Garcinol A Novel Inhibitor of Platelet Activation and Apoptosis.

Garcinol, an anti-inflammatory and anti-carcinogenic polyisoprenylated benzophenone isolated from Garcinia plants, stimulates tumor cell apoptosis and suicidal erythrocyte death, but supports the survival of hepatocytes and neurons. The present study explored whether the substance influences platelet function and/or apoptosis. To this end, we exposed murine blood platelets to garcinol (33 µM, 30 min) without and with activation by collagen-related peptide (CRP) (2-5 µg/mL) or thrombin (0.01 U/mL); flow cytometry was employed to estimate cytosolic Ca2+-activity ([Ca2+]i) from Fluo-3 fluorescence, platelet degranulation from P-selectin abundance, integrin activation from αIIbß3 integrin abundance, caspase activity utilizing an Active Caspase-3 Staining kit, phosphatidylserine abundance from annexin-V-binding, relative platelet volume from forward scatter, and aggregation utilizing staining with CD9-APC and CD9-PE. As a result, in the absence of CRP and thrombin, the exposure of the platelets to garcinol did not significantly modify [Ca2+]i, P-selectin abundance, activated αIIbß3 integrin, annexin-V-binding, cell volume, caspase activity, and aggregation. Exposure of platelets to CRP or thrombin was followed by a significant increase of [Ca2+]i, P-selectin abundance, αIIbß3 integrin activity, annexin-V-binding, caspase activity, and aggregation, as well as significant cell shrinkage. All effects of CRP were strong and significant; those of thrombin were only partially and slightly blunted in the presence of garcinol. In conclusion, garcinol blunts CRP-induced platelet activity, apoptosis and aggregation.

Inhibition of Erythrocyte Cell Membrane Scrambling Following Energy Depletion and Hyperosmotic Shock by Alectinib.

BACKGROUND/AIMS: The anaplastic lymphoma kinase (ALK) inhibitor alectinib is clinically used for the treatment of ALK positive non-small-cell lung cancer. At least in part the substance is effective by triggering suicidal death or apoptosis of tumor cells. Erythrocytes are lacking mitochondria and nuclei, key organelles of apoptosis but are, similar to apoptosis of nucleated cells, able to enter suicidal erythrocyte death or eryptosis. Stimulators of eryptosis include energy depletion, hyperosmotic shock, oxidative stress, and increase of cytosolic Ca2+ activity ([Ca2+]i). The present study explored, whether alectinib influences eryptosis. METHODS: Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding and cell volume from forward scatter. Measurements were made without or with energy depletion (glucose deprivation for 48 hours), hyperosmotic shock (+550mM sucrose for 6 hours), oxidative stress (50 min exposure to 0.3 mM tert-butylhydroperoxide), and Ca2+ loading (60 minutes treatment with 1 µM Ca2+ ionophore ionomycin). RESULTS: A 48 hours exposure of human erythrocytes to alectinib (150-600 ng/ml) did not significantly modify the percentage of annexin-V-binding cells and forward scatter. Energy depletion, hyperosmotic shock, oxidative stress and Ca2+ loading were each followed by profound and significant increase of the percentage annexin-V-binding erythrocytes and a significant decrease of forward scatter. The effects of energy depletion and hyperosmotic shock, but not of oxidative stress or Ca2+ loading on annexin-V-binding were significantly blunted in the presence of alectinib (150-600 ng/ml). In none of the conditions was forward scatter significantly modified by alectinib. CONCLUSION: Alectinib inhibits cell membrane scrambling following energy depletion and hyperosmotic shock.

Stimulation of Eryptosis, the Suicidal Erythrocyte Death, by Costunolide.

