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1 Department of Haematology, Barts and The London Queen Mary’s School of Medicine and Dentistry, London, United Kingdom, and 2 Tianjin Medical University, Tianjin, People’s Republic of China.
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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m). This study aimed to use the Bcl-2 inhibitors to sensitize CEM cells to TRAIL-induced apoptosis by switching on the mitochondrial apoptotic pathway. We found that a low dose of BH3I-2' or HA14-1, which did not induce cytochrome c release, greatly sensitized CEM cells to TRAIL-induced apoptosis. In a similar manner to the classical uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), both BH3I-2' and HA14-1 induced a reduction in m, a generation of reactive oxygen species (ROS), an increased mitochondrial respiration, and a decreased ATP synthesis. This uncoupling function of the Bcl-2 inhibitors was responsible for the synergy with TRAIL-induced apoptosis. CCCP per se did not induce apoptosis but again sensitized CEM cells to TRAIL-induced apoptosis by uncoupling mitochondrial respiration. The uncoupling effect facilitated TRAIL-induced Bax conformational change and cytochrome c release from mitochondria. Inhibition of caspases failed to block TRAIL-mediated cell death when mitochondrial respiration was uncoupled. We observed that BH3I-2', HA14-1, or CCCP can overcome resistance to TRAIL-induced apoptosis in TRAIL-resistant cell lines, such as CEM, HL-60, and U937. Our results suggest that the uncoupling of mitochondrial respiration can sensitize leukemic cells to TRAIL-induced apoptosis. However, caspase activation per se does not represent an irreversible point of commitment to TRAIL-induced cell death when mitochondrial respiration is uncoupled. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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m) and cytochrome c release into the cytosol. This facilitates assembly of the Apaf-1 apoptosome with recruitment and activation of caspase-9 and subsequently the effector caspases (3)
. Multidomain proapoptotic members of the Bcl-2 family, such as Bax and Bak, are counterbalanced by the antiapoptotic family members Bcl-2 or Bcl-XL (4)
. BH3-only proteins, such as Bid, interact with proapoptotic Bcl-2 family members to augment their activity. Once cleaved by caspase-8 during treatment with TRAIL, Bid translocates to the mitochondria and activates Bax, thus providing a mechanism for cross-talk between the extrinsic and intrinsic apoptotic pathways (5
, 6)
. The requirement for Bax activation in TRAIL-induced apoptosis is cell type dependent (7, 8, 9, 10) . Early events triggered by TRAIL, such as death-inducing signaling complex formation, caspase-8 activation, and Bid cleavage were not dependent on Bax; however, mitochondrial depolarization, cytochrome c release, and activation of caspase-9 were prevented in Bax-deficient cells (9 , 11) . Thus, in these cells, the intrinsic pathway was required for TRAIL-mediated apoptosis, with Bax being essential for induction of the mitochondrial events.
BH3I-2' and HA14-1 are small nonpeptidic organic compounds that interact with the surface pocket of Bcl-2 and can be used as cell-permeable agents to affect Bcl-2-regulated apoptotic pathways and are called inhibitors of Bcl-2 or BH3-mimetic compounds (12
, 13)
. These small compounds not only induce cytochrome c release from mitochondria but also dissipate m (12, 13, 14)
.
The mitochondria of healthy cells maintain an electrochemical gradient across the mitochondrial inner membrane (MIM) that is created by pumping protons from the matrix to the inter-membrane space of these organelles in conjugation with electron transport through the respiratory chain. The proton gradient and membrane potential are the proton-motive force that is used to drive ATP synthesis. Coupling of electron transport through the respiratory chain and ATP generation can be disrupted by some acidic aromatic substances such as carbonyl cyanide m-chlorophenylhydrazone (CCCP) and 2,4-dinitrophenol. These so-called uncouplers of oxidative phosphorylation carry protons across the inner mitochondrial membrane. This specific attack of oxidative phosphorylation leads to a reduction of m, to the cessation of ATP generation in the mitochondrion, and to the collapse of the pH gradient by shuttling protons back across the membrane (15)
.
