We have previously demonstrated that resveratrol (Resv)-induced cellular apoptosis occurs after formation of reactive oxygen species (ROS) but the role of GSH has not been well defined. Our experimental data enumerated that Resv treatment (50 μm) induced apoptosis in human leukemic monocyte lymphoma cells, which was preceded by cellular GSH efflux. High concentration of extracellular thiol (GSH, N-acetyl cysteine) and two specific inhibitors of carrier-mediated GSH extrusion, methionine or cystathionine, prevented the process of oxidative burst and cell death. This proved that GSH efflux could be a major molecular switch to modulate Resv-induced ROS generation. Spectrofluorometric data depicted that after 6 h of Resv treatment, ROS generation was evident. Pretreatment of cells with intracellular ROS scavenger (polyethylene glycol-superoxide dismutase and polyethylene glycol-catalase) efficiently reduced ROS generation but failed to prevent intracellular GSH depletion. Thus, it suggested that intracellular GSH depletion was independent of ROS production but dependent on GSH extrusion. Furthermore, to bridge the link between GSH efflux and ROS generation, we carried out confocal microscopy of the localization of Bax protein. Microscopic analysis and small interfering RNA treatment emphasized that cellular GSH efflux triggered Bax translocation to mitochondria, which resulted in the loss of mitochondrial membrane potential, ROS generation, and caspase 3 activation and thus triggered apoptosis.
Oxidants have been widely shown to initiate the cellular apoptotic cascade by perturbing the balance between cellular signals for survival and suicide. GSH is the most abundant intracellular low molecular weight thiol, and it is among the many detoxification processes that maintain cellular redox homeostasis. Its protective action is based on oxidation of the thiol group of its cysteine residue, resulting in the formation of oxidized glutathione (GSSG); this in turn is catalytically reversed to GSH by GSH reductase (Meister and Anderson, 1983). Under conditions of oxidative stress, GSSG may either recycle to GSH or exit from the cells, leading to an overall depletion of GSH (Reed, 1990). GSH can also be extruded in the reduced form, through specific carriers, by many cell types under physiological conditions.
Intracellular loss of GSH is an early hallmark in the progression of cell death in response to different apoptotic stimuli (Hammond et al., 2004; Franco and Cidlowski, 2006) and has been associated with activation of a plasma membrane transport mechanism rather than with its oxidation (van den Dobbelsteen et al., 1996; He et al., 2003). Several studies have also shown a correlation between cellular depletion of GSH and progression of apoptosis (Ghibelli et al., 1998; Franco and Cidlowski, 2006). We have already reported that treatment with resveratrol (Resv) stimulates production of reactive oxygen species (ROS), leading to a caspase-dependent apoptosis in human leukemic monocyte lymphoma (U937) cell line (Guha et al., 2010). However, the characterization of the nature of the ROS generated (hydrogen peroxide, peroxynitrite, singlet oxygen, or hydroxyl radical) and the effect of Resv on the intracellular level of GSH was not explored. At the same time, it has already been reported that generation of ROS is modulated by translocation of mitochondrial Bax (Kirkland and Franklin, 2007). Bax is a proapoptotic Bcl2 family of protein wherein apoptotic stimuli stimulate mitochondrial Bax translocation, leading to dissipation of the mitochondrial membrane potential (ΔΨm) and generation of ROS. Intracellular GSH depletion enhanced mitochondrial Bax translocation, resulting in apoptosis (Honda et al., 2004). Resv-induced Bax activation is an upstream event in apoptosis (Mohan et al., 2006), but the exact link between GSH depletion, ROS generation, and mitochondrial Bax translocation has not been specified. Hence, here we tried to draw a correlation among depletion of intracellular GSH, production of ROS, and translocation of mitochondrial Bax in Resv-induced apoptotic cell death.
Materials and Methods
Reagents and Kits.
