Glycolytic Inhibitor 2-deoxyglucose Research Articles

Dual Inhibition of Tumor Energy Pathway by 2-Deoxyglucose and Metformin Is Effective against a Broad Spectrum of Preclinical Cancer Models

Tumor cell proliferation requires both growth signals and sufficient cellular bioenergetics. The AMP-activated protein kinase (AMPK) pathway seems dominant over the oncogenic signaling pathway suppressing cell proliferation. This study investigated the preclinical efficacy of targeting the tumor bioenergetic pathway using a glycolysis inhibitor 2-deoxyglucose (2DG) and AMPK agonists, AICAR and metformin. We evaluated the in vitro antitumor activity of 2DG, metformin or AICAR alone, and 2DG in combination either with metformin or AICAR. We examined in vivo efficacy using xenograft mouse models. 2DG alone was not sufficient to promote tumor cell death, reflecting the limited efficacy showed in clinical trials. A combined use of 2DG and AICAR also failed to induce cell death. However, 2DG and metformin led to significant cell death associated with decrease in cellular ATP, prolonged activation of AMPK, and sustained autophagy. Gene expression analysis and functional assays revealed that the selective AMPK agonist AICAR augments mitochondrial energy transduction (OXPHOS) whereas metformin compromises OXPHOS. Importantly, forced energy restoration with methyl pyruvate reversed the cell death induced by 2DG and metformin, suggesting a critical role of energetic deprivation in the underlying mechanism of cell death. The combination of 2DG and metformin inhibited tumor growth in mouse xenograft models. Deprivation of tumor bioenergetics by dual inhibition of energy pathways might be an effective novel therapeutic approach for a broad spectrum of human tumors.

2-Deoxyglucose Induces Noxa-Dependent Apoptosis in Alveolar Rhabdomyosarcoma

Alveolar and embryonal rhabdomyosarcomas are childhood tumors that do not respond well to current chemotherapies. Here, we report that the glycolytic inhibitor 2-deoxyglucose (2-DG) can efficiently promote cell death in alveolar, but not embryonal, rhabdomyosarcoma cell lines. Notably, 2-DG also induced cell differentiation accompanied by downregulation of PAX3/FOXO1a, the chromosome translocation-encoded fusion protein that is a central oncogenic driver in this disease. Cell death triggered by 2-DG was associated with its ability to activate Bax and Bak. Overexpression of the antiapoptotic Bcl-2 homologues Bcl-x(L) and Mcl-1 prevented apoptosis, indicating that cell death proceeds through the mitochondrial pathway. Mechanistic investigations indicated that Mcl-1 downregulation and Noxa upregulation were critical for 2-DG-induced apoptosis. In addition, 2-DG promoted eIF2α phosphorylation and inactivation of the mTOR pathway. Mcl-1 loss and cell death were prevented by downregulation of the endoplasmic reticulum (ER) stress-induced protein ATF4 and by incubating cells in the presence of mannose, which reverted 2-DG-induced ER stress but not ATP depletion. Thus, energetic stresses created by 2-DG were not the primary cause of cell death. Together, our findings suggest that glycolysis inhibitors such as 2-DG may be highly effective in treating alveolar rhabdomyosarcoma and that Noxa could offer a prognostic marker to monitor the efficacy of such agents.

939 poster EFFECTS OF RADIATION-INDUCED ALPHAVBETA3 INTEGRIN MODULATION ON CILENGITIDE EFFICIENCY IN PEDIATRIC GLIOMA CELLS

