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Uptake and metabolic conversion of saturated and unsaturated fatty acids in Hep2 human larynx tumor cells
Prostaglandins, Leukotrienes and Essential FattyAcids (2001) 65(5&6), 295^300 & 2001 Harcourt Publishers Ltd doi:10.1054/plef.2001.0328, available online at http://www.idealibrary.com on
Uptake and metabolic conversion of saturated and unsaturated fatty acids in Hep2 human larynx tumor cells L. Albino, M. P. Polo, M. G. de Bravo,1 M. J.T. de Alaniz1 Instituto de Investigaciones Bioqu|¤ micas de La Plata (INIBIOLP), CONICET-UNLP, Facultad de Ciencias Me¤dicas, La Plata, Argentina
Summary Research on fatty acid metabolism in cultured human larynx tumor cells Hep2 was carried out.The cells were incubated with either a saturated (palmitic) or a polyunsaturated (linoleic, a-linolenic and eicosatrienoic (n-6)) radioactive fatty acid (0.66 mM, 24 h).The best incorporation capacity was observed in the linoleic acid followed by a-linolenic, palmitic and eicosatrienoic acids. All fatty acids tested were anabolized to higher derivatives within their own family. Palmitic acid was primarily monodesaturated rather than elongated, proving to have a very active D9 desaturase activity.With respect to polyunsaturated acid metabolism, the conversion of a-linolenic acid to higher homologs, although better than linoleic acid, occurred far less efficiently than that observed in other non-highly undifferentiated human tumor cells.This impairment in higher polyunsaturated fatty acid biosynthesis, reflected in the low levels of arachidonic acid in the fatty acid composition, would not reside in the D5 desaturation step since Hep2 cells can readily convert eicosatrienoic acid into arachidonic acid. Considering the potential regulatory role of specific polyunsaturated fatty acids in the cell proliferative control, the knowledge of the metabolism of fatty acids in this human tumor cell would be important for designing future experiments in order to clarify the mechanism involved in balance, proliferation and cell death. & 2001Harcourt Publishers Ltd
INTRODUCTION Most cellular functions depend on the structural and dynamic nature of cellular membranes. Fatty acids, particularly polyunsaturated fatty acids, are an integral part of the membranes. The acids regulate membrane functions through their contribution to membrane fluidity and their physicochemical properties.1–4 The specialized functions of differentiated mammalian cells require the utilization of various fatty acids that are selectively incorporated into the different classes of membrane phospholipids, after undergoing several metabolic pathways. Within the cells, the endoplasmic
Received 20 August 2001 Accepted 22 October 2001 Correspondence to: Dr M. J.T. de Alaniz.INIBIOLP, Fac. Ciencias Me¤dicas,Calle 60 y 120, (1900) La Plata, Argentina.Tel.: +54-0221-4834833; Fax: +54-02214258988; E-mail: [email protected] This work was supported in part by grants from CONICETand CIC, Argentina. 1 Members of the Carrera del Investigador Cient|¤ fico, Consejo Nacional de Investigaciones Cient|¤ ficas yTe¤cnicas, Argentina.
& 2001Harcourt Publishers Ltd
reticulum membrane is the site of residence of the elongating fatty acid enzymes and the D9, D6, and D5 desaturases5 that evoke unsaturated fatty acid biosynthesis and polyunsaturation, on which structure and fluidity of the membrane depends. Neoplastic cells require lipids in order to build their membrane structures at sufficient velocity to allow rapid growth. There exist important differences in lipid metabolism between normal and neoplastic tissue.6 One of these striking differences is a low level of D6 desaturase present in most cancer cells7–9 and there is a strong correlation between the degree of cell proliferation inhibition and the degree of unsaturation of 18-carbon fatty acids.10 However, the biosynthetic capacity of human tumor cells concerning the desaturation and elongation of fatty acids is still poorly understood. In order to elucidate the biochemical routes of polyunsaturated fatty acid biosynthesis in the human species in particular, we supplemented normal growth medium of the human laryngeal carcinoma (Hep2) cells with a chemically defined (i.e., serum-free) one. The chemically defined medium contained a given precursor fatty acid of
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which we examined the incorporation into various lipid classes along with its concomitant anabolism to higher polyenoic acid derivatives.
MATERIALS AND METHODS
Chemicals Radiochemicals were obtained from New England Nuclear (Boston, MA). Solvents and chemicals were obtained from Merk (Darmstadt, Germany) and Sigma Chemical Co. (St Louis, MO).
Culture conditions Stock cultures of human laryngeal carcinoma (Hep2) cell line were maintained and grown at 371C in confluent layers attached to 95 cm2 flasks on Eagle’s MEM supplemented with 10% calf serum (both from Gibco, Grand Island, NY). The cells were detached from the glass surface using a 0.25% trypsin solution, washed and suspended in isotonic saline solution and counted in a hemocytometer. Aliquots of 2.5 106 cells were seeded into 95 cm2 glass tissue culture and incubated in 15 ml of Eagle’s MEM containing 10% fetal calf serum for 48 h. Then, the medium was washed three times with 5 ml of cold PBS solution (295 mOsm/Kg H2O, pH 7.4) and incubated in 15 ml of serum-free MEM Zinc option (IMEM Zo)11 supplemented with 10 nmoles of palmitic[1-14C], linoleic[1-14C], a-linolenic[1-14C] or eicosatrienoic [1-14C] acids for 24 h. All radioactive fatty acids were added as their sodium salt bound to delipidated albumin according to Spector et al.12 in a ratio of 2 nmoles of fatty acid to 1 nmol of albumin. At the end of the experiment, the attached cells were washed three times with 5 ml of ice-cold saline solution, detached from the growing surface mechanically with a rubber-tipped spatula, and pelleted at 500g for 10 min. The sedimented cells were resuspended in 5 ml of the same solution. An aliquot of the resulting suspension was used to determine cellular protein content.13 The rest of the cell material was centrifuged in the same way, the supernatant discarded and the pellet processed for lipid analysis.
