Abstract 132: Cytoprotective induction of nitric oxide synthase in a cellular model of 5-aminolevulinic acid-based photodynamic therapy

Abstract

Abstract Photodynamic therapy (PDT) employs a photosensitizing agent, molecular oxygen, and visible light to produce reactive oxygen species that kill tumor and tumor vasculature cells. Nitric oxide (NO) produced by these cells could be pro-carcinogenic by inhibiting apoptosis or promoting angiogenesis and tumor growth. Our previous studies showed that NO from a chemical donor made COH-BR1 breast tumor cells hyperresistant to apoptotic photokilling induced by photoactivation of 5-aminolevulinic acid (ALA)-generated protoporphyrin IX localized in mitochondria. Hyperresistance was associated with inhibition of proapoptotic JNK and p38α MAP kinase phosphorylation-activation. The purpose of this study was to determine whether tumor cells upregulate NOS-generated NO as a cytoprotective measure during PDT.COH-BR1 cells sensitized in mitochondria with ALA-derived protoporphyrin IX died apoptotically following irradiation as measured by Hoechst staining. Western analysis revealed that inducible nitric oxide synthase (iNOS) was upregulated >3-fold within 4 h after ALA/light treatment, while other NOS isoforms were unaffected. Exposing cells to the iNOS inhibitor, 1400W during photochallenge enhanced caspase-3/7 activation and apoptotic killing up to 2-3-fold while substantially reducing chemiluminescence-assessed NO production, suggesting that this NO was cytoprotective. Consistently, the NO scavenger cPTIO enhanced ALA/light-induced caspase-3/7 activation and apoptotic kill by >2.5-fold. Of added significance, cells could be rescued from 1400W-exacerbated apoptosis by an exogenous NO donor, spermine-NONOate. Using Hoechst and TUNEL staining, we found that short hairpin RNA (shRNA)-induced knockdown of iNOS enhanced the apoptotic kill of ALA/light treated COH-BR1 cells. ALA/light-treated COH-BR1cells exhibited a transient post-irradiation activation of JNK and p38α as measured by Western blot analysis. Consistently, both effects were intensified and prolonged by 1400W. The survival MAP kinase ERK1/2 was deactivated more rapidly when 1400W was present during a PpIX/light challenge. Similar effects on MAP kinase activation/deactivation were observed for iNOS knockdown cells under photostress cinfirming iNOS's protective role. As demonstrated for non-photodynamic stress systems, NO could have interfered with apoptosis by inactivating participating MAP kinases and/or caspases. This is the first reported evidence for increased tumor cell resistance due to iNOS upregulation in a PDT model. Our findings indicate that stress-elicited NO in PDT-treated tumors could compromise therapeutic efficacy, and suggest NOS-based pharmacologic interventions for preventing this. (Supported by NIH Grant CA70823) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 132.