The efficacy of PARP inhibition under treatment conditions of RT plus temozolomide (TMZ) for glioblastoma (GBM) has not been previously reported, therefore we sought to determine the effects of PARP inhibition as well as the molecular determinants of response to PARP inhibitors in combination with standard treatment of GBM. Materials: In the present study, PJ-34, a highly potent PARP inhibitor, was used to investigate the effects of PTEN status on PARP inhibition in combination with RT and TMZ using in vitro and in vivo models of GBM. First, two primary GBM cell lines that lack PTEN expression and two primary GBM cells lines that express PTEN were utilized to determine if PTEN status affects the efficacy of PARP inhibition in combination with RT and TMZ. Next, DNA damage and ATP levels were measured after the addition of a PARP inhibitor to RT and TMZ with respect to PTEN status. The role of glucose metabolism and PI3K/ AKT signaling in the response to PARP inhibition was then characterized in correlation to PTEN status. Finally, a U87-MG xenograft model was used to validate our in vitro findings. Results: Cell lines lacking PTEN were sensitized to PJ-34 in the setting of RT plus TMZ, whereas PTEN expression was associated with resistance. AKT activation was higher in resistant cell lines and was associated with increased ATP levels. Additionally, forced expression of PTEN conferred resistance to PJ-34. Treatment with the glycolytic inhibitor 2-deoxyglucose (2-DG) or the phosphatidylinosotide-3 kinase (PI3K) inhibitor LY294002 sensitized resistant PTEN-expressing cell lines to PJ-34 suggesting PI3K/AKT signaling is likely responsible for resistance in PTEN positive cells. The therapeutic benefits of PARP inhibition observed in PTEN-null cell lines may be dependent on RT, as treatment with TMZ alone prevented sensitization. Conclusions: In summary, PTEN status may be a promising marker to identify tumors that will respond to PARP inhibition when administered with TMZ and RT. Our findings suggest that the ability of PARP inhibitors, such as PJ34, to enhance the anti-tumor effects of RT and TMZ is highly dependent on the equilibrium between PARP’s pro-survival (increased PI3K/AKT signaling) and pro-death (increased DNA damage) functions. To this end, we are the first to report that the ability of PARP inhibition to enhance TMZ and RT response depends on the proteomic profile of GBM tumors, especially PTEN status.

Some Leukemia Cells Are Dependent on Glutamine as Energy Source

AbstractAbstract 4861In cancer cells, glucose uptake is elevated and glycolysis persists even under aerobic conditions (Warburg effect). Glutamine metabolism is another target for alteration in cancer development. Glutaminolysis (catabolism of glutamine to generate ATP) is known to increase in tumors. We examined the dependency of the leukemia cells (Kasumi-1, THP-1, HL-60 and NB4) on glucose or glutamine by measuring the growth (MTS count) in glucose- or glutamine-deprived condition. Glucose withdrawal greatly suppressed the growth of all 4 cell lines. However, glutamine withdrawal showed different growth suppressive effects among the cell lines (Kasumi-1: 55% of control, THP-1: 60%, HL-60: 39%, NB4: 70%). HL-60 was most sensitive to glutamine deprivation. The growth suppression of HL-60 due to glutamine withdrawal was partially rescued by oxaloacetate (OAA), a TCA cycle metabolite, while the growth of other cell lines was not rescued by OAA. In the course of glutamine catabolism, ammonia is liberated. Although basal level of the ammonia concentration was not so different among each cell line, glycolysis inhibitor (2-deoxyglucose) treatment enhanced the ammonia generation in HL-60 (Kasumi-1: 2.8% increased, THP-1: 1.7%, HL-60: 6.1%, NB4: 2.8%). Glutaminase, an enzyme converting glutamine to glutamate, is most abundantly expressed in HL-60 in western blot analysis. In addition, HL-60 was most sensitive to the treatment with aminooxyacetate, an inhibitor of glutamate-dependent transaminases that convert glutamate into a-ketoglutarate in the glutaminolytic pathway (Kasumi-1: 86% of control, THP-1: 97%, HL-60: 79%, NB4: 83%). Taken together, HL-60 was considered as glutamine dependent cell line. Therapies targeting glutamine metabolism, such as glutamine depletion or use of inhibitor of glutaminolytic pathway, might be effective against some leukemia. Disclosures:No relevant conflicts of interest to declare.

Glycogen metabolism provides nutritional support to renal cancer cells under conditions of stress and may serve as a marker of response to antiangiogenic therapy with bevacizumab

