The specific PKC-α inhibitor chelerythrine blunts costunolide-induced eryptosis.

Costunolide, a natural sesquiterpene lactone, has multiple pharmacological activities such as neuroprotection or induction of apoptosis and eryptosis. However, the effects of costunolide on pro-survival factors and enzymes in human erythrocytes, e.g. glutathione and glucose-6-phosphate dehydrogenase (G6PDH) respectively, have not been studied yet. Our aim was to determine the mechanisms underlying costunolide-induced eryptosis and to reverse this process. Phosphatidylserine exposure was estimated from annexin-V-binding, cell volume from forward scatter in flow cytometry, and intracellular glutathione [GSH]i from high performance liquid chromatography. The oxidized status of intracellular glutathione and enzyme activities were measured by spectrophotometry. Treatment of erythrocytes with costunolide dose-dependently enhanced the percentage of annexin-V-binding cells, decreased the cell volume, depleted [GSH]i and completely inhibited G6PDH activity. The effects of costunolide on annexin-V-binding and cell volume were significantly reversed by pre-treatment of erythrocytes with the specific PKC-α inhibitor chelerythrine. The latter, however, had no effect on costunolide-induced GSH depletion. Costunolide induces eryptosis, depletes [GSH]i and inactivates G6PDH activity. Furthermore, our study reveals an inhibitory effect of chelerythrine on costunolide-induced eryptosis, indicating a relationship between costunolide and PKC-α. In addition, chelerythrine acts independently of the GSH depletion. Understanding the mechanisms of G6PDH inhibition accompanied by GSH depletion should be useful for development of anti-malarial therapeutic strategies or for synthetic lethality-based approaches to escalate oxidative stress in cancer cells for their sensitization to chemotherapy and radiotherapy.

Eryptosis in Haemochromatosis: Implications for rheology.

BACKGROUND: Haemochromatosis is an iron-storage disease with different genetic mutations, characterized by an increased intestinal absorption of iron, resulting in a deposition of excessive amounts of iron in parenchymal cells. When the iron is released in the blood, it is left in an unliganded form, where it can participate in Haber-Weiss and Fenton reactions, creating hydroxyl radicals. Erythrocytes (RBCs) are particularly vulnerable to hydroxyl radical damage, which can result in eryptosis (programmed cell death similar to apoptosis). STUDY DESIGN AND METHODS: Here, we used flow cytometry to study the presence of eryptosis in the main genotypic variations of HFE (heterozygous and homozygous C282Y; H63D; C282Y/H63D). We also viewed RBCs from the different mutations using super-resolution Airyscan confocal microscopy. RESULTS: Flow cytometry showed significant changes in membrane biochemistry, indicated by the presence of phosphatidylserine (PS) proteins on the outer leaflet of the membrane, as well as increased intracellular calpain. This was found in all of the studied mutations. Airyscan fluorescence revealed PS flip and also microparticles from RBCs. Such microparticles are known to be pro-inflammatory. CONCLUSION: We conclude that RBC pathology is present in all the studied HFE mutations, even in low penetrance mutations, and this might affect rheology in these individuals.

Eryptosis: An Erythrocyte's Suicidal Type of Cell Death.

Erythrocytes play an important role in oxygen and carbon dioxide transport. Although erythrocytes possess no nucleus or mitochondria, they fulfil several metabolic activities namely, the Embden-Meyerhof pathway, as well as the hexose monophosphate shunt. Metabolic processes within the erythrocyte contribute to the morphology/shape of the cell and important constituents are being kept in an active, reduced form. Erythrocytes undergo a form of suicidal cell death called eryptosis. Eryptosis results from a wide variety of contributors including hyperosmolarity, oxidative stress, and exposure to xenobiotics. Eryptosis occurs before the erythrocyte has had a chance to be naturally removed from the circulation after its 120-day lifespan and is characterised by the presence of membrane blebbing, cell shrinkage, and phosphatidylserine exposure that correspond to nucleated cell apoptotic characteristics. After eryptosis is triggered there is an increase in cytosolic calcium (Ca2+) ion levels. This increase causes activation of Ca2+-sensitive potassium (K+) channels which leads to a decrease in intracellular potassium chloride (KCl) and shrinkage of the erythrocyte. Ceramide, produced by sphingomyelinase from the cell membrane's sphingomyelin, contributes to the occurrence of eryptosis. Eryptosis ensures healthy erythrocyte quantity in circulation whereas excessive eryptosis may set an environment for the clinical presence of pathophysiological conditions including anaemia.

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

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).

The effects of obesity on CD47 expression in erythrocytes.

BACKGROUND: To investigate the effects of obesity on CD47, phosphatidylserine (PS) exposure, and Caspase-8 and Caspase-3 activities in erythrocytes. METHODS: The study included 25 morbidly obese patients and 20 healthy people as the control group. We evaluated CD47 expression on the red blood cell (RBC) membrane surface and eryptosis markers such as PS externalization and caspase activity using flow cytometric analyses. RESULTS: CD47 expression on the RBC surface was significantly lower in obese patients than in the control group (P = 0.000001). We did not find significant differences in the Caspase-3 and Caspase-8 activities between the obese and nonobese control groups. Additionally, we did not find differences in PS exposure on erythrocyte membranes. The fibrinogen levels were higher in the obese group than they were in the control group (P = 0.00002). Correlations between CD47 expression and body mass index (r = -0.65; P = 0.0004), waist circumference (r = -0.54; P = 0.0052), and fibrinogen (r = 0.57; P = 0.0024) were found. Univariate analyses revealed that body mass index, waist circumference, hip circumference, and fibrinogen levels were potential predictors of CD47 expression. Multivariate analyses found that fibrinogen levels (ß = 0.4708; P = 0.045) independently predicted CD47 expression. CONCLUSIONS: The study demonstrated that CD47 expression is decreased on the surface of RBCs in obese subjects. These changes in CD47 expression on the RBC surface may be an adaptive response to hyperfibrinogenemia associated with obesity. © 2015 International Clinical Cytometry Society.