All chemicals were obtained from Sigma-Aldrich (St. Louis, MO) except primary antibodies (Bax, β-actin, COX IV) and polyclonal secondary antibody were obtained from Cell Signaling Technology (Danvers, MA); Apo-direct terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay kit and caspase protease assay kit were purchased from Millipore Corporation (Billerica, MA); Alexa Fluor 488-AnnexinV/propidium iodide (PI) kit, CM-H2DCFDA, Singlet Oxygen Sensor Green dye, RPMI 1640 medium, fetal bovine serum (FBS), penicillin-streptomycin, and trypsin-EDTA were obtained from Invitrogen (Carlsbad, CA); and mitochondria/cytosol fractionation kit and GSH assay kit were purchased from BioVision (Mountain View, CA). Revert Aid H minus first strand cDNA synthesis kit and Dynamo SYBR Green quantitative PCR kit were obtained from MBI Fermentas (Hanover, MD).
U937 cell line was obtained from National Centre for Cell Science (Pune, India). U937 was cultured in RPMI 1640 medium, at pH 7.4. Media were supplemented with 10% FBS and antibiotics (100 U/ml penicillin-G, 100 μg/ml streptomycin, and 6 μg/ml gentamicin). The cells were incubated at 37°C in a humidified atmosphere containing 5% CO2. Cells were subcultured weekly, and cells from passages six to eight were used for experiments.
CellTiter-Glo Luminescent Cell Viability Assay.
The CellTiter-Glo Luminescent Cell Viability Assay was used to determine cell viability based on quantitation of the ATP present, which signals the presence of metabolically active cells. In brief, cells (5 × 104 cells/100 μl of medium/well) were seeded in 96-well plates and incubated for 12, 24, 48, and 72 h in the presence of Resv (dissolved in 0.02–0.07% dimethyl sulfoxide). Each well was then treated with a volume of CellTiter-Glo Reagent equal to the volume of cell culture medium present, mixed for 2 min on an orbital shaker to induce cell lysis, and incubated at room temperature for 10 min, after which the stabile luminescent signal was reordered in a luminometer (FLX800; BioTek Instruments, Winooski, VT).
Detection of Apoptosis by Annexin V/PI Double Staining Method.
Perturbations in the cellular membrane occur during the early stage of apoptosis and lead to a flip of phosphatidyl serine to the external aspect of the cell membrane. Annexin V selectively binds to phosphatidyl serine and helps to identify cells undergoing apoptosis. Cells were treated with different concentrations of Resv for 24 h, and apoptosis was measured using AnnexinV Alexa Fluor 488 and PI Apoptosis Detection Kit. In brief, U937 cells were harvested and PI (5 μl) and Annexin V Alexa Fluor 488 (5 μl) was added, incubated for 15 min at room temperature in the dark, and then analyzed on a flow cytometer (equipped with an argon laser light source at a wavelength of 488 nm, a band-pass filter at a wavelength of 515 nm for Alexa Fluor 488, and a band-pass filter at a wavelength of 623 nm for PI fluorescence) using CellQuest software. A total of 104 events were acquired.
To confirm the nature of tumor killing by Resv on U937, cells were fixed, permeabilized, and incubated with terminal deoxynucleotidyl transferase enzyme and fluorescein isothiocyanate-dUTP per the manufacturer's instructions. Cells were then washed, incubated with RNase solution, and acquired on a flow cytometer (BD Biosciences, San Diego, CA) that was equipped with an argon laser light source at a wavelength of 488 nm, and a band-pass filter at a wavelength of 515 nm for Alexa Fluor 488 FL1-H and analyzed using CellQuest software (BD Biosciences).
Measurement of Mitochondrial Membrane Potential.