Cellular lipid extraction and separation Lipids were extracted from the pellets using the method of Folch et al.14 An aliquot of the organic phase was used to separate phospholipids and neutral lipids following the method of Wren.15 Different classes of phospholipids were further partitioned by TLC on 0.25 mm thick silica gel G-60 plates by means of a solvent mixture containing chloroform : methanol : acetic acid : water (50 : 37.5 : 3.5 : 2, v : v : v : v).16 The neutral lipids were also partitioned by
TLC system using as a mobile phase petroleum ether : ethyl ether : acetic acid (80 : 20 : 1, v : v : v).17 The different subclasses of lipids were identified by means of standard mixture run in parallel with the samples. After development, TLC plates were dried and scanned for radioactivity (radio-TLC) using a TLC-Proportional Radioactivity Scanner, (Berthold LB-2832 Wildbard, Germany), equipped with a Hewlett-Packard 3396-A Data Station. Another aliquot from the cell organic phase was methylated and analyzed by gas–liquid chromato graphy.18 The distribution of radioactivity among the fatty acid methyl esters (FAME) was analyzed using the radiochromatograph (Varian Star 3400 CX) in tandem with a proportional gas detector (GC-Ram; Inus System Inc. Tampa, FL). A 6-foot glass column packed with 10% (w/w) SP-2330 on 100–200 mesh chromosorb WAWDMCS (Supelco Inc., Bellefonte, PA). After 1 min initial hold, the oven was programmed from 145 to 2201C at 11C/min. The fatty-acid methyl esters (FAME) were identified by comparison of their relative retention times with authentic standards, while the radioactivity distribution was calculated electronically by quantification of the peak areas. An aliquot of the organic phase was also used to measure total radioactivity incorporated into the cells, using a Wallac 1214 Rackbeta liquid scintillation counter (Pharmacia, Turku, Finland) with a 97% efficiency for 14C, interfaced to an Olivetti M-240 computer system. Data are reported as the mean and standard error calculated from 3–5 independent analyses. RESULTS The analysis of the total fatty acid composition of human laryngeal carcinoma (Hep2) cells maintained on Eagle’s MEM supplemented with 10% (v/v) fetal bovine serum is presented in Table 1. It is hallmarked by high levels of saturated and monounsaturated fatty acids together with low levels of linoleic and polyunsaturated fatty acids. Table 1 also shows the fatty acid composition of serum fraction of the culture medium on which the cells were grown. This serum fraction constitutes the cells’ only source of exogenous lipids. As can be seen, the profile of this fatty acid composition strongly differs from that of the cells. In order to test the ability of Hep2 cells to incorporate and metabolize labeled fatty acid precursors from the medium, cultures were incubated with specific radio labeled fatty acid precursors, and the cellular lipids were further analyzed for the distribution of radioactivity among the endogenous polyunsaturated fatty acids (PUFAs). Under such conditions all labeled fatty acids tested (16 : 0, 18 : 2 (n-6), 18 : 3 (n-3) and 20 : 3 (n-6)) were taken up by the cells (Fig. 1). As seen, linoleic acid showed
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Table 1 Percentage distribution of fatty acid within the total lipids of Hep2 human larynx cells Fatty acids
Medium
Cells
14 : 0 15 : 0 16 : 0 16 :1 18 : 0 18 :1 (n-9) 18 :1 (n-7) 18 : 2 (n-6) 18 : 4 (n-3) 20 : 0 20 : 3 (n-6) 20 : 4 (n-6) 20 : 5 (n-3) 22 : 2 (n-6) 22 : 3 (n-6) 22 : 5 (n-3) 22 : 6 (n-3)
0.7 ND 27.1 3.0 14.4 18.4 3.3 5.4 2.8 ND 2.6 9.7 1.1 2.1 ND 4.3 5.1
1.470.1 1.870.4 21.271.8 7.670.9 10.470.4 21.373.8 11.072.5 3.870.2 ND 1.270.5 1.370.3 3.370.6 ND 7.372.5 1.971.1 1.670.4 4.970.8
Cells were grown at 371C in MEM containing10% fetal bovine serum. Results are given as means7SE (n=4) in one of three similar experiments. ND: Not Detected.
the best incorporation capacity followed by a-linolenic, palmitic and eicosatrienoic acids. These incorporated fatty acids were subsequently actively metabolized. Palmitic acid (Fig. 2) was desaturated at the D9 position to palmitoleic (16 : 1), and this acid was in turn further elongated to give oleic acid (18 : 1). However, 16 : 0 might be firstly elongated to 18 : 0, and then desaturated at the 9 position giving rise to oleic acid. Figure 3A shows that in these tumor cells, despite the large uptake of [1-14C]linoleic acid, very small amounts of arachidonic acid were formed. Besides, the linoleate chain elongation product, eicosa-11,14-dienoate and its de saturated derivative, eicosatrienoic acid, could be detected. In spite of these observations, a different pattern of anabolic conversion was carried out with a-linolenate[1-14C] as a metabolic precursor: the conversion to the elongated-plus-desaturated product (eicosa-8, 11, 14, 17) occurred more readily, followed by a small amount of its further D5 desaturation analogue eicosapentaenoic acid (Fig. 3B). Figure 4 shows that Hep2 cells can actively convert eicosatrienoic acid (n-6) into arachidonate, demonstrating that this human line has a very active D5 desaturase. The percentage distribution of radioactivity found in the major lipid class of the Hep2 cells after their incubation with [14C]palmitic or [14C]eicosatrienoic acids is presented in Table 2. Under such conditions both precursors and their corresponding metabolic products were predominantly incorporated into the phospholipid fraction, preferentially into phosphatidyl-choline and phosphatidyl-ethanolamine. Palmitic acid and its deriva& 2001Harcourt Publishers Ltd
Fig. 1 Uptake of radioactivity in Hep2 cells incubated in the presence of various [1-14C] fatty acids. Results are expressed as dpm per mg of cellular protein (mean7SE). All fatty acids were added at 0.66 mM concentration as sodium salt bound to delipidated albumin for 24 h. Technical details are described in Methods.
