Stimulation of erythrocyte cell membrane scrambling by mitotane.
background: Mitotane (1,1-dichloro-2-[o-chlorophenyl]-2-[p-chlorophenyl]ethane), a cytostatic drug used for the treatment of adrenocortical carcinomas, is effective by triggering tumor cell apoptosis. In analogy to apoptosis of nucleated cells, eryptosis is the suicidal death of erythrocytes, which is typically paralleled by cell shrinkage and breakdown of cell membrane phosphatidylserine asymmetry with subsequent phosphatidylserine exposure at the erythrocyte surface. Eryptosis may be triggered by increase of cytosolic Ca(2+) concentration ([Ca(2+)]i). The present study tested, whether treatment of human erythrocytes with mitotane is followed by eryptosis.


METHODS
[Ca(2+)]i was estimated from Fluo3 fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin V binding, and hemolysis from hemoglobin release.


RESULTS
Exposure to mitotane (≥ 5 µg/ml ≈ 16 µM) significantly increased [Ca(2+)]i, increased annexin V binding and triggered hemolysis, but did not significantly modify forward scatter. The effect on annexin V binding was significantly blunted in the absence of extracellular Ca(2+). Within 30 min Ca(2+) ionophore ionomycin (1 µM) decreased forward scatter, an effect virtually abolished in the presence of mitotane (15 µg/ml).


CONCLUSIONS
Mitotane increases [Ca(2+)]i with subsequent phosphatidylserine translocation. By the same token mitotane inhibits Ca(2+) induced cell shrinkage.
Department of Physiology, University of Tuebingen, Tuebingen, Germany.
Janin
J
Jacobi
Elisabeth
E
Lang
Rosi
R
Bissinger
Leonie
L
Frauenfeld
Paola
P
Modicano
Caterina
C
Faggio
Majed
M
Abed
Florian
F
Lang
D002118:Calcium / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D008939:Mitotane / Q000494:pharmacology*; D013329:Structure-Activity Relationship
D008939:Mitotane; D002118:Calcium