Babaodan overcomes cisplatin resistance in cholangiocarcinoma via inhibiting YAP1.

CONTEXT: Cholangiocarcinoma with highly heterogeneous, aggressive, and multidrug resistance has a poor prognosis. Although babaodan (BBD) combined with cisplatin improved non-small cell lung cancer efficacy, its impact on overcoming resistance in cholangiocarcinoma remains unexplored. OBJECTIVE: This study explored the role and mechanism of BBD on cisplatin resistance in cholangiocarcinoma cells (CCAs). MATERIALS AND METHODS: Cisplatin-resistant CCAs were exposed to varying concentrations of cisplatin (25-400 µg/mL) or BBD (0.25-1.00 mg/mL) for 48 h. IC50 values, inhibition ratios, apoptosis levels, DNA damage, glutathione (GSH) levels, oxidized forms of GSH, total GSH content, and glutaminase relative activity were evaluated using the cell counting kit 8, flow cytometry, comet assay, and relevant assay kits. RESULTS: BBD-reduced the cisplatin IC50 in CCAs from 118.8 to 61.83 µg/mL, leading to increased inhibition rate, apoptosis, and DNA damage, and decreased expression of B-cell lymphoma-2, p-Yes-associated protein 1/Yes-associated protein 1, solute carrier family 1 member 5, activating transcription factor 4, and ERCC excision repair 1 in a dose-dependent manner with maximum reductions of 78.97%, 51.98%, 54.03%, 56.59%, and 63.22%, respectively; bcl2-associated X and gamma histone levels were increased by 0.43-115.77% and 22.15-53.39%. The impact of YAP1 knockdown on cisplatin-resistant CCAs resembled BBD. GSH, oxidized GSH species, total GSH content, and glutaminase activity in cisplatin-resistant CCAs with BBD treatment also decreased, while YAP1 overexpression countered BBD's effects. DISCUSSION AND CONCLUSION: This study provides a scientific basis for BBD clinical application and provides a new direction for BBD biological mechanism research.

Improved Photodynamic Therapy Based on Glutaminase Blockage via Tumor Membrane Coated CB-839/IR-780 Nanoparticles.

Photodynamic therapy (PDT) has promising applications. However, the lethal function of reactive oxygen species (ROS) produced during PDT is typically limited. This restriction is induced by oxygen shortage in the tumor microenvironment due to tumor cell hypermetabolism and reductive chemicals overexpression in tumor tissues. Glutamine (Gln) metabolism is crucial for malignancy development and is closely associated with redox. Herein, a novel nanoparticle (NP) named IRCB@M is constructed to boost PDT through dual effects. This NP simultaneously blocks aerobic respiration and inhibits cellular reduced substances by blocking the Gln metabolic pathway. Within the nanocomplex, a photosensitizer (IR-780) and a glutaminase inhibitor (CB-839) are self-assembled and then encapsulated by cancer cell membranes for homologous targeting. The Gln metabolism intervention relieves hypoxia and decreases the levels of nicotinamide adenine dinucleotide phosphate (NADPH) as well as reduced glutathione (GSH) in vitro and in vivo, which are the dual amplification effects on the IR-780-mediated lethal PDT. The antitumor effects against gastric cancer are ultimately evoked in vivo, thus offering a novel concept for enhancing PDT and other ROS-dependent therapeutic approaches.

LncRNA PVT1 Confers Cisplatin Resistance of Esophageal Cancer Cells through Modulating the miR-181a-5p-Glutaminase (GLS) Axis.

Esophageal carcinoma (ESCA) is one of the prevalent malignancies worldwide. Cisplatin (CDDP) is a conventional chemotherapy drug. However, the acquired cisplatin resistance limits its extensively clinical applications. In this study, the roles and underlying mechanisms of lncRNA PVT1 in cisplatin-resistant ESCA are investigated. PVT1 was significantly upregulated in ESCA patient specimens and cell lines. Higher PVT1 level was associated with a poor survival rate of ESCA patients. Silencing PVT1 effectively increased cisplatin sensitivity of ESCA cells. We established cisplatin-resistant ESCA cell line (EC109 CDDP Res) and detected that PVT1 and glutamine metabolism were remarkedly elevated in CDDP-resistant esophageal cancer cells. Bioinformatical analysis and luciferase assay illustrated that PVT1 sponged miR-181a-5p to form a ceRNA network, resulting in the downregulation of miR-181a-5p expression in ESCA cells. Glutaminase (GLS), which is a key enzyme in the glutamine metabolism, was identified and validated as a direct target of miR-181-5p in ESCA cells. Inhibiting glutamine metabolism effectively re-sensitized CDDP-resistant cells. Rescue experiments demonstrated that restoration of miR-181a-5p in PVT1-overexpressing CDDP-resistant ESCA cells successfully overcame the PVT1-promoted cisplatin resistance through targeting GLS. Summarily, our study revealed molecular mechanisms of the lncRNA PVT1-promoted cisplatin resistance in ESCA by modulating the miR-181a-5p-GLS axis.

