centrifugation at 15,000 x g for 20 min, pellets were air-dried and dissolved in 20 µL of TE buffer (10 mmol/L Tris and 1 mmol/L EDTA, pH 7.4). The extracted DNA was electrophoresed in a 2.0% agarose gel in a mixture of 90 mmol/L Tris, 90 mmol/L boric acid, and 2 mmol/L EDTA buffer (pH 8.4) at 100 V. Each gel was stained with ethidium bromide and photographed under UV light.

    DNA fragmentation assay. The outer part of the solid tumor was transferred into a glass tube, suspended in lysis buffer (5 mmol/L Tris, 20 mmol/L EDTA, 0.5% Triton X-100, pH 8.0), and incubated for 30 min at 4°C. After incubation, the tube was centrifuged at 15000 x g for 20 min to separate the intact chromatin from the DNA fragments. After centrifugation, 1 mL of lysis buffer was added to the pellets. Both the pellets and supernatants were assayed for DNA concentration using diphenylamine (16). The results were expressed as the ratio of DNA concentration in the supernatant to the total DNA concentration recovered in both the pellet and the supernatant.

    Determination of phospholipid hydroperoxides. Membranous phospholipid hydroperoxides, i.e., phosphatidylcholine hydroperoxide (PCOOH) and phosphatidylethanolamine hydroperoxide (PEOOH), in tumor tissues, liver, and plasma were determined by a chemiluminescence detection-HPLC (CL-HPLC) method, as described by Miyazawa et al. (17–19). The CL-HPLC system consisted of a Jasco HPLC system (Japan Spectroscopic) combined with a CLD-100 chemiluminescence detector (Tohoku Electronic Industries) and a Jasco UV detector (UV-970) equipped with a Jasco Finepak SIL NH2–5 column (n-propylamine-bound silica column, 5-µm particle size, 250 x 4.6 mm). The mobile phase consisted of 2-propanol:methanol:water (1350:450:200, by vol), and the flow rate was 1.0 mL/min. The luminescent reagent was prepared by dissolving cytochrome c (from horse heart, type 4; Sigma) and luminol (3-aminophytaloyl hydrazine; Wako Pure Chemical) in an alkaline borate buffer (pH 10) and was added at a flow rate of 1.2 mL/min. Tumor and liver tissues were homogenized with 4 volume equivalents of ice-cold saline. The total lipids of the plasma and the tissue homogenates were extracted by Folch’s procedure (20), and the total lipid extracts from the tumor tissue, liver tissue, and plasma were used as samples. A calibration curve was made for PCOOH prepared by photooxidation of synthetic phosphatidylcholine (1-hexadecanoyl-2-[9-cis-octadecadienoyl]-sn-glycero-3-phosphocholine, Sigma).

    TBARS assay. Tumor tissues, liver tissues, and plasma were assayed for TBARS as a conventional index for lipid peroxidation (21,22). Tumor and liver tissues were homogenized with 4 volume equivalents of ice-cold saline. Plasma (20 µL) and 20% tissue homogenate samples (20 µL) were transferred to a glass tube, and 4.5 mL of a 0.67% TBA solution was added. Then the tubes were screw-capped and centrifuged at 3000 x g for 5 min. Next, 1.5 mL of the supernatant was transferred to another tube and centrifuged at 15000 x g for 5 min at 4°C. The supernatant fluorescence was measured at 553 nm with excitation at 515 nm in a Jasco FP-750. Fluorescence intensity was converted to nmol of malondialdehyde equivalents based on a standard curve generated with 1,1,3,3-tetraethoxypropane.

    Statistical analysis. Statistical analysis was performed using one-way ANOVA, followed by a Newman-Keuls test for multiple comparisons among several groups. A difference was considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
    DLD-1 cell-transplanted tumor growth in mice. The DLD-1 cells that were transplanted into the back of the mice grew to a lump after 4 wk. The tumor growth was greatly suppressed in the CEPA group compared with the other groups (P < 0.05) (Fig. 2). Hence, CEPA had the strongest suppressive effect on tumor growth, followed by CLA, and then EPA. Based on the change in the tumor weight, the tumor growth in the CEPA group was suppressed on d 16 compared with other groups (P < 0.05) (Fig. 2). After d 16, there was a further gradual increase in the difference in the tumor growth rate between the CEPA group and the other groups. CLA and EPA also had a suppressive effect on tumor growth, but the control group and the CLA or EPA groups did not differ. Consequently, the suppressive effect of CEPA on tumor cell growth was confirmed in vivo, and the effect of CEPA was extremely strong compared with that of EPA and CLA.



