title: Camalexin-Induced Cell Membrane Scrambling and Cell Shrinkage in Human Erythrocytes.
abstract: OBJECTIVE
  The thaliana phytoalexin Camalexin has been proposed for the treatment of malignancy. Camalexin counteracts tumor growth in part by stimulation of suicidal death or apoptosis of tumor cells. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Cellular mechanisms contributing to the complex machinery executing eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, protein kinase C and caspases. The present study explored, whether Camalexin induces eryptosis and, if so, to shed light on mechanisms involved.
  
  
  METHODS
  Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo-3 fluorescence, ROS formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies.
  
  
  RESULTS
  A 48 hours exposure of human erythrocytes to Camalexin significantly increased the percentage of annexin-V-binding cells (≥ 10 µg/ml), significantly decreased forward scatter (≥ 5 µg/ml) and significantly increased Fluo-3-fluorescence (≥ 10 µg/ml), but did not significantly modify DCFDA fluorescence or ceramide abundance. The effect of Camalexin on annexin-V-binding was significantly blunted by removal of extracellular Ca2+, by kinase inhibitors staurosporine (1 µM) and chelerythrine (10 µM), as well as by caspase inhibitors zVAD (10 µM) and zIETD-fmk (50 µM).
  
  
  CONCLUSIONS
  Camalexin triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part depending on Ca2+ entry, as well as staurosporine and chelerythrine sensitive kinase(s) as well as zVAD and zIETD-fmk sensitive caspase(s).
authors.forename: Mustafa
authors.initials: M
authors.lastname: Almasry
authors.forename: Mohamed
authors.initials: M
authors.lastname: Jemaà
authors.forename: Morena
authors.initials: M
authors.lastname: Mischitelli
authors.forename: Florian
authors.initials: F
authors.lastname: Lang
authors.forename: Caterina
authors.initials: C
authors.lastname: Faggio
mesh_terms: D053119:Benzophenanthridines / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D061945:Caspase Inhibitors / Q000494:pharmacology; D048429:Cell Size / Q000187:drug effects; D002518:Ceramides / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D000072817:Eryptosis / Q000187:drug effects; D004910:Erythrocyte Membrane / Q000187:drug effects*; D004912:Erythrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D007211:Indoles / Q000494:pharmacology*; D009842:Oligopeptides / Q000494:pharmacology; D010718:Phosphatidylserines / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology*; D000094162:Phytoalexins
chemical_list: D053119:Benzophenanthridines; D002118:Calcium; D061945:Caspase Inhibitors; D002518:Ceramides; D007211:Indoles; D009842:Oligopeptides; D010718:Phosphatidylserines; D017382:Reactive Oxygen Species; D019311:Staurosporine; D013844:Thiazoles; C517629:benzyloxycarbonyl-valyl-alanyl-aspartic acid; C102405:camalexin; D000094162:Phytoalexins; C016299:chelerythrine
keywords: Calcium; Cell volume; Chelerythrine; Eryptosis; Phosphatidylserine; Staurosporine; zVAD