A Dual-Mechanism Based Nutrient Partitioning Nanoregulator for Enhanced Immunotherapy against Anti-PD-1 Resistant Tumors.

Competitive consumption of nutrients between rapidly proliferating cancer cells and T cells results in an immunosuppressive tumor microenvironment (TME) and nutrient deprivation of T cells, which can cause low response rate and resistance to immunotherapies. In this study, we proposed a dual-mechanism based nutrient partitioning nanoregulator (designated as DMNPN), which can simultaneously regulate the immunosuppressive TME and enhance T cell nutrient availability. DMNPN consists of a charge-reversal biodegradable mesoporous silica, encapsulating glycolysis inhibitor lonidamine, and small interfering RNA against glutaminase. Through inhibiting glycolysis to decrease the lactic acid production and downregulating glutaminase expression to reduce the uptake of glutamine by tumor cells, DMNPN enables effective remodeling of metabolism and nutrient partitioning, which alleviates the immunosuppressive TME and boosts nutrient availability for T cells with enhanced antitumor immunity. Such a nutrient partitioning nanoregulator can effectively inhibit the growth of anti-programmed death receptor 1 (anti-PD-1) resistant tumors and prevent tumor metastasis and recurrence. Overall, this dual-mechanism based nutrient reallocation strategy provides a promising approach for cancer therapy.

Emodin promotes hepatic stellate cell senescence and alleviates liver fibrosis via a nuclear receptor (Nur77)-mediated epigenetic regulation of glutaminase 1.

BACKGROUND AND PURPOSE: Senescence in hepatic stellate cells (HSCs) limits liver fibrosis. Glutaminolysis promotes HSC activation. Here, we investigated how emodin affected HSC senescence involving glutaminolysis. EXPERIMENTAL APPROACH: Senescence, glutaminolysis metabolites, Nur77 nuclear translocation, glutaminase 1 (GLS1) promoter methylation and related signalling pathways were examined in human HSC-LX2 cells using multiple cellular and molecular approaches. Fibrotic mice with shRNA-mediated knockdown of Nur77 were treated with emodin-vitamin A liposome for investigating the mechanisms in vivo. Human fibrotic liver samples were examined to verify the clinical relevance. KEY RESULTS: Emodin upregulated several key markers of senescence and inhibited glutaminolysis cascade in HSCs. Emodin promoted Nur77 nuclear translocation, and knockdown of Nur77 abolished emodin blockade of glutaminolysis and induction of HSC senescence. Mechanistically, emodin facilitated Nur77/DNMT3b interaction and increased GLS1 promoter methylation, leading to inhibited GLS1 expression and blockade of glutaminolysis. Moreover, the glutaminolysis intermediate α-ketoglutarate promoted extracellular signal-regulated kinase (ERK) phosphorylation, which in turn phosphorylated Nur77 and reduced its interaction with DNMT3b. This led to decreased GLS1 promoter methylation and increased GLS1 expression, forming an ERK/Nur77/glutaminolysis positive feedback loop. However, emodin repressed ERK phosphorylation and interrupted the feedback cascade, stimulating senescence in HSCs. Studies in mice showed that emodin-vitamin A liposome inhibited glutaminolysis and induced senescence in HSCs, and consequently alleviated liver fibrosis; but knockdown of Nur77 abrogated these beneficial effects. Similar alterations were validated in human fibrotic liver tissues. CONCLUSIONS AND IMPLICATIONS: Emodin stimulated HSC senescence through interruption of glutaminolysis. HSC-targeted delivery of emodin represented a therapeutic option for liver fibrosis.

Targeting glutaminase is therapeutically effective in ibrutinib-resistant mantle cell lymphoma.