Abstract Purpose: The clear cell type of renal cell carcinoma (ccRCC) derives its histologic appearance from accumulation of glycogen, but the functional significance of this phenomenon is yet unexplored. In physiological conditions, liver and muscle are the main tissues capable of producing and storing glycogen, which is mobilized to produce energy through glycolysis under stress conditions or limited nutrient supply. Tumors frequently experience fluctuations in nutrient availability, uptake and metabolic stress. The purpose of this study is to test the hypothesis that renal tumors express active glycogen synthase (GYS1) to produce glycogen, which may provide with an alternative energy source to support growth and survival under conditions of energetic stress. Agents blocking glycogen utilization (glycogen phosphorylase inhibitors) may contribute to beneficial therapeutic strategies for renal cell carcinoma. In addition, glycogen levels may determine response or resistance to therapeutic strategies that alter nutrient availability (antiangiogenic therapy). Methods: Cultured 786-0 renal cancer cells, clear-cell renal tumors and normal renal cortex samples were subjected to protein extraction or homogenization with sterile water. To detect the levels of GYS1, protein lysates were tested using reverse-phase protein arrays and Western blotting. Lysates in water were used to detect glycogen with the glucoamylase chromogenic method. We tested the functional significance of GYS1 by knocking down its levels with GYS1-specific siRNA, and measured cell numbers and proliferation under normal culture conditions or under metabolic stress mediated by the glycolysis inhibitor 2-deoxyglucose (2DG) or an inhibitor of oxidative phosphorylation (metformin). In order to determine whether GYS1 correlates with responses to antiangiogenic therapy, we tested phospho and total GYS1 expression in renal tumors from bevacizumab-treated patients who participated in a previously published study (Tsavachidou-Fenner et al., Annals of Oncol, 2010). Results: ccRCC tissues and renal cancer cell lines contained glycogen at significantly higher levels than the normal renal cortex. They also expressed higher levels of GYS1. We show that the activation status of GYS1 is controlled by the PI3K/AKT/GSK3α/β cascade via phosphorylation at serine 640, which is considered to be an inactivating event. siRNA-mediated knockdown of GYS1 expression abrogated glycogen synthesis in 786-0 cells. It also suppressed cell growth under conditions of energetic stress induced by 2DG. Blocking glycogen utilization with a glycogen phosphorylase inhibitor had a similar growth suppressing effect on 786-0 cells in vitro, in the presence of metabolic stress mediated by 2DG or metformin. Tumors from patients responding to bevacizumab expressed the inactive form of GYS1 (GYS1-pSer640). In particular, renal tumors from patients with long progression-free survival exhibited higher levels of phospho-GYS1 (p value = 0.00075), consistent with the notion that tumors with lower glycogen production may respond more favorably to anti-angiogenic therapy. Conclusions: Clear-cell renal carcinomas and renal cancer cells produce large amounts of glycogen as a consequence of increased GYS1 levels. Increased glycogen serves as an alternative energy source, enabling cell growth under conditions of metabolic stress. Consequently, glycogen may be responsible for bypassing limitations in nutrient supply imposed by angiogenic therapies, as evidenced by the increased levels of inactive GYS1 in bevacizumab responders. The glycogen pathway may prove a valuable therapeutic target as well as a marker of response to therapies. This talk is also presented as Poster B18.

Bioenergetic characterization of mouse podocytes

Mitochondrial dysfunction contributes to podocyte injury, but normal podocyte bioenergetics have not been characterized. We measured oxygen consumption rates (OCR) and extracellular acidification rates (ECAR), using a transformed mouse podocyte cell line and the Seahorse Bioscience XF24 Extracellular Flux Analyzer. Basal OCR and ECAR were 55.2 +/- 9.9 pmol/min and 3.1 +/- 1.9 milli-pH units/min, respectively. The complex V inhibitor oligomycin reduced OCR to approximately 45% of baseline rates, indicating that approximately 55% of cellular oxygen consumption was coupled to ATP synthesis. Rotenone, a complex I inhibitor, reduced OCR to approximately 25% of the baseline rates, suggesting that mitochondrial respiration accounted for approximately 75% of the total cellular respiration. Thus approximately 75% of mitochondrial respiration was coupled to ATP synthesis and approximately 25% was accounted for by proton leak. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), which uncouples electron transport from ATP generation, increased OCR and ECAR to approximately 360% and 840% of control levels. FCCP plus rotenone reduced ATP content by 60%, the glycolysis inhibitor 2-deoxyglucose reduced ATP by 35%, and 2-deoxyglucose in combination with FCCP or rotenone reduced ATP by >85%. The lactate dehydrogenase inhibitor oxamate and 2-deoxyglucose did not reduce ECAR, and 2-deoxyglucose had no effect on OCR, although 2-deoxyglucose reduced ATP content by 25%. Mitochondrial uncoupling induced by FCCP was associated with increased OCR with certain substrates, including lactate, glucose, pyruvate, and palmitate. Replication of these experiments in primary mouse podocytes yielded similar data. We conclude that mitochondria play the primary role in maintaining podocyte energy homeostasis, while glycolysis makes a lesser contribution.

