Bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl Sulfide Research Articles

Necroptosis-Mediated Synergistic Photodynamic and Glutamine-Metabolic Therapy Enabled by a Biomimetic Targeting Nanosystem for Cholangiocarcinoma.

Targeted delivery of glutamine metabolism inhibitors holds promise for cholangiocarcinoma therapy, yet effective delivery vehicles remain a challenge. This study reports the development of a biomimetic nanosystem, termed R-CM@MSN@BC, integrating mesoporous organosilicon nanoparticles with reactive oxygen species-responsive diselenide bonds for controlled release of the glutamine metabolism inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (BPTES) and the photosensitizer Ce6. Erythrocyte membrane coating, engineered with Arg-Gly-Asp (RGD) peptides, not only enhanced biocompatibility but also improved tumor targeting and tissue penetration. Upon laser irradiation, R-CM@MSN@BC executed both photodynamic and glutamine-metabolic therapies, inducing necroptosis in tumor cells and triggering significant immunogenic cell death. Time-of-flight mass cytometry analysis revealed that R-CM@MSN@BC can remodel the immunosuppressive tumor microenvironment by polarizing M1-type macrophages, reducing infiltration of M2-type and CX3CR1+ macrophages, and decreasing T cell exhaustion, thereby increasing the effectiveness of anti-programmed cell death ligand 1 immunotherapy. This strategy proposed in this study presents a viable and promising approach for the treatment of cholangiocarcinoma.

Exploration of mechanism of total triterpenoids from fruits of Chaenomeles speciosa against senescent GES-1 cells induced by D-galactose based on Gln/GLS1/α-KG metabolic axis and mitochondrial apoptosis signaling pathway

Total triterpenoids from the fruits of Chaenomeles speciosa(TCS) are active components in the prevention and treatment of gastric mucosal damage, which have potential anti-aging effects. However, it is still unclear whether TCS can improve gastric aging, especially its molecular mechanism against gastric aging. On this basis, this study explored the effect and mechanism of TCS on senescent GES-1 cells induced by D-galactose(D-gal) to provide scientific data for the clinical use of TCS to prevent gastric aging. GES-1 cells cultured in vitro and those transfected with overexpression GLS1(GLS1-OE) plasmid of glutaminase 1(GLS1) were induced to aging by D-gal, and then TCS and or GLS1 inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide(BPTES) were given. Cell survival rate, positive rate of β-galactosidase(SA-β-gal) staining, mitochondrial membrane potential(MMP), and apoptosis were investigated. GLS1 activity, levels of glutamine(Gln), glutamate(Glu), α-ketoglutarate(α-KG), urea, and ammonia in supernatant and cells were detected by enzyme-linked immunosorbent assay(ELISA) and colorimetric methods. The mRNA and protein expressions of GLS1 and the related genes of the mitochondrial apoptosis signaling pathway were measured by real-time fluorescence quantitative PCR and Western blot. The results manifested that compared with the D-gal model group and GLS1-OE D-gal model group, TCS significantly decreased the SA-β-gal staining positive cell rate and MMP of D-gal-induced senescent GES-1 cells and GLS1-OE senescent GES-1 cells, inhibited the survival of senescent cells, and promoted their apoptosis(P<0.01). It decreased the activity of GLS1 and the content of Gln, Glu, α-KG, urea, and ammonia in supernatant and cell(P<0.01), reduced the concentration of cytochrome C(Cyto C) in mitochondria and the mRNA and protein expressions of GLS1 and proliferating nuclear antigen in cells(P<0.01). The mRNA expression of Bcl-2 and Bcl-xl, the protein expression of pro-caspase-9 and pro-caspase-3, and the ratio of Bcl-2/Bax and Bcl-xl/Bad in cells were decreased(P<0.01). Cyto C concentration in the cytoplasm, the mRNA expressions of Bax, Bad, apoptosis protease activating factor 1(Apaf-1), and protein expressions of cleaved-caspase-9, cleaved-caspase-3, cleaved-PARP-1 were increased(P<0.01). The aforementioned results indicate that TCS can counteract the senescent GES-1 cells induced by D-gal, and its mechanism may be closely related to suppressing the Gln/GLS1/α-KG metabolic axis, activating the mitochondrial apoptosis pathway, and thereby accelerating the apoptosis of the senescent cells and eliminating senescent cells.

