Deciphering glutamine metabolism patterns for malignancy and tumor microenvironment in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer characterized by metabolic reprogramming. Glutamine metabolism is pivotal in metabolic reprogramming, contributing to the significant heterogeneity observed in ccRCC. Consequently, developing prognostic markers associated with glutamine metabolism could enhance personalized treatment strategies for ccRCC patients. This study obtained RNA sequencing and clinical data from 763 ccRCC cases sourced from multiple databases. Consensus clustering of 74 glutamine metabolism related genes (GMRGs)- profiles stratified the patients into three clusters, each of which exhibited distinct prognosis, tumor microenvironment, and biological characteristics. Then, six genes (SMTNL2, MIOX, TMEM27, SLC16A12, HRH2, and SAA1) were identified by machine-learning algorithms to develop a predictive signature related to glutamine metabolism, termed as GMRScore. The GMRScore showed significant differences in clinical prognosis, expression profile of immune checkpoints, abundance of immune cells, and immunotherapy response of ccRCC patients. Besides, the nomogram incorporating the GMRScore and clinical features showed strong predictive performance in prognosis of ccRCC patients. ALDH18A1, one of the GRMGs, exhibited elevated expression level in ccRCC and was related to markedly poorer prognosis in the integrated cohort, validated by proteomic profiling of 232 ccRCC samples from Fudan University Shanghai Cancer Center (FUSCC). Conducting western blotting, CCK-8, transwell, and flow cytometry assays, we found the knockdown of ALDH18A1 in ccRCC significantly promoted apoptosis and inhibited proliferation, invasion, and epithelial-mesenchymal transition (EMT) in two human ccRCC cell lines (786-O and 769-P). In conclusion, we developed a glutamine metabolism-related prognostic signature in ccRCC, which is tightly linked to the tumor immune microenvironment and immunotherapy response, potentially facilitating precision therapy for ccRCC patients. Additionally, this study revealed the key role of ALDH18A1 in promoting ccRCC progression for the first time.

Curcumin synergistically enhances the efficacy of gemcitabine against gemcitabine-resistant cholangiocarcinoma via the targeting LAT2/glutamine pathway.

Cholangiocarcinoma (CCA) is often diagnosed late, leading to incomplete tumor removal, drug resistance and reduced chemotherapy efficacy. Curcumin has the potential for anti-cancer activity through various therapeutic properties and can improve the efficacy of chemotherapy. We aimed to investigate the synergistic effect of a combination of curcumin and gemcitabine against CCA, targeting the LAT2/glutamine pathway. This combination synergistically suppressed proliferation in gemcitabine-resistant CCA cells (KKU-213BGemR). It also resulted in a remarkable degree of CCA cell apoptosis and cell cycle arrest, characterized by a high proportion of cells in the S and G2/M phases. Knockdown of SLC7A8 decreased the expressions of glutaminase and glutamine synthetase, resulting in inhibited cell proliferation and sensitized CCA cells to gemcitabine treatment. Moreover, in vivo experiments showed that a combination curcumin and gemcitabine significantly reduced tumor size, tumor growth rate and LAT2 expression in a gemcitabine-resistant CCA xenograft mouse model. Suppression of tumor progression in an orthotopic CCA hamster model provided strong support for clinical application. In conclusion, curcumin synergistically enhances gemcitabine efficacy against gemcitabine-resistant CCA by induction of apoptosis, partly via inhibiting LAT2/glutamine pathway. This approach may be an alternative strategy for the treatment of gemcitabine-resistant in CCA patients.

Genome-scale metabolic network of human carotid plaque reveals the pivotal role of glutamine/glutamate metabolism in macrophage modulating plaque inflammation and vulnerability.

