Ca2+ Entry, Oxidative Stress, Ceramide and Suicidal Erythrocyte Death Following Diosgenin Treatment.
OBJECTIVE
The bioactive steroid sapogenin diosgenin is considered for a wide variety of applications including treatment of malignancy. The substance counteracts tumor growth in part by stimulating apoptosis of tumor cells. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the stimulation of eryptosis includes increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and ceramide. The present study explored, whether diosgenin induces eryptosis and, if so, to decipher 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 DCF dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was quantified by determination of haemoglobin concentration in the supernatant.


RESULTS
A 48 hours exposure of human erythrocytes to diosgenin significantly increased the percentage of annexin-V-binding cells (≥ 5 µM), significantly decreased forward scatter (15 µM), significantly increased Fluo3-fluorescence (≥ 10 µM), significantly increased DCF fluorescence (15 µM), significantly increased ceramide abundance (15 µM) and significantly increased hemolysis (15 µM). The effect of diosgenin (15 µM) on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca2+.


CONCLUSIONS
Diosgenin stimulates eryptosis with erythrocyte shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect paralleled by and 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*; D004144:Diosgenin / Q000494:pharmacology*; 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; D018384:Oxidative Stress; D017382:Reactive Oxygen Species / Q000819:agonists / Q000378:metabolism; D012503:Saponins / Q000494:pharmacology*; D014966:Xanthenes
C110400:2',7'-dichlorodihydrofluorescein diacetate; D000814:Aniline Compounds; D002413:Cations, Divalent; D002518:Ceramides; D005452:Fluoresceins; D005456:Fluorescent Dyes; D017382:Reactive Oxygen Species; D012503:Saponins; D014966:Xanthenes; C059715:Fluo-3; D004144:Diosgenin; D002118:Calcium