Removal of gemcitabine-induced senescent cancer cells by targeting glutaminase1 improves the therapeutic effect in pancreatic ductal adenocarcinoma.

Insufficient cancer treatment can induce senescent cancer cell formation and treatment resistance. The characteristics of induced senescent cancer (iSnCa) cells remain unclear. Pancreatic ductal adenocarcinoma (PDAC) has a low and nondurable response rate to current treatments. Our study aimed to analyze the properties of iSnCa cells and the relationship between cellular senescence and prognosis in PDAC. We evaluated the characteristics of gemcitabine-induced senescent cancer cells and the effect of senescence-associated secretory phenotype (SASP) factors released by iSnCa cells on surrounding PDAC cells. The relationship between cellular senescence and the prognosis was investigated in 50 patients with PDAC treated with gemcitabine-based neoadjuvant chemotherapy. Exposure to 5 ng/mL gemcitabine-induced senescence, decreased proliferation and increased senescence-associated β-galactosidase-cell staining without cell death in PDAC cells; the expression of glutaminase1 (GLS1) and SASP factors also increased and caused epithelial-mesenchymal transition in surrounding PDAC cells. iSnCa cells were selectively removed by the GLS1 inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) through apoptosis induction. Cellular senescence was induced in PDAC cells via insufficient gemcitabine in subcutaneous tumor model mice. GLS1 expression was an independent prognostic factor in patients with PDAC who received gemcitabine-based neoadjuvant chemotherapy. This is the first study to identify the relationship between senescence and GLS1 in PDAC. Low-dose gemcitabine-induced senescence and increased GLS1 expression were observed in PDAC cells. Cellular senescence may contribute to treatment resistance of PDAC, hence targeting GLS1 in iSnCa cells may improve the therapeutic effect.

The Glutaminase-1 Inhibitor [11C-carbony]BPTES: Synthesis and Positron Emission Tomography Study in Mice.

Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) is a selective inhibitor of glutaminase-1 (GLS1), consequently inhibiting glutaminolysis. BPTES is known for its potent antitumor activity and plays a significant role in senescent cell removal. In this study, we synthesized [11C-carbonyl]BPTES ([11C]BPTES) as a positron emission tomography (PET) probe for the first time and assessed its biodistribution in mice using PET. [11C]BPTES was synthesized by the reaction of an amine precursor () with [11C-carbonyl]phenylacetyl acid anhydride ([11C]2), which was prepared from [11C]CO2 and benzyl magnesium chloride, followed by in situ treatment with isobutyl chloroformate. The decay-corrected isolated radiochemical yield of [11C]BPTES was 9.5% (based on [11C]CO2) during a synthesis time of 40 min. A PET study with [11C]BPTES showed high uptake levels of radioactivity in the liver, kidney, and small intestine of mice.

A photodynamic-mediated glutamine metabolic intervention nanodrug for triple negative breast cancer therapy.

"Glutamine addiction" is a unique feature of triple negative breast cancer (TNBC), which has a higher demand for glutamine and is more susceptible to glutamine depletion. Glutamine can be hydrolyzed to glutamate by glutaminase (GLS) for synthesis of glutathione (GSH), which is an important downstream of glutamine metabolic pathways in accelerating TNBC proliferation. Consequently, glutamine metabolic intervention suggests potential therapeutic effects against TNBC. However, the effects of GLS inhibitors are hindered by glutamine resistance and their own instability and insolubility. Therefore, it is of great interest to harmonize glutamine metabolic intervention for an amplified TNBC therapy. Unfortunately, such nanoplatform has not been realized. Herein, we reported a self-assembly nanoplatform (BCH NPs) with a core of the GLS inhibitor Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (BPTES) and photosensitizer Chlorin e6 (Ce6) and a shell of human serum albumin (HSA), enabling effective harmonization of glutamine metabolic intervention for TNBC therapy. BPTES inhibited the activity of GLS to block the glutamine metabolic pathways, thereby inhibiting the production of GSH to amplify the photodynamic effect of Ce6. While Ce6 not only directly killed tumor cells by producing excessive reactive oxygen species (ROS), but also deplete GSH to destroy redox balance, thus enhancing the effects of BPTES when glutamine resistance occurred. BCH NPs effectively eradicated TNBC tumor and suppressed tumor metastasis with favorable biocompatibility. Our work provides a new insight for photodynamic-mediated glutamine metabolic intervention against TNBC.