Fig. 2 Percentage distribution of radioactive metabolic products obtained after incubation of Hep2 cells with [1-14C]palmitic acid. Each bar represents the mean7SE (n¼5).The substrate was added as sodium salt bound to delipidated albumin at a final concentration of 0.66 mM, and incubated with the cells for 24 h. Radiochromatographic analyses of the total cellular lipids were performed as indicated in Methods.
tives were better incorporated into neutral lipids compared to those from eicosatrienoic acid. Within the different phospholipid subclasses, 16 : 0 as well as 20 : 3 acids were incorporated mainly into phosphatidyl-choline and phosphatidyl-ethanolamine fractions. However, in spite of the fact that both substrates were found in considerable amounts in
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Fig. 4 Percent distribution of radioactive metabolic products obtained after incubation of Hep2 cellswith [1-14C]eicosatrienoic acid. Each bar represents the mean7SE (n¼5).The substrate was added as sodium salt bound to delipidated albumin at a final concentration of 0.66 mM, and incubated with the cells for 24 h. Radiochromatographic analyses of the total cellular lipids were performed as indicated in Methods. Fig. 3 Percentage distribution of radioactive metabolic products obtained after incubation of Hep2 cells with [1-14C]linoleic (A) or [1-14C]a-linolenic (B) fatty acids. Each bar represents the mean7SE (n¼5).The substrate was added as sodium salt bound to delipidated albumin at a final concentration of 0.66 mM, and incubated with the cells for 24 h. Radiochromatographic analyses of the total cellular lipids were performed as indicated in Methods.
phosphatidyl-inositol, eicosatrienoic and its D5 desaturation product arachidonic acid were mostly incorporated into this phospholipid fraction rather than palmitic acid and its metabolic products. Conversely, the radioactive products resulting from palmitic acid incubation of the cells were largely incorporated into neutral lipids compared to those from eicosatrienoic acid supplementation. DISCUSSION It has been shown frequently that the fatty acid composition of tumor cells differs from that of normal cells. In this respect, the low levels of polyene content, particularly the high levels of monounsaturated fatty acids found in human laryngeal carcinoma (Hep2) cells (Table 1), are in accordance with the most frequently reported abnormalities in lipids in neoplasms.19–22 The
Table 2 Distribution of radiactivity within the lipid subclasses of Hep2 after incubation in the presence of various [14C] fatty acids [14C] Fatty acid incubated
PS PI SM PE PC DAG FFA TAG CHOE [14C]Phospholipids/ [14C]Neutral lipids
16 : 0
20 : 3 (n-6)
2.570.3 6.971.1 7.672.4 34.074.8 35.371.1 5.470.7 0.970.1 5.871.1 1.370.5
5.070.2 14.973.0 10.371.5 26.072.3 36.371.9 2.470.1 0.670.04 3.670.1 0.970.3
6.970.9
12.671.1
Results are expressed as the percentage distribution of the total radioactivity incorporated into the cells (mean of four independent determinations7SEM).The cells were incubated in the presence of 1 mM [14C]fatty acid for 24 h and then processed as indicated in Materials and Methods.
differences observed in the fatty acid composition between medium (lipids of calf serum) and Hep2 cells demonstrate that these cells not only take up the fatty acids from the medium, but also modify their
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composition by means of a selective incorporation or metabolization. After 24 h of culture in the presence of 0.66 mM of the different labeled fatty acids, all of them were taken up by the cells (Fig. 1), linoleic acid being the bestincorporated, followed by a-linolenic, palmitic and eicosatrienoic acids. It is generally accepted that neoplastic cells are able to synthesize monounsaturated fatty acids.19,22–24 Moreover, several authors reported that the monoenoic/ saturated fatty acid ratio proved to be a useful marker reflecting both growth rate and malignancy in different human neoplasms.25–27 In this kind of tumor, palmitic acid was primarily desaturated rather than elongated proving to have a very active D9 desaturase (Fig. 2). Palmitoleic acid was further elongated to give oleic acid. However, 16 : 0 might be firstly elongated to 18 : 0, and then desaturated at the 9 position giving rise to oleic acid. These results markedly differ from those reported in other highly undifferentiated neoplastic cells19 in which palmitic acid is mainly elongated rather than desaturated. The metabolic conversions of essential fatty acids linoleic (n-6 series) and a-linolenic acids (n-3 series) in neoplastic cells are of particular interest since they are the precursors of arachidonic (n-6), eicosapentaenoic and docosahexaenoic acids (n-3) related to cell proliferation inhibition and induction of apoptosis.28 Despite the fact that linoleic acid was the bestincorporated precursor, its conversion to higher homologs was very low and the acid was mainly elongated rather than desaturated (Fig. 3A). However, when a-linolenic acid was the substrate, the conversion into higher homologs occurred far more readily (Fig. 3B), indicating that these cells possess a D6 desaturase capacity for a-linolenic acid despite their largely dimi nished ability to perform the analogous reaction with linoleate. As stated before in human liver microsomes,29 as well as in different cell-culture lines,19,23,30–32 the D6 desaturase enzyme exhibits a marked preference for a-linolenic acid over linoleate as a substrate. In consequence, given an even greater diminution in the conversion of the latter, as in this work, the level of formation of higher metabolic products becomes extremely low. However, the conversion of both linoleic and a-linolenic acids into higher-chain polyunsaturated fatty acids, initiated by a desaturation at the D6 position, occurred far less efficiently than that observed in other non-highly undifferentiated human tumor cells.22 As described previously,7–9 very low levels of D6 desaturase are present in cancer cells. Insufficient D6 desaturase may cause a decrease in g-linolenic acid and subsequently arachidonic acid (AA) levels. It has also been postulated that the loss of proliferative control in tumor cells is the consequence of a depletion of cellular & 2001Harcourt Publishers Ltd
