IU Indianapolis ScholarWorks :: Browsing by Author "Wang, Mu"

Browsing by Author "Wang, Mu"

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Attraction and Compaction of Migratory Breast Cancer Cells by Bone Matrix Proteins through Tumor-Osteocyte Interactions
(Nature Publishing Group, 2018-04-03) Chen, Andy; Wang, Luqi; Liu, Shengzhi; Wang, Yue; Liu, Yunlong; Wang, Mu; Nakshatri, Harikrishna; Li, Bai-Yan; Yokota, Hiroki; Biomedical Engineering, School of Engineering and Technology
Bone is a frequent site of metastasis from breast cancer. To understand the potential role of osteocytes in bone metastasis, we investigated tumor-osteocyte interactions using two cell lines derived from the MDA-MB-231 breast cancer cells, primary breast cancer cells, and MLO-A5/MLO-Y4 osteocyte cells. When three-dimensional (3D) tumor spheroids were grown with osteocyte spheroids, tumor spheroids fused with osteocyte spheroids and shrank. This size reduction was also observed when tumor spheroids were exposed to conditioned medium isolated from osteocyte cells. Mass spectrometry-based analysis predicted that several bone matrix proteins (e.g., collagen, biglycan) in conditioned medium could be responsible for tumor shrinkage. The osteocyte-driven shrinkage was mimicked by type I collagen, the most abundant organic component in bone, but not by hydroxyapatite, a major inorganic component in bone. RNA and protein expression analysis revealed that tumor-osteocyte interactions downregulated Snail, a transcription factor involved in epithelial-to-mesenchymal transition (EMT). An agarose bead assay showed that bone matrix proteins act as a tumor attractant. Collectively, the study herein demonstrates that osteocytes attract and compact migratory breast cancer cells through bone matrix proteins, suppress tumor migration, by Snail downregulation, and promote subsequent metastatic colonization.
Biomarker Discovery in Early Stage Breast Cancer Using Proteomics Technologies
(2009-06-24T12:49:22Z) Qi, Guihong; Wang, Mu
Cell model of DJ-1-associated Parkinson’s Disease
(2017-10-31) Madison, Mackenzie; Hoang, Quyen; Wang, Mu; Hudmon, Andy
Parkinson’s disease (PD) is a neurodegenerative disorder characterized by progressive loss of motor function resulting from dopaminergic neuronal death in the substantia nigra pars compacta leading to subsequent decreased striatal dopamine levels. The majority of PD cases are diagnosed as sporadic in nature, however 10% - 15% of patients show a positive family history of the disease. While many genes have been found to be implicated in the familial form of PD, early-onset autosomal recessive PD has been associated with mutations in PARK7, a gene which codes for the protein DJ-1. While there are many proposed roles of DJ-1 across numerous systems, the function of DJ-1 in relation to the development and progression of PD remains largely unclear. A first step towards determining this function is the creation of biologically relevant cell models of PD. The goal of this work was to design a representative cell model of DJ-1-associated PD in order to further study DJ-1 with the intention of elucidating its relevant function in relation of PD pathogenesis.
Characterization of the Mitochondrial Proteome in Pyruvate Dehydrogenase Kinase 4 Wild-Type and Knockout Mice
(2009-06-24T12:51:58Z) Ringham, Heather Nicole; Wang, Mu; Harris, Robert; Witzmann, Frank
The goal of this study was to determine the effect of a PDK4 (pyruvate dehydrogenase kinase isoenzyme 4) knock-out on mitochondrial protein expression. A 2-D gel based mass spectrometry approach was used to analyze the mitochondrial proteomes of PDK4 wild-type and knockout mice. Mitochondria were isolated from the kidneys of mice in both well-fed and starved states. Previous studies show PDK4 increases greatly in the kidney in response to starvation and diabetes suggesting its significance in glucose homeostasis. The mitochondrial fractions of the four experimental groups (PDK4+/+ fed, PDK4+/+ starved, PDK4-/- fed, and PDK4-/- starved) were separated via large- format, high resolution two-dimensional gel electrophoresis. Gels were scanned, image analyzed, and ANOVA performed followed by a pair-wise multiple comparison procedure (Holm-Sidak method) for statistical analysis. The abundance of a total of 87 unique protein spots was deemed significantly different (p<0.01). 22 spots were up- or down-regulated in the fed knockout vs. fed wild-type; 26 spots in the starved knockout vs. starved wild-type; 61 spots in the fed vs. starved wild-types; and 44 in the fed vs. starved knockouts. Altered protein spots were excised from the gel, trypsinized, and identified via tandem mass spectrometry (LC-MS/MS). Differentially expressed proteins identified with high confidence include ATP synthase proteins, fatty acid metabolism proteins, components of the citric acid cycle and electron transport chain. Proteins of interest were analyzed with Ingenuity Pathway Analysis (IPA) to examine relationships among the proteins and analyze biological pathways, as well as ontological analysis with Generic Gene Ontology (GO) Term Mapper. IPA found a number of canonical pathways, biological functions, and functional networks associated with the 87 proteins. Oxidative phosphorylation was the pathway associated with a majority of the proteins, while the largest network of proteins involved carbohydrate metabolism and energy production. Overall, the effects of starvation were more extensive on mitochondrial protein expression than the PDK4 knockout.