Alteration in mitochondrial function can change the sensitivity of tumor cells to apoptosis mediated by death receptors. Increase in mitochondrial respiration sensitizes leukemic cells to tumor necrosis factor-mediated apoptosis (16) . Depletion in mitochondrial DNA renders tumor cells resistant to apoptosis induced by TRAIL (17) . The uncoupler CCCP can enhance the Fas death signal, although CCCP alone does not have an apoptotic effect (18) . However, the precise mechanism by which the mitochondrial function contributes to death receptor-mediated apoptosis is still unclear.
In this study, we used BH3I-2' or HA14-1 as a sensitizer for overcoming the resistance of leukemic cells to TRAIL-induced apoptosis. It was found that both BH3I-2' and HA14-1 showed an uncoupling effect on the oxidative phosphorylation when they were used at the concentrations that could not induce cytochrome c release and apoptosis. CCCP, which does not induce apoptosis, also showed a large synergistic effect on TRAIL-induced apoptosis in leukemic cells. Our data showed that the synergistic effect of uncoupling agents on TRAIL-induced apoptosis is via the intrinsic apoptotic pathway (i.e., to enhance Bax conformational change, cytochrome c release, and caspase-3 activation).
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture and the Treatment of Cells.
The human T-lymphoblastic leukemia CEM, the promyelocytic leukemia HL-60, the promonocytic leukemia U937, and the chronic erythroleukemia K562 cell lines were used in this study, and cell culture was performed as described previously (16)
. For the single treatment of cells, 106 cells/ml were treated with 500 ng/ml TRAIL, 30 µM BH3I-2', 10 µM HA14-1, or 10 µM CCCP for up to 24 h. For the cotreatment, cells were pretreated with 30 µM BH3I-2', 10 µM HA14-1, or 10 µM CCCP for 1 h and then treated together with 500 ng/ml TRAIL for up to 24 h.
Apoptosis Assay by Flow Cytometry.
DNA content was measured by flow cytometry. Cells were permeabilized with 70% ethanol and stained with 100 µg/ml PI. PI fluorescence of nuclei was measured by a FACScan flow cytometer (Becton Dickinson, Cambridge, United Kingdom). Data analysis was carried out on cells gated on an FL2-Area channel versus FL2-Width channel display to exclude cell debris and clumped cells. DNA content distribution (PI fluorescence) was analyzed on the FL2-Area histogram, and cells with a DNA content less than G0/G1 (hypodiploid) were defined as apoptotic cells (7)
.
Soft Agar Assay for Colony Formation.
Two percent of gum agar was melted in a microwave and cooled to 50–60°C in a water bath. Eagle’s Minimum Essential Medium (EMEM) and 20% FCS were prewarmed to 40°C in water bath. Agar was mixed with medium and FCS to give 0.5% agar and 10% FCS. Two ml of 0.5% agar were added to each 35-mm Petri dish and allowed to set. The top agar was prepared with 2% agar, EMEM, and FCS to give 0.3% agar and 10% FCS. Agar (1.8 ml of 0.3%) was mixed with 0.2 ml of CEM cell suspension (containing 2 x 104 cells) by vortexing the contents vigorously until the cells were evenly suspended. The cell-containing mixture was plated in a 2-ml volume on the top agar. The dish was overlaid with 1 ml of EMEM containing supplements and with or without 30 µM BH3I-2' or 10 µM HA14-1. Cells were incubated for 1 week at 37°C in 5% CO2 before counting colonies (19)
.
Detection of m and ROS Generation by Flow Cytometry.
For measuring m, CEM cells were stained with 20 nM tetramethylrhodamine methylester for 30 min at 37°C. The fluorescent intensities were measured in the FL3-H channel with a FACScan flow cytometer. The intracellular accumulation of ROS was determined using the fluorescent probe dihydroethidium to measure O 
Measurement of Caspase-3 Activation.
