Triggering of Programmed Erythrocyte Death by Alantolactone

The sesquiterpene alantolactone counteracts malignancy, an effect at least in part due to stimulation of suicidal death or apoptosis of tumor cells. Signaling of alantolactone induced apoptosis involves altered gene expression and mitochondrial depolarization. Erythrocytes lack mitochondria and nuclei but may enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the erythrocyte surface. Cellular mechanisms involved in triggering of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i) and oxidative stress. The present study explored, whether alantolactone stimulates eryptosis. To this end, erythrocyte volume was estimated from forward scatter, phosphatidylserine-exposure at the erythrocyte surface from FITC-annexin-V-binding, [Ca2+]i from Fluo3-fluorescence, ceramide abundance from binding of fluorescent antibodies, and oxidative stress from 2',7'-dichlorodihydrofluorescein-diacetate (DCFDA) fluorescence. As a result, a 48 h exposure of human erythrocytes to alantolactone (≥20 μM) significantly decreased erythrocyte forward scatter and increased the percentage of annexin-V-binding cells. Alantolactone significantly increased Fluo3 fluorescence (60 μM), ceramide abundance (60 μM) and DCFDA fluorescence (≥40 μM). The effect of alantolactone (60 μM) on annexin-V-binding was not significantly modified by removal of extracellular Ca2+. In conclusion, alantolactone stimulates suicidal erythrocyte death or eryptosis, an effect paralleled by increase of [Ca2+]i, ceramide abundance and oxidative stress.

The present study explored, whether eryptosis is stimulated by alantolactone. To this end, human erythrocytes drawn from healthy volunteers were exposed to alantolactone and cell volume, phosphatidylserine abundance at the cell surface, [Ca 2+ ]i and reactive oxygen species (ROS) determined.

Results and Discussion
In order to test whether the sesquiterpene alantolactone triggers eryptosis, the suicidal erythrocyte death, human erythrocytes were exposed for 48 h to Ringer solution without or with alantolactone (10-60 μM) and cell volume as well as phosphatidylserine translocation to the erythrocyte surface were determined.
Forward scatter was determined utilizing flow cytometry in order to estimate alterations of cell volume. As shown in Figure 1, a 48 h exposure to alantolactone-containing Ringer was followed by a decrease of forward scatter, an effect reaching statistical significance at 20 μM alantolactone concentration.  forward scatter of erythrocytes following exposure for 48 h to Ringer solution without  (grey area) and with (black line) presence of 60 μM alantolactone; (B) Arithmetic means ± SEM (n = 15) of the normalized erythrocyte forward scatter (FSC) following incubation for 48 h to Ringer solution without (white bar) or with (black bars) alantolactone (10-60 μM). For comparison, the effect of 1 μL DMSO/mL Ringer is shown (grey bar). * (p < 0.05), *** (p < 0.001) indicates significant difference from the absence of alantolactone (ANOVA); (C) Arithmetic means ± SEM (n = 4) of the normalized erythrocyte forward scatter (FSC) following incubation for 48 h to Ringer solution without alantolactone (white bar), or following 48 h treatment with 60 μM alantolactone (black bar) or following 1 h treatment with 1 μM ionomycin (grey bar). *** (p < 0.001) indicates significant difference from the absence of treatment (ANOVA).

C
Phosphatidylserine translocation to the erythrocyte surface was quantified from binding of FITC-labelled annexin-V as determined in flow cytometry. As shown in Figure 2, a 48 h exposure to alantolactone was followed by an increase of the percentage of erythrocytes binding FITC-labelled annexin-V, an effect reaching statistical significance at 20 μM alantolactone concentration.
Hemolysis was estimated by determination of hemoglobin in the supernatant. As shown in Figure 2F, alantolactone tended to slightly increase the percentage of hemolysed erythrocytes, an effect, however, not reaching statistical significance.
Both, cell shrinkage and phosphatidylserine translocation to the cell surface could have resulted from an increase of cytosolic Ca 2+ activity ([Ca 2+ ]i). Thus, additional experiments explored the effect of alantolactone on [Ca 2+ ]i. Following a 48 h incubation in Ringer solution without or with alantolactone (10-60 µM), the erythrocytes were loaded with Fluo3-AM and the Fluo3 fluorescence determined by flow cytometry. As shown in Figure 3, exposure of the erythrocytes to alantolactone was followed by an increase of Fluo3 fluorescence, an effect reaching statistical significance at 60 µM alantolactone concentration.  In order to test whether the alantolactone induced cell membrane scrambling required entry of extracellular Ca 2+ , erythrocytes were exposed for 48 h to 60 µM alantolactone in the presence or nominal absence of extracellular Ca 2+ . As illustrated in Figure 4A, the effect of alantolactone on annexin-V-binding was not significantly modified by removal of extracellular Ca 2+ . Thus, the effect of alantolactone on annexin-V-binding did not depend on Ca 2+ entry. To ascertain that the high calcium content (5 mM) in the staining solution did not affect the results, the effect ionomycin was studied using the same protocol. As illustrated in Figure 4B, the effect of ionomycin on annexin-V-binding was completely abrogated by removal of extracellular Ca 2+ .

