Nazneen Shaik

Sunitinib-sensitive suicidal erythrocyte death.

Sunitinib, a multikinase inhibitor, stimulates apoptosis and is thus utilized for the treatment of malignancy. Even though lacking mitochondria and nuclei, critical elements in apoptosis of nucleated cells, erythrocytes may undergo eryptosis, an apoptosis-like suicidal death, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserineexposure at the cell surface. Triggers of eryptosis include activation of Ca2+ permeable cation channels with subsequent increase of cytosolic Ca2+-activity ([Ca2+]i), ceramide formation, ATP-depletion, stimulation of p38 kinase and caspase activation. The present study explored, whether sunitinib stimulates eryptosis. [Ca2+]i was estimated from Fluo-3-fluorescence, cell volume from forward scatter, phosphatidylserine-exposure from annexin-V-binding, hemolysis from hemoglobin release, ceramide abundance from anti-ceramide antibody binding, and cytosolic ATP from luciferin–luciferase activity. A 48 h exposure to sunitinib (10 µM) significantly decreased forward scatter and increased annexin-V-binding, effects paralleled by significant increase of [Ca2+]i. Sunitinib exposure was followed by a slight but significant increase of hemolysis. Sunitinib induced annexin-V-binding was slightly, but significantly blunted by removal of extracellular Ca2+, by p38 kinase inhibitor SB203580 (10 µM) and by the pancaspase inhibitor zVAD (10 µM). Sunitinib, however, did not significantly modify cytosolic ATP and ceramide abundance. The present observations reveal that sunitinib is able to trigger suicidal death in erythrocytes even in the absence of nuclei and mitochondria.

Inhibition of Ca2+ 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 Ca2+ activity, e.g. following energy depletion or oxidative stress. The present study thus explored whether naringin interferes with eryptosis. To this end, the cytosolic Ca2+ 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 Ca2+ concentration, the shrinkage, the cell membrane scrambling and thus the suicidal death of erythrocytes following energy depletion or oxidative stress.

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.

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 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.

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.

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 death of erythrocytes by rifampicin

The antibiotic rifampicin is widely used in the treatment of tuberculosis. Side effects of rifampicin include hemolytic anemia. Loss of circulating erythrocytes resembling hemolytic anemia could result from stimulation of eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine (PS) exposure at the cell surface. Stimulators of eryptosis include increase of cytosolic Ca(2+) activity ([Ca(2+)](i)) and formation of ceramide. The present study explored, whether and, if so, how rifampicin triggers eryptosis. To this end, [Ca(2+)](i) was estimated from Fluo3 fluorescence, cell volume from forward scatter in flow cytometry, PS exposure from annexin binding, ceramide formation from binding of fluorescent antibodies and hemolysis from hemoglobin release. As a result, a 48 h exposure to rifampicin (≥ 24 μg/ml) significantly increased Fluo3 fluorescence, ceramide abundance and annexin binding, and significantly decreased forward scatter. Rifampicin triggered slight, but significant hemolysis. Removal of extracellular Ca(2+) significantly blunted, but did not fully abolish rifampicin induced annexin binding. In conclusion, exposure of human erythrocytes to rifampicin is followed by suicidal erythrocyte death or eryptosis, an effect at least partially due to increase of cytosolic Ca(2+) concentration and stimulation of ceramide formation.

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 Ca2+-activity ([Ca2+]i), ceramide formation, ATP-depletion and caspase activation. The present study explored, whether gambogic acid triggers eryptosis of human erythrocytes. [Ca2+]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 [Ca2+]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 Ca2+ 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 Ca2+-entry, ceramide formation, cell shrinkage and phosphatidylserine-exposure.

Inhibition of suicidal erythrocyte death by probucol.

Abstract: Probucol, an antioxidant and anti-inflammatory agent counteracting atherosclerosis and restenosis, is partially effective by influencing suicidal cell death or apoptosis. In analogy to apoptosis of nucleated cells, suicidal death of erythrocytes or eryptosis is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the erythrocyte surface. Eryptosis is stimulated by increase in cytosolic Ca2+ activity, for example, after energy depletion or oxidative stress. The present study explored whether probucol influences eryptosis. Phosphatidylserine exposure was estimated from annexin-V-binding, cell volume from forward scatter (FSC), and cytosolic Ca2+ concentration from fluo-3 fluorescence in flow cytometry. As a result, energy depletion (48-hour glucose removal) increased annexin-V-binding, decreased FSC, and increased fluo-3 fluorescence. Probucol (⩽30 &mgr;M) did not significantly modify annexin-V-binding, FSC, or fluo-3 fluorescence in the presence of glucose but (at ≥5 &mgr;M) blunted the effect of glucose depletion on annexin-V-binding. Probucol (≥20 &mgr;M) only slightly blunted the effects of glucose depletion on FSC and fluo-3 fluorescence. Ca2+ ionophore ionomycin (1 &mgr;M) and oxidative stress (30-minute exposure to 0.3 mM of tert-butylhydroperoxide) increased annexin-V-binding, effects again blunted by 30 &mgr;M of probucol. In conclusion, probucol blunts cell membrane scrambling after energy depletion and oxidative stress, effects primarily because of interference with the scrambling effects of increased cytosolic Ca2+ concentration.

Stimulation of suicidal erythrocyte death by trans-cinnamaldehyde

Trans-cinnamaldehyde, a component of leaves from Cinnamomum osmophloeum kaneh, has been shown to counteract tumor growth. The substance exerts its effect at least in part by triggering apoptosis. The propapoptotic signaling involves altered gene expression and mitochondrial depolarization. In analogy to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the 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). The present study explored, whether trans-cinnamaldehyde triggers eryptosis. Cell volume has been estimated from forward scatter, phosphatidylserine-exposure from annexin-V-binding, hemolysis from hemoglobin release, and [Ca(2+)]i from Fluo3-fluorescence. A 48 h exposure to trans-cinnamaldehyde (30 μM) significantly decreased forward scatter and increased annexin-V-binding, effects paralleled by increase of [Ca(2+)]i. Trans-cinnamaldehyde exposure was followed by a slight but significant increase of hemolysis. Removal of extracellular Ca(2+) virtually abolished the effect of trans-cinnamaldehyde (30 μM) on annexin-V-binding. The present observations show that trans-cinnamaldehyde triggers suicidal death of erythrocytes, i.e. cells devoid of mitochondria and gene expression.