Arsenic Trioxide Induces Apoptosis in Peripheral Blood T Lymphocyte Subsets by Inducing Oxidative Stress: A Role of Bcl-2

Arsenic trioxide (As₂O₃) has been used successfully in the treatment of acute promyelocytic leukemia (1, 2). In vitro, arsenic trioxide induces apoptosis in malignant cells from acute promyelocytic leukemia, non-Hodgkin’s lymphoma, multiple myeloma, and chronic lymphocytic leukemia (3–5). Arsenic compounds also inhibit certain immune functions (6, 7). However, the mechanism underlying arsenic-induced immune suppression is unclear. Furthermore, the effects of As₂O₃ on apoptosis in normal lymphocytes have not been studied in detail.

Apoptosis is a physiological process that plays an important role in the maintenance of cellular homeostasis and the elimination of self-reactive lymphocytes (8). There are two major pathways of apoptosis, the death receptor pathway (9–12) and the mitochondrial pathway (12, 13). The mitochondrial pathway of apoptosis can be triggered by a variety of stimuli, including chemotherapeutic agents, UV radiation, and oxidative stress. Activation of mitochondria results in dissipation of membrane PTP,² resulting in decreased (depolarization) mitochondrial membrane potential (Δψ_m) and release of intermembrane contents including cytochrome c and AIF from the mitochondria into cytoplasm. Cytochrome c binds to an adapter protein, Apaf-1 (apoptotic protease-activating factor), in the presence of ATP or dATP, which then recruits procaspase-9 to form an apoptosome. This results in the autolytic activation of procaspase-9 to active caspase-9, which in turn cleaves and activates effector procaspases to active effector caspases, including caspase-3. These effector caspases cleave a number of substrates, resulting in morphological and biochemical changes of apoptosis (8, 14). The function of PTP and release of intermembrane contents of mitochondria are under regulatory control of a number of proteins of the Bcl-2 family (8, 14).

In this investigation, we show that As₂O₃-induces apoptosis in both CD4+ and CD8+ T cells via the mitochondrial pathway by inducing oxidative stress and that Bcl-2 inhibits As₂O₃-induced apoptosis.

MNCs were separated from peripheral blood obtained from healthy young volunteers (approved by the Institutional Review Board, University of California, Irvine, CA). MNCs were incubated in the presence or absence of various concentrations (1.0–5.0 μm) of As₂O₃ (Sigma, St. Louis, MO) for specified duration and then analyzed.

Apoptosis was measured by TUNEL assay. Cells (1 × 10⁶ cells/ml) were incubated for 48 h with or without various concentrations of As₂O₃ and stained with PerCp-labeled anti-CD4 and phycoerythrin-labeled anti-CD8 (BD BioSciences, San Jose, CA). Cells were washed with PBS containing 1% BSA and 01% sodium azide and fixed in 2% paraformaldehyde for 30 min at room temperature. Cells were washed with PBS and permeabilized with sodium citrate buffer containing Triton X-100 for 2 min on ice. After washing, cells were incubated with FITC-dUTP in the presence of terminal deoxynucleotidyl transferase enzyme solution containing 1 μm potassium cacodylate and 125 mm Tris-HCl (pH 6.6; In Situ Death Detection Kit; Boehringer Mannheim, Indianapolis, IN) for 1 h at 37°C. After incubation, cells were washed with PBS, and 5000 cells were acquired and analyzed by FACScan.

MNCs (2 × 10⁶ cells/ml) were incubated for 18 h with or without 5 μm As₂O₃ at 37°C and then incubated for 10 min at 37°C in medium containing 50 nm TMRE (Molecular Probes, Eugene, OR) alone or in the presence of 10 μm carbonyl cyanide p-trifluoromethoxy phenyl-hydrazone (Sigma) as an internal control. Cells were stained with PerCp-labeled anti-CD4 and FITC-labeled anti-CD8 monoclonal antibodies, and changes in Δψ_m were measured by FACScan. Data were acquired and analyzed by FACScan using Cell Quest software (Becton Dickinson, Menlo Park, CA).

