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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Cancer Res. Author manuscript; available in PMC 2010 July 21.
Published in final edited form as:
PMCID: PMC2907516
NIHMSID: NIHMS89146

Silencing of Elongation Factor-2 Kinase Potentiates the Effect of 2-Deoxy-D-Glucose against Human Glioma Cells through Blunting of Autophagy*

Abstract

2-Deoxy-D-glucose (2-DG), a synthetic glucose analog that acts as a glycolytic inhibitor, is currently being evaluated in the clinic as an anticancer agent. In this study, we observed that treatment of human glioma cells with 2-DG activated autophagy, a highly conserved cellular response to metabolic stress and a catabolic process of self-digestion of intracellular organelles for energy utilization and survival in stressed cells. The induction of autophagy by 2-DG was associated with activation of elongation factor-2 kinase (eEF-2 kinase), a structurally and functionally unique enzyme that phosphorylates eEF-2 leading to loss of affinity of this elongation factor for the ribosome and to termination of protein elongation. We also showed that inhibition of eEF-2 kinase by RNA interference blunted the 2-DG-induced autophagic response, resulted in a greater reduction of cellular ATP contents, and increased the sensitivity of tumor cells to the cytotoxic effect of 2-DG. Furthermore, the blunted autophagy and enhanced 2-DG cytotoxicity were accompanied by augmentation of apoptosis in cells in which eEF-2 kinase expression was knocked down. The results of this study indicate that the energy stress and cytotoxicity caused by 2-DG can be accelerated by inhibition of eEF-2 kinase, and suggest that targeting eEF-2 kinase – regulated autophagic survival pathway may represent a novel approach to sensitizing cancer cells to glycolytic inhibitors.

Keywords: Elongation factor-2 kinase, 2-Deoxy-D-Glucose, Glycolysis, Autophagy, Protein synthesis, Glioblastoma

Introduction

Cellular metabolism of malignant cells differs significantly from that of normal cells. While normal cells rely on respiration, a process that consumes oxygen and glucose to produce energy-storing molecule ATP, malignant cells mainly depend on glycolysis, the anaerobic metabolism of glucose into ATP, even in the presence of sufficient oxygen. This increased dependency of malignant cells on glycolysis for ATP production is known as the so-called Warburg effect (1, 2). A number of molecular pathways have been revealed to be associated with this metabolic phenotype, including hexokinase 2 (3, 4), p53 (5), c-Myc (6, 7), HIF-1 (7), and defect in mitochondrial respiration (8). Due to the high dependence of malignant cells on glycolysis, interfering with this metabolic process has recently been proposed as a potentially useful approach for developing new selective cancer therapy (9). Treatment of cancer cells with 2-deoxy-D-glucose (2-DG), a synthetic glucose analog that acts as a glycolytic inhibitor, has been shown to inhibit growth and viability of cancer cells (1012), and enhance the efficacy of cancer chemotherapeutics and radiation regiments (1318). In both in vitro and in vivo models, 2-DG was effective in the treatment of a variety of solid tumors (1921). The pharmacologic basis of anti-tumor action of 2-DG is believed to be the high dependence of malignant cells, especially those hypoxic cells on glycolysis, the preferred ingestion and retention of 2-DG by tumor cells, and the blocking effect of 2-DG on glucose metabolic pathways. In addition, 2-DG causes oxidative stress through increasing pro-oxidant production and disrupting thiol metabolism, as evidenced by alterations in total glutathione content (16, 22). In the treatment of human brain malignancies, 2-DG has been shown to be effective in sensitizing tumor cells to radiation therapy (17, 23). Despite the demonstrations of the antitumor activity of 2-DG, large doses are usually needed to achieve a therapeutic effect, and cancer cells quickly become refractory to this agent. Therefore, approaches that can enhance the efficacy of 2-DG may make this agent more useful in the treatment of cancers.

Elongation factor-2 kinase (eEF-2 kinase; a.k.a. calmodulin-dependent protein kinase III), a unique calmodulin/calcium - dependent enzyme that inhibits protein synthesis, is overexpressed in several types of malignancies including gliomas (24, 25). eEF-2 kinase phosphorylates elongation factor-2, a 100 kDa protein that mediates the translocation step in peptide-chain elongation by inducing the transfer of peptidyl-tRNA from the ribosomal A to P site. Phosphorylation of EF-2 at Thr56 by eEF-2 kinase decreases the affinity of this elongation factor for ribosomes and terminates elongation, thereby inhibiting protein synthesis. Since protein synthesis requires a large proportion of cellular energy (26, 27), inhibition of protein synthesis by terminating elongation through activating eEF-2 kinase decreases energy utilization, and provides a survival mechanism against energy stress.

