glutaminase and Ovarian-Neoplasms

Metabolomics, the newest of the omics sciences that also include genomics, transcriptomics and proteomics, has matured into a reliable high-throughput technology. Gas chromatography combined with time-of-flight mass spectrometry (GC-TOFMS) is a suitable method to analyze the central metabolism in fresh frozen tumor tissue samples. Bioinformatics methods, including the PROFILE clustering developed by us, permit integrated analysis and fast interpretation of metabolomics data in the context of enzymatic reactions and metabolic pathways. The metabolome analyses of three solid tumor types presented here, together with the results of other authors, show that metabolites are suitable as biomarkers and provide diverse options for translation into the clinical setting.

Topics: 4-Aminobutyrate Transaminase; beta-Alanine; Biomarkers, Tumor; Breast; Breast Neoplasms; Colonic Neoplasms; Female; Gas Chromatography-Mass Spectrometry; Glutamic Acid; Glutaminase; Glutamine; Humans; Metabolome; Neoplasms; Neoplasms, Hormone-Dependent; Ovarian Neoplasms; Ovary

Approximately 50%-55% of high-grade serous ovarian carcinoma (HGSOC) patients have MYC oncogenic pathway activation. Because MYC is not directly targetable, we have analyzed molecular pathways enriched in MYC-high HGSOC tumors to identify potential therapeutic targets. Here, we report that MYC-high HGSOC tumors show enrichment in genes controlled by NRF2, an antioxidant signaling pathway, along with increased thioredoxin redox activity. Treatment of MYC-high HGSOC tumors cells with US Food and Drug Administration (FDA)-approved thioredoxin reductase 1 (TrxR1) inhibitor auranofin resulted in significant growth suppression and apoptosis in MYC-high HGSOC cells in vitro and also significantly reduced tumor growth in an MYC-high HGSOC patient-derived tumor xenograft. We found that auranofin treatment inhibited glycolysis in MYC-high cells via oxidation-induced GAPDH inhibition. Interestingly, in response to auranofin-induced glycolysis inhibition, MYC-high HGSOC cells switched to glutamine metabolism for survival. Depletion of glutamine with either glutamine starvation or glutaminase (GLS1) inhibitor CB-839 exerted synergistic anti-tumor activity with auranofin in HGSOC cells and OVCAR-8 cell line xenograft. These findings suggest that applying a combined therapy of GLS1 inhibitor and TrxR1 inhibitor could effectively treat MYC-high HGSOC patients.

Topics: Auranofin; Cell Line, Tumor; Female; Genes, myc; Glutaminase; Glutamine; Humans; Ovarian Neoplasms; Thioredoxin-Disulfide Reductase; Thioredoxins

Increasing demand for glutaminase (GLS) due to high rates of glutamine metabolism is considered one of the hallmarks of malignancy. In parallel, cancer antigen 125 (CA-125) is a commonly used ovarian tumor marker. This study aimed to compare the roles of GLS and CA-125 in distinguishing between benign and malignant ovarian tumors. The research was conducted as a comparative study, enrolling 156 patients with ovarian tumors. Preoperative serum CA-125 and GLS levels were analyzed to evaluate their effectiveness in distinguishing between benign and malignant ovarian tumors. The results revealed that the mean levels of CA-125 and GLS were significantly higher in malignant ovarian tumors compared with benign ones (389.54 ± 494.320 vs. 193.15 ± 529.932 (U/mL) and 17.37 ± 12.156 vs. 7.48 ± 4.095 (μg/mL), respectively). The CA-125 and GLS cutoff points of 108.2 U/mL and 18.32 μg/mL, respectively, were associated with malignant ovarian tumors. Multivariate analyses showed that GLS had higher predictive capabilities compared with CA-125 (odds ratio 9.4 vs. 2.1). The accuracy of using GLS combined with CA-125 was higher than using CA-125 alone (73.1% vs. 68.8%). In conclusion, higher levels of CA-125 and GLS are associated with malignant ovarian tumors. GLS outperforms CA-125 in distinguishing between benign and malignant ovarian tumors. The combination of GLS and CA-125 demonstrated improved accuracy for distinguishing benign and malignant ovarian tumors when compared with using CA-125 alone.

Topics: Biomarkers, Tumor; CA-125 Antigen; Female; Glutaminase; Humans; Ovarian Neoplasms

Targeting glutamine metabolism has emerged as a novel therapeutic strategy for several human cancers, including ovarian cancer. The primary target of this approach is the kidney isoform of glutaminase, glutaminase 1 (GLS1), a key enzyme in glutamine metabolism that is overexpressed in several human cancers. A first-in-class inhibitor of GLS1, called CB839 (Telaglenastat), has been investigated in several clinical trials, with promising results. The first clinical trial of CB839 in platinum-resistant ovarian cancer patients is forthcoming.

