Doxorubicin (Dox) is an anthracycline drug that is effectively used as a chemotherapeutic agent for the treatment of a variety of cancers. Its dose-dependent cardiotoxicity, however, has limited its chemoprotective properties in clinical practice. One of the primary mechanisms of Dox-induced cardiotoxicity is the generation of intracellular reactive oxygen species (ROS) by an iron-catalyzed reaction. We hypothesized that the consequent protein oxidation under oxidative stress can be monitored and correlated with cellular outcome of survival or death. First, to understand how Dox-induced oxidative stress is related to the cardiotoxicity, we identified specific cardiac proteins that are oxidatively modified under Dox-induced oxidative stress in a spontaneously hypertensive rat model of acute toxicity. Next we characterized the type(s) of modifications and the amino acid residues within the protein that undergo modification under oxidative stress. Finally, we investigated the functional consequences of such oxidative modifications. Based on our findings, we focused on protein carbonylation as an indicator of severe oxidative damage. We detected a highly carbonylated protein in the cardiac tissue after Dox-treatment. The Dox-induced carbonylated protein was identified as a cardiac myosin binding protein C (MyBPC) using mass spectrometry. To determine the carbonylated sites and effect of such modification in the protein structure and function, we expressed MyBPC in E. coli and purified recombinant MyBPC. Consistent with the in vivo results, recombinant MyBPC showed an increase in carbonylation under excessive oxidative conditions. The degree of carbonylation was time- and concentration-dependent. Excessive carbonylation was associated with the degradation of MyBPC. Identification of carbonylated sites and the effect of such carbonylation in the protein functions will help to better understand the Dox-induced cardiac toxicity. Our findings also indicate the potential for an oxidative biomarker for monitoring the Dox-induced cardiotoxicity.

Citation Format: Baikuntha P. Aryal, Jinsook Jeong, V. Ashutosh Rao. Identification and functional characterization of a novel oxidatively modified protein in the cardiac tissue after doxorubicin-induced cardiotoxicity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2496. doi:10.1158/1538-7445.AM2013-2496