Vitamin E Analogues Inhibit Angiogenesis by Selective Induction of Apoptosis in Proliferating Endothelial Cells: The Role of Oxidative Stress

Under physiologic conditions, endothelial cells are suspended in G₀ and proliferate only in response to endothelial injury. In growing tumors, malignant cells secrete a cocktail of mitogens, such as vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2), which diffuse from the tumor cell and interact with cognate receptors on preexisting endothelial cells, triggering their proliferation (1). A prerequisite for tumor progression beyond a small initial carcinoma is formation of new blood vessels, a process known as angiogenesis. The stage at which tumor growth stimulates the “switch-to-the-angiogenic-phenotype” is not known. It has been assumed that this occurs at the stage when the diffusion of oxygen and nutrients across the cells is hampered so that a hypoxic core within the growing tumor is formed (2, 3). Because angiogenesis is essential for tumor progression, its inhibition makes a plausible anticancer strategy (4–6). Several approaches have been used to inhibit tumor angiogenesis and prevent tumor progression, including gene therapy, immunotherapy, or chemotherapy (7–9).

A novel approach to inhibit angiogenesis is selective induction of apoptosis in endothelial cells (10). Hogg and colleagues (11) recently observed that targeting angiogenic endothelial cells by an arsenic derivative efficiently suppressed their proliferation and drove them into apoptosis. This translated to suppression of tumor progression and regression of tissue vascularization (10, 11). The authors showed that the drug interfered with the function of the adenine nucleotide translocator within the mitochondrial inner membrane. This is consistent with the notion that mitochondria are emerging as novel targets for cancer therapy (11–13).

We have defined a class of compounds, mitocans, which suppresses cancer by inducing apoptosis via targeting mitochondria (13). These drugs include a number of cytotoxic agents, such as the above-mentioned arsenic derivative, as well as a group of vitamin E analogues, epitomized by the redox-silent α-tocopheryl succinate (α-TOS), a selective inducer of apoptosis in cancer cells and a potent anticancer agent (14–20). α-TOS and its analogues have been shown to efficiently induce apoptosis by targeting mitochondria, a process involving generation of reactive oxygen species (ROS; refs. 16–19), which also suppresses cancer cell proliferation (18). Recently, we have shown that α-TOS causes ROS generation in cancer cells by displacing ubiquinone in its binding sites on complex II (13). This activity of vitamin E analogues translates to cancer suppression in a variety of preclinical models (15, 20–23).

We observed earlier that α-TOS specifically caused apoptosis in proliferating endothelial cells, being nontoxic to arrested endothelial cells, although the mechanism was not resolved (24). Because this finding is suggestive of selective toxicity of α-TOS for angiogenic endothelial cells, we explored this phenomenon in more detail. Here we present data on the mechanism of greater susceptibility of angiogenic endothelial cells to α-TOS as well as to the ether analogue and link this efficacy to inhibition of tumor angiogenesis in vivo in a preclinical model of breast cancer using mice with spontaneous breast carcinomas.

Cell culture and treatment. The endothelial-like EAhy926 cells (25) were maintained in DMEM supplemented with 100 μmol/L hypoxanthine, 0.4 μmol/L aminopterin, and 16 μmol/L thymidine. These cells retain properties of endothelial cells, including expression of factor VIII (25), tube-forming activity, and the propensity to persist in confluent cultures (7). EAhy926 cells deficient in mtDNA (ρ⁰ phenotype) were prepared as detailed elsewhere (16). Acquisition of the ρ⁰ phenotype was confirmed by lack of expression of cytochrome c oxidase subunit II but not subunit IV (data not shown).

The cells were treated under conditions of high, medium, or nil proliferation (40–50%, ∼70%, and 100% confluency, respectively). The drugs used (Supplementary Scheme 1) were α-tocopherol (α-TOH), the ester analogue α-TOS (both Sigma), and the orally applicable ether analogue 2,5,7,8-tetramethyl-2R-(4R,8R,12-trimethyltridecyl)-chroman-6-yloxyacetic acid (α-tocopheryloxyacetic acid, α-TEA; ref. 22).

Assessment of apoptosis and mitochondrial potential. Apoptosis was estimated routinely by the Annexin V binding method based on phosphatidyl serine externalization at the relatively early phases of programmed cell death, essentially as described elsewhere (16).

