Triclosan inhibition of membrane enzymes and glycolysis of Streptococcus mutans in suspensions and biofilms

Triclosan was found to be a potent inhibitor of the F(H+)-ATPase of the oral pathogen Streptococcus mutans and to increase proton permeabilities of intact cells. Moreover, it acted additively with weak-acid transmembrane proton carriers, such as fluoride or sorbate, to sensitize glycolysis to acid inhibition. Even at neutral pH, triclosan could inhibit glycolysis more directly as an irreversible inhibitor of the glycolytic enzymes pyruvate kinase, lactic dehydro genase, aldolase, and the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Cell glycolysis in suspensions or biofilms was inhibited in a pH-dependent manner by triclosan at a concentration of about 0.1 mmol/L at pH 7, approximately the lethal concentration for S. mutans cells in suspensions. Cells in intact biofilms were almost as sensitive to triclosan inhibition of glycolysis as were cells in suspensions but were more resistant to killing. Targets for irreversible inhibition of glycolysis included the PTS and cytoplasmic enzymes, specifically pyruvate kinase, lactic dehydrogenase, and to a lesser extent, aldolase. General conclusions are that triclosan is a multi-target inhibitor for mutans streptococci, which lack a triclosan-sensitive FabI enoyl-ACP reductase, and that inhibition of glycolysis in dental plaque biofilms, in which triclosan is retained after initial or repeated exposure, would reduce cariogenicity.     

Proteomic and metabonomic analysis uncovering Enterovirus A71 reprogramming host cell metabolic pathway

Enterovirus A71 (EV71) infection can cause hand, foot, and mouth disease (HFMD) and severe neurological complications in children. However, the biological processes regulated by EV71 remain poorly understood. Herein, proteomics and metabonomics studies were conducted to uncover the mechanism of EV71 infection in rhabdomyosarcoma (RD) cells and identify potential drug targets. Differential expressed proteins from enriched membrane were analyzed by isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics technology. Twenty-six differential proteins with 1.5-fold (p < 0.05) change were detected, including 14 upregulated proteins and 12 downregulated proteins. The upregulated proteins are mainly involved in metabolic process, especially in the glycolysis pathway. Alpha-enolase (ENO1) protein was found to increase with temporal dependence following EV71 infection. The targeted metabolomics analysis revealed that glucose absorption and glycolysis metabolites were increased after EV71 infection. The glycolysis pathway was inhibited by knocking down ENO1 or the use of a glycolysis inhibitor (dichloroacetic acid [DCA]); and we found that EV71 infection was inhibited by depleting ENO1 or using DCA. Our study indicates that EV71 may reprogram glucose metabolism by activating glycolysis, and EV71 infection can be inhibited by interrupting the glycolysis pathway. ENO1 may be a potential target against EV71, and DCA could act as an inhibitor of EV71.     

Bioenergetic properties of human sarcoma cells help define sensitivity to metabolic inhibitors

Sarcomas represent a diverse group of malignancies with distinct molecular and pathological features. A better understanding of the alterations associated with specific sarcoma subtypes is critically important to improve sarcoma treatment. Renewed interest in the metabolic properties of cancer cells has led to an exploration of targeting metabolic dependencies as a therapeutic strategy. In this study, we have characterized key bioenergetic properties of human sarcoma cells in order to identify metabolic vulnerabilities between sarcoma subtypes. We have also investigated the effects of compounds that inhibit glycolysis or mitochondrial respiration, either alone or in combination, and examined relationships between bioenergetic parameters and sensitivity to metabolic inhibitors. Using 2-deoxy-D-glucose (2-DG), a competitive inhibitor of glycolysis, oligomycin, an inhibitor of mitochondrial ATP synthase, and metformin, a widely used anti-diabetes drug and inhibitor of complex I of the mitochondrial respiratory chain, we evaluated the effects of metabolic inhibition on sarcoma cell growth and bioenergetic function. Inhibition of glycolysis by 2-DG effectively reduced the viability of alveolar rhabdomyosarcoma cells vs. embryonal rhabdomyosarcoma, osteosarcoma, and normal cells. Interestingly, inhibitors of mitochondrial respiration did not significantly affect viability, but were able to increase sensitivity of sarcomas to inhibition of glycolysis. Additionally, inhibition of glycolysis significantly reduced intracellular ATP levels, and sensitivity to 2-DG-induced growth inhibition was related to respiratory rates and glycolytic dependency. Our findings demonstrate novel relationships between sarcoma bioenergetics and sensitivity to metabolic inhibitors, and suggest that inhibition of metabolic pathways in sarcomas should be further investigated as a potential therapeutic strategy.     