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  		FIGURE 2 Changes in the relative tumor weight in mice transplanted with DLD-1 cells that were fed the control, CLA, EPA, or CEPA diet for 4 wk. Values are means ± SD, n = 10. Values at a time not sharing a letter differ, P < 0.05.

 
    Body, tumor, and liver weights in DLD-1 cell-transplanted mice. The body weight of the CEPA group tended to be the heaviest, followed in order by the CLA (P = 0.15), EPA (P = 0.10), and control groups (P = 0.07) (Fig. 3). Conversely, tumor weight in the control group generally was greater than in the EPA (P = 0.08), CLA (P = 0.06), and CEPA groups (P < 0.001), suggesting that tumor growth inhibited the normal growth of the mice. Tumor weights in the CLA, EPA, and CEPA groups were 60, 80, and 30% that of the control group, and the tumor weight was significantly suppressed in the CEPA group compared with other 3 groups. Liver weights did not differ among the 4 groups.



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  		FIGURE 3 Body weight, tumor weight, and liver weight changes in mice transplanted with DLD-1 cells that were fed a control, CLA, EPA, or CEPA diet for 4 wk. Values are mean ± SD, n = 10. Values not sharing a letter differ, P < 0.05.

 
    DNA fragmentation and the presence of a DNA ladder in DLD-1 cell-transplanted tumors. The DNA ladder was highly accentuated in the CEPA group (Fig. 4A), and DNA fragmentation was significantly increased in the CEPA group (Fig. 4B). In the CLA, EPA, and CEPA groups, the DNA fragmentation was 2.2-, 2-, and 4-fold, respectively, that of the control group, which suggested that CEPA-induced apoptosis in tumor cells suppressed tumor growth.



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  		FIGURE 4 DNA ladders (A) and DNA fragmentation (B) in tumor tissue taken from mice transplanted with DLD-1 cells that were fed a control, CLA, EPA, or CEPA diet for 4 wk. (A) Agarose gel electrophoresis of low-molecular-weight DNA extracted from tumor tissue. (B) DNA fragmentation of DNA extracted from tumor tissue. Values are mean ± SD, n = 10. Values not sharing a letter differ, P < 0.05. M, molecular weight markers.

 
    Membrane phospholipid peroxidation. The PCOOH level in the tumor tissue was greater in the CEPA group compared with the other 3 groups (P < 0.001) (Table 2). The PEOOH level in the tumor tissue did not differ among the 4 groups. The PCOOH levels in the tumor tissue were also increased slightly in the CLA (P = 0.1) and EPA groups (P = 0.08) compared with the control group. The PCOOH and PEOOH levels in the plasma and liver did not differ among the 4 groups. Hence, the TBARS level in the tumor tissue was increased in the CEPA group, compared with the other 3 groups (P < 0.001) (Table 3). The TBARS level in the plasma and liver did not differ among the 4 groups. These results suggest that CEPA induced apoptosis in tumor cells via lipid peroxidation. They further suggest that CEPA had a specific effect only on tumor cells, because no effects were observed on normal liver tissue, or in the plasma.


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  		TABLE 2 Phospholipid hydroperoxide concentrations of plasma, liver and tumor tissues of mice fed a control, CLA, EPA, or CEPA diet for 4 wk1

 

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  		TABLE 3 TBARS concentrations of plasma, liver and tumor tissues of mice fed a control, CLA, EPA, or CEPA diet for 4 wk1

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
This study was performed to evaluate the antitumor effect of CEPA, an (n-3) highly unsaturated fatty acid with conjugated double bonds. The effect of CEPA was compared with that of CLA and EPA in nude mice into which DLD-1 human colon tumor cells were transplanted. It was reported previously that EPA and DHA, both of which are (n-3) highly unsaturated fatty acids, have antitumor effects (9) and other reports have confirmed the antitumor effect of CLA (1,8). Therefore, EPA and CLA were used as positive controls in the evaluation of the antitumor effect of CEPA.