Abstract LB-107: Targeting the glycolytic pathway as an effective therapeutic approach for triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with estrogen receptor-negative, progesterone receptor-negative, and HER-2-negative phenotype. TNBC often exhibits poor prognosis due in part to their resistance to therapeutic agents. Thus, development of novel therapeutic approaches and effective agents are urgently needed to improve the clinical outcomes of this disease. In our recent study of metabolic alterations in cancer cells, we observed a striking metabolic pattern in TNBC cells manifested by lower mitochondrial respiration and elevated glycolysis. Using the extracellular flux analyzer XF24, we compared 4 TNBC cell lines (BT-20, MDA-231, MDA-468, MDA-436) with other subtypes of breast cancer cells (BT-474, MCF7, T47D, ZR-751) for their oxygen consumption rate (OCR) as an indicator of mitochondrial respiration and extracellular acidification rate (ECAR) as an indication of lactic acid production from glycolysis. All TNBC cell lines consistently exhibited lower OCAR and higher ECAR. These metabolic alterations were further confirmed using several conventional biochemical analyses including Clark-type electrode detection of oxygen consumption, [3H]-glucose uptake, and direct lactate measurement. In triple-positive breast cancer cells (BT474), blockage of ER-signaling by 4-hydroxy-tamoxifen (4-HOT), progestin signaling by RU486, or the HER-2 pathway by herceptin caused a moderate decrease in mitochondrial respiration when each inhibitor was used alone. Simultaneous inhibition of the three receptors by a combination of 4-HOT, RU486 and herceptin significantly down-regulated mitochondrial respiration (40%), suggesting a possible cross-talk among the three pathways in regulating mitochondrial metabolism. Western blot and mitochondrial functional analyses revealed that TNBC cells had altered expression of certain respiratory chain components and corresponding defect in the respiratory complex activity. Based on these novel observations, we further tested the hypothesis that TNBC cells might be more dependent on glycolysis for ATP generation and thus more vulnerable to glycolytic inhibitors. Indeed, our results demonstrated that TNBC cells were significantly more sensitive to the glycolytic inhibitors 3-bromopyruvate and 2-deoxyglucose than other breast cancer subtypes. Inhibition of glycolysis caused a massive cell death in TNBC cells, as evidenced by positive annexin-V staining and loss of membrane potential. Our study suggest that TNBC cells have profound metabolic alterations characterized by decreased mitochondrial respiration and increased glycolysis, and that glycolytic inhibition may represent a novel therapeutic strategy to effective kill the drug-resistant TNBC cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-107.

Enhanced Phrenic Long‐Term Facilitation Following Repetitive Acute Intermittent Hypoxia: Role of Glycolytic Flux

Acute intermittent hypoxia (AIH) elicits a form of respiratory plasticity known as phrenic long‐term facilitation (pLTF). pLTF is enhanced by pre‐treatment with repetitive AIH (3xwAIH: 10.5% O2; 10, 5min episodes/day, 3 days/week for 4 weeks; Vinit et al. ibid), and this enhancement (metaplasticity) is associated with increased expression of key molecules necessary for pLTF (Satriotomo et al. ibid). Since cell glycolysis affects transcriptional regulation of the same key molecules (Carriga‐Canut et al. 2006), we tested the hypothesis that the glycolytic inhibitor 2‐deoxyglucose (2‐DG) prevents the metaplasticity associated with pLTF following 3xwAIH. In anesthetized rats, phrenic nerve activity was increased (ie. pLTF 47%, p<0.05) 60min post‐AIH (3, 5min episodes, 10.5% O2) and this was enhanced by pre‐treatment with 3xwAIH (110%, p<0.05). Further, the enhanced pLTF was prevented in rats that also received 2‐DG in their drinking water (80mg/kg/day) during 3xwAIH, but similar to normoxic‐treated rats (50%). Acute 2‐DG administration (240mg/kg i.p.) had no effect on pLTF indicating glycolytic flux is necessary for the metaplasticity underlying 3xwAIH‐induced pLTF. Our data suggest a novel link between glycolysis and pLTF induced by repetitive AIH and that genes regulating respiratory metaplasticity may be linked to cellular energy metabolism (NIH HL‐80209 and Francis Family Foundation).