BACKGROUND: Metabolism is increasingly recognized as a key regulator of the function and phenotype of the primary cellular constituents of the atherosclerotic vascular wall, including endothelial cells, smooth muscle cells, and inflammatory cells. However, a comprehensive analysis of metabolic changes associated with the transition of plaque from a stable to a hemorrhaged phenotype is lacking. METHODS: In this study, we integrated two large mRNA expression and protein abundance datasets (BIKE, n = 126; MaasHPS, n = 43) from human atherosclerotic carotid artery plaque to reconstruct a genome-scale metabolic network (GEM). Next, the GEM findings were linked to metabolomics data from MaasHPS, providing a comprehensive overview of metabolic changes in human plaque. RESULTS: Our study identified significant changes in lipid, cholesterol, and inositol metabolism, along with altered lysosomal lytic activity and increased inflammatory activity, in unstable plaques with intraplaque hemorrhage (IPH+) compared to non-hemorrhaged (IPH-) plaques. Moreover, topological analysis of this network model revealed that the conversion of glutamine to glutamate and their flux between the cytoplasm and mitochondria were notably compromised in hemorrhaged plaques, with a significant reduction in overall glutamate levels in IPH+ plaques. Additionally, reduced glutamate availability was associated with an increased presence of macrophages and a pro-inflammatory phenotype in IPH+ plaques, suggesting an inflammation-prone microenvironment. CONCLUSIONS: This study is the first to establish a robust and comprehensive GEM for atherosclerotic plaque, providing a valuable resource for understanding plaque metabolism. The utility of this GEM was illustrated by its ability to reliably predict dysregulation in the cholesterol hydroxylation, inositol metabolism, and the glutamine/glutamate pathway in rupture-prone hemorrhaged plaques, a finding that may pave the way to new diagnostic or therapeutic measures.

MRPL35 Induces Proliferation, Invasion, and Glutamine Metabolism in NSCLC Cells by Upregulating SLC7A5 Expression.

BACKGROUND: Mitochondrial ribosomal protein L35 (MRPL35) has been reported to contribute to the growth of non-small cell lung cancer (NSCLC) cells. However, the functions and mechanisms of MRPL35 on glutamine metabolism in NSCLC remain unclear. METHODS: The detection of mRNA and protein of MRPL35, ubiquitin-specific protease 39 (USP39), and solute carrier family 7 member 5 (SLC7A5) was conducted using qRT-PCR and western blotting. Cell proliferation, apoptosis, and invasion were evaluated using the MTT assay, EdU assay, flow cytometry, and transwell assay, respectively. Glutamine metabolism was analyzed by detecting glutamine consumption, α-ketoglutarate level, and glutamate production. Cellular ubiquitination analyzed the deubiquitination effect of USP39 on MRPL35. An animal experiment was conducted for in vivo analysis. RESULTS: MRPL35 was highly expressed in NSCLC tissues and cell lines, and high MRPL35 expression predicted poor outcome in NSCLC patients. In vitro analyses suggested that MRPL35 knockdown suppressed NSCLC cell proliferation, invasion, and glutamine metabolism. Moreover, MRPL35 silencing hindered tumor growth in vivo. Mechanistically, USP39 stabilized MRPL35 expression by deubiquitination and then promoted NSCLC cell proliferation, invasion, and glutamine metabolism. In addition, MRPL35 positively affected SLC7A5 expression in NSCLC cells in vitro and in vivo. Moreover, the anticancer effects of MRPL35 silencing could be rescued by SLC7A5 overexpression in NSCLC cells. CONCLUSION: MRPL35 expression was stabilized by USP39-induced deubiquitination in NSCLC cells, and knockdown of MRPL35 suppressed NSCLC cell proliferation, invasion, and glutamine metabolism in vitro and impeded tumor growth in vivo by upregulating SLC7A5, providing a promising therapeutic target for NSCLC.

Therapeutic resurgence of 6-diazo-5-oxo-l-norleucine (DON) through tissue-targeted prodrugs.

