Apoptosis typically proceeds by either the intrinsic pathway, in which cytochrome c is released from mitochondria in a Bax/Bak- dependent manner, activating apoptosomes in the cytosol, or the extrinsic pathway, in which caspases are activated directly downstream of death receptors (reviewed by Salvesen & Riedl [11]). Often there is cross-talk between these two pathways. Because ABT binds mitochondrial proteins, ABT-induced apop- tosis is thought to occur through the intrinsic pathway, but we do not know exactly how this is done. The critical step in the intrinsic pathway of apoptosis is when cytochrome c is released from mitochondria because once cytochrome c is released into cytosol, it stimulates the formation of apoptosomes, the death executioner. For this reason, cyto- chrome c release is often called the point of no return [12]. Until several years ago, it was thought that calcium-activated mito- chondria permeability transition pores (mPTP) may be required for cytochrome c release during the intrinsic pathway of apoptosis. However, it was shown that tBid-induced cytochrome c release and subsequent apoptosis could take place in the absence of VDAC [13] and cyclophilin D [14,15], two essential components of the mPTP (reviewed in Yamaguchi & Perkins [16]). Instead, the prevailing dogma is that cytochrome c release occurs through mitochondrial outermembrane pores (MOMPs). Although MOMP is a well-established concept, its existence remains hypothetical because unlike nuclear pores or mPTP, there are no EM pictures of MOMPs. Likewise, MOMPs have not been isolated as a biological entity. Thus, it is not known whether Bak or Bax reside in MOMPs. The most detailed analysis of MOMP formation during apoptosis, came from studying lipid vesicles assembled with mitochondrial outermembrane proteins. In these studies, tBid-activated Bax was used to stimulate MOMP assembly on the vesicles [17], stimulating the release of proteins as large as 2000 kDa [18] through pores with diameters of 25–100 nm [19]. If MOMPs on mitochondria are about the same size, one would expect to see them using electron microscopy (EM), but they haven’t been imaged. Thus, the existence of MOMPs remains hypothetical. Nevertheless, from a mechanistic viewpoint, the existence of MOMPs comprised either wholly or in part by Bax and Bak makes sense. BH3-only proteins such as tBid, BimS are called ‘‘activators’’ because they directly interact with Bax or Bak, changing their conformation and inducing oligomerization, in other words ‘‘activating’’ them, leading to cytochrome c release and apoptosis. More recent findings, however, suggest another scenario in which apoptosis takes place in the absence of activator proteins. First, Huang and colleagues showed that Bak is normally sequestered by Mcl-1 and Bcl-xL, and Bak must be released from both before it can form oligomers [20]. The idea that inactivating some anti- apoptotic proteins without the help from some activator can induce apoptosis, is not without controversy [21,22]. We favor the idea because Huang and colleagues found that inactivating both Bcl-xL and Mcl-1 was enough to induce apoptosis in Bid and Bim knockout MEFs [23], and Hayashi and colleagues found the same in Bid and Bim deficient platelet cells [24]. Assuming that Bim and Bid are not involved in ABT-induced apoptosis, we can speculate reasons for varied sensitivities to ABT- 737. Since ABT-737 binds to Bcl-xL, inactivating it, but it does not bind to Mcl-1, if there are less Mcl-1 proteins present, then the efficacy of ABT-737 must increase. Indeed, Huang and colleagues found that by depleting Mcl-1 from HeLa cells with siRNA, they became sensitive to ABT-737 [25]. Many groups also reported inverse correlations between cancer cell sensitivity to ABT-737 and Mcl-1 expression levels in many cancer types (see Information S1). However, there are exceptions; HL-60 human promyerlocytic 2DG-ABT Combination Therapy leukemia cells express reasonably large amounts of Mcl-1, but they are nevertheless, sensitive to ABT-737 [26]. Thus how ABT-737 induces apoptosis in some cancer cells and not in others, remains poorly understood. We initiated this project in order to search for agents that can increase the efficacy of ABT. When we co-treated human tumor cell lines with ABT and drugs that interfere with cancer specific cellular metabolism, we found that 2DG pre-treatment vastly improved the efficacy of ABT without harming healthy cells. We show that 2DG-ABT induces apoptosis without altering Mcl-1 levels in ABT-resistant cells. When cancer cells resistant to ABT are pre-treated with 2DG, the protein levels of Mcl-1 remain the same, but Bak-Mcl-1 association is lost, sensitizing cells for ABT- induced apoptosis. How 2DG treatment of cancer cells disrupts Bak-Mcl-1 is not known. Thus, cancer cell sensitivity to ABT seems to be determined not only by the protein levels of Mcl-1, but also by how much Bak is sequestered by its association with Mcl-1. Results 2DG-ABT efficiently induces apoptosis of cancer cells Applied as a single agent, 30 nM of ABT-737 killed 90% of RS(4;11) non-Hodgkin’s B-cell lymphoma cells in 9 hours (Fig. 1a). However, a 1-hour pre-incubation of RS(4;11) cells with 5 to 10 mM 2DG in medium containing 10 mM glucose increased the efficiency of ABT dramatically, killing 90% of cells with just 3 nM ABT-737. 2DG alone did not cause cell death during the 10-hour incubation. When we tested for caspase activation, it was found that as little as 1 nM of ABT- 737 could activate caspases in 3 hours as long as the cells were pre-incubated with 2DG for 3 hours. No caspase activation beyond the background was observed in cells that were incubated with 2DG alone. Thus, it can be concluded that 2DG sensitizes RS(4;11) cells to ABT-induced apoptosis. We repeated the experiment using various concentrations of ABT- 263 and counted cells by trypan-blue dye to score for dead cells 24 hours after ABT addition (Fig. S1a). The result also showed a synergistic effect of 2DG and ABT. Next, we tested whether 2DG could sensitize ABT-resistant tumor cells to ABT-737 or ABT-263. Initial experiments were performed using HeLa cervical cancer cells. HeLa cells were pre- treated for 1 hour with 2DG before the addition of ABT-737. Nine hours after the addition of 1 mM of ABT-737, approximately 35% of HeLa cells that were pre-treated with 2DG, were Annexin V-positive (Fig. 1b). This rate of cell death was comparable to that seen after the treatment of HeLa cells with 1 mM staurosporine (STS), a well-known inducer of apoptosis in HeLa cells In another set of experiments, when HeLa cells were pre-treated for 3 hours with 10 mM 2DG in a medium containing 25 mM glucose, robust caspase activation was observed within 3 hours at concentrations of ABT-737 as low as 0.1 mM (Fig. 1c). Without 2DG pre- treatment, even 10 mM ABT-737 could not activate caspases during the same period. This experiment was also repeated using ABT-263 and scoring dead cells with trypan-blue dye incorpora- tion (Fig. S1b). Again, it showed a synergistic effect of ABT-263 and 2DG. ABT-737 and ABT-263 were compared side-by-side using three-hour 2DG-pretreatment of HeLa cells. ABT-263 was slightly better than ABT-737 in inducing apoptosis (Fig. S2a). Conditions for 2DG-ABT induced apoptosis Kinetic and dose-response studies with HeLa cells showed the following: (i) the presence of glucose in the medium is necessary for 2DG-ABT induced apoptosis (Fig. S2b), and the optimal molar PLoS ONE | www.plosone.org 2 September 2011 | Volume 6 | Issue 9 | e24102