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Mitoxantrone-Induced Suicidal Erythrocyte Death

Background/Aims: Mitoxantrone, a cytotoxic drug used for the treatment of malignancy and multiple sclerosis, is at least in part effective by triggering apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, a type of suicidal cell death. Hallmarks of eryptosis are cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signalling involved in eryptosis include Ca2+-entry, ceramide formation and oxidative stress. Methods: Cell volume was estimated from forward scatter, phosphatidylserine-exposure from annexin V binding, formation of reactive oxidant species (ROS) from 2′,7′-dichlorodihydrofluorescein-diacetate fluorescence, and ceramide abundance from binding of fluorescent antibodies in flow cytometry. Results: A 48 hours exposure to mitoxantrone was followed by significant decrease of forward scatter (≥ 5 μg/ml mitoxantrone) and increase of annexin-V-binding (≥ 10 μg/ml mitoxantrone), effects paralleled by significant increases of ROS formation (25 μg/ml mitoxantrone) and ceramide abundance (25 μg/ml mitoxantrone). The effect of mitoxantrone was not significantly modified by nominal absence of extracellular Ca2+ but significantly blunted by the antioxidant N-acetylcysteine (1 mM). Conclusions: Mitoxantrone triggers cell membrane scrambling, an effect not requiring entry of extracellular Ca2+ but at least partially due to formation of ROS and ceramide.


Introduction
Mitoxantrone, a synthetic antineoplastic cytotoxic drug [1], is used for the treatment of both, aggressive multiple sclerosis [1, 2] and malignancy [3][4][5][6][7][8][9][10]. The antineoplastic effects result from triggering of tumor cell apoptosis [11][12][13][14][15][16][17], the efficacy of mitoxantrone in multiple sclerosis has been attributed to apoptosis of lymphocytes and dendritic cells [18]. Determination of ceramide formation For the determination of ceramide, a monoclonal antibody-based assay was used. After incubation, cells were stained for 1 hour at 37°C with 0.1 µg/ml anti ceramide antibody (clone MID 15B4, Alexis, Grünberg, Germany) diluted in PBS containing 0.1% bovine serum albumin (BSA). The samples were washed twice with PBS-BSA. Subsequently, the cells were stained for 30 minutes with polyclonal FITC-conjugated goat anti-mouse IgG and IgM specific antibody (Pharmingen, Hamburg, Germany) diluted 1:50 in PBS-BSA. Unbound secondary antibody was removed by repeated washing with PBS-BSA. The samples were then analyzed by flow cytometric analysis with an excitation wavelength of 488 nm and an emission wavelength of 530 nm.

Measurement of hemolysis
For the determination of hemolysis, the samples were centrifuged (3 min at 1600 rpm, room temperature) after incubation under the above mentioned experimental condition, the supernatants were harvested. As a measure of hemolysis, the hemoglobin (Hb) concentration of the supernatant was determined photometrically at 405 nm. The absorption of the supernatant of erythrocytes lysed in distilled water was defined as 100% hemolysis.

Statistics
Data are expressed as arithmetic means ± SEM. As indicated in the figure legends, statistical analysis was made using one-way ANOVA with Tukey's test as post-test and two-tailed t-test as appropriate. The number of different erythrocyte specimens studied is given as n. Since different erythrocyte specimens used in distinct experiments are differently susceptible to triggers of eryptosis, the same erythrocyte specimens have been used for control and experimental conditions.

Results
The present study explored, whether mitoxantrone triggers eryptosis, the suicidal erythrocyte death. The hallmarks of eryptosis include cell membrane scrambling with phosphatidylserine translocation to the cell surface. Annexin-V-binding was quantified by flow cytometry in order to identify phosphatidylserine exposing erythrocytes. As illustrated in Fig. 1, a 48 hours treatment with mitoxantrone was followed by an increase of the percentage annexin-V-binding erythrocytes, an effect reaching statistical significance at 10 µg/ml mitoxantrone concentration.
Eryptosis is further characterized by cell shrinkage. Accordingly, cell volume was estimated from forward scatter in flow cytometry. As illustrated in Fig. 2, a 48 hours treatment with mitoxantrone decreased forward scatter, an effect reaching statistical significance at 5 µg/ml mitoxantrone concentration.  Cell membrane scrambling and cell shrinkage following mitoxantrone treatment could have been due to increase of cytosolic Ca 2+ activity ([Ca 2+ ] i ) resulting from entry of extracellular Ca 2+ . Attempts to quantify [Ca 2+ ] i in erythrocytes utilizing Fluo3 fluorescence failed, as the cells apparently lost the dye following mitoxantrone treatment (data not shown). Possibly, mitoxantrone compromizes cell membrane integrity leading to hemolysis on the one hand and loss of Fluo3 on the other. In order to test, whether the effect of mitoxantrone on cell membrane scrambling required the entry of extracellular Ca 2+ , erythrocytes were exposed for 48 hours to 25 µg/ml mitoxantrone in the presence or nominal absence of extracellular Ca 2+ . As shown in Fig. 3, the effect of mitoxantrone on annexin-V-binding was not significantly different between the nominal absence and the presence of Ca 2+ . Mitoxantrone significantly increased the percentage of annexin-V-binding erythrocytes in both, the absence and presence of extracellular Ca 2+ . This result indicates that the effect of mitoxantrone on cell membrane scrambling did not require Ca 2+ entry.
Further experiments addressed alternative mechanisms possibly involved in the effect of mitoxantrone on eryptosis. The ROS-formation was determined utilizing DCFDA. As illustrated in Fig. 4A,B, a 48 hours exposure to 25 µg/ml mitoxantrone markedly and significantly increased the DCFDA fluorescence, a finding pointing to induction of oxidative stress. In order to test, whether ROS was required for the full effect of mitoxantrone on cell membrane scrambling, erythrocytes were exposed for 48 hours to 25 µg/ml mitoxantrone in the absence and presence of the antioxidant N-acetylcysteine (1 mM). As illustrated in Fig.   Fig. 4 4C, the effect of mitoxantrone on annexin-V-binding was significantly blunted in the presence of N-acetylcysteine. This result indicates that the effect of mitoxantrone on cell membrane scrambling was at least partially due to oxidative stress. In order to test, whether the effect of mitoxantrone required activation of p38 kinase, experiments were performed in the absence and presence of the p38 kinase inhibitor SB203580 (2 µM). As a result, a 48 hours exposure to 25 µg/ml mitoxantrone increased the percentage of annexin-V-binding erythrocytes significantly from 1.7 ± 0.3 % to 11.3 ± 0.8 % in the absence of SB203580 and significantly from 1.9 ± 0.4 % to 11.2 ± 0.8 % in the presence of SB203580. The increase was similar in the absence and presence of SB203580 indicating that activation of p38 kinase was not required for the stimulation of eryptosis by mitoxantrone.
As cell membrane scrambling could further be triggered by ceramide, additional experiments tested, whether mitoxantrone increases the ceramide abundance at the erythrocyte surface. The abundance of ceramide at the erythrocyte surface was quantified utilizing anti-ceramide antibodies. As illustrated in Fig. 5, a 48 hours exposure to 25 µg/ ml mitoxantrone was followed by a significant increase of ceramide abundance at the erythrocyte surface.

