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Zeaxanthin and α-tocopherol have been previously shown to efficiently protect liposomal membrane lipids against photosensitized peroxidation, and to protect cultured RPE cells against photodynamic killing. Here the protective action of combined zeaxanthin and α-tocopherol was analyzed in ARPE-19 cells subjected to photodynamic (PD) stress mediated by rose Bengal (RB) or merocyanine-540 (MC-540) at sub-lethal levels. Stress-induced cytotoxicity was analyzed by the MTT assay. The peroxidation of membrane lipids was determined by HPLC-EC (Hg) measurements of cholesterol hydroperoxides using cholesterol as a mechanistic reporter molecule. The specific phagocytosis of FITC-labeled photoreceptor outer segments (POS) isolated from bovine retinas was measured by flow cytometry, and the levels of phagocytosis receptor proteins αv integrin subunit, β5 integrin subunit and MerTK were quantified by Western blot analysis. Cytotoxicity measures confirmed that PD stress levels used for phagocytosis analysis were sub-lethal and that antioxidant supplementation protected against higher, lethal PD doses. Sub-lethal PD stress mediated by both photosensitizers induced the accumulation of 5α-OOH and 7α/β-OOH cholesterol hydroperoxides and the addition of the antioxidants substantially inhibited their accumulation. Antioxidant delivery prior to PD stress also reduced the inhibitory effect of stress on POS phagocytosis and partially reduced the stress-induced diminution of phagocytosis receptor proteins. The use of a novel model system where oxidative stress was induced at sub-lethal levels enable observations that would not be detectable using lethal stress models. Moreover, novel observations about the protective effects of zeaxanthin and α-tocopherol on photodynamic damage to ARPE-19 cell membranes and against reductions in the abundance of receptor proteins involved in POS phagocytosis, a process essential for photoreceptor survival, supports the importance of the antioxidants in protecting of the retina against photooxidative injury.

Using a 5-aminolevulinic acid (ALA)-photodynamic therapy model, we have discovered a new effect of nitric oxide (NO)-the ability to accommodate apoptosis. When sensitized by disseminated ALA-generated protoporphyrin IX, COH-BR1 tumor cells in glucose-containing medium died mainly by necrosis with a low level of apoptosis. Introduced before light at a nontoxic concentration, the NO donor SPNO inhibited necrosis, but supported apoptosis such that the latter became predominant in the remaining cell death. Accompanying this was a large increase in caspase-3/7 activation. SPNO-supported apoptosis was more pronounced when glucose-deprived cells were compared with glucose-replenished, SPNO-treated counterparts. SPNO plus glucose also suppressed plasma membrane-damaging lipid peroxidation and loss of cellular ATP under photostress. The NO effect is attributed to membrane protection with maintenance of sufficient glycolytic ATP to sustain apoptosis.

Relatively little is known about how nitric oxide (NO) generated by tumor vascular cells or tumor cells themselves might affect the outcome of photodynamic therapy (PDT). Using a breast tumor epithelial line (COH-BR1) metabolically sensitized with protoporphyrin IX (PpIX) by pre-treating with 5-aminolevulinic acid (ALA), we have recently shown that NO from chemical donors can elicit both an immediate (NO-now) and delayed (NO-then) hyperresistance to photokilling. Cell death was mainly apoptotic when PpIX was confined to mitochondria, but mainly necrotic when it was allowed to diffuse to the cell periphery. We found that NO-now operates primarily by scavenging lipid-derived free radicals, whereas NO-then "preconditions" cells by some other mechanism. In addressing this, we have used a biologically relevant NO donor/tumor target model, viz . RAW 264.7 macrophages grown on microporous membrane inserts and COH-BR1 cells grown in culture plate wells. The RAW cells were activated with lipopolysaccharide, and 15 h later (when NO output was ~ 2 mM/h) placed over the tumor cells for 20 h, after which the latter were ALA-treated and then irradiated. Prior exposure to activated RAW macrophages reduced tumor cell photokilling by >50 %. This effect was completely lost when the RAW cells were pre-treated with the nitric oxide synthase inhibitor L-NAME, confirming that NO was involved in the hyperresistance. Results from other experiments suggest that heme oxygenase-1 and ferritin play a role in the preconditioning effect described. These studies provide new insights into how NO might modulate PDT efficacy.

Of the numerous biological activities attributed to nitric oxide ((*)NO), relatively little is known about its ability to intercept lipid-derived free radicals and thus protect cells against the damaging effects of lipid peroxidation, particularly in photodynamic settings. To address this, we asked how the (*)NO donor spermine-NONOate (SPER/NO) would affect porphyrin (PpIX)-photosensitized, iron/ascorbate-amplified chain peroxidation in cholesterol (Ch)/phospholipid (0.8:1.0, mol/mol) liposomes. Several Ch oxidation products (ChOX) were monitored by high performance chromatographic techniques. When added immediately before irradiation, SPER/NO (0.4 mM) had no effect on accumulation of 5alpha-hydroperoxide, a primary singlet oxygen-derived ChOX, but strongly suppressed the secondary species arising from postphotooxidation chain reactions, including 7alpha/7beta-hydroperoxides, 7alpha/7beta-hydroxides, and 5,6-epoxides. Metabolism of exogenous 5-aminolevulinate to PpIX in COH-BR1 tumor cells sensitized them to ChOX photogeneration and necrotic photokilling. When present during irradiation, active (but not decomposed) SPER/NO strongly inhibited both effects. These findings support the hypothesis that suitably presented NO, by intercepting lipid-derived radicals, can antagonize the antitumor effects of photodynamic therapy and other oxidative therapies.

In the presence of exciting light, iron and reductants, the singlet oxygen (1O2)-generating sensitizer protoporphyrin IX (PpIX) induces free radical lipid peroxidation in membranes, but gradually degrades in the process. We postulated that NO, acting as a chain-breaking antioxidant, would protect PpIX against degradation and consequently prolong its ability to produce 1O2. This idea was tested by irradiating PpIX-containing liposomes (LUVs) in the presence of iron and ascorbate, and monitoring the cholesterol hydroperoxides 5α-OOH and 7α/β-OOH as respective 1O2 and free radical reporters. 5α-OOH accumulation, initially linear with light fluence, slowed progressively after prolonged irradiation, whereas 7α/β-OOH accumulation only accelerated after an initial lag. The active, but not spent, NO donor spermine NONOate (0.4 mM) virtually abolished 7α/β-OOH buildup as well as 5α-OOH slowdown. Increasing membrane phospholipid unsaturation hastened the onset of rapid chain peroxidation and 5α-OOH slowdow...

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.