Chain-breaking Antioxidant and Cytoprotective Action of Nitric Oxide on Photodynamically Stressed Tumor Cells¶

Abstract

Nitric oxide (.NO) has a multitude of physiological roles, including the ability to protect cells against oxidant-induced killing, e.g. by inhibiting caspase-mediated apoptosis or by intercepting damaging free radicals derived from membrane lipids. The purpose of this study was to test the hypothesis that low flux .NO acting in the latter fashion can enhance tumor-cell resistance to photodynamic killing, specifically that sensitized by 5-aminolevulinic acid (ALA)-derived protoporphyrin IX (PpIX). Preliminary model experiments with iron-ascorbate-treated, PpIX-sensitized liposomes showed that spermine NONOate (SPER/NO)-derived .NO had no effect on photoinduced accumulation of primary singlet oxygen adducts, e.g. the cholesterol hydroperoxide 5 alpha-OOH, but dose-dependently inhibited the buildup of free radical-generated oxidation products arising from one-electron turnover of primary peroxides. In subsequent studies, breast tumor COH-BR1 cells in serum-free medium were treated with 1 mM ALA for 15 min and then without ALA for 3.75 h, allowing biogenerated PpIX to diffuse to extramitochondrial sites, including plasma membrane. Cells were irradiated in the absence or presence of SPER/NO and compared for peroxidative damage and Hoechst-assessed viability after 5 h in the dark. Iron-stimulated necrotic photo-killing and accumulation of chain lipid peroxidation products were observed, and this was inhibited strongly by SPER/NO, but not by decomposed SPER/NO, confirming that .NO was the active agent. When introduced after irradiation, .NO became progressively less inhibitory, consistent with ongoing but waning free-radical activity. These findings provide new insights into the possible role of .NO in tumor resistance to ALA-photodynamic therapy and other photo-dynamic treatments.

Cholesterol as a natural probe for free radical-mediated lipid peroxidation in biological membranes and lipoproteins

“…On the other hand, SPNO added immediately before irradiation reduced all [ 14 C]ChOX levels (Fig. 5A,B), consistent with OLOO · /OLO · interception by NO [29,30]. …”

Nitric oxide-mediated resistance to photodynamic therapy in a human breast tumor xenograft model: Improved outcome with NOS2 inhibitors

“…These include: (a) whether antagonistic NO derives from the tumor cells themselves or whether stromal cells such as endothelial cells and tumor-associated macrophages might contribute, either before or after PDT stress develops; (b) which NOS isoform is most important as an endogenous NO donor; (c) whether pre-existing NO at some low steady state level is sufficient for PDT resistance or whether NOS/NO induction by photodynamic stress plays s more important role; (d) whether NO might act cytoprotectively by scavenging damaging intermediates generated by PDT, e.g. oxyl/peroxyl radicals from membrane lipid peroxidation [26,27]; and (e) the types of pro-survival signaling pathways that may be activated by pre-existing or stress-upregulated NO [28–30]. Some of these crucial questions have been addressed in the present study.…”

Modulation of the Anti-Tumor Efficacy of Photodynamic Therapy by Nitric Oxide

“…Using breast cancer COH-BR1 cells, Niziolek et al [46,47] were the first to investigate this in the context of ALA-PDT. Cells were sensitized with ALA-induced PpIX, which was allowed to diffuse from mitochondria to plasma membrane.…”

“…Cells were sensitized with ALA-induced PpIX, which was allowed to diffuse from mitochondria to plasma membrane. When irradiation was carried out in the presence of a catalytic iron chelate, chain lipid peroxidation induced by one-electron reduction of 1 O 2 -generated LOOHs occurred and the cells died mainly by plasma membrane-rending necrosis [47]. Chain peroxidation and necrosis were strongly suppressed by SPNO-derived NO acting as a LO∙/LOO · quencher.…”