We have recently reported the critical role of eEF-2 kinase in the regulation of autophagy, a highly conserved cellular process that is activated in times of metabolic or environmental stress and leads to large-scale degradation of proteins (28). The process of autophagy involves formation of a double-membrane vesicle (“autophagosome”) in the cytosol that engulfs organelles and cytoplasm, then fuses with the lysosome to form the autolysosme, where the contents are degraded and recycled for protein and ATP synthesis (29). The formation of the autophagosome is mediated by a series of autophagy specific genes (ATGs). This form of self-digestion leads to self-preservation in times of nutrient deprivation; however, if left unchecked autophagy has the potential of producing terminal self-consumption. While apoptosis is known as type I cell death, autophagy is referred to as type II cell death. We demonstrated in human glioma cells that the activity of eEF-2 kinase is closely associated with the mammalian macroautophagy pathway that is activated in response to nutrient, growth factor or oxygen deprivation. Furthermore, inhibition of eEF-2 kinase blunts autophagy and has deleterious effects on cell viability under nutrient starvation condition (28). These observations raise the possibility that blocking the activation of eEF-2 kinase may represent a potential therapeutic strategy to promote cell death induced by metabolic stress. As 2-DG inhibits cell growth and causes death of tumor cells through antagonizing glucose (20), we sought to determine the effects of this glycolytic inhibitor on eEF-2 kinase activity and autophagic cell survival pathway, and the impact of inhibiting eEF-2 kinase on sensitivity of tumor cells to 2-DG. We found that treatment of human glioma cells with 2-DG activated an eEF-2 kinase - dependent autophagic response, and inhibiting eEF-2 kinase by RNA interference (RNAi) blocked the induction of autophagy by 2-DG and increased the sensitivity of tumor cells to the cytotoxic effect of this glycolytic inhibitor. Our results suggest that targeting the eEF-2 kinase - regulated autophagic pathway could be an effective approach to augmenting the activity of 2-DG, and thus may have clinical implication in cancer treatment.

Materials and Methods

Cell lines and culture

The human glioblastoma cell lines, T98G and LN-229, were purchased from American Type Culture Collection (Manassas,VA). T98G cells were cultured in Ham’s F-10 : DMEM (10:1) medium, and LN-229 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin and 100 µg/ml streptomycin. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2/95% air.

Reagents and antibodies

2-Deoxy-D-Glucose, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and anti-α-tubulin antibodies were purchased from Sigma Chemical Co. (St. Louis, MO). All cell culture media and other products were purchased from Invitrogen Corporation (Carlsbad, California). Chemiluminescence Western blot reagents were obtained from Pierce Biotechnology, Inc. (Rockford, IL). The following antibodies were used in this study: anti-caspase-3 antibody, anti-eEF-2 kinase, anti-eEF-2, anti-phospho-eEF-2 (Thr56), anti-phospho-AMPKα (Thr172), and anti-AMPKα anti-S6 kinase, anti-phospho-S6 kinase (Thr389) antibodies (Cell Signaling Technologies, Beverly, MA); monoclonal anti-LC3 antibody (nanoTools, though Axxora, LLC, San Diego, CA).

siRNA preparation and transfection

siRNA sequence targeting eEF-2 kinase mRNA corresponded to the coding region 144–164 (5'-AAGCTCGAACCAGAATGTC-3') relative to the start codon (28). siRNA duplexes were prepared by Dharmacon Research, Inc. (Lafayette, CO). For transfection, cells in exponential phase of growth were plated in 60-mm tissue culture dishes at 5 × 105 cells per dish, grown for 24 hours, then transfected with siRNA (100 nM) using Oligofectamine and OPTI-MEM I-reduced serum medium, according to the protocol of the manufacturer. The concentrations of siRNAs were chosen based on dose-response studies. Twenty-four hours following transfection, the cells were harvested for further experiments.

Measurement of protein synthesis

The rate of protein synthesis was measured as described previously (30). Briefly, cells were seeded in 60-mm tissue culture dishes and labeled with 25 µCi/ml of EasyTag EXPRESS [35S] protein labeling mix (PerkinElmer, Boston, MA) in RPMI 1640 medium. After incubation at 37°C for 15 min, cells were washed 4 times with 4 ml of ice-cold PBS and lysed in 200 µl of Complete Lysis-M lysis reagent containing the Mini Protease Inhibitor Cocktail (Roche Diagnostics, Indianapolis, IN). Lysates were collected in a microfuge tube and clarified by centrifugation at 13000 × g for 10 min at 4°C. The supernatants were precipitated with 20% of trichloracetic acid and collected on GF/C filters (Millipore, Bedford, MA). The filters were washed 4 times with 1ml of 10% trichloracetic acid and subject to liquid scintillation counting. The specific activity of protein synthesis was determined by the amount of incorporated 35S-methionine/cysteine per mg of total protein per min.

Measurement of cellular ATP

Cells were plated in 96-well plates at 2.5 × 103 cells per well and treated with 2-DG for 24h. ATP contents were determined using the ATPlite™ Luminescence Assay Kit (PerkinElmer, Boston, MA) according to the manufacturer’s protocol. The luminescence was measured by a Victor3 Multi Label plate reader (PerkinElmer, Boston, MA).

