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through Blunting of Autophagy Effect of Deoxy Glucose against

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									Silencing of Elongation Factor-2 Kinase Potentiates the
Effect of 2-Deoxy-D-Glucose against Human Glioma Cells
through Blunting of Autophagy
Hao Wu, Hua Zhu, David X. Liu, et al.

Cancer Res Published OnlineFirst February 24, 2009.



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                Published Online First on February 24, 2009 as 10.1158/0008-5472.CAN-08-2872

                                                                                                                                          Research Article

Silencing of Elongation Factor-2 Kinase Potentiates the Effect of
2-Deoxy-D-Glucose against Human Glioma Cells through
Blunting of Autophagy
Hao Wu, Hua Zhu, David X. Liu, Ting-Kuang Niu, Xingcong Ren, Rajesh Patel,
William N. Hait, and Jin-Ming Yang
Departments of Pharmacology, Neural and Behavioral Sciences, and The Penn State Cancer Institute, Pennsylvania State University College
of Medicine and Milton S. Hershey Medical Center, Hershey, Pennsylvania


Abstract                                                                                    respiration (8). Due to the high dependence of malignant cells on
2-Deoxy-D-glucose (2-DG), a synthetic glucose analogue that                                 glycolysis, interfering with this metabolic process has recently been
acts as a glycolytic inhibitor, is currently being evaluated in                             proposed as a potentially useful approach for developing new
the clinic as an anticancer agent. In this study, we observed                               selective cancer therapy (9). Treatment of cancer cells with
that treatment of human glioma cells with 2-DG activated                                    2-deoxy-D-glucose (2-DG), a synthetic glucose analogue that acts
autophagy, a highly conserved cellular response to metabolic                                as a glycolytic inhibitor, has been shown to inhibit growth and
stress and a catabolic process of self-digestion of intracellular                           viability of cancer cells (10–12), and enhance the efficacy of cancer
organelles for energy use and survival in stressed cells. The                               chemotherapeutics and radiation regiments (13–18). In both
induction of autophagy by 2-DG was associated with activa-                                  in vitro and in vivo models, 2-DG was effective in the treatment
tion of elongation factor-2 kinase (eEF-2 kinase), a structurally                           of a variety of solid tumors (19–21). The pharmacologic basis of
and functionally unique enzyme that phosphorylates eEF-2,                                   antitumor action of 2-DG is believed to be the high dependence of
leading to loss of affinity of this elongation factor for the                               malignant cells, especially those hypoxic cells on glycolysis, the
ribosome and to termination of protein elongation. We also                                  preferred ingestion and retention of 2-DG by tumor cells, and the
showed that inhibition of eEF-2 kinase by RNA interference                                  blocking effect of 2-DG on glucose metabolic pathways. In addition,
blunted the 2-DG–induced autophagic response, resulted in a                                 2-DG causes oxidative stress through increasing pro-oxidant
greater reduction of cellular ATP contents, and increased the                               production and disrupting thiol metabolism, as evidenced by
sensitivity of tumor cells to the cytotoxic effect of 2-DG.                                 alterations in total glutathione content (16, 22). In the treatment of
Furthermore, the blunted autophagy and enhanced 2-DG                                        human brain malignancies, 2-DG has been shown to be effective in
cytotoxicity were accompanied by augmentation of apoptosis                                  sensitizing tumor cells to radiation therapy (17, 23). Despite the
in cells in which eEF-2 kinase expression was knocked down.                                 demonstrations of the antitumor activity of 2-DG, large doses are
The results of this study indicate that the energy stress and                               usually needed to achieve a therapeutic effect, and cancer cells
cytotoxicity caused by 2-DG can be accelerated by inhibition                                quickly become refractory to this agent. Therefore, approaches that
of eEF-2 kinase, and suggest that targeting eEF-2 kinase–                                   can enhance the efficacy of 2-DG may make this agent more useful
regulated autophagic survival pathway may represent a novel                                 in the treatment of cancers.
approach to sensitizing cancer cells to glycolytic inhibitors.                                  Elongation factor-2 kinase (eEF-2 kinase; aka calmodulin-
[Cancer Res 2009;69(6):OF1–8]                                                               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
Introduction
                                                                                            kinase phosphorylates elongation factor-2, a 100-kDa protein that
   Cellular metabolism of malignant cells differs significantly from                        mediates the translocation step in peptide-chain elongation by
that of normal cells. Whereas normal cells rely on respiration, a                           inducing the transfer of peptidyl-tRNA from the ribosomal A to P
process that consumes oxygen and glucose to produce energy-                                 site. Phosphorylation of EF-2 at Thr56 by eEF-2 kinase decreases
storing molecule ATP, malignant cells mainly depend on glycolysis,                          the affinity of this elongation factor for ribosomes and terminates
the anaerobic metabolism of glucose into ATP, even in the presence                          elongation, thereby inhibiting protein synthesis. Because protein
of sufficient oxygen. This increased dependency of malignant cells                          synthesis requires a large proportion of cellular energy (26, 27),
on glycolysis for ATP production is known as the so-called Warburg                          inhibition of protein synthesis by terminating elongation through
effect (1, 2). A number of molecular pathways have been revealed to                         activating eEF-2 kinase decreases energy use, and provides a
be associated with this metabolic phenotype, including hexokinase                           survival mechanism against energy stress.
2 (3, 4), p53 (5), c-Myc (6, 7), HIF-1 (7), and defect in mitochondrial                         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
   Note: Current address for H. Wu, H. Zhu, and R. Patel: The Cancer Institute of
New Jersey, University of Medicine and Dentistry, New Jersey-Robert Wood Johnson            to large-scale degradation of proteins (28). The process of
Medical School, 195 Little Albany Street, New Brunswick, NJ 08903; current address          autophagy involves formation of a double-membrane vesicle
for W.N. Hait: OrthoBiotech Research and Development, 920 Route 202, Raritan, NJ
08869.
                                                                                            (‘‘autophagosome’’) in the cytosol that engulfs organelles and
   Requests for reprints: Jin-Ming Yang, Penn State College of Medicine, Department         cytoplasm, then fuses with the lysosome to form the autolysosme,
of Pharmacology, H072, 500 University Drive, P.O. Box 850, Hershey, PA 17033-0850.          where the contents are degraded and recycled for protein and ATP
Phone: 717-531-0003, ext. 281124; Fax: 717-531-0002; E-mail: jyang2@hmc.psu.edu.
   I2009 American Association for Cancer Research.                                          synthesis (29). The formation of the autophagosome is mediated
   doi:10.1158/0008-5472.CAN-08-2872                                                        by a series of autophagy specific genes (ATG). This form of


