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Eryptosis, the suicidal erythrocyte death, is characterized by erythrocyte shrinkage and phosphatidylserine translocation to the erythrocyte surface. Eryptosis is triggered by cell stress such as energy depletion and oxidative stress, by Ca(2+)-entry, ceramide, caspases, calpain and/or altered activity of several kinases. Phosphatidylserine-exposing erythrocytes adhere to the vascular wall and may thus impede microcirculation. Eryptotic cells are further engulfed by phagocytes and thus rapidly cleared from circulation.Stimulation of eryptosis contributes to anemia of several clinical conditions such as metabolic syndrome, diabetes, malignancy, hepatic failure, heart failure, uremia, hemolytic uremic syndrome, sepsis, fever, dehydration, mycoplasma infection, malaria, iron deficiency, sickle cell anemia, thalassemia, glucose-6-phosphate dehydrogenase deficiency and Wilson's disease. On the other hand, eryptosis with subsequent clearance of infected erythrocytes in malaria may counteract parasitemia.In theory, anemia due to excessive eryptosis could be alleviated by treatment with small molecules inhibiting eryptosis. In malaria, stimulators of eryptosis may accelerate death of infected erythrocytes and thus favorably influence the clinical course of the disease. Many small molecules inhibit or stimulate eryptosis. Several stimulators favorably influence murine malaria. Further preclinical and subsequent clinical studies are required to elucidate the therapeutic potential of stimulators or inhibitors of eryptosis.

Background: Phloretin, a natural component of apples, pears and strawberries, has previously been shown to stimulate apoptosis of nucleated cells. Erythrocytes may similarly enter suicidal death or eryptosis, which is characterized by cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i), ceramide, ATP depletion, and activation of protein kinase C (PKC) as well as p38 mitogen activated protein kinase (p38 kinase). Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, and ceramide abundance from binding of specific antibodies. Results: A 48 h exposure of human erythrocytes to phloretin significantly increased the percentage of annexin-V-binding cells (≥100 µM) without significantly influencing forward scatter. Phloretin did not significantly modify [Ca2+]i and the stimulation of annexin-V-binding by phloretin (300 µM) did not require presence of extracellular Ca2+. Phloretin did not significantly modify erythrocyte ATP levels, and the effect of phloretin on annexin-V-binding was not significantly altered by PKC inhibitor staurosporine (1 µM) or p38 kinase inhibitor SB2203580 (2 µM). However, phloretin significantly increased the ceramide abundance at the cell surface. Conclusions: Phloretin stimulates phospholipid scrambling of the erythrocyte cell membrane, an effect at least partially due to up-regulation of ceramide abundance.

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.

Background/Aims: Gedunin, an inhibitor of heat shock protein HSP90, triggers apoptosis of tumor cells and is thus effective against malignancy. Moreover, the drug has antimalarial potency. In analogy to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and by phosphatidylserine translocation to the erythrocyte surface. Eryptosis may be triggered by increase of cytosolic Ca2+-activity ([Ca2+]i). The present study explored whether gedunin stimulates eryptosis. Methods: Forward scatter was determined to estimate cell volume, annexin V binding to identify phosphatidylserine-exposing erythrocytes, hemoglobin release to depict hemolysis, and Fluo3-fluorescence to quantify [Ca2+]i. Results: A 48 h exposure of human erythrocytes to gedunin significantly increased [Ca2+]i (12 µM), significantly decreased forward scatter (24 µM) and significantly increased annexin-V-binding (12 µM). The effect of gedunin (24 µM) on annexin-V-binding was virtually abrogated by removal of extracellular Ca2+. Conclusion: Gedunin stimulates suicidal erythrocyte death or eryptosis, an effect mainly if not exclusively due to stimulation of Ca2+ entry.

Background: Novobiocin, an aminocoumarin antibiotic, interferes with heat shock protein 90 and hypoxia inducible factor dependent gene expression and thus compromises cell survival. Similar to survival of nucleated cells, erythrocyte survival could be disrupted by eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and by phospholipd scrambling of the cell membrane with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i). The Ca2+ sensitivity of phospholipid scrambling is enhanced by ceramide. The present study explored, whether novobiocin elicits eryptosis. Methods: [Ca2+]i was estimated from Fluo3-fluorescence, ceramide abundance utilizing fluorescent antibodies, cell volume from forward scatter, phosphatidylserine-exposure from annexin V binding. Results: A 48 hours exposure to novobiocin (500 µM) was followed by a significant increase of [Ca2+]i, decrease of forward scatter, increase of annexin-V-binding and enhanced ceramide formation. Removal of extracellular Ca2+ virtually abrogated the increase of annexin-V-binding following novobiocin exposure. Conclusions: Novobiocin stimulates eryptosis, an effect at least in part due to entry of extracellular Ca2+ and formation of ceramide.

