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 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 Sclareol on Suicidal Death of Human Erythrocytes.

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

Micafungin-Induced Suicidal Erythrocyte Death.

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

Ceranib-2-induced suicidal erythrocyte death.

Ceramide is known to trigger apoptosis of nucleated cells and eryptosis of erythrocytes. Eryptosis is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Besides ceramide, stimulators of eryptosis include increase of cytosolic Ca(2+) -activity ([Ca(2+) ]i ) and oxidative stress. Ceramide is degraded by acid ceramidase and inhibition of the enzyme similarly triggers apoptosis. The present study explored, whether ceramidase inhibitor Ceranib-2 induces eryptosis. Flow cytometry was employed to quantify phosphatidylserine-exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca(2+) ]i from Fluo3-fluorescence, reactive oxygen species (ROS) from DCF dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was estimated from hemoglobin concentration in the supernatant. A 48 h exposure of human erythrocytes to Ceranib-2 significantly increased the percentage of annexin-V-binding cells (≥50 µM) and the percentage of hemolytic cells (≥10 µM) without significantly modifying forward scatter. Ceranib-2 significantly increased Fluo3-fluorescence, DCF fluorescence and ceramide abundance. The effect of Ceranib-2 on annexin-V-binding was not significantly blunted by removal of extracellular Ca(2+) . Ceranib-2 triggers phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to increase of ceramide abundance and induction of oxidative stress, but not dependent on Ca(2+) entry. Copyright © 2016 John Wiley & Sons, Ltd.

Stimulation of Eryptosis, the Suicidal Erythrocyte Death by Piceatannol.

BACKGROUND/AIMS: Piceatannol, an analog and metabolite of resveratrol, is effective against various disorders including malignancy. It is in part effective by triggering suicidal death or apoptosis of tumor cells. Cellular mechanisms mediating the proapoptotic effect of Piceatannol include mitochondrial depolarization and cytochrome c release. Erythrocytes lack mitochondria but may nevertheless enter suicidal 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 and ceramide formation. The present study explored, whether Piceatannol induces eryptosis and, if so, to shed some 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, reactive oxygen species (ROS) formation from 2',7'-dichlorodihydrofluorescein (DCF) diacetate-dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemoglobin concentration in the supernatant was taken as measure of hemolysis. RESULTS: A 48 hours exposure of human erythrocytes to Piceatannol (10 - 20 µM) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter, significantly increased DCFDA-fluorescence, significantly increased ceramide abundance, but did not significantly increase Fluo3-fluorescence. Removal of extracellular Ca2+ slightly blunted but did not abolish the effect of Piceatannol on annexin-V-binding and forward scatter. Piceatannol (20 µM) significantly augmented the increase of annexin-V-binding, but significantly blunted the decrease of forward scatter following treatment with the Ca2+ ionophore ionomycin. CONCLUSIONS: Piceatannol triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part downstream of Ca2+ and involving oxidative stress and ceramide formation.

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+.

Stimulating Effect of Elvitegravir on Suicidal Erythrocyte Death.

BACKGROUND/AIMS: The antiviral drug Elvitegravir is used for the treatment of Human Immunodeficiency Virus (HIV) infections. The present study explored whether the drug is able to trigger eryptosis, the suicidal death of erythrocytes. Eryptosis is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, activated p38 kinase and activated caspases. The present study explored, whether Elvitegravir induces eryptosis and, if so, to shed light on the mechanisms involved. METHODS: Phosphatidylserine abundance at the erythrocyte surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, abundance of reactive oxygen species (ROS) from DCFDA dependent fluorescence, and ceramide abundance at the erythrocyte surface utilizing specific antibodies. RESULTS: A 48 hours exposure of human erythrocytes to Elvitegravir (≥ 1.5 µg/ml) significantly increased the percentage of annexin-V-binding cells, and significantly decreased forward scatter. Elvitegravir (2.5 µg/ml) significantly increased Fluo3-fluorescence, but did not significantly modify DCFDA fluorescence or ceramide abundance. The effect of Elvitegravir on annexin-V-binding was significantly blunted by removal of extracellular Ca2+, but not in the presence of p38 kinase inhibitor SB203580 (2 µM) or in the presence of pancaspase inhibitor zVAD (10 µM). CONCLUSIONS: Elvitegravir triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to entry of extracellular Ca2+.

Stimulation of Eryptosis by Combretastatin A4 Phosphate Disodium (CA4P).

BACKGROUND/AIMS: Combretastatin A4 phosphate disodium (CA4P) is utilized for the treatment of malignancy. The substance has previously been shown to trigger suicidal cell death or apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, 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), ceramide, oxidative stress and ATP depletion. The present study explored, whether CA4P induces eryptosis and, if so, to gain insight into mechanisms involved. METHODS: Flow cytometry has been 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 DCF fluorescence, glutathione (GSH) abundance from CMF fluorescence and ceramide abundance from fluorescent antibodies. In addition cytosolic ATP levels were quantified utilizing a luciferin-luciferase-based assay and hemolysis was estimated from hemoglobin concentration in the supernatant. RESULTS: A 48 hours exposure of human erythrocytes to CA4P (≥ 50 µM) significantly increased the percentage of annexin-V-binding cells and significantly decreased forward scatter. CA4P did not appreciably increase hemolysis. Hundred µM CA4P significantly increased Fluo3-fluorescence. The effect of CA4P (100 µM) on annexin-V-binding was significantly blunted, but not abolished, by removal of extracellular Ca2+. CA4P (≥ 50 µM) significantly decreased GSH abundance and ATP levels but did not significantly increase ROS or ceramide. CONCLUSIONS: CA4P triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to entry of extracellular Ca2+ and energy depletion.