BACKGROUND/AIMS: The sesquiterpene lactone Costunolide is effective against various disorders including inflammation and malignancy. The substance is effective in part by triggering suicidal death or apoptosis of tumor cells. Mechanisms involved include altered function of transcription factors and mitochondria. Erythrocytes lack nuclei and mitochondria but are - in analogy to apoptosis of nucleated cells - able to 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 Costunolide induces eryptosis and, if so, to shed light on the mechanisms involved. METHODS: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) formation from 2',7'-dichlorodihydrofluorescein (DCF)-dependent fluorescence, and ceramide abundance utilizing specific antibodies. RESULTS: A 48 hours exposure of human erythrocytes to Costunolide (15 µg/ml) significantly enhanced the percentage of annexin-V-binding cells, significantly decreased forward scatter and significantly increased Fluo3-fluorescence, DCF-fluorescence, and ceramide abundance. The effect of Costunolide on annexin-V-binding was significantly blunted by removal of extracellular Ca2+. CONCLUSION: Costunolide triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry and paralleled by oxidative stress and ceramide formation.

Taurolidine Sensitivity of Eryptosis, the Suicidal Erythrocyte Death.

BACKGROUND/AIMS: The taurine derivative Taurolidine is effective against diverse bacteria and tumor growth. In the treatment of cancer, the substance is effective in part by triggering suicidal death or apoptosis of tumor cells. The Taurolidine-induced apoptosis involves mitochondria. Erythrocytes lack mitochondria but are nevertheless able to enter suicidal erythrocyte 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), oxidative stress and ceramide. The present study explores, whether Taurolidine induces eryptosis and, if so, which cellular mechanisms are involved. METHODS: Phosphatidylserine exposure at the cell surface was estimated using annexin-V-binding, cell volume using forward scatter, [Ca2+]i using Fluo3-fuorescence, reactive oxygen species (ROS) formation using 2',7'-dichlorodihydrofuorescein (DCF)-dependent fluorescence, and ceramide abundance using specific antibodies. RESULTS: A 48 hours exposure of human erythrocytes to Taurolidine (60 µg/ml) significantly enhanced the percentage of annexin-V-binding cells, significantly decreased forward scatter and significantly increased Fluo3-fluorescence and ceramide abundance, but not DCF-fluorescence. The effect of Taurolidine on annexin-V-binding was virtually abrogated by removal of extracellular Ca2+. CONCLUSION: Taurolidine triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry and paralleled by increase of ceramide abundance.

Sonidegib, a Novel Inhibitor of Suicidal Erythrocyte Death.

BACKGROUND/AIMS: The Hedgehog pathway disrupting drug sonidegib is used in the treatment of basal cell carcinoma. Side effects of sonidegib include anemia, which could result either from impaired erythropoiesis or from loss of erythrocytes e.g. due to suicidal erythrocyte death or eryptosis, which is characterized by cell membrane scrambling with phosphatidylserine translocation to the cell surface and by cell shrinkage. Eryptosis is stimulated by cell stress, including energy depletion, hyperosmotic shock, oxidative stress and excessive increase of cytosolic Ca2+ activity ([Ca2+]i). The present study explored, whether sonidegib exerts an effect on eryptosis. METHODS: Human erythrocytes have been treated with energy depletion (glucose withdrawal for 48 hours), hyperosmotic shock (addition of 550 mM sucrose for 6 hours), oxidative stress (addition of 0.3 mM tert-butylhydroperoxide [tBOOH] for 50 min) or Ca2+ ionophore ionomycin (1 µM for 60 min) in absence and presence of sonidegib (2-6 µg/ ml). After treatment flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, and cell volume from forward scatter. Hemolysis was estimated from the hemoglobin concentration in the supernatant. RESULTS: In the absence of cell stress exposure to sonidegib did not significantly modify annexin-V-binding or forward scatter, but triggered hemolysis. Energy depletion, hyperosmotic shock, oxidative stress and ionomycin, all markedly and significantly increased the percentage of annexin-V-binding erythrocytes, and decreased the forward scatter. Sonidegib significantly blunted the effect of energy depletion, hyperosmotic shock, and oxidative stress, but not of ionomycin on annexin-V-binding. Sonidegib further significantly blunted the effect of energy depletion, but not of hyperosmotic shock, oxidative stress, and ionomycin on forward scatter. CONCLUSIONS: Sonidegib is a novel inhibitor of erythrocyte cell membrane scrambling following energy depletion, hyperosmotic shock and oxidative stress.

