Sensitization of Glioblastoma Cells to Irradiation by Modulating the Glucose Metabolism Han Shen1, Eric Hau1,2, Swapna Joshi1, Pierre J

Published OnlineFirst June 10, 2015; DOI: 10.1158/1535-7163.MCT-15-0247

Small Molecule Therapeutics Molecular Cancer Therapeutics Sensitization of Glioblastoma Cells to Irradiation by Modulating the Glucose Metabolism Han Shen1, Eric Hau1,2, Swapna Joshi1, Pierre J. Dilda3, and Kerrie L. McDonald1

Abstract

Because radiotherapy significantly increases median survival in combination. In vitro, an enhanced inhibition of clonogenicity of patients with glioblastoma, the modulation of radiation resis- a panel of glioblastoma cells was observed when dichloroacetate tance is of significant interest. High glycolytic states of tumor cells was combined with radiotherapy. Further mechanistic investiga- are known to correlate strongly with radioresistance; thus, the tion revealed that dichloroacetate sensitized glioblastoma cells to concept of metabolic targeting needs to be investigated in com- radiotherapy by inducing the cell-cycle arrest at the G2–M phase, bination with radiotherapy. Metabolically, the elevated glycolysis reducing mitochondrial reserve capacity, and increasing the oxi- in glioblastoma cells was observed postradiotherapy together dative stress as well as DNA damage in glioblastoma cells together with upregulated hypoxia-inducible factor (HIF)-1a and its target with radiotherapy. In vivo, the combinatorial treatment of dichlor- pyruvate dehydrogenase kinase 1 (PDK1). Dichloroacetate, a PDK oacetate and radiotherapy improved the survival of orthotopic inhibitor currently being used to treat lactic acidosis, can modify glioblastoma-bearing mice. In conclusion, this study provides the tumor metabolism by activating mitochondrial activity to force proof of concept that dichloroacetate can effectively sensitize glycolytic tumor cells into oxidative phosphorylation. Dichlor- glioblastoma cells to radiotherapy by modulating the metabolic oacetate alone demonstrated modest antitumor effects in both in state of tumor cells. These findings warrant further evaluation of vitro and in vivo models of glioblastoma and has the ability to the combination of dichloroacetate and radiotherapy in clinical reverse the radiotherapy-induced glycolytic shift when given in trials. Mol Cancer Ther; 14(8); 1794–804. 2015 AACR.

Introduction stabilize the chemical composition of the DNA damage by react- ing with the free radicals, such that O chemically "fixes" DNA Glioblastoma is the most malignant form of primary brain 2 damage. Unlike the balance achieved in normal tissues, the tumor in adults. Despite increasing attention on targeted thera- consumption of O by tumor tissue is much higher than the peutics in the treatment of glioblastoma, radiotherapy remains 2 O supply from the surrounding blood vessels. Malignant solid the most clinically effective treatment modality (1). However, 2 tumors with inadequate blood supply and inconsistent perfusion radiotherapy only offers palliation, the efficacy of which is often therefore contain large portions of hypoxic cells which exhibit a limited by the occurrence of radioresistance, reflected as a dimin- high degree of resistance to chemoradiotherapy due, in part, to an ished susceptibility of the irradiated cells to undergo cell death increase of hypoxia-inducible factor-1a (HIF1a) and expression (2). To enhance the tumor cell sensitivity to radiotherapy, the of other cellular survival molecules (3). Radiation itself, has been mechanisms underlying radioresistance need to be further eluci- shown to stabilize the activity of HIF1a, which in turn regulates a dated and strategies developed to overcome them. plethora of genes involved in angiogenesis, invasion, metabolism, When ionizing radiation passes the living tissue, the ionization and protection against oxidative stress (4). The residual tumor of H O leads to the production of reactive oxygen species (ROS) 2 cells surviving after chemoradiotherapy eventually proliferate and that contain chemically active oxygen molecules leading to oxi- lead to cancer relapse. dative stress and DNA damage. Oxygen molecules (O ) can 2 It has long been known that the metabolism of solid tumors is radically different from that in the corresponding normal tissues. Numerous studies have demonstrated that tumor cells predom- 1Cure Brain Cancer Neuro-Oncology Group, Adult Cancer Program, inantly utilize glycolysis even in the presence of ample oxygen, Lowy Cancer Research Centre, Prince of Wales Clinical School, Uni- versity of New South Wales, Sydney, New South Wales, Australia. referred as the Warburg effect. Using glycolysis provides a growth 2Cancer Care Centre, St George Hospital, Kogarah, New South Wales, advantage for tumor cells and leads to malignant progression (5). Australia. 3Tumour Metabolism Group, Adult Cancer Program, Lowy Glioblastoma, like most malignant solid tumors, is highly glyco- Cancer Research Centre, Prince of Wales Clinical School, University of lytic, producing large amounts of lactic acid as a metabolic New South Wales, Sydney, New South Wales, Australia. byproduct. It has been shown that tumors with high levels of Note: Supplementary data for this article are available at Molecular Cancer glycolysis are less responsive to radiotherapy and behave more Therapeutics Online (http://mct.aacrjournals.org/). aggressively (6). More recent reports have identified the Warburg H. Shen and E. Hau share first authorship of this article. effect to be implicated in resistance to cytotoxic stress induced by Corresponding Author: Kerrie L. McDonald, Cure Brain Cancer Neuro-Oncology either chemotherapy or radiotherapy (7). In this way, treatment Group, Adult Cancer Program, Lowy Cancer Research Centre, Prince of Wales methods which block or reduce glycolytic metabolism may Clinical School, University of New South Wales, Sydney, NSW 2052, Australia. increase tumor cell sensitivity to radiotherapy. Phone: 61-2-93851471; Fax: 61-2-93851510; E-mail: [email protected] Under hypoxic conditions, HIF1a causes an increase in its doi: 10.1158/1535-7163.MCT-15-0247 target gene pyruvate dehydrogenase kinase 1 (PDK1), which acts 2015 American Association for Cancer Research. to limit the amount of pyruvate entering the citric acid cycle,

