Redox-sensitive high-mobility group box-1 isoforms contribute to liver fibrosis progression and resolution in mice.

BACKGROUND & AIMS: High-mobility group box-1 (HMGB1) significantly increases and undergoes post-translational modifications (PTMs) in response to liver injury. Since oxidative stress plays a major role in liver fibrosis and induces PTMs in proteins, we hypothesized that redox-sensitive HMGB1 isoforms contribute to liver fibrosis progression and resolution. METHODS: We used ESI-LC-MS (electrospray ionization-liquid chromatography-mass spectrometry) to study PTMs of HMGB1 during fibrosis progression and resolution. Conditional knockout mice were used for functional analyses. RESULTS: We identified that disulfide ([O]) and sulfonated ([SO3]) HMGB1 increase during carbon tetrachloride-induced liver fibrosis progression, however, while [O] HMGB1 declines, [SO3] HMGB1 drops but remains, during fibrosis resolution. Conditional knockout of Hmgb1 revealed that production of [O] and [SO3] HMGB1 occurs mostly in hepatocytes. Co-injection of [O] HMGB1 worsens carbon tetrachloride-induced liver fibrosis more than co-injection of [H] HMGB1. Conversely, ablation of [O] Hmgb1 in hepatocytes reduces liver fibrosis. Moreover, ablation of the receptor for advanced-glycation end-products (Rage) reveals that the profibrogenic effect of [O] HMGB1 is mediated by RAGE signaling in hepatic stellate cells (HSCs). Notably, injection of [SO3] HMGB1 accelerates fibrosis resolution due to RAGE-dependent stimulation of HSC apoptosis. Importantly, gene signatures activated by redox-sensitive HMGB1 isoforms in mice, classify patients with fibrosis according to fibrosis and inflammation scores. CONCLUSION: Dynamic changes in hepatocyte-derived [O] and [SO3] HMGB1 signal through RAGE-dependent mechanisms on HSCs to drive their profibrogenic phenotype and fate, contributing to progression and resolution of liver fibrosis. IMPACT AND IMPLICATIONS: Since oxidative stress plays a major role in liver fibrosis and induces post-translational modifications of proteins, we hypothesized that redox-sensitive HMGB1 isoforms contribute to liver fibrosis progression and resolution. This study is significant because a rise in [H] HMGB1 could flag 'patient at risk', the presence of [O] HMGB1 could suggest 'disease in progress or active scarring', while the appearance of [SO3] HMGB1 could point at 'resolution under way'. The latter could be used as a readout for response to pharmacological intervention with anti-fibrotic agents.

Identification of 2-Sulfonyl/Sulfonamide Pyrimidines as Covalent Inhibitors of WRN Using a Multiplexed High-Throughput Screening Assay.

Werner syndrome protein (WRN) is a multifunctional enzyme with helicase, ATPase, and exonuclease activities that are necessary for numerous DNA-related transactions in the human cell. Recent studies identified WRN as a synthetic lethal target in cancers characterized by genomic microsatellite instability resulting from defects in DNA mismatch repair pathways. WRN's helicase activity is essential for the viability of these high microsatellite instability (MSI-H) cancers and thus presents a therapeutic opportunity. To this end, we developed a multiplexed high-throughput screening assay that monitors exonuclease, ATPase, and helicase activities of full-length WRN. This screening campaign led to the discovery of 2-sulfonyl/sulfonamide pyrimidine derivatives as novel covalent inhibitors of WRN helicase activity. The compounds are specific for WRN versus other human RecQ family members and show competitive behavior with ATP. Examination of these novel chemical probes established the sulfonamide NH group as a key driver of compound potency. One of the leading compounds, H3B-960, showed consistent activities in a range of assays (IC50 = 22 nM, KD = 40 nM, KI = 32 nM), and the most potent compound identified, H3B-968, has inhibitory activity IC50 ∼ 10 nM. These kinetic properties trend toward other known covalent druglike molecules. Our work provides a new avenue for screening WRN for inhibitors that may be adaptable to different therapeutic modalities such as targeted protein degradation, as well as a proof of concept for the inhibition of WRN helicase activity by covalent molecules.

Identification of Osteoporosis-Associated Protein Biomarkers from Ovariectomized Rat Urine.

