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.

Trifluoperazine-Induced Suicidal Erythrocyte Death and S-Nitrosylation Inhibition, Reversed by the Nitric Oxide Donor Sodium Nitroprusside.

BACKGROUND AND PURPOSE: The high potency antipsychotic drug trifluoperazine (10-[3-(4-methyl-1-piperazinyl)-propyl]-2-(trifluoromethyl)-(10)H-phenothiazine dihydrochloride; TFP) may either counteract or promote suicidal cell death or apoptosis. Similar to apoptosis, erythrocytes may enter eryptosis, characterized by phosphatidylserine exposure at the cell surface and cell shrinkage. Eryptosis can be stimulated by an increase in cytoplasmic Ca2+ concentration ([Ca2+]i) and inhibited by nitric oxide (NO). We explored whether TFP treatment of erythrocytes induces phosphatidylserine exposure, cell shrinkage, and calcium influx, whether it impairs S-nitrosylation and whether these effects are inhibited by NO. METHODS: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, and protein nitrosylation from fluorescence switch of the Bodipy-TMR/Sypro Ruby signal. RESULTS: Exposure of human erythrocytes to TFP significantly enhanced the percentage of annexin-V-binding cells, raised [Ca2+]i, and decreased S-nitrosylation. The effect of TFP on annexin-V-binding was not affected by removal of extracellular Ca2+ alone, but was significantly inhibited by pre-treatment with sodium nitroprusside (SNP), an effect significantly augmented by additional removal of extracellular Ca2+. A 3 hours treatment with 0.1 µM Ca2+ ionophore ionomycin triggered annexin-V-binding and cell shrinkage, effects fully reversed by removal of extracellular Ca2+. CONCLUSIONS: TFP induces eryptosis and decreases protein S-nitrosylation, effects blunted by nitroprusside. The effect of nitroprusside is attenuated in the presence of extracellular Ca2+.

Age sensitivity of NFκB abundance and programmed cell death in erythrocytes induced by NFκB inhibitors.

BACKGROUND/AIMS: Erythrocytes may enter eryptosis, a suicidal death characterized by cell shrinkage and phosphatidylserine exposure at the erythrocyte outer membrane. Susceptibility to eryptosis is enhanced in aged erythrocytes and stimulated by NFκB-inhibitors Bay 11-7082 and parthenolide. Here we explored whether expression of NFκB and susceptibility to inhibitor-induced eryptosis is sensitive to erythrocyte age. METHODS: Human erythrocytes were separated into five fractions, based on age-associated characteristics cell density and volume. NFκB compared to ß-actin protein abundance was estimated by Western blotting and cell volume from forward scatter. Phosphatidylserine exposure was identified using annexin-V binding. RESULTS: NFκB was most abundant in young erythrocytes but virtually absent in aged erythrocytes. A 24h or 48h exposure to Ringer resulted in spontaneous decrease of forward scatter and increase of annexin V binding, effects more pronounced in aged than in young erythrocytes. Both, Bay 11-7082 (20 µM) and parthenolide (100 µM) triggered eryptosis, effects again most pronounced in aged erythrocytes. CONCLUSION: NFκB protein abundance is lowest and spontaneous eryptosis as well as susceptibility to Bay 11-7082 and parthenolide highest in aged erythrocytes. Thus, inhibition of NFκB signalling alone is not responsible for the stimulation of eryptosis by parthenolide or Bay 11-7082.

The impact of erythrocyte age on eryptosis.

Mature, circulating erythrocytes undergo senescence, which limits their life span to approximately 120 d. Upon injury, erythrocytes may undergo suicidal erythrocyte death or eryptosis, which may accelerate senescence and shorten their survival. Eryptosis is defined as cell shrinkage and exposure of phosphatidylserine at the cell surface. Triggers of eryptosis include oxidative stress. The present study addresses the impact of erythrocyte age on the relative susceptibility to eryptosis. Erythrocytes were separated into five fractions, based on age-associated differences in density and volume. Cell membrane scrambling was estimated from binding of annexin V to phosphatidylserine at the erythrocyte surface, the cell volume from forward scatter, and the Ca(2+) level from Fluo-3-dependent fluorescence. In addition, glutathione (GSH) concentrations were measured by an enzymatic/colourimetric method. After 48 h incubation in Ringer solution, Annexin V binding increased significantly with erythrocyte age. The differences were not accompanied by altered GSH concentrations, but were reversed by addition of the antioxidant N-acetyl-L-cysteine in vitro. Also, N-acetyl-L-cysteine significantly prolonged the half-life of circulating mouse erythrocytes in vivo. Thus, the susceptibility to eryptosis increases with the age of the erythrocytes, and this effect is at least partially due to enhanced sensitivity to oxidative stress.

Eryptosis: Programmed Death of Nucleus-Free, Iron-Filled Blood Cells.