The caspase-3 activity was measured by a fluorogenic method using Z-DEVD-AFC as a substrate. The fluorescence of AFC at 400/505 nm was measured with a TD-700 fluorometer (Turner Design, Sunnyvale, CA). Caspase activity was defined as micromolar AFC release per hour per milligram protein (µM/h/mg protein; Ref. 19
).
Measurement of Mitochondrial Respiration.
CEM cells were suspended in the respiratory medium [250 mM sucrose, 20 mM HEPES, 10 mM MgCl2, 5 mm KH2PO4, 0.1% BSA, 1 mM ADP (pH 7.4)]. Oxygen consumption of leukemic cells was measured with the rank oxygen electrode (Rank Brothers, Cambridge, United Kingdom) in a thermojacked sample chamber stirred with a magnetic flea. One ml of cell suspension containing 107 cells was added to the 1-ml sample chamber (16)
. The respiratory rate was monitored for 3 min and then 10 µM CCCP, 30 µM BH3I-2', or 10 µM HA14-1 was added to the suspension and recorded for another 3 min. Oxygen consumption was calibrated with air-saturated respiratory medium assuming 390 ng atoms O2/ml. The respiratory rate was expressed as nanogram atoms of O2 per minute per 107 cells.
ATP Measurement.
Intracellular ATP content was determined using a bioluminescence assay kit according to the Calbiochem manufacturer’s suggestions. Cells (1 x 106) were treated with drugs as described. Cells (104) were taken out at 4 h. After washing once with Hanks buffer, cells were lysed with releasing reagent. Immediately after solubilization, the intracellular ATP content was measured with the luciferin-luciferase assay in a TD-20/20 Luminometer (Turner Design, Sunnyvale, CA). ATP content was calculated using an ATP standard curve and expressed as nanomolar ATP released from 107 cells (nM/107 cells; Ref. 20
).
Determination of Intracellular pH Value.
CEM cells were suspended in HEPES-buffered medium (without serum) and incubated with 5 µM SNARF-1 acetate for 30 min at 37°C. After centrifuge, cells were resuspended in fresh medium. A pH calibration curve was generated by preloading cells with 5 µM SNARF-1 acetate, followed by incubation for 30 min in different pH buffers (from 6.5 to 8) in the presence of permeabilizing agent nigrericin (10 µM) in a high-K+ HEPES buffer (135 mM KH2PO4/K2HPO4, 20 mM HEPES, 20 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, and 10 mM glucose). Fluorescence intensities were measured in both FL2-H and FL3-H channels. The ratio of FL3-H/FL2-H fluorescence intensity was proportional to the intracellular pH value (21)
.
Bax Conformational Change.
Cells were washed with PBS and lysed with Chaps buffer [10 mM HEPES (pH 7.4), 150 mM NaCl, 1% Chaps, 1 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride, 3 µg/ml aprotinin, 25 µg/ml leupeptin, and 25 µg/ml pepstain]. One µg of anti-Bax (6A7) monoclonal antibody was preincubated with 20 µl of Dynabeads (M-450 rat antimouse IgG1) at 4°C on the rotor for 3 h. The cell lysates were normalized for protein content, and 1000 µg of total protein in 300 µl Chaps lysis buffer were then added to the immuno-precipitation tube containing Bax antibody (6A7)-loaded Dynabeads and incubated at 4°C on the rotor overnight. After rinsing four times with Chaps buffer, beads were collected with a Dynal Magnetic Particle Concentrator (Dynal). Conformationally changed Bax protein was eluted with 25 µl of sample buffer for Western blotting by the monoclonal anti-Bax antibody, clone 2D2 (22)
.
Immunofluorescence Analysis of Cytochrome c Release.
