Stimulation of Erythrocyte Cell Membrane Scrambling by Gedunin

Gedunin ((1S,3aS,4aR,4bS,5R,6aR,10aR,10bR,12aS)-5-(Acetyloxy)-1-(3-furanyl)- 1,5,6,6a,7, 10a,10b,11,12,12a,decahydro-4b,7,7,10a,12a,-pentamethyloxireno[c]phenanthro [1,2-d]pyran-3,8 (3aH,4bH)-dione, Fig. 1), a component of oleum azadirachti from dried seeds of Azadirachta indica A. Juss. (family: Meliaceae) [1], has been shown to inhibit heat shock protein HSP90 thus triggering apoptosis of tumor cells [2,3,4,5,6]. The substance has thus been considered for the treatment of malignancy [3,4,6,7]. Mechanisms invoked in the triggering of tumor cell apoptosis following gedunin treatment include inhibition of the antiapoptotic protein HSP90 and downregulation of the anti-apoptotic proteins HSP70 and HSP27 [3], activation of the caspases 3, 8, and 9 [8] and inhibition of sphingomyelin biosynthesis [9]. Gedunin has further been shown to be effective in vitro against Plasmodium falciparum [10,11,12,13,14]. The target accounting for the effects of gedunin on parasites remained illdefined.

In analogy to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell membrane scrambling and cell shrinkage [15]. Triggers of eryptosis include increase of cytosolic Ca²⁺ concentration ([Ca²⁺]_i), e.g. following Ca²⁺ entry through Ca²⁺-permeable cation channels [15]. Increased [Ca²⁺]_i activates Ca²⁺-sensitive K⁺ channels leading to cell shrinkage due to K⁺ exit, hyperpolarization, Cl^- exit and thus cellular loss of KCl and osmotically obliged water [16]. Increased [Ca²⁺]_i further triggers phospholipid scrambling of the cell membrane with translocation of phosphatidylserine to the erythrocyte surface [15]. Signaling in the regulation of eryptosis includes in addition ceramide formation [15], caspase activation [17,18,19,20,21] and deranged regulation of several kinases such as AMP activated kinase AMPK [22], casein kinase 1α [23,24], cGMP-dependent protein kinase [25], Janus-activated kinase JAK3 [26], protein kinase C [27], p38 kinase [28], PAK2 kinase [29] as well as sorafenib [30] and sunitinib [31] sensitive kinases.

Eryptosis may be stimulated by a wide variety of xenobiotics [15,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66] and excessive eryptosis is further observed in several clinical conditions including diabetes, renal insufficiency, hemolytic uremic syndrome, sepsis, malaria, sickle cell disease, Wilson's disease, iron deficiency, malignancy, phosphate depletion, and metabolic syndrome [15].

Erythrocytes have previously been shown to express HSP90 [67,68] and eryptosis could be stimulated by the HSP90 inhibitor geldanamycin [60].

The present study thus explored, whether and how gedunin influences eryptosis. To this end, [Ca²⁺]_i, cell volume and phosphatidylserine translocation to the erythrocyte surface were determined in the absence and presence of gedunin.

Erythrocytes were kindly provided by the blood bank of the University of Tübingen. For retriveal of leukocyte-free erythrocyte concentrates, citrate was used as anticoagulant. Prior to experiments, the concentrates were stored at 4°C in the commonly-used SAG mannitol solution (0.41 - 0.26 ml/ml concentrate) with CPD stabilisator solution (0.015 - 0.007 ml/ml concentrate). 100 ml SAG mannitol solution contained 0.877 g NaCl, 0.9 g glucose, 0.0169 g adenosine, 0.525 g mannitol. Hundred ml CPD stabilisator solution contained 0.327 g citric acid monohydrate, 2.63 g sodium citrate, 2.55 g glucose monohydrate, 0.251 g sodium dihydrogenphosphate. The erythrocyte concentrates were 7 - 20 days old when starting the experiment. The study is approved by the ethics committee of the University of Tübingen (184/2003V). 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 gedunin (Tocris, Bristol, U.K.) at the indicated concentrations. In Ca²⁺-free Ringer solution, 1 mM CaCl₂ was substituted by 1 mM glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA). Gedunin was added from a stock solution (10 mM) dissolved in DMSO. The maximal final concentration of solvent was 2.4 µl[.]

After incubation under the respective experimental condition, 50 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). Alterations of forward scatter are paralleled by similar alterations of mean erythrocyte volume (MCV) [69]. No attempt was made to calculate mean erythrocyte cell volume from forward scatter.

