scholarly journals Diverse Effect of Vitamin C and N-Acetylcysteine on Aluminum-Induced Eryptosis

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Ali Reza Zangeneh ◽  
Mohammad Ali Takhshid ◽  
Reza Ranjbaran ◽  
Mahsa Maleknia ◽  
Mohammad Hassan Meshkibaf
Keyword(s):  
Oxidative Stress ◽  
Vitamin C ◽  
Ex Vivo ◽  
Annexin V ◽  
Dependent Manner ◽  
Ros Production ◽  
Cell Shrinkage ◽  
Forward Scatter ◽  
C And N ◽  
Vit C

Purpose. The role of oxidative stress in Aluminum (Al)-induced apoptotic effects has been investigated and suicidal death of erythrocytes, eryptosis, is characterized by cell shrinkage and phosphatidylserine externalization (PSE) at the surface of the erythrocyte cell membrane. Eryptosis is stimulated by an increase in cytosolic Ca2+ concentration and reactive oxygen species (ROS). This ex vivo study was conducted to evaluate the effect of well-known antioxidants including vitamin C (vit C) and N-acetylcysteine (NAC), against Al-induced hemolysis and eryptosis. Methods. Isolated erythrocytes from the healthy volunteers were partitioned into various groups (6 replicates/group) and treated by various concentrations of Al (3–100 µM) in the presence and absence of vit C (0.6 mM) and NAC (1 mM). After 24 hours of treatment, hemolysis was determined from hemoglobin levels in the supernatant. Flowcytometric methods were applied to measure PSE, cell shrinkage, Ca2+ content, and ROS abundance using annexin V-binding, forward scatter, Fluo3-fluorescence, and DCFDA dependent fluorescence, respectively. Reduced glutathione (GSH) was measured by the ELISA method. Results. The results showed that a 24 hours’ exposure of the erythrocytes to Al (10–100 µM) significantly increased hemolysis in a dose and Ca2+dependent manner. Al also dramatically decreased forward scatter. The percentage of PSE cells, Fluo3-fluorescence, and DCFDA fluorescence were increased by Al. Furthermore, cotreatment with NAC inhibited the effect of Al on hemolysis, eryptosis, and ROS production. Vit C decreased Al-induced ROS production. However, increased Al-induced eryptosis. There were no significant changes in glutathione after the ALCL3 treatment. Conclusions. Al-induced eryptosis and hemolysis through triggering oxidative stress, while NAC could diverse this effect. In contrast, vit C might intensify Al-induced eryptosis at particular doses through a less known mechanism.

scholarly journals Triggering of Suicidal Erythrocyte Death by Fascaplysin

2016 ◽  
Vol 39 (4) ◽  
pp. 1638-1647 ◽  
Author(s):  
Morena Mischitelli ◽  
Mohamed Jemaà ◽  
Mustafa Almasry ◽  
Caterina Faggio ◽  
Florian Lang
Keyword(s):  
Oxidative Stress ◽  
Cell Membrane ◽  
Indole Alkaloid ◽  
Annexin V ◽  
Hemoglobin Concentration ◽  
Cell Shrinkage ◽  
Cellular Mechanisms ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Phosphatidylserine Translocation

Background/Aims: The bis-indole alkaloid Fascaplysin is effective against malignancy, an effect at least partially due to stimulation of tumor cell apoptosis. Similar to apoptosis of nucleated cells, erythrocytes could enter suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and ceramide. The present study explored, whether Fascaplysin induces eryptosis and, if so, to shed light on the cellular mechanisms involved. Methods: Flow cytometry was employed to estimate phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was quantified from the hemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to Fascaplysin (≥ 5 µM) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter, and significantly increased Fluo3-fluorescence, DCFDA fluorescence as well as ceramide abundance. The effect of Fascaplysin on annexin-V-binding and forward scatter was significantly blunted but not abolished by removal of extracellular Ca2+. Conclusions: Fascaplysin triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry, oxidative stress and ceramide.

