Search Results (64)

Cantharidin (CTD), the principal active constituent of the traditional Chinese medicine (TCM) Mylabris, exhibits potent antitumor activity. However, its clinical application is limited by organ toxicity (especially nephrotoxicity), and the underlying mechanisms remain incompletely defined. This research endeavored to elucidate the nephrotoxic effects and molecular mechanisms of CTD in rats using serum biochemical assays, histopathological examinations, and a multi-omics strategy. CTD treatment significantly increased levels of urea nitrogen and creatinine and induced histopathological injury in the kidneys. Transcriptomics, proteomics, and network pharmacology integrated analysis have revealed 14 common targets, mainly involved in the glutathione metabolic pathway. Further verification showed that CTD markedly upregulated protein expression of glutaminase (GLS), while downregulating homogentisate 1,2-dioxygenase (HGD), glutamate-cysteine ligase catalytic subunit (GCLC), and regulatory subunit (GCLM), thereby suppressing the glutathione metabolic pathway and exacerbating kidney injury. Our results indicate that CTD induces oxidative stress and consequent nephrotoxicity by inhibiting the glutathione metabolic pathway, providing a mechanistic basis for optimizing clinical strategies to mitigate CTD-induced kidney injury. Full article

Cancer progression is influenced by the dynamic interplay between tumor cells and the surrounding stromal microenvironment. Therapy-induced senescence (TIS) of stromal fibroblasts represents a common outcome of anticancer treatments, contributing to tumor progression through the senescence-associated secretory phenotype (SASP). While SASP cytokines promote cancer malignancy, the contribution of secreted metabolites from senescent cells remains poorly understood. Here, we investigate the role of senescent stromal metabolism in regulating prostate and ovarian cancer cell invasion. Conditioned media (CM) from TIS-induced human prostate (HPFs) and ovarian fibroblasts (HOFs) promote enhanced invasion of cancer cells. Invasion is partially preserved after exposure to boiled CM, suggesting a role for heat-stable metabolic factors. Metabolomic profiling of senescent fibroblasts-derived CM reveals a significant increase in Glutamine (Gln) levels, identifying senescent stromal fibroblasts as a previously unrecognized source of extracellular Gln in the tumor microenvironment (TME). Exposure of cancer cells to senescent CM increases Gln uptake, together with upregulation of the transporter SLC1A5 and increased intracellular Gln. This metabolic adaptation is associated with increased malignant phenotype including epithelial-to-mesenchymal transition (EMT) and stemness features. Extracellular Gln depletion, pharmacological inhibition of glutaminase-1 (GLS1) in cancer cells, or Gln synthetase (GS) silencing in fibroblasts markedly impair senescent fibroblasts CM-induced invasion, EMT markers expression, and stemness features in cancer cells. Stromal-derived Gln is associated with increased cancer cell invasion through activation of a redox-dependent NRF2/ETS1 signaling axis. Analysis of patient-derived transcriptomic datasets further suggests chemotherapy-associated upregulation of Gln metabolism and ETS1 expression. These findings identify senescent stromal-derived Gln as a key metabolic driver of prostate and ovarian cancer aggressiveness and reveal a TIS-associated metabolic vulnerability that could be explored in future preclinical studies. Full article

Background/Objectives: To evaluate the immunohistochemical expression of hexokinase-2 (HK2), glutaminase-1 (GLS1), and fatty acid synthase (FASN) and its prognostic significance in diffuse gastric adenocarcinoma. Materials and Methods: Formalin-fixed paraffin-embedded tissue samples from 92 patients with diffuse gastric adenocarcinoma were analyzed. Immunohistochemistry (IHC) was performed to assess the expression of HK2, GLS1 and FASN. Expression levels were evaluated semi-quantitatively based on staining intensity and the percentage of positive cells. Associations between enzyme expression and clinicopathological features were assessed using the Chi-square test. Kaplan–Meier survival analysis was employed to evaluate progression-free survival (PFS) and overall survival (OS) and the log-rank test and Cox proportional hazards models were used for statistical analysis. Results: HK2 and FASN were overexpressed in 20.7% and 22.8% of patients, respectively, and were significantly associated with advanced tumor stage. In contrast, GLS1 expression, found in 30.4% of patients, did not independently correlate with clinicopathological characteristics. Furthermore, HK2 expression and co-expression of HK2/FASN (10.9%) and HK2/GLS1/FASN (8.7%) were associated with progressive disease. In the univariate analysis, stage, HK2 overexpression, and co-expression of HK2/FASN and HK2/GLS1/FASN were associated with shorter survival. However, only stage retained prognostic value in the multivariate analysis. Conclusions: Co-expression of these key metabolic enzymes remains a promising candidate as prognostic markers and therapeutic targets. Concurrent targeting of these metabolic pathways may offer novel therapeutic opportunities for patients with advanced-stage gastric cancer. Full article

Upregulation of glutaminase enzymatic activity promotes tumour cell proliferation. Its overexpression correlates with poor disease outcome in patients, including those with breast cancer. A selective glutaminase inhibitor, CB-839, which targets cancer cells by blocking glutamine conversion to glutamate, has shown promising preclinical results as a therapeutic target in triple-negative breast cancer treatment. The current study aimed to determine the importance of glutaminase in Oestrogen Receptor positive/luminal breast cancer to potentially identify therapeutic targets to treat this subtype. In vitro studies using luminal breast cancer cells were performed to investigate the effects of siRNA knockdown of glutaminase genes (GLS and GLS2) and inhibition using CB-839 on functional assays. Silencing GLS in luminal breast cancer cells significantly reduced cell proliferation whilst inducing apoptosis. A similar impact on cell proliferation was observed when silencing GLS2 in luminal B cells, but there was no observed effect on cell apoptosis and cell cycle. There was little effect of GLS inhibition using CB-839 in luminal breast cancer. This study demonstrates that glutaminase is necessary for luminal breast cancer growth and survival. Co-targeting GLS and GLS2 might be a novel approach for the treatment of this subclass. Further functional studies to evaluate the underlying molecular mechanisms of this process are warranted. Full article

Glutamine is a known regulator of vascular smooth muscle cell (VSMC) function, but the molecular pathways underlying this response remain incompletely understood. This study investigated how glutamine metabolism influences VSMC behavior and identified the responsible enzymes and metabolites. Glutamine deprivation markedly reduced VSMC proliferation, migration, and collagen synthesis, while modestly decreasing viability. Pharmacological inhibition of glutaminase-1 (GLS1) or aminotransferases (AT) similarly suppressed these cellular functions, whereas inhibiting glutamate dehydrogenase 1 (GLUD1) had no effect. Metabolite analysis revealed that glutamine deprivation or AT inhibition, but not GLUD1 inhibition, reduced intracellular α-ketoglutarate (αKG) concentrations, establishing AT as the primary enzyme converting glutamine-derived glutamate to αKG. To identify which metabolite drives VSMC responses, glutamine-starved cells were supplemented with various glutamine-derived molecules. The cell-permeable αKG analog dimethyl-αKG significantly restored VSMC proliferation, migration, collagen synthesis, and survival, while ammonia only enhanced viability, demonstrating αKG’s primary role in mediating glutamine-dependent functions. These findings establish that glutamine metabolism via the GLS1-AT-αKG pathway is a critical driver of VSMC activation and survival. Targeting this glutamine-αKG metabolic axis through GLS1 inhibition, AT blockade, or downstream αKG disruption offers a compelling therapeutic strategy for ameliorating fibroproliferative vascular diseases, including atherosclerosis, post-angioplasty restenosis, and pulmonary hypertension. Full article

Background: Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer and is marked by pronounced intra-tumoural heterogeneity that complicates therapeutic response. Patient-derived organoids offer a physiologically relevant model to capture this diversity and evaluate treatment effects. When integrated with spatial transcriptomics, they might enable the mapping of spatially resolved transcriptional and isoform-level changes within the tumour microenvironment. Methods: We established a robust workflow for generating patient-derived ccRCC organoids, that are not passaged and retain original cellular components. These retain key features of the original tumours, including cancer cell, stromal, and immune components. Results: Spatial transcriptomic profiling revealed multiple transcriptionally distinct regions within and across organoids, reflecting the intrinsic heterogeneity of ccRCC. Isoform-level analysis identified spatially variable expression of glutaminase (GLS) isoforms, with heterogeneous distributions of both the GAC and KGA variants. Treatment with NUC-7738, a phosphoramidate derivative of 3′-deoxyadenosine, induced marked transcriptional remodelling of organoids, including alterations in ribosomal and mitochondrial gene expression. Conclusions: This study demonstrates that combining long-read spatial transcriptomics with patient-derived organoid models provides a powerful and scalable approach for dissecting gene and isoform-level heterogeneity in ccRCC and for elucidating spatially resolved transcriptional responses to novel therapeutics. Full article

The extremophile yeast Rhodotorula mucilaginosa can grow in extremely poor environments. Glutamine (Gln) is an important anaplerotic substrate for gluconeogenesis and pentose synthesis. Glutaminase (Glnase) produces glutamate which in turn undergoes transamination to produce the Krebs cycle intermediate α-keto-glutarate. The yeast enzyme has low similarity with human GLS1, although the active site is partially conserved. Also, antibodies against GLS1 cross react with the yeast enzyme. Glnase is a therapeutic mark in tumor treatments, where endogenous Glnase is inhibited with different pharmaceutical agents. Another proposed approach is to add exogenous fungal Glnase to deplete Gln pools, thus starving the tumor. Using Gln as the sole carbon source, R. mucilaginosa grew better than Debaryomyces hansenii, while Saccharomyces cerevisiae did not grow. In addition, the Gln-dependent growth of R. mucilaginosa was inhibited by two different Gln metabolism inhibitors used in cancer therapy, namely 6-diazo-5-oxo-L-norleucine (DON) and Telaglenastat (CB-839). In cell homogenates from R. mucilaginosa DON inhibited Gln metabolism at similar concentrations as those used in mammals. The ability of R. mucilaginosa to grow on Gln as the sole carbon source is exceptional and it may be used as a suitable tool to evaluate agents targeting tumoral Gln metabolism. It is proposed that R. mucilaginosa may be a valuable source of exogenous Glnase. Full article

Enterococcus spp. produce diverse bioactive molecules used for biotechnological purposes or as probiotic agents for livestock and human health. The main aim of this study was to decipher the genetic traits using whole-genome sequencing (WGS) of a bacteriocinogenic Enterococus faecium 1702 strain showing diverse probiotic traits. Genetic traits of the strain were determined by performing WGS using the NovaSeq6000 platform followed by consecutive sequence analysis using appropriate software. WGS showed that the genome of the E. faecium 1702 strain has a size of 2,621,416 bp, with a GC content of 38.03%. The strain belonged to the sequence type ST722 not known as a human clonal lineage. The strain was free of genes encoding clinically relevant antibiotic resistance; in addition, genes encoding sensu stricto virulence factors, plasmids, and prophages were not detected. Annotations through the Prokaryotic Genomes Automatic Annotation Pipeline (PGAP) tool revealed 2413 coding sequencing entries (CDC) out of 2521 predicted chromosomal genes. The functional annotation of the whole genome through the KEGG database using KofaScan revealed several genes related to several biological activities, including metabolic process, carbohydrate metabolism, amino acid metabolism, and nucleotide metabolism. The strain harbored three entero-bacteriocins (enterocins) encoded by entA, entB, and entX (enterocin X-alpha and X-beta) genes. Interestingly, the strain harbored the ansB, glsA, and arcA genes encoding L-asparaginase, L-glutaminase, and arginine deiminase, respectively, known for their anticancer activities. E. faecium 1702 harbored the gadB, gadC, and gadR genes implicated in gamma(γ)-aminobutyric acid (GABA) production, which is known for its analgesic, anti-anxiety, hypotensive, diuretic, and antidiabetic effects. The WGS findings and phenotypic traits of E. faecium 1702 revealed significant features that allow for its use as a probiotic or for biotechnological and pharmaceutical applications. Full article

Background/Objectives: Metabolic reprogramming is an essential feature of tumors. Mitochondrial sirtuins SIRT3 and SIRT5 differently regulate glutamine metabolism with SIRT5 inhibiting glutaminase (GLS) and SIRT3 increasing glutamate dehydrogenase (GDH). Considering the important and interconnected role of glutamine, SIRT3 and SIRT5 for cancer growth and progression, our hypothesis is that a simultaneous modulation of SIRT3 and SIRT5 could represent a valid anti-tumoral strategy. Methods: wt and GLS1-silenced triple negative breast cancer spheroids were treated with 3-TYP, a selective SIRT3 inhibitor, and with MC3138, a new selective SIRT5 activator, both alone and in combination. The effects of such treatments on hypoxia, autophagy and mitophagy markers were determined by immunofluorescence and Western blot. Mitochondria morphology was studied by transmission electron microscopy (TEM) and mitochondrial ROS production by confocal analysis. Results: We observed that 3-TYP+MC3138 treatment decreased the size of spheroids by affecting HIF-1α, c-Myc, glutamine transporter SLC1A5 and autophagy (LC3II) and mitophagy (BNIP3) markers. Moreover, such treatments altered the morphology and conformation of the mitochondria. Finally, we also documented an increase in mitochondria reactive oxygen species (mtROS). Conclusions: The combined inhibition of SIRT3 and activation of SIRT5 greatly reduces the size of spheroids through the inhibition of hypoxic response, which is then followed by the alteration of the autophagic and mitophagic process and the toxic accumulation of mitochondrial ROS, representing a new anti-tumoral strategy. Full article

Glutamine addiction in human melanoma is a premier example of the cancer hallmark of metabolic reprogramming. In the present study, we investigate the presence of metabotropic glutamate receptor 1 (mGluR1/GRM1) and glutaminase (GLS1/GLS) in canine oral malignant melanoma (OMM) and those of low malignant potential, termed histologically well-differentiated melanocytic neoplasm of the lips and oral mucosa (HWDMN). We used immunohistochemistry (IHC) and qPCR to evaluate mGluR1 and GLS1 protein expression and RNA expression, respectively. Nearly 20% of OMM cases had an mGluR1 IHC score ≥ 1, while none of the HWDMN cases had any expression. Due to low IHC expression, only 10 cases were selected for determination of GRM1 RNA expression, and none were positive. GLS RNA expression did not differ between OMM and HWDMN. A GLS1 IHC score ≥ 1 was significantly higher in OMM cases and highly specific (95%) for correctly identifying tumors with a Ki67 index ≥ 19.5. These results may have been negatively impacted by use of a brown chromogen for IHC labeling among background pigment, particularly in HWDMN. Ultimately, these findings suggest that canine OMM does not heavily rely on mGluR1 for tumorigenesis or progression. Differential GLS1 protein expression warrants further investigation with protein quantification. Full article

Hepatocellular carcinoma (HCC) is the most prevalent primary liver cancer, with a poor prognosis due to late diagnosis, limited curative therapies, and underlying liver disease. Glutamine metabolism, a crucial pathway in cancer, remains poorly understood in HCC, which develops in an already metabolically dynamic organ. This study aimed to characterize glutamine metabolism in HCC. Glutamine metabolism in HCC was explored through in vitro analysis of neoplastic characteristics, experimental hepatocarcinogenesis in C57BL/6 mice, and examination of liver samples from patients with HCC, cirrhosis, and non-diseased liver. The evaluation included metabolite abundance and mRNA/protein expressions. In mice, tumors exhibited hyperactive glutaminolysis compared to adjacent tissue. Notably, glutaminase expression shifted from the liver isoform (GLS2) in normal and cirrhotic livers to the kidney isoform (GLS1) in HCC. In samples from patients, HCC tumors showed overexpression of glutamine synthetase and GLS1 along with a loss of GLS2 expression, providing excellent discrimination of HCC lesions from cirrhotic and normal liver samples. Inhibiting GLS1 with CB-839 significantly impacted glutamine metabolism in HCC cells while showing limited activity on normal hepatocytes. HCC tumors show reprogramming of GLS2 to GLS1, with a concomitant increase in glutamine synthetase. These characteristics can discriminate HCC from cirrhotic and normal liver tissues. Overexpressed GLS1 and loss of GLS2 within tumors convey an unfavorable prognosis in patients with HCC. Pharmacological inhibition of GLS1 in HCC cells successfully harnesses glutamine metabolism, representing an attractive target for novel therapeutic approaches. Full article

This research assessed the efficacy of glutamate (Glu) supplementation to feed in counteracting growth restriction and intestinal stress-induced injury in juvenile groupers (Epinephelus coioides; initial weight 15.11 ± 0.03 g). The study comprised five isonitrogenous and iso-lipidic diets: a fish-meal-based (FM) diet, a soya-meal-based (SBM) diet, and SBM diets containing varying Glu levels of 1.0% (G-1), 2.0% (G-2), or 3.0% (G-3). The trial employed a randomized design with five treatment groups. Each group was housed in triplicate aquariums and received assigned diets for 56 consecutive days. Supplementation with Glu resulted in dose-dependent enhancements in weight gain, specific growth rate, serum high-density lipoprotein cholesterol, intestinal superoxide dismutase activity, digestive enzyme activity (trypsin, lipase, amylase), amino acid metabolic enzyme activity (glutaminase, GLS; glutamine synthetase), and intestinal mRNA levels of GLS, IL-10, and TGF-β1. Maximal values of the G-3 diet were restored to the levels of the FM diet (p > 0.05). Serum total cholesterol, intestinal total antioxidant capacity, and catalase activity followed a similar increasing trend with Glu level, attaining maxima in diet G-3, yet these values remained markedly lower than those of the FM diet (p < 0.05). Conversely, intestinal malondialdehyde content and mRNA levels of genes IL-8, IL-12, IL-1β, and TNF-α exhibited a significant dose-dependent decrease, reaching minimal levels in diet G-3 that were restored to the levels of the FM diet (p > 0.05). The results above demonstrate that Glu addition enhances nutritional status and intestinal structural integrity by augmenting antioxidant and digestive capacity and mitigating inflammatory responses, consequently enhancing growth performance and intestinal health. Full article

Glutamine metabolism is essential for infectious spleen and kidney necrosis virus (ISKNV) replication. Glutaminase 1 (GLS1), the key enzyme of the glutamine metabolism, and c-Myc positively regulate ISKNV infection, while c-Myc is closely correlated with GLS1. However, the regulatory mechanism among ISKNV, c-Myc and glutamine metabolism remains unclear. Here, we indicated that c-Myc increased glutamine uptake by increasing the GLS1, glutamate dehydrogenase (GDH) and isocitrate dehydrogenase (IDH2) expression of glutamine metabolism. ISKNV ORF102R, ORF093R and ORF118L co-located with c-Myc in CPB cells. Co-IP results showed that ISKNV ORF102R and ORF093R interacted with c-Myc, while ORF118L did not interact with c-Myc. The expression levels of c-Myc, GLS1 and IDH2 were increased in ISKNV ORF093R expression cells, and the mRNA and protein levels of GLS1 were upregulated in ISKNV 102R-expressing cells. These results indicated that ISKNV reconstructed glutamine metabolism to satisfy the energy and macromolecule requirements for virus proliferation by ORF093R and ORF102R interacting with c-Myc, which provides the foundation for innovative antiviral strategies. Full article

Glutaminase controls the first step in glutaminolysis, impacting bioenergetics, biosynthesis and oxidative stress. Two isoenzymes exist in humans, GLS and GLS2. GLS is considered prooncogenic and overexpressed in many tumours, while GLS2 may act as prooncogenic or as a tumour suppressor. Glioblastoma cells usually lack GLS2 while they express high GLS. We investigated how GLS2 expression modifies the metabolism of glioblastoma cells, looking for changes that may explain GLS2’s potential tumour suppressive role. We developed LN-229 glioblastoma cells stably expressing GLS2 and performed isotope tracing using U-¹³C-glutamine and metabolomic quantification to analyze metabolic changes. Treatment with GLS inhibitor CB-839 was also included to concomitantly inhibit endogenous GLS. GLS2 overexpression resulted in extensive metabolic changes, altering the TCA cycle by upregulating part of the cycle but blocking the synthesis of the 6-carbon intermediates from acetyl-CoA. Expression of GLS2 caused downregulation of PDH activity through phosphorylation of S293 of PDHA1. GLS2 also altered nucleotide levels and induced the accumulation of methylated metabolites and S-adenosyl methionine. These changes suggest that GLS2 may be a key regulator linking glutamine and glucose metabolism, also impacting nucleotides and epigenetics. Future research should ascertain the mechanisms involved and the generalizability of these findings in cancer or physiological conditions. Full article

Thermal condition affects the development and growth of ectotherms. The stenothermic honeybee brood, particularly the prepupae, are sensitive to low rearing temperature. The fat body plays important roles in energy reserve and metabolism during the honeybee brood development. To date, the fat body metabolic changes in prepupae responding to cold stress have not been completely understood. In this study, the ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS)-based non-target metabolome was analyzed between the cold-treated (CT, 20 °C, 36 h) and control (CK, 35 °C) fat body in prepupal honeybees. The fat body metabolomic data showed that the levels of 1860 and 254 metabolites were significantly increased and decreased, respectively, in cold-stressed prepupae. These altered metabolites, glutamine, glutamic acid, pyroglutamic acid, and oxidized glutathione, were significantly enriched into glutamine metabolism and glutathione metabolism pathways. Furthermore, the expression levels of glutamine metabolism-related genes, glutaminase (GLS), glutamate dehydrogenase (GDH), and gamma-glutamyl transferase (GGT-1 and GGT-7), were significantly decreased in cold-exposed prepupae compared with the control groups. Meanwhile, the oxidized glutathione (GSSG), but not the reduced glutathione (GSH) content, was increased in the cold-exposed group compared with controls. Collectively, our data revealed the fat body metabolomic changes in larva-to-pupa transition when exposed to cold stress. Our data provided new insights into stenothermic honeybee sensitivity to cold, characterized by perturbation of glutamine metabolism and oxidative stress. Full article