title: Clofazimine Induced Suicidal Death of Human Erythrocytes.
abstract: OBJECTIVE
  The antimycobacterial riminophenazine clofazimine has previously been shown to up-regulate cellular phospholipase A2 and to induce apoptosis. In erythrocytes phospholipase A2 stimulates eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Phospholipase A2 is in part effective by fostering formation of prostaglandin E2, 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 A2 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.
  
  
  CONCLUSIONS
  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 A2 inhibitor quinacrine.
authors: Department of Physiology, University of Tx00FC;bingen, Tx00FC;bingen, Germany.
authors: Arbace
authors: A
authors: Officioso
authors: Kousi
authors: K
authors: Alzoubi
authors: Caterina
authors: C
authors: Manna
authors: Florian
authors: F
authors: Lang
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D017304:Annexin A5 / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects*; D048429:Cell Size / Q000187:drug effects; D002991:Clofazimine / Q000494:pharmacology*; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism; D004912:Erythrocytes / Q000187:drug effects* / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D018384:Oxidative Stress / Q000187:drug effects; D010718:Phosphatidylserines / Q000378:metabolism; D054467:Phospholipases A2 / Q000378:metabolism; D011796:Quinacrine / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D017304:Annexin A5; D006454:Hemoglobins; D010718:Phosphatidylserines; D017382:Reactive Oxygen Species; D000255:Adenosine Triphosphate; D002991:Clofazimine; D054467:Phospholipases A2; D011796:Quinacrine; D002118:Calcium