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Journal of Neuroimmune Pharmacology :... Mar 2015We previously have shown that cerebellar fastigial nucleus (FN) modulates immune function, but pathways or mechanisms underlying this immunomodulation require...
We previously have shown that cerebellar fastigial nucleus (FN) modulates immune function, but pathways or mechanisms underlying this immunomodulation require clarification. Herein, an anterograde and retrograde tracing of nerve tracts between the cerebellar FN and hypothalamus/thalamus was performed in rats. After demonstrating a direct cerebellar FN-hypothalamic/thalamic glutamatergic projection, 6-diazo-5-oxo-L-norleucine (DON), an inhibitor of glutaminase that catalyzes glutamate synthesis, was injected bilaterally in the cerebellar FN and simultaneously, D,L-threo-β-hydroxyaspartic acid (THA), an inhibitor of glutamate transporters on cell membrane, was bilaterally injected in the lateral hypothalamic area (LHA) or the ventrolateral (VL) thalamic nucleus. DON treatment in the FN alone decreased number of glutamatergic neurons that projected axons to the LHA and also diminished glutamate content in both the hypothalamus and the thalamus. These effects of DON were reduced by combined treatment with THA in the LHA or in the VL. Importantly, DON treatment in the FN alone attenuated percentage and cytotoxicity of natural killer (NK) cells and also lowered percentage and cytokine production of T lymphocytes. These DON-caused immune effects were reduced or abolished by combined treatment with THA in the LHA, but not in the VL. Simultaneously, DON treatment elevated level of norepinephrine (NE) in the spleen and mesenteric lymphoid nodes, and THA treatment in the LHA, rather than in the VL, antagonized the DON-caused NE elevation. These findings suggest that glutamatergic neurons in the cerebellar FN regulate innate and adaptive immune functions and the immunomodulation is conveyed by FN-hypothalamic glutamatergic projections and sympathetic nerves that innervate lymphoid tissues.
Topics: Animals; Aspartic Acid; Axons; Cerebellar Nuclei; Diazooxonorleucine; Enzyme Inhibitors; Female; Glutamic Acid; Glutaminase; Hypothalamic Area, Lateral; Hypothalamus; Immunity; Injections; Killer Cells, Natural; Male; Rats; Rats, Sprague-Dawley; Sympathetic Nervous System; T-Lymphocytes; Thalamus
PubMed: 25649846
DOI: 10.1007/s11481-014-9572-y -
Clinical Cancer Research : An Official... Oct 2019Atypical teratoid/rhabdoid tumors (AT/RT) are aggressive infantile brain tumors with poor survival. Recent advancements have highlighted significant molecular...
PURPOSE
Atypical teratoid/rhabdoid tumors (AT/RT) are aggressive infantile brain tumors with poor survival. Recent advancements have highlighted significant molecular heterogeneity in AT/RT with an aggressive subgroup featuring overexpression of the proto-oncogene. We perform the first comprehensive metabolic profiling of patient-derived AT/RT cell lines to identify therapeutic susceptibilities in high MYC-expressing AT/RT.
EXPERIMENTAL DESIGN
Metabolites were extracted from AT/RT cell lines and separated in ultra-high performance liquid chromatography mass spectrometry. Glutamine metabolic inhibition with 6-diazo-5-oxo-L-norleucine (DON) was tested with growth and cell death assays and survival studies in orthotopic mouse models of AT/RT. Metabolic flux analysis was completed to identify combination therapies to act synergistically to improve survival in high MYC AT/RT.
RESULTS
Unbiased metabolic profiling of AT/RT cell models identified a unique dependence of high MYC AT/RT on glutamine for survival. The glutamine analogue, DON, selectively targeted high MYC cell lines, slowing cell growth, inducing apoptosis, and extending survival in orthotopic mouse models of AT/RT. Metabolic flux experiments with isotopically labeled glutamine revealed DON inhibition of glutathione (GSH) synthesis. DON combined with carboplatin further slowed cell growth, induced apoptosis, and extended survival in orthotopic mouse models of high MYC AT/RT.
CONCLUSIONS
Unbiased metabolic profiling of AT/RT identified susceptibility of high MYC AT/RT to glutamine metabolic inhibition with DON therapy. DON inhibited glutamine-dependent synthesis of GSH and synergized with carboplatin to extend survival in high MYC AT/RT. These findings can rapidly translate into new clinical trials to improve survival in high MYC AT/RT.
Topics: Animals; Antimetabolites, Antineoplastic; Apoptosis; Cell Proliferation; Diazooxonorleucine; Female; Glutamine; Humans; Metabolome; Mice; Mice, Nude; Proto-Oncogene Mas; Proto-Oncogene Proteins c-myc; Rhabdoid Tumor; Teratoma; Tumor Cells, Cultured; Xenograft Model Antitumor Assays
PubMed: 31300448
DOI: 10.1158/1078-0432.CCR-19-0189 -
Proceedings of the National Academy of... Oct 2015The most deadly complication of Plasmodium falciparum infection is cerebral malaria (CM) with a case fatality rate of 15-25% in African children despite effective...
