Anti-erbB2 treatment induces cardiotoxicity by interfering with cell survival pathways

Abstract

Introduction: Cardiac dysfunction is among the serious side effects of therapy with recombinant humanized anti-erbB2 monoclonal antibody. The antibody blocks ErbB-2, a receptor tyrosine kinase and co-receptor for other members of the ErbB and epidermal growth factor families, which is over-expressed on the surface of many malignant cells. ErbB-2 and its ligands neuregulin and ErbB-3/ErbB-4 are involved in survival and growth of cardiomyocytes in both postnatal and adult hearts, and therefore the drug may interrupt the correct functioning of the ErbB-2 pathway.

Methods: The effect of the rat-anti-erbB2 monoclonal antibody B-10 was studied in spontaneously beating primary myocyte cultures from rat neonatal hearts. Gene expression was determined by RT-PCR (reverse transcription polymerase chain reaction) and by rat stress-specific microarray analysis, protein levels by Western blot, cell contractility by video motion analysis, calcium transients by the FURA fluorescent method, and apoptosis using the TUNEL (terminal uridine nick-end labelling) assay.

Results: B-10 treatment induces significant changes in expression of 24 out of 207 stress genes analyzed using the microarray technique. Protein levels of ErbB-2, ErbB-3, ErbB-4 and neuregulin decreased after 1 day. However, both transcription and protein levels of ErbB-4 and gp130 increased several fold. Calreticulin and calsequestrin were overexpressed after three days, inducing a decrease in calcium transients, thereby influencing cell contractility. Apoptosis was induced in 20% cells after 24 hours.

Conclusion: Blocking ErbB-2 in cultured rat cardiomyocytes leads to changes that may influence the cell cycle and affects genes involved in heart functions. B-10 inhibits pro-survival pathways and reduces cellular contractility. Thus, it is conceivable that this process may impair the stress response of the heart.

Figure 1

RT-PCR analysis of control and treated (B-10) cells. Primers used are described in Table 1. RT-PCR, reverse transcription polymerase chain reaction.

Figure 2

Western blot analysis of control and treated (B-10) cells after 24/72 hours.

Figure 3

Contraction, as assessed by percentage shortening. The values (mean ± standard deviation) are as follows: control 0.3037 ± 0.0096 and B-10 treatment 0.1842 ± 0.0205. P < 0.001.

Figure 4

Calcium transient analysis. The values (relative absorbance; mean ± standard deviation) are as follows: control 0.803 ± 0.04 and B-10 0.644 ± 0.04. P < 0.001.

Figure 5

TUNEL assay, 24 hours after addition of B-10 or doxorubicin. The first row shows untreated control cells. The second row shows doxorubicin (10 μmol/l) treated positive control cells. The third row shows B-10 treated cells. TUNEL, terminal uridine nick-end labeling.

Figure 6

Percentage of apoptotic cells 24 hours after addition of B-10 or doxorubicin. The TUNEL method was used to determine the percentage of apoptotic cells. The values (mean ± standard deviation) are as follows: control cells 2.35 ± 0.02%; doxorubicin treated cells 98 ± 0.09%; and B-10 treated cells 20.4 ± 0.09%.