OBJECTIVE: Osteoporotic fracture is one of the most common health risks and aggravates the quality of life among postmenopausal women worldwide. In this study, osteoporosis-associated protein biomarkers were identified from urine of osteoporotic female Sprague-Dawley rats developed by ovariectomy. METHOD: Four months after the operation, the bone mineral density of the femur of ovariectomized rats was significantly lowered in comparison with that of the sham operated rats. The protein profiles of the urine samples collected from the sham, ovariectomized (OVX) and 2 month-old non-operated (Young) rats were compared by 2-D gel and MS spectrometry. RESULTS: Proteins consistently expressed between Young and sham but differentially expressed in OVX rats were selected and identified. One down-regulated 21 kDa protein, superoxide dismutase (SOD), and 1 up-regulated 53-54 kDa protein, alph-1-antitrypsin (A1AT), were selected from urine of the ovariectomized rats by 2-D gel analysis. Further, a total of 30 with 19 up-regulated and 11-down-regulated proteins were selected by LC-MS analysis with more than 2-fold differences in spectral counts. The fact that SOD and A1AT are also listed in the 30 differential proteins suggests that our biomarker isolation procedure suitably represents osteoporosis-associated proteins in urine. CONCLUSION: Supporting the facts, the differential expressions of SOD and A1AT in urine could be validated by Western blotting. These urinary osteoporosis-associated proteins have high potentials to become candidates for non-invasive diagnosis of osteoporosis from urine.

Homeostasis of iron and hepcidin in erythropoietic protoporphyria.

BACKGROUND: Erythropoietic protoporphyria (EPP) and X-linked protoporphyria (XLP) are genetic abnormalities of heme synthesis that result in excess production of protoporphyrin and that manifest as severe photosensitivity. These disorders are often associated with iron deficiency anaemia (IDA). Our aim was to determine whether hepcidin is increased in EPP/XLP patients, resulting in decreased enteral iron absorption and IDA. MATERIAL AND METHODS: Eight subjects with EPP, one with XLP and nine controls had baseline blood and urine samples collected, and thereafter were given oral ferrous sulphate (660 mg). Post-iron blood and urine samples were collected at 2, 4, 6 and 8 h. Blood counts, serum cytokines, ferritin and iron studies were analysed at baseline. Serum iron studies, serum and urine hepcidin, and erythropoietin (Epo) were analysed at baseline and subsequent time points. RESULTS: At baseline, EPP-XLP subjects had lower mean blood haemoglobin (13·9/15·3 g/dL) and serum ferritin (31·6/115 ng/mL) than controls. Serum iron levels increased markedly in both cohorts. Mean serum and urine hepcidin levels were significantly lower in the EPP-XLP group at 4 and 8 h post-iron (serum - 4 h, 3·79/26·6, 8 h, 5·79/34·6 nM; urine - 4 h, 0·85/2·50, 8 h, 1·44/6·63 nM/mM creatinine). Serum cytokines and Epo were normal and not different between groups. CONCLUSIONS: We conclude that serum and urine hepcidin are not inappropriately increased in EPP/XLP subjects at baseline and do not increase over time as serum iron increases after oral ferrous sulphate. Levels of serum cytokines and Epo are normal in EPP/XLP. The molecular basis for the iron-deficient phenotype in EPP/XLP remains unknown.

Proteomic analysis of active multiple sclerosis lesions reveals therapeutic targets.

Understanding the neuropathology of multiple sclerosis (MS) is essential for improved therapies. Therefore, identification of targets specific to pathological types of MS may have therapeutic benefits. Here we identify, by laser-capture microdissection and proteomics, proteins unique to three major types of MS lesions: acute plaque, chronic active plaque and chronic plaque. Comparative proteomic profiles identified tissue factor and protein C inhibitor within chronic active plaque samples, suggesting dysregulation of molecules associated with coagulation. In vivo administration of hirudin or recombinant activated protein C reduced disease severity in experimental autoimmune encephalomyelitis and suppressed Th1 and Th17 cytokines in astrocytes and immune cells. Administration of mutant forms of recombinant activated protein C showed that both its anticoagulant and its signalling functions were essential for optimal amelioration of experimental autoimmune encephalomyelitis. A proteomic approach illuminated potential therapeutic targets selective for specific pathological stages of MS and implicated participation of the coagulation cascade.

Proteomic strategy for probing complementary lethality of kinase inhibitors against pancreatic cancer.

