Chapter 10
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Divergent Cytotoxic Effects of Conjugated Linoleic Acid Isomers on NCI-N87 Cells Sook J. Park, Cherl W. Park, Seek J. Kim, Jung K. Kim, Young R. Kim, Young S. Kim, and Yeong L. Ha Division of Applied Life Science, Graduate School, Institute of Agriculture and Life Sciences, Gyeongsang National University, Chinju 660-701, Korea
Cytotoxic effects of four conjugated linoleic acid ( C L A ) isomers (trans-10,cis-12 C L A ; cis-9,trans-11 C L A ; trans10,trans-12 C L A ; and trans-9,trans-11 C L A ) were evaluated in NCI-N87 gastric cancer cells. NCI-N87 cells were incubated in serum-free medium with 20 μΜ CLA isomers and linoleic acid over a period of 6 days. A l l CLA isomers inhibited the proliferation of the cells, however, linoleic acid did not. Efficacy of trans-9trans-11 C L A in inhibiting the cell proliferation was similar to that of trans-10,trans-12 C L A and was much higher than that of trans-10,cis-12 CLA or cis9,trans-11 C L A . These results indicate that cytotoxicity of trans-9,trans-11 and trans-10,trans-12 C L A (designated trans/trans C L A ) isomers for NCI-N87 cells was stronger than that of their corresponding cis-9,trans-11 CLA or trans-10,cis12 CLA (designated cis/trans C L A ) isomers.
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Conjugated linoleic acid (CLA) is a collective term for positional (9,11 and 10,12) and geometric (trans,cis; cis,trans; cis,cis; and trans,trans) isomers of octadecadienoic acid (C18:2) with a conjugated double bond. Synthetic C L A , prepared from linoleic acid by alkaline isomerization, exhibits potent anticarcinogenic activities in several carcinogen-induced animal models (1-4) and cytotoxicities for several cancer cell lines (5-8). Other biological activities, such as immune stimulation (9), body fat reduction (10,11), modulation of cholesterol content (12) and growth stimulation (13) were also reported. Predominant C L A isomers in the synthetic C L A are trans-\0,cis-\2 C L A and cis-9,trans-\ 1 C L A ; small amounts of trans-\Q,trans-\2 C L A and trans9,trms-\ 1 C L A isomers also occur. There is evidence that C L A isomers exhibit different biological activities in an animal study; Park et al. reported that the trans-\0,cis-\2 C L A isomer is more effective for the reduction of mice body fat than the cis-9,trans-\l C L A isomer (14). However, to date, most studies were accomplished with this C L A isomer mixture, but the activity of each isomer was not well understood. In the present study we examined the cytotoxic effects of four C L A isomers (trans-\0,cis-\2\ cis-9,trans-\\\ trans-lQ,trans-l2 and trans-9,trans-\\ C L A ) on NCI-N87 cells.
MATERIALS AND METHODS
Reagents Linoleic acid and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma Chemical Co. (St. Louis, M O ) . Dulbeccos Modified Eagle's Medium/Hams F-12 nutrient mixture (DMEM/F12) containing 2 μΜ linoleic acid, fetal bovine serum (FBS), trypsin-EDTA, penicillin-streptomycin, selenium and transferrin were purchased from Gibco B R L (Rockville, M D ) . Other chemicals used were of reagent grade.
Synthesis of C L A Mixture
C L A , mainly composed of 47% trans-\0,cis-l2 C L A and 45% cis-9,trans11 C L A , was synthesized from linoleic acid by alkaline isomerization and methylated with 4% sulfuric acid/methanol solution as described by Park et al.
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(15). A round bottom flask (3 L) containing ethylene glycol (1 L ) was maintained at 190 °C for 10 min, followed by cooling down to 165 °C. After carefully adding K O H (250 g) to the round bottom flask, the bottle was heated to 180 °C. The linoleic acid (500 mL) was slowly added and maintained at 180 °C with frequent swirling. One hour later the reactant was cooled to room temperature by a string of running tap water. A l l of the steps were performed under nitrogen. Methanol (500 mL) and 6 N HC1 (1 L) were added to the reactant in order. Synthetic C L A was extracted with hexane (500 mL χ 2) and washed with distilled water (250 mL χ 3). The hexane extract was dried over anhydrous N a S 0 . Synthetic C L A was obtained by removing the hexane solvent under vacuum. 2
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Preparation of Individual C L A Isomers The trans-\0,cis-\2 or cis-9 trans-ll C L A - M e isomer fraction was separated from the synthetic C L A - M e in acetone (100 g/1200 mL) by lowtemperature precipitation at -68 °C and -71 °C. These fractions were refluxed in acetone (1200 mL) containing urea (100 g) for 2 hrs and then crystallized at 4 °C over night (16). The purified trans-\Q,cis-\2 and cis-9,trans-l 1 C L A - M e isomer fractions were saponified by conventional method, followed by isomerizing to their corresponding transJrans isomers by methylation in a boiling water bath for 30 min with 14% BF /methanol. The trans-l0,trans-\2 and trans-9,trans-l 1 C L A - M e fractions recovered by conventional method were further purified by both low-temperature precipitation at -68 °C and urea treatment described by K i m et al. (16) and Park et al. (17). 9
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Analysis of C L A - M e Isomers The purity of C L A isomers was analyzed by G C (Hewlett Packard 5890, Avondale, PA) equipped with a flame ionization detector (FID) and a Supelcowax-10 capillary column (60 m χ 0.32 mm, i.d., 25 μ film thickness). The carrier gas used was N . The oven temperature was increased from 170 °C to 200 °C at a rate of 2 °C/min. The injector and detector temperatures were 240 °C and 260 °C, respectively. The percentage of individual C L A isomers was calculated by the peak area ratio of a given C L A - M e isomer to that of total C L A - M e isomers (Hewlett Packard 3396 Series III, Avondale, PA). C L A - M e isomers of samples were identified by comparison with the relative retention time of standard C L A - M e isomers and by the reported method of H a et al. (/). The purity of trans, trans C L A - M e isomer fractions was analyzed by A g - H P L C (Young-Lin M930 solvent delivery system; Anyang, 2
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Korea), equipped with a U V detector (M720) and ChromSpher 5 analytical silver-impregnated column (4.6 mm i.d. χ 250 mm stainless steel; 5 μ particle size; Chrompack, Bridgewater, NJ). The wavelength of the U V used was 233 nm. The mobile phase was 0.1% acetonitrile in hexane and operated isocratically at a flow rate of 1.0 mL/min by the report of Sehat et al. (18). Composition of trans, trans C L A - M e isomers was calculated by the area ratio of individual isomers.
