scholarly journals Micafungin-Induced Suicidal Erythrocyte Death

2016 ◽  
Vol 39 (2) ◽  
pp. 584-595 ◽  
Author(s):  
Thomas Peter ◽  
Rosi Bissinger ◽  
Elena Signoretto ◽  
Andreas F. Mack ◽  
Florian Lang
Keyword(s):  
Cell Membrane ◽  
Kinase Inhibitor ◽  
Fungal Infections ◽  
Haemoglobin Concentration ◽  
Annexin V ◽  
Casein Kinase ◽  
Cell Shrinkage ◽  
P38 Kinase ◽  
Forward Scatter ◽  
Erythrocyte Surface

Background/Aims: The antifungal drug Micafungin is used for the treatment of diverse fungal infections including candidiasis and aspergillosis. Side effects of Micafungin treatment include microangiopathic hemolytic anemia and thrombocytopenia with microvascular thrombosis. The development of thrombosis may be fostered by stimulation of eryptosis, the suicidal death of erythrocytes 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), oxidative stress, ceramide, activated protein kinase C (PKC), casein kinase 1α or p38 kinase and activated caspases. The present study explored, whether Micafungin induces eryptosis. Methods: Flow cytometry was employed to estimate phosphatidylserine abundance at the erythrocyte surface 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 at the erythrocyte surface utilizing specific antibodies. Hemolysis was quantified by measuring haemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to Micafungin (10 - 25 µg/ml) significantly increased hemolysis and the percentage of annexin-V-binding cells, and significantly decreased forward scatter. Micafungin (25 µg/ml) did not significantly modify Fluo3-fluorescence, DCFDA fluorescence, or ceramide abundance. The effect of Micafungin on annexin-V-binding was not significantly modified by removal of extracellular Ca2+, by PKC inhibitor staurosporine (1 µM), p38 kinase inhibitor SB203580 (2 µM), casein kinase 1α inhibitor D4476 (10 µM) or pancaspase inhibitor zVAD (10 µM). Conclusions: Micafungin triggers hemolysis and eryptosis with cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane.

scholarly journals Anidulafungin-Induced Suicidal Erythrocyte Death

2016 ◽  
Vol 38 (6) ◽  
pp. 2272-2284 ◽  
Author(s):  
Thomas Peter ◽  
Rosi Bissinger ◽  
Guilai Liu ◽  
Florian Lang
Keyword(s):  
Cell Membrane ◽  
Cell Volume ◽  
Fungal Infections ◽  
Annexin V ◽  
Casein Kinase ◽  
Antifungal Drug ◽  
Cell Shrinkage ◽  
P38 Kinase ◽  
Forward Scatter ◽  
Erythrocyte Surface

Background/Aims: The novel antifungal drug Anidulafungin is used for the treatment of diverse fungal infections including candidiasis and aspergillosis. The traditional antifungal drug amphotericin B has previously been shown to trigger eryptosis, the suicidal death of erythrocytes 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), oxidative stress, ceramide, activated protein kinase C (PKC), casein kinase 1α or p38 kinase and activated caspases. Inhibitors of eryptosis include nitric oxide (NO). The present study explored, whether Anidulafungin induces eryptosis. Methods: Flow cytometry was employed to estimate phosphatidylserine abundance at the erythrocyte surface 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 at the erythrocyte surface utilizing specific antibodies. Hemolysis was quantified by measuring haemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to Anidulafungin (1.5 - 6 µg/ml) significantly increased hemolysis and the percentage of annexin-V-binding cells, and significantly decreased forward scatter. Anidulafungin (6 µg/ml) slightly, but significantly inceased Fluo3-fluorescence and the effect of Anidulafungin on annexin-V-binding was slightly, but significantly blunted by removal of extracellular Ca2+. The effect of Anidulafungin on annexin-V-binding was further significantly blunted by the p38 kinase inhibitor SB203580 (2 µM) and NO donor nitroprusside (1 µM). An increase of extracellular K+ concentration significantly blunted the effect of Anidulafungin on cell volume but not on annexin-V-binding. Anidulafungin rather decreased DCFDA fluorescence and the effect of Anidulafungin on annexin-V-binding was not significantly blunted by the antioxidant N-acetylcysteine (1 mM). Moreover, the effect of Anidulafungin on annexin-V-binding was not paralleled by significant increase of ceramide abundance and was not significantly blunted by PKC inhibitor staurosporine (1 µM), casein kinase 1α inhibitor D4476 (10 µM) or pancaspase inhibitor zVAD (10 µM). Conclusions: Anidulafungin triggers hemolysis and eryptosis with cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to Ca2+ entry and activation of p38 kinase.

