glutaminase and Carcinoma

Although glycolysis is substantially elevated in many tumors, therapeutic targeting of glycolysis in cancer patients has not yet been successful, potentially reflecting the metabolic plasticity of tumor cells. In various cancer cells exposed to a continuous glycolytic block, we identified a recurrent reprogramming mechanism involving sustained mTORC1 signaling that underlies escape from glycolytic addiction. Active mTORC1 directs increased glucose flux via the pentose phosphate pathway back into glycolysis, thereby circumventing a glycolysis block and ensuring adequate ATP and biomass production. Combined inhibition of glycolysis and mTORC1 signaling disrupted metabolic reprogramming in tumor cells and inhibited their growth in vitro and in vivo. These findings reveal novel combinatorial therapeutic strategies to realize the potential benefit from targeting the Warburg effect.

Topics: Adenosine Triphosphate; Animals; Carcinoma; Cell Line, Tumor; Citric Acid Cycle; Combined Modality Therapy; Cytokines; Deoxyglucose; Drug Resistance, Neoplasm; Drug Synergism; Energy Metabolism; Everolimus; Female; Glucose-6-Phosphate Isomerase; Glutaminase; Glutamine; Glycolysis; Hep G2 Cells; Humans; Mechanistic Target of Rapamycin Complex 1; Metabolomics; Mice; Mice, Nude; Molecular Targeted Therapy; Multiprotein Complexes; Neoplasm Proteins; Neoplasms; Ovarian Neoplasms; Pentose Phosphate Pathway; Ribosomal Protein S6 Kinases, 70-kDa; RNA Interference; RNA, Small Interfering; TOR Serine-Threonine Kinases; Tumor Stem Cell Assay; Xenograft Model Antitumor Assays

It has been assumed that oxidative phosphorylation (OxPhos) in solid tumors is severely reduced due to cytochrome c oxidase substrate restriction, although the measured extracellular oxygen concentration in hypoxic areas seems not limiting for this activity. To identify alternative hypoxia-induced OxPhos depressing mechanisms, an integral analysis of transcription, translation, enzyme activities and pathway fluxes was performed on glycolysis and OxPhos in HeLa and MCF-7 carcinomas. In both neoplasias exposed to hypoxia, an early transcriptional response was observed after 8h (two times increased glycolysis-related mRNA synthesis promoted by increased HIF-1alpha levels). However, major metabolic remodeling was observed only after 24h hypoxia: increased glycolytic protein content (1-5-times), enzyme activities (2-times) and fluxes (4-6-times). Interestingly, in MCF-7 cells, 24h hypoxia decreased OxPhos flux (4-6-fold), and 2-oxoglutarate dehydrogenase and glutaminase activities (3-fold), with no changes in respiratory complexes I and IV activities. In contrast, 24h hypoxia did not significantly affect HeLa OxPhos flux; neither mitochondria related mRNAs, protein contents or enzyme activities, although the enhanced glycolysis became the main ATP supplier. Thus, prolonged hypoxia (a) targeted some mitochondrial enzymes in MCF-7 but not in HeLa cells, and (b) induced a transition from mitochondrial towards a glycolytic-dependent energy metabolism in both MCF-7 and HeLa carcinomas.

Topics: Breast Neoplasms; Carcinoma; Electron Transport Complex I; Energy Metabolism; Female; Glutaminase; Glycolysis; HeLa Cells; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Ketoglutarate Dehydrogenase Complex; Mitochondria; Oxidative Phosphorylation; Uterine Cervical Neoplasms

Previous work has indicated that the PDZ domain Tax interacting protein 1 (Tip-1) is a target of the HTLV1 Tax protein and is a potential RhoA effector. We have used the yeast two-hybrid system to show that Tip-1 also interacts with the HPV16 E6 protein. This interaction was confirmed by co-immunoprecipitation from E6 expressing C33A cervical carcinoma cells (C33A-E6) which showed that Tip-1 was not degraded by interaction with the HPV16 E6 oncoprotein. During routine passage we observed that C33A-E6 had a less compact morphology and were less adherent than control vector transfected cells C33A-V cells - a known effect of GTP-RhoA. Comparison of C33A-E6 to C33A-V demonstrated that E6 expressing cells had higher levels of phosphorylated myosin light chains (MLC) and increased cell motility, which was inhibited by antisense silencing of Tip-1 expression and by the RhoA kinase (ROCK) inhibitor Y27632. Both C33A-E6 and C33A-V cells were shown to express GTP activated RhoA. Since ROCKs can be activated by GTP RhoA these data indicate that E6 may increase cell motility by augmenting GTP RhoA mediated activation of ROCKs and that this is dependent on the expression of the Tip-1 protein.