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AA, and that exogenous AA and n-6 fatty acid may restore control of proliferation.9,33 The last step in the synthesis of AA from linoleate is catalyzed by D5 desaturase. This enzyme was shown to be present not only in Hep2 cells (Fig. 4), but also in all cells tested, including those that were deficient in D6 desaturase.34 This observation supports the conclusion that the metabolic block in arachidonic acid biosynthesis from linoleate as precursor, must reside in a prior anabolic step. The analysis of the percentage distribution of radioactivity found in the major lipid class of the Hep2 cells after incubation with labeled palmitic or eicosatrienoic acid presented in Table 2, revealed that both precursors were predominantly incorporated in one phospholipid fraction. However, the ratio of the total radioactivity found in the phospholipid classes (Table 2, rows 1–5), compared to that detected in the neutral-lipid species (Table 2, rows 6–9) and present in row 10 of this Table, was shown to be 2-fold higher when the cells were incubated with the polyunsaturated precursor eicosatrienoic acid than when the saturated acid (palmitic) was incubated. These observations, similar to those reported in human hepatoma cells in culture,19 are consistent with what might be expected from the known requirement for polyunsaturated acids within the phospholipid of cellular membranes for the maintenance of normal fluidity within their lipid phases. The differences observed between our results and those previously reported by Colquhoun et al. with Hep2 cells35 may be due to the incubation time used by these authors (only 6 h). In conclusion, we have shown that human larynx tumor cells Hep2 are able to take up both saturated and unsaturated fatty acids from the medium. These species and their metabolic products were incorporated into each basic lipid class as well as into each of the principal phospholipid subclasses in cellular membranes in a similar rate to that observed in other kinds of cells in culture. Radioactive precursors were anabolized to higher homologs and analogs within their own family, demonstrating the presence of D9, D6, and D5 desaturases. However, as shown in other undifferentiated cells,7,19 low levels of D6 desaturase were detected, and they were reflected in the low levels of AA in the fatty acid composition. If AA mediates ceramide-induced apoptosis, the absence of AA in tumor cells may make these cells resistant to apoptosis induction. This impairment in the biosynthesis of AA does not arise from the D5 desaturation step since Hep2 cells can readily convert eicosatrienoic into arachidonic acid. In consequence, we can assume that the impairment in the biosynthesis of polyunsaturated fatty acid would be an important feature in the mechanism related to the loss of proliferative control.
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ACKNOWLEDGMENTS The authors are grateful to Elsa Claverie for her technical assistance and to Norma Tedesco for language revision.
REFERENCES 1. Stubbs C. D., Smith A. D. The modification of mammalian membrane polyunsaturated fatty aid composition in relation to membrane fluidity and functions. Biochim Biophys Acta 1984; 779: 89–137. 2. Spector A. A., York M. A. Membrane lipid composition and cellular function. J Lipid Res 1985; 26: 1015–1035. 3. Brenner R. R. Effect of unsaturated acids on membrane structure and enzyme kinetics. Prog Lipid Res 1984; 23: 69–96. 4. Leger C. L. Physicochemical approach of the function of the fatty acid incorporation in biological membranes. Prostaglandins Leukot Essent Fatty Acids 1993; 48: 17–21. 5. Brenner R. R. The oxidative desaturation of unsaturated fatty acids in animals. Mol Cell Biochem 1974; 3: 41–52. 6. Weinhouse S., Langan J., Saton J. A., Morris H. P. Fatty acids as metabolic fuels of cancer cells. In: Wood R, ed. Tumor Lipids: Biochemistry and metabolism. Champaign IL: A.O.C.S. 1973; 14–20. 7. Dunbar L. M., Bailey J. M. Enzyme deletion and essential fatty acid metabolism in cultured cells. J Biol Chem 1975; 250: 1152. 8. de Alaniz M. J. T., de Gomez Dumm I. N. T., Brenner R. R. Biosynthesis of polyunsaturated fatty acid from linoleic acid family in cultured cells. In: Baza´n N. G., Brenner R. R., Giusto N., eds. Adv Exp Med Biol New York: Plennum Press, 1977; 617–624. 9. Horrobin D. F. Essential fatty acids, lipid peroxidation and cancer. In: Horrobin D. F., ed. Omega-6, Essential Fatty Acids. New York: Alan R. Liss Inc., 1990; 351–377. 10. Colquhoun A. Effects of saturated and polyunsaturated fatty acids on human tumor-cell proliferation. Gen Pharmacol 1998; 30: 191–194. 11. Richter A., Sanford R. R., Evans V. J. Influence of oxygen and culture media on plating efficiency of some mammalian tissue cells. J Natl Cancer Inst 1972; 49: 1705–1712. 12. Spector A. A., Steinberg O. S., Tanaka A. Uptake of free fatty acids by Erlich ascites tumor cells. J Biol Chem 1965; 240: 1032–1041. 13. Lowry O. H., Rosebrough N. J., Farr A. L., Randall R. J. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265–275. 14. Folch J., Lees M., Stanley S. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 1957; 226: 497–503. 15. Wren J. J. Chromatography of lipids on silicic acid. J Chromatogr 1960; 4: 173–195. 16. Holub J. B., Skeaff C. M. Nutritional regulation of cellular phosphatidylinositol. Method Enzymol 1987; 141: 234–244. 17. Malins D. C., Mangold H. K. Analysis of complex lipid mixtures by thin layer chromatographic and complementary methods. J Am Oil Chrom Soc 1960; 37: 576–582.