scholarly journals Triggering of Eryptosis, the Suicidal Erythrocyte Death by Mammalian Target of Rapamycin (mTOR) inhibitor Temsirolimus

2017 ◽  
Vol 42 (4) ◽  
pp. 1575-1591 ◽  
Author(s):  
Abdulla Al Mamun Bhuyan ◽  
Hang Cao ◽  
Florian Lang
Keyword(s):  
Oxidative Stress ◽  
Cell Membrane ◽  
Mtor Inhibitor ◽  
Mammalian Target Of Rapamycin ◽  
Annexin V ◽  
Target Of Rapamycin ◽  
Cell Shrinkage ◽  
Forward Scatter ◽  
Casein Kinase 1 ◽  
Erythrocyte Surface

Background/Aims: The mammalian target of rapamycin (mTOR) inhibitor temsirolimus is utilized for the treatment of malignancy. Temsirolimus is at least in part effective by triggering suicidal tumor cell death. The most common side effect of temsirolimus treatment is anemia. At least in theory, the anemia following temsirolimus treatment could result from stimulation of eryptosis, the suicidal erythrocyte death. Hallmarks of eryptosis include cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the orchestration of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, as well as activation of staurosporine and chelerythrine sensitive protein kinase C, SB203580 sensitive p38 kinase, D4476 sensitive casein kinase 1, and zVAD sensitive caspases. The purpose of the present study was to test whether temsirolimus influences eryptosis and, if so, to shed light on the signaling involved. Methods: Flow cytometry was employed to estimate cell volume from forward scatter, phosphatidylserine exposure at the cell surface from annexin-V-binding, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) abundance from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was determined from hemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to temsirolimus (5 – 20 µg/ml) significantly decreased forward scatter and significantly increased the percentage of annexin-V-binding cells. Temsirolimus significantly increased Fluo3-fluorescence, DCFDA fluorescence and ceramide abundance at the erythrocyte surface. The effect of temsirolimus on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca2+ and by addition of staurosporine (1 µM) or chelerythrine (10 µM) but not significantly modified by addition of SB203580 (2 µM), D4476 (10 µM), or zVAD (10 µM). Chelerythrine (10 µM) further significantly blunted the effect of temsirolimus on DCFDA fluorescence but not ceramide formation. Removal of extracellular Ca2+ had no effect on temsirolimus induced ROS formation or ceramide abundance. Conclusions: Temsirolimus triggers eryptosis with cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry, oxidative stress, ceramide and activation of staurosporine/Chelerythrine sensitive kinase(s).

scholarly journals Stimulation of Eryptosis, the Suicidal Erythrocyte Death, by Costunolide

2018 ◽  
Vol 50 (6) ◽  
pp. 2283-2295 ◽  
Author(s):  
Madeline Fink ◽  
Abdulla Al Mamun Bhuyan ◽  
Nefeli Zacharopoulou ◽  
Florian Lang
Keyword(s):  
Oxidative Stress ◽  
Cell Membrane ◽  
Annexin V ◽  
Cell Shrinkage ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation ◽  
Ros Formation ◽  
Binding Cell

Background/Aims: The sesquiterpene lactone Costunolide is effective against various disorders including inflammation and malignancy. The substance is effective in part by triggering suicidal death or apoptosis of tumor cells. Mechanisms involved include altered function of transcription factors and mitochondria. Erythrocytes lack nuclei and mitochondria but are – in analogy to apoptosis of nucleated cells – able to enter suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and ceramide. The present study explored, whether Costunolide induces eryptosis and, if so, to shed light on the mechanisms involved. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) formation from 2’,7’-dichlorodihydrofluorescein (DCF)-dependent fluorescence, and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to Costunolide (15 µg/ml) significantly enhanced the percentage of annexin-V-binding cells, significantly decreased forward scatter and significantly increased Fluo3-fluorescence, DCF-fluorescence, and ceramide abundance. The effect of Costunolide on annexin-V-binding was significantly blunted by removal of extracellular Ca2+. Conclusion: Costunolide triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry and paralleled by oxidative stress and ceramide formation.