Mantle cell lymphoma (MCL) is an incurable B-cell non-Hodgkin lymphoma characterized by frequent relapses. The development of resistance to ibrutinib therapy remains a major challenge in MCL. We previously showed that glutaminolysis is associated with resistance to ibrutinib. In this study, we confirmed that glutaminase (GLS), the first enzyme in glutaminolysis, is overexpressed in ibrutinib-resistant MCL cells, and that its expression correlates well with elevated glutamine dependency and glutaminolysis. Furthermore, we discovered that GLS expression correlates with MYC expression and the functioning of the glutamine transporter ASCT2. Depletion of glutamine or GLS significantly reduced cell growth, while GLS overexpression enhanced glutamine dependency and ibrutinib resistance. Consistent with this, GLS inhibition by its specific inhibitor telaglenastat suppressed MCL cell growth both in vitro and in vivo. Moreover, telaglenastat showed anti-MCL synergy when combined with ibrutinib or venetoclax in vitro, which was confirmed using an MCL patient-derived xenograft model. Our study provides the first evidence that targeting GLS with telaglenastat, alone or in combination with ibrutinib or venetoclax, is a promising strategy to overcome ibrutinib resistance in MCL.

Glutamine promotes the proliferation of epithelial cells via mTOR/S6 pathway in oral lichen planus.

BACKGROUND: Although abnormal cell proliferation and apoptosis are associated with the pathogenesis of oral lichen planus (OLP), the exactly mechanism of which is not yet known. It has been reported that glutamine (Gln) can promote cell proliferation and inhibit apoptosis of various tumor cells. This study aims to evaluate the effect of Gln metabolism on the balance of proliferation and apoptosis in epithelial cells of OLP. METHODS: Thirty human OLP specimens and 11 normal controls were stained by immunohistochemistry to detect the levels of proliferation and Gln metabolism related proteins. Then, the critical role of Gln in cell proliferation and apoptosis was determined by Gln deprivation or treatment with glutaminase inhibitor (CB-839) to intervene Gln metabolism in human gingival epithelial cells. Cell proliferation was detected using CCK8, p-mTOR and p-S6 proteins were detected using Western Blot, cell apoptosis and cell cycle were detected using flow cytometry, and cell stress was detected using immunofluorescence. RESULTS: Compared with normal controls, OLP specimens showed higher levels of Ki-67 and Gln metabolism-related proteins, including Gln transporter (ASCT2), glutaminase (GLS), and pathway proteins (p-mTOR and p-S6). In vitro, Gln promoted cell proliferation and simultaneously upregulated the activity of mTOR/S6 pathway. Moreover, rapamycin, an mTOR pathway inhibitor, could effectively block the Gln-induced cell proliferation. MHY1485, an mTOR pathway agonist, could effectively reverse the decline of cell proliferation under Gln deprivation. In addition, inhibiting Gln metabolism caused the accumulation of intracellular radical oxygen species (ROS) and induced cell apoptosis. However, N-acetylcysteine reversed this state and then decreased cell apoptosis by eliminating intracellular ROS. CONCLUSION: Gln metabolism is essential to maintain the balance of proliferation and apoptosis in oral epithelial cells, and inhibition of Gln metabolism may have a beneficial effect on OLP treatment.

Targeting Glutaminolysis to Treat Multiple Myeloma: An In Vitro Evaluation of Glutaminase Inhibitors Telaglenastat and Epigallocatechin-3-gallate.

BACKGROUND: Cancer is associated with metabolic changes from increased cell proliferation and growth. Compared to normal differentiated cells, MM cells use the glycolytic pathway even when adequate oxygen is present triggering "Glutamine addiction". OBJECTIVE: To investigate the single and combined effects of epigallocatechin-3-gallate (EGCG) and telaglenastat, a glutaminase inhibitor, on the proliferation and apoptosis of the multiple myeloma cell line KM3/BTZ. METHODS: KM3/BTZ cells were treated with different concentrations of telaglenastat and EGCG alone or in combination to investigate their effect on proliferation and apoptosis using the CCK8 assay, flow cytometry, and western blotting. The Chou-Talalay combination index analysis was used to explore the effect of telaglenastat combined with EGCG, while the Combination Index (CI) was calculated to analyze whether the combination of the two drugs had a synergistic effect. RESULTS: Telaglenastat and EGCG alone as well as in combination (5 µmol/L telaglenastat + 120 µmol/L EGCG) significantly inhibited the proliferation of KM3/BTZ cells compared to the inhibition effect of the control. Additionally, the combined treatment increased the proportion of KM3/BTZ cells in the G2 phase and decreased the proportion of cells in the G1 phase. The apoptosis rate of EGCG alone and the combined treatment was significantly higher than that of the control group. Bax protein expression was highest in the combined treatment group, whereas Bcl-2 expression was lowest, with the combined treatment group having the highest ratio of Bax/Bcl-2. CONCLUSION: Telaglenastat and EGCG act synergistically to inhibit cell proliferation and promote apoptosis in KM3/BTZ cells, possibly by targeting glutamine metabolism and glycolysis.