www.aacrjournals.org                                                                  OF1                                Cancer Res 2009; 69: (6). March 15, 2009
Cancer Research


self-digestion leads to self-preservation in times of nutrient                   blot reagents were obtained from Pierce Biotechnology, Inc. The following
deprivation; however, if left unchecked, autophagy has the                       antibodies were used in this study: anti–caspase-3 antibody, anti–eEF-2
potential of producing terminal self-consumption. Although                       kinase, anti–eEF-2, anti–phospho-eEF-2 (Thr56), anti–phospho-AMPKa
apoptosis is known as type I cell death, autophagy is called type                (Thr172); and anti-AMPKa, anti-S6 kinase, anti–phospho-S6 kinase
                                                                                 (Thr389) antibodies (Cell Signaling Technologies); monoclonal anti-LC3
II cell death. We showed in human glioma cells that the activity
                                                                                 antibody (nanoTools, though Axxora; LLC).
of eEF-2 kinase is closely associated with the mammalian macro-                      siRNA preparation and transfection. siRNA sequence targeting eEF-2
autophagy pathway that is activated in response to nutrient,                     kinase mRNA corresponded to the coding region 144 to 164 (5¶-
growth factor or oxygen deprivation. Furthermore, inhibition of                  AAGCTCGAACCAGAATGTC-3¶) relative to the start codon (28). siRNA
eEF-2 kinase blunts autophagy and has deleterious effects on cell                duplexes were prepared by Dharmacon Research, Inc. For transfection, cells
viability under nutrient starvation condition (28). These observa-               in exponential phase of growth were plated in 60-mm tissue culture dishes
tions raise the possibility that blocking the activation of eEF-2 kinase         at 5 Â 105 cells per dish, grown for 24 h, then transfected with siRNA
may represent a potential therapeutic strategy to promote cell death             (100 nmol/L) using Oligofectamine and OPTI-MEM I–reduced serum
induced by metabolic stress. As 2-DG inhibits cell growth and causes             medium, according to the protocol of the manufacturer. The concentrations
death of tumor cells through antagonizing glucose (20), we sought to             of siRNAs were chosen based on dose-response studies. Twenty-four hours
                                                                                 after transfection, the cells were harvested for further experiments.
determine the effects of this glycolytic inhibitor on eEF-2 kinase
                                                                                     Measurement of protein synthesis. The rate of protein synthesis was
activity and autophagic cell survival pathway, and the effect of                 measured as described previously (30). Briefly, cells were seeded in 60-mm
inhibiting eEF-2 kinase on sensitivity of tumor cells to 2-DG. We                tissue culture dishes and labeled with 25 ACi/mL of EasyTag EXPRESS [35S]
found that treatment of human glioma cells with 2-DG activated an                protein labeling mix (PerkinElmer) in RPMI 1640. After incubation at 37jC
eEF-2 kinase–dependent autophagic response, and inhibiting eEF-2                 for 15 min, cells were washed 4 times with 4 mL of ice-cold PBS and lysed
kinase by RNA interference (RNAi) blocked the induction of                       in 200 AL of Complete Lysis-M lysis reagent containing the Mini Protease
autophagy by 2-DG and increased the sensitivity of tumor cells to                Inhibitor Cocktail (Roche Diagnostics). Lysates were collected in a microfuge
the cytotoxic effect of this glycolytic inhibitor. Our results suggest           tube and clarified by centrifugation at 13,000 Â g for 10 min at 4jC. The
that targeting the eEF-2 kinase–regulated autophagic pathway could               supernatants were precipitated with 20% of trichloracetic acid and collected
be an effective approach to augmenting the activity of 2-DG, and                 on GF/C filters (Millipore). The filters were washed 4 times with 1 mL of 10%
                                                                                 trichloracetic acid and subject to liquid scintillation counting. The specific
thus may have clinical implication in cancer treatment.
                                                                                 activity of protein synthesis was determined by the amount of incorporated
                                                                                 35
                                                                                    S-methionine/cysteine per mg of total protein per min.
                                                                                     Measurement of cellular ATP. Cells were plated in 96-well plates at
Materials and Methods                                                            2.5 Â 103 cells per well and treated with 2-DG for 24 h. ATP contents were
