This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pastorino, J. G. Articles by Shulga, N. Search for Related Content PubMed PubMed Citation Articles by Pastorino, J. G. Articles by Shulga, N.

Activation of Glycogen Synthase Kinase 3ß Disrupts the Binding of Hexokinase II to Mitochondria by Phosphorylating Voltage-Dependent Anion Channel and Potentiates Chemotherapy-Induced Cytotoxicity

Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania

Requests for reprints: John G. Pastorino, Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Room 272, Jefferson Alumni Hall, Philadelphia, PA 19107. Phone: 215-503-5022; Fax: 215-923-2218; E-mail: John.Pastorino{at}jefferson.edu.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Transformed cells are highly glycolytic and overexpress hexokinase II (HXK II). HXK II is capable of binding to the mitochondria through an interaction with the voltage-dependent anion channel (VDAC), an abundant outer mitochondrial membrane protein. The binding of HXK II to mitochondria has been shown to protect against loss of cell viability. Akt activation inhibits apoptosis partly by promoting the binding of HXK II to the mitochondria, but the mechanism through which Akt accomplishes this has not been characterized. The present report shows that Akt mediates the binding of HXK II to the mitochondria by negatively regulating the activity of glycogen synthase kinase 3ß (GSK3ß). On inhibition of Akt, GSK3ß is activated and phosphorylates VDAC. HXK II is unable to bind VDAC phosphorylated by GSK3ß and dissociates from the mitochondria. Inhibition of Akt potentiates chemotherapy-induced cytotoxicity, an effect that is dependent on GSK3ß activation and its attendant ability to disrupt the binding of HXK II to the mitochondria. Moreover, agents that can force the detachment of HXK II from mitochondria in the absence of Akt inhibition or GSK3ß activation promoted a synergistic increase in cell killing when used in conjunction with chemotherapeutic drugs. Such findings indicate that interference with the binding of HXK II to mitochondria may be a practicable modality by which to potentiate the efficacy of conventional chemotherapeutic agents.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Over 70 years ago, Warburg emphasized that poorly differentiated and rapidly growing tumors tend to have higher glycolytic rates (1). An increased rate of glycolysis would be beneficial to a tumor cell, giving it the ability to generate adequate supplies of ATP anaerobically. However, the production of ATP may not be the only or primary necessity for the increased glycolysis seen in tumor cells, because high rates of glycolysis occur in such cells even under conditions where oxygen is plentiful. Indeed, measurements of the rate of glycolysis indicate that a large proportion of the glycolytic flux is coupled to mitochondrial oxidation, a circumstance called aerobic glycolysis (2).

The high glycolytic rate is now known to be due in part to the greatly increased expression of hexokinase II (HXK II) in transformed cells (3–6). In an ATP-dependent process, HXK II mediates the first and a rate-controlling step of glycolysis, phosphorylating glucose to produce glucose-6-phosphate. HXK I and HXK II are capable of binding to the mitochondria (7, 8). Both of these isoforms possess a hydrophobic NH₂-terminal sequence of 15 amino acids that has features compatible with an amphipathic -helical structure. This NH₂-terminal domain is essential for the mitochondrial binding of the proteins. The specificity of the high-affinity binding of HXK I and HXK II to the outer mitochondrial membrane is due to their interaction with the voltage-dependent anion channel (VDAC) also known as porin. VDAC is a 30-kDa ß-barrel pore protein that spans the mitochondrial outer membrane and mediates the permeation of metabolites into and out of the mitochondrial intermembrane space (9, 10).

In recent years, it has become apparent that mitochondria are positioned at a critical juncture in the control of apoptosis where proapoptotic signals from diverse upstream pathways converge (11, 12). Mitochondria can play this role due to the fact that a cadre of proapoptotic proteins is located in the mitochondrial intermembrane space. Sundry modes of protein release from the intermembrane space are possible and the control of the escape pathways is an ongoing area of controversy.

There is evidence that VDAC is capable of modulating the entry of intermembrane space proteins to the cytosol during apoptosis. Significantly, it has been shown that, under certain circumstances, VDAC can assume a configuration that promotes the release of cytochrome c. For instance, superoxide anions have been shown to cause a massive release of cytochrome c, which was suppressed by agents that promote VDAC closure, such as Koning's polyanion and antibodies that bind to VDAC (13). Proapoptotic proteins can also promote the release of mitochondrial intermembrane space constituents through an interaction with VDAC. Electrophysiologic studies in reconstituted systems have shown that Bax, Bak, and Bim can interact with VDAC and induce the formation of a novel large pore (14–16). Conversely, the antiapoptotic protein Bcl-x_L prevented Bax from inducing VDAC-dependent pore formation.

Studies suggest that the binding of HXK II to mitochondria may prevent mitochondrial dysfunction during cell stress and injury through its interaction with VDAC. Antibodies against VDAC prevent Bax-induced cytochrome c release in isolated mitochondria and intact cells (17). Such a result suggests that a protein that can bind to VDAC, like HXK II, may also prevent proapoptotic proteins from interacting with the mitochondria. Indeed, when expressed in NIH3T3 and Rat1a cell lines, HXK I, an isoform that binds to VDAC like HXK II, was found to increase cell proliferation and inhibit apoptosis (18). By contrast, a decrease in the level of mitochondrial bound HXK I worsened the apoptosis of Rat1a cells induced by growth factor withdrawal or UV irradiation. Mitochondrial bound HXK II was also shown to inhibit the binding of Bax to mitochondria and its accompanying damage to mitochondrial function (19). Similarly, Akt can protect against Bid-induced cell killing by promoting the binding of HXK II to the mitochondria, wherein the oligomerization Bak is prevented (20).

Akt promotes the binding of HXK II to the mitochondria, which may in part account for some of Akt's antiapoptotic properties (18, 21, 22). However, the mechanism by which Akt regulates the binding of HXK II to the mitochondria is uncharacterized. Akt is known to localize to the mitochondria along with one of its targets, glycogen synthase kinase 3ß (GSK3ß; refs. 23, 24). Akt phosphorylates GSK3ß on Ser⁹ resulting in an inhibition of the kinase's activity (25). Activation of GSK3ß is proapoptotic in several systems and can provoke mitochondrial injury (26–29).