Glutamine metabolism targeting liposomes for synergistic chemosensitization and starvation therapy in ovarian cancer.

Platinum-based chemotherapy is a first-line therapeutic regimen against ovarian cancer (OC); however, the therapeutic potential is always reduced by glutamine metabolism. Herein, a valid strategy of inhibiting glutamine metabolism was proposed to cause tumor starvation and chemosensitization. Specifically, reactive oxygen species-responsive liposomes were developed to co-deliver cisplatin (CDDP) and bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (BPTES) [C@B LPs]. The C@B LPs induced effective tumor cell starvation and significantly sensitized OC cells to CDDP by reducing glutathione generation to prevent CDDP detoxification, suppressing ATP production to avoid CDDP efflux, hindering nucleotide synthesis to aggravate DNA damage induced by CDDP, and blocking mammalian target of rapamycin (mTOR) signaling to promote cell apoptosis. More importantly, C@B LPs remarkably inhibited tumor growth in vivo and reduced the side effects. Taken together, this study provided a successful strategy of synergistic chemosensitization and starvation therapy escalating the rate of therapeutic success in OCs. STATEMENT OF SIGNIFICANCE: This work proposed a valid strategy of inhibiting glutamine metabolism to cause tumor starvation and chemosensitization. Specifically, ROS-responsive liposomes were developed to co-deliver cisplatin CDDP and BPTES [C@B LPs]. The C@B LPs induced effective tumor cell starvation and significantly sensitized OC cells to cisplatin by reducing glutathione generation to prevent cisplatin detoxification, suppressing ATP production to avoid cisplatin efflux, hindering nucleotide synthesis to aggravate DNA damage induced by cisplatin, and blocking mTOR signaling to promote cell apoptosis. More importantly, C@B LPs remarkably inhibited tumor growth in vivo and reduced the side effects. Taken together, this study provided a successful strategy of synergistic chemosensitization and starvation therapy escalating the rate of therapeutic success in OCs.

Endogenous glutamine is rate-limiting for anti-CD3 and anti-CD28 induced CD4+ T-cell proliferation and glycolytic activity under hypoxia and normoxia

To meet the demand for energy and biomass, T lymphocytes (T cells) activated to proliferation and clonal expansion, require uptake and metabolism of glucose (Gluc) and the amino acid (AA) glutamine (Gln). Whereas exogenous Gln is converted to glutamate (Glu) by glutaminase (GLS), Gln is also synthesized from the endogenous pool of AA through Glu and activity of glutamine synthase (GS). Most of this knowledge comes from studies on cell cultures under ambient oxygen conditions (normoxia, 21% O2). However, in vivo, antigen induced T-cell activation often occurs under moderately hypoxic (1–4% O2) conditions and at various levels of exogenous nutrients. Here, CD4+ T cells were stimulated for 72 h with antibodies targeting the CD3 and CD28 markers at normoxia and hypoxia (1% O2). This was done in the presence and absence of the GLS and GS inhibitors, Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (BPTES) and methionine sulfoximine (MSO) and at various combinations of exogenous Gluc, Gln and pyruvate (Pyr) for the last 12 h of stimulation. We found that T-cell proliferation, viability and levels of endogenous AA were significantly influenced by the availability of exogenous Gln, Gluc and Pyr as well as inhibition of GLS and GS. Moreover, inhibition of GLS and GS and levels of oxygen differentially influenced oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Finally, BPTES-dependent down-regulation of ECAR was associated with reduced hexokinase (HK) activity at both normoxia and hypoxia. Our results demonstrate that Gln availability and metabolism is rate-limiting for CD4+ T-cell activity.

