bims-mascan 2019-06-16 papers

bims-mascan

Biomed News

on Mass spectrometry in cancer research

Issue of 2019–06–16
twenty-one papers selected by
Giovanny Rodríguez Blanco, The Beatson Institute for Cancer Research

Connections between metabolism and epigenetics in Cancers.
High extracellular lactate causes reductive carboxylation in breast tissue cell lines grown under normoxic conditions.
Leptin promotes fatty acid oxidation and OXPHOS via the c-Myc/PGC-1 pathway in cancer cells.
Identification of potential metabolic biomarkers of polycystic ovary syndrome in follicular fluid by SWATH mass spectrometry.
Hypoxia and Metabolism in Metastasis.
Characterization of Bulk Phosphatidylcholine Compositions in Human Plasma Using Side-Chain Resolving Lipidomics.
Cancer cell derived small extracellular vesicles contribute to recipient cell metastasis through promoting HGF/c-Met pathway.
Fatty Acids Corrupt Neutrophils in Cancer.
Pyruvate Kinase M2: a Metabolic Bug in Re-Wiring the Tumor Microenvironment.
XNet: A Bayesian approach to Extracted Ion Chromatogram Clustering for Precursor Mass Spectrometry Data.
Cytosine-5 RNA methylation links protein synthesis to cell metabolism.
Ultrafast Micro-Electrophoresis: Behind Direct Mass Spectrometry Measurements of Proteins and Metabolites in Living Cell/Cells.
Cardiolipin-induced activation of pyruvate dehydrogenase links mitochondrial lipid biosynthesis to TCA cycle function.
Measurement of Subcellular Metabolite Concentrations in Relation to Phloem Loading.
Lipid metabolism and Calcium signaling in epithelial ovarian cancer.
Metabolomic Analysis of Liver Tissues for Characterization of Hepatocellular Carcinoma.
LC-MS-based lipid profile in colorectal cancer patients: TAGs are the main disturbed lipid markers of colorectal cancer progression.
Profiling of Polar Metabolites in Mouse Feces Using Four Analytical Platforms to Study the Effects Of Cathelicidin-Related Antimicrobial Peptide in Alcoholic Liver Disease.
Combining Glucose Units, m/z and Collision Cross Section values: Multi-attribute data for increased accuracy in automated glycosphingolipid glycan identifications and its application in Triple Negative Breast Cancer.
Generating fatty acid profiles in the gas-phase: Fatty acid identification and relative quantitation using ion/ion charge inversion chemistry.
α-Ketoglutarate inhibits autophagy.

Semin Cancer Biol. 2019 Jun 08. pii: S1044-579X(18)30082-8. [Epub ahead of print]

Connections between metabolism and epigenetics in Cancers.

Chitra Thakur, Fei Chen.

  In the past half century, our version on cancer, from tumor initiation, growth, to metastasis, is dominated by genetic mutation. The importance of metabolism and epigenetics was not recognized until most recently. Extensive cell proliferation is one of the hallmarks of cancers. To support the energetic and anabolic demands of enhanced proliferation, tumors reprogram the pathways of nutrient procurement and metabolism. In this context, a new link between metabolic alterations and cancer progression has been unraveled over the last decade by the studies conducted in the area of cancer cell metabolism. Cancer cells are known to alter their metabolic profile during the course of tumorigenesis and metastasis thereby exhibiting a tightly regulated program of metabolic plasticity. Noteworthy, certain metabolic alteration are known to occur at the epigenetic level, thus making epigenetics and metabolism highly interwoven in a reciprocal manner. Metabolites that are generated during metabolic pathways, such as in glycolytic cycle and oxidative phosphorylation, serve as cofactors or substrates for the enzymatic reactions that catalyze the epigenetic modifications and transcriptional regulation. Several studies also indicate that the epigenome is sensitive to cellular metabolism. Since many of the metabolic alterations and consequently aberrated epigenetic regulation are common to a wide range of cancer types, they serve as promising targets for anti-cancer therapies. Here we discuss the latest findings in cancer cell metabolism, elucidating the major anabolic, catabolic and energetic demands required for sustaining cancer growth, and the influence of altered metabolism on epigenetics and vice versa. A comprehensive research pertaining to metabolomic profiling and epigenome interactors/mediators in malignant neoplasias is imperative in deciphering the potential targets that can be exploited for the development of robust anti-cancer therapies.

Keywords:  DNA and histone methylation; cancer cell metabolism; epigenetics; warburg effect

DOI:  https://doi.org/10.1016/j.semcancer.2019.06.006
PLoS One. 2019 ;14(6): e0213419

High extracellular lactate causes reductive carboxylation in breast tissue cell lines grown under normoxic conditions.

Arthur Nathan Brodsky, Daniel C Odenwelder, Sarah W Harcum.

