TY - JOUR
T1 - Mitoxantrone impairs proteasome activity and prompts early energetic and proteomic changes in HL-1 cardiomyocytes at clinically relevant concentrations
AU - Costa, Vera Marisa
AU - Capela, João Paulo
AU - Sousa, Joana R.
AU - Eleutério, Rute P.
AU - Rodrigues, Patrícia R.S.
AU - Dores-Sousa, José Luís
AU - Carvalho, Rui A.
AU - Lourdes Bastos, Maria
AU - Duarte, José Alberto
AU - Remião, Fernando
AU - Almeida, M. Gabriela
AU - Varner, Kurt J.
AU - Carvalho, Félix
N1 - Publisher Copyright:
© 2020, Springer-Verlag GmbH Germany, part of Springer Nature.
PY - 2020/12
Y1 - 2020/12
N2 - 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 10 µM) during short (2, 4, 6, or 12 h) or longer incubation periods (24 or 48 h). At earlier time points, (6 and 12 h) cytotoxicity was already observed for 1 and 10 µM MTX. Proteomic analysis of total protein extracts found 14 proteins with higher expression and 26 with lower expression in the cells exposed for 12 h 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 10 µM MTX, respectively. At earlier time-points, 10 µM MTX increased intracellular ATP levels, while decreasing media lactate levels. At later stages (24 and 48 h), 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 24 h. Regarding cellular redox status, total glutathione increased in 1 µM MTX (24 h), and that increase was dependent on gamma-glutamylcysteine synthetase activity. Meanwhile, for both 1 and 10 µM MTX, oxidized glutathione was significantly higher than control at 48 h. 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.
AB - 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 10 µM) during short (2, 4, 6, or 12 h) or longer incubation periods (24 or 48 h). At earlier time points, (6 and 12 h) cytotoxicity was already observed for 1 and 10 µM MTX. Proteomic analysis of total protein extracts found 14 proteins with higher expression and 26 with lower expression in the cells exposed for 12 h 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 10 µM MTX, respectively. At earlier time-points, 10 µM MTX increased intracellular ATP levels, while decreasing media lactate levels. At later stages (24 and 48 h), 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 24 h. Regarding cellular redox status, total glutathione increased in 1 µM MTX (24 h), and that increase was dependent on gamma-glutamylcysteine synthetase activity. Meanwhile, for both 1 and 10 µM MTX, oxidized glutathione was significantly higher than control at 48 h. 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.
KW - Cardiotoxicity
KW - Caspases
KW - Glutathione
KW - HL-1 cells
KW - Mitoxantrone
KW - Proteomics
UR - http://www.scopus.com/inward/record.url?scp=85090302163&partnerID=8YFLogxK
U2 - 10.1007/s00204-020-02874-4
DO - 10.1007/s00204-020-02874-4
M3 - Article
C2 - 32894303
AN - SCOPUS:85090302163
SN - 0340-5761
VL - 94
SP - 4067
EP - 4084
JO - Archives of Toxicology
JF - Archives of Toxicology
IS - 12
ER -