scholarly journals Impaired anaplerosis is a major contributor to glycolysis inhibitor toxicity in glioma

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Sunada Khadka ◽  
Kenisha Arthur ◽  
Yasaman Barekatain ◽  
Eliot Behr ◽  
Mykia Washington ◽  
...  
Keyword(s):  
Culture Media ◽  
Single Agent ◽  
Glioma Cells ◽  
Tca Cycle ◽  
Redox Status ◽  
Metabolomic Profiling ◽  
The Tca Cycle ◽  
Selective Sensitivity

Abstract Background Reprogramming of metabolic pathways is crucial to satisfy the bioenergetic and biosynthetic demands and maintain the redox status of rapidly proliferating cancer cells. In tumors, the tricarboxylic acid (TCA) cycle generates biosynthetic intermediates and must be replenished (anaplerosis), mainly from pyruvate and glutamine. We recently described a novel enolase inhibitor, HEX, and its pro-drug POMHEX. Since glycolysis inhibition would deprive the cell of a key source of pyruvate, we hypothesized that enolase inhibitors might inhibit anaplerosis and synergize with other inhibitors of anaplerosis, such as the glutaminase inhibitor, CB-839. Methods We analyzed polar metabolites in sensitive (ENO1-deleted) and resistant (ENO1-WT) glioma cells treated with enolase and glutaminase inhibitors. We investigated whether sensitivity to enolase inhibitors could be attenuated by exogenous anaplerotic metabolites. We also determined the synergy between enolase inhibitors and the glutaminase inhibitor CB-839 in glioma cells in vitro and in vivo in both intracranial and subcutaneous tumor models. Results Metabolomic profiling of ENO1-deleted glioma cells treated with the enolase inhibitor revealed a profound decrease in the TCA cycle metabolites with the toxicity reversible upon exogenous supplementation of supraphysiological levels of anaplerotic substrates, including pyruvate. ENO1-deleted cells also exhibited selective sensitivity to the glutaminase inhibitor CB-839, in a manner rescuable by supplementation of anaplerotic substrates or plasma-like media PlasmaxTM. In vitro, the interaction of these two drugs yielded a strong synergistic interaction but the antineoplastic effects of CB-839 as a single agent in ENO1-deleted xenograft tumors in vivo were modest in both intracranial orthotopic tumors, where the limited efficacy could be attributed to the blood-brain barrier (BBB), and subcutaneous xenografts, where BBB penetration is not an issue. This contrasts with the enolase inhibitor HEX, which, despite its negative charge, achieved antineoplastic effects in both intracranial and subcutaneous tumors. Conclusion Together, these data suggest that at least for ENO1-deleted gliomas, tumors in vivo—unlike cells in culture—show limited dependence on glutaminolysis and instead primarily depend on glycolysis for anaplerosis. Our findings reinforce the previously reported metabolic idiosyncrasies of in vitro culture and suggest that cell culture media nutrient composition more faithful to the in vivo environment will more accurately predict in vivo efficacy of metabolism targeting drugs.

Impaired Anaplerosis Is a Major Contributor to Glycolysis Inhibitor Toxicity in Glioma

2020 ◽  
Author(s):  
Sunada Khadka ◽  
Kenisha Arthur ◽  
Mykia Washington ◽  
Yasaman Barekatain ◽  
Jeff Ackroyd ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Single Agent ◽  
Tca Cycle ◽  
Redox Status ◽  
Homozygous Deletion ◽  
Metabolomic Profiling ◽  
Selective Sensitivity ◽  
Homozygous Deletions