18. Angeletti C., de Alaniz M. J. T. Effect of ethanol on glycerolipid and fatty acid metabolism in HepG2 human hepatoma cells. Acta Physiol et Pharmacol Latinoam 1996; 46: 57–69. 19. Marra C. A., de Alaniz M. J. T. Incorporation and metabolic conversion of saturated and unsaturated fatty acids in SK-Hep 1 human hepatoma cells in culture. Mol Cell Biochem 1992; 117: 107–118. 20. Wood R., Falch J., Wiegand R. D. Lipids of cultured hepatoma cells. V. Distribution of isomeric monoene fatty acids in individual lipid classes. Lipids 1974; 9: 987–992. 21. de Alaniz M. J. T., de Go´mez Dumm I. N. T., Brenner R. R. Effect of fatty acids of !6 series on the biosynthesis of arachidonic acid in HTC cells. Mol Cell Biochem 1984; 64: 31–37. 22. Angeletti C., de Alaniz M. J. T. Fatty acid uptake and metabolism in HepG2 human hepatoma cells. Mol Cell Biochem 1995; 143: 99–105. 23. Gaspar G., de Alaniz M. J. T., Brenner R. R. Incorporation and metabolism of stearic, oleic, linoleic and a-linolenic acids in minimal deviation hepatoma 7288c cells. Mol Cell Biochem 1977; 16: 197–203. 24. De Antueno R. J., Niedfeld G., De Toma´s, M. E., Mercuri O., Montoro L. Microsomal fatty acid desaturation and elongation in a human lung carcinoma grown in nude mice. Biochem Int 1988; 16: 413–420. 25. Hartz J. M., Morton R. E., Waite M. M., Morris H. P. Correlation of fatty acyl composition of mitochondrial and microsomal phospholipids with growth rate of rat hepatomas. Lab Invest 1982; 46: 73–78. 26. Halib N. A., Wood C. B., Apostolob K., et al. Reduction in the stearic to oleic acid ratio in human malignant liver neoplasms. Eur J Surg Oncol 1985; 11: 347–348. 27. Halib N. A., Wood C. B., Apostolob K., et al. Stearic acid and carcinogenesis. Br J Cancer 1987; 56: 455–458. 28. Calviello G., Palozza P., Piccioni E., et al. Dietary supplementation with eicosapentaenoic and docosahexaenoic acid inhibits growth of Morris hepatocarcinoma 3924A in rats: effects on proliferation and apoptosis. Int J Cancer 1998; 75: 699–705. 29. de Go´mez Dumm I. N. T., Brenner R. R. Oxidative desaturation of a-linolenic, linoleic and stearic acids by human liver microsomes. Lipids 1975; 10: 315–317. 30. Rosenthal M. D. Fatty acid metabolism of isolated mammalian cells. Prog Lipid Res 1987; 26: 87–124. 31. Maeda M., Doi O., Akamatsu Y. Metabolic conversion of polyunsaturated fatty acids in mammalian cultured cells. Biochim Biophys Acta 1978; 530: 153–164. 32. Iturralde M., Gonza´lez B., Pin˜eiro A. Linoleate and linolenate desaturation by rat hepatoma cells. Biochem Int 1990; 20: 37–43. 33. Williams J. R., Leaver H. A., Ironside J. W., Miller E. P., Whittle I. R., Gregor A. Apoptosis in human primary brain tumours: actions of arachidonic acid. Prostaglandins Leukot Essent Fatty Acids 1998; 58: 193–200. 34. Spector A. A., Mathur S. N., Kaduce T. L., Hyman B. T. Lipid nutrition and metabolism of cultured mammalian cells. Prog Lipid Res 1981; 19: 155–186. 35. Colquhoun A., Curi R. Metabolic fate and effects of saturated and unsaturated fatty acids in Hep2 human larynx tumor cells. Biochem and Mol Biol Int 1997; 41: 597–607.
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Uptake and metabolic conversion of saturated and unsaturated fatty acids in Hep2 human larynx tumor cells L. Albino, M. P. Polo, M. G. de Bravo,1 M. J.T. de Alaniz1 Instituto de Investigaciones Bioqu|¤ micas de La Plata (INIBIOLP), CONICET-UNLP, Facultad de Ciencias Me¤dicas, La Plata, Argentina
Summary Research on fatty acid metabolism in cultured human larynx tumor cells Hep2 was carried out.The cells were incubated with either a saturated (palmitic) or a polyunsaturated (linoleic, a-linolenic and eicosatrienoic (n-6)) radioactive fatty acid (0.66 mM, 24 h).The best incorporation capacity was observed in the linoleic acid followed by a-linolenic, palmitic and eicosatrienoic acids. All fatty acids tested were anabolized to higher derivatives within their own family. Palmitic acid was primarily monodesaturated rather than elongated, proving to have a very active D9 desaturase activity.With respect to polyunsaturated acid metabolism, the conversion of a-linolenic acid to higher homologs, although better than linoleic acid, occurred far less efficiently than that observed in other non-highly undifferentiated human tumor cells.This impairment in higher polyunsaturated fatty acid biosynthesis, reflected in the low levels of arachidonic acid in the fatty acid composition, would not reside in the D5 desaturation step since Hep2 cells can readily convert eicosatrienoic acid into arachidonic acid. Considering the potential regulatory role of specific polyunsaturated fatty acids in the cell proliferative control, the knowledge of the metabolism of fatty acids in this human tumor cell would be important for designing future experiments in order to clarify the mechanism involved in balance, proliferation and cell death. & 2001Harcourt Publishers Ltd
INTRODUCTION Most cellular functions depend on the structural and dynamic nature of cellular membranes. Fatty acids, particularly polyunsaturated fatty acids, are an integral part of the membranes. The acids regulate membrane functions through their contribution to membrane fluidity and their physicochemical properties.1–4 The specialized functions of differentiated mammalian cells require the utilization of various fatty acids that are selectively incorporated into the different classes of membrane phospholipids, after undergoing several metabolic pathways. Within the cells, the endoplasmic
Received 20 August 2001 Accepted 22 October 2001 Correspondence to: Dr M. J.T. de Alaniz.INIBIOLP, Fac. Ciencias Me¤dicas,Calle 60 y 120, (1900) La Plata, Argentina.Tel.: +54-0221-4834833; Fax: +54-02214258988; E-mail: [email protected] This work was supported in part by grants from CONICETand CIC, Argentina. 1 Members of the Carrera del Investigador Cient|¤ fico, Consejo Nacional de Investigaciones Cient|¤ ficas yTe¤cnicas, Argentina.