Overcoming cisplatin resistance of human lung cancer by sinomenine through targeting the miR-200a-3p-GLS axis.

Lung cancer, a malignant disease, is one of the leading causes of patient death. Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. Currently, chemotherapeutic agents such as cisplatin are widely used against lung cancer. However, development of chemoresistance, which led to poor prognosis and low survival rate greatly limited the clinical applications of cisplatin. Sinomenine (SIN) is a bioactive component of sinomenium acutum. Accumulating evidence revealed SIN exhibits potential anti-tumor activities in various types of cancers. However, the precise molecular mechanisms for the sinomenine-induced anti-cancer effects have not been fully elucidated. Here, we assessed the effects of sinomenine on cisplatin sensitivity in NSCLC cells. The combination of SIN with cisplatin showed synergistically inhibitory effects on lung cancer cells by calculating the combination index (CI value) using the Calcusyn 2.0 software. Moreover, we detected that the glutamine metabolism was significantly suppressed by sinomenine treatments in lung cancer cells. Under low glutamine supply, A549 cells showed less sensitivity to sinomenine treatments. Meanwhile, miR-200a-3p was found to be significantly induced by SIN treatments. We demonstrated a suppressive role of miR-200a-3p on glutamine metabolism. Furthermore, miR-200a-3p was downregulated but the glutamine metabolism was significantly hyperactive in A549 cisplatin resistant cells compared with parental cells. Bioinformatical analysis and luciferase assay demonstrated the glutaminase (GLS), a key enzyme of glutamine metabolism, is the direct target of miR-200a-3p in lung cancer cells. Finally, rescue experiments demonstrated that recovery of GLS in miR-200a-3p overexpressing-cisplatin resistant cells successfully overrode the sinomenine-mediated cisplatin sensitization. In summary, this study revealed a new molecular mechanism for the sinomenine-promoted cisplatin sensitization, contributing to investigating the sinomenine-based therapeutic agents against chemoresistant NSCLC.

TGF-ß3 induces lactate production in Sertoli cell through inhibiting the Notch pathway.

BACKGROUNDS: In the testis, spermatocytes and spermatids rely on lactate produced by Sertoli cells (SCs) as energy source. Transforming growth factor-beta 3 (TGF-ß3) is one of the generally accepted paracrine regulatory factors of SC-created blood-testis barrier (BTB), yet its role in SC glycolysis and lactate production still remains unclear. OBJECTIVES: To investigate the effect of TGF-ß3 on glycolysis and lactate production in SCs and determine the role of lethal giant larvae 2 (Lgl2) and Notch signaling activity during this process. MATERIALS AND METHODS: Primary cultured rat SCs and TM4 cells were treated with different concentrations of TGF-ß3. In some experiments, cells were transfected with siRNA specifically targeting Lgl2 and then treated with TGF-ß3 or N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester. Lactate concentration, glucose and glutamine (Gln) consumption in the culture medium, activity of phosphofructokinase (PFK), lactate dehydrogenase (LDH), and glutaminase (Gls), ATP level, oxygen consumption, extracellular acidification, and mitochondrial respiration complex activity were detected using commercial kits. The protein level of Lgl2, LDH, monocarboxylate transporter 4 (MCT4), and activity of Akt, ERK, p38 MAPK, and Notch pathway were detected by Western blot. The stage-specific expression of Jagged1 was examined by immunohistochemistry (IHC) and qPCR after laser capture microdissection. Spermatogenesis in rat testis injected with recombinant Jagged1 (re-Jagged1) was observed by HE staining, and lactate concentration in testis lysate was measured at a different day point after re-Jagged1 treatment. RESULTS: Significant enhancement of lactate concentration was detected in a culture medium of both primary SCs and TM4 cells treated with TGF-ß3 at 3 or 5 ng/ml. Besides, other parameters of glycolysis, that is, glucose and Gln consumption, enzyme activity of PFK, LDH, and Gls displayed different levels of increment in primary SCs and TM4 cells after TGF-ß3 treatment. Mitochondria respiration of SCs was shown to decrease in response to TGF-ß3. Lgl2, MCT4, activity of ERK, and p38 MAPK were up-regulated, whereas Akt and Notch pathway activity were inhibited by TGF-ß3. Silencing of Lgl2 in SCs affected lactate production and attenuated the previous effects of TGF-ß3 on SC glycolysis except for Gln consumption, Gls activity, and activity of Akt, ERK, and p38. N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) treatment in SCs antagonized glycolysis suppression caused by Lgl2-silencing. In vivo analysis revealed a stage-specific expression of Jagged1 in contrary with TGF-ß3. Activating Notch signaling by re-Jagged1 resulted in restorable hypospermatogenesis and lowered lactate level in rat testis. CONCLUSION: TGF-ß3 induces lactate production in SC through up-regulating Lgl2, which weakened the Notch signaling activity and intensified glycolysis in SCs. Thus, besides the known function of TGF-ß3 as the BTB regulator, TGF-ß3-Lgl2-Notch may be considered an important pathway controlling SC glycolysis and spermatogenesis.

