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Background/Aims: The 3-hydroxy-3-methyl-glutaryl-Coenzyme A (HMG-CoA) reductase inhibitor simvastatin has been shown to trigger apoptosis of several cell types. The substance has thus been proposed as an additional treatment of malignancy. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal erythrocyte death. Hallmarks of eryptosis include cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the extracellular face of the erythrocyte cell membrane. Signaling contributing to stimulation of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), induction of oxidative stress, increase of ceramide abundance, and activation of SB203580-sensitive p38 kinase. The present study explored, whether simvastatin induces eryptosis and aimed to shed light on cellular mechanisms 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 DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was estimated from hemoglobin concentration in the supernatant. Results: A 48 h exposure of human erythrocytes to simvastatin (1 μg/ml) significantly decreased the forward scatter, significantly augmented the percentage of annexin-V-binding cells, significantly increased Fluo3-fluorescence, and significantly enhanced DCFDA fluorescence. Simvastatin tended to increase ceramide abundance, an effect, however, escaping statistical significance. The effect of simvastatin on annexin-V-binding was significantly blunted by removal of extracellular Ca2+ and by addition of SB203580 (2 μM). Conclusions: Simvastatin stimulates eryptosis, an effect at least in part due to Ca2+ entry, oxidative stress, and p38 kinase. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background/Aims: The neurodegenerative disease Chorea-Acanthocytosis (ChAc) is caused by loss-of-function-mutations of the chorein-encoding gene VPS13A. In ChAc neurons transcript levels and protein abundance of Ca2+ release activated channel moiety (CRAC) Orai1 as well as its regulator STIM1/2 are decreased, resulting in blunted store operated Ca2+-entry (SOCE) and enhanced suicidal cell death. SOCE is up-regulated and cell death decreased by lithium. The effects of lithium are paralleled by upregulation of serum & glucocorticoid inducible kinase SGK1 and abrogated by pharmacological SGK1 inhibition. In other cell types SGK1 has been shown to be partially effective by upregulation of NFκB, a transcription factor stimulating the expression of Orai1 and STIM. The present study explored whether pharmacological inhibition of NFκB interferes with Orai1/STIM1/2 expression and SOCE and their upregulation by lithium in ChAc neurons. Methods: Cortical neurons were differentiated from induced pluripotent stem cells generated from fibroblasts of ChAc patients and healthy volunteers. Orai1 and STIM1 transcript levels and protein abundance were estimated from qRT-PCR and Western blotting, respectively, cytosolic Ca2+-activity ([Ca2+]i) from Fura-2-fluorescence, SOCE from increase of [Ca2+]i following Ca2+ re-addition after Ca2+-store depletion with sarco-endoplasmatic Ca2+-ATPase inhibitor thapsigargin (1µM), as well as CRAC current utilizing whole cell patch clamp recording. Results: Orai1 and STIM1 transcript levels and protein abundance as well as SOCE and CRAC current were significantly enhanced by lithium treatment (2 mM, 24 hours). These effects were reversed by NFκB inhibitor wogonin (50 µM). Conclusion: The stimulation of expression and function of Orai1/STIM1/2 by lithium in ChAc neurons are disrupted by pharmacological NFκB inhibition. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background/Aims: The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor afatinib is used for the treatment of several malignancies. Afatinib is at least partially effective by triggering apoptosis of tumor cells. Platelets may similarly undergo apoptosis, which is characterized by caspase 3 activation, cell shrinkage and phosphatidylserine translocation. However, an effect of afatinib on platelets has never been reported. The present study explored whether treatment of platelets with afatinib modifies platelet activation and apoptosis in the absence and presence of platelet activators thrombin or collagen related peptide (CRP). Methods: Platelets isolated from wild-type mice were exposed for 30 minutes to afatinib (18 μg/ml) without or with subsequent treatment with thrombin (0.005 U/ml or 0.01 U/ml) or CRP (2 μg/ml or 5 μg/ml). Flow cytometry was employed to estimate Orai1 abundance at the platelet surface with specific antibodies, cytosolic Ca2+-activity ([Ca2+]i) from Fluo-3 fluorescence, platelet degranulation from P-selectin abundance, integrin activation from αIIbβ3 integrin abundance, caspase activity utilizing an Active Caspase-3 Staining kit, phosphatidylserine abundance from annexin-V-binding, platelet volume from forward scatter and aggregation utilizing staining with CD9-APC and CD9-PE. Results: In the absence of thrombin and CRP, the administration of afatinib (18 μg/ml) slightly, but significantly, increased [Ca2+]i and annexin-V-binding, but did not significantly modify Orai1 abundance, P-selectin abundance, activated αIIbβ3 integrin, cell volume, caspase activity and aggregation. Exposure of platelets to 0.005 U/ml or 0.01 U/ml thrombin or 2 μg/ml or 5 μg/ml CRP was followed by a significant increase of Orai1 abundance, increase of [Ca2+]i, P-selectin abundance, αIIbβ3 integrin activity, annexin-V-binding, caspase activity, and aggregation, as well as a significant decrease of forward scatter, all effects significantly blunted (thrombin) or virtually abolished (CRP) by afatinib. Conclusions: Afatinib is a powerful inhibitor of platelet activation, platelet apoptosis and platelet aggregation. [ABSTRACT FROM AUTHOR]