   Cell lines and culture. The human glioblastoma cell lines, T98G and           determined using the ATPlite Luminescence Assay kit (PerkinElmer)
LN-229, were purchased from American Type Culture Collection. T98G               according to the manufacturer’s protocol. The luminescence was measured
cells were cultured in Ham’s F-10: DMEM (10:1) medium, and LN-229 cells          by a Victor3 Multi Label plate reader (PerkinElmer).
were cultured in DMEM supplemented with 10% fetal bovine serum,                      Measurement of autophagy. Autophagy was monitored by measuring
100 units/mL penicillin, and 100 Ag/mL streptomycin. Cells were                  the formation of LC3-II in the absence or presence of lysosomal protease
maintained at 37jC in a humidified atmosphere containing 5% CO2/95% air.         inhibitors E64D (10 Ag/mL) and pepstatin A (10 Ag/mL), as described by
   Reagents and antibodies. 2-Deoxy-D-Glucose, 3-(4,5-dimethylthiazol-2-         Tanida and colleagues (31). For detection of LC3-II, cell lysates were
yl)-2,5-diphenyltetrazolium bromide (MTT), and anti–a-tubulin antibodies         prepared and 25 Ag of total proteins were subjected to Western blot analysis
were purchased from Sigma. All cell culture medium and other products            using a monoclonal anti-LC3 antibody. GFP-LC3 cleavage assay was
were purchased from Invitrogen Corporation. Chemiluminescence Western            performed as previously described (32). Briefly, cells (1 Â 106) were




                                                                                                                     Figure 1. Effects of 2-DG on the activity of
                                                                                                                     eEF-2 kinase (A ) and protein synthesis (B)
                                                                                                                     in glioma cells. T98G or LN-229 cells were
                                                                                                                     treated with the indicated concentrations
                                                                                                                     of 2-DG for 24 h. At the end of treatment,
                                                                                                                     (A) eEF-2 kinase activity was determined
                                                                                                                     by Western blot analysis of phospho-EF-2
                                                                                                                     using an anti–phospho-EF-2 (Thr56 )
                                                                                                                     antibody as described in Materials and
                                                                                                                     Methods. Tubulin was used as a loading
                                                                                                                     control. B, the rate of protein synthesis
                                                                                                                     was measured by labeling the cells with
                                                                                                                     25 ACi/mL of EasyTag EXPRESS [35S]
                                                                                                                     protein labeling mix and liquid scintillation
                                                                                                                     counting, as described in Materials and
                                                                                                                     Methods. The specific activity of protein
                                                                                                                     synthesis was determined by the amount of
                                                                                                                     incorporated 35S-methionine/cysteine per
                                                                                                                     mg of total protein per minute. Results
                                                                                                                     shown are the representative of three
                                                                                                                     similar experiments; columns, mean of
                                                                                                                     quadruplicate determinations; bars, SD.
                                                                                                                     **, P < 0.01.