A B
Mechanisms stimulating eryptosis without increase of [Ca 2+ ]i include ceramide. Thus, additional experiments were performed in order to quantify the effect of alantolactone on the ceramide abundance at the erythrocyte surface. To this end the ceramide abundance was determined utilizing a fluorescent anti-ceramide antibody. As shown in Figure 5, a 48 h exposure of erythrocytes to 60 µM alantolactone significantly increased the abundance of ceramide at the erythrocyte surface.
Eryptosis is further triggered by oxidative stress. Thus, DCFDA fluorescence was determined to estimate reactive oxygen species (ROS). As illustrated in Figure 6, a 48 h exposure to alantolactone (40 or 60 µM) was followed by a significant increase of DCFDA fluorescence pointing to induction of oxidative stress.
Apparently higher concentrations of alantolactone are required to appreciably increase [Ca 2+ ]i than the alantolactone concentrations required for stimulation of phosphatidylserine translocation or induction of cell shrinkage. Moreover, the removal of extracellular Ca 2+ did not appreciably modify alantolactone induced cell membrane scrambling. Thus, entry of extracellular Ca 2+ cannot account for the stimulation of eryptosis by alantolactone. Instead, stimulation of eryptosis by alantolactone presumably involves ceramide formation and induction of oxidative stress. NBD-PS and NBD-PC uptake studies point to the modification of flippase and scramblase activities.
The cell shrinkage, which is presumably in part due to due to activation of Ca 2+ sensitive K + channels and subsequent cellular loss of KCl and water [13], is only mild and much less pronounced than the cell shrinkage induced by the Ca 2+ ionophore ionomycin. Close inspection of the histogram in Figure 1 reveals that a small subpopulation of erythrocytes even rather swells. Possibly, alantolactone stimulated Na + entry, which may in some cells override K + exit. In contrast to its strong effect on eryptosis, alantolactone treatment tended to only slightly increase hemolysis, an effect not reaching statistical significance (Figure 2).
The cell shrinkage serves to counteract cell swelling and the phosphatidylserine exposure at the cell surface is an "eat me" signal leading to phagocytosis of eryptotic cells. The triggering of eryptosis thus counteracts hemolysis of defective erythrocytes. The hemolysis would otherwise be followed by release of hemoglobin, which may be filtered in renal glomeruli and subsequently precipitate in the acidic lumen of renal tubules [68]. The removal of eryptotic erythrocytes is an important host defence mechanism during infection with Plasmodia [69]. The intraerythrocytic parasite activates several ion channels in the host cell membrane including the Ca 2+ -permeable erythrocyte cation channels [70,71]. The subsequent Ca 2+ entry triggers eryptosis with subsequent clearance of the infected erythrocytes from circulating blood [69,72]. Accordingly, genetic disorders sensitizing erythrocytes to eryptosis, such as sickle-cell trait, beta-thalassemia-trait, homozygous Hb-C and G6PD-deficiency [12,[73][74][75], lead to accelerated eryptosis of infected erythrocytes thus counteracting parasitemia and a severe course of the disease [69]. Similarly, some clinical conditions fostering eryptosis, such as iron deficiency [72], and several eryptosis stimulating xenobiotics, such as lead [76], chlorpromazine [77] or NO synthase inhibitors [78] have been shown to favourably influence the clinical course of malaria. It remains to be tested, whether alantolactone influences the clinical course of malaria.
The alantolactone concentrations required for stimulation of eryptosis were similar to those effective in cancer cells [2][3][4][5][6][7][8][9][10][11]. In theory, enhanced eryptosis may thus limit the use of alantolactone in the treatment of tumors. It must be kept in mind that eryptosis is enhanced in malignancy [82], a complication presumably compounded by therapeutic use of eryptosis inducing substances.

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 rcf for 20 min at room temperature 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), 5 glucose, 1 CaCl2; pH 7.4 at 37 °C for 48 h. Where indicated, erythrocytes were exposed to alantolactone (Sigma Aldrich, Schnelldorf, Germany) at the indicated concentrations, solved in 1 µL/mL DMSO. For comparison, the effect of 1 µL DMSO/mL Ringer was tested.

Analysis of Annexin-V-Binding and Forward Scatter
After incubation under the respective experimental condition, 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 20 min under protection from light. In the following, the forward scatter (FSC) of the cells was determined, and annexin-V fluorescence intensity was measured with an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur (BD, Heidelberg, Germany). The incubation with Annexin-V-FITC required presence of 5 mM CaCl2 even in experiments on the effect of a 48 h incubation with alantolactone in the absence of Ca 2+ . In order to test whether the short incubation with 5 mM Ca 2+ could have biased the results, experiments were performed in erythrocytes treated with Ca 2+ ionophore ionomycin. As indicated in Figure 4, a 20 min exposure to extracellular Ca 2+ in the presence of Ca 2+ ionophore ionomycin (1 µM) was not sufficient to trigger significant annexin-V binding.

Determination of Ceramide Formation
To determine ceramide abundance, a monoclonal antibody-based assay was used. After incubation, cells were stained for 1 h at 37 °C with 1 μg/mL anti-ceramide antibody (clone MID 15B4; Alexis, Grünberg, Germany) in phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (BSA) at a dilution of 1:10. After two washing steps with PBS-BSA, cells were stained for 30 min with polyclonal fluorescein-isothiocyanate (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. Samples were then analyzed by flow cytometric analysis at an excitation wavelength of 488 nm and an emission wavelength of 530 nm.

Determination of Reactive Oxygen Species (ROS)
ROS production was determined utilizing 2',7'-dichlorodihydrofluorescein diacetate (DCFDA). Briefly, the cells were suspended in FACS buffer and the fluorescence was analysed with flow cytometry (FACS-Calibur from Becton Dickinson; Heidelberg, Germany). DCFDA fluorescence intensity was measured in FL-1 with an excitation wavelength of 488 nm and an emission wavelength of 530 nm.

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 and t test as appropriate. 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.

Conclusions
Exposure of human erythrocytes to alantolactone is followed by stimulation of eryptosis, characterized by erythrocyte shrinkage and phosphatidylserine translocation to the erythrocyte surface. Signaling involved includes increase of [Ca 2+ ]i, ceramide formation and induction of oxidative stress.