Background/aims: Geldanamycin, a benzoquinone ansamycin antibiotic, and its analogues induce apoptosis of tumor cells and are thus considered for the treatment of cancer. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and by cell membrane scrambling with phosphatidylserine-exposure at the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca2+-concentration ([Ca2+]i) and formation of ceramide. The present study explored, whether geldanamycin modifies [Ca2+]i, ceramide formation, cell volume and phosphatidylserine abundance at the erythrocyte surface. Methods: Erythrocyte volume was estimated from forward scatter, phosphatidylserine-abundance from annexin V binding, hemolysis from hemoglobin release, ceramide formation from binding of fluorescent antibodies and [Ca2+]i from Fluo3-fluorescence. Results: A 48 hours exposure to geldanamycin significantly decreased forward scatter (≥ 5 µM), significantly increased annexin-V-binding (≥ 25 µM), but did not significantly modify Fluo3-fluorescence (up to 50 µM). The annexin-V-binding following geldanamycin treatment was not significantly modified by removal of extracellular Ca2+ but was paralleled by significantly increased ceramide formation (50 µM). Conclusions: Geldanamycin stinulated eryptosis, an effect at least partially due to ceramide formation.

The selective cyclooxygenase-2 (COX-2) inhibitor celecoxib triggers apoptosis of tumor cells and is thus effective against malignancy. The substance is at least partially effective through mitochondrial depolarization. Even though lacking mitochondria, erythrocytes may enter apoptosis-like suicidal death or eryptosis, which is characterized by cell shrinkage and by phosphatidylserine translocation to the erythrocyte surface. Eryptosis may be triggered by increase of cytosolic Ca2+-activity ([Ca2+]i). The present study explored whether celecoxib stimulates eryptosis. Forward scatter was determined to estimate cell volume, annexin V binding to identify phosphatidylserine-exposing erythrocytes, hemoglobin release to depict hemolysis, and Fluo3-fluorescence to quantify [Ca2+]i. A 48 h exposure of human erythrocytes to celecoxib was followed by significant increase of [Ca2+]i (15 µM), significant decrease of forward scatter (15 µM) and significant increase of annexin-V-binding (10 µM). Celecoxib (15 µM) induced annexin-V-binding was blunted but not abrogated by removal of extracellular Ca2+. In conclusion, celecoxib stimulates suicidal erythrocyte death or eryptosis, an effect partially due to stimulation of Ca2+ entry.

Zearalenone, a cereal mycotoxin with mycoestrogen activity and effect on fertility, is known to trigger apoptosis of a variety of nucleated cell types including hematopoietic progenitor cells. In analogy to apoptosis of nucleated cells, eryptosis, the suicidal death of erythrocytes, leads to cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the erythrocyte surface. The most important stimulator of eryptosis is an increase in cytosolic Ca(2+) activity ([Ca(2+)]i). The present study explored whether zearalenone triggers eryptosis. Erythrocyte volume was estimated from forward scatter, phosphatidylserine exposure at the erythrocyte surface from annexin-V binding, hemolysis from hemoglobin release, and [Ca(2+)]i from Fluo3 fluorescence. A 48-h exposure to zearalenone (≥25 μM) was followed by a significant increase in [Ca(2+)]i and annexin-V binding, and a significant decrease in forward scatter. The effect on annexin-V binding was significantly blunted in the nominal absence of extracellular Ca(2+). Zearalenone stimulates the suicidal erythrocyte death, an effect at least partially due to stimulation of Ca(2+) entry.

Withaferin A, a triterpenoid component from Withania somnifera, counteracts malignancy, an effect attributed to stimulation of apoptosis. Withaferin A is partially effective through induction of oxidative stress, altered gene expression and mitochondrial depolarization. Erythrocytes lack mitochondria and nuclei but may enter apoptosis-like eryptosis, a suicidal cell death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Triggers of eryptosis include increase of cytosolic Ca2+-activity [ Ca 2 + ] i following activation of oxidant-sensitive Ca2+-permeable cation channels, ceramide formation and/or ATP-depletion. The present study explored, whether withaferin A triggers eryptosis. To this end, [ Ca 2 + ] i was estimated from Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin-V-binding, hemolysis from hemoglobin release, oxidative stress from DCFDA-fluorescence and ceramide abundance utilizing antibodies. A 48 h exposure to withaferin A significantly decreased forward scatter (at ⩾ 10 μM withaferin concentration) and increased [ Ca 2 + ] i (⩾5 μM), ROS-formation (⩾10 μM) ceramide-formation (⩾10 μM) as well as annexin-V-binding (⩾5 μM). Withaferin A treatment was followed by slight but significant increase of hemolysis. Extracellular Ca2+ removal, amiloride, and the antioxidant N-acetyl- l -cysteine significantly blunted withaferin A-triggered annexin-V-binding. The present observations reveal that withaferin A triggers suicidal erythrocyte death despite the absence of gene expression and key elements of apoptosis such as mitochondria.

