Following the screening of a battery of distinct small-interfering RNAs that target various components of the apoptotic machinery, we found that knockdown of the voltage-dependent anion channel 1 (VDAC1) was particularly efficient in preventing cell death induced by cisplatin (CDDP) in non-small cell lung cancer cells. Both the downregulation of VDAC1 and its chemical inhibition with 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid reduced the apoptosis-associated modifications induced by CDDP, including mitochondrial transmembrane potential dissipation and plasma membrane permeabilization. VDAC1 inhibition strongly reduced the CDDP-induced conformational activation of Bax, yet had no discernible effect on the activation of Bak, suggesting that VDAC1 acts downstream of Bak and upstream of Bax. Accordingly, knockdown of Bak abolished the activation of Bax, whereas Bax downregulation had no effect on Bak activation. In VDAC1-depleted cells, the failure of CDDP to activate Bax could be reversed by means of the Bcl-2/Bcl-XL antagonist ABT-737, which concomitantly restored CDDP cytotoxicity. Altogether, these results delineate a novel pathway for the induction of mitochondrial membrane permeabilization (MMP) in the course of CDDP-induced cell death that involves a hierarchical contribution of Bak, VDAC1 and Bax. Moreover, our data suggest that VDAC1 may act as a facultative regulator/effector of MMP, depending on the initial cytotoxic event.
Cisplatin (CDDP) is one of the principal chemotherapeutic agents used for the first-line treatment of non-small cell lung cancer (NSCLC), with deceptive results. After an initial, pyrrhic success leading to partial therapeutic responses or stabilization of the disease, chemotherapy-resistant tumor cells are selected, imposing successive changes in the chemotherapeutic regimen. Almost invariably, chemotherapy becomes ineffective and the patient eventually succumbs to the disease (Cosaert and Quoix, 2002; Seve and Dumontet, 2005). Thus, the comprehension of CDDP-induced cell death mechanisms may have direct clinical implications for the design of therapeutic regimens that overcome CDDP resistance.
As many other conventional chemotherapeutic agents, CDDP triggers the intrinsic (or ‘mitochondrial’) pathway of apoptosis. Through the induction of DNA damage, resulting in p53-dependent and/or p53-independent damage responses, CDDP elicits mitochondrial membrane permeabilization (MMP), which is the rate-limiting step of the intrinsic pathway (Perfettini et al., 2004; Zamzami and Kroemer, 2005; Kroemer et al., 2007). Moreover, recent work revealed that CDDP is able to cause MMP in enucleated cells, suggesting that CDDP can trigger cytoplasmic cell death pathways that are independent of its effects on nuclear DNA (Mandic et al., 2003; Berndtsson et al., 2007). These effects may be related to direct pro-oxidant activities and/or to the depletion of reduced (non-oxidized) glutathione or other reducing equivalents.
Mitochondrial membrane permeabilization is a common feature of many, if not most, apoptotic pathways, because distinct pro-apoptotic signal transducers including reactive oxygen species (ROS), oxidized lipids, Ca2+ ions, proteases, kinases as well as pro-apoptotic proteins from the Bcl-2 family share the ability to converge on mitochondria for triggering MMP. MMP is a lethal event as it results in (1) dissipation of the mitochondrial transmembrane potential (ΔΨm), (2) cessation of the bioenergetic and redox-detoxifying functions of mitochondria and (3) the release of catabolic hydrolases or hydrolase activators (Kroemer et al., 2007). In particular, intermembrane space (IMS) proteins that usually are inert (and serve vital roles) become pro-apoptotic once they have been liberated from mitochondria through the permeabilized outer membrane (OM) (Ravagnan et al., 2002). For instance, cytochrome c (Cyt c) is normally secluded in the IMS, where it serves as an electron shuttle in the respiratory chain. Once in the cytosol, Cyt c interacts with dATP and apoptotic peptidase-activating factor-1, a soluble monomeric protein that has been recently implicated in the regulation of cell cycle following DNA damage (Zermati et al., 2007), to form a heptameric complex (the so-called apoptosome) that recruits and allosterically activates caspase-9 and hence sets off the caspase cascade (Garrido et al., 2006). Among caspase-independent death effectors released upon MMP, apoptosis-inducing factor (AIF) translocates from mitochondria to the cytosol and eventually to the nucleus to participate in chromatin condensation (Lorenzo et al., 1999; Modjtahedi et al., 2006).