Human erythrocytes are organelle-free cells packaged with iron-containing hemoglobin, specializing in the transport of oxygen. With a total number of approximately 25 trillion cells per individual, the erythrocyte is the most abundant cell type not only in blood but in the whole organism. Despite their low complexity and their inability to transcriptionally upregulate antioxidant defense mechanisms, they display a relatively long life time, of 120 days. This ensures the maintenance of tissue homeostasis where the clearance of old or damaged erythrocytes is kept in balance with erythropoiesis. Whereas the regulatory mechanisms of erythropoiesis have been elucidated over decades of intensive research, the understanding of the mechanisms of erythrocyte clearance still requires some refinement. Here, we present the main pathways leading to eryptosis, the programmed death of erythrocytes, with special emphasis on Ca2+ influx, the generation of ceramide, oxidative stress, kinase activation, and iron metabolism. We also compare stress-induced erythrocyte death with erythrocyte ageing and clearance, and discuss the similarities between eryptosis and ferroptosis, the iron-dependent regulated death of nucleated blood cells. Finally, we focus on the pathologic consequences of deranged eryptosis, and discuss eryptosis in the context of different infectious diseases, e.g., viral or parasitic infections, and hematologic disorders.

Coronavirus disease 2019 (COVID-19), human erythrocytes and the PKC-alpha/-beta inhibitor chelerythrine -possible therapeutic implication.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19. Until now, diverse drugs have been used for the treatment of COVID-19. These drugs are associated with severe side effects, e.g. induction of erythrocyte death, named eryptosis. This massively affects the oxygen (O2) supply of the organism. Therefore, three elementary aspects should be considered simultaneously: (1) a potential drug should directly attack the virus, (2) eliminate virus-infected host cells and (3) preserve erythrocyte survival and functionality. It is known that PKC-α inhibition enhances the vitality of human erythrocytes, while it dose-dependently activates the apoptosis machinery in nucleated cells. Thus, the use of chelerythrine as a specific PKC-alpha and -beta (PKC-α/-ß) inhibitor should be a promising approach to treat people infected with SARS-CoV-2.

Stimulation of eryptosis by cadmium ions.

Cadmium ions are known to trigger apoptosis. Erythrocytes may similarly undergo suicidal death or eryptosis, which is characterized by exposure of phosphatidylserine at the erythrocyte surface. As macrophages are equipped with phosphatidylserine receptors, they bind, engulf and degrade phosphatidylserine exposing cells. Cellular mechanisms known to trigger cell membrane phospholipid scrambling include increased cytosolic Ca(2+) activity and activation of a sphingomyelinase with formation of ceramide. The present experiments were performed to explore whether cadmium ions (Cd(2+)) trigger phosphatidylserine exposure of erythrocytes and to possibly identify underlying mechanisms. Phosphatidylserine exposure was estimated from annexin V-binding as determined in fluorescence activated cell sorting (FACS) analysis. Exposure to Cd(2+) (>or= 5.5 microM Cd(2+)) indeed significantly increased annexin V-binding. This effect was paralleled by erythrocyte shrinkage as apparent from the decrease of forward scatter in FACS analysis. According to Fluo3 fluorescence, Cd(2+) increased the entry of Ca(2+) into erythrocytes. According to antibody binding, Cd(2+) did not stimulate the formation of ceramide. In the nominal absence of extracellular Ca(2+) and in the presence of cation channel inhibitor amiloride the effects of Cd(2+) on erythrocyte phosphatidylserine exposure and forward scatter were blunted. In conclusion, in human erythrocytes Cd(2+) stimulates entry of Ca(2+), which activates Ca(2+)-sensitive K(+) channels leading to erythrocyte shrinkage and triggers Ca(2+)-sensitive erythrocyte membrane scrambling leading to phosphatidylserine exposure.

Suicidal erythrocyte death in sepsis.

Sequelae of sepsis include anemia which presumably results from accelerated clearance of erythrocytes from circulating blood. The underlying mechanisms, however, remained hitherto elusive. Most recent studies disclosed that increased cytosolic Ca2+ activity and ceramide both trigger suicidal erythrocyte death (i.e., eryptosis), which is characterized by lipid scrambling of the cell membrane leading to phosphatidylserine exposure at the erythrocyte surface. Phosphatidylserine exposing erythrocytes may adhere to vascular walls or may be engulfed by macrophages equipped with phosphatidylserine receptors. To explore whether sepsis leads to eryptosis, erythrocytes from healthy volunteers were exposed to plasma of patients suffering from sepsis, or to supernatants from sepsis producing pathogens. Then, phosphatidylserine exposure (annexin V binding), cell volume (forward scatter), cytosolic Ca2+ activity (Fluo3 fluorescence), and ceramide formation (anti-ceramide antibody) were determined by flow cytometry. Challenge of erythrocytes with plasma from the patients but not with plasma from healthy individuals triggered annexin V binding. The effect of patient plasma on erythrocyte annexin V binding was paralleled by formation of ceramide and a significant increase of cytosolic Ca2+ activity. Exposure of erythrocytes to supernatant of pathogens similarly induced eryptosis, an effect correlating with sphingomyelinase activity. The present observations disclose a novel pathophysiological mechanism leading to anemia and derangement of microcirculation during sepsis. Exposure to plasma from septic patients triggers phosphatidylserine exposure leading to adherence to the vascular wall and clearance from circulating blood.

