Triggering of Eryptosis, the Suicidal Erythrocyte Death, by Perifosine.

BACKGROUND/AIMS: The alkylphospholipid perifosine is used for the treatment of malignancy. The substance is effective by triggering suicidal tumor cell death or apoptosis. Side effects of perifosine include anemia. At least in theory, perifosine-induced anemia could result from stimulation of suicidal erythrocyte death or eryptosis. Hallmarks of eryptosis are cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Cellular mechanisms participating in the orchestration of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, increase of ceramide abundance, as well as activation of staurosporine sensitive protein kinase C and/or of SB203580 sensitive p38 kinase. The present study explored, whether perifosine induces eryptosis and, if so, whether its effect involves and/or requires Ca2+ entry, oxidative stress, ceramide and kinase activation. 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 24 hours exposure of human erythrocytes to perifosine (2.5 µg/ml) significantly increased the percentage of annexin-V-binding cells, significantly decreased average forward scatter, significantly increased the percentage of shrunken erythrocytes, and significantly decreased the percentage of swollen erythrocytes. Perifosine significantly increased the percentage of hemolytic erythrocytes. Perifosine significantly increased Fluo3-fluorescence, but decreased DCFDA fluorescence and ceramide abundance. The effect of perifosine on annexin-V-binding was significantly blunted by removal of extracellular Ca2+ and by addition of staurosporine (1 µM), but not by addition of SB203580 (2 µM). CONCLUSIONS: Perifosine triggers eryptosis, an effect at least in part due to Ca2+ entry and activation of staurosporine sensitive kinases.

Stimulating Effect of Manumycin A on Suicidal Erythrocyte Death.

BACKGROUND/AIMS: The streptomycete derived farnesyltransferase inhibitor Manumycin A triggers apoptosis of tumor cells and is thus considered for the treatment of malignancy. The present study explored whether Manumycin A could similarly stimulate eryptosis, the suicidal death of erythrocytes characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include Ca2+ entry as well as activation of staurosporine sensitive protein kinase C and SB203580 sensitive p38 kinase. The present study explored, whether Manumycin A induces eryptosis and, if so, to shed some light on the mechanisms involved. METHODS: Phosphatidylserine abundance at the human erythrocyte surface was estimated from annexin-V-binding, cell volume from forward scatter, and hemolysis from hemoglobin concentration in the supernatant. RESULTS: A 48 hours exposure of human erythrocytes to Manumycin A (≥ 5 µg/ml) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter and significantly incrased hemolysis. The effect of Manumycin A on annexin-V-binding was significantly blunted by removal of extracellular Ca2+, by addition of staurosporine (1 µM) and by addition of SB203580 (2 µM). CONCLUSIONS: Manumycin A triggers hemolysis, cell shrinkage and phospholipid scrambling of the human erythrocyte cell membrane. The effect on cell membrane scrambling was in part but not fully dependent on entry of extracellular Ca2+, as well as activity of staurosporine and SB203580 sensitive kinases.

Licochalcone A Induced Suicidal Death of Human Erythrocytes.

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

Lapatinib Induced Suicidal Death of Human Erythrocytes.

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

Enhanced eryptosis following auranofin exposure.

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

Induction of Suicidal Erythrocyte Death by Cantharidin.

The natural phosphoprotein phosphatase inhibitor cantharidin, primarily used for topical treatment of warts, has later been shown to trigger tumor cell apoptosis and is thus considered for the treatment of malignancy. Similar to apoptosis of tumor cells, erythrocytes may undergo eryptosis, a suicidal cell death characterized by cell shrinkage and translocation of cell membrane phosphatidylserine to the erythrocyte surface. Signaling of eryptosis includes increase of cytosolic Ca2+-activity ([Ca2+]i), ceramide, oxidative stress and dysregulation of several kinases. Phosphatidylserine abundance at the erythrocyte surface was quantified utilizing annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ceramide from antibody binding, and reactive oxidant species (ROS) from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence. A 48 h treatment of human erythrocytes with cantharidin significantly increased the percentage of annexin-V-binding cells (≥10 mg/mL), significantly decreased forward scatter (≥25 mg/mL), significantly increased [Ca2+]i (≥25 mg/mL), but did not significantly modify ceramide abundance or ROS. The up-regulation of annexin-V-binding following cantharidin treatment was not significantly blunted by removal of extracellular Ca2+ but was abolished by kinase inhibitor staurosporine (1 mM) and slightly decreased by p38 inhibitor skepinone (2 mM). Exposure of erythrocytes to cantharidin triggers suicidal erythrocyte death with erythrocyte shrinkage and erythrocyte membrane scrambling, an effect sensitive to kinase inhibitors staurosporine and skepinone.

Triggering of programmed erythrocyte death by alantolactone.

The sesquiterpene alantolactone counteracts malignancy, an effect at least in part due to stimulation of suicidal death or apoptosis of tumor cells. Signaling of alantolactone induced apoptosis involves altered gene expression and mitochondrial depolarization. Erythrocytes lack mitochondria and nuclei but may enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the erythrocyte surface. Cellular mechanisms involved in triggering of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i) and oxidative stress. The present study explored, whether alantolactone stimulates eryptosis. To this end, erythrocyte volume was estimated from forward scatter, phosphatidylserine-exposure at the erythrocyte surface from FITC-annexin-V-binding, [Ca2+]i from Fluo3-fluorescence, ceramide abundance from binding of fluorescent antibodies, and oxidative stress from 2',7'-dichlorodihydrofluorescein-diacetate (DCFDA) fluorescence. As a result, a 48 h exposure of human erythrocytes to alantolactone (≥20 µM) significantly decreased erythrocyte forward scatter and increased the percentage of annexin-V-binding cells. Alantolactone significantly increased Fluo3 fluorescence (60 µM), ceramide abundance (60 µM) and DCFDA fluorescence (≥40 µM). The effect of alantolactone (60 µM) on annexin-V-binding was not significantly modified by removal of extracellular Ca2+. In conclusion, alantolactone stimulates suicidal erythrocyte death or eryptosis, an effect paralleled by increase of [Ca2+]i, ceramide abundance and oxidative stress.