Stimulation of Eryptosis by Afatinib.

BACKGROUND/AIMS: The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor afatinib is primarily utilized for the treatment of non-small cell lung carcinoma. The drug is at least partially effective by triggering suicidal tumor cell death. Side effects of afatinib treatment include anemia. At least in theory, afatinib induced anemia could be secondary to stimulation of suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling potentially stimulating eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), induction of oxidative stress, and increase of ceramide abundance. The present study explored, whether afatinib induces eryptosis and, if so, whether its effect involves Ca2+ entry, oxidative stress, and/or ceramide. METHODS: Flow cytometry was employed to quantify 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 DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. RESULTS: A 48 hours exposure of human erythrocytes to afatinib (≥ 4 µg/ml) significantly increased the percentage of annexin-V-binding cells and significantly decreased forward scatter. Afatinib significantly increased Fluo3-fluorescence, DCFDA fluorescence and ceramide abundance. The effect of afatinib on annexin-V-binding and forward scatter was significantly blunted by removal of extracellular Ca2+. CONCLUSIONS: Afatinib triggers phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry, oxidative stress, and ceramide.

Rhodomyrtone (Rom) is a membrane-active compound.

Particularly in Asia medicinal plants with antimicrobial activity are used for therapeutic purpose. One such plant-derived antibiotic is rhodomyrtone (Rom) isolated from Rhodomyrtus tomentosa leaves. Rom shows high antibacterial activity against a wide range of Gram-positive bacteria, however, its mode of action is still unclear. Reporter gene assays and proteomic profiling experiments in Bacillus subtilis indicate that Rom does not address classical antibiotic targets like translation, transcription or DNA replication, but acts at the cytoplasmic membrane. In Staphylococcus aureus, Rom decreases the membrane potential within seconds and at low doses, causes release of ATP and even the excretion of cytoplasmic proteins (ECP), but does not induce pore-formation as for example nisin. Lipid staining revealed that Rom induces local membrane damage. Rom's antimicrobial activity can be antagonized in the presence of a very narrow spectrum of saturated fatty acids (C15:0, C16:0, or C18:0) that most likely contribute to counteract the membrane damage. Gram-negative bacteria are resistant to Rom, presumably due to reduced penetration through the outer membrane and its neutralization by LPS. Rom is cytotoxic for many eukaryotic cells and studies with human erythrocytes showed that Rom induces eryptosis accompanied by erythrocyte shrinkage, cell membrane blebbing, and membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Rom's distinctive interaction with the cytoplasmic membrane reminds on the amphipathic, alpha-helical peptides, the phenol-soluble modulins (PSMs), and renders Rom an important tool for the investigation of membrane physiology.

Inhibition of Suicidal Erythrocyte Death by Volasertib.

BACKGROUND/AIMS: The Polo-like kinase 1 (Plk1) inhibitor volasertib is used in the treatment of malignancy. Volasertib is partially effective by triggering suicidal death or apoptosis of tumor cells. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal cell death or eryptosis, which is characterized by cell membrane scrambling with phosphatidylserine translocation to the cell surface and by cell shrinkage. Stimulators of eryptosis include energy depletion, hyperosmotic shock, oxidative stress and excessive increase of cytosolic Ca2+ activity ([Ca2+]i). The present study explored, whether volasertib impacts on eryptosis. METHODS: Human erythrocytes have been exposed to energy depletion (glucose withdrawal for 48 hours), hyperosmotic shock (addition of 550 mM sucrose for 6 hours), oxidative stress (addition of 0.3 mM tert-butylhydroperoxide [tBOOH] for 50 min) or Ca2+ ionophore ionomycin (1 µM for 60 min) in absence and presence of volasertib (0.5-1.5 µg/ml) and flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3 fluorescence, reactive oxygen species from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence and ceramide abundance utilizing antibodies. For comparison, annexin-V-binding and forward scatter were determined following a 48 hours exposure of human leukemic K562 cells in RPMI-1640 medium to volasertib. RESULTS: Treatment with volasertib alone did not significantly modify annexin-V-binding or forward scatter in mature erythrocytes. Energy depletion, hyperosmotic shock, oxidative stress and ionomycin, all markedly and significantly increased the percentage of annexin-V-binding erythrocytes, and decreased the forward scatter. Volasertib significantly blunted the effect of energy depletion and hyperosmotic shock, but not of oxidative stress and ionomycin on annexin-V-binding. Volasertib did not significantly influence the effect of any maneuver on forward scatter. In K562 cells, volasertib enhanced annexin-V-binding and decreased the forward scatter. CONCLUSIONS: Volasertib is a novel inhibitor of erythrocyte cell membrane scrambling following energy depletion and hyperosmotic shock, effects contrasting the stimulation of K562 cell apoptosis.

