Tumor growth suppression by {alpha}-eleostearic acid, a linolenic acid isomer with a conjugated triene system, via lipid peroxidation

Tsuyoshi Tsuzuki, Yoshiko Tokuyama, Miki Igarashi and Teruo Miyazawa1

Food and Biodynamic Chemistry Laboratory, Graduate School of Life Science and Agriculture, Tohoku University, 1-1 Tsutsumidori, Amamiyamachi, Sendai 981-8555, Japan

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have previously shown that conjugated linolenic acids (CLnA) prepared by alkaline isomerization have a stronger antitumor effect than conjugated linoleic acids (CLA). In this study we have compared the suppressive effect on tumor growth of {alpha}-eleostearic acid ({alpha}-ESA, 9Z11E13E-18:3) with those of the CLA isomers 9Z11E-CLA and 10E12Z-CLA, using nude mice into which DLD-1 human colon cancer cells were transplanted. The results showed that {alpha}-ESA, which is a CLnA that can be prepared from natural sources in bulk, had a stronger antitumor effect than CLA. DNA fragmentation was enhanced and lipid peroxidation was increased in tumor tissues of the {alpha}-ESA-fed mice, which suggested that {alpha}-ESA induced apoptosis via lipid peroxidation. Furthermore, treatment of DLD-1 cells with {alpha}-ESA, 9Z11E-CLA and 10E12Z-CLA confirmed that {alpha}-ESA had a stronger antitumor effect than CLA in cultured cell lines. The induction of apoptosis by {alpha}-ESA was consistent with enhanced DNA fragmentation, increased caspase activity and increased expression of caspase mRNA following {alpha}-ESA treatment. Addition of {alpha}-tocopherol, an antioxidant, suppressed oxidative stress and apoptosis, suggesting that these effects were associated with lipid peroxidation.

Abbreviations: CLA, conjugated linoleic acid; CL-HPLC, high performance liquid chromatography with chemiluminescence detection; CLnA, conjugated linolenic acid; ESA, eleostearic acid; FBS, fetal bovine serum; GC, gas chromatographic; LA, linoleic acid; PBS, phosphate-buffered saline; PCOOH, phosphatidylcholine hydroperoxide; PEOOH, phosphatidylethanolamine hydroperoxide; TBARS, thiobarbituric acid reactive substances


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Conjugated fatty acid is a generic term used for fatty acids with conjugated double bond systems, as exemplified by conjugated linoleic acid (CLA) (Figure 1) (1). Several CLA isomers exist due to positional and geometrical isomerism of the conjugated double bonds, the major naturally existing one of which is referred to as 9Z11E-18:2 (Figure 1) (1). CLA was first reported to have an anticarcinogenic effect and, subsequently, various physiological effects have also been shown, including an anti-arteriosclerotic effect and a role in regulation of lipid metabolism (15). These CLA activities are associated with the conjugated double bond system in the molecules and their level differs considerably between CLA isomers. For example, 10E12Z-CLA (Figure 1), a minor isomer in nature, shows a stronger physiological effect than 9Z11E-CLA (6).



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Fig. 1. Chemical structures of linoleic acid, conjugated linoleic acid, {alpha}-linolenic acid, {alpha}-eleostearic acid and ß-eleostearic acid.

 
CLA is found naturally and is especially present in ruminant fats such as beef tallow and milk fat (1). However, the CLA level in these foodstuffs is around 1%, and this does not allow natural fats to be used as a health-promoting food containing CLA. Therefore, at present, oils that include CLA are prepared by alkali isomerization of vegetable oils such as safflower oil and are marketed as health supplements. Conjugated fatty acids other than CLA exist in nature, but there have been only a few studies on the physiological function of these fatty acids (713). Seed oils of certain plants include conjugated triene fatty acids such as {alpha}-eleostearic acid ({alpha}-ESA, 9Z11E13E-18:3) and calendic acid (8E10E12Z-18:3) at levels of 30–80% (Figure 1) (7,8).

We have previously shown that conjugated linolenic acids (CLnA) have a stronger antitumor effect on human tumor cells than CLA (11) and that {alpha}-ESA (a CLnA) was converted to CLA in rats to which feed including 1% {alpha}-ESA was given for 4 weeks (13). In Okinawa, a region inhabited by the longest living people in Japan, which is in itself one of the leading countries in the world in terms of life expectancy, people often eat bitter gourds (Momordica charantia), whose seed oil contains 60% {alpha}-ESA and whose flesh contains a small amount of {alpha}-ESA.