Measurement of Autophagy

Autophagy was monitored by measuring the formation of LC3-II in the absence or presence of lysosomal protease inhibitors E64D (10 µg/ml) and pepstatin A (10 µg/ml), as described by Tanida et al. (31). For detection of LC3-II, cell lysates were prepared and 25 µg of total proteins were subjected to Western blot analysis using a monoclonal anti-LC3 antibody. GFP-LC3 cleavage assay was performed as previously described (32). Briefly, cells (1 × 106) were co-transfected with 3µg of GFP-LC3 plasmid DNA and an eEF-2 kinase - targeted siRNA or a non-targeting RNA in Opti-MEM reduced medium (Invitrogen) and incubated overnight at 37°C. The cells were then treated with 2-DG in the presence of lysosomal protease inhibitors E64d (10 µg/ml) and pepstatin A (10 µg/ml). At the end of treatment, cells were fixed with 4% formaldehyde for 15min and inspected at 60× magnification for numbers of GFP-LC3 puncta. Autophagy was also evaluated by electron microscopic examination of double or multi-membrane vacuoles in the cytoplasm, as described below.

Western blot analysis

Cells were pelleted at 500 × g for 5 minutes and were lysed in cold lysis buffer [20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton X-100, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L β-glycerolphosphate, 1 mmol/L Na3VO4, 1 µg/mL leupeptin, and 1 mmol/L PMSF] and sonicated for 5 seconds. The lysates were clarified by centrifugation at 12000 × g for 30 minutes at 4°C. Identical amounts (25 µg of protein) of cell lysates were resolved by 8% or 15% SDS-PAGE, and proteins were transferred onto nitrocellulose or PVDF. Membranes were incubated in blocking solution consisting of 5% powered milk in TBST [10 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, and 0.1% Tween 20] for 1 hour, then immunoblotted with the respective antibodies. Detection by enzyme-linked chemiluminescence was carried out according to the protocol of the manufacturer.

Electron microscopy

Cells were harvested by trypsinization, fixed in 2.5% gluteraldehyde/4% paraformaldehyde in 0.1 mol/L cacodylate buffer, and then post-fixed in 1% osmium tetroxide buffer. After dehydration in acetone, the cells were embedded in spur resin. Thin sections (90 nm) were cut on a Reichert Ultracut E microtome. Sectioned grids were stained with saturated solution of uranyl acetate and lead citrate. Sections were examined at 80 kV with a JEOL 1200EX transmission electron microscope.

Cellular viability

Cell viability was measured by MTT assay. Briefly, cells were plated at 5 × 103 per well in 96-well tissue culture plates and incubated at 37°C in a humidified atmosphere containing 5% CO2/95% air. The formazan product, formed after a 4-hour incubation with MTT, was dissolved in DMSO and read at 570 nm using a Victor3 Multi Label plate reader (PerkinElmer, Boston, MA).

Measurement of Apoptosis

Active caspase-3, which is present in cells undergoing apoptosis, was detected by Western blot using polyclonal anti- caspase-3 antibody that recognizes the cleaved, active caspase-3, as described previously (33). For apoptotic nuclei staining, pEGFP-transfected cells with different treatments were stained with Hoechst 33342 and observed under green fluorescence and UV. GFP-positive cells were scored for the presence of apoptotic nuclei, and apoptotic rates were presented as the percentage of apoptotic cells out of the total number of GFP-positive cells assessed (34).

Results

Treatment with 2-DG Activates eEF-2 Kinase and Decreases Protein Synthesis in Glioma Cells

We previously reported that nutrient depletion caused activation of eEF-2 kinase and autophagy in glioma cells, and blocking activation of eEF-2 kinase blunted the autophagic response (28). To determine the specific effect of glucose deprivation on the activity of eEF-2 kinase and autophagy, we first treated T98G and LN229 human glioma cells with varying concentrations of 2-DG, and then assayed the activity of the kinase. As shown in Figure 1A, treatment of these glioma cells with 2-DG for 24 h increased the activity of eEF-2 kinase in a dose-dependent manner, as evidenced by the increased phosphorylation of its substrate, EF-2. To confirm the stimulatory effect of 2-DG on eEF-2 kinase activity, we compared the rate of protein synthesis in the cells treated with 2-DG to that of the cells treated with vehicle. Figure 1B shows that protein synthesis in the cells treated with 2-DG was markedly inhibited as compared to that in vehicle - treated cells. These results were consistent with the role of eEF-2 kinase in regulating translation, i.e., activation of eEF-2 kinase inhibits elongation. Activation of eEF-2 kinase by 2-DG was accompanied by a reduction of cellular ATP contents (Figure 2A), an inactivation (decreased phosphorylation) of the key translational governor and downstream effector of mTOR, S6 kinase (Figure 2B), and an activation of AMPK, as demonstrated by the increased phosphorylation of this intracellular energy sensor (Figure 2C). These results indicate that treatment of glioma cells with 2-DG elicited an energy stress response.

