title: Anidulafungin-Induced Suicidal Erythrocyte Death.
abstract: OBJECTIVE
  The novel antifungal drug Anidulafungin is used for the treatment of diverse fungal infections including candidiasis and aspergillosis. The traditional antifungal drug amphotericin B has previously been shown to trigger eryptosis, the suicidal death of erythrocytes characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, activated protein kinase C (PKC), casein kinase 1α or p38 kinase and activated caspases. Inhibitors of eryptosis include nitric oxide (NO). The present study explored, whether Anidulafungin induces eryptosis.
  
  
  METHODS
  Flow cytometry was employed to estimate phosphatidylserine abundance at the erythrocyte surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, abundance of reactive oxygen species (ROS) from DCFDA dependent fluorescence, and ceramide abundance at the erythrocyte surface utilizing specific antibodies. Hemolysis was quantified by measuring haemoglobin concentration in the supernatant.
  
  
  RESULTS
  A 48 hours exposure of human erythrocytes to Anidulafungin (1.5 - 6 µg/ml) significantly increased hemolysis and the percentage of annexin-V-binding cells, and significantly decreased forward scatter. Anidulafungin (6 µg/ml) slightly, but significantly inceased Fluo3-fluorescence and the effect of Anidulafungin on annexin-V-binding was slightly, but significantly blunted by removal of extracellular Ca2+. The effect of Anidulafungin on annexin-V-binding was further significantly blunted by the p38 kinase inhibitor SB203580 (2 µM) and NO donor nitroprusside (1 µM). An increase of extracellular K+ concentration significantly blunted the effect of Anidulafungin on cell volume but not on annexin-V-binding. Anidulafungin rather decreased DCFDA fluorescence and the effect of Anidulafungin on annexin-V-binding was not significantly blunted by the antioxidant N-acetylcysteine (1 mM). Moreover, the effect of Anidulafungin on annexin-V-binding was not paralleled by significant increase of ceramide abundance and was not significantly blunted by PKC inhibitor staurosporine (1 µM), casein kinase 1α inhibitor D4476 (10 µM) or pancaspase inhibitor zVAD (10 µM).
  
  
  CONCLUSIONS
  Anidulafungin triggers hemolysis and eryptosis with cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to Ca2+ entry and activation of p38 kinase.
authors.forename: Thomas
authors.initials: T
authors.lastname: Peter
authors.forename: Rosi
authors.initials: R
authors.lastname: Bissinger
authors.forename: Guilai
authors.initials: G
authors.lastname: Liu
authors.forename: Florian
authors.initials: F
authors.lastname: Lang
mesh_terms: D000077612:Anidulafungin; D000935:Antifungal Agents / Q000009:adverse effects*; D002118:Calcium / Q000378:metabolism; D048429:Cell Size / Q000187:drug effects; D054714:Echinocandins / Q000009:adverse effects*; D000072817:Eryptosis / Q000187:drug effects*; D004912:Erythrocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D006461:Hemolysis / Q000187:drug effects*; D006801:Humans; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D000935:Antifungal Agents; D054714:Echinocandins; D017382:Reactive Oxygen Species; D000077612:Anidulafungin; D002118:Calcium