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leading to decreased mitochondrial oxygen consumption (8). manufacturer's instructions. After staining, cells were trypsinized, These findings suggest that inhibition of PDK1 could alter the centrifuged, and resuspended in HBSS (1 mL). Sytox Blue (Invi- glucose metabolism and increase oxygen consumption of tumor trogen; 1 mmol/L) was added to counterstain for nonviable cells. cells, which would resensitize the tumor cells to radiotherapy. MitoSOX Red fluorescence was analyzed using BD FACSCanto II Dichloroacetate, a PDK inhibitor that has the potential for such flow cytometer. metabolic targeting, has been shown to reverse the Warburg effect by shifting glucose metabolism from glycolysis to mitochondrial Extracellular flux assay oxidation and to inhibit tumor cell growth (9, 10). By combining The oxygen consumption rate (OCR) and extracellular acidi- with radiotherapy, dichloroacetate has been demonstrated to fication rate (ECAR) of glioblastoma cells were determined using in vitro – enhance the radiosensitivity of several tumor types (11 the XF Extracellular Flux Analyzer (Seahorse Bioscience). XF 13). Interestingly, a previous study using 2 human cancer cell lines 24-well plates were coated with Matrigel (BD biosciences) to (colon adenocarcinoma and glioblastoma) demonstrated that allow cells to attach for this assay. Briefly, each well of the 24- fi in vitro dichloroacetate sensitized the ef cacy of radiotherapy but well plate was coated with 50 mL diluted Matrigel (1:50 in PBS) in vivo attenuated radiotherapy-induced tumor growth delay overnight. The next day, cells were plated at a density of 30,000 (colon adenocarcinoma model; ref. 13). This paradoxical effect cells (U87) and 50,000 cells (RN1) per well and allowed to attach fi of dichloroacetate drove us to further investigate the ef cacy and overnight in culture media. The following day, the adherent cells – the mechanism of action of radiotherapy dichloroacetate com- were treated as indicated. On the assay day, cells were washed and in vitro in vivo bination in both and glioblastoma models. There- fresh assay media were added. The cartridge was loaded to fi fore, in the present study, we rst examined the hypothesis that dispense 3 metabolic inhibitors sequentially as specific time radiotherapy promotes glycolytic metabolism and then tested points: oligomycin (inhibitor of ATP synthase, 1 mmol/L), whether a reversal of the glycolytic phenotype will resensitize followed by FCCP (a protonophore and uncoupler of mitochon- glioblastoma cells to radiotherapy using dichloroacetate. The drial oxidative phosphorylation, 1 mmol/L), followed by the fi ndings of this study may have important implications for clinical combination of rotenone (mitochondrial complex I inhibitor, trials aimed at preventing postradiotherapy metabolic changes 1 mmol/L) and antimyxin (inhibitor of complex III, 1 mmol/L). and increasing the therapeutic index of radiotherapy for patients Specifically, the addition of oligomycin is used to measure the rate with glioblastoma. of proton leak across the inner mitochondrial membrane. The injection of FCCP is used to measure the rate of uncoupled Materials and Methods respiration and is determined from the maximum respiration Cell culture and chemical rate. The addition of rotenone–antimycin combination is to Glioblastoma cell lines (U87, U251, LN229, DBTRG) were inhibit the transfer of electrons from iron–sulfur centers in com- purchased from ATCC. U87 and U251 were cultured in MEM plex I to ubiquinone and the oxidation of ubiquinol in the (Gibco) with 10% FBS and 2 mmol/L L-glutamine. LN229 was electron transport system (ETS), thereby inhibiting the oxidative cultured in DMEM (Gibco) with 10% FBS and 2 mmol/L phosphorylation. Basal OCR and ECAR were measured, as well as L-glutamine. DBTRG was cultured in RPMI-1640 (Gibco) supple- the changes in OCR caused by the addition of the metabolic mented with 10% FBS, 2 mmol/L L-glutamine, 25 mmol/L inhibitors described above. Several parameters were deducted HEPES, and 1 mmol/L sodium pyruvate. Cell lines were obtained from the changes in OCR and mitochondrial reserve capacity within the past 5 years and authentication was not performed. A was calculated by subtracting basal respiration from maximal patient-derived glioblastoma cell line RN1 (unmethylated, respiratory capacity as described previously (14). A mirror plate rs16906252 wild-type, and p53 mutant) was kindly provided by was set up and treated identically in parallel with the assay plate. our collaborative researchers at the Queensland Institute of Med- At the end of the assay, cells from the mirror plate were harvested, ical Research (Brisbane, Queensland, Australia) and was cultured and the numbers of viable cells determined by flow cytometry was in advanced DMEM/F12 (Gibco) mixed with Neurobasal-A medi- used to normalize the measurements. um (Gibco; 1:1) supplemented with B-27 (1), FGF (20 ng/mL), and EGF (20 ng/mL). Sodium dichloroacetate was purchased Western blotting from Sigma. Cells were seeded in 6-well plates, followed by treatments as indicated. Cells were lysed with RIPA buffer (Life Technologies), Cell-cycle analysis sonicated and centrifuged (14,000 rpm, 10 minutes, 4 C). Protein Cells were seeded in 6-well plates, followed by treatments as concentration was measured using BCA Assay Kit (Pierce). Pro- indicated. After treatments, cells were harvested and fixed in cold teins (50 mg/sample) were separated via reducing 10% SDS-PAGE, 70% v/v ethanol for at least 2 hours. Fixed cells were washed with and standard Western blotting procedures (15) were used to PBS and stained in the dark with a solution containing propidium detect proteins of interest with the following primary antibodies: iodide (10 mg/mL), Triton X-100 (0.1%), and RNAse (100 mg/mL) g-H2AX (Cell Signaling Technology, CST #9718), HIF1a (CST, for 20 minutes at room temperature. DNA content was analyzed #3716), PDK-1 (CST, #3820), and b-actin (Abcam, ab8227). using a BD FACSCanto II flow cytometer and data analysis was Images and densitometry were acquired by using ImageQuant performed using FlowJo (TreeStar Inc). TL software.

Mitochondrial superoxide production assays Glucose metabolism PCR array Cells were seeded in 6-well plates, followed by treatments as Cells were plated in 6-well plates, followed by treatments as indicated. MitoSOX Red (Invitrogen) was used to measure the indicated. Total RNA from cells was extracted using RNeasy Mini level of mitochondrial superoxide production according to the Kit (Qiagen). About 0.8 mg of total RNA was used for generating

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cDNA with RT2 First Strand Kits (Qiagen) and was analyzed by a using the target retrieval solution (DAKO) at 95C for 20 minutes. glucose metabolism PCR array according to the manufacturer's Sections were stained for proliferating tumor cells as follows: instructions using ViiA 7 Real-Time PCR System (Life Technolo- sections were incubated with primary Ki-67 antibody (DAKO) at gies). All data were normalized to expression of untreated control 1:75 dilution for 1 hour at room temperature and secondary in the corresponding sample. antibody (DAKO) at 1:300 dilution for 1 hour at room temperature. The proliferative index of tumor cells was determined by Colony survival assay counting Ki-67 positive cells in 3 random 40 magnification Cells were seeded into 6-well plates and were allowed 24 hours fields per slice. for attachment followed by treatments as indicated. Culture medium was changed after 24-hour treatment. Plates were incu- Statistical analyses bated for 2 weeks undisturbed. Colonies were gently washed with All analyses were performed using GraphPad Prism. Each PBS followed by staining and fixation with crystal violet solution independent experiment was performed with at least triplicate (0.5% in H O:methanol, 1:1) for 15 minutes. Stained colonies 2 samples per treatment group. Results are expressed as mean SD consisting of >50 cells were counted and the number was of replicate values from 3 independent experiments or represen- recorded. Plating efficiency was calculated as the number of tative of 3 independent experiments presented as means SD colonies counted divided by number of cells seeded and normal- of triplicate measurements. Statistical analysis was performed by ized to the average plating efficiency of untreated samples. The 2-way ANOVA corrected by the Dunnett or Student t tests. average of these values was reported as "surviving fraction." For Kaplan–Meier survival was compared using log-rank (Mantel– the combination therapy of dichloroacetate and radiotherapy, the Cox) test. All tests of statistical significance were 2-sided and P < surviving fraction was normalized to dichloroacetate treatment 0.05 was considered statistically significant. alone.

