Role Of Gln Research Articles

Glutamine Metabolism and Prostate Cancer.

Glutamine (Gln) is a non-essential amino acid that is involved in the development and progression of several malignancies, including prostate cancer (PCa). While Gln is non-essential for non-malignant prostate epithelial cells, PCa cells become highly dependent on an exogenous source of Gln. The Gln metabolism in PCa is tightly controlled by well-described oncogenes such as MYC, AR, and mTOR. These oncogenes contribute to therapy resistance and progression to the aggressive castration-resistant PCa. Inhibition of Gln catabolism impedes PCa growth, survival, and tumor-initiating potential while sensitizing the cells to radiotherapy. Therefore, given its significant role in tumor growth, targeting Gln metabolism is a promising approach for developing new therapeutic strategies. Ongoing clinical trials evaluate the safety and efficacy of Gln catabolism inhibitors in combination with conventional and targeted therapies in patients with various solid tumors, including PCa. Further understanding of how PCa cells metabolically interact with their microenvironment will facilitate the clinical translation of Gln inhibitors and help improve therapeutic outcomes. This review focuses on the role of Gln in PCa progression and therapy resistance and provides insights into current clinical trials.

Targeting of glutamine transporter ASCT2 and glutamine synthetase suppresses gastric cancer cell growth

PurposeGlutamine (Gln) is essential for the proliferation of most cancer cells, making it an appealing target for cancer therapy. However, the role of Gln in gastric cancer (GC) metabolism is unknown and Gln-targeted therapy against GC remains scarce. The aim of this study was to investigate the relevance of Gln in GC growth and targeting.MethodsExpression of Gln transporter ASCT2 and glutamine synthetase (GS) in the parental and molecularly engineered GC cells or in human GC specimens was determined by RT-PCR and western blot analysis or immunohistochemistry. Cell proliferation and survival was assessed by CCK-8 assay and colony formation assay. Intracellular Gln content was measured by a HPLC system. Effects of ASCT2 and/or GS inhibitor on tumor growth were investigated in xenograft models.ResultsA significant heterogeneity of GC cells was observed with respect to their response to the treatment of ASCT2 inhibitor benzylserine (BenSer). Gln deprivation did not affect the BenSer-resistant cell growth due to endogenous GS expression, whose inhibition remarkably reduced cell proliferation. The differential in vitro sensitivity correlated with overall intracellular Gln content. Combined therapy with both ASCT2 and GS inhibitors produced a greater therapeutic efficacy than the treatment of either inhibitor alone. Furthermore, 77% human GC tissues were found to express moderate and high levels of ASCT2, 12% of which also co-expressed relatively high levels of GS.ConclusionGln mediates GC growth and the therapeutic efficacy of Gln-targeted treatment relies on distinct ASCT2 and GS expression pattern in specific gastric cancer groups.

Reactivity and Dynamics of H2S, NO, and O2 Interacting with Hemoglobins from Lucina pectinata

Hemoglobin HbI from the clam Lucina pectinata is involved in H2S transport, whereas homologous heme protein HbII/III is involved in O2 metabolism. Despite similar tertiary structures, HbI and HbII/III exhibit very different reactivity toward heme ligands H2S, O2, and NO. To investigate this reactivity at the heme level, we measured the dynamics of ligand interaction by time-resolved absorption spectroscopy in the picosecond to nanosecond time range. We demonstrated that H2S can be photodissociated from both ferric and ferrous HbI. H2S geminately rebinds to ferric and ferrous out-of-plane iron with time constants (τgem) of 12 and 165 ps, respectively, with very different proportions of photodissociated H2S exiting the protein (24% in ferric and 80% in ferrous HbI). The Gln(E7)His mutation considerably changes H2S dynamics in ferric HbI, indicating the role of Gln(E7) in controling H2S reactivity. In ferric HbI, the rate of diffusion of H2S from the solvent into the heme pocket (kentry) is 0.30 μM(-1) s(-1). For the HbII/III-O2 complex, we observed mainly a six-coordinate vibrationally excited heme-O2 complex with O2 still bound to the iron. This explains the low yield of O2 photodissociation and low koff from HbII/III, compared with those of HbI and Mb. Both isoforms behave very differently with regard to NO and O2 dynamics. Whereas the amplitude of geminate rebinding of O2 to HbI (38.5%) is similar to that of myoglobin (34.5%) in spite of different distal heme sites, it appears to be much larger for HbII/III (77%). The distal Tyr(B10) side chain present in HbII/III increases the energy barrier for ligand escape and participates in the stabilization of bound O2 and NO.

Abstract 1887: Bone marrow-derived stromal cells and hypoxia promote glutamine dependency in leukemias.

