Drug Repurposing of Asparaginase and Vitamin C Targeting Glutamine Synthetase Improves Anticancer Effect in Metastatic Castration-resistant Prostate Cancer

Background: Although in North America or Europe early dignosis of prostate cancer could sucessfully improves the therapeutic outcome. However, about 70-80% of patients still suffer from metastatic castration-resistant prostate cancer (mCRPC), because of the disproportionate medical care in China. Lutetium-177 (Lu-177) or Radium-223 (Ra-223) has been suggested as the most effective therapy for mCRPC. Unfortunately, they are either not been approved in a few countries or too expensive for patients with the financial issue. Drug repurposing has been recognized as a cost-effective and relatively low-risk alternative, gains a lot interesting recently. In this study, we explored the combined treatment with asparaginase (ASNase) and/or vitamin Cas an alternative therapeutic option for mCRPC management.Methods: Prostate cancer cell lines PC3 and DU145 were used to observe the therapeutic effect of ASNase and/or vitamin C on mCRPC in vitro and in vivo. Change of cell proliferation, cell death as well as expression of glutamine synthetase eunder different treatment conditions were detected to analyzed anticancer effect of combined therapy with ASNase and vitamin C on mCRPC. Intracellular oxidation was also observed with NADPH and NADP+ assay. Male BALB/c nude mice bearing prostate carcinoma xenografts (PC3 or DU145) were used to assess treatment response to vitamin C with or without ASNase through tumor growth, small animal PET/CT scans as well as Immunohistochemistry in vivo.Results: Our in vitro studies demonstrate that ASNase synergizes with vitamin C targeting expression of glutamine synthetase enhances redox imbalance and induces anticancer effect in mCRPC cells through regulation the glutamine synthetase (GS) expression. In vivo, combination of ASnase and vitamin C could provide a significant better therapeutic outcome in comparison with controls or single treated mice. 18F-FDG PET imaging illustrated that the treatment with combined therapy could significantly reduce the 18F-FDG uptake in tumor.Conclusions: In this current study, we suggest that ASNase combined with vitamin C could be as a cost-effective strategy to manage mCRPC.18F-FDG PET/CT imaging could indicate the therapeutic response of treatment for mCRPC.

Characterization of glutamine synthetase from the ammonium-excreting strain HM053 of Azospirillum brasilense

Glutamine synthetase (GS), encoded by glnA, catalyzes the conversion of L-glutamate and ammonium to L-glutamine. This ATP hydrolysis driven process is the main nitrogen assimilation pathway in the nitrogen-fixing bacterium Azospirillum brasilense. The A. brasilense strain HM053 has poor GS activity and leaks ammonium into the medium under nitrogen fixing conditions. In this work, the glnA genes of the wild type and HM053 strains were cloned into pET28a, sequenced and overexpressed in E. coli. The GS enzyme was purified by affinity chromatography and characterized. The GS of HM053 strain carries a P347L substitution, which results in low enzyme activity and rendered the enzyme insensitive to adenylylation by the adenilyltransferase GlnE.

Identification of glutamine synthetase as a contryphan-Bt binding protein by His-tag pull-down

Background: Contryphan-Bt is a D-tryptophan-containing disulfide-constrained decapeptide recently isolated from the venom of Conus betulinus. The molecular targets of contryphans are controversial, and the identification of its interacting proteins may be of great importance. Methods: His-tag pull-down assays were performed to investigate intracellular binding proteins of contryphan-Bt from rat brain lysate. Bt-Acp-[His]6, a contryphan-Bt derivative containing hexahistidine tag, was synthesized and used as the bait. As a control, Acp-[His]6 was used to exclude nonspecific bindings. Results: Glutamine synthetase was identified as a potential contryphan-Bt binding protein by pull-down assays and subsequent LC-MS/MS. The binding of contryphan-Bt to glutamine synthetase was confirmed and determined using microscale thermophoresis, with a Kd of 74.02 ± 2.8 μM. The binding did not affect glutamine synthetase activity, suggesting that the interaction site was distinct from the catalytic center. Conclusions: Glutamine synthetase was identified as a novel contryphan-Bt binding protein. This is the first report in which the conopeptide binds to an intracellular protein.