In cancer tumors, lactate accumulation was initially attributed to high glucose consumption associated with the Warburg Effect. Now it is evident that lactate can also serve as an energy source in cancer cell metabolism. Additionally, lactate has been shown to promote metastasis, generate gene expression patterns in cancer cells consistent with "cancer stem cell" phenotypes, and result in treatment resistant tumors. Therefore, the goal of this work was to quantify the impact of lactate on metabolism in three breast cell lines (one normal and two breast cancer cell lines-MCF 10A, MCF7, and MDA-MB-231), in order to better understand the role lactate may have in different disease cell types. Parallel labeling metabolic flux analysis (13C-MFA) was used to quantify the intracellular fluxes under normal and high extracellular lactate culture conditions. Additionally, high extracellular lactate cultures were labelled in parallel with [U-13C] lactate, which provided qualitative information regarding the lactate uptake and metabolism. The 13C-MFA model, which incorporated the measured extracellular fluxes and the parallel labeling mass isotopomer distributions (MIDs) for five glycolysis, four tricarboxylic acid cycle (TCA), and three intracellular amino acid metabolites, predicted lower glycolysis fluxes in the high lactate cultures. All three cell lines experienced reductive carboxylation of glutamine to citrate in the TCA cycle as a result of high extracellular lactate. Reductive carboxylation previously has been observed under hypoxia and other mitochondrial stresses, whereas these cultures were grown aerobically. In addition, this is the first study to investigate the intracellular metabolic responses of different stages of breast cancer progression to high lactate exposure. These results provide insight into the role lactate accumulation has on metabolic reaction distributions in the different disease cell types while the cells are still proliferating in lactate concentrations that do not significantly decrease exponential growth rates.

DOI: https://doi.org/10.1371/journal.pone.0213419
Acta Biochim Biophys Sin (Shanghai). 2019 Jun 12. pii: gmz058. [Epub ahead of print]

Leptin promotes fatty acid oxidation and OXPHOS via the c-Myc/PGC-1 pathway in cancer cells.

Qianqian Liu, Yang Sun, Zaiyi Fei, Zhibin Yang, Ke Duan, Jiaji Zi, Qinghua Cui, Min Yu, Wei Xiong.

  Alteration in cellular energy metabolism plays a critical role in the development and progression of cancer. Leptin is a hormone secreted by adipose tissue. Recent reports have shown that leptin can induce cancer cell proliferation and regulate cell energy metabolism, but the regulatory mechanism is still unclear. Here, we showed that leptin could promote cell proliferation and maintain high adenosine triphosphate levels in HCT116 and MCF-7 cells. The expression levels of carnitine palmitoyl transferase 1A (CPT1A), pyruvate dehydrogenase, succinate dehydrogenase subunit A and mitochondrial respiratory chain-associated proteins NADH dehydrogenase 1 (ND1), NADH:ubiquinone oxidoreductase subunit B8, and mitochondrial transcription factor A (TFAM) were distinctly increased in leptin-treated HCT116 and MCF-7 cells, while fatty acid synthase and lactate dehydrogenase expression were downregulated. Simultaneously, we found that c-Myc and peroxisome proliferator-activated receptor gamma co-activator 1 (PGC-1) protein expression levels were significantly increased. These results indicated that leptin boosted fatty acid β-oxidation and the tricarboxylic acid cycle, enhanced oxidative phosphorylation (OXPHOS) activity, and inhibited fatty acid synthesis and glycolysis in tumor cells. Gene transfection experiments revealed that leptin could induce the expression of c-Myc. Moreover, the expressions of PGC-1, CPT1A, and TFAM proteins were downregulated in HCT116 cells with low expression of c-Myc, and the expression levels of these proteins were increased in HCT116 cells overexpressing c-Myc. These findings suggest that leptin plays an important role in the regulation of energy metabolism in tumor cells. It may regulate fatty acid oxidation and OXPHOS of tumor cells by regulating the c-Myc/PGC-1 pathway. Targeting metabolic pathways for cancer treatment has been investigated as potential preventive or therapeutic methods. This study has important implications for the clinical therapy of tumor cell metabolism through hormone regulation.

Keywords:  OXPHOS; c-Myc/PGC-1 pathway; cancer cells; fatty acid oxidation; leptin

DOI:  https://doi.org/10.1093/abbs/gmz058
Reprod Biol Endocrinol. 2019 Jun 11. 17(1): 45

Identification of potential metabolic biomarkers of polycystic ovary syndrome in follicular fluid by SWATH mass spectrometry.

Zhengao Sun, Hsun-Ming Chang, Aijuan Wang, Jingyan Song, Xingxing Zhang, Jiayin Guo, Peter C K Leung, Fang Lian.

