Mitoxantrone impairs proteasome activity and prompts early energetic and proteomic changes in HL-1 cardiomyocytes at clinically relevant concentrations.

Abstract

Mitoxantrone (MTX) is used to treat several types of cancers and to improve neurological disability in multiple sclerosis. Unfortunately, cardiotoxicity is a severe and common adverse effect in MTX-treated patients. Herein, we aimed to study early and late mechanisms of MTX-induced cardiotoxicity using murine HL-1 cardiomyocytes. Cells were exposed to MTX (0.1, 1 or 10µM) during short (2, 4, 6, or 12h) or longer incubation periods (24 or 48h). At earlier time points, (6 and 12h) cytotoxicity was already observed for 1 and 10µM MTX. Proteomic analysis of total protein extracts found 14 proteins with higher expression and 26 with lower expression in the cells exposed for 12h to MTX (pH gradients 4-7 and 6-11). Of note, the expression of the regulatory protein 14-3-3 protein epsilon was increased by a factor of two and three, after exposure to 1 and 10µM MTX, respectively. At earlier time-points, 10µM MTX increased intracellular ATP levels, while decreasing media lactate levels. At later stages (24 and 48h), MTX-induced cytotoxicity was concentration and time-dependent, according to the MTT reduction and lactate dehydrogenase leakage assays, while caspase-9, -8 and -3 activities increased at 24h. Regarding cellular redox status, total glutathione increased in 1µM MTX (24h), and that increase was dependent on gamma-glutamylcysteine synthetase activity. Meanwhile, for both 1 and 10µM MTX, oxidized glutathione was significantly higher than control at 48h. Moreover, MTX was able to significantly decrease proteasomal chymotrypsin-like activity in a concentration and time-independent manner. In summary, MTX significantly altered proteomic, energetic and oxidative stress homeostasis in cardiomyocytes at clinically relevant concentrations and our data clearly demonstrate that MTX causes early cardiotoxicity that needs further study.