-
Journal of Neuro-oncology Feb 2020Glioblastoma is an aggressive central nervous system tumor with a 5-year survival rate of < 10%. The standard therapy for glioblastoma is maximal safe resection,...
PURPOSE
Glioblastoma is an aggressive central nervous system tumor with a 5-year survival rate of < 10%. The standard therapy for glioblastoma is maximal safe resection, followed by radiation therapy and chemotherapy with temozolomide. New approaches to treatment of glioblastoma, such as targeting metabolism, have been studied. The object of this study is to evaluate whether asparagine could be a new target for treatment of glioblastoma.
METHODS
We investigated a potential treatment for glioblastoma that targets the amino acid metabolism. U251, U87, and SF767 glioblastoma cells were treated with L-asparaginase and/or 6-diazo-5-oxo-L-norleucine (DON). L-asparaginase hydrolyzes asparagine into aspartate and depletes asparagine. L-asparaginase has been used for the treatment of acute lymphoblastic leukemia. DON is a glutamine analog that inhibits several glutamine-utilizing enzymes, including asparagine synthetase.
RESULTS
Cell viability was measured after 72 h of treatment. MTS assay showed that L-asparaginase suppressed the proliferation of U251, U87, and SF767 cells in a dose-dependent manner. DON also inhibited the proliferation of these cell lines in a dose-dependent manner. Combined treatment with these drugs had a synergistic antiproliferative effect in these cell lines. Exogenous asparagine rescued the effect of inhibition of proliferation by L-asparaginase and DON. The expression of asparagine synthetase mRNA was increased in cells treated with a combination of L-asparaginase and DON. This combined treatment also induced greater apoptosis and autophagy than did single-drug treatment.
CONCLUSION
The results suggest that the combination of L-asparaginase and DON could be a new therapeutic option for patients with glioblastoma.
Topics: Antineoplastic Agents; Asparaginase; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Diazooxonorleucine; Glioblastoma; Humans
PubMed: 32020477
DOI: 10.1007/s11060-019-03351-4 -
Reproduction (Cambridge, England) Dec 2019The developmental competence of in vitro-matured oocytes is still lower than that of the in vivo-matured oocytes due to precocious meiotic resumption and inappropriate...
The developmental competence of in vitro-matured oocytes is still lower than that of the in vivo-matured oocytes due to precocious meiotic resumption and inappropriate cytoplasmic maturation. Although numerous efforts have been attempted to accomplish better in vitro maturation (IVM) condition, only limited progress has been achieved. Thus, a current study was conducted to examine the effects of 6-diazo-5-oxo-l-norleucine (DON, an inhibitor of hyaluronan synthesis) during the first half period of IVM on nuclear/cytoplasmic maturation of porcine oocytes and subsequent embryonic development. Based on the observation of the nucleus pattern, metaphase II (MII) oocyte production rate in 1 µM DON group was significantly higher than other groups at 44 h of IVM. The 1 µM of DON was suggested to be optimal for porcine IVM and was therefore used for further investigation. Meiotic arrest effect of DON was maximal at 6 h of IVM, which was supported by the maintenance of significantly higher intra-oocyte cAMP level. In addition, increased pERK1/2 levels and clear rearrangement of cortical granules in membrane of MII oocytes matured with DON provided the evidence for balanced meiosis progression between nuclear and cytoplasmic maturation. Subsequently, DON significantly improved blastocyst formation rate, total cell numbers, and cellular survival in blastocysts after parthenogenetic activation, in vitro fertilization, and somatic cell nuclear transfer. Altogether, our results showed for the first time that 1 µM DON can be used to increase the yield of developmentally competent MII oocytes by synchronizing nuclear/cytoplasmic maturation, and it subsequently improves embryo developmental competence.
Topics: Animals; Antibiotics, Antineoplastic; Cell Nucleus; Cytoplasm; Diazooxonorleucine; Embryonic Development; Female; Fertilization in Vitro; In Vitro Oocyte Maturation Techniques; Meiosis; Nuclear Transfer Techniques; Oocytes; Pregnancy; Swine
PubMed: 31652418
DOI: 10.1530/REP-19-0235 -
The Journal of Clinical Investigation Jan 2020Pancreatic ductal adenocarcinoma (PDAC) is considered to be a highly immunosuppressive and heterogenous neoplasm. Despite improved knowledge regarding the genetic...