Abstract Reprogramming of metabolic pathways is crucial to satisfy the bioenergetic and biosynthetic demands and maintain the redox status of rapidly proliferating cancer cells. In tumors, the tricarboxylic acid (TCA) cycle generates biosynthetic intermediates by oxidation of anaplerotic substrates, such as glucose-derived pyruvate and glutamine20 derived glutamate. We have previously documented that a subset of tumors with 1p36 homozygous deletion exhibit co-deletion of ENO1, in turn becoming extremely dependent on its redundant isoform ENO2 and sensitive to an overall enzymatic deficiency of enolase. Metabolomic profiling of ENO1-deleted glioma cells treated with an enolase inhibitor revealed a profound decrease in TCA cycle metabolites, which correlated with cell-line specific sensitivity to enolase inhibition, highlighting the importance of glycolysis derived pyruvate for anaplerosis. Correspondingly, the toxicity of the enolase inhibitor was significantly attenuated by exogenous supplementation of supraphysiological levels of anaplerotic substrates including pyruvate. These findings led us to hypothesize that cancer cells with ENO1 homozygous deletions treated with an enolase inhibitor might show exceptional sensitivity to inhibition of glutaminolysis because of reduced anaplerotic flow from glycolysis. We found that ENO1-deleted cells indeed exhibited selective sensitivity to the glutaminase inhibitor CB-839, and this sensitivity was also attenuated by exogenous supplementation of anaplerotic substrates including pyruvate. Despite these promising in vitro results, the antineoplastic effects of CB-839 as a single agent in ENO1-deleted xenograft tumors in vivo were modest in both intracranial orthotopic tumors, where the limited efficacy could be attributed to the blood brain barrier (BBB), and subcutaneous xenografts, where BBB penetration is not an issue. This contrasts with the enolase inhibitor HEX, which, despite its negative charge, achieved antineoplastic effects in both intracranial and subcutaneous tumors. Together, these data suggest that at least for 1p36-deleted gliomas, tumors in vivo—unlike cells in culture—show limited dependence on glutaminolysis and instead primarily depend on glycolysis for anaplerosis. Our findings reinforce the previously reported metabolic idiosyncrasies of the in vitro and in vivo environments as the potential reasons for the differential efficacy of metabolism targeted therapies in in vitro and in vivo systems.

scholarly journals Bovine Oviduct Epithelial Cell-Derived Culture Media and Exosomes Improve Mitochondrial Health by Restoring Metabolic Flux during Pre-Implantation Development

2020 ◽  
Vol 21 (20) ◽  
pp. 7589
Author(s):  
Tabinda Sidrat ◽  
Abdul Aziz Khan ◽  
Myeon-Don Joo ◽  
Yiran Wei ◽  
Kyeong-Lim Lee ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Embryonic Development ◽  
Metabolic Flux ◽  
Culture Media ◽  
Tca Cycle ◽  
Fine Tuning ◽  
The Tca Cycle ◽  
Oviduct Epithelial Cell

Oviduct flushing is enriched by a wide variety of nutrients that guide the 3–4 days journey of pre-implantation embryo through the oviduct as it develops into a competent blastocyst (BL). However, little is known about the specific requirement and role of these nutrients that orchestrate the early stages of embryonic development. In this study, we aimed to characterize the effect of in vitro-derived bovine oviduct epithelial cell (BOECs) secretion that mimics the in vivo oviduct micro-fluid like environment, which allows successful embryonic development. In this study, the addition of an in vitro derived BOECs-condition media (CM) and its isolated exosomes (Exo) significantly enhances the quality and development of BL, while the hatching ability of BLs was found to be high (48.8%) in the BOECs-Exo supplemented group. Surprisingly, BOECs-Exo have a dynamic effect on modulating the embryonic metabolism by restoring the pyruvate flux into TCA-cycle. Our analysis reveals that Exo treatment significantly upregulates the pyruvate dehydrogenase (PDH) and glutamate dehydrogenase (GLUD1) expression, required for metabolic fine-tuning of the TCA-cycle in the developing embryos. Exo treatment increases the influx into TCA-cycle by strongly suppressing the PDH and GLUD1 upstream inhibitors, i.e., PDK4 and SIRT4. Improvement of TCA-cycle function was further accompanied by higher metabolic activity of mitochondria in BOECs-CM and Exo in vitro embryos. Our study uncovered, for the first time, the possible mechanism of BOECs-derived secretion in re-establishing the TCA-cycle flux by the utilization of available nutrients and highlighted the importance of pyruvate in supporting bovine in vitro embryonic development.

scholarly journals Control of topoisomerase II activity and chemotherapeutic inhibition by TCA cycle metabolites

2021 ◽  
Author(s):  
Joyce H. Lee ◽  
Eric P. Mosher ◽  
Young-Sam Lee ◽  
Namandjé N. Bumpus ◽  
James M. Berger
Keyword(s):  
Topoisomerase Ii ◽  
Tca Cycle ◽  
Chemical Fractionation ◽  
Dna Breaks ◽  
Double Strand Dna Breaks ◽  
The Tca Cycle ◽  
Cellular Sensitivity ◽  
Topo Ii