We were particularly interested in seed oils including CLnA, which is the only conjugated fatty acid that can be prepared from natural sources in bulk. Hence, in this study we used tung oil and karela (bitter gourd) seed oil, which contain a large amount of {alpha}-ESA, a natural CLnA, to determine the cytotoxic effect of {alpha}-ESA in several kinds of human tumor cells and compared this effect with those of 9Z11E-CLA and 10E12Z-CLA. DLD-1 human colon cancer cells were then transplanted into mice and the suppressive effect of tung oil, which had a strong activity in cell culture, on tumor growth was investigated. The tumor growth suppression mechanism in mice and cell culture was analyzed and it was shown that {alpha}-ESA has a strong suppressive effect on tumor growth through induction of apoptosis via lipid peroxidation.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials
RPMI 1640 medium (containing 0.3 mg/ml L-glutamine and 2.0 mg/ml sodium bicarbonate) and linoleic acid (18:2, n-6) were obtained from Sigma (St Louis, MO). Fetal bovine serum (FBS) was purchased from Dainippon Pharmaceutical (Osaka). Penicillin and streptomycin were products of Gibco BRL (Rockville, MD). Tung oil was a kind gift from Nippon Oil and Fats Co. Ltd (Tokyo). Authentic karela seeds (Momordica charantia) were obtained from the local market in Sendai, Japan. 9Z11E-CLA (9Z11E-18:2, 80% purity), 10E12Z-CLA (10E12Z-18:2, 80% purity) and safflower oil were kindly provided by Rinoru Oil Mills (Nagoya, Japan). 9Z11E-CLA (99% purity) and 10E12Z-CLA (99% purity) were obtained from Cayman Chemical Co. (Ann Arbor, MI). {alpha}-Eleostearic acid (99% purity) was obtained from Larodan Fine Chemicals AB (Malmö, Sweden).

Preparation of tung oil fatty acid, karela seed oil fatty acid and safflower oil fatty acids
The karela seeds were crushed into fine particles and the karela seed oil was extracted by Folch's procedure (14). Tung oil, karela seed oil and safflower oil fatty acids were prepared from tung oil, karela seed oil and safflower oil as also previously reported (11). The three oil fatty acids were stored at –20°C under nitrogen gas after preparation.

Cells and cell cultures
DLD-1 (colorectal adenocarcinoma), HepG2 (hepatoma), A549 (lung adenocarcinoma) and HL-60 (acute promyelocytic leukemia) human tumor cells were obtained from the Cell Resource Center for Biochemical Research at Tohoku University (Sendai, Japan). All tumor cells were cultured in RPMI 1640 medium (containing 0.3 mg/ml L-glutamine and 2.0 mg/ml sodium bicarbonate) supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO2.

Cell proliferation assay using tung oil and karela seed oil fatty acids
Proliferation of the cells was assessed by the WST-1 method (12,15). The cells (DLD-1, HepG2, A549 and HL-60) were seeded onto 96-well culture plates at a density of 1 x 105 cells/well in 100 µl of RPMI 1640 containing 10% FBS. Stock solutions of tung oil fatty acids and karela seed oil fatty acids were prepared in ethanol at a concentration of 25 mg/ml. For the experiments, the tung oil fatty acids and karela seed oil fatty acids were prepared from the stock solution and diluted to final concentrations of 0–25 µg/ml in FBS-free RPMI 1640. The final concentration of ethanol never exceeded 0.1% (v/v). After incubation for 24 h at 37°C, the cells were placed in 100 µl of fresh FBS-free RPMI 1640 medium with various concentrations of tung oil fatty acids and karela seed oil fatty acids. Twenty-four hours later, 10 µl of WST-1 solution was added to each well in order to evaluate cell proliferation. After incubation for 3 h at 37°C, cell proliferation was measured using a microplate reader (Model 550; Bio-Rad, Tokyo, Japan) at a wavelength of 450 nm and a reference wavelength of 655 nm.

Animals and treatments
Male athymic nude mice (BALB/cA Jcl-nu nu/nu, 4 weeks of age) were obtained from Japan Clea (Tokyo, Japan) and maintained in a clean environment. A commercial diet (CL-2) for the mice was purchased from Japan Clea. After acclimatization for 1 week, DLD-1 tumor cells were s.c. inoculated into nude mice. DLD-1 cells in culture were detached by trypsinization and washed with phosphate-buffered saline (PBS). Cell suspensions of 5 x 106 cells in 100 µl of PBS were injected into the posterior region of each mouse using an 18 gauge needle. Two days after tumor cell inoculation, mice were randomly divided into four groups: the control group (n = 10), the 9Z11E-CLA group (n = 10), the 10E12Z-CLA group (n = 10) and the tung group (n = 10), which were subsequently administered safflower oil fatty acid, 9Z11E-CLA, 10E12Z-CLA and tung oil fatty acid, respectively (Table I). Each mouse received 50 mg of test oil administered orally and forcibly once every 2 days. 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 cycle. All procedures were performed in accordance with the Animal Experimentation Guidelines of Tohoku University. After experimental feeding for 32 days, the mice were killed by decapitation and the tumor and liver were excised, weighed and stored at –80°C until assayed.


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Table I. Fatty acid composition of the test oils (9Z11E-CLA, 10E12Z-CLA, tung oil, karela seed oil and safflower oil)

 
Cell proliferation assay with {alpha}-ESA
Proliferation of the cells was assessed by the WST-1 method (see Cell proliferation assay using tung oil and karela seed oil fatty acids). The DLD-1 cells were seeded onto 96-well culture plates. Stock solutions of fatty acids were prepared in ethanol at a concentration of 100 mM. For the experiments, solutions of the fatty acids were prepared from the stock solution and diluted to final concentrations of 0–100 µM in FBS-free RPMI 1640. The final concentration of ethanol never exceeded 0.1% (v/v). After incubation for 24 h at 37°C, the cells were placed in 100 µl of fresh FBS-free RPMI 1640 with various concentrations of linoleic acid (99% purity), 9Z11E-CLA (99% purity), 10E12Z-CLA (99% purity) or {alpha}-ESA (99% purity). Twelve hours later, WST-1 solution was added to each well in order to evaluate cell proliferation. After incubation for 3 h at 37°C, cell proliferation was measured using a microplate reader.