In the present study, proteomic analysis was performed to discover combinational molecular targets for therapy and chemoresistance by comparing differential protein expression from Panc-1 cells treated with FDA-approved drugs such as sunitinib, imatinib mesylate, dasatinib, and PD184352. A total of 4041 proteins were identified in the combined data from all of the treatment groups by nano-electrospray ultra-performance LC and MS/MS analysis. Most of the proteins with significant changes are involved in apoptosis, cytoskeletal remodeling, and epithelial-to-mesenchymal transition. These processes are associated with increased chemoresistance and progression of pancreatic cancer. Among the differentially expressed proteins, heme oxygenase-1 (HO-1) was found in the sunitinib and imatinib mesylate treatment groups, which possibly acts as a specific target for synthetic lethality in combinational treatment. HO-1 was found to play a key role in sensitizing the chemoresistant Panc-1 cell line to drug therapy. Viability was significantly decreased in Panc-1 cells cotreated with sunitinib and imatinib mesylate at low doses, compared to those treated with sunitinib or imatinib mesylate alone. The results suggest that induction of chemosensitization by manipulating specific molecular targets can potentiate synergistic chemotherapeutic effects at lower, better tolerated doses, and in turn reduce the toxicity of multidrug treatment of pancreatic cancer.

Circadian rhythms in acute intermittent porphyria--a pilot study.

BACKGROUND: Acute intermittent porphyria (AIP) is an inherited disorder of haem synthesis wherein a partial deficiency of porphobilinogen (PBG) deaminase (PBGD) with other factors may give rise to biochemical and clinical manifestations of disease. The biochemical hallmarks of active AIP are relative hepatic haem deficiency and uncontrolled up-regulation of hepatic 5-aminolevulinic acid (ALA) synthase-1 (ALAS1) with over-production of ALA and PBG. The treatment of choice is intravenous haem, which restores the deficient regulatory haem pool of the liver and represses ALAS1. Recently, haem has been shown to influence circadian rhythms by controlling their negative feedback loops. We evaluated whether subjects with AIP exhibited an altered circadian profile. MATERIALS AND METHODS: Over a 21-h period, we measured levels of serum cortisol, melatonin, ALA, PBG and mRNA levels (in peripheral blood mononuclear cells) of selected clock-controlled genes and genes involved in haem synthesis in 10 Caucasian (European-American) women who were either postmenopausal or had been receiving female hormone therapy, six of whom have AIP and four do not and are considered controls. RESULTS: Four AIP subjects with biochemical activity exhibited higher levels of PBG and lower levels and dampened oscillation of serum cortisol, and a trend for lower levels of serum melatonin, than controls or AIP subjects without biochemical activity. Levels of clock-controlled gene mRNAs showed significant increases over baseline in all subjects at 5 a.m. and 11 p.m., whereas mRNA levels of ALAS1, ALAS2 and PBGD were increased only at 11 p.m. in subjects with active AIP. CONCLUSIONS: This pilot study provides evidence for disturbances of circadian markers in women with active AIP that may trigger or sustain some common clinical features of AIP.

Catechins in dietary supplements and hepatotoxicity.

BACKGROUND: Many herbal dietary supplements (HDS) contain green tea extract (GTE) and its component catechins, although their presence may not always be indicated on the product label. PURPOSE: Because GTE and catechins have been implicated in human hepatotoxicity in several case reports, our objective was to determine whether catechins were present in HDS that were implicated in hepatotoxicity, even if not identified among the labeled ingredients, and whether these compounds could be associated with liver injury. METHODS: We assayed 97 HDS implicated in human hepatotoxicity for catechins. RESULTS: We found that 29 of 73 HDS (39.7%) that did not identify GTE or any of its component catechins on their label contained catechins. Among patients with confirmed hepatotoxicity, there was no statistically significant association between the presence of catechin or the dose consumed and liver injury causality score, severity, or pattern of liver injury. Catechin levels tended to be highest in products used for weight loss, although catechin concentrations were low in most products. CONCLUSIONS: Many HDS commonly contain catechins that are implicated in hepatotoxicity, although their presence may not be indicated on the product label. Although our results did not establish an association between GTE or catechins with hepatotoxicity, they highlight some of the many complexities and uncertainties that surround the attribution of drug-induced liver injury (DILI) to HDS.

Human myelin proteome and comparative analysis with mouse myelin.

