Hypoxia-inducible Factor 1 Activation by Aerobic Glycolysis Implicates the Warburg Effect in Carcinogenesis

Exosomes are small membrane vesicles secreted into the extracellular compartment by exocytosis. Tumor exosomes may be involved in the sampling of antigens to antigen presenting cells or as decoys allowing the tumor to escape immune-directed destruction. The proteins present in exosomes secreted by tumor cells have been poorly defined. This study describes the protein composition of mesothelioma cell-derived exosomes in more detail. After electrophoresis of exosome preparations, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) was used to characterize the protein spots. MHC class I was found to be present together with the heat shock proteins HSC70 and HSP90. In addition, we found annexins and PV-1, proteins involved in membrane transport and function. Cytoskeleton proteins and their associated proteins ezrin, moesin, actinin-4, desmoplakin, and fascin were also detected. Besides the molecular motor kinesin-like protein, many enzymes were detected revealing the cytoplasmic orientation of exosomes. Most interesting was the detection of developmental endothelial locus-1 (DEL-1), which can act as a strong angiogenic factor and can increase the vascular development in the neighborhood of the tumor. In conclusion, mesothelioma cells release exosomes that express a discrete set of proteins involved in antigen presentation, signal transduction, migration, and adhesion. Exosomes may play an important role in the interaction between tumor cells and their environment.

/pdf/proteomic-analysis-of-exosomes-secreted-by-human-1pmovn8lgq.pdf

Proteomic Analysis of Exosomes Secreted by Human Mesothelioma Cells

The estimation of the intracellular fluxes of mammalian cells using only the mass balances of the relevant metabolites is not possible because the set of linear equations defined by these mass balances is underdetermined. Either additional experimental flux data or additional theoretical constraints are required to find one unique flux distribution out of the solution space that is bound by the mass balances. Here, a method is developed using the latter approach. The uptake and production rates of amino acids, glucose, lactate, O(2), CO(2), NH(4), MAB, and the intracellular amino acid pools have been determined for two different steady-states. The cellular composition {total protein and protein composition, total lipids and fatty acid distribution, total carbohydrates, DNA and RNA} has been measured to calculate the requirements for biosynthesis. It is shown to be essential to determine the uptake/production rates of ammonia and either carbon dioxide or oxygen. In mammalian cells these are cometabolites of cyclic metabolic pathways. The flux distribution that is found using the Euclidean minimum norm as the additional theoretical constraint and taking either the CO(2) or the NAD(P)H mass balance into account is shown to be in agreement with the measured O(2) and CO(2) metabolic rates.The metabolic fluxes in hybridoma cells in continuous culture at a specific growth rate of 0.83 day(-1) are estimated for a medium with (optimal medium) and without (suboptimal medium) Primatone RL, an enzymatic hydrolysate of animal tissue that causes a more than twofold increase in cell density. It is concluded that (i)The majority of the consumed glucose (>90%) is channeled through the pentose-phosphate pathway in rapidly proliferating cells.(ii)Pyruvate oxidation and tricarboxylic acid (TCA) cycle activity are relatively low, i.e., 8% of the glucose uptake in suboptimal and 14% in optimal medium, respectively. Under both conditions, only a small fraction of pyruvate is further oxidized to CO(2).(iii)The flux from glutamate to alpha-ketoglutarate (catalyzed by glutamate dehydrogenase) is almost zero in medium with and even slightly reversed in medium without Primatone RL. Almost all glutamate enters the TCA cycle due to the action of transaminases.(iv)Transhydrogenation plays a significant role in hybridoma cells under our experimental conditions. NADPH is produced at relatively high rates (11 x 10(-12) to 13 x 10(-12) mol . cell(-1) . day(-1)) compared to other fluxes in both culture media.

Metabolic flux analysis of hybridoma cells in different culture media using mass balances.

Cardiolipin and electron transport chain abnormalities in mouse brain tumor mitochondria: lipidomic evidence supporting the Warburg theory of cancer

Brain tumours lack metabolic versatility and are dependent largely on glucose for energy. This contrasts with normal brain tissue that can derive energy from both glucose and ketone bodies. We examined for the first time the potential efficacy of dietary therapies that reduce plasma glucose and elevate ketone bodies in the CT-2A syngeneic malignant mouse astrocytoma. C57BL/6J mice were fed either a standard diet unrestricted (SD-UR), a ketogenic diet unrestricted (KD-UR), the SD restricted to 40% (SD-R), or the KD restricted to 40% of the control standard diet (KD-R). Body weights, tumour weights, plasma glucose, β-hydroxybutyrate (β-OHB), and insulin-like growth factor 1 (IGF-1) were measured 13 days after tumour implantation. CT-2A growth was rapid in both the SD-UR and KD-UR groups, but was significantly reduced in both the SD-R and KD-R groups by about 80%. The results indicate that plasma glucose predicts CT-2A growth and that growth is dependent more on the amount than on the origin of dietary calories. Also, restriction of either diet significantly reduced the plasma levels of IGF-1, a biomarker for angiogenesis and tumour progression. Owing to a dependence on plasma glucose, IGF-1 was also predictive of CT-2A growth. Ketone bodies are proposed to reduce stromal inflammatory activities, while providing normal brain cells with a nonglycolytic high-energy substrate. Our results in a mouse astrocytoma suggest that malignant brain tumours are potentially manageable with dietary therapies that reduce glucose and elevate ketone bodies.

/pdf/role-of-glucose-and-ketone-bodies-in-the-metabolic-control-2eadruqqht.pdf

Role of glucose and ketone bodies in the metabolic control of experimental brain cancer.