   BACKGROUND: Polycystic ovary syndrome (PCOS) is a complex disorder associated with multiple metabolic disturbance, including defective glucose metabolism and insulin resistance. The altered metabolites caused by the related metabolic disturbance may affect ovarian follicles, which can be reflected in follicular fluid composition. The aim of this study is to investigate follicular fluid metabolic profiles in women with PCOS using an advanced sequential window acquisition of all theoretical fragment-ion spectra (SWATH) mass spectrometry.
MATERIALS AND METHODS: Nineteen women with PCOS and twenty-one healthy controls undergoing IVF/ET were recruited, and their follicular fluid samples were collected for metabolomic study. Follicular fluid metabolic profiles, including steroid hormones, free fatty acids, bioactive lipids, and amino acids were analyzed using the principal component analysis (PCA) and partial least squares to latent structure-discriminant analysis (PLS-DA) model.
RESULTS: Levels of free fatty acids, 3-hydroxynonanoyl carnitine and eicosapentaenoic acid were significantly increased (P < 0.05), whereas those of bioactive lipids, lysophosphatidylcholines (LysoPC) (16:0), phytosphingosine, LysoPC (14:0) and LysoPC (18:0) were significantly decreased in women with PCOS (P < 0.05). Additionally, levels of steroid hormone deoxycorticosterone and two amino acids, phenylalanine and leucine were higher in the PCOS patients (P < 0.05).
CONCLUSION: Women with PCOS display unique metabolic profiles in their follicular fluid, and this data may provide us with important biochemical information and metabolic signatures that enable a better understanding of the pathogenesis of PCOS.

Keywords:  Follicular fluid; IVF-ET; Metabolomics; PCOS; SWATH

DOI:  https://doi.org/10.1186/s12958-019-0490-y
Adv Exp Med Biol. 2019 ;1136 87-95

Hypoxia and Metabolism in Metastasis.

Tong Zhang, Caixia Suo, Chenyang Zheng, Huafeng Zhang.

  The hypoxic microenvironment is one of the major features of solid tumors, which regulates cell malignancy in multiple ways. As a response to hypoxia, a large number of target genes involved in cell growth, metabolism, metastasis and immunity are activated in cancer cells. Hypoxia-inducible factor 1 (HIF-1), as a heterodimeric DNA-binding complex, is comprised of a constitutively expressed HIF-1β subunit and an oxygen sensitive HIF-1α subunit, thus, adapts to decreased oxygen availability as a transcriptional factor. HIF-1 regulates many genes involved in tumorigenesis. Here, we focus on cancer cell metabolism and metastasis regulated by hypoxia.

Keywords:  EMT; Glycogen synthesis; Glycolysis; HIF1; Hypoxia; Lipid metabolism; Metabolic enzymes; Metastasis; Metastatic niche; Mitochondria

DOI:  https://doi.org/10.1007/978-3-030-12734-3_6
Metabolites. 2019 Jun 08. pii: E109. [Epub ahead of print]9(6):

Characterization of Bulk Phosphatidylcholine Compositions in Human Plasma Using Side-Chain Resolving Lipidomics.

Jan D Quell, Werner Römisch-Margl, Mark Haid, Jan Krumsiek, Thomas Skurk, Anna Halama, Nisha Stephan, Jerzy Adamski, Hans Hauner, Dennis Mook-Kanamori, Robert P Mohney, Hannelore Daniel, Karsten Suhre, Gabi Kastenmüller.

  Kit-based assays, such as AbsoluteIDQTM p150, are widely used in large cohort studies and provide a standardized method to quantify blood concentrations of phosphatidylcholines (PCs). Many disease-relevant associations of PCs were reported using this method. However, their interpretation is hampered by lack of functionally-relevant information on the detailed fatty acid side-chain compositions as only the total number of carbon atoms and double bonds is identified by the kit. To enable more substantiated interpretations, we characterized these PC sums using the side-chain resolving LipidyzerTM platform, analyzing 223 samples in parallel to the AbsoluteIDQTM. Combining these datasets, we estimated the quantitative composition of PC sums and subsequently tested their replication in an independent cohort. We identified major constituents of 28 PC sums, revealing also various unexpected compositions. As an example, PC 16:0_22:5 accounted for more than 50% of the PC sum with in total 38 carbon atoms and 5 double bonds (PC aa 38:5). For 13 PC sums, we found relatively high abundances of odd-chain fatty acids. In conclusion, our study provides insights in PC compositions in human plasma, facilitating interpretation of existing epidemiological data sets and potentially enabling imputation of PC compositions for future meta-analyses of lipidomics data.

Keywords:  fatty acid composition; harmonization; imputation; isobaric phosphatidylcholines; lipid species; lipidomics; metabolomics; phospholipids; platform comparison

DOI:  https://doi.org/10.3390/metabo9060109
Mol Cell Proteomics. 2019 Jun 13. pii: mcp.RA119.001502. [Epub ahead of print]

Cancer cell derived small extracellular vesicles contribute to recipient cell metastasis through promoting HGF/c-Met pathway.

Zhi Qiao, Yan Zhang, Maolin Ge, Sha Liu, Xiaoteng Jiang, Zhi Shang, Han Liu, Chengxi Cao, Hua Xiao.

