Anthracycline-induced cardiotoxicity : the role of proteolytic pathways

[摘要] ENGLISH ABSTRACT: Introduction: The anthracyclines (ACs), daunorubicin (DNR) and doxorubicin (DXR)are two of the most effective drugs known for the treatment of systemic neoplasmsand solid tumours. However, their clinical use is often hampered by their dosedependentcumulative cardiotoxicity, which leads to irreversible and fatal druginducedcongestive heart failure. The mechanism by which ACs induces heartdamage is not fully understood. Recent reports have indicated that DXR activatesautophagy and ubiquitin proteasome-mediated degradation of specific transcriptionfactors, however, no reports exists on the effect of ACs on the E3 ubiquitin ligases,MuRF-1 and MAFbx. The aim of the first part of the study was therefore toinvestigate the effect of DNR treatment on the protein and organelle degradationsystems in the heart and to elucidate the signalling mechanisms involved.Although this model was ideal in allowing the investigation of the signalling pathwayswhich are affected by DNR, it did not allow for further exploration or manipulation ofsignalling pathways that may be of potential benefit in this context. The in vitro modelwas therefore used to validate the hypothesis that increased autophagy alleviatesAC-induced cardiotoxicity and delays the onset of cardiomyocyte death. The aims forthe second part of the study were (i) to characterize the effect of DXR in H9C2 cells,(ii) to determine whether the induction/inhibition of autophagy in combination withDXR alleviates cytotoxicity and (iii) to investigate the influence ofincreased/decreased autophagy in combination with DXR on reactive oxygenspecies (ROS) production, mitochondrial function, endoplasmic reticulum (ER) stressand the ubiquitin proteasome pathway. In the final part of this study, an in vivo modelwas used to assess the potential benefit of autophagy in a novel GFP-LC-3 tumourbearing mouse model of acute DXR-induced cardiotoxicity.Material and Methods: Adult rats were divided into two groups where one groupreceived six intraperitoneal injections of 2 mg/kg DNR on alternate days and theother group received saline injections as control. Hearts were excised and perfused on a working heart system the day after the last injection and freeze clamped forbiochemical analysis.H9C2s were cultured and treated with Bafilomycin A1 (10 nM, inhibitor of autophagy)for 6 hrs, Rapamycin (50 μM, inducer of autophagy) for 24 hrs, DXR (3 μM) for 24hrs or a combination of these drugs. Following treatment, cells were harvested andassessed for cell death, proteolytic activity and oxidative stress using westernblotting, fluorescence microscopy and flow cytometry.In the final phase of the study, twenty-four female mice were injected at 8 weeks witha mouse breast cancer cell line (EO771) and after observation of tumour growth,animals were either treated with one injection (i.p.) of Rapamycin (4 mg/kg), twoinjections (i.p.) of DXR (10 mg/kg) or a combination of the two drugs. After theexperimental protocol, mice were terminated and their hearts were rapidly excised.The hearts were divided cross-sectionally and utilized for biochemical andhistological analyses.Results and Discussion: DNR treatment significantly attenuated myocardialfunction and increased apoptosis in the ex vivo heart model. DNR-induced cardiaccytotoxicity was associated with the upregulation of two E3 ubiquitin ligases, MuRF-1and MAFbx as well as a significant increase in two markers of autophagy, beclin-1and LC-3. These changes observed in the heart were also associated withattenuation of the PI3-kinase/Akt signalling pathway.The augmentation of autophagy with rapamycin before DXR treatment significantlyreduced cell death in the in vitro model. Indeed, rapamycin treatment demonstratedto be a vital survival mechanism for acute DXR-induced cardiotoxicity as itdecreased cellular ROS production, improved mitochondrial function and preventednuclear translocation of DXR. Moreover, these changes in cardiomyocytes were alsoassociated with a reduction in the ubiquitin-proteasome pathway (UPP). In the final part of this study, a novel tumour bearing GFP-LC3 mouse model wasdeveloped to confirm the results obtained in the in vitro study. It was demonstratedthat acute DXR-induced cardiotoxicity resulted in increased apoptosis, the inhibitionof autophagy and increased proteolysis via the UPP. These findings were associatedwith a reduction in body weight and cardiomyocyte cross-sectional area. Thecardiotoxic effects of DXR were substantially reduced when autophagy was inducedwith rapamycin. Taken together, our data strongly indicates that it is possible toattenuate the cardiotoxic effects of doxorubicin in cancer patients by carefullycontrolling the levels of autophagy using rapamycin as adjuvant therapy.

[发布机构] Stellenbosch University