After incubation erythrocytes were washed in Ringer solution and then loaded with Fluo-3/AM (Biotium, Hayward, USA) in Ringer solution containing 5 mM CaCl₂ and 5 µM Fluo-3/AM. The cells were incubated at 37°C for 30 min and washed twice in Ringer solution containing 5 mM CaCl₂. The Fluo-3/AM-loaded erythrocytes were resuspended in 200 µl Ringer. Then, Ca²⁺-dependent fluorescence intensity was measured with an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur. As shown in previous papers [70], alterations of Ca²⁺ uptake leading to respective alterations of Fluo3 fluorescence are paralleled by similar alterations of radioactive Ca²⁺ fluxes. Alterations of Fluo3 fluorescence thus reflect alterations of cytosolic Ca²⁺ activity. However, the dye does not allow ratiometric measurements and thus does not allow calculating cytosolic Ca²⁺ activity.

For the determination of ceramide, a monoclonal antibody-based assay was used. After incubation, cells were stained for 1 hour at 37°C with 1 µg/ml anti ceramide antibody (clone MID 15B4, Alexis, Grünberg, Germany) in PBS containing 0.1% bovine serum albumin (BSA) at a dilution of 1:10. The samples were washed twice with PBS-BSA. Subsequently, the cells were stained for 30 minutes 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. The samples were then analyzed by flow cytometric analysis with an excitation wavelength of 488 nm and an emission wavelength of 530 nm.

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.

The present study explored the putative effect of gedunin on eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling, both events stimulated by increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i).

In a first series of experiments, forward scatter was determined in flow cytometry as a measure of cell volume. Prior to measurements, erythrocytes were incubated in Ringer solution without or with gedunin (2-24 µM). As shown in Fig. 2, a 48 h exposure to gedunin was followed by a slight decrease of forward scatter, an effect reaching statistical significance at 24 µM gedunin concentration. Thus, exposure of erythrocytes to gedunin was followed by erythrocyte shrinkage.

The decrease of forward scatter could have resulted from activation of Ca²⁺-sensitive K⁺ channels with subsequent cell shrinkage due to exit of KCl and osmotically obliged water. Thus, the effect of gedunin on [Ca²⁺]_i was tested. Human erythrocytes were loaded with Fluo3-AM and the Fluo3 fluorescence determined by flow cytometry. Prior to determination of Fluo3-fluorescence, erythrocytes were incubated in Ringer solution without or with gedunin (2-24 µM). As shown in Fig. 3, a 48 h exposure of human erythrocytes to gedunin increased Fluo3 fluorescence, an effect reaching statistical significance at 12 µM gedunin concentration. Thus, gedunin increased cytosolic Ca²⁺ concentration.

An increase of [Ca²⁺]_i is known to stimulate cell membrane phospholipid scrambling leading to phosphatidylserine translocation to the erythrocyte surface. In order to identify phosphatidylserine exposing erythrocytes, annexin-V-binding was determined in flow cytometry. As shown in Fig. 4, a 48 h exposure to gedunin increased the percentage of annexin-V-binding erythrocytes, an effect reaching statistical significance at 12 µM gedunin. Thus, gedunin triggered erythrocyte cell membrane scrambling with phosphatidylserine translocation to the cell surface.

In order to quantify the effect of gedunin on hemolysis, the percentage of hemolysed erythrocytes was estimated from hemoglobin concentration in the supernatant. As a result, the percentage of hemolysed erythrocytes approached 0.21 ± 0.07%, 0.86± 0.50%, 1.2 ± 0.85%, 3.4 ± 1.6% and 5.2 ± 1.6% following exposure of erythrocytes for 48 h to 0, 2, 6, 12 and 24 µM gedunin (n = 4).

A further series of experiments tested whether the gedunin induced cell membrane scrambling required entry of extracellular Ca²⁺. To this end, erythrocytes were exposed to 24 µM gedunin for 48 h in the presence and in the nominal absence of extracellular Ca²⁺. As shown in Fig. 5, the effect of gedunin on annexin-V-binding was virtually abrogated in the nominal absence of Ca²⁺. Thus, the effect of gedunin was mainly if not exclusively due to Ca²⁺ entry.

Additional experiments were made on the effect of gedunin on ceramide formation. Following a 48 hour incubation with or without 24 µM gedunin, the ceramide abundance at the erythrocyte surface was 13.4 ± 3.1 (n = 4) in the absence and 13.8 ± 2.6 (n = 4) in the presence of gedunin, values not significantly different.

