Physiological Role of Glutamate Dehydrogenase in Cancer Cells.

NH 4 + increased growth rates and final densities of several human metastatic cancer cells. To assess whether glutamate dehydrogenase (GDH) in cancer cells may catalyze the reverse reaction of NH 4 + fixation, its covalent regulation and kinetic parameters were determined under near-physiological conditions. Increased total protein and phosphorylation were attained in NH 4 + -supplemented metastatic cells, but total cell GDH activity was unchanged. Higher V max values for the GDH reverse reaction vs. forward reaction in both isolated hepatoma (HepM) and liver mitochondria [rat liver mitochondria (RLM)] favored an NH 4 + -fixing role. GDH sigmoidal kinetics with NH 4 + , ADP, and leucine fitted to Hill equation showed n H values of 2 to 3. However, the K 0.5 values for NH 4 + were over 20 mM, questioning the physiological relevance of the GDH reverse reaction, because intracellular NH 4 + in tumors is 1 to 5 mM. In contrast, data fitting to the Monod-Wyman-Changeux (MWC) model revealed lower K m values for NH 4 + , of 6 to 12 mM. In silico analysis made with MWC equation, and using physiological concentrations of substrates and modulators, predicted GDH N-fixing activity in cancer cells. Therefore, together with its thermodynamic feasibility, GDH may reach rates for its reverse, NH 4 + -fixing reaction that are compatible with an anabolic role for supporting growth of cancer cells.

Modeling cancer glycolysis under hypoglycemia, and the role played by the differential expression of glycolytic isoforms.

UNLABELLED: The effect of hypoglycemia on the contents of glycolytic proteins, activities of enzymes/transporters and flux of HeLa and MCF-7 tumor cells was experimentally analyzed and modeled in silico. After 24 h hypoglycemia (2.5 mm initial glucose), significant increases in the protein levels of glucose transporters 1 and 3 (GLUT 1 and 3) (3.4 and 2.1-fold, respectively) and hexokinase I (HKI) (2.3-fold) were observed compared to the hyperglycemic standard cell culture condition (25 mm initial glucose). However, these changes did not bring about a significant increase in the total activities (Vmax ) of GLUT and HK; instead, the affinity of these proteins for glucose increased, which may explain the twofold increased glycolytic flux under hypoglycemia. Thus, an increase in more catalytically efficient isoforms for two of the main controlling steps was sufficient to induce increased flux. Further, a previous kinetic model of tumor glycolysis was updated by including the ratios of GLUT and HK isoforms, modified pyruvate kinase kinetics and an oxidative phosphorylation reaction. The updated model was robust in terms of simulating most of the metabolite levels and fluxes of the cells exposed to various glycemic conditions. Model simulations indicated that the main controlling steps were glycogen degradation > HK > hexosephosphate isomerase under hyper- and normoglycemia, and GLUT > HK > glycogen degradation under hypoglycemia. These predictions were experimentally evaluated: the glycolytic flux of hypoglycemic cells was more sensitive to cytochalasin B (a GLUT inhibitor) than that of hyperglycemic cells. The results indicated that cancer glycolysis should be inhibited at multiple controlling sites, regardless of external glucose levels, to effectively block the pathway. DATABASE: The mathematical models described here have been submitted to the JWS Online Cellular Systems Modelling Database and can be accessed at http://jjj.mib.ac.uk/database/achcar/index.html. [Database section added 21 July 2014 after original online publication].

HIF-1alpha modulates energy metabolism in cancer cells by inducing over-expression of specific glycolytic isoforms.

To develop new and more efficient anti-cancer strategies it will be important to characterize the products of transcription factor activity essential for tumorigenesis. One such factor is hypoxia-inducible factor-1alpha (HIF-1alpha), a transcription factor induced by low oxygen conditions and found in high levels in malignant solid tumors, but not in normal tissues or slow-growing tumors. In fast-growing tumors, HIF-1alpha is involved in the activation of numerous cellular processes including resistance against apoptosis, over-expression of drug efflux membrane pumps, vascular remodeling and angiogenesis as well as metastasis. In cancer cells, HIF-1alpha induces over-expression and increased activity of several glycolytic protein isoforms that differ from those found in non-malignant cells, including transporters (GLUT1, GLUT3) and enzymes (HKI, HKII, PFK-L, ALD-A, ALD-C, PGK1, ENO-alpha, PYK-M2, LDH-A, PFKFB-3). The enhanced tumor glycolytic flux triggered by HIF-1alpha also involves changes in the kinetic patterns of expressed isoforms of key glycolytic enzymes. The HIF-1alpha induced isoforms provide cancer cells with reduced sensitivity to physiological inhibitors, lower affinity for products and higher catalytic capacity (Vmax(f)) in forward reactions because of marked over-expression compared to those isoforms expressed in normal tissues. Some of the HIF1alpha-induced glycolytic isoforms also participate in survival pathways, including transcriptional activation of H2B histone (by LDH-A), inhibition of apoptosis (by HKII) and promotion of cell migration (by ENO-alpha). HIF-1alpha action may also modulate mitochondrial function and oxygen consumption by inactivating the pyruvate dehydrogenase complex in some tumor types, or by modulating cytochrome c oxidase subunit 4 expression to increase oxidative phosphorylation in other cancer cell lines. In this review, the roles of HIF-1alpha and HIF1alpha-induced glycolytic enzymes are examined and it is concluded that targeting the HIF1alpha-induced glucose transporter and hexokinase, important to glycolytic flux control, might provide better therapeutic targets for inhibiting tumor growth and progression than targeting HIF1alpha itself.