  Cancer progression is frequently caused by metastasis and leads to significantly increased mortality. Cell derived extracellular vesicles, including exosomes, in the microenvironment play key roles in cellular signal transduction, while their biological function in cancer metastasis and progression needs in-depth investigation. Here, we initially demonstrate that the small extracellular vesicles (sEVs) derived from highly metastatic lung cancer cells exhibited great capacity to promote the progression of recipient cells. Quantitative proteomics was employed to comprehensively decipher the proteome of cell derived sEVs and more than 1400 sEVs proteins were identified. Comparison analysis indicates that sEVs-HGF is a potential metastasis related protein and our verification data from clinical lung cancer plasma samples and in vivo experiments further confirmed the association. We found that sEVs-HGF could induce epithelial-mesenchymal transition and the coordination between HGF and c-Met was confirmed through corresponding target knockdown and kinase inhibition. Our data collectively demonstrate that cancer cell derived sEVs contribute to recipient cell metastasis through promoting HGF/c-Met pathway, which are potential targets for the prevention and treatment of cancer metastasis.

Keywords:  Exosomes; HGF/c-Met pathway; Lung cancer; Mass Spectrometry; Metastasis; Proteomics; Subcellular analysis

DOI:  https://doi.org/10.1074/mcp.RA119.001502
Cancer Cell. 2019 Jun 10. pii: S1535-6108(19)30249-1. [Epub ahead of print]35(6): 827-829

Fatty Acids Corrupt Neutrophils in Cancer.

Max D Wellenstein, Karin E de Visser.

Understanding how tumors escape from immune attack may offer novel therapeutic opportunities. Veglia et al. (2019) demonstrate in Nature that fatty acid transport protein 2 (FATP2) endows neutrophils with immunosuppressive capabilities that promote cancer growth. This receptor can be targeted to unleash anti-tumor immunity and to potentiate immune checkpoint blockade.

DOI: https://doi.org/10.1016/j.ccell.2019.05.007
Cancer Microenviron. 2019 Jun 10.

Pyruvate Kinase M2: a Metabolic Bug in Re-Wiring the Tumor Microenvironment.

Mohd Rihan, Lakshmi Vineela Nalla, Anil Dharavath, Amit Shard, Kiran Kalia, Amit Khairnar.

  Metabolic reprogramming is a newly emerged hallmark of cancer attaining a recent consideration as an essential factor for the progression and endurance of cancer cells. A prime event of this altered metabolism is increased glucose uptake and discharge of lactate into the cells surrounding constructing a favorable tumor niche. Several oncogenic factors help in promoting this consequence including, pyruvate kinase M2 (PKM2) a rate-limiting enzyme of glycolysis in tumor metabolism via exhibiting its low pyruvate kinase activity and nuclear moon-lightening functions to increase the synthesis of lactate and macromolecules for tumor proliferation. Not only its role in cancer cells but also its role in the tumor microenvironment cells has to be understood for developing the small molecules against it which is lacking with the literature till date. Therefore, in this present review, the role of PKM2 with respect to various tumor niche cells will be clarified. Further, it highlights the updated list of therapeutics targeting PKM2 pre-clinically and clinically with their added limitations. This upgraded understanding of PKM2 may provide a pace for the reader in developing chemotherapeutic strategies for better clinical survival with limited resistance.

Keywords:  Aerobic glycolysis; Metabolic reprogramming; PKM2; Tumor metabolism; Tumor microenvironment

DOI:  https://doi.org/10.1007/s12307-019-00226-0
J Proteome Res. 2019 Jun 10.

XNet: A Bayesian approach to Extracted Ion Chromatogram Clustering for Precursor Mass Spectrometry Data.

Matthew Gutierrez, Kyle Handy, Rob Smith.

Liquid chromatography mass spectrometry is a popular technique for high throughput analysis of biological samples. Identification and quantification of molecular species via mass spectrometry output requires post-experimental computational analysis of the raw instrument output. While tandem mass spectrometry remains a primary method for identification and quantification, species-resolved precursor data provides a rich source of un-exploited information. Several algorithms have been proposed to resolve raw precursor signals into species-resolved isotopic envelopes. Many methods are particularly dependent on user parameters, and because they lack a means to optimize parameters, tend to perform poorly. To this end we present XNet, a parameter-less Bayesian machine learning approach to isotopic envelope extraction through the clustering of extracted ion chromatograms. We evaluate the performance of XNet and other prevalent methods on a quantitative ground truth data set. XNet is publicly available with an Apache license.

DOI: https://doi.org/10.1021/acs.jproteome.9b00068
PLoS Biol. 2019 Jun 14. 17(6): e3000297

Cytosine-5 RNA methylation links protein synthesis to cell metabolism.