In the present report, it is shown that GSK3ß activation induces mitochondrial injury in transformed cells in part by disrupting the binding of HXK II to the mitochondria. When activated, GSK3ß induces the dissociation of HXK II from the mitochondria by phosphorylating VDAC. Thus, Akt promotes the binding of HXK II to mitochondria by inhibiting GSK3ß. Importantly, inhibition of Akt activity potentiates chemotherapy-induced cell killing through the subsequent activation of GSK3ß and the ensuing disruption in the binding of HXK II to mitochondria. Moreover, agents that induce the detachment of HXK II from the mitochondria bring about a synergistic increase in cytotoxicity when used in combination with chemotherapeutic drugs.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Treatment of cells. After an overnight incubation, HeLa cells were washed once with PBS and placed in fresh DMEM. The cells were then either left untreated or pretreated for the period of time as indicated in the figures with 100 nmol/L wortmannin or 1 µmol/L Akt inhibitor IV. Alternatively, the cells were exposed to the indicated concentrations of doxorubicin or paclitaxel in the presence or absence of either wortmannin or Akt inhibitor IV.

Measurements of viability. Cell viability was determined by the ability of the cells to exclude trypan blue. Ten microliters of a 0.5% solution of trypan blue were added to the wells. Both viable and nonviable cells were counted for each data point in a total of eight microscopic fields. Cell viability was also monitored using the plasma membrane–impermeable, dimeric cyanine dye, YOYO-1. YOYO-1 fluoresces brightly only when bound to nucleic acids. After treatments, cells are incubated with YOYO-1 before and after the addition of digitonin. Fluorescence before digitonin addition originates from dying cells that have lost plasma membrane integrity and take up YOYO-1. At the end of the experiments, digitonin was added to a final concentration of 100 µg/mL, which permeabilized 100% of the cells.

Western blot analysis. Samples were separated on 12% SDS-PAGE gels and electroblotted onto nitrocellulose membranes. Phospho-GSK3ß and total GSK3ß were detected with rabbit polyclonal antibodies at a dilution of 1:500 (Cell Signaling Technology, Beverly, MA). HXK II was detected by a rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Total VDAC was detected and immunoprecipitated with a mouse monoclonal antibody (Calbiochem, La Jolla, CA). Phosphothreonine-reactive VDAC was detected by first immunoprecipitating VDAC using the mouse monoclonal antibody followed by running the immunoprecipitate out on an SDS-PAGE gel and electroblotting onto a nitrocellulose membrane. The Western blot was then probed with an anti-phosphothreonine-reactive antibody (Zymed Laboratories, Carlsbad, CA). In all cases, the relevant protein was visualized by staining with the appropriate secondary horseradish peroxidase–labeled antibody (1:10,000) and was detected by enhanced chemiluminescence.

RNA interference. The Dharmacon (Lafayette, CO) SMART selection and SMART pooling technologies are used for the RNA interference studies of GSK3ß. The small interfering RNAs (siRNA) 1, 2, and 3 all decreased GSK3ß expression, with siRNA 3 giving the most consistent results of 95% suppression. Therefore, siRNA 3 was used in subsequent experiments to assess the effects of decreasing GSK3ß levels on the sensitivity of the cells to chemotherapeutic agents. The siRNA was delivered by a lipid-based method supplied from a commercial vendor (Gene Therapy Systems, San Diego, CA) at a final siRNA concentration of 100 nmol/L. After 24 hours, the GSK3ß levels were decreased by 95% and were maintained at that level for 1 week.

Isolation of mitochondria fraction. Following treatment, the cells were harvested by trypsinization and centrifuged at 600 x g for 10 minutes at 4°C. The cell pellets were washed once in PBS and then resuspended in 3 volumes of isolation buffer in 250 mmol/L sucrose. The cells were disrupted by 40 strokes of a glass homogenizer. The homogenate was centrifuged twice at 1,500 x g at 4°C to remove unbroken cells and nuclei. The mitochondria-enriched fraction was then pelleted by centrifugation at 12,000 x g for 30 minutes. The mitochondria fractions were normalized for protein content (25 µg/lane) and run on 12% SDS-PAGE gels followed by electroblotting onto nitrocellulose membranes.

Akt and glycogen synthase kinase 3ß activity assay. Akt or GSK3ß protein was immunoprecipitated from total cell extracts. Akt substrate or GSK3ß substrate peptides were added and the kinase reaction was started by adding ATP. Samples were incubated for 15 minutes at 37°C. After centrifugation, equal aliquots of the supernatants were transferred to Pierce (Rockford, IL) phosphocellulose unit and washed thrice with binding buffer. The level of phosphorylation of the peptides was then detected with an anti-phosphoserine antibody in an ELISA colorimetric assay.

In vitro phosphorylation of voltage-dependent anion channel. VDAC was obtained by in vitro transcription and translation followed by purification on a nickel resin. Purified VDAC was coincubated with recombinant and active GSK3ß (10 ng). Magnesium/ATP cocktail was then added. The reaction was incubated for 30 minutes at 30°C. VDAC was immunoprecipitated with anti-VDAC antibody. Phosphorylated VDAC was then detected with a phosphothreonine-reactive antibody. In some experiments, HXK II was added to the incubation reaction to assess its interaction with VDAC. The reaction was incubated for 30 minutes at 30°C as above followed by immunoprecipitation with anti-VDAC antibody and immunoblotting with anti-HXK II antibody.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Inhibition of Akt promotes the activation of glycogen synthase kinase 3ß resulting in a disruption of the binding of hexokinase II to the mitochondria. HeLa cells were treated with either wortmannin, an inhibitor of phosphatidylinositol-3-kinase (PI3-kinase), the upstream activator of Akt, or a selective Akt inhibitor (Akt inhibitor IV). Akt-1 was immunoprecipitated from HeLa cell lysates and its activity is determined by its ability to phosphorylate a substrate peptide containing the Akt phosphorylation consensus sequence. As can be seen in Fig. 1A (left), treatment of HeLa cells with 100 nmol/L wortmannin caused a decrease in basal Akt activity that reached a minimum by 4 hours. Treatment with the selective Akt inhibitor IV caused a more rapid decrease of Akt activity than wortmannin, reaching a minimum after only 2 hours of exposure. When phosphorylated on Ser⁹ by Akt, GSK3ß is inactivated (30). Therefore, the activity of GSK3ß was measured in cells when Akt was inhibited. GSK3ß was immunoprecipitated and its activity was determined by its ability to phosphorylate the GSK3ß peptide substrate. As shown in Fig. 1A (right), as anticipated, GSK3ß activity was markedly increased in wortmannin- and Akt inhibitor IV–treated cells, reaching a peak 4-fold increase over that of control levels at 2 hours. Importantly, the activation of GSK3ß was associated with its translocation to the mitochondria. As shown in Fig. 1B, treatment of HeLa cells with wortmannin or Akt inhibitor IV resulted in a decrease of inactive Ser⁹ phosphorylated GSK3ß in the cytosol and a concomitant increase of active nonphosphorylated GSK3ß at the mitochondria.