The loss of mitochondrial membrane potential is a hallmark for apoptosis. It is an early event preceding phosphatidyl serine externalization and coinciding with activation of caspases. ΔΨm was measured using a JC-1 kit following the manufacturer's protocol. The ΔΨm detection kit uses a unique fluorescent cationic dye, JC-1 (5,5′6,6′-tetracholoro-1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanine iodide) (excitation at 488 nm and emission at 525 nm) to signal the loss of ΔΨm. Cells were harvested at different time points (0.5, 1, 3, 6, 12, 24, and 48 h) during Resv treatment. Then mitochondrial permeability transition was determined by staining the cells with JC-1. In brief, cells (106) were incubated with JC-1 (2.5 μg/ml in 1 ml of PBS) for 30 min at 37°C with moderate shaking. Cells were then centrifuged (1000g, 4°C for 5 min), washed twice with ice-cold PBS, and finally resuspended in 200 μl of PBS. Mitochondrial permeability transition was subsequently quantified on a spectrofluorometer (FP6300; Jasco, Tokyo, Japan), and data are given in the ratio of 590/530.
Preparation of Cytosolic and Mitochondrial Fractions.
U937 cells were harvested after treatment with Resv. Isolation of a highly enriched mitochondrial and cytosolic fraction of cells was performed using a mitochondria/cytosol fractionation kit. In brief, cells (5 × 107) were washed twice in ice-cold PBS, resuspended in 1.0 ml of cytosol extraction buffer mix containing dithiothreitol and protease inhibitors, and incubated on ice for 10 min. The cells were homogenized on ice and centrifuged (700g for 10 min at 4°C), and the resultant supernatant was then centrifuged at 10,000g for 30 min at 4°C. The supernatant along with the pellet that was resuspended in 0.1 ml of mitochondrial extraction buffer mix containing dithiothreitol and protease inhibitors were vortexed for 10 s and stored as the cytosolic and mitochondrial fraction, respectively, and subjected to Western blot analysis.
Caspase Activation Assay.
Caspase-3 was assayed by flow cytometry using CaspaTag Caspase-3 In Situ Assay Kit, Fluorescein (Millipore Corporation, Billerica, MA) according to the manufacturer's instructions.
Western Blot Analysis.
Cell lysates were prepared using a mammalian cell lysis kit MCL1 (Sigma-Aldrich) containing 5× Tris-EDTA buffer, 5× NaCl buffer, 5× lauryl sulfate buffer, 5× deoxycholic acid buffer, 5× Igepal CA 630 buffer, and protease inhibitor cocktail. To prepare 1 ml of cell lysis buffer for the extraction of 106–107 cells, each of the buffers was taken in 200 μl of volume, and protease inhibitor cocktail was used at a 1:100 dilution in the cell lysis buffer. In brief, cells were immersed in freshly prepared lysis buffer with protease inhibitor cocktail, sonicated, and centrifuged (12,000g for 10 min), and the protein concentration of the supernatant was measured (Bradford, 1976). Cellular proteins (50 μg) were resolved by 10% nonreducing lauryl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked for 2 h at room temperature in 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.02% Tween 20-containing 3% bovine serum albumin followed by overnight incubation at 4°C in 1:500 dilution of the respective antibodies for Bax, β-actin, and COX IV. The membranes were washed three times with Tween 20, incubated with alkaline phosphatase-conjugated secondary antibody, and the bands visualized using a 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate.
Confocal Imaging of Localization of Bax on Mitochondria.
U937 cells were fixed with 4% paraformaldehyde for 15 min, followed by two steps of washing with PBS. Cells were then permeabilized with 0.1% Triton X-100 for 4 min, followed by two washings with PBS. Mitochondria were stained using COX IV Alexa Fluor 488 monoclonal antibody (diluted 1:100) along with Bax primary antibody (1:100), followed by Alexa Fluor 594 antimouse secondary antibody (1:300). After washing, the cells were mounted by using gold antifade mounting medium (Invitrogen) and examined under a confocal microscope (Nikon, Melville, NY).