Mitochondrial inner transmembrane potential (ΔΨ_m) was estimated using the polychromatic probe 5,5′,6,6‘-tetrachloro-1,1’,3,3′-tetraethylbenz-imidoazolyl-carbocyanino iodide (JC-1; Molecular Probes) as detailed elsewhere (16).

Evaluation of ROS accumulation, cell proliferation, and cell cycle distribution. Cellular ROS were detected indirectly by flow cytometry using dihydroethidinium (DHE; Molecular Probes; ref. 18) and directly by electron paramagnetic resonance (EPR) spectroscopy (16), after treatment of cells with α-TOS or α-TEA. In some experiments, the cells were pretreated for 1 h with 2 μmol/L mitochondrially targeted coenzyme Q (MitoQ; ref. 26) or coincubated with superoxide dismutase [polyethylene glycol-superoxide dismutase (PEG-SOD); Sigma S4636] at 750 units/mL.

Cell proliferation was determined using an ELISA colorimetric kit (Roche) to determine the number of cells in S phase of the cell cycle, based on DNA incorporation of 5-bromo-2-deoxyuridine (BrdUrd) using the manufacturer's protocol. For cell cycle analysis, cells were plated in 24-well plates so that they reached ∼50%, 70%, and 100% confluency after 24-h recuperation. Cells were then harvested and resuspended in buffer containing sodium citrate (1%), Triton X-100 (0.1%), RNase A (0.05 μg/mL), and propidium iodide at 5 μg/mL, incubated in the dark for 30 min at 4°C and analyzed by flow cytometry.

Assessment of complex II (succinate dehydrogenase) activity. Solutions of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma) were prepared before use by dissolving 2.5 mg/mL of MTT in PBS containing 20 mmol/L succinic acid (pH 7.4) as the sole substrate. Stock solutions of thenoyltrifluoroacetone (TTFA; Sigma) in DMSO, and α-TOS and MitoQ in ethanol were prepared. The drugs were tested on EAhy926 cells in the exponential growth phase in 96-well microtiter plates using four to eight replicate wells per drug dilution assayed. To assess the ability of MitoQ to restore MTT reduction, cells were preincubated for 3 h with 3 μmol/L MitoQ. Cells were allowed to reduce MTT for 2 h at 37°C, and absorbance was measured at 570 nm.

Wound-healing and tube-forming activity assessment. Endothelial cells were seeded and cultured to complete confluence. The central region of a monolayer of cells was “wounded” by scraping away cells, generating a denuded 0.5-mm wide stripe. Regrowth of cells was assessed by following the kinetics of filling the gap, visualized under a microscope equipped with a grid in the eyepiece. The healing rate was expressed in μm/h.

For the tube-forming activity of endothelial cells, formation of capillary-like structures in a three-dimensional setting was assessed, essentially as described elsewhere (7). In brief, 300 μL of cold Matrigel (BD Biosciences) per well were transferred with a cold tip using a 24-well plate. Matrigel was overlaid with a suspension of EAhy926 cells, so that a total of 200 μL of complete medium with 5 × 10⁵ cells were added to each well. After 6 h in the incubator, the polygonal structures, made by a network of EAhy926 capillaries was established. The cells were treated and tube-forming activity was estimated by counting the number of complete capillaries connecting individual points of the polygonal structures in a light microscope 24 h after transferring the cells to Matrigel. Three fields in the central area were chosen randomly in every well. The number of capillaries in control cultures was considered 100%.

Cell transfections. EAhy926 cells were transfected with a plasmid harboring the Bcl-x_L-EGFP gene (27) as described elsewhere (16). The cells were maintained in the selection medium for at least five passages, after which they were assessed for expression of the protein by inspection for green fluorescence using a fluorescence microscope or by Western blotting, revealing >95% transfected cells (data not shown).

VE-cadherin expression. Western blotting was performed as reported earlier (16) using anti–VE-cadherin IgG (Santa Cruz). For immunofluorescence microscopy, EAhy926 cells at ∼40% or 100% confluence were incubated with anti–VE-cadherin IgG, followed by secondary FITC-conjugated IgG and mounting with 4′,6-diamidino-2-phenylindole–containing Vectashield. The cells were inspected in the Leica DMIRE2 fluorescence microscope fitted with deconvolution software.

