姜黄素对食管癌KYSE410细胞的生长抑制与氧化调节

通过四氮唑蓝盐化合物(MTS)和重要氧化还原酶及其代谢物的试剂盒检测等方法, 考察了姜黄素对食管癌KYSE410细胞生长以及对细胞氧化还原状态和代谢的影响. 结果表明, 姜黄素对KYSE410细胞具有较强的抑制作用, 其IC₅₀=17.9 μmol/L. 进一步研究发现, 姜黄素可引起细胞培养上层清液中超氧化物歧化酶(SOD)和丙二醛(MDA)水平发生变化. 当姜黄素浓度为40 μmol/L时, MDA水平比对照组提高了125.1%, 而SOD水平则降低了43.2%; 同时, 乳酸水平降低了44.4%, 乳酸脱氢酶的活性下降了58.2%, 丙酮酸激酶的活性升高了216.7%. 姜黄素可能通过干扰氧化还原途径, 致使发生脂质过氧化反应, 并抑制肿瘤细胞糖酵解作用, 进而抑制食管癌KYSE410细胞增殖.     

同位素示踪技术定量分析肿瘤细胞中的代谢重编

肿瘤代谢在最近几年重新获得重视,讨论肿瘤代谢我们无法避开瓦伯格效应,尽管瓦伯格效应在20世纪20年代就提出来了。瓦伯格效应也称需氧糖酵解,是指在有氧的条件下癌细胞过量摄取葡萄糖,产生更多的乳酸。此外,癌细胞也可摄取其他物质,如谷氨酰胺、丝氨酸和甘氨酸等作为能量需求与营养来源。为了描述癌细胞中改变了的代谢通路,同位素标记示踪技术提供了一种新的全局观点,让我们更好地理解在癌基因或抑癌基因调控下的代谢切换。色谱与质谱联用是一种重要的分析工具,可定量检测带有同位素标记的代谢物,从而剖析肿瘤细胞中的生物化学反应。在本文中,我们总结了基于质谱的肿瘤代谢分析的研究概况并提出未来的研究方向。重点讨论碳-13标记与氘代标记的示踪技术分析中间代谢产物,包括还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)。目前生物化学教科书中认为NADPH主要来源于磷酸戊糖途径。然而,氘代示踪技术分析表明,包括一碳代谢在内的其他代谢通路对产生NADPH也很重要。以上内容都在本文中进行了讨论。     

Metabolic Studies of Tumor Cells Using [1-13C] Pyruvate Hyperpolarized by Means of PHIP-Side Arm Hydrogenation

The kinetics of metabolic processes can be assessed, in real time by means of MR hyperpolarized (HP) metabolites. [1-¹³C]pyruvate, hyperpolarized by means of d-DNP, is, by far, the substrate most widely applied to the investigation of several pathologies characterized by deregulated glycolytic metabolic networks, including cancer. Hyperpolarization of [1-¹³C]pyruvate by means of the cost effective, fast and easy to handle PHIP-SAH (para-hydrogen induced polarization-side arm hydrogenation) method opens-up a pathway for the application of HP metabolites to a wide range of cancer-related studies. Herein, we report the first application of PHIP-SAH hyperpolarized [1-¹³C]pyruvate in the investigation of upregulated glycolysis in two murine breast cancer cell lines (168FARN and 4T1). The results obtained using HP pyruvate have been validated with a conventional biochemical assay and are coherent with previously-reported lactate dehydrogenase activity measured in those cells.     

A pairwise chemical genetic screen identifies new inhibitors of glucose transport

