title: Withaferin A-stimulated Ca2+ entry, ceramide formation and suicidal death of erythrocytes.
abstract: Withaferin A, a triterpenoid component from Withania somnifera, counteracts malignancy, an effect attributed to stimulation of apoptosis. Withaferin A is partially effective through induction of oxidative stress, altered gene expression and mitochondrial depolarization. Erythrocytes lack mitochondria and nuclei but may enter apoptosis-like eryptosis, a suicidal cell death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Triggers of eryptosis include increase of cytosolic Ca(2+)-activity [Ca(2+)](i) following activation of oxidant-sensitive Ca(2+)-permeable cation channels, ceramide formation and/or ATP-depletion. The present study explored, whether withaferin A triggers eryptosis. To this end, [Ca(2+)](i) was estimated from Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin-V-binding, hemolysis from hemoglobin release, oxidative stress from DCFDA-fluorescence and ceramide abundance utilizing antibodies. A 48 h exposure to withaferin A significantly decreased forward scatter (at ≥ 10 μM withaferin concentration) and increased [Ca(2+)](i) (≥ 5 μM), ROS-formation (≥ 10 μM) ceramide-formation ( ≥ 10 μM) as well as annexin-V-binding ( ≥ 5 μM). Withaferin A treatment was followed by slight but significant increase of hemolysis. Extracellular Ca(2+) removal, amiloride, and the antioxidant N-acetyl-l-cysteine significantly blunted withaferin A-triggered annexin-V-binding. The present observations reveal that withaferin A triggers suicidal erythrocyte death despite the absence of gene expression and key elements of apoptosis such as mitochondria.
authors.affiliation: Department of Physiology, University of Tuebingen, Gmelinstraße 5, 72076 Tuebingen, Germany.
authors.forename: Kashif
authors.initials: K
authors.lastname: Jilani
authors.forename: Adrian
authors.initials: A
authors.lastname: Lupescu
authors.forename: Mohanad
authors.initials: M
authors.lastname: Zbidah
authors.forename: Nazneen
authors.initials: N
authors.lastname: Shaik
authors.forename: Florian
authors.initials: F
authors.lastname: Lang
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D002518:Ceramides / Q000378:metabolism*; D004912:Erythrocytes / Q000187:drug effects* / Q000378:metabolism; D006461:Hemolysis / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects; D054358:Withanolides / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; D002518:Ceramides; D054358:Withanolides; C009684:withaferin A; D002118:Calcium