Doxorubicin-based combination treatments to enhance antitumour effects and reduce cardiotoxicity

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the La Trobe Institute for Molecular Science, Faculty of Science, Health, and Engineering, La Trobe University, Bundoora.

Doxorubicin (DOX) is a chemotherapeutic agent clinically used against a wide range of solid and blood neoplasms. However the cumulative lifetime dose of DOX is limited by dose-dependent cardiotoxicity. A drug treatment combination of DOX and the formaldehyde-releasing prodrug AN7 exhibits cardioprotection, while maintaining antitumour efficacy. The present study investigated the response of breast cancer and cardiomyocyte cell lines to DOX/AN7 combination treatments in vitro, in terms of the formation of DOX-DNA covalent adducts and the effect adduct levels had on cell death. The cell lines encompassed a range of species; human, mouse and rat. High throughput gene expression analyses were conducted in cardiomyocyte and breast cancer cells in vitro to identify pathways involved in adduct response in cells. Cardiac effects of DOX/AN7 were also assessed in mice, employing damage markers and ultrastructural changes at 72 hours after acute DOX treatment. Gene expression levels were also assessed in ventricles of treated mice to investigate pathways or biomarkers associated with cardiac damage or cardioprotection. In the in vitro studies it was apparent that higher levels of adducts formed in cardiomyocyte compared to breast cancer cells. Adducts were associated with increased mitochondrial dysfunction and cell death in the breast cancer cells, while adducts in cardiomyocyte cells exhibited no mitochondrial dysfunction nor increase in cell death. Mice treated with the DOX/AN7 combination showed reduced plasma levels of cardiac troponinT compared to DOX alone. Furthermore, these mice displayed amelioration of DOX-induced ultrastructural disorganization in electron microscope analysis of heart ventricle sections. Preliminary gene expression analyses indicated that DOX/AN7 cardioprotection was associated with a p53-independent induction of p21, corresponding Sirt1 up-regulation, and increase in expression of mitochondrial biogenesis factor PGC1α. The results of this study indicate the addition of AN7 alters the response of cardiomyocytes to DOX while enhancing its anticancer efficacy in vitro. The whole organ protection of DOX/AN7 in vivo opens the possibility for similar formaldehyde-releasing drugs to be used in combination with DOX to reduce dose-dependent cardiotoxicity, making DOX a far more valuable clinical tool in breast cancer regimes.