Rutin prevents seizures in kainic acid-treated rats: evidence of glutamate levels, inflammation and neuronal loss modulation.

Rutin, a naturally derived flavonoid molecule with known neuroprotective properties, has been demonstrated to have anticonvulsive potential, but the mechanism of this effect is still unclear. The current study aimed to investigate the probable antiseizure mechanisms of rutin in rats using the kainic acid (KA) seizure model. Rutin (50 and 100 mg kg-1) and carbamazepine (100 mg kg-1) were administered daily by oral gavage for 7 days before KA (15 mg kg-1) intraperitoneal (i.p.) injection. Seizure behavior, neuronal cell death, glutamate concentration, excitatory amino acid transporters (EAATs), glutamine synthetase (GS), glutaminase, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluA1 and GluA2, N-methyl-D-aspartate (NMDA) receptor subunits GluN2A and GluN2B, activated astrocytes, and inflammatory and anti-inflammatory molecules in the hippocampus were evaluated. Supplementation with rutin attenuated seizure severity in KA-treated rats and reversed KA-induced neuronal loss and glutamate elevation in the hippocampus. Decreased glutaminase and GluN2B, and increased EAATs, GS, GluA1, GluA2 and GluN2A were observed with rutin administration. Rutin pretreatment also suppressed activated astrocytes, downregulated the protein levels of inflammatory molecules [interleukin-1ß (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), high mobility group Box 1 (HMGB1), interleukin-1 receptor 1 (IL-1R1), and Toll-like receptor-4 (TLR-4)] and upregulated anti-inflammatory molecule interleukin-10 (IL-10) protein expression. Taken together, the results indicate that the preventive treatment of rats with rutin attenuated KA-induced seizures and neuronal loss by decreasing glutamatergic hyperactivity and suppressing the IL-1R1/TLR4-related neuroinflammatory cascade.

The glutamate/GABA system in the retina of male rats: effects of aging, neurodegeneration, and supplementation with melatonin and antioxidant SkQ1.

Glutamate and -aminobutyric acid (GABA) are the most abundant amino acids in the retina. An imbalance of the glutamate/GABA system is involved in the pathogenesis of various neurodegenerative disorders. Here we for the first time analyzed alterations of expression of glutamate- and GABA-synthesizing enzymes, transporters, and relevant receptors in the retina with age in Wistar rats and in senescence-accelerated OXYS rats who develop AMD-like retinopathy. We noted consistent age-dependent expression changes of GABAergic-system proteins (GAD67, GABA-T, and GAT1) in OXYS and Wistar rats: upregulation by age 3 months and downregulation at age 18 months. At a late stage of AMD-like retinopathy in OXYS rats (18 months), there was significant upregulation of glutaminase and downregulation of glutamine synthetase, possibly indicating an increasing level of glutamate in the retina. AMD-like-retinopathy development in the OXYS strain was accompanied by underexpression of glutamate transporter GLAST. Prolonged supplementation with both melatonin and SkQ1 (separately) suppressed the progression of the AMD-like pathology in OXYS rats without affecting the glutamate/GABA system but worsened the condition of the Wistar rat's retina during normal aging. We observed decreasing protein levels of glutamine synthetase, GLAST, and GABAAR1 and an increasing level of glutaminase in Wistar rats. In summary, both melatonin and mitochondrial antioxidant SkQ1 had different effect on the retinal glutamate / GABA in healthy Wistar and senescence-accelerated OXYS rats.

Phosphomimetic Dicer S1016E triggers a switch to glutamine metabolism in gemcitabine-resistant pancreatic cancer.