The molecular regulation of MMP constitutes the battleground where opposing forces (anti- and pro-apoptotic signals) determine the fate of the cell. Among the most important mediators of MMP, there are the pro-apoptotic proteins of the Bcl-2 family as well as the permeability transition pore complex (PTPC) (Grimm and Brdiczka, 2007; Kroemer et al., 2007). Within the Bcl-2 family, the two multi-domain proteins Bax and Bak have been reported to act either in a redundant (independent) or in a sequential (interdependent) fashion to mediate MMP, by forming supramolecular complexes in the OM that allow for the release of IMS proteins (Reed, 2006). To this aim, Bak (which is constitutively associated with the mitochondria) would fully insert into and oligeromerize within the OM (Griffiths et al., 1999). On the other hand, Bax is normally located in the cytosol in a monomeric form. The activation of Bax involves the formation of an active dimer that translocates to OM to promote the assembly of large oligomeric pores (Lalier et al., 2007). Obviously, both Bax and Bak have to change their overall conformation to become activated, and this can be detected with conformation-specific monoclonal antibodies.
The PTPC is a large supramolecular complex assembled at the junctions between the inner and outer mitochondrial membranes by the interaction of multiple proteins located in the cytosol (for example, hexokinase), the OM (for example, voltage-dependent anion channel (VDAC)), the inner mitochondrial membrane (for example, adenine nucleotide translocase) or the matrix (for example, cyclophilin D). The exact composition of the PTPC is rather variable between different cells and is influenced by the existence of several isoforms of its constituents (such as hexokinase-1, hexokinase-2, VDAC1–3, adenine nucleotide translocase-1 to -4) (Brenner and Grimm, 2006; Grimm and Brdiczka, 2007; Kroemer et al., 2007). Although several studies have insisted on the fact that Bax/Bak-mediated MMP and PTPC-dependent MMP would occur in a completely independent, mechanistically distinct fashion, other reports suggest that proteins from the Bcl-2 family can functionally and physically interact with the PTPC (Marzo et al., 1998; Brenner et al., 2000; Belzacq et al., 2002). In this context, it has been suggested that VDAC2 would exert antiapoptotic functions by sequestering Bak (Cheng et al., 2003; Chandra et al., 2005) whereas other VDAC isoforms (possibly VDAC1 and VDAC3) would interact cooperatively with Bax to induce apoptosis (Shimizu et al., 1999, 2001). Recently, one study performed on genetically manipulated mouse cells purported that none of the VDAC isoforms would be required for apoptotic MMP (Baines et al., 2007; Galluzzi and Kroemer, 2007). In contrast, it has been proposed that VDAC2 and VDAC3 may be required for the cytotoxic action of the experimental anticancer agent erastin (Yagoda et al., 2007).
On the basis of these premises, we decided to re-evaluate the relative contribution of Bcl-2 family members and PTPC components to CDDP-induced apoptosis of NSCLC cells. Surprisingly, we found that VDAC1 played a major role in this system, by occupying an intermediate position in the hierarchy of events leading to MMP, downstream of Bak but upstream of Bax.
Results and discussion
Identification of VDAC1 as a modulator of CDDP-induced apoptosis
To identify molecules that influence the susceptibility to CDDP-induced cell death, A549 NSCLC cells were transfected with a battery of small-interfering RNAs (siRNAs) that knock down distinct proteins involved in the regulation of apoptosis-related MMP, following a previously reported approach (de La Motte Rouge et al., 2007). Then, the cells were treated with an apoptogenic dose of CDDP and subsequently assessed for residual viability and cytotoxicity, by measuring the reduction of the water-soluble tetrazolium salt 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) by adherent cells (Figure 1a) or the release of the cytosolic enzyme lactate dehydrogenase (LDH) into the culture supernatant (Figure 1b), respectively. Among 56 distinct siRNA transfections (54 single and 2 cotransfections), one siRNA species that depleted VDAC1 was consistently (in five out of five experiments) the most cytoprotective one. In this experimental setting, VDAC1 depletion was more efficient in maintaining viability and/or in inhibiting cell death than the knockdown of any other confirmed pro-apoptotic protein known to participate in CDDP-elicited lethal signaling, including breast cancer 1 (BRCA1) (Bartz et al., 2006), p53 (Siddik, 2003), apoptotic peptidase-activating factor-1 (Zermati et al., 2007), Bak and Bax (alone and in combination) (Wang et al., 2001; Kepp et al., 2007) as well as excision repair cross-complementing rodent repair deficiency, complementation group 1 (ERCC1) (Olaussen et al., 2006). VDAC1 knockdown exhibited greater cytoprotective effects against CDDP-induced cell death than the depletion of VDAC2 and VDAC3 (which were only marginally effective or totally ineffective, respectively) or of any other constituent of the PTPC such as the three adenine nucleotide translocase isoforms, the two hexokinase isoforms or cyclophilin D (all of which are implicated in CDDP-induced cell death) (Figure 1).
