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// Rosi Bissinger 1, * , Carla Schumacher 2, * , Syed M. Qadri 3 , Sabina Honisch 1 , Abaid Malik 1 , Friedrich Gotz 4 , Hans-Georg Kopp 2, # , Florian Lang 1, # 1 Department of Physiology, University of Tubingen, Tubingen, Germany 2 Department of Internal Medicine, University of Tubingen, Tubingen, Germany 3 Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Canada 4 Department of Microbial Genetics, University of Tubingen, Tubingen, Germany * These authors have contributed equally and thus share first authorship # These authors have contributed equally and thus share last authorship Correspondence to: Florian Lang, e-mail: florian.lang@uni-tuebingen.de Keywords: anemia, eryptosis, ceramide, cell shrinkage, carcinoma Received: November 16, 2015 Accepted: January 29, 2016 Published: February 9, 2016 ABSTRACT Objectives: Anemia is a common complication of malignancy, which could result from either compromised erythropoiesis or decreased lifespan of circulating erythrocytes. Premature suicidal erythrocyte death, characterized by cell shrinkage and phosphatidylserine (PS) externalization, decreases erythrocyte lifespan and could thus cause anemia. Here, we explored whether accelerated eryptosis participates in the pathophysiology of anemia associated with lung cancer (LC) and its treatment. Methods: Erythrocytes were drawn from healthy volunteers and LC patients with and without cytostatic treatment. PS exposure (annexin V-binding), cell volume (forward scatter), cytosolic Ca 2+ (Fluo3 fluorescence), reactive oxygen species (ROS) production (DCFDA fluorescence) and ceramide formation (anti-ceramide antibody) were determined by flow cytometry. Results: Hemoglobin concentration and hematocrit were significantly lower in LC patients as compared to healthy controls, even though reticulocyte number was higher in LC (3.0±0.6%) than in controls (1.4±0.2%). The percentage of PS-exposing erythrocytes was significantly higher in LC patients with (1.4±0.1%) and without (1.2±0.3%) cytostatic treatment as compared to healthy controls (0.6±0.1%). Erythrocyte ROS production and ceramide abundance, but not Fluo3 fluorescence, were significantly higher in freshly drawn erythrocytes from LC patients than in freshly drawn erythrocytes from healthy controls. PS exposure of erythrocytes drawn from healthy volunteers was significantly more pronounced following incubation in plasma from LC patients than following incubation in plasma from healthy controls. Conclusion: Anemia in LC patients with and without cytostatic treatment is paralleled by increased eryptosis, which is triggered, at least in part, by increased oxidative stress and ceramide formation.

The K(+),H(+) ionophore and antibiotic nigericin has been shown to trigger apoptosis and is thus considered for the treatment of malignancy. Cellular mechanisms involved include induction of oxidative stress, which is known to activate erythrocytic Ca(2+)-permeable unselective cation channels leading to Ca(2+) entry, increase in cytosolic Ca(2+) activity ([Ca(2+)]i) and subsequent stimulation of eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. This study explored whether and how nigericin induces eryptosis. Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, [Ca(2+)]i from Fluo3 fluorescence, pHi from BCECF fluorescence, ceramide abundance utilizing antibodies and reactive oxygen species (ROS) formation from DCFDA-dependent fluorescence. A 48-hr exposure of human erythrocytes to nigericin significantly increased the percentage of annexin-V-binding cells (0.1-10 nM), significantly decreased forward scatter (0.1-1 nM), significantly decreased cytosolic pH (0.1-1 nM) and significantly increased Fluo3 fluorescence (0.1-10 nM). Nigericin (1 nM) slightly, but significantly, increased ROS, but did not significantly modify ceramide abundance. The effect of nigericin on annexin V binding was significantly blunted, but not abolished by removal of extracellular Ca(2+). The nigericin-induced increase in [Ca(2+)]i and annexin V binding was again significantly blunted but not abolished by the Na(+)/H(+) exchanger inhibitor cariporide (10 μM). Nigericin triggers eryptosis, an effect paralleled by ROS formation, in part dependent on stimulation of Ca(2+) entry, and involving the cariporide-sensitive Na(+)/H(+) exchanger.

Background: Mitotane (1,1-dichloro-2-[o-chlorophenyl]-2-[p-chlorophenyl]ethane), a cytostatic drug used for the treatment of adrenocortical carcinomas, is effective by triggering tumor cell apoptosis. In analogy to apoptosis of nucleated cells, eryptosis is the suicidal death of erythrocytes, which is typically paralleled by cell shrinkage and breakdown of cell membrane phosphatidylserine asymmetry with subsequent phosphatidylserine exposure at the erythrocyte surface. Eryptosis may be triggered by increase of cytosolic Ca 2+ concentration ([Ca 2+ ] i ). The present study tested, whether treatment of human erythrocytes with mitotane is followed by eryptosis. Methods: [Ca 2+ ] i was estimated from Fluo3 fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin V binding, and hemolysis from hemoglobin release. Results: Exposure to mitotane (≥ 5 µg/ml ≈ 16 µM) significantly increased [Ca 2+ ] i , increased annexin V binding and triggered hemolysis, but did not significantly modify forward scatter. The effect on annexin V binding was significantly blunted in the absence of extracellular Ca 2+ . Within 30 min Ca 2+ ionophore ionomycin (1 µM) decreased forward scatter, an effect virtually abolished in the presence of mitotane (15 µg/ml). Conclusions: Mitotane increases [Ca 2+ ] i with subsequent phosphatidylserine translocation. By the same token mitotane inhibits Ca 2+ induced cell shrinkage.