The present study discloses a novel effect of gedunin, i.e. stimulation of erythrocyte cell membrane scrambling leading to phosphatidylserine translocation to the erythrocyte surface. Treatment of human erythrocytes with gedunin further decreased cell volume and increased cytosolic Ca²⁺ activity ([Ca²⁺]_i). The concentrations required for those effects were similar to those encountered in vivo [14].

Erythrocytes express HSP 90 [67,68] and the HSP 90 inhibitor geldanamycin has previously been shown to trigger eryptosis [60]. To the best of our knowledge this is the first demonstration that gedunin increases cytosolic [Ca²⁺]_i. Notably, both, HSP90 and transient receptor potential channels, interact with XPORT (exit protein of rhodopsin and TRP) [71] and HSP90 may thus, at least in theory, modify the function of the erythrocyte cation channel, which involves TRPC6 [15]. The increase of [Ca²⁺]_i presumably accounts for the erythrocyte shrinkage. Increased [Ca²⁺]_i activates Ca²⁺ sensitive K⁺ channels [15] followed by K⁺ exit, cell membrane hyperpolarisation, Cl^- exit and thus cellular loss of KCl with osmotically obliged water [16]. The decrease of cell volume during eryptosis counteracts swelling of defective erythrocytes, which may otherwise lead to rupture of the erythrocyte membrane with subsequent cellular release, glomerular filtration and intratubular precipitation of hemoglobin [72].

Stimulation of Ca²⁺ entry may possibly contribute to the stimulation of apoptosis by gedunin. Increased [Ca²⁺]_i is a well known trigger of apoptosis in nucleated cells [73,74,75]. Further experiments in nucleated cells are warranted to explore whether gedunin increases [Ca²⁺]_i similarly in tumor cells and, if so, whether this effect participates in the triggering of apoptosis.

The stimulation of cell membrane scrambling with phosphatidylserine exposure at the cell surface may be favourable during infection with the malaria pathogen Plasmodium falciparum. In view of its effect on noninfected cells gedunin may accelerate suicidal death of infected erythrocytes [76]. The intraerythrocytic parasite activates several channels including Ca²⁺-permeable erythrocyte cation channels [77,78]. The channels provide the intraerythrocytic pathogen with nutrients, Na⁺ and Ca²⁺ and allow the disposal of waste products [78]. Intraerythrocyte survival of the pathogen thus requires activation of those channels [77,78]. The Ca²⁺ entry through the Ca²⁺-permeable cation channels triggers, however, eryptosis [76], thus leading to rapid clearance of the infected erythrocytes from circulating blood [15]. Accordingly, Ca²⁺ entry and subsequent eryptosis limits the life span of infected erythrocytes and thus counteracts parasitemia [76]. Along those lines several genetic disorders predisposing to accelerated eryptosis, such as sickle-cell trait, beta-thalassemia-trait, homozygous Hb-C and G6PD-deficiency [15] are known to confer partial resistance to malaria [79,80,81], even though the partial resistance may involve further mechanisms other than eryptosis [82,83,84,85].

In addition to genetic erythrocyte disorders, several clinical conditions and drugs stimulating eryptosis have been shown to favourably influence the clinical course of malaria, such as iron deficiency [86], lead [87], chlorpromazine [88] and inhibition of NO synthase [89].

On the other hand, stimulation of eryptosis may lead to anemia. Phosphatidylserine at the surface of eryptotic cells binds to the respective receptors of phagocytosing cells thus leading to rapid removal of the suicidal erythrocytes from circulating blood [15]. As soon as accelerated loss of erythrocytes during stimulated eryptosis cannot be matched by similarly enhanced formation of new erythrocytes, anemia develops [15]. Phosphatidylserine exposing erythrocytes may further impede microcirculation [90,91,92,93,94,95], as they adhere to endothelial CXCL16/SR-PSO [91]. Phosphatidylserine exposing erythrocytes may further stimulate blood clotting and thrombosis [90,96,97].

Whether or not gedunin and/or HSP90 inhibition leads to anemia and/or deranged microcirculation, remains to be established. In HSP90 knockout mice enhanced antibody production to a T-dependent antigen and enhanced MHC class II antigen presentation to T helper cells by dendritic cells has been observed [98]. To the best of our knowledge, nothing is hitherto known about anemia, microcirculation and thrombosis in those mice.

Gedunin stimulates Ca²⁺ entry leading to cell shrinkage and cell membrane scrambling of erythrocytes, hallmarks of suicidal erythrocyte death or eryptosis.

The authors acknowledge the meticulous preparation of the manuscript by Tanja Loch. The study was supported by the Deutsche Forschungsgemeinschaft and the Open Access Publishing Fund of Tuebingen University.