Figure 1
Effects of 2-DG on the activity of eEF-2 kinase (A) and protein synthesis (B) in glioma cells
Figure 2
Effect of 2-DG on ATP content (A), S6 kinase activity (B) and AMP kinase activity (C) in glioma cells

Treatment with 2-DG Induces Autophagy in Glioma Cells

Given the effects of 2-DG on the activities of eEF-2 kinase (Figure 1), S6 kinase, AMPK and the cellular level of ATP (Figure 2), we next determined whether treatment of tumor cells with this glycolytic inhibitor induced autophagy. LC3-II, a cleaved product of microtubule-associated protein 1 light chain 3, was used as a marker for autophagy. We found that both steady – state level (Figure 3A) and turnover (Figure 3B) of LC3-II were increased in the glioma cells treated with 2-DG, as compared to the cells treated with the vehicle. The induction of autophagy by 2-DG was confirmed by electron microscopy (Figure 3C), which visualized abundant double or multi-membrane vacuoles in the cytoplasm of the cells treated with 2-DG. By contrast, these vacuoles were rarely observed in glioma cells treated with the vehicle (Figure 3C). Although the basal level of autophagy in cells cultured in medium containing high concentration of glucose (25 mM) was much lower than that in cells cultured in medium with low concentration of glucose (5.6 mM), under either condition 2–DG activated autophagy in these tumor cells, and the levels of autophagy were correlated to the activity of eEF-2 kinase (Figure 3D).

Figure 3
Effect of 2-DG on autophagy in glioma cells

Silencing of EF-2 Kinase Expression Blunts the 2-DG-induced Autophagy

To further investigate whether induction of autophagy by 2-DG was mediated through eEF-2 kinase, we silenced the expression of eEF-2 kinase using RNAi approach and then determined the effect of eEF-2 kinase inhibition on the autophagic response to 2-DG treatment. Tumor cells were transfected with an eEF-2 kinase - targeted siRNA or non-targeting RNA for 24 h, and then treated with various concentrations of 2-DG. Figure 4A shows that knockdown of eEF-2 kinase expression decreased the activity of the enzyme, as indicated by the dramatic decreases in the phospo-EF-2 at Thr56, and blunted the autophagic response in the cells treated with 2-DG, as manifested by the decreased formation of LC3-II. Electron microscopy and GFP-LC3 cleavage assay also demonstrated that silencing of eEF-2 kinase by siRNA blocked the 2-DG – induced autophagy, as evidenced by the decreased formation of double-membrane vacuoles in the cytoplasm (Figure 4B) and decreased numbers of cells with > 20 GFP-LC3 punctae (Figure 4C). Moreover, silencing of eEF-2 kinase also diminished the inhibitory effect of 2-DG on protein synthesis (Figure 5A) and accelerated the 2-DG - induced reduction of cellular ATP contents (Figure 5B), further indicating that inhibition of this kinase weakens the adaptive response of cells to metabolic stress and worsens the energy supply.

Figure 4
Silencing of eEF-2 kinase expression blunts 2-DG – activated autophagy
Figure 5
Silencing of eEF-2 kinase expression mitigates the inhibition of protein synthesis (A) and worsened the energy stress (B) in the 2-DG-treated glioma cells

Silencing of eEF-2 Kinase Expression Enhances the Sensitivity of Glioma Cells to 2-DG

It has been shown that autophagy promotes cell survival under conditions of nutrient deprivation and that inhibiting eEF-2 kinase can abrogate this pro-survival response (28). To test whether blockade of 2-DG – activated autophagy via inhibiting eEF-2 kinase could enhance the cytotoxicity of this glycolytic inhibitor, we transfected tumor cells with a non-targeting RNA or siRNA targeting eEF-2 kinase, then exposed the cells to various concentrations of 2-DG. As shown in Figure 6A, the cytotoxicity of 2-DG was significantly increased by silencing eEF-2 kinase expression in T98G and LN-229 glioma cells. To explore the mechanism underlying the increased 2-DG cytotoxicity in cells with decreased eEF-2 kinase activity, we compared apoptosis in tumor cells with and without silencing of the enzyme following 2-DG treatment. Figure 6B demonstrates that treatment of T98G cells with 2-DG not only activated autophagy, as measured by the formation of LC3-II, but also triggered apoptosis in a dose – dependent manner, as measured by the activation of caspase-3. More notably, inhibition of autophagy by silencing of eEF-2 kinase augmented apoptosis triggered by 2-DG, as evidenced by the increased appearance of the cleaved form of caspase-3 (Figure 6B) and increased apoptotic nuclei (Figure 6C), suggesting an increased death in cells with inhibition of eEF-2 kinase.

Figure 6
Silencing of eEF-2 kinase expression increases the sensitivity of glioma cells to the cytotoxicity of 2-DG

Discussion

eEF-2 kinase is a calcium/calmodulin - dependent enzyme that regulates protein elongation (35), and has been observed to be up-regulated in human and rat gliomas (25, 36). This kinase was subsequently found to have several unique characteristics including the ability to phosphorylate serines and threonines within alpha helical turns (37). Further work by Proud’s group demonstrated the exquisite regulation of eEF-2 kinase by multiple enzymes involved in energy sensing and utilization, including mTOR, S6 kinase, and AMP kinase (3840). The role of eEF-2 kinase as an energy sensor was also demonstrated in a study showing that this enzyme participates in the AMPK-mediated cardio-protection in response to metabolic stress (41). When exploring the role of eEF-2 kinase in malignant cells, we found that the activity of eEF-2 kinase and autophagy were rapidly increased by nutrient deprivation and that inhibiting the enzyme markedly diminished autophagic cell survival (28), suggesting that this apparent relationship between eEF-2 kinase activity and cell survival might be linked directly to its role in protein synthesis.