PKM2 depletion induces the compensation of glutaminolysis through β-catenin/c-Myc pathway in tumor cells

The metabolic activity in cancer cells primarily rely on aerobic glycolysis. Besides glycolysis, some tumor cells also exhibit excessive addition to glutamine, which constitutes an advantage for tumor growth. M2-type pyruvate kinase (PKM2) plays a pivotal role in sustaining aerobic glycolysis, pentose phosphate pathway and serine synthesis pathway. However, the participation of PKM2 in glutaminolysis is little to be known. Here we demonstrated that PKM2 depletion could provoke glutamine metabolism by enhancing the β-catenin signaling pathway and consequently promoting its downstream c-Myc-mediated glutamine metabolism in colon cancer cells. Treatment with 2-deoxy-d-glucose (2-DG), a glycolytic inhibitor, got consistent results with the above. In addition, the dimeric form of PKM2, which lacks the pyruvate kinase activities, plays a critical role in regulating β-catenin. Moreover, we found that overexpression of PKM2 negatively regulated β-catenin through miR-200a. These insights supply evidence that glutaminolysis plays a compensatory role for cell survival upon glucose metabolism impaired.     

A Membrane Protease Regulates Energy Production in Macrophages by Activating Hypoxia-inducible Factor-1 via a Non-proteolytic Mechanism

Most cells produce ATP in the mitochondria by oxidative phosphorylation. However, macrophages, which are major players in the innate immune system, use aerobic glycolysis to produce ATP. HIF-1 (hypoxia-inducible factor-1) regulates expression of glycolysis-related genes and maintains macrophage glycolytic activity. However, it is unclear how HIF-1 activity is maintained in macrophages during normoxia. In this study, we found that macrophages lacking membrane type 1 matrix metalloproteinase (MT1-MMP/MMP-14), a potent invasion-promoting protease, exhibited considerably lower ATP levels than wild-type cells. HIF-1 was activated by an unanticipated function of MT1-MMP, which led to the stimulation of ATP production via glycolysis. The cytoplasmic tail of MT1-MMP bound to FIH-1 (factor inhibiting HIF-1), which led to the inhibition of the latter by its recently identified inhibitor, Mint3/APBA3. We have thus identified a new function of MT1-MMP to mediate production of ATP so as to support energy-dependent macrophage functions by a previously unknown non-proteolytic mechanism.     

TRIM59, amplified in ovarian cancer,promotes tumorigenesis through the MKP3/ERK pathway

Tripartite motif containing 59 (TRIM59) functions as an oncoprotein in various human cancers including ovarian cancer. In this study, we found that TRIM59 gene amplification was prevalent in ovarian cancer tissues, and its amplification was significantly correlated with poorer overall survival. Moreover, knockdown of TRIM59 in SKOV3 and OVCAR3 cells, which had relatively high level of TRIM59, suppressed glucose uptake and lactate production. TRIM59 knockdown also decreased the expression of c-Myc and lactate dehydrogenase A, and the phosphorylation of extracellular signal-regulated kinase (ERK). TRIM59 overexpression in A2780 cells, which expressed low level of TRIM59, showed reverse effects. Notably, treatment with an ERK inhibitor (PD98059) completely abolished the oncogenic effects of TRIM59 overexpression. Interestingly, TRIM59 increased the ubiquitination of MAP kinase phosphatase 3 (MKP3), which may dephosphorylate and inactivate ERK. Ectopic expression of MKP3 inhibited the promoting effects of TRIM59 on glycolysis and the phosphorylation of ERK. TRIM59 protein expression was negatively correlated with MKP3 protein expression in ovarian cancer tissues. Finally, TRIM59 amplification potently affected the anticancer effect of 3-bromopyruvate, an inhibitor of glycolysis, in ovarian cancer cells and patient-derived xenograft. In conclusion, these results suggest that TRIM59 may regulate glycolysis in ovarian cancer via the MKP3/ERK pathway.     