Oxidative phosphorylation (OXPHOS) and glycolysis are the two main pathways that control energy metabolism of a cell. The Warburg effect, in which glycolysis remains active even under aerobic conditions, is considered a key driver for cancer cell proliferation, malignancy, metastasis, and therapeutic resistance. To target aerobic glycolysis, we exploited the complementary roles of OXPHOS and glycolysis in ATP synthesis as the basis for a chemical genetic screen, enabling rapid identification of novel small-molecule inhibitors of facilitative glucose transport. Blocking mitochondrial electron transport with antimycin A or leucascandrolide A had little effect on highly glycolytic A549 lung carcinoma cells, but adding known glycolytic inhibitors 2-deoxy-D-glucose, iodoacetate or cytochalasin B, rapidly depleted intracellular ATP, displaying chemical synthetic lethality. Based on this principle, we exposed antimycin A-treated A549 cells to a newly synthesized 955 member diverse scaffold small-molecule library, screening for compounds that rapidly depleted ATP levels. Two compounds potently suppressed ATP synthesis, induced G1 cell-cycle arrest and inhibited lactate production. Pathway analysis revealed that these novel probes inhibited GLUT family of facilitative transmembrane transporters but, unlike cytochalasin B, had no effect on the actin cytoskeleton. Our work illustrated the utility of a pairwise chemical genetic screen for discovery of novel chemical probes, which would be useful not only to study the system-level organization of energy metabolism but could also facilitate development of drugs targeting upregulation of aerobic glycolysis in cancer.     

通过调控肿瘤代谢增强T细胞抗肿瘤活性

肿瘤免疫治疗是一种新型的癌症治疗方法。 然而,现阶段肿瘤免疫疗法的临床响应率低等问题严重制约了其进一步应用。 其根本原因在于肿瘤组织免疫抑制微环境限制了T细胞的抗肿瘤活性,而肿瘤组织代谢改变在肿瘤免疫抑制微环境形成过程中起关键作用。 本文重点总结了通过调控肿瘤的有氧糖酵解、氨基酸代谢和脂肪酸代谢增强T细胞的抗肿瘤活性。 最后,提出当前肿瘤代谢调控研究方面仍然存在的问题,并对纳米载体在肿瘤代谢领域的发展前景进行了展望。     

Direct coupling of intracerebral dialysis with flow injection analysis based on enzymatic/fluorescence detection of lactic acid

Rapid, specific measurement of the lactic acid content of the extracellular fluid of a conscious, freely-moving rat can be accomplished by direct combination of intracerebral dialysis and flow injection analysis with enzymatic/fluorescence detection. Basal levels of lactic acid, the end product of anaerobic glycolysis, can be measured by using lactate dehydrogenase to convert lactate to pyruvate with consequent generation of NADH, which is monitored fluorimetrically. The rapid response time of this system, 40 s, allows real-time measurement of transient changes in anaerobic metabolism in the striatum of the rat, as illustrated after electrically-induced seizures.     

MS analysis reveals O-methylation of L-lactate dehydrogenase from pancreatic ductal adenocarcinoma cells

L-lactate dehydrogenase (LDH) converts pyruvate to lactate when oxygen is absent or in short supply, and the enzyme plays a crucial role in cancer metabolism. The functions of many mammalian proteins are modulated by posttranslational modifications (PTMs), and it has been reported that LDH was subjected to several PTMs, including phosphorylation, acetylation, and methylation. In this present work, we characterized the PTMs of LDH from pancreatic ductal adenocarcinoma (PDAC) cells by electrophoresis and mass spectrometry, and identified 13 O-methylated residues from the enzyme. In addition, our qualitative analysis revealed differential methylation of LDH from normal duct cells. The preliminary findings from this study provide important biochemical information toward further understanding of the LDH modifications and their functional significance in pathophysiological processes of pancreatic cancer.     

Molecular recognition and signal processing in biosensors

The specificity of molecular recognition is reflected to a high degree by the selectivity of the respective biosensor response. Therefore, the application of highly specific enzymes offers advantages for analytical purposes. Using lactate monooxygenase in combination with lactate dehydrogenase and pyruvate kinase, sequentially acting enzyme electrodes for lactate, pyruvate, ADP and enzyme activities, e.g. lactate dehydrogenase, creatine kinase and aminotransferases were developed. A high sensitivity was achieved based on the cycling enzyme pairs hexokinase/pyruvate kinase for ATP and ADP, and lactate monooxygenase/malate dehydrogenase for malate and oxaloacetate, respectively. On the other hand, in a sensor for a large group of substances, the unspecific microsomal cytochrome P-450 system was applied.     

