Mechanisms of Cancer-related Cardiomyopathy67Protection against chemotherapy cardiotoxicity by the human amniotic fluid stem cell secretome: a new tool for future paracrine therapy68Hyperlipidaemia reduces mortality in breast, prostate, lung and bowel cancer69DNA-repair in cardiomyocytes is critical for maintaining cardiac function

Introduction: Advances in diagnosis and therapy have made cancer curable in a substantial proportion of patients. As a consequence, long-term side effects of oncological drugs, such as anthracyclines, may become the major health problem in cancer survivors. Chemotherapy-derived cardiotoxicity is a chronic complication with a dramatically relevant clinical impact in the development of late onset cardiomyopathy. Unfortunately, no truly effective way to prevent anthracycline cardiotoxicity currently exists. Recent work has identified human AFS (hAFS) and their conditioned medium (hAFS-CM) as a novel tool to significantly reduce myocardial infarct damage. Purpose: The aim of this study is to validate the cardioprotective potential of the hAFS secretome in a doxorubicin (Dox)-derived cardiotoxicity model in vitro. Methods: c-kit+ hAFS were isolated from left over samples of II trimester amniotic fluid diagnostic amniocenteses with normal karyotype, following informed consent. Cells were cultured for 24h without serum in normoxia (20% O2) or hypoxia (1% O2) to enrich the hAFS-CM with cardioactive factors. The cardioprotective potential of the hAFS-CM on Dox-induced senescence and apoptosis was assessed on rat H9c2 cardiomyoblasts, primary mouse neonatal cardiomyocytes (mNVCM) and human c-kit+ cardiac progenitor cells. Senescence was evaluated by staining for β-galactosidase and p16INK4a, whereas apoptosis was assessed by cleaved caspase-3 expression. The activation of the DNA damage response (DDR) was measured by γH2AX immunostaining. Gene expression profiling was performed to identify pathways activated by the hAFS-CM. The role of PI3K/Akt signaling was investigated by western blot and via functional experiments using the PI3K inhibitor LY294002. Results: Both Dox-induced senescence and apoptosis were considerably antagonized by the hAFS-CM, in particular by the hypoxic hAFS-CM (hAFS-CMHypo). The hAFS-CMHypo also limited the DDR activation by Dox in mNVCM. Gene expression analysis on treated mNVCM revealed substantial up-regulation of pro-survival cytokines, such as Il6 and Cxcl1 and of the Abcb1b gene, encoding for a protein involved in Dox efflux. Akt phosphorylation was promptly activated by the hAFS-CMHypo, while LY294002 prevented its beneficial effect in counteracting Dox-triggered senescence and apoptosis. Conclusions: The paracrine potential of the hAFS secretome protects cardiomyocytes and cardiac progenitor cells against doxorubicin negative side effects, suggesting a novel therapeutic strategy to tackle the cardiotoxicity of anthracyclines during oncological therapy.