The recognition that rapidly proliferating cancer cells rely heavily on glutamine for their survival and growth has renewed interest in the development of glutamine antagonists for cancer therapy. Glutamine plays a pivotal role as a carbon source for synthesizing lipids and metabolites through the TCA cycle, as well as a nitrogen source for synthesis of amino acid and nucleotides. Numerous studies have explored the significance of glutamine metabolism in cancer, providing a robust rationale for targeting this metabolic pathway in cancer treatment. The glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON) has been explored as an anticancer therapeutic for nearly six decades. Initial investigations revealed remarkable efficacy in preclinical studies and promising outcomes in early clinical trials. However, further advancement of DON was hindered due to dose-limiting gastrointestinal (GI) toxicities as the GI system is highly dependent on glutamine for regulating growth and repair. In an effort to repurpose DON and mitigate gastrointestinal (GI) toxicity concerns, prodrug strategies were utilized. These strategies aimed to enhance the delivery of DON to specific target tissues, such as tumors and the central nervous system (CNS), while sparing DON delivery to normal tissues, particularly the GI tract. When administered at low daily doses, optimized for metabolic inhibition, these prodrugs exhibit remarkable effectiveness without inducing significant toxicity to normal tissues. This approach holds promise for overcoming past challenges associated with DON, offering an avenue for its successful utilization in cancer treatment.

Glutamine maintains the stability of alveolar structure and function after lung transplantation by inhibiting autophagy.

Excessive autophagy may lead to degradation and damage of alveolar epithelial cells after lung transplantation, eventually leading to alveolar epithelial cell loss, affecting the structural integrity and function of alveoli. Glutamine (Gln), a nutritional supplement, regulates autophagy through multiple signaling pathways. In this study, we explored the protective role of Gln on alveolar epithelial cells by inhibiting autophagy. In vivo, a rat orthotopic lung transplant model was carried out to evaluate the therapeutic effect of glutamine. Ischemia/reperfusion (I/R) induced alveolar collapse, edema, epithelial cell apoptosis, and inflammation, which led to a reduction of alveolar physiological function, such as an increase in peak airway pressure, and a decrease in lung compliance and oxygenation index. In comparison, Gln preserved alveolar structure and function by reducing alveolar apoptosis, inflammation, and edema. In vitro, a hypoxia/reoxygenation (H/R) cell model was performed to simulate IR injury on mouse lung epithelial (MLE) cells and human lung bronchus epithelial (Beas-2B) cells. H/R impaired the proliferation of epithelial cells and triggered cell apoptosis. In contrast, Gln normalized cell proliferation and suppressed I/R-induced cell apoptosis. The activation of mTOR and the downregulation of autophagy-related proteins (LC3, Atg5, Beclin1) were observed in Gln-treated lung tissues and alveolar epithelial cells. Both in vivo and in vitro, rapamycin, a classical mTOR inhibitor, reversed the beneficial effects of Gln on alveolar structure and function. Taken together, Glnpreserved alveolar structure and function after lung transplantation by inhibiting autophagy.

Quercetin alleviates inflammation induced by porcine reproductive and respiratory syndrome virus in MARC-145 cells through the regulation of arachidonic acid and glutamine metabolism.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) infection causes severe inflammatory response, respiratory disease and sow reproductive failure. Quercetin is among the widely occurring polypheno found abundantly in nature. Quercetin has anti-inflammatory, anti-oxidative and anti-viral properties. OBJECTIVES: This study aimed to explore the effect and mechanism of quercetin on PRRSV-induced inflammation in MARC-145 cells. METHODS: Observing the cytopathic effect and measurements of inflammatory markers in MARC-145 cells collectively demonstrate that quercetin elicits a curative effect on PRRSV-induced inflammation. Liquid chromatography-mass spectrometry was further used for a non-targeted metabolic analysis of the role of quercetin in the metabolic regulation of PRRSV inflammation in MARC-145 cells. RESULTS: It was shown that quercetin attenuated PRRSV-induced cytopathy in MARC-145 cells. Quercetin treatment inhibited PRRSV replication in MARC-145 cells in a dose-dependent manner. We also found that quercetin inhibited PRRSV-induced mRNA expression and secretion levels of tumour necrosis factor-α, interleukin 1ß and interleukin 6. Metabolomics analysis revealed that quercetin ameliorated PRRSV-induced inflammation. Pathway analysis results revealed that PRRSV-induced pathways including arachidonic acid metabolism, linoleic acid, glycerophospholipid and alanine, aspartate and glutamate metabolism were suppressed by quercetin. Moreover, we confirmed that quercetin inhibited the activation of NF-κB/p65 pathway, probably by attenuating PLA2, ALOX and COX mRNA expression. CONCLUSIONS: These results provide a crucial insight into the molecular mechanism of quercetin in alleviating PRRSV-induced inflammation.

