Reactive oxygen species-independent apoptosis in doxorubicin-treated H9c2 cardiomyocytes : role for heme oxygenase-1 down-modulation

Increased oxidative stress and apoptosis have been implicated in the cardiotoxicity that limits the clinical use of doxorubicin (DOX) as an anti-tumoral drug, but the mechanism of DOX-mediated apoptosis remains unclear. We examined the interplay between oxidative stress and cell death in cardiac-derived H9c2 myocytes exposed to DOX doses in the range of the plasma levels found in patients undergoing chemotherapy. A low DOX concentration (0.25muM) induced apoptosis, whereas the cells treated with the high dose of 2muM also showed necrosis. The production of reactive oxygen species (ROS) and induction of oxidative stress markers was increased in the cells treated with 2muM DOX but not in those treated with the low dose. Surprisingly, heme oxygenase (HO-1) expression was down-modulated in the cells exposed to 0.25muM DOX, and its Bach 1 transcriptional repressor was induced. In line with the role of HO-1 as an anti-apoptotic protein, inhibiting HO-1 activity with SnPPIX was sufficient to induce apoptosis and increased DOX-mediated apoptosis, whereas hemin-induced HO-1 activation prevented DOX-mediated apoptotic cell death. In brief, our findings do not support the hypothesis that oxidative stress plays a role in the apoptotic cell death occurring in cardiomyocytes exposed to low concentrations of DOX, but suggest that DOX may facilitate the apoptosis of cardiomyocytes by inhibiting the anti-apoptotic HO-1.