Triggering of Suicidal Erythrocyte Death by Regorafenib

Abstract

Background/Aims: The multikinase inhibitor regorafenib is utilized for the treatment of malignancy. The substance is effective in part by triggering suicidal death or apoptosis of tumor cells. Side effects of regorafenib include anemia. At least in theory, regorafenib induced anemia could result from stimulated suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i), oxidative stress and ceramide. The present study explored, whether regorafenib induces eryptosis and, if so, whether it is effective up- and/or downstream of Ca²⁺. Methods: To this end, phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo3-fluorescence, ROS formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to regorafenib (≥ 0.5 µg/ml) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter (≥ 1.25 µg/ml), but did not significantly increase Fluo3-fluorescence, DCFDA fluorescence or ceramide abundance. The effect of regorafenib on annexin-V-binding and forward scatter was not significantly blunted by removal of extracellular Ca²⁺. Regorafenib (5 µg/ml) significantly augmented the increase of annexin-V-binding, but significantly blunted the decrease of forward scatter following treatment with the Ca²⁺ ionophore ionomycin. Conclusions: Regorafenib triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part downstream of Ca²⁺.