Gas chromatographic (GC) analysis
A known amount of heptadecanoic acid (17:0) (Sigma) was added to each test oil fatty acid as an internal standard and the mixture was methylated with 4% HCl–methanol for 20 min at 60°C to prepare the fatty acid methylesters (13). These were then subjected to gas chromatography (GC 353B gas chromatograph; GL Sciences Inc., Tokyo) using a FID detector and a Supelcowax-10 fused silica capillary column (60 m x 0.32 mm i.d.; Supelco, Bellefonte, PA). The GC conditions were programmed as described previously (13). The total fatty acid compositions of the CLA isomers, tung oil fatty acids, karela seed oil fatty acids and safflower oil fatty acids are shown in Table I.

The tumor and liver were homogenized with four volumes of ice-cold saline. Total lipids from the tumor and liver homogenate were extracted by Folch's procedure, methylated and subjected to gas chromatography. CLA and CLnA were determined by comparison with an internal standard.

DNA fragmentation assay
Tumor tissue was transferred to a glass tube. Next, 1 ml of lysis buffer (5 mM Tris, 20 mM EDTA, 0.5% Triton X-100, pH 8.0) was added and lysis was allowed to occur for 30 min at 4°C. After incubation, the tube was spun at 15 000 r.p.m. for 20 min to separate the intact chromatin from the DNA fragments. Following centrifugation, 1 ml of lysis buffer was added to the pellets. Both the pellets and supernatants were assayed for DNA content using diphenylamine (16). 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.

DLD-1 cells (1 x 105/ml) were seeded onto 10 cm dishes and treated with 20 µM linoleic acid, 9Z11E-CLA, 10E12Z-CLA or {alpha}-ESA for 12 h. For the experiments, the fatty acids were diluted to final concentrations of 0–20 µM in FBS-free RPMI 1640. The final concentration of ethanol never exceeded 0.1% (v/v). DLD-1 cells were transferred to a glass tube and the DNA fragmentation assay was performed as described above.

Determination of phospholipid hydroperoxides
Membranous phospholipid hydroperoxides, i.e. phosphatidylcholine hydroperoxide (PCOOH) and phosphatidylethanolamine hydroperoxide (PEOOH), in mouse tumor and liver were determined by a high performance liquid chromatography with chemiluminescence detection (CL-HPLC) method, as described by Miyazawa et al. (1719). The CL-HPLC system consisted of a Jasco HPLC system (Japan Spectroscopic Co., Tokyo, Japan) combined with a CLD-100 chemiluminescence detector (Tohoku Electronic Industries, Sendai, Japan) 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 v/v/v) and the flow rate was 1.0 ml/min. The luminescent reagent was prepared by dissolving cytochrome c (from horse heart, type IV; Sigma, Tokyo, Japan) and luminol (3-aminophytaloyl hydrazine; Wako Pure Chemicals) in an alkaline borate buffer (pH 10) and was added at a flow rate of 1.2 ml/min. The total lipid extract from the tumor and liver was used as sample. A calibration curve was prepared for PCOOH by photooxidation of synthetic phosphatidylcholine (1-hexadecanoyl-2-[9-cis-octadecadienoyl]-sn-glycero-3-phosphocholine; Sigma).

DLD-1 cells (1 x 105/ml) were seeded onto 10 cm dishes and treated with 0–20 µM {alpha}-ESA, 20 µM linoleic acid, 20 µM 9Z11E-CLA and 20 µM 10E12Z-CLA for 12 h. For the experiments, the fatty acids were diluted to final concentrations of 0–20 µM in FBS-free RPMI 1640. The final concentration of ethanol never exceeded 0.1% (v/v). Total lipid from the DLD-1 cells was extracted by Folch's procedure. The total lipid extract of the cells was used as samples. PCOOH and PEOOH levels in DLD-1 cells were determined using the CL-HPLC method.

TBARS assay
Tumor and liver homogenates were assayed for thiobarbituric acid reactive substances (TBARS) as a conventional index for lipid peroxidation (20,21). Twenty percent tissue homogenate samples (20 µl) were transferred to a glass tube and 4.5 ml of a 0.67% TBA solution was added. The tubes were then screw-capped and spun at 3000 r.p.m. for 5 min. Next, 1.5 ml of the supernatant was transferred to another tube and spun at 15 000 r.p.m. for 5 min at 4°C. The supernatant fluorescence was measured at 553 nm with excitation at 515 nm using a Jasco FP-750. Fluorescence intensity was converted to nmol MDA equivalence based on a standard curve generated with 1,1,3,3-tetraethoxypropane.

DLD-1 cells (1 x 105/ml) were seeded onto 10 cm dishes and treated with 0–20 µM {alpha}-ESA, 20 µM linoleic acid, 20 µM 9Z11E-CLA and 20 µM 10E12Z-CLA for 12 h. For the experiments, the fatty acids were diluted to final concentrations of 0–20 µM in FBS-free RPMI 1640. The final concentration of ethanol never exceeded 0.1% (v/v). The DLD-1 cells were homogenized with four volumes of ice-cold saline and the cell homogenates were assayed for TBARS.