Pharmacological targeting of glucose-6-phosphate dehydrogenase in human erythrocytes by Bay 11-7082, parthenolide and dimethyl fumarate.

In mature erythrocytes, glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) yield NADPH, a crucial cofactor of the enzyme glutathione reductase (GR) converting glutathione disulfide (GSSG) into its reduced state (GSH). GSH is essential for detoxification processes in and survival of erythrocytes. We explored whether the anti-inflammatory compounds Bay 11-7082, parthenolide and dimethyl fumarate (DMF) were able to completely deplete a common target (GSH), and to impair the function of upstream enzymes of GSH recycling and replenishment. Treatment of erythrocytes with Bay 11-7082, parthenolide or DMF led to concentration-dependent eryptosis resulting from complete depletion of GSH. GSH depletion was due to strong inhibition of G6PDH activity. Bay 11-7082 and DMF, but not parthenolide, were able to inhibit the GR activity. This approach "Inhibitors, Detection of their common target that is completely depleted or inactivated when pharmacologically relevant concentrations of each single inhibitor are applied, Subsequent functional analysis of upstream enzymes for this target" (IDS), can be applied to a broad range of inhibitors and cell types according to the selected target. The specific G6PDH inhibitory effect of these compounds may be exploited for the treatment of human diseases with high NADPH and GSH consumption rates, including malaria, trypanosomiasis, cancer or obesity.

Stimulation of eryptosis by aluminium ions.

Aluminium salts are utilized to impede intestinal phosphate absorption in chronic renal failure. Toxic side effects include anemia, which could result from impaired formation or accelerated clearance of circulating erythrocytes. Erythrocytes may be cleared secondary to suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and exposure of phosphatidylserine (PS) at the erythrocyte surface. As macrophages are equipped with PS receptors, they bind, engulf and degrade PS-exposing cells. The present experiments have been performed to explore whether Al(3+) ions trigger eryptosis. The PS exposure was estimated from annexin binding and cell volume from forward scatter in FACS analysis. Exposure to Al(3+) ions (> or =10 microM Al(3+) for 24 h) indeed significantly increased annexin binding, an effect paralleled by decrease of forward scatter at higher concentrations (> or =30 microM Al(3+)). According to Fluo3 fluorescence Al(3+) ions (> or =30 microM for 3 h) increased cytosolic Ca(2+) activity. Al(3+) ions (> or =10 microM for 24 h) further decreased cytosolic ATP concentrations. Energy depletion by removal of glucose similarly triggered annexin binding, an effect not further enhanced by Al(3+) ions. The eryptosis was paralleled by release of hemoglobin, pointing to loss of cell membrane integrity. In conclusion, Al(3+) ions decrease cytosolic ATP leading to activation of Ca(2+)-permeable cation channels, Ca(2+) entry, stimulation of cell membrane scrambling and cell shrinkage. Moreover, Al(3+) ions lead to loss of cellular hemoglobin, a feature of hemolysis. Both effects are expected to decrease the life span of circulating erythrocytes and presumably contribute to the development of anemia during Al(3+) intoxication.

Stimulation of erythrocyte phosphatidylserine exposure by paclitaxel.

Side effects of cytostatic treatment include development of anemia resulting from either decreased generation or accelerated clearance of circulating erythrocytes. Recent experiments revealed a novel kind of stress-induced erythrocyte death, i.e. eryptosis, which is characterized by enhanced cytosolic Ca(2+) levels, increased ceramide formation and exposure of phosphatidylserine at the cell surface. The present study explored whether cytostatic treatment with paclitaxel (Taxol) triggers eryptosis. Blood was drawn from cancer patients before and after infusion of 175 mg/m2 Taxol. The treatment significantly decreased the hematocrit and significantly increased the percentage of annexin-V-binding erythrocytes in vivo (by 37%). In vitro incubation of human erythrocytes with 10 microM paclitaxel again significantly increased annexin-V-binding (by 129%) and augmented the increase of annexin-V-binding following cellular stress. The enhanced phosphatidylserine exposure was not dependent on caspase-activity but paralleled by erythrocyte shrinkage, increase of cytosolic Ca(2+) activity, ceramide formation and activation of calpain. Phosphatidylserine exposure was similarly induced by docetaxel but not by carboplatin or doxorubicin. Moreover, eryptosis was triggered by the Ca(2+) ionophore ionomycin (10 microM). In mice, ionomycin-treated eryptotic erythrocytes were rapidly cleared from circulating blood and sequestrated into the spleen. In conclusion, our data strongly suggest that paclitaxel-induced anemia is at least partially due to induction of eryptosis.