Transfection of siRNA of Bax.
siRNA for Bax and negative control siRNA were purchased from Ambion (Austin, TX), and transfection of 5 nM siRNA were carried out using an amine transfection assay kit from Ambion. All Bax depletions were carried out using Silencer Select Validated siRNA for Bax ID s1889 (Applied Biosystems, Foster City, CA) 16 h after transfection. Silencer Select Negative Control 2 siRNA (Applied Biosystems) was included in each experiment. Cells were washed in medium containing serum and adjusted to 5 × 104 cells/ml in RPMI 1640 supplemented with 10% FBS. Cells (5 × 104) in 400 μl of volume were transfected with siRNA by amine transfection method.
RNA Isolation and TaqMan Real Time-PCR to Validate the Transfection of Bax siRNA.
After extracting the total RNA using Gen Elute Mammalian Total RNA miniprep kits (Sigma-Aldrich) and checking its integrity by electrophoresis, the cDNA was synthesized from 5 mg of purified total RNA using Revert Aid H minus first strand cDNA synthesis kit (MBI Fermentas). Quantitative real-time PCR was performed on ABI prism 7900HT sequence detection system (Applied Biosystems) using TaqMan gene master mix and pre-developed TaqMan assay specific to human Bax (Hs00751844_s1; 6-carboxyfluorescein/minor groove binder; Primer Ltd., Carlsbad, CA). 18s (4319413E; VIC/minor groove binder; Primer Ltd.) was used as the housekeeping gene. The reaction was performed at a final volume of 10 μl amplification condition consisting of uracil N-glycosidase activation at 50°C for 2 min and initial DNA polymerase activation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 s and annealing/extension at 60°C for 1 min. Total time required was 1 h and 48 min. The samples were quantified for all of the above genes using the comparative Ct (ΔΔCt) method, as described in the Assays-on-Demand Users Manual (Applied Biosystems).
Measurement of Intracellular GSH.
Intracellular GSH was measured spectrofluorometrically using ApoGSH Glutathione Detection Kit (BioVision). In brief, the treated and control cells (106) were collected into 1.5-ml microcentrifuge tubes and centrifuged at 700g for 5 min to remove the supernatant. Then the cell pellets were lysed in 100 μl of ice-cold cell lysis buffer. They were then incubated on ice for 10 min and centrifuged at top speed in an Eppendorf centrifuge for 10 min, and the cell lysate was transferred into new tubes for GSH assay. Assay samples were diluted with cell lysis buffer to total volume of 100 μl. Two microliters of the 50 U/ml GST reagent and 2 μl of monochlorobimane dye were added into each sample, and the reaction was incubated at 37°C for 30 min. Then the fluorescence value was measured in a fluorescence plate reader at excitation/emission wavelengths of 380/460 nm. The result was expressed in nanograms per milliliter of sample.
Detection of ROS.
ROS was measured by CM H2DCFDA (Invitrogen) (excitation at 490 nm, emission at 527 nm) (Guha et al., 2010), and singlet oxygen was measured by Singlet Oxygen Sensor Green dye (excitation at 480 nm, emission at 525 nm). The cells were preloaded with these dyes, and their reactivity with ROS was analyzed spectrofluorometrically.
Data were expressed as mean ± S.D. unless otherwise mentioned. Comparisons were made between different treatments (analysis of variance) using InStat software (GraphPad Software Inc., San Diego, CA), where an error protecting the multiple comparison procedure (Tukey-Kramer multiple comparison test) was applied for the analysis of significance of all data.
Resv Treatment-Induced Apoptosis in U937 Cell Line.
The antiproliferative effect of Resv was examined and evaluated on U937 cell lines using the CellTiter-Glo Luminescent Cell Viability Assay kit. Our study revealed that a significant (p < 0.001) reduction in cell viability was evident at 48 h of Resv treatment (Fig. 1A). The IC50 values of Resv at a dose of 50 μM exerted profound cytotoxic effects after 48 h of treatment on U937 cell line. Therefore, further experiments were performed at the IC50 dose of Resv.