Fumagillin, a cyclohexane isolated from fungus Aspergillus fumigatus, has anti-infective and anti-cancer potency. Fumagillin is at least partially effective by inducing suicidal death or apoptosis. In analogy to apoptosis of nucleated cells, eryptosis is the suicidal death of erythrocytes characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Stimulators of eryptosis include increase of cytosolic Ca(2+)-activity ([Ca(2+)](i)) and ceramide. The present study explored whether fumagillin (5-100 μM) could stimulate eryptosis. To this end, [Ca(2+)](i) was estimated from Fluo3 fluorescence, ceramide by utilizing specific antibodies, cell volume from forward scatter, phosphatidylserine exposure from annexin V binding and haemolysis from haemoglobin release. As a result, a 48-hr exposure to fumagillin significantly increased [Ca(2+)](i) (≥10 μM), enhanced ceramide abundance (100 μM), triggered annexin V binding (≥10 μM) and decreased forward scatter (≥10 μM). Fumagillin exposure was followed by slight but significant increase of haemolysis. Removal of extracellular Ca(2+) significantly blunted but did not abolish the effect of fumagillin (100 μM) on annexin V binding. The present observations disclose a novel effect of fumagillin, that is, stimulation of eryptosis, paralleled by Ca(2+) entry, ceramide formation, phosphatidylserine exposure and decrease of cell volume.

Carbon monoxide (CO) intoxication severely interferes with the oxygen-transporting function of haemoglobin. Beyond that, CO participates in the regulation of apoptosis. CO could be generated from CO-releasing molecules (CORM), such as the tricarbonyl-dichlororuthenium (II) dimer (CORM-2), which is presently considered for the treatment of vascular dysfunction, inflammation, tissue ischaemia and organ rejection. CORM-2 is at least partially effective by modifying gene expression and mitochondrial potential. Erythrocytes lack nuclei and mitochondria but may undergo suicidal cell death or eryptosis, characterized by cell shrinkage and phospholipid scrambling of the cell membrane. Eryptosis is triggered by the increase in cytosolic Ca2+ activity ([ Ca2+ ]i). The present study explored whether CORM-2 influences eryptosis. To this end, [ Ca2+ ]i was estimated from Fluo-3-fluorescence, cell volume from forward scatter, phospholipid scrambling from annexin-V-binding and haemolysis from haemoglobin release. CO-binding haemoglobin (COHb) was estimated utilizing a blood gas analyser. As a result, exposure of erythrocytes for 24 hr to CORM-2 (≥5 μM) significantly increased COHb, [ Ca2+ ]i, forward scatter, annexin-V-binding and haemolysis. Annexin-V-binding was significantly blunted by 100% oxygen and was virtually abolished in the nominal absence of Ca2+ . In conclusion, CORM-2 stimulates cell membrane scrambling of erythrocytes, an effect largely due to Ca2+ entry and partially reversed by O2 .

Loss-of-function mutations in human adenomatous polyposis coli (APC) lead to multiple colonic adenomatous polyps eventually resulting in colonic carcinoma. Similarly, heterozygous mice carrying defective APC (apc(Min/+)) suffer from intestinal tumours. The animals further suffer from anaemia, which in theory could result from accelerated eryptosis, a suicidal erythrocyte death triggered by enhanced cytosolic Ca(2+) activity and characterized by cell membrane scrambling and cell shrinkage. To explore, whether APC-deficiency enhances eryptosis, we estimated cell membrane scrambling from annexin V binding, cell size from forward scatter and cytosolic ATP utilizing luciferin-luciferase in isolated erythrocytes from apc(Min/+) mice and wild-type mice (apc(+/+)). Clearance of circulating erythrocytes was estimated by carboxyfluorescein-diacetate-succinimidyl-ester labelling. As a result, apc(Min/+) mice were anaemic despite reticulocytosis. Cytosolic ATP was significantly lower and annexin V binding significantly higher in apc(Min/+) erythrocytes than in apc(+/+) erythrocytes. Glucose depletion enhanced annexin V binding, an effect significantly more pronounced in apc(Min/+) erythrocytes than in apc(+/+) erythrocytes. Extracellular Ca(2+) removal or inhibition of Ca(2+) entry with amiloride (1 mM) blunted the increase but did not abrogate the genotype differences of annexin V binding following glucose depletion. Stimulation of Ca(2+) -entry by treatment with Ca(2+) -ionophore ionomycin (10 μM) increased annexin V binding, an effect again significantly more pronounced in apc(Min/+) erythrocytes than in apc(+/+) erythrocytes. Following retrieval and injection into the circulation of the same mice, apc(Min/+) erythrocytes were more rapidly cleared from circulating blood than apc(+/+) erythrocytes. Most labelled erythrocytes were trapped in the spleen, which was significantly enlarged in apc(Min/+) mice. The observations point to accelerated eryptosis and subsequent clearance of apc(Min/+) erythrocytes, which contributes to or even accounts for the enhanced erythrocyte turnover, anaemia and splenomegaly in those mice.