Knockdown of VDAC1 with two different siRNAs (inset in Figure 2a) reduced CDDP-induced signs of apoptosis such as ΔΨm dissipation or the permeabilization of plasma membrane, as determined by the simultaneous assessment of ΔΨm (by means of the ΔΨm-sensitive fluorochrome 3,3′dihexiloxalocarbocyanine iodide (DiOC6(3))) and viability (with the vital dye propidium iodide, PI) (Figures 2a and b). VDAC1 knockdown reduced CDDP-induced apoptosis not only in A549 NSCLC cells (Figures 1, 2a and b) but also in HeLa cervix carcinoma cells (Figure 2c), AGS gastric cancer cells and (much less efficiently) in HCT116 and RKO colon carcinoma cells (data not shown). The cytoprotective effect of VDAC1 depletion was particularly pronounced when CDDP was employed to induce cell death. On the contrary, no (or minor) antiapoptotic effects were provided by the downregulation of VDAC1 when cells were killed by microtubule poisons (for example, paclitaxel, docetaxel) or pro-oxidant agents (for example, hydrogen peroxide, that is, H2O2; tert-butylhydroperoxide) (Figure 2d).
VDAC1 downregulation strongly reduced multiple biochemical hallmarks of the intrinsic pathway to apoptosis activated by CDDP. For instance, siRNA-mediated depletion of VDAC1 inhibited the release of IMS proteins from mitochondria, as determined by immunofluorescence staining to visualize the redistribution from a punctuate, mitochondrial pattern to a more diffuse cytosolic or nuclear (in the case of Cyt c and AIF, respectively) staining (Figures 3a–d). This applied to both Cyt c (Figures 3a and b) and AIF (Figures 3c and d), which were liberated from mitochondria upon CDDP treatment in control cells, yet were retained in the IMS of VDAC1-depleted cells. In addition, the CDDP-mediated proteolytic maturation of caspase-3 was decreased after VDAC1 knockdown, as determined by cytofluorometric quantification of cells staining positively with an antibody that specifically recognizes the active form of caspase-3 (Figure 3e). Similar results were obtained by immunoblotting with antibodies that recognize the active fragments of caspase-3 (that is, p17 and p19) and the caspase-3 substrate poly(ADP-ribose) polymerase-1. VDAC1 depletion reduced caspase-3 activation induced by CDDP (Figure 3f).
Altogether, these results identify VDAC1 as a prominent mediator of CDDP-induced apoptosis.
Hierarchical relationship between VDAC1, Bax and Bak
The siRNA-mediated knockdown of either Bax or Bak reduced CDDP-induced signs of apoptosis such as ΔΨm dissipation and plasma membrane permeabilization (Figure 4a). As both Bax and Bak undergo conformational changes to get incorporated into mitochondrial membranes and form mitochondrion-permeabilizing pores (Kroemer et al., 2007), we wondered whether VDAC1 depletion would affect the activation status of Bax and Bak. To this aim, we immunoprecipitated CDDP-activated Bax or Bak molecules by means of suitable conformation-specific antibodies (6A7 for Bax and Ab-1 for Bak). Depletion of VDAC1 strongly suppressed the CDDP-induced activation of Bax, yet had no discernible effect on the activation of Bak, suggesting that VDAC1 acts downstream of Bak and upstream of Bax (Figure 4b). This was corroborated by the fact that the knockdown of Bak reduced the activation of Bax to minimal levels (Figure 4c), whereas Bax depletion had no effect on Bak activation (Figure 4d). Similar results were obtained when Bax activation was assessed by a distinct technique, that is, immunofluorescence microscopy. In this experimental setting, the knockdown of VDAC1 strongly diminished the percentage of cells exhibiting a positive staining for active Bax (Figures 4e and f).
Cisplatin-induced cell death is known to involve the depletion of reduced (non-oxidized) glutathione and other reducing equivalents (Zhang et al., 2001; Rudin et al., 2003). Thus, addition of the antioxidant N-acetylcysteine (NAC) dramatically inhibited CDDP-induced cell death (Figures 5a and b), concomitantly with a strong inhibition of CDDP-triggered generation of ROS (as quantified with the superoxide-sensitive probe hydroethidine, which is non-fluorescent and can be oxidized to ethidium, emitting in red) (Figure 5c). In contrast with VDAC1 downregulation, NAC inhibited the activation of both Bax and Bak (Figure 5d). Moreover, VDAC1 knockdown failed to inhibit the CDDP-induced superoxide overgeneration (Figure 5e), indicating that VDAC1 depletion does not create an antioxidant condition. In contrast to NAC, the effects of which do not coincide with those induced by the downregulation of VDAC1, we found that 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid, a chemical inhibitor of VDAC1 (Thinnes et al., 1990; Shoshan-Barmatz et al., 1996; Shafir et al., 1998; Tarze et al., 2007), can faithfully mimic VDAC1 depletion and hence inhibit CDDP-induced cell death (Figures 6a and b) as well as the activation of Bax (but not Bak) and caspase-3 (Figures 6c and d).
Taken together, these observations point to a role for VDAC1 in the CDDP-induced pathway of apoptosis, downstream of Bak but upstream of Bax.