Cancer Res 2009; 69: (6). March 15, 2009                                   OF2                                                       www.aacrjournals.org
                                                                                                    Elongation Factor-2 Kinase and 2-Deoxy-D-Glucose




Figure 2. Effect of 2-DG on ATP content
(A), S6 kinase activity (B ), and AMP kinase
activity (C ) in glioma cells. T98G or
LN-229 cells were treated with the
indicated concentrations of 2-DG for 24 h.
At the end of treatment, (A ) ATP
content was measured using the ATPlite
Luminescence Assay kit. B, S6 kinase
activity was determined by Western blot
analysis of phospho-S6 kinase using an
anti–phospho-S6 kinase antibody.
C, AMPK activity was determined by
Western blot analysis of phospho-AMPK
using an anti–phospho-AMPK antibody,
as described in Materials and Methods.
Tubulin was used as a loading control.
Results shown are the representative of
three similar experiments; columns, mean
of quadruplicate determinations; bars, SD.
*, P < 0.05; **, P < 0.01.




cotransfected with 3 Ag of GFP-LC3 plasmid DNA and an eEF-2 kinase–                    Electron microscopy. Cells were harvested by trypsinization, fixed in
targeted siRNA or a nontargeting RNA in Opti-MEM reduced medium                     2.5% gluteraldehyde/4% paraformaldehyde in 0.1 mol/L cacodylate buffer,
(Invitrogen) and incubated overnight at 37jC. The cells were then treated           and then postfixed in 1% osmium tetroxide buffer. After dehydration in
with 2-DG in the presence of lysosomal protease inhibitors E64d (10 Ag/mL)          acetone, the cells were embedded in spur resin. Thin sections (90 nm) were
and pepstatin A (10 Ag/mL). At the end of treatment, cells were fixed with          cut on a Reichert Ultracut E microtome. Sectioned grids were stained with
4% formaldehyde for 15 min and inspected at Â60 magnification for                   saturated solution of uranyl acetate and lead citrate. Sections were
numbers of GFP-LC3 puncta. Autophagy was also evaluated by electron                 examined at 80 kV with a JEOL 1200EX transmission electron microscope.
microscopic examination of double or multimembrane vacuoles in the                     Cellular viability. Cell viability was measured by MTT assay. Briefly, cells
cytoplasm, as described below.                                                      were plated at 5 Â 103 per well in 96-well tissue culture plates and
   Western blot analysis. Cells were pelleted at 500 Â g for 5 min and were         incubated at 37jC in a humidified atmosphere containing 5% CO2/95% air.
lysed in cold lysis buffer [20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl,           The formazan product, formed after a 4-h incubation with MTT, was
1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton X-100, 2.5 mmol/L sodium PPi,               dissolved in DMSO and read at 570 nm using a Victor3 Multi Label plate
1 mmol/L h-glycerolphosphate, 1 mmol/L Na3VO4, 1 Ag/mL leupeptin, and               reader (PerkinElmer).
1 mmol/L phenylmethylsulfonyl fluoride] and sonicated for 5 s. The lysates             Measurement of apoptosis. Active caspase-3, which is present in cells
were clarified by centrifugation at 12,000 Â g for 30 min at 4jC. Identical         undergoing apoptosis, was detected by Western blot using polyclonal anti–
amounts (25 Ag of protein) of cell lysates were resolved by 8% or 15% SDS-          caspase-3 antibody that recognizes the cleaved, active caspase-3, as
PAGE, and proteins were transferred onto nitrocellulose or polyvinylidene           described previously (33). For apoptotic nuclei staining, pEGFP-transfected
difluoride. Membranes were incubated in blocking solution consisting of 5%          cells with different treatments were stained with Hoechst 33342 and
powered milk in TBST [10 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, and             observed under green fluorescence and UV. Green fluorescent protein
0.1% Tween 20] for 1 h, then immunoblotted with the respective antibodies.          (GFP)-positive cells were scored for the presence of apoptotic nuclei, and
Detection by enzyme-linked chemiluminescence was carried out according              apoptotic rates were presented as the percentage of apoptotic cells out of
to the protocol of the manufacturer.                                                the total number of GFP-positive cells assessed (34).