Abstract Glutamine (Gln) is a versatile non-essential amino acid that plays a unique role in the metabolism of proliferating cancer cells; it provides cells with a variety of essential molecules to sustain cell proliferation, regulates redox homeostasis and modulates activity of signal transduction pathways. Recent data suggest that cancer cells employ multiple mechanisms that regulate Gln and glucose metabolism in a coordinated fashion. Microenvironment is recognized as one of the major factors that modulates glucose, Gln and lipid metabolism in cancer cells; however mechanisms that couple survival of leukemic cells to metabolic processes under different microenvironment conditions have not been elucidated. Leukemic bone marrow exhibits heterogeneous microenvironment niches reminiscent to microenvironment of solid tumor characterized by low oxygen tension, reduced nutrient availability and presence of supporting stromal cells. Our published data demonstrated that co-culture of leukemic cells with bone marrow-derived mesenchymal stromal cells (MSC) promotes mitochondrial dysfunction in myeloid leukemia cells, accumulation of HIF-1α and switch to glycolytic metabolism, all associated with chemoresistance (Cancer Biol Ther 13:858, 2012). Here we report in vitro studies aimed to determine the role of Gln in contributing to the core metabolism of proliferating leukemic cells under hypoxia and upon interaction with MSC. Genome-wide gene expression profiling of OCI-AML3 and REH leukemic cells co-cultured with BM-MSC under hypoxic conditions demonstrated that hypoxia alters metabolic patterns through increased rate of glycolysis and down-regulation of oxidative phosphorylation (oxphos), while MSC co-culture under hypoxia further promoted glycolysis in leukemic cells and induced genes regulating oxphos, TCA cycle and Gln utilization (e.g. GLS1, GOT). GLS1 protein level was also increased under hypoxia. Our in vitro findings further indicate that both acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL) cell lines rely on Gln as a carbon source under normoxia, while AML cells are more tolerant to Gln deprivation under hypoxia. In turn, inhibition of glutaminase (GLS1), the initial enzyme in glutaminolysis, with a small molecule BPTES decreased leukemia cell growth and increased apoptosis under both, normoxic and hypoxic conditions. MSC reduced BPTES-induced apoptosis in floating AML cells, however failed to protect firmly attached hypoxic AML cells, which are otherwise resistant to chemotherapy-induced cytotoxicity. Unexpectedly, GLS blockade negatively affected growth of MSC under co-culture conditions with leukemia cells. In summary, our results indicate the carbon source flexibility that fuels cancer cell growth in response to changes in microenvironment, and support the key role of glutamine utilization pathway for the survival of hypoxic marrow-resident leukemic cells. Citation Format: Polina Matre, Rodrigo O. Jacamo, Ying Wang, Jing Wang, R Eric Davis, Michael Andreeff, Aaron D. Schimmer, Marina Konopleva. Bone marrow-derived stromal cells and hypoxia promote glutamine dependency in leukemias. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1887. doi:10.1158/1538-7445.AM2013-1887

Abstract LB-259: The gluconeogenic pathway is operative in lung cancer cells

Abstract Accelerated aerobic glycolysis has been a hallmark of cancer metabolism since Otto Warburg's seminal discovery [1]. Glucose oxidation via glycolysis and the pentose phosphate pathway (PPP) not only produces energy and reducing equivalents but also anabolic precursors to meet the demands of proliferation and many essential functions of cancer cells. Although many of the anabolic precursors can be obtained from alternative fuel substrates, the de novo synthesis of ribose and therefore nucleotides normally requires glucose metabolism. Thus, under glucose deficiency, cancer cells will cease proliferation due to a lack of new synthesis of nucleic acids. This can occur in poorly vascularized areas of a tumor. We now have direct evidence that lung cancer cells can circumvent this problem by making ribose from Gln using part of the gluconeogenic pathway. By using U-13C5,15N2-Gln as tracer, we tracked the fate of the 13C and 15N atoms simultaneously into various metabolites of A549 and H1299 cells using FT-ICR-MS and NMR. We found that both labels were incorporated into ATP and other nucleotides. Although the expected 15N labeling patterns were found based on the purine biosynthetic pathway, the 13C labeling patterns show that the adenine ring and/or ribosyl unit of ATP were also labeled. This can only occur through the metabolic sequence of glutaminolysis, Krebs cycle, pyruvate carboxylation, PEP carboxykinase (PEPCK) reaction, gluconeogenesis, and PPP. We believe this is the first time that gluconeogenesis is demonstrated for cancer cell lines not known to be gluconeogenic, which could have important implications for how cancer cells adapt to a glucose-limited microenvironment and the role of Gln in such adaptations. 1. Warburg, O., On the origin of cancer cells. Science, 1956. 123: p. 309–314. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-259. doi:10.1158/1538-7445.AM2011-LB-259

Biochemical and biological properties of DNA photolyases derived from utraviolet-sensitive rice cultivars

Class I and class II CPD photolyases are enzymes which repair pyrimidine dimers using visible light. A detailed characterization of class I CPD photolyases has been carried out, but little is known about the class II enzymes. Photolyases from rice are suitable for functional analyses because systematic breeding for long periods in Asian countries has led to the selection of naturally occurring mutations in the CPD photolyase gene. We report the biochemical characterization of rice mutant CPD photolyases purified as GST-form from Escherichia coli. We identified three amino acid changes, Gln126Arg, Gly255Ser, and Gln296His, among which Gln but not His at 296 is important for complementing phr-defective E. coli, binding UV-damage in E. coli, and binding thymine dimers in vitro. The photolyase with Gln at 296 has an apoenzyme:FAD ratio of 1 : 0.5 and that with His at 296 has an apoenzyme:FAD ratio of 1 : 0.12-0.25, showing a role for Gln at 296 in the binding of FAD not in the binding of thymine dimer. Concerning Gln or Arg at 126, the biochemical activity of the photolyases purified from E. coli and complementing activity for phr-defective E. coli are similarly proficient. However, the sensitivity to UV of cultivars differs depending on whether Gln or Arg is at 126. The role of Gln and Arg at 126 for photoreactivation in rice is discussed.