Cells (2 × 10⁶ cells/ml) were resuspended in RPMI 1640 containing 5 μm of cell permeable oxidation-dependent fluorescence dye 2′,7′-dichlorofluorescein diacetate (Molecular Probes) for 20 min at 37°C. Then cells were cultured with or without 5 μm As₂O₃ for an additional 3 h at 37°C, stained with PerCp-labeled anti-CD4 and phycoerythrin-labeled anti-CD8 monoclonal antibodies, and analyzed by FACScan.

Intracellular levels of GSH were measured with monobrombiamine (Molecular Probes) by a flow cytometric method described by Hedley and Chow (15).

The release of cytochrome c and AIF from the mitochondria was analyzed by confocal microscopy. In brief, MNCs were incubated with 5 μm As₂O₃ for 18 h, washed three times with PBS, and then loaded with 150 nm Mitotracker Orange (Molecular Probes) for 15 min at 37°C. After fixation with 4% (w/v) freshly prepared paraformaldehyde in PBS for 30 min at room temperature and washing three times in PBS, cells were permeabilized with 0.25% (w/v) saponin in PBS for 5 min and washed three times in blocking buffer [0.05% saponin, 3% BSA in PBS (pH 7.4)]. Cells were incubated overnight with a 1:100 dilution of the primary mouse antihuman cytochrome c antibody (PharMingen, San Diego, CA) or rabbit antihuman AIF antibody (ProSci Inc., Poway, CA), washed, and then incubated with FITC-labeled goat antimouse IgG antibody (Antibody Inc., Davis, CA) or FITC-labeled goat antirabbit IgG antibody (Oncogene, Boston, MA) for 1 h at room temperature. Cells were washed three times with blocking buffer and cytospun onto slides. Coverslips were mounted with an anti-fade reagent (Bio-Rad, Hercules, CA), slides were imaged using a laser-based confocal microscope, and the percentages of the cells that have released cytochrome c or AIF were determined.

Cells were incubated with varying concentrations of As₂O₃ at 37°C for 18 h. Caspase-3, caspse-8, and caspase-9 activities were assessed using a colorimetric assay kit (Biovision Research Products, Palo Alto, CA) according to the protocol provided by the manufacturer and by flow cytometry.

The colorimetric assay is based on the spectrophotometric detection of the chromophore pNA after cleavage from the labeled substrate. Briefly, 1 × 10⁶ cells were resuspended in chilled lysis buffer for 10 min, and cytosolic extracts were isolated by centrifugation of lysed cells at 10,000 × g for 2 min. Cytosolic extracts were incubated with conjugated caspase-3 substrate DEVD-pNA, caspase-8 substrate IETD-pNA, or caspase-9 substrate LEHD-pNA in the presence of reaction buffer for 1 h at 37°C. The chromophore pNA was detected using a microtiter plate reader at 405 nm. Background readings from cell lysates incubated in the absence of substrates were subtracted from sample readings before determination of fold increase in caspase activity.

The activation of caspases in CD4+ and CD8+ T cells was analyzed with carboxyfluorescein-labeled cell-permeable peptide substrates (Intergen Co., Purchase, NY) that recognize cleaved caspase-9 (FAM-LEHD-FMK), caspase-8 (FAM-IETD-FMK), and caspase 3 (FAM-DEVD-FMK), by triple color analysis using FACScan.

MNCs were activated with α-CD3 monoclonal antibody for 48 h, cultured in interleukin 2-containing culture medium for an additional 24 h, and transfected with pBcl-2 plasmid (pcDNA3 Bcl-2; Science Reagent, El Cajon, CA) using the Lipofectin reagent (Life Technologies, Inc., Gaithersburg, MD). Briefly, 0.8 ml of the cell suspension (3 × 10⁶ cells/ml) was added to each well in 6-well plates. Two μg of pBcl-2 plasmid DNA were diluted in 98 μl of OPTI-MEM1 medium, and 16 μl of Lipofectin reagent were diluted in 84 μl of OPTI-MEM1 medium. The vector plasmid without bcl-2 gene was used as a negative control. After a 45-min incubation at room temperature, the DNA and Lipofectin diluents were combined and incubated for 15 min at room temperature. Then, 200 μl of the DNA/Lipofectin mixture were added to each well, and cells were incubated for 12 h at 37°C. Three ml of interleukin 2-containing culture medium were added to each well, and cells were allowed to express Bcl-2 for 2 days. Bcl-2 expression was confirmed by real-time RT-PCR. Total cellular RNA (0.2 μg) was used for reverse transcription, and subsequent PCR amplification was done with specific primers and TaqMan probes for bcl-2 and glyceraldehyde-3-phosphate dehydrogenase cDNAs.

