title: Doxorubicin-induced apoptosis is associated with increased transcription of endothelial nitric-oxide synthase. Effect of antiapoptotic antioxidants and calcium.
abstract: The clinical efficacy of the antitumor antibiotic drug doxorubicin (DOX) is severely limited by its dose-limiting cardiotoxicity in cancer patients. DOX-induced generation of reactive oxygen species was proposed to be a major mechanism of its cardiotoxicity. Previously, we showed that DOX undergoes a reductive activation at the reductase domain of endothelial nitric-oxide synthase (eNOS) forming the semiquinone and superoxide (Vásquez-Vivar, J., Martasek, P., Hogg, N., Masters, B. S. S., Pritchard, K. A., Jr., and Kalyanaraman, B. (1997) Biochemistry 36, 11293-11297). In this report, we provide evidence for DOX-induced increase in eNOS transcription and protein expression in bovine aortic endothelial cells (BAEC). We propose that DOX-induced hydrogen peroxide formation is responsible for the increased transcription of eNOS. BAEC treated with antisense eNOS oligonucleotide inhibits DOX-induced endothelial apoptosis. Treatment with antioxidants restored the levels of antiapoptotic proteins (Hsp70 and Bcl-2) in DOX-treated BAEC. DOX-induced intracellular oxidative stress, as measured by oxidation of dichlorodihydrofluorescein diacetate to dichlorofluorescein and hydroethidium to ethidium, was inhibited by antisense eNOS oligonucleotide and antioxidant treatment. Furthermore, antiapoptotic antioxidants (e.g. FeTBAP, ebselen, and alpha-phenyl-tert-butyl nitrone) inhibited DOX-induced eNOS transcription. We conclude that DOX-induced apoptosis is linked to the redox activation of DOX by eNOS.
authors.affiliation: Biophysics Research Institute and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
authors.forename: S V
authors.initials: SV
authors.lastname: Kalivendi
authors.forename: S
authors.initials: S
authors.lastname: Kotamraju
authors.forename: H
authors.initials: H
authors.lastname: Zhao
authors.forename: J
authors.initials: J
authors.lastname: Joseph
authors.forename: B
authors.initials: B
authors.lastname: Kalyanaraman
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D000975:Antioxidants / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D002417:Cattle; D002460:Cell Line; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000494:pharmacology*; D004727:Endothelium / Q000201:enzymology; D004730:Endothelium, Vascular / Q000166:cytology; D004789:Enzyme Activation; D005452:Fluoresceins / Q000494:pharmacology; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D008954:Models, Biological; D008956:Models, Chemical; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D009566:Nitrates / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics* / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D009573:Nitrites / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism; D017434:Protein Structure, Tertiary; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014158:Transcription, Genetic
chemical_list: D000970:Antineoplastic Agents; D000975:Antioxidants; D005452:Fluoresceins; D018840:HSP70 Heat-Shock Proteins; D009566:Nitrates; D009573:Nitrites; D016376:Oligonucleotides, Antisense; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; C037631:2',7'-dichlorofluorescein; D004317:Doxorubicin; D006861:Hydrogen Peroxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D009251:NADPH-Ferrihemoprotein Reductase; D010100:Oxygen; D002118:Calcium