To study the nature of cell death, cells were treated for 24 h, stained with Anexin V Alexa Fluor 488/PI, and analyzed by fluorescence-activated cell sorting (FACS). Although the cell membrane remains almost intact in the early stages of apoptosis, PI cannot permeate the cells and remains unable to stain the DNA. However, phosphatidyl serine, to which Annexin V binds specifically, is translocated to the extracellular leaflet of the membrane in the early phase of apoptosis. In contrast, during necrosis, because the cell membrane is ruptured, these cells take up only PI. Our flow cytometric data revealed that, in comparison with vehicle-treated control (0.02% dimethyl sulfoxide), Resv-treated unfixed U937 cells showed Annexin V-Alexa Fluor 488 binding, but PI staining was insignificant (Fig. 1B), indicating that the mode of cell death was apoptosis, not necrosis. Furthermore, DNA fragmentation by means of TUNEL assay and caspase 3 activation after 48 h of Resv treatment proved that the mode of cell death was predominantly apoptosis (Fig. 1, C and D).
Resv Treatment Modulated Dissipation of Mitochondrial Membrane Potential and Induced ROS Generation.
Caspase 3 activation indicated the involvement of mitochondrial death cascade in apoptosis. We therefore investigated the dissipation of ΔΨm in Resv-treated U937 cells. Spectrofluorometric data indicated that ΔΨm dissipation was evident from 1 h of treatment and increased in a time-dependent manner (Fig. 2A). Change in ΔΨm is concomitantly related to intracellular ROS production (Macho et al., 1997). Our spectrofluorometric study elucidated that ROS generation was significantly evident after 6 h of Resv treatment and maintained up to 12 h (Fig. 2B).
Resv Treatment Triggered Oxidative Burst in Mitochondria, and Production of H2O2 Resulted in Deleterious Effects.
According to Schimmel and Bauer (2002), extracellular ROS exhibits a strong apoptosis-inducing potential in different cancer cells. Membrane-associated NADPH oxidase seems to be the key enzyme responsible for generation of ROS. Here, we tried to delineate whether extracellular ROS would exhibit proapoptotic functions in Resv-induced cell death. The pretreatment of cells with the specific NADPH oxidase inhibitor apocynin (200 μM) failed to impart any significant effects on Resv-induced ROS generation and cell death (Fig. 3A), thus confirming that the extracellular ROS-generating system did not contribute to Resv-induced ROS production. The effect of apocynin was also evaluated in a dose-dependent manner, but no significant change was noticed (data not shown). The characterization of the type of ROS was also evaluated consequently. The increased fluorescence intensity of CM-H2DCFDA proved that significant production of H2O2 occurred in Resv-treated U937 cells, which was efficiently scavenged by cell-permeable polyethylene glycol (PEG)-SOD, PEG-catalase, and N-acetyl cysteine (NAC), as shown in Fig. 3B. Simultaneous treatment with PEG-SOD reduced the CM-H2DCFDA fluorescence level in a dose-dependent fashion (Supplemental Fig. 2), thus proving the role of superoxide. Superoxide can produce H2O2 and hydroxyl radicals (•OH). The dose-dependent study (Supplemental Fig. 3) of DMTU (•OH scavenger) at a dose of 6 mM efficiently prevented cell death after Resv treatment (Fig. 3C), thus proving that Resv treatment instigated mitochondrial superoxide production, which consequently produced H2O2 and •OH through downstream reactions. However, we failed to detect singlet oxygen (1O2; assessed with the Singlet Oxygen Sensor Green dye) (Fig. 3D), and the pretreatment of NOS inhibitor Nω-nitro-l-arginine methyl ester (l-NAME; 1 mM) also signified no role of peroxynitrate after Resv treatment (Fig. 3E). The effect of l-NAME was also evaluated in a dose-dependent manner, but no significant change was noticed (data not shown). Thus, the data characterized mitochondria as the important ROS-producing machinery, and the type of ROS was predominantly superoxide, which in turn produced ROS (•OH) and H2O2.