DNA ladder assay in DLD-1 cells
DLD-1 cells (1 x 105/ml) were seeded onto 10 cm dishes treated with 20 µM linoleic acid, 9Z11E-CLA, 10E12Z-CLA or {alpha}-ESA for 12 h and then transferred to a glass tube. For the experiments, the fatty acids were diluted to final concentrations of 0–100 µM in FBS-free RPMI 1640. The final concentration of ethanol never exceeded 0.1% (v/v). Pellets were suspended in lysis buffer (5 mM Tris, 20 mM EDTA, 0.5% Triton X-100, pH 8.0) and incubated for 30 min at 4°C. After incubation, the tube was spun at 15 000 r.p.m. for 20 min to separate the intact chromatin from DNA fragments. Following centrifugation, 2 µl of RNase A (1 mg/ml) was added to the supernatant and the mixture was incubated at 37°C for 1 h. Proteinase K (2 µl, 1 mg/ml) was then added and incubation was continued for an additional 1 h. DNA was precipitated with a mixture of 20 µl of 5 M NaCl and 120 µl of 2-propanol overnight at –20°C. Following centrifugation, pellets were air dried and dissolved in 20 µl of TE buffer (10 mM Tris and 1 mM EDTA, pH 7.4). Extracted DNA was electrophoresed in a 2.0% agarose gel in a mixture of 90 mM Tris, 90 mM boric acid and 2 mM EDTA buffer (pH 8.4) at 100 V. Each gel was stained with ethidium bromide and photographed under UV light.

Determination of caspase enzymatic activity
DLD-1 cells (1 x 105/ml) were seeded onto 10 cm dishes and treated with 20 µM linoleic acid, 9Z11E-CLA, 10E12Z-CLA or {alpha}-ESA for 12 h. For the experiments, the fatty acids were diluted to final concentrations of 0–100 µM in FBS-free RPMI 1640. The final concentration of ethanol never exceeded 0.1% (v/v). The enzymatic activities of caspases 3, 8 and 9 were determined using a Caspase Assay kit (Oncogene Research Products, San Diego, CA, USA).

RNA extraction
For real-time quantitative RT–PCR, total RNA was isolated from 1 x 106 DLD-1 cells 12 h after injection of 0, 10 or 20 µM {alpha}-ESA using an RNeasy Mini Kit (Qiagen, Valencia, CA). For the experiments, the fatty acids were diluted to final concentrations of 0–100 µM in FBS-free RPMI 1640. The final concentration of ethanol never exceeded 0.1% (v/v). The total RNA was eluted with 30 µl of RNase-free water and immediately stored at –70°C. The amount of total RNA was spectrophotometrically determined at 260 and 280 nm. RNA integrity was confirmed by visualizing intact 28S and 18S rRNAs on a denaturing formaldehyde–agarose gel.

Real-time quantitative RT–PCR
The gene expression levels of caspase 3, caspase 8 and caspase 9 mRNA in DLD-1 cells 12 h after injection of 0, 10 or 20 µM {alpha}-ESA were determined with a real-time PCR system (DNA Engine OpticonTM 2 System; MJ Research Inc., Waltham, MA), which allows real-time quantitative detection of the PCR product by measuring the increase in fluorescence caused by the binding of SYBR green to double-stranded DNA. The cDNA was made using a Ready-To-Go T-Primed First-Strand Kit (Amersham Pharmacia Biotech, Piscataway, NJ) from the total RNA in DLD-1 cells 12 h after injection of 0, 10 or 20 µM {alpha}-ESA. The cDNA was subjected to PCR amplification using a DyNAmo SYBY Green qPCR kit (Finnzymes, Espoo, Finland) and gene-specific primers for caspase 3, caspase 8, caspase 9 or for GAPDH (Table II). The real-time PCR was conducted under conditions suitable for the primers, based on the work of Engle et al. (22). The PCR conditions used were 95°C for 5 min and then 95°C for 10 s and 59°C for 50 s over 40 cycles for each gene. Melt curve analysis was performed following each reaction to confirm the presence of only a single reaction product. In addition, representative PCR products were electrophoresed on a 2.0% agarose gel to verify that only a single band was present. The threshold cycle (CT) represents the PCR cycle at which an increase in reporter fluorescence above a baseline signal can first be detected. The ratio between the GAPDH content in standard samples and test samples was defined as the normalization factor.


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Table II. Primer pairs used for the real-time quantitative RT–PCR reaction

 
Antioxidant assay
DLD-1 cells (1 x 105/ml) were seeded onto 96-well dishes and treated with 20 µM {alpha}-ESA, linoleic acid, 9Z11E-CLA or 10E12Z-CLA and 0, 5 or 20 µM {alpha}-tocopherol for 12 h. For the experiments, the fatty acids and {alpha}-tocopherol were diluted to final concentrations of 0–20 µM in FBS-free RPMI 1640. The final concentration of ethanol never exceeded 0.1% (v/v). Cell proliferation was assessed by the WST-1 method (see Cell proliferation assay with {alpha}-ESA), DNA fragmentation was assessed as described above (see DNA fragmentation assay) and caspase 3 mRNA expression 12 h after injection of 20 µM {alpha}-ESA, linoleic acid, 9Z11E-CLA or 10E12Z-CLA and 0, 5 or 20 µM {alpha}-tocopherol was determined using real-time PCR (see RNA extraction and Real-time quantitative RT–PCR).

