Doxorubicin-induced apoptosis in endothelial cells and cardiomyocytes is ameliorated by nitrone spin traps and ebselen - Role of reactive oxygen and nitrogen species

Doxorubicin (DOX) is a broad spectrum anthracycline antibiotic used to trea t a variety of cancers. Redox activation of DOX to form reactive oxygen spe cies has been implicated in DOX-induced cardiotoxicity, In this work we inv estigated DOX-induced apoptosis in cultured bovine aortic endothelial cells and cardiomyocytes isolated from adult rat heart. Exposure of bovine aorti c endothelial cells or myocytes to submicromolar levels of DOX induced sign ificant apoptosis as measured by DNA fragmentation and terminal deoxynucleo tidyltransferase-mediated nick-end labeling assays, Pretreatment of cells w ith 100 muM nitrone spin traps, N-tert-butyl-alpha -phenylnitrone (PBN) or alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) dramatically inhibited DOX-induced apoptosis, Ebselen (20-50 muM), a glutathione peroxidase mimet ic, also significantly inhibited apoptosis, DOX (0.5-1 muM) inactivated mit ochondrial complex I by a superoxide-dependent mechanism. PEN (100 muM), PO BN (100 muM), and ebselen (50 muM) restored complex I activity. These compo unds also inhibited DOX-induced caspase-3 activation and cytochrome c relea se. PEN and ebselen also restored glutathione levels in DOX-treated cells. We conclude that nitrone spin traps and ebselen inhibit the DOX-induced apo ptotic signaling mechanism and that this antiapoptotic mechanism may be lin ked in part to the inhibition in formation or scavenging of hydrogen peroxi de. Therapeutic strategies to mitigate DOX cardiotoxicity should be reexami ned in light of these emerging antiapoptotic mechanisms of antioxidants.