Mouse xenograft orthotopic model Results The procedures for developing an orthotopic brain tumor Radiotherapy shifts the glucose metabolism from oxidative model were performed according to a protocol approved by the phosphorylation to glycolysis and the addition of Animal Care and Ethics Committee of University of New South dichloroacetate blocks radiotherapy-induced glycolytic Wales (Sydney, New South Wales, Australia). Briefly, female phenotype in glioblastoma cells athymic nude mice (Balb/c, 8 weeks) were intracranially injected We first examined the expression of a panel of key genes 4 with 5 10 U87 cells stereotactically in the right caudate involved in the regulation and enzymatic pathways of glucose putamen using the coordinates: 1 mm anterior, 1.5 mm lateral, and glycogen metabolism. Compared with untreated control, we and 3.0 mm below from the bregma. observed that most of the glycolytic genes were upregulated in U87 cells irradiated with 6 Gy at 4 hours after radiotherapy. In Dichloroacetate treatment and radiotherapy particular, all the isozymes of PDK (PDK 1-4) were upregulated The U87-bearing mice were randomly divided into 4 groups of from 1.26- to 3.38-fold (Fig. 1A and Table 1). To determine 8 to 10: vehicle-treated control, radiation alone, dichloroacetate whether HIF1a might be involved in this radiotherapy-induced alone, and combination treatment. Dichloroacetate was admin- glycolysis, Western blotting was performed to examine the protein istered by oral gavage at 150 mg/kg/d from day 3 after tumor level of HIF1a and its target PDK1. Compared with untreated inoculation to the end of experiment. Mice in vehicle-treated control, treatment of U87 cells with 6 Gy radiotherapy signifi- control and radiation alone arms received distilled water by oral cantly increased HIF1a and PDK1 expression up to 2.47- and gavage. Radiation was delivered to the whole brain of the mouse 1.42-fold, respectively (Fig. 1B). To further confirm the radiother- using a self-contained X-ray system (X-RAD 320) from day 13 after apy-induced glycolysis, 2 metabolic parameters, OCR and ECAR, tumor implantation. During radiotherapy, mice were placed in a were measured using an extracellular flux assay. The bioenergetic customized lead box shielding the body to allow radiation to be profiles were obtained 4 hours postradiotherapy with increasing delivered directly to the entire brain. The total radiation dose radiotherapy doses. As shown in Fig. 1C, the ECAR values of U87 administered was 20 Gy at a clinically relevant 2 Gy/fraction cells after radiotherapy increased dose dependently, whereas a schedule on 10 consecutive days. Two mice were randomly dose-dependent decrease in OCR was observed. By combining selected from each arm and culled on the day of radiotherapy 10 mmol/L dichloroacetate with radiotherapy, the radiotherapy- completion. Their brains were harvested for hematoxylin and induced glycolytic rate of U87 cells was attenuated significantly eosin (H&E) staining and immunohistochemical examination. (P < 0.01) compared with cells treated with radiotherapy alone Mice were euthanized when they exhibited symptoms indicative (Fig. 1D). Similar results were also obtained for the primary of significant compromise to neurologic function and/or a greater glioblastoma cell line RN1 (Fig. 1E). than 20% body weight loss. Animal survival was defined as the time euthanasia and survival curves were established by Kaplan– – Meier estimator. Dichloroacetate treatment induces G2 M cell-cycle arrest in glioblastoma cells Immunohistochemistry We next further examined whether the proliferation arrest from Tumor cell proliferation was estimated after H&E staining and dichloroacetate treatment was associated with induction of cell- by immunostaining for Ki-67. Excised tumors were fixed in cycle arrest. Cells were treated with 25 and 50 mmol/L dichlor- formalin and embedded in paraffin and 5-mm sections were cut oacetate for 24 hours, and cell-cycle profiles were analyzed using and mounted on Superfrost slides. Briefly, sections were depar- flow cytometry. Dichloroacetate treatment induced changes in the affinized in xylene and rehydrated in PBS using an ethanol cell-cycle profiles of all tested glioblastoma cells (Fig. 2A and B). gradient and a heat-mediated antigen retrieval step was performed Specifically, after 24 hours of treatment with 25 mmol/L

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Figure 1. Radiotherapy (RT)-induced glycolytic phenotype is blocked by the addition of dichloroacetate (DCA). A, a panel of glycolytic genes in U87 cells was upregulated 4 hours after 6 Gy radiotherapy. B, 6 Gy radiotherapy induced HIF1 expression as well as its target, PDK1. Western blot analyses are representative of 3 independent experiments. C, radiotherapy induced a decrease in OCR whereas an increase in ECAR dose dependently. The addition of dichloroacetate signiﬁcantly inhibited the radiotherapy-induced acidiﬁcation rate in U87 (D) and RN1 (E) cells. Because of a high cell seeding density (30,000–50,000 cells per well) used in this assay, relatively high doses of radiotherapy (6–20 Gy) were used here to induce ECAR in a short timeframe. Results are representative of 3 independent experiments and presented as means SD of 3 to 5 measurements. , P < 0.05; , P < 0.01; , P < 0.001; #, P < 0.01 versus 20 Gy radiotherapy .

dichloroacetate, there was a slight increase (not signiﬁcant) in the 45.5%, respectively. A corresponding decrease in cells in G1 and cells in G2–M phase in U87 and U251 cells and a 1.2-fold increase S phases in all glioblastoma cell lines was also observed (Fig. 2B). (P < 0.001) in RN1 cells (Fig. 2B). Signiﬁcant increase in the mean – percentage of all 3 tested cell lines in G2 M phase was observed Dichloroacetate treatment depletes mitochondrial reserve when dichloroacetate dose was increased to 50 mmol/L (Fig. 2B). capacity in glioblastoma cells When compared with untreated control (U251, 8.8%; U87, Because ionizing radiation exerts its cytotoxic effects predom- 15.2%; RN1, 14.1%), dichloroacetate treatment increased the inantly through the generation of free radicals and subsequent – proportion of cells at G2 M phase to 35.5%, 34.7%, and oxidative stress, we investigated whether the mitochondrial

