Decreased Lactate Production Research Articles

FSMP-02. CHANGES IN GLUTAMINE METABOLISM INDUCED BY OXALOACETATE IN GLIOBLASTOMA

Anhydrous Enol-Oxaloacetate (AEO) has been shown to significantly increase survival and decrease tumor growth rates in animal models of glioblastoma and hepatocellular carcinoma. In the body, AEO is metabolized to “oxaloacetate” (OAA). Earlier, we demonstrated that AEO drastically reduced Warburg glycolysis in glioblastoma cells which was determined by the increase in pyruvate to lactate ratio and a 48.8% decrease in lactate production in 13C-labeled glucose metabolism studies. We have expanded this previous work to examine 13C-labeled glutamine metabolism. Cultured solid tumor cancer cells strongly rely on both glucose and glutamine to synthesize carbon intermediates for anaplerotic reactions. With treatment of OAA, we hypothesize that glutamine-derived OAA may be reduced which can be tracked through the use GC-MS based 13C-labeled glutamine isotopomer experiments. Patient-derived glioblastoma cells were grown in 15 mM glucose and 2 mM glutamine containing DMEM medium supplemented with 2 mM OAA for 10 days. 24 hours prior to harvesting the cells, 4 mM of [U-13C]glutamine was introduced to the medium. OAA treated cells showed significant decrease in the protein levels of lactate dehydrogenase A and C which indicates the switching off of glycolysis to support the utilization of elevated OAA levels during glutamine metabolism in the TCA cycle. 13C mass distribution analysis showed significant decrease in malate, aspartate and citrate pools (malate: 8.8%, p = 0.0098; aspartate: 9.2%, p = 0.0064; citrate: 9.5%, p = 0.0036) in their M+4 isotopomer labeling in OAA treated group compared to the control group. Decrease in lactate generation may reduce cancer proliferation, migration and invasion. Together, these data provide alternative way to modulate energy metabolism in glioblastoma using AEO treatment. Similarly, AEO treatment in other solid tumors (e.g. pancreatic ductal adenocarcinoma) may produce altered glutamine metabolism which may be of therapeutic value.

NAMPT promotes hepatitis B virus replication and liver cancer cell proliferation through the regulation of aerobic glycolysis.

Nicotinamide phosphoribosyltransferase (NAMPT) is a critical rate-limiting enzyme involved in NAD synthesis that has been shown to contribute to the progression of liver cancer. However, the potential role and mechanism of NAMPT in hepatitis B virus (HBV)-associated liver cancer remain unclear. The present study assessed the expression of NAMPT in HBV-positive and -negative liver cancer cells, and investigated whether HBV-induced NAMPT expression is dependent on HBV X protein (HBx). In addition, the role of NAMPT in HBV replication and transcription, and in HBV-mediated liver cancer cell growth was explored. The effects of NAMPT on the glycolytic pathway were also evaluated. Reverse transcription-quantitative PCR and western blotting results revealed that NAMPT expression levels were significantly higher in HBV-positive liver cancer cells than in HBV-negative liver cancer cells, and this effect was HBx-dependent. Moreover, the activation of NAMPT was demonstrated to be required for HBV replication and transcription. The NAMPT inhibitor FK866 repressed cell survival and promoted cell death in HBV-expressing liver cancer cells, and these effects were attenuated by nicotinamide mononucleotide. Furthermore, the inhibition of NAMPT was associated with decreased glucose uptake, decreased lactate production and decreased ATP levels in HBV-expressing liver cancer cells, indicating that NAMPT may promote the aerobic glycolysis. Collectively, these findings reveal a positive feedback loop in which HBV enhances NAMPT expression and the activation of NAMPT promotes HBV replication and HBV-mediated malignant cell growth in liver cancer. The present study highlights the important role of NAMPT in the regulation of aerobic glycolysis in HBV-mediated liver cancer, and suggests that NAMPT may be a promising treatment target for patients with HBV-associated liver cancer.

LAMC1 is related to the poor prognosis of patients with gastric cancer and facilitates cancer cell malignancies.

Gastric cancer is a common malignancy in the alimentary system. The laminin subunit gamma 1 (LAMC1) is an important oncogene in human cancers. However, how and whether LAMC1 takes part in gastric cancer progression is largely uncertain. This study analyzed the association between clinical factors of patients and LAMC1 expression and explored the influence of LAMC1 silencing on cell proliferation, migration, invasion, the Warburg effect, protein kinase B (AKT) pathway, and mitogen-activated protein kinase (MEK)/extracellular regulated protein kinase (ERK) pathway in gastric cancer cells. Our results showed LAMC1 abundance was enhanced in gastric cancer samples and cells. LAMC1 was related to the clinical stage, tumor depth, lymph node metastasis, and distant metastasis of patients. LAMC1 silencing inhibited cell proliferation, migration, and invasion. Moreover, LAMC1 knockdown suppressed the Warburg effect via decreasing lactate production, lactate dehydrogenase (LDH) activity, and glucose uptake. LAMC1 interference blocked the activation of the AKT and MEK/ERK signaling. Collectively, LAMC1 knockdown constrained cell proliferation, migration, invasion, and the Warburg effect in gastric cancer cells via inactivating the AKT and MEK/ERK pathway.

Identification of a novel non-ATP-competitive protein kinase inhibitor of PGK1 from marine nature products

Phosphoglycerate kinase 1 (PGK1) acts as both a glycolytic enzyme and a protein kinase playing critical roles in cancer progression, thereby being regarded as an attractive therapeutic target for cancer treatment. However, no effective inhibitor of PGK1 has been reported. Here, we demonstrate that GQQ-792, a thiodiketopiperazine derivative from marine nature products, is a non-ATP-competitive inhibitor of PGK1 with the disulfide group within the structure of GQQ-792 as a key pharmacophore. The disulfide group of GQQ-792 binds to Cys379 and Cys380 of PGK1, resulting in occlusion of ATP from binding to PGK1. GQQ-792 treatment blocks hypoxic condition- and EGF stimulation-enhanced protein kinase activity of PGK1 that phosphorylates PDHK1 at T338 in glioblastoma cells; this treatment leads to decreased lactate production and glucose uptake, and subsequent apoptosis of glioblastoma cells. Animal studies reveal that GQQ-792 significantly inhibits the growth of tumor derived from glioblastoma cells. These findings underscore the potential of GQQ-792 as a promising anticancer agent and pave an avenue to further optimize the structure of GQQ-792 basing on its target molecule and pharmacophore in future.

Nanoparticle-Mediated Delivery of 2-Deoxy-D-Glucose Induces Antitumor Immunity and Cytotoxicity in Liver Tumors in Mice.

Background & AimsImmune checkpoint inhibitors have shed light on the importance of antitumor immunity as a therapeutic strategy for hepatocellular carcinoma (HCC). The altered glucose metabolism known as the Warburg effect recently has gained attention as a cancer immune-resistance mechanism. Considering glycolysis inhibitors as therapeutic agents, their specific delivery to cancer cells is critical not to induce adverse effects. Thus, we investigated antitumor effects of a glycolysis inhibitor, consisting of 2-deoxy-D-glucose (2DG)-encapsulated poly(lactic-co-glycolic acid) (PLGA) nanoparticles (2DG-PLGA-NPs), against hepatocellular carcinoma in mice.MethodsThe antitumor effects of 2DG-PLGA-NPs were examined using hepatoma cell lines, xenograft tumors, and hepatocarcinogenic and syngeneic mouse models.ResultsThe 2DG-PLGA-NPs induced cytotoxic effects and antitumor immunity through enhanced T-cell trafficking. In addition, 2DG-PLGA-NPs induced decreased lactate production and increased interferon-γ–positive T cells in liver tumors. Human CD8+ T cells cocultured with 2DG-PLGA-NP–treated Huh7 cells showed their increased interferon-γ production and glucose uptake compared with the CD8+ T cells co-cultured with PLGA-NP–treated Huh7 cells. Chemotaxis of CD8+ T cells was suppressed by lactate and enhanced by glucose. Interferon-γ enhanced CD8+ T-cell chemotaxis in both an autocrine and paracrine manner. Notably, the 2DG-PLGA-NPs augmented chemokine (CXCL9/CXCL10) production in liver tumors via interferon-γ–Janus kinase–signal transducers and activator of transcription pathway and 5' adenosine monophosphate-activated protein kinase–mediated suppression of histone H3 lysine 27 trimethylation. These 2DG-PLGA-NPs not only amplified antitumor effects induced by sorafenib or an anti–programmed death-1 antibody, but also suppressed anti–programmed death-1–resistant tumors.ConclusionsThe newly developed 2DG-PLGA-NPs showed antitumor immunity and cytotoxicity in liver tumors in mice, suggesting the potential of 2DG-PLGA-NPs for future clinical applications.

KD025 Shifts Pulmonary Endothelial Cell Bioenergetics and Decreases Baseline Lung Permeability.

KD025 is a ROCK2 inhibitor currently being tested in clinical trials for the treatment of fibrotic lung diseases. The therapeutic effects of KD025 are partly due to its inhibition of profibrotic pathways and fat metabolism. However, whether KD025 affects pulmonary microvascular endothelial cell (PMVEC) function is unknown, despite evidence that alveolar-capillary membrane disruption constitutes major causes of death in fibrotic lung diseases. We hypothesized that KD025 regulates PMVEC metabolism, pH, migration, and survival, a series of interrelated functional characteristics that determine pulmonary barrier integrity. We used PMVECs isolated from Sprague Dawley rats. KD025 dose-dependently decreased lactate production and glucose consumption. The inhibitory effect of KD025 was more potent compared with other metabolic modifiers, including 2-deoxy-glucose, extracellular acidosis, dichloroacetate, and remogliflozin. Interestingly, KD025 increased oxidative phosphorylation, whereas 2-deoxy-glucose did not. KD025 also decreased intracellular pH and induced a compensatory increase in anion exchanger 2. KD025 inhibited PMVEC migration, but fasudil (nonspecific ROCK inhibitor) did not. We tested endothelial permeability in vivo using Evans Blue dye in the bleomycin pulmonary fibrosis model. Baseline permeability was decreased in KD025-treated animals independent of bleomycin treatment. Under hypoxia, KD025 increased PMVEC necrosis as indicated by increased lactate dehydrogenase release and propidium iodide uptake and decreased ATP; it did not affect Annexin V binding. ROCK2 knockdown had no effect on PMVEC metabolism, pH, and migration, but it increased nonapoptotic caspase-3 activity. Together, we report that KD025 promotes oxidative phosphorylation; decreases glycolysis, intracellular pH, and migration; and strengthens pulmonary barrier integrity in a ROCK2-independent manner.

Down regulating PHGDH affects the lactate production of sertoli cells in varicocele

BackgroundAlthough varicocele is considered to be one of the leading causes of male infertility, the precise mechanism underlying how varicocele leads to male infertility is not completely understood. We found the lactate concentration on the varicocele side of the patients was decreased compare with peripheral venous blood. In the testicles, the lactate produced by the sertoli cells through the glycolysis pathway provides most of the energy needed for spermatogenesis, the reduction of lactate will affect spermatogenesis. The objective of this study was to investigate the mechanism of this abnormal energy metabolism phenomenon in varicocele.MethodsIn this study, we collected the testicular tissue from patients with varicocele, the glycolysis related proteins PHGDH was identified by iTRAQ proteomics technology. Experimental rat varicocele model was constructed according to our new clip technique, the mRNA and protein expression levels of PHGDH were examined with qRT-PCR and Western blotting. We constructed a sertoli cell of PHGDH down-regulation model, and then detected the glucose consumption, LDH activities and lactate production in the sertoli cells. Western blot was conducted to investigate the effects of PHGDH on the expression of phosphoserine phosphatase (PSPH) and Pyruvate kinase M2 (PKM2). Flow cytometry was used to detect the cell apoptosis and cell cycle in sertoli cells.ResultsThe results showed that testicular protein PHGDH was down-regulated in patients with varicocele and in experimental rat varicocele model. Down-regulation of PHGDH in sertoli cells significantly decreased the glucose consumption, LDH activities and lactate production in the sertoli cells, indicating that the low expression of PHGDH ultimately led to a decrease in lactate production by affecting the glycolysis. The Western blot results showed that the down-regulation of PHGDH significantly reduced the expression of pathway protein PSPH and PKM2, leading to the reduction of lactate production. Moreover, PHGDH knockdown can promote apoptosis and inhibit cell cycle to affect cell growth.ConclusionsOverall, we conformed that varicocele lead to the decreasing of testis lactate production. Down-regulation of PHGDH in sertoli cells may mediate the process of abnormal glucose metabolism. Our study provide new insight into the mechanisms underlying metabolism-associated male infertility and suggests a novel therapeutic target for male infertility.

Testosterone improves muscle fiber asset and exercise performance in a metabolic syndrome model.

Lifestyle modifications, including physical exercise (PhyEx), are well-known treatments for metabolic syndrome (MetS), a cluster of metabolic and cardiovascular risk factors often associated to hypogonadism. Given the trophic role of testosterone on skeletal muscle (SkM), this study was aimed at evaluating the effects of testosterone treatment on SkM metabolism and exercise performance in male rabbits with high-fat diet (HFD)-induced MetS. HFD rabbits, treated or not with testosterone (30 mg/kg/week) for 12 weeks, were compared to regular diet animals (RD). A subset of each group was exercise-trained for 12 weeks. HFD increased type-II (fast, glycolytic) and decreased type-I (slow, oxidative) muscle fibers compared to RD as evaluated by RT-PCR and histochemistry. Testosterone reverted these effects, also inducing the expression of mitochondrial respiration enzymes and normalizing HFD-induced mitochondrial cristae reduction. Moreover, testosterone significantly increased the expression of myogenic/differentiation markers and genes related to glucidic/lipid metabolism. At the end of the PhyEx protocol, when compared to RD, HFD rabbits showed a significant reduction of running distance and running time, while testosterone counteracted this effect, also decreasing lactate production. In the trained groups, muscle histology showed a significant reduction of oxidative fibers in HFD compared to RD and the positive effect of testosterone in maintaining oxidative metabolism, as also demonstrated by analyzing mitochondrial ultrastructure, succinate dehydrogenase activity and ATP production. Our results indicate that testosterone could be useful to promote oxidative muscle metabolism altered by MetS, thus improving exercise performance. Conversely, testosterone administration to otherwise eugonadal rabbits (RD) only increased muscle fiber diameter but not endurance performance.

Combining lipoic acid to methylene blue reduces the Warburg effect in CHO cells: From TCA cycle activation to enhancing monoclonal antibody production.

The Warburg effect, a hallmark of cancer, has recently been identified as a metabolic limitation of Chinese Hamster Ovary (CHO) cells, the primary platform for the production of monoclonal antibodies (mAb). Metabolic engineering approaches, including genetic modifications and feeding strategies, have been attempted to impose the metabolic prevalence of respiration over aerobic glycolysis. Their main objective lies in decreasing lactate production while improving energy efficiency. Although yielding promising increases in productivity, such strategies require long development phases and alter entangled metabolic pathways which singular roles remain unclear. We propose to apply drugs used for the metabolic therapy of cancer to target the Warburg effect at different levels, on CHO cells. The use of α-lipoic acid, a pyruvate dehydrogenase activator, replenished the Krebs cycle through increased anaplerosis but resulted in mitochondrial saturation. The electron shuttle function of a second drug, methylene blue, enhanced the mitochondrial capacity. It pulled on anaplerotic pathways while reducing stress signals and resulted in a 24% increase of the maximum mAb production. Finally, the combination of both drugs proved to be promising for stimulating Krebs cycle activity and mitochondrial respiration. Therefore, drugs used in metabolic therapy are valuable candidates to understand and improve the metabolic limitations of CHO-based bioproduction.

McArdle Disease: Clinical, Biochemical, Histological and Molecular Genetic Analysis of 60 Patients.

A clinical, biochemical, histological and molecular genetic analysis of 60 McArdle patients (33 males and 27 females; mean age at diagnosis: 37 years) was performed. The objective of this study was to identify a possible genotype–phenotype correlation in McArdle disease. All patients complained of exercise-induced myalgia and fatigue; permanent weakness was present in 47% of the patients. Five percent of patients conveyed of masticatory muscle weakness. Age of onset was <15 years in 92% patients. Serum creatine kinase was elevated 5 to13-fold. Forearm ischemic test showed decreased lactate production but excessively increased ammonia upon exercise (n = 16). Muscle biopsies revealed highly reduced or missing myophosphorylase activity (n = 20) (mean: 0.17 ± 0.35 U/g tissue; normal: 12–61) and histologically, sub-sarcolemmal glycogen accumulation (n = 9). Molecular genetic analysis revealed the common p.Arg50Ter mutation in 68% of the patients. Other rather frequent mutations were p.Arg270Ter (allele frequency: 5%) followed by c.2262delA and p.Met1Val (allele frequencies: 3%). Twenty-four other rare mutations were also identified. No genotype–phenotype correlation was observed. The analysis highlights that testing of the p.Arg50Ter mutation could be performed first in molecular genetic testing of patients with exercise intolerance possibly due to McArdle disease. However, there is enormous mutation heterogeneity in McArdle disease thus sequencing of the myophosphorylase gene is needed in patients highly suspicious of McArdle disease.

Hypoxia-inducible Factor-1α Mediates Hyperglycemia-induced Pancreatic Cancer Glycolysis

Recent studies have suggested that 85% of pancreatic cancer patients accompanied with impaired glucose tolerance or even Diabetes Mellitus (DM) and the invasive and migratory abilities of pancreatic cancer could be enhanced by high glucose. This study aimed to investigate whether Hypoxia- Inducible Factor-1α (HIF-1α) mediates hyperglycemia-induced pancreatic cancer glycolysis. The cellular glycolytic activity was assessed by determining lactate production, glucose uptake and lactate dehydrogenase enzymatic activity. Pancreatic cancer cells (BxPC-3 cells) were transfected with short hairpin RNA targeting the HIF-1α. Hyperglycemia promotes pancreatic cancer glycolysis. Lactate dehydrogenase A (LDHA) activity and hexokinase 2 (HK2), platelet-type of phosphofructokinase (PFKP) expression were significantly upregulated under hyperglycemic conditions. HIF-1α knockdown prominently down-regulated the activity of LDHA and the expression of HK2, PFKP and decreased lactate production in BxPC-3 cells. Under hypoxia condition, hyperglycemia induced pancreatic glycolysis by mechanisms that are both dependent on HIF-1α and independent of it. The accumulation of HIF-1α induced by hyperglycemia increases LDHA activity and HK2, PFKP expression, thereby promoting pancreatic glycolysis to facilitate cancer progression.

Reversal of the Warburg phenomenon in chemoprevention of prostate cancer by sulforaphane.

Inhibition of metabolic re-programming represents an attractive approach for prevention of prostate cancer. Studies have implicated increased synthesis of fatty acids or glycolysis in pathogenesis of human prostate cancers. We have shown previously that prostate cancer prevention by sulforaphane (SFN) in Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model is associated with inhibition of fatty acid metabolism. This study utilized human prostate cancer cell lines (LNCaP, 22Rv1 and PC-3), two different transgenic mouse models (TRAMP and Hi-Myc) and plasma specimens from a clinical study to explore the glycolysis inhibition potential of SFN. We found that SFN treatment: (i) decreased real-time extracellular acidification rate in LNCaP, but not in PC-3 cell line; (ii) significantly downregulated expression of hexokinase II (HKII), pyruvate kinase M2 and/or lactate dehydrogenase A (LDHA) in vitro in cells and in vivo in neoplastic lesions in the prostate of TRAMP and Hi-Myc mice; and (iii) significantly suppressed glycolysis in prostate of Hi-Myc mice as measured by ex vivo1H magnetic resonance spectroscopy. SFN treatment did not decrease glucose uptake or expression of glucose transporters in cells. Overexpression of c-Myc, but not constitutively active Akt, conferred protection against SFN-mediated downregulation of HKII and LDHA protein expression and suppression of lactate levels. Examination of plasma lactate levels in prostate cancer patients following administration of an SFN-rich broccoli sprout extract failed to show declines in its levels. Additional clinical trials are needed to determine whether SFN treatment can decrease lactate production in human prostate tumors.

Reprogramming AA catabolism in CHO cells with CRISPR/Cas9 genome editing improves cell growth and reduces byproduct secretion

Chinese hamster ovary (CHO) cells are the preferred host for producing biopharmaceuticals. Amino acids are biologically important precursors for CHO metabolism; they serve as building blocks for proteogenesis, including synthesis of biomass and recombinant proteins, and are utilized for growth and cellular maintenance. In this work, we studied the physiological impact of disrupting a range of amino acid catabolic pathways in CHO cells. We aimed to reduce secretion of growth inhibiting metabolic by-products derived from amino acid catabolism including lactate and ammonium. To achieve this, we engineered nine genes in seven different amino acid catabolic pathways using the CRISPR-Cas9 genome editing system. For identification of target genes, we used a metabolic network reconstruction of amino acid catabolism to follow transcriptional changes in response to antibody production, which revealed candidate genes for disruption. We found that disruption of single amino acid catabolic genes reduced specific lactate and ammonium secretion while specific growth rate and integral of viable cell density were increased in many cases. Of particular interest were Hpd and Gad2 disruptions, which show unchanged AA uptake rates, while having growth rates increased up to 19%, and integral of viable cell density as much as 50% higher, and up to 26% decrease in specific ammonium production and to a lesser extent (up to 22%) decrease in lactate production. This study demonstrates the broad potential of engineering amino acid catabolism in CHO cells to achieve improved phenotypes for bioprocessing.

4'-Bromo-resveratrol, a dual Sirtuin-1 and Sirtuin-3 inhibitor, inhibits melanoma cell growth through mitochondrial metabolic reprogramming.

Sirtuin-1 and -3 (SIRT1 and SIRT3) are important nicotinamide adenine dinucleotide (NAD+ )-dependent deacetylases known to regulate a variety of cellular functions. Studies have shown that SIRT1 and SIRT3 were overexpressed in human melanoma cells and tissues and their inhibition resulted in a significant antiproliferative response in human melanoma cells and antitumor response in a mouse xenograft model of melanoma. In this study, we determined the antiproliferative efficacy of a newly identified dual small molecule inhibitor of SIRT1 and SIRT3, 4'-bromo-resveratrol (4'-BR), in human melanoma cell lines (G361, SK-MEL-28, and SK-MEL-2). Our data demonstrate that 4'-BR treatment of melanoma cells resulted in (a) decrease in proliferation and clonogenic survival; (b) induction of apoptosis accompanied by a decrease in procaspase-3, procaspase-8, and increase in the cleavage of caspase-3 and poly (ADP-ribose) polymerase (PARP); (c) marked downregulation of proliferating cell nuclear antigen (PCNA); and (d) inhibition of melanoma cell migration. Further, 4'-BR caused a G0/G1 phase arrest of melanoma cells that was accompanied by an increase in WAF-1/P21 and decrease in Cyclin D1/Cyclin-dependent kinase 6 protein levels. Furthermore, we found that 4'-BR causes a decrease in lactate production, glucose uptake, and NAD+ /NADH ratio. These responses were accompanied by downregulation in lactate dehydrogenase A and glucose transporter 1 in melanoma cells. Collectively, our data suggest that dual inhibition of SIRT1 and SIRT3 using 4'-BR imparted antiproliferative effects in melanoma cells through a metabolic reprogramming and affecting the cell cycle and apoptosis signaling. Therefore, concomitant pharmacological inhibition of SIRT1 and SIRT3 needs further investigation for melanoma management.

CD146 contributes the metastatic properties and antitumor immunity of human colon adenocarcinoma cells

Abstract CD146 (MCAM) expressed on not only vascular endothelial and smooth muscle cells but also various malignant tumor cells. It has been shown that overexpression of CD146 predicts poor prognosis of solid tumor. Several reports by using transfectants or gene silencing technique have shown that CD146 is involved in cell adhesion and inflammatory cell migration. However, contribution of CD146 in tumor metastasis and antitumor immunity are still unknown. To investigate the effect of CD146 on tumor metastatic properties, we establish two subclones from DLD-1 (human colon adenocarcinoma cells) expressing CD146 or not. Both cell lines express epithelial tumor marker EpCAM and TROP2 equally, while CD146 (+) DLD-1 could only express CD44 variant v8–v10 and EphA2. Using metabolic assay, we found enhanced metabolism and decreased lactate production in CD146 (+) DLD-1. Consist with the morphological changes to mesenchymal features CD146 (+) DLD-1, the expression of E-cadherin and claudin-3 in tight junction were decreased in CD146 (+) DLD-1. In addition, CD146 (+) DLD-1 significantly increased the production of VEGF and the induction of various genes (slug, twist and zeb1) which are involved in epithelial to mesenchymal transition (EMT). Interestingly, the expression of immune checkpoint molecules including B7-H1, B7-DC (both PD-1 ligands), CD155 and IDO were up-regulated in CD146 (+) DLD-1 inducing the resistance of cytotoxic effect of activated lymphocytes. These results indicate that CD146 (+) DLD-1 strongly induces immune escape mechanism, thus demonstrating a new relationship between CD146 and EMT and anti-tumor immunity, which would be a new treatment and diagnostic target for immunotherapy.

Antihypertensive and cardioprotective effects of new compound 1-(β-phenylethyl)-4-amino-1,2,4-triazolium bromide (Hypertril)

Despite the success which was achieved in the treatment of arterial hypertension, the problem remains actual. At the departments of pharmaceutical chemistry and pharmacology of the Zaporozhye State Medical Institute (Ukraine), our research team isolated the compound 1-(β-phenylethyl)-4-amino-1,2,4-triazolium bromide (Hypertril) as derivative of 4-amino-1,2,4-triazole. The objectives of this investigation were the study of cardioprotective and antihypertensive activities of this new compound Hypertril and we used the Spontaneously hypertensive rats (SHR) as an experimental model. We discovered that Hypertril has a reliable dose-dependent antihypertensive effect in the dose range 5–20 mg/kg after 30-day administration and this antihypertensive effect of Hypetril competes or significantly exceeds Metoprolol (20 mg/kg). Our studies obtained evidence of a dose-dependent improvement of myocardial energy metabolism. Hypertril reduces the manifestations of secondary mitochondrial dysfunction due to arterial hypertension. Hypertril can prevent oxidative modification of the protein; also Hypertril reduces the insufficiency of mitochondrial pores. As a result, Hypertril increases the content of ATP in the myocardium of SHR, normalizes the activity of mitochondrial enzymes, decreases lactate production and increases pyruvate. Hypertril enhances the cardioprotective effects of NO and increases the resistance of the cardiomyocytes to ischemia. The use of Hypertril leads to a dose-dependent increase of the density of cardiomyocyte nuclei, significant increase RNA content in nuclei and the cytoplasm of cardiomyocytes, and an increase of the nuclear-cytoplasmic index. These changes indicate a decrease of myocardial hypertrophy.

Sex‐Specific Adverse Effects of Chronic Dichloroacetate Administration in Mice

Dichloroacetate (DCA) is a small, orally available inhibitor of pyruvate dehydrogenase kinase (PDK). Consequently, DCA stimulates the activity of the pyruvate dehydrogenase complex (PDC), leading to glucose oxidation in the mitochondria. Therapeutically, DCA is employed to decrease lactate production and has been used in the acute treatment of certain metabolic diseases, such as lactic acidosis. More recently, DCA has been reported to have anti‐cancer efficacy and has been suggested as a chemotherapeutic. However, DCA as a therapeutic agent may have adverse effects, as it has been reported to cause hepatocellular and peripheral nerve toxicity. As it may have further adverse effects when used chronically, there is a need for further investigation of the effects and mechanisms triggered by DCA. We aimed at assessing the effects of chronic DCA administration in the mouse heart. We hypothesized that long‐term treatment of mice with DCA will lead to cardiac toxicity, ultimately contributing to heart failure development, and that the severity of these effects will differ between the sexes. Fourteen‐month old male and female C57Bl6 mice (n = 12/group) were treated with vehicle or DCA (0.8 g/L in drinking water leading to 80 mg/kg/d, similar to clinically used dose) for 7 months. All mice underwent transthoracic 2D echocardiography under anesthesia (isoflurane 2%) following established procedures. The study was conducted in conformance with the FASEB Statement of Principles for the use of Animals in Research and Education. Following the 7‐mo DCA treatment, echocardiographic measurements revealed no major functional effect in female mice. In contrast, male mice treated with DCA exhibited a significant systolic dysfunction associated with impaired contractility compared with control mice (ejection fraction: 36% vs. 47% respectively, P &lt; 0.001). Ultrastructural analysis of left ventricular samples by transmission electron microscopy revealed increased numbers of heavily damaged and swollen mitochondria in male mice treated with DCA, while female mice treated with DCA were not affected. Further cellular characterization revealed no major effects on apoptosis, as assessed by the TUNEL assay, or fibrosis, as assessed by Sirius‐red staining. Transcriptome analysis of left ventricles from vehicle‐ and DCA‐treated mice by genome‐wide expression profiling (n = 5/group, P &lt; 0.001) revealed decreased levels of genes involved in ion channel activity and metabolism in male DCA‐treated mice. In contrast, the levels of genes with antioxidant activity were increased in female DCA‐treated mice. In conclusion, chronic DCA treatment leads to male‐specific cardiac toxicity and dysfunction. These findings highlight the role of biological sex in therapeutic and adverse effects of pharmacological agents.Support or Funding InformationDZHK (German Centre for Cardiovascular Research) and BMBF (Federal Ministry of Education and Research).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

TIGAR promotes neural stem cell differentiation through acetyl-CoA-mediated histone acetylation

Cellular metabolism plays a crucial role in controlling the proliferation, differentiation, and quiescence of neural stem cells (NSCs). The metabolic transition from aerobic glycolysis to oxidative phosphorylation has been regarded as a hallmark of neuronal differentiation. Understanding what triggers metabolism reprogramming and how glucose metabolism directs NSC differentiation may provide new insight into the regenerative potential of the brain. TP53 inducible glycolysis and apoptosis regulator (TIGAR) is an endogenous inhibitor of glycolysis and is highly expressed in mature neurons. However, its function in embryonic NSCs has not yet been explored. In this study, we aimed to investigate the precise roles of TIGAR in NSCs and the possible involvement of metabolic reprogramming in the TIGAR regulatory network. We observed that TIGAR is significantly increased during brain development as neural differentiation proceeds, especially at the peak of NSC differentiation (E14.5–E16.5). In cultured NSCs, knockdown of TIGAR reduced the expression of microtubule-associated protein 2 (MAP2), neuron-specific class III beta-tubulin (Tuj1), glial fibrillary acidic protein (GFAP), Ngn1, and NeuroD1, and enhanced the expression of REST, suggesting that TIGAR is an important regulator of NSC differentiation. Furthermore, TIGAR enhanced the expression of lactate dehydrogenase B (LDHB) and the mitochondrial biogenesis and oxidative phosphorylation (OXPHOS) markers, peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1α), nuclear respiratory factor (NRF1), and MitoNEET during NSC differentiation. TIGAR can decrease lactate production and accelerate oxygen consumption and ATP generation to maintain a high rate of OXPHOS in differentiated NSCs. Interestingly, knockdown of TIGAR decreased the level of acetyl-CoA and H3K9 acetylation at the promoters of Ngn1, Neurod1, and Gfap. Acetate, a precursor of acetyl-CoA, increased the level of H3K9 acetylation and rescued the effect of TIGAR deficiency on NSC differentiation. Together, our data demonstrated that TIGAR promotes metabolic reprogramming and regulates NSC differentiation through an epigenetic mechanism.

Downregulated NOX4 underlies a novel inhibitory role of microRNA-137 in prostate cancer.

Prostate cancer is the second highest caused by cancer-related death among males. microRNAs (miRs) have been reported to participate in carcinogenesis, yet their roles in prostate cancer are rarely studied or investigated. Therefore, the present study attempted to explore the effect of miR-137 in prostate cancer via regulating NADPH oxidase 4 (NOX4). Initially, microarray analysis was performed to obtain prostate cancer-related differentially expressed genes and miRs that regulated NOX4, followed by detecting the expression of miR-137 and NOX4 and its target relationship. Moreover, PC-3 cells were transfected with small interfering RNA (siNOX4) and miR-137 mimic for exploring the effect of miR-137 on glycolysis, cell proliferation, and apoptosis in prostate cancer by evaluating lactate production, glucose uptake, adenosine triphosphate (ATP) production, viability rate, and expression of cleaved caspases 3, 8, and 9, cytochrome c, cleaved poly ADP ribose polymerase (PARP), Bax, and Bcl-2. miR-137 was vital to prostate cancer progression via regulating NOX4. Besides, miR-137 expressed poorly while NOX4 expressed highly in prostate cancer. NOX4 was the target gene of miR-137. Additionally, overexpression of miR-137 and silencing of NOX4 were observed to decrease NOX4 and Bcl-2 protein expression, but increase cleaved caspases 3, 8, and 9, cytochrome c, cleaved-PARP, and Bax protein expression. Furthermore, miR-137 overexpression and NOX4 silencing contributed to decreased lactate production, glucose uptake, ATP production, and cell proliferation, but increased apoptosis rate. Collectively, the present study showed that miR-137 repressed glycolysis in prostate cancer through knockdown of NOX4, which might be a potential theoretical target for prostate cancer treatment.

Exposure to Pb and Cd alters MCT4/CD147 expression and MCT4/CD147-dependent lactate transport in mice Sertoli cells cultured in vitro

Sertoli cells (SCs) provide lactate as an energy substrate to develop germ cells during spermatogenesis. Lead (Pb) and cadmium (Cd) can induce SC toxicity. However, the mechanisms remain unclear. This study aimed to investigate the molecular mechanisms by which Pb and Cd alter lactate transport and production by SCs. Mouse SC line (15P-1 cells) were cultured in the absence and presence of lead acetate (PbAc, 1, 10, 20 and 30 μM) or cadmium chloride (CdCl2, 0.5, 5, 10 and 15 μM) for 24 h. The results showed that PbAc exposure significantly decreased lactate dehydrogenase (LDH) activity and mRNA level, intracellular and extracellular lactate, and MCT4 and CD147 protein levels but increased MCT4 and CD147 mRNA levels. However, PbAc did not alter the glucose uptake, glucose transporters 1 (GLUT1) and 3 (GLUT3) mRNA expression of SCs. Thus, PbAc mainly decreased lactate production by inhibiting LDH activity. In CdCl2-treated SCs, intracellular lactate content increased but extracellular lactate content decreased significantly, P < .05. The glucose uptake, LDH activity, and mRNA expression of GLUT1, GLUT3 and LDH, all significantly increased. But the mRNA and protein levels of MCT4 and CD147 significantly decreased. Moreover, the fluorescence intensity of co-localizations of the MCT4–CD147 complex dose-dependently decreased in the cell membrane. Thus, CdCl2 may reduce lactate export by suppressing MCT4 and CD147 expression. These results suggest that PbAc and CdCl2 disrupt lactate production and transport in mouse SCs by disturbing glycolysis or inhibiting MCT4–CD147 transporter expression and co-localizations.