scholarly journals Stimulating Effect of Sclareol on Suicidal Death of Human Erythrocytes

2016 ◽  
Vol 39 (2) ◽  
pp. 554-564 ◽  
Author(s):  
Elena Signoretto ◽  
Stefan A. Laufer ◽  
Florian Lang
Keyword(s):  
Cell Membrane ◽  
Kinase Inhibitor ◽  
Haemoglobin Concentration ◽  
Annexin V ◽  
Casein Kinase ◽  
Human Erythrocytes ◽  
P38 Kinase ◽  
Cellular Mechanisms ◽  
Forward Scatter ◽  
Erythrocyte Surface

Background/Aims: The diterpene alcohol Sclareol has been proposed for the treatment of malignancy. In analogy to apoptosis of nucleated cells, erythrocytes may enter eryptosis, a suicidal cell death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Cellular mechanisms involved in the triggering of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, p38 kinase and casein kinase 1α. The present study explored, whether Sclareol induces eryptosis and, if so, shed light on the mechanisms involved. Methods: Phosphatidylserine abundance at the erythrocyte surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, abundance of reactive oxygen species (ROS) from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA)-dependent fluorescence, and ceramide abundance at the erythrocyte surface utilizing specific antibodies. Hemolysis was estimated from haemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to Sclareol (≥ 50 µM) significantly increased the percentage of annexin-V-binding cells without significantly modifying the average forward scatter, DCF-fluorescence or ceramide abundance. Sclareol (≥ 50 µM) further triggered hemolysis. Sclareol (100 µM) significantly increased Fluo3-fluorescence, but the effect of Sclareol on annexin-V-binding was not significantly blunted by removal of extracellular Ca2+. Instead, the effect of Sclareol on annexin-V-binding was significantly blunted in the presence of p38 kinase inhibitor skepinone (2 µM) and in the presence of casein kinase 1α inhibitor D4476 (10 µM). Conclusions: Sclareol triggers phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to activation of p38 kinase and casein kinase 1α.

scholarly journals Stimulation of Eryptosis by Caspofungin

2016 ◽  
Vol 39 (3) ◽  
pp. 939-949 ◽  
Author(s):  
Thomas Peter ◽  
Rosi Bissinger ◽  
Florian Lang
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Membrane ◽  
Kinase Inhibitor ◽  
Annexin V ◽  
Casein Kinase ◽  
Cell Shrinkage ◽  
P38 Kinase ◽  
Forward Scatter ◽  
Kinase C

Background/Aims: The echinocandin antifungal agent caspofungin has been shown to trigger apoptosis of fungal cells. Beyond that, caspofungin is toxic for host mitochondria. Even though lacking mitochondria, erythrocytes may enter apoptosis-like suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in triggering of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, caspase activation and/or activation of p38 kinase, protein kinase C, and casein kinase. The present study explored, whether caspofungin induces eryptosis and, if so, to shed some light on the cellular mechanisms involved. 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, ROS formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was quantified from hemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to caspofungin (≥ 30 µg/ml) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter, significantly enhanced hemolysis, but did not significantly increase Fluo3-fluorescence, DCFDA fluorescence or ceramide abundance. The effect of caspofungin on annexin-V-binding was not significantly blunted by removal of extracellular Ca2+, by inhibition of caspases with pancaspase inhibitor zVAD (10 µM), or by addition of the antioxidant N-acetyl-cysteine (1 mM), p38 kinase inhibitor SB203580 (2 µM) or protein kinase C inhibitor staurosporine (1 µM). The effect of caspofungin on annexin-V-binding was, however, significantly blunted in the presence of casein kinase inhibitor D4476 (10 µM). Conclusions: Caspofungin triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect possibly involving activation of casein kinase.

