Autophagy (but not metabolism) is a key event in mitoxantrone-induced cytotoxicity in differentiated AC16 cardiac cells.

Abstract

Mitoxantrone (MTX) is an antineoplastic agent used to treat advanced breast cancer, prostate cancer, acute leukemia, lymphoma and multiple sclerosis. Although it is known to cause cumulative dose-related cardiotoxicity, the underlying mechanisms are still poorly understood. This study aims to compare the cardiotoxicity of MTX and its' pharmacologically active metabolite naphthoquinoxaline (NAPHT) in an in vitro cardiac model, human-differentiated AC16 cells, and determine the role of metabolism in the cardiotoxic effects. Concentration-dependent cytotoxicity was observed after MTX exposure, affecting mitochondrial function and lysosome uptake. On the other hand, the metabolite NAPHT only caused concentration-dependent cytotoxicity in the MTT reduction assay. When assessing the effect of different inhibitors/inducers of metabolism, it was observed that metyrapone (a cytochrome P450 inhibitor) and phenobarbital (a cytochrome P450 inducer) slightly increased MTX cytotoxicity, while 1-aminobenzotriazole (a suicide cytochrome P450 inhibitor) decreased fairly the MTX-triggered cytotoxicity in differentiated AC16 cells. When focusing in autophagy, the mTOR inhibitor rapamycin and the autophagy inhibitor 3-methyladenine exacerbated the cytotoxicity caused by MTX and NAPHT, while the autophagy blocker, chloroquine, partially reduced the cytotoxicity of MTX. In addition, we observed a decrease in p62, beclin-1, and ATG5 levels and an increase in LC3-II levels in MTX-incubated cells. In conclusion, in our in vitro model, neither metabolism nor exogenously given NAPHT are major contributors to MTX toxicity as seen by the residual influence of metabolism modulators used on the observed cytotoxicity and by NAPHT's low cytotoxicity profile. Conversely, autophagy is involved in MTX-induced cytotoxicity and MTX seems to act as an autophagy inducer, possibly through p62/LC3-II involvement.