View larger version (32K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Inhibition of Akt promotes the activation of GSK3ß resulting in a disruption of the binding of HXK II to the mitochondria. A, HeLa cells were treated with 100 nmol/L wortmannin (Wort.) or 1 µmol/L Akt inhibitor IV for the periods of time indicated. The cells were then harvested and the activity of Akt and GSK3ß was determined as described in Materials and Methods. B, HeLa cells were incubated with either 100 nmol/L wortmannin or 1 µmol/L Akt inhibitor IV for 4 hours. The cells were then harvested and mitochondria were isolated. The mitochondrial and cytosolic fractions were then run out on an SDS-PAGE gel and electroblotted onto nitrocellulose membrane. The Western blot was probed with antibody against total GSK3ß or GSK3ß phosphorylated on Ser9. Hsp90 and cytochrome oxidase were used as loading controls for the cytosolic and mitochondrial fractions, respectively. C, HeLa cells were either transfected with 100 nmol/L nontargeting siRNA (A) or 100 nmol/L siRNA against GSK3ß (C). At 24 hours after transfection, the cells were either left untreated or treated with 100 nmol/L wortmannin or 1 µmol/L Akt inhibitor IV for 4 hours. Alternatively, nontransfected HeLa cells were pretreated for 30 minutes with 5 µmol/L GSK3ß inhibitor peptide before the addition of either 100 nmol/L wortmannin or 1 µmol/L Akt inhibitor IV (B). Mitochondria (Mito.) were isolated and the level of bound HXK II was detected by Western blotting.

Glycogen synthase kinase 3ß phosphorylates voltage-dependent anion channel and prevents it from binding to hexokinase II. VDAC was immunoprecipitated from HeLa cells and its phosphorylation state was determined by immunoblotting with a phosphothreonine-reactive antibody. As shown in Fig. 2A (1, lane 1), control untreated HeLa cells displayed a low level of threonine phosphorylated VDAC. However, in cells where Akt activity was inhibited by treatment with wortmannin (lane 2) or the Akt inhibitor (lane 3), there was a marked elevation in the levels of threonine phosphorylated VDAC. The phosphorylation of VDAC brought about by inhibiting Akt was dependent on GSK3ß activation. As shown in Fig. 2A (lanes 4 and 5), inhibition of GSK3ß activity through the use of siRNA against GSK3ß resulted in an abrogation of VDAC phosphorylation brought about by treatment with wortmannin or Akt inhibitor IV. Importantly, none of the treatments altered the total level of VDAC (Fig. 2A, 2). Purified VDAC was incubated with active recombinant GSK3ß. As seen in Fig. 2B (1, lane 2), recombinant GSK3ß phosphorylated purified VDAC. Thr⁵¹ of VDAC lies in a GSK3ß phosphorylation consensus sequence. In Fig. 2B (1, lane 4), incubation of a mutant VDAC, where Thr⁵¹ was mutated to an alanine residue, abolished the phosphorylation of VDAC by GSK3ß. Importantly, the ability of GSK3ß to phosphorylate VDAC was inhibited by coincubation with active Akt-1 or the GSK3ß inhibitor peptide (Fig. 2B, 1, lanes 5 and 6). In another series of experiments, purified HXK II was added to the incubation mixture containing VDAC and GSK3ß followed by immunoprecipitation of VDAC. As seen in Fig. 2B (2, lane 1), HXK II became bound to VDAC when the two where incubated in kinase buffer. However, as shown in Fig. 2B (2, lane 2), HXK II was unable to bind to VDAC when GSK3ß was present. Conversely, VDAC mutated at Thr⁵¹ to alanine was able to bind HXK II even in the presence of GSK3ß (Fig. 2B, 2, lane 4), thus illustrating the importance of VDAC phosphorylation in the ability of HXK II to bind VDAC. Addition of active Akt-1 or the GSK3ß inhibitor peptide, agents that inhibit the kinase activity of GSK3ß, restored the binding of HXK II to VDAC in the presence of GSK3ß (Fig. 2B, 2, lanes 5 and 6).

View larger version (50K): [in this window] [in a new window] [Download PPT slide]   Figure 2. GSK3ß phosphorylates VDAC in vitro and prevents it from binding to HXK II. A, HeLa cell lysates were prepared followed by immunoprecipitation with a monoclonal antibody against VDAC. The immunoprecipitates were run out on SDS-PAGE gels and transferred to nitrocellulose membranes. The membranes were probed with an anti-phosphothreonine-reactive antibody or with an anti-VDAC antibody. B, histidine-tagged wild-type VDAC (WT-VDAC) or VDAC mutated at Thr51 to alanine (VDACT51A) was generated by in vitro transcription and translation and purified on a nickel chelating resin. The VDACs were then added to kinase buffer in the presence or absence of purified and active GSK3ß. In lanes 5 and 6, active Akt-1 or the GSK3ß inhibitor peptide was added to the incubation. The VDACs were then immunoprecipitated. The immunoprecipitate was run out on an SDS-PAGE gel and blotted onto nitrocellulose. The membrane was then probed with a phosphothreonine-reactive antibody. 2, purified HXK II was added to the VDAC-GSK3ß incubation mixture followed by immunoprecipitation of VDAC and immunoblotting with HXK II antibody.