scholarly journals Efavirenz Induced Suicidal Death of Human Erythrocytes

2015 ◽  
Vol 37 (6) ◽  
pp. 2496-2507 ◽  
Author(s):  
Rosi Bissinger ◽  
Ghada Bouguerra ◽  
Abdulla Al Mamun Bhuyan ◽  
Sabrina Waibel ◽  
Salem Abbès ◽  
...  
Keyword(s):  
Kinase Inhibitor ◽  
Annexin V ◽  
Casein Kinase ◽  
Human Erythrocytes ◽  
Cell Shrinkage ◽  
P38 Kinase ◽  
Mitochondrial Potential ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Phosphatidylserine Translocation

Background/Aims: The reverse transcriptase inhibitor efavirenz utilized for the treatment of human immunodeficiency virus (HIV)-1 infection, triggers suicidal cell death or apoptosis, an effect in part due to interference with mitochondrial potential. Side effects of efavirenz include anemia. Causes of anemia include accelerated clearance of circulating erythrocytes. Even though lacking mitochondria, erythrocytes may enter suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include Ca2+ entry and increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, as well as activation of p38 kinase, casein kinase 1α and/or cyclooxygenase. The present study explored, whether and how efavirenz 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 selective antibodies. Results: A 48 hours exposure of human erythrocytes to efavirenz (≥ 2 µg/ml) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter (2 µg/ml), significantly increased Fluo3-fluorescence (≥ 2 µg/ml), but did not significantly modify DCFDA fluorescence or ceramide abundance. The effect of efavirenz on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+. The effect of efavirenz on annexin-V-binding was further significantly blunted by p38 kinase inhibitor SB203580 (2 µM) and casein kinase 1α inhibitor D4476 (10 µM), but not by cyclooxygenase inhibitor aspirin (50 µM). Conclusions: Efavirenz triggers cell shrinkage and phosphatidylserine translocation to the erythrocyte surface, an effect in part due to stimulation of Ca2+ entry as well as activation of p38 kinase and casein kinase 1α.

scholarly journals Stimulating Effect of Elvitegravir on Suicidal Erythrocyte Death

2016 ◽  
Vol 38 (3) ◽  
pp. 1111-1120 ◽  
Author(s):  
Rosi Bissinger ◽  
Abdulla Al Mamun Bhuyan ◽  
Elena Signoretto ◽  
Florian Lang
Keyword(s):  
Cell Membrane ◽  
Kinase Inhibitor ◽  
Antiviral Drug ◽  
Annexin V ◽  
Hiv Infections ◽  
Cell Shrinkage ◽  
P38 Kinase ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Suicidal Death

Background/Aims: The antiviral drug Elvitegravir is used for the treatment of Human Immunodeficiency Virus (HIV) infections. The present study explored whether the drug is able to trigger eryptosis, the suicidal death of erythrocytes. Eryptosis is 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, ceramide, activated p38 kinase and activated caspases. The present study explored, whether Elvitegravir induces eryptosis and, if so, to shed light on the mechanisms involved. Methods: Phosphatidylserine abundance at the erythrocyte 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 at the erythrocyte surface utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to Elvitegravir (≥ 1.5 µg/ml) significantly increased the percentage of annexin-V-binding cells, and significantly decreased forward scatter. Elvitegravir (2.5 µg/ml) significantly increased Fluo3-fluorescence, but did not significantly modify DCFDA fluorescence or ceramide abundance. The effect of Elvitegravir on annexin-V-binding was significantly blunted by removal of extracellular Ca2+, but not in the presence of p38 kinase inhibitor SB203580 (2 µM) or in the presence of pancaspase inhibitor zVAD (10 µM). Conclusions: Elvitegravir triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to entry of extracellular Ca2+.

scholarly journals Embelin-Induced Phosphatidylserine Translocation in the Erythrocyte Cell Membrane

2015 ◽  
Vol 37 (4) ◽  
pp. 1629-1640 ◽  
Author(s):  
Ghada Bouguerra ◽  
Omar Aljanadi ◽  
Rosi Bissinger ◽  
Salem Abbès ◽  
Florian Lang
Keyword(s):  
Cell Membrane ◽  
Kinase Inhibitor ◽  
Annexin V ◽  
P38 Kinase ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation ◽  
Surface Signaling ◽  
Binding Cell