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-Vbinding, 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. [ABSTRACT FROM AUTHOR]

Background/Aims: The anaplastic lymphoma kinase (ALK) inhibitor ASP3026 is in clinical development for the treatment of ALK expressing non-small cell lung carcinoma (NSCLC). ASP3026 is in part effective by inducing apoptosis of tumor cells. Erythrocytes lack mitochondria and nuclei, key organelles in the execution of apoptosis, but are nevertheless able to enter suicidal death or eryptosis, which is characterized by cell membrane scrambling with phosphatidylserine translocation to the cell surface and by cell shrinkage. Eryptosis is triggered by cell stress, such as energy depletion, hyperosmotic shock, oxidative stress and excessive increase of cytosolic Ca2+ activity ([Ca2+]i). The present study explored, whether ASP3026 impacts on eryptosis. Methods: Human erythrocytes have been exposed to energy depletion (glucose withdrawal for 48 hours), oxidative stress (addition of 0.3 mM tert-butylhydroperoxide [tBOOH] for 50 min) or Ca2+ loading with Ca2+ ionophore ionomycin (1 μM for 60 min) in absence and presence of ASP3026 (1-4 μg/ml). Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, and cell volume from forward scatter. Results: Treatment with ASP3026 alone did not significantly modify annexin- V-binding or forward scatter. Energy depletion, oxidative stress and ionomycin, all markedly and significantly increased the percentage of annexin-V-binding erythrocytes, and decreased the forward scatter. ASP3026 significantly blunted the effect of energy depletion and oxidative stress, but not of ionomycin on annexin-V-binding. ASP3026 did not significantly influence the effect of any maneuver on forward scatter. Conclusions: ASP3026 is a novel inhibitor of erythrocyte cell membrane scrambling following energy depletion and oxidative stress. [ABSTRACT FROM AUTHOR]

