Piperlongumine-Induced Phosphatidylserine Translocation in the Erythrocyte Membrane

Background: Piperlongumine, a component of Piper longum fruit, is considered as a treatment for malignancy. It is effective by inducing apoptosis. Mechanisms involved in the apoptotic action of piperlongumine include oxidative stress and activation of p38 kinase. In analogy to apoptosis of nucleated cells, erythrocytes may undergo eryptosis, the suicidal death of erythrocytes characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine-exposure at the erythrocyte surface. Signaling involved in eryptosis include increase of cytosolic Ca²⁺-activity ([Ca²⁺]_i), formation of ceramide, oxidative stress and activation of p38 kinase. Methods: Cell volume was estimated from forward scatter, phosphatidylserine-exposure from annexin V binding, [Ca²⁺]_i from Fluo3 fluorescence, reactive oxygen species from 2'',7''-dichlorodihydrofluorescein-diacetate fluorescence, and ceramide abundance from binding of fluorescent antibodies in flow cytometry. Results: A 48 h exposure to piperlongumine (30 µM) was followed by significant decrease of forward scatter and increase of annexin-V-binding. Piperlongumine did not significantly modify [Ca²⁺]_i and the effect was not dependent on presence of extracellular Ca²⁺. Piperlongumine significantly increased ROS formation and ceramide abundance. Conclusions: Piperlongumine triggers cell membrane scrambling, an effect independent from entry of extracellular Ca²⁺ but at least partially due to ROS and ceramide formation.     

In Vitro Sensitization of Erythrocytes to Programmed Cell Death Following Baicalein Treatment

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.     

Stimulation of Erythrocyte Cell Membrane Scrambling by Mushroom Tyrosinase

Background: Mushroom tyrosinase, a copper containing enzyme, modifies growth and survival of tumor cells. Mushroom tyrosinase may foster apoptosis, an effect in part due to interference with mitochondrial function. Erythrocytes lack mitochondria but are able to undergo apoptosis-like suicidal cell death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine-exposure at the erythrocyte surface. Signaling involved in the triggering of eryptosis include increase of cytosolic Ca²⁺-activity ([Ca²⁺]_i) and activation of sphingomyelinase with subsequent formation of ceramide. The present study explored, whether tyrosinase stimulates eryptosis. Methods: Cell volume has been 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 24 h exposure to mushroom tyrosinase (7 U/mL) was followed by a significant increase of [Ca²⁺]_i, a significant increase of ceramide abundance, and a significant increase of annexin-V-binding. The annexin-V-binding following tyrosinase treatment was significantly blunted but not abrogated in the nominal absence of extracellular Ca²⁺. Tyrosinase did not significantly modify forward scatter. Conclusions: Tyrosinase triggers cell membrane scrambling, an effect, at least partially, due to entry of extracellular Ca²⁺ and ceramide formation.     

Triggering of Programmed Erythrocyte Death by Alantolactone

The sesquiterpene alantolactone counteracts malignancy, an effect at least in part due to stimulation of suicidal death or apoptosis of tumor cells. Signaling of alantolactone induced apoptosis involves altered gene expression and mitochondrial depolarization. Erythrocytes lack mitochondria and nuclei but may enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the erythrocyte surface. Cellular mechanisms involved in triggering of eryptosis include increase of cytosolic Ca²⁺-activity ([Ca²⁺]_i) and oxidative stress. The present study explored, whether alantolactone stimulates eryptosis. To this end, erythrocyte volume was estimated from forward scatter, phosphatidylserine-exposure at the erythrocyte surface from FITC-annexin-V-binding, [Ca²⁺]_i from Fluo3-fluorescence, ceramide abundance from binding of fluorescent antibodies, and oxidative stress from 2'',7''-dichlorodihydrofluorescein-diacetate (DCFDA) fluorescence. As a result, a 48 h exposure of human erythrocytes to alantolactone (≥20 μM) significantly decreased erythrocyte forward scatter and increased the percentage of annexin-V-binding cells. Alantolactone significantly increased Fluo3 fluorescence (60 μM), ceramide abundance (60 μM) and DCFDA fluorescence (≥40 μM). The effect of alantolactone (60 μM) on annexin-V-binding was not significantly modified by removal of extracellular Ca²⁺. In conclusion, alantolactone stimulates suicidal erythrocyte death or eryptosis, an effect paralleled by increase of [Ca²⁺]_i, ceramide abundance and oxidative stress.     