COMPARATIVE ANALYSIS OF THE DISCORDANCE BETWEEN THE GLOBAL TRANSCRIPTIONAL AND PROTEOMIC RESPONSE OF THE YEAST SACCHAROMYCES CEREVISIAE TO DELETION OF THE F-BOX PROTEIN, GRR1
(2010-05) Heyen, Joshua William; Goebl, Mark, 1958-; Roach, Peter J.; Clemmer, David E.; Wang, Mu; Chen, Jake
The Grr1 (Glucose Repression Resistant) protein in Saccharomyces cerevisiae is an F-box protein for the E3 ubiquitin ligase protein complex known as the SCFGrr1 (Skp, Cullin, F-box). F-box proteins serve as substrate receptors for this complex and in this capacity Grr1 serves to promote the ubiquitylation and subsequent proteasomal degradation of a number of intracellular protein substrates. Substrates of SCFGrr1 include the G1-S phase cyclins, Cln1 and Cln2, the Cdc42 effectors and cell polarity proteins, Gic1 and Gic2, the FCH-bar domain protein, Hof1, required for cytokinesis, the meiosis activating serine/threonine protein kinase, Ime2, the transcriptional regulators of glucose transporters, Mth1 and Std1, and the mitochondrial retrograde response inhibitor Mks1. Stabilization of these substrates lead to pleiotrophic phenotypic defects in grr1Δ strains including resistance to glucose repression, accumulation of grr1Δ cells in G2 and M phase of the cell cycle, sensitivity to osmotic stress, and resistance to divalent cations. However, many of these phenotypes are not reflected at the gene expression level. We conducted a quantitative genomic vii and proteomic comparison of 914 loci in a grr1Δ and wild-type strain grown to early log-phase in glucose media. These loci encompassed 16.7% of the Saccharomyces proteome of which 22.3% exhibited discordance between gene and protein expression. GO process enrichment analysis revealed that discordant loci were enriched in the processes of “trafficking”, “mitosis”, and “carbon/energy” metabolism. Here we show that these instances of discordance are biologically relevant and in fact reflect phenotypes of grr1Δ strains not evident at the transcriptional level. Additionally, through combined biochemical and network analysis of discordant loci among “carbon and energy metabolism” we were able to not only construct a model for central carbon metabolism in grr1Δ strains but also were able to elucidate a novel molecular event that may serve to regulate glucose repression of genes needed for respiration in response to changes in glucose concentration.
Development and Application of a Mass Spectrometry-Based Quantitative Assay for Apolipoprotein M in Human and Mouse Serum
(2008-10-13T19:13:53Z) Copeland, Marci Lynn; Wang, Mu
Apolipoprotein M (apoM) is necessary for the formation of lipid-poor preβ-HDL particles, the initial precursor of HDL and acceptors of cholesterol efflux from peripheral cells. An assay to quantify apoM in serum is not widely-available, hampering the efforts to further understand apoM and to develop therapeutic methods to increase circulating levels of apoM. An antibody-free, high throughput mass spectrometry (MS)-based assay was developed to quantitatively measure apoM from a variety of species including human, mouse, and rat. Apolipoproteins were enriched by selectively binding to Liposorb, an affinity resin, followed by enzymatic digestion. This peptide mixture was separated by HPLC coupled in-line with tandem MS/ MS. Signal intensities from the MS/ MS fragmentation of apoM-specific peptides were measured simultaneously in a targeted method spanning many commonly used species. The same amount of purified human apolipoprotein A-IV uniformly labeled with 15N was spiked into all samples and was used as an internal standard to correct for any variation in sample handling and recovery. Assay variability and accuracy was statistically validated in a three-day spike recovery experiment to determine the working range of the assay. The concentration range for quantification of apoM using this assay was 11.2-500 nM, whereas average concentration of human apoM measured from a large sampling (n>100) was 370 nM. This assay was used to measure changes in apoM in mouse serum from a pre-clinical study that was designed to evaluate the effects of a microsomal triglyceride transfer protein (MTTP) inhibitor. All measured lipoproteins and apolipoproteins showed a dose-dependent decrease in concentration and the response of apoM closely followed the response of HDL. In a clinical application of the assay, apoM was measured in human serum to evaluate the effects of two cholesterol-lowering compounds, a statin drug and an experimental PPAR-α agonist. ApoM levels did not change with PPAR-α agonist or combination treatments, but significantly decreased with atorvastatin. The measurement of apoM provided additional information on the effects of these drug treatments that previously could not be measured. The availability of a quantitative assay for apoM provides a valuable tool in the development of cardio-protective therapeutics and understanding the mechanisms of these drugs.
