2DG-ABT Combination Therapy Figure 1. Annexin V-staining of 2DG-ABT treated RS(4;11) and HeLa cells. a, RS(4;11) cells pre-treated with 20 mM fructose or 0–10 mM 2- deoxy-D-glucose in regular RPMI medium containing 12 mM glucose, for 1 hour before the addition of ABT-737 at indicated concentrations. 9 hours later, cells were analyzed by FACS for Annexin-V positivity. Chi Square Test of Independence for two variables, 2DG and ABT, was P(x˜12.193.35) = 0.000000. Since the combination was clearly better than expected values, the statistic suggests synergistic interaction between 2DG and ABT-737. b, HeLa cells were pre-treated for 1 hour either with or without 10 mM deoxyglucose in DMEM containing 12 mM glucose, before adding ABT-737. Six hours later, cells were analyzed by FACS for Annexin-V staining. c, HeLa cells were pre-treated for 3 hours with 2DG before the addition of ABT-737. Cells were assayed for caspase activity at 3 hours after ABT-737 addition. d, HeLa, PPC-1 and MCF-7 cells were treated with 10 mM 2DG for 3 hours before adding 1 mM ABT-263 for overnight incubation. Trypan blue-negative (viable) cells were counted and graphed (mean 6 std dev; n = 3). e, Combination therapy reduces clonogenic survival of PPC-1 cells. PPC-1 cells were treated just once for 24 hrs with 5 mM 2DG, 1 mM ABT-263, both, or were left untreated. 250–1000 cells were plated per 30 mm dish, and 12 days later, surviving cell colonies were stained. doi:10.1371/journal.pone.0024102.g001 ratio for apoptosis induction with 2DG:glucose is approximately 0.4 to 1.0; (ii) 2DG should be added 3 to 4 hours prior to the addition of ABT (Fig. S2c & d); and (iii) caspase activation is observed within 3 hours of ABT addition. These conditions may reflect the stability of ABT-263/737 in solution (approximately 4 hours [9]), as well as the nature of intercellular signals generated by 2DG; it must take 3 to 4 hours for 2DG to generate a strong enough signal to be effective. Under these conditions, the reduction in cellular ATP concentration is very slight (Fig. 5a), suggesting that ATP insufficiency does not provide an explanation for the improved sensitivity to ABT-737 and ABT-263. All subsequent experiments were performed using the following conditions: Add 5–10 mM 2DG to cell culture. Three hours later, add 1–3 mM ABT. Assay for caspase activity and cytochrome c release from mitochondria three hours after ABT-treatment. Six hours after ABT-treatment, assay for Annexin V staining. 24 hours later, count viable cells by trypan blue dye exclusion assay. The response to the protocol varied from cell line to cell line. Among cancer cells that responded well were breast cancer cell line MCF-7 and hormone resistant prostate cancer cell line PPC-1. In both cases, over 95% of cells were eliminated in 24 hours by the combination treatment (Fig. 1d). A single agent application of 2DG caused some death (31.3% for HeLa, none observed in MCF-7, and 21.5% for PPC-1). Since all cells have varied amounts of glycogen as stored source of glucose, the rates of cell death by 2DG was largely dependent on the previous history of cultured conditions. Cultured cells normally overcome 2DG- induced growth-arrest and start to grow in 24–48 hours (data not shown). Furthermore, when 2DG was injected into cancer-bearing mice, it did not cause tumor regression, but delayed tumor growth by 5–6 days. After this short delay, tumors started to grow. Other groups had made similar observations [4]. The reason for the acquired resistance to 2DG is not known. As a single agent, the effect of ABT-263 was also marginal (13.7% for HeLa, 3.1% for PLoS ONE | www.plosone.org 3 September 2011 | Volume 6 | Issue 9 | e24102