Modification of Eryptosis, the Suicidal Erythrocyte Death, by Afatinib, Ceritinib, and Volasertib

Afatinib, Ceritinib und Volasertib werden zur Behandlung von Malignomen eingesetzt. Eine Nebenwirkung ist Anämie, die das Resultat einer übermäßigen Eryptose sein könnte. Die Eryptose, der suizidale Erythrozytentod, ist durch Zusammenbrechen der Phosphatidylserin (PS) - Asymmetrie in der Zellmembran, Zellschrumpfung und Bildung von Membranbläschen gekennzeichnet. Stimuli wie z.B. Energieentzug, hyperosmotischer Schock, oxidativer Stress sowie verschiedene Xenobiotika/Erkrankungen sind bekannte Stimulatoren der Eryptose. Die Signalmechanismen, die der Eryptose zu Grunde liegen, umfassen einen Überschuss an intrazellulärem Kalzium, Ceramid und reaktiven Sauerstoffspezies (ROS) sowie die Beteiligung verschiedener Kinasen und Caspasen. Um eine Wirkung der Substanzen auf Eryptose aufzudecken, wurden gesunde humane Erythrozyten (0,4% Hämatokrit) verschiedenen Konzentrationen der Substanzen ausgesetzt und für 48 Stunden bei 37°C inkubiert. Die Durchflusszytometrie wurde zur Bestimmung der PS-Translokation auf der Erythrozytenoberfläche anhand der Annexin-V-Bindung, der Zellschrumpfung durch das Vorwärtsstreulicht (FSC), des zytosolischen Kalziumgehalts anhand der Fluo3-Fluoreszenz, der Bildung von ROS anhand der DCFDA-Fluoreszenz sowie der Ceramid-Abundanz anhand von spezifischen Antikörpern eingesetzt. Die Ergebnisse des ersten Teils der Studie zeigen, dass Afatinib (≥8,2 µM) den Prozentsatz an eryptotischen Erythrozyten nach der jeweiligen Inkubationszeit signifikant erhöht und das FSC signifikant senkt. Afatinib erhöhte zudem den intrazellulären Kalziumspiegel, die Ceramid-Abundanz und die ROS-Produktion signifikant. Die Wirkung von Afatinib auf die PS-Translokation war nach Entfernen von extrazellulärem Kalzium signifikant verringert. Es konnte kein signifikanter Unterschied in der PS-Translokation nach Applikation der Inhibitoren von Caspasen oder Proteinkinase C (PKC), p38-Kinase, casein kinase 1α (CK1α) und Janus-Kinase 3 (JAK3) beobachtet werden. Afatinib löste ab einer Konzentration von 16,4 µM eine signifikant erhöhte Hämolyse aus. Für diese Wirkung sind jedoch Afatinib-Konzentrationen erforderlich, die weit über der Plasmakonzentration an freiem Afanitinib in behandelten Patienten liegen. Das Auftreten Afatinib-assoziierter Anämie ist daher wahrscheinlich nicht Folge einer übermäßigen Eryptose und Hämolyse. Der zugrunde liegende Signalmechanismus der durch Afatinib ausgelösten Eryptose umfasst die Erhöhung des intrazellulären Kalziums sowie eine erhöhte Produktion von Ceramid und ROS. Die Ergebnisse des zweiten Teils des Forschungsprojektes zeigen, dass Ceritinib (1,8 µM) den suizidalen Erythrozytentod signifikant erhöht und das Zellvolumen signifikant senkt. Ceritinib steigerte die zytosolische Kalziumkonzentration, nicht jedoch den ROS- oder Ceramid-Spiegel. Die Wirkung von Ceritinib auf die Annexin-V-Bindung wurde in Abwesenheit von extrazellulärem Kalzium sowie in Gegenwart des nicht-selektiven Kationenkanalblockers Amilorid (1mM), AKT1/2-Inhibitor A6730 (58 nM), PKC-Blocker Staurosporin (1 μM), p38-Kinase-Inhibitor SB203580 (2 μM), CK1α-Blocker D4476 (10 μM) und durch den Caspase-Inhibitor zVAD (10 μM) signifikant verringert. Ceritinib erhöhte ebenfalls signifikant die Hämolyse. Zusammenfassend lässt sich festhalten, dass Ceritinib den suizidalen Erythrozytentod stimuliert. Für diese Wirkung sind jedoch Ceritinib-Konzentrationen erforderlich, die weit über der Plasmakonzentration an freiem Ceritinib in behandelten Patienten liegen. Das Auftreten Ceritinib-assoziierter Anämie ist daher wahrscheinlich nicht Folge einer übermäßigen Eryptose. Die Ceritinib-induzierte Eryptose wird durch einen Kalziumanstieg, durch Aktivierung des AKT1/2-Signalwegs sowie Aktivierung von Caspasen und Kinasen wie PKC, p38 oder CK1α begleitet. Schließlich deckt der dritte Teil der Studie auf, dass Volasertib keinen suizidalen Erythrozytentod stimuliert. Nachdem Erythrozyten verschiedenen eryptotischen Stimuli ausgesetzt wurden, zeigte Volasertib vielmehr eine anti-eryptotische Wirkung. Humane Erythrozyten wurden einem Energiemangel 48 Stunden lang, einer hyperosmotischen Ringer-Lösung (550 mM Saccharose wurde zugegeben) für 6 Stunden, einem Oxidans (0,3 mM tert.-Butylhydroperoxid [Tbooh] für 50 Minuten, sowie dem Calcium-Ionophor Ionomycin (1 uM) für 60 min in Abwesenheit und Anwesenheit von Volasertib (0,8 - 2,4 µM) ausgesetzt. Um einen Vergleich der Wirkung von Volasertib auf kernhaltige und kernlose Zellen zu erhalten, wurde die Erythrozyten-Vorläuferzelllinie (K562-Zellen) mit RPMI-1640-Medium 48 Stunden lang mit Volasertib (0,8 - 2,4 µM) inkubiert. Anschließend wurden die PS-Externalisierung und das FSC quantifiziert. Glukosemangel, oxidativer Stress, hyperosmotischer Schock sowie ein Kalziumüberschuss erhöhten den Prozentsatz an PS-exponierenden Erythrozyten und verringerten das FSC. Volasertib verminderte den suizidalen Erythrozytentod nach Energieentzug oder osmotischem Schock signifikant, nicht jedoch nach oxidativem Stress oder einer Ionomycin-Behandlung. Volasertib zeigte keinen Einfluss auf das FSC oder den Kalziumeinstrom eines jeglichen Manövers. Die ROS- und Ceramidproduktion wurde nach Energieentzug in Gegenwart von Volasertib nicht verändert. Volasertib stimulierte die