In this study, we utilized the glycolytic inhibitor, 2-DG, to further explore the role of eEF-2 kinase in the autophagic response to energy stress. We demonstrate in human glioma cells that treatment with 2-DG activates autophagy (Figure 3); the induction of autophagy by 2-DG appears to be dependent on the activity of eEF-2 kinase, as treatment with 2-DG also activates this kinase (Figure 1); and knockdown of eEF-2 kinase expression blunts the autophagic response induced by 2-DG (Figure 4). Furthermore, we show that silencing of eEF-2 kinase expression increases the cytotoxcity of 2-DG (Figure 6A) and augments apoptosis (Figure 6B and C). Since autophagy is known to favor cell survival under environmental and metabolic stress conditions (4244), 2-DG – induced autophagy may represent an adaptive response to the reduction of energy supply caused by this glycolytic inhibitor, and explain, at least in part, the decreased sensitivity of tumor cells after exposure to this agent. Although the two glioma cell lines used in this study, T98G and LN-229, harbor aberrant and wild-type PTEN, respectively (45, 46), our results from the two cell lines were consistent, suggesting that different expression of PTEN does not affect the autophagic response to 2-DG treatment and the effect of inhibiting eEF-2 kinase on sensitivity to 2-DG.

The results showing that 2-DG – induced autophagy is associated with the activity of eEF-2 kinase are consistent with our previous observation that induction of autophagy by metabolic stress is regulated by this kinase (28), although the precise mechanism underlying this regulation is still unclear. The association of eEF-2 kinase activity with the regulation of autophagy is consistent with the function of this kinase, which acts as an inhibitor of peptide elongation. Protein synthesis is a process that accounts for a major percentage of energy consumption (26, 27); therefore, inhibition of protein synthesis may decrease cellular energy utilization to withstand nutrient starvation such as glucose deprivation. The activity of eEF-2 kinase is tightly regulated by signaling pathways involved in nutrient utilization and energy monitoring in the cell. For example, Proud’s group reported that eEF-2 kinase is inhibited by mTOR and S6 kinase signaling pathways (38). They also show that eEF-2 kinase is activated by AMP kinase, an intracellular energy sensor that regulates cell metabolism (39, 40). Thus, the activation of eEF-2 kinase in 2-DG – treated cells could be a consequence of energy stress caused by this glycolytic inhibitor, as treatment of this agent leads to reduction of cellular ATP contents (Figure 2A), decreased activity of S6 kinase (Figure 2B) and increased activity of AMP kinase (Figure 2C). A recent study in breast cancer cells also demonstrates the activation of AMP kinase and deactivation of S6 kinase by 2-DG (47). These results support our hypothesis that induction of autophagy by 2-DG is a type of metabolic adaptation to energy stress, since autophagy can increase ATP production through autophagic recycling of amino acids produced from digestion of cellular organelles and proteins, thus favoring cell survival in times of cellular stress. The stimulatory effect of 2-DG on autophagy may also contribute to the phenomenon that this agent protects glioma cells from glucose withdrawal – induced cell death (48). Additionally, our results showing that basal level of autophagy is higher in cells cultured in medium containing low concentration of glucose than in cells cultured in medium containing high concentration of glucose (Figure 3D) also support the role of autophagy as a pro-survival mechanism in cells stressed by nutrient deprivation or metabolic alteration. Silencing of eEF-2 kinase does not completely suppress the autophagic response activated by 2-DG (Figure 4), probably due to the fact that 2-DG treatment also causes oxidative stress (49) and this type of stress is known to be a trigger for autophagy via other pathways (50). Thus, inhibiting multiple autophagic pathways might result in a more profound suppression of 2-DG - activated autophagy and hence increase the anticancer activity of this agent. It has been reported that inhibition of glutamate cysteine ligase, an enzyme involved in the glutathione synthetic pathway, sensitizes tumor cells to the cytotoxicity of 2-DG (22). We recognize that autophagy may also lead to cell death that is accompanied by caspase activation (51); however, based on our results, the role of autophagy is pro-survival but not pro-death in response to 2-DG treatment, as we observed that while silencing of eEF-2 kinase expression further increased caspase-3 activation in glioma cells treated with 2-DG (Figure 6B), autophagy was inhibited. Moreover, cell viability was also decreased in siRNA-treated cells in comparison to the non-targeting RNA-treated cells (Figure 6A).