View larger version (39K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Activation of GSK3ß potentiates doxorubicin- and paclitaxel-induced cytotoxicity by promoting the dissociation of HXK II from the mitochondria. A, 1 and 2, mitochondria isolated from HeLa cells were incubated with 20 µmol/L N-HXK II-peptide or active GSK3ß in kinase buffer for 10 minutes. The mitochondria were then recovered by centrifugation and HXK II was detected in the supernatant or mitochondrial fraction. Additionally, the level of VDAC phosphorylation was determined by first immunoprecipitating VDAC from mitochondrial lysates with an anti-VDAC antibody. The immunoprecipitates were run out on SDS-PAGE gels and transferred to nitrocellulose membranes. The membranes were probed with an anti-phosphothreonine-reactive antibody (3). Alternatively, in 4 and 5, mitochondria were resuspended in respiratory buffer and recombinant Bax was then added at a final concentration of 1 µmol/L. After 15 minutes, the mitochondria were pelleted. The resulting supernatant was filtered through a 0.2- and 0.1-µm Ultrafree Millipore (Billerica, MA) membrane filter. Samples were normalized for protein content and 25 µg protein/lane were run out on SDS-PAGE gels and electroblotted onto nitrocellulose. Cytochrome c (Cyto. C) was detected by a monoclonal antibody and enhanced chemiluminescence. B, 1 and 2, HeLa cells were either left untreated or pretreated for 30 minutes with 100 nmol/L wortmannin or 1 µmol/L Akt inhibitor IV. The cells were then treated with 1 µmol/L doxorubicin (Dox.) or 10 nmol/L paclitaxel in the presence or absence of wortmannin or Akt inhibitor IV. Cell viability was determined at the indicated time points. 3 and 4, HeLa cells were transfected with siRNA against GSK3ß. After 24 hours of incubation, the cells were pretreated for 30 minutes with either 100 nmol/L wortmannin (Wort.) or 1 µmol/L Akt inhibitor IV. The cells were then treated with 1 µmol/L doxorubicin or 10 nmol/L paclitaxel (Pac.). Alternatively, the cells were exposed to 20 µmol/L N-HXK II-peptide for 1 hour before pretreatment with wortmannin or Akt inhibitor IV followed by the addition of 1 µmol/L doxorubicin or 10 nmol/L paclitaxel. Cell viability was determined at the time points indicated. C, left and right, HeLa cells were treated with 1 µmol/L doxorubicin, 100 nmol/L wortmannin, 20 µmol/L N-HXK II-peptide, or the combination of doxorubicin + wortmannin or doxorubicin + N-HXK II-peptide. At the indicated time points, the cell lysates were assayed for the level of DNA fragmentation using ELISA detection.

Cell killing induced by doxorubicin or paclitaxel treatment alone is dependent on activation of glycogen synthase kinase 3ß and a disruption in the binding of hexokinase II to the mitochondria. We next wanted to determine if doses of doxorubicin or paclitaxel that are capable of inducing extensive cell killing by themselves also required activation of GSK3ß to bring about a loss of cell viability. This required a 10-fold increase of the doxorubicin or paclitaxel concentrations compared with that used previously to show a potentiation of cell killing induced by inhibition of Akt or dissociation of HXK II from the mitochondria. As shown in Fig. 4 (left), treatment of HeLa cells with 10 µmol/L doxorubicin resulted in a progressive loss of cell viability, with 84% of the cells dead at 24 hours of treatment. Similarly, 100 nmol/L paclitaxel resulted in a 67% loss of cell viability after 24 hours of exposure (Fig. 4, right). The cell killing induced by doxorubicin and paclitaxel was accompanied by a disruption in the binding of HXK II to the mitochondria. As shown in Fig. 4B (1), mitochondria were progressively depleted of HXK II over a 4-hour time course following exposure to 10 µmol/L doxorubicin. The depletion of mitochondrial bound HXK II induced by doxorubicin treatment was accompanied by a parallel accumulation of GSK3ß at the mitochondria (Fig. 4B, 2) and phosphorylation of VDAC (Fig. 4B, 3). Importantly, the depletion of mitochondrial bound HXK II occurred at a time point before the extensive loss of cell viability that followed. Similar results were seen with paclitaxel. The doxorubicin- and paclitaxel-induced cell killing was preceded by a decrease of Akt activity. As shown in Fig. 4C (left), basal Akt activity dropped in the first 6 hours of exposure to 10 µmol/L doxorubicin or 100 nmol/L paclitaxel. Moreover, Akt activity in the chemotherapeutic drug–treated cells became resistant to stimulation by growth factors. HeLa cells were exposed to 10 µmol/L doxorubicin or 100 nmol/L paclitaxel for 4 hours followed by stimulation with epidermal growth factor (EGF). As shown in Fig. 4C (right), the doxorubicin- and paclitaxel-treated cells exhibited a severely blunted Akt activation in response to EGF stimulation compared with nontreated control cells.

View larger version (30K): [in this window] [in a new window] [Download PPT slide]   Figure 4. Doxorubicin- and paclitaxel-induced cytotoxicity is accompanied by inhibition of Akt, accumulation of active GSK3ß at the mitochondria, phosphorylation of VDAC, and disruption in the binding of HXK II to the mitochondria. A, HeLa cells were treated with 10 µmol/L doxorubicin or 100 nmol/L paclitaxel and cell viability was determined at the indicated time points. B, HeLa cells were treated with 10 µmol/L doxorubicin (0-4 hours). The cells were harvested at the indicated time points and mitochondria were isolated. 1 and 2, a mitochondrial extract was run out on SDS-PAGE gel and blotted onto a nitrocellulose membrane. The membrane was probed with antibodies for HXK II or GSK3ß. 3, the mitochondrial extract was immunoprecipitated with anti-VDAC antibody. The immunoprecipitate was run out on an SDS-PAGE gel and blotted onto nitrocellulose. The membrane was then probed with anti-phosphothreonine antibody. C, left, HeLa cells were either left alone or treated with 10 µmol/L doxorubicin or 100 nmol/L paclitaxel. At the time points indicated, the level of Akt activity was determined. Right, HeLa cells were treated with 10 µmol/L doxorubicin or 100 nmol/L paclitaxel for 4 hours. The cells were then stimulated with 20 nmol/L EGF and the level of Akt activity was determined at the indicated time points.

View larger version (30K): [in this window] [in a new window] [Download PPT slide]   Figure 5. Inhibition of GSK3ß and the consequent preservation of the binding of HXK II to the mitochondria prevent doxorubicin- and paclitaxel-induced cytotoxicity. A, HeLa cells were treated with 10 µmol/L doxorubicin or 100 nmol/L paclitaxel for the indicated time periods. Cell extracts were then prepared and GSK3ß activity was measured as described in Materials and Methods. B, HeLa cells were transfected with siRNA targeting GSK3ß. After 24 hours of incubation, the cells were treated with 10 µmol/L doxorubicin (left) or 100 nmol/L paclitaxel (right). Alternatively, cells in which GSK3ß levels were suppressed by siRNA were first pretreated with 20 µmol/L N-HXK II-peptide to force dissociation of HXK II from the mitochondria followed by treatment with 10 µmol/L doxorubicin or 100 nmol/L paclitaxel. Cell viability was determined at the time points indicated. C, HeLa cells transfected with nontargeting siRNA or siRNA targeting GSK3ß were treated with 10 µmol/L doxorubicin or 100 nmol/L paclitaxel. Alternatively, the cells were first pretreated with 20 µmol/L N-HXK II-peptide to detach HXK II from the mitochondria followed by treatment with 10 µmol/L doxorubicin or 100 nmol/L paclitaxel. After 4 hours of exposure, the mitochondria were isolated. Mitochondrial extracts were run out on SDS-PAGE gels and electroblotted onto nitrocellulose membranes. The membranes were probed with anti-HXK II antibody.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In highly malignant tumors, the level of HXK II expression may be elevated by up to 10-fold compared with normal tissue expression (31). Due to poor vascularization, tumors become hypoxic resulting in a decrease in mitochondrial oxidative phosphorylation. Therefore, the increased levels of HXK II and glycolysis would benefit tumor cell survival by providing ATP through anaerobic glycolysis. However, tumor cells exhibit increased glycolytic flux even when oxygen supplies are plentiful, a condition known as aerobic glycolysis. Aerobic glycolysis depends on the binding of HXK I or HXK II to the mitochondria through an interaction with VDAC (32, 33).

