Concentration- and time-dependenteffects of γ-linolenic acidsupplementation to tumor cells inculture
S. Hrelia,1 A. Pession,2 R. Buda,2 A. Lorenzini,1 D. F. Horrobin,3 P. L. Biagi,1 A. Bordoni1
1Department of Biochemistry ‘G. Moruzzi’, University of Bologna, via Irnerio, 48 40126 Bologna, Italy2Department of Experimental Pathology, University of Bologna, Bologna, Italy3Laxdale Ltd., Stirling, UK
Summary Gamma-linolenic acid (GLA) supplemented to neuroblastoma SK-N-BE, tubal carcinoma TG and coloncarcinorna SW-620 cells was incorporated into phospholipids in all the cell lines (although to different extents), in aconcentration- and time-dependent manner. All the cell lines were able to metabolize GLA to arachidonic acid, SK-N-BE being the most active. Supplementation with low GLA concentrations for short periods was not sufficient to impaircell proliferation; only higher amounts of GLA had an anti-proliferative effect also in short times. In these conditions,the antiproliferative effect of GLA is probably due to cellular dysfunction caused by fatty acid modifications.
The membrane fatty acid composition of cancer cells canbe modified either in culture or during growth in animalswithout disrupting basic membrane or cellular integrity.1
No changes occur in membrane cholesterol, phospholipidor protein content, and only fatty acids are affected; thefatty acid composition of several tumors can be modi-fied sufficiently to alter some of cell functions.2 Thesemodifications in fatty acid composition can be producedin culture by adding specific fatty acids to the growthmedium. The administration of certain polyunsaturatedfatty acids (PUFAs) in the concentration range of 5–60µg/ml has been reported to kill selectively many tumorcell lines in culture.3–6 The mechanism of action of PUFAsis unknown, but may involve increased lipid peroxida-tion, altered rate of prostaglandin synthesis and increasedmembrane fluidity.3,7–10 Each of these changes results indamage to the cells which leads to cell death. Since hasbeen reported that, among n-6 PUFAs, gamma-linolenicacid (18:3 n-6, GLA) and dihomo-gamma linolenic acid
(20:3 n-6) are the most effective cytotoxic agents and
Received 27 January 1999Accepted 1 February 1999
Correspondence to: S. Hrelia
show the greatest selectivity,11 in the present study, wehave evaluated the effects of the supplementation ofthree GLA concentrations on three tumor cell lines in culture after different time of exposure. The aim of thestudy was to point out a relationship between the modifi-cations of the fatty acid composition of the tumor cellphospholipids and alterations of cell proliferation and toverify the presence of concentration- and time-dependenteffects of GLA supplementation.
MATERIALS AND METHODS
Materials
GLA was a kind gift of Callanish Ltd. (Breasclete, UK). Itwas supplied as an oil and the purity was 99%. All otherunlabeled fatty acids were obtained from Nu. Chek.(Elysian, MN, USA) and culture media and sera were fromSigma (St Louis, MN, USA) Methyl-[3H]-thymidine wasfrom the Radiochemical Center (Amersham, UK). All otherchemicals and solvents were of the highest analyticalgrade.
Cells and culture conditions
The three cell lines used were derived from human
235
tumors: the SK-N-BE cell line (kindly provided by
236 Hrelia et al.
Table 1 Fatty acid composition (mol/100 mol) of phospholipids ofSW-620, SK-N-BE, and TG cells
Fatty acid composition analysis (as methyl esters) was performedby gas chromatography as described in Methods. Data are means± S.D. of four different cell cultures. UI=Unsaturation Index.
Professor P. Paolucci, Bologna, Italy) was from a neuro-blastoma, the TG cell line (kindly provided by Dr D. Hernandez-Verdun, Paris, France) was from a tubal carcinoma and the SW-620 cell line (from ATCC,Rockville, Maryland, USA) was from a colon carcinoma.All cell lines were maintained as monolayer cultures inRPMI 1640 (SK-N-BE and TG cell lines) or in Leibowitz-15medium, (SW-620 cell line), supplemented with nonessential amino acids, 100 U/ml penicillin, 100 µg/mlstreptomycin and 10% fetal calf serum. Cells were incu-bated at 37°C in a humidified atmosphere of 5% CO2,95% air. Cells of each line were seeded in 6-well plates(2.5 × 105 cells/well) and after 24 h they were treated withGLA dissolved in ethanol and diluted in medium contain-ing 5% fetal calf serum to give final GLA concentrations of10, 20 and 50 µg/ml. The final ethanol concentration waskept constant at 0.2% and was also added to control wells.After 30, 60, 120 or 180 min of incubation, DNA synthesisand fatty acid analysis were performed.