The BH3 mimetic ABT-737 restores apoptosis in VDAC1-depleted cells
The absence of VDAC1 apparently interrupts CDDP-triggered signaling at the level of, or before, Bax activation. We therefore explored the possibility to restore the sensitivity of VDAC1-depleted cells to CDDP by activating Bax with the small pharmacological Bcl-2 homology domain 3 (BH3) mimetic ABT-737, which competitively displaces Bax from inhibitory interactions with Bcl-2/Bcl-XL (Oltersdorf et al., 2005). Immunoprecipitation studies revealed that CDDP alone failed to activate Bax in cells transfected with a VDAC1-targeting siRNA whereas the combination of CDDP and ABT-737 activated Bax as efficiently in VDAC1-depleted as in control cells (Figure 7a). Thus, ABT-737 can overcome the blockade of Bax activation resulting from the loss of VDAC1. Accordingly, ABT-737 restored (though not completely, as compared to control cells) the deficient apoptotic response of VDAC1-depleted cells to CDDP. This observation was corroborated at several levels, including ΔΨm dissipation and plasma membrane permeabilization (Figure 7b), mitochondrial Cyt c release (Figure 7c) and caspase-3 activation (Figure 7d). It is worth noting that siRNA-mediated Bax downregulation totally abolished the effects of ABT-737, hindering it from restoring deficient responses of VDAC1-depleted cells to CDDP (Figure 7b).
Altogether, these results indicate that ABT-737 resensitizes VDAC1-deficient cells to apoptosis via its capacity to inhibit Bcl-2/Bcl-XL, and hence to indirectly facilitate Bax activation.
On the basis of the systematic screening of siRNAs targeting a panel of direct and indirect regulators of apoptosis, we found that VDAC1 knockdown has a robust effect on apoptosis induced by CDDP, but not by microtubule poisons and pro-oxidant agents.
This result underscores that MMP is regulated or mediated by an array of distinct proteins that comes into action in specific (yet partially overlapping) patterns as a result of the activation of diverse pro-apoptotic signal transduction pathways by different death stimuli. This is an important notion because it has been thought that inducer-specific signaling cascades (that are biochemically heterogeneous) would converge on mitochondrial membranes to induce MMP as a common final event of apoptotic demise. Although MMP is characterized by an uniform outcome (a structural and functional catastrophe of mitochondria), it appears that distinct mechanisms can trigger MMP, with or without the involvement of VDAC1. Although CDDP-induced MMP and apoptosis clearly require VDAC1, this component of the PTPC is not implicated in cell death induced by taxanes or pro-oxidants.
The present work sheds some light on the mechanism by which VDAC1 might contribute to CDDP-triggered MMP. Previous work has suggested that VDAC2 would act as an endogenous inhibitor of Bak (but not Bax) (Cheng et al., 2003), whereas other VDAC isoforms would cooperate with Bax to promote apoptosis (Shimizu et al., 1999, 2000a, 2001). Our results point to a role for VDAC1 as an activator of Bax (but not Bak). Serial siRNA experiments and epistatic analyses suggest that CDDP triggers the conformational activation of Bak in a VDAC1-independent (but ROS-dependent) fashion. In contrast, Bax activation (that occurs downstream of Bak) requires both ROS and the expression of VDAC1. Accordingly, in the absence of VDAC1, only Bak adopts the active conformation, whereas Bax remains inactive. However, pharmacological inhibition of Bcl-2/Bcl-XL with the BH3 mimetic ABT-737 was able to revert the defect in Bax activation caused by VDAC1 depletion. It is worth noting that in the conditions used here, ABT-737 was not able to induce Bax activation per se; to do so, it had to be combined with CDDP.
Altogether, our data delineate an MMP-inducing pathway in which Bak, VDAC1 and Bax participate in a strictly hierarchal fashion that has never been described before (Figure 8). Although it is known that Bak can function upstream of Bax, at least in some paradigms of apoptosis (Castedo et al., 2003; Kepp et al., 2007), it has never been reported that VDAC1 may be required at an intermediate step, between Bak and Bax activation. Future work must address the biochemical mechanisms underlying the cooperation between Bax and VDAC1. Interestingly, it has been shown that Bax and VDAC1 can form a large, multimeric channel that allows for the release of proteins through artificial membranes (Shimizu et al., 1999, 2000b). Hence, it will be important to investigate the putative physical and functional interactions between Bax and VDAC1 in the context of CDDP-triggered cell death.
Materials and methods
Cell cultures, reagents and siRNA transfections
A549 NSCLC, AGS gastric cancer and HeLa cervix carcinoma cells were grown in Dulbecco's modified Eagle's medium/F12 (1:1), RPMI or Dulbecco's modified Eagle's medium, respectively, supplemented with 10% fetal calf serum, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 100 U ml−1 penicillin G sodium and 100 μg ml−1 streptomycin sulfate. HCT116 and RKO colon carcinoma cells were maintained in McCoy's 5a medium supplemented with 10% fetal calf serum, 10 mM HEPES, 1 mM sodium pyruvate, 100 U ml−1 penicillin G sodium and 100 μg ml−1 streptomycin sulfate. For proliferation and cytotoxicity assays in 96-well plates, A549 cells were cultured in Dulbecco's modified Eagle's medium/F12 (1:1) with L-glutamine but no phenol red supplemented with 10% fetal calf serum and antibiotics (as above). Media and supplements were purchased from Gibco-Invitrogen (Carlsbad, CA, USA).