To colocalize cytochrome c in mitochondria, intact cells were first labeled with the mitochondrion-specific dye, MitoTracker red CMXRos. Cells in culture medium were incubated with MitoTracker (100 nM) at 37°C for 30 min. After washing, cells were fixed/permeabilized on slides. Cells were incubated with the anticytochrome c antibody 6H2.B4 (1:400 dilution) for 2 h and then incubated with FITC-conjugated antimouse secondary antibody (Sigma) at a 1:20 dilution. Slides were air dried at 4°C in the dark and stained with 4',6-diamidino-2-phenylindole before being viewed under a Zeiss Axioskop fluorescence microscope (Carl Zeiss, Oberkochen, Germany; Ref. 23
).
Measurement of Cell Membrane Integrity by PI Dye Exclusion.
After treatment, cells were stained with 10 µg/ml PI for 5 min at room temperature. The PI fluorescence was measured by flow cytometry in the FL3-H channel. Intact cells were PI negative, and PI-positive cells were defined as necrotic cells with a broken cell membrane.
Measurement of Cytotoxicity by the MTT Test.
MTT can be cleaved by mitochondrial enzymes, mainly by succinate dehydrogenase, to form a dark blue crystalline product, formazan. Reduced formation of formazan is caused by decreased mitochondrial dehydrogenase activity, inhibited cell proliferation, or cell death (14)
. Cells (105/ml) were treated for 24 h; MTT dye was added to each well to reach the final concentration of 50 µg/ml and allowed to incubate for 4 h at 37°C. Plates were centrifuged and media replaced with 150 µl of isopropyl alcohol containing 0.4 N HCl to solubilize the insoluble formazan complex. Absorbance at 540 nm was determined with an ELISA plate reader.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The cell- permeable inhibitors for Bcl-2, BH3I-2', and HA14-1 were used to test the synergistic effect on TRAIL-induced apoptosis. BH3I-2' did not significantly induce apoptosis at concentrations <50 µM within 4 h. The concentration of BH3I-2', which showed a significant synergistic effect on TRAIL-induced apoptosis (t test, P < 0.001), started at 30 µM (Fig. 1Ba)
. Therefore, 30 µM BH3I-2' were used to study the synergistic effect on TRAIL kinetically within 6 h, and it was shown (Fig. 1Bb)
that BH3I-2' significantly increased the sensitivity of CEM cells to TRAIL-induced apoptosis compared with cells treated with TRAIL alone (ANOVA, P < 0.001). The concentration of HA14-1, which showed a significant synergy (t test, P < 0.001) with TRAIL, started at about 6 µM (Fig. 1Ca)
. Ten µM HA14-1 were used to assess the sensitization effect on TRAIL kinetically (Fig. 1Cb)
, and again it showed a significant synergistic effect on TRAIL-induced apoptosis (ANOVA, P < 0.0001). The colony formation assay in soft agar was used to examine whether 30 µM BH3I-2' or 10 µM HA14-1 alone have an inhibitory effect on cell proliferation. It was observed that 30 µM BH3I-2' or 10 µM HA14-1 only slightly but not significantly inhibited colony formation (Fig. 1D)
. These results imply that Bcl-2 inhibitors can sensitize TRAIL-induced apoptosis, and this sensitization is not a simple additive effect.
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m at the concentration of 70 µM, and these can be inhibited by Bcl-2/Bcl-XL (14)
. In this study, we used 30 µM BH3I-2' or 10 µM HA14-1, which could not induce cytochrome c release nor apoptosis within 12 h (data not shown). We found that both BH3I-2' (30 µM) and HA14-1 (10 µM) induced a collapse in m in the CEM cell line after treatment for 2 h (Fig. 2A)
, as measured by flow cytometry when cells were stained with tetramethylrhodamine methylester dye. Generation of ROS, O
). CCCP was initially used as a positive control for the reduction of m and the generation of ROS. Results indicated that Bcl-2 inhibitors showed some similar effects to the uncoupler CCCP on the MIM (i.e., the reduction of m and the generation of ROS). It had been shown previously that the CEM cell line is relatively resistant to TRAIL-induced reduction in m (24)
. When combined with BH3I-2', HA14-1, or CCCP, TRAIL-mediated dissipation in m was greatly enhanced (Fig. 2A)
. TRAIL alone did not induce production of ROS, and it did not show synergistic effect on BH3I-2', HA14-1, or CCCP-induced generation of ROS (Fig. 2B)
.