Metabolic Tumor Imaging with Rapidly Signal-Enhanced 1-13C-Pyruvate-d3

The metabolism of malignant cells differs significantly from that of healthy cells and thus, it is possible to perform metabolic imaging to reveal not only the exact location of a tumor, but also intratumoral areas of high metabolic activity. Herein, we demonstrate the feasibility of metabolic tumor imaging using signal-enhanced 1-¹³C-pyruvate-d₃, which is rapidly enhanced via para-hydrogen, and thus, the signal is amplified by several orders of magnitudes in less than a minute. Using as a model, human melanoma xenografts injected with signal-enhanced 1-¹³C-pyruvate-d3, we show that the conversion of pyruvate into lactate can be monitored along with its kinetics, which could pave the way for rapidly detecting and monitoring changes in tumor metabolism.     

Surface Immobilization of Redox‐Labile Fluorescent Probes: Enabling Single‐Cell Co‐Profiling of Aerobic Glycolysis and Oncogenic Protein Signaling Activities

An analytical method is described for profiling lactate production in single cells via the use of coupled enzyme reactions on surface‐grafted resazurin molecules. The immobilization of the redox‐labile probes was achieved through chemical modifications on resazurin, followed by bio‐orthogonal click reactions. The lactate detection was demonstrated to be sensitive and specific. The method was incorporated into a single‐cell barcode chip for simultaneous quantification of aerobic glycolysis activities and oncogenic signaling phosphoproteins in cancer. The interplay between glycolysis and oncogenic signaling activities was interrogated on a glioblastoma cell line. Results revealed a drug‐induced oncogenic signaling reliance accompanying shifted metabolic paradigms. A drug combination that exploits this induced reliance exhibited synergistic effects in growth inhibition.     

Real‐Time Monitoring of Cancer Cell Metabolism and Effects of an Anticancer Agent using 2D In‐Cell NMR Spectroscopy

Altered metabolism is a critical part of cancer cell properties, but real‐time monitoring of metabolomic profiles has been hampered by the lack of a facile method. Here, we propose real‐time metabolomic monitoring of live cancer cells using ¹³C₆‐glucose and heteronuclear two‐dimensional (2D) NMR. The method allowed for metabolomic differentiation between cancer and normal cells on the basis of time‐dependent changes in metabolite concentrations. Cancer cells were found to have large in‐ and out‐flux of pyruvate as well as increased net production of alanine and acetate. The method also enabled evaluation of the metabolic effects of galloflavin whose anticancer effects have been attributed to its specific inhibition of lactate dehydrogenase. Our approach revealed previously unknown functional targets of galloflavin, which were further confirmed at the protein levels. Our method is readily applicable to the study of metabolic alterations in other cellular disease model systems.     

Metabolomics of colorectal cancer: past and current analytical platforms

Metabolomics is coming of age as an important area of investigation which may help reveal answers to questions left unanswered or only partially understood from proteomic or genomic approaches. Increased knowledge of the relationship of genes and proteins to smaller biomolecules (metabolites) will advance our ability to diagnose, treat, and perhaps prevent cancer and other diseases that have eluded scientists for generations. Colorectal tumors are the second leading cause of cancer mortality in the USA, and the incidence is rising. Many patients present late, after the onset of symptoms, when the tumor has spread from the primary site. Once metastases have occurred, the prognosis is significantly worse. Understanding alterations in metabolic profiles that occur with tumor onset and progression could lead to better diagnostic tests as well as uncover new approaches to treat or even prevent colorectal cancer (CRC). In this review, we explore the various analytical technologies that have been applied in CRC metabolomics research and summarize all metabolites measured in CRC and integrate them into metabolic pathways. Early studies with nuclear magnetic resonance and gas-chromatographic mass spectrometry suggest that tumor cells are characterized by aerobic glycolysis, increased purine metabolism for DNA synthesis, and protein synthesis. Liquid chromatography, capillary electrophoresis, and ion mobility, each coupled with mass spectrometry, promise to advance the field and provide new insight into metabolic pathways used by cancer cells. Studies with improved technology are needed to identify better biomarkers and targets for treatment or prevention of CRC.

