scholarly journals Lapatinib Induced Suicidal Death of Human Erythrocytes

2015 ◽  
Vol 37 (6) ◽  
pp. 2275-2287 ◽  
Author(s):  
Jens Zierle ◽  
Rosi Bissinger ◽  
Jasmin Egler ◽  
Florian Lang
Keyword(s):  
Cell Membrane ◽  
Kinase Inhibitor ◽  
Annexin V ◽  
Human Erythrocytes ◽  
Cell Shrinkage ◽  
Forward Scatter ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation ◽  
Theory Result ◽  
Phospholipid Scrambling

Background/Aims: The human epidermal growth factor receptors tyrosine kinase inhibitor lapatinib has been shown to trigger suicidal death or apoptosis of tumor cells and is thus used for the treatment of malignancy. Side effects of lapatinib include anemia, which could, at least in theory, result from stimulation of eryptosis, the suicidal death of erythrocytes which is characterized by cell shrinkage and phospholipid scrambling of the cell membrane leading to phosphatidylserine translocation to the erythrocyte surface. Mechanisms involved in the triggering of eryptosis include oxidative stress, increase of cytosolic Ca2+ activity ([Ca2+]i), and ceramide. The present study explored, whether lapatinib induces eryptosis. Methods: Phosphatidylserine exposure at the cell surface was estimated 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 utilizing labelled specific antibodies. Results: A 48 hours exposure of human erythrocytes to lapatinib (≥ 1 µg/ml) significantly increased the percentage of annexin-V-binding cells, and significantly decreased forward scatter. Lapatinib (7.5 µg/ml) did not significantly modify DCFDA fluorescence and ceramide abundance. Lapatinib slightly, but significantly decreased Fluo3-fluorescence (≥ 5 µg/ml). Lapatinib (7.5 µg/ml) enhanced the annexin-V-binding in the presence of the Ca2+ ionophore ionomycin (1 µM) without significantly modifying Fluo3 fluorescence in the presence of ionomycin. The effect of lapatinib on forward scatter but not on annexin-V-binding was significantly blunted by removal of extracellular Ca2+. Conclusion: Lapatinib triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect occurring despite decrease of cytosolic Ca2+ activity.

scholarly journals Edelfosine Induced Suicidal Death of Human Erythrocytes

2015 ◽  
Vol 37 (6) ◽  
pp. 2221-2230 ◽  
Author(s):  
Marilena Briglia ◽  
Antonella Fazio ◽  
Elena Signoretto ◽  
Caterina Faggio ◽  
Florian Lang
Keyword(s):  
Cell Membrane ◽  
Annexin V ◽  
Human Erythrocytes ◽  
Cell Shrinkage ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation ◽  
Phospholipid Scrambling ◽  
Binding Cell

Background/Aims: The anti-inflammatory, anti-autoimmune, antiparasitic, and anti-viral ether phospholipid edelfosine (1-O-octadecyl-2-O-methylglycero-3-phosphocholine) stimulates apoptosis of tumor cells and is thus considered for the treatment of malignancy. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and phospholipid scrambling of the cell membrane with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include Ca2+ entry with increase of cytosolic Ca2+ activity ([Ca2+]i) and oxidative stress. The present study explored, whether and how edelfosine induces eryptosis. Methods: Flow cytometry and photometry, respectively, were employed to estimate phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, hemolysis from hemoglobin release, [Ca2+]i from Fluo3-fluorescence, and abundance of reactive oxygen species (ROS) from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence. Results: A 6 hours exposure of human erythrocytes to edelfosine (5 µM) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter, and significantly increased Fluo3-fluorescence, but did not significantly modify DCFDA fluorescence. The effect of edelfosine on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+. Conclusions: Edelfosine triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to stimulation of Ca2+ entry.

scholarly journals Oxaliplatin Induced Suicidal Death of Human Erythrocytes

2015 ◽  
Vol 37 (6) ◽  
pp. 2393-2404 ◽  
Author(s):  
Antonella Fazio ◽  
Marilena Briglia ◽  
Caterina Faggio ◽  
Kousi Alzoubi ◽  
Florian Lang
Keyword(s):  
Cell Membrane ◽  
Annexin V ◽  
Human Erythrocytes ◽  
Cell Shrinkage ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation ◽  
Ros Formation ◽  
Binding Cell