Malignant cells utilize glucose at a higher rate than normal cells, and become more dependent on aerobic and anaerobic glycolysis. Hypoxic tumor cells are particularly dependent on anaerobic glycolysis, and are therefore more sensitive to 2-DG - induced cell cycle inhibition and cytotoxicity (12). Moreover, due to the up-regulation of glucose transporters in tumor cells and high affinity of 2-DG for glucose transporters, malignant cells show an enhanced uptake and retention of this agent (52). These factors are believed to contribute to the preferential toxicity of 2-DG in cancer cells. In this study, we demonstrate that targeting eEF-2 kinase, an inhibitor of protein elongation, can enhance the sensitivity of glioma cells to 2-DG (Figure 6A). The potentiation of 2-DG cytotoxicity by inhibition of eEF-2 kinase appears to result from the metabolic catastrophe caused by the glycolytic inhibitor, as autophagy is markedly attenuated and ATP level is further decreased (due to recovery of protein synthesis) in tumor cells with silencing of eEF-2 kinase in comparison with the cells without the silencing of the enzyme (Figure 4 and Figure 5). Inducing metabolic catastrophe in cancer cells has been proposed as a new therapeutic approach awaiting for further investigation (53).

2-DG has entered into phase I clinical trials, but results of the clinical studies have not been well documented in literatures. However, a recent study reported that 2-DG significantly prolongs survival of the mice bearing aggressive lymphoma with defective laforin expression (21). The results of the current study demonstrate that the cytotoxic effect of 2-DG on tumor cells can be potentiated by suppressing an eEF-2 kinase - dependent autophagic survival pathway, suggesting that 2-DG treatment in combination with the inhibition of eEF-2 kinase may stand for a new therapeutic strategy to improve the efficacy of this glycolytic inhibitor against malignant tumors. We have identified the inhibitors of eEF-2 kinase and reported their effects on cancer cell lines (54), and are currently evaluating the in vivo effectiveness and pharmacokinetic properties of those compounds through NCI’s Rapid Access to Intervention Development (RAID) program. Since eEF-2 kinase is up-regulated in several types of cancers, we expect that development and use of inhibitors of the kinase with favorable pharmacokinetic characteristics would increase the selectivity and effectiveness of glycolytic inhibitors such as 2-DG. Taken together, our study underscores the potential of eEF-2 kinase as a complementary target for sensitizing tumor cells to the glycolysis - targeted therapy.

The abbreviations used are

2-DG
2-deoxy-D-glucose
eEF-2
eukaryotic elongation factor-2
AMPK
AMP-activated protein kinase
RNAi
RNA interference
siRNA
small interfering RNA
MAP-LC3
Microtubule-associated protein 1 light chain 3
MTT
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

Footnotes

*Supported by grant from the US Public Health Service CA43888, the philanthropic fund from the Marks family (Howard Marks and Nancy Marks), and the grant from American Cancer Society.