OBJECTIVE: Dicer is an enzyme that processes microRNAs (miRNAs) precursors into mature miRNAs, which have been implicated in various aspects of cancer progressions, such as clinical aggressiveness, prognosis, and survival outcomes. We previously showed that high expression of Dicer is associated with gemcitabine (GEM) resistance in pancreatic ductal adenocarcinoma (PDAC); thus, in this study, we aimed to focus on how Dicer is involved in GEM resistance in PDAC, including cancer prognosis, cell proliferation, and metabolic regulation. METHODS: We generated stable shRNA knockdown of Dicer in GEM-resistant PANC-1 (PANC-1 GR) cells and explored cell viability by MTT and clonogenicity assays. Metabolomic profiling was employed to investigate metabolic changes between parental cells, PANC-1, and PANC-1 GR cells, and further implied to compare their sensitivity to the glutaminase inhibitor, CB839, and GEM treatments. To identify putative phosphorylation site involves with Dicer and its effects on GEM resistance in PDAC cells, we further generated phosphomimetic or phosphomutant Dicer at S1016 site and examined the changes in drug sensitivity, metabolic alteration, and miRNA regulation. RESULTS: We observed that high Dicer levels in pancreatic ductal adenocarcinoma cells were positively correlated with advanced pancreatic cancer and acquired resistance to GEM. Metabolomic analysis indicated that PANC-1 GR cells rapidly utilised glutamine as their major fuel and increased levels of glutaminase (GLS): glutamine synthetase (GLUL) ratio which is related to high Dicer expression. In addition, we found that phosphomimetic Dicer S1016E but not phosphomutant Dicer S1016A facilitated miRNA maturation, causing an imbalance in GLS and GLUL and resulting in an increased response to GLS inhibitors. CONCLUSION: Our results suggest that phosphorylation of Dicer on site S1016 affects miRNA biogenesis and glutamine metabolism in GEM-resistant pancreatic cancer.

L-Glutaminase Synthesis by Marine Halomonas meridiana Isolated from the Red Sea and Its Efficiency against Colorectal Cancer Cell Lines.

L-glutaminase is an important anticancer agent that is used extensively worldwide by depriving cancer cells of L-glutamine. The marine bacterium, Halomonas meridian was isolated from the Red Sea and selected as the more active L-glutaminase-producing bacteria. L-glutaminase fermentation was optimized at 36 h, pH 8.0, 37 °C, and 3.0% NaCl, using glucose at 1.5% and soybean meal at 2%. The purified enzyme showed a specific activity of 36.08 U/mg, and the molecular weight was found to be 57 kDa by the SDS-PAGE analysis. The enzyme was highly active at pH 8.0 and 37 °C. The kinetics' parameters of Km and Vmax were 12.2 × 10-6 M and 121.95 µmol/mL/min, respectively, which reflects a higher affinity for its substrate. The anticancer efficiency of the enzyme showed significant toxic activity toward colorectal adenocarcinoma cells; LS 174 T (IC50 7.0 µg/mL) and HCT 116 (IC50 13.2 µg/mL). A higher incidence of cell death was observed with early apoptosis in HCT 116 than in LS 174 T, whereas late apoptosis was observed in LS 174 T more than in HCT 116. Also, the L-glutaminase induction nuclear fragmentation in HCT 116 was more than that in the LS 174T cells. This is the first report on Halomonas meridiana as an L-glutaminase producer that is used as an anti-colorectal cancer agent.

Purification, characterization, and anticancer and antioxidant activities of L-glutaminase from Aspergillus versicolor Faesay4.