SUMMARYTopoisomerase II (topo II) is essential for disentangling newly replicated chromosomes. DNA unlinking involves the physical passage of one DNA duplex through another and depends on the transient formation of double-strand DNA breaks, a step exploited by frontline chemotherapeutics to kill cancer cells. Although anti-topo II drugs are efficacious, they also elicit cytotoxic side effects in normal cells; insights into how topo II is regulated in different cellular contexts is essential to improve their targeted use. Using chemical fractionation and mass spectrometry, we have discovered that topo II is subject to metabolic control through the TCA cycle. We show that TCA metabolites stimulate topo II activity in vitro and that levels of TCA flux modulate cellular sensitivity to anti-topo II drugs in vivo. Our works reveals an unanticipated connection between the control of DNA topology and cellular metabolism, a finding with important ramifications for the clinical use of anti-topo II therapies.

scholarly journals Impact of Global Transcriptional Regulation by ArcA, ArcB, Cra, Crp, Cya, Fnr, and Mlc on Glucose Catabolism in Escherichia coli

2005 ◽  
Vol 187 (9) ◽  
pp. 3171-3179 ◽  
Author(s):  
Annik Perrenoud ◽  
Uwe Sauer
Keyword(s):  
Escherichia Coli ◽  
Transcriptional Regulation ◽  
Redox Regulation ◽  
Tca Cycle ◽  
Batch Cultures ◽  
E Coli ◽  
Glucose Catabolism ◽  
The Tca Cycle

ABSTRACT Even though transcriptional regulation plays a key role in establishing the metabolic network, the extent to which it actually controls the in vivo distribution of metabolic fluxes through different pathways is essentially unknown. Based on metabolism-wide quantification of intracellular fluxes, we systematically elucidated the relevance of global transcriptional regulation by ArcA, ArcB, Cra, Crp, Cya, Fnr, and Mlc for aerobic glucose catabolism in batch cultures of Escherichia coli. Knockouts of ArcB, Cra, Fnr, and Mlc were phenotypically silent, while deletion of the catabolite repression regulators Crp and Cya resulted in a pronounced slow-growth phenotype but had only a nonspecific effect on the actual flux distribution. Knockout of ArcA-dependent redox regulation, however, increased the aerobic tricarboxylic acid (TCA) cycle activity by over 60%. Like aerobic conditions, anaerobic derepression of TCA cycle enzymes in an ArcA mutant significantly increased the in vivo TCA flux when nitrate was present as an electron acceptor. The in vivo and in vitro data demonstrate that ArcA-dependent transcriptional regulation directly or indirectly controls TCA cycle flux in both aerobic and anaerobic glucose batch cultures of E. coli. This control goes well beyond the previously known ArcA-dependent regulation of the TCA cycle during microaerobiosis.

Distinct Metabolic Dependency of Normal and Leukemic Cells in a Mouse Model

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 759-759
Author(s):  
Rushdia Z. Yusuf ◽  
Sanket S. Acharya ◽  
Vionnie Yu ◽  
Borja Saez ◽  
Mildred Duvet ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Tca Cycle ◽  
Leukemic Cells ◽  
Mouse Bone Marrow ◽  
Metabolomic Profiling ◽  
Glycolysis Pathway ◽  
Rate Limiting

Abstract Abstract 759 We hypothesized that metabolic differences between leukemia initiating cells and their normal counterparts represent a vulnerability in the leukemia initiating cell, which can be therapeutically exploited. To test this hypothesis, we used the MLL-AF9 acute myeloid leukemia (AML) model in mice. Actin-DsRed mouse bone marrow transduced with MLL-AF9 expressing retrovirus was used to produce serially transplantable leukemia. Leukemic granulocyte-monocyte precursors (L-GMPs), defined by others to be the leukemia initiating cells were flow sorted from secondary recipient mice and compared with normal GMPs (N-GMPs) from actin Ds-Red mice. Gene expression profiling, metabolomic profiling via liquid chromatography- mass spectrometry and an in vitro shRNA screen were used to identify metabolic pathways preferentially activated in leukemia initiating cells. Of 1574 defined metabolic enzymes, 44 were found to be differentially expressed between L-GMPs and their normal counterparts (N-GMPs). These together with 117 classic rate limiting enzymes were subjected to shRNA knockdown in vitro. Metabolomic profiling of both cell populations was used to corroborate findings from shRNA knockdowns. L-GMPs and N-GMPs were transduced with lentivirus expressing shRNAs of interest (5 shRNAs per gene) in a 384 well format, selected with puromycin and cultured for 72–96 hours in the presence of GFP-positive primary bone marrow stroma. The number of cells in each well at the end of this experiment was quantitated using an Image Xpress microscope. Genes, the knockdown of which by at least two independent shRNAs produced a two fold or more decrease in L-GMPs as compared to control wells and did not similarly decrease N-GMPs, were chosen for in vivo validation. Ten genes in the glycolysis pathway and TCA cycle, fatty acid metabolism and detoxification, and ketohexokinase were identified. Ketohexokinase, a rate-limiting enzyme in fructose metabolism was particularly potent and of interest given its potential to be exploited therapeutically. In vivo assessment of its relative ability to inhibit malignant versus normal hematopoietic cells is ongoing. These studies provide preliminary support for the hypothesis that specific metabolic circuits are differentially active in leukemia initiating cells in MLL-AF9 AML and may represent unique points of vulnerability that can be targeted therapeutically. Authors 1 and 2 contributed equally. Authors 3 and 4 contributed equally. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The hexokinase ″HKDC1″ interaction with the mitochondria is essential for hepatocellular carcinoma progression