Generation of ROS Was Modulated by Efflux of GSH.
GSH depletion is an early hallmark observed in H2O2-dependent apoptosis. We therefore investigated the GSH status after the treatment of Resv. Time-dependent study revealed that GSH depletion was significant from almost the initial hours of treatment and increased over time (Fig. 4A).
Most studies suggest that GSH depletion during apoptosis is an indicator for ROS formation and oxidative stress. Such findings have led to the widespread assumption that GSH depletion is a byproduct of the apoptotic process and that ROS formation and GSH depletion in apoptosis are coupled as cause and consequence phenomena. We next analyzed the role of ROS on the modulation of GSH depletion through the use of a variety of antioxidants. H2O2 formation induced by Resv was prevented in the presence of PEG-SOD and PEG-Cat (Fig. 3B, top), which acts as a potent intracellular scavenger for H2O2. However, scavenging of the ROS generated upon Resv treatment by these antioxidants did not significantly affect GSH depletion, whereas pretreatment of cells with NAC (8 mM) or supplementation of the media with GSH (5 mM) prevented intracellular GSH depletion (Fig. 4B). To gain better insight into the control of GSH depletion, we pretreated the cells with two specific inhibitors of carrier-mediated GSH extrusion, cystathionine (1 mM) and methionine (1 mM), which significantly prevented intracellular GSH depletion (Fig. 4C). As indicated in Fig. 4, D to F, pretreatment of cells with extracellular thiol (8 mM NAC and 5 mM GSH) or cystathionine (1 mM) not only inhibited GSH efflux, but also prevented the loss of ΔΨm and decreased cell death number. These results suggested a causative role for GSH efflux in Resv-induced apoptosis.
Resv-Induced Bax Translocation to Mitochondria Was Modulated by GSH Depletion.
GSH efflux was independent of ROS production, but intracellular ROS formation could be a sequential effect after GSH extrusion. After Resv treatment, total Bax expression remained unchanged compared with control; however, Western blot analysis of cytosolic and mitochondrial lysate clearly emphasized the phenomenon of mitochondrial Bax translocation (Fig. 5A). This was further validated by confocal microscopy (color picture of Bax translocation is given in Supplemental Fig. 4). Our confocal imaging study enumerated a significant amount of Bax translocation in mitochondria within 6 h of Resv treatment (Fig. 5B). To establish a connection between GSH efflux and mitochondrial Bax translocation, we initially treated the cells with BSO (1 mM), an irreversible inhibitor of γ-glutamylcysteine synthetase. BSO and Resv treatment stimulated the mitochondrial Bax translocation after 6 h, but treatment of NAC and cystathionine significantly prevented translocation of Bax to mitochondria in Resv-treated cells (Fig. 5); however, further addition of BSO reversed this protective effect, thus suggesting that GSH depletion stimulated mitochondrial Bax translocation in the Resv-treated U937 cell line.
Bax Translocation Triggered Mitochondrial Death Signal.
To bridge the link between GSH extrusion, Bax translocation, and mitochondrial apoptotic signal, the cells were treated with siRNA of Bax protein. The activity of siRNA was validated by real-time-PCR data (Fig. 6A). From Fig. 6B, it was evident that Bax siRNA treatment minimized the amount of cell death by 47%, which was well evident with the inhibition of caspase 3 activation (Fig. 6C). To gain better insight into the contribution of Bax in the oxidative burst and depletion of ΔΨm, we analyzed the amount of ROS generation and loss of ΔΨm in the Bax siRNA-treated cells. Experimental data spell out that in Resv-treated cells Bax siRNA significantly reduced ROS production (Fig. 6D) and prevented the dissipation of ΔΨm (Fig. 6E).