Circ-CREBBP promotes cell tumorigenesis and glutamine catabolism in glioma by regulating miR-375/glutaminase axis

Circular RNA CREB-binding protein (circ-CREBBP) has been reported to involve in the tumorigenesis of glioma. However, the role and underlying molecular mechanism of circ-CREBBP in glioma glutamine catabolism remain unclear. The expression of circ-CREBBP, microRNA (miR)-375 and glutaminase (GLS) was detected using quantitative real-time polymerase chain reaction and western blot. The 3‑(4, 5‑dimethylthiazol‑2‑y1)‑2, 5‑diphenyl tetrazolium bromide (MTT), colony formation, flow cytometry and transwell assays were used to determine the effects of them on glioma cell malignant biological behaviors in vitro. Glutamine metabolism was analyzed using assay kits. Murine xenograft model was established to investigate the role of circ-CREBBP in vivo. The binding interactions between miR-375 and circ-CREBBP or GLS were confirmed by the dual-luciferase reporter assay. Circ-CREBBP was highly expressed in glioma tissues and cells, and high expression of circ-CREBBP predicted poor prognosis. Circ-CREBBP knockdown suppressed cell proliferation, migration, invasion and glutamine metabolism while expedited cell apoptosis in glioma in vitro, as well as impeded tumor growth in vivo. Circ-CREBBP directly targeted miR-375, which was demonstrated to restrain glioma cell growth, motility and glutamine metabolism. Moreover, miR-375 inhibition reverted the anticancer effects of circ-CREBBP knockdown on glioma cells. GLS was a target of miR-375, GLS silencing or the treatment of GLS inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) impaired glioma cell malignant phenotypes and glutamine metabolism. Importantly, GLS up-regulation weakened the tumor-suppressive functions of miR-375 on glioma cells. Mechanistically, circ-CREBBP indirectly regulated GLS expression through sponging miR-375. In all, circ-CREBBP expedited glioma tumorigenesis and glutamine metabolism through miR-375/GLS axis, suggesting a promising target for combined glioma therapy.

α-ketoglutarate promotes the specialization of primordial germ cell-like cells through regulating epigenetic reprogramming.

There is growing evidence that cellular metabolism can directly participate in epigenetic dynamics and consequently modulate gene expression. However, the role of metabolites in activating the key gene regulatory network for specialization of germ cell lineage remains largely unknown. Here, we identified some cellular metabolites with significant changes by untargeted metabolomics between mouse epiblast-like cells (EpiLCs) and primordial germ cell-like cells (PGCLCs). More importantly, we found that inhibition of glutaminolysis by bis-2- (5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (BPTES) impeded PGCLC specialization, but the impediment could be rescued by addition of α-ketoglutarate (αKG), the intermediate metabolite of oxidative phosphorylation and glutaminolysis. Moreover, adding αKG alone to the PGCLC medium accelerated the PGCLC specialization through promoting H3K27me3 demethylation. Thus, our study reveals the importance of metabolite αKG in the germ cell fate determination and highlights the essential role of cellular metabolism in shaping the cell identities through epigenetic events.

Insulin Promotes Mitochondrial Respiration and Survival through PI3K/AKT/GSK3 Pathway in Human Embryonic Stem Cells.

SummaryInsulin is an essential growth factor for the survival and self-renewal of human embryonic stem cells (hESCs). Although it is best known as the principal hormone promoting glycolysis in somatic cells, insulin's roles in hESC energy metabolism remain unclear. In this report, we demonstrate that insulin is essential to sustain hESC mitochondrial respiration that is rapidly decreased upon insulin removal. Insulin-dependent mitochondrial respiration is stem cell specific, and mainly relies on pyruvate and glutamine, while glucose suppresses excessive oxidative phosphorylation. Pharmacologic and genetic manipulations reveal that continuous insulin signal sustains mitochondrial respiration through PI3K/AKT activation and downstream GSK3 inhibition. We further show that insulin acts through GSK3 inhibition to suppress caspase activation and rescue cell survival. This study uncovers a critical role of the AKT/GSK3 pathway in the regulation of mitochondrial respiration and cell survival, highlighting insulin as an essential factor for accurate assessment of mitochondrial respiration in hESCs.