2021 ◽  
Author(s):  
Md. Wasim Khan ◽  
Alexander Terry ◽  
Medha Priyadarshini ◽  
Grace Guzman ◽  
Jose Cordoba-Chacon ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Mitochondrial Dysfunction ◽  
Tca Cycle ◽  
Metabolic Reprogramming ◽  
Selective Approach ◽  
Glucose Flux ◽  
The Tca Cycle

Hepatocellular carcinoma (HCC) is a leading cause of death from cancer malignancies. Recently, hexokinase domain containing 1 (HKDC1), was shown to have significant overexpression in HCC compared to healthy tissue. Using in vitro and in vivo tools, we examined the role of HKDC1 in HCC progression. Importantly, HKDC1 ablation stops HCC progression by promoting metabolic reprogramming by shifting glucose flux away from the TCA cycle. Next, HKDC1 ablation leads to mitochondrial dysfunction resulting in less cellular energy which cannot be compensated by enhanced glucose uptake. And finally, we show that the interaction of HKDC1 with the mitochondria is essential for its role in HCC progression, and without this mitochondrial interaction mitochondrial dysfunction occurs. In sum, HKDC1 is highly expressed in HCC cells compared to normal hepatocytes, therefore targeting HKDC1, specifically its interaction with the mitochondria, reveals a highly selective approach to target cancer cells in HCC.

scholarly journals Thioredoxin, a master regulator of the tricarboxylic acid cycle in plant mitochondria

2015 ◽  
Vol 112 (11) ◽  
pp. E1392-E1400 ◽  
Author(s):  
Danilo M. Daloso ◽  
Karolin Müller ◽  
Toshihiro Obata ◽  
Alexandra Florian ◽  
Takayuki Tohge ◽  
...  
Keyword(s):  
Tricarboxylic Acid Cycle ◽  
Plant Mitochondria ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Mitochondrial Enzymes ◽  
Atp Citrate Lyase ◽  
Cycle Enzyme ◽  
The Tca Cycle

Plant mitochondria have a fully operational tricarboxylic acid (TCA) cycle that plays a central role in generating ATP and providing carbon skeletons for a range of biosynthetic processes in both heterotrophic and photosynthetic tissues. The cycle enzyme-encoding genes have been well characterized in terms of transcriptional and effector-mediated regulation and have also been subjected to reverse genetic analysis. However, despite this wealth of attention, a central question remains unanswered: “What regulates flux through this pathway in vivo?” Previous proteomic experiments withArabidopsisdiscussed below have revealed that a number of mitochondrial enzymes, including members of the TCA cycle and affiliated pathways, harbor thioredoxin (TRX)-binding sites and are potentially redox-regulated. We have followed up on this possibility and found TRX to be a redox-sensitive mediator of TCA cycle flux. In this investigation, we first characterized, at the enzyme and metabolite levels, mutants of the mitochondrial TRX pathway inArabidopsis: theNADP-TRX reductasea and b double mutant (ntra ntrb) and the mitochondrially locatedthioredoxin o1(trxo1) mutant. These studies were followed by a comparative evaluation of the redistribution of isotopes when13C-glucose,13C-malate, or13C-pyruvate was provided as a substrate to leaves of mutant or WT plants. In a complementary approach, we evaluated the in vitro activities of a range of TCA cycle and associated enzymes under varying redox states. The combined dataset suggests that TRX may deactivate both mitochondrial succinate dehydrogenase and fumarase and activate the cytosolic ATP-citrate lyase in vivo, acting as a direct regulator of carbon flow through the TCA cycle and providing a mechanism for the coordination of cellular function.

scholarly journals Tracing Nutrient Flux Following Monocarboxylate Transporter-1 Inhibition with AZD3965