Effect of Thioridazine on Erythrocytes

Background: Thioridazine, a neuroleptic phenothiazine with antimicrobial efficacy is known to trigger anemia. At least in theory, the anemia could result from stimulation of suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and by phospholipid scrambling of the cell membrane with phosphatidylserine exposure at the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca²⁺-concentration ([Ca²⁺]_i) and activation of p38 kinase. The present study explored, whether thioridazine elicits eryptosis. Methods: [Ca²⁺]_i has been estimated from Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin-V-binding, and hemolysis from hemoglobin release. Results: A 48 hours exposure to thioridazine was followed by a significant increase of [Ca²⁺]_i (30 µM), decrease of forward scatter (30 µM), and increase of annexin-V-binding (≥12 µM). Nominal absence of extracellular Ca²⁺ and p38 kinase inhibitor SB203580 (2 µM) significantly blunted but did not abolish annexin-V-binding following thioridazine exposure. Conclusions: Thioridazine stimulates eryptosis, an effect in part due to entry of extracellular Ca²⁺ and activation of p38 kinase.     

Induction of Suicidal Erythrocyte Death by Cantharidin

The natural phosphoprotein phosphatase inhibitor cantharidin, primarily used for topical treatment of warts, has later been shown to trigger tumor cell apoptosis and is thus considered for the treatment of malignancy. Similar to apoptosis of tumor cells, erythrocytes may undergo eryptosis, a suicidal cell death characterized by cell shrinkage and translocation of cell membrane phosphatidylserine to the erythrocyte surface. Signaling of eryptosis includes increase of cytosolic Ca²⁺-activity ([Ca²⁺]_i), ceramide, oxidative stress and dysregulation of several kinases. Phosphatidylserine abundance at the erythrocyte surface was quantified utilizing annexin-V-binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo3-fluorescence, ceramide from antibody binding, and reactive oxidant species (ROS) from 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence. A 48 h treatment of human erythrocytes with cantharidin significantly increased the percentage of annexin-V-binding cells (≥10 μg/mL), significantly decreased forward scatter (≥25 μg/mL), significantly increased [Ca²⁺]_i (≥25 μg/mL), but did not significantly modify ceramide abundance or ROS. The up-regulation of annexin-V-binding following cantharidin treatment was not significantly blunted by removal of extracellular Ca²⁺ but was abolished by kinase inhibitor staurosporine (1 μM) and slightly decreased by p38 inhibitor skepinone (2 μM). Exposure of erythrocytes to cantharidin triggers suicidal erythrocyte death with erythrocyte shrinkage and erythrocyte membrane scrambling, an effect sensitive to kinase inhibitors staurosporine and skepinone.     

Effect of miltefosine on erythrocytes

BackgroundMiltefosine, an alkylphosphocholine drug with antiparasite, antibacterial, antifungal and antineoplastic potency, is the only oral drug that can be used to treat visceral and cutaneous leishmaniasis. The effect of miltefosine is at least partially due to triggering of apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and by cell membrane scrambling with phosphatidylserine-exposure at the erythrocyte surface. Eryptosis may be triggered following increase of cytosolic Ca²⁺-level ([Ca²⁺]_i). The present study explored, whether miltefosine elicits eryptosis.MethodsCell volume has been estimated from forward scatter, phosphatidylserine-exposure from annexin-V-binding, hemolysis from hemoglobin release, [Ca²⁺]_i from Fluo3-fluorescence.ResultsA 48 h exposure to miltefosine (?4.9 μM) was followed by significant decrease of forward scatter and significant increase of annexin-V-binding. The effect was paralleled by significant increase of [Ca²⁺]_i. The annexin-V-binding following miltefosine treatment was significantly blunted in the nominal absence of extracellular Ca²⁺.ConclusionMiltefosine stimulates eryptosis, an effect at least partially due to stimulation of Ca²⁺ entry.     

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 Ca²⁺-activity ([Ca²⁺]_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, [Ca²⁺]_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 [Ca²⁺]_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 Ca²⁺. In conclusion, honokiol triggers suicidal erythrocyte death or eryptosis, an effect at least in part due to stimulation of Ca²⁺ entry and ceramide formation.     

Enhanced Eryptosis Following Gramicidin Exposure

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.     

Effect of Dermaseptin on Erythrocytes

Dermaseptin, an antimicrobial peptide participating in the host defence against pathogens, interacts with the membrane of target cells, leading to membrane permeabilization and eventual cell lysis. Dermaseptin has previously been shown to trigger haemolysis. Prior to haemolysis, 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. Triggers of eryptosis include increase in cytosolic Ca²⁺ activity [(Ca²⁺)]_i and formation of ceramide. This study explored whether dermaseptin modifies [Ca²⁺]_i and elicits eryptosis. Cell volume has been estimated from forward scatter, phosphatidylserine exposure from annexin‐V binding, haemolysis from haemoglobin release, ceramide formation from binding of fluorescent antibodies and [Ca²⁺]_i from Fluo3‐fluorescence. A 48‐hr exposure to dermaseptin (50 μM) was followed by a significant increase in [Ca²⁺]_i, a significant increase ceramide abundance, a significant decrease in forward scatter and a significant increase in annexin‐V binding. The annexin‐V binding after dermaseptin treatment was significantly blunted but not abrogated in the nominal absence of extracellular Ca²⁺. Dermaseptin triggers eryptosis, an effect at least partially due to entry of extracellular Ca²⁺.     