References

1. Warburg O. On respiratory impairment in cancer cells. Science. 1956;124:269–270. [PubMed]
2. Warburg O. On the origin of cancer cells. Science. 1956;123:309–314. [PubMed]
3. Bustamente E, Morris HP, Pedersen PL. Hexokinase: the direct link between mitochondrial and glycolytic reactions in rapidly growing cancer cells. Adv Exp Med Biol. 1977;92:363–380. [PubMed]
4. Pedersen PL. Voltage dependent anion channels (VDACs): a brief introduction with a focus on the outer mitochondrial compartment's roles together with hexokinase-2 in the "Warburg effect" in cancer. J Bioenerg Biomembr. 2008;40:123–126. [PubMed]
5. Matoba S, Kang JG, Patino WD, et al. p53 regulates mitochondrial respiration. Science. 2006;312:1650–1653. [PubMed]
6. Kim JW, Gao P, Liu YC, Semenza GL, Dang CV. Hypoxia-inducible factor 1 and dysregulated c-Myc cooperatively induce vascular endothelial growth factor and metabolic switches hexokinase 2 and pyruvate dehydrogenase kinase 1. Mol Cell Biol. 2007;27:7381–7393. [PMC free article] [PubMed]
7. Dang CV, Kim JW, Gao P, Yustein J. The interplay between MYC and HIF in cancer. Nat Rev Cancer. 2008;8:51–56. [PubMed]
8. Pelicano H, Xu RH, Du M, et al. Mitochondrial respiration defects in cancer cells cause activation of Akt survival pathway through a redox-mediated mechanism. J Cell Biol. 2006;175:913–923. [PMC free article] [PubMed]
9. Pan JG, Mak TW. Metabolic targeting as an anticancer strategy: dawn of a new era? Sci STKE. 2007:pe14. [PubMed]
10. Zhang XD, Deslandes E, Villedieu M, et al. Effect of 2-deoxy-D-glucose on various malignant cell lines in vitro. Anticancer Res. 2006;26:3561–3566. [PubMed]
11. Aft RL, Zhang FW, Gius D. Evaluation of 2-deoxy-D-glucose as a chemotherapeutic agent: mechanism of cell death. Br J Cancer. 2002;87:805–812. [PMC free article] [PubMed]
12. Maher JC, Krishan A, Lampidis TJ. Greater cell cycle inhibition and cytotoxicity induced by 2-deoxy-D-glucose in tumor cells treated under hypoxic vs aerobic conditions. Cancer Chemother Pharmacol. 2004;53:116–122. [PubMed]
13. Dwarakanath BS, Khaitan D, Ravindranath T. 2-deoxy-D-glucose enhances the cytotoxicity of topoisomerase inhibitors in human tumor cell lines. Cancer Biol Ther. 2004;3:864–870. [PubMed]
14. Maschek G, Savaraj N, Priebe W, et al. 2-deoxy-D-glucose increases the efficacy of adriamycin and paclitaxel in human osteosarcoma and non-small cell lung cancers in vivo. Cancer Res. 2004;64:31–34. [PubMed]
15. Simons AL, Ahmad IM, Mattson DM, Dornfeld KJ, Spitz DR. 2-Deoxy-D-glucose combined with cisplatin enhances cytotoxicity via metabolic oxidative stress in human head and neck cancer cells. Cancer Res. 2007;67:3364–3370. [PubMed]
16. Lin X, Zhang F, Bradbury CM, et al. 2-Deoxy-D-glucose-induced cytotoxicity and radiosensitization in tumor cells is mediated via disruptions in thiol metabolism. Cancer Res. 2003;63:3413–3417. [PubMed]
17. Mohanti BK, Rath GK, Anantha N, et al. Improving cancer radiotherapy with 2-deoxy-D-glucose: phase I/II clinical trials on human cerebral gliomas. Int J Radiat Oncol Biol Phys. 1996;35:103–111. [PubMed]
18. Hernlund E, Ihrlund LS, Khan O, et al. Potentiation of chemotherapeutic drugs by energy metabolism inhibitors 2-deoxyglucose and etomoxir. Int J Cancer. 2008;123:476–483. [PubMed]
19. Kern KA, Norton JA. Inhibition of established rat fibrosarcoma growth by the glucose antagonist 2-deoxy-D-glucose. Surgery. 1987;102:380–385. [PubMed]
20. Zhu Z, Jiang W, McGinley JN, Thompson HJ. 2-Deoxyglucose as an energy restriction mimetic agent: effects on mammary carcinogenesis and on mammary tumor cell growth in vitro. Cancer Res. 2005;65:7023–7030. [PubMed]
21. Wang Y, Liu Y, Wu C, McNally B, Liu Y, Zheng P. Laforin confers cancer resistance to energy deprivation-induced apoptosis. Cancer Res. 2008;68:4039–4044. [PMC free article] [PubMed]
22. Andringa KK, Coleman MC, Aykin-Burns N, et al. Inhibition of glutamate cysteine ligase activity sensitizes human breast cancer cells to the toxicity of 2-deoxy-D-glucose. Cancer Res. 2006;66:1605–1610. [PubMed]
23. Dwarkanath BS, Zolzer F, Chandana S, et al. Heterogeneity in 2-deoxy-D-glucose-induced modifications in energetics and radiation responses of human tumor cell lines. Int J Radiat Oncol Biol Phys. 2001;50:1051–1061. [PubMed]
24. Parmer TG, Ward MD, Yurkow EJ, Vyas VH, Kearney TJ, Hait WN. Activity and regulation by growth factors of calmodulin-dependent protein kinase III (elongation factor 2-kinase) in human breast cancer. Br J Cancer. 1999;79:59–64. [PMC free article] [PubMed]
25. Bagaglio DM, Cheng EH, Gorelick FS, Mitsui K, Nairn AC, Hait WN. Phosphorylation of elongation factor 2 in normal and malignant rat glial cells. Cancer Res. 1993;53:2260–2264. [PubMed]
26. Szaflarski W, Nierhaus KH. Question 7: optimized energy consumption for protein synthesis. Orig Life Evol Biosph. 2007;37:423–428. [PubMed]
27. Buttgereit F, Brand MD. A hierarchy of ATP-consuming processes in mammalian cells. Biochem J. 1995;312(Pt 1):163–167. [PubMed]