scholarly journals Enhanced Eryptosis Following Exposure to Dolutegravir

2016 ◽  
Vol 39 (2) ◽  
pp. 639-650 ◽  
Author(s):  
Abdulla Al Mamun Bhuyan ◽  
Elena Signoretto ◽  
Rosi Bissinger ◽  
Florian Lang
Keyword(s):  
Oxidative Stress ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Annexin V ◽  
Cell Shrinkage ◽  
P38 Kinase ◽  
Cellular Mechanisms ◽  
Forward Scatter ◽  
Kinase C

Background/Aims: The viral integrase enzyme inhibitor dolutegravir is utilized for the treatment of immunodeficiency virus (HIV) infection. Knowledge on cytotoxicity of dolutegravir is limited. The present study thus explored, whether dolutegravir is able to trigger suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Cellular mechanisms involved in the triggering of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, and activation of protein kinase C, p38 kinase, casein kinase, and caspases. The present study explored, whether Dolutegravir induces eryptosis and, if so, to gain insight into cellular mechanisms involved. Methods: Utilizing flow cytometry, phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was quantified from haemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to dolutegravir significantly increased the percentage of annexin-V-binding cells (≥ 4.8 µM), significantly increased hemolysis (19.1 µM), but did not significantly modify forward scatter. Dolutegravir significantly increased Fluo3-fluorescence (≥ 4.8 µM), DCFDA fluorescence (19.1 µM) and ceramide abundance (19.1 µM). The effect of dolutegravir on annexin-V-binding was significantly blunted by removal of extracellular Ca2+, but was not significantly modified by protein kinase C inhibitor staurosporine (1 µM), p38 kinase inhibitor SB203580 (2 µM), casein kinase inhibitor D4476 (10 µM) or pancaspase inhibitor zVAD (10 µM). Conclusions: Dolutegravir triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry, ceramide formation and oxidative stress.

scholarly journals Stimulation of Suicidal Erythrocyte Death by Tafenoquine

2016 ◽  
Vol 39 (6) ◽  
pp. 2464-2476 ◽  
Author(s):  
Abdulla Al Mamun Bhuyan ◽  
Rosi Bissinger ◽  
Katja Stockinger ◽  
Florian Lang
Keyword(s):  
Oxidative Stress ◽  
Cell Membrane ◽  
Annexin V ◽  
Casein Kinase ◽  
Cell Shrinkage ◽  
Cellular Mechanisms ◽  
Forward Scatter ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation ◽  
Stimulation Of

Background/Aims: The 8-aminoquinoline tafenoquine has been shown to be effective against Plasmodia, Leishmania and Trypanosoma. The substance is at least in part effective by triggering apoptosis of the parasites. Similar to apoptosis, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the regulation of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, zVAD sensitive caspases, SB203580 sensitive p38 kinase, staurosporine sensitive protein kinase C as well as D4476 sensitive casein kinase. The present study explored, whether tafenoquine induces eryptosis and aimed to possibly identify cellular mechanisms involved. Methods: Flow cytometry was employed to estimate phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) dependent fluorescence, and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to tafenoquine (500 ng/ml) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter, significantly increased Fluo3-fluorescence, and significantly increased DCFDA fluorescence. Tafenoquine did not significantly modify ceramide abundance. The effect of tafenoquine on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca2+. The effect of tafenoquine on annexin-V-binding was not significantly blunted by zVAD (10 µM), SB203580 (2 µM) or staurosporine (1 µM). The effect of tafenoquine on annexin-V-binding was significantly blunted but not abolished by D4476 (10 µM). Conclusions: Tafenoquine triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to stimulation of Ca2+ entry, oxidative stress and possibly activation of casein kinase.