Background/Aims: The mammalian target of rapamycin (mTOR) inhibitor temsirolimus is utilized for the treatment of malignancy. Temsirolimus is at least in part effective by triggering suicidal tumor cell death. The most common side effect of temsirolimus treatment is anemia. At least in theory, the anemia following temsirolimus treatment could result from stimulation of eryptosis, the suicidal erythrocyte death. Hallmarks of eryptosis include cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the orchestration of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, as well as activation of staurosporine and chelerythrine sensitive protein kinase C, SB203580 sensitive p38 kinase, D4476 sensitive casein kinase 1, and zVAD sensitive caspases. The purpose of the present study was to test whether temsirolimus influences eryptosis and, if so, to shed light on the signaling involved. Methods: Flow cytometry was employed to estimate cell volume from forward scatter, phosphatidylserine exposure at the cell surface from annexin-V-binding, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) abundance from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was determined from hemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to temsirolimus (5 - 20 μg/ml) significantly decreased forward scatter and significantly increased the percentage of annexin-V-binding cells. Temsirolimus significantly increased Fluo3-fluorescence, DCFDA fluorescence and ceramide abundance at the erythrocyte surface. The effect of temsirolimus on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca2+ and by addition of staurosporine (1 μM) or chelerythrine (10 μM) but not significantly modified by addition of SB203580 (2 μM), D4476 (10 μM), or zVAD (10 μM). Chelerythrine (10 μM) further significantly blunted the effect of temsirolimus on DCFDA fluorescence but not ceramide formation. Removal of extracellular Ca2+ had no effect on temsirolimus induced ROS formation or ceramide abundance. Conclusions: Temsirolimus triggers eryptosis with cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry, oxidative stress, ceramide and activation of staurosporine/ Chelerythrine sensitive kinase(s). [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background/Aims: The endogenous retinoid 9-cis-retinoic acid has previously been shown to trigger apoptosis in a wide variety of cells including several tumor cells and has thus been suggested for the treatment of malignancy. Similar to apoptosis of nucleated cells, 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. Cellular mechanisms participating in the accomplishment of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i) and formation of ceramide. The present study explored, whether 9-cis-retinoic acid induces eryptosis and whether the effect involves Ca2+ and/or ceramide. Methods: 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, and ceramide abundance utilizing specific antibodies. Hemolysis was quantified from hemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to 9-cis-retinoic acid (⩾ 0.5 μg/ml) significantly increased the percentage of annexin-V-binding cells and significantly decreased forward scatter. Exposure to 9-cis-retinoic acid (⩾ 0.5 μg/ml) significantly increased Fluo3-fluorescence, and the effect of 9-cis-retinoic acid on annexin-V-binding was significantly blunted by removal of extracellular Ca2+. Exposure to 9-cis-retinoic acid (1 μg/ml) further significantly increased the ceramide abundance at the erythrocyte surface and significantly increased hemolysis. Conclusions: 9-cis-retinoic acid triggers phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part downstream of Ca2+ and ceramide. [ABSTRACT FROM AUTHOR]

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 fluorescence, 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. [ABSTRACT FROM AUTHOR]

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. Moreover, tafenoquine has been shown to trigger eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. The effect of tafenoquine on eryptosis is in part due to stimulation of Ca2+ entry and oxidative stress. Ca2+ entry is a critical event in the activation of blood platelets by thrombin and collagen related peptide (CRP). The present study explored, whether tafenoquine influences Ca2+ entry, activation and apoptosis of blood platelets. Methods: Platelets isolated from wild-type mice were exposed for 30 minutes to tafenoquine (2.5 μg/ml) without or with an additional treatment with thrombin (0.01 U/ml) or CRP (2 μg/ml or 5 μg/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 αIIbβ3 integrin abundance, phosphatidylserine abundance from annexin-V-binding, relative platelet volume from forward scatter, reactive oxygen species (ROS) from DCF fluorescence, caspase 3 activity with an active caspase-3 Staining kit, and aggregation utilizing staining with CD9-APC and CD9-PE. Results: Both, thrombin (0.01 U/ml) and CRP (2 μg/ml or 5 μg/ml), significantly increased [Ca2+]i, P-selectin abundance, active αIIbβ3 integrin, and annexin-V-binding, and both significantly decreased platelet volume, activated caspase 3 and stimulated aggregation. Administration of tafenoquine (2.5 μg/ml, 30 min) significantly decreased [Ca2+]i both, in the absence and presence of thrombin and CRP. Tafenoquine significantly blunted the effect of thrombin and CRP on [Ca2+]i, P-selectin abundance, and active αIIbβ3 integrin, but significantly increased ROS and annexin-V-binding, significantly augmented the effect of thrombin on caspase 3 activity and platelet volume and significantly enhanced platelet aggregation. Conclusions: Tafenoquine counteracts thrombin and CRP induced increase of cytosolic Ca2+ activity and platelet activation, but enhances platelet apoptosis and platelet aggregation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 memb [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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-Vbinding 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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+. [ABSTRACT FROM AUTHOR]

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+. [ABSTRACT FROM AUTHOR]

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 1a 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 Mg/ml) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter (2 Mg/ml), significantly increased Fluo3-fluorescence (≥ 2 Mg/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 MM) and casein kinase 1α inhibitor D4476 (10 MM), but not by cyclooxygenase inhibitor aspirin (50 MM). 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. [ABSTRACT FROM AUTHOR]

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 Mg/ml), significantly decreased forward scatter (>15 Mg/ml), significantly increased hemolysis (≥ 15 Mg/ml), significantly increased Fluo3- fluorescence (20 Mg/ml), and significantly increased DCFDA fluorescence (20 Mg/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. [ABSTRACT FROM AUTHOR]