Programmed Erythrocyte Death Following in Vitro Treosulfan Treatment

Abstract

Background/Aims: The cytotoxic drug Treosulfan is clinically used for the treatment of malignancy. A common side effect of Treosulfan treatment is anemia. Treosulfan is at least partially effective by triggering apoptosis of tumor cells. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, a suicidal death characterized by cell shrinkage and translocation of phosphatidylserine from the inner to the outer leaflet of the plasma membrane. Triggers of eryptosis include oxidative stress, Ca²⁺-entry and increase of cytosolic Ca²⁺-activity ([Ca²⁺]_i). The present study explored whether Treosulfan stimulates eryptosis, which may contribute to development of anemia. Methods: Erythrocyte volume was estimated from forward scatter, phosphatidylserine abundance at the erythrocyte surface from Fluorescein isothiocyanate (FITC)-annexin-V-binding, [Ca²⁺]_i from Fluo3 fluorescence and reactive oxygen species (ROS) from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA)-fluorescence. Results: A 48 hours exposure of human erythrocytes to Treosulfan (800 µg/ml) significantly decreased erythrocyte forward scatter, increased the percentage of annexin-V-binding cells, increased [Ca²⁺]_i, and increased ROS. The effect of Treosulfan on annexin-V-binding was virtually abrogated by removal of extracellular Ca²⁺. Conclusion: Treosulfan stimulates suicidal erythrocyte death or eryptosis at least in part by inducing oxidative stress and stimulating Ca²⁺ entry.