Nikoletta A Gkatza, Cecilia Castro, Robert F Harvey, Matthias Heiß, Martyna C Popis, Sandra Blanco, Susanne Bornelöv, Abdulrahim A Sajini, Joseph G Gleeson, Julian L Griffin, James A West, Stefanie Kellner, Anne E Willis, Sabine Dietmann, Michaela Frye.

Posttranscriptional modifications in transfer RNA (tRNA) are often critical for normal development because they adapt protein synthesis rates to a dynamically changing microenvironment. However, the precise cellular mechanisms linking the extrinsic stimulus to the intrinsic RNA modification pathways remain largely unclear. Here, we identified the cytosine-5 RNA methyltransferase NSUN2 as a sensor for external stress stimuli. Exposure to oxidative stress efficiently repressed NSUN2, causing a reduction of methylation at specific tRNA sites. Using metabolic profiling, we showed that loss of tRNA methylation captured cells in a distinct catabolic state. Mechanistically, loss of NSUN2 altered the biogenesis of tRNA-derived noncoding fragments (tRFs) in response to stress, leading to impaired regulation of protein synthesis. The intracellular accumulation of a specific subset of tRFs correlated with the dynamic repression of global protein synthesis. Finally, NSUN2-driven RNA methylation was functionally required to adapt cell cycle progression to the early stress response. In summary, we revealed that changes in tRNA methylation profiles were sufficient to specify cellular metabolic states and efficiently adapt protein synthesis rates to cell stress.

DOI: https://doi.org/10.1371/journal.pbio.3000297
Anal Chem. 2019 Jun 13.

Ultrafast Micro-Electrophoresis: Behind Direct Mass Spectrometry Measurements of Proteins and Metabolites in Living Cell/Cells.

Yuting Chen, Gongyu Li, Siming Yuan, Yang Pan, Yang-Zhong Liu, Guangming Huang.

Direct chemical profiling and protein identification from living single cells using mass spectrometry (MS) have been demonstrated to further our understanding of biological variability and differential susceptibility to several diseases and treatments. Despite the great challenge from extremely complicated cytoplasm, we recently proposed a versatile MS strategy to achieve direct mass spectrometric characterization of both proteins and metabolite-like small molecules directly from living cells or single cells. Although the capability to directly handle cell cytoplasm was presumably attributed to micro-electrophoresis in our previous studies, the assumption had only been partially explored by some preliminary experiments. To better understand the mechanism, herein, we systematically characterized its separation behavior with a series of model compounds covering a wide range of molecular size. With the merit of in situ separation, micro-electrophoresis herein has been further demonstrated as an attractive and alternative tool, which can potentially contribute to direct MS measurements of more protein interactions or metabolic pathways in living single cells or few cells.

DOI: https://doi.org/10.1021/acs.analchem.9b00716
J Biol Chem. 2019 Jun 11. pii: jbc.RA119.009037. [Epub ahead of print]

Cardiolipin-induced activation of pyruvate dehydrogenase links mitochondrial lipid biosynthesis to TCA cycle function.

Yiran Li, Wenjia Lou, Vaishnavi Raja, Simone Denis, Wenxi Yu, Michael W Schmidtke, Christian A Reynolds, Michael Schlame, Riekelt H Houtkooper, Miriam L Greenberg.

  Cardiolipin[MS1] (CL) is the signature phospholipid of mitochondrial membranes. Although it has long been known that CL plays an important role in mitochondrial bioenergetics, recent evidence in the yeast model indicates that CL is also essential for intermediary metabolism. To gain insight into the function of CL in energy metabolism in mammalian cells, here we analyzed the metabolic flux of [U-13C]glucose in a mouse C2C12 myoblast cell line, TAZ-KO, which is CL-deficient because of a CRISPR/Cas9-mediated knockout of the CL-remodeling enzyme tafazzin (TAZ). TAZ-KO cells exhibited decreased flux of [U-13C]glucose to [13C]acetyl-CoA and M2 and M4 isotopomers of TCA cycle intermediates. Activity of pyruvate carboxylase (PC), the predominant enzyme for anaplerotic replenishing of the TCA cycle, was elevated in the TAZ-KO cells, which also exhibited increased sensitivity to the PC inhibitor phenylacetate. We attributed a decreased carbon flux from glucose to acetyl-CoA in the TAZ-KO cells to a ~50% decrease in pyruvate dehydrogenase (PDH) activity, which was observed in both TAZ-KO cells and cardiac tissue from TAZ-KO mice. Protein-lipid overlay experiments revealed that PDH binds to CL, and supplementing digitonin-solubilized TAZ-KO mitochondria with CL restored PDH activity to wildtype levels. Mitochondria from TAZ-KO cells exhibited an increase in phosphorylated PDH, levels of which were reduced in the presence of supplemented CL. These findings indicate that CL is required for optimal PDH activation, generation of acetyl-CoA, and TCA cycle function, findings that link the key mitochondrial lipid CL to TCA cycle function and energy metabolism.