The coupling of the adenine nucleotide translocator and VDAC with HXK II occurs at contact sites, regions where the inner and outer mitochondrial membranes appose one another (34, 35). Contact sites may be critical in the antiapoptotic effects of HXK II. Bax translocates to the mitochondria in a biphasic manner, first localizing to mitochondrial contact sites, whereupon an interaction between Bax and VDAC occurs (36). Similarly, Bid has also been found to localize to mitochondrial contact sites (37). HXK II prevents mitochondrial injury induced by both Bax and Bid (19, 20, 38). Like Bax, the BH3-only subfamily proapoptotic protein Bim interacts with VDAC (39). Significantly, both Bax and Bim have been implicated in the cell killing brought about by doxorubicin and paclitaxel, respectively (40–43). Such findings reinforce the notion that the binding of HXK II to VDAC can interfere with the killing mechanisms engendered by doxorubicin and paclitaxel.

Akt/protein kinase B controls a vast array of cellular functions and can exert antiapoptotic effects by several mechanisms. One of these mechanisms may be through promotion of HXK II binding to mitochondria. It was shown recently that Akt activity increases the basal levels of mitochondrial bound HXK I and HXK II and that forced displacement of HXK II from the mitochondria reduces the ability of Akt to inhibit apoptosis (21). Indeed, Akt localizes to the mitochondria on growth factor stimulation (23). However, the exact mechanism whereby Akt promotes the binding of HXK II to the mitochondria was uncharacterized.

Akt constrains the activity of GSK3ß by phosphorylating it on Ser⁹ (30). When Akt is inhibited, GSK3ß becomes stimulated. Inhibition of GSK3ß has been shown to protect neuronal cells against apoptosis brought about by several conditions (44, 45). A large proportion of GSK3ß is found in the cytosol, but pools are also found in the nucleus and mitochondria. GSK3ß has been implicated in mediating mitochondrial dysfunction. DNA damage induced an association between GSK3ß and p53 at the mitochondria, where inhibition of GSK3ß blocked release of cytochrome c and caspase activation (46). GSK3ß has also been shown to phosphorylate Bax and promote its localization to the mitochondria during neuronal apoptosis (47). Inhibition of GSK3ß activity protects cardiomyocytes from hypoxia/reoxygenation injury by suppressing onset of the mitochondrial permeability transition (28). Intriguingly, HXK II has been shown to inhibit opening of the permeability transition pore (35).

VDAC possesses a GSK3ß phosphorylation consensus motif at amino acids 51 to 55. Mutation of Thr⁵¹ to alanine abolished the ability of GSK3ß to phosphorylate VDAC. Significantly, this region is thought to be on the cytoplasmic side of the outer mitochondrial membrane where it could interact with GSK3ß. This region is highly conserved in mitochondrial porins and is in close proximity to Glu⁷², a residue identified as being critical to the ability of VDAC to bind HXK I and HXK II.

The present report shows the synergistic increase in cytotoxicity induced by chemotherapeutic drugs when the binding of HXK II to mitochondria is disrupted. The Akt signaling pathway preserves the interaction of HXK II with VDAC by restraining the activity of GSK3ß (Fig. 6). When the PI3-kinase/Akt pathway is inhibited, this triggers the activation of GSK3ß resulting in phosphorylation of VDAC with an attendant disruption in the binding of HXK II to the mitochondria. Inhibition of GSK3ß prevented the cell killing induced by treatment with doxorubicin or paclitaxel. Moreover, the ability of GSK3ß inhibition to prevent cytotoxicity was dependent on a preservation of HXK II binding to mitochondria, as forced displacement of HXK II abrogated the protective effect imparted from a restraining of GSK3ß activity. Moreover, forced detachment of HXK II from the mitochondria also potentiated the cell killing brought about by suboptimal doses of doxorubicin and paclitaxel.

View larger version (19K): [in this window] [in a new window] [Download PPT slide]   Figure 6. Potentiation of doxorubicin- and paclitaxel-induced cytotoxicity by disruption of HXK II binding to VDAC. Inhibition of Akt causes the activation of GSK3ß that phosphorylates VDAC. VDAC phosphorylated by GSK3ß is unable to bind HXK II, thus increasing the susceptibility of VDAC to the binding of proapoptotic proteins resulting in increased outer mitochondrial membrane permeability. The cytotoxicity of doxorubicin and paclitaxel is potentiated by detachment of HXK II from VDAC, mediated either by inhibition of Akt and the subsequent activation of GSK3ß or the forced detachment of HXK II from VDAC by treatment with the N-HXK II-peptide.

