Comparative analysis of stress responses of H9c2 rat cardiomyoblasts following treatment with doxorubicin and tBOOH

Abstract

Cardiotoxicity is the major dose-limiting adverse effect of anthracyclines and is hypothesized to result from damage induced by reactive oxygen species (ROS) or inhibition of topoisomerase II. Here, we comparatively analyzed the effect of doxorubicin and the organic peroxide tertiary-butylhydroperoxide (tBOOH) on stress responses of rat cardiomyblast cells (H9c2). Moreover, we investigated the impact of serum factors and the novel prototypical protein kinase CK2 inhibitor resorufin on the sensentivity of H9c2 cells exposed to doxorubicin or tBOOH. Measuring cell viability by use of the WST assay as well as cell cycle progression and apoptotic death by FACS-based methods, we found that the sensitivity of H9c2 cells to doxorubicin and tBOOH was differently affected by both serum factors and resorufin. Formation of reactive oxygen species was observed after exposure of H9c2 cells to high doses (i.e. ≥5 μM) of doxorubicin only. Moreover, the antioxidant N-acetylcysteine protected H9c2 cells from cytotoxicity provoked by tBOOH but not doxorubicin. Analyzing the phosphorylation level of genotoxic stress responsive protein kinases and histone H2AX, which is indicative of an activated DNA damage response (DDR), we found that resorufin modulates doxorubicin- and tBOOH-induced responses in an agent specific manner. Taken together, the data indicate that (i) oxidative injury is not the most relevant type of damage triggering cell death of H9c2 cells following doxorubicin treatment, (ii) serum factors differently influence the sensitivity of cardiomyoblasts to doxorubicin and tBOOH and (iii) inhibition of CK2 unequally affects doxorubicin- and tBOOH-induced DDR of rat cardiomyoblasts.