The most deadly complication of Plasmodium falciparum infection is cerebral malaria (CM) with a case fatality rate of 15-25% in African children despite effective antimalarial chemotherapy. There are no adjunctive treatments for CM, so there is an urgent need to identify new targets for therapy. Here we show that the glutamine analog 6-diazo-5-oxo-L-norleucine (DON) rescues mice from CM when administered late in the infection a time at which mice already are suffering blood-brain barrier dysfunction, brain swelling, and hemorrhaging accompanied by accumulation of parasite-specific CD8(+) effector T cells and infected red blood cells in the brain. Remarkably, within hours of DON treatment mice showed blood-brain barrier integrity, reduced brain swelling, decreased function of activated effector CD8(+) T cells in the brain, and levels of brain metabolites that resembled those in uninfected mice. These results suggest DON as a strong candidate for an effective adjunctive therapy for CM in African children.
Topics: Animals; Antimalarials; Blood-Brain Barrier; Diazooxonorleucine; Glutamine; Malaria, Cerebral; Malaria, Falciparum; Mice
PubMed: 26438846
DOI: 10.1073/pnas.1516544112 -
Scientific Reports May 2018Bacteria in nature often reside in differentiated communities termed biofilms, which are an active interphase between uni-cellular and multicellular life states for...
Bacteria in nature often reside in differentiated communities termed biofilms, which are an active interphase between uni-cellular and multicellular life states for bacteria. Here we demonstrate that the development of B. subtilis biofilms is dependent on the use of glutamine or glutamate as a nitrogen source. We show a differential metabolic requirement within the biofilm; while glutamine is necessary for the dividing cells at the edges, the inner cell mass utilizes lactic acid. Our results indicate that biofilm cells preserve a short-term memory of glutamate metabolism. Finally, we establish that drugs that target glutamine and glutamate utilization restrict biofilm development. Overall, our work reveals a spatial regulation of nitrogen and carbon metabolism within the biofilm, which contributes to the fitness of bacterial complex communities. This acquired metabolic division of labor within biofilm can serve as a target for novel anti-biofilm chemotherapies.
Topics: Aminooxyacetic Acid; Anti-Bacterial Agents; Bacillus subtilis; Bacterial Proteins; Biofilms; Carbon; Diazooxonorleucine; Gene Expression Regulation, Bacterial; Glutamic Acid; Glutamine; Mutant Proteins; Nitrogen; Repressor Proteins
PubMed: 29740028
DOI: 10.1038/s41598-018-25401-z -
Aging Cell Jun 2016Alzheimer's disease (AD) is characterized clinically by memory loss and cognitive decline. Protein kinase A (PKA)-CREB signaling plays a critical role in learning and...
Alzheimer's disease (AD) is characterized clinically by memory loss and cognitive decline. Protein kinase A (PKA)-CREB signaling plays a critical role in learning and memory. It is known that glucose uptake and O-GlcNAcylation are reduced in AD brain. In this study, we found that PKA catalytic subunits (PKAcs) were posttranslationally modified by O-linked N-acetylglucosamine (O-GlcNAc). O-GlcNAcylation regulated the subcellular location of PKAcα and PKAcβ and enhanced their kinase activity. Upregulation of O-GlcNAcylation in metabolically active rat brain slices by O-(2-acetamido-2-deoxy-d-glucopyranosylidenamino) N-phenylcarbamate (PUGNAc), an inhibitor of N-acetylglucosaminidase, increased the phosphorylation of tau at the PKA site, Ser214, but not at the non-PKA site, Thr205. In contrast, in rat and mouse brains, downregulation of O-GlcNAcylation caused decreases in the phosphorylation of CREB at Ser133 and of tau at Ser214, but not at Thr205. Reduction in O-GlcNAcylation through intracerebroventricular injection of 6-diazo-5-oxo-l-norleucine (DON), the inhibitor of glutamine fructose-6-phosphate amidotransferase, suppressed PKA-CREB signaling and impaired learning and memory in mice. These results indicate that in addition to cAMP and phosphorylation, O-GlcNAcylation is a novel mechanism that regulates PKA-CREB signaling. Downregulation of O-GlcNAcylation suppresses PKA-CREB signaling and consequently causes learning and memory deficits in AD.