DNA synthesis
Evaluation of DNA synthesis was performed as previouslyreported.12 Briefly, the samples used to evaluate DNA syn-thesis were pulsed for 15 min with methyl-[3H]-thymidine(0.5 µCi/ml, specific activity 25 µCi/mmol), washed withcold PBS containing 0.1 mM thymidine and treated with0.3 N perchloric acid. Pellets were solubilized with 0.3 NKOH for 60 min at 37°C. Samples were collected and pre-cipitated with 0.6 N perchloric acid, followed by centrifu-gation at 1000 g; pellets were washed twice, and DNAwere extracted with 7% perchloric acid for 15 min at 70°C.The incorporated radioactivity was measured in a beta-counter (Beckman) after addition of scintillation fluid (ReadGel, Beckman). Each experiment was carried out in triplicate.
Fatty acid composition analysis
After GLA supplementation for 30–180 min cells werewashed three times with medium supplemented with 5%fetal calf serum and two times with NaCl 0.9%. Prelimi-nary experiments demonstrated that GLA supplementedwith the medium was completely removed by thesewashes, and that medium was not carried over into theexcized cells. Cells were scraped off in methanol, totallipids were extracted according to Folch et al.13 and phos-pholipids separated by TLC (solvent system:hexane:diethylether:formic acid, 80:20:1, v/v). Phospholipids, visualizedunder UV light after spraying with 2′,7′-dichlorofluores-cein (0.2% w/v in ethanol) were identified by comparisonwith co-chromatographed standards. Fatty acid methylesters were prepared according to Stoffel et al.14 The fattyacid composition of total phospholipids was determined
by gas chromatography as previously reported.15
Prostaglandins, Leukotrienes and Essential Fatty Acids (1999) 60(4), 23
All experiments were done in quadruplicate; data aremeans ± S.D. and statistical analysis was performed usingthe analysis of variance and the Student’s t-test.
RESULTS
The baseline phospholipid fatty acid compositions of SW-620, SK-N-BE and TG cell lines are reported in Table 1.All three cell lines showed a high relative molar contentof saturated fatty acids and monounsaturated fatty acids(particularly oleic acid) and a low content of PUFAs. GLAwas present only in traces (Table 1). GLA supplementationto the culture medium caused significant modifications in the phospholipid fatty acid composition in all cancercell lines, although each of them showed a differentbehaviour. GLA incorporation into phospholipids of thethree tumor cell lines at each concentration used and forall the times of exposure is reported in Figure 1. Althoughin all cell lines GLA content was significantly increased at each concentration used, the amount of GLA incor-porated was dependent on GLA concentration in themedium and on the time of exposure. SW-620 cells wereless sensitive to the supplementation, their GLA relativemolar content being about 50% lower than in the othercell lines after 3 h of 50 µg/ml GLA exposure.
Figure 2 shows the amount of arachidonic acid (AA),the major elongated and desaturated metabolite of GLA,in phospholipids of the three cell lines after GLA supple-mentation. Notwithstanding the significant increase inAA content detected in all cell lines, the cells had not thesame ability to metabolize GLA. In comparison to controlconditions, SK-N-BE cells were able to increase their AA
levels in a dose- and time-dependent manner, while the
Fig. 1 Time-dependent levels of GLA in phospholipids derived fromtumor cell lines supplemented with different GLA concentrations.Data on GLA relative molar content were obtained by gaschromatographic analysis as reported in Methods. Data aremeans±S.D. of four different cell cultures. Statistical analysis wasby the analysis of variance: P< 0.001 at all the GLA concentrations
Fig. 2 Time-dependent levels of AA in phospholipids derived fromtumor cell lines supplemented with different GLA concentrations.Data on AA relative molar content were obtained by gaschromatographic analysis as reported in Methods. Data are means±S.D. of four different cell cultures. Statistical analysis was by theanalysis of variance: P< 0.001 at all the GLA concentrations used
conversion of GLA in SW-620 and TG cells seemed to bedependent on the time of exposure, but not on theamount of GLA supplemented and/or incorporated intophospholipids.