Triggering of Suicidal Erythrocyte Death by Exemestane.

BACKGROUND/AIMS: The steroidal aromatase inactivator exemestane blocks estrogen biosynthesis and is thus employed for the prevention and treatment of breast cancer. Exemestane is in part effective by stimulation of suicidal cell death or apoptosis. Side effects of exemestane treatment include anemia. At least in theory, exemestane induced anemia could be secondary to stimulation of suicidal erythrocyte death or eryptosis, 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, ceramide, several kinases and caspases. The present study explored, whether exemestane is able to trigger eryptosis and, if so, to shed some light on the signaling involved. METHODS: Flow cytometry was employed to quantify 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 DCF fluorescence, and ceramide abundance utilizing specific antibodies. RESULTS: A 48 hours exposure of human erythrocytes to exemestane (≥ 10 µg/ml) significantly increased the percentage of annexin-V-binding cells without significantly modifying forward scatter. Exemestane significantly increased Fluo3-fluorescence (10 and 20, but not 40 µg/ml), DCF fluorescence (40 µg/ml), and ceramide abundance (40 µg/ml). The effect of exemestane (40 µg/ml) on annexin-V-binding was significantly blunted by antioxidant N-acetylcysteine (1mM), but was not significantly modified by removal or increase of extracellular Ca2+, by p38 kinase inhibitor SB203580 (2 µM), casein kinase inhibitor D4476 (10 µM) and caspase inhibitor zVAD (10 µM). CONCLUSIONS: Exemestane triggers phospholipid scrambling of the erythrocyte cell membrane, an effect paralleled by enhanced [Ca2+]i, oxidative stress, and increased ceramide abundance.

Triggering of Suicidal Erythrocyte Death by Gefitinib.

BACKGROUND/AIMS: The epidermal growth factor receptor-tyrosine kinase inhibitor gefitinib is effective against several malignancies and is mainly utilized in the treatment of epidermal growth factor receptor mutation positive non-small cell lung cancer. The anti-cancer effect of the drug involves stimulation of apoptosis. Side effects of gefitinib include anemia. At least in theory, the development of anemia during gefitinib treatment could result from triggering of eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and by cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling potentially stimulating eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i) and generation of oxidative stress. The present study explored, whether gefitinib stimulates eryptosis and, if so, whether its effect involves Ca2+ entry and/or oxidative stress. METHODS: Flow cytometry was employed to quantify cell volume from forward scatter, phosphatidylserine exposure at the cell surface from annexin-V-binding, [Ca2+]i from Fluo3-fluorescence, and reactive oxygen species (ROS) abundance from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) dependent fluorescence. RESULTS: A 48 hours exposure of human erythrocytes to gefitinib (≥ 2 µg/ml) significantly decreased forward scatter and significantly increased the percentage of annexin-V-binding cells. Gefitinib did not significantly increase Fluo3-fluorescence but the effect of gefitinib on annexin-V-binding was significantly blunted by removal of extracellular Ca2+. Gefitinib further significantly increased DCFDA fluorescence. CONCLUSIONS: Gefitinib triggers erythrocyte shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part dependent on extracellular Ca2+ and paralleled by oxidative stress.