Cytotoxicity Assay The NCI-N87 cells were maintained in DMEM/F12 medium supplemented with 10% F B S , 100 units/ml penicillin and 100 μg/mL streptomycin in a C 0 incubator (5% C 0 and 95% air, 37 °C). The cells (5 χ 104 cells/1 mL/well) were incubated in a 12-well plate for 24 hrs, followed by replacing the DMEM/F12 medium with serum-free DMEM/F12 medium (SFM) containing 5 μg/mL transferrin and 5 ng/mL selenium. Twenty-four hours later, the medium was replaced again with S F M alone or S F M containing each C L A isomer or linoleic acid, a positive control agent, at a concentration of 20 μΜ. The incubation was continued over a period of 6 days. The number of cells was determined every two days by M T T assay (19). 2
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Results and Discussion The purity of trans-\0,cis-\2, cis-9,trans-ll, trans-\0,trans-\2 and trans9,trans-ll C L A - M e isomers used was 99.0, 90.3, 95.0 and 96.0%, respectively, when analyzed by G C (20). The purity of trans-lQ,cis-l2 and cis-9,trans-\\ C L A - M e fractions separated from the synthetic C L A in acetone at -68 °C and then -71 °C was 92.5 and 87.3%, respectively, but the purity was further increased to 99.0 and 90.3%, respectively, by urea treatment. Impurities, such as saturated fatty acids, oleic acid, oxidation by-products, trans trans-CLA isomers and cis,cis-CLA isomers, were easily removed from the trans-\0,cis-\2 C L A - M e fraction by urea treatment, but not from the cis-9,trans-\l C L A - M e fraction. Interestingly, the trans-\Q,trans-\2 or trans-9,trans-l 1 C L A - M e fraction was not a single compound, but contained a mixture of positional isomers of trans,trans C L A - M e , when analyzed by Ag -HPLC. The trans-\0 trans-\2 CLA-Me fraction contained 2.8% trans-\2,trans-\4, 14.9% trans-\\,trans-\2>, 34.3% trans-lOjrans-12, 33.8% trans-9,trans-\\, 11.6% trans-%,trans-\Q, and 2.6% trans-1 ,trans-9 C L A - M e , whereas the trans-9,trans-\ \ C L A - M e contained 1.5% trans-l2,trans-U, 10.2% trans-\\,trans-\3, 33.8% trans-10, trans-12, 38.5% trans-9,trans-\\ 13.8% trans-8,trans-\0, and 2.2% trans-ΊJrans-9 C L A - M e . 9
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Linoleic acid (20 μΜ) treatment enhanced the proliferation of NCI-N87 cells relative to control. This effect was maximized by 4 days of incubation (142% of control). In contrast, all C L A isomers tested significantly inhibited the proliferation of the cells over a period of 6 days. The efficacy of trans-9, trans-X 1 C L A on inhibition was very similar to that of trans-\0,trans-\2 C L A , but much higher than that of trans-lQ cis-l2 C L A or cis-9,trans-\\ C L A . No significant difference was seen in the efficacy of trans-10,cis-\2 C L A from that of cis9,trans-\ 1 C L A . At day 6, the proliferation rate of the cells treated with 20 μΜ trans-\0,trans-\2 C L A or trans-9,trans-\\ C L A isomer was found to be 83% (17% inhibition) and 62% (38% inhibition), respectively, relative to control. These results suggest that the C L A isomers showed a divergent cytotoxicity against NCI-N87 cells; trans/trans isomers exhibited a stronger effect than their corresponding cis/trans isomers. Many studies have shown that a mixture of C L A isomers inhibits the growth of human cancer cells (5-8), but they did not clarify the inhibitory activity of individual C L A isomers. The mechanistic action of anti-proliferative activity of the mixture of C L A isomers is poorly understood. However, it is, in part, attributed to the alterations in eicosanoid metabolism (21) and/or peroxidation of C L A (7). We believe that eicosanoid metabolism is more strongly altered by trans/trans C L A isomers than cis/trans C L A isomers. The chemical structure of trans/trans C L A isomers closely resembles the straight chain of stearic acid, which is, in turn, preferentially incorporated into the SN-2 position of membrane phospholipids. This might inhibit the process of elongation and desaturation of linoleic acid required for the biosynthesis of arachidonic acid, and thus, alter the eicosanoid metabolisms and/or inhibiting phospholipaseA activity. Further research looking for the mechanistic action of anti-proliferative activity of individual C L A isomers is under way. 9
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Acknowledgements This study was supported by a grant from K O S E F (2000-1-22000-001-3).
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