SCOPE Enniatin A, a peptide antibiotic and common food contaminant, triggers mitochondrial dysfunction and apoptosis. Even though lacking mitochondria, erythrocytes may similarly undergo suicidal cell death or eryptosis. Eryptosis is characterized by cell shrinkage and cell membrane phospholipid scrambling. Triggers of phospholipid scrambling include energy depletion and increase in cytosolic Ca(2+) activity ([Ca(2+) ](i) ). The present study explored whether enniatin A triggers phospholipid scrambling. METHODS AND RESULTS Phospholipid scrambling was estimated from annexin-V-binding, cell volume from forward scatter (FSC), [Ca(2+) ](i) from Fluo3-fluorescence, cytosolic ATP-concentration ([ATP](i) ) using a luciferase assay and hemolysis from hemoglobin release. Exposure of erythrocytes for 48 h to enniatin A (≥ 2.5 μM) significantly increased [Ca(2+) ](i) , decreased [ATP](i) , decreased FSC, triggered annexin-V-binding and elicited hemolysis. Annexin-V-binding affected 25%, and hemolysis 2% of treated erythrocytes. Decreased [ATP](i) by glucose depletion for 48 h was similarly followed by increased [Ca(2+) ](i) , decreased FSC and annexin-V-binding. Enniatin A augmented the effect on [Ca(2+) ](i) and annexin-V-binding, but not on FSC. Annexin-V-binding was blunted by Ca(2+) removal, by the cation channel inhibitor amiloride (1 mM), by the protein kinase C inhibitor staurosporine (500 nM) but not by the pancaspase inhibitor zVAD (10 μM). CONCLUSION The food contaminant enniatin A triggers ATP depletion and increases cytosolic Ca(2+) activity, effects resulting in suicidal erythrocyte death.

Oridonin triggers apoptosis of cancer cells and was suggested as anticancer agent. Oridonin is partially effective through mitochondrial depolarization and partially by modifying gene expression. Erythrocytes lack mitochondria and nuclei but may undergo eryptosis, a suicidal cell death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Triggers of eryptosis include increase of cytosolic Ca(2+)-activity, ATP depletion and ceramide formation. The present study explored, whether oridonin triggers eryptosis. Cytosolic Ca(2+)-concentration was estimated from Fluo3-fluorescence, cell volume from forward scatter in FACS analysis, phosphatidylserine exposure from binding of fluorescent annexin V, hemolysis from hemoglobin release, ATP concentration utilizing a luciferin-luciferase assay and ceramide abundance utilizing fluorescent anti-ceramide antibodies. A 48 h exposure to oridonin (≥25μM) significantly increased cytosolic Ca(2+)-concentration, increased ceramide formation, decreased forward scatter and triggered annexin V-binding (the latter in >20% of the erythrocytes). Oridonin didn't decrease ATP concentration and hemolysed

The mitogen- and stress-activated kinase MSK1/2 plays a decisive role in apoptosis. In analogy to apoptosis of nucleated cells, suicidal erythrocyte death called eryptosis is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine (PS) externalization. Here, we explored whether MSK1/2 participates in the regulation of eryptosis. To this end, erythrocytes were isolated from mice lacking functional MSK1/2 (msk−/−) and corresponding wild-type mice (msk+/+). Blood count, hematocrit, hemoglobin concentration and mean erythrocyte volume were similar in both msk−/− and msk+/+ mice, but reticulocyte count was significantly increased in msk−/− mice. Cell membrane PS exposure was similar in untreated msk−/− and msk+/+ erythrocytes, but was enhanced by pathophysiological cell stressors ex vivo such as hyperosmotic shock or energy depletion to significantly higher levels in msk−/− erythrocytes than in msk+/+ erythrocytes. Cell shrinkage following hyperosmotic shock and energy depletion, as well as hemolysis following decrease of extracellular osmolarity was more pronounced in msk−/− erythrocytes. The in vivo clearance of autologously-infused CFSE-labeled erythrocytes from circulating blood was faster in msk−/− mice. The spleens from msk−/− mice contained a significantly greater number of PS-exposing erythrocytes than spleens from msk+/+ mice. The present observations point to accelerated eryptosis and subsequent clearance of erythrocytes leading to enhanced erythrocyte turnover in MSK1/2-deficient mice.

Juglone, a quinone isolated from Juglans mandshurica Maxim, has previously been shown to be effective against malignancy. The effect is at least partially due to stimulation of suicidal death or apoptosis of tumour cells. On the other hand, juglone has been shown to counteract apoptosis, for example, of neurons. In analogy to apoptosis of nucleated cells, erythrocytes may enter eryptosis, a suicidal death characterized by cell shrinkage and breakdown of phosphatidylserine asymmetry of the cell membrane with phosphatidylserine exposure at the erythrocyte surface. Stimulators of eryptosis include increase in cytosolic Ca(2+) activity [(Ca(2+) )i]. This study explored whether juglone stimulates eryptosis. To this end, erythrocyte volume was estimated from forward scatter, phosphatidylserine exposure at the erythrocyte surface from FITC annexin V binding, ceramide abundance from binding of fluorescent antibodies in flow cytometry and cytosolic ATP with a luciferin-luciferase-based assay. As a result, a 24-hr exposure of human erythrocytes to juglone (5 μM) significantly decreased erythrocyte forward scatter. Juglone (1-5 μM) significantly increased the percentage of annexin V binding cells. Juglone (5 μM) significantly increased ceramide abundance at the erythrocyte surface and decreased erythrocyte ATP concentration. The effect of juglone (10 μM) on annexin V binding was slightly but significantly blunted by removal of extracellular Ca(2+) and by addition of protein kinase C (PKC) inhibitor staurosporine (1 μM). In conclusion, juglone stimulates suicidal erythrocyte death or eryptosis at least in part by upregulation of ceramide abundance, energy depletion and activation of PKC.