Direct control of mitochondrial function by mTOR

mTOR is a central regulator of cellular growth and metabolism. Using metabolic profiling and numerous small-molecule probes, we investigated whether mTOR affects immediate control over cellular metabolism by posttranslational mechanisms. Inhibiting the FKBP12/rapamycin-sensitive subset of mTOR functions in leukemic cells enhanced aerobic glycolysis and decreased uncoupled mitochondrial respiration within 25 min. mTOR is in a complex with the mitochondrial outer-membrane protein Bcl-xl and VDAC1. Bcl-xl, but not VDAC1, is a kinase substrate for mTOR in vitro, and mTOR regulates the association of Bcl-xl with mTOR. Inhibition of mTOR not only enhances aerobic glycolysis, but also induces a state of increased dependence on aerobic glycolysis in leukemic cells, as shown by the synergy between the glycolytic inhibitor 2-deoxyglucose and rapamycin in decreasing cell viability.

Glioblastoma Cells Require Glutamate Dehydrogenase to Survive Impairments of Glucose Metabolism or Akt Signaling

Oncogenes influence nutrient metabolism and nutrient dependence. The oncogene c-Myc stimulates glutamine metabolism and renders cells dependent on glutamine to sustain viability ("glutamine addiction"), suggesting that treatments targeting glutamine metabolism might selectively kill c-Myc-transformed tumor cells. However, many current or proposed cancer therapies interfere with the metabolism of glucose, not glutamine. Here, we studied how c-Myc-transformed cells maintained viability when glucose metabolism was impaired. In SF188 glioblastoma cells, glucose deprivation did not affect net glutamine utilization but elicited a switch in the pathways used to deliver glutamine carbon to the tricarboxylic acid cycle, with a large increase in the activity of glutamate dehydrogenase (GDH). The effect on GDH resulted from the loss of glycolysis because it could be mimicked with the glycolytic inhibitor 2-deoxyglucose and reversed with a pyruvate analogue. Furthermore, inhibition of Akt signaling, which facilitates glycolysis, increased GDH activity whereas overexpression of Akt suppressed it, suggesting that Akt indirectly regulates GDH through its effects on glucose metabolism. Suppression of GDH activity with RNA interference or an inhibitor showed that the enzyme is dispensable in cells able to metabolize glucose but is required for cells to survive impairments of glycolysis brought about by glucose deprivation, 2-deoxyglucose, or Akt inhibition. Thus, inhibition of GDH converted these glutamine-addicted cells to glucose-addicted cells. The findings emphasize the integration of glucose metabolism, glutamine metabolism, and oncogenic signaling in glioblastoma cells and suggest that exploiting compensatory pathways of glutamine metabolism can improve the efficacy of cancer treatments that impair glucose utilization.

The Glycolytic Inhibitor 2-Deoxyglucose Activates Multiple Prosurvival Pathways through IGF1R

Recent molecular studies indicate that aerobic glycolysis plays an important role in tumorigenesis and is a valid target for cancer therapy. Although 2-deoxyglucose (2-DG) is well characterized as a glycolytic inhibitor, we recently discovered that it activates a prosurvival oncoprotein, AKT, through PI3K. In this study, we discovered that 2-DG treatments disrupted the binding between insulin-like growth factor 1 (IGF-1) and IGF-binding protein 3 (IGFBP3) so that the free form of IGF-1 could be released from the IGF-1.IGFBP3 complex to activate IGF-1 receptor (IGF1R) signaling. Because IGF1R signaling is involved, PI3K/AKT constitutes only one of the prosurvival pathways that are activated by 2-DG treatment; we validated that MEK-ERK signaling was also induced in an IGF1R-dependent manner in some cancer cell lines. Furthermore, our phospho-specific antibody microarray analysis indicated that 2-DG up-regulated the phosphorylation of 64 sites within various signaling pathways in H460 cells. Chemical inhibition of IGF1R reduced 57 of these up-regulations. These data suggest that 2-DG-induced activation of many survival pathways can be jointly attenuated through IGF1R inhibition. Our in vitro analysis demonstrated that treatment with a combination of subtoxic doses of 2-DG and the IGF1R inhibitor II reduced cancer cell proliferation 90% and promoted significant apoptosis.

