Background/Aims: The cytotoxic drug Treosulfan is clinically used for the treatment of malignancy. A common side effect of Treosulfan treatment is anemia. Treosulfan is at least partially effective by triggering apoptosis of tumor cells. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, a suicidal death characterized by cell shrinkage and translocation of phosphatidylserine from the inner to the outer leaflet of the plasma membrane. Triggers of eryptosis include oxidative stress, Ca²⁺-entry and increase of cytosolic Ca²⁺-activity ([Ca²⁺]_i). The present study explored whether Treosulfan stimulates eryptosis, which may contribute to development of anemia. Methods: Erythrocyte volume was estimated from forward scatter, phosphatidylserine abundance at the erythrocyte surface from Fluorescein isothiocyanate (FITC)-annexin-V-binding, [Ca²⁺]_i from Fluo3 fluorescence and reactive oxygen species (ROS) from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA)-fluorescence. Results: A 48 hours exposure of human erythrocytes to Treosulfan (800 µg/ml) significantly decreased erythrocyte forward scatter, increased the percentage of annexin-V-binding cells, increased [Ca²⁺]_i, and increased ROS. The effect of Treosulfan on annexin-V-binding was virtually abrogated by removal of extracellular Ca²⁺. Conclusion: Treosulfan stimulates suicidal erythrocyte death or eryptosis at least in part by inducing oxidative stress and stimulating Ca²⁺ entry.

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.

Background/Aims: The novel antifungal drug Anidulafungin is used for the treatment of diverse fungal infections including candidiasis and aspergillosis. The traditional antifungal drug amphotericin B has previously been shown to trigger 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. Inhibitors of eryptosis include nitric oxide (NO). The present study explored, whether Anidulafungin 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 Anidulafungin (1.5 - 6 µg/ml) significantly increased hemolysis and the percentage of annexin-V-binding cells, and significantly decreased forward scatter. Anidulafungin (6 µg/ml) slightly, but significantly inceased Fluo3-fluorescence and the effect of Anidulafungin on annexin-V-binding was slightly, but significantly blunted by removal of extracellular Ca²⁺. The effect of Anidulafungin on annexin-V-binding was further significantly blunted by the p38 kinase inhibitor SB203580 (2 µM) and NO donor nitroprusside (1 µM). An increase of extracellular K⁺ concentration significantly blunted the effect of Anidulafungin on cell volume but not on annexin-V-binding. Anidulafungin rather decreased DCFDA fluorescence and the effect of Anidulafungin on annexin-V-binding was not significantly blunted by the antioxidant N-acetylcysteine (1 mM). Moreover, the effect of Anidulafungin on annexin-V-binding was not paralleled by significant increase of ceramide abundance and was not significantly blunted by PKC inhibitor staurosporine (1 µM), casein kinase 1α inhibitor D4476 (10 µM) or pancaspase inhibitor zVAD (10 µM). Conclusions: Anidulafungin triggers hemolysis and eryptosis with cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to Ca²⁺ entry and activation of p38 kinase.

Background/Aims: The echinocandin antifungal agent caspofungin has been shown to trigger apoptosis of fungal cells. Beyond that, caspofungin is toxic for host mitochondria. Even though lacking mitochondria, erythrocytes may enter apoptosis-like suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in triggering of eryptosis include increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i), oxidative stress, ceramide, caspase activation and/or activation of p38 kinase, protein kinase C, and casein kinase. The present study explored, whether caspofungin induces eryptosis and, if so, to shed some light on the cellular mechanisms involved. Methods: Flow cytometry was employed to determine phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo3-fluorescence, ROS formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was quantified from hemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to caspofungin (≥ 30 µg/ml) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter, significantly enhanced hemolysis, but did not significantly increase Fluo3-fluorescence, DCFDA fluorescence or ceramide abundance. The effect of caspofungin on annexin-V-binding was not significantly blunted by removal of extracellular Ca²⁺, by inhibition of caspases with pancaspase inhibitor zVAD (10 µM), or by addition of the antioxidant N-acetyl-cysteine (1 mM), p38 kinase inhibitor SB203580 (2 µM) or protein kinase C inhibitor staurosporine (1 µM). The effect of caspofungin on annexin-V-binding was, however, significantly blunted in the presence of casein kinase inhibitor D4476 (10 µM). Conclusions: Caspofungin triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect possibly involving activation of casein kinase.

The ionophore antibiotic nonactin permeabilizes cell membranes to NH þ 4 and K + . Treatment of erythrocytes with nonactin is expected to trigger cellular K + loss with subsequent cell shrinkage, which in turn is known to trigger suicidal death of a wide variety of cells including erythrocytes. This study explored whether nonactin exposure induces eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and translocation of cell membrane phosphatidylserine to the erythrocyte surface. Signalling of eryptosis includes increase in cytosolic Ca 2+ activity [(Ca 2+ ) i ] and stimulation of protein kinase C (PKC) as well as p38 mitogen-activated protein kinase. Phosphatidylserine abundance at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter (FSC) and (Ca 2+ ) i from Fluo3-fluorescence. A 48-hr treatment of human erythrocytes with nonactin significantly decreased FSC (≥10 ng/ml) and significantly increased the percentage of annexin-V-binding cells (≥10 ng/ml), effects paralleled by increase in (Ca 2+ ) i (≥50 ng/ml) and virtually abrogated by increase in extracellular K + concentration to 120 mM at the expense of Na + . The up-regulation of annexin-V-binding after nonactin treatment was significantly blunted but not abolished by the removal of extracellular Ca 2+ and by addition of either PKC inhibitor staurosporine (0.4 lM) or p38 kinase inhibitor SB203580 (2 lM). In conclusion, exposure of erythrocytes to the K + ionophore nonactin induces erythrocyte shrinkage and subsequent erythrocyte membrane scrambling, effects involving cellular K + loss, Ca 2+ entry and activation of staurosporine as well as SB203580-sensitive kinases.Nonactin is the parent compound of macrotetrolides, a group of ionophore antibiotics produced by This study explored whether nonactin stimulates eryptosis and, if so, whether the stimulation of eryptosis by nonactin requires cellular K + loss. To this end, erythrocytes from healthy volunteers were exposed to nonactin at low and high extracellular K + concentrations and phosphatidylserine abundance at the erythrocyte surface as well as cell volume determined. To gain insight into the signalling involved, (Ca 2+ ) i was determined and experiments were performed in the absence of extracellular Ca 2+ as well as in the absence or presence of PKC inhibitor staurosporine or p38 kinase inhibitor SB203580. Materials and MethodsErythrocytes, solutions and chemicals. Fresh lithium-heparinanticoagulated blood samples were kindly provided by the blood bank of the University of T€ ubingen. The study was approved by the ethics committee of the University of T€ ubingen (184/2003 V). The blood was centrifuged at 120 9 g for 20 min. at 20°C and the platelets and leucocytes-containing supernatant was disposed. Erythrocytes were incubated in vitro for 48 hr at a haematocrit of 0.4% in Ringer solution containing (in mM) 125 NaCl, 5 KCl, 1 MgSO 4 , 32 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 5 glucose and 1 CaCl 2 ; the pH was adjuste...