Topics: Carcinoma; Cell Movement; Female; Glutaminase; Humans; Immunoprecipitation; Intracellular Signaling Peptides and Proteins; Myosin Light Chains; Oncogene Proteins, Viral; Phosphorylation; Repressor Proteins; rhoA GTP-Binding Protein; Tumor Cells, Cultured; Uterine Cervical Neoplasms; Yeasts

Phosphate dependent glutaminase was purified from ascites fluid of ovarian cancer patients. The purified enzyme showed a final specific activity of 110 unit / mg protein with 72 fold purification and 21% yield. Purified enzyme gives one dark band of Mr approximately 65.5 KD and two light bands of Mr approximately 47.5 KD and approximately 45 KD respectively on 10% SDS-PAGE. One major immunoreactive band was found in trans-immunoblot analysis using antibodies against rat kidney and ascites fluid glutaminase raised in rabbit and mice respectively. Phosphate dependent glutaminase enzyme purified from mitochondria of malignant and non malignant ovarian tissue also showed bands of same molecular weight on 10% SDS-PAGE and gave same immunoreactive bands in trans-immunoblot like the purified glutaminase from ascites fluid. This result was confirmed by using the specific activity stain for glutaminase, which indicates that same enzyme activity is probably due to leakage of the same enzyme from malignant tissue into the ascites fluid. The purified enzyme from human peritoneal fluid showed a high specificity toward glutamine, therefore is a true glutaminase. Moreover, ascites fluid taken from patients of different age group with different stages of ovarian carcinoma revealed the presence of same glutaminase on 10% SDS-PAGE, and exhibited immunoreaction on ELISA, trans-immunoblot and dot immunoblot analysis.

Topics: Adult; Animals; Ascites; Blotting, Western; Carcinoma; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Female; Glutaminase; Humans; Mice; Ovarian Neoplasms; Rabbits; Rats

The three kinds of glutamineses in rat kidney and liver were defined in comparative terms by their properties and were separately purified. Substantial purification was obtained by polymerization and depolymerization of the kidney isozyme that is activated by phosphate, These isozymes differ most strikingly in the activators of their reactions:maleate and a high concentration of phosphate, respectively, for the two kidney isozymes, and a low concentration of phosphate for the liver isozyme. The kidney isozyme that is activated by phosphate was also activated by a much lower concentration of ATP and by other complex phosphates. They also differ in physical properties: the maleate-activated isozyme was heat resistant (50 degrees C) and associated with insoluble submitochondrial particles; both phosphate-activated isozymes were heat sensitive and could be solubilized from their respective mitochondria; the phosphate-activated isozyme of kidney polymerized in phosphate-borate solution while that of liver did not. The characteristics of the kidney isozyme that was activated by high phosphate were shared by the glutaminases in adult brain, transplanted tumors, and in fetal liver and kidney. The similarity to the kidney enzyme was confirmed by the use of polymerization in phosphate borate of the isozyme from a mammary carcinoma to effect its purification.

Topics: Age Factors; Animals; Brain; Carcinoma; Enzyme Activation; Female; Glutaminase; Isoenzymes; Kidney; Kidney Neoplasms; Liver; Neoplasms, Experimental; Phosphates; Pregnancy; Rats

Topics: Alcaligenes; Ammonia-Lyases; Animals; Asparaginase; Carboxypeptidases; Carcinoma; Carcinoma 256, Walker; Carcinoma, Ehrlich Tumor; Carcinoma, Hepatocellular; Cell Line; Enzyme Therapy; Glutaminase; Humans; Leukemia, Experimental; Liver Neoplasms; Mice; Neoplasms; Neoplasms, Experimental; Sarcoma, Experimental

Topics: Adenocarcinoma; Adenocarcinoma, Papillary; Animals; Carcinoma; Carcinoma, Squamous Cell; Glutaminase; Humans; Hydrogen-Ion Concentration; Isoenzymes; Kidney; Kinetics; Liver; Lung; Lung Neoplasms; Mitosis; Neoplasm Metastasis; Organometallic Compounds; Phosphates; Rats; Species Specificity