Cells treated with or without various concentrations of As₂O₃ were centrifuged, and whole cell extracts were prepared by lysing the cell pellet in 50 μl of cold TGNT buffer with protease and phosphatase inhibitors [100 mm Tris-Cl (pH 7.4), 20% glycerol, 100 mm NaCl, 2% Triton X-100, 20 mm EGTA, 100 mm NaF, 4 mm phenylmethylsulfonyl fluoride, 4 mm sodium orthovanadate, and 2 mm p-nitrophenol phosphate] and clarified by centrifugation at 4°C for 20 min. Protein concentration of the lysates was determined by Bradford assay (Bio-Rad, Richmond, CA). Aliquots of cell lysates containing 25 μg of total protein were resolved by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes by electroblotting. The membranes were blocked for 2 h at room temperature in TBS-T buffer with 5% nonfat dried milk and sequentially probed by overnight incubation at 4°C with primary antibodies diluted in TBS-T buffer with 5% nonfat dried milk. These antibodies include anti-XIAP, anti-Bcl-x_L, anti-Bax, anti-Bcl-2 (1:2000 dilution; Transduction Laboratory, San Diego, CA), and anti-VDAC (Affinity Bioreagent, Golden, CO). The blots were washed three times for 15 min with TBS-T buffer and then incubated with horseradish peroxidase-conjugated antimouse secondary antibody (1:2,000 dilution; Cell Signaling Technology, Beverly, MA) for 1 h at room temperature. After washing three times for 20 min in TBS-T buffer, blots were developed with ECL Plus detection system (Amersham Pharmacia Biotech Inc., Piscataway, NJ). Before each cycle of reprobing, blots were incubated at 50°C for 45 min in stripping buffer [62.5 mm Tris (pH 6.7), 2% SDS, and β-mercaptoethanol]. To normalize protein loading and transfer efficiency, the blots were probed with antiactin antibody (1:20,000 dilution; Chemicon, Temecula, CA).

Statistical analysis was performed by Student’s t test.

In vitro As₂O₃ induces apoptosis in malignant lymphocytes (3–5). In this study, we examined the effect of As₂O₃ on apoptosis of peripheral blood T-cell subsets. Peripheral blood MNCs from healthy subjects were incubated with various concentrations of As₂O₃ (1.0–5.0 μm) for 48 h, and apoptosis was measured by triple-color flow cytometry using TUNEL assay and specific monoclonal antibodies against CD4 and CD8 and isotype controls. Fig. 1A shows that As₂O₃, in a concentration-dependent manner, induced apoptosis in both CD4+ and CD8+ T cells.

We then examined the effect of As₂O₃ on the activation of caspase-9, caspase-3, and caspase-8 in CD4+ and CD8+ T-cell subsets by intracellular staining. As₂O₃ activated caspase-8 (Fig. 1B), caspase-9 (Fig. 1C), and caspase-3 (Fig. 1D). Fig. 1E shows As₂O₃-induced activation of caspases by colorimetric assay. To demonstrate a role of caspases in As₂O₃-induced apoptosis, we examined the effect of caspase inhibitors on As₂O₃-induced apoptosis. The inhibitors of caspase-8 (Z-IETD-FMK), caspase-9 (Z-LEHD-FMK), and caspase-3 (DEVD-FMK) partially but significantly (P < 0.05) suppressed As₂O₃-induced apoptosis in both CD4+ and CD8+ T cells (Fig. 1F).