Table 1. The fold change of glucose/glycogen metabolism-related genes in irradiated U87 cells in comparison with untreated control 12345 6 7 8 9 101112 A ACLY ACO1 ACO2 AGL ALDOA ALDOB ALDOC BPGM CS DLAT DLD DLST 2.17 1.09 2.69 1.12 1.98 1.34 2.62 1.48 1.27 1.06 1.15 2.67 B ENO1 ENO2 ENO3 FBP1 FBP2 FH G6PC G6PC3 G6PD GALM GBE1 GCK 1.94 2.58 1.52 3.05 2.87 1.51 5.38 1.35 2.69 1.7 1.46 1.56 C GPI GSK3A GSK3B GYS1 GYS2 H6PD HK2 HK3 IDH1 IDH2 IDH3A IDH3B 1.29 3.4 1.09 1.28 1.07 4.29 1.78 1.82 1.49 2.16 1.59 1.96 D IDH3G MDH1 MDH1B MDH2 OGDH PC PCK1 PCK2 PDHA1 PDHB PDK1 PDK2 1.92 1.04 1.54 1.52 2.17 1.56 7.76 2.63 1.75 1.38 1.34 3.38 E PDK3 PDK4 PDP2 PDPR PFKL PGAM2 PGK1 PGK2 PGLS PGM1 PGM2 PGM3 1.31 1.26 1.49 1.39 3.48 5.52 1.76 2.37 2.01 1.6 1.52 1.79 F PHKA1 PHKB PHKG1 PHKG2 PKLR PRPS1 PRPS1L1 PRPS2 PYGL PYGM RBKS RPE 1.99 1.85 2.75 1.31 1.12 2.14 1.8 1.5 1.58 1.58 1.42 1.55 G RPIA SDHA SHDB SDHC SDHD SUCLA2 SUCLG1 SUCLG2 TALDO1 TKT TPI1 UGP2 1.65 1.77 1.64 1.04 2.18 1.61 1.33 1.78 1.39 1.76 1.6 1.51 NOTE: This table shows the corresponding results of the heatmap (Fig. 1A). Glucose metabolism—Glycolysis: ALDOA, ALDOB, ALDOC, BPGM, ENO1, ENO2, ENO3, GALM, GCK, GPI, HK2, HK3, PFKL, PGAM2, PGK1, PGK2, PGM1, PGM2, PGM3, PKLR, TPI1; Gluconeogenesis: FBP1, FBP2, G6PC, G6PC3, PC, PCK1, PCK2; Regulation: PDK1, PDK2, PDK3, PDK4, PDP2, PDPR. TCA cycle: ACLY, ACO1, ACO2, CS, DLAT, DLD, DLST, FH, IDH1, IDH2, IDH3A, IDH3B, IDH3G, MDH1, MDH1B, MDH2, OGDH, PC, PCK1, PCK2, PDHA1, PDHB, SDHA, SDHB, SDHC, SDHD, SUCLA2, SUCLG1, SUCLG2; Pentose Phosphate Pathway: G6PD, H6PD, PGLS, PRPS1, PRPS1L1, PRPS2, RBKS, RPE, RPIA, TALDO1, TKT. Glycogen metabolism - Synthesis: GBE1, GYS1, GYS2, UGP2; Degradation: AGL, PGM1, PGM2, PGM3, PYGL, PYGM; Regulation: GSK3A, GSK3B, PHKA1, PHKB, PHKG1, PHKG2.

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Figure 2. Dichloroacetate (DCA) induces cell-cycle arrest in glioblastoma cells. A, representative ﬂow cytometric histograms showing cell-cycle distribution in untreated control and 24-hour dichloroacetate-treated RN1 cells. B, cell-cycle analysis showing the percentage of cells in G1, S-, and G2–M phases in untreated and dichloroacetate-treated glioblastoma cells (U87, U251, and RN1). Results are presented as means SD of 3 experiments performed in triplicates. , P < 0.05; , P < 0.01; , P < 0.001.

reserve capacity of glioblastoma cells would be altered by dichlor- radiotherapy alone, and the combination of 2 therapies fol- oacetate treatment (Fig. 3A). After 24-hour treatment with 10 lowed by mitochondrial ROS level detection. For U251, 24- mmol/L dichloroacetate, the basal OCR of dichloroacetate-trea- hour dichloroacetate treatment (25 and 50 mmol/L) led to a ted cells increased from about 4,200 pmole/min/106 cells dose-dependent increase in mitochondrial ROS levels by 1.5- to (untreated control) to about 5,400 pmole/min/106 cells (Fig. 2-fold compared with untreated control (Fig. 4A). On the other 3B). By sequentially injecting oligomycin, FCCP, the mixture of hand, the mitochondrial ROS level of U251 cells treated with antimycin and rotenone, complete mitochondrial profile was radiotherapy increased 1.3- (12 Gy) and 1.5-fold (18 Gy) further revealed in U87 and RN1 cells (Fig. 3B and C). As compared with that of control cells (Fig. 4A). These values dichloroacetate treatment did not obviously affect the maximal further increased when the dichloroacetate treatment was com- respiratory capacity of tumor cells, the mitochondrial reserve bined with radiotherapy. Specifically, the combination of capacity of dichloroacetate-treated cells significantly decreased dichloroacetate (25 and 50 mmol/L) and radiotherapy (12 (P < 0.05; Fig. 3D). By increasing dichloroacetate concentration to and 18 Gy) dose dependently boosted the mitochondrial ROS 20 mmol/L, the reserve respiratory capacity was further signifi- level of U251 up to about 3.5-fold as opposed to control cells cantly reduced to about 1,960 and 490 pmole/min/106 cells in (Fig. 4A). Compared with U251, U87 cells are more resistant U87 and RN1 cells, respectively (P < 0.001; Fig. 3D). to dichloroacetate treatment and radiotherapy. Significant elevation of the MitoSox Red fluorescence was only induced Radiotherapy in combination with dichloroacetate induces with high-dose dichloroacetate treatment (50 mmol/L) and ROS production in glioblastoma cell radiotherapy (18 Gy) alone (P < 0.01; Fig. 4B). A further As dichloroacetate increased oxidative stress by directing increase in mitochondrial ROS production was observed when pyruvate into mitochondria, increasing electron transport chain combined treatment was administered (Fig. 4B). Similar activity, and thus generating more ROS, we proposed that this results were obtained from RN1 cells (Fig. 4C). These data increased ROS could potentially further increase the ROS suggest a higher capacity of the combination to induce mito- induced by radiotherapy. To test this hypothesis, glioblastoma chondrial ROS generation in comparison with individual cells (U251, U87 and RN1) were treated with dichloroacetate, treatments alone.

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Figure 3. Dichloroacetate (DCA) depletes the mitochondrial reserve capacity of glioblastoma cells. A, schematic diagram extracted from Seahorse Bioscience demonstrating the use of specific inhibitors to determine the mitochondrial profile is shown. After 3 to 5 measurements of baseline OCR, oligomycin, FCCP, and the mixture of antimycin A and rotenone were injected sequentially with measurements of OCR recorded after each injection. ATP-linked OCR and the OCR due to proton leak can be calculated using the basal and the oligomycin-sensitive rate. Injection of FCCP is used to determine the maximal respiratory capacity. Injection of antimycin A and rotenone allows for the measurement of OCR independent of complex IV. The spare respiratory capacity (mitochondrial reserve capacity) is calculatedby subtracting the basal from the maximal respiration. Bioenergetic profiles of (B) U87 and (C) RN1 were measured using sequential injection of oligomycin (1 mmol/L), FCCP (1 mmol/L), and the mixture of antimycin A (1 mmol/L) and rotenone (1 mmol/L). D, mitochondrial reserve capacity of untreated control and dichloroacetate- treated glioblastoma cells (U87 and RN1) is shown. Seahorse XF24 analyzer protocol included 3 minutes of mixing, 2 minutes of waiting, and 3 minutes of measurement for each measurement. Results are representative of 3 independent experiments and presented as means SD of 3 to 5 measurements. , P < 0.05; , P < 0.001.