28. Wu H, Yang JM, Jin S, Zhang H, Hait WN. Elongation factor-2 kinase regulates autophagy in human glioblastoma cells. Cancer Res. 2006;66:3015–3023. [PubMed]
29. Kroemer G, Jaattela M. Lysosomes and autophagy in cell death control. Nat Rev Cancer. 2005;5:886–897. [PubMed]
30. Welsh GI, Proud CG. Regulation of protein synthesis in Swiss 3T3 fibroblasts. Rapid activation of the guanine-nucleotide-exchange factor by insulin and growth factors. Biochem J. 1992;284(Pt 1):19–23. [PubMed]
31. Tanida I, Minematsu-Ikeguchi N, Ueno T, Kominami E. Lysosomal turnover, but not a cellular level, of endogenous LC3 is a marker for autophagy. Autophagy. 2005;1:84–91. [PubMed]
32. Brady NR, Hamacher-Brady A, Yuan H, Gottlieb RA. The autophagic response to nutrient deprivation in the hl-1 cardiac myocyte is modulated by Bcl-2 and sarco/endoplasmic reticulum calcium stores. Febs J. 2007;274:3184–3197. [PubMed]
33. Yang JM, O'Neill P, Jin W, et al. Extracellular matrix metalloproteinase inducer (CD147) confers resistance of breast cancer cells to Anoikis through inhibition of Bim. J Biol Chem. 2006;281:9719–9727. [PubMed]
34. Liu DX, Nath N, Chellappan SP, Greene LA. Regulation of neuron survival and death by p130 and associated chromatin modifiers. Genes Dev. 2005;19:719–732. [PubMed]
35. Ryazanov AG. Ca2+/calmodulin-dependent phosphorylation of elongation factor 2. FEBS Lett. 1987;214:331–334. [PubMed]
36. Bagaglio DM, Hait WN. Role of calmodulin-dependent phosphorylation of elongation factor 2 in the proliferation of rat glial cells. Cell Growth Differ. 1994;5:1403–1408. [PubMed]
37. Ryazanov AG, Ward MD, Mendola CE, et al. Identification of a new class of protein kinases represented by eukaryotic elongation factor-2 kinase. Proc Natl Acad Sci U S A. 1997;94:4884–4889. [PubMed]
38. Wang X, Li W, Williams M, Terada N, Alessi DR, Proud CG. Regulation of elongation factor 2 kinase by p90(RSK1) and p70 S6 kinase. Embo J. 2001;20:4370–4379. [PubMed]
39. Horman S, Browne G, Krause U, et al. Activation of AMP-activated protein kinase leads to the phosphorylation of elongation factor 2 and an inhibition of protein synthesis. Curr Biol. 2002;12:1419–1423. [PubMed]
40. Browne GJ, Finn SG, Proud CG. Stimulation of the AMP-activated protein kinase leads to activation of eukaryotic elongation factor 2 kinase and to its phosphorylation at a novel site, serine 398. J Biol Chem. 2004;279:12220–12231. [PubMed]
41. Terai K, Hiramoto Y, Masaki M, et al. AMP-activated protein kinase protects cardiomyocytes against hypoxic injury through attenuation of endoplasmic reticulum stress. Mol Cell Biol. 2005;25:9554–9575. [PMC free article] [PubMed]
42. Degenhardt K, Mathew R, Beaudoin B, et al. Autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis. Cancer Cell. 2006;10:51–64. [PMC free article] [PubMed]
43. Jin S, White E. Role of autophagy in cancer: management of metabolic stress. Autophagy. 2007;3:28–31. [PMC free article] [PubMed]
44. Hait WN, Jin S, Yang JM. A matter of life or death (or both): understanding autophagy in cancer. Clin Cancer Res. 2006;12:1961–1965. [PubMed]
45. Adachi J, Ohbayashi K, Suzuki T, Sasaki T. Cell cycle arrest and astrocytic differentiation resulting from PTEN expression in glioma cells. J Neurosurg. 1999;91:822–830. [PubMed]
46. Wick W, Furnari FB, Naumann U, Cavenee WK, Weller M. PTEN gene transfer in human malignant glioma: sensitization to irradiation and CD95L-induced apoptosis. Oncogene. 1999;18:3936–3943. [PubMed]
47. Jiang W, Zhu Z, Thompson HJ. Modulation of the activities of AMP-activated protein kinase, protein kinase B, and mammalian target of rapamycin by limiting energy availability with 2-deoxyglucose. Mol Carcinog. 2008;47:616–628. [PubMed]
48. Jelluma N, Yang X, Stokoe D, Evan GI, Dansen TB, Haas-Kogan DA. Glucose withdrawal induces oxidative stress followed by apoptosis in glioblastoma cells but not in normal human astrocytes. Mol Cancer Res. 2006;4:319–330. [PubMed]
49. Coleman MC, Asbury CR, Daniels D, et al. 2-deoxy-D-glucose causes cytotoxicity, oxidative stress, and radiosensitization in pancreatic cancer. Free Radic Biol Med. 2008;44:322–331. [PubMed]
50. Moore MN. Autophagy as a second level protective process in conferring resistance to environmentally-induced oxidative stress. Autophagy. 2008;4:254–256. [PubMed]
51. Gozuacik D, Bialik S, Raveh T, et al. DAP-kinase is a mediator of endoplasmic reticulum stress-induced caspase activation and autophagic cell death. Cell Death Differ. 2008;15:1875–1886. [PubMed]
52. Cao X, Fang L, Gibbs S, et al. Glucose uptake inhibitor sensitizes cancer cells to daunorubicin and overcomes drug resistance in hypoxia. Cancer Chemother Pharmacol. 2007;59:495–505. [PubMed]
53. Jin S, DiPaola RS, Mathew R, White E. Metabolic catastrophe as a means to cancer cell death. J Cell Sci. 2007;120:379–383. [PMC free article] [PubMed]
54. Arora S, Yang JM, Kinzy TG, et al. Identification and characterization of an inhibitor of eukaryotic elongation factor 2 kinase against human cancer cell lines. Cancer Res. 2003;63:6894–6899. [PubMed]