Ceranib-2-induced suicidal erythrocyte death

Ceramide is known to trigger apoptosis of nucleated cells and eryptosis of erythrocytes. Eryptosis is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Besides ceramide, stimulators of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i) and oxidative stress. Ceramide is degraded by acid ceramidase and inhibition of the enzyme similarly triggers apoptosis. The present study explored, whether ceramidase inhibitor Ceranib-2 induces eryptosis. Flow cytometry was employed to quantify phosphatidylserine-exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) from DCF dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was estimated from hemoglobin concentration in the supernatant. A 48 h exposure of human erythrocytes to Ceranib-2 significantly increased the percentage of annexin-V-binding cells ( 6550 \u3bcM) and the percentage of hemolytic cells ( 6510 \u3bcM) without significantly modifying forward scatter. Ceranib-2 significantly increased Fluo3-fluorescence, DCF fluorescence and ceramide abundance. The effect of Ceranib-2 on annexin-V-binding was not significantly blunted by removal of extracellular Ca2+. Ceranib-2 triggers phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to increase of ceramide abundance and induction of oxidative stress, but not dependent on Ca2+ entry