Apoptose in kernhaltigen K562-Zellen signifikant und verringerte das Zellvolumen erheblich. So besitzt Volasertib nach Energieentzug oder hyperosmotischem Schock eine anti-eryptotische Wirkung im Gegensatz zur Stimulation der Apoptose in K562-Zellen nach Volasertib-Behandlung.The thesis explored the effect of the cytostatic molecules afatinib, ceritinib, and volasertib on eryptosis, i.e., the suicidal death of erythrocytes characterised by the breakdown of phosphatidylserine (PS) asymmetry, the shrinkage of the cells, and membrane blebbing. Stimuli of eryptosis include ATP depletion, oxidative stress, hyperosmotic shock, as well as different xenobiotics/diseases. Underlying signalling mechanisms include an excess of intracellular calcium, ceramide, and reactive oxygen species (ROS) generation, as well as the involvement of different kinases and caspases. Healthy human erythrocytes (0.4% hematocrit) were treated with different concentrations of afatinib, ceritinib, and volasertib at 37˚C for 48 hours. Flow cytometry was employed to quantify the PS translocation on the erythrocyte surface from annexin-V-binding, cell shrinkage from forward scatter (FSC), cytosolic calcium from Fluo3-fluorescence, ROS from DCFDA fluorescence, and ceramide using ceramide-specific antibodies. The first part of the investigation shows that after the respective incubation period, afatinib (≥8.2 µM) significantly enhanced the percentage of eryptotic erythrocytes and significantly decreased the FSC. Afatinib also significantly elevated the intracellular calcium level, ceramide abundance, and ROS production. The effect of afatinib on PS translocation significantly decreased after the removal of extracellular calcium. No significant distinction was observed in PS translocation after application of the inhibitors of caspases or protein kinase C (PKC), p38 kinase, casein kinase 1α (CK1α), and Janus kinase 3 (JAK3). Afatinib (≥16.4 µM) significantly enhanced haemolysis. The afatinib concentration required for induction of eryptosis is by far higher than the plasma concentration of free afatinib in treated patients and, thus, afatinib-triggered eryptosis cannot explain the drug-induced anaemia. Signalling mechanisms of afatinib-triggered eryptosis include the increase of cytosolic calcium, enhanced production of ceramide, and ROS. The results of the second part of the study indicate that ceritinib (1.8 µM) significantly increased the suicidal erythrocyte death and significantly decreased cell volume. Ceritinib elevated the cytosolic calcium concentration, but not the ROS or ceramide level. The effect of ceritinib on annexin-V-binding was significantly inhibited in the absence of extracellular calcium, in the presence of the non-selective cation channel inhibitor amiloride (1mM), AKT1/2 inhibitor A6730 (58 nM), PKC blocker staurosporine (1 µM), p38 kinase inhibitor SB203580 (2 µM), CK1α blocker D4476 (10 µM), and caspase inhibitor zVAD (10 µM). Ceritinib also significantly triggered haemolysis. In conclusion, ceritinib stimulates suicidal erythrocyte death. The ceritinib concentration required for inducing eryptosis is by far higher than the plasma concentration of free ceritinib in treated patients and ceritinib-triggered eryptosis cannot explain the drug-induced anaemia. Signalling of ceritinib-induced eryptosis includes calcium entry, activation of AKT1/2 signalling, and the activation of caspases and kinases such as PKC, p38, and CK1α. Finally, the third part of the study reveals that volasertib did not stimulate suicidal erythrocyte death, but rather showed an anti-eryptotic property following exposure of erythrocyte concentrates to various eryptotic stimuli. Human erythrocytes were exposed to energy-depleted Ringer’s solution for 48 hours, hyperosmotic Ringer’s solution (550 mM sucrose was added) for six hours, oxidative stress (0.3 mM tert-butylhydroperoxide [t-booh] was added) for 50 minutes, or to calcium ionophore ionomycin (1 µM) for 60 minutes in the absence and presence of volasertib (0.8 - 2.4 µM). For a comparative study of volasertib on nucleated and anucleated cells, the erythrocyte progenitor cell line (K562 cells) with RPMI-1640 medium was exposed to volasertib (0.8 – 2.4 µM) for 48 hours and then PS externalisation and FSC were quantified. Glucose depletion, oxidative stress, hyperosmotic shock, and calcium overload increased the percentage of PS-exposing erythrocytes and decreased FSC. Volasertib significantly blunted the suicidal erythrocyte death following energy depletion or osmotic shock, but not after oxidative stress or ionomycin treatment. Volasertib did not show any influence on FSC or calcium entry following any manoeuvre. The ROS generation or ceramide production following energy depletion was not changed in the presence of volasertib. Volasertib significantly stimulated apoptosis in nucleated K562 cells, as well as considerably decreased the cell volume. Thus, volasertib possesses an anti-eryptotic power following energy depletion or hyperosmotic shock, observations contrasting stimulation of apoptosis in K562 cells. Finally, collective data from the above investigations show that afatinib and ceritinib stimulate eryptosis whereas volasertib shows a protective effect against mature erythrocyte death but triggered apoptosis in the erythrocyte progenitor K562 cell line