The relative molar content of saturated fatty acids
used and in all the cell lines.
(mainly palmitic and stearic acid) significantly decreased
in the phospholipids of the three cell lines; even oleic acidrelative molar content slightly but significantly decreased(data not shown).
As an effect of the reported modifications in the fattyacid composition, the unsaturation index (UI) significantly
and in all the cell lines.
increased in all cell lines (Fig. 3). In SW-620 cells, the
ndins, Leukotrienes and Essential Fatty Acids (1999) 60(4), 235–241
238 Hrelia et al.
Fig. 3 Time-dependent modifications of the unsaturation index (UI)in phospholipids derived from tumor cell lines supplemented withdifferent GLA concentrations. UI values were obtained by data ongas chromatographic analysis. Data are means±S.D. of fourdifferent cell cultures. Statistical analysis was by the analysis ofvariance: P< 0.001 at all the GLA concentrations used and in all the
Fig. 4 Time-dependent DNA synthesis in tumor cell linessupplemented with different GLA concentrations. DNA synthesiswas evaluated by means of [3H]-thymidine incorporation asdescribed in Methods. The control incorporation (100%) of [3H]-thymidine was 8156±991 d.p.m./µg DNA for SK-N-BE cells,2911±280 d.p.m./µg DNA for TG cells and 2558±169 d.p.m./µgDNA for SW-620 cells. Data are means±S.D. of four different cellcultures. Statistical analysis was by the Student’s t-test: a = P< 0.05; b = P< 0.01; c = P< 0.001, compared to the
increase in the UI was lower than in the other cell lines,due to the lower GLA incorporation and metabolism. InTG cells the increase of UI was mainly due to GLA accu-mulation into phospholipids, while in SK-N-BE cells it was due not only to GLA incorporation but also to its
cell lines.
conversion to AA.
Prostaglandins, Leukotrienes and Essential Fatty Acids (1999) 60(4), 2
The effect of GLA administration on DNA synthesis,evaluated as [3H]-thymidine incorporation is shown inFigure 4. The control incorporation of [3H]-thymidine was2558±169 d.p.m./µg DNA for SW-620 cells, 8156±991 d.p.m./
corresponding unsupplemented cells.
µg DNA for SK-N-BE cells, and 2911±280 d.p.m./µg DNA
for TG cells. In SW-620 cells, supplementation of 10 and20 µg/ml GLA to the medium did not exert any effect oncell growth over one hour incubation; with a longer incu-bation time, GLA supplementation had a stimulatoryeffect on cell growth. The addition of GLA at the highestconcentration (50 µg/ml) resulted in a significant decreasein cell growth, thymidine incorporation being less than40% after the first 30 min incubation in comparison tocontrols. In SK-N-BE cells the behaviour was similar toSW-620 cells for the lowest GLA concentration (10 µg/ml)but the pro-proliferative effect was observed after only 3 h incubation. At 20 µg/ml GLA concentration, SK-N-BEcells showed a lower thymidine incorporation over60 min, then the effect was the opposite of becoming astimulatory one at 180 min. At 50 µg/ml GLA, thymidineincorporation was highly reduced, being less than 15% atthe first 30 min in comparison to unsupplemented cells.Even in TG cells, 10 µg/ml GLA had no effect over 2 hincubation, and a stimulatory effect at 3 h incubation wasobserved. The supplementation of 20 µg/ml GLA to themedium caused a lower thymidine incorporation overone hour, but the effect was reversed at the longest incu-bation time. As in the other cell lines, the most remark-able changes in cell proliferation were observed with 50 µg/ml GLA: the addition of GLA at the highest concen-tration resulted in a general decrease in cell growth inde-pendent of the incubation time, tnymidine incorporationbeing less than 15% at the first 30 min in comparison tocontrol conditions.