Triggering of Suicidal Erythrocyte Death by Pazopanib.

BACKGROUND/AIMS: The multi-targeted kinase inhibitor pazopanib, a drug employed for the treatment of a wide variety of malignancies, has previously been shown to trigger apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Mechanisms involved in the triggering of eryptosis include Ca2+ entry, oxidative stress and ceramide. The present study explored, whether pazopanib induces eryptosis and, if so, whether it is effective by Ca2+ entry, oxidative stress and/or ceramide. METHODS: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, reactive oxygen species (ROS) formation from DCF dependent fluorescence, and ceramide abundance utilizing specific antibodies. RESULTS: A 48 hours exposure of human erythrocytes to pazopanib significantly increased the percentage of annexin-V-binding (≥ 25 µg/ml) and of shrunken erythrocytes (≥ 50 µg/ml). Pazopanib treatment further resulted in significant hemolysis (≥ 25 µg/ml). The effect of pazopanib on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca2+. Pazopanib significantly increased DCF fluorescence (50 µg/ml) and ceramide abundance (50 µg/ml). CONCLUSIONS: Pazopanib triggers eryptosis, an effect involving Ca2+ entry, oxidative stress and ceramide.

Nocodazole Induced Suicidal Death of Human Erythrocytes.

BACKGROUND: The microtubule assembly inhibitor nocodazole has been shown to trigger caspase-independent mitotic death and caspase dependent apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may undergo eryptosis, the suicidal erythrocyte death 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 and ceramide. The present study explored, whether and how nocodazole induces eryptosis. METHODS: Flow cytometry was employed to determine phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, the abundance of reactive oxygen species (ROS) from 2',7'-dichlorodihydrofluorescein (DCF) diacetate dependent fluorescence as well as ceramide surface abundance utilizing specific antibodies. Tubulin abundance was quantified by TubulinTracker™ Green reagent and visualized by confocal microscopy. RESULTS: A 48 hours exposure of human erythrocytes to nocodazole (≥ 30 µg/ml) significantly increased the percentage of annexin-V-binding cells without significantly modifying average forward scatter. Nocodazole significantly increased Fluo3-fluorescence, significantly increased DCF fluorescence and significantly increased ceramide surface abundance. The effect of nocodazole on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+ and was not modified in the presence of Caspase 3 inhibitor zVAD (1 µM). Nocodazole treatment reduced the content of total tubulin. CONCLUSIONS: Nocodazole triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to stimulation of Ca2+ entry, oxidative stress and ceramide.

Fucoxanthin Induced Suicidal Death of Human Erythrocytes.

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

Edelfosine Induced Suicidal Death of Human Erythrocytes.

BACKGROUND/AIMS: The anti-inflammatory, anti-autoimmune, antiparasitic, and anti-viral ether phospholipid edelfosine (1-O-octadecyl-2-O-methylglycero-3-phosphocholine) stimulates 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 phospholipid scrambling of the cell membrane with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include Ca2+ entry with increase of cytosolic Ca2+ activity ([Ca2+]i) and oxidative stress. The present study explored, whether and how edelfosine induces eryptosis. METHODS: Flow cytometry and photometry, respectively, were employed to estimate phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, hemolysis from hemoglobin release, [Ca2+]i from Fluo3-fluorescence, and abundance of reactive oxygen species (ROS) from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence. RESULTS: A 6 hours exposure of human erythrocytes to edelfosine (5 µM) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter, and significantly increased Fluo3-fluorescence, but did not significantly modify DCFDA fluorescence. The effect of edelfosine on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+. CONCLUSIONS: Edelfosine triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to stimulation of Ca2+ entry.

Dictyostelium Nramp1, which is structurally and functionally similar to mammalian DMT1 transporter, mediates phagosomal iron efflux.

The Nramp (Slc11) protein family is widespread in bacteria and eukaryotes, and mediates transport of divalent metals across cellular membranes. The social amoeba Dictyostelium discoideum has two Nramp proteins. Nramp1, like its mammalian ortholog (SLC11A1), is recruited to phagosomal and macropinosomal membranes, and confers resistance to pathogenic bacteria. Nramp2 is located exclusively in the contractile vacuole membrane and controls, synergistically with Nramp1, iron homeostasis. It has long been debated whether mammalian Nramp1 mediates iron import or export from phagosomes. By selectively loading the iron-chelating fluorochrome calcein in macropinosomes, we show that Dictyostelium Nramp1 mediates iron efflux from macropinosomes in vivo. To gain insight in ion selectivity and the transport mechanism, the proteins were expressed in Xenopus oocytes. Using a novel assay with calcein, and electrophysiological and radiochemical assays, we show that Nramp1, similar to rat DMT1 (also known as SLC11A2), transports Fe(2+) and manganese, not Fe(3+) or copper. Metal ion transport is electrogenic and proton dependent. By contrast, Nramp2 transports only Fe(2+) in a non-electrogenic and proton-independent way. These differences reflect evolutionary divergence of the prototypical Nramp2 protein sequence compared to the archetypical Nramp1 and DMT1 proteins.