Topics: Adenocarcinoma, Clear Cell; Animals; DNA-Binding Proteins; Female; Glutaminase; Glutamine; Humans; Mice; Ovarian Neoplasms; Protective Factors; Transcription Factors

Programmed cell death 1 ligand (PD-L1) blockade has been used therapeutically in the treatment of ovarian cancer, and potential combination treatment approaches are under investigation to improve the treatment response rate. The increased dependence on glutamine is widely observed in various type of tumors, including ovarian cancer. Kidney-type glutaminase (GLS), as one of the isotypes of glutaminase, is found to promote tumorigenesis. Here, we have demonstrated that the combined treatment with GLS inhibitor 968 and PD-L1 blockade enhances the immune response against ovarian cancer. Survival analysis using the Kaplan-Meier plotter dataset from ovarian cancer patients revealed that the expression level of GLS predicts poor survival and correlates with the immunosuppressive microenvironment of ovarian cancer. 968 inhibits the proliferation of ovarian cancer cells and enhances granzyme B secretion by CD8

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Benzophenanthridines; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Synergism; Female; Gene Expression Regulation, Neoplastic; Glutaminase; Humans; Immune Checkpoint Inhibitors; Mice; Ovarian Neoplasms; Treatment Outcome; Tumor Microenvironment; Xenograft Model Antitumor Assays

Cytoreduction has an important role in improving the survival rate of epithelial ovarian cancer (EOC) patients. This study aimed to assess the ability of preoperative serum CA125, FASN and GLS as predictors of cytoreductive surgery for epithelial ovarian cancer (EOC).. The average values of serum CA-125, FASN, and GLS in the suboptimal cytoreduction group were higher than those in optimal cytoreduction group. The cut off point (COP) was 248.55 (p = 0.0001) with 73.2% sensitivity and 73.6% specificity for CA-125, 0.445 (p = 0.017) with 62.5% sensitivity and 60.4% specificity for FASN, and 22.895 (p = 0.0001) with 73.2% sensitivity and 75.5% specificity for GLS. The COP of CA-125 and GLS combined was 29.16 (p = 0.0001) with sensitivity 82.1% and specificity 73.6%, while the COP of CA-125, GLS, and FASN combined was 0.83 (p = 0.0001) with 87.5% sensitivity and 73.6% specificity.

Topics: CA-125 Antigen; Carcinoma, Ovarian Epithelial; Cytoreduction Surgical Procedures; Fatty Acid Synthase, Type I; Female; Glutaminase; Humans; Membrane Proteins; Neoplasm Staging; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Benzeneacetamides; Cell Line, Tumor; Cell Survival; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Glutaminase; Glutamine; Glutathione; Humans; Mice, Nude; Ovarian Neoplasms; Phthalazines; Piperazines; Poly(ADP-ribose) Polymerase Inhibitors; Proto-Oncogene Proteins c-myc; Thiadiazoles; Xenograft Model Antitumor Assays

miR-145 has been found to be a participant in cancer metastasis and glucose metabolism in ovarian cancer. However, the role of glutamine metabolism in ovarian cancer remains unclear. In this study, we aim to elucidate the molecular mechanism underlying the regulation of glutamine metabolism by miR-145 in ovarian cancer cells. The messenger RNA (mRNA) levels of miR-145 and glutaminase 1 (GLS1) were examined by quantitative real-time polymerase chain reaction (qRT-PCR). The protein levels of c-myc and GLS1 were detected by western blot analysis. Luciferase reporter assays were used to validate c-myc was a target of miR-145. Glutamine metabolism was analyzed using assay kits. In addition, we performed luciferase reporter assays and chromatin immunoprecipitation assay to validate c-myc transcription activated GLS1 and promoted GLS1 expression. The qRT-PCR demonstrated that the mRNA level of miR-145 and GLS1 was negatively correlated in ovarian cancer tissues and cell lines. Kaplan-Meier survival analysis and the log-rank test showed that patients with high miR-145 expression had significantly increased the overall survival. The overexpression of miR-145 inhibited glutamine consumption, α-ketoglutarate production, and cellular ATP levels. Furthermore, we found miR-145 inhibited glutamine metabolism by targeting c-myc. Moreover, c-myc could promote GLS1 expression by transcription activated. Together, our results revealed that miR-145 inhibited glutamine metabolism through c-myc/GLS1 pathways in ovarian cancer cells, which may improve the current strategy of ovarian cancer diagnosis and therapy.

