Estramustine-Induced Suicidal Erythrocyte Death

Abstract

Background: The nitrogen mustard derivative of estradiol-17β-phosphate estramustine is used for the treatment of prostate cancer. Estramustine may trigger suicidal death of cancer cells. Side effects of estramustine include anemia. At least in theory, estramustine could cause anemia by stimulation of eryptosis, the suicidal death of erythrocytes. Hallmarks of eryptosis include cell shrinkage, increased cytosolic Ca²⁺ activity ([Ca²⁺]), ceramide formation and phosphatidylserine translocation to the outer leaflet of the cell membrane with phosphatidylserine exposure at the erythrocyte surface. Eryptosis is stimulated by increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i). The present study explored whether estramustine triggers eryptosis. Methods: [Ca²⁺]_i was estimated from Fluo3 fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin V binding, and hemolysis from hemoglobin release. Results: A 24 h exposure to estramustine (≤ 100 µM) significantly increased [Ca²⁺]_i, increased annexin V binding and increased hemoglobin release. The effect of estramustine on annexin V binding was significantly blunted by removal of extracellular Ca²⁺. Conclusions: Estramustine stimulates both, eryptosis and hemolysis. The estramustine induced translocation of phosphatidylserine to the cell surface is at least partially due to increase of cytosolic Ca²⁺ activity.