Clofazimine induced suicidal death of human erythrocytes

Abstract

Background/Aims: The antimycobacterial riminophenazine clofazimine has previously been shown to up-regulate cellular phospholipase A 2 and to induce apoptosis. In erythrocytes phospholipase A 2 stimulates eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Phospholipase A 2 is in part effective by fostering formation of prostaglandin E 2 , which triggers Ca 2+ entry. Stimulators of Ca 2+ entry and eryptosis further include oxidative stress and energy depletion. The present study tested, whether and how clofazimine induces eryptosis. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, hemolysis from hemoglobin release, cytosolic Ca 2+ activity ([Ca 2+ ] i ) from Fluo3-fluorescence, reactive oxygen species (ROS) from 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, and cytosolic ATP level utilizing a luciferin-luciferase assay kit. Results: A 24-48 hours exposure of human erythrocytes to clofazimine (≥1.5 μg/ml) significantly increased the percentage of annexin-V-binding cells without appreciably modifying forward scatter. Clofazimine significantly increased [Ca 2+ ] i , significantly decreased cytosolic ATP, but did not significantly modify ROS. The effect of clofazimine on annexin-V-binding was significantly blunted, but not fully abolished by removal of extracellular Ca 2+ , and by phospholipase A 2 inhibitor quinacrine (25 μM). Clofazimine further augmented the effect of Ca 2+ ionophore ionomycin (0.1 μM) on eryptosis. The clofazimine induced annexin-V-binding was, however, completely abrogated by combined Ca 2+ removal and addition of quinacrine. Conclusion: Clofazimine stimulates phospholipid scrambling of the erythrocyte cell membrane, an effect in part dependent on entry of extracellular Ca 2+ , paralleled by cellular energy depletion and sensitive to phospholipase A 2 inhibitor quinacrine.