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Cancer Research




                                                                          Figure 3. Effect of 2-DG on autophagy in glioma cells. A, T98G or LN-229
                                                                          cells were treated with the indicated concentrations of 2-DG for 24 h. At the
                                                                          end of treatment, formation of autophagy marker LC3-II was detected by
                                                                          immunoblotting with an anti–MAP-LC3 antibody as described in Materials and
                                                                          Methods. B, T98G cells were treated with the indicated concentrations of
                                                                          2-DG for 24 h in the presence of lysosomal protease inhibitors E64D
                                                                          (10 Ag/mL) and pepstatin A (10 Ag/mL). At the end of treatment, formation
                                                                          of autophagy marker LC3-II was detected by immunoblotting with an
                                                                          anti–MAP-LC3 antibody as described in Materials and Methods. C, T98G
                                                                          treated with 2-DG (12.5 mmol/L) or vehicle were harvested by trypsinization,
                                                                          fixed, and embedded in spur resin. Ninety-nanometer-thin sections were
                                                                          cut and examined at 80 Kv with a JEOL 1200EX transmission electron
                                                                          microscope. Arrows, autophagic vacuoles. D, T98G cells cultured in medium
                                                                          containing low or high concentration of glucose were treated with the
                                                                          indicated concentrations of 2-DG for 24 h. LC3-II, phospho-EF-2, EF-2,
                                                                          and eEF-2 kinase were detected by Western blot using the respective
                                                                          antibodies. Tubulin was used as a loading control. Results shown are the
                                                                          representative of three similar experiments.




Results                                                                          Treatment with 2-DG induces autophagy in glioma cells.
                                                                              Given the effects of 2-DG on the activities of eEF-2 kinase (Fig. 1),
   Treatment with 2-DG activates eEF2 kinase and decreases
protein synthesis in glioma cells. We previously reported that                S6 kinase, AMPK, and the cellular level of ATP (Fig. 2), we next
nutrient depletion caused activation of eEF2 kinase and autophagy             determined whether treatment of tumor cells with this glycolytic
in glioma cells, and blocking activation of eEF-2 kinase blunted              inhibitor induced autophagy. LC3-II, a cleaved product of
the autophagic response (28). To determine the specific effect of             microtubule-associated protein 1 light chain 3, was used as
glucose deprivation on the activity of eEF-2 kinase and autophagy,            a marker for autophagy. We found that both steady-state level
we first treated T98G and LN229 human glioma cells with varying               (Fig. 3A) and turnover (Fig. 3B) of LC3-II were increased in the
concentrations of 2-DG, and then assayed the activity of the kinase.          glioma cells treated with 2-DG, compared with the cells treated
As shown in Fig. 1A, treatment of these glioma cells with 2-DG for            with the vehicle. The induction of autophagy by 2-DG was
24 hours increased the activity of eEF-2 kinase in a dose-dependent           confirmed by electron microscopy (Fig. 3C), which visualized
manner, as evidenced by the increased phosphorylation of its                  abundant double or multimembrane vacuoles in the cytoplasm of
substrate, EF2. To confirm the stimulatory effect of 2-DG on eEF-2            the cells treated with 2-DG. By contrast, these vacuoles were rarely
kinase activity, we compared the rate of protein synthesis in the             observed in glioma cells treated with the vehicle (Fig. 3C). Although
cells treated with 2-DG to that of the cells treated with vehicle.            the basal level of autophagy in cells cultured in medium containing
Figure 1B shows that protein synthesis in the cells treated with 2-           high concentration of glucose (25 mmol/L) was much lower than
DG was markedly inhibited compared with that in vehicle-treated               that in cells cultured in medium with low concentration of glucose
cells. These results were consistent with the role of eEF-2 kinase            (5.6 mmol/L), under either condition 2-DG activated autophagy in
in regulating translation, i.e., activation of eEF-2 kinase inhibits          these tumor cells, and the levels of autophagy were correlated to
elongation. Activation of eEF-2 kinase by 2-DG was accompanied                the activity of eEF-2 kinase (Fig. 3D).
by a reduction of cellular ATP contents (Fig. 2A), an inactivation               Silencing of EF-2 kinase expression blunts the 2-DG–
(decreased phosphorylation) of the key translational governor and             induced autophagy. To further investigate whether induction of
downstream effector of mammalian target of rapamycin (mTOR),                  autophagy by 2-DG was mediated through eEF-2 kinase, we
S6 kinase (Fig. 2B), and an activation of AMPK, as shown by                   silenced the expression of eEF-2 kinase using RNAi approach and
the increased phosphorylation of this intracellular energy sensor             then determined the effect of eEF-2 kinase inhibition on the
(Fig. 2C). These results indicate that treatment of glioma cells with         autophagic response to 2-DG treatment. Tumor cells were trans-
2-DG elicited an energy stress response.                                      fected with an eEF-2 kinase–targeted siRNA or nontargeting RNA