Background/Aims: The alkylating drug oxaliplatin is widely used for chemotherapy of malignancy. Oxaliplatin is effective by inducing both, necrosis and apoptosis. Similar to necrosis or apoptosis of nucleated cells, erythrocytes may enter hemolysis, which is apparent from hemoglobin release or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include oxidative stress and/or Ca2+ entry with increase of cytosolic Ca2+ activity ([Ca2+]i). The present study explored, whether and how oxaliplatin induces eryptosis. Methods: Phosphatidylserine exposure at the cell surface was quantified utilizing annexin-V-binding, cell volume estimated from forward scatter, hemolysis deduced from hemoglobin release, [Ca2+]i determined utilizing Fluo-3 fluorescence, and reactive oxygen species (ROS) abundance visualized using 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) dependent fluorescence. Results: A 48 hours exposure of human erythrocytes to oxaliplatin (10 µg/ml) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter, significantly increased Fluo-3 fluorescence, and significantly increased DCFDA fluorescence. The effect of oxaliplatin on annexin-V-binding and forward scatter was rather augmented by removal of extracellular Ca2+, but was significantly blunted in the presence of the antioxidant N-acetyl-cysteine (1 mM). Conclusions: Oxaliplatin triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect partially dependent on ROS formation.

scholarly journals Enhanced Eryptosis Following Exposure to Lopinavir

2015 ◽  
Vol 37 (6) ◽  
pp. 2486-2495 ◽  
Author(s):  
Rosi Bissinger ◽  
Sabrina Waibel ◽  
Ghada Bouguerra ◽  
Abdulla Al Mamun Bhuyan ◽  
Salem Abbès ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Haemoglobin Concentration ◽  
Annexin V ◽  
Hiv Infections ◽  
Cell Shrinkage ◽  
Forward Scatter ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation ◽  
Phospholipid Scrambling ◽  
Stimulation Of

Background/Aims: The protease inhibitor lopinavir, used for the treatment of HIV infections, triggers suicidal death or apoptosis of nucleated cells. Side effects of lopinavir include anemia, which could in theory result from stimulation of suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and by phospholipid scrambling of the cell membrane leading to phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include oxidative stress, increase of cytosolic Ca2+ activity ([Ca2+]i), and ceramide. The present study explored, whether lopinavir induces eryptosis. Methods: Flow cytometry was employed to estimate phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) abundance from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, reduced glutathione (GSH) from mercury orange fluorescence and ceramide abundance utilizing labelled specific antibodies. Hemolysis was estimated from haemoglobin concentration of the supernatant. Results: A 48 hours exposure of human erythrocytes to lopinavir significantly increased the percentage of annexin-V-binding cells (≥ 10 µg/ml), significantly decreased forward scatter (≥15 µg/ml), significantly increased hemolysis (≥ 15 µg/ml), significantly increased Fluo3-fluorescence (20 µg/ml), and significantly increased DCFDA fluorescence (20 µg/ml) but did not significantly modify ceramide abundance. The effect of lopinavir on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+. Conclusion: Lopinavir treatment of erythrocytes from healthy volunteers is followed by cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to stimulation of ROS formation and Ca2+ entry.

scholarly journals Enhanced Eryptosis Following Exposure to Carnosic Acid

2015 ◽  
Vol 37 (5) ◽  
pp. 1779-1791 ◽  
Author(s):  
Katja Stockinger ◽  
Rosi Bissinger ◽  
Ghada Bouguerra ◽  
Salem Abbès ◽  
Florian Lang
Keyword(s):  
Cell Membrane ◽  
Annexin V ◽  
Human Erythrocytes ◽  
Carnosic Acid ◽  
Cell Shrinkage ◽  
Cellular Mechanisms ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation

Background/Aims: The phenolic abietane diterpene component of rosemary and sage, carnosic acid, may either induce or inhibit apoptosis of nucleated cells. The mechanisms involved in the effects of carnosic acid include altered mitochondrial function and gene expression. Human erythrocytes lack mitochondria and nuclei but are nevertheless able to enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Cellular mechanisms involved in the stimulation of eryptosis include oxidative stress, increase of cytosolic Ca2+ activity ([Ca2+]i), and ceramide formation. The present study explored, whether and how carnosic acid induces eryptosis. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCFDA dependent fluorescence and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to carnosic acid significantly increased the percentage of annexin-V-binding cells (2.5 µg/ml), significantly decreased forward scatter (10 µg/ml), significantly increased Fluo3 fluorescence (10 µg/ml), significantly increased ceramide abundance (10 µg/ml), significantly increased hemolysis (10 µg/ml), but significantly decreased DCFDA fluorescence (10 µg/ml). The effect of carnosic acid on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+. Conclusion: Carnosic acid triggers cell shrinkage and phospholipid scrambling of the human erythrocyte cell membrane, an effect paralleled by and/or in part due to Ca2+ entry and increased ceramide abundance.

scholarly journals Saquinavir Induced Suicidal Death of Human Erythrocytes

2015 ◽  
Vol 37 (5) ◽  
pp. 1973-1982 ◽  
Author(s):  
Sabrina Waibel ◽  
Rosi Bissinger ◽  
Ghada Bouguerra ◽  
Salem Abbès ◽  
Florian Lang
Keyword(s):  
Cell Membrane ◽  
Annexin V ◽  
Hiv Infections ◽  
Human Erythrocytes ◽  
Cell Shrinkage ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation ◽  
Ros Formation

Background/Aims: The antiretroviral protease inhibitor saquinavir is used for the treatment of HIV infections. Effects of saquinavir include induction of apoptosis, the suicidal death of nucleated cells. Saquinavir treatment may further lead to anemia. In theory, anemia could result from accelerated erythrocyte loss by enhanced suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include Ca2+ entry with increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress with increase of reactive oxygen species (ROS) and ceramide. The present study explored, whether and how saquinavir induces eryptosis. Methods: To this end, flow cytometry was employed to estimate erythrocyte volume from forward scatter, phosphatidylserine exposure at the cell surface from annexin-V-binding, [Ca2+]i from Fluo3-fluorescence, ROS abundance from DCFDA fluorescence and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to saquinavir significantly decreased forward scatter (≥ 5 µg/ml), significantly increased the percentage of annexin-V-binding cells (≥ 10 µg/ml), significantly increased Fluo3-fluorescence (15 µg/ml), significantly increased DCFDA fluorescence (15 µg/ml), but did not significantly modify ceramide abundance. The effect of saquinavir on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+. Conclusions: Saquinavir triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to stimulation of ROS formation and Ca2+ entry.

scholarly journals Stimulation of Suicidal Erythrocyte Death by Phosphatase Inhibitor Calyculin A

2016 ◽  
Vol 40 (1-2) ◽  
pp. 163-171 ◽  
Author(s):  
Mustafa Almasry ◽  
Mohamed Jemaà ◽  
Morena Mischitelli ◽  
Caterina Faggio ◽  
Florian Lang
Keyword(s):  
Cell Membrane ◽  
Annexin V ◽  
Calyculin A ◽  
Cell Shrinkage ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation ◽  
Phospholipid Scrambling ◽  
Binding Cell

Background/Aims: The serine/threonine protein phosphatase 1 and 2a inhibitor Calyculin A may trigger suicidal death or apoptosis of tumor cells. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal erythrocyte death 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). Eryptosis is fostered by activation of staurosporine sensitive protein kinase C, SB203580 sensitive p38 kinase, and D4476 sensitive casein kinase. Eryptosis may further involve zVAD sensitive caspases. The present study explored, whether Calyculin A induces eryptosis and, if so, whether its effect requires Ca2+ entry, kinases and/or caspases Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, and [Ca2+] i from Fluo-3 fluorescence, as determined by flow cytometry. Results: A 48 hours exposure of human erythrocytes to Calyculin A (≥ 2.5 nM) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter and significantly increased Fluo-3 fluorescence. The effect of Calyculin A on annexin-V-binding was significantly blunted by removal of extracellular Ca2+, by staurosorine (1 µM), SB203580 (2 µM), D4476 (10 µM), and zVAD (10 µM). Conclusions: Calyculin A triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part requiring Ca2+ entry, kinase activity and caspase activation.

scholarly journals Camalexin-Induced Cell Membrane Scrambling and Cell Shrinkage in Human Erythrocytes

