MCF-7 and 36.7% for PPC-1). The effects of 2DG-ABT combination far exceeded the additive effects of 2DG and ABT in all three cell lines (HeLa expected viable cells 59%, observed 25%, MCF7 expected viable cells 115%, observed 3.8%, and PPC1 expected viable cells 49.7%, observed 3.9%). The Clonogenic Cell Survival assay also showed that over 95% of PPC-1 cells were eliminated by just one treatment while single agent applications of ABT or 2DG were largely ineffective (Fig. 1e). Given that p53 is inactive in some of the highly responsive tumor lines including PPC-1 [27,28,29], we conclude that the combina- tion-induced apoptosis is p53-independent. The possible reasons for varied responses will be discussed later. In order to understand exactly what molecular events take place when ABT is added to 2DG treated cells, we decided first to investigate what type of apoptosis is being induced by the combination of 2DG and ABT. Combination treatment with 2DG and ABT induces apoptosis through the intrinsic pathway It has been reported that the application of 10 nM ABT-737 for just 2 hours causes the mitochondrial outer membrane to rupture in chronic lymphocytic leukemia (CLL) cells [30]. This is not a normal phenomenon in apoptosis. Cytochrome c is usually released through mitochondrial outermembrane pores in orderly fashion [31]. The destruction of mitochondria takes place long after cytochrome c release by the activities of fully activated cell death machineries. When we examined HeLa cells treated with the 2DG-ABT combination using EM, rupture of mitochondrial outer membranes was not detected (Fig. 2a). In 2DG-ABT Combination Therapy ABT-treated cells, however, slight shortening of mitochondria were observed (Fig. 2b&c); from 0.717+/20.05 mm for untreat- ed to 0.530+/20.05 mm for ABT only, 0.553+/20.06 mm for 2DG+ABT, and slight lengthening of mitochondria in 2DG- treated cells at 0.844+/20.05 mm. We speculate that since Bcl- xL and Bcl-w are known to be involved in mitochondrial fusion/ fission [32], ABT may have inhibited their activities. As for the lengthening of mitochondria under 2DG, it may have created increased demand for mitochondrial respiration, and to meet that burden, mitochondria may have become longer. Even though 2DG and ABT had opposite effects on the length of mitochondria, they both influenced the physiologies of mito- chondria. Even though some speculates that mitochondria fusion/fission machinery may also play a role in apoptosis, the idea remains controversial [33]. To determine if 2DG-ABT-induced apoptosis is through the intrinsic pathway, we used wild-type (wt) mouse embryo fibroblasts (MEFs) and Bax/Bak double knockout (DKO) MEFs. These DKO cells are lacking the intrinsic pathway. In wild-type MEFs pre-treated with 2DG, caspase activation was induced within 3 hours of ABT addition (Fig. 3a). However, there was no rapid caspase activation in Bak/Bax DKO MEFs. Therefore, we concluded that 2DG-ABT-induced apoptosis occurs by the Bak/ Bax-dependent intrinsic pathway (see also Fig. S5a). To test whether 2DG also sensitizes tumor cells to the extrinsic pathway of apoptosis, we used HT1080 cells, in which there is no cross talk between the intrinsic and extrinsic pathways [34]. Pre- treatment of HT1080 cells with 2DG for 3 hours did not enhance Fas-induced apoptosis but lowered caspase activation (Fig. 3b). Thus, 2DG did not enhance the extrinsic pathway. Figure 2. Combination of 2DG and ABT-263 does not cause mitochondrial outer membrane rupture. HeLa cells treated with either 10 mM 2DG for 6 hours, 1 mM ABT-263 for 3 hours, or 2DG for 3 hours before adding ABT-263 for an additional 3 hours, or left untreated. a, Cells were fixed for electron microscopy (see Method). Three cells from each treatment group were examined closely for the length of mitochondria. We did not observe a single instance of ruptured mitochondrial outermembranes. b, From each cell, a minimum of 12 mitochondria were randomly chosen for measurement. c, Average lengths of mitochondria became shorter with treatments containing ABT-263 (mean +/2 std dev ). Since ABT- 263/737 binds to Bcl-2 family members, which are known to be involved in mitochondrial fusion/fission, ABT may have altered the balance of mitochondrial fusion/fission to the fission side (p = 0.0001, 95% confidence interval of this difference: from 14.5407 to 37.2513; unpaired t-test). doi:10.1371/journal.pone.0024102.g002 PLoS ONE | www.plosone.org 4 September 2011 | Volume 6 | Issue 9 | e24102