Dysregulated glycolysis underpins high-fat-associated endometrial decidualization impairment during early pregnancy in mice

Pregnancy complications are more likely to occur in obese women because of defective decidualization. However, the specific mechanism of glycolysis in decidual modulation associated with obesity remains unknown. Therefore, we explored the role of glycolysis in the endometrium of obese pregnant mice during decidualization. C57BL/6J mice were fed a high-fat diet (HFD) to induce obesity. All obesity related parameters were significantly higher in the HFD mice than control. Furthermore, the HFD mice had fewer implantation sites, a smaller decidual area growth, and decreased decidualization marker protein expression than control. The HFD mice also had significantly decreased lactate production and glycolytic enzyme expression. To confirm the functional role of glycolysis during the decidual period in obese pregnant mice, we extracted endometrial stromal cells (ESCs) and treated them with oleic acid (OA) and palmitic acid (PA) to mimic a high-fat environment. Decidualization and glycolysis were significantly restricted in the OA-and PA-treated groups. Moreover, we administered a glycolytic inhibitor, 2-DG, and an agonist, pioglitazone. 2-DG treatment considerably decreased the cells' glycolysis and decidualization. However, pioglitazone treatment improved glycolysis and alleviated defective decidualization. In conclusion, obesity-induced endometrial glycolysis modifications and key glycolytic enzyme downregulation during early pregnancy might cause abnormal decidualization, leading to an unsustainable pregnancy.     

Sperm mitochondrial integrity is not required for hyperactivated motility, zona binding, or acrosome reaction in the rhesus macaque

Whether the main energy source for sperm motility is from oxidative phosphorylation or glycolysis has been long-debated in the field of reproductive biology. Using the rhesus monkey as a model, we examined the role of glycolysis and oxidative phosphorylation in sperm function by using alpha-chlorohydrin (ACH), a glycolysis inhibitor, and pentachlorophenol (PCP), an oxidative phosphorylation uncoupler. Sperm treated with ACH showed no change in percentage of motile sperm, although sperm motion was impaired. The ACH-treated sperm did not display either hyperactivity- or hyperactivation-associated changes in protein tyrosine phosphorylation. When treated with PCP, sperm motion parameters were affected by the highest level of PCP (200 microM); however, PCP did not cause motility impairments even after chemical activation. Sperm treated with PCP were able to display hyperactivity and tyrosine phosphorylation after chemical activation. In contrast with motility measurements, treatment with either the glycolytic inhibitor or the oxidative phosphorylation inhibitor did not affect sperm-zona binding and zona-induced acrosome reaction. The results suggest glycolysis is essential to support sperm motility, hyperactivity, and protein tyrosine phosphorylation, while energy from oxidative phosphorylation is not necessary for hyperactivated sperm motility, tyrosine phosphorylation, sperm-zona binding, and acrosome reaction in the rhesus macaque.     

Energy metabolism in osteoclast formation and activity

Osteoclastogenesis and osteolysis are energy-consuming processes supported by high metabolic activities. In human osteoclasts derived from the fusion of monocytic precursors, we found a substantial increase in the number of mitochondria with differentiation. In mature osteoclasts, mitochondria were also increased in size, rich of cristae and arranged in a complex tubular network. When compared with immature cells, fully differentiated osteoclasts showed higher levels of enzymes of the electron transport chain, a higher mitochondrial oxygen consumption rate and a lower glycolytic efficiency, as evaluated by extracellular flux analysis and by the quantification of metabolites in the culture supernatant. Thus, oxidative phosphorylation appeared the main bioenergetic source for osteoclast formation. Conversely, we found that bone resorption mainly relied on glycolysis. In fact, osteoclast fuelling with galactose, forcing cells to depend on Oxidative Phosphorylation by reducing the rate of glycolysis, significantly impaired Type I collagen degradation, whereas non-cytotoxic doses of rotenone, an inhibitor of the mitochondrial complex I, enhanced osteoclast activity. Furthermore, we found that the enzymes associated to the glycolytic pathway are localised close to the actin ring of polarised osteoclasts, where energy-demanding activities associated with bone degradation take place. In conclusion, we demonstrate that the energy required for osteoclast differentiation mainly derives from mitochondrial oxidative metabolism, whereas the peripheral cellular activities associated with bone matrix degradation are supported by glycolysis. A better understanding of human osteoclast energy metabolism holds the potential for future therapeutic interventions aimed to target osteoclast activity in different pathological conditions of bone.     

