Ritonavir-Induced Suicidal Death of Human Erythrocytes.
The antiviral drug ritonavir has been shown to trigger suicidal death or apoptosis of tumour cells and has thus been considered for the treatment of malignancy. In analogy to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include Ca(2+) entry with increase in cytosolic Ca(2+) activity ([Ca(2+) ]i ), oxidative stress and ceramide. The present study explored whether and how ritonavir induces eryptosis. To this end, flow cytometry was employed to estimate cell volume from forward scatter, phosphatidylserine exposure at the cell surface from Annexin-V binding, [Ca(2+) ]i from Fluo3 fluorescence, abundance of reactive oxygen species (ROS) from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence and ceramide abundance utilizing specific antibodies. As a result, a 48-hr exposure of human erythrocytes to ritonavir significantly increased the percentage of Annexin-V-binding cells (≥5 μg/ml), significantly decreased forward scatter (≥5 μg/ml), significantly increased Fluo3 fluorescence (20 μg/ml), slightly, but significantly increased DCFDA fluorescence (20 μg/ml) and slightly, but significantly increased ceramide abundance (20 μg/ml). The effect of ritonavir on Annexin-V binding was significantly blunted, but not fully abolished by the removal of extracellular Ca(2+) . In conclusion, ritonavir triggers erythrocyte shrinkage and phosphatidylserine translocation at the erythrocyte cell membrane, an effect in part due to the stimulation of Ca(2+) entry, oxidative stress and ceramide.
Department of Physiology, University of Tübingen, Tübingen, Germany.
Sabrina
S
Waibel
Department of Physiology, University of Tübingen, Tübingen, Germany.
Rosi
R
Bissinger
Department of Physiology, University of Tübingen, Tübingen, Germany.|Molecular & Cellular Hematology Laboratory, Pasteur Institute of Tunis, University of Tunis-El Manar, El Manar, Tunisia.
Ghada
G
Bouguerra
Molecular & Cellular Hematology Laboratory, Pasteur Institute of Tunis, University of Tunis-El Manar, El Manar, Tunisia.
Salem
S
Abbès
Department of Physiology, University of Tübingen, Tübingen, Germany.
Florian
F
Lang
D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism; D048429:Cell Size / Q000187:drug effects; D002518:Ceramides / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism; D004912:Erythrocytes / Q000166:cytology / Q000187:drug effects*; D005452:Fluoresceins / Q000378:metabolism; D006801:Humans; D018384:Oxidative Stress / Q000187:drug effects; D010718:Phosphatidylserines / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D019438:Ritonavir / Q000633:toxicity*
C110400:2',7'-dichlorodihydrofluorescein diacetate; D017304:Annexin A5; D002518:Ceramides; D005452:Fluoresceins; D010718:Phosphatidylserines; D017382:Reactive Oxygen Species; D019438:Ritonavir; D002118:Calcium