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ABSTRACT: BACKGROUND: Alzheimer's disease is the most common type of dementia affecting people over 65 years of age. The hallmarks of AD are the extracellular deposits known as amyloid beta plaques and the intracellular neurofibrillary tangles, both of which are the principal players involved in synaptic loss and neuronal cell death. Tau protein and Abeta fragment 1--42 have been investigated so far in cerebrospinal fluid as a potential AD biomarkers. However, an urgent need to identify novel biomarkers which will capture disease in the early stages and with better specificity remains. High-throughput proteomic and pathway analysis of hippocampal tissue provides a valuable source of disease-related proteins and biomarker candidates, since it represents one of the earliest affected brain regions in AD. RESULTS: In this study 2954 proteins were identified (with at least 2 peptides for 1203 proteins) from both control and AD brain tissues. Overall, 204 proteins were exclusively detected in AD and 600 proteins in control samples. Comparing AD and control exclusive proteins with CSF literature-based proteome, 40 out of 204 AD related proteins and 106 out of 600 control related proteins were also present in CSF. As most of these proteins were extracellular/secretory origin, we consider them as a potential source of candidate biomarkers that need to be further studied and verified in CSF samples. CONCLUSIONS: Our semiquantitative proteomic analysis provides one of the largest human hippocampal proteome databases. The lists of AD and control related proteins represent a panel of proteins potentially involved in AD pathogenesis and could also serve as prospective AD diagnostic biomarkers.
Clinical Proteomics 05/2013; 10(1):5.
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ABSTRACT: The cancer invasion front (CIF), a spatially-recognized area due to the frequent presence of peritumoral desmoplastic reaction, represents a cancer site where many hallmarks of cancer metastasis occur. It is now strongly suggested that the desmoplastic microenvironment holds crucial information for determining tumor development and progression. Despite extensive research on tumor-host cell interactions at CIFs, the exact paracrine molecular network that is hardwired into the proteome of the stromal and cancer subpopulations remains partially understood. Here, we interrogated the signaling pathways and the molecular functional signatures across the proteome of a desmoplastic coculture model system of colorectal cancer progression. We discovered a group of bone morphogenetic protein (BMP) antagonists that coordinates major biological programs in CIFs, including cell proliferation, invasion, migration and differentiation processes. Using a mathematical model of cancer cell progression, coupled to in vitro cell migration assays, we demonstrated that the prominent BMP antagonist gremlin-1 (GREM1) may trigger motility of cancer cell cohorts. Our data collectively demonstrate that the desmoplastic CIFs deploy a microenvironmental signature, based on BMP antagonism, in order to regulate the motogenic fates of cancer cell cohorts invading the adjacent stroma.
Molecular oncology 04/2013; · 4.10 Impact Factor
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ABSTRACT: BACKGROUND: Down syndrome (DS), caused by an extra copy of chromosome 21, affects 1 in 750 live births and is characterized by cognitive impairment and a constellation of congenital defects. Currently, little is known about the molecular pathogenesis and no direct genotype-phenotype relationship has yet been confirmed. Since DS amniocytes are expected to have a distinct biological behaviour compared to normal amniocytes, we hypothesize that relative quantification of proteins produced from trisomy and euploid (chromosomally normal) amniocytes will reveal dysregulated molecular pathways. RESULTS: Chromosomally normal- and Trisomy 21-amniocytes were quantitatively analyzed by using Stable Isotope Labeling of Amino acids in Cell culture and tandem mass spectrometry. A total of 4919 unique proteins were identified from the supernatant and cell lysate proteome. More specifically, 4548 unique proteins were identified from the lysate, and 91% of these proteins were quantified based on MS/MS spectra ratios of peptides containing isotope-labeled amino acids. A total of 904 proteins showed significant differential expression and were involved in 25 molecular pathways, each containing a minimum of 16 proteins. Sixty of these proteins consistently showed aberrant expression from trisomy 21 affected amniocytes, indicating their potential role in DS pathogenesis. Nine proteins were analyzed with a multiplex selected reaction monitoring assay in an independent set of Trisomy 21-amniocyte samples and two of them (SOD1 and NES) showed a consistent differential expression. CONCLUSIONS: The most extensive proteome of amniocytes and amniotic fluid has been generated and differentially expressed proteins from amniocytes with Trisomy 21 revealed molecular pathways that seem to be most significantly affected by the presence of an extra copy of chromosome 21.