Background/Aims: Cryptotanshinone, a component of Salvia miltiorrhiza Bunge roots, may trigger suicidal death or apoptosis of tumor cells and has thus been recommended for the prevention and treatment of malignancy. On the other hand, Cryptotanshinone has been shown to counteract apoptosis of neurons and hepatocytes. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, a suicidal death characterized by cell shrinkage and phosphatidylserine translocation to the erythrocyte surface. Eryptosis may be triggered by increase of cytosolic Ca2+-activity ([Ca2+]i). The present study explored whether Cryptotanshinone stimulates eryptosis. Methods: Forward scatter was taken as measure of cell volume, annexin V binding for identification of phosphatidylserine-exposing erythrocytes and Fluo3-fluorescence for determination of [Ca2+]i. Results: A 48 h exposure of human erythrocytes to Cryptotanshinone (10 µM) was followed by significant decrease of forward scatter, significant increase of the percentage annexin-V-binding cells and significant increase of [Ca2+]i. The effect of Cryptotanshinone (1 µM) on annexin-V-binding was virtually abrogated by removal of extracellular Ca2+. Conclusion: Cryptotanshinone is a powerful stimulator of suicidal erythrocyte death or eryptosis, which is effective mainly, if not exclusively, by stimulation of Ca2+ entry.

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+).

Background: Patulin, the most common mycotoxin in apples and apple-derived products, triggers apoptosis and has thus been considered for the treatment of cancer. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and by cell membrane scrambling leading to phosphatidylserine-exposure at the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i). The present study explored, whether exposure of human erythrocytes to patulin is followed by eryptosis. Methods: Forward scatter was measured to estimate cell volume, annexin V binding to detect phosphatidylserine-exposure, hemoglobin release to quantify hemolysis, and Fluo3-fuorescence to determine [Ca2+]i. Results: A 48 h exposure to patulin significantly increased [Ca2+]I (5 µM), significantly decreased forward scatter (5 µM) and significantly increased annexin-V-binding (2.5 µM). Patulin (10 µM) induced annexin-V-binding was virtually abrogated by removal of extracellular Ca2+. Conclusion: Patulin stimulates Ca2+ entry into erythrocytes, an effect triggering suicidal erythrocyte death or eryptosis.

Honokiol ((3,5-di-(2-propenyl)-1,1-biphenyl-2,2-diol), a component of Magnolia officinalis, stimulates apoptosis and is thus considered for the treatment of malignancy. In analogy to apoptosis of nucleated cells, erythrocytes may enter eryptosis, a suicidal death characterized by cell shrinkage and by breakdown of cell membrane phosphatidylserine asymmetry with phosphatidylserine-exposure at the erythrocyte surface. Eryptosis may be triggered following increase of cytosolic Ca(2+)-activity ([Ca(2+)]i). The present study explored, whether honokiol elicits eryptosis. Cell volume has been estimated from forward scatter, phosphatidylserine-exposure from annexin V binding, hemolysis from hemoglobin release, [Ca(2+)]i from Fluo3-fluorescence, and ceramide from fluorescent antibodies. As a result, a 48 h exposure to honokiol was followed by a slight but significant increase of [Ca(2+)]i (15 μM), significant decrease of forward scatter (5 μM), significant increase of annexin-V-binding (5 μM) and significant increase of ceramide formation (15 μM). Honokiol further induced slight, but significant hemolysis. Honokiol (15 μM) induced annexin-V-binding was significantly blunted but not abrogated in the nominal absence of extracellular Ca(2+). In conclusion, honokiol triggers suicidal erythrocyte death or eryptosis, an effect at least in part due to stimulation of Ca(2+) entry and ceramide formation.

Background: Sulindac sulfide, a non-steroidal anti-inflammatory drug (NSAID), stimulates apoptosis of tumor cells and is thus effective against malignancy. In analogy to apoptosis of nucleated cells, erythrocytes may undergo eryptosis, an apoptosis-like suicidal erythrocyte death, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine-exposure at the cell surface. Stimulators of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i) and ceramide formation. The present study explored, whether sulindac sulfide stimulates eryptosis. Methods: [Ca2+]i was estimated from Fluo-3 fluorescence, cell volume from forward scatter, phosphatidylserine-exposure from binding of fluorescent annexin-V, hemolysis from hemoglobin release, and ceramide abundance utilizing fluorescent antibodies. Results: A 48 h exposure to sulindac sulfide (≤ 20 µM) was followed by significant increase of [Ca2+]i, enhanced ceramide abundance, decreased forward scatter and increased percentage of annexin-V-binding erythrocytes. Sulindac sulfide triggered slight but significant hemolysis. Removal of extracellular Ca2+ significantly blunted, but did not abrogate the effect of sulindac sulfide (20 µM) on annexin-V-binding. Conclusion: Sulindac sulfide stimulates the suicidal death of erythrocytes or eryptosis, an effect paralleled by Ca2+-entry, ceramide formation, cell shrinkage and phosphatidylserine-exposure.