Cancer Res 2009; 69: (6). March 15, 2009                                OF4                                                    www.aacrjournals.org
                                                                                            Elongation Factor-2 Kinase and 2-Deoxy-D-Glucose


for 24 hours, and then treated with various concentrations of 2-DG.          tumor cells with and without silencing of the enzyme after 2-DG
Figure 4A shows that knockdown of eEF-2 kinase expression                    treatment. Figure 6B shows that treatment of T98G cells with 2-DG
decreased the activity of the enzyme, as indicated by the dramatic           not only activated autophagy, as measured by the formation of
decreases in the phospo-EF-2 at Thr56, and blunted the autophagic            LC3-II, but also triggered apoptosis in a dose-dependent manner,
response in the cells treated with 2-DG, as manifested by the                as measured by the activation of caspase-3. More notably,
decreased formation of LC3-II. Electron microscopy and GFP-LC3               inhibition of autophagy by silencing of eEF-2 kinase augmented
cleavage assay also showed that silencing of eEF-2 kinase by siRNA           apoptosis triggered by 2-DG, as evidenced by the increased
blocked the 2-DG–induced autophagy, as evidenced by the                      appearance of the cleaved form of caspase-3 (Fig. 6B) and
decreased formation of double-membrane vacuoles in the cyto-                 increased apoptotic nuclei (Fig. 6C), suggesting an increased death
plasm (Fig. 4B) and decreased numbers of cells with >20 GFP-LC3              in cells with inhibition of eEF-2 kinase.
punctae (Fig. 4C). Moreover, silencing of eEF-2 kinase also
diminished the inhibitory effect of 2-DG on protein synthesis
(Fig. 5A) and accelerated the 2-DG–induced reduction of cellular
ATP contents (Fig. 5B), further indicating that inhibition of this           Discussion
kinase weakens the adaptive response of cells to metabolic stress               eEF-2 kinase is a calcium/calmodulin-dependent enzyme that
and worsens the energy supply.                                               regulates protein elongation (35), and has been observed to be up-
   Silencing of eEF-2 kinase expression enhances the sensitivity             regulated in human and rat gliomas (25, 36). This kinase was
of glioma cells to 2-DG. It has been shown that autophagy                    subsequently found to have several unique characteristics includ-
promotes cell survival under conditions of nutrient deprivation and          ing the ability to phosphorylate serines and threonines within a
that inhibiting eEF-2 kinase can abrogate this prosurvival response          helical turns (37). Further work by Proud’s group (38) showed the
(28). To test whether blockade of 2-DG–activated autophagy via               exquisite regulation of eEF-2 kinase by multiple enzymes involved
inhibiting eEF-2 kinase could enhance the cytotoxicity of this               in energy sensing and use, including mTOR, S6 kinase, and AMP
glycolytic inhibitor, we transfected tumor cells with a nontargeting         kinase (39, 40). The role of eEF-2 kinase as an energy sensor was
RNA or siRNA targeting eEF-2 kinase, then exposed the cells to               also shown in a study showing that this enzyme participates in the
various concentrations of 2-DG. As shown in Fig. 6A, the                     AMPK-mediated cardioprotection in response to metabolic stress
cytotoxicity of 2-DG was significantly increased by silencing eEF-           (41). When exploring the role of eEF-2 kinase in malignant cells, we
2 kinase expression in T98G and LN-229 glioma cells. To explore              found that the activity of eEF-2 kinase and autophagy were rapidly
the mechanism underlying the increased 2-DG cytotoxicity in cells            increased by nutrient deprivation and that inhibiting the enzyme
with decreased eEF-2 kinase activity, we compared apoptosis in               markedly diminished autophagic cell survival (28), suggesting that




                                                                                Figure 4. Silencing of eEF-2 kinase expression blunts 2-DG–activated
                                                                                autophagy. T98G cells were transfected with a nontargeting RNA or an
                                                                                eEF-2 kinase–targeted siRNA (100 nmol/L) for 24 h, and then treated
                                                                                with the indicated concentrations of 2-DG. A, phopsho-EF-2, EF-2,
                                                                                eEF-2 kinase, and the autophagy marker, LC3-II, were detected
                                                                                as described in Fig. 3. B, electron microscopic examination of
                                                                                autophagosomes was performed as described in Fig. 3C . Arrows,
                                                                                autophagic vacuoles. C, GFP-LC3–expressing cells were transfected
                                                                                with an eEF-2 kinase–targeted siRNA or a nontargeting RNA, and
                                                                                then treated with indicated concentration of 2-DG in the presence
                                                                                of lysosomal protease inhibitors E64d (10 Ag/mL) and pepstatin A
                                                                                (10 Ag/mL). At the end of treatment, cells were fixed with 4%
                                                                                formaldehyde for 15 min and inspected at Â60 magnification for numbers
                                                                                of GFP-LC3 puncta. At least 200 cells were scored in each treatment.
                                                                                Columns, mean of quadruplicate determinations; bars, SD. *, P < 0.01.
                                                                                Results shown are the representative of three similar experiments.




