[Lani]

Discussion

Abstract
Introduction
Materials and methods
Results
Discussion
Conclusion
Abbreviations
Competing interests
Authors' contributions
Acknowledgements
References

Targeted monoclonal antibody therapy as a biological treatment for different cancers appears to have many advantages over conventional chemo/radiotherapy. In the case of ErbB-2 overexpressing breast cancer cells, it was postulated that antibody therapy would specifically eliminate only tumour cells without affecting any other ErbB-2 expressing cells [23-25]. Although erbB-2 is expressed ubiquitously, it was thought that those levels are sufficiently low not to be affected by anti-erbB-2 antibodies. However, ErbB-2 plays a vital role both in developing and in adult murine cardiomyocytes [14,26-28]. ErbB-2 null mutant mice exhibit an absence of trabecules in the heart ventricle and die at midgestation [14]. Adult mice carrying a conditional ErbB-2 mutation develop severe dilated cardiomyopathy, usually in the second month of life [28]. As shown by Özcelik and coworkers [29], loss of ErbB-2 in cardiomyocytes leads to physiological stress on the heart, which over time induces cardiac decompensation and dilated cardiomyopathy.

The effect of trastuzumab on cells on the molecular level has repeatedly been analyzed on human breast cancer cell lines [30,31]. However, to our knowledge very few molecular studies on the effect of the antibody on cardiomyocytes have been reported. In vitro cultures of primary human cardiomyocytes are not readily attainable for both ethical and technical reasons. Therefore, in our study we opted to use the generally accepted in vitro model of neonatal rat ventricular heart muscle cells. Trastuzumab, being a humanized monoclonal antibody, does not bind to rat cells, consequently we used B-10 instead, which was shown previously to have biologic activity similar to that of trastuzumab [16,32]. Our results reveal a substantial decrease in the level of all ErbB family members analyzed after treatment with B-10. ErbB-2 by itself has no tyrosine kinase activity and needs to form heterodimers with one of its ligands to be functional [33]. All the ligands tested in our system were downregulated, thereby affecting the central role played by ErbB-2 in the heart. Moreover, gp130, which is thought to bind to ErbB-2 after biomechanical stress (hypoxia, haemodynamic overload and myocardial injury, among other insults) and activates a survival pathway [34], is strongly affected by B-10. Our results imply that the turnover of gp130 is augmented, as reflected in the several fold increase in transcription; nevertheless, the decrease in gp130 cannot be completely compensated.

It was previously demonstrated that overexpression of calreticulin modulates protein kinase B/Akt signalling, promoting apoptosis during cardiac differentiation of cardiomyoblasts [35]. In our system B-10 induced an increase in both calreticulin and calsequestrin levels three days after antibody treatment. The high levels of both of these calcium storage proteins in the sarcoplasmic reticulum may modify calcium release and subsequently lead to reduced cell contractility, decreasing the percentage shortening by 40%. This finding corroborates the studies described in ErbB-2 mutant mice and calsequestrin overexpressing mice [36,37]. Sawyer and coworkers [38], when comparing anthracycline- and ErbB2-induced myofibrillar disarray, reported no effect of B-10 on either akt or p-akt in cultures of adult rat ventricular myocytes, which is consistent with our data.

Finally, apoptosis may be a factor in the loss of ventricular function that is part of the cardiotoxic side effect profile of anti-erbB2 monoclonal antibody therapy. In our system B-10-induced apoptosis was three times greater than that recently described by Grazette and coworkers [39]. However, those investigators used a different antibody (clone 9G6), which may account for the difference in intensity.



Conclusion


In this molecular analysis of the effects of B-10 on neonatal cardiomyocytes, we demonstrated that the rat-anti-erbB2 monoclonal antibody B-10 severely affects ErbB-2 and its major ligands ErbB-3/4, neuregulin and gp130. In addition, the drug promotes increased expression of calreticulin and calsequestrin, which are involved in the sarcoplasmatic reticulum calcium pump and decrease cell contractility, which in vivo may lead to dilated cardiomyopathy in mice and humans as described previously [22,28,34,40]. Despite being present at a low level, in cardiomyocytes ErbB-2 plays a central role in the correct functioning of the cell. Blocking ErbB-2 by a monoclonal antibody disrupts the cell survival pathways and the ErbB-signalling network that depends on the relative stoichiometry between all of the players involved. We therefore speculate that this process may impair the stress response of the heart.

Problems with this study, among others:

done in mice(may not necessarily apply to humans)
done in neonatal mice heart-cells (adult cells may not rely on her2 to as great an extent)
didn't particularly look at mitochondrial function (as articles cited by Dr. Dennis Slamon did)
other mechanisms of toxicity may also apply