Abstract:
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.