In vivo experiments. A colony of transgenic FVB/N202 c-neu mice carrying the rat HER-2/neu proto-oncogene driven by the mouse mammary tumor virus promoter on the H-2^q FVB/N background (28) was established at the Griffith University Animal Facility and maintained under strict inbreeding conditions. Approximately 70% of the female mice develop spontaneous mammary carcinomas with a mean latency of ∼7 months. Formation of tumors was monitored by ultrasound imaging using the Vevo770 instrument (VisualSonics) equipped with the VisualSonics RMV704 scan head (mean frequency, 60 MHz; resolution, 40 μm), allowing noninvasive scanning of tumor tissue and tumor volume quantification. Scanning was performed on mice anesthetized using isoflurane with continuous monitoring of the heart rate as reported elsewhere (23). As soon as tumors were detected, the mice were treated by i.p. administration every 3 days of α-TOS dissolved in corn oil/4% ethanol (15 μmol α-TOS per injection per mouse). The kinetics of tumor growth was followed by ultrasound imaging every 3rd day.

The Vevo770 ultrasound imaging device is equipped with the Power Doppler function, which makes it possible to follow circulation in blood vessels. This was applied to assess the extent of angiogenesis in the FVB/N c-neu mice carcinomas. The control and treated mice were assessed by ultrasound imaging using the same respiratory gating, and the volume of blood vessels within tumors was expressed as percentage of vascularization of individual tumors.

We first exposed EAhy926 cells at different levels of proliferation to α-TOS and α-TEA. As shown in Fig. 1A and B, endothelial cells at the lowest confluency were most susceptible to apoptosis, whereas arrested endothelial cells showed much greater resistance. Proliferating endothelial cells neither accumulated ROS nor underwent apoptosis when exposed to the redox-active α-TOH (Supplementary Table S1). Both α-TOS and α-TEA also suppressed proliferation of endothelial cells (Fig. 1C and D). The differences in proliferation of endothelial cells were assessed by cell cycle distribution (Supplementary Fig. S1A) revealing only a small S phase subpopulation in the arrested cells, whereas a high number of cells in S phase were found for the 50% proliferating cultures. Supplementary Fig. S1B documents high levels of expression in the confluent endothelial cells of the cell surface protein VE-cadherin involved in cell-cell contact of endothelial cells.

The fact that angiogenic endothelial cells are susceptible to the mitocans α-TOS and α-TEA raises the question of the role of mitochondria in apoptosis induced by vitamin E analogues, with α-TOS known to cause accumulation of ROS in cancer cells. We therefore assessed endothelial cells under conditions of proliferation and arrest for ROS generation as a response to α-TOS or α-TEA. Indeed, proliferating but not arrested endothelial cells responded to the challenge by early accumulation of high levels of ROS, as assessed by flow cytometry (Fig. 2A and B) and EPR spectroscopy (Supplementary Fig. S2). MitoQ and SOD suppressed ROS generation and apoptosis induction by the vitamin E analogues in angiogenic endothelial cells (Fig. 2C and D).

Next, the role of mitochondria in apoptosis of endothelial cells exposed to α-TOS and α-TEA was investigated. Supplementary Fig. S3A shows that the majority (>97%) of control cells had high ΔΨ_m, whereas exposing proliferating endothelial cells to either agent resulted in early (within 24 h) dissipation of ΔΨ_m, occurring in 15% to 20% of the population. Mitochondrial destabilization is generally followed by caspase activation. Supplementary Fig. S3B reveals that caspases were activated in apoptosis induced by the two vitamin E analogues because the pan-caspase inhibitor z-VAD-fmk efficiently inhibited apoptosis in proliferating endothelial cells. We also show here that overexpression in endothelial cells of the antiapoptotic protein Bcl-x_L protected the cells from α-TOS– and α-TEA–induced apoptosis (Supplementary Fig. S3C).

We have shown that vitamin E analogues cause generation of ROS with ensuing induction of apoptosis in cancer cells by interfering with coenzyme Q binding in the mitochondrial complex II. We therefore tested the possibility that α-TOS triggers ROS generation in endothelial cells by interfering with the complex II SDH activity. Table 1 documents that α-TOS and α-TEA inhibited SDH activity similarly, as shown for TTFA, a compound known to displace coenzyme Q in complex II. Inhibition of SDH activity by α-TOS, α-TEA, or TTFA was rescued by MitoQ, which is known to interact with complex II. These data strongly suggest that α-TOS and α-TEA act by interfering with ubiquinone binding of complex II.