Development, validation, and comparison of four methods to simultaneously quantify L-arginine, citrulline, and ornithine in human plasma using hydrophilic interaction liquid chromatography and electrospray tandem mass spectrometry
(Elsevier, 2015-11) Lai, Xianyin; Kline, Jeffrey A.; Wang, Mu; Department of Biochemistry and Molecular Biology, IU School of Medicine
To understand the role of l-arginine depletion in impaired nitric oxide synthesis in disease, it is important to simultaneously quantify arginine, citrulline, and ornithine in the plasma. Because the three amino acids are endogenous analytes, true blank matrix for them is not available. It is necessary and valuable to compare the performance of different approaches due to lack of regulatory clarity for validation. A two-step sample preparation method using methanol as protein precipitation reagent was developed in this study is used for sample preparation. Because true blank matrix for endogenous analytes is not available, water as blank matrix, 1% BSA in PBS as blank matrix, surrogate analyte, and background subtraction were designed to establish successful quantification methods. Four methods to simultaneously quantify arginine, citrulline, and ornithine in human plasma using hydrophilic interaction liquid chromatography and electrospray tandem mass spectrometry were developed, validated, and compared. The developed two-step sample preparation method using methanol as protein precipitation reagent in this study needs less time and provides higher recovery comparing with other approaches. Three of the four methods, water as blank matrix, 1% BSA in PBS as blank matrix, and surrogate analyte, have been successful in fulfilling all the criteria, while background subtraction has failed. Results of the measured concentrations in 97 human plasma samples using the three methods show that the difference between any two methods or among the three methods presents 100% of samples with less than 20% for all the three amino acids and majority of them are under 10%. The developed two-step sample preparation method using methanol as protein precipitation reagent is simple and convenient. Three of the four methods are fully validated and the validation is successful. The BSA functioned effectively as a blank matrix for these three amino acids, considering cost, data quality, matrix similarity, and practicality.
The effects of dietary polyunsaturated fatty acids on prostate cancer-proteomic and phosphoproteomic studies
(2016-05) Zhao, Heng; Wang, Mu
This dissertation studies the effects of fatty acids on prostate cancer. Prostate cancer is one of the most common malignant diseases in males in the U.S. Because of the slow progression of this disease, early intervention methods, especially, dietary fatty acid interventions are considered very important to control the disease in early stages. This study describes how the depletion of the enzyme for endogenous fatty acid synthesis, fatty acid synthase, influences the expression of enzymes that metabolize dietary fatty acids and show how dietary fatty acids affect prostate cancer protein expression and function. Fatty acid synthase is an oncoprotein overexpressed in prostate cancer and its expression is suppressed with omega-3 fatty acid treatment. This study finds that the depletion of fatty acid synthase by siRNA knockdown induces suppression of cyclooxygenase-2 and fatty acid desaturase-1. Our results also show that fish oil (omega-3 fatty acid), but not oleic acid (omega-9 fatty acid), suppresses prostate cancer cell viability. Assessment of fatty acid synthesis activity indicates that oleic acid is a more potent inhibitor than fish oil of de novo fatty acid biosynthesis. In addition, the inhibition of its activity occurs over several days while its effects on cell viability occur within 24 hours. To better understand this relationship, label free LC-MS/MS based mass spectrometry was carried out to determine global proteomic and phosphoproteomic profiles of the prostate cell line PC3, with longitudinal treatment with fish oil or oleic acid. With short-term fish oil treatment, sequestosome-1was elevated. Prolonged treatment induced downregulation of microseminoprotein, a proinflammation factor, as well as proteins in the glycolysis pathway. In the phosphoproteomics study, we confidently identified 828 phosphopeptides from 361 phosphoproteins. Quantitative comparison between fish oil or oleic acid treated groups and the untreated group suggests that the fish oil induces changes in phosphorylation of proteins involved in the pathways associated with cell viability and metabolic processes, with fish oil inducing significant decreases in the levels of phospho-PDHA1Ser232 and phospho-PDHA1Ser300 and they were accompanied by an increase in PDH activity, suggesting a role for n-3 polyunsaturated fatty acids in controlling the balance between lipid and glucose oxidation.