Accumulation of mitochondrial DNA damage and bioenergetic dysfunction in CSB defective cells

Cockayne syndrome (CS) is a complex, progressive disease that involves neurological and developmental impairment and premature aging. The majority of CS patients have mutations in the CSB gene. The CSB protein is involved in multiple DNA repair pathways and CSB mutated cells are sensitive to a broad spectrum of genotoxic agents. We tested the hypothesis that sensitivity to such genotoxins could be mediated by mitochondrial dysfunction as a consequence of the CSB mutation. mtDNA from csb(m/m) mice accumulates oxidative damage including 8-oxoguanine, and cells from this mouse are hypersensitive to the mitochondrial oxidant menadione. Inhibitors of mitochondrial complexes and the glycolysis inhibitor 2-deoxyglucose kill csb(m/m) cells more efficiently than wild-type cells, via a mechanism that does not correlate with mtDNA damage formation. Menadione depletes cellular ATP, and recovery after depletion is slower in csb(m/m) cells. The bioenergetic alteration in csb(m/m) cells parallels the simpler organization of supercomplexes consisting of complexes I, III and IV in addition to partially disassembled complex V in the inner mitochondrial membrane. Exposing wild-type cells to DNA intercalating agents induces complex alterations, suggesting a link between mtDNA integrity, respiratory complexes and mitochondrial function. Thus, mitochondrial dysfunction may play a role in the pathology of CS.

3-Bromopyruvate as inhibitor of tumour cell energy metabolism and chemopotentiator of platinum drugs

Tumour cells depend on aerobic glycolysis for adenosine triphosphate (ATP) production, making energy metabolism an interesting therapeutic target. 3-Bromopyruvate (BP) has been shown by others to inhibit hexokinase and eradicate mouse hepatocarcinomas. We report that similar to the glycolysis inhibitor 2-deoxyglucose (DG), BP rapidly decreased cellular ATP within hours, but unlike DG, BP concomitantly induced mitochondrial depolarization without affecting levels of reducing equivalents. Over 24 h, and at equitoxic doses, DG reduced glucose consumption more than did BP. The observed BP-induced loss of ATP is therefore largely due to mitochondrial effects. Cell death induced over 24 h by BP, but not DG, was blocked by N-acetylcysteine, indicating involvement of reactive oxygen species. BP-induced cytotoxicity was independent of p53. When combined with cisplatin or oxaliplatin, BP led to massive cell death. The anti-proliferative effects of low-dose platinum were strikingly potentiated also in resistant p53-deficient cells. Together with the reported lack of toxicity, this indicates the potential of BP as a clinical chemopotentiating agent.

Modulation of Glucose Metabolism Inhibits Hypoxic Accumulation of Hypoxia-Inducible Factor-1α (HIF-1α)

The hypoxic accumulation of the transcription factor subunit hypoxia-inducible factor-1alpha (HIF-1alpha), a potential endogenous hypoxia marker and therapeutic target, has recently been shown to strongly depend on glucose availability. The aim of this study was to investigate the underlying mechanism of this effect. HIF-1alpha protein levels were studied by Western blotting in HT 1080 human fibrosarcoma cells and in a hypoxia-responsive element green fluorescent protein (HRE-GFP) reporter assay in stably transfected HT 1080 cells treated with hypoxia (0.1% O(2), 12 h) and glycolysis inhibitors 2-deoxyglucose (2-DG) or iodoacetate (IAA). HIF-1alpha mRNA expression was quantified via real-time polymerase chain reaction (RT-PCR). Both inhibitors drastically reduced hypoxic HIF-1alpha accumulation (2-DG + hypoxia 2% mean HIF-1alpha protein level vs. 59% hypoxia alone; IAA + hypoxia 13% mean HIF-1alpha protein level vs. 96% hypoxia alone), an effect not rescued by the addition of pyruvate and confirmed in an HRE-GFP reporter assay in stably transfected HT 1080 cells. RT-PCR under identical conditions showed no effect of glycolysis inhibition on HIF-1alpha mRNA levels, suggesting a translational or posttranslational mechanism. The effect of glycolysis modulation on the HIF-1alpha levels in tumor cells may provide a novel approach to therapeutically target HIF-1alpha.