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone, 1), a natural product from plants with potential anticancer potency, induces apoptosis. Mechanisms involved in 1-induced apoptosis include mitochondrial depolarization, inactivation of NF-κB, and altered expression of anti- and proapoptotic Bcl proteins. Similar to nucleated cells, erythrocytes may undergo suicidal death or eryptosis, which, like apoptosis, results in cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Triggers of eryptosis include increase of cytosolic Ca(2+) activity ([Ca(2+)]i) and ceramide formation. The present study explored whether 1 stimulates eryptosis. Cell volume was estimated from forward scatter, phosphatidylserine exposure from annexin-V-binding, hemolysis from hemoglobin release, [Ca(2+)]i from Fluo-3 fluorescence, and ceramide abundance utilizing antibodies. A 48 h exposure to 1 (2 μM) decreased forward scatter and increased annexin-V-binding significantly, events paralleled by increased [Ca(2+)]i and ceramide formation. Exposure to 1 was followed by a slight but significant increase of hemolysis. Removal of extracellular Ca(2+) slightly, but significantly blunted the effect of 1 (2 μM) on annexin-V-binding. The present observations demonstrate that 1 may trigger suicidal death of erythrocytes, cells devoid of mitochondria and nuclei.

Background: Sorafenib (Nexavar®), a polytyrosine kinase inhibitor, stimulates apoptosis and is thus widely used for chemotherapy in hepatocellular carcinoma (HCC). Hematological side effects of Nexavar® chemotherapy include anemia. Erythrocytes may undergo apoptosis-like suicidal death or eryptosis, which is characterized by cell shrinkage and phosphatidylserine-exposure at the cell surface. Signaling leading to eryptosis include increase in cytosolic Ca2+activity ([Ca2+]i), formation of ceramide, ATP-depletion and oxidative stress. The present study explored, whether sorafenib triggers eryptosis in vitro and in vivo. Methods: [Ca2+]i was estimated from Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine-exposure from annexin-V-binding, hemolysis from hemoglobin release, ceramide with antibody binding-dependent fluorescence, cytosolic ATP with a luciferin–luciferase-based assay, and oxidative stress from 2’,7’ dichlorodihydrofluorescein diacetate (DCFDA) fluorescence. Results: A 48 h exposure of erythrocytes to sorafenib (≥0.5 µM) significantly increased Fluo 3 fluorescence, decreased forward scatter, increased annexin-V-binding and triggered slight hemolysis (≥5 µM), but did not significantly modify ceramide abundance and cytosolic ATP. Sorafenib treatment significantly enhanced DCFDA-fluorescence and the reducing agents N-acetyl-L-cysteine and tiron significantly blunted sorafenib-induced phosphatidylserine exposure. Nexavar® chemotherapy in HCC patients significantly enhanced the number of phosphatidylserine-exposing erythrocytes. Conclusions: The present observations disclose novel effects of sorafenib, i.e. stimulation of suicidal erythrocyte death or eryptosis, which may contribute to the pathogenesis of anemia in Nexavar®-based chemotherapy.

Contains fulltext : 109681.pdf (Publisher’s version ) (Open Access) Tanshinone IIA, an antimicrobial, antioxidant, antianaphylactic, antifibrotic, vasodilating, antiatherosclerotic, organo-protective and antineoplastic component from the rhizome of Salvia miltiorrhiza, is known to trigger apoptosis of tumor cells. Tanshinone IIA is effective in part through mitochondrial depolarization and altered gene expression. Erythrocytes lack mitochondria and nuclei but may undergo eryptosis, an apoptosis-like suicidal cell death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Eryptosis is triggered by increase of cytosolic Ca(2+) activity, ATP depletion and ceramide formation. The present study explored, whether tanshinone IIA elicits eryptosis. Cytosolic Ca(2+)-concentration was determined from Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine exposure from binding of fluorescent annexin V, hemolysis from hemoglobin concentration in the supernatant, ATP concentration utilizing luciferin-luciferase and ceramide formation utilizing fluorescent anticeramide antibodies. Clearance of circulating erythrocytes was estimated by CFSE-labeling. A 48 h exposure to tanshinone IIA (>/=10 microM) significantly increased cytosolic Ca(2+)-concentration, decreased ATP concentration (25 microM), increased lactate concentration (25 microM), increased ceramide formation (25 microM), decreased forward scatter, increased annexin-V-binding and increased (albeit to a much smaller extent) hemolysis. The effect of 25 microM tanshinone IIA on annexin-V binding was partially reversed in the nominal absence of Ca(2+). Labelled tanshinone IIA-treated erythrocytes were more rapidly cleared from the circulating blood in comparison to untreated erythrocytes. The present observations reveal a completely novel effect of tanshinone IIA, i.e. triggering of Ca(2+) entry, ATP depletion and ceramide formation in erythrocytes, events eventually leading to eryptosis with cell shrinkage and cell membrane scrambling.