Arsenic compounds have been shown to induce generation of ROS in tumor cell lines (16, 17), and oxidative stress is one of the mediators of apoptosis (18, 19). Therefore, we examined the effect of As₂O₃ on intracellular levels of ROS. MNCs were incubated with 5 μm As₂O₃ for 3 h, and intracellular ROS was measured with 2′,7′-dichlorofluorescein diacetate by triple-color analysis using FACScan. As₂O₃ significantly enhanced intracellular levels of ROS in both CD4+ and CD8+ T cells (Fig. 2, A and B).

GSH, an antioxidant, protects cells from undergoing stress-induced apoptosis (20), and reduced intracellular levels of GSH are associated with increased susceptibility to apoptosis (21). Therefore, we analyzed the effect of As₂O₃ on intracellular concentration of GSH. MNCs were incubated with 5 μm As₂O₃, and intracellular GSH contents were measured by flow cytometry using FACS. As₂O₃ reduced intracellular GSH levels in MNCs (Fig. 2C).

To determine a role of decreased intracellular level of GSH in As₂O₃-induced apoptosis, MNCs were pre-exposed to 5.0 mm GSH for 2 h and then treated with 5.0 μm As₂O₃ for 48 h, and apoptosis was measured by TUNEL assay. GSH inhibited As₂O₃-induced apoptosis in both CD4+ and CD8+ T cells (Fig. 2D), suggesting that As₂O₃ induces apoptosis by altering intracellular redox status.

Because apoptosis mediated via the mitochondrial pathway is associated with sequential reduction of Δψ_m and generation of ROS (19), we examined the effect of As₂O₃ on Δψ_m. MNCs were incubated with 5.0 μm As₂O₃ for 18 h, and Δψ_m was measured with TMRE dye using FACScan. As₂O₃ induced depolarization of Δψ_m in both CD4+ and CD8+ T cells (Fig. 3).

The depolarization of mitochondrial membrane is associated with release of cytochrome c and AIF (13, 22, 23). Therefore, we examined the effect of As₂O₃ on the release of cytochrome c and AIF. MNCs were incubated with 5 μm As₂O₃ for 18 h, and release of cytochrome c and AIF was analyzed by confocal microscopy using mitochondrial dye Mitotracker Orange and anti-cytochrome c and anti-AIF antibodies. Hoechst 33342 dye was also used to examine chromatin fragmentation as an indication of apoptosis. In Fig. 4, a and b, A is MNCs alone, and D is MNCs treated with As₂O₃. As₂O₃ induced the release of cytochrome c (Fig. 4a, D) in 30% of cells and AIF (Fig. 4b, D) in 45% of cells from the mitochondria into the cytoplasm.

The release of cytochrome c and AIF is under regulatory control of Bcl-2 family proteins; Bcl-2 and Bcl-x_L block the release of cytochrome c and AIF, whereas Bax promotes the release of cytochrome c and AIF (24, 25). Because As₂O₃ increases intracellular ROS, and Bcl-2 has been shown to serve as an antioxidant (26, 27), we examined the effect of As₂O₃ on the expression of Bcl-2 family proteins. As₂O₃ down-regulated the expression of both Bcl-2 and Bcl-x_L; however, the expression of Bax was up-regulated (Fig. 5). One of the components of the PTP complex is VDAC, which is present in the outer membrane of the mitochondria. It has been suggested that Bcl-2 family proteins bind to VDAC and regulate the release of cytochrome c and apoptosis (26). Therefore, we also examined the effect of As₂O₃ on VDAC expression by Western blotting. As₂O₃ down-regulated the expression of VDAC (Fig. 5).