	Acknowledgments

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 6/ 2/05. Revised 8/ 1/05. Accepted 8/25/05.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Warburg O. On the origin of cafncer cells. Science 1956;123:309–14.[Free Full Text]
2. Elstrom RL, Bauer DE, Buzzai M, et al. Akt stimulates aerobic glycolysis in cancer cells. Cancer Res 2004;64:3892–9.[Abstract/Free Full Text]
3. Bustamante E, Morris HP, Pedersen PL. Energy metabolism of tumor cells. Requirement for a form of hexokinase with a propensity for mitochondrial binding. J Biol Chem 1981;256:8699–704.[Abstract/Free Full Text]
4. Erdal E, Ozturk N, Cagatay T, Eksioglu-Demiralp E, Ozturk M. Lithium-mediated downregulation of PKB/Akt and cyclin E with growth inhibition in hepatocellular carcinoma cells. Int J Cancer 2005;115:903–10.[CrossRef][Medline]
5. Golshani-Hebroni SG, Bessman SP. Hexokinase binding to mitochondria: a basis for proliferative energy metabolism. J Bioenerg Biomembr 1997;29:331–8.[CrossRef][Medline]
6. Oudard S, Boitier E, Miccoli L, et al. Gliomas are driven by glycolysis: putative roles of hexokinase, oxidative phosphorylation and mitochondrial ultrastructure. Anticancer Res 1997;17:1903–11.[Medline]
7. Wilson JE. Hexokinases. Rev Physiol Biochem Pharmacol 1995;126:65–198.[Medline]
8. Wilson JE. Isozymes of mammalian hexokinase: structure, subcellular localization and metabolic function. J Exp Biol 2003;206:2049–57.[Abstract/Free Full Text]
9. Colombini M. VDAC: the channel at the interface between mitochondria and the cytosol. Mol Cell Biochem 2004;256-257:107–15.
10. Mannella CA. Conformational changes in the mitochondrial channel protein, VDAC, and their functional implications. J Struct Biol 1998;121:207–18.[CrossRef][Medline]
11. Kroemer G, Dallaporta B, Resche-Rigon M. The mitochondrial death/life regulator in apoptosis and necrosis. Annu Rev Physiol 1998;60:619–42.[CrossRef][Medline]
12. Green DR, Reed JC. Mitochondria and apoptosis. Science 1998;281:1309–12.[Abstract/Free Full Text]
13. Madesh M, Hajnoczky G. VDAC-dependent permeabilization of the outer mitochondrial membrane by superoxide induces rapid and massive cytochrome c release. J Cell Biol 2001;155:1003–15.[Abstract/Free Full Text]
14. Shimizu S, Ide T, Yanagida T, Tsujimoto Y. Electrophysiological study of a novel large pore formed by Bax and the voltage-dependent anion channel that is permeable to cytochrome c. J Biol Chem 2000;275:12321–5.[Abstract/Free Full Text]
15. Shimizu S, Narita M, Tsujimoto Y. Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC. Nature 1999;399:483–7.[CrossRef][Medline]
16. Shimizu S, Narita M, Tsujimoto Y. Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC [see comments]. Nature 1999;399:483–7.
17. Shimizu S, Matsuoka Y, Shinohara Y, Yoneda Y, Tsujimoto Y. Essential role of voltage-dependent anion channel in various forms of apoptosis in mammalian cells. J Cell Biol 2001;152:237–50.[Abstract/Free Full Text]
18. Gottlob K, Majewski N, Kennedy S, et al. Inhibition of early apoptotic events by Akt/PKB is dependent on the first committed step of glycolysis and mitochondrial hexokinase. Genes Dev 2001;15:1406–18.[Abstract/Free Full Text]
19. Pastorino JG, Shulga N, Hoek JB. Mitochondrial binding of hexokinase II inhibits Bax-induced cytochrome c release and apoptosis. J Biol Chem 2002;277:7610–8.[Abstract/Free Full Text]
20. Majewski N, Nogueira V, Robey RB, Hay N. Akt inhibits apoptosis downstream of BID cleavage via a glucose-dependent mechanism involving mitochondrial hexokinases. Mol Cell Biol 2004;24:730–40.[Abstract/Free Full Text]
21. Majewski N, Nogueira V, Bhaskar P, et al. Hexokinase-mitochondria interaction mediated by Akt is required to inhibit apoptosis in the presence or absence of Bax and Bak. Mol Cell 2004;16:819–30.[CrossRef][Medline]
22. Azoulay-Zohar H, Israelson A, Abu-Hamad S, Shoshan-Barmatz V. In self-defence: hexokinase promotes voltage-dependent anion channel closure and prevents mitochondria-mediated apoptotic cell death. Biochem J 2004;377:347–55.[CrossRef][Medline]
23. Bijur GN, Jope RS. Rapid accumulation of Akt in mitochondria following phosphatidylinositol 3-kinase activation. J Neurochem 2003;87:1427–35.[Medline]
24. Bijur GN, Jope RS. Glycogen synthase kinase-3ß is highly activated in nuclei and mitochondria. Neuroreport 2003;14:2415–9.[CrossRef][Medline]
25. Jope RS, Johnson GV. The glamour and gloom of glycogen synthase kinase-3. Trends Biochem Sci 2004;29:95–102.[CrossRef][Medline]
26. Bijur GN, De Sarno P, Jope RS. Glycogen synthase kinase-3ß facilitates staurosporine- and heat shock-induced apoptosis. Protection by lithium. J Biol Chem 2000;275:7583–90.[Abstract/Free Full Text]
27. Turenne GA, Price BD. Glycogen synthase kinase 3ß phosphorylates serine 33 of p53 and activates p53's transcriptional activity. BMC Cell Biol 2001;2:12.[CrossRef][Medline]
28. Juhaszova M, Zorov DB, Kim SH, et al. Glycogen synthase kinase-3ß mediates convergence of protection signaling to inhibit the mitochondrial permeability transition pore. J Clin Invest 2004;113:1535–49.[CrossRef][Medline]
29. Macanas-Pirard P, Yaacob NS, Lee PC, et al. Glycogen synthase kinase-3 mediates acetaminophen-induced apoptosis in human hepatoma cells. J Pharmacol Exp Ther 2005;313:780–9.[Abstract/Free Full Text]
30. Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 1995;378:785–9.[CrossRef][Medline]
31. Mathupala SP, Rempel A, Pedersen PL. Glucose catabolism in cancer cells: identification and characterization of a marked activation response of the type II hexokinase gene to hypoxic conditions. J Biol Chem 2001;13:13.
32. Brand K. Aerobic glycolysis by proliferating cells: protection against oxidative stress at the expense of energy yield. J Bioenerg Biomembr 1997;29:355–64.[CrossRef][Medline]
33. Brand KA, Hermfisse U. Aerobic glycolysis by proliferating cells: a protective strategy against reactive oxygen species. FASEB J 1997;11:388–95.[Abstract]
34. Ardail D, Privat JP, Egret-Charlier M, et al. Mitochondrial contact sites. Lipid composition and dynamics. J Biol Chem 1990;265:18797–802.[Abstract/Free Full Text]
35. Brdiczka D, Beutner G, Ruck A, Dolder M, Wallimann T. The molecular structure of mitochondrial contact sites. Their role in regulation of energy metabolism and permeability transition. Biofactors 1998;8:235–42.[Medline]
36. Capano M, Crompton M. Biphasic translocation of Bax to mitochondria. Biochem J 2002;367:169–78.[CrossRef][Medline]
37. Lutter M, Perkins GA, Wang X. The pro-apoptotic Bcl-2 family member tBid localizes to mitochondrial contact sites. BMC Cell Biol 2001;2:22.[CrossRef][Medline]
38. Vyssokikh MY, Zorova L, Zorov D, et al. Bax releases cytochrome c preferentially from a complex between porin and adenine nucleotide translocator. Hexokinase activity suppresses this effect. Mol Biol Rep 2002;29:93–6.[CrossRef][Medline]
39. Sugiyama T, Shimizu S, Matsuoka Y, Yoneda Y, Tsujimoto Y. Activation of mitochondrial voltage-dependent anion channel by a pro-apoptotic BH3-only protein Bim. Oncogene 2002;21:4944–56.[CrossRef][Medline]
40. Panaretakis T, Pokrovskaja K, Shoshan MC, Grander D. Activation of Bak, Bax, and BH3-only proteins in the apoptotic response to doxorubicin. J Biol Chem 2002;277:44317–26.[Abstract/Free Full Text]
41. Bosanquet AG, Sturm I, Wieder T, et al. Bax expression correlates with cellular drug sensitivity to doxorubicin, cyclophosphamide and chlorambucil but not fludarabine, cladribine or corticosteroids in B cell chronic lymphocytic leukemia. Leukemia 2002;16:1035–44.[CrossRef][Medline]
42. Li R, Moudgil T, Ross HJ, Hu HM. Apoptosis of non-small-cell lung cancer cell lines after paclitaxel treatment involves the BH3-only proapoptotic protein Bim. Cell Death Differ 2005;12:292–303.[CrossRef][Medline]
43. Sunters A, Fernandez de Mattos S, Stahl M, et al. FoxO3a transcriptional regulation of Bim controls apoptosis in paclitaxel-treated breast cancer cell lines. J Biol Chem 2003;278:49795–805.[Abstract/Free Full Text]
44. Jope RS, Bijur GN. Mood stabilizers, glycogen synthase kinase-3ß and cell survival. Mol Psychiatry 2002;7 Suppl 1:S35–45.[CrossRef]
45. Morrison RS, Kinoshita Y, Johnson MD, et al. Neuronal survival and cell death signaling pathways. Adv Exp Med Biol 2002;513:41–86.[Medline]
46. Watcharasit P, Bijur GN, Song L, et al. Glycogen synthase kinase-3ß (GSK3ß) binds to and promotes the actions of p53. J Biol Chem 2003;278:48872–9.[Abstract/Free Full Text]
47. Linseman DA, Butts BD, Precht TA, et al. Glycogen synthase kinase-3ß phosphorylates Bax and promotes its mitochondrial localization during neuronal apoptosis. J Neurosci 2004;24:9993–10002.[Abstract/Free Full Text]

