Doxorubicin-induced cytotoxicity in rat myocardial H9c2 cells: the roles of reactive oxygen species and redox balance

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
Ho Young Lee (Creator)
The University of North Carolina at Greensboro (UNCG )
Web Site:
Zhenquan Jia

Abstract: Doxorubicin (Dox) is one of the most potent anti-neoplastic agents approved by the Food and Drug Administration. Its efficacy, however, is limited due to its well-documented cardiotoxic side effect. Since the first observation of this dosage-dependent side effect, the mechanisms and events leading to cardiotoxicity following exposure to doxorubicin have received much attention. However, the exact pathogenesis of Dox-induced cardiotoxicity remains to be elucidated. Although increased production of reactive oxygen species (ROS) from the redox cycling of Dox has been recognized as the primary mechanism of Dox-induced cardiotoxicity, it must be noted that many of the studies supporting the oxidative stress-induced cardiotoxicity hypothesis were conducted with supraclinical drug concentrations. This study examined the effect of clinically-relevant concentrations of Dox on H9c2 rat cardiomyoblasts. Through MTT-reduction cell viability assay, it was determined that exposure of H9c2 cells to Dox concentrations above 0.5 µM for more than 12 hours resulted in significant reduction in cell viability. To verify the role of oxidative stress on the development of cytotoxicity, ROS levels after exposure to low concentrations of Dox (less than 2 µM) were measured. Quantitative measurements of both cellular and mitochondrial ROS levels revealed no significant changes to superoxide presence while exhibiting significant decrease in hydrogen peroxide presence. However, despite the decreased presence of two major types of ROS, the potency of antioxidant responses from the H9c2 cells were found to have significantly increased. Also, exposure to Dox at clinically relevant concentrations led to significant increase in the gene expressions of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1(ICAM-1), two key adhesion molecules that have been implicated in Dox-induced cardiotoxicity. These results suggest that lower concentrations of Dox can stimulate intracellular anti-oxidative response that may thwart intracellular ROS levels required for maintaining of proper cell functions, ultimately leading to redox imbalance and inflammation in cardiomyocytes. While attempting to further investigate into the specific mode of cell death induced by Dox treatment, it was found that the innate fluorescence of Dox may be potent enough to be recognized by various fluorescence-based detection methods. Dox was found to exhibit fluorescence spectra consisting of a maximum excitation wavelength of 493 nm and a maximum emission wavelength of 592 nm, which were similar to the fluorescence characteristics of common fluorescent markers such as FITC, PI, MitoSOX, and DCF-DA which are widely used to assess cell viability, as well as ROS production. Furthermore, nuclear accumulation of Dox was confirmed by fluorescence microscopy, and spectrofluorometric measurements which detected the cellular uptake of Dox. This suggests that the innate fluorescence of Dox can be a valid probe used for future investigations for the uptake, release and distribution of Dox both in vitro and in vivo. Altogether, this study demonstrated for the first time that exposure to Dox at clinically relevant plasma concentrations significantly decreased hydrogen peroxide levels below the basal levels in both intact H9c2 rat cardiomyocytes and in isolated mitochondria. Cells treated with Dox showed a significant increase in the expression of genes associated with anti-oxidative response and inflammation. Utilizing the intrinsic fluorescence of Dox, it was found that incubation of H9c2 cells with Dox resulted in time-dependent intracellular uptake of Dox. This study may contribute in advancing our understanding of mechanisms responsible for Dox-induced cardiotoxicity and thereby improving the efficacy of Dox, one of the most prominent components of many chemotherapy regimens.

Additional Information

Language: English
Date: 2017
Cardiotoxicity, Cardiovascular Diseases, Doxorubicin, Fluorescence, H9c2, Redox
Doxorubicin $x Toxicology
Heart $x Effect of drugs on
Cardiovascular toxicology
Active oxygen

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