Bcl-2 regulates Δψ_m, generation of ROS, and release of cytochrome c and AIF (13, 22, 23) and serves as an antioxidant (27). Because As₂O₃ down-regulated Bcl-2 expression, enhanced generation of ROS, reduced GSH, and induced the release of cytochrome c and AIF, we determined whether Bcl-2 plays a major role in As₂O₃-induced apoptosis in T-cell subsets. T cells were transfected with control plasmid and Bcl-2 expression plasmid. The transfection was confirmed by real-time RT-PCR (Fig. 6a). Cells were incubated with 5.0 μm As₂O₃ and examined for apoptosis, Δψ_m, ROS, and release of cytochrome c and AIF. The transfection of T cells with Bcl-2 resulted in an inhibition of As₂O₃-induced apoptosis (Fig. 6B) and an inhibition of activation of caspases (Fig. 6C). In addition, overexpression of Bcl-2 resulted in a resistance of T-cell subsets to As₂O₃-induced depolarization of Δψ_m (Fig. 6D) and generation of ROS (Fig. 6E). Bcl-2 overexpression also inhibited cytochrome c and AIF release. Ten percent of Bcl-2-overexpressing cells release cytochrome c (Fig. 4a, F) and AIF (Fig. 4b, F), whereas 30% of control plasmid-transfected cells released cytochrome c (Fig. 4a, E), and 45% of control plasmid-transfected cells released AIF (Fig. 4b, E). These data strongly suggest that Bcl-2 plays a major role in As₂O₃-induced apoptosis in normal T-cell subsets.

In this study we have demonstrated that As₂O₃, at a clinically achievable concentration, induces apoptosis in normal peripheral blood CD4+ and CD8+ T cells via mitochondrial pathway by enhancing the generation of oxidative stress and by regulating the expression of Bcl-2 family proteins.

The changes in Δψ_m, generation of ROS, and release of cytochrome c and AIF are major elements of the mitochondrial pathway of apoptosis (8, 10, 13, 23, 28). Therefore, we examined the effect of As₂O₃ on Δψ_m. As₂O₃, in a concentration-dependent manner, decreased Δψ_m, suggesting depolarization of the mitochondrial membrane. Similar depolarization of the mitochondrial membrane has been observed with As₂O₃ in various tumor cell lines (17, 29).

Mitochondria are major generators of ROS, which predominantly include superoxide anion, hydrogen peroxide, and the hydroxyl radical (29, 30). The major source of ROS in most cell types is probably the electron leakage from mitochondrial electron transport chain that reduces molecular oxygen to superoxide ion. Elevated intracellular ROS are sufficient to trigger apoptosis, and it has been suggested that ROS are biochemical mediators of apoptosis (18). In this study, we observed that As₂O₃ increased intracellular levels of ROS in both CD4+ and CD8+ T cells.

Cells contain several antioxidant systems to limit the damage caused by increased ROS, and GSH is an antioxidant that protects cells from oxidative stress-induced apoptosis (20). Glutathione depletion is associated with an increased proportion of cells undergoing apoptosis, and glutathione monoethyl ester decreases apoptosis (21, 31). Furthermore, GSH depletion enforces mitochondrial PTP to induce apoptosis (32), and extrusion of GSH causes cytochrome c release (33, 34). In this study, we observed that As₂O₃ reduced intracellular levels of GSH, and in vitro glutathione protected T-cell subsets from As₂O₃-induced apoptosis. As₂O₃ has also been shown to reduce GSH levels in tumor cells, and in vitro depletion of GSH renders tumor cells more susceptible to As₂O₃-induced apoptosis (21, 29).

The PTP is a channel spanning the inner mitochondrial and OM membrane. The intermembrane space contains a number of molecules including cytochrome c and AIF. The permeabilization of the OM, therefore, results in the release of these molecules. Our data show that As₂O₃ induces release of both cytochrome c release and AIF from the mitochondria. AIF, once released from mitochondria, is transported into the nucleus, where it stimulates (ATP-independent and caspase-independent) large DNA fragmentation and condensation of chromatin (22). Because As₂O₃ also causes AIF release from the mitochondria, it is possible that, in addition to cytochrome c-mediated caspase-dependent apoptosis, As₂O₃ might also induce apoptosis in a caspase-independent manner. This would be consistent with our finding that As₂O₃-induced apoptosis was only partially blocked by caspase inhibitors.