Triggering of Suicidal Erythrocyte Death by Celecoxib

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.     

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 Ca²⁺‐activity ([Ca²⁺]_i) and ceramide. The present study explored whether fumagillin (5–100 μM) could stimulate eryptosis. To this end, [Ca²⁺]_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 [Ca²⁺]_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 Ca²⁺ 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 Ca²⁺ entry, ceramide formation, phosphatidylserine exposure and decrease of cell volume.     

Effect of Nitazoxanide on Erythrocytes

Nitazoxanide, a drug effective against a variety of pathogens, triggers apoptosis and is thus considered to be employed against malignancy. Similar to nucleated cells, erythrocytes may undergo an apoptosis‐like suicidal cell death or eryptosis. Hallmarks of eryptosis include cell shrinkage and phospholipid scrambling of the cell membrane with translocation of phosphatidylserine to the erythrocyte surface. Stimulators of eryptosis include increase in cytosolic Ca²⁺‐activity ([Ca²⁺]_i). The Ca²⁺‐sensitivity of eryptosis is increased by ceramide. This study explored whether nitazoxanide triggers eryptosis. [Ca²⁺]_i was estimated from Fluo3‐fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin‐V‐binding, ceramide abundance utilizing fluorescent antibodies and haemolysis from haemoglobin release. A 48‐hr exposure to nitazoxanide (1–50 μg/ml) did not significantly modify [Ca²⁺]_i but significantly increased ceramide formation, decreased forward scatter (≥10 μg/ml), increased the percentage of annexin‐V‐binding erythrocytes (≥10 μg/ml) and, at higher concentrations (≥20 μg/ml), stimulated haemolysis. The stimulation of annexin‐V‐binding was significantly blunted in the absence of calcium. Nitazoxanide thus stimulates eryptosis, an effect in part due to ceramide formation.     

Induction of Suicidal Erythrocyte Death by Nelfinavir

The HIV protease inhibitor, nelfinavir, primarily used for the treatment of HIV infections, has later been shown to be effective in various infectious diseases including malaria. Nelfinavir may trigger mitochondria-independent cell death. Erythrocytes may undergo eryptosis, a mitochondria-independent suicidal cell death characterized by cell shrinkage and phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include oxidative stress and increase of cytosolic Ca²⁺-activity ([Ca²⁺]_i). During malaria, accelerated death of infected erythrocytes may decrease parasitemia and thus favorably influence the clinical course of the disease. In the present study, phosphatidylserine abundance at the cell surface was estimated from annexin V binding, cell volume from forward scatter, reactive oxidant species (ROS) from 2'',7''-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, and [Ca²⁺]_i from Fluo3-fluorescence. A 48 h treatment of human erythrocytes with nelfinavir significantly increased the percentage of annexin-V-binding cells (≥5µg/mL), significantly decreased forward scatter (≥2.5µg/mL), significantly increased ROS abundance (10 µg/mL), and significantly increased [Ca²⁺]_i (≥5 µg/mL). The up-regulation of annexin-V-binding following nelfinavir treatment was significantly blunted, but not abolished by either addition of the antioxidant N-acetylcysteine (1 mM) or removal of extracellular Ca²⁺. In conclusion, exposure of erythrocytes to nelfinavir induces oxidative stress and Ca²⁺ entry, thus leading to suicidal erythrocyte death characterized by erythrocyte shrinkage and erythrocyte membrane scrambling.     

Stimulation of Suicidal Erythrocyte Death by Sulforaphane

Sulforaphane, an isothiocyanate from cruciferous vegetable, counteracts malignancy. The effect is at least in part due to the stimulation of suicidal death or apoptosis of tumour cells. Mechanisms invoked in sulforaphane‐induced apoptosis include mitochondrial depolarization and altered gene expression. Despite the lack of mitochondria and nuclei, erythrocytes may, similar to apoptosis of nucleated cells, enter eryptosis, a suicidal cell death characterized by cell shrinkage and phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca²⁺‐activity ([Ca²⁺]_i). This study explored whether sulforaphane stimulates eryptosis. Cell volume was estimated from forward scatter, phosphatidylserine exposure at the cell surface from annexin V binding and [Ca²⁺]_i from Fluo‐3 fluorescence. A 48‐hr treatment of human erythrocytes with sulforaphane (50–100 μM) significantly decreased forward scatter, significantly increased the percentage of annexin V binding cells and significantly increased [Ca²⁺]_i. The effect of sulforaphane (100 μM) on annexin V binding was significantly blunted but not abrogated by the removal of extracellular Ca²⁺. Sulforaphane (100 μM) significantly increased ceramide formation. In conclusion, sulforaphane stimulates suicidal erythrocyte death or eryptosis, an effect at least partially, but not exclusively, due to the stimulation of Ca²⁺ entry and ceramide formation.     