Inhibition of Erythrocyte Cell Membrane Scrambling Following Energy Depletion and Hyperosmotic Shock by Alectinib

Background/Aims: The anaplastic lymphoma kinase (ALK) inhibitor alectinib is clinically used for the treatment of ALK positive non-small-cell lung cancer. At least in part the substance is effective by triggering suicidal death or apoptosis of tumor cells. Erythrocytes are lacking mitochondria and nuclei, key organelles of apoptosis but are, similar to apoptosis of nucleated cells, able to enter suicidal erythrocyte death or eryptosis. Stimulators of eryptosis include energy depletion, hyperosmotic shock, oxidative stress, and increase of cytosolic Ca2+ activity ([Ca2+]i). The present study explored, whether alectinib influences eryptosis. Methods: Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding and cell volume from forward scatter. Measurements were made without or with energy depletion (glucose deprivation for 48 hours), hyperosmotic shock (+550mM sucrose for 6 hours), oxidative stress (50 min exposure to 0.3 mM tert-butylhydroperoxide), and Ca2+ loading (60 minutes treatment with 1 µM Ca2+ ionophore ionomycin). Results: A 48 hours exposure of human erythrocytes to alectinib (150-600 ng/ml) did not significantly modify the percentage of annexin-V-binding cells and forward scatter. Energy depletion, hyperosmotic shock, oxidative stress and Ca2+ loading were each followed by profound and significant increase of the percentage annexin-V-binding erythrocytes and a significant decrease of forward scatter. The effects of energy depletion and hyperosmotic shock, but not of oxidative stress or Ca2+ loading on annexin-V-binding were significantly blunted in the presence of alectinib (150-600 ng/ml). In none of the conditions was forward scatter significantly modified by alectinib. Conclusion: Alectinib inhibits cell membrane scrambling following energy depletion and hyperosmotic shock

