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Nitric oxide-induced resistance to lethal photooxidative damage in a breast tumor cell line
Magdalena Niziolek1, Witold Korytowski, Albert W Girotti
1Department of Biochemistry, Medical College of Wisconsin, Milwaukee, 53226, USA.
Abstract:
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).
Insights
Nitric oxide (NO) enhances cancer cell resistance to photodynamic therapy by upregulating protective proteins. This finding offers new strategies for improving antitumor photodynamic therapy (PDT) efficacy.
Area of Science:
- Biochemistry
- Cell Biology
- Oncology
Background:
- Photodynamic therapy (PDT) is a cancer treatment utilizing photosensitizers and light to generate reactive oxygen species.
- Nitric oxide (NO) is a signaling molecule with complex roles in cell physiology and disease.
- Understanding NO's impact on PDT is crucial for optimizing treatment strategies.
Purpose of the Study:
- To investigate the long-term effects of nitric oxide (NO) on the susceptibility of human breast tumor cells (COH-BR1) to photodynamic killing.
- To elucidate the molecular mechanisms underlying NO-mediated changes in cellular resistance to photooxidative stress.
Main Methods:
- COH-BR1 cells were treated with spermine NONOate (SPNO) to generate NO, followed by sensitization with protoporphyrin IX (PpIX) and photooxidative challenge.
- Cellular resistance was assessed via tert-butyl hydroperoxide challenge and oxidant accumulation measurements using 2",7"-dichlorofluorescein.
- Intracellular free iron levels were determined using calcein, and protein expression (heme oxygenase-1, ferritin) was analyzed via immunoblotting.
- Co-culture experiments with RAW 264.7 macrophages and L-NAME treatment were used to investigate NO's role in induced photoresistance.
Main Results:
- Active NO significantly increased COH-BR1 cell resistance to photodynamic killing and tert-butyl hydroperoxide-induced damage.
- This hyperresistance, observed ~8-20 hours post-NO exposure, correlated with diminished oxidant accumulation.
- NO treatment led to early increases in heme oxygenase-1 and gradual increases in ferritin, suggesting a cytoprotective mechanism involving iron mobilization.
- NO generated by macrophages also induced long-term photoresistance in underlying tumor cells, dependent on nitric oxide synthase activity.
Conclusions:
- Nitric oxide confers long-term resistance to photooxidative stress in human breast tumor cells.
- The observed cytoprotective effects involve the upregulation of heme oxygenase-1 and ferritin, potentially through iron signaling pathways.
- These findings reveal novel mechanisms by which NO influences cellular responses to oxidative stress and have significant implications for the application of photodynamic therapy in cancer treatment.