L-Glutaminase is an amidohydrolase which can act as a vital chemotherapeutic agent against various malignancies. In the present work, L-glutaminase productivity from Aspergillus versicolor Faesay4 was significantly increased by 7.72-fold (from 12.33 ± 0.47 to 95.15 ± 0.89 U/mL) by optimizing submerged fermentation parameters in Czapek's Dox (CZD) medium including an incubation period from 3 (12.33 ± 0.47 U/mL) to 6 days (23.36 ± 0.58 U/mL), an incubation temperature from 30 °C (23.36 ± 0.49 U/mL) to 25 °C (31.08 ± 0.60 U/mL), initial pH from pH 5.0 (8.49 ± 0.21 U/mL)  to pH 7.0 (32.18 ± 0.57 U/mL), replacement of glucose (30.19 ± 0.52 U/mL) by sucrose (48.97 ± 0.67 U/mL) as the carbon source at a concentration of 2.0% (w/v), increasing glutamine concentration as the nitrogen source from 1.0% (w/v, 48.54 ± 0.48 U/mL) to 1.5% (w/v, 63.01 ± 0.60 U/mL), and addition of a mixture of KH2PO4 and NaCl (0.5% w/v for both) to SZD as the metal supplementation (95.15 ± 0.89 U/mL). Faesay4 L-glutaminase was purified to yield total activity 13,160 ± 22.76 (U), specific activity 398.79 ± 9.81 (U/mg of protein), and purification fold 2.1 ± 3.18 with final enzyme recovery 57.22 ± 2.17%. The pure enzyme showed a molecular weight of 61.80 kDa, and it was stable and retained 100.0% of its activity at a temperature ranged from 10 to 40 °C and pH 7.0. In our trials, to increase the enzyme activity by optimizing the assay conditions (which were temperature 60 °C, pH 7.0, substrate glutamine, substrate concentration 1.0%, and reaction time 60 min), the enzyme activity increased by 358.8% after changing the assay temperature from 60 to 30 °C and then increased by 138% after decreasing the reaction time from 60 to 40 min. However, both pH 7.0 and glutamine as the substrate remain the best assay parameters for the L-glutaminase activity. When the glutamine in the assay as the reaction substrate was replaced by asparagine, lysine, proline, methionine, cysteine, glycine, valine, phenylalanine, L-alanine, aspartic acid, tyrosine, and serine, the enzyme lost 23.86%, 29.0%, 31.0%, 48.3%, 50.0%, 73.6%, 74.51%, 80.42%, 82.5%, 83.43%, 88.36%, and 89.78% of its activity with glutamine, respectively. Furthermore, Mn2+, K+, Na+, and Fe3+ were enzymatic activators that increased the L-glutaminase activity by 25.0%, 18.05%, 10.97%, and 8.0%, respectively. Faesay4 L-glutaminase was characterized as a serine protease enzyme as a result of complete inhibition by all serine protease inhibitors (PMSF, benzamidine, and TLCK). Purified L-glutaminase isolated from Aspergillus versicolor Faesay4 showed potent DPPH scavenging activities with IC50 = 50 µg/mL and anticancer activities against human liver (HepG-2), colon (HCT-116), breast (MCF-7), lung (A-549), and cervical (Hela) cancer cell lines with IC50 39.61, 12.8, 6.18, 11.48, and 7.25 µg/mL, respectively.

Insights into Potent Therapeutical Antileukemic Agent L-glutaminase Enzyme Under Solid-state Fermentation: A Review.

AIM AND OBJECTIVE: To review the applications and production studies of reported antileukemic drug L-glutaminase under Solid-state Fermentation (SSF). OVERVIEW: An amidohydrolase that gained economic importance because of its wide range of applications in the pharmaceutical industry, as well as the food industry, is L-glutaminase. The medical applications utilized it as an anti-tumor agent as well as an antiretroviral agent. L-glutaminase is employed in the food industry as an acrylamide degradation agent, as a flavor enhancer and for the synthesis of theanine. Another application includes its use in hybridoma technology as a biosensing agent. Because of its diverse applications, scientists are now focusing on enhancing the production and optimization of L-glutaminase from various sources by both Solid-state Fermentation (SSF) and submerged fermentation studies. Of both types of fermentation processes, SSF has gained importance because of its minimal cost and energy requirement. L-glutaminase can be produced by SSF from both bacteria and fungi. Single-factor studies, as well as multi-level optimization studies, were employed to enhance L-glutaminase production. It was concluded that L-glutaminase activity achieved by SSF was 1690 U/g using wheat bran and Bengal gram husk by applying feed-forward artificial neural network and genetic algorithm. The highest L-glutaminase activity achieved under SSF was 3300 U/gds from Bacillus sp., by mixture design. Purification and kinetics studies were also reported to find the molecular weight as well as the stability of L-glutaminase. CONCLUSION: The current review is focused on the production of L-glutaminase by SSF from both bacteria and fungi. It was concluded from reported literature that optimization studies enhanced L-glutaminase production. Researchers have also confirmed antileukemic and anti-tumor properties of the purified L-glutaminase on various cell lines.

Purification, characterization and anticancer efficiency of L-glutaminase from Aspergillus flavus.