4,4′-Diisothiocyanostilbene-2,2′-disulfonic acid, cis-diammineplatinum(II) dichloride (cisplatin, CDDP), NAC, H2O2 and tert-butylhydroperoxide were purchased from Sigma-Aldrich (St Louis, MO, USA). NAC solution was prepared extemporarily by dilution in complete medium at the concentration of 5 mM followed by pH adjustment of 7.3–7.4 with NaOH.
A549 and HeLa cells were transfected in six-well plates with Oligofectamine reagent (Invitrogen, Carlsbad, CA, USA) whereas HCT116 and RKO cells were transfected in 12-well plates by means of the HiPerFect reagent from Qiagen (Hilden, Germany), following the manufacturer's instructions in both the cases. For the siRNA battery screening, A549 were subjected to reverse transfection in 96-well plates, as previously described (de La Motte Rouge et al., 2007) and reported in the ‘Supplementary materials and methods’ section (where detailed information on the siRNAs that constituted the battery can also be found). The following siRNAs (included or not in the battery) were employed all along the study: siRNAs targeting VDAC1 (siVDAC1.1, sense 5′-IndexTermGUACGGCCUGACGUUUACAdTdT-3′, antisense 5′-IndexTermUGUAAACGUCAGGCCGUACdTdT-3′; siVDAC1.2, sense 5′-IndexTermGCUGCGACAUGGAUUUCGAdTdT-3′, antisense 5′-IndexTermUCGAAAUCCAUGUCGCAGCdTdT-3′) and a control siRNA with an irrelevant sequence (siUNR, sense 5′-IndexTermGCCGGUAUGCCGGUUAAGUdTdT-3′, antisense 5′-IndexTermACUUAACCGGCAUACCGGCdTdT-3′) were purchased from Sigma-Proligo (The Woodlands, TX, USA). siRNAs for the downregulation of Bak and Bax (Hs_BAK1_5 and Hs_BAX_10 HP Validated siRNAs, respectively) were purchased from Qiagen.
Cell proliferation and cytotoxicity assays in 96-well plates
Cell proliferation was quantified by means of a colorimetric assay based on the reduction of the water-soluble colorless tetrazolium salt WST-1 (from Roche Applied Science, Basel, Switzerland) to formazan (which exhibits an absorbance peak around 450 nm), according to the manufacturer's instructions. The release of the cytosolic enzyme LDH in the culture supernatant was monitored by means of the Cytotoxicity Detection Kit (Roche Applied Science) following the manufacturer's instructions. Experiments were performed in triplicates, and repeated at least twice. Data are reported as means±s.e.m. Statistical significance was evaluated by means of paired Student's t-test. ΔWST-1 and ΔLDH were introduced as assay-independent indicators to evaluate the effects of siRNAs per se separately from their influence on CDDP-induced cell death and cytotoxicity, respectively, as well as to allow for normalization among different assays. A precise mathematical definition of ΔWST-1 and ΔLDH may be found in ‘Supplementary materials and methods’.
Cytofluorometry and immunofluorescence microscopy
The following probes were employed to assess apoptosis-associated parameters: PI (1 μg ml−1; Sigma-Aldrich) or 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, 1 μg ml−1; Molecular Probes-Invitrogen, Eugene, OR, USA) for plasma membrane breakdown, DiOC6(3) (40 nM; Molecular Probes-Invitrogen) for ΔΨm dissipation, hydroethidine (25 μM; Molecular Probes-Invitrogen) for ROS generation (Zamzami et al., 1995; Zamzami and Kroemer, 2004; Galluzzi et al., 2007). Only intact (DAPI−) cells were analysed for ROS production. The activation of caspase-3 was monitored by staining the cells with a fluorescein isothiocyanate-conjugated monoclonal antibody specific for active caspase-3 (Becton Dickinson, Franklin Lankes, NJ, USA). Cytofluorometric analyses were performed using a FACSCalibur or a FACSVantage equipped with Cell Quest Pro software (Becton Dickinson).