Lactate and pyruvate levels correlation with lactate dehydrogenase gene expression and glucose consumption in Tamoxifen-resistant MCF-7 cells using capillary electrophoresis with contactless conductivity detection (CE-C4D)

Breast cancer is the second leading cause of cancer death in women after lung cancer. The first-line treatment of metastatic breast cancer in premenopausal women relies on tamoxifen. The development of tamoxifen resistance is not fully understood. In this study, capillary electrophoresis with capacitively coupled contactless conductivity detector was developed to monitor the changes in lactate and pyruvate levels in supernatant media of three models of developed MCF-7 tamoxifen-resistant cells and correlate these metabolites changes with lactate dehydrogenase genes expression and glucose consumption. The electrophoretic separation was achieved under reversed electroosmotic flow conditions. The linear ranges were 0.15–5 and 0.01–1 mM with a correlation coefficient of 0.9966 and 0.9971 and the limits of detection were 0.01 and 0.02 μM for lactate and pyruvate, respectively. Inter- and intrarun accuracy were in the range of 96.88–105.94% with precision (CV, %) of ≤7.35%. The method was completely validated and the results were in agreement with those obtained using the lactate and glucose assay kits. The results revealed a significant increase in both lactate and pyruvate production in the three tamoxifen-resistant MCF-7 cells models compared to control cells. This increase was correlated with the increase of lactate dehydrogenase genes expression and the increase of glucose consumption.     

Antiglioma pseurotin A from marine Bacillus sp. FS8D regulating tumour metabolic enzymes

Pseurotin A was isolated from a culture of marine Bacillus sp. FS8D and showed to be active against the proliferation of four different glioma cells with IC₅₀ values of 0.51–29.3 μM. It has been found that pseurotin A downregulated the expression of tumour glycolytic enzymes pyruvate kinase M2 (PKM2) and lactate dehydrogenase 5 (LDH5) and upregulated the expression of pyruvate dehydrogenase beta (PDHB), adenosine triphosphate synthase beta (ATPB) and cytochrome C (Cyto-C), the important regulators for tricarboxylic acid cycle and oxidative phosphorylation. The data suggested that targeting multiple metabolic enzymes might be one of the antiglioma mechanisms of pseurotin A.     

Probing lactate dehydrogenase activity in tumors by measuring hydrogen/deuterium exchange in hyperpolarized l-[1-(13)C,U-(2)H]lactate

(13)C magnetic resonance spectroscopy and spectroscopic imaging measurements of hyperpolarized (13)C label exchange between exogenously administered [1-(13)C]pyruvate and endogenous lactate, catalyzed by lactate dehydrogenase (LDH), has proved to be a powerful approach for probing tissue metabolism in vivo. This experiment has clinical potential, particularly in oncology, where it could be used to assess tumor grade and response to treatment. A limitation of the method is that pyruvate must be administered in vivo at supra-physiological concentrations. This problem can be avoided by using hyperpolarized [1-(13)C]lactate, which can be used at physiological concentrations. However, sensitivity is limited in this case by the relatively small pyruvate pool size, which would result in only low levels of labeled pyruvate being observed even if there was complete label equilibration between the lactate and pyruvate pools. We demonstrate here a more sensitive method in which a doubly labeled lactate species can be used to measure LDH-catalyzed exchange in vivo. In this experiment exchange of the C2 deuterium label between injected hyperpolarized l-[1-(13)C,U-(2)H]lactate and endogenous unlabeled lactate is observed indirectly by monitoring phase modulation of the spin-coupled hyperpolarized (13)C signal in a heteronuclear (1)H/(13)C spin-echo experiment.     

Pyruvate cellular uptake and enzymatic conversion probed by dissolution DNP‐NMR: the impact of overexpressed membrane transporters

Pyruvate membrane crossing and its lactate dehydrogenase‐mediated conversion to lactate in cells featuring different levels of expression of membrane monocarboxylate transporters (MCT4) were probed by dissolution dynamic nuclear polarization‐enhanced NMR. Hyperpolarized ¹³C‐1‐labeled pyruvate was transferred to suspensions of rodent tumor cell carcinoma, cell line 39. The pyruvate‐to‐lactate conversion rate monitored by dissolution dynamic nuclear polarization‐NMR in carcinoma cells featuring native MCT4 expression level was lower than the rate observed for cells in which the human MCT4 gene was overexpressed. The enzymatic activity of lactate dehydrogenase was also assessed in buffer solutions, following the real‐time pyruvate‐to‐lactate conversion speeds at different enzyme concentrations. Copyright © 2016 John Wiley & Sons, Ltd.     