Targeting Endothelial Cell Metabolism by Inhibition of Pyruvate Dehydrogenase Kinase and Glutaminase-1.

Targeting endothelial cell (EC) metabolism should impair angiogenesis, regardless of how many angiogenic signals are present. The dependency of proliferating ECs on glucose and glutamine for energy and biomass production opens new opportunities for anti-angiogenic therapy in cancer. The aim of the present study was to investigate the role of pyruvate dehydrogenase kinase (PDK) inhibition with dichloroacetate (DCA), alone or in combination with the glutaminase-1 (GLS-1) inhibitor, Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (BPTES), on Human umbilical vein endothelial cells (HUVECs) metabolism, proliferation, apoptosis, migration, and vessel formation. We demonstrated that both drugs normalize HUVECs metabolism by decreasing glycolysis for DCA and by reducing glutamate production for BPTES. DCA and BPTES reduced HUVECs proliferation and migration but have no impact on tube formation. While DCA increased HUVECs respiration, BPTES decreased it. Using both drugs in combination further reduced HUVECs proliferation while normalizing respiration and apoptosis induction. Overall, we demonstrated that DCA, a metabolic drug under study to target cancer cells metabolism, also affects tumor angiogenesis. Combining DCA and BPTES may reduce adverse effect of each drug alone and favor tumor angiogenesis normalization.

Allosteric kidney-type glutaminase (GLS) inhibitors with a mercaptoethyl linker

A series of allosteric kidney-type glutaminase (GLS) inhibitors possessing a mercaptoethyl (SCH2CH2) linker were synthesized in an effort to further expand the structural diversity of chemotypes derived from bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES), a prototype allosteric inhibitor of GLS. BPTES analog 3a with a mercaptoethyl linker between the two thiadiazole rings was found to potently inhibit GLS with an IC50 value of 50 nM. Interestingly, the corresponding derivative with an n-propyl (CH2CH2CH2) linker showed substantially lower inhibitory potency (IC50 = 2.3 μM) while the derivative with a dimethylsulfide (CH2SCH2) linker showed no inhibitory activity at concentrations up to 100 μM, underscoring the critical role played by the mercaptoethyl linker in the high affinity binding to the allosteric site of GLS. Additional mercaptoethyl-linked compounds were synthesized and tested as GLS inhibitors to further explore SAR within this scaffold including derivatives possessing a pyridazine as a replacement for one of the two thiadiazole moiety.

Embryonic Heat Conditioning Induces TET-Dependent Cross-Tolerance to Hypothalamic Inflammation Later in Life.

Early life encounters with stress can lead to long-lasting beneficial alterations in the response to various stressors, known as cross-tolerance. Embryonic heat conditioning (EHC) of chicks was previously shown to mediate resilience to heat stress later in life. Here we demonstrate that EHC can induce cross-tolerance with the immune system, attenuating hypothalamic inflammation. Inflammation in EHC chicks was manifested, following lipopolysaccharide (LPS) challenge on day 10 post-hatch, by reduced febrile response and reduced expression of LITAF and NFκB compared to controls, as well as nuclear localization and activation of NFκB in the hypothalamus. Since the cross-tolerance effect was long-lasting, we assumed that epigenetic mechanisms are involved. We focused on the role of ten-eleven translocation (TET) family enzymes, which are the mediators of active CpG demethylation. Here, TET transcription during early life stress was found to be necessary for stress resilience later in life. The expression of the TET family enzymes in the midbrain during conditioning increased in parallel to an elevation in concentration of their cofactor α-ketoglutarate. In-ovo inhibition of TET activity during EHC, by the α-ketoglutarate inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (BPTES), resulted in reduced total and locus specific CpG demethylation in 10-day-old chicks and reversed both thermal and inflammatory resilience. In addition, EHC attenuated the elevation in expression of the stress markers HSP70, CRHR1, and CRHR2, during heat challenge on day 10 post-hatch. This reduction in expression was reversed by BPTES. Similarly, the EHC-dependent reduction of inflammatory gene expression during LPS challenge was eliminated in BPTES-treated chicks. Thus, TET family enzymes and CpG demethylation are essential for the embryonic induction of stress cross-tolerance in the hypothalamus.