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


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell proliferation assay with tung oil and karela seed oil fatty acids
To determine which tumor cells and CLnA to use in an in vivo study of tumor growth suppression, four kinds of human tumor cells (DLD-1, HepG2, A549 and HL-60) and two oil fatty acids (tung oil fatty acid and karela seed oil fatty acid) were used in the cell growth study. Tung oil fatty acid and karela seed oil fatty acid both contain high levels of the CLnA {alpha}-ESA (~60–80%) (Table I), and {alpha}-ESA was therefore the major component of the two oil fatty acids. For comparison with the oils containing CLnA, two CLAs containing either 9Z11E-18:2 or 10E12Z-18:2 at a level of ~80%, which are referred to as 9Z11E-CLA and 10E12Z-CLA, respectively, were used.

Fatty acids of tung oil and karela seed oil showed a stronger dose-dependent antitumor effect on cultured human tumor cells, compared with the two CLAs (Figure 2). Compared with karela seed oil fatty acid, tung oil fatty acid contains a higher concentration of {alpha}-ESA and showed a stronger cytotoxic effect on all cells, while 10E12Z-CLA had a stronger cytotoxic effect than 9Z11E-CLA on all cells. Hence, the order of cytotoxic effects was tung oil fatty acid > karela seed oil fatty acid > 10E12Z-CLA > 9Z11E-CLA. Some differences were found between cell lines and both tung oil fatty acid and karela seed oil fatty acid had their strongest effect on DLD-1 and HL-60 cells, exhibiting effects at a concentration as low as 5 µg/ml. Based on these results, it was decided that DLD-1 cells should be transplanted into mice and that tung oil fatty acid would be used as the in vivo test oil.



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Fig. 2. Cell proliferation assay using 9Z11E-CLA (A), 10E12Z-CLA (B), tung oil fatty acid (C) and karela seed oil fatty acid (D) in various human tumor cells. Human tumor cells (A549, DLD-1, HepG2 and HL-60 Cells) were incubated for 24 h after treatment with the fatty acids. Values shown are means ± SD (n = 6). a,b,c,dValues with different superscripts are significantly different at P < 0.05.

 
Effect of tung oil on transplanted DLD-1 cell tumor growth in mice
To evaluate tumor growth suppression by CLnAs in vivo, DLD-1 cells were transplanted into nude mice and four test oils (safflower oil fatty acid, 9Z11E-CLA, 10E12Z-CLA and tung oil fatty acid) were administered to the animals. Each test oil group included 10 mice. Safflower oil fatty acids containing a large amount of linoleic acid was fed to mice of the control group and oils containing 9Z11E-18:2, 10E12Z-18:2 and {alpha}-ESA at a level of ~80% were fed to mice of the 9Z11E-CLA, 10E12Z-CLA and tung groups, respectively (Table I). Test oils were forcibly given once every 2 days for 32 days at a dose of 50 mg/animal. The diet plus 50 mg of the test oil for each mouse is equivalent to a 7–8% fat diet. This is not a high fat diet and is within the normal limits of lipid content. Mice were fed conjugated fatty acids at a level accounting for an average of 1% of their daily diet, and we have previously shown that intake of feed including 1% conjugated fatty acids (CLA and CLnA) had no negative effects in rats (13). Hence, we have used a similar feed including 1% conjugated fatty acids to investigate the antitumor effects of the fatty acids.

DLD-1 cells transplanted into the back of mice had formed a lump 32 days after transplantation (Figure 3A–D). As shown in the photographs, tumor growth was strongly suppressed in mice of the tung group, compared with the other three groups. The extent of growth suppression was tung > 10E12Z-CLA > 9Z11E-CLA, which was similar to the cell culture results. There were no significant differences in body weight between the groups 32 days after oil administration, but the trend for body weight was tung > 10E12Z-CLA > 9Z11E-CLA > control (Figure 3E). This trend was the reverse of tumor weight, i.e. control > 9Z11E-CLA > 10E12Z-CLA > tung group, which suggested that tumor growth suppressed growth of the mouse itself. Based on a tumor weight in the control group of 100%, the weights of the 9Z11E-CLA, 10E12Z-CLA and tung groups were ~90, 60 and 30%, respectively. Hence, the tumor weight of the tung group was significantly less than that of the other three groups. There was no significant difference in liver weight between the four groups. Consequently, the suppressive effect of {alpha}-ESA on tumor growth was confirmed in vivo. This effect was very strong and considerably higher than that of CLA.



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Fig. 3. Backs of nude mice transplanted with DLD-1 cells that received forcible fatty acid medication for 32 days. (A) Transplanted mice fed the control (safflower oil fatty acid) diet; (B) transplanted mice fed the 9Z11E-CLA diet; (C) transplanted mice fed the 10E12Z-CLA diet; (D) transplanted mice fed the tung (tung oil fatty acid) diet. (E) Tumor weight, body weight and liver weight of mice transplanted with DLD-1 cells that were fed a control, 9Z11E-CLA, 10E12Z-CLA or tung diet. Values shown are means ± SD (n = 10). a,b,cValues with different superscripts are significantly different at P < 0.05.

