Stimulation of Suicidal Erythrocyte Death by Garcinol Open Access

The benzophenone garcinol [1], a component of the dried fruit rind of Garcinia indica [1,2,3], has been shown to counteract oxidative stress [2,3,4], inflammation [2,4,5] and malignancy [2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17]. Garcinol is effective against malignancy at least in part by triggering suicidal cell death or apoptosis [8,11,12,14,16,18,19,20,21,22]. On the other hand, garcinol could counteract apoptosis [23]. Cellular mechanisms involved in the effects of garcinol include inhibition of histone acetyltransferases and thus modification of gene expression [10,13,15,18,24,25,26,27], down-regulation of nuclear factor NF-κB [5,7,8,16,18,19,23], signal transducer and activator of transcription 3 (STAT3) [5,6,28], cyclin-dependent kinase 2 (CDK2) [4],p38 kinase [4], and phosphoinositide 3 kinase (PI3K)/Akt signaling [21]. Garcinol treatment leads to growth arrest and expression of DNA damage-inducible gene 153 (GADD153) [29]. Garcinol further triggers mitochondrial depolarisation [29].

Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis [30], a suicidal death characterized by cell shrinkage [31] and cell membrane scrambling with phosphatidylserine translocation to the cell surface [30]. Triggers of eryptosis include Ca²⁺ entry through Ca²⁺ permeable unselective cation channels with increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i). Eryptosis is further stimulated by ceramide [32], oxidative stress [30], energy depletion [30], activated caspases [30,33,34], casein kinase 1α, Janus-activated kinase JAK3, protein kinase C and p38 kinase [30]. Eryptosis is inhibited by AMP activated kinase AMPK, cGMP-dependent protein kinase, PAK2 kinase, and sorafenib/sunitinib sensitive kinases [30]. Eryptosis may be stimulated by a wide variety of xenobiotics [30,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60].

The present study explored whether and how garcinol triggers eryptosis. To this end, human erythrocytes from healthy volunteers were exposed to garcinol and phosphatidylserine surface abundance, cell volume, [Ca²⁺]_i and abundance of reactive oxygen species (ROS) determined by flow cytometry.

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 24 h. Where indicated, erythrocytes were exposed to garcinol (Tocris bioscience, Bristol, UK) at the indicated concentrations.

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. The annexin V abundance at the erythrocyte surface was subsequently determined on a FACS Calibur (BD, Heidelberg, Germany). A dot plot of forward scatter (FSC) vs. side scatter (SSC) was set to linear scale for both parameters. The threshold of forward scatter was set at the default value of “52”.

For the determination of hemolysis, the samples were centrifuged (3 min at 1600 rpm, room temperature) after incubation under the respective experimental conditions 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.

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 once 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.

Oxidative stress was determined utilizing 2', 7'-dichlorodihydrofluorescein diacetate (DCFDA). After incubation, a 100 µl suspension of erythrocytes was washed in Ringer solution and then stained with DCFDA (Sigma, Schnelldorf, Germany) in PBS containing DCFDA at a final concentration of 10 µM. Erythrocytes were incubated at 37°C for 30 min in the dark and then washed in PBS. The DCFDA-loaded erythrocytes were resuspended in 200 µl Ringer solution, and ROS-dependent fluorescence intensity was measured at an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur (BD).

For the determination of intracellular erythrocyte ATP, 80 µl of erythrocyte pellets were incubated for 24 h at 37°C in Ringer solution (final hematocrit 4.7%). All subsequent manipulations were performed at 4°C to avoid ATP degradation. Cells were lysed in distilled water, and proteins were precipitated by addition of HClO₄ (6%). After centrifugation, an aliquot of the supernatant (400 µl) was adjusted to pH 7.7 by addition of saturated KHCO₃ solution. After dilution of the supernatant, the ATP concentrations of the aliquots were determined utilizing the luciferin-luciferase assay kit (Roche Diagnostics) on a luminometer (Berthold Biolumat LB9500, Bad Wildbad, Germany) according to the manufacturer's protocol. ATP concentrations are expressed in mmol/l cytosol of erythrocytes.

