In Vitro Sensitization of Erythrocytes to Programmed Cell Death Following Baicalein Treatment

The polyphenolic flavonoid Baicalein has been shown to trigger suicidal death or apoptosis of tumor cells and is thus considered for the prevention and treatment of malignancy. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i) and ceramide. The present study explored whether Baicalein stimulates eryptosis. To this end, forward scatter was taken for measurement of cell volume, annexin-V-binding for phosphatidylserine-exposure, Fluo3 fluorescence for [Ca2+]i and fluorescent antibodies for ceramide abundance. As a result, a 48 h exposure of human erythrocytes to Baicalein was followed by significant decrease of forward scatter (≥10 µM), significant increase of the percentage of annexin-V-binding cells (≥25 µM), significant increase of [Ca2+]i (50 µM) and significant increase of ceramide abundance (50 µM). The effect of Baicalein (50 µM) on annexin-V-binding was significantly blunted but not abrogated by removal of extracellular Ca2+. In conclusion, at the concentrations employed, Baicalein stimulates suicidal erythrocyte death or eryptosis, an effect at least in part due to the combined effects of Ca2+ entry and ceramide formation.

Eryptosis is elicited by a wide variety of xenobiotics [24, and is observed in several clinical conditions including sepsis, malaria, sickle cell disease, Wilson's disease, iron deficiency, malignancy, metabolic syndrome, diabetes, renal insufficiency, hemolytic uremic syndrome, hyperphosphatemia and phosphate depletion [22,69]. However, to the best of our knowledge, experiments exploring an effect of the polyphenolic flavonoid Baicalein on eryptosis have never been reported.
The present study thus tested whether Baicalein stimulates eryptosis. To this end, human erythrocytes were incubated in Ringer with or without presence of Baicalein and cell volume, phosphatidylserine abundance at the cell surface, [Ca 2+ ] i , as well as ceramide abundance determined utilizing flow cytometry.