The Pyruvate Dehydrogenase Multienzyme Complex

The three enzymes pyruvate dehydrogenase, dihydrolipoamide transacetylase, and dihydrolipoamide dehydrogenase constitute the pyruvate dehydrogenase multienzyme complex of E. coli; in mammals the complex also contains a kinase and a phosphatase. Multienzyme complexes are structural, functional, and regulatory units enabling the organism to operate more economically than with single enzymes. The pyruvate dehydrogenase multienzyme complex may stand at the switch-point between energy metabolism and gluconeogenesis.     

Nanoscale metal organic frameworks inhibition of pyruvate kinase of M2

Tumor cells usually show abnormally high glycolysis rate to maintain the dynamic balance of energy. The growth of tumor cells can be affected by inhibiting the activity of pyruvate kinase (especially M2-type isozyme, PKM2), the rate limiting enzyme of glycolysis. This is helpful to the treatment of tumor. Herein, metal organic frameworks (MOFs) were found to inhibit the activity of PKM2. Nanoscale ZIF-8 was synthesized by standing and ultrasonic method, respectively. The ZIF-8 has the performance of inhibiting PKM2. Further research showed that the inhibition ability was attributed to zinc ion in ZIF-8. Interestingly, the IC₅₀ of ZIF-8 on PKM2 was one percent of that of zinc ion. This novel enzyme inhibitor is expected to be used in cancer therapy.     

Metabolic Inertia in Contracting Skeletal Muscle: The Expanding Role of the Carnitine Pool

The ability of the muscular carnitine pool to accept and temporally donate acetyl groups (from and towards the coenzyme A pool) is an important functional role of carnitine within biological systems that is often overlooked within the scientific literature. The present review will discuss recent research demonstrating the existence of a period of inadequate acetyl-CoA delivery towards the tricarboxylic acid cycle (the so-called ‘acetyl group deficit’), which occurs as a consequence of the impaired integration of cytosolic (glycolysis) and mitochondrial energy producing pathways at the onset of muscular contraction; due to a lag in the activation of the pyruvate dehydrogenase complex. During this period of inadequate acetyl-CoA delivery, acetyl groups can be sequestered from the limited muscular acetylcarnitine reserve in an attempt to sustain continued tricarboxylic acid cycle flux. Following on from this, the present review will highlight the metabolic and functional benefits to be gained by overcoming this period of metabolic inertia, through elevating the concentration of acetylcarnitine prior to physical exercise; in the presence and absence of pyruvate dehydrogenase complex activation and through appropriately timed ‘warm-up’ exercise.     

Metabolic Inertia in Contracting Skeletal Muscle: The Expanding Role of the Carnitine Pool

Summary. The ability of the muscular carnitine pool to accept and temporally donate acetyl groups (from and towards the coenzyme A pool) is an important functional role of carnitine within biological systems that is often overlooked within the scientific literature. The present review will discuss recent research demonstrating the existence of a period of inadequate acetyl-CoA delivery towards the tricarboxylic acid cycle (the so-called ‘acetyl group deficit’), which occurs as a consequence of the impaired integration of cytosolic (glycolysis) and mitochondrial energy producing pathways at the onset of muscular contraction; due to a lag in the activation of the pyruvate dehydrogenase complex. During this period of inadequate acetyl-CoA delivery, acetyl groups can be sequestered from the limited muscular acetylcarnitine reserve in an attempt to sustain continued tricarboxylic acid cycle flux. Following on from this, the present review will highlight the metabolic and functional benefits to be gained by overcoming this period of metabolic inertia, through elevating the concentration of acetylcarnitine prior to physical exercise; in the presence and absence of pyruvate dehydrogenase complex activation and through appropriately timed ‘warm-up’ exercise.