 
To investigate whether conjugated fatty acids given to the animals were distributed throughout the body, 9Z11E-CLA, 10E12Z-CLA and {alpha}-ESA concentrations in the liver and tumors were determined using GC analysis (Table III). Conjugated fatty acid was not given to mice of the control group. 9Z11E-18:2 and 10E12Z-18:2 were detected in the 9Z11E-CLA and 10E12Z-CLA groups, respectively, but the amount of 10E12Z-18:2 detected in the 10E12Z-CLA group was less than the amount of 9Z11E-18:2 in the 9Z11E-CLA group, although the administered amounts were almost the same in the two groups. Both {alpha}-ESA and 9Z11E-CLA were detected in the tung group. However, most {alpha}-ESA had been converted to 9Z11E-CLA in the tung group, with the converted material accounting for ~90% of the total conjugated fatty acid concentration. Hence, it was confirmed that the administered conjugated fatty acids were absorbed and reached the tumor tissue, although the conjugated fatty acid content differed among the groups.


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Table III. Amount of CLA and CLnA in a tumor from and the liver of mice fed the test oil for 32 days

 
To investigate the effect of {alpha}-ESA on transplanted tumor cells, the DNA fragmentation rate was determined in tumor cells that had been transplanted into mice. The DNA fragmentation rate of the tung group was increased significantly compared with those of the other three groups (Figure 4). The fragmentation rate of the 10E12Z-CLA group showed a tendency to increase without showing a significant difference from the control. The rate of the tung group was approximately double that of the control group, which suggested that {alpha}-ESA induced apoptosis in tumor cells, causing suppression of tumor growth.



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Fig. 4. DNA fragmentation in cancer cells from mice transplanted with DLD-1 cells that were fed a control (safflower oil fatty acid), 9Z11E-CLA (9Z11E), 10E12Z-CLA (10E12Z) or tung (tung oil fatty acid) diet. Values shown are means ± SD (n = 10). a,bValues with different superscripts are significantly different at P < 0.05.

 
To clarify the mechanism by which {alpha}-ESA suppressed tumor growth, the amount of membrane phospholipid peroxidation and the TBARS level, both of which are measures of oxidative stress, were determined. The amounts of PCOOH and PEOOH in tumor tissues were significantly increased in the tung group compared with those of the other three groups (Table IV). A slight increase in PCOOH and PEOOH was also found in the 10E12Z-CLA group. There was little difference among the four groups in both the PCOOH and PEOOH levels in the liver. Similar tendencies were found for the TBARS levels (Table V). Hence, the TBARS level in tumor tissues was significantly increased in the tung group compared with those of the other three groups, but there was little difference among the groups in terms of TBARS levels in the liver. From these results it was concluded that {alpha}-ESA induced apoptosis in tumor cells via lipid peroxidation and its effect was specific to tumor cells because normal liver tissue showed no effects.


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Table IV. Phospholipid hydroperoxide concentrations in liver and tumor tissue of mice after oral test oil administration

 

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Table V. TBARS contents of liver and tumor tissue of test oil-administered mice

 
Effect of {alpha}-ESA in DLD-1 cells
It was hypothesized that {alpha}-ESA, a CLnA, induced apoptosis in tumor cells of mice via lipid peroxidation. To verify the hypothesis, a study was conducted to confirm apoptosis in DLD-1 cells and to clarify the relationship between apoptosis and lipid peroxidation. DLD-1 cells were treated with 0–100 µM fatty acids (linoleic acid, 9Z11E-CLA, 10E12Z-CLA or {alpha}-ESA) at a purity of 99% and cell growth 12 h after administration was measured using the WST-1 method. Significant apoptosis was induced in the cells treated with {alpha}-ESA (Figure 5A). After administration of 9Z11E-CLA and 10E12Z-CLA at a dose of 100 µM, the cell survival rates were 80 and 60%, respectively, compared with the control. This was due to growth suppression, rather than to apoptosis. The {alpha}-ESA concentration for 50% suppression of cell growth was ~20 µM. The DNA fragmentation rate at a dose of 20 µM was determined from a DNA ladder, which indicated that DNA fragmentation was advanced in the {alpha}-ESA-treated DLD-1 cells (Figure 5B and C). It was also confirmed that DNA fragmentation was somewhat advanced in the 10E12Z-CLA-treated DLD-1 cells. In addition, the activity of caspases, which are apoptosis-promoting factors, was determined at the same {alpha}-ESA concentration. Increased activities of caspase 3 and caspase 9 was observed in the {alpha}-ESA-treated DLD-1 cells at a dose of 20 µM, compared with those of the other three fatty acid groups (Figure 6A). The activity of caspase 8 also showed a tendency to increase in the {alpha}-ESA-treated cells, but without a significant difference from the other three groups. In the linoleic acid-treated and CLA-treated DLD-1 cells little change was found in caspase activity. The amount of caspase mRNA in the {alpha}-ESA-treated DLD-1 cells was determined using a quantitative PCR and showed a dose-dependent increase (Figure 6B). In particular, the amount of caspase 3 and caspase 9 mRNA was significantly increased by {alpha}-ESA administration. Consequently, it was verified that {alpha}-ESA induced apoptosis via the caspase pathway.