& 2001Harcourt Publishers Ltd
reticulum membrane is the site of residence of the elongating fatty acid enzymes and the D9, D6, and D5 desaturases5 that evoke unsaturated fatty acid biosynthesis and polyunsaturation, on which structure and fluidity of the membrane depends. Neoplastic cells require lipids in order to build their membrane structures at sufficient velocity to allow rapid growth. There exist important differences in lipid metabolism between normal and neoplastic tissue.6 One of these striking differences is a low level of D6 desaturase present in most cancer cells7–9 and there is a strong correlation between the degree of cell proliferation inhibition and the degree of unsaturation of 18-carbon fatty acids.10 However, the biosynthetic capacity of human tumor cells concerning the desaturation and elongation of fatty acids is still poorly understood. In order to elucidate the biochemical routes of polyunsaturated fatty acid biosynthesis in the human species in particular, we supplemented normal growth medium of the human laryngeal carcinoma (Hep2) cells with a chemically defined (i.e., serum-free) one. The chemically defined medium contained a given precursor fatty acid of
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which we examined the incorporation into various lipid classes along with its concomitant anabolism to higher polyenoic acid derivatives.
MATERIALS AND METHODS
Chemicals Radiochemicals were obtained from New England Nuclear (Boston, MA). Solvents and chemicals were obtained from Merk (Darmstadt, Germany) and Sigma Chemical Co. (St Louis, MO).
Culture conditions Stock cultures of human laryngeal carcinoma (Hep2) cell line were maintained and grown at 371C in confluent layers attached to 95 cm2 flasks on Eagle’s MEM supplemented with 10% calf serum (both from Gibco, Grand Island, NY). The cells were detached from the glass surface using a 0.25% trypsin solution, washed and suspended in isotonic saline solution and counted in a hemocytometer. Aliquots of 2.5 106 cells were seeded into 95 cm2 glass tissue culture and incubated in 15 ml of Eagle’s MEM containing 10% fetal calf serum for 48 h. Then, the medium was washed three times with 5 ml of cold PBS solution (295 mOsm/Kg H2O, pH 7.4) and incubated in 15 ml of serum-free MEM Zinc option (IMEM Zo)11 supplemented with 10 nmoles of palmitic[1-14C], linoleic[1-14C], a-linolenic[1-14C] or eicosatrienoic [1-14C] acids for 24 h. All radioactive fatty acids were added as their sodium salt bound to delipidated albumin according to Spector et al.12 in a ratio of 2 nmoles of fatty acid to 1 nmol of albumin. At the end of the experiment, the attached cells were washed three times with 5 ml of ice-cold saline solution, detached from the growing surface mechanically with a rubber-tipped spatula, and pelleted at 500g for 10 min. The sedimented cells were resuspended in 5 ml of the same solution. An aliquot of the resulting suspension was used to determine cellular protein content.13 The rest of the cell material was centrifuged in the same way, the supernatant discarded and the pellet processed for lipid analysis.
Cellular lipid extraction and separation Lipids were extracted from the pellets using the method of Folch et al.14 An aliquot of the organic phase was used to separate phospholipids and neutral lipids following the method of Wren.15 Different classes of phospholipids were further partitioned by TLC on 0.25 mm thick silica gel G-60 plates by means of a solvent mixture containing chloroform : methanol : acetic acid : water (50 : 37.5 : 3.5 : 2, v : v : v : v).16 The neutral lipids were also partitioned by
TLC system using as a mobile phase petroleum ether : ethyl ether : acetic acid (80 : 20 : 1, v : v : v).17 The different subclasses of lipids were identified by means of standard mixture run in parallel with the samples. After development, TLC plates were dried and scanned for radioactivity (radio-TLC) using a TLC-Proportional Radioactivity Scanner, (Berthold LB-2832 Wildbard, Germany), equipped with a Hewlett-Packard 3396-A Data Station. Another aliquot from the cell organic phase was methylated and analyzed by gas–liquid chromato graphy.18 The distribution of radioactivity among the fatty acid methyl esters (FAME) was analyzed using the radiochromatograph (Varian Star 3400 CX) in tandem with a proportional gas detector (GC-Ram; Inus System Inc. Tampa, FL). A 6-foot glass column packed with 10% (w/w) SP-2330 on 100–200 mesh chromosorb WAWDMCS (Supelco Inc., Bellefonte, PA). After 1 min initial hold, the oven was programmed from 145 to 2201C at 11C/min. The fatty-acid methyl esters (FAME) were identified by comparison of their relative retention times with authentic standards, while the radioactivity distribution was calculated electronically by quantification of the peak areas. An aliquot of the organic phase was also used to measure total radioactivity incorporated into the cells, using a Wallac 1214 Rackbeta liquid scintillation counter (Pharmacia, Turku, Finland) with a 97% efficiency for 14C, interfaced to an Olivetti M-240 computer system. Data are reported as the mean and standard error calculated from 3–5 independent analyses. RESULTS The analysis of the total fatty acid composition of human laryngeal carcinoma (Hep2) cells maintained on Eagle’s MEM supplemented with 10% (v/v) fetal bovine serum is presented in Table 1. It is hallmarked by high levels of saturated and monounsaturated fatty acids together with low levels of linoleic and polyunsaturated fatty acids. Table 1 also shows the fatty acid composition of serum fraction of the culture medium on which the cells were grown. This serum fraction constitutes the cells’ only source of exogenous lipids. As can be seen, the profile of this fatty acid composition strongly differs from that of the cells. In order to test the ability of Hep2 cells to incorporate and metabolize labeled fatty acid precursors from the medium, cultures were incubated with specific radio labeled fatty acid precursors, and the cellular lipids were further analyzed for the distribution of radioactivity among the endogenous polyunsaturated fatty acids (PUFAs). Under such conditions all labeled fatty acids tested (16 : 0, 18 : 2 (n-6), 18 : 3 (n-3) and 20 : 3 (n-6)) were taken up by the cells (Fig. 1). As seen, linoleic acid showed
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Table 1 Percentage distribution of fatty acid within the total lipids of Hep2 human larynx cells Fatty acids
Medium
Cells
14 : 0 15 : 0 16 : 0 16 :1 18 : 0 18 :1 (n-9) 18 :1 (n-7) 18 : 2 (n-6) 18 : 4 (n-3) 20 : 0 20 : 3 (n-6) 20 : 4 (n-6) 20 : 5 (n-3) 22 : 2 (n-6) 22 : 3 (n-6) 22 : 5 (n-3) 22 : 6 (n-3)
0.7 ND 27.1 3.0 14.4 18.4 3.3 5.4 2.8 ND 2.6 9.7 1.1 2.1 ND 4.3 5.1
1.470.1 1.870.4 21.271.8 7.670.9 10.470.4 21.373.8 11.072.5 3.870.2 ND 1.270.5 1.370.3 3.370.6 ND 7.372.5 1.971.1 1.670.4 4.970.8
Cells were grown at 371C in MEM containing10% fetal bovine serum. Results are given as means7SE (n=4) in one of three similar experiments. ND: Not Detected.
