2DG-ABT Combination Therapy Figure 3. 2DG-ABT induced apoptosis is a non-canonical mitochondria-dependent form of apoptosis. a, Wt, and Bax/Bak DKO MEFs were treated with 10 mM 2DG for 3 hours before the addition of 3 mM ABT-263 for 3 hours. 3 hours later, cells were harvested for caspase activity assays. (See also Fig. S5a). b, HT1080 cells were treated with or without 2DG for 3 hours. Then cells were treated with either 1 mM STS or anti-Fas (CD95) antibody at indicated concentrations (mg/ml). Cells were harvested 3 hours after, and assayed for caspase activity. Interestingly, 2DG seems to have interfered with Fas-induced apoptosis. In repeated experiments, we observed consistently lower rates of caspase activation in 2DG pre-treated cells, but the degrees of inhibitory activity varied from experiment to experiment. We are not sure of its cause. c, For the first 3 hours, HeLa cells were either treated with 10 mM 2DG or left untreated and then treated with 10 mM z-VAD, 1 mM ABT-737, both, or neither for 3 more hours. The cells were fixed and stained for DNA (red) and cytochrome c (green) and examined for loss of cytochrome c staining, as described in Methods. Bar indicates 100 mm. The representative images are shown in Fig. S3. This graph shows the percentage of cells with loss of cytochrome c staining. The combination treatment had higher rates of cells with released cytochrome c (p = 0.008 by unpaired t-test ). d, Cells treated as in c were fractionated and cytosolic fractions were analyzed by western blots. doi:10.1371/journal.pone.0024102.g003 2DG-ABT induces cytochrome c release through a caspase-dependent mechanism A critical step in the intrinsic pathway is the release of cytochrome c from mitochondria. When 1 mM of ABT-737 was applied to HeLa cells that had been pre-treated with 2DG for 3 hours, we observed the release of cytochrome c in over 30% of the cells within the next 3 hours but not in untreated cells or cells treated with either 2DG or ABT alone (Fig. 3c and Fig. S3). In canonical mitochondria-dependent forms of apoptosis such as staurosporine-, etoposide- or UV-induced cell death, the release of cytochrome c from the mitochondria occurs upstream of caspase activation and can proceed in the presence of caspase inhibitors [35,36]. By contrast, during death receptor-induced apoptosis, caspases activated by these receptors can also cleave the Bid protein, producing truncated Bid (tBid), with tBid then inducing Bak/Bax oligomerization and the formation of pores at the mitochondrial outer membranes through which cytochrome c escapes. Thus, a pan-caspase inhibitor such as z-VAD blocks death receptor-induced cytochrome c release from mitochondria. We found that 2DG-ABT induced cytochrome c release was also blocked by z-VAD when added synchronously with ABT (Fig. 3c & d, Fig. S2), yet we showed that the 2DG-ABT induced apoptosis is Bax/Bak-dependent. This conundrum could be explained by a Bid-dependent amplification loop in which a small amount of cytochrome c released by ABT could activate a cascade of caspases (such as caspases 9,3,6 and 8) in the cytosol, cleaving Bid to generate tBid, with tBid in turn activating Bak/Bax and causing further release of cytochrome c from the mitochondria [37,38]. The effects of Bid depletion on 2DG-ABT-induced apoptosis We explored whether the hypothesized Bid-based amplification loop plays a role in 2DG-ABT-induced apoptosis. First, we determined whether Bid is cleaved during 2DG-ABT induced apoptosis. In both HeLa and PPC-1 cells, 2DG pre-treatment followed by the addition of 1 mM ABT-263/737 induced cytochrome c release from the mitochondria in 3 hours. In both cell types, the release of cytochrome c was blocked by z-VAD-fmk. With the PPC-1 line, almost 100% of the cells released cytochrome c, causing nearly 100% apoptosis. With HeLa cells, the proportion of cells displaying cytochrome c release within the first three hours was approximately 30% (Fig. 3c). Truncated tBid was seen 3 hours after the addition of ABT in PPC-1 and HeLa cells (Fig. 4a, data not shown). tBid was absent in cells treated with 2DG alone. When z-VAD-fmk was added with ABT, the cleavage of Bid was blocked. To determine what effect, if any, Bid has on 2DG-ABT-induced apoptosis, we depleted the Bid protein by siRNA. 2DG-ABT- induced apoptosis was largely blocked in Bid-depleted PPC-1 cells. Bid depletion increased the percentages of viable cells by ,8-fold PLoS ONE | www.plosone.org 5 September 2011 | Volume 6 | Issue 9 | e24102