Background/Aims: The anaplastic lymphoma kinase (ALK) inhibitor ASP3026 is in clinical development for the treatment of ALK expressing non-small cell lung carcinoma (NSCLC). ASP3026 is in part effective by inducing apoptosis of tumor cells. Erythrocytes lack mitochondria and nuclei, key organelles in the execution of apoptosis, but are nevertheless able to enter suicidal death or eryptosis, which is characterized by cell membrane scrambling with phosphatidylserine translocation to the cell surface and by cell shrinkage. Eryptosis is triggered by cell stress, such as energy depletion, hyperosmotic shock, oxidative stress and excessive increase of cytosolic Ca2+ activity ([Ca2+]i). The present study explored, whether ASP3026 impacts on eryptosis. Methods: Human erythrocytes have been exposed to energy depletion (glucose withdrawal for 48 hours), oxidative stress (addition of 0.3 mM tert-butylhydroperoxide [tBOOH] for 50 min) or Ca2+ loading with Ca2+ ionophore ionomycin (1 µM for 60 min) in absence and presence of ASP3026 (1-4 µg/ml). Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, and cell volume from forward scatter. Results: Treatment with ASP3026 alone did not significantly modify annexin-V-binding or forward scatter. Energy depletion, oxidative stress and ionomycin, all markedly and significantly increased the percentage of annexin-V-binding erythrocytes, and decreased the forward scatter. ASP3026 significantly blunted the effect of energy depletion and oxidative stress, but not of ionomycin on annexin-V-binding. ASP3026 did not significantly influence the effect of any maneuver on forward scatter. Conclusions: ASP3026 is a novel inhibitor of erythrocyte cell membrane scrambling following energy depletion and oxidative stress.

The anaplastic lymphoma kinase (ALK) originally detected in anaplastic large cell lymphomas (ALCL) [1] plays a decisive pathophysiological role in several solid tumors [1], including ALK expressing non-small cell lung carcinoma (NSCLC). Inhibitors of ALK include ASP3026 [2, 3], which is in clinical development for the treatment of ALK expressing NSCLC [2-5]. ASP3026 is in part effective by inducing apoptotic tumor cell death [6].

ALK expressing cells include eythrocytes [7], which may enter eryptosis, an apoptosis-like suicidal erythrocyte death [8]. Eryptosis is characterized by cell membrane scrambling with phosphatidylserine translocation to the cell surface [8]. Another hallmark of eryptosis is cell shrinkage [9]. Cellular mechanisms orchestrating the triggering of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i) [8], ceramide [10], caspases [8, 11, 12], G-protein Gαi2 [13], casein kinase 1α [8], Janus-activated kinase JAK3 [8], protein kinase C [8], and p38 kinase [8]. Eryptosis is inhibited by several kinases, including AMP activated kinase AMPK [8], cGMP-dependent protein kinase [8], mitogen and stress activated kinase MSK1/2 [14], PAK2 kinase [8] and sorafenib/sunitinib sensitive kinases [8]. Triggers of eryptosis include hyperosmotic shock [8], oxidative stress [8], energy depletion [8], exposure to a plethora of xenobiotics [8, 14-70], and diverse clinical conditions including iron deficiency [8], dehydration [71], hyperphosphatemia [72], vitamin D excess [73], chronic kidney disease (CKD) [74-78], hemolytic-uremic syndrome [79], diabetes [80], hepatic failure [45, 81], malignancy [78, 82], arteritis [83], sepsis [84], sickle-cell disease [8], beta-thalassemia [8], Hb-C and G6PD-deficiency [8], lung cancer [85], Wilsons disease [86], as well as advanced age [87]. Eryptosis is further observed following blood storage for transfusion [88]. Eryptosis may be inhibited by several substances [8].