Topics: DNA-Binding Proteins; Female; Glutaminase; Glutamine; Humans; MicroRNAs; Ovarian Neoplasms; Transcription Factors; Tumor Cells, Cultured

Altered metabolism is a reemerging hallmark of tumorigenesis. Increased cell proliferation results in metabolic reprogramming to facilitate the needs of the rapidly dividing tumor cells. In addition to increased glucose uptake, tumors also take up increased levels of glutamine. Some cancers develop a reliance on glutamine, and are referred to as "glutamine addicted." These tumors over express the enzyme glutaminase which is involved in the first step of glutaminolysis. The goal of this study was to determine the effects of combined treatment of the glutaminase inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (BPTES) with chemotherapy on drug resistant ovarian cancer cells. We found that ovarian cancer cells show different dependencies on exogenous glutamine. However, regardless of glutamine dependence status, treatment with BPTES sensitized both paclitaxel, and cisplatin resistant cancer cell lines to chemotherapy by inhibiting cell proliferation. Monotherapy with BPTES alone resulted in a significant reduction in the ability of glutamine dependent cancer cells to form colonies in a clonogenic assay. In addition, glutamine dependent, metastatic cancer cells expressed higher levels of glutaminase 1 (GLS1) isoforms, KGA and GAC, than untransformed cells. Moreover, dual targeting of both isoforms using siRNA was more effective at sensitizing the cancer cells to cisplatin than targeting either GAC or KGA alone. Our results suggest that both GLS1 isoforms are important for glutamine dependent ovarian cancer survival, hence, both GLS1 isoforms should be targeted for therapy in metastatic ovarian cancer therapy.

Topics: Antineoplastic Agents; Cell Proliferation; Drug Resistance, Neoplasm; Female; Glutaminase; Glutamine; Humans; Molecular Targeted Therapy; Ovarian Neoplasms; Sulfides; Thiadiazoles; Tumor Cells, Cultured

The PI3K/Akt/mTOR axis in ovarian cancer is frequently activated and implicated in tumorigenesis. Specific targeting of this pathway is therefore an attractive therapeutic approach for ovarian cancer. However, ovarian cancer cells are resistant to PP242, a dual inhibitor of mTORC1 and mTORC2. Interestingly, blockage of GLS1 with a selective inhibitor, CB839, or siRNA dramatically sensitized the PP242-induced cell death, as evident from increased PARP cleavage. The anti-cancer activity of CB-839 and PP242 was abrogated by the addition of the TCA cycle product α-ketoglutarate, indicating the critical function of GLS1 in ovarian cancer cell survival. Finally, glutaminolysis inhibition activated apoptosis and synergistically sensitized ovarian cancer cells to priming with the mTOR inhibitor PP242. GLS1 inhibition significantly reduced phosphorylated STAT3 expression in ovarian cancer cells. These findings show that targeting glutamine addiction via GLS1 inhibition offers a potential novel therapeutic strategy to overcome resistance to PI3K/Akt/mTOR inhibition.

Topics: Antineoplastic Agents; Apoptosis; Benzeneacetamides; Blotting, Western; Cell Line, Tumor; Drug Resistance, Neoplasm; Female; Flow Cytometry; Gene Knockdown Techniques; Glutaminase; Glutamine; Humans; Indoles; Ovarian Neoplasms; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Purines; Real-Time Polymerase Chain Reaction; Signal Transduction; STAT3 Transcription Factor; Thiadiazoles; TOR Serine-Threonine Kinases

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

Ovarian cancer is characterized by an increase in cellular energy metabolism, which is predominantly satisfied by glucose and glutamine. Targeting metabolic pathways is an attractive approach to enhance the therapeutic effectiveness and to potentially overcome drug resistance in ovarian cancer. In platinum-sensitive ovarian cancer cell lines the metabolism of both, glucose and glutamine was initially up-regulated in response to platinum treatment. In contrast, platinum-resistant cells revealed a significant dependency on the presence of glutamine, with an upregulated expression of glutamine transporter ASCT2 and glutaminase. This resulted in a higher oxygen consumption rate compared to platinum-sensitive cell lines reflecting the increased dependency of glutamine utilization through the tricarboxylic acid cycle. The important role of glutamine metabolism was confirmed by stable overexpression of glutaminase, which conferred platinum resistance. Conversely, shRNA knockdown of glutaminase in platinum resistant cells resulted in re-sensitization to platinum treatment. Importantly, combining the glutaminase inhibitor BPTES with platinum synergistically inhibited platinum sensitive and resistant ovarian cancers in vitro. Apoptotic induction was significantly increased using platinum together with BPTES compared to either treatment alone. Our findings suggest that targeting glutamine metabolism together with platinum based chemotherapy offers a potential treatment strategy particularly in drug resistant ovarian cancer.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cisplatin; Drug Resistance, Neoplasm; Female; Glutaminase; Glutamine; Humans; Metabolic Networks and Pathways; Ovarian Neoplasms; Proteome