scholarly journals Triggering of Suicidal Erythrocyte Death by Pazopanib

2016 ◽  
Vol 38 (3) ◽  
pp. 926-938 ◽  
Author(s):  
Elena Signoretto ◽  
Jens Zierle ◽  
Rosi Bissinger ◽  
Michela Castagna ◽  
Elena Bossi ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Kinase Inhibitor ◽  
Annexin V ◽  
Cell Shrinkage ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation ◽  
Ros Formation ◽  
Binding Cell

Background/Aims: The multi-targeted kinase inhibitor pazopanib, a drug employed for the treatment of a wide variety of malignancies, has previously been shown to trigger apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Mechanisms involved in the triggering of eryptosis include Ca2+ entry, oxidative stress and ceramide. The present study explored, whether pazopanib induces eryptosis and, if so, whether it is effective by Ca2+ entry, oxidative stress and/or ceramide. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, reactive oxygen species (ROS) formation from DCF dependent fluorescence, and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to pazopanib significantly increased the percentage of annexin-V-binding (≥ 25 µg/ml) and of shrunken erythrocytes (≥ 50 µg/ml). Pazopanib treatment further resulted in significant hemolysis (≥ 25 µg/ml). The effect of pazopanib on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca2+. Pazopanib significantly increased DCF fluorescence (50 µg/ml) and ceramide abundance (50 µg/ml). Conclusions: Pazopanib triggers eryptosis, an effect involving Ca2+ entry, oxidative stress and ceramide.

scholarly journals Enhanced Eryptosis Following Auranofin Exposure

2015 ◽  
Vol 37 (3) ◽  
pp. 1018-1028 ◽  
Author(s):  
Kousi Alzoubi ◽  
Jasmin Egler ◽  
Majed Abed ◽  
Florian Lang
Keyword(s):  
Oxidative Stress ◽  
Cell Membrane ◽  
Annexin V ◽  
Cell Shrinkage ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation ◽  
Phospholipid Scrambling ◽  
Binding Cell

Background/Aims: The antiinflammatory, antimicrobial and anticancer drug auranofin has previously been shown to trigger apoptosis, the suicidal death of nucleated cells. Side effects of the drug include anaemia. At least in theory the anaemia could result from stimulation of suicidal death of erythrocytes or eryptosis, which involves cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Methods: Stimulators of eryptosis include oxidative stress and increase of cytosolic Ca2+-activity ([Ca2+]i). In the present study, phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, hemolysis from hemoglobin release, reactive oxygen species (ROS) from 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, and [Ca2+]i from Fluo3-fluorescence. Results: A 24 hours exposure of human erythrocytes to auranofin (≥5 µg/ml) significantly increased the percentage of annexin-V-binding cells (from 2.2 ± 0.5 to 17.4 ± 1.5%), significantly decreased forward scatter and significantly enhanced ROS. At higher concentrations (10 µg/ml) auranofin triggered slight hemolysis (from 2.1 ± 0.2 to 3.2 ± 0.3%). Conclusions: Auranofin stimulates cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least partially due to induction of oxidative stress.

scholarly journals Nocodazole Induced Suicidal Death of Human Erythrocytes

2016 ◽  
Vol 38 (1) ◽  
pp. 379-392 ◽  
Author(s):  
Elena Signoretto ◽  
Sabina Honisch ◽  
Marilena Briglia ◽  
Caterina Faggio ◽  
Michela Castagna ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Membrane ◽  
Annexin V ◽  
Human Erythrocytes ◽  
Microtubule Assembly ◽  
Cell Shrinkage ◽  
Forward Scatter ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation ◽  
Mitotic Death