Norovirus NS1/2 protein increases glutaminolysis for efficient viral replication.

Viruses are obligate intracellular parasites that rely on host cell metabolism for successful replication. Thus, viruses rewire host cell pathways involved in central carbon metabolism to increase the availability of building blocks for successful propagation. However, the underlying mechanisms of virus-induced alterations to host metabolism are largely unknown. Noroviruses (NoVs) are highly prevalent pathogens that cause sporadic and epidemic viral gastroenteritis. In the present study, we uncovered several strain-specific and shared host cell metabolic requirements of three murine norovirus (MNV) strains, MNV-1, CR3, and CR6. While all three strains required glycolysis, glutaminolysis, and the pentose phosphate pathway for optimal infection of macrophages, only MNV-1 relied on host oxidative phosphorylation. Furthermore, the first metabolic flux analysis of NoV-infected cells revealed that both glycolysis and glutaminolysis are upregulated during MNV-1 infection of macrophages. Glutamine deprivation affected the viral lifecycle at the stage of genome replication, resulting in decreased non-structural and structural protein synthesis, viral assembly, and egress. Mechanistic studies further showed that MNV infection and overexpression of the non-structural protein NS1/2 increased the enzymatic activity of the rate-limiting enzyme glutaminase. In conclusion, the inaugural investigation of NoV-induced alterations to host glutaminolysis identified NS1/2 as the first viral molecule for RNA viruses that regulates glutaminolysis either directly or indirectly. This increases our fundamental understanding of virus-induced metabolic alterations and may lead to improvements in the cultivation of human NoVs.

Including glutamine in a resource allocation model of energy metabolism in cancer and yeast cells.

Energy metabolism is crucial for all living cells, especially during fast growth or stress scenarios. Many cancer and activated immune cells (Warburg effect) or yeasts (Crabtree effect) mostly rely on aerobic glucose fermentation leading to lactate or ethanol, respectively, to generate ATP. In recent years, several mathematical models have been proposed to explain the Warburg effect on theoretical grounds. Besides glucose, glutamine is a very important substrate for eukaryotic cells-not only for biosynthesis, but also for energy metabolism. Here, we present a minimal constraint-based stoichiometric model for explaining both the classical Warburg effect and the experimentally observed respirofermentation of glutamine (WarburQ effect). We consider glucose and glutamine respiration as well as the respective fermentation pathways. Our resource allocation model calculates the ATP production rate, taking into account enzyme masses and, therefore, pathway costs. While our calculation predicts glucose fermentation to be a superior energy-generating pathway in human cells, different enzyme characteristics in yeasts reduce this advantage, in some cases to such an extent that glucose respiration is preferred. The latter is observed for the fungal pathogen Candida albicans, which is a known Crabtree-negative yeast. Further, optimization results show that glutamine is a valuable energy source and important substrate under glucose limitation, in addition to its role as a carbon and nitrogen source of biomass in eukaryotic cells. In conclusion, our model provides insights that glutamine is an underestimated fuel for eukaryotic cells during fast growth and infection scenarios and explains well the observed parallel respirofermentation of glucose and glutamine in several cell types.

HuR controls glutaminase RNA metabolism.

