Endothelial Nitric Oxide Synthase-Dependent Superoxide Generation from Adriamycin

Abstract

Adriamycin (or doxorubicin) is an active and broad spectrum chemotherapeutic agent. Unfortunately, its clinical use is severely restricted by a dose-limiting cardiotoxicity which has been linked to the formation of superoxide. Enzymatic one-electron reduction of adriamycin forms adriamycin semiquinone radical, which rapidly reacts with oxygen to form superoxide and adriamycin. In this way, adriamycin provides a kinetic mechanism for the one-electron reduction of oxygen by flavoenzymes such as NADPH-cytochrome P450 reductase and mitochondrial NADH dehydrogenase. We demonstrate here that the endothelial isoform of nitric oxide synthase (eNOS) reduces adriamycin to the semiquinone radical. As a consequence, superoxide formation is enhanced and nitric oxide production is decreased. Adriamycin binds to eNOS with a Km of approximately 5 microM, as calculated from both eNOS-dependent NADPH consumption and superoxide generation. Adriamycin stimulated superoxide formation is not affected by calcium/calmodulin and is abolished by the flavoenzyme inhibitor, diphenyleneiodonium. This strongly suggests that adriamycin undergoes reduction at the reductase domain of eNOS. A consequence of eNOS-mediated reductive activation of adriamycin is the disruption of the balance between nitric oxide and superoxide. This may lead eNOS to generate peroxynitrite and hydrogen peroxide, potent oxidants implicated in several vascular pathologies.

Doxorubicin-induced chronic dilated cardiomyopathy—the apoptosis hypothesis revisited

“…ROS is usually thought to be elevated in cardiomyocyte by DOX binding to cardiolipin at the inner mitochondrial membrane, impairing mitochondrial respiration [72]. ROS after DOX exposure can also be induced by altering endothelial nitric oxidase signaling leading further to reduced production of the cardiac vasodilator NO [73]. Both, increased ROS and lack NO can hence directly impair the myocardium and heart muscle compliance even in the absence of apoptosis [74].…”

p38 MAPK downregulates phosphorylation of Bad in doxorubicin-induced endothelial apoptosis

“…It is known that doxorubicin treatment of cultured cells leads to production of reactive oxygen species (ROS) [39,40], and that oxidative stress activates p38 MAPK [41], thus we wanted to elucidate the role of p38 MAPK in doxorubicin-induced apoptosis. To this end, we used lentiviral transduction of EA.hy926 cells to express a dominant negative mutant Flag-p38α MAPK harboring T180A and Y182 F amino acid substitutions [37].…”

Phenylbutyrate, a histone deacetylase inhibitor, protects against Adriamycin-induced cardiac injury

“…Oxidative stress is generally held as the mediating mechanism in the multiple biological processes leading to ADR cardiotoxicity, e.g. redox mediated superoxide radical production [5,6], tissue-specific mitochondrial DNA damage [7], and disturbances of calcium [8,9] or iron homeostasis [10,11]. Structurally, ADR is a quinone, which can generate a large amount of O 2 • -via a redox cycling reaction catalyzed by several endogenous reductases [5] and endothelial isoform of nitric oxide synthase [6].…”

“…redox mediated superoxide radical production [5,6], tissue-specific mitochondrial DNA damage [7], and disturbances of calcium [8,9] or iron homeostasis [10,11]. Structurally, ADR is a quinone, which can generate a large amount of O 2 • -via a redox cycling reaction catalyzed by several endogenous reductases [5] and endothelial isoform of nitric oxide synthase [6]. O 2 • -in turn gives rise to a variety of more active reactive oxygen species (ROS), including H 2 O 2 , •OH and ONOO -., which trigger further oxidation of bio-molecules [12].…”