BACKGROUND/AIMS: Mitoxantrone, a cytotoxic drug used for the treatment of malignancy and multiple sclerosis, is at least in part effective by triggering apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, a type of suicidal cell death. Hallmarks of eryptosis are cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signalling involved in eryptosis include Ca(2+)-entry, ceramide formation and oxidative stress. METHODS: Cell volume was estimated from forward scatter, phosphatidylserine-exposure from annexin V binding, formation of reactive oxidant species (ROS) from 2',7'-dichlorodihydrofluorescein-diacetate fluorescence, and ceramide abundance from binding of fluorescent antibodies in flow cytometry. RESULTS: A 48 hours exposure to mitoxantrone was followed by significant decrease of forward scatter (≥ 5 μg/ml mitoxantrone) and increase of annexin-V-binding (≥ 10 μg/ml mitoxantrone), effects paralleled by significant increases of ROS formation (25 μg/ml mitoxantrone) and ceramide abundance (25 μg/ml mitoxantrone). The effect of mitoxantrone was not significantly modified by nominal absence of extracellular Ca(2+) but significantly blunted by the antioxidant N-acetylcysteine (1 mM). CONCLUSIONS: Mitoxantrone triggers cell membrane scrambling, an effect not requiring entry of extracellular Ca(2+) but at least partially due to formation of ROS and ceramide.
BACKGROUND/AIMS: Mitoxantrone, a cytotoxic drug used for the treatment of malignancy and multiple sclerosis, is at least in part effective by triggering apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, a type of suicidal cell death. Hallmarks of eryptosis are cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signalling involved in eryptosis include Ca(2+)-entry, ceramide formation and oxidative stress. METHODS: Cell volume was estimated from forward scatter, phosphatidylserine-exposure from annexin V binding, formation of reactive oxidant species (ROS) from 2',7'-dichlorodihydrofluorescein-diacetate fluorescence, and ceramide abundance from binding of fluorescent antibodies in flow cytometry. RESULTS: A 48 hours exposure to mitoxantrone was followed by significant decrease of forward scatter (≥ 5 μg/ml mitoxantrone) and increase of annexin-V-binding (≥ 10 μg/ml mitoxantrone), effects paralleled by significant increases of ROS formation (25 μg/ml mitoxantrone) and ceramide abundance (25 μg/ml mitoxantrone). The effect of mitoxantrone was not significantly modified by nominal absence of extracellular Ca(2+) but significantly blunted by the antioxidant N-acetylcysteine (1 mM). CONCLUSIONS:Mitoxantrone triggers cell membrane scrambling, an effect not requiring entry of extracellular Ca(2+) but at least partially due to formation of ROS and ceramide.