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Cancer Research


this apparent relationship between eEF-2 kinase activity and cell               the fact that 2-DG treatment also causes oxidative stress (49) and
survival might be linked directly to its role in protein synthesis.             this type of stress is a trigger for autophagy via other pathways (50).
   In this study, we used the glycolytic inhibitor, 2-DG, to further            Thus, inhibiting multiple autophagic pathways might result in a
explore the role of eEF-2 kinase in the autophagic response to                  more profound suppression of 2-DG–activated autophagy and
energy stress. We show in human glioma cells that treatment with                hence increase the anticancer activity of this agent. It has been
2-DG activates autophagy (Fig. 3); the induction of autophagy by                reported that inhibition of glutamate cysteine ligase, an enzyme
2-DG seems to be dependent on the activity of eEF-2 kinase, as                  involved in the glutathione synthetic pathway, sensitizes tumor cells
treatment with 2-DG also activates this kinase (Fig. 1); and                    to the cytotoxicity of 2-DG (22). We recognize that autophagy may
knockdown of eEF-2 kinase expression blunts the autophagic                      also lead to cell death that is accompanied by caspase activation
response induced by 2-DG (Fig. 4). Furthermore, we show that                    (51); however, based on our results, the role of autophagy is
silencing of eEF-2 kinase expression increases the cytotoxicity of              prosurvival but not prodeath in response to 2-DG treatment, as we
2-DG (Fig. 6A) and augments apoptosis (Fig. 6B and C). Because                  observed that although silencing of eEF-2 kinase expression further
autophagy is known to favor cell survival under environmental and               increased caspase-3 activation in glioma cells treated with 2-DG
metabolic stress conditions (42–44), 2-DG–induced autophagy may                 (Fig. 6B), autophagy was inhibited. Moreover, cell viability was also
represent an adaptive response to the reduction of energy supply                decreased in siRNA-treated cells compared with the nontargeting
caused by this glycolytic inhibitor, and explain, at least in part, the         RNA-treated cells (Fig. 6A).
decreased sensitivity of tumor cells after exposure to this agent.                 Malignant cells use glucose at a higher rate than normal cells,
Although the two glioma cell lines used in this study, T98G and LN-             and become more dependent on aerobic and anaerobic glycolysis.
229, harbor aberrant and wild-type PTEN, respectively (45, 46), our             Hypoxic tumor cells are particularly dependent on anaerobic
results from the two cell lines were consistent, suggesting that                glycolysis, and are therefore more sensitive to 2-DG–induced cell
different expression of PTEN does not affect the autophagic                     cycle inhibition and cytotoxicity (12). Moreover, due to the up-
response to 2-DG treatment and the effect of inhibiting eEF-2                   regulation of glucose transporters in tumor cells and high affinity
kinase on sensitivity to 2-DG.                                                  of 2-DG for glucose transporters, malignant cells show an enhanced
   The results showing that 2-DG–induced autophagy is associated                uptake and retention of this agent (52). These factors are believed
with the activity of eEF-2 kinase are consistent with our previous              to contribute to the preferential toxicity of 2-DG in cancer cells. In
observation that induction of autophagy by metabolic stress is                  this study, we show that targeting eEF-2 kinase, an inhibitor of
regulated by this kinase (28), although the precise mechanism                   protein elongation, can enhance the sensitivity of glioma cells to
underlying this regulation is still unclear. The association of eEF-2           2-DG (Fig. 6A). The potentiation of 2-DG cytotoxicity by inhibition
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 use to withstand
nutrient starvation such as glucose deprivation. The activity of eEF-
2 kinase is tightly regulated by signaling pathways involved in
nutrient use and energy monitoring in the cell. For example, Proud’s
group (38) reported that eEF-2 kinase is inhibited by mTOR and
S6 kinase signaling pathways. 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 (Fig. 2A), decreased
activity of S6 kinase (Fig. 2B), and increased activity of AMP kinase
(Fig. 2C). A recent study in breast cancer cells also shows 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 because
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            Figure 5. Silencing of eEF-2 kinase expression mitigates the inhibition of protein
cultured in medium containing low concentration of glucose than                 synthesis (A ) and worsened the energy stress (B ) in the 2-DG–treated glioma
                                                                                cells. T98G cells were transfected with a nontargeting RNA (NT) or an eEF-2
in cells cultured in medium containing high concentration of                    kinase–targeted siRNA (100 nmol/L) for 24 h, and then treated with the indicated
glucose (Fig. 3D) also support the role of autophagy as a prosurvival           concentrations of 2-DG. At the end of treatment, (A ) protein synthesis activity
mechanism in cells stressed by nutrient deprivation or metabolic                was determined as described in Fig. 1B. B, ATP content was measured
                                                                                as described in Fig. 2A . Results shown are the representative of three
alteration. Silencing of eEF-2 kinase does not completely suppress              similar experiments; points, mean of quadruplicate determinations; bars, SD.
the autophagic response activated by 2-DG (Fig. 4), probably due to             *, P < 0.05.