Background/Aims: The antihelminthic, contraceptive, anti-inflammatory and anticancer phytochemical embelin is at least in part effective against malignancy by inducing suicidal death or apoptosis of tumor cells. Erythrocytes are similarly able to enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling of eryptosis includes increase of cytosolic Ca2+-activity ([Ca2+]i), ceramide formation, oxidative stress as well as activation of p38 kinase and protein kinase C (PKC). The present study tested, whether and how embelin 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, ceramide abundance utilizing specific antibodies and reactive oxygen species (ROS) from 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence. Results: A 48 hours exposure of human erythrocytes to embelin (≥25 µM) significantly increased the percentage of annexin-V-binding cells and hemolysis. Embelin did not significantly modify [Ca2+]i. The effect of embelin on annexin-V-binding was not blunted by removal of extracellular Ca2+, by p38 kinase inhibitor SB203580 (2 µM) or by PKC inhibitor staurosporine (1 µM). Embelin did, however, significantly increase the ceramide abundance. Conclusions: Embelin stimulates phospholipid scrambling of the erythrocyte cell membrane, an effect involving ceramide formation.

scholarly journals Ca2+ Entry, Oxidative Stress, Ceramide and Suicidal Erythrocyte Death Following Diosgenin Treatment

2016 ◽  
Vol 39 (4) ◽  
pp. 1626-1637 ◽  
Author(s):  
Morena Mischitelli ◽  
Mohamed Jemaà ◽  
Mustafa Almasry ◽  
Caterina Faggio ◽  
Florian Lang
Keyword(s):  
Oxidative Stress ◽  
Cell Membrane ◽  
Haemoglobin Concentration ◽  
Annexin V ◽  
Cellular Mechanisms ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation ◽  
Surface Signaling

Background/Aims: The bioactive steroid sapogenin diosgenin is considered for a wide variety of applications including treatment of malignancy. The substance counteracts tumor growth in part by stimulating 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. Signaling involved in the stimulation of eryptosis includes increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and ceramide. The present study explored, whether diosgenin induces eryptosis and, if so, to decipher cellular mechanisms involved. 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, ROS formation from DCF dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was quantified by determination of haemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to diosgenin significantly increased the percentage of annexin-V-binding cells (≥ 5 µM), significantly decreased forward scatter (15 µM), significantly increased Fluo3-fluorescence (≥ 10 µM), significantly increased DCF fluorescence (15 µM), significantly increased ceramide abundance (15 µM) and significantly increased hemolysis (15 µM). The effect of diosgenin (15 µM) on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca2+. Conclusions: Diosgenin stimulates eryptosis with erythrocyte shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect paralleled by and at least in part due to Ca2+ entry, oxidative stress and ceramide.

scholarly journals Triggering of Suicidal Erythrocyte Death by Fascaplysin

2016 ◽  
Vol 39 (4) ◽  
pp. 1638-1647 ◽  
Author(s):  
Morena Mischitelli ◽  
Mohamed Jemaà ◽  
Mustafa Almasry ◽  
Caterina Faggio ◽  
Florian Lang
Keyword(s):  
Oxidative Stress ◽  
Cell Membrane ◽  
Indole Alkaloid ◽  
Annexin V ◽  
Hemoglobin Concentration ◽  
Cell Shrinkage ◽  
Cellular Mechanisms ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Phosphatidylserine Translocation

Background/Aims: The bis-indole alkaloid Fascaplysin is effective against malignancy, an effect at least partially due to stimulation of tumor cell apoptosis. Similar to apoptosis of nucleated cells, erythrocytes could enter suicidal erythrocyte death or eryptosis, 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), oxidative stress and ceramide. The present study explored, whether Fascaplysin induces eryptosis and, if so, to shed light on the cellular mechanisms involved. 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, ROS formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was quantified from the hemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to Fascaplysin (≥ 5 µM) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter, and significantly increased Fluo3-fluorescence, DCFDA fluorescence as well as ceramide abundance. The effect of Fascaplysin on annexin-V-binding and forward scatter was significantly blunted but not abolished by removal of extracellular Ca2+. Conclusions: Fascaplysin triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry, oxidative stress and ceramide.

