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Sensitivity of K562 and HL-60 Cells to Edelfosine, an Ether Lipid Drug, Correlates with Production of Reactive Oxygen Species¹

Departments of Medicine [B. A. W., C. P. B.] and Radiology/Radiation Research Laboratory [G. R. B., L. W. O.], and the Electron Spin Resonance Facility [G. R. B.], The University of Iowa College of Medicine, The University of Iowa Cancer Center, Iowa City, Iowa 52242

Edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine; ET-18-OCH₃), a membrane-targeting anticancer ether lipid drug has been shown previously in vitro to be capable of initiating oxidative processes in cells. Here we study two human leukemia cell lines (HL-60 and K562) that have different sensitivities to edelfosine; HL-60 cells are more sensitive than K562 cells. To determine whether edelfosine alters the sensitivity of these lines to an oxidative stress, cells were subjected to the oxidative stress of iron(II) plus ascorbate and then monitored for free radical formation, membrane integrity, and cytotoxicity. The HL-60 cell was sensitive to the ether lipid drug in clonogenic and dye exclusion assays; a lipid-derived free radical was generated by this sensitive cell in the presence of small amounts of Fe²⁺ and ascorbate as detected by electron paramagnetic resonance and the spin trap -(4-pyridyl-1-oxide)-N-tert-butylnitrone. There was also simultaneous generation of an ascorbate-free radical, which has been shown to estimate cellular oxidative flux. In contrast, the K562 cell was resistant to edelfosine cytotoxicity in all assays and did not generate either lipid-derived or ascorbate-free radicals. Subcellular homogenates of the HL-60 cell generated both radicals when exposed to the drug, but homogenates of K562 did not generate either, suggesting that differential drug uptake or intracellular drug localization is not the cause of the difference in oxidation. Trypan blue uptake by the HL-60, but not the K562 cells, measured under the same conditions as the oxidation experiments, demonstrated a loss of membrane impermeability with similar time and concentration dependence, suggesting a causal relationship of membrane damage and radical generation. Complementary studies of HL-60 cell membrane integrity with propidium iodide impermeability and light scatter using the flow cytometer showed a concentration dependence that was similar to radical generation. Biochemical studies of the fatty acids of the HL-60 cell revealed more highly polyun-saturated lipids in the cells. Cellular antioxidant enzymes and vitamin E contents of the two cell lines were similar. We conclude that there is a time- and concentration-dependent generation of important oxidations by the sensitive HL-60 cells exposed to the membrane-targeted ether lipid, but the resistant K562 cells are oxidatively silent. This may be due in part to the differences in fatty acid polyunsaturation of the cellular membranes. The difference in oxidative susceptibility could be the basis for drug resistance to this membrane-specific anticancer agent.

¹ This investigation was supported by Grant P01 CA66081 awarded by the National Cancer Institute; Grant HL49264 awarded by the National Heart Lung and Blood Institute, Department of Health and Human Services; The Mamie C. Hopkins Fund; The Richard O. Emmons Memorial Fund; The Koch Bequest Fund; and The Iowa Leukemia and Cancer Research Fund.

² To whom requests for reprints should be addressed, at Department of Medicine, University Hospitals, Iowa City, IA 52242. Phone: (319) 356-2038; Fax: (319) 353-8383; E-mail: c-burns@uiowa.edu.

Received 12/ 8/97. Accepted 5/ 1/98.

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