Clinical Proteomics 02/2013; 10(1):2.
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ABSTRACT: Breast-cancer subtypes present with distinct clinical characteristics. Therefore, characterization of subtype-specific proteins may augment the development of targeted therapies and prognostic biomarkers. To address this issue, mass spectrometry-based secretome analysis of eight breast cancer cell lines, corresponding to the three main breast cancer subtypes was performed. More than 5,200 non-redundant proteins were identified with twenty-three, four, and four proteins identified uniquely in basal, HER2-neu amplified and luminal breast cancer cells, respectively. An in silico mRNA analysis using publicly available breast cancer tissue microarray data was carried out as a preliminary verification step. In particular, the expression profiles of 15 out of 28 proteins included in the microarray (from a total of 31 in our subtype-specific signature) showed significant correlation with estrogen receptor expression. A mass spectrometry-based analysis of breast cancer tissues was undertaken to verify the results at the proteome level. Eighteen out of 31 proteins were quantified in the proteomes of estrogen receptor positive and negative breast cancer tissues. Survival analysis using microarray data was performed to examine the prognostic potential of these selected candidates. Three proteins correlated with estrogen receptor status at both mRNA and protein levels: ABAT, PDZK1 and PTX3, with the former showing significant prognostic potential.
Proteomics 02/2013; · 4.43 Impact Factor
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ABSTRACT: Abstract Background: Ovarian cancer is the leading cause of death among all gynecological disorders. Aberrant glycosylation, or more specifically, increased sialylation of proteins has been observed in ovarian cancer. Several sialyltransferase genes have been shown to be up-regulated at both the mRNA and protein levels in a number of cancers, including that of the ovary. ST6GAL1 (β-galactosamide α2,6-sialyltranferase 1) gene expression has previously been shown to be upregulated in ovarian cancers of all major subtypes. Methods: We have identified the sialome (i.e., sialic acid containing glycoproteins) of biological fluids from ovarian cancer patients and ovarian cancer cell lines utilizing tandem mass spectrometry as a potential pool of novel biomarker candidates. The sialoglycopeptides from four ovarian cancer cell lines, pooled ascites (n=13) and ovarian cyst (n=14) fluids from ovarian cancer patients were enriched utilizing affinity to agarose-immobilized Elderberry lectin (Sambucus nigra agglutinin) and magnetic hydrazide beads folowing periodate-mediated oxidation of sialic acids. Benign ovarian cyst (n=10) and peritoneal effusion (n=20) fluids were analyzed in the same fashion to serve as controls. PNGase F deglycosylated peptides were identified using electrospray ionization-LTQ Orbitrap tandem mass spectrometry. Results: In all of the samples analyzed in the glycoproteomic portion of the study, we have identified 579 glycosylation sites on 333 proteins. Of these, 13 were exclusively identified in biological fluids from ovarian cancer patients, and another eight were common to these fluids and the ovarian cancer cell line supernatants. Conclusions: The proteins identified in the present study could form the basis for future studies examining and quantifying their sialylation status as biomarkers of ovarian cancer.
Clinical Chemistry and Laboratory Medicine 12/2012; · 2.15 Impact Factor
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ABSTRACT: To investigate the quantitative response of energy metabolic pathways in human MCF-7 breast cancer cells to hypoxia, glucose deprivation, and estradiol stimulation, we developed a targeted proteomics assay for accurate quantification of protein expression in glycolysis/gluconeogenesis, TCA cycle, and pentose phosphate pathways. Cell growth conditions were selected to roughly mimic the exposure of cells in the cancer tissue to the intermittent hypoxia, glucose deprivation, and hormonal stimulation. Targeted proteomics assay allowed for reproducible quantification of 76 proteins in four different growth conditions after 24 and 48 h of perturbation. Differential expression of a number of control and metabolic pathway proteins in response to the change of growth conditions was found. Elevated expression of the majority of glycolytic enzymes was observed in hypoxia. Cancer cells, as opposed to near-normal MCF-10A cells, exhibited significantly increased expression of key energy metabolic pathway enzymes (FBP1, IDH2, and G6PD) that are known to redirect cellular metabolism and increase carbon flux through the pentose phosphate pathway. Our quantitative proteomic protocol is based on a mass spectrometry-compatible acid-labile detergent and is described in detail. Optimized parameters of a multiplex selected reaction monitoring (SRM) assay for 76 proteins, 134 proteotypic peptides, and 401 transitions are included and can be downloaded and used with any SRM-compatible mass spectrometer. The presented workflow is an integrated tool for hypothesis-driven studies of mammalian cells as well as functional studies of proteins, and can greatly complement experimental methods in systems biology, metabolic engineering, and metabolic transformation of cancer cells.
