2DG-ABT Combination Therapy Figure 4. The Bid-based feedback loop is required for efficient 2DG-ABT induced apoptosis. a, Bid is activated during 2DG-ABT induced apoptosis. Western blots of PPC-1 cell lysates treated with 10 mM 2DG for 0–6 hours (left panel), with either 1 mM ABT-263 alone or ABT- 263+10 mM z-VAD for the last 3 hours (right panel), probed with the anti-tBid antibody (upper panels), anti-Opa1 antibody (middle panels), and anti- alpha-tubulins (lower panels). Note: the loss of Opa-1-L was used as a marker for remodeled mitochondrial cristae during cytochrome c release from the mitochondria [34]. b, The effects of Bid depletion on 2DG-AB-induced apoptosis. Insert: western blots of PPC-1 cell lysates, mock transfected (left lane) or transfected with Bid siRNA (right lane). Bid (+) and Bid (2) PPC-1 cells were treated with either 2DG, ABT-263 (1 mM), both, 10 mM ABT-263, or left untreated for 24 hours. The viable cells were counted and the increase/decrease over the input numbers were graphed. c, In PPC-1 cells either pre-treated with 2DG for 3 hours or without pre-treatment, then 1 mM ABT-263 or 1 mM STS was added for three hours, and cytosolic fractions were analyzed by western blots. In parallel experiments, Bid-depleted PPC-1 cells were treated with the combination of 2DG and ABT-263 or with STS and analyzed (designated as Bid kd, last 2 lanes). Cells were harvested 3 hour after ABT/STS treatment, and cytosolic fractions were analyzed with western blots. doi:10.1371/journal.pone.0024102.g004 (p,0.0077; unpaired t-test) (Fig. 4b). The amounts of released cytochrome c correlated with apoptotic rates (Fig. 4c). Thus, the Bid-dependent amplification loop is required for efficient 2DG- ABT induced apoptosis. Bid depletion had a similar impact on apoptosis induced by 10 mM ABT-263 but the impact was less on STS-induced apoptosis (data not shown and Fig. 4b & c). Lastly, Bid KO MEFs also displayed reduced sensitivity to 2DG-ABT treatments (Fig. S5a). We concluded that Bid plays a significant role in 2DG-ABT-induced apoptosis. Search for 2DG effectors Since 2DG-ABT combination-induced apoptosis is Bax/Bak- dependent, we examined the effects of 2DG on Bax and Bak during the three-hour pre-incubation. Some cells cultured under hypoxic conditions or in glucose-depleted media for extended periods, become sensitized to apoptosis [39,40,41]. Although no universal marker exists for cellular sensitivity to apoptosis, Bax is known to translocate to the mitochondria prior to apoptosis and necrosis in some cases [41]. Bax translocation also seems to be a distinct step, and not necessarily concurrent with its activation [42]. However, in HeLa cells treated with 2DG alone, Bax translocation was not induced for at least 16 hours (Fig. S4a). Thus, unlike in cells cultured in glucose-depleted media, Bax translocation does not take place during the 2DG pre-incubation period. Furthermore Bax knockout MEFs responded to the 2DG- ABT-combination treatment (Fig. S5a), suggesting that Bax may not play a critical role in priming of cancer cells by 2DG. Of course, in the absence of Bak, Bax is already be at mitochondria, playing the role of Bak, and Bax may be activated by a different mechanism when it is at mitochondria [43]. Because 2DG can affect glycosylation, 2DG-treatment might induce ER stress. Indeed, expression of the ER stress marker, Grp74, was observed in HeLa cells treated with 2DG for 2 to 4 hours (Fig. 5b and Fig. S4b). We hypothesized that if 2DG is acting through ER stress, we might be able to sensitize HeLa cells for ABT-induced apoptosis by pre-treating them with other ER stress inducers such as thapsigargin. However, this was not the case (Fig. S4c). Thus, 2DG appears to sensitize tumor cells to Bcl-2 antagonists through mechanisms that do not depend on glucose- depletion or ER stress. 2DG disrupts Bak-Mcl-1 association, priming cells for ABT-induced apoptosis It has been reported that culturing cells in glucose-depleted medium or in the presence of 2DG for extended time (24 hours or longer), could lower cellular ATP concentrations and prime various cells for apoptosis [39,40,44]. During this induced ATP insufficiency, translation of anti-apoptotic Mcl-1 is hampered, lowering protein levels of Mcl-1 and priming cells for apoptosis. We tested whether 2DG lowers Mcl-1 protein levels. As mentioned earlier, the decreases in cellular ATP concentration is very slight during the first 8 hours with 2DG (Fig. 5a). Furthermore, no significant changes were observed in the levels of Mcl-1 and several other anti- and pro-apoptotic proteins during the first eight hours of 2DG exposure (Fig. 5b). The levels of Mcl-1 also remained unchanged after ABT addition in HeLa, PPC-1 and other cells (Fig. 5c and Fig. S4d). Altogether, these experiments suggest that ABT-263/737 induces apoptosis without depleting the Mcl-1 protein. We cannot exclude the possibility that more subtle changes such as phosphorylation or other post-translational modifications of Mcl-1 and other anti-apoptotic proteins occurred during 2DG-incubation, which may be responsible for the sensitizing effect of 2DG. Indeed, we found that even though Mcl-1 protein levels seem unchanged, Mcl-1-Bak association was lost during 2DG pre-incubation, and remained absent during the subsequent incubation with 2DG and ABT (Fig. 5d). Since both PLoS ONE | www.plosone.org 6 September 2011 | Volume 6 | Issue 9 | e24102