2DG-ABT Combination Therapy Figure 6. Model of 2DG-ABT Induced Apoptosis. a, 2DG is specifically taken up by cancer cells through a glucose transporter. Once inside, it is phosphorylated and becomes a hexokinase inhibitor. At the same time, it activates several signal transduction cascades. One of them is IGFR-PI3K- mTOR-AKT pro-survival pathway. Through another cascade activation, Bak-Mcl-1 association is lost. When ABT is added 3 hours later, it can bind to Bcl-xL and disrupts its association with Bak. b, Freed Bak oligomerizes, releasing a small amount of cytochrome c into the cytosol. It activates cascades of caspases 9-3-6-8 and caspase 8 cleaves Bid, generating BH3 only tBid protein. Since tBid can dissociate Bak-Mcl-1 and Bak-Bcl-xL association, it induces mitochondria outermembrane pore formation through which more cytochrome can be released. Released cytochrome c recruits Apaf1, caspase 9 and dATP, forming a ‘‘death executioner’’ apotosome complex. doi:10.1371/journal.pone.0024102.g006 activities was recorded in 2DG-ABT treated HeLa cells (Fig. S5b), confirming that 2DG generates a pro-survival signal through PI3K in HeLa cells. 2DG also induced phosphorylation of ATK in HeLa cells (data not shown). Thus, 2DG seems to be sending complicated and often conflicting signals (Fig. 6). The way in which cells respond to 2DG might determine if they will become sensitized or resistant to apoptosis. We thus decided to examine how commonly available cell lines and primary cells would respond to the 2DG-ABT combination treatment. Most cancer cells responded to the 2DG-ABT treatment Most cancer cells tested responded well to the combination treatment; apoptosis was induced in most cells with just 1 mM ABT-263/737. The list of 2DG-ABT sensitive cells includes cells from leukemia (RS(4;11), BP3), cervical cancer (HeLa), prostate cancer (PPC-1/PC-3), hepatocarcinoma (HepG2), breast cancer (MCF-7), and most small lung carcinomas. However, cells with low glucose transporter activities such as confluent NIH3T3 cells and CLL cells that do not grow in culture could not be sensitized by 2DG for ABT-induced apoptosis. Small-cell lung carcinoma- derived cell lines, on the other hand, grow well in culture, and most of them are sensitive to ABT [8,45]. The NIC H82 cell line is a well-studied exception to this rule, but why it is resistant to ABT remains unclear [8,46]. However, we now know that activation of the Bid protein is necessary for efficient apoptosis induction by ABT, and the CASP8 gene encoding the Bid-activating capsase-8 enzyme is deleted in NCI H82 cells [47,48]. Thus, it seems likely that the absence of caspase-8 makes NCI H82 cells resistant to ABT-induced apoptosis. Pancreatic cancer cells are highly resistant to apoptosis. Despite our repeated attempts at inducing apoptosis in PANC-1 pancreatic cancer cells, the 2DG-ABT treatment failed to induce apoptosis. PANC-1 cells express Bid, Bcl-2 and Bak, and cleaved Bid is observed in TNF-treated cells, and these cells have active glucose transporter(s) - all the conditions necessary for 2DG-ABT induced apoptosis. Thus, these conditions are clearly not sufficient. We are trying to discover why these cells are so highly resistant. We are also setting up screens to identify apoptosis inducers specific for 2DG-treated pancreatic cancer cells. 2DG improves anti-tumor activity of ABT-263 in a xenograft model of human prostate cancer Previous work demonstrated that either 1 g/kg of 2DG [49,50] or 100 mg/kg of ABT-263 [9] could safely be injected into mice. To test the safety of the combination, we treated wild-type C57BL/6 mice with intraperitoneal injection of 0.5 g/kg of 2DG+/20.5 g/kg of glucose in the morning, and 100 mg/kg of ABT-263 in the afternoon. The combination treatment produced only a few side effects, including mild lymphopenia and reversible thrombopenia, similar to ABT-263 alone (data not shown) [9,51,52]. For xenograft implants, we chose a highly chemorefractory tumor line PPC-1 [27,28,29,53,54,55,56]. Five million PPC-1 cells were injected into each athymic nude mouse, and the mice were treated three times a week for 3 weeks from day 9 to day 27 with the following regimens: Group A mice received buffer only, Group B mice received 2DG only, Group C mice received ABT-263 only, Group D mice received 2DG in the morning and ABT-263 in the PLoS ONE | www.plosone.org 8 September 2011 | Volume 6 | Issue 9 | e24102