Results and Discussion
The present study explored the influence of the polyphenolic flavonoid Baicalein on eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and phosphatidylserine translocation to the cell surface.
In a first step, cell volume was estimated from forward scatter determined in flow cytometry following an incubation of human erythrocytes for 48 h in Ringer solution without or with Baicalein (5-50 µM). As shown in Figure 1, Baicalein treatment was followed by a decrease of average erythrocyte forward scatter reflecting cell shrinkage, an effect reaching statistical significance at 10 µM Baicalein concentration. The histogram reveals that Baicalein increases forward scatter in a subpopulation of erythrocytes. . ** (p < 0.01), *** (p < 0.001) indicate significant difference from the absence of Baicalein (ANOVA); (C) Arithmetic means ± SEM (n = 5) of forward scatter (arbitrary units) in erythrocytes exposed for 6-48 h to Ringer solution without (white squares) or with 50 µM Baicalein (black squares). * (p < 0.05) indicates significant difference from the absence of Baicalein.
In a second step, cell membrane phospholipid scrambling with phosphatidylserine translocation to the erythrocyte surface was quantified utilizing annexin-V-binding in flow cytometry following a 48 h incubation in Ringer solution without or with Baicalein (5-50 µM). As illustrated in Figure 2, a 48 h treatment with Baicalein increased the percentage of annexin-V-binding erythrocytes, an effect reaching statistical significance at 25 µM Baicalein concentration.
The effect of Baicalein on phosphatidylserine exposure is paralleled by hemolysis, which, however, affects fewer erythrocytes than cell membrane scrambling ( Figure 2). The phosphatidylserine exposure was not modified by inhibition of caspases with the pancaspase inhibitor zVAD (10 µM).
Cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the cell surface are both known to be stimulated by increase of cytosolic Ca 2+ activity ([Ca 2+ ] i ). Thus, a further series of experiments was performed to elucidate the effect of Baicalein on [Ca 2+ ] i . Erythrocytes were loaded with Fluo3-AM and the Fluo3 fluorescence determined by flow cytometry following incubation for 48 h in Ringer solution without or with Baicalein (5-50 µM). As illustrated in Figure 3, exposure of the erythrocytes to Baicalein increased the Fluo3 fluorescence, an effect reaching statistical significance at 50 µM Baicalein concentration.   Exposure of the erythrocytes to the Ca 2+ ionophore ionomycin was followed by a strong increase of annexin-V-binding ( Figure 4). In order to test, whether the Baicalein-induced cell membrane scrambling required entry of extracellular Ca 2+ , erythrocytes were exposed for 48 h to 50 µM Baicalein in the presence or nominal absence of extracellular Ca 2+ . As illustrated in Figure 4, the effect of Baicalein on annexin-V-binding was significantly blunted in the nominal absence of Ca 2+ . Nevertheless, even in the nominal absence of extracellular Ca 2+ , the percentage of annexin-V-binding erythrocytes was significantly higher in the presence than in the absence of Baicalein. Thus, Baicalein was effective partially, but not exclusively, through stimulation of Ca 2+ entry. In search for an additional mechanism triggering eryptosis following Baicalein treatment, further experiments were performed to possibly disclose an effect of Baicalein on ceramide formation. Ceramide abundance at the erythrocyte surface was quantified utilizing an anti-ceramide antibody. As illustrated in Figure 5, exposure of erythrocytes to 50 µM Baicalein significantly increased the ceramide abundance at the erythrocyte surface. The present study discloses a novel effect of Baicalein, i.e., stimulation of eryptosis, the suicidal death of erythrocytes. Incubation of human erythrocytes with Baicalein is followed by cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface, the most important hallmark of eryptosis. The Baicalein induced cell membrane scrambling affected only a subpopulation of the erythrocytes, an observation pointing to variable vulnerability of the erythrocytes. According to an earlier study, the susceptibility against several triggers of eryptosis is enhanced in aged erythrocytes [44].
On average, Baicalein further decreases cell volume, another hallmark of eryptosis. However, Baicalein exposure increases cell volume of an erythrocyte subpopulation. This observation again points to heterogeneity among the erythrocytes, which may be similarly due to differences in erythrocyte age. The Baicalein concentrations (10-25 µM) required for those effects were in the range of the peak concentrations reported in Baicalein treated rats [70]. However, Baicalein was not immediately effective but a 24 h exposure of erythrocytes to the substance was required in order to trigger eryptosis. Whether or not those high concentrations could be maintained in vivo for 24 h remains uncertain. Baicalein increased cytosolic Ca 2+ activity ([Ca 2+ ] i ), an effect presumably due to stimulation of cation channels in the cell membrane. Earlier studies revealed that the erythrocyte cation channels involve TRPC6 [22].
The cell shrinkage following Baicalein treatment was presumably the result of Ca 2+ entry with subsequent increase of [Ca 2+ ] i , activation of Ca 2+ sensitive K + channels, K + exit, cell membrane hyperpolarisation, Cl − exit and thus cellular loss of KCl accompanied by osmotically driven water [23]. The cellular loss of KCl with water serves to counteract the swelling and subsequent hemolysis of injured erythrocytes. Hemolysis leads to release of hemoglobin, which is subject to glomerular filtration with subsequent precipitation in the acidic lumen of renal tubules [71]. The swelling of some erythrocytes following Baicalein exposure may result from Na + entry through the unselective cation channel.
The stimulation of cell membrane scrambling by Baicalein is similarly in part due to increase of [Ca 2+ ] i . Accordingly, the effect of Baicalein on phosphatidylserine translocation is in part dependent on entry of extracellular Ca 2+ .
However, even in the absence of extracellular Ca 2+ , Baicalein treatment is still followed by a significant increase of phosphatidylserine exposure. The residual effect is in part due to stimulation of ceramide formation. Ceramide is a well-known stimulator of eryptosis [22]. Similar to what has been shown for several other stimulators of eryptosis [22], the effect of Baicalein was not sensitive to the pancaspase inhibitor zVAD and thus did not require activation of caspases.
Consequences of excessive eryptosis include anemia, since phosphatidylserine exposing eryptotic erythrocytes are phagocytosed and thus rapidly cleared from circulating blood [22]. Anemia is prevented as long as accelerated clearance of erythrocytes during stimulated eryptosis is compensated by a similarly accelerated formation of new erythrocytes [22].
Elimination of phosphatidylserine exposing erythrocytes may protect against untoward effects of hemolysis [22]. The removal of phosphatidylserine exposing erythrocytes further impacts on the clinical course of malaria [80]. Infected erythrocytes undergo eryptosis [80], since the intraerythrocytic pathogen activates ion channels including the Ca 2+ -permeable erythrocyte cation channels [81,82]. Subsequent clearance of phosphatidylserine exposing infected erythrocytes from circulating blood decreases parasitemia and by the same token precedes and thus prevents hemolysis of the parasitized erythrocytes [80]. Accordingly, the clinical course of malaria is ameliorated by genetic disorders sensitizing erythrocytes to eryptosis, such as sickle-cell trait, beta-thalassemia-trait, homozygous Hb-C and G6PD-deficiency, [22,[83][84][85], by conditions with enhanced eryptosis, such as iron deficiency [86], and by eryptosis stimulating xenobiotics, such as lead [87], chlorpromazine [88] or NO synthase inhibitors [89]. In theory, Baicalein may similarly decrease parasitemia in malaria.

Analysis of Annexin-V-Binding and Forward Scatter
After incubation under the respective experimental condition, 50 µL cell suspension was washed in Ringer solution containing 5 mM CaCl 2 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).

Measurement of Intracellular Ca 2+
After incubation, erythrocytes were washed in Ringer solution and then loaded with Fluo-3/AM (Biotium, Hayward, CA, USA) in Ringer solution containing 5 mM CaCl 2 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 CaCl 2 . The Fluo-3/AM-loaded erythrocytes were resuspended in 200 µL Ringer. Then, Ca 2+ -dependent 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).

Determination of Ceramide Formation
For the determination of 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 PBS containing 0.1% bovine serum albumin (BSA) at a dilution of 1:5. The samples were washed twice with PBS-BSA. Subsequently, the 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. The samples were then analyzed by flow cytometric analysis 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
In conclusion, the polyphenolic flavonoid Baicalein stimulates Ca 2+ entry and ceramide formation thus leading to subsequent erythrocyte shrinkage and erythrocyte cell membrane scrambling. Accordingly, Baicalein stimulates eryptosis, the suicidal erythrocyte death. The concentrations required for those effects are 10-50 µM.