Discussion
The present study discloses a novel effect of mitoxantrone, i.e. stimulation of eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and breakdown of phosphatidylserine asymmetry of the erythrocyte cell membrane. The mitoxantrone concentrations required for the stimulation of eryptosis are within the range of mitoxantrone concentrations required for the antineoplastic activity of the substance [46]. At least in theory, the erythrocytes could be sensitized to the effect of mitoxantrone by parallel exposure to further eryptosis triggering xenobiotics [28,39,. Moreover, the sensitivity to mitoxantrone may be increased in patients suffering from disorders with enhanced eryptosis [28], such as diabetes [34,74,75], dehydration [76], renal insufficiency [77,78], hemolytic uremic syndrome [79], sepsis [80], malaria [81], sickle cell disease [81], Wilson's disease [82], iron deficiency [83], malignancy [84], phosphate depletion [85], and metabolic syndrome [65].
The effect of mitoxantrone did not require Ca 2+ entry. Whether or not mitoxantrone modifies cytosolic Ca 2+ activity ([Ca 2+ ] i ), cannot be answered with certainty. The exposure to  , the effect appears not to be relevant for the triggering of eryptosis. We thus explored further putative mechanisms. As evident from DCFDA fluorescence, mitoxantrone induces oxidative stress, a well known trigger of eryptosis [28]. Mitoxantrone has previously been shown to similarly induce oxidative stress of nucleated cells [8,[86][87][88]. On the other hand, mitoxantrone may strengthen the antioxidative defence [89,90]. The latter effect presumably requires expression of the respective proteins, an effect not possible in erythrocytes. In erythrocytes oxidative stress activates Ca 2+ permeable cation channels with subsequent Ca 2+ entry [91], an effect apparently not critically important for the stimulation of cell membrane scrambling by mitoxantrone. Oxidative stress further activates erythrocyte Clchannels and aspartyl and cysteinyl proteases [91]. Additional experimentation will be required to fully elucidate the link between mitoxantrone induced oxidative stress and cell membrane scrambling. Mitoxantrone further increased the abundance of ceramide, another powerful stimulator of eryptosis [28]. Mitoxantrone may stimulate ceramide formation by activation of sphingomyelinase in nucleated cells [92]. Mitoxantrone has been shown to stimulate phospholipase C with subsequent hydrolysis of phosphatidylcholine [92]. In erythrocytes, sphingomyelinase and thus ceramide formation are stimulated by platelet activating factor [93,94]. Ceramide has been shown to trigger cell membrane scrambling in part by sensibilisation against Ca 2+ [93]. The present observations rather suggest Ca 2+ independence of mitoxantroneinduced eryptosis.
Eryptosis is a physiological mechanism accomplishing removal of defective erythrocytes from circulating blood. Phosphatidylserine exposing erythrocytes bind to respective receptors of phagocyting cells which engulf and degrade the defective erythrocytes [28]. Accordingly, phosphatidylserine exposing erythrocytes are rapidly cleared from circulating blood [28]. Stimulation of eryptotic cell shrinkage [29] counteracts erythrocyte swelling, which could, at least in theory, result in hemolysis with subsequent rupture of the cell membrane and release of cellular hemoglobin. Hemoglobin released from hemolytic erythrocytes may be filtered in renal glomeruli, undergo subsequent precipitation in renal tubules, occlude tubular lumina and thus lead to obstructive kidney injury [95].
Excessive eryptosis may be similarly harmful. Removal of phosphatidylserine exposing erythrocytes from circulating blood may result in anemia, as soon as the loss of eryptotic erythrocytes exceeds the parallel formation of new erythrocytes [28]. Phosphatidylserine exposing erythrocytes furher adhere to the vascular wall by binding of erythrocytic phosphatidylserine to endothelial CXCL16 [96]. The binding of eryptotic erythrocytes to the vascular wall impedes microcirculation [96][97][98][99][100][101]. Phosphatidylserine exposing erythrocytes may further foster blood clotting and thus thrombosis [97,102,103].
In conclusion, mitoxantrone triggers eryptosis with erythrocyte shrinkage and cell membrane scrambling. Cellular mechanisms involved in the pro-eryptotic effect of mitoxantrone include oxidative stress and ceramide formation.