Lipopeptide-Induced Suicidal Erythrocyte Death Correlates with the Degree of Acylation.

BACKGROUND/AIMS: Consequences of bacterial infection include anemia, which could result from stimulation of suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Bacterial components known to stimulate eryptosis include lipopeptides. Signaling mediating the triggering of eryptosis include increased cytosolic Ca2+ activity ([Ca2+]_i), oxidative stress and cellular accumulation of ceramide. The present study aimed to define the molecular requirements for lipopeptide-induced cell membrane scrambling. METHODS: Human erythrocytes were incubated for 48 hours in the absence and presence of 1 or 5 µg/ml of the synthetic lipopeptides Pam1 (lipopeptide with one fatty acid), Pam2 (lipopeptide with two fatty acids), or Pam3 (lipopeptide with three fatty acids). In the following 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 DCF dependent fuorescence, and ceramide abundance utilizing specific antibodies. RESULTS: Pam1 (5 µg/ml), Pam2 (5 µg/ml) and Pam3 (1 and 5 µg/ml) significantly increased the percentage of annexin-V-binding to erythrocytes in a dose dependent manner, which was largely independent of Ca2+. Pam1-3 increased the percentage of both, swollen and shrunken erythrocytes without significantly modifying the average forward scatter. They also increased reactive oxygen species (ROS) and ceramide abundance. In all assays the effect on eryptosis increased with increasing number of fatty acids, with Pam3 showing always the strongest effect. In contrast, a comparison of the effect of Pam1-3 on TLR2 dependent immune stimulation showed that not Pam3 but Pam2 displayed the strongest activity, and that immune stimulation was triggered at much lower concentrations than eryptosis. CONCLUSIONS: Lipopeptides are not only important activators of the immune system; at higher concentrations they also drive host cells into apoptosis thus aggravating a bacterial infection.

Stimulation of Suicidal Erythrocyte Death by Ceritinib-Treatment of Human Erythrocytes.

BACKGROUND/AIMS: The anaplastic lymphoma kinase (ALK) inhibitor ceritinib is utilized for the treatment of ALK positive non-small cell lung carcinoma. Side effects of the drug include decrease of blood hemoglobin concentration. Possible causes of anemia include stimulation of suicidal erythrocyte 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), oxidative stress, ceramide, staurosporine sensitive protein kinase C, SB203580 sensitive p38 kinase, D4476 sensitive casein kinase 1, and zVAD sensitive caspases. The present study explored, whether ceritinib induces eryptosis and, if so, to shed light on the cellular mechanisms involved. 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 ceritinib (1 µg/ml) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter, significantly increased Fluo3-fluorescence, but did not significantly modify DCFDA fluorescence or ceramide abundance. The effect of ceritinib on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca2+, by the kinase inhibitors staurosporine (1 µM), SB203580 (2 µM) and D4476 (10 µM), as well as by caspase inhibitor zVAD (10 µM). CONCLUSIONS: Ceritinib triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry, as well as activation of kinases and Caspases.

Triggering of Suicidal Erythrocyte Death by Bexarotene.

BACKGROUND/AIMS: The retinoid X receptor agonist bexarotene is utilized for the treatment of cutaneous T-cell lymphoma and is effective in several further malignancies. The substance counteracts tumor growth in part by triggering suicidal death or apoptosis of tumor cells. Side effects of bexarotene treatment include anemia. Theoretically, bexarotene induced anemia could be secondary to stimulation of suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling potentially stimulating eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), induction of oxidative stress, increase of ceramide abundance, as well as activation of staurosporine sensitive protein kinase C, SB203580 sensitive p38 kinase, D4476 sensitive casein kinase 1, and zVAD sensitive caspases. The present study explored, whether bexarotene induces eryptosis and, if so, whether its effect involves Ca2+ entry, oxidative stress, ceramide, kinases and/or caspases. METHODS: Flow cytometry was employed to quantify 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 DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was estimated from hemoglobin concentration in the supernatant. RESULTS: A 48 hours exposure of human erythrocytes to bexarotene (≥ 0.4 µg/ml) significantly increased the percentage of annexin-V-binding cells without significantly modifying forward scatter. Bexarotene significantly increased Fluo3-fluorescence and DCFDA fluorescence. Bexarotene tended to increase ceramide abundance, an effect, however, not reaching statistical significance. The effect of bexarotene on annexin-V-binding was significantly blunted by removal of extracellular Ca2+ and by addition of D4476 (10 µM), but not by addition of staurosporine (1 µM), SB203580 (2 µM), or zVAD (10 µM). CONCLUSIONS: Bexarotene triggers phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry, oxidative stress, and activation of D4476 sensitive casein kinase.

Stimulation of Suicidal Erythrocyte Death by Tafenoquine.

BACKGROUND/AIMS: The 8-aminoquinoline tafenoquine has been shown to be effective against Plasmodia, Leishmania and Trypanosoma. The substance is at least in part effective by triggering apoptosis of the parasites. Similar to apoptosis, 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 regulation of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, zVAD sensitive caspases, SB203580 sensitive p38 kinase, staurosporine sensitive protein kinase C as well as D4476 sensitive casein kinase. The present study explored, whether tafenoquine induces eryptosis and aimed to possibly identify 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 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) dependent fluorescence, and ceramide abundance utilizing specific antibodies. RESULTS: A 48 hours exposure of human erythrocytes to tafenoquine (500 ng/ml) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter, significantly increased Fluo3-fluorescence, and significantly increased DCFDA fluorescence. Tafenoquine did not significantly modify ceramide abundance. The effect of tafenoquine on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca2+. The effect of tafenoquine on annexin-V-binding was not significantly blunted by zVAD (10 µM), SB203580 (2 µM) or staurosporine (1 µM). The effect of tafenoquine on annexin-V-binding was significantly blunted but not abolished by D4476 (10 µM). CONCLUSIONS: Tafenoquine triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to stimulation of Ca2+ entry, oxidative stress and possibly activation of casein kinase.

Triggering of Suicidal Erythrocyte Death by Psammaplin A.

BACKGROUND/AIMS: Psammaplin A, a natural product isolated from marine sponges, triggers apoptosis of tumor cells and is thus considered for the treatment of malignancy. In analogy to apoptosis of nucleated tumor cells, erythrocytes may enter eryptosis, a suicidal death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Cellular mechanisms stimulating eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and ceramide. The present study explored, whether Psammaplin A induces eryptosis and to possibly shed some light on the underlying mechanisms. METHODS: Phosphatidylserine exposing erythrocytes were identified utilizing annexin-V-binding, cell volume was estimated from forward scatter, [Ca2+]i determined utilizing Fluo3-fluorescence, the abundance of reactive oxygen species (ROS) quantified with DCFDA dependent fluorescence, and ceramide abundance at the erythrocyte surface detected with specific antibodies. RESULTS: A 48 hours exposure of human erythrocytes to Psammaplin A (2-8 µg/ml) significantly decreased forward scatter and significantly increased the percentage of annexin-V-binding cells. Psammaplin A significantly increased Fluo3-fluorescence, the effect of Psammaplin A on annexin-V-binding and forward scatter was, however, not significantly blunted by removal of extracellular Ca2+. Psammaplin A significantly increased DCFDA fluorescence and ceramide abundance. CONCLUSIONS: Psammaplin A triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect paralleled by increase of [Ca2+]i, induction of oxidative stress and enhanced appearance of ceramide.

Enhanced Eryptosis Following Exposure to Dolutegravir.

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.

Stimulating Effect of Terfenadine on Erythrocyte Cell Membrane Scrambling.

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+.

Efavirenz Induced Suicidal Death of Human Erythrocytes.

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.

Enhanced Eryptosis Following Exposure to Lopinavir.

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.