Patulin-induced suicidal erythrocyte death.

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 Ca(2+)-activity ([Ca(2+)]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 [Ca(2+)]i. RESULTS: A 48 h exposure to patulin significantly increased [Ca(2+)]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 Ca(2+). CONCLUSION: Patulin stimulates Ca(2+) entry into erythrocytes, an effect triggering suicidal erythrocyte death or eryptosis.

Effect of honokiol on erythrocytes.

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.

Withaferin A-stimulated Ca2+ entry, ceramide formation and suicidal death of erythrocytes.

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 Ca(2+)-activity [Ca(2+)](i) following activation of oxidant-sensitive Ca(2+)-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 Ca(2+) 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.

Stimulation of suicidal erythrocyte death by fumagillin.

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.

Sulindac sulfide--induced stimulation of eryptosis.

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 Ca(2+)-activity ([Ca(2+)](i)) and ceramide formation. The present study explored, whether sulindac sulfide stimulates eryptosis. METHODS: [Ca(2+)](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 [Ca(2+)](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 Ca(2+) 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 Ca(2+)-entry, ceramide formation, cell shrinkage and phosphatidylserine-exposure.

Enhanced Ca2+ entry, ceramide formation, and apoptotic death of erythrocytes triggered by plumbagin.

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.

Gossypol-induced suicidal erythrocyte death.

Side effects of gossypol, a polyphenolic component of Gossypium, with male contraceptive, anticancer, antimicrobial and antiviral activities include anemia due to accelerated demise of erythrocytes. Erythrocytes may be cleared from circulating blood following apoptosis-like suicidal death or eryptosis. Hallmarks of eryptosis are cell shrinkage and cell membrane scrambling with subsequent phosphatidylserine-exposure at the cell surface. Stimulators of eryptosis include increase of cytosolic Ca(2+)-activity ([Ca(2+)](i)). The present study explored, whether gossypol stimulates eryptosis of human erythrocytes. Utilizing flow cytometry, [Ca(2+)](i) was estimated from Fluo-3 fluorescence, cell volume from forward scatter, phosphatidylserine-exposure from annexin-V-binding, and hemolysis from hemoglobin release. A 48 h exposure to gossypol (0.75 µM) significantly increased [Ca(2+)](i), decreased forward scatter and increased annexin-V-binding. Gossypol exposure was followed by a slight but significant increase of hemolysis. Removal of extracellular Ca(2+) significantly blunted the effect of gossypol (1 µM) on annexin-V-binding. The present observations reveal a novel effect of gossypol on human erythrocytes, which contributes to or even accounts for the triggering of anemia by this substance.

Enhanced apoptotic death of erythrocytes induced by the mycotoxin ochratoxin A.

BACKGROUND: The mycotoxin ochratoxin A, an agent responsible for endemic Balkan nephropathy is known to trigger apoptosis and thus being toxic to several organs including the kidney. The mechanisms involved in ochratoxin A induced apoptosis include oxidative stress. Sequelae of ochratoxin intoxication include anemia. Similar to apoptosis of nucleated cells, erythrocytes may undergo suicidal cell death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling resulting in phosphatidylserine-exposure at the cell surface. Eryptosis could be triggered by Ca2+ -entry through oxidant sensitive unspecificcation channels increasing cytosolic Ca2+ activity ([Ca2+]i). The Ca2+ -sensitivity of cell membrane scrambling could be enhanced and eryptosis thus triggered by ceramide. The removal of suicidal erythrocytes may lead to anemia. Moreover, eryptotic erythrocytes could adhere to the vascular wall thus impeding microcirculation. The present study explored, whether ochratoxin A stimulates eryptosis. METHODS: Fluo3-fluorescence was utilized to determine [Ca2+]i, forward scatter to estimate cell volume, annexin-V-binding to identify phosphatidylserine-exposing cells, fluorescent antibodies to detect ceramide formation and hemoglobin release to quantify hemolysis. Moreover, adhesion to human vascular endothelial cells (HUVEC) was determined utilizing a flow chamber. RESULTS: A 48 h exposure to ochratoxin A was followed by significant increase of Fluo3-fluorescencei (≥ 2.5 µM), increase of ceramide abundance (10 µM), decrease of forward scatter (≥ 5 µM) and increase of annexin-V-binding (≥ 2.5 µM). Ochratoxin A exposure slightly but significantly enhanced hemolysis (10 µM). Ochratoxin (10 µM) enhanced erythrocyte adhesion to HUVEC. Removal of extracellular Ca2+ significantly blunted, but did not abrogate ochratoxin A-induced annexin V binding. CONCLUSIONS: Ochratoxin A triggers suicidal erythrocyte death or eryptosis, an effect partially but not fully due to stimulation of Ca2+ -entry.

Inhibition of Ca(2+) entry and suicidal erythrocyte death by naringin.

Naringin is a dietary flavonoid from citrus fruits with antioxidant and antiapoptotic activity. Similar to apoptosis of nucleated cells, suicidal death of erythrocytes or eryptosis is paralleled by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the erythrocyte surface. Eryptosis is triggered by increased cytosolic Ca(2+) activity, e.g. following energy depletion or oxidative stress. The present study thus explored whether naringin interferes with eryptosis. To this end, the cytosolic Ca(2+) concentration was estimated from Fluo3 fluorescence, phosphatidylserine exposure from annexin-V-binding and cell volume from forward scatter in FACS analysis. As a result, energy depletion (48 h glucose removal) and oxidative stress (30 min exposure to 0.3 mM tert-butylhydroperoxide) increased Fluo-3 fluorescence, decreased the erythrocyte forward scatter and enhanced the percentage of annexin-V-binding erythrocytes. Naringin (up to 40 µM) did not significantly modify Fluo-3 fluorescence, erythrocyte forward scatter or annexin-V-binding in the presence of glucose and absence of oxidative stress. Naringin, however, significantly blunted the effect of glucose depletion and oxidative stress on Fluo-3 fluorescence, erythrocyte forward scatter or annexin-V-binding. In conclusion, naringin blunts the increase of cytosolic Ca(2+) concentration, the shrinkage, the cell membrane scrambling and thus the suicidal death of erythrocytes following energy depletion or oxidative stress.

Enhanced erythrocyte membrane exposure of phosphatidylserine following sorafenib treatment: an in vivo and in vitro study.

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 Ca(2+)activity ([Ca(2+)](i)), formation of ceramide, ATP-depletion and oxidative stress. The present study explored, whether sorafenib triggers eryptosis in vitro and in vivo. METHODS: [Ca(2+)](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.

Induction of programmed erythrocyte death by gambogic acid.

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.

Induction of apoptotic erythrocyte death by rotenone.

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.

Hexavalent chromium-induced erythrocyte membrane phospholipid asymmetry.

Hexavalent (VI) chromium is a global contaminant with cytotoxic activity. Chromium (VI) induces oxidative stress, inflammation, cell proliferation, malignant transformation and may trigger carcinogenesis and at the same time apoptosis. The toxic effects of chromium (VI) at least partially result from mitochondrial injury and DNA damage. Erythrocytes lack mitochondria and nuclei but may experience an apoptosis-like suicidal cell death, i.e. eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Eryptosis may result from increase of cytosolic Ca(2+) activity, ATP depletion and/or ceramide formation. The present study explored, whether chromium (VI) triggers eryptosis. Fluo-3-fluorescence was employed to determine cytosolic Ca(2+)-concentration, forward scatter to estimate cell volume, binding of fluorescent annexin V to detect phosphatidylserine exposure, hemoglobin concentration in the supernatant to quantify hemolysis, luciferin-luciferase to determine cytosolic ATP concentration and fluorescent anti-ceramide antibodies to uncover ceramide formation. A 48 h exposure to chromium (VI) (≥10 µM) significantly increased cytosolic Ca(2+)-concentration, decreased ATP concentration (20 µM), decreased forward scatter, increased annexin V-binding and increased (albeit to a much smaller extent) hemolysis. Chromium (VI) did not significantly modify ceramide formation. The effect of 20 µM chromium (VI) on annexin V binding was partially reversed in the nominal absence of Ca(2+). The present observations disclose a novel effect of chromium (VI), i.e. Ca(2+) entry and cytosolic ATP depletion in erythrocytes, effects resulting in eryptosis with cell shrinkage and cell membrane scrambling.

Apigenin-induced suicidal erythrocyte death.

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.