The present study explored, whether ASP3026 influences eryptosis. To this end, human erythrocytes from healthy volunteers were exposed to ASP3026 without or with additional exposure to energy depletion, oxidative stress, and Ca2+ loading with Ca2+ ionophore ionomycin. Phosphatidylserine surface abundance and cell volume were subsequently determined utilizing flow cytometry

Erythrocytes, solutions and chemicals

Fresh Li-Heparin-anticoagulated blood samples were kindly provided by the blood bank of the University of Tübingen. The study is approved by the ethics committee of the University of Tübingen (184/2003 V). The blood was centrifuged at 120 g for 20 min at 21 °C and the platelets and leukocytes-containing supernatant was disposed. Erythrocytes were incubated in vitro at a hematocrit of 0.4% in Ringer solution containing (in mM) 125 NaCl, 5 KCl, 1 MgSO4, 32 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES; pH 7.4), 5 glucose, 1 CaCl2, at 37°C for 48 hours. Where indicated, erythrocytes were exposed for 48 hours to glucose depleted Ringer solution, for 50 minutes to the oxidant tert-butyl-hydroperoxide (0.3 mM, Sigma Aldrich, Hamburg, Germany), or for 60 minutes to Ca2+ ionophore ionomycin (1 µM, Merck Millipore, Darmstadt, Germany), each in the absence and presence of ASP3026 (MedChem Express, Princeton, USA) or, as control, the solvent DMSO alone. As ASP3026 plasma concentrations higher than 1 µg/ml ASP3026 have been observed in humans, ASP3026 concentrations of 1-4 µg/ml have been tested.

Annexin-V-binding and forward scatter

After incubation under the respective experimental condition, a 150 µl cell suspension was washed in Ringer solution containing 5 mM CaCl2 and then stained with Annexin-V-FITC (1: 200 dilution; ImmunoTools, Friesoythe, Germany) in this solution at 37°C for 15 min under protection from light. The annexin-V-abundance at the erythrocyte surface was subsequently determined on a FACS Calibur (BD, Heidelberg, Germany). Annexin-V-binding was measured with an excitation wavelength of 488 nm and an emission wavelength of 530 nm. A marker (M1) was placed to set an arbitrary threshold between annexin-V-binding cells and control cells. The same threshold was used for untreated and ASP3026 treated erythrocytes. A dot plot of forward scatter (FSC) vs. side scatter (SSC) was set to linear scale for both parameters.

Statistics

Data are expressed as arithmetic means ± SEM. As indicated in the figure legends, statistical analysis was made using ANOVA with Tukey’s test as post-test; n denotes the number of different erythrocyte specimens studied. 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.

In order to test, whether ASP3026 influences eryptosis, eryptotic erythrocytes were identified from the two hallmarks of eryptosis, i.e. phospholipid scrambling of the cell membrane and decrease of cell volume. Cell membrane scrambling was evidenced by annexin-V-binding to phosphatidylserine at the erythrocyte surface, and cell shrinkage was quantified utilizing forward scatter in flow cytometry.

The percentage of human erythrocytes binding annexin-V after 48 hours incubation in standard, glucose containing, Ringer solution was low (1.86 ± 0.17%, n=14) and not significantly modified by the presence of 4 µg/ml ASP3026 (2.92 ± 0.53%, n=14). Thus, in standard glucose containing Ringer solution, ASP3026 did not appreciably modify erythrocyte cell membrane scrambling. As illustrated in Fig. 1, glucose removal was followed by a marked increase of the percentage of annexin-V-binding erythrocytes. The addition of ASP3026 (1-4 µg/ml) blunted the increase of the percentage of annexin-V-binding erythrocytes following glucose deprivation, an effect reaching statistical significance at 4 µg/ml ASP3026 (Fig. 1). Nevertheless, even in the presence of ASP3026, energy depletion significantly increased the percentage of phosphatidylserine exposing erythrocytes (Fig. 1). ASP3026 thus partially prevented cell membrane scrambling following energy depletion,

Fig. 1.