Changes in the intracellular redox environment of cells have been reported as being critical for the activation of apoptotic enzymes and the progression of programmed cell death. Small thiols, including GSH, are viewed as protective antioxidants acting as free radical scavengers in response to oxidative damage, thus playing important roles in the redox balance of the cell. GSH depletion has been shown to occur in apoptosis induced by a wide variety of stimuli, and it has been shown to be mediated by its extrusion and oxidation. However, Ghibelli et al. (1998) and Franco et al. (2007) have emphasized that GSH efflux is one of the important events in apoptosis but that GSH oxidation is not.
It has already been proven that Resv-induced apoptosis is mostly ROS-driven (Guha et al., 2010); however, the exact role of GSH in ROS generation and cell death had not yet been explored. Therefore, here we tried to investigate the effect of Resv on modulation of GSH level and its probable role in Resv-induced apoptosis in U937 cells.
A luminometric cell viability study demonstrated that Resv showed its antiproliferative effects in a time-dependent manner, and the IC50 value came at ∼48 h of treatment at a dose of 50 μM (Fig. 1A). Furthermore, flow cytometric analysis for Annexin V and TUNEL assay proved the apoptotic mode of cell death, which was also supported by activation of caspase 3 (Fig. 1, B–D). Activation of caspase 3 helped to presume the role of mitochondria in Resv-treated apoptosis.
Fluorometric investigations suggested that Resv treatment started to dissipate ΔΨm rapidly within 1 h of treatment and continued in a time-dependent manner (Fig. 2A). However, time-dependent study illustrated that ROS generation was significant after 6 h of Resv treatment and was maintained for up to 12 h (Fig. 2B). Within 3 h of Resv treatment, a trend of oxidative burst was evident, but pretreatment of cells with PEG-SOD or PEG-Cat failed to prevent the dissipation of ΔΨm (Supplemental Data 1). From these data, it is clear that loss of ΔΨm was independent of ROS generation.
Although mitochondria are considered to be the major source of ROS, extracellular superoxide anion generation through membrane-associated NADPH oxidase cannot be ignored (Schimmel and Bauer, 2002). However, experimental data demonstrated that treatment of NADPH oxidase inhibitor apocynin remained unable to protect Resv-treated cells (Fig. 3A). It is noteworthy that PEG-SOD treatment reduced both cell death (Supplemental Data 2B) and CM-H2DCFDA (Fig. 3B) fluorescence after Resv treatment, which confirmed mitochondrial superoxide production. Furthermore, pretreatment of cells with DMTU (•OH radical scavenger) gave significant protection to cells (p < 0.001) after Resv treatment, thus proving the production of •OH and H2O2 (Fig. 3C). Therefore, the overall data signified that Resv treatment stimulated mitochondrial superoxide production, which further engendered H2O2 formation and •OH radical production through downstream reaction.
Generation of ROS usually oxidizes GSH to GSSG and ultimately reduces the total GSH level. However, in vitro studies have also shown that a reduction in the intracellular GSH is necessary for the formation of ROS (Franco et al., 2007). GSH depletion has been shown to directly modulate both the loss of ΔΨm and the activation of executioner caspases (e.g., caspase 3) (Armstrong and Jones, 2002; Franco et al., 2007). Hence, we sought to clarify the precise mechanism of GSH loss in Resv-induced cell death. Time-dependent study illustrated that Resv treatment depleted intracellular GSH content within the first hour, and it continued over time (Fig. 4A). However, cell-permeable potent ROS scavengers (PEG-SOD and PEG-Cat) failed to prevent the intracellular GSH depletion (Fig. 4B), thus emphasizing that intracellular GSH depletion was independent of ROS generation.
To gain better insight into the control of GSH depletion in Resv-treated cells, we analyzed two compounds, cystathionine and methionine, that are known to inhibit specific (sinusoidal-type) carriers responsible for reduced GSH efflux from hepatocytes and U937 cells (Chow et al., 1995; Lu et al., 1996). As indicated in Fig. 4C, both compounds minimized GSH depletion. Moreover, pretreatment of cells with extracellular thiols (NAC, GSH) and cystathionine prevented the loss of ΔΨm (Fig. 4D) and ROS generation (Fig. 4E) and simultaneously protected the cells after Resv treatment (Fig. 4F). GSH efflux was one of the major events leading to the intracellular GSH depletion and apoptosis in Resv-treated cells.