Pancreatic ductal adenocarcinoma (PDAC) is considered to be a highly immunosuppressive and heterogenous neoplasm. Despite improved knowledge regarding the genetic background of the tumor and better understanding of the tumor microenvironment, immune checkpoint inhibitor therapy (targeting CTLA4, PD1, PDL1) has not been very successful against PDAC. The robust desmoplastic stroma, along with an extensive extracellular matrix (ECM) that is rich in hyaluronan, plays an integral role in this immune evasion. Hexosamine biosynthesis pathway (HBP), a shunt pathway of glycolysis, is a metabolic node in cancer cells that can promote survival pathways on the one hand and influence the hyaluronan synthesis in the ECM on the other. The rate-limiting enzyme of the pathway, glutamine-fructose amidotransferase 1 (GFAT1), uses glutamine and fructose 6-phosphate to eventually synthesize uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). In the current manuscript, we targeted this glutamine-utilizing enzyme by a small molecule glutamine analog (6-diazo-5-oxo-l-norleucine [DON]). Our results showed that DON decreased the self-renewal potential and metastatic ability of tumor cells. Further, treatment with DON decreased hyaluronan and collagen in the tumor microenvironment, leading to an extensive remodeling of the ECM and an increased infiltration of CD8+ T cells. Additionally, treatment with DON sensitized pancreatic tumors to anti-PD1 therapy, resulting in tumor regression and prolonged survival.
Topics: Animals; CD8-Positive T-Lymphocytes; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Diazooxonorleucine; Female; Hexosamines; Humans; Mice; Mice, Nude; Neoplasm Metastasis; Neoplasm Proteins; Pancreatic Neoplasms; Programmed Cell Death 1 Receptor; Xenograft Model Antitumor Assays
PubMed: 31613799
DOI: 10.1172/JCI127515 -
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 -
Communications Biology 2019Glioblastoma (GBM) is an aggressive primary human brain tumour that has resisted effective therapy for decades. Although glucose and glutamine are the major fuels that...
Glioblastoma (GBM) is an aggressive primary human brain tumour that has resisted effective therapy for decades. Although glucose and glutamine are the major fuels that drive GBM growth and invasion, few studies have targeted these fuels for therapeutic management. The glutamine antagonist, 6-diazo-5-oxo-L-norleucine (DON), was administered together with a calorically restricted ketogenic diet (KD-R) to treat late-stage orthotopic growth in two syngeneic GBM mouse models: VM-M3 and CT-2A. DON targets glutaminolysis, while the KD-R reduces glucose and, simultaneously, elevates neuroprotective and non-fermentable ketone bodies. The diet/drug therapeutic strategy killed tumour cells while reversing disease symptoms, and improving overall mouse survival. The therapeutic strategy also reduces edema, hemorrhage, and inflammation. Moreover, the KD-R diet facilitated DON delivery to the brain and allowed a lower dosage to achieve therapeutic effect. The findings support the importance of glucose and glutamine in driving GBM growth and provide a therapeutic strategy for non-toxic metabolic management.
Topics: Animals; Body Weight; Brain; Brain Neoplasms; Caloric Restriction; Cell Line, Tumor; Cell Proliferation; Diazooxonorleucine; Diet, Ketogenic; Disease Models, Animal; Female; Fermentation; Glioblastoma; Glucose; Glutamine; Humans; Immunohistochemistry; Ketone Bodies; Ketones; Male; Mice; Mice, Inbred C57BL; Neoplasm Transplantation
PubMed: 31149644
DOI: 10.1038/s42003-019-0455-x -
Scientific Reports Apr 2019Glutamine metabolism and the mechanistic target of rapamycin (mTOR) pathway are activated cooperatively in the differentiation and activation of inflammatory immune...
Glutamine metabolism and the mechanistic target of rapamycin (mTOR) pathway are activated cooperatively in the differentiation and activation of inflammatory immune cells. But the combined inhibition of both pathways was rarely investigated. This study investigated how inhibiting both glutamine metabolism with 6-diazo-5-oxo-L-norleucine (DON) and mTOR with rapamycin affects immune cells and the arthritis in a mouse model. We revealed that rapamycin and DON additively suppressed CD4 T cell proliferation, and both of them inhibited Th17 cell differentiation. While DON inhibited the differentiation of dendritic cells and macrophages and facilitated that of Ly6G granulocytic (G)-MDSCs more strongly than did rapamycin, G-MDSCs treated with rapamycin but not DON suppressed CD4 T cell proliferation in vitro. The combination of rapamycin and DON significantly suppressed the arthritis in SKG mice more strongly than did each monotherapy in vivo. The numbers of CD4 T and Th17 cells in the spleen were lowest in mice treated with the combination therapy. Thus, combined treatment with rapamycin and DON additively ameliorated the arthritis in SKG mice, possibly by suppressing CD4 T cell proliferation and Th17 differentiation. These results suggest the combination of rapamycin and DON may be a potential novel therapy for arthritis.
Topics: Animals; Arthritis; Bone Marrow Cells; Cell Differentiation; Cell Proliferation; Dendritic Cells; Diazooxonorleucine; Female; Glutamine; Immunosuppression Therapy; Macrophages; Mice, Inbred BALB C; Myeloid-Derived Suppressor Cells; Sirolimus; TOR Serine-Threonine Kinases; Th17 Cells
PubMed: 31011190
DOI: 10.1038/s41598-019-42932-1 -
Journal of Medicinal Chemistry Apr 20196-Diazo-5-oxo-l-norleucine (DON) is a glutamine antagonist with robust anticancer efficacy; however, its therapeutic potential was hampered by its biodistribution and...