ASP3026 sensitivity of phosphatidylserine exposure following energy depletion. A. Original histograms of annexin-V-binding of erythrocytes following exposure for 48 hours to glucose containing Ringer solution (grey area), Ringer solution without glucose (black solid line) and Ringer solution without glucose and presence of ASP3026 (4 µg/ml) (black dashed line). B. Arithmetic means ± SEM (n = 15) of the percentage of annexin-V-binding erythrocytes after a 48 hours treatment with Ringer solution with glucose (white bar) or without glucose in the absence (black bar) and presence (grey bars) of ASP3026 (1-4 µg/ml). For comparison, the value in absence of glucose and presence of the solvent DMSO is shown (striped bar). ***(p<0.001) indicates significant difference from the presence of glucose, ##(p<0.01) indicates significant difference from the absence of ASP3026 (ANOVA).

Fig. 1.

ASP3026 sensitivity of phosphatidylserine exposure following energy depletion. A. Original histograms of annexin-V-binding of erythrocytes following exposure for 48 hours to glucose containing Ringer solution (grey area), Ringer solution without glucose (black solid line) and Ringer solution without glucose and presence of ASP3026 (4 µg/ml) (black dashed line). B. Arithmetic means ± SEM (n = 15) of the percentage of annexin-V-binding erythrocytes after a 48 hours treatment with Ringer solution with glucose (white bar) or without glucose in the absence (black bar) and presence (grey bars) of ASP3026 (1-4 µg/ml). For comparison, the value in absence of glucose and presence of the solvent DMSO is shown (striped bar). ***(p<0.001) indicates significant difference from the presence of glucose, ##(p<0.01) indicates significant difference from the absence of ASP3026 (ANOVA).

Close modal

Erythrocyte forward scatter was, following a 48 hours exposure to Ringer solution in the presence of glucose, again similar in the absence (508.40 ± 6.40, n=14) and presence (503.40 ± 5.12, n=14) of ASP3026 (4 µg/ml). Accordingly, in standard glucose containing Ringer solution, ASP3026 did not appreciably modify erythrocyte volume. As illustrated in Fig. 2, a 48 hours exposure to glucose-depleted Ringer solution was followed by a marked decrease of forward scatter, reflecting erythrocyte shrinkage. The decline of erythrocyte forward scatter was, following a 48 hours exposure to Ringer solution in the absence of glucose, virtually identical in the absence and presence of ASP3026 (1–4 µg/ml) (Fig. 2). Accordingly, neither in energy repleted nor in energy depleted erythrocytes, ASP3026 did significantly modify erythrocyte volume.

Fig. 2.

ASP3026 sensitivity of erythrocyte shrinkage following energy depletion. A. Original histograms of forward scatter of erythrocytes following exposure for 48 hours to glucose containing Ringer solution (grey area), Ringer solution without glucose (black solid line) and Ringer solution without glucose and presence of ASP3026 (4 µg/ml) (black dashed line). B. Arithmetic means ± SEM (n = 15) of the erythrocyte forward scatter after a 48 hours treatment with Ringer solution with glucose (white bar) or without glucose in the absence (black bar) and presence (grey bars) of ASP3026 (1-4 µg/ml). For comparison, the value in absence of glucose and presence of the solvent DMSO is shown (striped bar). ***(p<0.001) indicates significant difference from the presence of glucose (ANOVA).

Fig. 2.

ASP3026 sensitivity of erythrocyte shrinkage following energy depletion. A. Original histograms of forward scatter of erythrocytes following exposure for 48 hours to glucose containing Ringer solution (grey area), Ringer solution without glucose (black solid line) and Ringer solution without glucose and presence of ASP3026 (4 µg/ml) (black dashed line). B. Arithmetic means ± SEM (n = 15) of the erythrocyte forward scatter after a 48 hours treatment with Ringer solution with glucose (white bar) or without glucose in the absence (black bar) and presence (grey bars) of ASP3026 (1-4 µg/ml). For comparison, the value in absence of glucose and presence of the solvent DMSO is shown (striped bar). ***(p<0.001) indicates significant difference from the presence of glucose (ANOVA).

Close modal

A next series of experiments addressed the effect of oxidative stress due to addition of 0.3 mM tert-butylhydroperoxide (tBOOH) for 50 minutes. As illustrated in Fig. 3, oxidative stress was followed by a marked increase of the percentage of annexin-V-binding erythrocytes. The addition of ASP3026 (1-4 µg/ml) blunted the increase of the percentage of annexin-V-binding erythrocytes following oxidative stress, an effect reaching statistical significance at 2 µg/ml ASP3026 (Fig. 3). Again, even in the presence of ASP3026, oxidative stress significantly increased the percentage of phosphatidylserine exposing erythrocytes (Fig. 3). ASP3026 thus partially blunted cell membrane scrambling following oxidative stress.

Fig. 3.

ASP3026 sensitivity of phosphatidylserine exposure following oxidative stress. A. Original histograms of annexin-V-binding of erythrocytes following exposure for 50 min to Ringer solution without tert-butylhydroperoxide (grey area), or Ringer solution with tert-butylhydroperoxide (0.3 mM) and absence (solid black line) or presence (black dashed line) of ASP3026 (4 µg/ml). B. Arithmetic means ± SEM (n = 15) of the percentage of annexin-V-binding erythrocytes after a 50 min treatment with Ringer solution without tert-butylhydroperoxide (white bar) or with tert-butylhydroperoxide (0.3 mM) in the absence (black bar) and presence (grey bars) of ASP3026 (1-4 µg/ml). For comparison, the value in presence of tert-butylhydroperoxide and DMSO is shown. ***(p<0.001) indicates significant difference from the absence of tert-butylhydroperoxide, #(p<0.05) indicates significant difference from the absence of ASP3026 (ANOVA).

Fig. 3.

ASP3026 sensitivity of phosphatidylserine exposure following oxidative stress. A. Original histograms of annexin-V-binding of erythrocytes following exposure for 50 min to Ringer solution without tert-butylhydroperoxide (grey area), or Ringer solution with tert-butylhydroperoxide (0.3 mM) and absence (solid black line) or presence (black dashed line) of ASP3026 (4 µg/ml). B. Arithmetic means ± SEM (n = 15) of the percentage of annexin-V-binding erythrocytes after a 50 min treatment with Ringer solution without tert-butylhydroperoxide (white bar) or with tert-butylhydroperoxide (0.3 mM) in the absence (black bar) and presence (grey bars) of ASP3026 (1-4 µg/ml). For comparison, the value in presence of tert-butylhydroperoxide and DMSO is shown. ***(p<0.001) indicates significant difference from the absence of tert-butylhydroperoxide, #(p<0.05) indicates significant difference from the absence of ASP3026 (ANOVA).

Close modal

Oxidative stress was further followed by a marked decrease of forward scatter, reflecting erythrocyte shrinkage (Fig. 4). The erythrocyte shrinkage following oxidative stress was virtually identical in the absence and presence of ASP3026 (1–4 µg/ml) (Fig. 4). Accordingly, ASP3026 did not significantly interfere with the effect of oxidative stress on erythrocyte volume.

Fig. 4.

ASP3026 sensitivity of erythrocyte shrinkage following oxidative stress. A. Original histograms of erythrocyte forward scatter following exposure for 50 min to Ringer solution without tert-butylhydroperoxide (grey area), or Ringer solution with tert-butylhydroperoxide (0.3 mM) and absence (solid black line) or presence (black dashed line) of ASP3026 (4 µg/ml). B. Arithmetic means ± SEM (n = 15) of erythrocyte forward scatter after a 50 min treatment with Ringer solution without tert-butylhydroperoxide (white bar) or with tert-butylhydroperoxide (0.3 mM) in the absence (black bar) and presence (grey bars) of ASP3026 (1-4 µg/ml). For comparison, the value in presence of tert-butylhydroperoxide and DMSO is shown. ***(p<0.001) indicates significant difference from the absence of tert-butylhydroperoxide (ANOVA).

Fig. 4.