The aim of this work was to purify L-glutaminase from Aspergillus flavus. The enzyme was purified 12.47-fold from a cell-free extract with a final specific activity of 613.3 U/mg and the yield was 51.11%. The molecular weight of the enzyme, as estimated by SDS-PAGE, was found to be 69 kDa. The maximal activity of L-glutaminase was recorded at pH 8 and 40°C. The highest activity was reported towards L-glutamine as substrate, with an apparent Km value of 4.5 mmol and Vmax was 20 Uml-1. The enzyme was activated by Na+ and Co2+, while it was greatly suppressed by iodoacetate, NEM, Zn2+ and Hg2+ at 10 mM. L-glutaminase activity increased with a gradual increase of sodium chloride concentration up to 15%. In vivo, the median lethal dose (LD50) was approximately 39.4 mg/kg body weight after intraperitoneal injection in Sprague Dawley rats. Also, L-glutaminase showed no observed changes in liver and kidney functions and hematological parameters on rates. Purified A. flavus L-glutaminase had neither a cognizable effect on human platelet aggregation nor hemolytic activity. In addition, MTT assay showed that the purified L-glutaminase has a high toxic impact on Hela and Hep G2 cell lines with an IC50 value 18 and 12 µg/ml, respectively, and a moderate cytotoxic effect on HCT-116 and MCF7 cells, with an IC50 value 44 and 58 µg/ml, respectively.

Purification and characterization of l-glutaminase enzyme from camel liver: Enzymatic anticancer property.

l-Glutaminase has gained an important attention as glutamine-depleting enzyme in treatment of various cancers. Therefore, this study aimed to purify, characterize and investigate antitumor activity of l-glutaminase from camel liver mitochondria (CL-Glu), since no available information about CL-Glu from camel. CL-Glu was purified using cell fractionation, ultrafiltration, DEAE-and CM-cellulose chromatography columns. The purified CL-Glu was a monomer with a molecular weight of 70 ± 3 kDa, isoelectric point of 7.2, optimum temperature of 70 °C and it was active over a broad pH range with a pH optimum at pH 8.0. Its activity had a clear dependence on phosphate ions. The studied enzyme showed sigmoidal kinetics, indicated its allosteric behavior with Km of 36 ± 4 mM and Hill coefficient of 1.5 which suggested a positive cooperatively of active sites. The purified l-glutaminase exerted antitumor activity against different cell lines with the highest cytotoxic activity against Hepatocellular carcinoma cell line (HepG-2) with an IC50 value of 152 µg/ml. In conclusion, l-glutaminase was purified from camel liver using simple methods and its unique properties such as stability at both wide pH range and at high temperature along with its relatively low molecular weight, facilitated its usage in medical applications as antitumor drug.

Production, optimization, purification, characterization, and anti-cancer application of extracellular L-glutaminase produced from the marine bacterial isolate.

L-glutaminase from bacterial sources has been proven to be effective and economical agents in cancer therapy, food industry and high-value chemicals like threonine. In the present study, a newly isolated bacterial strain was potentially producing extracellular L-glutaminase, it identified as Bacillus subtilis OHEM11 (MK389501) using the 16S rRNA gene. L-glutaminase production optimized and the optimum factors for production under submerged fermentation were at pH 6.5-7.0 and 35 °C after 28 hr using rhamnose and glutamine as carbon and nitrogen sources, respectively, while bagasse was the best inducer for the production under solid-state fermentation. Ethanol precipitation and ion-exchange chromatography using QFF are the purification steps. L-glutaminase was purified to 2-fold with specific activity 89.78 U/mg and its molecular weight about 54.8 kDa with the alkaline property of the enzyme makes it clear having carcinostatic property; maximum enzyme activity at pH 8.2 and 40 °C and retained about 90% activity for 1 hr. The cytotoxicity effect of L-glutaminase indicated a significant safety on Vero cells with high anticancer activity against NFS-60, HepG-2, and MCF-7 cancer cell lines. The outcomes demonstrated that L-glutaminase could be applied in many biotechnological applications such as pharmaceutical and food processing.

Recent advances in microbial glutaminase production and applications-a concise review.

This article focuses on significant advances in the production and applications of microbial glutaminases and provides insight into the structures of different glutaminases. Glutaminases catalyze the deamidation of glutamine to glutamic acid, and this unique ability forms the basis of their applications in various industries such as pharmaceutical and food organizations. Microbial glutaminases from bacteria, actinomycetes, yeast, and fungi are of greater significance than animal glutaminases because of their stability, affordability, and ease of production. Owing to these notable benefits, they are considered to possess considerable potential in anticancer and antiviral therapy, flavor enhancers in oriental foods, biosensors and in the production of a nutraceutical theanine. This review also aims to fully explore the potential of microbial glutaminases and to set the pace for future prospects.