DISCUSSION
Membranes are a plausible target for antineoplastic therapy. Previous findings provided a rationale for direct-ing such an approach specifically to membrane fatty acylgroups.1 The membrane fatty acid composition in a neo-plastic cell is not stringently regulated, and substantialmodifications can be produced, both in vivo and in vitro.In cultured cancer cells, the supplementation of PUFAs tothe medium can cause the replacement of the saturatedand monounsaturated fatty acyl groups by polyunsatu-rated groups. The resulting structural changes can modu-late the membrane function and, therefore, influence the response of the neoplastic cell to certain stimuli orperturbations. In vitro inhibition of cell proliferation byPUFAs in several tumor cell lines have been previouslyreported.3–6 GLA has been reported to be able to kill arange of malignant cells,16–18 one key observation beingthat cell death usually did not occur until after 5 days ofincubation.
In order to better clarify GLA effect on tumor cellgrowth, in this study we have verified whether: (1) theGLA cytotoxic effect may be detectable after short time;
(2) the effect is dose-dependent; (3) the effect is accompa-
nied by modifications in the fatty acid composition of cellphospholipids; and (4) the effect is different in differenttumor cell lines.
Robert et al.19 reported that fatty acids supplemented tothe medium of cultured tumor cells were preferentiallyincorporated in the phospholipid fraction, and only asmall amount of added fatty acids was found in the neu-tral lipid fraction, mainly in diacylglycerol. Therefore, we examined GLA incorporation and metabolism in thephospholipid fraction of the three different tumor celllines. Our data clearly show that GLA incorporation intocell phospholipids was dose-dependent, although theamount of GLA incorporated was different among thethree cell lines, SW-620 cells incorporating the fatty acidless than SK-N-BE and TG cells. All the cell lines were ableto elongate/desaturate the incorporated GLA, although todifferent extents, as indicated by the increase in AA level.Similar observations were made by Cantrill et al.20 in v-Kiras transformed NIH-3T3 cells supplemented with GLA.The above described modifications in the phospholipidfatty acid composition, together with the observeddecrease in the saturated and monounsaturated fatty acidcontent, led to modifications in the UI, which increased inall the cell lines. The modifications of the UI value wereless remarkable in SW-620 cells, which incorporated andmetabolized GLA to a lesser extent.
When using 50 µg/ml GLA, a high degree of inhibitionof cell growth was clearly detected in all the cell lines; for this concentration the anti-proliferative effect wasalready significant after 30 min incubation. SW-620 cellsappeared to be less sensitive to this anti-proliferativeeffect; the differences among the three cells lines mayrelate to their different abilities to incorporate and meta-bolize the supplemented GLA. Consequently, as indicatedby the UI, the lipid bilayer was less altered in the SW-620cell line, and so structural changes within the hydro-phobic core of the membrane could exert less influencein the growth of the tumor cells. At low concentrations(10 and 20 µg/ml), after a short incubation time, GLA didnot exert any effect on cell proliferation, apart from aslight inhibition in TG cells; at longer incubation times,GLA exerted a stimulatory effect on cell growth. In ahuman breast cancer cell line, Rose et al.21 showed a stimulatory effect on cell growth at 0.5 µg/ml GLA, while 10 µg/ml GLA produced a mild degree of inhibition onlyover a 6 day incubation period. Possibly, one reason forthe pro-prolifelative effect of low concentrations of GLAmay be the very low content of PUFAs characteristic ofcancer cells, partly due to their low delta-6-desaturaseactivity.22 Supplementation with GLA for short periods is not sufficient to lead to the events which produce its cytotoxicity, but could only by-pass the enzymaticblock, replenishing the phospholipid pool of fatty acids
necessary for cell metabolism and growth. Only higher
ndins, Leukotrienes and Essential Fatty Acids (1999) 60(4), 235–241
240 Hrelia et al.
amounts of GLA result in an anti-proliferative effectwhich also can occur in a short time. In these conditions,the antitumor effect of GLA is probably due to cellulardysfunction caused by fatty acid modifications after GLAincorporation. Whether the short-term increase in cyto-toxicity is correlated with increased lipid peroxidation,altered prostaglandin metabolism and/or perturbation ofthe lipid bilayer leading to modifications in the propertiesof enzymes, receptors and carriers remains to be explored.Jiang et al.23,24 have reported a range of short-term effectsof GLA relatively to membrane properties and activities ofanti-metastatic proteins.