Glutaminase 1 Inhibition Reduces Glycolysis and Ameliorates Lupus-like Disease in MRL/lpr Mice and Experimental Autoimmune Encephalomyelitis.

Glutaminase 1 (Gls1) is the first enzyme in glutaminolysis. The selective Gls1 inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) suppresses Th17 development and ameliorates experimental autoimmune encephalomyelitis (EAE). The present study was undertaken to investigate whether inhibition of glutaminolysis is beneficial for the treatment of systemic lupus erythematosus (SLE), and the involved mechanisms. MRL/lpr mice were treated with BPTES or vehicle control, and disease activity was examined. Then naive CD4+ T cells from patients with SLE were cultured under Th17-polarizing conditions with BPTES or vehicle. Furthermore, using newly generated Gls1 conditional-knockout mice, in vitro Th17 differentiation was examined, and EAE was induced in the mice. Glutaminolysis and glycolysis were measured with an extracellular flux analyzer. The expression of hypoxia-inducible factor 1α (HIF-1α) was examined by Western blotting. Treatment of MRL/lpr mice with BPTES improved autoimmune pathology in a Th17-dependent manner. T cells from patients with SLE treated with BPTES displayed decreased Th17 differentiation (P < 0.05). Using the conditional-knockout mice, we demonstrated that both in vitro Th17 differentiation (P < 0.05) and the development of EAE were dependent on Gls1. Gls1 inhibition reduced glycolysis and the expression of HIF-1α protein, which induces glycolysis. We demonstrated that inhibition of glutaminolysis represents a potential new treatment strategy for patients with SLE and Th17-related autoimmune diseases. Mechanistically, we have shown that inhibition of glutaminolysis affects the glycolysis pathway by reducing HIF-1α protein in Th17 cells.

Spontaneous colitis in IL-10-deficient mice was ameliorated via inhibiting glutaminase1.

Immunity imbalance and barrier damage in the intestinal mucosa are the main pathogenic factors of Crohn's disease (CD). Bis‐2‐(5‐phenylacetamido‐1,2,4‐thiadiazol‐2‐yl) ethyl sulfide (BPTES) is a glutaminase 1 (Gls1) inhibitor with the dual functions of increasing glutamine levels and immune regulation. In this study, we focused on the role of BPTES in CD‐like enteritis and the possible mechanisms. We found that Gls1 expression was significantly increased in CD intestinal tissue compared with control tissue. Bis‐2‐(5‐phenylacetamido‐1,2,4‐thiadiazol‐2‐yl) ethyl sulfide treatment significantly ameliorated chronic colitis in the IL‐10−/−, as manifested by decreased disease activity index, body weight change, histological inflammatory degree and inflammatory cytokine expression. Bis‐2‐(5‐phenylacetamido‐1,2,4‐thiadiazol‐2‐yl) ethyl sulfide treatment exerted protective effects on CD that were associated with the maintenance of intestinal barrier integrity and the Th/Treg balance. Bis‐2‐(5‐phenylacetamido‐1,2,4‐thiadiazol‐2‐yl) ethyl sulfide treatment may act in part through TCR‐mediated mammalian target of rapamycin complex 1 (mTORC1) signalling activation. In conclusion, inhibition of Gls1 expression attenuated chronic colitis by maintaining intestinal barrier integrity and the Th/Treg balance, thereby ameliorating CD‐like colitis.

LincRNA-p21 suppresses glutamine catabolism and bladder cancer cell growth through inhibiting glutaminase expression.

Long intergenic non-coding RNA p21 (lincRNA-p21) is down-regulated in some solid tumors. Glutamine catabolism plays an important role in cancer development. However, the role of lincRNA-p21 and its association with glutamine catabolism remain unknown in bladder cancer (BC). In the present study, we investigated the involvement of lincRNA-p21 and glutamine catabolism in BC cell growth and found that ectopic linRNA-p21 expression reduced the proliferation and growth of BIU87 and 5637 cells. Opposite results were observed in lincRNA-p21 silenced J82 and T24 cells. The expression of glutaminase (GLS), intracellular level of glutamate and α-Ketoglutarate (α-KG) were negatively regulated by lincRNA-p21. GLS overexpression reversed the suppressive function of lincRNA-p21 on BC cell growth and proliferation. In contrast, GLS reduction by siRNA blunted the viability of lincRNA-p21 lowly expressed BC cells. Furthermore, lincRNA-p21 and GLS abundance dictated the sensitivity of BC cells to bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) treatment. Importantly, reduced lincRNA-p21 expression and increased GLS mRNA level were observed in BC tissues compared with the normal tissues. Our results demonstrate that lincRNA-p21 suppresses the BC cell growth through inhibiting GLS and glutamine catabolism. Targeting this cascade may be a promising treatment strategy for BC patients.

Targeting cellular metabolism to reduce head and neck cancer growth

Head and neck squamous cell carcinoma (HNSCC) presents a major public health concern because of delayed diagnosis and poor prognosis. Malignant cells often reprogram their metabolism in order to promote their survival and proliferation. Aberrant glutaminase 1 (GLS1) expression enables malignant cells to undergo increased glutaminolysis and utilization of glutamine as an alternative nutrient. In this study, we found a significantly elevated GLS1 expression in HNSCC, and patients with high expression levels of GLS1 experienced shorter disease-free periods after therapy. We hypothesized that the GLS1 selective inhibitor, bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES), which curtails cells’ glutamine consumption, may inhibit HNSCC cell growth. Our results support the idea that BPTES inhibits HNSCC growth by inducing apoptosis and cell cycle arrest. Considering that metformin can reduce glucose consumption, we speculated that metformin would enhance the anti-neoplasia effect of BPTES by suppressing malignant cells’ glucose utilization. The combination of both compounds exhibited an additive inhibitory effect on cancer cell survival and proliferation. All of our data suggest that GLS1 is a promising therapeutic target for HNSCC treatment. Combining BPTES with metformin might achieve improved anti-cancer effects in HNSSC, which sheds light on using novel therapeutic strategies by dually targeting cellular metabolism.

Glutamine Deprivation Enhances Acetyl-CoA Carboxylase Inhibitor-induced Death of Human Pancreatic Cancer Cells.

Acetyl-CoA carboxylase (ACC) is a rate-limiting enzyme in fatty acid synthesis. In this study, we investigated the effect of ACC inhibition on survival of pancreatic cancer cells. AsPC-1, BxPC-3 and PANC-1 were used as human pancreatic cancer cell lines. 5-(etradecyloxy)-2-furoic acid (TOFA) and bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl) ethyl sulfide (BPTES) were used as inhibitors of ACC and glutaminase (GLS) respectively. Apoptotic and live cells were distinguished by annexin-V staining. The activity of caspase-3 was evaluated by measuring the fluorescence intensity of the degradation product of the substrate, N-acetyl-Asp-Glu-Val-Asp-7-amido-4-trifluoromethylcoumarin. TOFA increased the number of annexin V-positive cells and enhanced caspase-3 activity in AsPC-1 and BxPC-3, but not in PANC-1 cells. The number of PANC-1 cells increased after 48 h in Earle's balanced salt solution. Interestingly, proliferation of PANC-1 cells was drastically suppressed by glutamine deprivation, but not by inhibition of glycolysis. BPTES also induced cell death to the same extent as glutamine deprivation. In addition, TOFA induced cell death of PANC-1 cells, both in the presence of BPTES and with glutamine deprivation, suggesting that inhibition of glutaminolysis causes cell death and enhances the effect of TOFA in PANC-1 cells. These findings suggest that glutaminolysis is important for the survival of pancreatic cancer cells showing tolerance to nutrient starvation such as PANC-1 cells, and use of a combination of inhibitors of ACC and GLS may be a new strategy for treatment of pancreatic cancer.

