Comparative cardiac toxicity of anthracyclines in vitro and in vivo in the mouse

Abstract

Purpose: The antineoplastic efficacy of anthracyclines is limited by their cardiac toxicity. In this study, we evaluated the toxicity of doxorubicin, non-pegylated liposomal-delivered doxorubicin, and epirubicin in HL-1 adult cardiomyocytes in culture as well as in the mouse in vivo.

Methods: The cardiomyocytes were incubated with the three anthracyclines (1 µM) to assess reactive oxygen generation, DNA damage and apoptotic cell death. CF-1 mice (10/group) received doxorubicin, epirubicin or non-pegylated liposomal-doxorubicin (10 mg/kg) and cardiac function was monitored by Doppler echocardiography to measure left ventricular ejection fraction (LVEF), heart rate (HR) and cardiac output (CO) both prior to and 10 days after drug treatment.

Results: In HL-1 cells, non-pegylated liposomal-doxorubicin generated significantly less reactive oxygen species (ROS), as well as less DNA damage and apoptosis activation when compared with doxorubicin and epirubicin. Cultured breast tumor cells showed similar sensitivity to the three anthracyclines. In the healthy mouse, non-pegylated liposomal doxorubicin showed a minimal and non-significant decrease in LVEF with no change in HR or CO, compared to doxorubicin and epirubicin.

Conclusion: This study provides evidence for reduced cardiac toxicity of non-pegylated-liposomal doxorubicin characterized by attenuation of ROS generation, DNA damage and apoptosis in comparison to epirubicin and doxorubicin.

Figure 1. Effects of anthracyclines on reactive oxygen species production.

Cells were treated with the three anthracyclines for 20 minutes prior to determination of reactive oxygen species (ROS). The graph represents the fluorescence units recorded following each treatment. Abbreviations list: controlsCtrl; epirubicinEpi; doxorubicinDox; non-pegylated liposomal-doxorubicinLDox. Results are reported as Mean±Standard Error.

Figure 2. Comparative analysis of anthracyclines effects on DNA double strand breaks: phosphorylation of serine 139 of H2AX (γH2AX).

Cells were treated with the anthracyclines for 2 hours. Panel A presents representative images of HL-1 cell nuclei stained for γH2AX. The nuclei were counterstained with DAPI to detect specific signals and images were collected through confocal microscopy. Light microscopy images were taken for the field analyzed. Panel B presents the absolute value of the average number of foci detected in each nucleus for each treatment. Experiment was performed in quadruplicate. Abbreviations list: controlsCtrl; epirubicinEpi; doxorubicinDox; non-pegylated liposomal-doxorubicinLDox. Results are reported as Mean±Standard Error.

Figure 3. Effects of anthracyclines on apoptotic markers in HL-1 cardiomyocytes in vitro.

Cells were treated with the anthracyclines for 24 hours. Panel A shows caspase-3 cleavage and a representative densitometric analysis of the active/cleaved caspase-3. Panel B presents a comparison of the enzymatic activity associated with the cleavage of a substrate common to caspase-3 and caspase-7. Panel C shows the percentage of annexin V positive cardiomyocytes following anthracycline treatment. Abbreviations list: controlsCtrl; epirubicinEpi; doxorubicinDox; non-pegylated liposomal-doxorubicinLDox. Results are reported as Mean±Standard Error.

Figure 4. Effects of anthracyclines on growth of MCF-7 breast cancer cells (MTT assay).

MCF-7 cells were exposed to increasing doses (100 nM, 250 nM, 500 nM, 1 µM and 5 µM) of anthracyclines for 24 hours. Absorbance values indicated are proportional to viable cell number. Abbreviations list: controlsCtrl; epirubicinEpi; doxorubicinDox; non-pegylated liposomal-doxorubicinLDox. Results are reported as Mean±Standard Error.

Figure 5. Interval changes in left ventricular ejection fraction (LVEF), heart rate (HR) and cardiac output (CO) in the hearts of mice treated with anthracyclines.

CF-1 mice (N = 10/group) were treated with a single intraperitoneal dose of 10 mg/kg of epirubicin, doxorubicin, non-pegylated liposomal-doxorubicin (L-Doxorubicin) or a matching volume of vehicle-solution (0.9% NaCl). The changes in LVEF, HR and CO were recorded 10 days following treatment and the results are reported as percentage change compared to baseline values and expressed as Mean±Standard Error.