Nitric oxide-induced resistance to lethal photooxidative damage in a breast tumor cell line

The long-term effects of nitric oxide (NO) on cell susceptibility to photodynamic killing have been studied, using a human breast tumor line (COH-BR1). Subconfluent cells were exposed to a nonlethal dose of spermine NONOate (SPNO, 0.2 mM) and 20 h later were metabolically sensitized with protoporphyrin IX (PpIX) by incubating with 5-aminolevulinic acid. PpIX overproduced in mitochondria was allowed to diffuse to peripheral sites, including plasma membrane, after which a photooxidative challenge was imposed. Active (but not decomposed) SPNO made cells substantially more resistant to necrotic photokilling than non-SPNO-treated controls. A similar response to a tert-butyl hydroperoxide challenge was observed. Hyperresistance was detected approximately 8 h post-SPNO, maximized after approximately 20 h, and reflected diminished oxidant accumulation, as determined with 2',7'-dichlorofluorescein. Intracellular free iron determined with the fluorescent probe calcein rose to approximately 160% of the control level 6 h after SPNO, but declined to approximately 70% after 24 h. Immunoblot analyses revealed a rapid early (approximately 2 h post-NO) increase in heme oxygenase-1 level, followed by a gradual (4-20 h post-NO) increase in ferritin. Upregulation of these proteins is consistent with a cytoprotective mechanism involving mobilization of "signaling" iron. Preactivated RAW 264.7 macrophages on microporous inserts also induced a long-term photoresistance in underlying PpIX-sensitized COH-BR1 cells. This response was abolished by L-NAME, indicating that NO from induced nitric oxide synthase was involved. The NO effects described are entirely novel in the context of photooxidative stress and provide new insights into how NO might affect antitumor photodynamic therapy (PDT).