2DG-ABT Combination Therapy Figure 5. 2DG disrupts Bak-Mcl-1 association, priming cancer cells without lowering protein levels of Mcl-1. a, The effect of 2DG on cellular ATP levels. The amounts of ATP were measured during HeLa cell culture with 10 mM 2DG in the presence of 25 mM glucose in DMEM supplemented with 10% serum. b, Protein profiles during 2DG incubation of HeLa cells. Samples were taken from HeLa cells treated as in a, and various protein contents of pro- and anti-apoptotic proteins were analyzed by Western blots. In this experiment, we see increases in Bcl-xL for the first hour. When we repeated the experiment twice, Bcl-xL levels remained the same. c, Mcl-1 levels remained unchanged during 2DG-ABT- induced apoptosis. PPC-1 cells were treated with 10 mM 2DG for 6 hours, 1 mM ABT-263 for 3 hours, or 10 mM 2DG for 6 hours of which the last 3 hours were incubated with 1 mM ABT-263. d, Bak and Mcl-1 do not co-precipitate in 2DG treated cells. PPC-1 cells were treated as in c. Bak and Mcl-1 were immunoprecipitated by specific antibodies conjugated to Protein G-Sepharose and the co-precipitated proteins were analyzed by Western blots. Left Upper Panels, Bak co-precipitates, Right Upper Panels, Mcl-1 co-precipitates, and Left Lower Panels, Protein G-Sepharose precipitates. For technical reasons, we could not immunoprecipitate Bcl-xL and analyze its content. doi:10.1371/journal.pone.0024102.g005 Mcl-1 and Bcl-xL can sequester Bak from forming oligomers, loss of Mcl-1-Bak association during 2DG incubation can sensitize cells because ABT can now effectively bind Bcl-xL and free Bak. Earlier we noted that HL-60 is sensitive to ABT-737 despite having large amounts of Mcl-1 expressed. It was also shown that anti-Bak antibody failed to co-precipitate Mcl-1 even though it can co- precipitate Bcl-2 [26]. Thus the reason for its sensitivity might be the absence of Bak-Mcl-1 complex. We do not know why Bak does not bind to Mcl-1 in HL-60 cells. Currently we are investigating the possible molecular changes to Bcl-xL and Mcl-1 disruptive to their association. Taken together, we propose the following model for 2DG-ABT induced apoptosis in cancer cells (Fig. 6): 2DG induces changes in Bak or Mcl-1 so that they can no longer associate. ABT binds to the BH3 pocket of Bcl-xL, breaking its hold on Bak. Released Bak forms oligomers, releasing a small amount of cytochrome c. Released cytochrome c now activates caspase cascades, generating tBid which can induce full-scale outer membrane pore formation on mitochondria. A large-scale release of cytochrome c triggers full-scale apoptosome formation and cell death. This model is supported by our findings. 2DG activates PI3K and generates a pro-survival signal It has been reported that 2DG can activate multiple signal transduction cascades, activating well-known kinases such as PI3K, MAPK and IKK that phosphorylate over 30 proteins, some of which are known to be involved in apoptosis [5,6]. Thus it is possible that one of these signals is responsible for the loss of Bak-Mcl-1 association. However, these reports caution that 2DG can also activate a pro-survival pathway via PI3K and the phosphorylation of AKT; thus, adding 2DG could hinder, rather than facilitate apoptosis, depending on the cell type. Indeed, we have consistently observed lower rates of STS-induced apoptosis when cells were pre-incubated with 2DG (Fig. 3a and Fig. S2c). We thus tested whether pro-survival signals are generated in HeLa cells during 2DG-ABT induced apoptosis by using LY294002, a PI3K inhibitor, to ascertain if we could block this signal. With the addition of LY294002, an almost three-fold increase in caspase PLoS ONE | www.plosone.org 7 September 2011 | Volume 6 | Issue 9 | e24102