title: Cytoprotective signaling associated with nitric oxide upregulation in tumor cells subjected to photodynamic therapy-like oxidative stress.
abstract: Photodynamic therapy (PDT) employs photoexcitation of a sensitizer to generate tumor-eradicating reactive oxygen species. We recently showed that irradiating breast cancer COH-BR1 cells after treating with 5-aminolevulinic acid (ALA, a pro-sensitizer) resulted in rapid upregulation of inducible nitric oxide (NO) synthase (iNOS). Apoptotic cell killing was strongly enhanced by an iNOS inhibitor (1400W), iNOS knockdown (kd), or a NO scavenger, suggesting that NO was acting cytoprotectively. Stress signaling associated with these effects was examined in this study. ALA/light-stressed COH-BR1 cells, and also breast adenocarcinoma MDA-MB-231 cells, mounted an iNOS/NO-dependent resistance to apoptosis that proved to be cGMP-independent. Immunocytochemistry and subcellular Western analysis of photostressed COH-BR1 cells revealed a cytosol-to-nucleus translocation of NF-κB which was negated by the NF-κB activation inhibitor Bay11. Bay11 also enhanced apoptosis and prevented iNOS induction, consistent with NF-κB involvement in the latter. JNK and p38 MAP kinase inhibitors suppressed apoptosis, implicating these kinases in death signaling. Post-irradiation extent and duration of JNK and p38 phosphorylation were dramatically elevated by 1400 W or iNOS-kd, suggesting that these activations were suppressed by NO. Regarding pro-survival stress signaling, rapid activation of Akt was unaffected by 1400 W, but prevented by Wortmannin, which also enhanced apoptosis. Thus, a link between upstream Akt activation and iNOS induction was apparent. Furthermore, p53 protein expression under photostress was elevated by iNOS-kd, whereas robust Survivin induction was abolished, consistent with p53 and Survivin being negatively and positively regulated by NO, respectively. Collectively, these findings enhance our understanding of cytoprotective signaling associated with photostress-induced NO and suggest iNOS inhibitor-based approaches for improving PDT efficacy.
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin, USA. rbhowmic@mcw.edu
authors.forename: Reshma
authors.initials: R
authors.lastname: Bhowmick
authors.forename: Albert W
authors.initials: AW
authors.lastname: Girotti
mesh_terms: D000230:Adenocarcinoma / Q000378:metabolism / Q000628:therapy; D000622:Aminolevulinic Acid / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D001943:Breast Neoplasms / Q000188:drug therapy / Q000378:metabolism; D045744:Cell Line, Tumor; D019610:Cytoprotection; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors; D020935:MAP Kinase Signaling System; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism*; D009570:Nitriles / Q000494:pharmacology; D018384:Oxidative Stress; D010778:Photochemotherapy; D021381:Protein Transport / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species / Q000378:metabolism; D013450:Sulfones / Q000494:pharmacology; D000077022:Survivin; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors
chemical_list: C434003:3-(4-methylphenylsulfonyl)-2-propenenitrile; D051017:Apoptosis Regulatory Proteins; C107489:BIRC5 protein, human; D004791:Enzyme Inhibitors; D051034:Inhibitor of Apoptosis Proteins; D016328:NF-kappa B; D009570:Nitriles; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D013450:Sulfones; D000077022:Survivin; D009569:Nitric Oxide; D000622:Aminolevulinic Acid; C496319:NOS2 protein, human; D052247:Nitric Oxide Synthase Type II; D051057:Proto-Oncogene Proteins c-akt; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases