glutaminase and Liver-Neoplasms

Topics: Acid Phosphatase; Alcohol Oxidoreductases; Aldehyde Oxidoreductases; Alkaline Phosphatase; Animals; Carcinoma, Hepatocellular; DNA Nucleotidyltransferases; Esterases; Fructose-Bisphosphatase; Fructose-Bisphosphate Aldolase; Glucosyltransferases; Glutaminase; Glycogen Synthase; Hexokinase; Humans; Isocitrate Dehydrogenase; Isoenzymes; L-Lactate Dehydrogenase; Liver Neoplasms; Malate Dehydrogenase; Mice; Neoplasms; Phosphotransferases; Pyruvate Kinase; Rats; Terminology as Topic; Thymidine Kinase; Transaminases; tRNA Methyltransferases; Uridine

Topics: Animals; Biological Evolution; Carcinoma, Hepatocellular; DNA, Neoplasm; Glutaminase; Isoenzymes; Kidney; Liver; Liver Neoplasms; Liver Regeneration; Nitrogen; Rats; RNA, Neoplasm; Urea

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Ferroptosis; Glutaminase; Humans; Liver Neoplasms; Tumor Suppressor Protein p53

The pathological diagnosis and prognosis prediction of hepatocellular carcinoma (HCC) is challenging due to the lack of specific biomarkers. This study aimed to validate the diagnostic and prognostic efficiency of Kidney-type glutaminase (GLS1) for HCC in prospective cohorts with a large sample size.. A total of 1140 HCC patients were enrolled in our prospective clinical trials. Control cases included 114 nontumour tissues. The registered clinical trial (ChiCTR-DDT-14,005,102, chictr.org.cn) was referred to for the exact protocol. GLS1 immunohistochemistry was performed on the whole tumour section. The diagnostic and prognostic performances of GLS1 was evaluated by the receiver operating characteristic curve and Cox regression model.. The sensitivity, specificity, positive predictive value, negative predictive value, Youden index, and area under the curve of GLS1 for the diagnosis of HCC were 0.746, 0.842, 0.979, 0.249, 0.588, and 0.814, respectively, which could be increased to 0.846, 0.886, 0.987,0.366, 0.732, and 0.921 when combined with glypican 3 (GPC3) and alpha-fetoprotein (AFP), indicating better diagnostic performance. Further, we developed a nomogram with GPC3 and GLS1 for identifying HCC which showed good discrimination and calibration. GLS1 expression was also related with age, T stage, TNM stage, Edmondson-Steiner grade, microvascular invasion, Ki67, VEGFR2, GPC3, and AFP expression in HCC. GLS1 expression was negatively correlated with disease-free survival (P < 0.001) probability of patients with HCC.. It was validated that GLS1 was a sensitive and specific biomarker for pathological diagnosis of HCC and had prognostic value, thus having practical value for clinical application.

Topics: alpha-Fetoproteins; Biomarkers, Tumor; Carcinoma, Hepatocellular; Glutaminase; Glypicans; Humans; Kidney; Liver Neoplasms; Prognosis; Prospective Studies

Glutaminolysis is a critical metabolic process that promotes cancer cell proliferation, including hepatocellular carcinoma (HCC). Delineating the molecular control of glutaminolysis could identify novel targets to ameliorate this oncogenic metabolic pathway. Here, we evaluated the role of general control of amino acid synthesis 5 like 1 (GCN5L1), a regulator of mitochondrial protein acetylation, in modulating the acetylation and activity of glutaminase to regulate HCC development.. Cell proliferation was determined by MTT, 2D and soft agar clone formation assays and orthotopic tumour assays in nude mice. GLS1/2 acetylation and activities were measured in cells and tumours to analyse the correlation with GCN5L1 expression and mTORC1 activation.. Hepatic GCN5L1 ablation in mice markedly increased diethylnitrosamine (DEN)-induced HCC, and conversely, the transduction of mitochondrial-restricted GCN5L1 protected wild-type mice against HCC progression in response to DEN and carbon tetrachloride (CCl. Our study identified that glutaminase activity, rather than GLS1 or GLS2 expression, is the key factor in HCC development that activates mTORC1 and promotes HCC. In the Kaplan-Meier analysis of liver cancer, we found that HCC patients with high GCN5L1 expression survived longer than those with low GCN5L1 expression. Collectively, GCN5L1 functions as a tumour regulator by modulating glutaminase acetylation and activity in the development of HCC.

Topics: Acetylation; Animals; Carcinoma, Hepatocellular; Glutaminase; Humans; Liver Neoplasms; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Nude; Mitochondria, Liver; Mitochondrial Proteins; Nerve Tissue Proteins

Hepatocellular carcinoma (HCC) is one of the primary liver malignancies with a poor prognosis. Glutamic-oxaloacetic transaminase 2 (GOT2) is a highly tissue-specific gene in the liver, but the roles GOT2 plays in the progression of HCC remain unclear. Here, we report that GOT2 is downregulated in HCC tumor tissues and that low expression of GOT2 is associated with advanced progression and poor prognosis. In HCC cells, knockdown of GOT2 promoted proliferation, migration, and invasion. In mouse models of HCC, loss of GOT2 promoted tumor growth as well as hematogenous and intrahepatic metastasis. Mechanistically, silencing of GOT2 enhanced glutaminolysis, nucleotide synthesis, and glutathione synthesis by reprogramming glutamine metabolism to support the cellular antioxidant system, which activated the PI3K/AKT/mTOR pathway to contribute to HCC progression. Furthermore, HCC with low expression of GOT2 was highly dependent on glutamine metabolism and sensitive to the glutaminase inhibitor CB-839 in vitro and in vivo. Overall, GOT2 is involved in glutamine metabolic reprogramming to promote HCC progression and may serve as a therapeutic and diagnostic target for HCC.. Altered glutamine metabolism induced by GOT2 loss supports HCC growth and metastasis but confers a targetable vulnerability to glutaminase inhibitors.

Topics: Animals; Antioxidants; Aspartate Aminotransferase, Mitochondrial; Aspartate Aminotransferases; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Glutaminase; Glutamine; Glutathione; Humans; Liver Neoplasms; Mice; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; TOR Serine-Threonine Kinases

Glutamine synthase 2 (GLS2) is a key regulator of glutaminolysis and has been previously implicated in activities consistent with tumor suppression. Here we generated Gls2 knockout (KO) mice that develop late-occurring B-cell lymphomas and hepatocellular carcinomas (HCC). Further, Gls2 KO mice subjected to the hepatocarcinogenic Stelic Animal Model (STAM) protocol produce larger HCC tumors than seen in wild-type (WT) mice. GLS2 has been shown to promote ferroptosis, a form of cell death characterized by iron-dependent accumulation of lipid peroxides. In line with this, GLS2 deficiency, either in cells derived from Gls2 KO mice or in human cancer cells depleted of GLS2, conferred significant resistance to ferroptosis. Mechanistically, GLS2, but not GLS1, increased lipid reactive oxygen species (ROS) production by facilitating the conversion of glutamate to α-ketoglutarate (αKG), thereby promoting ferroptosis. Ectopic expression of WT GLS2 in a human hepatic adenocarcinoma xenograft model significantly reduced tumor size; this effect was nullified by either expressing a catalytically inactive form of GLS2 or by blocking ferroptosis. Furthermore, analysis of cancer patient datasets supported a role for GLS2-mediated regulation of ferroptosis in human tumor suppression. These data suggest that GLS2 is a bona fide tumor suppressor and that its ability to favor ferroptosis by regulating glutaminolysis contributes to its tumor suppressive function.. This study demonstrates that the key regulator of glutaminolysis, GLS2, can limit HCC in vivo by promoting ferroptosis through αKG-dependent lipid ROS, which in turn might lay the foundation for a novel therapeutic approach.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Ferroptosis; Glutamates; Glutaminase; Glutamine; Humans; Iron; Ketoglutaric Acids; Lipid Peroxides; Liver Neoplasms; Mice; Reactive Oxygen Species

Metabolic activities of tumor cells lead to a depletion of nutrients within the tumor microenvironment, which results in the dysfunction of infiltrating T cells. Here, we explored how glutamine (gln) metabolism, which is essential for biosynthesis and cellular function, can affect the functions of cytotoxic T lymphocytes (CTLs).. Activated CTLs were co-cultured with hepatoma cells. Western blot was used to analyze changes of proteins and ELISA was used to analyze changes of effector. RNA-sequencing was used to detect differentially expressed genes in CTLs. The status of the endoplasmic reticulum (ER) was investigated using transmission electron microscopy experiments.. Co-culturing CTLs and hepatoma cells revealed that CTLL-2 cells in the co-culture group expressed high levels of PD-1 (Programmed cell death protein 1), TIM-3 (T cell immunoglobulin and mucin domain-containing protein-3), GRP78 (Glucose regulated protein 78), and P-PERK (phosphorylated protein kinase RNA-activated-like endoplasmic reticulum kinase) and secreted low levels of Granzyme B and perforin. Additionally, the substructure of the ER was severely damaged. When CTLs were treated with an inhibitor of ER stress, their functions were restored. Next, complete medium without Gln was used to culture cells, causing CTLs to display dysfunction and ER stress. WB results revealed decreased expression levels of GLS2 and SLC1A5 (Solute carrier family 1 member 5) in CTLs in the co-culture group. Subsequently, glutaminase (GLS) inhibitors were added to the cultures. As expected, CTLs treated with a GLS2 inhibitor had increased protein content of PD-1 and TIM-3, decreased secretion of Granzyme B and perforin, and an enhanced ER stress response.. In summary, CTLs are functionally downregulated induced by hepatoma cells through the Gln-GLS2-ERS pathway.

Topics: Amino Acid Transport System ASC; Carcinoma, Hepatocellular; Endoplasmic Reticulum Stress; Glutaminase; Granzymes; Hepatitis A Virus Cellular Receptor 2; Humans; Liver Neoplasms; Minor Histocompatibility Antigens; Perforin; Programmed Cell Death 1 Receptor; RNA; Signal Transduction; T-Lymphocytes, Cytotoxic; Tumor Microenvironment

Topics: Animals; Antineoplastic Agents; Camelus; Carcinoma, Hepatocellular; Glutaminase; HCT116 Cells; Hep G2 Cells; Humans; Liver; Liver Neoplasms; MCF-7 Cells; Mitochondria, Liver

Plasticity of cancer metabolism can be a major obstacle to efficient targeting of tumour-specific metabolic vulnerabilities. Here, we identify the compensatory mechanisms following the inhibition of major pathways of central carbon metabolism in c-MYC-induced liver tumours. We find that, while inhibition of both glutaminase isoforms (Gls1 and Gls2) in tumours considerably delays tumourigenesis, glutamine catabolism continues, owing to the action of amidotransferases. Synergistic inhibition of both glutaminases and compensatory amidotransferases is required to block glutamine catabolism and proliferation of mouse and human tumour cells in vitro and in vivo. Gls1 deletion is also compensated for by glycolysis. Thus, co-inhibition of Gls1 and hexokinase 2 significantly affects Krebs cycle activity and tumour formation. Finally, the inhibition of biosynthesis of either serine (Psat1-KO) or fatty acid (Fasn-KO) is compensated for by uptake of circulating nutrients, and dietary restriction of both serine and glycine or fatty acids synergistically suppresses tumourigenesis. These results highlight the high flexibility of tumour metabolism and demonstrate that either pharmacological or dietary targeting of metabolic compensatory mechanisms can improve therapeutic outcomes.

Topics: Animals; Cell Proliferation; Glucose; Glutaminase; Glutamine; Humans; Liver Neoplasms; Mice; Proto-Oncogene Proteins c-myc

The dependency of cancer cells on glutamine may be exploited therapeutically as a new strategy for treating cancers that lack druggable driver genes. Here we found that human liver cancer was dependent on extracellular glutamine. However, targeting glutamine addiction using the glutaminase inhibitor CB-839 as monotherapy had a very limited anticancer effect, even against the most glutamine addicted human liver cancer cells. Using a chemical library, we identified V-9302, a novel inhibitor of glutamine transporter ASCT2, as sensitizing glutamine dependent (GD) cells to CB-839 treatment. Mechanically, a combination of CB-839 and V-9302 depleted glutathione and induced reactive oxygen species (ROS), resulting in apoptosis of GD cells. Moreover, this combination also showed tumor inhibition in HCC xenograft mouse models in vivo. Our findings indicate that dual inhibition of glutamine metabolism by targeting both glutaminase and glutamine transporter ASCT2 represents a potential novel treatment strategy for glutamine addicted liver cancers.

Topics: Amino Acid Transport System ASC; Animals; Antineoplastic Agents; Apoptosis; Benzeneacetamides; Carrier Proteins; Cell Line, Tumor; Drug Synergism; Glutaminase; Glutamine; Humans; Liver Neoplasms; Mice; Minor Histocompatibility Antigens; Reactive Oxygen Species; Thiadiazoles; Xenograft Model Antitumor Assays

Kidney-type glutaminase [KGA/isoenzyme glutaminase C (GAC)] is becoming an important tumor metabolism target in cancer chemotherapy. Its allosteric inhibitor, CB839, showed early promise in cancer therapeutics but limited efficacy in in vivo cancer models. To improve the in vivo activity, we explored a bioisostere replacement of the sulfur atom in bis-2-(5-phenylacetamido-1,2,4-thiadiazol)ethyl sulfide and CB839 analogues with selenium using a novel synthesis of the selenadiazole moiety from carboxylic acids or nitriles. The resulting selenadiazole compounds showed enhanced KGA inhibition, more potent induction of reactive oxygen species, improved inhibition of cancer cells, and higher cellular and tumor accumulation than the corresponding sulfur-containing molecules. However, both CB839 and its selenium analogues show incomplete inhibition of the tested cancer cells, and a partial reduction in tumor size was observed in both the glutamine-dependent HCT116 and aggressive H22 liver cancer xenograft models. Despite this, tumor tissue damage and prolonged survival were observed in animals treated with the selenium analogue of CB839.

Topics: Allosteric Regulation; Animals; Antineoplastic Agents; Apoptosis; Azoles; Cell Line, Tumor; Cell Proliferation; Enzyme Inhibitors; Glutaminase; Humans; Kidney; Liver Neoplasms; Mice; Mice, Inbred ICR; Mice, Nude; Reactive Oxygen Species; Selenium; Structure-Activity Relationship; Thiadiazoles; Transplantation, Heterologous

Hepatocellular carcinoma (HCC) is an aggressive malignant disease with poor prognosis. Recent advances suggest the existence of cancer stem cells (CSCs) within liver cancer, which are considered to be responsible for tumor relapse, metastasis, and chemoresistance. However, novel therapeutic approaches for eradicating CSCs are yet to be established. Here, we aimed to identify the role of glutaminase 1 (GLS1) in stemness, and the feasibility that GLS1 serves as a therapeutic target for elimination CSCs as well as the possible mechanism.. Publicly-available data from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) was mined to unearth the association between GLS1 and stemness phenotype. Using big data, human tissues and multiple cell lines, we gained a general picture of GLS1 expression in HCC progression. We generated stable cell lines by lentiviral-mediated overexpression or CRISPR/Cas9-based knockout. Sphere formation assays and colony formation assays were employed to analyze the relationship between GLS1 and stemness. A series of bioinformatics analyses and molecular experiments including qRT-PCR, immunoblotting, flow cytometry, and immunofluorescence were employed to investigate the role of GLS1 in regulating stemness in vitro and in vivo.. We observed GLS1 (both KGA and GAC isoform) is highly expressed in HCC, and that high expression of GAC predicts a poor prognosis. GLS1 is exclusively expressed in the mitochondrial matrix. Upregulation of GLS1 is positively associated with advanced clinicopathological features and stemness phenotype. Targeting GLS1 reduced the expression of stemness-related genes and suppressed CSC properties in vitro. We further found GLS1 regulates stemness properties via ROS/Wnt/β-catenin signaling and that GLS1 knockout inhibits tumorigenicity in vivo.. Targeting GLS1 attenuates stemness properties in HCC by increasing ROS accumulation and suppressing Wnt/β-catenin pathway, which implied that GLS1 could serve as a therapeutic target for elimination of CSCs.

Topics: Adult; Aged; Animals; Big Data; Carcinoma, Hepatocellular; Cell Line, Tumor; CRISPR-Cas Systems; Female; Gene Expression Regulation, Neoplastic; Gene Knockout Techniques; Glutaminase; Hep G2 Cells; Humans; Liver Neoplasms; Male; Mice; Middle Aged; Neoplasm Transplantation; Neoplastic Stem Cells; Prognosis; Reactive Oxygen Species; Up-Regulation; Wnt Signaling Pathway

Topics: Biomarkers; Energy Metabolism; Glutamic Acid; Glutaminase; Humans; Liver Neoplasms; Neoplastic Stem Cells; Reactive Oxygen Species; Signal Transduction

Topics: Animals; Autophagy; Carcinoma, Hepatocellular; Cell Proliferation; Cell Survival; Drug Resistance, Neoplasm; Drug Therapy, Combination; Glutaminase; Hep G2 Cells; Hepatocyte Growth Factor; Humans; Liver Neoplasms; Male; Mice; Mice, Nude; Phosphorylation; Proto-Oncogene Proteins c-met; Pyruvate Dehydrogenase Complex; Signal Transduction; TOR Serine-Threonine Kinases; Transplantation, Heterologous

Glutamine metabolism is an important metabolic pathway for cancer cell survival, and there is a critical connection between tumor growth and glutamine metabolism. However, the role of GLS1 in hepatocellular carcinoma (HCC) progression remains to be elucidated. In this study, we reported that GLS1 expression was significantly increased in HCC tissues and correlated with serum AFP, tumor differentiation, lymphatic metastasis, TNM stage, and poorer patient outcome. We further showed that GLS1 promoted colony formation and cell proliferation of HCC cells. Furthermore, our data showed that GLS1 inhibitor compound 968 inhibited the proliferation of HCC cells in a dose-dependent manner. Importantly, we found that GLS1 overexpression increased p-AKT, p-GSK3β and cyclinD1 expression, and had no influence on total AKT and GSK3β protein level, indicating that GLS1 was involved in AKT/GSK3β/CyclinD1 pathway. It is suggested that GLS1 promotes proliferation in HCC cells probably via AKT/GSK3β/CyclinD1 pathway and may be a potential target for anti-hepatocellular carcinoma cancer.

Topics: Animals; Benzophenanthridines; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Disease Progression; Drug Delivery Systems; Female; Glutaminase; Glycogen Synthase Kinase 3 beta; Humans; Liver Neoplasms; Male; Mice; Mice, Nude; Middle Aged; Oncogenes; Proto-Oncogene Proteins c-akt; Retrospective Studies; Signal Transduction; Up-Regulation

Based on molecular profiling, several prognostic markers for HCC are also used in clinic, but only a few genes have been identified as useful. We collected 72 post-operative liver cancer tissue samples. Genes expression were tested by RT-PCR. Multilayer perceptron and discriminant analysis were built, and their ability to predict the prognosis of HCC patients were tested. Receiver operating characteristic (ROC) analysis was performed and multivariate analysis with Cox's Proportional Hazard Model was used for confirming the markers'predictive efficiency for HCC patients'survival. A simple risk scoring system devised for further predicting the prognosis of liver tumor patients. Multilayer perceptron and discriminant analysis showed a very strong predictive value in evaluating liver cancer patients'prognosis. Cox multivariate regression analysis demonstrated that DUOX1, GLS2, FBP1 and age were independent risk factors for the prognosis of HCC patients after surgery. Finally, the risk scoring system revealed that patients whose total score >1 and >3 are more likely to relapse and die than patients whose total score ≤1 and ≤3. The three genes model proposed proved to be highly predictive of the HCC patients' prognosis. Implementation of risk scoring system in clinical practice can help in evaluating survival of HCC patients after operation.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Hepatocellular; Dual Oxidases; Female; Fructose-Bisphosphatase; Glutaminase; Humans; Liver Neoplasms; Male; Middle Aged; Models, Theoretical; NADPH Oxidases; Prognosis; Young Adult

Various tumors develop addiction to glutamine to support uncontrolled cell proliferation. Here we identify the nuclear receptor liver receptor homolog 1 (LRH-1) as a key regulator in the process of hepatic tumorigenesis through the coordination of a noncanonical glutamine pathway that is reliant on the mitochondrial and cytosolic transaminases glutamate pyruvate transaminase 2 (GPT2) and glutamate oxaloacetate transaminase 1 (GOT1), which fuel anabolic metabolism. In particular, we show that gain and loss of function of hepatic LRH-1 modulate the expression and activity of mitochondrial glutaminase 2 (GLS2), the first and rate-limiting step of this pathway. Acute and chronic deletion of hepatic LRH-1 blunts the deamination of glutamine and reduces glutamine-dependent anaplerosis. The robust reduction in glutaminolysis and the limiting availability of α-ketoglutarate in turn inhibit mTORC1 signaling to eventually block cell growth and proliferation. Collectively, these studies highlight the importance of LRH-1 in coordinating glutamine-induced metabolism and signaling to promote hepatocellular carcinogenesis.

Topics: Animals; Carcinogenesis; Diethylnitrosamine; Gene Expression Regulation, Neoplastic; Glutaminase; Glutamine; Liver; Liver Neoplasms; Male; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Knockout; Mitochondria; Multiprotein Complexes; Receptors, Cytoplasmic and Nuclear; Signal Transduction; TOR Serine-Threonine Kinases

Glutaminolysis that catabolizes glutamine to glutamate plays a critical role in cancer progression. Glutaminase 2 (GLS2) has been reported as a tumor suppressor. Recent studies implied that GLS2 may display its multifunction besides classical metabolic feature by different localizations and potential protein binding domains. Here, we showed that GLS2 expression correlates inversely with stage, vascular invasion, tumor size and poor prognosis in human hepatocellular carcinoma (HCC) tissues. We found that GLS2 significantly represses cell migration, invasion and metastasis of HCC through downregulation of Snail in vitro and in vivo. Moreover, our results demonstrated that GLS2 interacts with Dicer and stabilizes Dicer protein to facilitate miR-34a maturation and subsequently represses Snail expression in a glutaminase activity independent manner. Our findings indicate that non-glutaminolysis function of GLS2 inhibits migration and invasion of HCC cells by repressing the epithelial-mesenchymal transition via the Dicer-miR-34a-Snail axis.

Topics: Animals; Carcinoma, Hepatocellular; Cell Movement; DEAD-box RNA Helicases; Down-Regulation; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Glutaminase; HEK293 Cells; Hep G2 Cells; Humans; Kaplan-Meier Estimate; Liver Neoplasms; Male; Mice, Inbred NOD; Mice, SCID; MicroRNAs; Neoplasm Invasiveness; Neoplasm Staging; Protein Stability; Ribonuclease III; RNA Interference; Signal Transduction; Snail Family Transcription Factors; Time Factors; Transfection; Tumor Burden

The lack of sensitive and specific biomarkers hinders pathological diagnosis and prognosis for hepatocellular carcinoma (HCC). Since glutaminolysis plays a crucial role in carcinogenesis and progression, we sought to determine if the expression of kidney-type and liver-type glutaminases (GLS1 and GLS2) were informative for pathological diagnosis and prognosis of HCC. We compared the expression of GLS1 and GLS2 in a large set of clinical samples including HCC, normal liver, and other liver diseases. We found that GLS1 was highly expressed in HCC; whereas, expression of GLS2 was mainly confined to non-tumor hepatocytes. The sensitivity and specificity of GLS1 for HCC were 96.51% and 75.21%, respectively. A metabolic switch from GLS2 to GLS1 was observed in a series of tissues representing progressive pathologic states mimicking HCC oncogenic transformation, including normal liver, fibrotic liver, dysplasia nodule, and HCC. We found that high expression of GLS1 and low expression of GLS2 in HCC correlated with survival time of HCC patients. Expression of GLS1 and GLS2 were independent indexes for survival time; however, prognosis was predominantly determined by the level of GLS1 expression. These findings indicate that GLS1 expression is a sensitive and specific biomarker for pathological diagnosis and prognosis of HCC.

Topics: Biomarkers; Carcinoma, Hepatocellular; Cell Line, Tumor; Female; Glutaminase; Humans; Liver Neoplasms; Male; Prognosis; Tissue Array Analysis

Accumulated evidence demonstrated that long non-coding RNAs (lncRNAs) play a pivotal role in tumorigenesis. However, it is still largely unknown how these lncRNAs were regulated by small ncRNAs, such as microRNAs (miRNAs), at the post-transcriptional level. We here use lncRNA HOTTIP as an example to study how miRNAs impact lncRNAs expression and its biological significance in hepatocellular carcinoma (HCC). LncRNA HOTTIP is a vital oncogene in HCC, one of the deadliest cancers worldwide. In the current study, we identified miR-192 and miR-204 as two microRNAs (miRNAs) suppressing HOTTIP expression via the Argonaute 2 (AGO2)-mediated RNA interference (RNAi) pathway in HCC. Interaction between miR-192 or miR-204 and HOTTIP were further confirmed using dual luciferase reporter gene assays. Consistent with this notion, a significant negative correlation between these miRNAs and HOTTIP exists in HCC tissue specimens. Interestingly, the dysregulation of the three ncRNAs was associated with overall survival of HCC patients. In addition, the posttranscriptional silencing of HOTTIP by miR-192, miR-204 or HOTTIP siRNAs could significantly suppress viability of HCC cells. On the contrary, antagonizing endogenous miR-192 or miR-204 led to increased HOTTIP expression and stimulated cell proliferation. In vivo mouse xenograft model also support the tumor suppressor role of both miRNAs. Besides the known targets (multiple 5' end HOX A genes, i.e. HOXA13), glutaminase (GLS1) was identified as a potential downstream target of the miR-192/-204-HOTTIP axis in HCC. Considering glutaminolysis as a crucial hallmark of cancer cells and significantly inhibited cell viability after silencingGLS1, we speculate that the miR-192/-204-HOTTIP axis may interrupt HCC glutaminolysis through GLS1 inhibition. These results elucidate that the miR-192/-204-HOTTIP axis might be an important molecular pathway during hepatic cell tumorigenesis. Our data in clinical HCC samples highlight miR-192, miR-204 and HOTTIP with prognostic and potentially therapeutic implications.

Topics: Animals; Argonaute Proteins; Carcinoma, Hepatocellular; Female; Glutaminase; Hep G2 Cells; Humans; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Middle Aged; RNA, Long Noncoding

The tumor suppressor p53 and its signaling pathway play a critical role in tumor prevention. As a direct p53 target gene, the role of glutaminase 2 (GLS2) in tumorigenesis is unclear. In this study, we found that GLS2 expression is significantly decreased in majority of human hepatocellular carcinoma (HCC). Restoration of GLS2 expression in HCC cells inhibits the anchorage-independent growth of cells and reduces the growth of HCC xenograft tumors. Interestingly, we found that GLS2 negatively regulates the PI3K/AKT signaling, which is frequently activated in HCC. Blocking the PI3K/AKT signaling in HCC cells largely abolishes the inhibitory effect of GLS2 on the anchorage-independent cell growth and xenograft tumor growth. The GLS2 promoter is hypermethylated in majority of HCC samples. CpG methylation of GLS2 promoter inhibits GLS2 transcription, whereas reducing the methylation of GLS2 promoter induces GLS2 expression. Taken together, our results demonstrate that GLS2 plays an important role in tumor suppression in HCC, and the negative regulation of PI3K/AKT signaling contributes greatly to this function of GLS2. Furthermore, hypermethylation of GLS2 promoter is an important mechanism contributing to the decreased GLS2 expression in HCC.

Topics: Animals; Blotting, Western; Carcinoma, Hepatocellular; Cell Adhesion; Cell Movement; Cell Proliferation; DNA Methylation; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Glutaminase; Humans; Immunoenzyme Techniques; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Phosphatidylinositol 3-Kinases; Promoter Regions, Genetic; Proto-Oncogene Proteins c-akt; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Glutaminase 2 (Gls2) is a p53 target gene and is known to play an important role in energy metabolism. Gls2 has been reported to be downregulated in human hepatocellular carcinomas (HCC). However, the underlying mechanism responsible for its downregulation is still unclear. Here, we investigated Gls2 expression and its promoter methylation status in human liver and colon cancers.. mRNA expression of Gls2 was determined in human liver and colon cancer cell lines and HCC tissues by real-time PCR and promoter methylation was analyzed by methylation-specific PCR (MSP) and validated by bisulfite genome sequencing (BGS). Cell growth was determined by colony formation assay and MTS assay. Statistical analysis was performed by Wilcoxon matched-pairs test or non-parametric t test.. First, we observed reduced Gls2 mRNA level in a selected group of liver and colon cancer cell lines and in the cancerous tissues from 20 HCC and 5 human colon cancer patients in comparison to their non-cancerous counter parts. Importantly, the lower level of Gls2 in cancer cells was closely correlated to its promoter hypermethylation; and chemical demethylation treatment with 5-aza-2'-deoxycytidine (Aza) increased Gls2 mRNA level in both liver and colon cancer cells, indicating that direct epigenetic silencing suppressed Gls2 expression by methylation. Next, we further examined this correlation in human HCC tissues, and 60% of primary liver tumor tissues had higher DNA methylation levels when compared with adjacent non-tumor tissues. Detailed methylation analysis of 23 CpG sites at a 300-bp promoter region by bisulfite genomic sequencing confirmed its methylation. Finally, we examined the biological function of Gls2 and found that restoring Gls2 expression in cancer cells significantly inhibited cancer cell growth and colony formation ability through induction of cell cycle arrest.. We provide evidence showing that epigenetic silencing of Gls2 via promoter hypermethylation is common in human liver and colon cancers and Gls2 appears to be a functional tumor suppressor involved in the liver and colon tumorigenesis.

Topics: Adult; Aged; Base Sequence; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Colon; Colonic Neoplasms; DNA Methylation; Down-Regulation; Enzyme Repression; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Glutaminase; Humans; Liver; Liver Neoplasms; Male; Middle Aged; Molecular Sequence Data; Promoter Regions, Genetic; Sequence Analysis, DNA

Diphenylarsinic acid [DPAA(V)] was detected in ground water used as drinking water after a poisonous incident in Kamisu, Japan. An approach to define the target molecules of DPAA(V) with a high throughput analysis of proteins from cultured human cells demonstrated down-regulation of glutaminase C (GAC). GAC is a splicing variant of the kidney-type glutaminase (KGA) gene and has the enzyme activity of phosphate-activated glutaminase (PAG). To gain some insights into the mechanism of arsenic intoxication in Kamisu, the effects of various arsenic compounds, including arsenicals that were detected in ground water ([DPAA(V)], phenylarsonic acid [PAA(V)] and bis(diphenylarsine)oxide [BDPAO(III)]) and rice (phenylmethylarsinic acid [PMAA(V)]), were investigated for the expression of GAC and PAG activity. When cultured human HepG2 cells were incubated with arsenicals for 24h, the pentavalent phenylarsenic form of PAA(V) and PMAA(V) as well as DPAA(V) suppressed the expression of GAC protein and PAG activity in a concentration-dependent manner. On the other hand, the trivalent phenylarsenic form of BDPAO(III) had no suppressive effect on GAC and PAG. In addition, trivalent phenylarsenic compounds, such as the glutathione (GSH) conjugate of DPAA(V) [DPA-GS (III)] and triphenylarsine [TPA(III)], and the inorganic arsenics, iAs(V) and iAs(III), and methylated metabolites of inorganic arsenics, dimethylarsinic acid [DMA(V)] and dimethylarsinous acid [DMA(III)], had no suppressive effect on glutaminase. Likewise, methyl substituents of the hydroxyl groups of DPAA(V), PAA(V) and PMAA(V), diphenylmethylarsine oxide [DPMAO(V)] and phenyldimethylarsine oxide [PDMAO(V)], did not have any suppressive effects. These results suggest that pentavalent arsenic compounds with both phenyl groups and hydroxyl groups are effective in the suppression of glutaminase. In addition, the fact that it was only the arsenicals detected in Kamisu that were effective in suppressing glutaminase provides insights into the cause of the arsenic intoxication at Kamisu.

Topics: Arsenicals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Down-Regulation; Glutaminase; Hepatocytes; Humans; Liver Neoplasms; Molecular Structure; Time Factors

Dynamic nuclear polarization (DNP) is an emerging technique for increasing the sensitivity of (13)C MR spectroscopy (MRS). [5-(13)C(1)]Glutamine was hyperpolarized using this technique by up to 5%, representing a 6000-fold increase in sensitivity. The conversion of hyperpolarized glutamine to glutamate by mitochondrial glutaminase was demonstrated using (13)C-MRS measurements in cultured human hepatoma cells (HepG2). These results represent the first step in developing an imaging technique for detecting glutamine metabolism in vivo. Furthermore, since glutamine utilization has been correlated with cell proliferation, the study suggests a new technique for detecting changes in tumor cell proliferation.

Topics: Biomarkers, Tumor; Carbon Radioisotopes; Carcinoma, Hepatocellular; Cell Line, Tumor; Diagnosis, Computer-Assisted; Enzyme Activation; Glutaminase; Glutamine; Humans; Liver Neoplasms; Magnetic Resonance Spectroscopy; Neoplasm Proteins

The authors determined the effects of growth inhibition on glutamine transport and metabolism in human hepatoma cells.. Hepatoma cells exhibit markedly higher (10- to 30-fold) glutamine uptake than normal human hepatocytes, via a disparate transporter protein with a higher affinity for glutamine. Currently, little is known about the effects of growth arrest on glutamine transport and metabolism in hepatoma cells.. The authors determined proliferation rates, glutamine transport, and glutaminase activities in the human hepatoma cell lines HepG2, Huh-7, and SK-Hep, both in the presence and absence of the chemotherapeutic agents novobiocin and sodium butyrate. The transport activities for alanine, arginine, and leucine also were determined in both treated and untreated cells. Glutaminase activity was determined in normal human liver tissue and compared with that present in hepatoma cells.. Glutaminase activities were similar in all three cell lines studied, despite differences in proliferation rates, and were sixfold higher than the activity in normal human liver. In contrast to normal hepatocytes, which expressed the liver-specific glutaminase, hepatomas expressed the kidney-type isoform. Sodium butyrate (1 mmol/L) and novobiocin (0.1 mmol/L) inhibited cellular proliferation and reduced both glutamine transport and glutaminase activity by more than 50% after 48 hours in the faster-growing, less differentiated SK-Hep cells. In contrast, the agents required 72 hours to attenuate glutamine uptake by 30% and 50% in the slower-growing, more differentiated HepG2 and Huh-7 cell lines, respectively. Treatment of all three cell lines with novobiocin/butyrate also resulted in a 30% to 60% attenuation of the transport of alanine, arginine, and leucine, and glutamine, indicating that inhibition of cellular proliferation similarly affects disparate amino acid transporters.. Hepatocellular transformation is characterized by a marked increase in glutamine transport and metabolism. Inhibition of cellular proliferation attenuates glutamine transport and metabolism, especially in fast-growing, relatively undifferentiated hepatoma cells. Because the uptake of other amino acids is similarly reduced under cytostatic conditions, plasma membrane amino acid transport activity in hepatoma cells is regulated by the proliferation state of the cells.

Topics: Amino Acid Transport Systems; Amino Acids; Animals; Biological Transport; Butyrates; Butyric Acid; Carcinoma, Hepatocellular; Carrier Proteins; Cell Division; Female; Glutaminase; Glutamine; Humans; Liver Neoplasms; Male; Novobiocin; Rats; Rats, Inbred F344; Time Factors; Tumor Cells, Cultured

Glutamine synthetase and glutaminase activities in human cirrhotic liver tissues and hepatocellular carcinomas were determined for comparison with normal liver tissues. In hepatocellular carcinoma, glutamine synthetase activity was approximately one-third of that in normal liver, whereas no detectable change in the enzyme activity was observed in cirrhotic liver. Phosphate-dependent and phosphate-independent glutaminase activities were increased approximately 20-fold and 6-fold, respectively, both in the carcinoma and cirrhotic liver compared with those from normal liver, Oxypolarographic tests showed that the rate of glutamine oxidation in the tumor and cirrhotic liver mitochondria was about 5-fold higher than that in the liver mitochondria. The rate of glutamate oxidation in the liver mitochondria was comparable to that in the cirrhotic liver and tumor mitochondria. Glutamine oxidation was inhibited by prior incubation of the mitochondria with 6-diazo-5-oxo-L-norleucine, which inhibited mitochondrial glutaminase. These results indicate that the product of glutamine hydrolysis, glutamate, is catabolized in the tumor and cirrhotic liver mitochondria to supply ATP. In the liver and cirrhotic liver mitochondria, glutamate was oxidized via the routes of transamination and deamination. On the other hand, glutamate oxidation was initiated preferentially via a transamination pathway in the tumor mitochondria.

Topics: Carcinoma, Hepatocellular; Glutamate-Ammonia Ligase; Glutaminase; Glutamine; Humans; Liver; Liver Cirrhosis; Liver Neoplasms; Mitochondria, Liver; Oxygen Consumption

Glutamine synthetase and glutaminase activities in a series of hepatoma cells of human and rat origins were determined for comparison with normal liver tissues. Marked decrease in glutamine synthetase activity was observed in the tumor cells. Phosphate-dependent and phosphate-independent glutaminase activities were increased compared with those from normal liver tissues. Well coupled mitochondria were isolated from HuH 13 line of human hepatoma cells and human liver. Oxypolarographic tests showed that glutamine oxidation was prominent in the tumor mitochondria, while mitochondria from the liver showed a feeble glutamine oxidation. Glutamine oxidation was inhibited by prior incubation of the mitochondria with DON (6-diazo-5-oxo-L-norleucine), which inhibited mitochondrial glutaminase. These results indicate that the product of glutamine hydrolysis, glutamate, is catabolized in the tumor mitochondria to supply ATP.

Topics: Animals; Carcinoma, Hepatocellular; Glutamate-Ammonia Ligase; Glutaminase; Humans; In Vitro Techniques; Liver; Liver Neoplasms; Liver Neoplasms, Experimental; Rats

Topics: Adenosine Triphosphate; Adenylyl Imidodiphosphate; Ammonium Chloride; Animals; Bicarbonates; Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); Carbamyl Phosphate; Carcinoma, Hepatocellular; Cells, Cultured; Glutamates; Glutaminase; Glycine; Kinetics; Liver Neoplasms; Magnesium; Phosphotransferases; Rats

Topics: Animals; Antibiotics, Antineoplastic; Asparagine; Aspartate Aminotransferases; Aspartic Acid; Ataxia; Azo Compounds; Carbon Radioisotopes; Carboxy-Lyases; Carcinoma, Hepatocellular; Chemical and Drug Induced Liver Injury; Diarrhea; DNA; Formates; Glutamate Dehydrogenase; Glutamate-Ammonia Ligase; Glutamates; Glutaminase; Lethal Dose 50; Ligases; Liver Neoplasms; Malate Dehydrogenase; Mice; Mice, Inbred Strains; Protein Biosynthesis; Seizures; Spleen; Time Factors

Topics: Alcaligenes; Ammonia-Lyases; Animals; Asparaginase; Carboxypeptidases; Carcinoma; Carcinoma 256, Walker; Carcinoma, Ehrlich Tumor; Carcinoma, Hepatocellular; Cell Line; Enzyme Therapy; Glutaminase; Humans; Leukemia, Experimental; Liver Neoplasms; Mice; Neoplasms; Neoplasms, Experimental; Sarcoma, Experimental

Topics: 5-Aminolevulinate Synthetase; Acetamides; Animals; Arginase; Carcinoma, Hepatocellular; DNA Nucleotidyltransferases; Enzyme Induction; Female; Fetus; Glucose; Glutamate-Ammonia Ligase; Glutaminase; Hydrocortisone; Kidney; Kidney Neoplasms; Liver; Liver Neoplasms; Male; Mixed Function Oxygenases; Ornithine; Oxidoreductases; Phosphotransferases; Pyruvate Kinase; Rats; Transaminases; Tryptophan; Tryptophan Oxygenase

Topics: Ammonia; Animals; Carbon Dioxide; Carbon Isotopes; Carcinoma, Ehrlich Tumor; Carcinoma, Hepatocellular; Cyanides; Glutamates; Glutaminase; Glutamine; Hydrazones; In Vitro Techniques; Ketoglutaric Acids; Kinetics; Liver Neoplasms; Mice; Mitochondria; NADP; Neoplasms, Experimental; Oxygen Consumption; Pyruvates; Rats; Rotenone

Topics: Animals; Carcinoma, Hepatocellular; Fetus; Glutaminase; Isoenzymes; Kidney; Liver Neoplasms; Male; Neoplasms, Experimental; Rats

Topics: Adrenalectomy; Animal Feed; Animals; Animals, Laboratory; Carcinoma, Hepatocellular; Cell Line; Chromosomes; Dactinomycin; Dietary Proteins; Enzyme Activation; Glutaminase; Glutamine; Hydrocortisone; Ligases; Liver Neoplasms; Neoplasm Transplantation; Neoplasms, Experimental; Rats; Thyroxine; Transaminases; Transplantation, Homologous

Topics: Animals; Carcinoma, Hepatocellular; Glutaminase; Kidney; Liver Neoplasms; Neoplasm Transplantation; Rats

Topics: Acetates; Animals; Brain; Carcinoma, Hepatocellular; Centrifugation, Density Gradient; Chloromercuribenzoates; Female; Fetus; Glutaminase; Glutamine; Hydrogen-Ion Concentration; Intestine, Small; Isoenzymes; Kidney; Kinetics; Liver; Liver Neoplasms; Liver Regeneration; Male; Mitochondria; Phosphates; Pyruvates; Rats; Vibration

Topics: Animals; Asparaginase; Carcinoma, Hepatocellular; Glutaminase; Humans; Liver; Liver Neoplasms; Mice; Neoplasms, Experimental; Rats; Rectal Neoplasms; Rhabdomyosarcoma; Sarcoma, Experimental