Topics: Acetylglucosamine; Alzheimer Disease; Animals; Brain; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinase Catalytic Subunits; Diazooxonorleucine; Down-Regulation; Glycosylation; HEK293 Cells; HeLa Cells; Humans; Learning; Male; Memory; Memory Disorders; Mice, Inbred C57BL; Phosphorylation; Protein Transport; Rats, Sprague-Dawley; Subcellular Fractions; tau Proteins
PubMed: 26840030
DOI: 10.1111/acel.12449 -
Journal of Neurovirology Apr 2015Recovery from encephalomyelitis induced by infection with mosquito-borne alphaviruses is associated with a high risk of lifelong debilitating neurological deficits....
Recovery from encephalomyelitis induced by infection with mosquito-borne alphaviruses is associated with a high risk of lifelong debilitating neurological deficits. Infection of mice with the prototypic alphavirus, Sindbis virus, provides an animal model with which to study disease mechanisms and examine potential therapeutics. Infectious virus is cleared from the brain within a week after infection, but viral RNA is cleared slowly and persists for the life of the animal. However, no studies have examined the effect of infection on neurocognitive function over time. In the present study, we examined neurocognitive function at different phases of infection in 5-week-old C57BL/6 mice intranasally inoculated with Sindbis virus. At the peak of active virus infection, mice demonstrated hyperactivity, decreased anxiety, and marked hippocampal-dependent memory deficits, the latter of which persisted beyond clearance of infectious virus and resolution of clinical signs of disease. Previous studies indicate that neuronal damage during alphavirus encephalomyelitis is primarily due to inflammatory cell infiltration and glutamate excitotoxicity rather than directly by virus infection. Therefore, mice were treated with 6-diazo-5-oxo-l-norleucine (DON), a glutamine antagonist that can suppress both the immune response and excitotoxicity. Treatment with DON decreased inflammatory cell infiltration and cell death in the hippocampus and partially prevented development of clinical signs and neurocognitive impairment despite the presence of infectious virus and high viral RNA levels. This study presents the first report of neurocognitive sequelae in mice with alphavirus encephalomyelitis and provides a model system for further elucidation of the pathogenesis of virus infection and assessment of potential therapies.
Topics: Alphavirus Infections; Animals; Antimetabolites, Antineoplastic; Behavior, Animal; Diazooxonorleucine; Disease Models, Animal; Encephalitis, Viral; Enzyme-Linked Immunosorbent Assay; Glutamine; Immunohistochemistry; Male; Mice; Mice, Inbred C57BL; Motor Activity; Sindbis Virus
PubMed: 25645378
DOI: 10.1007/s13365-015-0314-6 -
Journal of Medicinal Chemistry Sep 2016The glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON, 1) has shown robust anticancer efficacy in preclinical and clinical studies, but its development was halted due...
The glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON, 1) has shown robust anticancer efficacy in preclinical and clinical studies, but its development was halted due to marked systemic toxicities. Herein we demonstrate that DON inhibits glutamine metabolism and provides antitumor efficacy in a murine model of glioblastoma, although toxicity was observed. To enhance DON's therapeutic index, we utilized a prodrug strategy to increase its brain delivery and limit systemic exposure. Unexpectedly, simple alkyl ester-based prodrugs were ineffective due to chemical instability cyclizing to form a unique diazo-imine. However, masking both DON's amine and carboxylate functionalities imparted sufficient chemical stability for biological testing. While these dual moiety prodrugs exhibited rapid metabolism in mouse plasma, several provided excellent stability in monkey and human plasma. The most stable compound (5c, methyl-POM-DON-isopropyl-ester) was evaluated in monkeys, where it achieved 10-fold enhanced cerebrospinal fluid to plasma ratio versus DON. This strategy may provide a path to DON utilization in glioblastoma multiforme patients.
Topics: Animals; Antimetabolites, Antineoplastic; Brain Neoplasms; Diazooxonorleucine; Female; Glioblastoma; Glutamine; Haplorhini; Humans; Mice; Mice, Nude; Prodrugs
PubMed: 27560860
DOI: 10.1021/acs.jmedchem.6b01069 -
Molecular Carcinogenesis Jun 2019Platinum anticancer agents are essential components in chemotherapeutic regimens for non-small-cell lung cancer (NSCLC) patients ineligible for targeted therapy....