the best incorporation capacity followed by a-linolenic, palmitic and eicosatrienoic acids. These incorporated fatty acids were subsequently actively metabolized. Palmitic acid (Fig. 2) was desaturated at the D9 position to palmitoleic (16 : 1), and this acid was in turn further elongated to give oleic acid (18 : 1). However, 16 : 0 might be firstly elongated to 18 : 0, and then desaturated at the 9 position giving rise to oleic acid. Figure 3A shows that in these tumor cells, despite the large uptake of [1-14C]linoleic acid, very small amounts of arachidonic acid were formed. Besides, the linoleate chain elongation product, eicosa-11,14-dienoate and its de saturated derivative, eicosatrienoic acid, could be detected. In spite of these observations, a different pattern of anabolic conversion was carried out with a-linolenate[1-14C] as a metabolic precursor: the conversion to the elongated-plus-desaturated product (eicosa-8, 11, 14, 17) occurred more readily, followed by a small amount of its further D5 desaturation analogue eicosapentaenoic acid (Fig. 3B). Figure 4 shows that Hep2 cells can actively convert eicosatrienoic acid (n-6) into arachidonate, demonstrating that this human line has a very active D5 desaturase. The percentage distribution of radioactivity found in the major lipid class of the Hep2 cells after their incubation with [14C]palmitic or [14C]eicosatrienoic acids is presented in Table 2. Under such conditions both precursors and their corresponding metabolic products were predominantly incorporated into the phospholipid fraction, preferentially into phosphatidyl-choline and phosphatidyl-ethanolamine. Palmitic acid and its deriva& 2001Harcourt Publishers Ltd
Fig. 1 Uptake of radioactivity in Hep2 cells incubated in the presence of various [1-14C] fatty acids. Results are expressed as dpm per mg of cellular protein (mean7SE). All fatty acids were added at 0.66 mM concentration as sodium salt bound to delipidated albumin for 24 h. Technical details are described in Methods.
Fig. 2 Percentage distribution of radioactive metabolic products obtained after incubation of Hep2 cells with [1-14C]palmitic acid. Each bar represents the mean7SE (n¼5).The substrate was added as sodium salt bound to delipidated albumin at a final concentration of 0.66 mM, and incubated with the cells for 24 h. Radiochromatographic analyses of the total cellular lipids were performed as indicated in Methods.
tives were better incorporated into neutral lipids compared to those from eicosatrienoic acid. Within the different phospholipid subclasses, 16 : 0 as well as 20 : 3 acids were incorporated mainly into phosphatidyl-choline and phosphatidyl-ethanolamine fractions. However, in spite of the fact that both substrates were found in considerable amounts in
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Fig. 4 Percent distribution of radioactive metabolic products obtained after incubation of Hep2 cellswith [1-14C]eicosatrienoic acid. Each bar represents the mean7SE (n¼5).The substrate was added as sodium salt bound to delipidated albumin at a final concentration of 0.66 mM, and incubated with the cells for 24 h. Radiochromatographic analyses of the total cellular lipids were performed as indicated in Methods. Fig. 3 Percentage distribution of radioactive metabolic products obtained after incubation of Hep2 cells with [1-14C]linoleic (A) or [1-14C]a-linolenic (B) fatty acids. Each bar represents the mean7SE (n¼5).The substrate was added as sodium salt bound to delipidated albumin at a final concentration of 0.66 mM, and incubated with the cells for 24 h. Radiochromatographic analyses of the total cellular lipids were performed as indicated in Methods.
phosphatidyl-inositol, eicosatrienoic and its D5 desaturation product arachidonic acid were mostly incorporated into this phospholipid fraction rather than palmitic acid and its metabolic products. Conversely, the radioactive products resulting from palmitic acid incubation of the cells were largely incorporated into neutral lipids compared to those from eicosatrienoic acid supplementation. DISCUSSION It has been shown frequently that the fatty acid composition of tumor cells differs from that of normal cells. In this respect, the low levels of polyene content, particularly the high levels of monounsaturated fatty acids found in human laryngeal carcinoma (Hep2) cells (Table 1), are in accordance with the most frequently reported abnormalities in lipids in neoplasms.19–22 The
Table 2 Distribution of radiactivity within the lipid subclasses of Hep2 after incubation in the presence of various [14C] fatty acids [14C] Fatty acid incubated
PS PI SM PE PC DAG FFA TAG CHOE [14C]Phospholipids/ [14C]Neutral lipids
16 : 0
20 : 3 (n-6)
2.570.3 6.971.1 7.672.4 34.074.8 35.371.1 5.470.7 0.970.1 5.871.1 1.370.5
5.070.2 14.973.0 10.371.5 26.072.3 36.371.9 2.470.1 0.670.04 3.670.1 0.970.3
6.970.9
12.671.1
Results are expressed as the percentage distribution of the total radioactivity incorporated into the cells (mean of four independent determinations7SEM).The cells were incubated in the presence of 1 mM [14C]fatty acid for 24 h and then processed as indicated in Materials and Methods.
differences observed in the fatty acid composition between medium (lipids of calf serum) and Hep2 cells demonstrate that these cells not only take up the fatty acids from the medium, but also modify their
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composition by means of a selective incorporation or metabolization. After 24 h of culture in the presence of 0.66 mM of the different labeled fatty acids, all of them were taken up by the cells (Fig. 1), linoleic acid being the bestincorporated, followed by a-linolenic, palmitic and eicosatrienoic acids. It is generally accepted that neoplastic cells are able to synthesize monounsaturated fatty acids.19,22–24 Moreover, several authors reported that the monoenoic/ saturated fatty acid ratio proved to be a useful marker reflecting both growth rate and malignancy in different human neoplasms.25–27 In this kind of tumor, palmitic acid was primarily desaturated rather than elongated proving to have a very active D9 desaturase (Fig. 2). Palmitoleic acid was further elongated to give oleic acid. However, 16 : 0 might be firstly elongated to 18 : 0, and then desaturated at the 9 position giving rise to oleic acid. These results markedly differ from those reported in other highly undifferentiated neoplastic cells19 in which palmitic acid is mainly elongated rather than desaturated. The metabolic conversions of essential fatty acids linoleic (n-6 series) and a-linolenic acids (n-3 series) in neoplastic cells are of particular interest since they are the precursors of arachidonic (n-6), eicosapentaenoic and docosahexaenoic acids (n-3) related to cell proliferation inhibition and induction of apoptosis.28 Despite the fact that linoleic acid was the bestincorporated precursor, its conversion to higher homologs was very low and the acid was mainly elongated rather than desaturated (Fig. 3A). However, when a-linolenic acid was the substrate, the conversion into higher homologs occurred far more readily (Fig. 3B), indicating that these cells possess a D6 desaturase capacity for a-linolenic acid despite their largely dimi nished ability to perform the analogous reaction with linoleate. As stated before in human liver microsomes,29 as well as in different cell-culture lines,19,23,30–32 the D6 desaturase enzyme exhibits a marked preference for a-linolenic acid over linoleate as a substrate. In consequence, given an even greater diminution in the conversion of the latter, as in this work, the level of formation of higher metabolic products becomes extremely low. However, the conversion of both linoleic and a-linolenic acids into higher-chain polyunsaturated fatty acids, initiated by a desaturation at the D6 position, occurred far less efficiently than that observed in other non-highly undifferentiated human tumor cells.22 As described previously,7–9 very low levels of D6 desaturase are present in cancer cells. Insufficient D6 desaturase may cause a decrease in g-linolenic acid and subsequently arachidonic acid (AA) levels. It has also been postulated that the loss of proliferative control in tumor cells is the consequence of a depletion of cellular & 2001Harcourt Publishers Ltd
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AA, and that exogenous AA and n-6 fatty acid may restore control of proliferation.9,33 The last step in the synthesis of AA from linoleate is catalyzed by D5 desaturase. This enzyme was shown to be present not only in Hep2 cells (Fig. 4), but also in all cells tested, including those that were deficient in D6 desaturase.34 This observation supports the conclusion that the metabolic block in arachidonic acid biosynthesis from linoleate as precursor, must reside in a prior anabolic step. The analysis of the percentage distribution of radioactivity found in the major lipid class of the Hep2 cells after incubation with labeled palmitic or eicosatrienoic acid presented in Table 2, revealed that both precursors were predominantly incorporated in one phospholipid fraction. However, the ratio of the total radioactivity found in the phospholipid classes (Table 2, rows 1–5), compared to that detected in the neutral-lipid species (Table 2, rows 6–9) and present in row 10 of this Table, was shown to be 2-fold higher when the cells were incubated with the polyunsaturated precursor eicosatrienoic acid than when the saturated acid (palmitic) was incubated. These observations, similar to those reported in human hepatoma cells in culture,19 are consistent with what might be expected from the known requirement for polyunsaturated acids within the phospholipid of cellular membranes for the maintenance of normal fluidity within their lipid phases. The differences observed between our results and those previously reported by Colquhoun et al. with Hep2 cells35 may be due to the incubation time used by these authors (only 6 h). In conclusion, we have shown that human larynx tumor cells Hep2 are able to take up both saturated and unsaturated fatty acids from the medium. These species and their metabolic products were incorporated into each basic lipid class as well as into each of the principal phospholipid subclasses in cellular membranes in a similar rate to that observed in other kinds of cells in culture. Radioactive precursors were anabolized to higher homologs and analogs within their own family, demonstrating the presence of D9, D6, and D5 desaturases. However, as shown in other undifferentiated cells,7,19 low levels of D6 desaturase were detected, and they were reflected in the low levels of AA in the fatty acid composition. If AA mediates ceramide-induced apoptosis, the absence of AA in tumor cells may make these cells resistant to apoptosis induction. This impairment in the biosynthesis of AA does not arise from the D5 desaturation step since Hep2 cells can readily convert eicosatrienoic into arachidonic acid. In consequence, we can assume that the impairment in the biosynthesis of polyunsaturated fatty acid would be an important feature in the mechanism related to the loss of proliferative control.
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ACKNOWLEDGMENTS The authors are grateful to Elsa Claverie for her technical assistance and to Norma Tedesco for language revision.
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