Keywords:  cardiolipin; mitochondria; pyruvate carboxylase (PC); pyruvate dehydrogenase complex (PDC); tricarboxylic acid cycle (TCA cycle) (Krebs cycle)

DOI:  https://doi.org/10.1074/jbc.RA119.009037
Methods Mol Biol. 2019 ;2014 235-251

Measurement of Subcellular Metabolite Concentrations in Relation to Phloem Loading.

Gertrud Lohaus.

  The key step of carbon export from green leaves is the loading of sugars into the phloem. To fully understand and quantify this process, it is essential to know the concentration of sugars in the different compartments of the cells along the phloem loading pathway. However, determining subcellular metabolite concentrations has been technically challenging. This paper describes a technique to measure metabolite levels in the chloroplast, the cytosol, and the vacuole of mesophyll cells with high accuracy. The nonaqueous fractionation (NAF) technique is arguably the method of choice to analyze the subcellular metabolite distributions as it minimizes the risk of metabolite interconversions or redistribution during the process. The principle of NAF is the separation of small subcellular particles, which are obtained by homogenization, lyophilization, and sonication, in a nonaqueous density gradient. Due to the varying composition-dependent density of the fragments, their segregation reflects compartmental distributions throughout the gradient. By determining marker enzymes for chloroplast stroma, cytosol, and vacuole in gradient fractions the proportions of each subcellular compartment in each gradient fraction can be analyzed. The measured distribution of marker enzymes and of metabolites in each fraction of the gradient can be used to calculate the subcellular distribution of the metabolites.

Keywords:  Amino acid; Cell metabolism; Nonaqueous fractionation; Subcellular metabolite concentration; Sugar

DOI:  https://doi.org/10.1007/978-1-4939-9562-2_20
Cell Calcium. 2019 Jun 05. pii: S0143-4160(19)30068-5. [Epub ahead of print]81 38-50

Lipid metabolism and Calcium signaling in epithelial ovarian cancer.

Sana Kouba, Lobna Ouldamer, Céline Garcia, Delphine Fontaine, Aurélie Chantome, Christophe Vandier, Caroline Goupille, Marie Potier-Cartereau.

Epithelial Ovarian cancer (EOC) is the deadliest gynecologic malignancy and represents the fifth leading cause of all cancer-related deaths in women. The majority of patients are diagnosed at an advanced stage of the disease that has spread beyond the ovaries to the peritoneum or to distant organs (stage FIGO III-IV) with a 5-year overall survival of about 29%. Consequently, it is necessary to understand the pathogenesis of this disease. Among the factors that contribute to cancer development, lipids and ion channels have been described to be associated to cancerous diseases particularly in breast, colorectal and prostate cancers. Here, we reviewed the literature data to determine how lipids or lipid metabolites may influence EOC risk or progression. We also highlighted the role and the expression of the calcium (Ca2+) and calcium-activated potassium (KCa) channels in EOC and how lipids might regulate them. Although lipids and some subclasses of nutritional lipids may be associated to EOC risk, lipid metabolism of LPA (lysophosphatidic acid) and AA (arachidonic acid) emerges as an important signaling network in EOC. Clinical data showed that they are found at high concentrations in EOC patients and in vitro and in vivo studies referred to them as triggers of the Ca2+entry in the cancer cells inducing their proliferation, migration or drug resistance. The cross-talk between lipid mediators and Ca2+ and/or KCa channels needs to be elucidated in EOC in order to facilitate the understanding of its outcomes and potentially suggest novel therapeutic strategies including treatment and prevention.

DOI: https://doi.org/10.1016/j.ceca.2019.06.002
J Proteome Res. 2019 Jun 12.

Metabolomic Analysis of Liver Tissues for Characterization of Hepatocellular Carcinoma.

Alessia Ferrarini, Cristina Di Poto, Shisi He, Chao Tu, Rency S Varghese, Abdalla Kara Balla, Meth Jayatilake, Zhenzhi Li, Kian Ghaffari, Ziling Fan, Zaki A Sherif, Deepak Kumar, Alexander Kroemer, Mahlet G Tadesse, Habtom W Ressom.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer causing more than half a million annual deaths world-wide. Understanding the molecular mechanisms contributing to HCC development and progression is highly desirable for improved surveillance, diagnosis and treatment. Liver tissue metabolomics has the potential to reflect the physiological changes behind HCC development. Also, it allows identification of candidate biomarkers for future evaluation in bio fluids and investigation of racial disparities in HCC. Tumor and non-tumor tissues from 40 patients were analyzed by both gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) platforms to increase the metabolome coverage. The levels of the metabolites extracted from solid liver tissue of the HCC area and adjacent non-HCC area were compared. Among the analytes detected by GC-MS and LC-MS with significant alterations, 18 were selected based on biological relevance and confirmed metabolite identification. These metabolites belong to TCA cycle, glycolysis, purines, and lipid metabolism, and have been previously reported in liver metabolomic studies where high correlation with HCC progression is implied. We demonstrated that metabolites related to HCC pathogenesis can be identified through liver tissue metabolomic analysis. Additionally, this study has led to the identification of race-specific metabolites associated with HCC.