Dichloroacetate augments the DNA double-strand breaks showed similar levels of DNA damage with 1.29- and 1.82-fold induced by radiotherapy g-H2AX production. When the 2 treatments were combined, As we observed that the combination of radiotherapy and signiﬁcantly higher g-H2AX production (2.41-fold) was observed dichloroacetate induced higher mitochondrial levels of ROS than (Fig. 4D). Similar results were also obtained from RN1 with 2.08-, each treatment alone, a further experiment was conducted to 1.52-, and 3.48-fold increase for radiotherapy, dichloroacetate, investigate the change in g-H2AX levels, a hallmark of DNA and the combination, respectively (Fig. 4D). damage, after treatment of radiotherapy and dichloroacetate. Western blotting for g-H2AX production was measured 30 min- Dichloroacetate treatment synergizes with radiotherapy to utes after radiotherapy (4 Gy), 24-hour dichloroacetate treatment impair the clonogenicity of glioblastoma cells (50 mmol/L), and the combination (pretreatment with dichlor- Clonogenic survival assays were conducted to examine whether oacetate for 24 hours followed by radiotherapy). For U251, both dichloroacetate is a radiosensitizing agent for glioblastoma cells. radiotherapy and dichloroacetate treatment increased g-H2AX U251, LN229, and DBTRG were irradiated with 2, 4, and 6 Gy production (4.75- and 2.71-fold) compared with untreated con- after 6 hours of pretreatment with dichloroacetate (U87 and RN1 trol, whereas the combination further induced g-H2AX produc- were not selected for this assay as they were not able to form tion up to 8.93-fold (Fig. 4D). Compared with U251, U87 is more clear colonies to be counted). As a single agent, dichloroacetate resistant to the radiotherapy and dichloroacetate treatment. Either treatment induced a dose-dependent clonogenic inhibition in radiotherapy or dichloroacetate treatment as monotherapy these glioblastoma cell lines (Supplementary Table S1). The

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Figure 4. The combination of radiotherapy (RT) and dichloroacetate (DCA) induces oxidative stress and DNA double-strand breaks. Radiotherapy, dichloroacetate, and combination induced a dose-dependent increase in the ﬂuorescence of MitoSOX Red in U251 (A), U87 (B), and RN1 (C). D, expressions of g-H2AX, a hallmark of DNA double-strand breaks, in glioblastoma cells (U251, U87, and RN1) are shown after treatment with radiotherapy, dichloroacetate, and their combination. Western blot analyses are representative of 3 independent experiments. Results are presented as means SD of 3 experiments. , P < 0.01; , P < 0.001; #, P < 0.05; ##, P < 0.01 versus untreated control; $, P < 0.05; $$, P < 0.01 versus untreated control.

clonogenicity of these cells was further impeded when treated orthotopic model by 2 days (P < 0.01), whereas a more significant with combination compared with dichloroacetate and radiother- prolongation of median survival (P < 0.001) was found in apy alone (Fig. 5A–C). For U251 and DBTRG cells, dichloroace- radiotherapy alone (MS, 38 days) arm (Fig. 5D). Moreover, an tate significantly synergized with all of the 3 tested radiotherapy even longer survival benefit was observed in radiotherapy– doses to reduce the colony formation (Fig. 5A and C), whereas for dichloroacetate combination arm (MS, 43 days) compared with LN229 cells, we observed an additive effect when dichloroacetate each treatment alone (P < 0.001 vs. radiotherapy arm; Fig. 5D). was combined with 2 and 4 Gy radiotherapy and synergy with 6 The histologic analysis of the vehicle and treated tumors at day 23 Gy (Fig. 5B). confirmed tumor formation (Fig. 5E) and indicated significant reductions in the proliferation of the tumor cells, as reflected in The combination therapy extends the median survival of number of cells staining for the proliferation marker Ki-67 (Fig. 5F glioblastoma orthotopic model and G). We determined the efficacy of radiotherapy–dichloroacetate combination using a xenograft orthotopic mouse model bearing Discussion U87 cells. The dosage of radiotherapy (20 Gy in 10 fractions) and dichloroacetate (150 mg/kg/d) were selected on the basis of our In the current study, several distinct lines of evidence indicate previous studies showing either of them significantly delayed the that the efficacy of radiotherapy was significantly enhanced by growth of orthotopic U87 xenograft (data not shown). No toxicity dichloroacetate treatment in glioblastoma cells. Our findings first was noticed in any of the treatment arms. Compared with vehicle- confirmed an initial finding from others that a shift of glucose treated control (median survival time; MS, 29 days), dichloroa- metabolism from oxidative phosphorylation to glycolysis cetate alone (MS, 31 days) prolonged the median survival of occurred in glioblastoma cells following radiotherapy, despite

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Figure 5. Efficacy of radiotherapy (RT) in combination with dichloroacetate (DCA) in vitro and in vivo. Clonogenic survival assay showing the combination of dichloroacetate and radiotherapy impairs the clonogenicity of U251 (A), LN229 (B), and DBTRG (C) cells. The surviving fraction curves of radiotherapy/dichloroacetate combination have been normalized to the curves of dichloroacetate-only. Any combination curve overlapping with the radiotherapy-only curve indicates additive effect, whereas any combination curve under the radiotherapy-only curve suggests synergistic effect. Results are representative of 3 experiments and presented as means SD of triplicate measurements. D, Kaplan–Meier survival analysis of xenograft orthotopic U87 model treated with dichloroacetate (150 mg/kg/d), radiotherapy (20 Gy in 10 fractions), and radiotherapy–dichloroacetate combination. E, representative images from H&E staining of mouse brain containing U87 tumor. b, normal brain parenchyma; t, tumor. F, representative images (40) from Ki-67 staining of U87 orthotopic tumor. G, quantification of Ki-67–positive cells in U87 orthotopic tumors treated with 150 mg/kg/d dichloroacetate, 20 Gy radiotherapy, and the combination treatment. Results are expressed as the means SD, with n ¼ 6 in each group. ns, not significant; , P < 0.01; , P < 0.001. #, P < 0.001 versus 20 Gy radiotherapy; ns, not significant.

