The Aqueous Extract of TA Bark Reduces Mitochondrial Oxidative Stress Induced by Doxorubicin in H9c2 cells

Abstract

Increased oxidative stress (OS) is one of mechanisms responsible for cardiotoxicity induced by doxorubicin (DOX), a commonly-used anticancer chemotherapeutic agent. We have found that the aqueous extract of TA bark (TAAqE) has a carditonic effect on adult ventricular myocytes. This study is to investigate effects of TAAqE on DOX-induced OS in H9c2 cells, a cell-line model derived from embryonic rat heart. Fluorescence microscopy (FM) with OS-sensitive probes and immunocytochemistry (ICC) were used to determine the level of oxidative status and target proteins. H9c2 cells were treated in the absence or presence of TAAqE without or with exposure to 1 μM DOX for 2 to 24 hours. FM of dihydroethidium showed that TAAqE decreased DOX-induced increase in O2-• production in a concentration-dependent manner (1-100 μg/ml) after 2 hr treatment. Similar inhibitory effects of TAAqE were observed after 24 hr treatment of DOX. In contrast, FM of dichlorodihydro-fluorescein showed that TAAqE had no significant effect on DOX-induced increase in the H2O2-associated OS, suggestive of various reactive oxygen species involved in increased OS induced by DOX. Moreover, FM of Mitotracker-Orange showed that TAAqE also attenuated DOX-induced increase in mitochondrial OS in a concentration-dependent manner, while TAAqE (100 μg/ml) per se induced 20% reduction of mitochondrial OS in cells not treated with DOX. In addition, ICC detection of Cytochrome C (CytC) showed that TAAqE attenuated DOX-induced increase in mitochondrial release of CytC after 24-h treatment. In conclusion, TAAqE appears to protect primarily mitochondria from increased O2-• production caused by DOX.