DOI: https://doi.org/10.1021/acs.jproteome.9b00185
Anal Bioanal Chem. 2019 Jun 14.

LC-MS-based lipid profile in colorectal cancer patients: TAGs are the main disturbed lipid markers of colorectal cancer progression.

Tong Liu, Feng Peng, Jing Yu, Zhirong Tan, Tai Rao, Yao Chen, Yicheng Wang, Zhaoqian Liu, Honghao Zhou, Jingbo Peng.

  Colorectal cancer (CRC) is one of the most common causes of cancer-related death worldwide. Emerging evidence has shown that lipid metabolism plays important roles in the occurrence and progression of CRC. The identification of potential biomarkers for CRC progression is critical for precise diagnosis and treatment. Therefore, the aim of this study is to explore the potential lipid markers in relation to CRC progression. The plasma of patients with stage I/II CRC (n = 20) and stage III/IV CRC (n = 20) was collected. Lipidomic screening was performed by ultrahigh-performance liquid chromatography-mass spectrometry. After multivariate data analysis, including orthogonal partial least squares discriminant analysis, determination of the fold change, and the Mann-Whitney U test, eight lipid species with altered levels with p < 0.05 and fold change greater than 2 were selected as potential lipid biomarkers. Compared with patients with early-stage CRC, patients with advanced-stage CRC showed significantly higher levels of cholesteryl ester (20:4) and some triglycerides with a saturated fatty acid chain and a lower level of fatty acid ester of hydroxy fatty acid 27:1 (9:0-18:1) in plasma. Furthermore, the receiver operating characteristic including these potential lipid biomarkers yielded a sensitivity of 85% and specificity of 80% for separation of early-stage CRC patients from advanced-stage CRC patients. In all, this is the first report showing that the levels of triglycerides, the major contents of lipid droplets, increase in plasma of advanced-stage CRC patients compared with early-stage CRC patients. These data indicate that lipid droplets may be target organelles for the study of CRC progression and treatment. Graphical abstract.

Keywords:  Biomarkers; Colorectal cancer; Lipid profile; Triglyceride lipids; Ultrahigh-performance liquid chromatography–mass spectrometry

DOI:  https://doi.org/10.1007/s00216-019-01872-5
J Proteome Res. 2019 Jun 12.

Profiling of Polar Metabolites in Mouse Feces Using Four Analytical Platforms to Study the Effects Of Cathelicidin-Related Antimicrobial Peptide in Alcoholic Liver Disease.

Liqing He, Fengyuan Li, Xinmin Yin, Patrick Bohman, Seongho Kim, Craig J McClain, Wenke Feng, Xiang Zhang.

  Alterations in gut bacterial homeostasis result in changes in intestinal metabolites. To investigate the effects of alcohol on fecal metabolites and the role of cathelicidin-related antimicrobial peptide (CRAMP) in alcoholic liver disease (ALD), CRAMP knockout (KO) and their control wild type (WT) mice were fed a Lieber-DeCarli liquid diet with or without alcohol. Polar metabolites in mouse feces were analyzed by GC × GC-MS and 2DLC-MS, and the concentrations of short chain fatty acids (SCFAs) were measured by GC-MS. A total of 95 and 190 metabolites were detected by GC × GC-MS and 2DLC-MS, respectively. Among the significantly changed metabolites, taurine and nicotinic acid were decreased in WT mice fed alcohol, which were also down-regulated in KO mice fed without alcohol. Interestingly, these two metabolites were increased in KO mice fed alcohol compared to them in WT controls. Additionally, SCFAs were significantly decreased in WT mice fed alcohol and in KO mice fed without alcohol, whereas two branched-chain SCFAs were increased by alcohol treatment in KO mice. In summary, the analytical platforms employed in this study successfully dissected the alterations of polar metabolites and SCFAs in fecal samples, which helped understand the effects of alcohol consumption and CRAMP in intestinal metabolism and alcohol-induced liver injury.

Keywords:  alcoholic liver disease; cathelicidin-related antimicrobial peptide; metabolomics; nicotinic acid; short chain fatty acids; taurine

DOI:  https://doi.org/10.1021/acs.jproteome.9b00181
Anal Chem. 2019 Jun 09.

Combining Glucose Units, m/z and Collision Cross Section values: Multi-attribute data for increased accuracy in automated glycosphingolipid glycan identifications and its application in Triple Negative Breast Cancer.

Katherine Wongtrakul-Kish, Ian Walsh, Lyn Chiin Sim, Amelia Mak, Brian Liau, Vanessa Ding, Noor Hayati, Han Wang, Andre Boon-Hwa Choo, Pauline M Rudd, Terry Nguyen-Khuong.