Apart from these hypothesis, our data demonstrate that the effect of GLA supplementation to cancer cells isaccompanied by modifications in the phospholipid fattyacid composition and depends on three variables: thetumor cell line, the concentration of GLA used and thetime of exposure to GLA itself. Independent of the cellline, it is evident that only at a high concentration doesGLA have an inhibitory effect on cell growth after a veryshort exposure time; using lower GLA concentration, theexposure to the fatty acid must be prolonged, since it mayexert a stimulatory effect or no effect when used for shorttimes. These results may also have clinical importance. Inchemiotherapy of malignant tumors, it is desired thatagents attack only tumor cells without exerting anyadverse effect on normal cells. Moreover, resistance ofcancer cell to conventional anticancer drugs is a majorclinical problem, and development of methods by whichsuch resistance is overcome may lead to major clinicalimplications. A marked differential cytostatic effect ofGLA against tumor cells compared to its effects againstnormal cells has been reported,3–5 suggesting that inappropriate conditions selective effects can be achieved.Weber et al.5 have also reported that even human multi-drug-resistant cancer cells are sensitive to treatment with GLA. While our data show the effects of GLA in vitro,recent studies have shown that GLA has a beneficialeffect in patients with cancer.25 Since effective anticancerconcentrations of GLA can be achieved in vivo,26,27 GLAtreatment may be a useful adjunct to certain currentlyavailable therapies when provided in the right modalities.
ACKNOWLEDGEMENTS
This work was supported by grants from M.U.R.S.T. (Italy) 60%and 40%.
REFERENCES
1. Spector A. A., Burns C. P. Biological and therapeutic potential ofmembrane lipid modification in tumors. Cancer Res 1987;47: 4529–4537.
2. Burns C. P., Spector A. A. Membrane fatty acid modification in
tumor cells: a potential therapeutic adjunct. Lipids 1987;22: 178–184.
Prostaglandins, Leukotrienes and Essential Fatty Acids (1999) 60(4), 23
3. Begin M. F., Ells G., Das U. N., Horrobin D. F. Polyunsaturatedfatty acids induced cytotoxicity against tumor cells and itsrelationship to lipid peroxidation. Natl Cancer Inst 1986;77: 1053–1062.
4. Horrobin D. F. Essential fatty acids, lipid peroxidation andcancer. In: Horrobin D. F. (Ed). Omega-6 essential fatty acids:pathophysiology and roles in clinical medicine. New York:Wiley-Liss, 1990: 351–377.
5. Weber J. M., Sircar S., Begin M. E. Greater sensitivity of humanmultidrug-resistant (mdr) cancer cells to polyunsaturated fattyacids than their non-mdr counterparts. J Natl Cancer Inst 1994;86: 638–639.
6. Jiang W. G., Hiscox S., Hallett M. B., Horrobin D. F., Mansel R. E.,Puntis C. A. Regulation of the expression of E-cadherin onhuman cancer cells by gamma-linolenic acid (GLA). Cancer Res1995; 55: 5043–5048.
7. Begin M. E. Fatty acids, lipid peroxidation and diseases. ProcNutr Soc 1990; 49: 261–267.
8. Takeda S., Horrobin D. F., Manku M., Sim P. G., Ells G., SimmonsW. Lipid peroxidation in human breast cancer cells in responseto gamma-linolenic acid and iron. Anticancer Res 1992;12: 329–333.
9. Takeda S., Sim P. G., Horrobin D. F., Sanford T., Chisholm K. A.,Simmons V. Mechanism of lipid peroxidation in cancer cells inresponse to gamma-linolenic acid (GLA) analyzed by GC-MS (I):conjungated dienes with peroxyl (or hydroperoxyl) groups andcell-killing effects. Anticancer Res 1993; 13: 193–199.
10. Chapman G. E., Wallace H. M. Spermine prevents lipidperoxidation induced by essential fatty acids in human breastcancer cells. Biochem Soc Transaction 1994; 22: 401S.