Next, we investigated the missing link between GSH efflux and mitochondrial apoptotic signal. According to Honda et al. (2004), intracellular GSH depletion could stimulate Bax-induced apoptosis in lung cancer. Here, we tried to determine in the case of Resv-treated cells whether GSH depletion could provoke mitochondrial Bax translocation, resulting in activation of mitochondrial death cascade. Initial Western blot data demonstrated mitochondrial translocation of Bax protein (Fig. 5A) that was further validated through confocal microscopic study. Confocal imaging signified mitochondrial Bax translocation as early as within 6 h of Resv treatment (Fig. 5B). However, pretreatment of cystathionine or NAC profoundly prevented Bax translocation, which was significantly reversed by BSO treatment (Fig. 5B). Thus, it revealed that mitochondrial Bax translocation, which is a major apoptotic inducer, was stimulated by GSH efflux in Resv-treated cells. In addition, Bax siRNA treatment revealed the importance of Bax in Resv-induced apoptosis. Bax siRNA treatment inhibited cell death by 47%, and it prevented the loss of ΔΨm, ROS generation, and the downstream caspase activation (Fig. 6). Therefore, it confirmed that mitochondrial Bax translocation in Resv-treated U937 cells stimulated the loss of ΔΨm followed by ROS generation and caspase activation, which finally triggered apoptosis. However, because BSO itself failed to instigate apoptosis despite mitochondrial Bax translocation and ROS accumulation, it could be concluded that mitochondrial Bax translocation and ROS generations are the important linker molecules in the Resv-induced apoptotic signal transduction cascade but not sufficient to induce Resveratrol-provoked caspase activation and apoptosis alone.
From these sets of data, a potential signal transduction cascade can be sketched in Resv-induced apoptosis in which GSH depletion played the central role. In the niche of GSH efflux, mitochondrial Bax translocation amplified the whole apoptotic signal. Initiated from dissipation of ΔΨm, ROS production ultimately led to the downstream caspase activation and cell death. Thus, Resv treatment provoked GSH extrusion and mitochondrial Bax translocation to accomplish apoptosis in the U937 cell line.
Participated in research design: Guha and Bandyopadhyay.
Conducted experiments: Guha and Dey.
Contributed new reagents or analytic tools: Sen, Chatterjee, and Chattopadhyay.
Performed data analysis: Guha, Dey, Chattopadhyay, and Bandyopadhyay.
Wrote or contributed to the writing of the manuscript: Guha, Dey, and Bandyopadhyay.
Other: Sen and Chatterjee performed FACS analysis.
We thank the reviewers for valuable suggestions on improving the manuscript and P. K. Mitra, (Director, Institute of Postgraduate Medical Education and Research) for providing infrastructural facilities and constant encouragement.
This work was supported by Life Science Research Board, Defense Research and Development Organisation, Government of India (to A.D.); and Department of Science and Technology, Government of India (to P.G.).
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- oxidized glutathione
- reactive oxygen species
- fetal bovine serum
- polymerase chain reaction
- propidium iodide
- terminal deoxynucleotidyl transferase dUTP nick-end labeling
- mitochondrial membrane potential
- 5,5′6,6′-tetracholoro-1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanine iodide
- phosphate-buffered saline
- 5-chloromethyl-2,7-dichlorodihydrofluorescein diacetate acetyl ester
- fluorescence-activated cell sorting
- polyethylene glycol
- superoxide dismutase
- N-acetyl cysteine
- buthionine sulfoximine
- small interfering RNA
- Nω-nitro-l-arginine methyl ester.
- Received June 24, 2010.
- Accepted September 22, 2010.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics