[CANCER RESEARCH 53. 711-713, February 15. 199.1] Advances in Brief Increased Generation of Lipid-derived and Ascorbate Free Radicals by LI210 Cells Exposed to the Ether Lipid Edelfosine1 Brett A. Wagner, Garry R. Buettner, and C. Patrick Burns Department of Medicine IB. A. W., C, P. B. I and the Electron Spin Resonance Facility [G. R. B.¡,The University of Iowa College of Medicine, Iowa City. Iowa 52242 Abstract Using the spin trap a-(4-pyridyl-l-oxide)-A'-fert-butylnitrone, we have detected a lipid-derived carbon-centered free radical generated from in tact 1,1210 lymphoblastic leukemia cells that were exposed to 1-O-octade- cyl-2-O-methyl-rac-glycero-3-phosphocholine (edelfosine or I I- IK-()( II,) and oxidative stress. The spectral characteristics, including hyperfine splitting constants of¡IN= 15.61G and a" = 2.65G, were consistent with the spin trapping of an alkyl radical. Radical detection required iron and prior enrichment of cellular components with the polyunsaturated fatty acid docosahexaenoic acid; unmodified cells failed to generate detectable free radical. Ascorbate further enhanced radical generation. The detection of lipid-derived free radicals when intact cells are exposed to edelfosine provides further evidence that oxidative stress may play an important role in the cytotoxic mechanism of this class of anticancer drug. Introduction The ether lipids are membrane-active anticancer drugs with a wide spectrum of antineoplastic and biological activity. Their mechanism of action remains unknown. We have recently reported that these agents enhance iron-induced lipid peroxidation of neoplastic cells (1). We have now detected a lipid-derived free radical from docosahexaenoic acid-enriched leukemia cells during oxidative stress and shown that edelfosine enhances the formation of this radical species. Materials and Methods Fatty Acid Modification. LI210 cells were grown for 48 h in RPMI 1640 containing 5% fetal bovine serum and 32 UM 22:62 (Nu Chek Prep, Inc., Elysian, MN) (2). We have shown previously that supplementation of growth media with 22:6 results in cells that contain 23-37% of 22:6 in cellular phospholipids (1,3, 4). For comparison, unmodified cells contain <l% 22:6 (1,5). Ascorbate and Lipid-derived Radicals from Intact Cells. Fatty acid- modified LI 210 cells were washed and placed in 0.9% NaCl. In initial studies, 10 HIMPOBN (Sigma Chemical Co.. St. Louis, MO), 20 UM FeSO4-7H,O (Fisher Scientific Co., Fair Lawn, NJ). 100 UMascorbic acid (Mallinckrodt, Inc., Paris. KY) and edelfosine (l-O-octadecyl-2-O-methyl-roc-glycero-3- phosphocholine or ET-18-OCH,; Medmark Pharma GmbH. Griinwald, Ger many; kindly supplied by Dr. R. Nordström) were added to the air-saturated cell suspension, in that order, and placed into an EPR quartz flat cell at room temperature and scanned by EPR. Time course studies were initiated by adding to L1210 cell suspensions, 50 m.MPOBN, 100 UMascorbic acid, and then 20 UM Fe2+. After monitoring by EPR for 190 s. edelfosine was added and scanning was continued. For studies of the general cellular oxidative state, as estimated by the ascorbate free radical signal intensity, no POBN was added. Quantita- Received 11/12/92; accepted 12/30/92. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 'This investigation was supported by Grants C A31526 awarded by the National Cancer Institute, Department of Health and Human Services. Data analysis utilized the Clinfo system. Grant RR59 from the General Clinical Research Centers Program. Division of Research Resources. NIH. 2 The abbreviations used are: 22:6. docosahexaenoic acid; POBN. a-(4-pyridyl-l- oxide)-/V-rrrr-butylnitn>ne; EPR. electron paramagnetic resonance. tion of ascorbate radical signal intensities from EPR scans were determined as described using 3-carboxyproxyl as a standard (6). Electron Paramagnetic Resonance. Spectra were measured using a Bruker ESP 300 spectrometer equipped with a TM,,,, cavity and operating at 9.77 GH/. microwave frequency and 100 kHz modulation frequency. Results Spin Trapping of Lipid-derived POBN-Spin Adducts from Cells. When LI210 cells enriched with 22:6 were incubated in the presence of 10 HIMPOBN, no spin adducts were detected (Fig. 1/4). Likewise, neither 22:6-enriched LI210 cells treated with 40 UMedel fosine and 10 HIMPOBN without any oxidant cofactors (Fig. Iß)nor unmodified L1210 cells treated with edelfosine and cofactors (not shown) produced detectable POBN spin adducts or other EPR detect able radical species. Repeated experiments with higher concentrations of edelfosine failed to produce any detectable POBN spin adducts. Therefore, edelfosine does not produce EPR-detectable free radicals in the absence of oxidative cofactors or in cells with low polyunsat urated fatty acid content. 22:6-enriched cells incubated with 100 UMascorbic acid produced a weak ascorbate radical signal (Fig. 1C), which dissipated with time. When 20 UMFe2^ was added to POBN-treated cells, a carbon-cen tered spin adduci was detected (Fig. ID). This adduci spectrum was stable in intensity for at least 30 min (data not shown). Cells treated with iron and ascorbic acid (Fig. IE) produced a carbon-centered POBN spin adduci similar to the one found in cells irealed wilh iron alone (Fig. ID), which is superimposed on an ascorbale radical signal. This POBN-spin adduci EPR signal was more intense than ihose from cells irealed wilh iron alone. The ascorbale radical spectrum, which was high in intensily al ine early lime poinls of 2-5 min, dissipaled wilh lime, and became undeleclable al 30 min (data noi shown). In general ¡herewas a inverse relationship over lime belween Ihe inten sity of the POBN-spin adduci and lhal of Ihe ascorbale radical. The addition of 40 UMedelfosine to 22:6-enriched cells with iron and ascorbic acid also caused the appearance of both the carbon- centered spin adduci and Ihe ascorbale radical (Fig. IF). The carbon- ceniered spin adduci and speclral characlerislics of the ascorbale radical were similar in configuration lo ihose observed in cells irealed with iron and ascorbic acid; however, in all experiments, cells trealed with edelfosine appeared lo have higher carbon-cenlered radical in tensities than those treated with FeSO4 and ascorbic acid, alone or in combination. Kinetics of Lipid-derived Radical Formation in Whole Cells: Augmentation by Edelfosine. In internally controlled EPR experi ments, 22:6-enriched L1210 cells were incubated with 50 ITIMPOBN spin irap, 20 UMFe2+, and 100 UMascorbale and monilored by EPR (Fig. 2). Al 190 s after Ihe inilialion of the experiment, varying concentralions of edelfosine were added lo Ihe cell suspension. The addilion of edelfosine resulted in intensification of the POBN-adduct formation in a concentraiion-dependenl manner beginning 20-60 s after addition of the drug. Analysis of covariance comparing controls 711