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ample oxygen availability, which strongly support a switch to citric acid cycle in addition to confirming that there is a aerobic glycolysis. By combining dichloroacetate with radiother- significant fraction of glucose that is shunted to lactate gener- apy, this glycolytic phenotype was reversed. We demonstrated ation. Most recently, the same group of investigators published that dichloroacetate alone moderately induced proliferation data in the orthotopic models and in patients demonstrating arrest at the G2–M phase and reduced the mitochondrial reserve that acetate is also being oxidized in the citric acid cycle, capacity, indicative of sensitizing glioblastoma cells to the effect of which further confirms the significant role of mitochondria in radiotherapy. In addition, dichloroacetate and radiotherapy glucose metabolism of glioblastoma cells. After exposing to worked synergistically to induce elevated mitochondrial ROS ionizing radiation, the uncoupling of oxidative phosphoryla- level as well as g-H2AX production, further confirming the mech- tion occurred (28), which in turn elevates the glycolytic rate, anism of action of this combination. Finally, the clonogenicity of providing cancer cells with numerous advantages via the War- glioblastoma cells was impaired by the combination of dichlor- burg effect (29). In this respect, reversal of the Warburg effect oacetate and radiotherapy, and the survival of an orthotopic offers dual therapeutic advantages by not only enhancing the mouse model was extended significantly by the combination effect of ionizing radiation through induction of oxidative therapy compared with each single treatment arm. All these stress but also lowering acid production resulted from the findings above provide the evidence that modulating the glucose uncoupling of oxidative phosphorylation. metabolism may serve as an effective approach to sensitize these Dichloroacetate is a small-molecule PDK inhibitor that can malignant cells to radiotherapy. penetrate blood–brain barrier and reverse the Warburg effect in Radiotherapy kills cancer cells primarily through free radical– cancer cells (30). It has been demonstrated with moderate induced DNA damage (16). However, it has been shown that this antitumor activity (31–33) and to induce apoptosis in tumors cornerstone of treatment acts as a double-edged sword. Radio- of patients with glioblastoma by restoring the mitochondrial therapy has been linked with HIF1 activation/stabilization, which activity (34). Several studies have also used it as a radiosensi- in turn activates transcription of numerous genes involved in tizer in lung (11), colorectal (13), and prostate cancer cells (12) angiogenesis, proliferation, pH regulation, and glycolytic metab- in vitro. In the present study, we employed dichloroacetate as a olism (17–19). In particular, HIF1 not only initiates transcription radiosensitizer in both in vitro and in vivo glioblastoma models. of genes that encode transporters and enzymes regulating glucose Dichloroacetate was selected for combination with radiother- metabolism but also activates the PI3K/AKT pathway that is apy, as we proposed that by reversing the glycolytic phenotype involved in the regulation of metabolic shift to aerobic glycolysis. with dichloroacetate and directing more pyruvate into mito- The underlying mechanism by which radiotherapy activates HIF1 chondrial oxidation (to produce more ROS), tumor cells was mostly investigated using in vivo preclinical model. It has been would be more sensitive to radiotherapy. We found that con- thought that HIF1 expression increases after radiotherapy due to current use of dichloroacetate with radiotherapy indeed pre- reoxygenation and ROS elevation that induce HIF1a stabilization ventedtheincreaseinglycolyticrateobservedfollowingradio- (20). We confirmed here using cultured glioblastoma cells that therapy alone. Our previous study demonstrated that dichlor- HIF1a was activated postradiotherapy as well as a panel of oacetate effectively induced a dose-dependent proliferation glycolytic genes. Interestingly, the upregulation of PDK1, a crucial arrest of in vitro cultures of glioblastoma cells but had no mitochondrial enzyme that plays an important role linking gly- significant effect on noncancerous cells (15). This result is in colysis to oxidative phosphorylation and a direct target of HIF1a line with most of the studies that investigated the efficacy (21), was also observed. To further confirm this finding, we of dichloroacetate demonstrating that a therapeutic window measured the glucose metabolism using an extracellular flux exists although suprapharmacologic level of dichloroacetate analyzer. A dose-dependent increase in the acid production was (5–50 mmol/L) is necessary to halt the growth of cancer cells observed and a decline in the oxygen consumption occurred in vitro (9, 32, 33, 35). In the current study, we further analyzed simultaneously. the cell-cycle distribution after dichloroacetate treatment show- It has been demonstrated that the glycolytic metabolism in ing that dichloroacetate induced G2–M arrest and decreased the malignancies highly correlates with radioresistance (22). cell proportion in S-phase. These findings are consistent with Tumor cells predominantly using glycolysis counter the direct some (35, 36), but not all studies (12, 37), indicating not all and indirect action of radiotherapy, that is radiotherapy- cancer types share the same mechanism of action after dichlor- induced free radical and oxidative stress, by upregulating the oacetate treatment. As cancer cells are most sensitive to radio- endogenous antioxidant capacity through accumulation of therapy in the G2–M phase of the cell cycle whereas most pyruvate, lactate, and the redox couples glutathione/glutathi- resistance in the S-phase (38–40), our results suggest that one disulfide and NAD(P)H/NAD(P)þ (23). These macromo- dichloroacetate treatment can sensitize glioblastoma cells to lecules are also the products of glycolytic pathway which radiotherapy by altering cell cycle-distribution. Moreover, the constitute an intracellular redox buffer network effectively mitochondrial reserve capacity, a measure of the ability of cells scavenging free radicals and ROS, thus blunting the efficacy to resist oxidative stress (41), was reduced dose dependently of radiotherapy. In this way, it is a potential strategy to over- after dichloroacetate treatment, which further confirms the role come radioresistance by modulating tumor glucose metabo- of dichloroacetate as a potential radiosensitizer to glioblastoma lism and the cellular redox status. Glioblastoma is a deadly cells, even if the inhibition of glycolysis may also partly brain tumor for which there are limited therapies and chemor- contribute to its role of radiosensitization in this setting. adioresistance remains a serious problem to be conquered. As ROS are synthesized during mitochondrial respiration, an Altered glucose metabolism in glioblastoma has been exten- increased rate of mitochondrial oxidation might exacerbate the sively investigated in vitro (5, 24, 25) and, more recently, in vivo ROS-induced DNA damage caused by radiotherapy (42, 43). It in patients (26) and in human orthotopic glioblastoma models has been reported that dichloroacetate treatment induced ROS (27). These studies established that glucose is oxidized in the production both in vitro and in vivo (44). Recent evidence also