ASP3026 sensitivity of erythrocyte shrinkage following oxidative stress. A. Original histograms of erythrocyte forward scatter following exposure for 50 min to Ringer solution without tert-butylhydroperoxide (grey area), or Ringer solution with tert-butylhydroperoxide (0.3 mM) and absence (solid black line) or presence (black dashed line) of ASP3026 (4 µg/ml). B. Arithmetic means ± SEM (n = 15) of erythrocyte forward scatter after a 50 min treatment with Ringer solution without tert-butylhydroperoxide (white bar) or with tert-butylhydroperoxide (0.3 mM) in the absence (black bar) and presence (grey bars) of ASP3026 (1-4 µg/ml). For comparison, the value in presence of tert-butylhydroperoxide and DMSO is shown. ***(p<0.001) indicates significant difference from the absence of tert-butylhydroperoxide (ANOVA).

Close modal

A next series of experiments explored the effect of Ca2+ entry, which was achieved by a 60 min exposure of erythrocytes to Ca2+ ionophore ionomycin (1 µM). As illustrated in Fig. 5, exposure of the erythrocytes for 60 minutes to 1 µM ionomycin was followed by a sharp increase of the percentage of annexin-V-binding erythrocytes. The effect tended to be slightly blunted by ASP3026, an effect, however, not reaching statistical significance (Fig. 5). Instead, even in the presence of ASP3026, ionomycin significantly increased the percentage of phosphatidylserine exposing erythrocytes (Fig. 5).

Fig. 5.

ASP3026 sensitivity of phosphatidylserine exposure following Ca2+ loading. A. Original histograms of annexin-V-binding of erythrocytes following exposure for 60 min to Ringer solution without Ca2+ ionophore ionomycin (grey area), or Ringer solution with ionomycin (1 µM) and absence (solid black line) or presence (black dashed line) of ASP3026 (4 µg/ml). B. Arithmetic means ± SEM (n = 10) of the percentage of annexin-V-binding erythrocytes after a 60 min treatment with Ringer solution without ionomycin (white bar) or with ionomycin (1 µM) in the absence (black bar) and presence (grey bars) of ASP3026 (1-4 µg/ml). For comparison, the value in presence of ionomycin and DMSO is shown. ***(p<0.001) indicates significant difference from the absence of ionomycin (ANOVA).

Fig. 5.

ASP3026 sensitivity of phosphatidylserine exposure following Ca2+ loading. A. Original histograms of annexin-V-binding of erythrocytes following exposure for 60 min to Ringer solution without Ca2+ ionophore ionomycin (grey area), or Ringer solution with ionomycin (1 µM) and absence (solid black line) or presence (black dashed line) of ASP3026 (4 µg/ml). B. Arithmetic means ± SEM (n = 10) of the percentage of annexin-V-binding erythrocytes after a 60 min treatment with Ringer solution without ionomycin (white bar) or with ionomycin (1 µM) in the absence (black bar) and presence (grey bars) of ASP3026 (1-4 µg/ml). For comparison, the value in presence of ionomycin and DMSO is shown. ***(p<0.001) indicates significant difference from the absence of ionomycin (ANOVA).

Close modal

Exposure of the erythrocytes for 60 minutes to 1 µM ionomycin was followed by a sharp decrease of forward scatter (Fig. 6). The cell shrinkage was not significantly modified by ASP3026 (Fig. 6).

Fig. 6.

ASP3026 sensitivity of erythrocyte shrinkage following Ca2+ loading. A. Original histograms of erythrocyte forward scatter following exposure for 60 min to Ringer solution without ionomycin (grey area), or Ringer solution with ionomycin (1 µM) and absence (solid black line) or presence (black dashed line) of ASP3026 (4 µg/ml). B. Arithmetic means ± SEM (n = 10) of erythrocyte forward scatter after a 60 min treatment with Ringer solution without ionomycin (white bar) or with ionomycin (1 µM) in the absence (black bar) and presence (grey bars) of ASP3026 (1-4 µg/ml). For comparison, the value in presence of ionomycin and DMSO is shown. ***(p<0.001) indicates significant difference from the absence of ionomycin (ANOVA).

Fig. 6.

ASP3026 sensitivity of erythrocyte shrinkage following Ca2+ loading. A. Original histograms of erythrocyte forward scatter following exposure for 60 min to Ringer solution without ionomycin (grey area), or Ringer solution with ionomycin (1 µM) and absence (solid black line) or presence (black dashed line) of ASP3026 (4 µg/ml). B. Arithmetic means ± SEM (n = 10) of erythrocyte forward scatter after a 60 min treatment with Ringer solution without ionomycin (white bar) or with ionomycin (1 µM) in the absence (black bar) and presence (grey bars) of ASP3026 (1-4 µg/ml). For comparison, the value in presence of ionomycin and DMSO is shown. ***(p<0.001) indicates significant difference from the absence of ionomycin (ANOVA).

Close modal

The present observations uncover a novel inhibitor of eryptosis, i.e. the anaplastic lymphoma kinase (ALK) inhibitor [2, 3] ASP3026. In the absence of eryptosis stimulating challenges, ASP3026 did not appreciably influence cell membrane scrambling or cell shrinkage, the two hallmarks of eryptosis [8]. However, the substance significantly blunted the stimulating effect of energy depletion and oxidative stress on cell membrane scrambling. The ASP3026 concentrations required for this effect were well in the range of concentrations encountered in the plasma of patients [89]. The effect of ASP3026 on oxidative stress induced cell membrane scrambling reached statistical significance at 2 but not at 1 and 4 µg/ml ASP3026. The data do, however, not allow the conclusion that ASP3026 was more effective at 2 than at 1 or 4 µg/ml. ASP3026 did not appreciably influence the cell membrane scrambling following Ca2+ loading with the Ca2+ ionophore ionomycin. Moreover, ASP3026 did not interfere appreciably with the erythrocyte shrinkage following treatment with energy depletion, oxidative stress, or ionomycin.

The present observations were compatible with the assumption that stimulation of cell membrane scrambling requires ALK activity. However, the effects of ASP3026 are not necessarily due to ALK inhibition but may result from unspecific side effects of the drug. Future experimental effort is required to disclose the cellular mechanisms linking ASP3026 to cell membrane scrambling during energy depletion and oxidative stress, but not following flooding the cells with Ca2+.

Irrespective of the underlying mechanisms, the inhibition of erythrocyte cell membrane scrambling by ASP3026 may counteract anemia in malignancy, a major clinical problem [90-98]. Following triggering of cell membrane scrambling, phosphatidylserine exposing erythrocytes are rapidly cleared from circulating blood and eryptosis leads to anemia as soon as the loss of eryptotic erythrocytes outcasts the formation of new erythrocytes by erythropoiesis [8]. The anemia of malignancy is at least in part due to stimulation of eryptosis [78, 82], and would thus be amenable to inhibitors of cell membrane scrambling.

As ASP3026 did not interfere with cell shrinkage, it does not disrupt the attempt of dying erythrocytes to avoid cell swelling with subsequent hemolysis. Hemoglobin released following hemolysis may otherwise pass the renal glomerular filter, precipitate in the acidic lumen of renal tubules, occlude nephrons and may thus lead to renal failure [99].

Besides the impact of eryptosis on anemia, phosphatidylserine exposing erythrocytes may adhere to the vascular wall [100], stimulate blood clotting/thrombosis [101-103], and interfere with microcirculation [10, 101, 104-107]. Those negative effects of eryptosis are presumably counteracted by inhibitors of cell membrane scrambling, such as ASP3026.

In conclusion, ASP3026 inhibits erythrocyte cell membrane scrambling following energy depletion and oxidative stress thus counteracting suicidal erythrocyte death and subsequent development of anemia.

The authors acknowledge the meticulous preparation of the manuscript by Lejla Subasic. The study was supported by the Deutsche Forschungsgemeinschaft and Open Access Publishing Fund of Tuebingen University. Work of R.B. is supported by the Institutional Strategy of the University of Tübingen (Deutsche Forschungsgemeinschaft, ZUK 63).

None.

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