Glutaminase (GLS) is directly related to cell growth and tumor progression, making it a target for cancer treatment. The RNA-binding protein HuR (encoded by the ELAVL1 gene) influences mRNA stability and alternative splicing. Overexpression of ELAVL1 is common in several cancers, including breast cancer. Here we show that HuR regulates GLS mRNA alternative splicing and isoform translation/stability in breast cancer. Elevated ELAVL1 expression correlates with high levels of the glutaminase isoforms C (GAC) and kidney-type (KGA), which are associated with poor patient prognosis. Knocking down ELAVL1 reduces KGA and increases GAC levels, enhances glutamine anaplerosis into the TCA cycle, and drives cells towards glutamine dependence. Furthermore, we show that combining chemical inhibition of GLS with ELAVL1 silencing synergistically decreases breast cancer cell growth and invasion. These findings suggest that dual inhibition of GLS and HuR offers a therapeutic strategy for breast cancer treatment.

Origin and Roles of Alanine and Glutamine in Gluconeogenesis in the Liver, Kidneys, and Small Intestine under Physiological and Pathological Conditions.

Alanine and glutamine are the principal glucogenic amino acids. Most originate from muscles, where branched-chain amino acids (valine, leucine, and isoleucine) are nitrogen donors and, under exceptional circumstances, a source of carbons for glutamate synthesis. Glutamate is a nitrogen source for alanine synthesis from pyruvate and a substrate for glutamine synthesis by glutamine synthetase. The following differences between alanine and glutamine, which can play a role in their use in gluconeogenesis, are shown: (i) glutamine appearance in circulation is higher than that of alanine; (ii) the conversion to oxaloacetate, the starting substance for glucose synthesis, is an ATP-consuming reaction for alanine, which is energetically beneficial for glutamine; (iii) most alanine carbons, but not glutamine carbons, originate from glucose; and (iv) glutamine acts a substrate for gluconeogenesis in the liver, kidneys, and intestine, whereas alanine does so only in the liver. Alanine plays a significant role during early starvation, exposure to high-fat and high-protein diets, and diabetes. Glutamine plays a dominant role in gluconeogenesis in prolonged starvation, acidosis, liver cirrhosis, and severe illnesses like sepsis and acts as a substrate for alanine synthesis in the small intestine. Interactions among muscles and the liver, kidneys, and intestine ensuring optimal alanine and glutamine supply for gluconeogenesis are suggested.

The potential therapeutic targets of glutamine metabolism in head and neck squamous cell carcinoma.

Targeting metabolic reprogramming may be an effective strategy to enhance cancer treatment efficacy. Glutamine serves as a vital nutrient for cancer cells. Inhibiting glutamine metabolism has shown promise in preventing tumor growth both in vivo and in vitro through various mechanisms. Therefore, this review collates recent scientific literature concerning the correlation between glutamine metabolism and cancer treatment. Novel treatment modalities based on amino acid transporters, metabolites, and glutaminase are discussed. Moreover, we demonstrate the relationship between glutamine metabolism and tumor proliferation, drug resistance, and the tumor immune microenvironment, offering new perspectives for the clinical treatment of head and neck squamous cell carcinoma, particularly for combined therapies. Identifying innovative approaches for enhancing the efficacy of glutamine-based metabolic therapy is crucial to improving HNSCC treatment.

How arginine inhibits substrate-binding domain 2 elucidated using molecular dynamics simulations.

The substrate-binding domain 2 (SBD2) is an important part of the bacterial glutamine (GLN) transporter and mediates binding and delivery of GLN to the transporter translocation subunit. The SBD2 consists of two domains, D1 and D2, that bind GLN in the space between domains in a closed structure. In the absence of ligand, the SBD2 adopts an open conformation with larger space between domains. The GLN binding and closing are essential for the subsequent transport into the cell. Arginine (ARG) can also bind to SBD2 but does not induce closing and inhibits GLN transport. We use atomistic molecular dynamics (MD) simulations in explicit solvent to study ARG binding in the presence of the open SBD2 structure and observed reversible binding to the native GLN binding site with similar contacts but no transition to a closed SBD2 state. Absolute binding free energy simulations predict a considerable binding affinity of ARG and GLN to the binding site on the D1 domain. Free energy simulations to induce subsequent closing revealed a strong free energy penalty in case of ARG binding in contrast to GLN binding that favors the closed SBD2 state but still retains a free energy barrier for closing. The simulations allowed the identification of the molecular origin of the closing penalty in case of bound ARG and suggested a mutation of lysine at position 373 to alanine that strongly reduced the penalty and allowed closing even in the presence of bound ARG. The study offers an explanation of the molecular mechanism of how ARG competitively inhibits GLN from binding to SBD2 and from triggering the transition to a closed conformation. The proposed Lys373Ala mutation shows promise as a potential tool to validate whether a conformational mismatch between open SBD2 and the translocator is responsible for preventing ARG uptake to the cell.

