ascorbic-acid and edelfosine

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 polyunsatu

Topics: Antineoplastic Agents; Ascorbic Acid; Cell Membrane; Fatty Acids; Flow Cytometry; Free Radicals; HL-60 Cells; Humans; Indicators and Reagents; Iron; Lipid Peroxidation; Nitrogen Oxides; Phospholipid Ethers; Propidium; Pyridines; Reactive Oxygen Species; Trypan Blue; Tumor Cells, Cultured; Vitamin E

Oxidizability of lipids in homogeneous solution varies linearly with the extent of their unsaturation. In vitro cellular, as well as in vivo, studies of oxidizability have generally relied upon chemical indicators of peroxidation such as thiobarbituric acid-reactive substances. To examine the oxidizability of lipids in cells, we have measured oxygen uptake and, using electron paramagnetic resonance spin trapping with alpha-(1-oxo-4-pyridyl)-N-tert-butylnitrone (POBN), the real time generation of lipid-derived free radicals. We have used our experimental in vitro cellular lipid modification model to examine the rate and extent of lipid peroxidation versus the degree of lipid unsaturation in L1210 murine leukemia cells. Lipid peroxidation was stimulated using the prooxidants iron, ascorbate, and the ether lipid compound 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine. We did a total cellular lipid analysis to determine the number of lipid carbon-carbon double bonds contained in L1210 cells enriched with eight fatty acids of different degrees of unsaturation. We found in cellular lipids that (i) lipid chain length had no apparent effect on the rate or extent of radical formation; (ii) the maximum amount of lipid radical generated increases with the total number of bis-allylic positions in the cellular lipids; and, most importantly, (iii) the rate of cellular lipid peroxidation increases exponentially with the number of bis-allylic positions. Our quantitative results clearly demonstrate, for the first time, that the number of bis-allylic positions contained in the cellular lipids of intact cells determines their susceptibility, i.e., oxidizability, to free radical-mediated peroxidative events.

Topics: Animals; Ascorbic Acid; Electron Spin Resonance Spectroscopy; Fatty Acids; Free Radicals; Hydrogen; Iron; Kinetics; Leukemia L1210; Lipid Peroxidation; Lipids; Mice; Nitrogen Oxides; Oxidation-Reduction; Oxygen; Phospholipid Ethers; Pyridines; Solutions; Spin Labels; Tumor Cells, Cultured

Using the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone, we have detected a lipid-derived carbon-centered free radical generated from intact L1210 lymphoblastic leukemia cells that were exposed to 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (edelfosine or ET-18-OCH3) and oxidative stress. The spectral characteristics, including hyperfine splitting constants of aN = 15.61G and aH = 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.

Topics: Animals; Antineoplastic Agents; Ascorbic Acid; Docosahexaenoic Acids; Dose-Response Relationship, Drug; Free Radicals; Iron; Leukemia L1210; Lipid Peroxidation; Nitrogen Oxides; Phospholipid Ethers; Pyridines; Spin Labels

The ether lipid antineoplastic agents have no known interaction with DNA, but rather they appear to target membranes. The primary mechanism of action is unknown but effects on membrane biology are documented. We have studied the effect of two ether lipids on membrane lipids and examined the hypothesis that membrane peroxidative damage may be involved in their mechanism of action. With the use of cells having membranes enriched in polyunsaturated fatty acids of the omega-3 family of fatty acids, we have demonstrated that the prototypical ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine and a thioether lipid analogue, 1-O-hexadecylmercapto-2-methoxymethyl-rac-glycero-3-phosphocholine , increase membrane lipid peroxidation and cytotoxicity in a time- and drug concentration-dependent manner. The oxidative cofactors Fe2+ and ascorbic acid were required. The pattern of cell death did not fully correspond to the peroxidation, since cofactors were required for peroxidation but not cytotoxicity. However, the rate of decrease in cell viability after exposure to the drug and cofactors corresponded to the peroxidation rate. In addition, when L1210 cells modified with the monounsaturated fatty acid oleic acid or unmodified cells were used, there was no ether lipid-enhanced peroxidation, and the cells were significantly less sensitive to the drug, with or without cofactors. The lipid-soluble antioxidant vitamin E inhibited 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine peroxidation and cytotoxicity in a concentration-dependent manner in the presence of cofactors but not consistently without them. Depletion of cellular glutathione content of L1210 cells using L-buthionine-(SR)-sulfoximine resulted in 40% augmentation of cofactor-facilitated cytotoxicity of 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine and a borderline effect on peroxidation. Another ether lipid, the thio compound 1-O-hexadecylmercapto-2-methoxymethyl-rac-glycero-3-phosphocholine , enhanced peroxidation in the presence of cofactors with kinetics corresponding to those of cytotoxicity. In the presence of ether lipid and cofactors the intensity of ascorbate free radical increased, consistent with oxidative stress. We conclude that the ether lipids stimulate membrane lipid peroxidation in a time- and drug concentration-dependent manner in the presence of oxidative cofactors. Even though peroxidation may not fully explain the cytotoxic effect of the ether lipid class of antica

Topics: Animals; Antineoplastic Agents; Ascorbic Acid; Cell Death; Drugs, Investigational; Fatty Acids; Free Radicals; Glutathione; Iron; Leukemia L1210; Lipid Peroxidation; Membrane Lipids; Phospholipid Ethers; Phospholipids; Vitamin E