Bcl-2 family proteins regulate the release of cytochrome c and AIF (35, 36). It has been demonstrated that Bax present in the cytoplasm is translocated to the mitochondrial membrane, where it undergoes conformational changes. The docking of Bax to the OM and its subsequent in situ clustering/multimerization cause gating of PTP and release of cytochrome c (25). This conformational change and insertion of Bax into mitochondrial membrane are assisted by Bid (37). Bcl-2 and Bcl-x_L inhibit conformational changes in Bax and therefore inhibit release of cytochrome c and apoptosis (38). However, a role of Bcl-2 family proteins in the regulation of mitochondrial outer member permeability is controversial. Recently Kuwana et al. (39) have demonstrated that Bid, Bax, and lipid form supramolecular openings in the OM membrane and that during apoptosis mitochondrial protein release through supramolecular openings in the OM is promoted by Bid/Bax and directly inhibited by Bcl-X_L. In the present study we have demonstrated that As₂O₃ increases the expression of Bax and decreases the expression of Bcl-2 and Bcl-x_L. Chen et al. (3) and Akao et al. (40) have reported an As₂O₃-induced down-regulation of Bcl-2 in tumor cell lines; however, the effect of As₂O₃ on Bcl-2 in normal lymphocytes and of Bax expression in any cell type has not been documented. Taken together, it appears that As₂O₃ induces apoptosis in T-cell subsets by the release of cytochrome c and AIF from the mitochondria by regulating Bcl-2 family proteins.

To determine a role of Bcl-2 in As₂O₃-induced apoptosis, we examined the effect of overexpression of Bcl-2 on various events in As₂O₃-induced apoptosis. An overexpression of Bcl-2 blocked As₂O₃-induced apoptosis, depolarization of Δψ_m, generation of ROS, and release of both cytochrome c and AIF, demonstrating that Bcl-2 plays a major role in As₂O₃-induced apoptosis in T-cell subsets.

The VDAC, which is present in the outer membrane of mitochondria, has been shown to play a role in apoptosis (26, 28). Furthermore, oxidative stress increases the gating potential of the voltage sensor of PTP (41, 42). Several Bcl-2 family members can bind to VDAC and regulate its channel activity (26). It has been suggested that VDAC undergoes significant conformational changes upon binding to proapoptotic members of Bcl-2 family. In this study, we show that As₂O₃ induces oxidative stress, up-regulates Bax expression, and down-regulates VDAC expression. It is unclear whether down-regulation of VDAC protein represents conformational change or its phosphorylation. The mechanism of down-regulation of VDAC and its role in As₂O₃-induced cytochrome c and/or AIF release remains to be investigated.

The release of cytochrome c triggers the assembly of Apaf-1 and procaspase-9 to form an apoptosome (8, 13). Procaspase-9 is then autolyticaly cleaved to active caspases-9, which then activates effector caspases including caspase-3, resulting in cleavage of its substrates and apoptosis. In this study, we have demonstrated that As₂O₃ not only activates caspase-9 and caspase-3 but also activates caspase-8. Furthermore, inhibitors of caspase-9, caspase-3, and caspase-8 partially inhibited As₂O₃-induced apoptosis in T-cell subsets. The activation of caspase-8 and partial inhibition of As₂O₃-induced apoptosis by caspase-8 inhibitor suggest that As₂O₃ may also induce apoptosis in T-cell subsets via the death receptor signaling pathway. This observation is in agreement with recent reports that show that As₂O₃-induced tumor necrosis factor α production (43), and tumor necrosis factor α-induced apoptosis is associated with translocation of tBid to the mitochondria (44). Furthermore, Bid/Bax form macro-openings in the OM and induce release of cytochrome c and apoptosis (44).

The IAP family of proteins is a novel family of antiapoptotic endogenous proteins (45). IAPs inhibit apoptosis by inhibiting both caspase activation and caspase activity via interaction with caspases-3, -7, and -9. As₂O₃ had no effect on XIAP expression (Fig. 5), demonstrating that XIAP does not play any significant role in As₂O₃-induced apoptosis. This is in contrast to mercury-induced apoptosis in T cells, in which XIAP plays an important role in apoptosis (46).

In summary, As₂O₃ induces apoptosis in human T cells via the mitochondrial pathway by inducing oxidative stress and by regulating Bcl-2 family protein expression. A role of VDAC in As₂O₃-induced apoptosis remains to be investigated. We propose the possibility that long-term treatment of malignant disorders with As₂O₃ may be associated with immune suppression and therefore should be investigated.