Chemical Proteomic Profiling of Protein Dopaminylation in Colorectal Cancer Cells.

Histone dopaminylation is a newly identified epigenetic mark that plays a role in the regulation of gene transcription, where an isopeptide bond is formed between the fifth amino acid of H3 (i.e., glutamine) and dopamine. Recently, we developed a chemical probe to specifically label and enrich histone dopaminylation via bioorthogonal chemistry. Given this powerful tool, we found that histone H3 glutamine 5 dopaminylation (H3Q5dop) was highly enriched in colorectal tumors, which could be attributed to the high expression level of its regulator, transglutaminase 2 (TGM2), in colon cancer cells. Due to the enzyme promiscuity of TGM2, nonhistone proteins have also been identified as dopaminylation targets; however, the dopaminylated proteome in cancer cells still remains elusive. Here, we utilized our chemical probe to enrich dopaminylated proteins from colorectal cancer cells in a bioorthogonal manner and performed the chemical proteomics analysis. Therefore, 425 dopaminylated proteins were identified, many of which are involved in nucleic acid metabolism and transcription pathways. More importantly, a number of dopaminylation sites were identified and attributed to the successful application of our chemical probe. Overall, these findings shed light on the significant association between cellular protein dopaminylation and cancer development, further suggesting that targeting these pathways may become a promising anticancer strategy.

PoRVA G9P[23] and G5P[7] infections differentially promote PEDV replication by reprogramming glutamine metabolism.

PoRVA and PEDV coinfections are extremely common in clinical practice. Although coinfections of PoRVA and PEDV are known to result in increased mortality, the underlying mechanism remains unknown. Here, we found that PoRVA infection promoted PEDV infection in vivo and in vitro and that PoRVA G9P[23] (RVA-HNNY strain) enhanced PEDV replication more significantly than did PoRVA G5P[7] (RVA-SXXA strain). Metabolomic analysis revealed that RVA-HNNY more efficiently induced an increase in the intracellular glutamine content in porcine small intestinal epithelial cells than did RVA-SXXA, which more markedly promoted ATP production to facilitate PEDV replication, whereas glutamine deprivation abrogated the effect of PoRVA infection on promoting PEDV replication. Further studies showed that PoRVA infection promoted glutamine uptake by upregulating the expression of the glutamine transporter protein SLC1A5. In SLC1A5 knockout cells, PoRVA infection neither elevated intracellular glutamine nor promoted PEDV replication. During PoRVA infection, the activity and protein expression levels of glutamine catabolism-related enzymes (GLS1 and GLUD1) were also significantly increased promoting ATP production through glutamine anaplerosis into the TCA cycle. Consistent with that, siRNAs or inhibitors of GLS1 and GLUD1 significantly inhibited the promotion of PEDV replication by PoRVA. Notably, RVA-HNNY infection more markedly promoted SLC1A5, GLS1 and GLUD1 expression to more significantly increase the uptake and catabolism of glutamine than RVA-SXXA infection. Collectively, our findings illuminate a novel mechanism by which PoRVA infection promotes PEDV infection and reveal that the modulation of glutamine uptake is key for the different efficiencies of PoRVA G9P[23] and PoRVA G5P[7] in promoting PEDV replication.

Transcranial electrical stimulation modulates emotional experience and metabolites in the prefrontal cortex in a donation task.