For immunofluorescence studies, cells were fixed with 4% paraformaldehyde (w/v)+0.19% picric acid (v/v) in phosphate-buffered saline, permeabilized with sodium dodecyl sulfate (1% w/v in phosphate-buffered saline) and stained with antibodies for the detection of active Bax (6A7 mouse monoclonal, no. ab5714; Abcam, Cambridge, UK), Cyt c (mouse monoclonal, no. 556432; BD Pharmingen, San Diego, CA, USA) and AIF (rabbit polyclonal, no. VPA16501; AbCys, Paris, France). Nuclear counterstain was obtained with 10 μg ml−1 Hoechst 33342 (Molecular Probes-Invitrogen). Primary antibodies were revealed either with goat anti-rabbit or anti-mouse IgG conjugated to Alexa 488 (green) from Molecular Probes-Invitrogen. Fluorescence microscopy determinations were done by means of a Leica IRE2 microscope equipped with a Leica DC300F camera.
Immunoprecipitation and analysis of protein expression
Semiquantitative analysis of the activation of Bax and Bak by immunoprecipitation was performed as previously described (Kepp et al., 2007). Briefly, cells were washed in phosphate-buffered saline and lysed in a buffer containing 150 mM NaCl, 10 mM HEPES, 1% CHAPS (buffer P, pH 7.4) supplemented with Complete Protease Inhibitor (Roche Applied Science). Thereafter, cleared lysates were incubated overnight with 2 μg of conformation-specific anti-Bak (Ab-1 mouse monoclonal, no. AM03; Calbiochem, San Diego, CA, USA) or anti-Bax (6A7 mouse monoclonal, no. ab5714; Abcam) antibody in an overhead rotator at 4 °C, followed by the addition of 20 μl protein G-sepharose beads (Amersham Pharmacia Biotech, Uppsala, Sweden) for additional 2 h. After extensive washing with buffer P, precipitates were incubated at 70 °C for 10 min and eventually analysed by immunoblotting, as described below.
For the analysis of protein expression, cells were lysed on ice in a buffer containing 1% NP-40, 20 mM HEPES pH 7.9, 10 mM KCl, 1 mM EDTA, 10% glycerol, 1 mM orthovanadate, 1 mM phenylmethanesulphonylfluoride, 1 mM dithiothreitol and 10 μg ml−1 aprotinin, leupeptin, pepstatin prior to centrifugation (20 min, 14 000 r.p.m.) and collection of supernatant. Upon separation on precast 4–12% polyacrylamide gradient gels (Invitrogen), total protein extracts were analysed by standard immunoblotting procedures (Criollo et al., 2007) by using primary antibodies specific for Bak (rabbit monoclonal, no. 06-536; Millipore, Bedford, MA, USA), Bax (rabbit monoclonal, no. 04-434; Millipore), cleaved caspase-3 (Asp175 rabbit polyclonal, no. 9661; Cell Signaling Technology, Beverly, MA, USA), poly(ADP-ribose) polymerase-1 (rabbit polyclonal, no. 9542; Cell Signaling Technology) or VDAC1 (rabbit polyclonal, no. 4866; Cell Signaling Technology). Immunoblotting with an antibody that specifically recognizes β-actin (mouse monoclonal, no. MAB1501; Chemicon International, Temecula, CA, USA) was performed to ensure equal loading of lanes.
mitochondrial transmembrane potential
Bcl-2 homology domain 3
breast cancer 1, early onset
- Cyt c:
excision repair cross-complementing rodent repair deficiency, complementation group 1
mitochondrial membrane permeabilization
non-small cell lung cancer
mitochondrial outer membrane
poly (ADP-ribose) polymerase 1
reactive oxygen species
voltage-dependent anion channel 1
Baines CP, Kaiser RA, Sheiko T, Craigen WJ, Molkentin JD . (2007). Voltage-dependent anion channels are dispensable for mitochondrial-dependent cell death. Nat Cell Biol 9: 550–555.
Bartz SR, Zhang Z, Burchard J, Imakura M, Martin M, Palmieri A et al. (2006). Small interfering RNA screens reveal enhanced cisplatin cytotoxicity in tumor cells having both BRCA network and TP53 disruptions. Mol Cell Biol 26: 9377–9386.
Belzacq AS, Vieira HL, Kroemer G, Brenner C . (2002). The adenine nucleotide translocator in apoptosis. Biochimie 84: 167–176.
Berndtsson M, Hagg M, Panaretakis T, Havelka AM, Shoshan MC, Linder S . (2007). Acute apoptosis by cisplatin requires induction of reactive oxygen species but is not associated with damage to nuclear DNA. Int J Cancer 120: 175–180.
Brenner C, Cadiou H, Vieira HL, Zamzami N, Marzo I, Xie Z et al. (2000). Bcl-2 and Bax regulate the channel activity of the mitochondrial adenine nucleotide translocator. Oncogene 19: 329–336.
Brenner C, Grimm S . (2006). The permeability transition pore complex in cancer cell death. Oncogene 25: 4744–4756.
Castedo M, Perfettini JL, Andreau K, Roumier T, Piacentini M, Kroemer G . (2003). Mitochondrial apoptosis induced by the HIV-1 envelope. Ann NY Acad Sci 1010: 19–28.