Myelin, formed by oligodendrocytes (OLs) in the CNS, is critical for axonal functions, and its damage leads to debilitating neurological disorders such as multiple sclerosis. Understanding the molecular mechanisms of myelination and the pathogenesis of human myelin disease has been limited partly by the relative lack of identification and functional characterization of the repertoire of human myelin proteins. Here, we present a large-scale analysis of the myelin proteome, using the shotgun approach of 1-dimensional PAGE and liquid chromatography/tandem MS. Three hundred eight proteins were commonly identified from human and mouse myelin fractions. Comparative microarray analysis of human white and gray matter showed that transcripts of several of these were elevated in OL-rich white matter compared with gray matter, providing confidence in their detection in myelin. Comparison with other databases showed that 111 of the identified proteins/transcripts also were expressed in OLs, rather than in astrocytes or neurons. Comparison with 4 previous myelin proteomes further confirmed more than 50% of the identified proteins and revealed the presence of 163 additional proteins. A select group of identified proteins also were verified by immunoblotting. We classified the identified proteins into biological subgroups and discussed their relevance in myelin biogenesis and maintenance. Taken together, the study provides insights into the complexity of this metabolically active membrane and creates a valuable resource for future in-depth study of specific proteins in myelin with relevance to human demyelinating diseases.

Subcellular tissue proteomics of hepatocellular carcinoma for molecular signature discovery.

Hepatocellular carcinoma (HCC) is one of the leading causes of mortality from solid organ malignancy worldwide. Because of the complexity of proteins within liver cells and tissues, the discovery of therapeutic targets of HCC has been difficult. To investigate strategies for decreasing the complexity of tissue samples for detecting meaningful protein mediators of HCC, we employed subcellular fractionation combined with 1D-gel electrophoresis and liquid chromatography-tandem mass spectrometry analysis. Moreover, we utilized a statistical method, namely, the Power Law Global Error Model (PLGEM), to distinguish differentially expressed proteins in a duplicate proteomic data set. Mass spectrometric analysis identified 3045 proteins in nontumor and HCC from cytosolic, membrane, nuclear, and cytoskeletal fractions. The final lists of highly differentiated proteins from the targeted fractions were searched for potentially translocated proteins in HCC from soluble compartments to the nuclear or cytoskeletal compartments. This analysis refined our targets of interest to include 21 potential targets of HCC from these fractions. Furthermore, we validated the potential molecular targets of HCC, MATR3, LETM1, ILF2, and IQGAP2 by Western blotting, immunohistochemisty, and immunofluorescent microscopy. Here we demonstrate an efficient strategy of subcellular tissue proteomics toward molecular target discovery of one of the most complicated human disease, HCC.

Two methods for assessment of choline status in a randomized crossover study with varying dietary choline intake in people: isotope dilution MS of plasma and in vivo single-voxel magnetic resonance spectroscopy of liver.

BACKGROUND: Choline deficiency has numerous negative health consequences; although the preponderance of the US population consumes less than the recommended Adequate Intake (AI), clinical assessment of choline status is difficult. Further, several pathways involved in primary metabolism of choline are estrogen-sensitive and the AI for premenopausal women is lower than that for men. OBJECTIVES: We sought to determine whether in vivo magnetic resonance spectroscopy (MRS) of liver and/or isotope-dilution MS of plasma could identify biomarkers reflective of choline intake (preregistered primary outcomes 1 and 2, secondary outcome 1). Determination of whether biomarker concentrations showed sex dependence was a post hoc outcome. This substudy is a component of a larger project to identify a clinically useful biomarker panel for assessment of choline status. METHODS: In a double-blind, randomized, crossover trial, people consumed 3 diets, representative of ∼100%, ∼50%, and ∼25% of the choline AI, for 2-wk periods. We measured the concentrations of choline and several metabolites using 1H single-voxel MRS of liver in vivo and using 2H-labeled isotope dilution MS of several choline metabolites in extracted plasma. RESULTS: Plasma concentrations of 2H9-choline, unlabeled betaine, and 2H9-betaine, and the isotopic enrichment ratio (IER) of betaine showed highly significant between-diet effects (q < 0.0001), with unlabeled betaine concentration decreasing 32% from highest to lowest choline intake. Phosphatidylcholine IER was marginally significant (q = 0.03). Unlabeled phosphatidylcholine plasma concentrations did not show between-diet effects (q = 0.34). 2H9 (trimethyl)-phosphatidylcholine plasma concentrations (q = 0.07) and MRS-measured total soluble choline species liver concentrations (q = 0.07) showed evidence of between-diet effects but this was not statistically significant. CONCLUSIONS: Although MRS is a more direct measure of choline status, variable spectral quality limited interpretation. MS analysis of plasma showed clear correlation of plasma betaine concentration, but not plasma phosphatidylcholine concentration, with dietary choline intake. Plasma betaine concentrations also correlate with sex status (premenopausal women, postmenopausal women, men).This trial was registered at clinicaltrials.gov as NCT03726671.