Understanding the neural, metabolic, and psychological mechanisms underlying human altruism and decision-making is a complex and important topic both for science and society. Here, we investigated whether transcranial Direct Current Stimulation (tDCS) applied to two prefrontal cortex regions, the ventromedial prefrontal cortex (vmPFC, anode) and the right dorsolateral prefrontal cortex (DLPFC, cathode) can induce changes in self-reported emotions and to modulate local metabolite concentrations. We employed in vivo quantitative MR Spectroscopy in healthy adult participants and quantified changes in GABA and Glx (glutamate + glutamine) before and after five sessions of tDCS delivered at 2 mA for 20 min (active group) and 1 min (sham group) while participants were engaged in a charitable donation task. In the active group, we observed increased levels of GABA in vmPFC. Glx levels decreased in both prefrontal regions and self-reported happiness increased significantly over time in the active group. Self-reported guiltiness in both active and sham groups tended to decrease. The results indicate that self-reported happiness can be modulated, possibly due to changes in Glx concentrations following repeated stimulation. Therefore, local changes may induce remote changes in the reward network through interactions with other metabolites, previously thought to be unreachable with noninvasive stimulation techniques.

Derivatives of D(-) glutamine-based MMP-2 inhibitors as an effective remedy for the management of chronic myeloid leukemia-Part-I: Synthesis, biological screening and in silico binding interaction analysis.

Chronic myeloid leukemia (CML) is a global issue and the available drugs such as tyrosine kinase inhibitors (TKIs) comprise various toxic effects as well as resistance and cross-resistance. Therefore, novel molecules targeting specific enzymes may unravel a new direction in antileukemic drug discovery. In this context, targeting gelatinases (MMP-2 and MMP-9) can be an alternative option for the development of novel molecules effective against CML. In this article, some D(-)glutamine derivatives were synthesized and evaluated through cell-based antileukemic assays and tested against gelatinases. The lead compounds, i.e., benzyl analogs exerted the most promising antileukemic potential showing nontoxicity in normal cell line including efficacious gelatinase inhibition. Both these lead molecules yielded effective apoptosis and displayed marked reductions in MMP-2 expression in the K562 cell line. Not only that, but both of them also revealed effective antiangiogenic efficacy. Importantly, the most potent MMP-2 inhibitor, i.e., benzyl derivative of p-tosyl D(-)glutamine disclosed stable binding interaction at the MMP-2 active site correlating with the highly effective MMP-2 inhibitory activity. Therefore, such D(-)glutamine derivatives might be explored further as promising MMP-2 inhibitors with efficacious antileukemic profiles for the treatment of CML in the future.

Dietary supplement enriched in antioxidants and omega-3 promotes retinal glutamine synthesis.

To prevent ocular pathologies, new generation of dietary supplements have been commercially available. They consist of nutritional supplement mixing components known to provide antioxidative properties, such as unsaturated fatty acid, resveratrol or flavonoids. However, to date, few data evaluating the impact of a mixture mainly composed of those components (Nutrof Total®) on the retina are available. Only one in-vivo preclinical study demonstrated that dietary supplementation (DS) prevents the retina from light-induced retinal degeneration; and only one in-vitro study on Müller cells culture showed that glutamate metabolism cycle was key in oxidative stress response. Therefore, we raised the question about the in-vivo effect of DS on glutamate metabolism in the retina. Herein, we showed that the dietary supplementation promotes in-vivo increase of retinal glutamine amount through a higher glutamine synthesis as observed in-vitro on Muller cells. Therefore, we can suggest that the promotion of glutamine synthesis is part of the protective effect of DS against retinal degeneration, acting as a preconditioning mechanism against retinal degeneration.

SIRT4 Has an Anti-Cancer Role in Cervical Cancer by Inhibiting Glutamine Metabolism via the MEK/ERK/C-Myc Axis.