Up‐regulation of plasma membrane‐associated redox activities in neuronal cells lacking functional mitochondria

Mitochondria-deficient cells (rho(o) cells) survive through enhanced glycolytic metabolism in the presence of pyruvate and uridine. The plasma membrane redox system (PMRS) contains several NAD(P)H-related enzymes and plays a key role in maintaining the levels of NAD(+)/NADH and reduced coenzyme Q. In this study, rho(o) cells were used to investigate how the PMRS is regulated under conditions of mitochondrial dysfunction. rho(o) cells exhibited a lower oxygen consumption rate and higher levels of lactate than parental cells, and were more sensitive to glycolysis inhibitors (2-deoxyglucose and iodoacetamide) than control cells. However, they were more resistant to H(2)O(2), consistent with increased catalase activity and decreased oxidative damage (protein carbonyls and nitrotyrosine). PM-associated redox enzyme activities were enhanced in rho(o) cells compared to those in control cells. Our data suggest that all PMRS enzymes and biomarkers tested are closely related to the ability of the PMs to maintain redox homeostasis. These results illustrate that an up-regulated PM redox activity can protect cells from oxidative stress as a result of an improved antioxidant capacity, and suggest a mechanism by which neurons adapt to conditions of impaired mitochondrial function.

Death of stoma guard cells in leaf epidermis under disturbance of energy provision.

Cyanide is an apoptosis inducer in stoma guard cells from pea leaf epidermis. Unlike CN-, the uncoupler of oxidative and photosynthetic phosphorylation carbonyl cyanide m-chlorophenylhydrazone (CCCP), the combination of CCCP, 3-(3 ,4 -dichlorophenyl)-1,1-dimethylurea (DCMU), benzylhydroxamate (BH), myxothiazol, antimycin A, and a glycolysis inhibitor 2-deoxyglucose (DG) did not induce destruction of guard cell nuclei for 20 h of incubation of epidermal peels in the light. DCMU prevented the effect of CN- as a programmed cell death (PCD) inducer. CCCP, the combination of DCMU and CCCP, or the combination of DCMU, CCCP, BH, myxothiazol, antimycin A, and DG supplemented by CN- caused destruction of cell nuclei; the number of the cells lacking nuclei in this case was higher than with CN- alone. DG and CCCP caused cell destruction after longer incubation of the isolated epidermis - after 2 days and to a greater degree after 4 days. The effect of DG and CCCP was reduced by illumination. Cell destruction during long-term incubation was prevented by the combination of DG and CCCP. From data of electron microscopy, DCMU and dinitrophenyl ester of iodonitrothymol (DNP-INT) prevented apoptotic changes of the nuclear ultrastructure induced by CN-. The suppression of the destruction of the guard cell nuclei under combined action of DG and CCCP was apparently caused by switching of cell death from PCD to necrosis. Thus, the type of cell death - via apoptosis or necrosis - is controlled by the level of energy provision.

CD13 in cell adhesion: aminopeptidase N (CD13) mediates homotypic aggregation of monocytic cells

Homotypic aggregation (HA) of cells plays key roles in physiological and pathological processes, such as embryogenesis, immune responses, angiogenesis, tumor cell invasion, and metastasis. Aminopeptidase N (CD13) has been implicated in most of these phenomena, although its participation has been attributed to its enzymatic activity, while its role as an adhesion molecule has been almost unexplored. Here, we show that certain anti‐CD13 monoclonal antibodies induce HA of monocytic U‐937 cells, independently of their effect on enzymatic activity. The phenomenon is related to binding to a specific site on the CD13 molecule and is independent of integrins. It is abrogated by low temperature, by the glycolysis inhibitor 2‐deoxyglucose, and by inhibitors of tyrosine and mitogen‐activated protein kinases. The inhibitor of microtubule polymerization colchicine has a synergistic effect on CD13‐mediated aggregation, suggesting an inhibitory role of microtubules in this process. Finally, during HA, CD13 actively redistributes to the zones of cell‐cell contact, as determined by live cell imaging studies, demonstrating a direct role of CD13 in the adhesion phenomenon. Together, these data show for the first time the participation of CD13 in monocytic cell adhesion.

Glucose deprivation decreases nitric oxide production via NADPH depletion in immunostimulated rat primary astrocytes.

