Objective
Although the combination of doxorubicin (Dox) and trastuzumab (Trz) reduces breast cancer progression and recurrence, it is limited by significant cardiotoxic side effects. Little is known about the utility of antioxidants in the prevention of this drug-induced cardiomyopathy. The aim of the study was to determine whether the antioxidant probucol (Prob) would be useful in attenuating Dox and Trz-mediated cardiotoxicity.
Methods
A total of 114 mice were randomized to treatment with Trz, Dox, or Dox+Trz. Within each arm, mice received prophylactic treatment with placebo or Prob. Serial murine echocardiography with tissue Doppler imaging was performed daily for 10 days. At 10 days posttreatment, the hearts were removed for histopathologic and Western blot analyses.
Results
Left ventricular cavity dimensions and systolic parameters were preserved in mice prophylactically treated with Prob after the administration of Dox+Trz. Although the combination of Dox+Trz demonstrated >80% mortality at day 5, prophylactic treatment with Prob reduced mortality to 40% at day 10. There was decreased histologic evidence of cardiac damage and reduced apoptosis due to Dox+Trz in mice pretreated with Prob.
Conclusion
The cardiotoxic effects of Dox+Trz are partially attenuated by the prophylactic administration of the antioxidant Prob.
In North America, it is estimated that more than 250,000 women will be newly diagnosed with breast cancer in 2010, leading to 45,000 deaths. Approximately 25% to 30% of breast cancer malignancies are positive for an overexpression of the human epidermal growth factor receptor 2 (HER2). HER2 overexpression is associated with aggressive neoplastic growth, which may be resistant to traditional anthracycline-based chemotherapy. Trastuzumab (Trz), a monoclonal antibody against HER2, is effective at reducing both progression and recurrence of breast cancer.
Despite the therapeutic benefits of Trz, the incidence of cardiotoxicity is increased, particularly when administered after anthracycline-based chemotherapy. Clinical trials in the adjuvant setting of breast cancer suggest that 5% to 10% of patients develop asymptomatic cardiac dysfunction, requiring discontinuation of Trz. Outside of clinical trials, recent studies suggest that up to 1 in 4 women undergoing treatment with doxorubicin (Dox) and adjuvant Trz may develop cardiotoxicity.
Trz may directly block anti-apoptotic signaling, leading to premature cardiac dysfunction. In an in vivo model of acute chemotherapy-induced cardiac dysfunction, Dox+Trz synergistically increased the degree of myocardial apoptosis. In vitro blockade of HER2 is associated with increased oxidative stress, which is attenuated by the administration of the antioxidant N-acetylcysteine. In addition, the antioxidant drug probucol (Prob) is cardioprotective against Dox-induced cardiotoxicity by reducing apoptosis. Little is known, however, about the role of antioxidant therapy in the prevention of cardiotoxicity due to Dox+Trz.
The aim of the current study was to determine whether prophylactic treatment with the antioxidant Prob is cardioprotective in an acute murine model of Dox and Trz-mediated cardiomyopathy.
Materials and Methods
Experimental Animals
All animal procedures were conducted in accordance with guidelines published by the Canadian Council on Animal Care. All procedures, including drug administration and longitudinal echocardiographic studies, were approved by the Animal Protocol Review Committee at the University of Manitoba.
A total of 114 wild-type C57Bl/6 mice were randomly assigned to one of eight groups. Mice received one of the following treatment drug regimens administered via intraperitoneal injection: (1) 0.9% saline control ( n = 5); (2) Prob (15 mg/kg; n = 5); (3) Dox (20 mg/kg; n = 25); (4) Trz (10 mg/kg; n = 8); (5) Dox+Trz ( n = 33); (6) Prob+Trz ( n = 11); (7) Prob+Dox ( n = 11); and (8) Prob+Dox+Trz ( n = 16) ( Figure 1 A). Prob (15 mg/kg) injections were given on alternative days, for 2 weeks before the experimental arm, and one injection was given during the experimental arm for a cumulative dose of 120 mg/kg ( Figure 1 B). Acute chemotherapeutic treatment injections were administered intraperitoneally at day 0 as single injections with Dox (20 mg/kg) or Trz (10 mg/kg) or the combination of Dox+Trz. All mice underwent daily echocardiography for 10 days, at which time the hearts were removed for histopathologic and Western blot analyses.