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 tested, whether garcinol modifies eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the cell surface. In order to uncover cell membrane scrambling, phosphatidylserine at the erythrocyte surface was visualized with annexin-V-binding, which was determined by flow cytometry. Prior to measurement, erythrocytes were incubated for 24 hours in Ringer solution without or with garcinol (1 - 5 µM). As shown in Fig. 1, a 24 hours exposure to garcinol increased the percentage of phosphatidylserine exposing erythrocytes, an effect reaching statistical significance at 2.5 µM garcinol. For comparison, hemolysis was quantified from the hemoglobin concentration in the supernatant. As a result, the percentage of hemolytic erythrocytes was significantly higher following exposure to 5 µM garcinol than in the absence of garcinol (Fig. 1). However, the percentage of hemolytic erythrocytes remained lower than the percentage of annexin-V-binding erythrocytes (Fig. 1).

Erythrocyte volume was estimated from forward scatter, which was determined utilizing flow cytometry. As illustrated in Fig. 2, a 24 hours incubation in Ringer solution with 1 and 2.5 µM garcinol was followed by a significantly lower erythrocyte forward scatter than a 24 hours incubation in Ringer solution without garcinol. In contrast, a 24 hours incubation in Ringer solution with 5 µM garcinol was followed by a significantly higher erythrocyte forward scatter than a 24 hours incubation in Ringer solution without garcinol. Thus, 1 µM and 2.5 µM garcinol shrank but 5 µM garcinol swelled erythrocytes.

In order to quantify cytosolic Ca²⁺ activity ([Ca²⁺]_i), Fluo3 fluorescence was measured. As illustrated in Fig. 3, a 24 hours exposure to garcinol increased the Fluo3 fluorescence, an effect reaching statistical significance at 5 µM garcinol.

In order to test whether garcinol -induced translocation of phosphatidylserine or erythrocyte shrinkage required entry of extracellular Ca²⁺, erythrocytes were incubated for 24 hours in the absence or presence of 5 µM garcinol in the presence or nominal absence of extracellular Ca²⁺. As shown in Fig. 4, removal of extracellular Ca²⁺ slightly, but significantly blunted the effect of garcinol on annexin-V-binding. However, garcinol significantly increased the percentage of annexin-V-binding erythrocytes even in the absence extracellular Ca²⁺. Garcinol-induced cell membrane scrambling was thus partially but not fully due to entry of extracellular Ca²⁺.

Removal of extracellular Ca²⁺ did not significantly blunt the effect of 5 µM garcinol on forward scatter. In the absence of garcinol the forward scatter was virtually identical in the presence (437 ± 8, n = 12) and in the absence (437 ± 5, n = 12) of extracellular Ca²⁺ and incubation with 5 µM garcinol increased the forward scatter to similar values in the presence (473 ± 6, n = 12) and in the absence (480 ± 5, n = 12) of extracellular Ca²⁺.

Additional experiments explored whether the effect of garcinol was modified by the NF-κB inhibitor Bay 11-7082. As a result, Bay 11-7082 (20 µM) tended to increase annexin-V-binding of erythrocytes in the presence and absence of 5 µM garcinol, an effect, however, not reaching statistical significance (Fig. 5).

In order to quantify oxidative stress, reactive oxygen species (ROS) was determined with 2′, 7′-dichlorodihydrofluorescein diacetate (DCFDA). As shown in Fig. 6, a 24 hours exposure to garcinol (1 - 5 µM) significantly increased the DCFDA fluorescence. Accordingly, garcinol induced oxidative stress.

Additional experiments explored whether the effect of garcinol was modified by the antioxidant N-acetylcysteine (3 mM). As a result, N-acetylcysteine did not significantly modify the effect of 5 µM garcinol on annexin-V-binding of erythrocytes (Fig. 7).

