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
alpha-(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 polyunsaturated
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.