title: Nitric Oxide-Releasing Aspirin Suppresses NF-κB Signaling in Estrogen Receptor Negative Breast Cancer Cells in Vitro and in Vivo.
abstract: Estrogen receptor negative (ER(-)) breast cancer is aggressive, responds poorly to current treatments and has a poor prognosis. The NF-κB signaling pathway is implicated in ER(-) tumorigenesis. Aspirin (ASA) is chemopreventive against ER(+) but not for ER(-) breast cancers. Nitric oxide-releasing aspirin (NO-ASA) is a safer ASA where ASA is linked to an NO-releasing moiety through a spacer. In vitro, we investigated anti-proliferation effects of NO-ASA (para- and meta-isomers) against ER(-) breast cancer cells MDA-MB-231 and SK-BR-23, effects on NF-κB signaling, and reactive oxygen species by standard techniques. In vivo, effects of NO-ASA were evaluated in a mouse xenograft model using MDA-MB-231 cells. p-NO-ASA inhibited the growth of MDA-MB-231 and SK-BR-3 cells at 24 h, the respective IC50s were 13 ± 2 and 17 ± 2 μM; ASA had an IC50 of >3000 μM in both cell lines. The IC50s for m-NO-ASA in MDA-MB-231 and SK-BR-3 were 173 ± 15 and 185 ± 12 μM, respectively, therefore, implying p-NO-ASA as a stronger inhibitor of growth p-NO-ASA reduced cell growth by inhibiting proliferation, inducing apoptosis and causing G0/G1 cell cycle block. Activation of NF-κB was inhibited by both isomers as demonstrated by decreases in NF-κB-DNA binding and luciferase activity at 24 h, However, m-NO-ASA produced transient effects at 3 h such as increased NF-κB-DNA-binding, increased levels of nuclear p50, even though both isomers inhibited IκB degradation. Increase in nuclear p50 by m-NO-ASA was associated with translocation of p50 in to the nucleus as observed by immunoflouresence at 3 h. NO-ASA induced reactive oxygen species (ROS) as evidenced by overall increases in both H2DCFDA (2',7'-dichlorodihydrofluorescein) and DHE (dihydroethidium)-derived fluorescence. Inhibition of ROS by N-acetyl-cysteine reversed the m-NO-ASA-mediated translocation of p50 in to the nucleus. In xenografts, p-NO-ASA inhibited tumor growth by inhibiting proliferation (PCNA and tumor volume), inducing apoptosis (TUNEL positive cells) and reducing NF-κB expression. Both isomers inhibit cancer cells, inhibit NF-κB pathway and induce ROS, and have potential as anticancer compounds.
authors.affiliation: Department of Physiology, Pharmacology and Neuroscience, Sophie Davis School of Biomedical Education, City University of New York Medical School, New York, NY 10031, USA. nnath@nyit.edu.|Department of Life Sciences, New York Institute of Technology, New York, NY 10023, USA. nnath@nyit.edu.
authors.forename: Niharika
authors.initials: N
authors.lastname: Nath
authors.affiliation: Department of Physiology, Pharmacology and Neuroscience, Sophie Davis School of Biomedical Education, City University of New York Medical School, New York, NY 10031, USA. mitali@med.cuny.edu.
authors.forename: Mitali
authors.initials: M
authors.lastname: Chattopadhyay
authors.affiliation: Department of Physiology, Pharmacology and Neuroscience, Sophie Davis School of Biomedical Education, City University of New York Medical School, New York, NY 10031, USA. debrodes@optonline.net.
authors.forename: Deborah B
authors.initials: DB
authors.lastname: Rodes
authors.affiliation: Department of Physiology, Pharmacology and Neuroscience, Sophie Davis School of Biomedical Education, City University of New York Medical School, New York, NY 10031, USA. meetannanazarenko@gmail.com.
authors.forename: Anna
authors.initials: A
authors.lastname: Nazarenko
authors.affiliation: Department of Physiology, Pharmacology and Neuroscience, Sophie Davis School of Biomedical Education, City University of New York Medical School, New York, NY 10031, USA. ravinder@med.cuny.edu.
authors.forename: Ravinder
authors.initials: R
authors.lastname: Kodela
authors.affiliation: Department of Physiology, Pharmacology and Neuroscience, Sophie Davis School of Biomedical Education, City University of New York Medical School, New York, NY 10031, USA. kashfi@med.cuny.edu.|Division of Cancer Prevention, Department of Medicine, Stony Brook University, Stony Brook, NY 11794, USA. kashfi@med.cuny.edu.
authors.forename: Khosrow
authors.initials: K
authors.lastname: Kashfi
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001241:Aspirin / Q000031:analogs & derivatives* / Q000494:pharmacology; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter; D006801:Humans; D020128:Inhibitory Concentration 50; D007279:Injections, Subcutaneous; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000819:agonists / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000172:deficiency* / Q000235:genetics; D011960:Receptors, Estrogen / Q000172:deficiency* / Q000235:genetics; D015398:Signal Transduction; D023041:Xenograft Model Antitumor Assays
chemical_list: D000970:Antineoplastic Agents; D001241:Aspirin; D008156:Luciferases; D016328:NF-kappa B; D018809:Proliferating Cell Nuclear Antigen; D017382:Reactive Oxygen Species; D018719:Erb-b2 Receptor Tyrosine Kinases; D011960:Receptors, Estrogen; C508053:ERBB2 protein, human; C102148:nitroaspirin
keywords: NF-κB; NO-NSAIDs; NSAIDs; breast cancer; estrogen receptor; oxidative stress