Branched-chain amino acid transport in Streptococcus agalactiae.

The transport of the branched-chain amino acids in Streptococcus agalactiae was characterized. Glucose-grown cells were able to utilize only glucose as an energy source for transport of L-leucine, whereas lactose-grown cells could utilize both glucose and lactose. It was determined from metabolic inhibitor studies that energy from glycolysis and substrate level phosphorylation was required for active transport. Energy was found to be coupled to transport by the action of adenosine triphosphatase and the generation of a proton motive force. The branched-chain amino acids were found to share a common transport system that may consist of multiple components.     

Branched-chain amino acid transport in Streptococcus agalactiae

The transport of the branched-chain amino acids in Streptococcus agalactiae was characterized. Glucose-grown cells were able to utilize only glucose as an energy source for transport of L-leucine, whereas lactose-grown cells could utilize both glucose and lactose. It was determined from metabolic inhibitor studies that energy from glycolysis and substrate level phosphorylation was required for active transport. Energy was found to be coupled to transport by the action of adenosine triphosphatase and the generation of a proton motive force. The branched-chain amino acids were found to share a common transport system that may consist of multiple components.     

艾氏乳腺癌腹水细胞质膜NADH-铁氰化钾氧化还原酶质子泵活性诱导细胞与脂质体融合

在本文中,我们用荧光能量共振转移分析和荧光显微技术证明,小鼠艾氏乳腺癌腹水细胞质膜NADH-铁氰化钾氧化还原反应的电子传递所偶联的质子泵活性能诱导细胞与人工脂质体融合。糖酵解代谢的抑制剂碘乙酸能抑制融合,同时融合过程是吸取质子的。近几年来,我们实验室已报道了多种生物膜质子泵均具有诱导膜融合的功能。因此,质子泵诱导膜融合可能具有比较广泛的生理意义。并为细胞中存在有受能量代谢控制的驱动膜融合的生理机制提供了实验证据。     

Structure-based development of small molecule PFKFB3 inhibitors: a framework for potential cancer therapeutic agents targeting the Warburg effect

Cancer cells adopt glycolysis as the major source of metabolic energy production for fast cell growth. The HIF-1-induced PFKFB3 plays a key role in this adaptation by elevating the concentration of Fru-2,6-BP, the most potent glycolysis stimulator. As this metabolic conversion has been suggested to be a hallmark of cancer, PFKFB3 has emerged as a novel target for cancer chemotherapy. Here, we report that a small molecular inhibitor, N4A, was identified as an initial lead compound for PFKFB3 inhibitor with therapeutic potential. In an attempt to improve its potency, we determined the crystal structure of the PFKFB3•N4A complex to 2.4 Å resolution and, exploiting the resulting molecular information, attained the more potent YN1. When tested on cultured cancer cells, both N4A and YN1 inhibited PFKFB3, suppressing the Fru-2,6-BP level, which in turn suppressed glycolysis and, ultimately, led to cell death. This study validates PFKFB3 as a target for new cancer therapies and provides a framework for future development efforts.     

13C-NMR spectroscopic studies on the glucose metabolism of Angiostrongylus cantonesis' eggs with special reference to the end-products and metabolic pathway

13C-nuclear magnetic resonance (NMR) spectroscopy was used to identify metabolites excreted by Angiostrongylus cantonensis eggs which had been maintained aerobically in the presence of D-[13C6] glucose. Using 13C-NMR we proved that lactate, acetate and alanine originated from glucose present in the medium via glycolysis. Aminooxyacetate, an inhibitor of alanine transferase, inhibited simultaneously alanine production and the ability to take up glutamate, aspartate and valine from the medium. In addition, we demonstrated that these amino acids can serve as amino group donors of the pyruvate to alanine transamination system in the eggs.     