Molecular targeting of glutaminase sensitizes ovarian cancer cells to chemotherapy.

Altered metabolism is a reemerging hallmark of tumorigenesis. Increased cell proliferation results in metabolic reprogramming to facilitate the needs of the rapidly dividing tumor cells. In addition to increased glucose uptake, tumors also take up increased levels of glutamine. Some cancers develop a reliance on glutamine, and are referred to as "glutamine addicted." These tumors over express the enzyme glutaminase which is involved in the first step of glutaminolysis. The goal of this study was to determine the effects of combined treatment of the glutaminase inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (BPTES) with chemotherapy on drug resistant ovarian cancer cells. We found that ovarian cancer cells show different dependencies on exogenous glutamine. However, regardless of glutamine dependence status, treatment with BPTES sensitized both paclitaxel, and cisplatin resistant cancer cell lines to chemotherapy by inhibiting cell proliferation. Monotherapy with BPTES alone resulted in a significant reduction in the ability of glutamine dependent cancer cells to form colonies in a clonogenic assay. In addition, glutamine dependent, metastatic cancer cells expressed higher levels of glutaminase 1 (GLS1) isoforms, KGA and GAC, than untransformed cells. Moreover, dual targeting of both isoforms using siRNA was more effective at sensitizing the cancer cells to cisplatin than targeting either GAC or KGA alone. Our results suggest that both GLS1 isoforms are important for glutamine dependent ovarian cancer survival, hence, both GLS1 isoforms should be targeted for therapy in metastatic ovarian cancer therapy.

Glutaminase 1 regulates the release of extracellular vesicles during neuroinflammation through key metabolic intermediate alpha-ketoglutarate

BackgroundExtracellular vesicles (EVs) are important in the intercellular communication of the central nervous system, and their release is increased during neuroinflammation. Our previous data demonstrated an increased release of EVs during HIV-1 infection and immune activation in glial cells. However, the molecular mechanism by which infection and inflammation increase EV release remains unknown. In the current study, we investigated the role of glutaminase 1 (GLS1)-mediated glutaminolysis and the production of a key metabolic intermediate α-ketoglutarate on EV release.MethodsHuman monocyte-derived macrophage primary cultures and a BV2 microglia cell line were used to represent the innate immune cells in the CNS. Transmission electron microscopy, nanoparticle tracking analysis, and Western blots were used to determine the EV regulation. GLS1 overexpression was performed using an adenovirus vector in vitro and transgenic mouse models in vivo. Data were evaluated statistically by ANOVA, followed by the Bonferroni post-test for paired observations.ResultsOur data revealed an increased release of EVs in GLS1-overexpressing HeLa cells. In HIV-1-infected macrophages and immune-activated microglia BV2 cells, treatment with bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) or CB839, two specific GLS inhibitors, significantly decreased EV release, suggesting a critical role of GLS1 in EV release. Furthermore, addition of α-ketoglutarate or ceramide rescued EV release during BPTES treatment, implicating α-ketoglutarate and ceramide as critical downstream effectors for GLS inhibitors. These findings were further corroborated with the investigation of brain tissues in GLS1-transgenic mice. The EV levels were significantly higher in GLS1 transgenic mice than those in control mice, suggesting that GLS1 increases EV release in vivo.ConclusionsThese findings suggest that GLS1-mediated glutaminolysis and its downstream production of α-ketoglutarate are essential in regulating EV release during HIV-1 infection and immune activation. These new mechanistic regulations may help understand how glutamine metabolism shapes EV biogenesis and release during neuroinflammation.