Echocardiographic Analysis
In vivo assessment of cardiac function was performed using murine echocardiography with two-dimensional and tissue Doppler imaging (TDI). All mice were examined at baseline and followed daily thereafter for 10 days. Murine echocardiography was performed using a 13-Mhz probe (Vivid 7, GE Medical Systems, Milwaukee, WI) and a 30-MHz MicroScan transducer (Vevo 2100, Visualsonics, Toronto, Canada), as previously described. Briefly, hearts were imaged in the two-dimensional parasternal short-axis view, and three different frames of a M-mode echocardiogram were recorded. LV end-diastolic diameter (LVEDD), LV end-systolic diameter, posterior end-diastolic wall thickness, and LV fractional shortening (FS) were measured. Parasternal long-axis views were obtained for measurement of LV end-systolic and diastolic volumes using the prolate ellipsoid geometric model, and the LV ejection fraction (EF) was calculated.
TDI was acquired on a parasternal short-axis view at the level of the papillary muscles, at a rate of 483 frames per second. For peak endocardial velocity (Vendo), a region of interest (0.2 × 0.2 mm) was manually positioned along the posterior wall of the endocardium. Radial strain rate (SR) was measured over an axial distance of 1 mm (width 0.6 mm) using Echopac PC (GE Medical, Milwaukee, WI). The temporal smoothing filters were turned off for all measurements. The values obtained in five consecutive cardiac cycles were averaged.
To test for intraobserver variability, 25 mice were randomly chosen from the following groups: (1) 0.9% saline control ( n = 2); (2) Prob ( n = 2); (3) Dox ( n = 3); (4) Trz ( n = 3); (5) Dox+Trz ( n = 3); (6) Prob+Trz ( n = 4); (7) Prob+Dox ( n = 3); and (8) Prob+Dox+Trz ( n = 5). The same individual (D.S.J.), blinded to the study groups, analyzed the TDI parameters at baseline in the 25 mice 1 week apart. For interobserver variability, 30 mice were randomly chosen from the following groups: (1) 0.9% saline control ( n = 3); (2) Prob ( n = 3); (3) Dox ( n = 4); (4) Trz ( n = 3); (5) Dox+Trz ( n = 5); (6) Prob+Trz ( n = 4); (7) Prob+Dox ( n = 4); and (8) Prob+Dox+Trz ( n = 4). Two individuals (J.R.W. and D.S.J.), again blinded to the study groups, analyzed the TDI parameters at baseline. Intraobserver and interobserver variability were defined as the absolute difference between the corresponding repeated measurements expressed in percent of their mean.
Histologic and Biochemical Analysis
Ten days posttreatment, all mice were euthanized and the hearts were removed and processed for histologic and biochemical analysis. For histology, the sections were stained with Masson’s trichrome to assess for the degree of cardiac fibrosis (Zeiss Axio-imager 2, Carl Zeiss MicroImaging GmbH, Jena, Germany), as previously described.
Protein Extraction and Immunoblotting
Quantification of Bax and Bcl-XL was performed as previously described. Briefly, the hearts were powdered in liquid nitrogen and homogenized in radioimmunoprecipitation assay buffer containing protease inhibitor cocktail (Sigma-Aldrich Corporation, St. Louis, MO). The lysates were centrifuged at 14,000 rpm for 10 minutes. The upper layer containing protein fraction was sonicated and stored at −75°C. Protein concentration was measured using the BioRad protein assay. The protein samples were thawed and subjected to one-dimensional 12% to 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis in a discontinuous system, as previously described. Equal loading of protein (30 mg/lane) was confirmed by Coomassie blue staining and the use of antiactin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) as an internal control. Separated proteins were transferred onto 0.45-mm nitrocellulose membrane and incubated overnight with Bax and Bcl-XL polyclonal antibodies (Cell Signaling Technology Inc, Beverly, MA). Primary antibodies were detected using a goat antirabbit immunoglobulin G horseradish peroxidase-conjugated secondary antibody (Bio-Rad Laboratories, Inc, Hercules, CA) using the BM Chemiluminescence kit (POD substrate; Roche Diagnostics, Laval, Canada). The protein bands were visualized with a Fluor-S-MultiImager MAX system (Bio-Rad Laboratories, Inc.) and quantified using image analysis software (Quantity One; Bio-Rad Laboratories, Inc.).