We next asked the question whether the propensity of angiogenic endothelial cells to undergo apoptosis, when challenged with vitamin E analogues, translates to inhibition of angiogenesis. We used two experimental approaches to assess angiogenesis in vitro, based on the wound-healing and the tube-forming activity assays. In the wound-healing assay, cells were grown to complete confluence and then a central strip of cells was removed, after which regrowth into the cleared space was evaluated (Supplementary Fig. S4). Figure 3A and B show that the healing rate was ∼20 μm/h for control cells, ∼8 and ∼3 μm/h for cells exposed to 25 and 37.5 μmol/L α-TOS, respectively. No regrowth was observed at 50 μmol/L α-TOS. A dose-dependent inhibition of wound healing was also observed for α-TEA (Fig. 3C and D). Importantly, inhibition of wound healing by the vitamin E analogues was associated with extensive apoptosis induction in the wound zone of the endothelium (Fig. 3B and D). Next, a dose-dependent inhibitory effect on endothelial cell tube-forming activity was observed for α-TOS and α-TEA (Fig. 4). The vitamin E analogues suppressed the tube-forming activity of the endothelial cells by way of apoptosis induction, as found by assessing the cells for apoptosis after their treatment and analysis after retrieval from the Matrigel cultures.

The role of mitochondria in susceptibility of angiogenic endothelial cells to α-TOS was confirmed in experiments in which mtDNA-deficient endothelial cells were exposed to the vitamin E analogue and assessed for ROS accumulation and apoptosis induction by flow cytometry. As documented in Fig. 5A, dividing populations of ρ⁰ cells, unlike their parental counterparts, showed reduced levels of ROS. The subconfluent ρ⁰ endothelial cells also failed to undergo efficient apoptosis when exposed to α-TOS (Fig. 5B). We were interested whether endothelial cells deficient in mtDNA maintain their wound-healing and tube-forming activity, and, if so, how this is affected by α-TOS. Figure 5C and D reveal that endothelial cells deficient in mtDNA are capable of wound healing and tube formation, although at a slower rate than the parental cells. Importantly, the ρ⁰ cells maintained the two features of in vitro angiogenesis (wound healing and tube formation) even in the presence of α-TOS. These results strongly suggest the importance of normal mitochondrial function for susceptibility of angiogenic endothelial cells to vitamin E analogues.

We next tested whether the inhibitory activity of α-TOS on angiogenesis in vitro translates to an in vivo situation. Hence, we used the transgenic FVB/N c-neu mice that form spontaneous breast ductal carcinomas at the age of ∼7 months due to mammary tissue–specific overexpression of the receptor tyrosine kinase erbB2 (HER2). In particular, we studied the effect of i.p. administered α-TOS on the kinetics of the tumor growth and vascularization of the carcinomas. This was performed using the Power Doppler function of the ultrasound instrument that allows for noninvasive and very precise quantification of the tumor volume and percentage tumor vascularization. Figure 6 documents that α-TOS suppressed growth of the tumor, decreasing the volume of the carcinomas by ∼30%. Importantly, the extent of tumor vascularization increased with time in the control mice, whereas it decreased significantly when the animals were treated with the vitamin E analogue. These data link the antitumor efficacy of α-TOS with its propensity to act as an antiangiogenic agent in vivo.

Our earlier data showed that proliferating endothelial cells, unlike their confluent counterparts, were susceptible to apoptosis induction when exposed to α-TOS (24), but the molecular mechanism had not been resolved. Recent data by Don et al. (10) are consistent with our findings because they showed that proliferating endothelial cells were susceptible to the mitocan arsenide, whereas growth arrested cells were resistant to the drug, although the reasons for protection of the growth-arrested endothelial cells were not identified.