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.
The synergistic effect of Bcl-2 inhibitors and CCCP on TRAIL-induced apoptosis was also examined on other leukemic cell lines. Both HL-60 (Fig. 3A)
and U937 (Fig. 3B)
cell lines were relatively resistant to TRAIL-induced apoptosis. However, TRAIL-induced apoptosis was significantly increased in both HL-60 and U937 cell lines when cells were pretreated with BH3I-2', HA14-1, or CCCP (Fig. 3, A and B)
. The K562 cell line is sensitive to TRAIL-induced apoptosis, as reported previously (19
, 24)
. Neither Bcl-2 inhibitors nor CCCP further sensitized K562 cells to TRAIL-induced apoptosis (Fig. 3C)
.
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. ATP content was evaluated by luciferin-luciferase after the treatment of cells for 4 h. As expected, BH3I-2', HA14-1, and CCCP also inhibited ATP synthesis (Fig. 4B)
. Treatment with TRAIL did not cause depletion of the intracellular ATP level but in contrast increased the ATP level. With the combination of TRAIL with BH3I-2', HA14-1, or CCCP, ATP levels were further decreased when compared with those treated with these reagents alone (Fig. 4B)
. This evidence suggests that the cell-permeable Bcl-2 inhibitors have a function in uncoupling mitochondrial respiration from oxidative phosphorylation.
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. This implies that the uncoupling and the acidification effects of Bcl-2 inhibitors are via a mechanism unrelated to the movement of protons across the MIM.
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. The determination of cytochrome c release was performed by an immunostaining of triple colors. The mitochondria were stained as a red fluorescence, cytochrome c was stained as a green color, and the nuclei were stained with 4',6-diamidino-2-phenylindole showing blue color (23)
. Cytochrome c in control cells (Fig. 7A)
or cells treated with BH3I-2' (Fig. 7C)
HA14-1 (Fig. 7E)
or CCCP (Fig. 7G)
alone localized to mitochondria as red and green-merged pixels appeared orange/yellow. TRAIL-induced cytochrome c release was weakly shown in the CEM cells as the green-labeled cytochrome c image separating from the mitochondria with a clearly diffuse pattern (Fig. 7B)
, and it was enhanced by cotreatment with BH3I-2' (Fig. 7D)
, HA14-1 (Fig. 7F)
, or CCCP (Fig. 7H)
.
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regardless of the lower levels of ATP. This suggests that the synergistic effect of uncoupling agents on TRAIL-induced apoptosis is caspase dependent.
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, or TRAIL combined with BH3I-2', HA14-1, or CCCP (Fig. 8B, d-f)
, kept impermeable to PI, indicating the integrity of the cell membrane. However, cotreatment of cells with TRAIL and BH3I-2', HA14-1, or CCCP in the presence of Z-VAD.fmk for 6 h, part of cells were permeable to PI, show the increased PI fluorescence in the FL3-H channel (Fig. 8B, g-i)
. In addition, BH3I-2', HA14-1, or CCCP alone did not induce necrosis in the presence of Z-VAD.fmk (data not shown). The MTT test confirmed that the inhibition of caspases failed to block the uncoupling effect-sensitized TRAIL-mediated killing (Fig. 8C)
. Z-VAD.fmk could nor inhibit dissipation of m or generation of ROS, which was induced by TRAIL when combined with BH3I-2', HA14-1, or CCCP (data not shown), indicating that they induce permanent damage on the MIM. These results imply that the uncoupling effect facilitates TRAIL-induced apoptosis when caspases are activated and leads to necrosis when caspases are inhibited. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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m, generation of ROS, stimulation in oxygen consumption, and reduction in ATP synthesis were identical to the uncoupler CCCP. These uncoupling functions were associated with a marked synergy with TRAIL-induced apoptosis via the mitochondrial apoptotic pathway. They also induced necrotic cell death when caspases were inhibited. The synergistic effect of both Bcl-2 inhibitors and CCCP on TRAIL-induced apoptosis was detected in TRAIL-resistant leukemic cell lines, such as CEM, HL-60, and U937 but not in TRAIL-sensitive K562 cell line. This is in agreement with our previous study that TRAIL-induced apoptosis in the K562 cell line is via the mitochondria-independent pathway (7)
. This study showed evidence, for the first time, that the uncoupling effect can overcome resistance of leukemic cells to TRAIL-induced apoptosis.
