Nocodazole Induced Suicidal Death of Human Erythrocytes

Background: The microtubule assembly inhibitor nocodazole has been shown to trigger caspase-independent mitotic death and caspase dependent apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may undergo eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell...

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Main Authors: Elena Signoretto, Sabina Honisch, Marilena Briglia, Caterina Faggio, Michela Castagna, Florian Lang
Format: Article
Language:English
Published: Cell Physiol Biochem Press GmbH & Co KG 2016-01-01
Series:Cellular Physiology and Biochemistry
Subjects:
Online Access:http://www.karger.com/Article/FullText/438638
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author Elena Signoretto
Sabina Honisch
Marilena Briglia
Caterina Faggio
Michela Castagna
Florian Lang
author_facet Elena Signoretto
Sabina Honisch
Marilena Briglia
Caterina Faggio
Michela Castagna
Florian Lang
author_sort Elena Signoretto
collection DOAJ
description Background: The microtubule assembly inhibitor nocodazole has been shown to trigger caspase-independent mitotic death and caspase dependent apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may undergo eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and ceramide. The present study explored, whether and how nocodazole induces eryptosis. 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, the abundance of reactive oxygen species (ROS) from 2′,7′-dichlorodihydrofluorescein (DCF) diacetate dependent fluorescence as well as ceramide surface abundance utilizing specific antibodies. Tubulin abundance was quantified by TubulinTracker™ Green reagent and visualized by confocal microscopy. Results: A 48 hours exposure of human erythrocytes to nocodazole (≥ 30 µg/ml) significantly increased the percentage of annexin-V-binding cells without significantly modifying average forward scatter. Nocodazole significantly increased Fluo3-fluorescence, significantly increased DCF fluorescence and significantly increased ceramide surface abundance. The effect of nocodazole on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+ and was not modified in the presence of Caspase 3 inhibitor zVAD (1 µM). Nocodazole treatment reduced the content of total tubulin. Conclusions: Nocodazole triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to stimulation of Ca2+ entry, oxidative stress and ceramide.
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spelling doaj.art-4a582acadbc4429c8d44e39e510ffa962022-12-21T23:20:24ZengCell Physiol Biochem Press GmbH & Co KGCellular Physiology and Biochemistry1015-89871421-97782016-01-0138137939210.1159/000438638438638Nocodazole Induced Suicidal Death of Human ErythrocytesElena SignorettoSabina HonischMarilena BrigliaCaterina FaggioMichela CastagnaFlorian LangBackground: The microtubule assembly inhibitor nocodazole has been shown to trigger caspase-independent mitotic death and caspase dependent apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may undergo eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and ceramide. The present study explored, whether and how nocodazole induces eryptosis. 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, the abundance of reactive oxygen species (ROS) from 2′,7′-dichlorodihydrofluorescein (DCF) diacetate dependent fluorescence as well as ceramide surface abundance utilizing specific antibodies. Tubulin abundance was quantified by TubulinTracker™ Green reagent and visualized by confocal microscopy. Results: A 48 hours exposure of human erythrocytes to nocodazole (≥ 30 µg/ml) significantly increased the percentage of annexin-V-binding cells without significantly modifying average forward scatter. Nocodazole significantly increased Fluo3-fluorescence, significantly increased DCF fluorescence and significantly increased ceramide surface abundance. The effect of nocodazole on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+ and was not modified in the presence of Caspase 3 inhibitor zVAD (1 µM). Nocodazole treatment reduced the content of total tubulin. Conclusions: Nocodazole triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to stimulation of Ca2+ entry, oxidative stress and ceramide.http://www.karger.com/Article/FullText/438638CalciumPhosphatidylserineCell volumeEryptosisOxidative stress
spellingShingle Elena Signoretto
Sabina Honisch
Marilena Briglia
Caterina Faggio
Michela Castagna
Florian Lang
Nocodazole Induced Suicidal Death of Human Erythrocytes
Cellular Physiology and Biochemistry
Calcium
Phosphatidylserine
Cell volume
Eryptosis
Oxidative stress
title Nocodazole Induced Suicidal Death of Human Erythrocytes
title_full Nocodazole Induced Suicidal Death of Human Erythrocytes
title_fullStr Nocodazole Induced Suicidal Death of Human Erythrocytes
title_full_unstemmed Nocodazole Induced Suicidal Death of Human Erythrocytes
title_short Nocodazole Induced Suicidal Death of Human Erythrocytes
title_sort nocodazole induced suicidal death of human erythrocytes
topic Calcium
Phosphatidylserine
Cell volume
Eryptosis
Oxidative stress
url http://www.karger.com/Article/FullText/438638
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