Our major interest was to understand the molecular mechanism of differential sensitivity of endothelial cells to the clinically interesting vitamin E analogues in relation to their proliferative status. Because in vivo endothelial cells in tumors show a high proliferative rate, whereas endothelial cells of normal blood vessels feature a very long half-life before dividing, it can be expected that vitamin E analogues will kill angiogenic endothelial cells in tumors but not endothelial cells of the normal vasculature. Similarly, as shown by Don et al. (10), we also found that proliferating endothelial cells responded to exposure to apoptogenic vitamin E analogues by accumulation of relatively high levels of ROS. In the case of agents such as α-TOS, the mechanism of generation of ROS in cancer cells is most likely due to displacement by the vitamin E analogue of ubiquinone in one or both of its binding pockets within the mitochondrial complex II (13). It is therefore probable that the absence of the electron acceptor, ubiquinone, will result in recombination of electrons with molecular oxygen to form superoxide. It has been shown that the absence of complex II [more specifically, the cytochrome B-large, CybL, or the SDH subunit C (SDHC)] due to mutations in SDHC results in lower levels of ROS generation when the cells are exposed to α-TOS or several unrelated inducers of apoptosis, and this is reflected by low levels of apoptosis induction (13, 29). We also assessed the possible role of complex II in apoptosis induction by vitamin E analogues in endothelial cells and found that α-TOS and α-TEA inhibited SDH activity, and that it was restored in the presence of the complex II–interacting MitoQ (30), similarly, as also observed for TTFA, an agent that displaces coenzyme Q in complex II (31). Our data suggest a general mechanism of ROS-mediated induction of apoptosis by vitamin E analogues in both cancer cells and proliferating endothelial cells, which is based on displacement of coenzyme Q in its binding pocket(s) in the mitochondrial complex II, which results in generation of superoxide. We propose that this site is also likely to be responsible for the antiangiogenic effect of vitamin E analogues by way of binding of vitamin E analogues that displace the natural electron acceptor, resulting in generation of apoptosis-inducing ROS levels.

Not only does the selectivity of vitamin E analogues as agents inducing apoptosis in angiogenic endothelial cells have significant clinical relevance in helping to arrest tumor progression, but also the molecular mechanism as defined here is of major interest and importance. Don et al. (10) reported on selective killing of proliferating endothelial cells by an arsenic derivative. They showed that the angiogenic cells accumulated high levels of ROS, unlike their arrested counterparts. We also observed this in angiogenic endothelial cells exposed to α-TOS and α-TEA. These differences in accumulation of ROS seem central to the susceptibility or resistance of the endothelial cells to apoptosis because eliminating ROS accumulation, such as by coexposure to antioxidants, also suppresses the extent of apoptosis. Numerous compounds have been reported to inhibit angiogenesis by way of killing proliferating endothelial cells or causing their cytostasis. These include anticancer drugs paclitaxel (32, 33) or vinblastine (34), the histone deacetylase inhibitor LBH589 (35), the vascular-targeting compound ZD6126 (36), or the proteasome inhibitor bortezomib (37). Although these agents have been shown to reduce tumors in experimental animals, neither of them has been reported for selective toxicity toward proliferating while being nontoxic to arrested endothelial cells. To the best of our knowledge, this intriguing paradigm has only been shown for the mitocans arsenide (10) and vitamin E analogues (reported here). The selectivity of apoptosis induction in angiogenic endothelial cells by vitamin E analogues is particularly interesting because vitamin E analogues have shown promise as anticancer agents in several animal models. This is further fueled by our clinical outcome from a mesothelioma patient who has been treated with transdermally applied α-TOS. The data reveal a significant clinical benefit with α-TOS therapy, causing a reduction in tumor volume and improved well-being of our subject who previously was suffering from a lethal neoplastic pathology (38, 39).

The molecular mechanism for the very low levels of ROS accumulation in arrested endothelial cells exposed to vitamin E analogues is not known at present. We can suggest at least two possibilities that could explain this. First, the arrested cells may respond to the stress imposed by vitamin E analogues by generating lower levels of ROS due to a difference in the cellular systems that cause formation of radicals. The other, probably more plausible possibility, is due to potential up-regulation of the antioxidant systems in the resistant, arrested endothelial cells. One enzyme that may be up-regulated is manganese superoxide dismutase (MnSOD). This idea is consistent with reports showing that cells deficient in mtDNA are resistant to apoptosis and show increased expression of MnSOD (40–42). We have also found elevated expression of MnSOD in arrested endothelial cells,⁷