Chandra D, Choy G, Daniel PT, Tang DG . (2005). Bax-dependent regulation of Bak by voltage-dependent anion channel 2. J Biol Chem 280: 19051–19061.
Cheng EH, Sheiko TV, Fisher JK, Craigen WJ, Korsmeyer SJ . (2003). VDAC2 inhibits BAK activation and mitochondrial apoptosis. Science 301: 513–517.
Cosaert J, Quoix E . (2002). Platinum drugs in the treatment of non-small-cell lung cancer. Br J Cancer 87: 825–833.
Criollo A, Galluzzi L, Chiara Maiuri M, Tasdemir E, Lavandero S, Kroemer G . (2007). Mitochondrial control of cell death induced by hyperosmotic stress. Apoptosis 12: 3–18.
de La Motte Rouge T, Galluzzi L, Olaussen KA, Zermati Y, Tasdemir E, Robert T et al. (2007). A novel epidermal growth factor receptor inhibitor promotes apoptosis in non-small cell lung cancer cells resistant to erlotinib. Cancer Res 67: 6253–6262.
Galluzzi L, Kroemer G . (2007). Mitochondrial apoptosis without VDAC. Nat Cell Biol 9: 487–489.
Galluzzi L, Zamzami N, de La Motte Rouge T, Lemaire C, Brenner C, Kroemer G . (2007). Methods for the assessment of mitochondrial membrane permeabilization in apoptosis. Apoptosis 12: 803–813.
Garrido C, Galluzzi L, Brunet M, Puig PE, Didelot C, Kroemer G . (2006). Mechanisms of cytochrome c release from mitochondria. Cell Death Differ 13: 1423–1433.
Griffiths GJ, Dubrez L, Morgan CP, Jones NA, Whitehouse J, Corfe BM et al. (1999). Cell damage-induced conformational changes of the pro-apoptotic protein Bak in vivo precede the onset of apoptosis. J Cell Biol 144: 903–914.
Grimm S, Brdiczka D . (2007). The permeability transition pore in cell death. Apoptosis 12: 841–855.
Kepp O, Rajalingam K, Kimmig S, Rudel T . (2007). Bak and Bax are non-redundant during infection- and DNA damage-induced apoptosis. EMBO J 26: 825–834.
Kroemer G, Galluzzi L, Brenner C . (2007). Mitochondrial membrane permeabilization in cell death. Physiol Rev 87: 99–163.
Lalier L, Cartron PF, Juin P, Nedelkina S, Manon S, Bechinger B et al. (2007). Bax activation and mitochondrial insertion during apoptosis. Apoptosis 12: 887–896.
Lorenzo HK, Susin SA, Penninger J, Kroemer G . (1999). Apoptosis inducing factor (AIF): a phylogenetically old, caspase-independent effector of cell death. Cell Death Differ 6: 516–524.
Mandic A, Hansson J, Linder S, Shoshan MC . (2003). Cisplatin induces endoplasmic reticulum stress and nucleus-independent apoptotic signaling. J Biol Chem 278: 9100–9106.
Marzo I, Brenner C, Zamzami N, Jurgensmeier JM, Susin SA, Vieira HL et al. (1998). Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. Science 281: 2027–2031.
Modjtahedi N, Giordanetto F, Madeo F, Kroemer G . (2006). Apoptosis-inducing factor: vital and lethal. Trends Cell Biol 16: 264–272.
Olaussen KA, Dunant A, Fouret P, Brambilla E, Andre F, Haddad V et al. (2006). DNA repair by ERCC1 in non-small-cell lung cancer and cisplatin-based adjuvant chemotherapy. N Engl J Med 355: 983–991.
Oltersdorf T, Elmore SW, Shoemaker AR, Armstrong RC, Augeri DJ, Belli BA et al. (2005). An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature 435: 677–681.
Perfettini JL, Kroemer RT, Kroemer G . (2004). Fatal liaisons of p53 with Bax and Bak. Nat Cell Biol 6: 386–388.
Ravagnan L, Roumier T, Kroemer G . (2002). Mitochondria, the killer organelles and their weapons. J Cell Physiol 192: 131–137.
Reed JC . (2006). Proapoptotic multidomain Bcl-2/Bax-family proteins: mechanisms, physiological roles, and therapeutic opportunities. Cell Death Differ 13: 1378–1386.
Rudin CM, Yang Z, Schumaker LM, VanderWeele DJ, Newkirk K, Egorin MJ et al. (2003). Inhibition of glutathione synthesis reverses Bcl-2-mediated cisplatin resistance. Cancer Res 63: 312–318.
Seve P, Dumontet C . (2005). Chemoresistance in non-small cell lung cancer. Curr Med Chem Anticancer Agents 5: 73–88.
Shafir I, Feng W, Shoshan-Barmataz V . (1998). Voltage-dependent anion channel proteins in synaptosomes of the torpedo electric organ: immunolocalization, purification, and characterization. J Bioenerg Biomembr 30: 499–510.