Tumor cells intensely utilize glutamine as the major source of respiratory fuel. Glutamine-analogue acivicin inhibits tumor growth and tumor-induced angiogenesis in Ehrlich ascites carcinoma. In the present study, antitumor properties of acivicin in combination with glutaminase enzyme is reported. Acivicin along with E. coli glutaminase synergistically reduced in vitro proliferation and matrigel invasion of human MCF-7 and OAW-42 cells. Effects of single and combined treatments with acivicin and glutaminase on angiogenic factors were also analyzed in these cell lines. Co-administration of the treatment agents inhibits the release of VEGF and MMP-9 by cells in culture supernatant significantly than single agent treatments. The result suggests that combination of acivicin with glutaminase may provide a better therapeutic option than either of them given separately for treating human breast and ovarian cancer. However, further studies are required to be conducted in vivo for its confirmation.

Topics: Antimetabolites, Antineoplastic; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Collagen; Drug Combinations; Female; Glutaminase; Glutamine; Humans; In Vitro Techniques; Isoxazoles; Laminin; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Ovarian Neoplasms; Proteoglycans; Tetrazolium Salts; Thiazoles; Vascular Endothelial Growth Factor A

In this study the enzyme glutaminase, purified from the ascites fluid of ovarian cancer patients, was analysed for its antiangiogenic activity. Intraperitoneal administration of this enzyme reduces the number of tumor directed capillaries in solid and ascites tumor bearing Swiss mice induced by transplantation of Ehrlich ascites cells. The enzyme has a critical role in regulating the secretion of vascular endothelial growth factor (VEGF) from tumor cell and in turn tumor growth. Glutamine analogue like 6-diazo, 5- oxo L-norleucine (DON) is also found to be effective in regulating vascular endothelial growth factor (VEGF) secretion from tumor cells in vitro. Treatment with enzyme reduced serum VEGF levels of the tumor induced animals. In vitro VEGF production by EAC cells was reduced in a concentration dependent manner in presence of glutamine analogue.

Topics: Animals; Antineoplastic Agents; Body Weight; Carcinoma, Ehrlich Tumor; Cell Line, Tumor; Cell Proliferation; Diazooxonorleucine; Dose-Response Relationship, Drug; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Glutaminase; Glutamine; Mice; Neoplasm Transplantation; Neoplasms; Neovascularization, Pathologic; Ovarian Neoplasms; Time Factors; Vascular Endothelial Growth Factor A

Glutamine is the major respiratory fuel and energy source of the rapidly proliferating tumor cells and that is why glutamine clearance by glutaminase therapy provides an opportunity to fight against the neoplasm. Glutaminase from bacterial source was tried on experimental models but had to be excluded because of its limited efficacy. Search for a better glutaminase continued exploiting the mammalian sources. In the present study, glutaminase purified from human ovarian cancer ascites fluid was used in experimental solid and ascites mice model alone and in combination with Cu-Sulphate and heparin. Cumulative findings indicate that the enzyme alone is quite effective in lowering tumor burden and reducing not only the tumor induced angiogenesis, but also an angiogenic inducer, heparin mediated angiogenesis. However, the presence of Cu with the enzyme, amplified the antineoplastic response by improving anti-angiogenic potential and hematological status of the tumor bearing host. Therefore, Cu-glutaminase combination strengthened the hypothesis that together they may provide a better therapeutic regimen in experimental mice tumor model.

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Agents; Ascitic Fluid; Blood Cell Count; Body Weight; Copper; Drug Therapy, Combination; Female; Glutaminase; Heparin; Humans; Male; Mice; Neoplasms, Experimental; Neovascularization, Pathologic; Ovarian Neoplasms; Spleen

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

Specific L-asparaginase activity and non-specific cytotoxicity of asparaginase-glutaminase preparation from Pseudomonas fluorescens were studied. Two cell lines, i.e. the asparaginase-dependent (Berkitt lymphoma cells) and the asparaginase-independent (the ovary cancer cells) were used as the test-system. Incorporation of 3H-timidine into DNA was used as the criterion of the drug effect on the cells. Krasnitin was used as the reference preparation. The preparation of asparaginase-glutaminase was inferior to krasnitine by its specific antitumour asparaginase activity and superior to it by the general cytotoxicity in the cells of CaOv. With the help of the above test-system it is possible to study the specific asparaginase activity of the drugs containing L-asparaginase. For studying the specific glutaminase properties it is necessary to develop another cell test-system.

Topics: Antibiotics, Antineoplastic; Asparaginase; Burkitt Lymphoma; Cell Line; Cells, Cultured; Drug Combinations; Drug Evaluation, Preclinical; Escherichia coli; Female; Glutaminase; Humans; Ovarian Neoplasms; Pseudomonas fluorescens