Because killing of angiogenic endothelial cells by α-TOS and α-TEA suggests that vitamin E analogues may possess antiangiogenic activity, thereby suppressing tumor progression, we studied the effect of the two agents on angiogenesis in vitro. In these experiments, we used the immortalized EAhy926 cells because these cells, unlike the primary endothelial cells with limited life span (usually four to five cell cycle transitions), can be used for up to 100 doublings, while preserving properties of primary endothelial cells including expression of von Willebrand factor or P selectin, as well as formation of tubes in a three-dimensional setting and persistent arrest (7, 25). Our results clearly document the efficacy of α-TOS and α-TEA in inhibiting angiogenesis in vitro, as assessed by both the wound-healing and tube-forming approach. Importantly, inhibition of angiogenesis in vitro was associated with the induction of apoptosis in the proliferating endothelial cells, which suggests a link between the efficacy of vitamin E analogues to induce apoptosis in proliferating endothelial cells with their antiangiogenic activity.

We also assessed endothelial cells deficient in mtDNA for their susceptibility to α-TOS because ρ⁰ cancer cells are resistant to apoptosis (16, 19, 40, 45) and feature an impaired mitochondrial electron redox chain, a major source of ROS generation (46, 47). We found that ρ⁰ endothelial cells were relatively resistant to α-TOS–induced apoptosis. We also observed that ρ⁰ endothelial cells retained the propensity of normal endothelial cells to undergo wound healing after injury as well as tube forming in Matrigel, and that neither the wound-healing nor the tube-forming activities were impaired by α-TOS. These are important findings that further support the importance of fully functional mitochondria to make angiogenic endothelial cells susceptible to apoptosis induction by vitamin E analogues.

Lastly, we evaluated the effect of α-TOS on angiogenesis in vivo. To do this, we used the transgenic FVB/N c-neu mice with spontaneous development of breast carcinomas (28). We have observed recently that analogues of vitamin E coupled to peptides binding the HER2 receptor suppressed progression of these tumors (23). The Vevo770 ultrasound device allows us to visualize and quantify blood vessels so that noninvasive assessment of the kinetics of angiogenesis in tumors treated with a potential inhibitor of angiogenesis can be assessed directly in real time in vivo. We found that α-TOS significantly suppressed tumor progression, consistent with our recent report (23), and that this was accompanied by inhibition of angiogenesis. Ultrasound imaging revealed that the percentage of the tumor mass occupied by blood vessels increased in control animals by as much as ∼8-fold over the 3 weeks of the experiment in the control animals, whereas it was suppressed by ∼50% in mice treated with α-TOS. These data unequivocally document the antiangiogenic property of α-TOS. This result is of clinical relevance because HER2-positive breast cancer is resistant to therapy (48) and because breast cancer accounts for >25% of female cancer patients in the USA, with almost 200,000 new cases and >40,000 deaths predicted for 2007 (49).

Our in vitro results suggest that apoptosis is a plausible factor by which vitamin E analogues inhibit angiogenesis. However, we cannot rule out other possibilities that may contribute to the overall antiangiogenic activity of the drugs. These include, in particular, the effect of α-TOS on expression of genes by which tumor cells promote angiogenesis, such as VEGF, as shown for breast cancer cells (50), and FGF2, as reported for mesothelioma cells (18, 51). Notwithstanding, apoptosis seems to be an important mechanism by which vitamin E analogues inhibit angiogenesis, thereby inhibiting tumor progression, which is clinically intriguing.

Grant support: Australian Research Council (J. Neuzil and P.K. Witting); the Queensland Cancer Fund; the National Breast Cancer Foundation; the Grant Agency of the Academy of Sciences of the Czech Republic KAN200520703, IAA5005220602, and IAA5005200602 (J. Neuzil); Concept Grant AV0Z50520514 awarded by the Academy of Sciences of the Czech Republic; and Grant from the Ministry of Agriculture of the Czech Republic (grant No. MZE 0002716201). J. Eliasson was a visiting student at the Apoptosis Research Group (Griffith University) supported by a scholarship from the University of Linkoping, Linkoping, Sweden; M. Gold was a visiting student at the Apoptosis Research Group (Griffith University) supported by the Bravo! Award from the University of Arizona, Tucson, AZ; L. Prochazka was a visiting student at and in part supported by the Apoptosis Research Group (Griffith University).

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We thank R. Youle (NIH, Bethesda, MD) for providing the Bcl-xL plasmid and R. Smith (Otago University, Duneedin, New Zealand) for the MitoQ.