Fucoxanthin Induced Suicidal Death of Human Erythrocytes.

BACKGROUND/AIMS: Fucoxanthin, a carotenoid isolated from brown seaweeds, induces suicidal death or apoptosis of tumor cells and is thus considered for the treatment or prevention of malignancy. In analogy to apoptosis of nucleated cell, 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 Ca2+ entry with increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and activation of p38 kinase or protein kinase C. The present study explored, whether and how fucoxanthin induces eryptosis. METHODS: 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 abundance of reactive oxygen species (ROS) from DCFDA dependent fluorescence and lipid peroxidation using BODIPY fluoresence. RESULTS: A 48 hours exposure of human erythrocytes to fucoxanthin significantly increased the percentage of annexin-V-binding cells (≥ 50 µM), significantly decreased average forward scatter (≥ 25 µM), significantly increased hemolysis (≥ 25 µM), significantly increased Fluo3-fluorescence (≥ 50 µM), significantly increased lipid peroxidation, but did not significantly modify DCFDA fluorescence. The effect of fucoxanthin on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+, and was insensitive to p38 kinase inhibitor skepinone (2 µM) and to protein kinase C inhibitor calphostin (100 nM). CONCLUSION: Fucoxanthin triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to stimulation of Ca2+ entry.

Triggering of Suicidal Erythrocyte Death Following Boswellic Acid Exposure.

BACKGROUND/AIMS: The antinflammatory natural product boswellic acid is effective against cancer at least in part by inducing tumor cell apoptosis. Similar to apoptosis of nucleated cells erythrocytes may enter eryptosis, a suicidal death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include oxidative stress, increase of cytosolic Ca(2+)-activity ([Ca(2+)]i), energy depletion, ceramide formation and p38 kinase activation. The present study tested, whether and how boswellic acid induces eryptosis. METHODS: Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, hemolysis from hemoglobin release, [Ca(2+)]i from Fluo3-fluorescence, ceramide abundance utilizing specific antibodies, reactive oxygen species (ROS) from 2',7'-dichlorodihydrofuorescein diacetate (DCFDA) fluorescence, and cytosolic ATP concentration utilizing a luciferin-luciferase assay kit. RESULTS: A 24 hours exposure of human erythrocytes to boswellic acid (5 µg/ml) significantly increased the percentage of annexin-V-binding cells (to 9.3 ± 0.9 %) and significantly decreased forward scatter. Boswellic acid did not significantly modify [Ca(2+)]i, cytosolic ATP, ROS, or ceramide abundance. The effect of boswellic acid on annexin-V-binding was significantly blunted, but not abolished by p38 kinase inhibitors skepinone (2 µM) and SB203580 (2 µM). CONCLUSIONS: Boswellic acid stimulates cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part dependent on p38 protein kinase activity.

Stimulation of suicidal erythrocyte death by PRIMA-1.

BACKGROUND: The anticarcinogenic drug PRIMA-1 (p53 reactivation and induction of massive apoptosis 1) induces suicidal death of tumor cells, an effect in large part attributed to the up-regulation of the proapoptotic transcription factor p53. Erythrocytes are lacking gene transcription but are nevertheless able to enter eryptosis, a 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 Ca(2+)-activity ([Ca(2+)]i) and ceramide formation. The present study tested whether PRIMA-1 stimulates eryptosis. METHODS: Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, [Ca(2+)]i from Fluo3-fluorescence, ceramide abundance from binding of specific antibodies, and ROS formation from DCFDA fluorescence. RESULTS: A 48 h exposure of human erythrocytes to PRIMA-1 (25 µM) significantly increased the percentage of annexin-V-binding cells without significantly influencing [Ca(2+)]i or forward scatter. PRIMA-1 (100 µM) induced annexin-V-binding was not significantly blunted by removal of extracellular Ca(2+) or by the caspase-3 inhibitor zVAD. PRIMA-1 (100 µM) further increased the ceramide abundance at the cell surface and ROS formation. CONCLUSIONS: PRIMA-1 stimulates phosphatidylserine translocation at the erythrocyte cell membrane, an effect at least partially due to up-regulation of ceramide abundance and ROS formation.

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 Ca(2+) activity ([Ca(2+)]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), [Ca(2+)]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 [Ca(2+)]i , and the stimulation of annexin V binding by ellipticine (5 µg/ml) did not require the presence of extracellular Ca(2+). 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.

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 Ca(2+)-activity ([Ca(2+)]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 [Ca(2+)]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 [Ca(2+)]i. The effect of sulforaphane (100 µM) on annexin V binding was significantly blunted but not abrogated by the removal of extracellular Ca(2+). 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 Ca(2+) entry and ceramide formation.

Enhanced eryptosis following juglone exposure.

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.

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.

Aristolochic acid induced suicidal erythrocyte death.

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 Ca(2+)-activity ([Ca(2+)]i) and formation of ceramide. METHODS: Cell volume was estimated from forward scatter, phosphatidylserine-exposure from annexin V binding, [Ca(2+)]i from Fluo3 fluorescence, and ceramide abundance from binding of fluorescent antibodies in flow cytometry. RESULTS: A 48 hours exposure to Aristolochic Acid (≥ 75 µg/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 [Ca(2+)]i and significantly blunted, but not abrogated by removal of extracellular Ca(2+). Aristolochic Acid further significantly increased ceramide abundance. CONCLUSIONS: Aristolochic Acid triggers eryptosis, an effect at least in part due to entry of extracellular Ca(2+) and ceramide formation.

Stimulation of suicidal erythrocyte death by artesunate.

BACKGROUND: The artemisinin derivative artesunate is effective in the treatment of severe malaria and is considered for the treatment of malignancy. Artesunate triggers tumor cell apoptosis, an effect at least in part mediated by mitochondria. Even though lacking mitochondria, erythrocytes may similarly enter suicidal death or eryptosis, which is characterized by cell shrinkage and breakdown of the phospholipid asymmetry of the cell membrane with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca(2+)-activity ([Ca(2+)]i), ceramide formation, and oxidative stress. The present study explored whether artesunate stimulates eryptosis. METHODS: Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, [Ca(2+)]i from Fluo3-fluorescence, ceramide abundance from binding of specific antibodies, and oxidative stress from 2',7'-dichlorodihydrofluorescein-diacetate fluorescence. RESULTS: A 48 h exposure of human erythrocytes to artesunate significantly increased the percentage of annexin-V-binding cells (≥ 9 µg/ml) without significantly influencing forward scatter. Artesunate significantly increased [Ca(2+)]i. The stimulation of annexin-V-binding by artesunate (15 µg/ml) was significantly blunted but not abolished by removal of extracellular Ca(2+). Artesunate increased the ceramide abundance at the cell surface and the 2',7'-dichlorodihydrofluorescein-diacetate fluorescence. CONCLUSIONS: Artesunate stimulates phosphatidylserine translocation at the erythrocyte cell membrane, an effect at least partially due to increase of [Ca(2+)]i, stimulation of ceramide formation and generation of oxidative stress.