Stimulation of Erythrocyte Cell Membrane Scrambling by Gedunin

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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 Ca²⁺-activity ([Ca²⁺]_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, [Ca²⁺]_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 [Ca²⁺]_i or forward scatter. PRIMA-1 (100 µM) induced annexin-V-binding was not significantly blunted by removal of extracellular Ca²⁺ 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.

Section: Discussionmentioning

confidence: 99%

Stimulation of Suicidal Erythrocyte Death by PRIMA-1

Faggio

Alzoubi

Calabrò

et al. 2015

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“…It is, therefore, possible that the CDK4 inhibitors II and III differentially target these mechanisms, independently of CDK4 kinase inhibition, in mitigating eryptosis due to oxidative stress or removal of extracellular Cl -ions. Eryptosis is enhanced by erythrocyte age [34], and is triggered by a wide variety of anemia-causing xenobiotics and endogeneous substances [35][36][37][38][39][40][41][42][43][44][45][46][47][48]. Furthermore, eryptosis participates in the pathophysiology of several clinical disorders such as iron deficiency, hepatic failure, chronic kidney disease, sepsis, malignancy, Wilson's disease and presumably metabolic syndrome [12,13,[49][50][51].…”

Section: +mentioning

confidence: 99%

Impact of Cyclin-Dependent Kinase CDK4 Inhibition on Eryptosis

Lang

Zelenak

Eberhard

et al. 2015

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Background/Aims: The cyclin-dependent kinase 4 (CDK4) participates in the regulation of apoptosis of nucleated cells by altering transcriptional regulation of genes governing cell proliferation and cell death. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine (PS) exposure at the cell surface. As mature erythrocytes lack nuclei, acute stimulation of eryptosis cannot result from altered gene expression. Eryptosis is triggered by isotonic cell shrinkage following Cl^- removal (replacement with the impermeant organic anion gluconate) or by oxidative stress (exposure to 0.3 mM tertbutyl-hydroperoxide [tBOOH]). The present study explored whether CDK4 is expressed in erythrocytes and whether the CDK4 inhibitors II (NSC625987) and III (ryuvidine) influence eryptosis. Methods: Western blotting was utilized for determination of the presence of CDK4 protein in human erythrocytes, and FACS analysis to determine Fluo3 fluorescence (reflecting cytosolic Ca²⁺), annexin-V-binding (reflecting PS-exposure) and forward scatter (reflecting cell volume). Results: CDK4 protein was present in human erythrocytes. Cl^- removal was followed by decrease of forward scatter and increase of both annexin-V-binding and Fluo3 fluorescence, an effect significantly curtailed by CDK4 inhibitors II and III. Furthermore, CDK4 inhibition blunted enhanced PS-exposure elicited by tBOOH treatment. Conclusions: The present observations disclose the presence of CDK4 protein in human erythrocytes and the suppression of suicidal erythrocyte death by pharmacological inhibition of CDK4.

“…Moreover, eryptosis is triggered by activated casein kinase 1α, Janus-activated kinase JAK3, protein kinase C, p38 kinase, and PAK2 kinase [25], as well as by inhibited or lacking AMP activated kinase AMPK, cGMP-dependent protein kinase, sorafenib sensitive kinases and sunitinib sensitive kinases [25]. Stimulators of eryptosis further include diverse xenobiotics [25,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57]. …”

Section: Introductionmentioning

confidence: 99%

Stimulation of Suicidal Erythrocyte Death by the Antimalarial Drug Mefloquine

Bissinger

Barking

Alzoubi

et al. 2015

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Background: The antimalarial drug mefloquine has previously been shown to stimulate apoptosis of nucleated cells. Similar to apoptosis, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include oxidative stress, increase of cytosolic Ca²⁺-activity ([Ca²⁺]_i), and ceramide. Methods: 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, [Ca²⁺]_i from Fluo3-fluorescence, and ceramide abundance from specific antibody binding. Results: A 48 h treatment of human erythrocytes with mefloquine significantly increased the percentage of annexin-V-binding cells (≥5 µg/ml), significantly decreased forward scatter (≥5 µg/ml), significantly increased ROS abundance (5 µg/ml), significantly increased [Ca²⁺]_i (7.5 µg/ml) and significantly increased ceramide abundance (10 µg/ml). The up-regulation of annexin-V-binding following mefloquine treatment was significantly blunted but not abolished by removal of extracellular Ca²⁺. Even in the absence of extracellular Ca²⁺, mefloquine significantly increased annexin-V-binding. Conclusions: Mefloquine treatment leads to erythrocyte shrinkage and erythrocyte membrane scrambling, effects at least partially due to induction of oxidative stress, increase of [Ca²⁺]_i and up-regulation of ceramide abundance.