Molecular & Cellular Proteomics 04/2012; 11(8):422-34. · 7.40 Impact Factor
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ABSTRACT: Cancer-associated fibroblasts (CAFs), represent a pivotal compartment of solid cancers (desmoplasia), and are causatively implicated in cancer development and progression. CAFs are recruited by growth factors secreted by cancer cells and they present a myofibroblastic phenotype, similar to the one obtained by resident fibroblasts during wound healing. Paracrine signaling between cancer cells and CAFs results in a unique protein expression profile in areas of desmoplastic reaction, which is speculated to drive metastasis. In an attempt to decipher large-scale proteomic profiles of the cancer invasive margins, we developed an in vitro coculture model system, based on tumor-host cell interactions between colon cancer cells and CAFs. Proteomic analysis of conditioned media derived from these cocultures coupled to mass spectrometry and bioinformatic analysis was performed to uncover myofibroblastic signatures of the cancer invasion front. Our analysis resulted in the identification and generation of a desmoplastic protein dataset (DPD), consisting of 152 candidate proteins of desmoplasia. By using monoculture exclusion datasets, a secretome algorithm and gene-expression meta-analysis in DPD, we specified a 22-protein "myofibroblastic signature" with putative importance in the regulation of colorectal cancer metastasis. Of these proteins, we investigated collagen type XII by immunohistochemistry, a fibril-associated collagen with interrupted triple helices (FACIT), whose expression has not been reported in desmoplastic lesions in any type of cancer. Collagen type XII was highly expressed in desmoplastic stroma by and around alpha-smooth muscle actin (α-SMA) positive CAFs, as well as in cancer cells lining the invasion front, in a small cohort of colon cancer patients. Other stromal markers, such as collagen type III, were also expressed in stromal collagen, but not in cancer cells. In a complementary fashion, gene expression meta-analysis revealed that COL12A1 is also an upregulated gene in colorectal cancer. Our proteomic analysis identified previously documented markers of tumor invasion fronts and our DPD could serve as a pool for future investigation of the tumor microenvironment. Collagen type XII is a novel candidate marker of myofibroblasts, and/or cancer cells undergoing dedifferentiation.
Oncotarget 03/2012; 3(3):267-85. · 4.78 Impact Factor
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ABSTRACT: Cardiac glycosides (e.g., digoxin, digitoxin) constitute a diverse family of plant-derived sodium pump inhibitors that have been in clinical use for the treatment of heart-related diseases (congestive heart failure, atrial arrhythmia) for many years. Recently though, accumulating in vitro and in vivo evidence highlight potential anticancer properties of these compounds. Despite the fact that members of this family have advanced to clinical trial testing in cancer therapeutics, their cytotoxic mechanism is not yet elucidated. In this study, we investigated the cytotoxic properties of cardiac glycosides against a panel of pancreatic cancer cell lines, explored their apoptotic mechanism, and characterized the kinetics of cell death induced by these drugs. Furthermore, we deployed a high-throughput kinome screening approach and identified several kinases of the Na-K-ATPase-mediated signal transduction circuitry (epidermal growth factor receptor, Src, pkC, and mitogen-activated protein kinases) as important mediators downstream of cardiac glycoside cytotoxic action. To further extend our knowledge on their mode of action, we used mass-spectrometry-based quantitative proteomics (stable isotope labeling of amino acids in cell culture) coupled with bioinformatics to capture large-scale protein perturbations induced by a physiological dose of digitoxin in BxPC-3 pancreatic cancer cells and identified members of the interferon family as key regulators of the main protein/protein interactions downstream of digitoxin action. Hence, our findings provide more in-depth information regarding the molecular mechanisms underlying cardiac glycoside-induced cytotoxicity.
Molecular Cancer Therapeutics 08/2011; 10(11):2083-93. · 5.23 Impact Factor