BACKGROUND/AIM: Glutamine metabolism is crucial in cell proliferation, aging, and apoptosis across various cancer types. Existing research indicates that Sirtuin 4 (SIRT4), primarily located in mitochondria, modulates this process. This study aimed to clarify the regulatory relationship between SIRT4 and glutamine metabolism in cervical cancer. MATERIALS AND METHODS: SIRT4 mRNA levels and their clinical correlation to cervical cancer were analyzed using the UALCAN database. Immunohistochemistry (IHC) was performed to assess SIRT4 protein expression in tissue samples from cervical cancer patients. Transient transfection was employed to create Hela and Siha cell lines with overexpressed SIRT4, mitogen-activated extracellular signal-regulated kinase (MEK), and glutaminase 1 (GLS1). The impact on cellular functions was studied using MTT, soft agar, transwell, and western blotting assays. Glutamate and ATP levels were also measured to evaluate metabolic changes. RESULTS: Low levels of SIRT4 mRNA in cervical cancer tissues correlated with tumor metastasis and poor survival rates. Overexpression of SIRT4 led to suppressed cell proliferation, colony growth, and motility, along with significant down-regulation of GLS expression, a key contributor to glutamine metabolism. Additionally, SIRT4 overexpression resulted in the inactivation of the MEK/ERK/c-myc signaling pathway, while overexpression of MEK reversed these effects. Notably, the inhibitory effects of SIRT4 on cell proliferation, colony formation, migration, and invasion in Hela and Siha cells were significantly attenuated following GLS1 overexpression. CONCLUSION: SIRT4 acts as an anti-cancer agent in cervical cancer by inhibiting glutamine metabolism through the MEK/ERK/c-myc signaling pathway, providing a novel sight for cervical cancer therapy.

Endoplasmic reticulum stress-dependent regulation of the expression of serine hydroxymethyltransferase 2 in glioblastoma cells.

Objective. Serine hydroxymethyltransferase (SHMT2) plays a multifunctional role in mitochondria (folate-dependent tRNA methylation, translation, and thymidylate synthesis). The endoplasmic reticulum stress, hypoxia, and glucose and glutamine supply are significant factors of malignant tumor growth including glioblastoma. Previous studies have shown that the knockdown of the endoplasmic reticulum to nucleus signaling 1 (ERN1) pathway of endoplasmic reticulum stress strongly suppressed glioblastoma cell proliferation and modified the sensitivity of these cells to hypoxia and glucose or glutamine deprivations. The present study aimed to investigate the regulation of the SHMT2 gene in U87MG glioblastoma cells by ERN1 knockdown, hypoxia, and glucose or glutamine deprivations with the intent to reveal the role of ERN1 signaling in sensitivity of this gene expression to hypoxia and nutrient supply. Methods. The control U87MG glioblastoma cells (transfected by an empty vector) and ERN1 knockdown cells with inhibited ERN1 endoribonuclease and protein kinase (dnERN1) or only ERN1 endoribonuclease (dnrERN1) were used. Hypoxia was introduced by dimethyloxalylglycine (500 ng/ml for 4 h). For glucose and glutamine deprivations, cells were exposed in DMEM without glucose and glutamine, respectively for 16 h. RNA was extracted from cells and reverse transcribed. The expression level of the SHMT2 gene was studied by real-time qPCR and normalized to ACTB. Results. It was found that inhibition of ERN1 endoribonuclease and protein kinase in glioblastoma cells led to a down-regulation of SHMT2 gene expression in U87MG cells. At the same time, the expression of this gene did not significantly change in cells with inhibited ERN1 endoribonuclease, but tunicamycin strongly increased its expression. Moreover, the expression of the SHMT2 gene was not affected in U87MG cells after silencing of XBP1. Hypoxia up-regulated the expression level of the SHMT2 gene in both control and ERN1 knockdown U87MG cells. The expression of this gene was significantly up-regulated in glioblastoma cells under glucose and glutamine deprivations and ERN1 knockdown significantly increased the sensitivity of the SHMT2 gene to these nutrient deprivation conditions. Conclusion. The results of the present study demonstrate that the expression of the SHMT2 gene responsible for serine metabolism and formation of folate one-carbon is controlled by ERN1 protein kinase and induced by hypoxia as well as glutamine and glucose deprivation conditions in glioblastoma cells and reflects the ERN1-mediated reprogramming of sensitivity this gene expression to nutrient deprivation.