Cancer Res 2009; 69: (6). March 15, 2009                                  OF6                                                         www.aacrjournals.org
                                                                                                             Elongation Factor-2 Kinase and 2-Deoxy-D-Glucose




Figure 6. Silencing of eEF-2 kinase expression increases the sensitivity of glioma cells to the cytotoxicity of 2-DG. A, T98G or LN-229 cells transfected with a
nontargeting RNA or an eEF-2 kinase–targeted siRNA were treated with the indicated concentrations of 2-DG for 60 h. At the end of treatment, cell viability was
measured using MTT assay. Points, mean of quadruplicate determinations; bars, SD. *, P < 0.05. B, T98G cells with or without silencing of eEF-2 kinase expression
were treated with the indicated concentrations of 2-DG for 24 h. Caspase-3, LC3-II, and eEF-2 kinase were detected by Western blot, as described in Materials and
Methods. Tubulin was used as a loading control. C, T98G cells were cotransfected with a pEGFP expression vector and a siRNA targeting eEF-2 kinase or a
nontargeting RNA, and then treated with the indicated concentrations of 2-DG. At the end of treatment, cells were fixed, 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. Columns, mean of triplicate determinations; bars, SD. *, P < 0.05; **, P < 0.01.



of eEF-2 kinase seems to result from the metabolic catastrophe                             properties of those compounds through National Cancer Institute’s
caused by the glycolytic inhibitor, as autophagy is markedly                               Rapid Access to Intervention Development program. Because eEF-
attenuated and ATP level is further decreased (due to recovery of                          2 kinase is up-regulated in several types of cancers, we expect that
protein synthesis) in tumor cells with silencing of eEF-2 kinase in                        development and use of inhibitors of the kinase with favorable
comparison with the cells without the silencing of the enzyme                              pharmacokinetic characteristics would increase the selectivity and
(Figs. 4 and 5). Inducing metabolic catastrophe in cancer cells has                        effectiveness of glycolytic inhibitors such as 2-DG. Taken together,
been proposed as a new therapeutic approach awaiting for further                           our study underscores the potential of eEF-2 kinase as a
investigation (53).                                                                        complementary target for sensitizing tumor cells to the glycoly-
   2-DG has entered into phase I clinical trials, but results of the                       sis-targeted therapy.
clinical studies have not been well-documented in literatures.
However, a recent study reported that 2-DG significantly prolongs
                                                                                           Disclosure of Potential Conflicts of Interest
survival of the mice bearing aggressive lymphoma with defective
laforin expression (21). The results of the current study show that                           No potential conflicts of interest were disclosed.
the cytotoxic effect of 2-DG on tumor cells can be potentiated by
suppressing an eEF-2 kinase–dependent autophagic survival                                  Acknowledgments
pathway, suggesting that 2-DG treatment in combination with
                                                                                           Received 7/25/2008; revised 11/18/2008; accepted 12/15/2008; published OnlineFirst
the inhibition of eEF-2 kinase may stand for a new therapeutic                             02/24/2009.
strategy to improve the efficacy of this glycolytic inhibitor against                         Grant support: US Public Health Service CA43888, the philanthropic fund from the
malignant tumors. We have identified the inhibitors of eEF-2 kinase                        Marks family (H. Marks and N. Marks), and the grant from American Cancer Society.
                                                                                              The costs of publication of this article were defrayed in part by the payment of page
and reported their effects on cancer cell lines (54), and are                              charges. This article must therefore be hereby marked advertisement in accordance
currently evaluating the in vivo effectiveness and pharmacokinetic                         with 18 U.S.C. Section 1734 solely to indicate this fact.




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Cancer Res 2009; 69: (6). March 15, 2009                                                 OF8                                                               www.aacrjournals.org

								
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