11. Begin M. E., Sircar S., Weber J. M. Differential sensitivity oftumorigenic and genetically related non-tumorigenic cells tocytotoxic polyunsaturated fatty acids. Anticancer Res 1989;9: 1049–1052.
12. Derenzini M., Pession A., Farabegoli F., Trerè D., Badiali M.,Dehan P. Relationship between interphasic nucleolar organizerregions and growth rate in two neuroblastoma cell lines.American J Path 1989; 134: 925–932.
13. Folch J, Lees M., Sloane-Stanley G. H. A simple method for theisolation and purification of total lipids from animal tissues. J Biol Chem 1957; 226: 497–509.
14. Stoffel W., Chu F., Ahrens E. H. Jr. Analysis of long chain fattyacids by gas liquid chromatography. Anal Chem 1959;31: 307–311.
15. Biagi P. L., Bordoni A., Hrelia S., Celadon M., Horrobin D. F.Gamma-linolenic acid dietary supplementation can reverse theaging influence on rat liver microsome delta-6-desaturaseactivity. Biochim Biophys Acta 1991; 1083: 187–192.
16. Ramanathan R., Das N. P., Tan C. H. Effects of gamma linolenicacid, flavonoids and vitamins on cytotoxicity and lipidperoxidation. Free Rad Biol Med 1994; 16: 43–48.
17. Booyens J., Dippenaar N., Fabri D. The effect of theprostaglandin precursor gamma-linolenic acid on the rate ofproliferation of human osteogenic sarcoma and oesophagealcarcinoma cells in culture. South Afr Med J 1984; 65: 240–242.
18. Girao L. A., Cantrill R. C., Rock A. C., Davidson B. C. Theinfluence of C18 fatty acids on the growth of a 3T6 derived cellline. Anticancer Res 1987; 7: 133–138.
19. Robert J., Montaudon D., Hugues P. Incorporation andmetabolism of exogenous fatty acids by cultured normal andtumoral glial cells. Biochim Biophys Acta 1983; 752: 383–395.
20. Cantrill R. C., Ells G. W., deAntueno R. J., Elliot M., Raha S. K.,Horrobin D. F. The effect of n-6 fatty acids on normal and v-Kiras transformed NIH-3T3 cells. Anticancer Res 1992;
21. Rose D. P., Connolly J. M., Liu X. H. Effects of linoleic acid andgamma-linolenic acid on the growth and metastasis of a humanbreast cancer cell line in nude mice and on its growth andinvasive capacity in vitro. Nutr Cancer 1995; 24: 33–45.
22. Hrelia S., Bordoni A., Biagi P. L., Galeotti T., Palombini G.,Masotti L. The role of delta-6 and delta-9-desaturase in the fattyacid metabolism of hepatomas with different growth rate.Biochem Mol Biol Int 1994; 34: 449–445.
23. Jiang W. G., Bryce R. P., Horrobin D. F., Mansel R. E. Gamma-linolenic acid blocks cell cycle progression by regulatingphosphorylation of p27kip1 and p57kip2 and their interactionswith other cycle regulators in cancel cells. Int J Oncol 1998;13: 611–617.
24. Jiang W. G., Hiscox S., Horrobin D. F., Bryce R. P., Mansel E.
Gamma linolenic acid regulates expression of maspin and themotility of cancer cells. Biochem Biophys Res Commun 1997;237: 639–644.
25. Fearon K. C. H., Falconer J. S., Ross J. A., Carter D. C., HunterJ. O., Reynolds P. D., Tuffnell O. An open-label phase I/II doseescalation study of the treament of pancreatic cancer usinglithium gammalinolenate. Anticancer Res 1996; 16: 867–874.
26. Matsuzaki A., Shimura H., Okuda A., Ohtsu M., Sazaki M.,Onodera K., Kimura G. Mechanism of selective killing bydilinoleoylglycerol of cells transformed by the E1A gene ofadenovirus type 12. Cancer Res 1998; 49: 5702–5707.
27. Naidu M. R., Das U. N., Kishan A. Intratumoral gamma-linolenicacid therapy of human gliomas. Prostaglandins Leukot Essent
Fatty Acids 1992; 45: 181–184.
ndins, Leukotrienes and Essential Fatty Acids (1999) 60(4), 235–241