In order to explore whether garcinol triggers energy depletion, ATP levels were determined utilizing a luciferin-luciferase assay. As illustrated in Fig. 8, a 24 hours exposure to garcinol decreased the cytosolic ATP levels, an effect reaching statistical significance at 5 µM garcinol concentration.

The present study reveals that exposure of human erythrocytes to garcinol is followed by stimulation of cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Moreover, lower concentrations of garcinol (1 and 2.5 µM) trigger cell shrinkage, the second hallmark of eryptosis, the suicidal erythrocyte death. The concentrations required for the effect are lower than those required to counteract growth of tumor cells [20]. In theory, lower garcinol concentrations may be required for triggering of eryptosis in clinical conditions associated with enhanced eryptosis susceptibility of the erythrocytes, such as dehydration [48], hyperphosphatemia [58] chronic kidney disease (CKD) [40,61,62,63], hemolytic-uremic syndrome [64], diabetes [65], hepatic failure [66], malignancy [30], sepsis [67], Sickle-cell disease [30], beta-thalassemia [30], Hb-C and G6PD-deficiency [30], as well as Wilsons disease [68]. In those disorders lower garcinol concentrations may be sufficient to trigger eryptosis.

The effect of garcinol on cell membrane scrambling was in small part due to increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i), which is known to trigger cell membrane scrambling by activating an illdefined scramblase [30]. Accordingly, removal of extracellular Ca²⁺ significantly blunted cell membrane scrambling. Ca²⁺ entered presumably through Ca²⁺ permeable cation channels, which are known to be activated by oxidative stress [30]. DCFDA fluorescence indeed revealed that high concentrations of garcinol increased the abundance of reactive oxidant species.

An increase of [Ca²⁺]_i could further trigger erythrocyte shrinkage by activation of Ca²⁺ sensitive K⁺ channels with subsequent cell shrinkage due to K⁺ exit, cell membrane hyperpolarization, Cl^- exit and thus cellular loss of KCl with water [31]. However, higher concentrations of garcinol are required to appreciably increase [Ca²⁺]_i and higher concentrations of garcinol increase rather than decrease erythrocyte volume. The increase of cell volume may result from the observed decline of ATP levels, which should compromise the function of the Na⁺/K⁺ ATPase with subsequent increase of cytosolic Na⁺, as well as decline of cytosolic K⁺ with subsequent depolarization and gain of Cl^- with osmotically obliged water [69].

The most important functional significance of eryptosis is removal of defective erythrocytes prior to hemolysis [30]. Hemolysis triggers release of hemoglobin, which is filtered in renal glomerula with subsequent precipitation in the acidic lumen of renal tubules and thus occlusion of nephrons [70]. Eryptosis further fosters the removal of erythrocytes infected with the malaria pathogen Plasmodium. Plasmodia induce oxidative stress with subsequent activation of Ca²⁺-permeable erythrocyte cation channels [30,71]. Several genetic erythrocyte disorders, such as sickle-cell trait, beta-thalassemia-trait, Hb-C and G6PD-deficiency enhance the susceptibility to triggers of eryposis and thus accelerate the removal of infected erythrocytes. As a result, the disorders blunt parasitemia and thus protect against a severe course of malaria [30,72,73,74]. Eryptosis is further fostered and thus increase of parasitemia slowed by iron deficiency [75] as well as treatment with lead [75], chlorpromazine [76] or NO synthase inhibitors [76]. In theory, garcinol may similarly foster eryptosis of plasmodium infected erythrocytes.

On the other hand, eryptosis may lead to anemia as soon as the loss of erythrocytes is not matched by a similar increase of erythrocyte formation [30]. Moreover, phosphatdylserine exposing erythrocytes may adhere to the vascular wall [77] and stimulate clotting as well as thrombosis [78,79,80]. Accordingly, excessive eryptosis may interfere with microcirculation [32,78,81,82,83,84].

Garcinol triggers cell membrane scrambling and cell shrinkage. At higher concentrations, garcinol leads to cell swelling, energy depletion, oxidative stress and increase of cytosolic Ca²⁺ activity.

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

The authors state that they have nothing to disclose.