Gambogic acid, a xanthone from Garcinia hanburyi, stimulates apoptosis and has thus anticancer potency. Similar to apoptosis of nucleated cells, erythrocytes may undergo apoptosis-like suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine-exposure at the cell surface. Eryptosis could be triggered by increase of cytosolic Ca(2+)-activity ([Ca(2+)](i)), ceramide formation, ATP-depletion and caspase activation. The present study explored, whether gambogic acid triggers eryptosis of human erythrocytes. [Ca(2+)](i )was estimated utilizing Fluo-3 fluorescence, cell volume from forward scatter, phosphatidylserine-exposure from annexin-V-binding, hemolysis from hemoglobin release, ceramide abundance utilizing antibodies, and cytosolic ATP with luciferin- luciferase. A 48 h exposure to gambogic acid (500 nM) significantly increased [Ca(2+)](i), stimulated ceramide formation, decreased forward scatter and increased annexin-V-binding. Gambogic acid exposure was followed by a slight but significant increase of hemolysis. Gambogic acid did not significantly modify cytosolic ATP-concentration. Removal of extracellular Ca(2+) slightly, but significantly blunted the effect of gambogic acid (500 nM) on annexin-V-binding. The present observations disclose a novel effect of gambogic acid, i.e. stimulation of suicidal death of human erythrocytes or eryptosis, paralleled by Ca(2+)-entry, ceramide formation, cell shrinkage and phosphatidylserine-exposure.

The pesticide rotenone stimulates apoptosis and rotenone intoxication has been considered a cause of Parkinson's disease. Rotenone further sensitizes tumor cells to cytotoxic drugs. The apoptotic effect of rotenone is at least partially due to mitochondrial injury. Even though lacking mitochondria and nuclei, erythrocytes may undergo eryptosis, an apoptosis-like suicidal death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine-exposure at the cell surface. Triggers of eryptosis include increase of cytosolic Ca 2+ -activity ([Ca 2+ ] i ) and enhanced ceramide formation. The present study explored, whether rotenone elicits eryptosis. To this end, [Ca 2+ ] i was estimated utilizing Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine-exposure from annexin-V-binding, ceramide utilizing fluorescence antibodies and hemolysis from hemoglobin release. A 48 h exposure to rotenone significantly increased Fluo3-fluorescence i (≥1 μM), increased ceramide abundance (10 μM), decreased forward scatter (≥2.5 μM) and increased annexin-V-binding (≥ 1 μM). Rotenone exposure was further followed by slight but significant hemolysis. Rotenone-induced cell membrane scrambling was significantly blunted, but not completely abrogated by removal of extracellular Ca 2+ . The present observations disclose a novel effect of rotenone, i.e. triggering of erythrocyte shrinkage and cell membrane scrambling, an effect paralleled by and partially dependent on Ca 2+ -entry.

Apigenin, a flavone in fruits and vegetables, stimulates apoptosis and thus counteracts cancerogenesis. Erythrocytes may similarly undergo suicidal cell death or eryptosis, characterized by cell shrinkage and phosphatidylserine exposure at the cell surface. Triggers of eryptosis include increase of cytosolic Ca(2+) activity ([Ca(2+)](i)), ceramide formation and ATP depletion. The present study explored the effect of apigenin on eryptosis. [Ca(2+)](i) was estimated from Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin V binding, hemolysis from hemoglobin release, ceramide utilizing antibodies, and cytosolic ATP with luciferin-luciferase. A 48 h exposure to apigenin significantly increased [Ca(2+)](i) (≥ 1 μM), increased ceramide formation (15 μM), decreased ATP concentration (15 μM), decreased forward scatter (≥ 1 μM), and increased annexin V binding (≥ 5 μM) but did not significantly modify hemolysis. The effect of 15 μM apigenin on annexin V binding was blunted by Ca(2+) removal. The present observations reveal novel effects of apigenin, i.e. stimulation of Ca(2+) entry, ceramide formation and ATP depletion in erythrocytes with subsequent triggering of suicidal erythrocyte death, paralleled by cell shrinkage and phosphatidylserine exposure.

p38 protein kinase is activated by hyperosmotic shock, participates in the regulation of cell volume sensitive transport and metabolism and is involved in the regulation of various physiological functions including cell proliferation and apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may undergo suicidal death or eryptosis, which is paralleled by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Triggers of eryptosis include hyperosmotic shock, which increases cytosolic Ca(2+) activity and ceramide formation. The present study explored whether p38 kinase is expressed in human erythrocytes, is activated by hyperosmotic shock and participates in the regulation of eryptosis. Western blotting was utilized to determine phosphorylation of p38 kinase, forward scatter to estimate cell volume, annexin V binding to depict phosphatidylserine exposure and Fluo3 fluorescence to estimate cytosolic Ca(2+) activity. As a result, erythrocytes express p38 kinase, which is phosphorylated upon osmotic shock (+550 mM sucrose). Osmotic shock decreased forward scatter, increased annexin V binding and increased Fluo3 fluorescence, all effects significantly blunted by the p38 kinase inhibitors SB203580 (2 μM) and p38 Inh III (1 μM). In conclusion, p38 kinase is expressed in erythrocytes and participates in the machinery triggering eryptosis following hyperosmotic shock.