Effects of HIV Protease Inhibitor Ritonavir on Akt-Regulated Cell Proliferation in Breast Cancer
(American Association for Cancer Research, 2006-03-15) Srirangam, Anjaiah; Mitra, Ranjana; Wang, Mu; Gorski, J. Christopher; Badve, Sunil; Baldridge, Lee Ann; Hamilton, Justin; Kishimoto, Hiromitsu; Hawes, John; Li, Lang; Orschell, Christie M.; Srour, Edward F.; Blum, Janice S.; Donner, David; Sledge, George W.; Nakshatri, Harikrishna; Potter, David A.
Purpose These studies were designed to determine whether ritonavir inhibits breast cancer in vitro and in vitro and, if so, how. Experimental Design Ritonavir effects on breast cancer cell growth were studied in the estrogen receptor (ER)-positive lines MCF7 and T47D and in the ER-negative lines MDA-MB-436 and MDA-MB-231. Effects of ritonavir on Rb-regulated and Akt-mediated cell proliferation were studied. Ritonavir was tested for inhibition of a mammary carcinoma xenograft. Results ER-positive estradiol-dependent lines (IC50, 12–24 µmol/L) and ER-negative (IC50, 45 µmol/L) lines exhibit ritonavir sensitivity. Ritonavir depletes ER-α levels notably in ER-positive lines. Ritonavir causes G1 arrest, depletes cyclin-dependent kinases 2, 4, and 6 and cyclin D1 but not cyclin E, and depletes phosphorylated Rb and Ser473 Akt. Ritonavir induces apoptosis independent of G1 arrest, inhibiting growth of cells that have passed the G1 checkpoint. Myristoyl-Akt, but not activated K-Ras, rescues ritonavir inhibition. Ritonavir inhibited a MDA-MB-231 xenograft and intratumoral Akt activity at a clinically attainable serum Cmax of 22 ± 8 µmol/L. Because heat shock protein 90 (Hsp90) substrates are depleted by ritonavir, ritonavir effects on Hsp90 were tested. Ritonavir binds Hsp90 (KD, 7.8 µmol/L) and partially inhibits its chaperone function. Ritonavir blocks association of Hsp90 with Akt and, with sustained exposure, notably depletes Hsp90. Stably expressed Hsp90α short hairpin RNA also depletes Hsp90, inhibiting proliferation and sensitizing breast cancer cells to low ritonavir concentrations. Conclusions Ritonavir inhibits breast cancer growth in part by inhibiting Hsp90 substrates, including Akt. Ritonavir may be of interest for breast cancer therapeutics and its efficacy may be increased by sustained exposure or Hsp90 RNA interference.
Endothelial disruptive proinflammatory effects of nicotine and e-cigarette vapor exposures
(American Physiological Society, 2015-07-15) Schweitzer, Kelly S.; Chen, Steven X.; Law, Sarah; Van Demark, Mary; Poirier, Christophe; Justice, Matthew J.; Hubbard, Walter C.; Kim, Elena S.; Lai, Xianyin; Wang, Mu; Kranz, William D.; Carroll, Clinton J.; Ray, Bruce D.; Bittman, Robert; Goodpaster, John V.; Petrache, Irina; Department of Biochemistry & Molecular Biology, IU School of Medicine
The increased use of inhaled nicotine via e-cigarettes has unknown risks to lung health. Having previously shown that cigarette smoke (CS) extract disrupts the lung microvasculature barrier function by endothelial cell activation and cytoskeletal rearrangement, we investigated the contribution of nicotine in CS or e-cigarettes (e-Cig) to lung endothelial injury. Primary lung microvascular endothelial cells were exposed to nicotine, e-Cig solution, or condensed e-Cig vapor (1-20 mM nicotine) or to nicotine-free CS extract or e-Cig solutions. Compared with nicotine-containing extract, nicotine free-CS extract (10-20%) caused significantly less endothelial permeability as measured with electric cell-substrate impedance sensing. Nicotine exposures triggered dose-dependent loss of endothelial barrier in cultured cell monolayers and rapidly increased lung inflammation and oxidative stress in mice. The endothelial barrier disruptive effects were associated with increased intracellular ceramides, p38 MAPK activation, and myosin light chain (MLC) phosphorylation, and was critically mediated by Rho-activated kinase via inhibition of MLC-phosphatase unit MYPT1. Although nicotine at sufficient concentrations to cause endothelial barrier loss did not trigger cell necrosis, it markedly inhibited cell proliferation. Augmentation of sphingosine-1-phosphate (S1P) signaling via S1P1 improved both endothelial cell proliferation and barrier function during nicotine exposures. Nicotine-independent effects of e-Cig solutions were noted, which may be attributable to acrolein, detected along with propylene glycol, glycerol, and nicotine by NMR, mass spectrometry, and gas chromatography, in both e-Cig solutions and vapor. These results suggest that soluble components of e-Cig, including nicotine, cause dose-dependent loss of lung endothelial barrier function, which is associated with oxidative stress and brisk inflammation.

Browsing by Author "Wang, Mu"

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