Efficient Elimination of Cancer Cells by Deoxyglucose-ABT-263/737 Combination Therapy Ryuji Yamaguchi1,4*, Edith Janssen2, Guy Perkins3, Mark Ellisman3, Shinichi Kitada1, John C. Reed1 1 Program of Cell Death and Apoptosis, Sanford-Burnham Medical Research Institute, La Jolla, California, United States of America, 2 Division of Molecular Immunology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, United States of America, 3 Department of Neurosciences, University of California San Diego, La Jolla, California, United States of America, 4 Department of Molecular Biology, Kyushu University Medical School, Fukuoka, Japan Abstract As single agents, ABT-263 and ABT-737 (ABT), molecular antagonists of the Bcl-2 family, bind tightly to Bcl-2, Bcl-xL and Bcl- w, but not to Mcl-1, and induce apoptosis only in limited cell types. The compound 2-deoxyglucose (2DG), in contrast, partially blocks glycolysis, slowing cell growth but rarely causing cell death. Injected into an animal, 2DG accumulates predominantly in tumors but does not harm other tissues. However, when cells that were highly resistant to ABT were pre- treated with 2DG for 3 hours, ABT became a potent inducer of apoptosis, rapidly releasing cytochrome c from the mitochondria and activating caspases at submicromolar concentrations in a Bak/Bax-dependent manner. Bak is normally sequestered in complexes with Mcl-1 and Bcl-xL. 2DG primes cells by interfering with Bak-Mcl-1 association, making it easier for ABT to dissociate Bak from Bcl-xL, freeing Bak to induce apoptosis. A highly active glucose transporter and Bid, as an agent of the mitochondrial apoptotic signal amplification loop, are necessary for efficient apoptosis induction in this system. This combination treatment of cancer-bearing mice was very effective against tumor xenograft from hormone-independent highly metastasized chemo-resistant human prostate cancer cells, suggesting that the combination treatment may provide a safe and effective alternative to genotoxin-based cancer therapies. Citation: Yamaguchi R, Janssen E, Perkins G, Ellisman M, Kitada S, et al. (2011) Efficient Elimination of Cancer Cells by Deoxyglucose-ABT-263/737 Combination Therapy. PLoS ONE 6(9): e24102. doi:10.1371/journal.pone.0024102 Editor: Andrei L. Gartel, University of Illinois at Chicago, United States of America Received April 16, 2011; Accepted July 31, 2011; Published September 19, 2011 Copyright: 2011 Yamaguchi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: EJ received National Institutes of Health(NIH)/National Cancer Institute Grant CA138617, ME received NIH grant P41RR004050, and JCR received CA113318, CA055164, and CA081534. Some of the work described here was conducted using facilities at the National Center for Microscopy and Imaging Research at the University of California San Diego, supported by the NIH NCRR through grant number P41RR004050 to ME. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: rudy.yamaguchi@gmail.com Introduction 2-Deoxy-D-glucose is a glucose molecule which has the 2- hydroxyl group replaced by hydrogen. 2DG is transported across the plasma membrane by a glucose transporter [1]. Once in the cytosol, 2DG is phosphorylated by hexokinase II and its product, 2-deoxyglucose 6-phosphate, is trapped in the cytosol and becomes an inhibitor of hexokinases, just as glucose becomes glucose 6-phosphate and becomes an inhibitor of hexokinases. However, as glucose 6-phosphate is hydrolyzed by glucose 6- phosphatase very rapidly, producing NADPH and generating energy, its counterpart, 2-deoxyglucose 6-phosphate, is a poorer substrate of glucose 6-phosphatase. Consequently 2-deoxyglucose 6-phosphate accumulates in the cytosol, inhibiting hexokinases and lowering cellular energy levels. It is estimated that the intracellular half-life of 2-deoxyglucose 6-phsophate is approxi- mately 50 min in cancer cells [2]. Thus 2DG acts as an inhibitor of the glycolytic pathway. The slower hydrolysis rates of 2-deoxyglucose 6-phsophate allow for glucose uptake to be measured in living animals by 18F- fluoro-2-deoxyglucose based Positron Emission Tomography (FDG-PET). Recent advances in FDG-PET has clearly shown much higher rates of glucose transporter activities in vivo, of almost all solid tumors compared to normal healthy tissues, confirming the Warburg Effect [1]. FDG-PET has also become a very sensitive way to detect tumors at a very early stage, e.g., early stage asymptomatic pancreatic cancer. Since 2DG accumulates predominantly in cancer cells and partially inhibits much-utilized glycolysis in these cells, administration of 2DG is a safe and effective way of slowing cancer growth [1]. However, the cytostatic activity of 2DG is generally short-lived, lasting only about a week [3]. Thus 2DG has been tried in combination with other chemotherapeutics, but again with mixed results. In some cases, it could lower the efficacy of other treatments [4]. One reason for decreased efficacy in these combination therapies might be that in some cells, 2DG activates an insulin-like growth factor receptor (IGFR), which could activate the PI3K-mTOR-AKT pro-survival pathway [5,6]. Various drugs that target Bcl-2 and Mcl-1 have been tested for their abilities to induce apoptosis in cancer cells [7]. The most prominent among them are the Bcl-2 antagonists ABT-737 and ABT-263 [8,9]. ABT-737 binds specifically and with high affinity to the Bcl-2 family of proteins, including Bcl-2, Bcl-xL and Bcl-w, but not to Mcl-1. ABT-737 was modified at three sites to create ABT-263 for improved oral bioavailability without the loss of affinity to the Bcl-2 family of proteins [9]. As a single agent, however, ABT-263/737 (ABT) induces apoptosis only in limited tumor types, such as lymphomas and some small cell lung carcinomas [8,9,10]. The reason for varied sensitivities to ABT is not entirely clear. PLoS ONE | www.plosone.org 1 September 2011 | Volume 6 | Issue 9 | e24102