Statistical Analysis
Data are expressed as mean ± standard error of the mean. All statistical analysis was done using SPSS 15.0 (SPSS Inc., Chicago, IL) and Graphpad Prism 5 software (GraphPad Software Inc., La Jolla, CA). Student t tests were used to compare continuous variables. Comparison of variables within each group versus baseline was performed using repeated-measures analysis of variance and Dunnett’s test. A P value < .05 was considered statistically significant.
Results
Survival
The survival probability of the mice in the various treatment regimens is shown in Figure 2 . Mice receiving Dox alone had a 50% survival rate at day 5. The addition of Prob to Dox (Prob+Dox) significantly increased the survival probability to 91% at day 10 ( Figure 2 ). Mice treated with the combination of Dox+Trz demonstrated >80% mortality at day 5. Mice treated with Prob+Dox+Trz demonstrated increased survival probability to 63% at day 10. Thus, the addition of Prob shifted the survival curve to the right and preserved the survival in mice receiving Dox alone or Dox+Trz.
Conventional Echocardiographic and TDI Indices
At baseline, the LV dimensions and systolic function, as determined by both conventional parameters (FS and EF) and TDI indices (Vendo and SR), were similar in all mice. Heart rates were within normal limits at baseline. There was no evidence of left ventricular (LV) hypertrophy comparing posterior end-diastolic wall thickness at baseline and day 10 of follow-up ( Table 1 ).
| Echo variable | Group | Baseline values | Day 10 values | P value |
|---|---|---|---|---|
| HR (beats/min) | ||||
| Saline | 734 ± 15 | 728 ± 11 | .82 | |
| Prob | 727 ± 8 | 715 ± 10 | .76 | |
| Trz | 744 ± 11 | 754 ± 9 | .58 | |
| Dox | 715 ± 12 | 725 ± 14 | .45 | |
| Dox+Trz | 731 ± 8 | 725 ± 10 | .67 | |
| Prob+Trz | 719 ± 10 | 734 ± 8 | .72 | |
| Prob+Dox | 724 ± 8 | 715 ± 7 | .41 | |
| Prob+Dox+Trz | 735 ± 8 | 715 ± 10 | .31 | |
| PWT (mm) | ||||
| Saline | 0.81 ± 0.02 | 0.84 ± 0.03 | .82 | |
| Prob | 0.83 ± 0.02 | 0.84 ± 0.02 | .83 | |
| Trz | 0.80 ± 0.04 | 0.81 ± 0.01 | .85 | |
| Dox | 0.82 ± 0.02 | 0.80 ± 0.02 | .78 | |
| Dox+Trz | 0.81 ± 0.02 | 0.83 ± 0.03 | .84 | |
| Prob+Trz | 0.83 ± 0.02 | 0.82 ± 0.02 | .88 | |
| Prob+Dox | 0.81 ± 0.03 | 0.83 ± 0.04 | .78 | |
| Prob+Dox+Trz | 0.82 ± 0.03 | 0.84 ± 0.04 | .76 | |
| LVEDD (mm) | ||||
| Saline | 3.2 ± 0.2 | 3.2 ± 0.1 | .84 | |
| Prob | 3.1 ± 0.1 | 3.2 ± 0.2 | .78 | |
| Trz | 3.2 ± 0.2 | 3.2 ± 0.1 | .87 | |
| Dox | 3.2 ± 0.1 | 3.8 ± 0.2 ∗ | <.05 | |
| Dox+Trz | 3.1 ± 0.1 | 4.1 ± 0.2 ∗ | <.05 | |
| Prob+Trz | 3.2 ± 0.1 | 3.2 ± 0.1 | 1.00 | |
| Prob+Dox | 3.2 ± 0.1 | 3.5 ± 0.1 ∗ | <.05 | |
| Prob+Dox+Trz | 3.1 ± 0.1 | 3.6 ± 0.1 ∗ | <.05 | |
| FS (%) | ||||
| Saline | 51 ± 3 | 53 ± 2 | .67 | |
| Prob | 50 ± 2 | 51 ± 3 | .72 | |
| Trz | 53 ± 2 | 52 ± 3 | .84 | |
| Dox | 51 ± 2 | 42 ± 2 ∗ | <.05 | |
| Dox+Trz | 52 ± 3 | 35 ± 3 ∗ | <.05 | |
| Prob+Trz | 51 ± 2 | 50 ± 1 | .81 | |
| Prob+Dox | 52 ± 2 | 47 ± 2 ∗ | <.05 | |
| Prob+Dox+Trz | 51 ± 1 | 44 ± 2 ∗ | <.05 |
∗ P < . 05 comparing day 10 with baseline values within the same group.