This article has been cited by other articles:

				Z. Wang, A. Havasi, J. Gall, R. Bonegio, Z. Li, H. Mao, J. H. Schwartz, and S. C. Borkan GSK3{beta} Promotes Apoptosis after Renal Ischemic Injury J. Am. Soc. Nephrol., February 1, 2010; 21(2): 284 - 294. [Abstract] [Full Text] [PDF]

				M. A. Omar, L. Wang, and A. S. Clanachan Cardioprotection by GSK-3 inhibition: role of enhanced glycogen synthesis and attenuation of calcium overload Cardiovasc Res, January 31, 2010; (2010): cvp421v2 - cvp421. [Abstract] [Full Text] [PDF]

				S. Bonnet, R. Paulin, G. Sutendra, P. Dromparis, M. Roy, K. O. Watson, J. Nagendran, A. Haromy, J. R.B. Dyck, and E. D. Michelakis Dehydroepiandrosterone Reverses Systemic Vascular Remodeling Through the Inhibition of the Akt/GSK3-{beta}/NFAT Axis Circulation, September 29, 2009; 120(13): 1231 - 1240. [Abstract] [Full Text] [PDF]

				P. T. Bhaskar, V. Nogueira, K. C. Patra, S.-M. Jeon, Y. Park, R. B. Robey, and N. Hay mTORC1 Hyperactivity Inhibits Serum Deprivation-Induced Apoptosis via Increased Hexokinase II and GLUT1 Expression, Sustained Mcl-1 Expression, and Glycogen Synthase Kinase 3{beta} Inhibition Mol. Cell. Biol., September 15, 2009; 29(18): 5136 - 5147. [Abstract] [Full Text] [PDF]

				P. Dromparis and E. D. Michelakis A redox-metabolic-electrical remodeling in the diseased left and right ventricle: direct clinical implications in heart disease and beyond. Focus on "Role of {gamma}-glutamyl transpeptidase in redox regulation of K+ channel remodeling in postmyocardial infarction rat hearts" Am J Physiol Cell Physiol, August 1, 2009; 297(2): C231 - C234. [Full Text] [PDF]

				D. B. Zorov, M. Juhaszova, Y. Yaniv, H. B. Nuss, S. Wang, and S. J. Sollott Regulation and pharmacology of the mitochondrial permeability transition pore Cardiovasc Res, July 15, 2009; 83(2): 213 - 225. [Abstract] [Full Text] [PDF]

				M. Juhaszova, D. B. Zorov, Y. Yaniv, H. B. Nuss, S. Wang, and S. J. Sollott Role of Glycogen Synthase Kinase-3{beta} in Cardioprotection Circ. Res., June 5, 2009; 104(11): 1240 - 1252. [Abstract] [Full Text] [PDF]

				E. Gurel, K. M. Smeele, O. Eerbeek, A. Koeman, C. Demirci, M. W. Hollmann, and C. J. Zuurbier Ischemic preconditioning affects hexokinase activity and HKII in different subcellular compartments throughout cardiac ischemia-reperfusion J Appl Physiol, June 1, 2009; 106(6): 1909 - 1916. [Abstract] [Full Text] [PDF]

				Y. Wang, W. Feng, W. Xue, Y. Tan, D. W. Hein, X.-K. Li, and L. Cai Inactivation of GSK-3{beta} by Metallothionein Prevents Diabetes-Related Changes in Cardiac Energy Metabolism, Inflammation, Nitrosative Damage, and Remodeling Diabetes, June 1, 2009; 58(6): 1391 - 1402. [Abstract] [Full Text] [PDF]

				S. Miyamoto, M. Rubio, and M. A. Sussman Nuclear and mitochondrial signalling Akts in cardiomyocytes Cardiovasc Res, May 1, 2009; 82(2): 272 - 285. [Abstract] [Full Text] [PDF]