Glutamine: Do the Data Support the Cause for Glutamine Supplementation in Humans?

This review examines the preclinical rationale for using glutamine supplements and reviews the prospective randomized trials using glutamine to improve outcomes in patients. A special role for glutamine in gut physiology and in management of a variety of serious illnesses has been suggested, because it is the most abundant extracellular amino acid, and is used at high rates by the gut, liver, central nervous system, and immune cells. A state of relative Gln deficiency has been postulated in humans based on the decrease in plasma Gln in acute critical illness, but the decrease in plasma Gln is not specific for that amino acid, predicts only poorer outcome, and has not been validated to identify a deficiency state. Current evidence does not necessarily predict a special need or role for Gln in critical illness. Clinical efficacy of supplemental Gln has been difficult to demonstrate, possibly related to the lack of a Gln deficiency state, the wide range of end points used that reflect the lack of certainty of the predicted effect of supplementation, the heterogeneous patient populations studied, the lack of stable clinical course during the study, the lack of adequate power, and the relatively short follow-up period. Prospective randomized clinical trials of Gln supplementation were reviewed in patients with short-bowel syndrome, during cancer chemotherapy and in bone marrow transplantation, and in surgical, burn, and intensive care unit patients. No firm recommendation can be made at this time. Future studies should seek to develop a more standard and stable design for intervention in sufficiently powered studies.

Clostridium difficile Toxin A Induces Intestinal Epithelial Cell Apoptosis and Damage: Role of Gln and Ala-Gln in Toxin A Effects

The aim of this study was to investigate the effect of Clostridium difficile toxin A (TxA) on intestinal epithelial cell migration, apoptosis, and transepithelial resistance and to evaluate the effect of glutamine (Gln) and its stable derivative, alanyl-glutamine (Ala-Gln), on TxA-induced damage. Migration was measured in rat intestinal epithelial cells (IEC-6) 6 and 24 hr after a razor scrape of the cell monolayer. Cell proliferation was indirectly measured utilizing the tetrazolium salt WST-1. The cells were incubated with TxA (1-100 ng/ml) in medium without Gln or medium containing Gln or Ala-Gln (1-30 mM). Apoptosis was quantified in IEC-6 cells using annexin V assay. Transepithelial resistance was measured using an epithelial voltohmmeter across T84 cells seeded on a transwell filter. TxA-induced a dose-dependent reduction of migration and also caused dose and time-dependent apoptosis in IEC-6 cells. Gln and Aln-Gln significantly enhanced IEC-6 cell migration and proliferation. Gln and Ala-Gln also prevented the inhibition of migration, apoptosis, and the initial drop in transepithelial resistance induced by TxA. In conclusion, both peptides reduced toxin-induced epithelial damage and thus might play an adjunctive role in C. difficile-induced colitis therapy.

Function of renal γ-glutamyltransferase: Significance of glutathione and glutamine interactions

The membrane bound γ-glutamyltransferase (γ-GT) is capable of utilizing both glutathione, GSH, and glutamine, gln, as the natural γ-glutamyl donor. The enzyme is oriented in the membrane to react with extracellular substrates and is present on both the brush border and peritubular capillaries. The reaction catalyzed by γ-GT is critically dependent upon the ratio of γ-glutamyl donor/γ-GT which under near physiological conditions results in the formation of γ-glutamyl peptide and glu on the blood and urine side respectively; this effectively establishes an osmotic gradient which could contribute to transepithelial and transcapillary water fluxes. Interestingly, utilization of extrarenal γ-glutamyl substrates are quantitatively more significant in the microvascular than brush border location. Delivery of gln to these enzyme sites is some 20 times greater than GSH. Gln utilization unlike GSH is limited by the reaction with the γ-glutamyl donor site; thus reactivity is greatly enhanced by the maleate like activator hippurate which may account for the acidosis-induced adaptation in ammonia formation from gln by this enzyme. Coupled to a role in ammoniagenesis the brush border enzyme appears to play a role in the reabsorption of filtered gln. The hydrolysis of filtered GSH as well as its utilization in transfer reactions involved in amino acid reabsorption at the brush border may reflect the role of the enzyme in eliminating osmotically active solutes from the urine and thereby facilitating water fluxes. However the role of gln as γ-glutamyl donor relative to GSH will depend upon the quantitative significance of tubular GSH synthesis and secretion.