Merocyanine 540-sensitized photokilling of leukemia cells: role of post-irradiation chain peroxidation of plasma membrane lipids as revealed by nitric oxide protection

The lipophilic dye merocyanine 540 (MC540) localizes primarily in the plasma membrane (PM) of tumor cells, where it can sensitize lethal photoperoxidative damage of potential therapeutic importance. We postulated (i) that chain peroxidation triggered by iron-catalyzed turnover of nascent hydroperoxides (LOOHs) generated by singlet oxygen (1O2) attack on PM lipids contributes significantly to overall cytolethality, and (ii) that nitric oxide (NO), a known scavenger of organic free radicals, would suppress this and, thus, act cytoprotectively. In accordance, irradiation of MC540-sensitized L1210 cells produced 5α-OOH, a definitive 1O2 adduct of PM cholesterol, which decayed during subsequent dark incubation with appearance of other signature peroxides, viz. free-radical-derived 7α/β-OOH. Whereas chemical donor (SPNO or SNAP)-derived NO had little or no effect on post-irradiation 5α-OOH disappearance, it dose-dependently inhibited 7α/β-OOH accumulation, consistent with interception of chain-carrying radicals arising from one-electron reduction of primary LOOHs. Using [14C]cholesterol as an L1210 PM probe, we detected additional after-light products of chain peroxidation, including diols (7α-OH, 7β-OH) and 5,6-epoxides, the yields of which were enhanced by iron supplementation, but strongly suppressed by NO. Correspondingly, photoinitiated cell killing was significantly inhibited by NO introduced either immediately before or after light exposure. These findings indicate that prooxidant LOOH turnover plays an important role in photokilling and that NO, by intercepting propagating radicals, can significantly enhance cellular resistance.