The LVEDD was within normal limits in all groups at baseline ( Table 1 ). In mice receiving Dox alone, the LVEDD increased significantly from 3.2 ± 0.1 mm at baseline to 3.8 ± 0.2 mm by day 10 ( P < .05). In mice receiving Dox+Trz, the LVEDD increased significantly from 3.1 ± 0.1 mm at baseline to 4.1 ± 0.2 mm by day 10 ( P < .05). The administration of Prob, however, attenuated the increase in LVEDD to only 3.5 ± 0.1 mm at day 10 in the Prob+Dox group and 3.6 ± 0.1 mm at day 10 in the Prob+Dox+Trz group, respectively ( Table 1 ).
LV FS was within normal limits in all groups at baseline ( Table 1 ). In mice receiving Dox alone, the FS decreased from 51% ± 2% at baseline to 42% ± 2% at day 10 ( P < . 05). In mice receiving Dox+Trz, the FS decreased from 52% ± 3% at baseline to 35% ± 3% at day 10 ( P < . 05). The administration of Prob, however, partially preserved LV systolic dysfunction in the Prob+Dox and Prob+Dox+Trz groups with FS of 47% ± 2% and 44% ± 2% at day 10, respectively ( Table1 ).
Similar to the FS, TDI indices of Vendo and SR revealed LV systolic dysfunction by day 10 ( Table 2 ). In mice receiving Dox alone, Vendo and SR decreased from 3.2 ± 0.1 cm/s and 23 ± 2 s −1 at baseline to 1.7 ± 0.1 cm/s and 13 ± 2 s −1 at day 10, respectively ( P < . 05). In mice receiving Dox+Trz, Vendo and SR decreased from 3.3 ± 0.1 cm/s and 24 ± 1 s −1 at baseline to 1.4 ± 0.1 cm/s and 10 ± 1 s −1 at day 10, respectively ( P < . 05) ( Figure 3 A). The administration of Prob, however, attenuated the decrease in Vendo and SR in the Prob+Dox group to only 2.1 ± 0.1 cm/s and 17 ± 2 s −1 , respectively. Similarly, the administration of Prob attenuated the decrease in Vendo and SR in the Prob+Dox+Trz group to only 2.0 ± 0.1 cm/s and 15 ± 1 s −1 , respectively ( Figure 3 B).
| Echo variable | Group | Baseline values | Day 10 values | P value |
|---|---|---|---|---|
| Vendo (cm/s) | ||||
| Saline | 3.2 ± 0.1 | 3.2 ± 0.2 | .92 | |
| Prob | 3.2 ± 0.1 | 3.2 ± 0.2 | .91 | |
| Trz | 3.3 ± 0.2 | 3.3 ± 0.1 | .88 | |
| Dox | 3.2 ± 0.1 | 1.7 ± 0.1 ∗ | <.05 | |
| Dox+Trz | 3.3 ± 0.1 | 1.4 ± 0.1 | <.05 | |
| Prob+Trz | 3.2 ± 0.1 | 3.2 ± 0.2 | .88 | |
| Prob+Dox | 3.3 ± 0.1 | 2.1 ± 0.1 ∗ | <.05 | |
| Prob+Dox+Trz | 3.2 ± 0.1 | 2.0 ± 0.1 ∗ | <.05 | |
| SR (S −1 ) | ||||
| Saline | 22 ± 2 | 22 ± 1 | .92 | |
| Prob | 23 ± 1 | 22 ± 2 | .81 | |
| Trz | 23 ± 1 | 23 ± 2 | .90 | |
| Dox | 23 ± 2 | 13 ± 2 ∗ | <.05 | |
| Dox+Trz | 24 ± 1 | 10 ± 1 ∗ | <.05 | |
| Prob+Trz | 23 ± 1 | 22 ± 1 | .81 | |
| Prob+Dox | 22 ± 1 | 17 ± 2 ∗ | <.05 | |
| Prob+Dox+Trz | 23 ± 1 | 15 ± 1 ∗ | <.05 |
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