Shimizu S, Ide T, Yanagida T, Tsujimoto Y . (2000a). Electrophysiological study of a novel large pore formed by Bax and the voltage-dependent anion channel that is permeable to cytochrome c. J Biol Chem 275: 12321–12325.
Shimizu S, Matsuoka Y, Shinohara Y, Yoneda Y, Tsujimoto Y . (2001). Essential role of voltage-dependent anion channel in various forms of apoptosis in mammalian cells. J Cell Biol 152: 237–250.
Shimizu S, Narita M, Tsujimoto Y . (1999). Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC. Nature 399: 483–487.
Shimizu S, Shinohara Y, Tsujimoto Y . (2000b). Bax and Bcl-xL independently regulate apoptotic changes of yeast mitochondria that require VDAC but not adenine nucleotide translocator. Oncogene 19: 4309–4318.
Shoshan-Barmatz V, Hadad N, Feng W, Shafir I, Orr I, Varsanyi M et al. (1996). VDAC/porin is present in sarcoplasmic reticulum from skeletal muscle. FEBS Lett 386: 205–210.
Siddik ZH . (2003). Cisplatin: mode of cytotoxic action and molecular basis of resistance. Oncogene 22: 7265–7279.
Tarze A, Deniaud A, Le Bras M, Maillier E, Molle D, Larochette N et al. (2007). GAPDH, a novel regulator of the pro-apoptotic mitochondrial membrane permeabilization. Oncogene 26: 2606–2620.
Thinnes FP, Schmid A, Benz R, Hilschmann N . (1990). Studies on human porin. III. Does the voltage-dependent anion channel ‘Porin 31HL’ form part of the chloride channel complex, which is observed in different cells and thought to be affected in cystic fibrosis? Biol Chem Hoppe Seyler 371: 1047–1050.
Wang GQ, Gastman BR, Wieckowski E, Goldstein LA, Gambotto A, Kim TH et al. (2001). A role for mitochondrial Bak in apoptotic response to anticancer drugs. J Biol Chem 276: 34307–34317.
Yagoda N, von Rechenberg M, Zaganjor E, Bauer AJ, Yang WS, Fridman DJ et al. (2007). RAS-RAF-MEK-dependent oxidative cell death involving voltage-dependent anion channels. Nature 447: 864–868.
Zamzami N, Kroemer G . (2004). Methods to measure membrane potential and permeability transition in the mitochondria during apoptosis. Methods Mol Biol 282: 103–115.
Zamzami N, Kroemer G . (2005). p53 in apoptosis control: an introduction. Biochem Biophys Res Commun 331: 685–687.
Zamzami N, Marchetti P, Castedo M, Decaudin D, Macho A, Hirsch T et al. (1995). Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death. J Exp Med 182: 367–377.
Zermati Y, Mouhamad S, Stergiou L, Besse B, Galluzzi L, Boehrer S et al. (2007). Non-apoptotic role for Apaf-1 in the DNA damage response. Mol Cell 28: 624–637.
Zhang K, Chew M, Yang EB, Wong KP, Mack P . (2001). Modulation of cisplatin cytotoxicity and cisplatin-induced DNA cross-links in HepG2 cells by regulation of glutathione-related mechanisms. Mol Pharmacol 59: 837–843.
This work has been supported by a special grant from Ligue National contre le cancer (équipe labellisée), as well as by grants from Agence Nationale de Recherche, Agence Nationale pour la Recherche sur le Sida, Cancéropôle Ile-de-France, Fondation pour la Recherche Médicale, Institut National du Cancer, European Commission (ApoSys, RIGHT, Active p53, Trans-Death, DeathTrain, ChemoRes) and Sidaction. NT is recipient of an FRM PhD fellowship. OK is recipient of an EMBO PhD fellowship. LS is funded by a DeathTrain PhD fellowship. EM is recipient of a DeathTrain PhD student fellowship.
About this article
Cite this article
Tajeddine, N., Galluzzi, L., Kepp, O. et al. Hierarchical involvement of Bak, VDAC1 and Bax in cisplatin-induced cell death. Oncogene 27, 4221–4232 (2008) doi:10.1038/onc.2008.63
- Bcl-2 family
- non-small cell lung cancer (NSCLC)
- reactive oxygen species (ROS)
A VDAC1-Derived N-Terminal Peptide Inhibits Mutant SOD1-VDAC1 Interactions and Toxicity in the SOD1 Model of ALS
Frontiers in Cellular Neuroscience (2019)
Store-Operated Calcium Entry Contributes to Cisplatin-Induced Cell Death in Non-Small Cell Lung Carcinoma
Cu(ii) phenanthroline–phenazine complexes dysregulate mitochondrial function and stimulate apoptosis
British Journal of Pharmacology (2019)
Cell Calcium (2018)