Cardiac protective effects of dexrazoxane on animal cardiotoxicity model induced by anthracycline combined with trastuzumab is associated with upregulation of calpain-2

Abstract

Cardiotoxicity is a well-recognized side effect induced by chemotherapeutic drugs such as anthracycline and trastuzumab through different mechanisms. Currently, accumulating evidence supports that dexrazoxane (DZR) can minimize the risk of cardiotoxicity. In this study, we investigated whether dexrzoxane could reduce cardiotoxicity in the treatment of anthracycline combined with trastuzumab. We randomly divided 90 experimental F344 rats into control group, chemotherapeutics and trastuzumab (doxorubicin [DOX] + herceptin [Her]) group, and chemotherapeutics, trastuzumab, and DZR (DOX + Her + DZR) group. Animal status and body weight, cardiac function, serum cardiac markers, cardiomyocyte apoptosis of the rats, and expression level of calpain-2 were evaluated. Left ventricular ejection fraction (LVEF) and fractional shortening (FS) of the left ventricle were observed. The serum levels of malondialdehyde (MDA) and cardiac troponin I (cTnI) and cardiomyocyte apoptosis were detected by enzyme linked immunosorbent assay and TdT-mediated dUTP nick end labeling assays. The mRNA and protein level of calpain-2 were measured by reverse transcriptase polymerase chain reaction and Western blot. We observed that the LVEF and FS of the left ventricle were significantly higher in the DOX + Her + DZR group than that in the DOX + Her group (P < 0.05). The serum levels of MDA and cTnI between DOX + Her group and DOX + Her + DZR group were significantly different. In addition, cardiomyocyte apoptosis in the DOX + Her + DZR group was significantly less severe than that in the DOX + Her group (P < 0.05). After DZR treatment, the calpain-2 mRNA and protein levels in the DOX + Her + DZR group were significantly higher than the DOX + Her group (P < 0.05). Our results suggest that DZR can effectively reduce the cardiotoxicity of combinatorial treatment of trastuzumab and anthracycline partly through upregulating calpain-2.

Figure 1

Effect of chemotherapy and DZR treatment on body weight of animals. Animals in DOX + Her group (n = 28) had significantly reduced body weight compared with the control group (n = 29) (all P < 0.05). DOX + Her + DZR group (n = 28) animals had increased body weight at week 2 (P = 0.018) and week 3 (P = 0.031) compared with DOX + Her group. Each point represents the mean ± SD. DOX + Her = chemotherapeutics and trastuzumab, DOX + Her + DZR = chemotherapeutics, trastuzumab, and dexrazoxane, SD = standard deviation.

Figure 2

Morphology of myocardial tissues in rats of different treatment groups at week 4. (A) HE staining of myocardial tissues in the control group, original magnification 400×. (B) HE staining of myocardial tissues in DOX + Her group, original magnification 400×. (C) HE staining of myocardial tissues in DOX + Her + DZR group, original magnification 400×. (D) Comparison of cardiomyocyte damage between different treatment groups. Each column represents the mean ± SD. The statistical analysis was performed with Student t test. DOX + Her vs control: ^∗∗P < 0.01; DOX + Her + DZR vs control: ^†P < 0.05; DOX + Her + DZR vs DOX + Her: ^‡P < 0.05. DOX + Her = chemotherapeutics and trastuzumab, DOX + Her + DZR = chemotherapeutics, trastuzumab, and dexrazoxane, HE = hematoxylin–eosin, SD = standard deviation.

Figure 3

Cardiomyocyte apoptosis changes in rats of different treatment groups at week 4. (A) Cardiomyocyte apoptosis in the control group. The nucleoli of apoptotic cells appeared as yellow granules. (B) Cardiomyocyte apoptosis in DOX + Her group. (C) Cardiomyocyte apoptosis in DOX + Her + DZR group. (D) Comparison of apoptotic indexes between different treatment groups. Each column represents the mean ± SD. The statistical analysis was performed with Student t test. DOX + Her vs control: ^∗P < 0.05; DOX + Her + DZR vs control: ^†P < 0.05; DOX + Her + DZR vs DOX + Her: ^‡P < 0.05. DOX + Her = chemotherapeutics and trastuzumab, DOX + Her + DZR = chemotherapeutics, trastuzumab, and dexrazoxane.

Figure 4

Analysis of calpain-1 and calpain-2 expression. (A) At week 4, the mRNA expression levels of calpain-1 and calpain-2 in cardiac tissue samples from different groups were analyzed by quantitative RT-PCR. (B) The expression of calpain-1 and calpain-2 proteins was analyzed by Western blot. (C) Quantification of calpain-1, relative to GAPDH expression. (D) Quantification of calpain-2, relative to GAPDH expression. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001. DOX + Her = chemotherapeutics and trastuzumab, DOX + Her + DZR = chemotherapeutics, trastuzumab, and dexrazoxane, NS = not significant, RT-PCR = reverse transcriptase-polymerase chain reaction.