Triggering of Eryptosis, the Suicidal Erythrocyte Death, by Perifosine

Abstract

Background/aims: The alkylphospholipid perifosine is used for the treatment of malignancy. The substance is effective by triggering suicidal tumor cell death or apoptosis. Side effects of perifosine include anemia. At least in theory, perifosine-induced anemia could result from stimulation of suicidal erythrocyte death or eryptosis. Hallmarks of eryptosis are cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Cellular mechanisms participating in the orchestration of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, increase of ceramide abundance, as well as activation of staurosporine sensitive protein kinase C and/or of SB203580 sensitive p38 kinase. The present study explored, whether perifosine induces eryptosis and, if so, whether its effect involves and/or requires Ca2+ entry, oxidative stress, ceramide and kinase activation.

Methods: 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) abundance from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was estimated from hemoglobin concentration in the supernatant.

Results: A 24 hours exposure of human erythrocytes to perifosine (2.5 µg/ml) significantly increased the percentage of annexin-V-binding cells, significantly decreased average forward scatter, significantly increased the percentage of shrunken erythrocytes, and significantly decreased the percentage of swollen erythrocytes. Perifosine significantly increased the percentage of hemolytic erythrocytes. Perifosine significantly increased Fluo3-fluorescence, but decreased DCFDA fluorescence and ceramide abundance. The effect of perifosine on annexin-V-binding was significantly blunted by removal of extracellular Ca2+ and by addition of staurosporine (1 µM), but not by addition of SB203580 (2 µM).

Conclusions: Perifosine triggers eryptosis, an effect at least in part due to Ca2+ entry and activation of staurosporine sensitive kinases.

Keywords: Calcium; Ceramide; Eryptosis; Oxidative stress; Phosphatidylserine; Staurosporine.