The cell-permeable small nonpeptide compounds, BH3I-2' and HA14-1, are inhibitors for Bcl-2/Bcl-XL, or "BH3 mimetics," and function through their ability to occupy the hydrophobic pocket of Bcl-2/Bcl-XL (12
, 13)
. It has been reported that BH3I-2' induces apoptosis by neither directly inducing Bax oligomerization and mitochondrial insertion nor by mediating pore formation by Bcl-XL (13)
. BH3I-2' and the other BH3Is induce apoptosis by inhibiting the heterodimerization of Bcl-2/Bcl-XL and releasing the proapoptotic Bcl-2 family members, which in turn initiate downstream apoptotic events (13)
. This presumed that "BH3 mimetic"-mediated apoptosis is indirectly achieved through activating Bax; however, Bax does not form channels on its own but interacts with and/or modulates a pre-existing mitochondrial outer membrane channel. Such a channel is termed the "permeability transition pore," which crosses both mitochondrial membranes at contacting sites and transports adenine nucleotides and other small molecules (27)
. Both BH3I-2' and HA14-1 induced rapid reduction in m and generation of ROS at concentrations that do not actively induce apoptosis. They have similarities to CCCP on the uncoupling function. In addition to the dissipation of m and the generation of ROS, they uncouple ATP synthesis from mitochondrial respiration and increase the oxygen consumption. The only difference we observed between the Bcl-2 inhibitors and CCCP is that both BH3I-2' and HA14-1 induce cytosolic acidification, but CCCP does not. It has been reported that overexpression of Bax induces cytosolic acidification, which can be prevented by Bcl-2, and CCCP does not alter cytosolic pH value (25)
. This may imply that the uncoupling effect of Bcl-2 inhibitors is not through dissipation of the H+ gradient across the MIM. The precise mechanism by which Bcl-2 inhibitors uncouple mitochondrial respiration is elusive. Bcl-2 protein has been identified in both MIM (28)
and mitochondrial outer membrane (29)
. Bcl-2 and Bcl-XL are proteins that maintain the integrity of both mitochondrial outer membrane and MIM (30)
. The cell-permeable property of these Bcl-2 inhibitors may enable them to bind to Bcl-2/Bcl-XL in the MIM and disrupt their role as a gatekeeper of the MIM.
TRAIL did not show the ability to uncouple mitochondrial respiration because it could not inhibit ATP synthesis. The reduction in m induced by TRAIL was caspase dependent. It has also been reported that neither tumor necrosis factor nor Fas uncouples mitochondrial respiration in the CEM cell line (16
, 18)
. The resistance of CEM cells to death receptor-mediated apoptosis may be largely associated with the lower expression of caspase-8 (18
, 24
, 31) . The requirement for a functional mitochondrial electron transport chain in TRAIL-induced apoptosis has not been widely investigated. Depletion of mitochondrial DNA rendered tumor cells resistant to TRAIL-induced apoptosis (17)
, and a deficiency in the mitochondrial electron transport chain, which confers resistance to TRAIL, is caused by reduction in both mitochondrial respiration and ATP synthesis. The uncoupling effect, which synergizes TRAIL-induced apoptosis, causes an increase in mitochondrial respiration and a decrease in ATP synthesis. These results imply that mitochondrial ATP synthesis is not crucial for the sensitivity of cells to TRAIL-induced apoptosis. We suggest that increased mitochondrial respiration and ROS generation may be responsible for the synergistic effect of the uncouplers to TRAIL-induced apoptosis. Cytochrome c-dependent activation of caspases requires ATP or dATP (26)
, but the inhibition of ATP synthesis by either Bcl-2 inhibitors or CCCP did not abrogate TRAIL-induced activation of caspase-3. This indicates that the residual levels of intracellular ATP are sufficient for the requirement of cytochrome c-induced caspase activation. However, following the inhibition of caspases, TRAIL induced necrosis in the presence of uncoupling agents. This suggests that caspases are not crucial for TRAIL-induced killing in the presence of uncoupler.