2017 ◽  
Vol 41 (2) ◽  
pp. 731-741 ◽  
Author(s):  
Mustafa Almasry ◽  
Mohamed Jemaà ◽  
Morena Mischitelli ◽  
Florian Lang ◽  
Caterina Faggio
Keyword(s):  
Cell Membrane ◽  
Kinase Inhibitors ◽  
Annexin V ◽  
Human Erythrocytes ◽  
Cell Shrinkage ◽  
Cellular Mechanisms ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation

Background/Aims: 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).

scholarly journals Licochalcone A Induced Suicidal Death of Human Erythrocytes

2015 ◽  
Vol 37 (5) ◽  
pp. 2060-2070 ◽  
Author(s):  
Jasmin Egler ◽  
Florian Lang
Keyword(s):  
Cell Membrane ◽  
Annexin V ◽  
Human Erythrocytes ◽  
Cell Shrinkage ◽  
Forward Scatter ◽  
Licochalcone A ◽  
Erythrocyte Surface ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation ◽  
Glycyrrhiza Inflata

Background: The anti-inflammatory, immunomodulatory, and antimicrobial Glycyrrhiza inflata extract component licochalcone A triggers apoptosis of tumor cells and is thus considered for the treatment of malignancy. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the triggering of eryptosis includes Ca2+ entry with increase of cytosolic Ca2+ activity ([Ca2+]i), and ceramide. The present study explored, whether and how licochalcone A induces eryptosis. Methods: Human erythrocytes drawn from healthy individuals were exposed for 24 hours to 1-10 µg/ml licochalcone A. Flow cytometry was subsequently employed to estimate phosphatidylserine exposure at the cell surface from annexin V binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, and ceramide utilizing specific antibodies. In addition, hemolysis was quantified from hemoglobin release. Results: Licochalcone A significantly increased the percentage of annexin-V-binding cells (≥ 5 µg/ml), significantly decreased forward scatter (2.5 - 5 µg/ml), significantly increased Fluo3-fluorescence (≥ 7.5 µg/ml), and significantly increased ceramide abundance (10 µg/ml). The effect of licochalcone on annexin-V-binding was not significantly modified, but hemolysis significantly enhanced by removal of extracellular Ca2+. Conclusions: Licochalcone triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect independent from Ca2+ entry and presumably in part due to ceramide.

scholarly journals Enhanced Eryptosis Following Auranofin Exposure

2015 ◽  
Vol 37 (3) ◽  
pp. 1018-1028 ◽  
Author(s):  
Kousi Alzoubi ◽  
Jasmin Egler ◽  
Majed Abed ◽  
Florian Lang
Keyword(s):  
Oxidative Stress ◽  
Cell Membrane ◽  
Annexin V ◽  
Cell Shrinkage ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation ◽  
Phospholipid Scrambling ◽  
Binding Cell

Background/Aims: The antiinflammatory, antimicrobial and anticancer drug auranofin has previously been shown to trigger apoptosis, the suicidal death of nucleated cells. Side effects of the drug include anaemia. At least in theory the anaemia could result from stimulation of suicidal death of erythrocytes or eryptosis, which involves cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Methods: Stimulators of eryptosis include oxidative stress and increase of cytosolic Ca2+-activity ([Ca2+]i). In the present study, phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, hemolysis from hemoglobin release, reactive oxygen species (ROS) from 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, and [Ca2+]i from Fluo3-fluorescence. Results: A 24 hours exposure of human erythrocytes to auranofin (≥5 µg/ml) significantly increased the percentage of annexin-V-binding cells (from 2.2 ± 0.5 to 17.4 ± 1.5%), significantly decreased forward scatter and significantly enhanced ROS. At higher concentrations (10 µg/ml) auranofin triggered slight hemolysis (from 2.1 ± 0.2 to 3.2 ± 0.3%). Conclusions: Auranofin stimulates cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least partially due to induction of oxidative stress.

scholarly journals Nocodazole Induced Suicidal Death of Human Erythrocytes

2016 ◽  
Vol 38 (1) ◽  
pp. 379-392 ◽  
Author(s):  
Elena Signoretto ◽  
Sabina Honisch ◽  
Marilena Briglia ◽  
Caterina Faggio ◽  
Michela Castagna ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Membrane ◽  
Annexin V ◽  
Human Erythrocytes ◽  
Microtubule Assembly ◽  
Cell Shrinkage ◽  
Forward Scatter ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation ◽  
Mitotic Death

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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