title: Triggering of programmed erythrocyte death by alantolactone.
abstract: The sesquiterpene alantolactone counteracts malignancy, an effect at least in part due to stimulation of suicidal death or apoptosis of tumor cells. Signaling of alantolactone induced apoptosis involves altered gene expression and mitochondrial depolarization. Erythrocytes lack mitochondria and nuclei but may enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the erythrocyte surface. Cellular mechanisms involved in triggering of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i) and oxidative stress. The present study explored, whether alantolactone stimulates eryptosis. To this end, erythrocyte volume was estimated from forward scatter, phosphatidylserine-exposure at the erythrocyte surface from FITC-annexin-V-binding, [Ca2+]i from Fluo3-fluorescence, ceramide abundance from binding of fluorescent antibodies, and oxidative stress from 2',7'-dichlorodihydrofluorescein-diacetate (DCFDA) fluorescence. As a result, a 48 h exposure of human erythrocytes to alantolactone (≥20 μM) significantly decreased erythrocyte forward scatter and increased the percentage of annexin-V-binding cells. Alantolactone significantly increased Fluo3 fluorescence (60 μM), ceramide abundance (60 μM) and DCFDA fluorescence (≥40 μM). The effect of alantolactone (60 μM) on annexin-V-binding was not significantly modified by removal of extracellular Ca2+. In conclusion, alantolactone stimulates suicidal erythrocyte death or eryptosis, an effect paralleled by increase of [Ca2+]i, ceramide abundance and oxidative stress.
authors.affiliation: Department of Physiology, University of Tübingen, Gmelinstr. 5, 72076 Tuebingen, Germany. kossai.z@gmail.com.
authors.forename: Kousi
authors.initials: K
authors.lastname: Alzoubi
authors.affiliation: Department of Physiology, University of Tübingen, Gmelinstr. 5, 72076 Tuebingen, Germany. cfaggio@unime.it.
authors.forename: Salvatrice
authors.initials: S
authors.lastname: Calabrò
authors.affiliation: Department of Physiology, University of Tübingen, Gmelinstr. 5, 72076 Tuebingen, Germany. jasmin.egler@t-online.de.
authors.forename: Jasmin
authors.initials: J
authors.lastname: Egler
authors.affiliation: Department of Biological and Environmental Sciences, University of Messina, Viale Ferdinando Stagno d'Alcontres 31, 98166 S. Agata-Messina, Italy. cfaggio@unime.it.
authors.forename: Caterina
authors.initials: C
authors.lastname: Faggio
authors.affiliation: Department of Physiology, University of Tübingen, Gmelinstr. 5, 72076 Tuebingen, Germany. florian.lang@uni-tuebingen.de.
authors.forename: Florian
authors.initials: F
authors.lastname: Lang
mesh_terms: D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane; D048429:Cell Size / Q000187:drug effects; D002518:Ceramides / Q000378:metabolism; D004912:Erythrocytes / Q000166:cytology / Q000187:drug effects*; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives / Q000378:metabolism; D005452:Fluoresceins / Q000378:metabolism; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D007783:Lactones / Q000633:toxicity*; D018384:Oxidative Stress / Q000187:drug effects; D010718:Phosphatidylserines / Q000378:metabolism; D011485:Protein Binding; D017382:Reactive Oxygen Species / Q000378:metabolism; D045787:Sesquiterpenes, Eudesmane / Q000633:toxicity*; D013499:Surface Properties
chemical_list: C110400:2',7'-dichlorodihydrofluorescein diacetate; D017304:Annexin A5; D002518:Ceramides; C492760:FITC-annexin A5; D005452:Fluoresceins; D007783:Lactones; D010718:Phosphatidylserines; D017382:Reactive Oxygen Species; D045787:Sesquiterpenes, Eudesmane; D016650:Fluorescein-5-isothiocyanate; C004363:alantolactone; D002118:Calcium