A New Glutamine Synthetase Index to Evaluate Hepatic Lobular Restoration in Advanced Fibrosis During Anti-HBV Therapy

Background Hepatic lobular architecture distortion is a deleterious turning point and crucial histological feature of advanced liver fibrosis in chronic liver diseases. Regression of fibrosis has been documented in chronic hepatitis B (CHB) patients. However, the restoration of lobular architecture after antiviral therapy is still unclear. Here, we propose a new glutamine synthetase (GS) index (GS-index) to evaluate the extent of architectural disruption and restoration. Methods We evaluated 43 pre-and post-treatment liver biopsies of CHB patients with advanced fibrosis (Ishak stage≥4). Glutamine synthetase (GS) is normally expressed by perivenular hepatocytes around hepatic veins (HV). When GS expression is observed in the vicinity of portal tracts (PT), it denotes parenchymal extinction and lobular collapse. We propose the new glutamine synthetase index (GS-index), defined as the percentage of GSHV/(GSHV+ GSPT), to evaluate the extent of architectural disruption and restoration. Results The median GS-index improved from 7% at baseline to 36% at week 78 (P<0.001). When GS-index78w≥50% used to define hepatic lobular restoration, 37% patients (16/43) achieved lobular restoration, with improvement in ALT and AST levels. More importantly, GS-index correlated with fibrosis regression, one stage fibrosis improvement in restored group and no change in non-restored patients (P=0.030). Conclusion In the era of antiviral therapy for CHB, restoration of hepatic lobular architecture is achievable. GS-index gives a new evaluation tool to quantitively assess hepatic lobular status and therapeutic benefits in CHB patients.

Characterizing the Role of Glutamine synthetase Gene on Ammonia Nitrogen Detoxification Metabolism of the Razor Clam Sinonovacula constricta

Ammonia nitrogen is a common toxic substance in the aquatic system, which seriously threatens the survival and growth of clams. However, less is known about the ammonia metabolism and detoxification strategy in razor clam. In this study, the polymorphism of the Glutamine synthetase gene from Sinonovacula constricta (Sc-GS) was found to be related to ammonia tolerance. By comparing the coding sequence (CDS) region of Sc-GS from two geographical populations, a total of 14 and 12 single nucleotide polymorphisms (SNPs) were identified, respectively, of which 10 loci were shared between the two populations. Among them, the locus c.1133T &gt; G exhibited an extremely significant and strong association with ammonia tolerance in both populations (P &lt; 0.01), and it was missense mutation, which led to the amino acid change from leucine (Leu) to arginine (Arg). Furthermore, the results about H&amp;E staining and immunohistochemistry of Sc-GS protein in gills and hepatopancreas revealed that it was specifically localized in the lateral cilia of gill filaments and the endothelial cells of hepatocytes. After inhibiting the Sc-GS expression by RNA interference (RNAi) technology, the transcript levels of Sc-GS were extremely significantly downregulated at 24, 48, 72, and 96 h (P &lt; 0.01) in the hepatopancreas. Taken together, these results indicated that the Sc-GS gene may participate in ammonia metabolism. In addition, these results will help to demonstrate the role of Sc-GS in ammonia nitrogen metabolism and provide markers related to ammonia nitrogen tolerance for molecular marker-assisted selection (MAS) of the razor clam.

Genome-Wide Characterization of Glutamine Synthetase Family Genes in Cucurbitaceae and Their Potential Roles in Cold Response and Rootstock-Scion Signaling Communication

Glutamine synthetase (GS; EC 6.3.1.2, L-glutamate: ammonia ligase ADP-forming) is the key enzyme responsible for the primary assimilation and reassimilation of nitrogen (N) in higher plants. There are two main isoforms of GS in higher plants, classified as cytosolic GS (GS1) and chloroplastic GS (GS2) by their size and subcellular localization. In order to improve the stress tolerance, quality, and yield of cucurbit crops such as cucumbers (Csa, Cucumis sativus L.), pumpkins (Cmo, Cucurbita moschata var. Rifu) are often used as rootstocks. Here, the GS family of the two species were comprehensively analyzed using bioinformatics in terms of aspects of the phylogenic tree, gene structure, chromosome location, subcellular localization, and evolutionary and expression patterns. Seven and four GS gene family members were screened in pumpkin and cucumber, respectively. GS family genes were divided into three groups (one for GS2 and two for GS1) according to their homology and phylogenetic relationships with other species. The analysis of gene ontology annotation of GS family genes, promoter regulatory elements, and tissue-specific expression patterns indicates the potential different biological roles of GS isoforms in Cucurbitaceae. In particular, we have identified a potentially available gene (GS1: CmoCh08G004920) from pumpkin that is relatively highly expressed and tissue-specifically expressed. RT-PCR analysis showed that most CmoGSs are induced by low temperature, and long-term (day 2 to day 9) cold stress has a more obvious effect on the RNA abundance of CmoGS. Our work presents the structure and expression patterns of all candidate members of the pumpkin and cucumber GS gene family, and to the best of our knowledge, this is the first time such work has been presented. It is worth focusing on the candidate genes with strong capacity for improving pumpkin rootstock breeding in order to increase nitrogen-use efficiency in cold conditions, as well as rootstock-scion communication.

