Triggering of Suicidal Erythrocyte Death by Penta-O-galloyl-β-d-glucose

The polyphenolic 1,2,3,4,6-penta-O-galloyl-beta-d-glucose from several medicinal herbs triggers apoptosis and has, thus, been proposed for treatment of malignancy. The substance is at least partially effective through caspase activation. In analogy to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and by phosphatidylserine translocation to the erythrocyte surface. Eryptosis is triggered by increase of cytosolic Ca2+-activity ([Ca2+]i). The sensitivity to [Ca2+]i is enhanced by ceramide. The present study explored whether penta-O-galloyl-β-d-glucose stimulates eryptosis. Cell volume was estimated from forward scatter, phosphatidylserine exposure from annexin V binding, hemolysis from hemoglobin-release, [Ca2+]i from Fluo3-fluorescence and ceramide abundance from fluorescent antibodies. A 48-h exposure of human erythrocytes to penta-O-galloyl-β-d-glucose significantly decreased forward scatter (50 µM) and significantly increased annexin V binding (10 µM). Up to 50 µM penta-O-galloyl-β-d-glucose did not significantly modify [Ca2+]i. However, the effect of penta-O-galloyl-β-d-glucose (25 µM) induced annexin V binding was slightly, but significantly, blunted by removal of extracellular Ca2+, pointing to sensitization of erythrocytes to the scrambling effect of Ca2+. Penta-O-galloyl-β-d-glucose (25 µM) further increased ceramide formation. In conclusion, penta-O-galloyl-β-d-glucose stimulates suicidal erythrocyte death or eryptosis, an effect partially due to stimulation of ceramide formation with subsequent sensitization of erythrocytes to Ca2+.

The present study explored the effect of penta-O-galloyl-β-D-glucose on cell volume and phosphatidylserine abundance at the erythrocyte surface. As a result, penta-O-galloyl-β-D-glucose is a powerful stimulator of eryptosis.

Results and Discussion
Flow cytometry was employed to explore whether penta-O-galloyl-β-D-glucose triggers eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling. Cell volume of human erythrocytes was estimated from forward scatter. As illustrated in Figure 1, a 48-h exposure to penta-O-galloyl-β-D-glucose led to a decrease of forward scatter, an effect reaching statistical significance at 25 µM penta-O-galloyl-β-D-glucose concentration. Accordingly, penta-O-galloyl-β-D-glucose treatment was followed by erythrocyte shrinkage.
In order to elucidate whether penta-O-galloyl-β-D-glucose stimulates cell membrane phospholipid scrambling with phosphatidylserine exposure at the erythrocyte surface, phosphatidylserine exposing erythrocytes were identified from annexin V binding determined in flow cytometry. As illustrated in Figure 2, a 48-h exposure to penta-O-galloyl-β-D-glucose increased the percentage of annexin V binding erythrocytes, an effect reaching statistical significance at a concentration of 10 µM penta-O-galloyl-β-D-glucose. Accordingly, penta-O-galloyl-β-D-glucose triggered erythrocyte cell membrane scrambling with phosphatidylserine exposure at the cell surface.  To explore whether penta-O-galloyl-β-D-glucose exposure triggers hemolysis, the percentage of hemolysed erythrocytes was estimated from hemoglobin concentration in the supernatant. As illustrated in Figure 3, the percentage erythrocytes releasing hemoglobin increased following a 48-h exposure to penta-O-galloyl-β-D-glucose concentration, an effect reaching statistical significance at 50 µM penta-O-galloyl-β-D-glucose concentration.  Even though penta-O-galloyl-β-D-glucose did not significantly modify cytosolic Ca 2+ activity, the effect of the substance could still be dependent on the presence of Ca 2+ . In order to test this possibility, erythrocytes were exposed to 25 µM penta-O-galloyl-β-D-glucose for 48 h in the presence and in the nominal absence of extracellular Ca 2+ . As illustrated in Figure 4, the effect of penta-O-galloyl-β-D-glucose on annexin V binding was slightly but significantly blunted in the nominal absence of Ca 2+ . Thus, the effect of penta-O-galloyl-β-D-glucose required in part the presence of Ca 2+ .
The sensitivity of cell membrane scrambling to cytosolic Ca 2+ could be enhanced by ceramide, which is known to trigger eryptosis even at constant [Ca 2+ ]i. In order to test, whether ceramide is enhanced by penta-O-galloyl-β-D-glucose, ceramide abundance at the cell surface was elucidated utilizing FITC-labeled anti-ceramide antibodies. As shown in Figure 5, penta-O-galloyl-β-D-glucose significantly increased ceramide-dependent fluorescence.
The present study discloses a novel xenobiotic triggering suicidal erythrocyte death or eryptosis. Treatment of human erythrocytes with penta-O-galloyl-β-D-glucose is followed by erythrocyte shrinkage and erythrocyte cell membrane scrambling, the two hallmarks of eryptosis. The concentrations required (10-50 µM) are similar to those inhibiting hepatocyte apoptosis [14].
Penta-O-galloyl-β-D-glucose is not paralleled by and thus not due to increase of cytosolic Ca 2+ activity. Most triggers of eryptosis exert their effect on annexin V binding and cell volume by activation of Ca 2+ permeable non-selective cation channels in erythrocytes, which involve the transient receptor potential channel TRPC6 [15]. Even though penta-O-galloyl-β-D-glucose treatment did not significantly modify cytosolic Ca 2+ activity, the full effect of penta-O-galloyl-β-D-glucose was dependent on the presence of extracellular Ca 2+ . Thus, penta-O-galloyl-β-D-glucose appeared to sensitize the erythrocytes to the effects of cytosolic Ca 2+ . A substance known to sensitize erythrocytes to the scrambling effect of enhanced cytosolic Ca 2+ activity is ceramide [15]. As a matter of fact, penta-O-galloyl-β-D-glucose did stimulate ceramide formation.  In nucleated cells, penta-O-galloyl-β-D-glucose has been shown to either stimulate [1][2][3][8][9][10][11][12][13] or inhibit apoptosis [6,7,10,14]. A powerful regulator of nuclear cell fate is ceramide, which stimulates death of a wide variety of cells [30]. To the best of our knowledge, penta-O-galloyl-β-D-glucose has never been shown before to stimulate the formation of ceramide. Thus, the present observations disclose a mechanism, which could well contribute to triggering of apoptosis.