Triggering of Erythrocyte Death by Triparanol

The cholesterol synthesis inhibitor Triparanol has been shown to trigger apoptosis in several malignancies. Similar to the apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include oxidative stress which may activate erythrocytic Ca²⁺ permeable unselective cation channels with subsequent Ca²⁺ entry and increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i). The present study explored whether and how Triparanol induces eryptosis. To this end, phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, hemolysis from hemoglobin release, [Ca²⁺]_i from Fluo3-fluorescence, and ROS formation from 2’,7’-dichlorodihydrofluorescein diacetate (DCFDA) dependent fluorescence. As a result, a 48 h exposure of human erythrocytes to Triparanol (20 µM) significantly increased DCFDA fluorescence and significantly increased Fluo3-fluorescence. Triparanol (15 µM) significantly increased the percentage of annexin-V-binding cells, and significantly decreased the forward scatter. The effect of Triparanol on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca²⁺. In conclusion, Triparanol leads to eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane. Triparanol is at least in part effective by stimulating ROS formation and Ca²⁺ entry.     

Fluoxetine Induced Suicidal Erythrocyte Death

The antidepressant fluoxetine inhibits ceramide producing acid sphingomyelinase. Ceramide is in turn known to trigger eryptosis the suicidal death of erythrocytes characterized by cell shrinkage and exposure of phosphatidylserine at the erythrocyte surface. Ceramide is effective through sensitizing the erythrocytes to the pro-eryptotic effect of increased cytosolic Ca²⁺ activity ([Ca²⁺]_i). In nucleated cells, fluoxetine could either inhibit or stimulate suicidal death or apoptosis. The present study tested whether fluoxetine influences eryptosis. To this end cell volume was estimated from forward scatter, phosphatidylserine exposure from annexin V binding, hemolysis from hemoglobin release and [Ca²⁺]_i from Fluo-3 fluorescence intensity. As a result, a 48 h exposure of erythrocytes to fluoxetine (≥25 µM) significantly decreased forward scatter, increased annexin V binding and enhanced [Ca²⁺]_i. The effect on annexin V binding was significantly blunted, but not abolished, in the absence of extracellular Ca²⁺. In conclusion, fluoxetine stimulates eryptosis, an effect at least in part due to increase of cytosolic Ca²⁺ activity.     

Triggering of Suicidal Erythrocyte Death by the Antibiotic Ionophore Nigericin

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 Ca²⁺‐permeable unselective cation channels leading to Ca²⁺ entry, increase in cytosolic Ca²⁺ activity ([Ca²⁺]_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, [Ca²⁺]_i from Fluo3 fluorescence, pH_i 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 Ca²⁺. The nigericin‐induced increase in [Ca²⁺]_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 Ca²⁺ entry, and involving the cariporide‐sensitive Na⁺/H⁺ exchanger.     

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.     

Stimulation of Suicidal Erythrocyte Death by Ellipticine

Ellipticine is a potent antineoplastic alkaloid effective in part by triggering apoptosis. Mechanisms involved in ellipticine‐induced apoptosis include mitochondrial depolarization and DNA damage. Erythrocytes lack mitochondria and nuclei but may nevertheless enter suicidal death or eryptosis, which is characterized by cell shrinkage and phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include increase in cytosolic Ca²⁺ activity ([Ca²⁺]_i), ceramide formation and oxidative stress. This study tested whether ellipticine stimulates eryptosis. Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter (FSC), [Ca²⁺]_i from Fluo‐3 fluorescence, ceramide abundance from binding of specific antibodies and reactive oxygen species from 2′,7′‐dichlorodihydrofluorescein diacetate fluorescence. A 24‐hr exposure of human erythrocytes to ellipticine (5 μg/ml) significantly increased the percentage of annexin V binding cells, ceramide abundance and oxidative stress. Ellipticine did not significantly modify [Ca²⁺]_i, and the stimulation of annexin V binding by ellipticine (5 μg/ml) did not require the presence of extracellular Ca²⁺. Ellipticine (5 μg/ml) did not significantly modify FSC. Ionomycin (1 μM, 1 hr) decreased FSC, an effect slightly but significantly blunted by ellipticine (5 μg/ml). Ellipticine thus stimulates phosphatidylserine translocation in the erythrocyte cell membrane, an effect at least partially due to stimulation of oxidative stress and ceramide formation.