The CLA and CEPA oils that were fed to the mice contained a large number of isomers, because these fatty acids were prepared by alkaline isomerization. The main natural form of CLA is 9Z11E-CLA (Fig. 1) (1), but CLA prepared by alkaline isomerization contains 2 major components, i.e., 9Z11E-CLA and 10E12Z-CLA; it was confirmed that the former has little antitumor effect, whereas the latter has a strong effect (23). GC analysis showed that the CLA used in this study consisted of 44.8% 9Z11E-CLA, 46.2% 10E12Z-CLA, 3.6% all-Z-CLA, and 5.4% all-E-CLA. The difference in activity among the isomers was not compared, but it is likely that a significant effect on tumor growth would have been found, compared with the control group, if the CLA consisted only of the 10E12Z-CLA isomer, which is considered to have strong activity. CEPA isomers such as 5Z7E9E14Z17Z-20:5 and 5E7E9E14Z17Z-20:5 are found in nature in red algae (Fig. 1) (4), but the amounts of these molecules are extremely small, and it is very difficult to extract and purify CEPA from red algae. CEPA prepared by alkaline isomerization contains more isomers than similarly prepared CLA; at present, it is difficult to identify all of the CEPA isomers. As shown for CLA, it is possible that some CEPA isomers produced by alkaline isomerization have little antitumor effect; therefore, the bioactivity of each isomer should be investigated.

The results of this study indicated that compared with EPA and CLA, CEPA had an extremely strong antitumor effect on tumor cells that were transplanted into nude mice (Fig. 2). Antitumor mechanisms of fatty acids were reported to include lipid peroxidation, modulated eicosanoid production due to changes in component fatty acids, and changes in membrane fluidity (9). There are also reports that the lipid peroxide itself can induce apoptosis and inhibition of cell growth (24–29). In the tumor cells of mice that were fed CEPA, the membrane phospholipid hydroperoxide and TBARS levels were increased (Tables 2, and 3), suggesting that apoptosis was induced via lipid peroxidation as we showed previously in cell culture (10).

CEPA had no effect on normal liver tissues or on plasma (Fig. 3, Tables 2, and 3). We reported previously that in rats fed {alpha}-eleostearic acid (9Z11E13E-18:3), a conjugated fatty acid purified from plant seed, as 1% of the total feed weight for 4 wk, no significant differences were found in the lipid components of the plasma and liver, and no change in oxidative stress occurred, compared with groups fed linoleic acid, {alpha}-linolenic acid, and CLA (30). These results suggest that conjugated fatty acids have little or no effect on normal tissues, even at a concentration at which they affect tumor cells. Hence, we concluded that the tumor cells were less resistant to oxidative stress than normal cells. Administration of an unsaturated fatty acid such as CEPA, which is subject to lipid peroxidation, creates oxidative stress in tumor cells, but not in normal tissue (Tables 2, and 3). The lack of CEPA effects on normal tissue could be very desirable for food applications.

It is difficult to separate the CLA isomers prepared by alkaline isomerization in bulk. Therefore, to test the safety of administration of CLA to humans, the CLA isomer mixture was examined in long-term safety studies and clinical trials in humans. At present, CLA prepared by alkaline isomerization is on the market as a health supplement. Here, we showed that a similar preparation of CEPA, an (n-3) highly unsaturated fatty acid with conjugated double bonds, had a stronger in vivo antitumor effect than EPA and CLA, and that the antitumor mechanism occurred through lipid peroxidation–induced apoptosis. Hence, the use of CEPA in food and medicine is likely to have great promise after its safety is confirmed in long-term safety studies and clinical trials similar to those performed for CLA. Further studies of CEPA are warranted to verify the effectiveness of long-term administration and to investigate the specific active form of CEPA.


   FOOTNOTES
 
2 Abbreviations used: CEPA, conjugated eicosapentaenoic acid (5Z7E9E14Z17Z-20:5); CLA, conjugated linoleic acid; CL-HPLC, chemiluminescence detection-HPLC; EPA, eicosapentaenoic acid (5Z8Z11Z14Z17Z-20:5); FBS, fetal bovine serum; PCOOH, phosphatidylcholine hydroperoxide; PEOOH, phosphatidylethanolamine hydroperoxide. Back

3 Approximate composition of CL-2 (g/kg diet): carbohydrate, 477; protein, 240; fat, 55; fiber, 46; moisture, 90; ash, 92. Back

Manuscript received 29 October 2003. Initial review completed 13 December 2003. Revision accepted 23 February 2004.


   LITERATURE CITED
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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