The peptide antibiotic and ionophore gramicidin has previously been shown to trigger apoptosis of nucleated cells. In analogy to apoptosis, the suicidal death of erythrocytes or eryptosis involves cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include oxidative stress, increase of cytosolic Ca2+ activity ([Ca2+]i), and ceramide. The present study explored, whether gramicidin triggers eryptosis. To this end phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, red blood cell distribution width (RDW) from electronic particle counting, reactive oxidant species (ROS) from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, [Ca2+]i from Fluo3- and Fluo4 fluorescence, and ceramide abundance from binding of specific antibodies. As a result, a 24 h exposure of human erythrocytes to gramicidin significantly increased the percentage of annexin-V-binding cells (≥1 µg/mL), forward scatter (≥0.5 µg/mL) and hemolysis. Gramicidin enhanced ROS activity, [Ca2+]i and ceramide abundance at the erythrocyte surface. The stimulation of annexin-V-binding by gramicidin was significantly blunted but not abolished by removal of extracellular Ca2+. In conclusion, gramicidin stimulates phospholipid scrambling of the erythrocyte cell membrane, an effect at least partially due to induction of oxidative stress, increase of [Ca2+]i and up-regulation of ceramide abundance. Despite increase of [Ca2+]i, gramicidin increases cell volume and slightly reduces RWD.

The polyphenolic flavonoid Baicalein has been shown to trigger suicidal death or apoptosis of tumor cells and is thus considered for the prevention and 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. Stimulators of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i) and ceramide. The present study explored whether Baicalein stimulates eryptosis. To this end, forward scatter was taken for measurement of cell volume, annexin-V-binding for phosphatidylserine-exposure, Fluo3 fluorescence for [Ca2+]i and fluorescent antibodies for ceramide abundance. As a result, a 48 h exposure of human erythrocytes to Baicalein was followed by significant decrease of forward scatter (≥10 µM), significant increase of the percentage of annexin-V-binding cells (≥25 µM), significant increase of [Ca2+]i (50 µM) and significant increase of ceramide abundance (50 µM). The effect of Baicalein (50 µM) on annexin-V-binding was significantly blunted but not abrogated by removal of extracellular Ca2+. In conclusion, at the concentrations employed, Baicalein stimulates suicidal erythrocyte death or eryptosis, an effect at least in part due to the combined effects of Ca2+ entry and ceramide formation.

Background/Aims: Aristolochic Acid, a component of Aristolochia plants, has been shown to cause acute kidney injury, renal aristolochic acid nephropathy, Balkan endemic nephropathy, and urothelial carcinoma. Aristolochic acid nephropathy may be associated with severe anemia. The anemia could theoretically be due to stimulation of eryptosis, the suicidal death of erythrocytes characterized by cell shrinkage and cell membrane scrambling with translocation of phosphatidylserine to the erythrocyte cell membrane surface. Signalling involved in the stimulation of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i) and formation of ceramide. Methods: Cell volume was estimated from forward scatter, phosphatidylserine-exposure from annexin V binding, [Ca2+]i from Fluo3 fluorescence, and ceramide abundance from binding of fluorescent antibodies in flow cytometry. Results: A 48 hours exposure to Aristolochic Acid (≥ 75 g/ml) was followed by a significant decrease of forward scatter and increase of annexin-V-binding. The effects were paralleled by a significant increase of [Ca2+]i and significantly blunted, but not abrogated by removal of extracellular Ca2+. Aristolochic Acid further significantly increased ceramide abundance. Conclusions: Aristolochic Acid triggers eryptosis, an effect at least in part due to entry of extracellular Ca2+ and ceramide formation. i 2014 S. Karger AG, Basel

Salinomycin, a polyether ionophore antibiotic effective against a variety of pathogens, has been shown to trigger apoptosis of cancer cells and cancer stem cells. The substance is thus considered for the treatment of malignancy. Salinomycin compromises tumour cell survival at least in part by interference with mitochondrial function. Erythrocytes lack mitochondria but may undergo apoptosis-like suicidal cell death or eryptosis, which is characterized by scrambling of the cell membrane with phosphatidylserine exposure at the erythrocyte surface. Signalling involved in the triggering of eryptosis includes activation of oxidant-sensitive Ca(2+) permeable cation channels with subsequent increase in cytosolic Ca(2+) activity ([Ca(2+)]i). This study explored whether salinomycin stimulates eryptosis. Phosphatidylserine-exposing erythrocytes were identified by measurement of annexin-V binding, cell volume was estimated from forward scatter, haemolysis determined from haemoglobin release, [Ca(2+)]i quantified utilizing Fluo3-fluorescence and oxidative stress from 2',7' dichlorodihydrofluorescein diacetate (DCFDA) fluorescence in flow cytometry. A 48-hr exposure to salinomycin (5-100 nM) was followed by a significant increase in Fluo3-fluorescence, DCFDA fluorescence and annexin-V binding, as well as a significant decrease in forward scatter (at 5-10 nM, but not at 50 and 100 nM). The annexin-V binding after salinomycin treatment was significantly blunted but not abrogated in the nominal absence of extracellular Ca(2+) or in the presence of antioxidant n-acetyl cysteine (1 mM). Salinomycin triggers cell membrane scrambling, an effect at least partially due to oxidative stress and entry of extracellular Ca(2+).