We have previously reported that the production of nitric oxide (NO) in immunostimulated astrocytes was markedly decreased under glucose-deprived conditions. The present study was undertaken to find the contributing factor(s) for the decreased NO production in glucose-deprived immunostimulated astrocytes. NO production in rat primary astrocytes was stimulated for 24-48 h by cotreatment with lipopolysaccharides (1 microg/ml) and interferon-gamma (100 U/ml). Decreased NO production in immunostimulated astrocytes by glucose deprivation was mimicked by the glycolytic inhibitor 2-deoxyglucose and reversed by addition of pyruvate and lactate. Glucose deprivation did not alter the expression of inducible nitric oxide synthase (iNOS) in immunostimulated astrocytes. Addition of beta-NADPH, but not tetrahydrobiopterine, both of which are essential cofactors for NOS function, completely restored the NO production that was decreased in glucose-deprived immunostimulated astrocytes. Glucose deprivation and immunostimulation synergistically reduced intracellular NADPH level in astrocytes. The results indicate that glucose deprivation decreases NO production in immunostimulated astrocytes by depleting intracellular NADPH, a cofactor of iNOS.

Arachidonate release and c-fos expression in various models of hypoxia and hypoxia–hypoglycemia in retinoic acid differentiated neuroblastoma cells

Hypoxia–hypoglycemia has played an important role in inducing both phospholipase A 2 activation and the expression of the early gene c-fos, in the neuroblastoma cell line SK-N-BE, after it has been differentiated by retinoic acid. Under hypoxic–hypoglycemic conditions, arachidonic acid release has found to be significant after 30 min, whereas c-fos expression has required at least 4 h. This model has been obtained by adding glycolytic inhibitor 2-deoxyglucose to the culture and by placing cells in an atmosphere containing 100% N 2 for different time periods. This condition has been compared with two different models: NaCN and nitrogen have been used as hypoxic stimuli, without inhibiting the glycolytic pathway, but the same cell cultures have been used. Cell viability and the fall of cellular ATP levels have been evaluated in all the models, in order to monitor and compare the hypoxic cellular damage. Phospholipase A 2 activation has been found to be significant in all conditions, even if to a different extent; but only hypoxia combined with the inhibition of the glycolytic pathway, has induced a significant expression of c-fos. It is very difficult to study hypoxic stimuli in ‘in vitro’ systems. Our study has compared three different models and the one combining gaseous hypoxia and hypoglycemic conditions seems to be very effective in stimulating early events involved in hypoxic fenomena such as phospholipase activation and the expression of the early gene c-fos.

Effects of Low Oxygen Concentrations and Metabolic Inhibitors on Proteoglycan and Protein Synthesis Rates in the Intervertebral Disc

The intervertebral disc is the largest asvacular structure in the body; consequently, there are steep gradients in O2 concentration, with PO2 falling to as low as 1% O2 in the centre of the disc. We investigated the effect of O2 concentration on the rates of O2 consumption, lactate production, and sulphate and proline incorporation in bovine caudal discs. We also investigated the effects of metabolic inhibitors of energy production pathways on tracer incorporation. Samples from the outer annulus and nucleus pulposus were incubated for 24 hours in 1-21% O2. Rates were measured during the last 4 hours of incubation. As O2 concentration was reduced from 10 to 1% O2, O2 consumption rates fell by around 75% and lactate production rates almost doubled; the bovine discs thus showed a positive progressive Pasteur effect. Incorporation rates of [3H]proline and [35S]sulphate were lowest at 1% O2. In the nucleus, but not in the outer annulus, the rate of [35S]incorporation peaked at 5% O2, where it was 30% greater than at 21% O2 and 150% greater than at 1% O2. The competitive glycolysis inhibitor 2-deoxyglucose, the oxidative phosphorylation uncoupler 2,4-dinitrophenol, and the oxidative phosphorylation inhibitor sodium azide all markedly reduced sulphate incorporation. These results, together with previous measurements of CO2 production rates, suggest that a functionally significant fraction of the disc's energy is supplied by oxidative phosphorylation. However, low levels of PO2, 2,4-dinitrophenol, and sodium azide have been reported to reduce sulphate incorporation in articular cartilage, a tissue that derives its energy almost entirely from glycolysis.