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indicated that dichloroacetate increases autophagy in association glioblastoma, suggesting metabolic modulation through PDK with increased ROS production in colorectal cancer cells (45). inhibition as a novel therapeutic strategy for the treatment of this Given ROS plays an important role in their individual mode of devastating brain tumor (34). This study also observed that apo- action of dichloroacetate and radiotherapy, we thus combined ptosis was further increased in the glioblastoma stem cells treated dichloroacetate with radiotherapy and demonstrated that the with dichloroacetate plus temozolomide, indicating dichloroace- combination treatment not only increased the ROS production tate may potentiate the effect of standard chemotherapy. Moreover, but also caused higher level of double-strand breaks than each the use of dichloroacetate reverses the postradiotherapy glycolytic treatment alone. These findings were further verified by our in vitro changes such that the efficacy of radiotherapy is enhanced without efficacy study demonstrating that the clonogenicity of glioblas- adding extra toxicity. Taken together, our findings warrant further toma cells was impaired synergistically when dichloroacetate was evaluation of the combination of dichloroacetate and radiothera- combined with radiotherapy. Ultimately, an orthotopic glioblas- py/temozolomide in clinical trials for newly diagnosed patients toma mouse model was used to test this hypothesis in vivo.A with glioblastoma. survival advantage was observed in the combination treatment arm compared with either single treatment arm and the results Disclosure of Potential Conflicts of Interest were confirmed with histologic examination showing a low No potential conflicts of interest were disclosed. percentage of Ki-67–positive cells in the combination treated tumors. These data again support our conclusion that dichlor- Authors' Contributions oacetate treatment may overcome the radioresistance of glioblas- Conception and design: H. Shen, E. Hau, P.J. Dilda, K.L. McDonald toma cells by reversing the glycolytic phenotype while synergizing Development of methodology: H. Shen, E. Hau, S. Joshi, K.L. McDonald with radiotherapy by stimulating the production of ROS. Notably, Acquisition of data (provided animals, acquired and managed patients, the discordance in the radiosensitizing effect of dichloroacetate provided facilities, etc.): H. Shen, E. Hau Analysis and interpretation of data (e.g., statistical analysis, biostatistics, was observed when it was tested in vitro and in vivo. In vitro, the computational analysis): H. Shen, E. Hau, K.L. McDonald effective radiosensitizing dose of dichloroacetate ranges from 25 Writing, review, and/or revision of the manuscript: H. Shen, E. Hau, P.J. Dilda, to 50 mmol/L evidenced from the result of colony surviving assay K.L. McDonald as well as our mechanistic studies. In contrast, the efficacy of Administrative, technical, or material support (i.e., reporting or organizing radiotherapy was sensitized by dichloroacetate with 150 mg/kg/d data, constructing databases): E. Hau, K.L. McDonald in vivo that gives much lower serum level of dichloroacetate. This Study supervision: E. Hau, K.L. McDonald finding is consistent with previous reports describing that this anti-metabolic compound has better in vivo efficacy than in vitro Acknowledgments activity (37, 46, 47). The reasons for the limited antitumor effect The authors thank Hayley Franklin for preparation of tumor sections; Robert in vitro Rapkins for assisting in PCR assay; Sylvia Chung for initial establishment of of dichloroacetate with clinically relevant doses may lie in orthotopic model; and Sheri Nixdorf for gathering the information of primary the complex cellular physiology and the immense excess of cultures. metabolites existing in cell culture media (46). Because of the in vivo in vitro fact that dichloroacetate has better activity than Grant Support activity, it also has been suggested that there might be unique This study was supported by the grant from Brain Cancer Discovery Collab- metabolic features and special growth pattern for solid tumors oration, Cure Brain Cancer Foundation, and the NHMRC scholarship. that are difficult to recapitulate in vitro and may be critical in K.L. McDonald received grants from the Brain Cancer Discovery Collaboration determining the efficacy of this class of drugs (46). and the Cure Brain Cancer Foundation. E. Hau received scholarship from Dichloroacetate has been used as an orphan drug for various NHMRC. The costs of publication of this article were defrayed in part by the payment of acquired and congenital disorders of mitochondrial metabolism page charges. This article must therefore be hereby marked advertisement in for decades and has recently been proved to be feasible and well- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. tolerated in patients with recurrent malignant gliomas in a recent phase I clinical trial (48). In addition, a recent study has tested the Received March 26, 2015; revised June 1, 2015; accepted June 3, 2015; efficacy of dichloroacetate in a small cohort of patients with published OnlineFirst June 10, 2015.

References 1. Sheehan JP, Shaffrey ME, Gupta B, Larner J, Rich JN, Park DM. Improving 6. Sattler UG, Meyer SS, Quennet V, Hoerner C, Knoerzer H, Fabian C, et al. the radiosensitivity of radioresistant and hypoxic glioblastoma. Future Glycolytic metabolism and tumour response to fractionated irradiation. Oncol 2010;6:1591–601. Radiother Oncol 2010;94:102–9. 2. Fedrigo CA, Grivicich I, Schunemann DP, Chemale IM, dos Santos D, 7. Pitroda SP, Wakim BT, Sood RF, Beveridge MG, Beckett MA, MacDermed Jacovas T, et al. Radioresistance of human glioma spheroids and expression DM, et al. STAT1-dependent expression of energy metabolic pathways links of HSP70, p53 and EGFr. Radiat Oncol 2011;6:156. tumour growth and radioresistance to the Warburg effect. BMC Med 3. Hsieh CH, Wu CP, Lee HT, Liang JA, Yu CY, Lin YJ. NADPH oxidase subunit 2009;7:68. 4 mediates cycling hypoxia-promoted radiation resistance in glioblastoma 8. Papandreou I, Cairns RA, Fontana L, Lim AL, Denko NC. HIF-1 mediates multiforme. Free Radic Biol Med 2012;53:649–58. adaptation to hypoxia by actively downregulating mitochondrial oxygen 4. Zhong J, Rajaram N, Brizel DM, Frees AE, Ramanujam N, Batinic-Haberle I, consumption. Cell Metab 2006;3:187–97. et al. Radiation induces aerobic glycolysis through reactive oxygen species. 9. Abildgaard C, Dahl C, Basse AL, Ma T, Guldberg P. Bioenergetic modula- Radiother Oncol 2013;106:390–6. tion with dichloroacetate reduces the growth of melanoma cells and 5. Zhou Y, Zhou Y, Shingu T, Feng L, Chen Z, Ogasawara M, et al. potentiates their response to BRAFV600E inhibition. J Transl Med Metabolic alterations in highly tumorigenic glioblastoma cells: prefer- 2014;12:247. ence for hypoxia and high dependency on glycolysis. J Biol Chem 10. Ruggieri V, Agriesti F, Scrima R, Laurenzana I, Perrone D, Tataranni T, et al. 2011;286:32843–53. Dichloroacetate, a selective mitochondria-targeting drug for oral squamous

www.aacrjournals.org Mol Cancer Ther; 14(8) August 2015 1803

Shen et al.

