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 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 Enhanced Eryptosis Following Exposure to Dolutegravir

2016 ◽  
Vol 39 (2) ◽  
pp. 639-650 ◽  
Author(s):  
Abdulla Al Mamun Bhuyan ◽  
Elena Signoretto ◽  
Rosi Bissinger ◽  
Florian Lang
Keyword(s):  
Oxidative Stress ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Annexin V ◽  
Cell Shrinkage ◽  
P38 Kinase ◽  
Cellular Mechanisms ◽  
Forward Scatter ◽  
Kinase C

Background/Aims: The viral integrase enzyme inhibitor dolutegravir is utilized for the treatment of immunodeficiency virus (HIV) infection. Knowledge on cytotoxicity of dolutegravir is limited. The present study thus explored, whether dolutegravir is able to trigger suicidal erythrocyte 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 triggering of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, and activation of protein kinase C, p38 kinase, casein kinase, and caspases. The present study explored, whether Dolutegravir induces eryptosis and, if so, to gain insight into cellular mechanisms involved. Methods: Utilizing flow cytometry, 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. Hemolysis was quantified from haemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to dolutegravir significantly increased the percentage of annexin-V-binding cells (≥ 4.8 µM), significantly increased hemolysis (19.1 µM), but did not significantly modify forward scatter. Dolutegravir significantly increased Fluo3-fluorescence (≥ 4.8 µM), DCFDA fluorescence (19.1 µM) and ceramide abundance (19.1 µM). The effect of dolutegravir on annexin-V-binding was significantly blunted by removal of extracellular Ca2+, but was not significantly modified by protein kinase C inhibitor staurosporine (1 µM), p38 kinase inhibitor SB203580 (2 µM), casein kinase inhibitor D4476 (10 µM) or pancaspase inhibitor zVAD (10 µM). Conclusions: Dolutegravir triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry, ceramide formation and oxidative stress.

scholarly journals Fucoxanthin Induced Suicidal Death of Human Erythrocytes

2015 ◽  
Vol 37 (6) ◽  
pp. 2464-2475 ◽  
Author(s):  
Marilena Briglia ◽  
Salvatrice Calabró ◽  
Elena Signoretto ◽  
Kousi Alzoubi ◽  
Stefan Laufer ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Membrane ◽  
Annexin V ◽  
Cell Shrinkage ◽  
P38 Kinase ◽  
Forward Scatter ◽  
Kinase C ◽  
Suicidal Death

Background/Aims: Fucoxanthin, a carotenoid isolated from brown seaweeds, induces suicidal death or apoptosis of tumor cells and is thus considered for the treatment or prevention of malignancy. In analogy to apoptosis of nucleated cell, erythrocytes may enter eryptosis, the suicidal death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include Ca2+ entry with increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and activation of p38 kinase or protein kinase C. The present study explored, whether and how fucoxanthin induces eryptosis. Methods: Phosphatidylserine exposure at the cell surface was estimated 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 DCFDA dependent fluorescence and lipid peroxidation using BODIPY fluoresence. Results: A 48 hours exposure of human erythrocytes to fucoxanthin significantly increased the percentage of annexin-V-binding cells (≥ 50 µM), significantly decreased average forward scatter (≥ 25 µM), significantly increased hemolysis (≥ 25 µM), significantly increased Fluo3-fluorescence (≥ 50 µM), significantly increased lipid peroxidation, but did not significantly modify DCFDA fluorescence. The effect of fucoxanthin on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+, and was insensitive to p38 kinase inhibitor skepinone (2 µM) and to protein kinase C inhibitor calphostin (100 nM). Conclusion: Fucoxanthin triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to stimulation of Ca2+ entry.

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 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 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 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 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+.

Role of protein kinase C in estrogen protection against apoptotic cerebellar cell death in ethanol-withdrawn rats

Alcohol ◽  
2003 ◽  
Vol 31 (1-2) ◽  
pp. 39-48 ◽  
Author(s):  
Marianna E. Jung ◽  
David G. Watson ◽  
Yi Wen ◽  
James W. Simpkins
Keyword(s):  
Cell Death ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Kinase C ◽  
Cerebellar Cell
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