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Fig. 5. Comparison of cytotoxicity (A) and DNA fragmentation (B and C) of linoleic acid (LA), 9Z11E-CLA (9Z11E), 10E12Z-CLA (10E12Z) and {alpha}-eleostearic acid ({alpha}-ESA) in DLD-1 cells. DLD-1 cells were incubated for 12 h after treatment with each fatty acid. (B) DLD-1 cells were incubated for 12 h after treatment with 20 µM LA, 9Z11E-CLA, 10E12Z-CLA and {alpha}-ESA. (C) Agarose gel electrophoresis of low molecular weight DNA extracted from DLD-1 cells. DLD-1 cells were exposed to 20 µM LA, 9Z11E-CLA, 10E12Z-CLA and {alpha}-ESA for 12 h. M, molecular weight markers. Values shown are means ± SD (n = 6). a,bValues with different superscripts are significantly different at P < 0.05.

 


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Fig. 6. Enzymatic activity (CASP3, CASP8 and CASP9) (A) of DLD-1 cells. DLD-1 cells were incubated for 12 h after treatment with 20 µM linoleic acid (LA), 9Z11E-CLA (9Z11E), 10E12Z-CLA (10E12Z) and {alpha}-ESA. mRNA expression (CASP3, CASP8 and CASP9) (B) of DLD-1 cells. DLD-1 cells were incubated for 12 h after treatment with 0–20 µM {alpha}-ESA. Values shown are means ± SD (n = 6). a,b,cValues with different superscripts are significantly different at P < 0.05. CASP3, caspase 3; CASP8, caspase 8; CASP9, caspase 9.

 
To investigate whether apoptosis was induced by {alpha}-ESA via lipid peroxidation, the amounts of membrane phospholipid peroxidation and TBARS, both of which are measures of oxidative stress, were determined. In addition, it was also investigated whether addition of {alpha}-tocopherol, a fat-soluble antioxidant, suppressed apoptosis. The amounts of PCOOH, PEOOH and TBARS were significantly and dose-dependently increased by addition of {alpha}-ESA (Table VI). Addition of linoleic acid, 9Z11E-CLA and 10E12Z-CLA had no effect on the PCOOH, PEOOH and TBARS levels (Tables IVGoVI). Simultaneous addition of both 20 µM {alpha}-ESA and 0–20 µM {alpha}-tocopherol suppressed {alpha}-ESA-induced apoptosis in a dose-dependent manner (Figure 7A) and led to dose-dependent decreases in DNA fragmentation and caspase 3 mRNA and TBARS levels (Figure 7B–D). Concomitantly added linoleic acid, 9Z11E-CLA or 10E12Z-CLA and {alpha}-tocopherol had no effect on the DLD-1 cells. From these results it was verified that {alpha}-ESA induced apoptosis in tumor cells via lipid peroxidation.


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Table VI. Phospholipid hydroperoxide and TBARS concentrations in DLD-1 cells supplemented with {alpha}-ESA

 


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Fig. 7. Comparison of cytotoxicity (A), DNA fragmentation (B), caspase 3 mRNA expression (C) and, TBARS concentrations (D) of DLD-1 cells treated with {alpha}-ESA and {alpha}-tocopherol. DLD-1 cells were incubated for 12 h after treatment with 20 µM {alpha}-ESA, linoleic acid, 9Z11E-CLA and 10E12Z-CLA, with or without 0–20 µM {alpha}-tocopherol. Values shown are means ± SD (n = 6). a,b,cValues with different superscripts are significantly different at P < 0.05. There were no significant differences in the values with or without {alpha}-tocopherol for all fatty acids except for {alpha}-ESA,. Toc, {alpha}-tocopherol; LA, 20 µM linoleic acid; 9Z11E, 20 µM 9Z11E-CLA; 10E12Z, 20 µM 10E12Z-CLA.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study we have taken particular note of the properties of tung oil, a vegetable oil that contains a CLnA that can be prepared in bulk from a natural source. The tumor growth suppression effect of the CLnA was investigated in nude mice into which DLD-1 human colon cancer cells were transplanted, and this effect was compared with that of CLA. The mechanism of tumor growth suppression was then clarified in mice and cultured cells. Consequently, it was confirmed that {alpha}-ESA (9Z11E13E-18:3), a CLnA present in tung oil in large amounts, had a strong suppressive effect on tumor growth and that this occurred through induction of apoptosis via lipid peroxidation. As far as we aware, this is the first study world wide to verify the inhibitory effect of {alpha}-ESA on tumor growth. {alpha}-ESA is much easier to purify than CLA and has a strong antitumor effect. Hence, it is likely to be very useful, particularly since this study and our previous work (13) have shown the safety of {alpha}-ESA in animals. In the Philippines and in Okinawa, a region of Japan known for its long lifespans, people often eat Goya (Momordica charantia), which contains {alpha}-ESA, hence suggesting that {alpha}-ESA might be of significance in promoting longevity.

Most CLA in nature is in the form of 9Z11E-CLA (Figure 1) (1). However, natural sources contain a minute amount of CLA and, therefore, CLA used experimentally is generally prepared from linoleic acid by alkali isomerization. CLA prepared by alkali isomerization consists of two isomers, 9Z11E-CLA and 10E12Z-CLA. Several studies have shown an antitumor effect of CLA prepared by alkali isomerization (1,4,5), but it was only recently verified in a comparative study of the isomers that 9Z11E-CLA has little effect and 10E12Z-CLA, the minor component in nature, has a strong effect (6). This result was confirmed by the current study, which showed that 10E12Z-CLA has a stronger tumor growth suppression effect than 9Z11E-CLA (Figures 2 and 3).