scholarly journals Triggering of Suicidal Erythrocyte Death by Zosuquidar

2015 ◽  
Vol 37 (6) ◽  
pp. 2355-2365 ◽  
Author(s):  
Marilena Briglia ◽  
Antonella Fazio ◽  
Caterina Faggio ◽  
Florian Lang
Keyword(s):  
Cell Death ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Annexin V ◽  
Tumor Cell Death ◽  
P38 Kinase ◽  
Forward Scatter ◽  
Kinase C ◽  
Erythrocyte Surface

Background: The P-glycoprotein inhibitor zosuquidar (LY335979) is clinically used to augment the effect of cytostatic drugs on suicidal tumor cell death or apoptosis. The present study explored whether the substance is cytotoxic to erythrocytes. Upon injury, erythrocytes may undergo suicidal cell death or eryptosis, which is characterized by cell shrinkage and translocation of cell membrane phosphatidylserine to the erythrocyte surface. Signaling of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i), oxidative stress and activation of several kinases, such as p38 kinase and protein kinase C. Methods: Phosphatidylserine abundance at the erythrocyte surface was quantified from binding of FITC-labelled annexin-V, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, and reactive oxygen species (ROS) from 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence. Results: A 48 h treatment of human erythrocytes with zosuquidar significantly increased the percentage of annexin-V-binding cells (2 and 4 µg/ml), significantly decreased forward scatter (4 µg/ml), significantly increased [Ca2+]i (4 µg/ml), but did not significantly modify ROS. The up-regulation of annexin-V-binding following zosuquidar (4 µg/ml) treatment was significantly blunted by removal of extracellular Ca2+, by presence of p38 kinase inhibitor SB203580 (2 µM) and by presence of protein kinase C inhibitor calphostin (100 nM). Conclusions: Exposure of erythrocytes to zosuquidar triggers suicidal erythrocyte death with erythrocyte shrinkage and erythrocyte membrane scrambling, an effect involving Ca2+ entry and requiring activity of SB203580 and calphostin sensitive kinases.

scholarly journals Stimulating Effect of Terfenadine on Erythrocyte Cell Membrane Scrambling

2016 ◽  
Vol 38 (4) ◽  
pp. 1425-1434 ◽  
Author(s):  
Elena Signoretto ◽  
Michela Castagna ◽  
Abdulla Al Mamun Bhuyan ◽  
Florian Lang
Keyword(s):  
Cell Membrane ◽  
Human Erythrocyte ◽  
Haemoglobin Concentration ◽  
Annexin V ◽  
Human Erythrocytes ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation ◽  
Surface Signaling

Background/Aims: The antihistaminic drug Terfenadine may trigger apoptosis of tumor cells, an effect unrelated to its effect on histamine receptors. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal death of erythrocytes characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling triggering eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, and ceramide. The present study explored, whether Terfenadine is capable to trigger eryptosis. Methods: Flow cytometry was employed to estimate phosphatidylserine abundance at the erythrocyte surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, abundance of reactive oxygen species (ROS) from 2′,7′-dichlorodihydrofluorescein (DCF) diacetate dependent fluorescence, and ceramide abundance at the human erythrocyte surface utilizing specific antibodies. Hemolysis was quantified from haemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to Terfenadine (≥ 5 µM) significantly increased the percentage of annexin-V-binding cells and triggered hemolysis without significantly modifying the average forward scatter. Terfenadine (7.5 µM) significantly increased Fluo3-fluorescence, but did not significantly modify DCF fluorescence or ceramide abundance. The effect of Terfenadine on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca2+. Exposure of human erythrocytes to Ca2+ ionophore ionomycin (1 µM, 15 min) triggered annexin-V-binding, an effect augmented by Terfenadine pretreatment (10 µM, 48 hours). Conclusions: Terfenadine triggers phospholipid scrambling of the human erythrocyte cell membrane, an effect in part due to entry of extracellular Ca2+ and in part due to sensitizing human erythrocyte cell membrane scrambling to Ca2+.

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