6-Diazo-5-oxo-l-norleucine (DON) is a glutamine antagonist with robust anticancer efficacy; however, its therapeutic potential was hampered by its biodistribution and toxicity to normal tissues, specifically gastrointestinal (GI) tissues. To circumvent DON's toxicity, we synthesized a series of tumor-targeted DON prodrugs designed to circulate inert in plasma and preferentially activate over DON in tumor. Our best prodrug 6 (isopropyl 2-(6-acetamido-2-(adamantane-1-carboxamido)hexanamido)-6-diazo-5-oxohexanoate) showed stability in plasma, liver, and intestinal homogenates yet was readily cleaved to DON in P493B lymphoma cells, exhibiting a 55-fold enhanced tumor cell-to-plasma ratio versus that of DON and resulting in a dose-dependent inhibition of cell proliferation. Using carboxylesterase 1 knockout mice that were shown to mimic human prodrug metabolism, systemic administration of 6 delivered 11-fold higher DON exposure to tumor (target tissue; AUC = 5.1 nmol h/g) versus GI tissues (toxicity tissue; AUC = 0.45 nmol h/g). In summary, these studies describe the discovery of a glutamine antagonist prodrug that provides selective tumor exposure.
Topics: Acetylation; Animals; Antineoplastic Agents; Area Under Curve; Carboxylic Ester Hydrolases; Cell Line, Tumor; Diazooxonorleucine; Drug Delivery Systems; Humans; Lysine; Mice; Mice, Knockout; Prodrugs; Swine
PubMed: 30892035
DOI: 10.1021/acs.jmedchem.8b02009 -
Physiological Reports Mar 2019Despite recent advances, acute respiratory distress syndrome (ARDS) remains a severe and often fatal disease for which there is no therapy able to reduce the underlying...
Despite recent advances, acute respiratory distress syndrome (ARDS) remains a severe and often fatal disease for which there is no therapy able to reduce the underlying excessive lung inflammation or enhance resolution of injury. Metabolic programming plays a critical role in regulating inflammatory responses. Due to their high metabolic needs, neutrophils, macrophages, and lymphocytes rely upon glutamine metabolism to support activation and function. Additionally, during times of physiologic stress, nearly all cells, including fibroblasts and epithelial cells, require glutamine metabolism. We hypothesized that inhibiting glutamine metabolism reduces lung inflammation and promotes resolution of acute lung injury. Lung injury was induced by instilling lipopolysaccharide (LPS) intratracheally. To inhibit glutamine metabolism, we administered a glutamine analogue, 6-diazo-5-oxo-L-norleucine (DON) that binds to glutamine-utilizing enzymes and transporters, after injury was well established. Treatment with DON led to less lung injury, fewer lung neutrophils, lung inflammatory and interstitial macrophages, and lower levels of proinflammatory cytokines and chemokines at 5 and/or 7 days after injury. Additionally, DON led to earlier expression of the growth factor amphiregulin and more rapid recovery of LPS-induced weight loss. Thus, DON reduced lung inflammation and promoted resolution of injury. These data contribute to our understanding of how glutamine metabolism regulates lung inflammation and repair, and identifies a novel target for future therapies for ARDS and other inflammatory lung diseases.
Topics: Acute Lung Injury; Amphiregulin; Animals; Anti-Inflammatory Agents; Antimetabolites; Cytokines; Diazooxonorleucine; Disease Models, Animal; Glutamine; Inflammation Mediators; Lipopolysaccharides; Lung; Macrophages; Mice, Inbred C57BL; Neutrophil Infiltration; Pneumonia; Time Factors
PubMed: 30821123
DOI: 10.14814/phy2.14019 -
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 -
Proceedings of the National Academy of... Dec 2018The deadliest complication of infection is cerebral malaria (CM), with a case fatality rate of 15 to 25% in African children despite effective antimalarial...
The deadliest complication of infection is cerebral malaria (CM), with a case fatality rate of 15 to 25% in African children despite effective antimalarial chemotherapy. No adjunctive treatments are yet available for this devastating disease. We previously reported that the glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON) rescued mice from experimental CM (ECM) when administered late in the infection, a time by which mice had already suffered blood-brain barrier (BBB) dysfunction, brain swelling, and hemorrhaging. Herein, we used longitudinal MR imaging to visualize brain pathology in ECM and the impact of a new DON prodrug, JHU-083, on disease progression in mice. We demonstrate in vivo the reversal of disease markers in symptomatic, infected mice following treatment, including the resolution of edema and BBB disruption, findings usually associated with a fatal outcome in children and adults with CM. Our results support the premise that JHU-083 is a potential adjunctive treatment that could rescue children and adults from fatal CM.
Topics: Adult; Animals; Antimalarials; Biomarkers; Blood-Brain Barrier; Brain; Brain Edema; Child; Diazooxonorleucine; Disease Models, Animal; Disease Progression; Female; Glutamine; Humans; Magnetic Resonance Imaging; Malaria, Cerebral; Malaria, Falciparum; Mice; Mice, Inbred C57BL; Plasmodium falciparum
PubMed: 30514812
DOI: 10.1073/pnas.1812909115