Triggering of Suicidal Erythrocyte Death by Fascaplysin.
OBJECTIVE
The bis-indole alkaloid Fascaplysin is effective against malignancy, an effect at least partially due to stimulation of tumor cell apoptosis. Similar to apoptosis of nucleated cells, erythrocytes could enter 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 Fascaplysin induces eryptosis and, if so, to shed light on the cellular mechanisms involved.


METHODS
Flow cytometry was employed to estimate phosphatidylserine exposure at the cell surface 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. Hemolysis was quantified from the hemoglobin concentration in the supernatant.


RESULTS
A 48 hours exposure of human erythrocytes to Fascaplysin (≥ 5 µM) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter, and significantly increased Fluo3-fluorescence, DCFDA fluorescence as well as ceramide abundance. The effect of Fascaplysin on annexin-V-binding and forward scatter was significantly blunted but not abolished by removal of extracellular Ca2+.


CONCLUSIONS
Fascaplysin triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry, oxidative stress and ceramide.
Departments of Cardiology, Vascular Medicine and Physiology, Eberhard-Karls-University of Tuebingen, Tuebingen, Germany.
Morena
M
Mischitelli
Mohamed
M
Jemaà
Mustafa
M
Almasry
Caterina
C
Faggio
Florian
F
Lang
D000814:Aniline Compounds; D002118:Calcium / Q000378:metabolism*; D002413:Cations, Divalent; D048429:Cell Size / Q000187:drug effects; D002518:Ceramides / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D000072817:Eryptosis / Q000187:drug effects*; D004912:Erythrocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005434:Flow Cytometry; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D007211:Indoles / Q000494:pharmacology*; D018384:Oxidative Stress; D010718:Phosphatidylserines / Q000378:metabolism; D010860:Pigments, Biological / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000819:agonists / Q000378:metabolism; D014966:Xanthenes; D000644:Quaternary Ammonium Compounds; D002243:Carbolines; D007212:Indolizines
D000814:Aniline Compounds; D002118:Calcium; D002413:Cations, Divalent; D002518:Ceramides; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007211:Indoles; D010718:Phosphatidylserines; D010860:Pigments, Biological; D017382:Reactive Oxygen Species; D014966:Xanthenes; C110400:2',7'-dichlorodihydrofluorescein diacetate; C069346:fascaplysine; D000644:Quaternary Ammonium Compounds; D002243:Carbolines; D007212:Indolizines; C059715:Fluo-3