cell carcinoma: a metabolic perspective of treatment. Oncotarget 2015; 29. Vander Heiden MG, Cantley LC, Thompson CB. Understanding the War- 6:1217–30. burg effect: the metabolic requirements of cell proliferation. Science 11. Shavit R, Ilouze M, Feinberg T, Lawrence YR, Tzur Y, Peled N. Mitochon- 2009;324:1029–33. drial induction as a potential radio-sensitizer in lung cancer cells - a short 30. Stacpoole PW. The pharmacology of dichloroacetate. Metabolism report. Cell Oncol 2015;38:247–52. 1989;38:1124–44. 12.CaoW,YacoubS,ShiverickKT,NamikiK,SakaiY,PorvasnikS,etal. 31. Liu D, Wang F, Yue J, Jing X, Huang Y. Metabolism targeting therapy of Dichloroacetate (DCA) sensitizes both wild-type and over expressing dichloroacetate-loaded electrospun mats on colorectal cancer. Drug Deliv Bcl-2 prostate cancer cells in vitro to radiation. Prostate 2008;68: 2015;22:136–43. 1223–31. 32. Delaney LM, Ho N, Morrison J, Farias NR, Mosser DD, Coomber BL. 13. Zwicker F, Kirsner A, Peschke P, Roeder F, Debus J, Huber PE, et al. Dichloroacetate affects proliferation but not survival of human colorectal Dichloroacetate induces tumor-specific radiosensitivity in vitro but attenu- cancer cells. Apoptosis 2015;20:63–74. ates radiation-induced tumor growth delay in vivo. Strahlenther Onkol 33. Haugrud AB, Zhuang Y, Coppock JD, Miskimins WK. Dichloroacetate 2013;189:684–92. enhances apoptotic cell death via oxidative damage and attenuates lactate 14. Ferrick DA, Neilson A, Beeson C. Advances in measuring cellular production in metformin-treated breast cancer cells. Breast Cancer Res bioenergetics using extracellular flux. Drug Discov today 2008;13: Treat 2014;147:539–50. 268–74. 34. Michelakis ED, Sutendra G, Dromparis P, Webster L, Haromy A, Niven E, 15. Shen H, Decollogne S, Dilda PJ, Hau E, Chung SA, Luk PP, et al. Dual- et al. Metabolic modulation of glioblastoma with dichloroacetate. Sci targeting of aberrant glucose metabolism in glioblastoma. J Exp Clin Transl Med 2010;2:31ra4. Cancer Res 2015;34:14. 35. Madhok BM, Yeluri S, Perry SL, Hughes TA, Jayne DG. Dichloroacetate 16. Ward JF, Evans JW, Limoli CL, Calabro-Jones PM. Radiation and hydrogen induces apoptosis and cell-cycle arrest in colorectal cancer cells. Br J Cancer peroxide induced free radical damage to DNA. Br J Cancer Suppl 2010;102:1746–52. 1987;8:105–12. 36. Duan Y, Zhao X, Ren W, Wang X, Yu KF, Li D, et al. Antitumor activity of 17. Rademakers SE, Span PN, Kaanders JH, Sweep FC, van der Kogel AJ, Bussink dichloroacetate on C6 glioma cell: in vitro and in vivo evaluation. Onco J. Molecular aspects of tumour hypoxia. Mol Oncol 2008;2:41–53. Targets Ther 2013;6:189–98. 18. Semenza GL. Defining the role of hypoxia-inducible factor 1 in cancer 37. Wong JY, Huggins GS, Debidda M, Munshi NC, De Vivo I. Dichloroacetate biology and therapeutics. Oncogene 2010;29:625–34. induces apoptosis in endometrial cancer cells. Gynecol Oncol 2008; 19. Lee JW, Bae SH, Jeong JW, Kim SH, Kim KW. Hypoxia-inducible factor 109:394–402. (HIF-1)alpha: its protein stability and biological functions. Exp Mol Med 38. Sinclair WK, Morton RA. X-ray sensitivity during the cell generation cycle of 2004;36:1–12. cultured Chinese hamster cells. Radiat Res 1966;29:450–74. 20. Dewhirst MW, Cao Y, Moeller B. Cycling hypoxia and free radicals 39. Sinclair WK. Cyclic x-ray responses in mammalian cells in vitro. Radiat Res regulate angiogenesis and radiotherapy response. Nat Rev Cancer 1968;33:620–43. 2008;8:425–37. 40. Pawlik TM, Keyomarsi K. Role of cell cycle in mediating sensitivity to 21. Kim JW, Tchernyshyov I, Semenza GL, Dang CV. HIF-1-mediated radiotherapy. Int J Radiat Oncol Biol Phys 2004;59:928–42. expression of pyruvate dehydrogenase kinase: a metabolic switch 41. Hill BG, Dranka BP, Zou L, Chatham JC, Darley-Usmar VM. Importance of required for cellular adaptation to hypoxia. Cell Metab 2006;3: the bioenergetic reserve capacity in response to cardiomyocyte stress 177–85. induced by 4-hydroxynonenal. Biochem J 2009;424:99–107. 22. Meijer TW, Kaanders JH, Span PN, Bussink J. Targeting hypoxia, HIF-1, and 42. Trachootham D, Alexandre J, Huang P. Targeting cancer cells by ROS- tumor glucose metabolism to improve radiotherapy efficacy. Clin Cancer mediated mechanisms: a radical therapeutic approach? Nat Rev Drug Res 2012;18:5585–94. Discov 2009;8:579–91. 23. Sattler UG, Mueller-Klieser W. The anti-oxidant capacity of tumour gly- 43. Colen CB, Seraji-Bozorgzad N, Marples B, Galloway MP, Sloan AE, Mathu- colysis. Int J Radiat Biol 2009;85:963–71. pala SP. Metabolic remodeling of malignant gliomas for enhanced sensi- 24. Vlashi E, Lagadec C, Vergnes L, Matsutani T, Masui K, Poulou M, et al. tization during radiotherapy: an in vitro study. Neurosurgery 2006;59: Metabolic state of glioma stem cells and nontumorigenic cells. Proc Natl 1313–23; discussion 1323–4. Acad Sci U S A 2011;108:16062–7. 44. Sutendra G, Michelakis ED. Pyruvate dehydrogenase kinase as a novel 25. Wolf A, Agnihotri S, Micallef J, Mukherjee J, Sabha N, Cairns R, et al. therapeutic target in oncology. Front Oncol 2013;3:38. Hexokinase 2 is a key mediator of aerobic glycolysis and promotes 45. Lin G, Hill DK, Andrejeva G, Boult JK, Troy H, Fong AC, et al. Dichlor- tumor growth in human glioblastoma multiforme. J Exp Med 2011;208: oacetate induces autophagy in colorectal cancer cells and tumours. Br J 313–26. Cancer 2014;111:375–85. 26. Maher EA, Marin-Valencia I, Bachoo RM, Mashimo T, Raisanen J, Hatanpaa 46. Papandreou I, Goliasova T, Denko NC. Anticancer drugs that target KJ, et al. Metabolism of [U-13 C]glucose in human brain tumors in vivo. metabolism: Is dichloroacetate the new paradigm? Int J Cancer 2011; NMR Biomed 2012;25:1234–44. 128:1001–8. 27. Marin-Valencia I, Yang C, Mashimo T, Cho S, Baek H, Yang XL, et al. 47. Kumar K, Wigfield S, Gee HE, Devlin CM, Singleton D, Li JL, et al. Analysis of tumor metabolism reveals mitochondrial glucose oxidation in Dichloroacetate reverses the hypoxic adaptation to bevacizumab and genetically diverse human glioblastomas in the mouse brain in vivo. Cell enhances its antitumor effects in mouse xenografts. J Mol Med Metab 2012;15:827–37. 2013;91:749–58. 28. Scaife JF, Hill B. Uncoupling of oxidative phosphorylation by ionizing 48. Dunbar EM, Coats BS, Shroads AL, Langaee T, Lew A, Forder JR, et al. Phase radiation. II. The stability of mitochondrial lipids and cytochrome c. Can J 1 trial of dichloroacetate (DCA) in adults with recurrent malignant brain Biochem Physiol 1963;41:1223–33. tumors. Invest New Drugs 2014;32:452–64.

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Sensitization of Glioblastoma Cells to Irradiation by Modulating the Glucose Metabolism

Han Shen, Eric Hau, Swapna Joshi, et al.

Mol Cancer Ther 2015;14:1794-1804. Published OnlineFirst June 10, 2015.

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