Glycan head-groups attached to glycosphingolipids (GSLs) found in the cell membrane bilayer can alter in response to external stimuli and disease, making them potential markers and/or targets for cellular disease states. To identify such markers, comprehensive analyses of glycan structures must be undertaken. Conventional analyses of fluorescently labelled glycans using hydrophilic interaction high performance liquid chromatography (HILIC) coupled with mass spectrometry (MS) provides relative quantitation and has the ability to perform automated glycan assignments using glucose unit (GU) and mass matching. The use of ion mobility (IM) as an additional level of separation can aid the characterisation of closely-related or isomeric structures through the generation of glycan Collision Cross Section (CCS) identifiers. Here, we present a workflow for the analysis of procainamide-labelled GSL glycans using HILIC IM-MS and a new, automated glycan identification strategy whereby multiple glycan attributes are combined to increase accuracy in automated structural assignments. For glycan matching and identification, an experimental reference database of GSL glycans containing GU, mass and CCS values for each glycan was created. To assess the accuracy of glycan assignments, a distance-based confidence metric was used. The assignment accuracy was significantly better compared to conventional HILIC-MS approaches (using mass and GU only). This workflow was applied to the study of two Triple Negative Breast Cancer (TNBC) cell lines and revealed potential GSL glycosylation signatures characteristic of different TNBC subtypes.

DOI: https://doi.org/10.1021/acs.analchem.9b01476
Anal Chem. 2019 Jun 14.

Generating fatty acid profiles in the gas-phase: Fatty acid identification and relative quantitation using ion/ion charge inversion chemistry.

Caitlin E Randolph, David J Foreman, Stephen J Blanksby, Scott A McLuckey.

Representing the most fundamental lipid class, fatty acids (FA) play vital biological roles serving as energy sources, cellular signaling molecules, and key architectural components of complex lipids. Direct infusion electrospray ionization spectrometry, also known as shotgun lipidomics, has emerged as a rapid and powerful toolbox for lipid analysis. While shotgun lipidomics can be a sensitive approach to FA detection, the diverse molecular structure of FA presents challenges for unambiguous identification and the relative quantification of isomeric contributors. In particular, pinpointing double bond position(s) in unsaturated FA and determining the relative contribution of double bond isomers has limited the application of the shotgun approach. Recently, we reported the use of gas-phase ion/ion reactions to facilitate the identification of FA. Briefly, singly deprotonated FA anions undergo charge inversion when reacted in the gas-phase with tris-phenanthroline magnesium dications by forming [FA - H + MgPhen]+ complex ions. These charge-inverted FA complex cations fragment upon ion-trap collision induced dissociation (CID) to generate product ion spectra unique to individual FA isomers. Herein, we report the development of a mass spectral library comprised of [FA - H + MgPhen]+ product ion spectra. The developed FA library permits confident FA identification, including polyunsaturated FA isomers. Furthermore, we demonstrate the ability to determine relative contributions of isomeric FA using multiple linear regression analysis paired with gas-phase ion/ion reactions. We successfully applied the presented method to generate a FA profile for bovine liver phospholipidome based entirely on gas-phase chemistries.

DOI: https://doi.org/10.1021/acs.analchem.9b01333
Aging (Albany NY). 2019 Jun 07.

α-Ketoglutarate inhibits autophagy.

Elisa Elena Baracco, Francesca Castoldi, Sylvère Durand, David P Enot, Jelena Tadic, Katharina Kainz, Frank Madeo, Alexis Chery, Valentina Izzo, Maria Chiara Maiuri, Federico Pietrocola, Guido Kroemer.

  The metabolite α-ketoglutarate is membrane-impermeable, meaning that it is usually added to cells in the form of esters such as dimethyl -ketoglutarate (DMKG), trifluoromethylbenzyl α-ketoglutarate (TFMKG) and octyl α-ketoglutarate (O-KG). Once these compounds cross the plasma membrane, they are hydrolyzed by esterases to generate α-ketoglutarate, which remains trapped within cells. Here, we systematically compared DMKG, TFMKG and O-KG for their metabolic and functional effects. All three compounds similarly increased the intracellular levels of α-ketoglutarate, yet each of them had multiple effects on other metabolites that were not shared among the three agents, as determined by mass spectrometric metabolomics. While all three compounds reduced autophagy induced by culture in nutrient-free conditions, TFMKG and O-KG (but not DMKG) caused an increase in baseline autophagy in cells cultured in complete medium. O-KG (but neither DMKG nor TFMK) inhibited oxidative phosphorylation and exhibited cellular toxicity. Altogether, these results support the idea that intracellular α-ketoglutarate inhibits starvation-induced autophagy and that it has no direct respiration-inhibitory effect.

Keywords:  Krebs cycle; acetyl CoA; aging; cell death; metabolomics; mitochondria

DOI:  https://doi.org/10.18632/aging.102001