New insights into the evolution of glutamine synthetase isoenzymes in plants

Glutamine synthetase (GS) is a key enzyme responsible for the incorporation of inorganic nitrogen in the form of ammonium into the amino acid glutamine. The genes encoding GS are among the oldest existing genes in living organisms. In plants, two groups of functional GS enzymes are found: eubacterial GSIIb (GLN2) and eukaryotic GSIIe (GLN1/GS). Phylogenetic analyses have shown that the GLN2 group originated from bacteria following horizontal gene transfer. Only GLN1/GS genes are found in vascular plants, which suggests that they are involved in the final adaptation of plants to terrestrial life. The present phylogenetic study reclassifies the different GS of seed plants into three clusters: GS1a, GS1b and GS2. The presence of genes encoding GS2 has been expanded to Cycadopsida gymnosperms, which suggests the origin of this gene in a common ancestor of Cycadopsida, Ginkgoopsida and angiosperms. GS1a genes have been identified in all gymnosperms, basal angiosperms and some Magnoliidae species. Previous studies in conifers and the gene expression profiles obtained in ginkgo and magnolia in the present work could explain the absence of GS1a in more recent angiosperm species (e.g., monocots and eudicots) due to the redundant roles of GS1a and GS2 in photosynthetic cells. Altogether, the results provide a better understanding of the evolution of plant GS isoenzymes and their physiological roles, which is valuable for improving crop nitrogen use efficiency and productivity.

Glutamine synthetase expression in the brain during experimental acute liver failure (immunohistochemical study)

The aim of the study was to determine the immunohistochemical level of glutamine synthetase (GS) expression in different brain regions in the conditions of experimental acute liver failure in rats. Materials and methods. The study was conducted in Wistar rats: 5 sham (control) animals and 10 rats with acetaminophen induced liver failure model (AILF). The immunohistochemical study of GS expression in the sensorimotor cortex, white matter, hippocampus, thalamus, caudate nucleus/putamen was carried out in the period of 12-24 h after acetaminophen treatment. Results. Beginning from the 6th hour after acetaminophen treatment all AILF-animals showed the progressive increase in clinical signs of acute brain disfunction finished in 6 rats by comatose state up to 24 h - they constituted subgroup AILF-B, “non-survived”. 4 animals survived until the 24 h - subgroup AILF-A, “survived”. In the AILF-B group, starting from 16 to 24 hours after treatment, a significant (relative to control) regionally-specific dynamic increase in the level of GS expression was observed in the brain: in the cortex – by 307.33 %, in the thalamus – by 249.47%, in the hippocampus – by 245.53%, in the subcortical white matter – by 126.08%, from 12th hour – in the caudate nucleus/putamen, by 191.66 %; with the most substantive elevation of GS expression in the cortex: by 4.07 times. Conclusion. Starting from the 16th hours after the acetaminophen treatment (from the 12th h in the caudate nucleus/putamen region) and up to 24 h, it is observed reliable compared to control dynamic increase in GS protein expression in the cortex, white matter, hippocampus, thalamus, caudate nucleus/putamen of the rat brain with the most significant elevation in the cortex among other regions. The heterogeneity in the degree of GS expression rising in different brain regions potentially may indicate regions more permeable for ammonia and/or other systemic toxic factors as well as heterogeneous sensitivity of brain regions to deleterious agents in conditions of AILF. Subsequently, revealed diversity in the GS expression reflects the specificity of reactive response of local astroglia in the condition of AILF-encephalopathy during specific time-period. The dynamic increase in the GS expression associated with impairment of animal state, indicates involvement of increased GS levels in the mechanisms of experimental acute hepatic encephalopathy.