In evaluating the suppressive effect of {alpha}-ESA on tumor growth, 9Z11E-CLA and 10E12Z-CLA were used as positive controls. The results showed that {alpha}-ESA has an extremely strong suppressive effect on tumor growth, compared with the CLAs (Figures 2, 3 and 5). The reported mechanisms underlying the antitumor effects of fatty acids include lipid peroxidation, modulation of eicosanoid production by changes in fatty acid composition and changes in membrane fluidity (23). Some studies have suggested that lipid peroxidation itself induces apoptosis and suppression of cell growth (2431). In tumor tissues of {alpha}-ESA-fed mice and {alpha}-ESA-treated DLD-1 cells, increased amounts of membrane phospholipid peroxidation and TBARS were observed (Tables IVGoVI) and addition of {alpha}-tocopherol, an antioxidant, suppressed oxidative stress and induction of apoptosis (Figure 7). Hence, it was concluded that {alpha}-ESA induced apoptosis in tumor cells via lipid peroxidation and this was consistent with the increased activity and expression of caspases, which are apoptosis-promoting factors (Figures 6 and 7). Therefore, {alpha}-ESA, which is more available and has a stronger physiological effect than CLA, is expected to be effective as a physiologically active lipid.

{alpha}-ESA also had no adverse effects on normal liver tissue (Figure 3 and Tables IV and V). We previously reported that when feed including 1% {alpha}-ESA was given to rats for 4 weeks, no significant differences were found in the lipid components of the plasma and liver or in oxidative stress, compared with rats receiving {alpha}-linolenic acid and CLA (13). These results suggested that {alpha}-ESA induced apoptosis selectively in tumor cells. Antioxidant enzyme expression in tumor cells is weaker than in normal cells and the amount of antioxidants in tumor cells is low (32,33), suggesting that the absence of an effect of {alpha}-ESA on normal tissues is due to normal cells having the capacity to recover from {alpha}-ESA-induced lipid peroxidation.

For application of {alpha}-ESA in humans, the dosage is important. At present, CLA is the only conjugated fatty acid that has been investigated in humans, but no reports show an inhibitory effect of CLA on tumor growth in humans. CLA administered to humans at a dose of 4 g/day for 9–12 weeks shows no serious negative effects (3436). The human dose equivalent to the dose given to mice in the current study is >4 g/day and, therefore, a study of {alpha}-ESA administered at a lower dose needs to be conducted. Furthermore, the combination of conjugated fatty acids with other agents may provide an even stronger antitumor effect, even at a lower dose, and this should also be investigated. It is difficult to obtain conjugated fatty acids (including CLA) from food at a dose of 4 g/day and, therefore, it may be desirable to obtain conjugated fatty acids from added dressings or supplements.

9Z11E-CLA was detected in the {alpha}-ESA-fed mice (Table III) and we have previously shown that {alpha}-ESA is metabolized and converted to 9Z11E-CLA in {alpha}-ESA-fed rats (13). Hence, it was concluded that {alpha}-ESA was also metabolized and converted to 9Z11E-CLA in mice and, similarly to the results of the study in rats, most {alpha}-ESA was metabolized to 9Z11E-CLA (Table III). We originally considered that {alpha}-ESA was converted to 9Z11E-CLA and that CLA suppressed tumor growth. However, although 9Z11E-CLA accumulated in tumor tissues of 9Z11E-CLA-fed mice to a greater extent than in {alpha}-ESA-fed mice, a much stronger suppression effect on tumor growth was observed in the {alpha}-ESA-fed mice (Figure 3). These results suggest that most absorbed {alpha}-ESA was metabolized to 9Z11E-CLA, but that non-absorbed {alpha}-ESA had a strong effect directly on tumor cells. Although approximately the same amounts of 9Z11E-CLA and 10E12Z-CLA were given to mice of the 9Z11E-CLA and 10E12Z-CLA groups, there were great differences in the contents of 9Z11E-CLA and 10E12Z-CLA in the liver and tumor tissues of the 9Z11E-CLA and 10E12Z-CLA groups (Table III), which suggests that there were differences in absorption and metabolism between 9Z11E-CLA and 10E12Z-CLA. It is also a possibility that 10E12Z-CLA was susceptible to various in vivo reactions and, therefore, had a stronger physiological activity than 9Z11E-CLA.

CLA is present in natural sources in only minute amounts, which makes it extremely difficult to purify CLA from such natural sources. Furthermore, it is difficult to separate CLA isomers prepared by alkali isomerization in bulk and, therefore, CLA mixtures are currently on the market only as health supplements. In contrast, CLnA can be purified relatively easily from tung oil and Goya. Furthermore, some seed oils contain CLnA as almost a single component and, therefore, it is easy to conduct studies using these oils (Table I). Therefore, {alpha}-ESA is expected to be a superior health supplement, after its safety is confirmed. {alpha}-ESA may also offer potential therapeutic applications, since our results suggest that it has a stronger suppressive effect on tumor growth.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received November 11, 2003; revised January 26, 2004; accepted February 3, 2004.





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