Enhanced Erythrocyte Membrane Exposure of Phosphatidylserine Following Sorafenib Treatment: An in vivo and in vitro Study

Abstract

Background: Sorafenib (Nexavar^®), a polytyrosine kinase inhibitor, stimulates apoptosis and is thus widely used for chemotherapy in hepatocellular carcinoma (HCC). Hematological side effects of Nexavar^® chemotherapy include anemia. Erythrocytes may undergo apoptosis-like suicidal death or eryptosis, which is characterized by cell shrinkage and phosphatidylserine-exposure at the cell surface. Signaling leading to eryptosis include increase in cytosolic Ca²⁺activity ([Ca²⁺]_i), formation of ceramide, ATP-depletion and oxidative stress. The present study explored, whether sorafenib triggers eryptosis in vitro and in vivo. Methods: [Ca²⁺]_iwas estimated from Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine-exposure from annexin-V-binding, hemolysis from hemoglobin release, ceramide with antibody binding-dependent fluorescence, cytosolic ATP with a luciferin–luciferase-based assay, and oxidative stress from 2’,7’ dichlorodihydrofluorescein diacetate (DCFDA) fluorescence. Results: A 48 h exposure of erythrocytes to sorafenib (≥0.5 µM) significantly increased Fluo 3 fluorescence, decreased forward scatter, increased annexin-V-binding and triggered slight hemolysis (≥5 µM), but did not significantly modify ceramide abundance and cytosolic ATP. Sorafenib treatment significantly enhanced DCFDA-fluorescence and the reducing agents N-acetyl-L-cysteine and tiron significantly blunted sorafenib-induced phosphatidylserine exposure. Nexavar^® chemotherapy in HCC patients significantly enhanced the number of phosphatidylserine-exposing erythrocytes. Conclusions: The present observations disclose novel effects of sorafenib, i.e. stimulation of suicidal erythrocyte death or eryptosis, which may contribute to the pathogenesis of anemia in Nexavar^®-based chemotherapy.