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A Novel Inhibitor of Glucose Uptake Sensitizes Cells to FAS-Induced Cell Death

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A Novel Inhibitor of Glucose Uptake Sensitizes Cells to FAS-Induced Cell Death 3546 A novel inhibitor of glucose uptake sensitizes cells to FAS-induced cell death Tabitha E. Wood,1,2 Shadi Dalili,1,2 transport as a mechanism to sensitize cells to death Craig D. Simpson,1 Rose Hurren,1 Xinliang Mao,1 receptor stimuli. Thus, fasentin is a novel inhibitor of Fernando Suarez Saiz,1 Marcela Gronda,1 glucose transport that blocks glucose uptake and high- Yanina Eberhard,1 Mark D. Minden,1 Philip J. Bilan,3 lights a new mechanism to sensitize cells to death ligands. Amira Klip,3 Robert A. Batey,2 [Mol Cancer Ther 2008;7(11):3546–55] and Aaron D. Schimmer1 1Princess Margaret Hospital, Ontario Cancer Institute; Introduction 2Department of Chemistry, University of Toronto; 3Cell Biology Resistance to stimuli of the death receptor pathway of Program, Hospital for Sick Children, Toronto, Ontario, Canada caspase activation can render malignant cells resistant to chemotherapy, immune surveillance, and anchorage-inde- Abstract pendent cell death, a process termed anoikis (1, 2). In addition, such resistance would also limit the therapeutic Evasion of death receptor ligand-induced apoptosis is an utility of agonistic monoclonal antibodies targeting tumor important contributor to cancer development and progres- necrosis factor (TNF) apoptosis-inducing ligand receptors sion. Therefore, molecules that restore sensitivity to death that are now in clinical trial (3). Therefore, small molecules receptor stimuli would be important tools to better that restore sensitivity of tumor cells to TNF family death understand this biological pathway and potential leads receptors would be useful probes to understand this for therapeutic adjuncts. Previously, the small-molecule pathway and potentially useful therapeutic adjuncts for N-[4-chloro-3-(trifluoromethyl)phenyl]-3-oxobutanamide the treatment of malignancy. (fasentin) was identified as a chemical sensitizer to the Diverse mechanisms can create roadblocks to apoptosis death receptor stimuli FAS and tumor necrosis factor triggered by stimuli of the death receptor pathway of apoptosis-inducing ligand, but its mechanism of action caspase activation (4). Documented resistance mechanisms was unknown. Here, we determined that fasentin alters relevant to the death receptor pathway include reduced expression of genes associated with nutrient and glucose expression of TNF family death receptors, shedding of deprivation. Consistent with this finding, culturing cells in soluble death receptors, expression of ligand-binding low-glucose medium recapitulated the effects of fasentin decoy receptors, reduced expression of caspase-8 and and sensitized cells to FAS. Moreover, we showed that caspase-10, and overexpression of intracellular caspase fasentin inhibited glucose uptake. Using virtual docking inhibitors such as the caspase-8 inhibitor c-FLIP (reviewed studies with a homology model of the glucose transport in ref. 1). protein GLUT1, fasentin interacted with a unique site in Previously, we used a cell-based high-throughput screen the intracellular channel of this protein. Additional chem- to identify compounds that selectively modulate the ical studies with other GLUT inhibitors and analogues of extrinsic pathway, sensitizing resistant tumor cells to fasentin supported a role for partial inhibition of glucose TNF family death receptors and death ligands (5). From this screen, we identified 5809354 that sensitized cells to death ligands by decreasing expression of FLIP mRNA. Received 6/17/08; revised 8/12/08; accepted 8/27/08. This screen also identified the novel FAS-sensitizing N Grant support: Canadian Institutes of Health Research, Canadian Cancer chemical compound ( -[4-chloro-3-(trifluoromethyl)- Society, and Prostate Cancer Research Foundation of Canada, and Ontario phenyl]-3-oxobutanamide; fasentin) that selectively sensi- Cancer Research Network through funding provided bythe Ministryof tized to death ligands but did not decrease FLIP Research and Innovation in the Province of Ontario. expression. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked The relatively unique 3-oxobutananilide structure of the advertisement in accordance with 18 U.S.C. Section 1734 solelyto compound fasentin provided little insight into the possible indicate this fact. intracellular targets of this molecule. Therefore, to better Note: Supplementarymaterial for this article is available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). understand the targets and pathways affected by this T.E. Wood is a Natural Sciences and Engineering Research Council of molecule, we analyzed changes in gene expression after Canada and Ontario Cancer Institute research fellow. A.D. Schimmer is a treatment with fasentin. These experiments showed that scholar in clinical research from the Leukemia and Lymphoma Society. fasentin altered expression of genes associated with Requests for reprints: Aaron D. Schimmer, Princess Margaret Hospital, nutrient and glucose metabolism. Based on this finding, Ontario Cancer Institute, 610 UniversityAvenue, Toronto, Ontario, Canada M5G 2M9. Phone: 416-946-2838; Fax: 416-946-6546. we explored the effects of fasentin on glucose metabolism E-mail: [email protected] and showed that this molecule rapidly inhibited glucose Copyright C 2008 American Association for Cancer Research. uptake. Subsequent studies supported that the inhibition of doi:10.1158/1535-7163.MCT-08-0569 glucose uptake of fasentin was functionally important for Mol Cancer Ther 2008;7(11). November 2008 Downloaded from mct.aacrjournals.org on October 2, 2021. © 2008 American Association for Cancer Research. Molecular Cancer Therapeutics 3547 its ability to sensitize cells to death receptor stimuli. Thus, (GenBank NM-002046; 5-GAAGGTGAAGGTCGGAGTC- fasentin is a novel inhibitor of glucose transport that blocks 3/5-GAAGATGGTGATGGGATTTC-3). Relative mRNA glucoseuptakeandhighlightsanewmechanismto expression was determined using the CTmethod as sensitize cells to death ligands. described previously (2). Gene Expression Profiling Gene expression profiling was done using Affymetrix Materials and Methods Human Genome Plus 2.0 with methods recommended by Reagents the manufacturer. Genes that were dysregulated at least The small-molecule death receptor sensitizer fasentin 2-fold between control and treatment were further validat- (Chembridge) was obtained as a powder and stored at ed by real-time quantitative PCR as described above. room temperature in a glass vial (Chembridge). Stock Identification of gene ontology for genes altered by solutions (100 mmol/L) of fasentin were prepared by treatment with fasentin was done using the Database for diluting fasentin in water with 16% DMSO and were stored Annotation, Visualization, and Integrated Discovery. This at 4jC in polystyrene tubes. Dipyridamole, phloretin, and tool identifies enrichment of gene ontologies comparing the cytochalasin B were purchased from Sigma-Aldrich. Indi- users’ gene list against the gene ontology database and navir was obtained from Merck & Co. CH-11 anti-Fas scoring for significance (7, 8). monoclonal antibody (FAS) to activate the Fas receptor CellViability (MBL) was obtained in glycerol and stored at À20jC. Cell viability was assessed using the 3-(4,5-dimethylth- 3 2-[ H]deoxy-D-glucose (20-50 Ci/mmol) was purchased iazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- from Perkin-Elmer. 2H-tetrazolium inner salt (MTS) reduction assay (Promega) Cell Cultures and Treatments according to the manufacturer’s protocols as described PPC-1 prostate cancer cells, DU145 prostate cancer cells, previously (2). The percent relative cell viability was and U937 leukemia cells were maintained in RPMI 1640. expressed as [(absorbance of treated cells) / (absorbance Primary human peripheral blood mononuclear cells were of controls)] Â 100%. isolated from fresh peripheral blood samples from healthy Apoptosis was measured by Annexin V-FITC and volunteers. Mononuclear cells were isolated from the propidium iodide (Biovision Research Products) staining samples by Ficoll density centrifugation. Primary cells and flow cytometry according to the manufacturer’s were cultured in IMDM. L6 myoblasts stably overexpress- instructions and as described previously (2). ing myc-tagged GLUT1 and GLUT4 (ref. 6; L6GLUT1myc Assessment of Glucose Uptake and L6GLUT4myc) were cultured in a-MEM. All cells were 2-Deoxy-D-glucose uptake was done as described previ- supplemented with 10% fetal bovine serum (Hyclone), ously (6). Briefly, PPC-1, U937, and DU145 cells were penicillin (500 IU/mL), and streptomycin (50 Ag/mL). All grown in RPMI 1640 with 10% (v/v) fetal bovine serum in cells were cultured at 37jC in a humid atmosphere with 24-well plates. Cells were then washed twice with HEPES- 5% CO2. buffered saline solution, which contained 140 mmol/L The collection and use of human tissue for this study was NaCl, 5 mmol/L KCl, 2.5 mmol/L MgSO4, 1 mmol/L approved by the local ethics review board (University CaCl2, and 20 mmol/L HEPES (pH 7.4). 2-Deoxy-D-glucose Health Network). uptake was initiated by addition of 10 Amol/L 2-deoxy-D- 3 Real-time ReverseTranscription-PCR glucose and 1 ACi/mL 2-[ H]deoxy-D-glucose in the above First-strand cDNA was synthesized from 1 Ag DNase- HEPES solution at room temperature. 2-Deoxy-D-glucose treated total cellular RNA from PPC-1 cells using random uptake was terminated by rinsing the cells three times primers and SuperScript II reverse transcriptase
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