				L. Arzoine, N. Zilberberg, R. Ben-Romano, and V. Shoshan-Barmatz Voltage-dependent Anion Channel 1-based Peptides Interact with Hexokinase to Prevent Its Anti-apoptotic Activity J. Biol. Chem., February 6, 2009; 284(6): 3946 - 3955. [Abstract] [Full Text] [PDF]

				A. Gimenez-Cassina, F. Lim, T. Cerrato, G. M. Palomo, and J. Diaz-Nido Mitochondrial Hexokinase II Promotes Neuronal Survival and Acts Downstream of Glycogen Synthase Kinase-3 J. Biol. Chem., January 30, 2009; 284(5): 3001 - 3011. [Abstract] [Full Text] [PDF]

				P. Zhai and J. Sadoshima Overcoming an Energy Crisis?: An Adaptive Role of Glycogen Synthase Kinase-3 Inhibition in Ischemia/Reperfusion Circ. Res., October 24, 2008; 103(9): 910 - 913. [Full Text] [PDF]

				E. D. Michelakis, M. R. Wilkins, and M. Rabinovitch Emerging Concepts and Translational Priorities in Pulmonary Arterial Hypertension Circulation, September 30, 2008; 118(14): 1486 - 1495. [Full Text] [PDF]

				S. Yuan, Y. Fu, X. Wang, H. Shi, Y. Huang, X. Song, L. Li, N. Song, and Y. Luo Voltage-dependent anion channel 1 is involved in endostatin-induced endothelial cell apoptosis FASEB J, August 1, 2008; 22(8): 2809 - 2820. [Abstract] [Full Text] [PDF]

				Y. Nishino, I. G. Webb, S. M. Davidson, A. I. Ahmed, J. E. Clark, S. Jacquet, A. M. Shah, T. Miura, D. M. Yellon, M. Avkiran, et al. Glycogen Synthase Kinase-3 Inactivation Is Not Required for Ischemic Preconditioning or Postconditioning in the Mouse Circ. Res., August 1, 2008; 103(3): 307 - 314. [Abstract] [Full Text] [PDF]

				S. Abu-Hamad, H. Zaid, A. Israelson, E. Nahon, and V. Shoshan-Barmatz Hexokinase-I Protection against Apoptotic Cell Death Is Mediated via Interaction with the Voltage-dependent Anion Channel-1: MAPPING THE SITE OF BINDING J. Biol. Chem., May 9, 2008; 283(19): 13482 - 13490. [Abstract] [Full Text] [PDF]

				S. J. Clarke, I. Khaliulin, M. Das, J. E. Parker, K. J. Heesom, and A. P. Halestrap Inhibition of Mitochondrial Permeability Transition Pore Opening by Ischemic Preconditioning Is Probably Mediated by Reduction of Oxidative Stress Rather Than Mitochondrial Protein Phosphorylation Circ. Res., May 9, 2008; 102(9): 1082 - 1090. [Abstract] [Full Text] [PDF]

				L. Sun, S. Shukair, T. J. Naik, F. Moazed, and H. Ardehali Glucose Phosphorylation and Mitochondrial Binding Are Required for the Protective Effects of Hexokinases I and II Mol. Cell. Biol., February 1, 2008; 28(3): 1007 - 1017. [Abstract] [Full Text] [PDF]

				Z. P. Shaik, E. K. Fifer, and G. Nowak Akt activation improves oxidative phosphorylation in renal proximal tubular cells following nephrotoxicant injury Am J Physiol Renal Physiol, February 1, 2008; 294(2): F423 - F432. [Abstract] [Full Text] [PDF]

				C. J. Zuurbier, P. J. M. Keijzers, A. Koeman, H. B. Van Wezel, and M. W. Hollmann Anesthesia's Effects on Plasma Glucose and Insulin and Cardiac Hexokinase at Similar Hemodynamics and Without Major Surgical Stress in Fed Rats Anesth. Analg., January 1, 2008; 106(1): 135 - 142. [Abstract] [Full Text] [PDF]

				W. Kim, J.-H. Yoon, J.-M. Jeong, G.-J. Cheon, T.-S. Lee, J.-I. Yang, S.-C. Park, and H.-S. Lee Apoptosis-inducing antitumor efficacy of hexokinase II inhibitor in hepatocellular carcinoma Mol. Cancer Ther., September 1, 2007; 6(9): 2554 - 2562. [Abstract] [Full Text] [PDF]

				J. Y. Lee, S. J. Yu, Y. G. Park, J. Kim, and J. Sohn Glycogen Synthase Kinase 3{beta} Phosphorylates p21WAF1/CIP1 for Proteasomal Degradation after UV Irradiation Mol. Cell. Biol., April 15, 2007; 27(8): 3187 - 3198. [Abstract] [Full Text] [PDF]

				A. Patenaude, R. G. Deschesnes, J. L.C. Rousseau, E. Petitclerc, J. Lacroix, M.-F. Cote, and R. C.-Gaudreault New Soft Alkylating Agents with Enhanced Cytotoxicity against Cancer Cells Resistant to Chemotherapeutics and Hypoxia Cancer Res., March 1, 2007; 67(5): 2306 - 2316. [Abstract] [Full Text] [PDF]

				S. Li, Y. Zhou, R. Wang, H. Zhang, Y. Dong, and C. Ip Selenium sensitizes MCF-7 breast cancer cells to doxorubicin-induced apoptosis through modulation of phospho-Akt and its downstream substrates Mol. Cancer Ther., March 1, 2007; 6(3): 1031 - 1038. [Abstract] [Full Text] [PDF]

				L. Huc, X. Tekpli, J. A. Holme, M. Rissel, A. Solhaug, C. Gardyn, G. Le Moigne, M. Gorria, M.-T. Dimanche-Boitrel, and D. Lagadic-Gossmann c-Jun NH2-Terminal Kinase-Related Na+/H+ Exchanger Isoform 1 Activation Controls Hexokinase II Expression in Benzo(a)Pyrene-Induced Apoptosis Cancer Res., February 15, 2007; 67(4): 1696 - 1705. [Abstract] [Full Text] [PDF]

				G. Kroemer, L. Galluzzi, and C. Brenner Mitochondrial Membrane Permeabilization in Cell Death Physiol Rev, January 1, 2007; 87(1): 99 - 163. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pastorino, J. G. Articles by Shulga, N. Search for Related Content PubMed PubMed Citation Articles by Pastorino, J. G. Articles by Shulga, N.