The rates of disappearance of glucose from the medium of 13 human glioma-derived cell lines and one cultured of normal human cortical astrocytes were determined by ftuorometric techniques. High-grade glioma-derived cultures showed a range of glucose consumption between 1 and 5 nmol/min/mg protein. Normal astrocyte cultures and cultures derived from grades I–III gliomas had a glucose consumption rate of 2–3 nmol/min/mg protein. Seven high-grade glioma lines were derived from surgical samples taken from patients who had been scanned by18F-2-deoxy-d-glucose positron computed tomography. The rate of glucose consumption in these high-grade glioma-derived lines was close to the maximum local cerebral metabolic rate for glucose (LCMRglc) measuredin situ in the tumors from which the cultures were derived. In cultured glioma-derived lines, approximately one-half of the glucose consumed was recovered as lactate and pyruvate, suggesting a reliance of glioma cells on aerobic glycolysis. ATP and phosphocreatine (PCr) levels were variable in the gliomaderived lines, and ATP was lower in the glioma-derived lines than in the normal astrocytes. Levels and regulation of glycogen differed significantly among the various glioma-derived cell lines. Glycogen content did not diminish as glucose was consumed, suggesting that glycogen utilization is not tightly regulated by the glucose metabolic rate. These results suggest that human glioma-derived cell cultures (1) adequately reflect the metabolic capacity of gliomasin situ and (2) are significantly altered in several aspects of their glycolytic metabolism.

Glucose metabolism in human gliomas: correspondence of in situ and in vitro metabolic rates and altered energy metabolism.

Several aspects of the regulation of the pentose phosphate pathway were examined in cultured normal human cortical astrocytes and gliomas of pathological grades I-IV. The generation of radiolabeled CO2 from [1-14C]glucose by the oxidative arm of the pentose phosphate pathway is a saturable process and has a maximum flux rate of 8-9 nmol/hr/mg cell protein. The flux can be blocked by the glycolytic inhibitor iodoacetamide but is unaffected by agents which inhibit oxidative phosphorylation. The magnitude of the pentose phosphate flux is directly related to the glioma grade. Grade IV gliomas (glioblastoma) show a pentose phosphate flux rate of approximately 4% of the total glucose flux. The flux rate can be increased by pharmacological agents which decrease the NADPH/NADP+ ratio. Both the activity and the regulation of glioma glucose-6-phosphate dehydrogenase (G6PDH) are altered in high-grade gliomas. While the affinity constants for cofactors in whole homogenates were not significantly different in glioma or normal astrocyte homogenates, normal astrocytes have a lower Km for glucose-6-phosphate and a G6PDH activity which is 10-fold greater than that of gliomas. NADPH is a powerful regulator of G6PDH activity in the normal astrocytes and in gliomas. At a NADPH/NADP+ ratio of 7:1 the normal astrocyte G6PDH is entirely inhibited, while the glioma enzyme is only 70% inhibited even at a ratio of 20:1. Increased metabolic flux through the oxidative arm of the pentose phosphate pathway is apparently due to an altered form of G6PDH.

Regulation of the pentose phosphate pathway in human astrocytes and gliomas.

The enzymes of glycolysis and selected enzymes of the pentose phosphate pathways were measured by fluorometric methods in extracts prepared from cultures of normal cortical human astrocytes and from cultures derived from low-grade (II) or high-grade (IV) gliomas. The hexokinase and phosphofructokinase levels of the low-grade glioma-derived line were not significantly different from those of the normal astrocyte cultures. However, the activities of hexokinase and phosphofructokinase were consistently and significantly increased in the high-grade glioma-derived lines. The activity of glucose-6-phosphate dehydrogenase was significantly decreased in all glioma-derived lines and by more than 90% in the high-grade-derived lines. Other enzymes of the glycolytic pathway were not significantly different from those of normal astrocytes, or they showed a variation inconsistently related to the neoplastic state. Glucose flux is not apparently regulated to a significant degree of hexokinase in glioma-derived lines, since the measured Vmax values are in substantial excess over the measured flux rates. Reversible binding of hexokinase to the particulate fraction was observed in both the normal astrocytes cultures and the high-grade glioma-derived lines. A twofold displacement of particulate hexokinase by ATP, ADP, 1-O-methylglucose, sorbitol-6-phosphate, and dibutyryl cyclic AMP was observed in the high-grade glioma-derived lines. The degree of displacement by various agents and the basal ratio of free/bound was not significantly different between the transformers and the nontransformants. The hexokinase from both the gliomas and the normal astrocytes was noncompetitively inhibited by the glucose analogue 2-deoxy-d-glucose. Phosphofructokinase activity is close to the observed glucose flux rates in both the normal astrocyte and the glioma-derived cultures. The phosphofructokinase activity of normal astrocytes is activated twofold or more by ADP, AMP, fructose-2,6-diphosphate, and Pi. However, these same ligands activate phosphofructokinase by less than twofold in a typical high-grade glioma-derived line. ATP, dibutyryl cyclic AMP, and citrate inhibit glioma and normal astrocytic phosphofructokinase, but the magnitude of the inhibition is much less than in the glioma-derived lines.

Enzymes of glucose metabolism in cultured human gliomas: neoplasia is accompanied by altered hexokinase, phosphofructokinase, and glucose-6-phosphate dehydrogenase levels.

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Glucose metabolism in human gliomas: correspondence of in situ and in vitro metabolic rates and altered energy metabolism.

Regulation of the pentose phosphate pathway in human astrocytes and gliomas.

Enzymes of glucose metabolism in cultured human gliomas: neoplasia is accompanied by altered hexokinase, phosphofructokinase, and glucose-6-phosphate dehydrogenase levels.