Dicoumarol, a widely used anticoagulant, may cause anemia, which may result from enhanced erythrocyte loss due to bleeding or due to accelerated erythrocyte death. Erythrocytes may undergo suicidal death or eryptosis, characterized by cell shrinkage and phospholipid scrambling of the cell membrane. Eryptosis may be triggered by increase of cytosolic Ca(2+)-activity ([Ca(2+)](i)). The present study explored, whether dicoumarol induces eryptosis. [Ca(2+)](i) was estimated from Fluo3-fluorescence, cation channel activity utilizing whole cell patch clamp, cell volume from forward scatter, phospholipid scrambling from annexin-V-binding, and hemolysis from haemoglobin release. Exposure of erythrocytes for 48 hours to dicoumarol (=10 μM) significantly increased [Ca(2+)](i), enhanced cation channel activity, decreased forward scatter, triggered annexin-V-binding and elicited hemolysis. Following exposure to 30 μM dicoumarol, annexin-V-binding affected approximately 15%, and hemolysis 2% of treated erythrocytes. The stimulation of annexin-V-binding by dicoumarol was abrogated in the nominal absence of Ca(2+). In conclusion, dicoumarol stimulates suicidal death of erythrocytes by stimulating Ca(2+) entry and subsequent triggering of Ca(2+) dependent cell membrane scrambling.

Ursolic acid (1), a triterpenoid with pleotropic effects including inhibition of tumor growth, is well known to trigger apoptosis of nucleated cells. The effect is at least partially due to altered gene expression and mitochondrial dysfunction. Erythrocytes lack nuclei and mitochondria but, similar to nucleated cells, may undergo suicidal cell death or eryptosis, which is characterized by cell shrinkage and phospholipid scrambling of the cell membrane. Triggers of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), ceramide formation and/or ATP depletion. The present study has investigated whether or not 1 induces eryptosis. [Ca2+]i was estimated from Fluo-3 fluorescence, cell volume from forward scatter, phospholipid scrambling from annexin V binding, hemolysis from hemoglobin release, and cytosolic ATP concentration ([ATP]i) utilizing a luciferase assay and ceramide-utilizing fluorescent antibodies in FACS analysis. As a result, exposure of erythrocytes for 48 h to 1 (≥5 μM) did not significantly modify [ATP]i, but significantly increased [Ca2+]i, stimulated ceramide formation, decreased forward scatter, triggered annexin V binding, and elicited hemolysis. At 5 μM, 1 stimulated phospholipid scrambling in 10% and hemolysis in 2% of treated erythrocytes. Annexin V binding was blunted in the nominal absence of Ca2+. In conclusion, the food component ursolic acid stimulates suicidal death of erythrocytes, i.e., cells devoid of nuclei and mitochondria.

Phlorhizin interferes with glucose transport. Glucose depletion triggers suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling. Eryptosis is further triggered by oxidative stress. The present study explored whether phlorhizin influences eryptosis following glucose depletion or oxidative stress. Cell membrane scrambling was estimated from annexin binding, cell volume from forward scatter (FSC), and cytosolic Ca(2+) concentration from Fluo-3 fluorescence. Phlorhizin (10-100 μM) added alone did not modify scrambling, FSC, or Fluo-3 fluorescence. Glucose depletion (48 h) significantly increased Fluo-3 fluorescence, decreased FSC, and increased annexin binding, effects in part significantly blunted by phlorhizin (annexin binding ≥ 10 μM, FSC ≥ 50 μM). Oxidative stress (30 min 0.3 mM tert-butylhydroperoxide) again significantly increased Fluo-3 fluorescence and triggered annexin binding, effects again in part significantly blunted by phlorhizin (Fluo-3 fluorescence ≥ 50 μM, annexin-binding ≥ 10 μM). Phlorhizin did not blunt the cell shrinkage induced by oxidative stress. The present observations disclose a novel effect of phlorhizin, that is, an influence on suicidal erythrocyte death following energy depletion and oxidative stress.

Benzethonium, an antimicrobial surfactant widely used as preservative of pharmaceuticals, topical wound care product and oral disinfectant, triggers apoptosis of several cell types. The apoptosis is preceded and possibly triggered by mitochondrial depolarization. Even though lacking mitochondria, erythrocytes may similarly undergo suicidal cell death or eryptosis. Hallmarks of eryptosis include cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Eryptosis may be triggered by energy depletion, which leads to increase of cytosolic Ca(2+)-activity with subsequent Ca(2+)-sensitive cell shrinkage and cell membrane scrambling. Ca(2+)-sensitivity is enhanced by ceramide. The present study explored the effect of benzethonium on eryptosis. Cell membrane scrambling was estimated from binding of fluorescent annexin V to phosphatidylserine, cell volume from forward scatter in FACS analysis, cytosolic Ca(2+)-concentration from Fluo3-fluorescence, hemolysis from hemoglobin release, lactate formation by colorimetry and ceramide utilizing fluorescent antibodies. A 48 hours exposure to benzethonium (=5μM) significantly increased cytosolic Ca(2+)-concentration, decreased forward scatter and triggered annexin V-binding affecting some 30% of the erythrocytes at 5 μM benzethonium. Only 5% of treated erythrocytes were hemolytic. The effects of benzethonium on annexin V binding were blunted in the nominal absence of Ca(2+) and in the presence of amiloride (1 mM) but not in the presence of the pancaspase inhibitor zVAD (10 μM). Benzethonium further significantly enhanced the effect of glucose depletion on cytosolic Ca(2+)-concentration and annexin V-binding, but significantly blunted the effect of glucose depletion on forward scatter. Benzethonium (5 μM) significantly enhanced lactic acid formation but not ceramide abundance. The present observations disclose a novel effect of benzethonium, i.e. triggering of suicidal death of erythrocytes.