Background: The microtubule assembly inhibitor nocodazole has been shown to trigger caspase-independent mitotic death and caspase dependent apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may undergo 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), oxidative stress and ceramide. The present study explored, whether and how nocodazole induces eryptosis. Methods: Flow cytometry was employed to determine phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, the abundance of reactive oxygen species (ROS) from 2′,7′-dichlorodihydrofluorescein (DCF) diacetate dependent fluorescence as well as ceramide surface abundance utilizing specific antibodies. Tubulin abundance was quantified by TubulinTracker™ Green reagent and visualized by confocal microscopy. Results: A 48 hours exposure of human erythrocytes to nocodazole (≥ 30 µg/ml) significantly increased the percentage of annexin-V-binding cells without significantly modifying average forward scatter. Nocodazole significantly increased Fluo3-fluorescence, significantly increased DCF fluorescence and significantly increased ceramide surface abundance. The effect of nocodazole on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+ and was not modified in the presence of Caspase 3 inhibitor zVAD (1 µM). Nocodazole treatment reduced the content of total tubulin. Conclusions: Nocodazole triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to stimulation of Ca2+ entry, oxidative stress and ceramide.

scholarly journals Zopolrestat Induced Suicidal Death of Human Erythrocytes

2015 ◽  
Vol 37 (4) ◽  
pp. 1537-1546 ◽  
Author(s):  
Ghada Bouguerra ◽  
Rosi Bissinger ◽  
Salem Abbès ◽  
Florian Lang
Keyword(s):  
Oxidative Stress ◽  
Cell Membrane ◽  
Annexin V ◽  
Human Erythrocytes ◽  
Cell Shrinkage ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation ◽  
Binding Cell

Background/Aims: The aldose reductase inhibitor zopolrestat has been shown to either decrease or increase apoptosis, the suicidal death of nucleated cells. Erythrocytes may similarly enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include oxidative stress, Ca2+ entry with increase of cytosolic Ca2+ activity ([Ca2+]i), and ceramide formation. The present study explored, whether and how zopolrestat induces eryptosis. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, oxidative stress from DCFDA dependent fluorescence, [Ca2+]i from Fluo3-fluorescence, and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to zopolrestat (≥ 150 µg/ml) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter (≥ 125 µg/ml), significantly increased Fluo3-fluorescence (200 µg/ml), significantly increased ceramide abundance (150 µg/ml), but did not significantly modify DCFDA fluorescence. The effect of zopolrestat on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+. Conclusions: Exposure of human erythrocytes to zopolrestat triggers cell shrinkage and cell membrane scrambling, an effect in part due to Ca2+ entry and ceramide.

scholarly journals Stimulation of Eryptosis, the Suicidal Erythrocyte Death by Piceatannol

2016 ◽  
Vol 38 (6) ◽  
pp. 2300-2310 ◽  
Author(s):  
Elena Signoretto ◽  
Michela Castagna ◽  
Florian Lang
Keyword(s):  
Oxidative Stress ◽  
Cell Membrane ◽  
Annexin V ◽  
Hemoglobin Concentration ◽  
Cell Shrinkage ◽  
Cellular Mechanisms ◽  
Cytochrome C Release ◽  
Forward Scatter ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation

Background/Aims: Piceatannol, an analog and metabolite of resveratrol, is effective against various disorders including malignancy. It is in part effective by triggering suicidal death or apoptosis of tumor cells. Cellular mechanisms mediating the proapoptotic effect of Piceatannol include mitochondrial depolarization and cytochrome c release. Erythrocytes lack mitochondria but may nevertheless enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Cellular mechanisms involved in the triggering of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and ceramide formation. The present study explored, whether Piceatannol induces eryptosis and, if so, to shed some light on the cellular mechanisms involved. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) formation from 2',7'-dichlorodihydrofluorescein (DCF) diacetate-dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemoglobin concentration in the supernatant was taken as measure of hemolysis. Results: A 48 hours exposure of human erythrocytes to Piceatannol (10 - 20 µM) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter, significantly increased DCFDA-fluorescence, significantly increased ceramide abundance, but did not significantly increase Fluo3-fluorescence. Removal of extracellular Ca2+ slightly blunted but did not abolish the effect of Piceatannol on annexin-V-binding and forward scatter. Piceatannol (20 µM) significantly augmented the increase of annexin-V-binding, but significantly blunted the decrease of forward scatter following treatment with the Ca2+ ionophore ionomycin. Conclusions: Piceatannol triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part downstream of Ca2+ and involving oxidative stress and ceramide formation.

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