title: Stimulation of Eryptosis by Caspofungin.
abstract: OBJECTIVE
  The echinocandin antifungal agent caspofungin has been shown to trigger apoptosis of fungal cells. Beyond that, caspofungin is toxic for host mitochondria. Even though lacking mitochondria, erythrocytes may enter apoptosis-like suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in triggering of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, caspase activation and/or activation of p38 kinase, protein kinase C, and casein kinase. The present study explored, whether caspofungin induces eryptosis and, if so, to shed some light on the cellular mechanisms involved.
  
  
  METHODS
  Flow cytometry was employed to determine phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was quantified from hemoglobin concentration in the supernatant.
  
  
  RESULTS
  A 48 hours exposure of human erythrocytes to caspofungin (≥ 30 µg/ml) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter, significantly enhanced hemolysis, but did not significantly increase Fluo3-fluorescence, DCFDA fluorescence or ceramide abundance. The effect of caspofungin on annexin-V-binding was not significantly blunted by removal of extracellular Ca2+, by inhibition of caspases with pancaspase inhibitor zVAD (10 µM), or by addition of the antioxidant N-acetyl-cysteine (1 mM), p38 kinase inhibitor SB203580 (2 µM) or protein kinase C inhibitor staurosporine (1 µM). The effect of caspofungin on annexin-V-binding was, however, significantly blunted in the presence of casein kinase inhibitor D4476 (10 µM).
  
  
  CONCLUSIONS
  Caspofungin triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect possibly involving activation of casein kinase.
authors: Department of Cardiology, Vascular Medicine and Physiology, Eberhard-Karls-University of Tuebingen, Tuebingen, Germany.
authors: Thomas
authors: T
authors: Peter
authors: Rosi
authors: R
authors: Bissinger
authors: Florian
authors: F
authors: Lang
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D000814:Aniline Compounds; D017304:Annexin A5; D000935:Antifungal Agents / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D047388:Casein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D020169:Caspases / Q000235:genetics / Q000378:metabolism; D000077336:Caspofungin; D002478:Cells, Cultured; D002518:Ceramides / Q000378:metabolism; D054714:Echinocandins / Q000494:pharmacology*; D000072817:Eryptosis / Q000187:drug effects*; D004912:Erythrocytes / Q000737:chemistry / Q000166:cytology / Q000187:drug effects*; D005434:Flow Cytometry; D005452:Fluoresceins; D005456:Fluorescent Dyes; D015870:Gene Expression; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D055666:Lipopeptides / Q000494:pharmacology*; D009842:Oligopeptides / Q000494:pharmacology; D018384:Oxidative Stress; D010718:Phosphatidylserines / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D014966:Xanthenes; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: D000814:Aniline Compounds; D017304:Annexin A5; D000935:Antifungal Agents; D002518:Ceramides; D054714:Echinocandins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D055666:Lipopeptides; D009842:Oligopeptides; D010718:Phosphatidylserines; D047428:Protein Kinase Inhibitors; D017382:Reactive Oxygen Species; D014966:Xanthenes; C517629:benzyloxycarbonyl-valyl-alanyl-aspartic acid; C029569:diacetyldichlorofluorescein; C059715:Fluo-3; D047388:Casein Kinases; D011493:Protein Kinase C; D048051:p38 Mitogen-Activated Protein Kinases; D020169:Caspases; D000077336:Caspofungin; D002118:Calcium; D000111:Acetylcysteine