Platinum anticancer agents are essential components in chemotherapeutic regimens for non-small-cell lung cancer (NSCLC) patients ineligible for targeted therapy. However, platinum-based regimens have reached a plateau of therapeutic efficacy; therefore, it is critical to implement novel approaches for improvement. The hexosamine biosynthesis pathway (HBP), which produces amino-sugar N-acetyl-glucosamine for protein glycosylation, is important for protein function and cell survival. Here we show a beneficial effect by the combination of cisplatin with HBP inhibition. Expression of glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme of HBP, was increased in NSCLC cell lines and tissues. Pharmacological inhibition of GFAT activity or knockdown of GFATimpaired cell proliferation and exerted synergistic or additive cytotoxicity to the cells treated with cisplatin. Mechanistically, GFAT positively regulated the expression of binding immunoglobulin protein (BiP; also known as glucose-regulated protein 78, GRP78), an endoplasmic reticulum chaperone involved in unfolded protein response (UPR). Suppressing GFAT activity resulted in downregulation of BiP that activated inositol-requiring enzyme 1α, a sensor protein of UPR, and exacerbated cisplatin-induced cell apoptosis. These data identify GFAT-mediated HBP as a target for improving platinum-based chemotherapy for NSCLC.
Topics: A549 Cells; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cisplatin; Diazooxonorleucine; Down-Regulation; Drug Synergism; Endoplasmic Reticulum Chaperone BiP; Gene Expression Regulation, Neoplastic; Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); Heat-Shock Proteins; Hexosamines; Humans; Lung Neoplasms
PubMed: 30790354
DOI: 10.1002/mc.22992 -
PloS One 2015Neuroblastomas (NBL) and Ewing's sarcomas (EWS) together cause 18% of all pediatric cancer deaths. Though there is growing interest in targeting the dysregulated...
Neuroblastomas (NBL) and Ewing's sarcomas (EWS) together cause 18% of all pediatric cancer deaths. Though there is growing interest in targeting the dysregulated metabolism of cancer as a therapeutic strategy, this approach has not been fully examined in NBL and EWS. In this study, we first tested a panel of metabolic inhibitors and identified the glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON) as the most potent chemotherapeutic across all NBL and EWS cell lines tested. Myc, a master regulator of metabolism, is commonly overexpressed in both of these pediatric malignancies and recent studies have established that Myc causes cancer cells to become "addicted" to glutamine. We found DON strongly inhibited tumor growth of multiple tumor lines in mouse xenograft models. In vitro, inhibition of caspases partially reversed the effects of DON in high Myc expressing cell lines, but not in low Myc expressing lines. We further showed that induction of apoptosis by DON in Myc-overexpressing cancers is via the pro-apoptotic factor Bax. To relieve inhibition of Bax, we tested DON in combination with the Bcl-2 family antagonist navitoclax (ABT-263). In vitro, this combination caused an increase in DON activity across the entire panel of cell lines tested, with synergistic effects in two of the N-Myc amplified neuroblastoma cell lines. Our study supports targeting glutamine metabolism to treat Myc overexpressing cancers, such as NBL and EWS, particularly in combination with Bcl-2 family antagonists.
Topics: Aniline Compounds; Animals; Antimetabolites, Antineoplastic; Apoptosis; Bone Neoplasms; Caspases; Cell Line, Tumor; Cell Proliferation; Diazooxonorleucine; Drug Synergism; Glutamine; Humans; Mice; Neuroblastoma; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; Sarcoma, Ewing; Sulfonamides; Xenograft Model Antitumor Assays
PubMed: 25615615
DOI: 10.1371/journal.pone.0116998 -
Oncology Reports Sep 2015There is a strong rationale for targeting the metabolic alterations of cancer cells. The most studied of these are the higher rates of glycolysis, glutaminolysis and...
There is a strong rationale for targeting the metabolic alterations of cancer cells. The most studied of these are the higher rates of glycolysis, glutaminolysis and de novo synthesis of fatty acids (FAs). Despite the availability of pharmacological inhibitors of these pathways, no preclinical studies targeting them simultaneously have been performed. In the present study it was determined whether three key enzymes for glycolysis, glutaminolysis and de novo synthesis of FAs, hexokinase-2, glutaminase and fatty acid synthase, respectively, were overexpressed as compared to primary fibroblasts. In addition, we showed that at clinically relevant concentrations lonidamine, 6-diazo-5-oxo-L-norleucine and orlistat, known inhibitors of the mentioned enzymes, exerted a cell viability inhibitory effect. Genetic downregulation of the three enzymes also reduced cell viability. The three drugs were highly synergistic when administered as a triple combination. Of note, the cytotoxicity of the triple combination was low in primary fibroblasts and was well tolerated when administered into healthy BALB/c mice. The results suggest the feasibility and potential clinical utility of the triple metabolic targeting which merits to be further studied by using either repositioned old drugs or newer, more selective inhibitors.
Topics: Animals; Apoptosis; Cell Survival; Diazooxonorleucine; Drug Synergism; Fatty Acid Synthases; Fatty Acids; Fibroblasts; Gene Expression Regulation, Neoplastic; Glutaminase; Glycolysis; Hexokinase; Humans; Indazoles; Lactones; Metabolic Networks and Pathways; Mice; Neoplasms; Orlistat
PubMed: 26134042
DOI: 10.3892/or.2015.4077