Despite the uncoupling effect, both Bcl-2 inhibitors and CCCP at the concentration used for this study did not induce Bax conformational change and cytochrome c release. However, they greatly sensitized TRAIL-induced Bax conformational change and cytochrome c release. It has been reported that Bax activation is associated with the loss of m (32)
or a rise in intracellular pH value (33)
. Our results showed that either Bcl-2 inhibitors or CCCP alone did not induce Bax conformational change after the treatment for 4 h. However, both BH3I-2' and HA14-1 could induce cytochrome c release if their concentrations or incubation period were increased. CCCP can trigger Bax translocation in the presence of the F1-F0-ATPase oligomycin, suggesting that the ATP level must be maintained for Bax activation, and the collapse in m is the cause of Bax translocation (32)
. The uncoupling effect of BH3I-2', HA14-1, or CCCP decreased the intracellular ATP content, and their ATP-depleting function was further facilitated in the presence of TRAIL. TRAIL-induced Bax activation and cytochrome c release were greatly enhanced under the uncoupling condition. An increase in the pH value can induce Bax conformational change in vivo and in vitro (33)
; however, the Bax conformational change-induced by TRAIL alone or combined with BH3I-2'/HA14-1 was under acidic conditions. In addition, CCCP that did not change the intracellular pH value also enhanced TRAIL-induced Bax conformational change. Our results suggest that the uncoupling effect on TRAIL-induced Bax conformational change may not be associated with changes in the pH value.
In conclusion, we have shown that Bcl-2 inhibitors, BH3I-2', and HA14-1 have an uncoupling function on mitochondrial respiration at the concentrations that do not induce cytochrome c release. Therefore, similar to the uncoupler CCCP, the uncoupling effect of these reagents greatly sensitized TRAIL-induced Bax conformational change, cytochrome c release. Increased mitochondrial respiration and dissipation in m appears to sensitize CEM cells to TRAIL-induced apoptosis. The uncoupling effect leads TRAIL to induce necrosis when caspases were inhibited. Both Bcl-2 inhibitors and mitochondria uncoupler showed great synergy with TRAIL to overcome leukemic cell resistance to TRAIL-induced killing.
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FOOTNOTES |
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Note:J-H. Hao and M. Yu contributed equally to this work.
Requests for reprints:Dr. Li Jia, Department of Haematology, Barts and The London Queen Mary’s School of Medicine and Dentistry, London, E1 2AD, United Kingdom. Phone: 0044-207-377-7076; Fax: 0044-207-377-7629; E-mail: L.jia{at}qmul.ac.uk
Received 11/21/03. Revised 1/23/04. Accepted 3/ 1/04.
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M. Konopleva, E. Elstner, T. J. McQueen, T. Tsao, A. Sudarikov, W. Hu, W. D. Schober, R.-Y. Wang, D. Chism, S. M. Kornblau, et al. Peroxisome proliferator-activated receptor {gamma} and retinoid X receptor ligands are potent inducers of differentiation and apoptosis in leukemias Mol. Cancer Ther., October 1, 2004; 3(10): 1249 - 1262. [Abstract] [Full Text] [PDF]
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