Combining p53 stabilizers with metformin induces synergistic apoptosis through regulation of energy metabolism in castration-resistant prostate cancer

Since altered energy metabolism is a hallmark of cancer, many drugs targeting metabolic pathways are in active clinical trials. The tumor suppressor p53 is often inactivated in cancer, either through downregulation of protein or loss-of-function mutations. As such, stabilization of p53 is considered as one promising approach to treat those cancers carrying wild type (WT) p53. Herein, SIRT1 inhibitor Tenovin-1 and polo-like kinase 1 (Plk1) inhibitor BI2536 were used to stabilize p53. We found that both Tennovin-1 and BI2536 increased the anti-neoplastic activity of metformin, an inhibitor of oxidative phosphorylation, in a p53 dependent manner. Since p53 has also been shown to regulate metabolic pathways, we further analyzed glycolysis and oxidative phosphorylation upon drug treatments. We showed that both Tennovin-1 and BI2536 rescued metformin-induced glycolysis and that both Tennovin-1 and BI2536 potentiated metformin-associated inhibition of oxidative phosphorylation. Of significance, castration-resistant prostate cancer (CRPC) C4-2 cells show a much more robust response to the combination treatment than the parental androgen-dependent prostate cancer LNCaP cells, indicating that targeting energy metabolism with metformin plus p53 stabilizers might be a valid approach to treat CRPC carrying WT p53.     

Irreversible paraben inhibition of glycolysis by Streptococcus mutans GS-5

Parabens were found to inhibit irreversibly glycolysis by the cariogenic dental plaque bacterium Streptococcus mutans GS-5 and to decrease the capacity of the bacterium to lower the pH in dense cell suspensions containing excess glucose. The hierarchy of effectiveness was butyl > propyl > ethyl > methyl paraben. Results of studies of the nature of glycolytic inhibition by butyl paraben indicated that it could act at millimolar concentrations as an irreversible inhibitor of the phosphotransferase system for sugar uptake and was lethal for the bacterium at these same levels. Butyl paraben acted also as a reversible inhibitor of the F-ATPase of the organism. Overall, it appeared that the lethal actions of parabens can be interpreted at least in part as due to irreversible damage to key enzymes, such as those of the phosphotransferase system.     

Evaluating the potential of vacuolar ATPase inhibitors as anticancer agents and multigram synthesis of the potent salicylihalamide analog saliphenylhalamide

The natural product salicylihalamide is a potent inhibitor of the Vacuolar ATPase (V-ATPase), a potential target for antitumor chemotherapy. We generated salicylihalamide-resistant tumor cell lines typified by an overexpansion of lysosomal organelles. We also found that many tumor cell lines upregulate tissue-specific plasmalemmal V-ATPases, and hypothesize that tumors that derive their energy from glycolysis rely on these isoforms to maintain a neutral cytosolic pH. To further validate the potential of V-ATPase inhibitors as leads for cancer chemotherapy, we developed a multigram synthesis of the potent salicylihalamide analog saliphenylhalamide.     

Tumor cell killing by macrophages activated in vitro with lymphocyte mediators. IV. Role of energy metabolism

The role of energy metabolism on tumor cell killing by in vitro activated macrophages was studied. Depletion of extracellular glucose had little effect on the cytotoxic capacity of mediator-activated macrophages. Respiratory antagonists did not inhibit cytotoxicity regardless of whether or not the assays were carried out in low-glucose-containing medium. Sodium fluoride, a known inhibitor of glycolysis, inhibited the killing of tumor cells by activated macrophages. 2-Deoxyglucose, an analog of glucose, was found to be an effective inhibitor of cytotoxicity. Three other analogs, 5-thio-d-glucose, 3-O-methylglucose, and 2-deoxy-d-galactose, were without effect. The concentrations of 2-DG that inhibited cytotoxicity did not lower cellular ATP levels to an appreciable extent. The combined addition of inhibitors of glycolysis and respiration resulted in a marked reduction in ATP levels. Under these experimental conditions, macrophage-mediated cytotoxicity was also significantly inhibited.     

Study on the regulatory role of fructose-1,6-diphosphate in the formation of AMP in rat skeletal muscle. A mechanism for synchronization of glycolysis and the purine nucleotide cycle.

Fructose-1,6-diphosphate strongly inhibited adenylosuccinate synthetase purified from rat skeletal muscle. This compound was found to be a non-competitive inhibitor of all substrates of the enzyme. No other glycolytic intermediates affected adenylosuccinate synthetase activity. From these findings, it was proposed that this inhibition might play an important role in the oscillation of glycolysis in skeletal muscle.