Stimulation of Eryptosis, the Suicidal Erythrocyte Death, by Costunolide

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

Taurolidine Sensitivity of Eryptosis, the Suicidal Erythrocyte Death

Background/Aims: The taurine derivative Taurolidine is effective against diverse bacteria and tumor growth. In the treatment of cancer, the substance is effective in part by triggering suicidal death or apoptosis of tumor cells. The Taurolidine-induced apoptosis involves mitochondria. Erythrocytes lack mitochondria but are nevertheless able to enter suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling of eryptosis includes increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and ceramide. The present study explores, whether Taurolidine induces eryptosis and, if so, which cellular mechanisms are involved. Methods: Phosphatidylserine exposure at the cell surface was estimated using annexin-V-binding, cell volume using forward scatter, [Ca2+]i using Fluo3-fuorescence, reactive oxygen species (ROS) formation using 2’,7’-dichlorodihydrofuorescein (DCF)-dependent fluorescence, and ceramide abundance using specific antibodies. Results: A 48 hours exposure of human erythrocytes to Taurolidine (60 µg/ml) significantly enhanced the percentage of annexin-V-binding cells, significantly decreased forward scatter and significantly increased Fluo3-fluorescence and ceramide abundance, but not DCF-fluorescence. The effect of Taurolidine on annexin-V-binding was virtually abrogated by removal of extracellular Ca2+. Conclusion: Taurolidine triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry and paralleled by increase of ceramide abundance

Inhibition of Lithium Sensitive Orai1/ STIM1 Expression and Store Operated Ca2+ Entry in Chorea-Acanthocytosis Neurons by NF-κB Inhibitor Wogonin

Background/Aims: The neurodegenerative disease Chorea-Acanthocytosis (ChAc) is caused by loss-of-function-mutations of the chorein-encoding gene VPS13A. In ChAc neurons transcript levels and protein abundance of Ca2+ release activated channel moiety (CRAC) Orai1 as well as its regulator STIM1/2 are decreased, resulting in blunted store operated Ca2+-entry (SOCE) and enhanced suicidal cell death. SOCE is up-regulated and cell death decreased by lithium. The effects of lithium are paralleled by upregulation of serum & glucocorticoid inducible kinase SGK1 and abrogated by pharmacological SGK1 inhibition. In other cell types SGK1 has been shown to be partially effective by upregulation of NFκB, a transcription factor stimulating the expression of Orai1 and STIM. The present study explored whether pharmacological inhibition of NFκB interferes with Orai1/STIM1/2 expression and SOCE and their upregulation by lithium in ChAc neurons. Methods: Cortical neurons were differentiated from induced pluripotent stem cells generated from fibroblasts of ChAc patients and healthy volunteers. Orai1 and STIM1 transcript levels and protein abundance were estimated from qRT-PCR and Western blotting, respectively, cytosolic Ca2+-activity ([Ca2+]i) from Fura-2-fluorescence, SOCE from increase of [Ca2+]i following Ca2+ re-addition after Ca2+-store depletion with sarco-endoplasmatic Ca2+-ATPase inhibitor thapsigargin (1µM), as well as CRAC current utilizing whole cell patch clamp recording. Results: Orai1 and STIM1 transcript levels and protein abundance as well as SOCE and CRAC current were significantly enhanced by lithium treatment (2 mM, 24 hours). These effects were reversed by NFκB inhibitor wogonin (50 µM). Conclusion: The stimulation of expression and function of Orai1/STIM1/2 by lithium in ChAc neurons are disrupted by pharmacological NFκB inhibition

Stimulation of Eryptosis by Afatinib

Background/Aims: The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor afatinib is primarily utilized for the treatment of non-small cell lung carcinoma. The drug is at least partially effective by triggering suicidal tumor cell death. Side effects of afatinib treatment include anemia. At least in theory, afatinib induced anemia could be secondary to stimulation of suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling potentially stimulating eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), induction of oxidative stress, and increase of ceramide abundance. The present study explored, whether afatinib induces eryptosis and, if so, whether its effect involves Ca2+ entry, oxidative stress, and/or ceramide. Methods: Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) abundance from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to afatinib (≥ 4 µg/ml) significantly increased the percentage of annexin-V-binding cells and significantly decreased forward scatter. Afatinib significantly increased Fluo3-fluorescence, DCFDA fluorescence and ceramide abundance. The effect of afatinib on annexin-V-binding and forward scatter was significantly blunted by removal of extracellular Ca2+. Conclusions: Afatinib triggers phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry, oxidative stress, and ceramide