Confocal Microscopy and Immunofluorescence
For the visualization of eryptotic erythrocytes, 20 μL erythrocytes were incubated under the respective experimental conditions and then stained with FITC-conjugated Annexin V (1:100 dilution; ImmunoTools, Friesoythe, Germany) in 200 μL Ringer solution containing 5 mM CaCl 2 . Then, the erythrocytes were washed twice and finally resuspended in 100 μL Ringer solution containing 5 mM CaCl 2 . Forty microliters were placed with Prolong Gold antifade reagent (Invitrogen, Darmstadt, Germany) onto a glass slide, covered with a coverslip, and images were subsequently taken on a Zeiss LSM 5 EXCITER confocal laser-scanning microscope (Carl Zeiss MicroImaging, Oberkochen, Germany) with a water immersion Plan-Neofluar 40/1.3 NA DIC.

Measurement of Hemolysis
For the determination of hemolysis the samples were centrifuged (3 min at 400 g, room temperature) after incubation, and 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.

Determination of Ceramide Formation
For the determination of ceramide, 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 1:10. 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 at an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur (BD, Heidelberg, Germany).

Confocal Microscopy and Immunofluorescence
For the visualization of eryptotic erythrocytes, 20 μL erythrocytes were incubated under the respective experimental conditions and then stained with FITC-conjugated Annexin V (1:100 dilution; ImmunoTools) in 200 μL Ringer solution containing 5 mM CaCl 2 . Then, the erythrocytes were washed twice and finally resuspended in 100 μL Ringer solution containing 5 mM CaCl 2 . Forty microliters were placed with Prolong Gold antifade reagent (Invitrogen, Darmstadt, Germany) onto a glass slide, covered with a coverslip, and images were subsequently taken on a Zeiss LSM 5 EXCITER confocal laser-scanning microscope (Carl Zeiss MicroImaging, Oberkochen, Germany) with a water immersion Plan-Neofluar 40/1.3 NA DIC.

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. As 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
Penta-O-galloyl-β-D-glucose triggers cell shrinkage and cell membrane scrambling of human erythrocytes and thus eryptosis, the suicidal death of erythrocytes. The effect is in part dependent on the presence of extracellular Ca 2+ and is at least in part due to stimulation of ceramide formation.