Cancers ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 1703
Author(s):  
Marta Braga ◽  
Maciej Kaliszczak ◽  
Laurence Carroll ◽  
Zachary T. Schug ◽  
Kathrin Heinzmann ◽  
...  
Keyword(s):  
Cell Metabolism ◽  
B Cell Lymphoma ◽  
Tca Cycle ◽  
Monocarboxylate Transporter ◽  
Monocarboxylate Transporter 1 ◽  
Large B Cell Lymphoma ◽  
The Tca Cycle ◽  
18F Fdg

The monocarboxylate transporter 1 (MCT1) is a key element in tumor cell metabolism and inhibition of MCT1 with AZD3965 is undergoing clinical trials. We aimed to investigate nutrient fluxes associated with MCT1 inhibition by AZD3965 to identify possible biomarkers of drug action. We synthesized an 18F-labeled lactate analogue, [18F]-S-fluorolactate ([18F]-S-FL), that was used alongside [18F]fluorodeoxyglucose ([18F]FDG), and 13C-labeled glucose and lactate, to investigate the modulation of metabolism with AZD3965 in diffuse large B-cell lymphoma models in NOD/SCID mice. Comparative analysis of glucose and lactate-based probes showed a preference for glycolytic metabolism in vitro, whereas in vivo, both glucose and lactate were used as metabolic fuel. While intratumoral L-[1-13C]lactate and [18F]-S-FL were unchanged or lower at early (5 or 30 min) timepoints, these variables were higher compared to vehicle controls at 4 h following treatment with AZD3965, which indicates that inhibition of MCT1-mediated lactate import is reversed over time. Nonetheless, AZD3965 treatment impaired DLBCL tumor growth in mice. This was hypothesized to be a consequence of metabolic strain, as AZD3965 treatment showed a reduction in glycolytic intermediates and inhibition of the TCA cycle likely due to downregulated PDH activity. Glucose ([18F]FDG and D-[13C6]glucose) and lactate-based probes ([18F]-S-FL and L-[1-13C]lactate) can be successfully used as biomarkers for AZD3965 treatment.

Radiosensitivity of glioma to Gamma Knife treatment enhanced in vitro and in vivo by RNA interfering Ku70 that is mediated by a recombinant adenovirus

2010 ◽  
Vol 113 (Special_Supplement) ◽  
pp. 228-235 ◽  
Author(s):  
Qiang Jia ◽  
Yanhe Li ◽  
Desheng Xu ◽  
Zhenjiang Li ◽  
Zhiyuan Zhang ◽  
...  
Keyword(s):  
Western Blot ◽  
Gamma Knife ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Glioma Cells ◽  
C6 Glioma ◽  
C6 Glioma Cells ◽  
C6 Cells

Object The authors sought to evaluate modification of the radiation response of C6 glioma cells in vitro and in vivo by inhibiting the expression of Ku70. To do so they investigated the effect of gene transfer involving a recombinant replication-defective adenovirus containing Ku70 short hairpin RNA (Ad-Ku70shRNA) combined with Gamma Knife treatment (GKT). Methods First, Ad-Ku70shRNA was transfected into C6 glioma cells and the expression of Ku70 was measured using Western blot analysis. In vitro, phenotypical changes in C6 cells, including proliferation, cell cycle modification, invasion ability, and apoptosis were evaluated using the MTT (3′(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay, Western blot analysis, and cell flow cytometry. In vivo, parental C6 cells transfected with Ad-Ku70shRNA were implanted stereotactically into the right caudate nucleus in Sprague-Dawley rats. After GKS, apoptosis was analyzed using the TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling) method. The inhibitory effects on growth and invasion that were induced by expression of proliferating cell nuclear antigen and matrix metalloproteinase–9 were determined using immunohistochemical analyses. Results The expression of Ku70 was clearly inhibited in C6 cells after transfection with Ad-Ku70shRNA. In vitro following transfection, the C6 cells showed improved responses to GKT, including suppression of proliferation and invasion as well as an increased apoptosis index. In vivo following transfection of Ad-Ku70shRNA, the therapeutic efficacy of GKT in rats with C6 gliomas was greatly enhanced and survival times in these animals were prolonged. Conclusions Our data support the potential for downregulation of Ku70 expression in enhancing the radiosensitivity of gliomas. The findings of our study indicate that targeted gene therapy–mediated inactivation of Ku70 may represent a promising strategy in improving the radioresponsiveness of gliomas to GKT.

Sign in / Sign up

Export Citation Format

Share Document