4'-Epidoxorubicin to re-explore anthracycline degradation in cardiomyocytes

Abstract

Cardiotoxicity limits the clinical use of doxorubicin (DOX) and other quinone-hydroquinone antitumor anthracyclines. One-electron reduction of the quinone moiety is followed by the formation of reactive oxygen species (ROS) that have been proposed to induce cardiotoxicity through an oxidative stress; conversely, one-electron oxidation of the hydroquinone moiety by hydrogen peroxide (H(2)O(2)) and oxyferrous myoglobin (Mb(II)O(2)) is followed by an anthracycline degradation process that has been proposed to limit cardiotoxicity. We previously reported that tert-butoxycarbonyl-alanine (t-BA) impeded DOX oxidation/degradation by H(2)O(2)/Mb(II)O(2) in a cell-free system; accordingly, t-BA increased the levels of DOX, its conversion to ROS, and its concentration-related toxicity in cardiomyocytes. To re-explore methodological and toxicological aspects of anthracycline degradation, we used 4'-epidoxorubicin (EPI), an anthracycline analogue that is very similar to DOX but undergoes protonation-sequestration in cytoplasmic acidic organelles. t-BA lacked an effect on H9c2 cardiomyocytes exposed to EPI; however, blocking the protonation-sequestration mechanism with the vacuolar H(+)-ATPase inhibitor, bafilomycin A1 (BFL), enabled t-BA to increase the cellular levels of EPI, its conversion to ROS, and its concentration-related toxicity. This suggested that t-BA was specific enough to increase the cellular levels and toxicity of only those anthracyclines that were liable to oxidation/degradation by H(2)O(2)/Mb(II)O(2). By exposing cardiomyocytes to nontoxic concentrations of DOX or EPI and by increasing their cellular levels by means of appropriate combinations with t-BA, BFL, or t-BA+BFL, we nonetheless found that the loss of cardiomyocyte viability correlated with the accumulation of undegraded anthrayclines but not with their ability to form ROS or to induce lipid peroxidation. This suggested that an accumulation of undegraded anthracyclines might induce cardiotoxicity also by mechanisms independent of ROS and oxidative stress. Thus, EPI proved useful to refine the value of t-BA in the studies of anthracycline degradation and to reappraise the role of anthracycline degradation in cardiotoxicity.