Role of spectral counting in quantitative proteomics.

Spectral count, defined as the total number of spectra identified for a protein, has gained acceptance as a practical, label-free, semiquantitative measure of protein abundance in proteomic studies. In this review, we discuss issues affecting the performance of spectral counting relative to other label-free methods, as well as its limitations. Possible consequences of modifications, which are commonly applied to raw spectral counts to improve abundance estimations, are considered. The use of spectral counting for different types of quantitation studies is explored and critiqued. Different statistical methods and underlying frameworks that have been applied to spectral count analysis are described and compared, and problem areas that undermine confident statistical analysis are considered. Finally, the issue of accurate estimation of false-discovery rates is addressed and identified as a major current challenge in quantitative proteomics.

Automating Complex, Multistep Processes on a Single Robotic Platform to Generate Reproducible Phosphoproteomic Data.

Mass spectrometry-based phosphoproteomics holds promise for advancing drug treatment and disease diagnosis; however, its clinical translation has thus far been limited. This is in part due to an unstandardized and segmented sample preparation process that involves cell lysis, protein digestion, peptide desalting, and phosphopeptide enrichment. Automating this entire sample preparation process will be key in facilitating standardization and clinical translation of phosphoproteomics. While peptide desalting and phosphopeptide enrichment steps have been individually automated, integrating these two extractions and, further, the entire process requires more advanced robotic platforms as well as automation-friendly extraction tools. Here we describe a fully automated peptide desalting and phosphopeptide enrichment method using IMCStips on a Hamilton STAR. Using our established automated method, we identified more than 10,000 phosphopeptides from 200 µg of HCT116 cell lysate without fractionation with >85% phosphopeptide specificities. Compared with titania-based Spin Tip products, the automated IMCStips-based method gave 50% higher phosphopeptide identifications. The method reproducibility was further assessed using multiple reaction monitoring (MRM) to show >50% phosphopeptide recoveries after the automated phosphopeptide extraction with coefficients of variation (CVs) of <20% over a 3-week period. The established automated method is a step toward standardization of the sample preparation of phosphopeptide samples and could be further expanded upon to create a fully automated "cells to phosphopeptides" method.

Analysis of virion associated host proteins in vesicular stomatitis virus using a proteomics approach.

BACKGROUND: Vesicular stomatitis virus (VSV) is the prototypic rhabdovirus and the best studied member of the order Mononegavirales. There is now compelling evidence that enveloped virions released from infected cells carry numerous host (cellular) proteins some of which may play an important role in viral replication. Although several cellular proteins have been previously shown to be incorporated into VSV virions, no systematic study has been done to reveal the host protein composition for virions of VSV or any other member of Mononegavirales. RESULTS: Here we used a proteomics approach to identify cellular proteins within purified VSV virions, thereby creating a "snapshot" of one stage of virus/host interaction that can guide future experiments aimed at understanding molecular mechanisms of virus-cell interactions. Highly purified preparations of VSV virions from three different cell lines of human, mouse and hamster origin were analyzed for the presence of cellular proteins using mass spectrometry. We have successfully confirmed the presence of several previously-identified cellular proteins within VSV virions and identified a number of additional proteins likely to also be present within the virions. In total, sixty-four cellular proteins were identified, of which nine were found in multiple preparations. A combination of immunoblotting and proteinase K protection assay was used to verify the presence of several of these proteins (integrin beta1, heat shock protein 90 kDa, heat shock cognate 71 kDa protein, annexin 2, elongation factor 1a) within the virions. CONCLUSION: This is, to our knowledge, the first systematic study of the host protein composition for virions of VSV or any other member of the order Mononegavirales. Future experiments are needed to determine which of the identified proteins have an interaction with VSV and whether these interactions are beneficial, neutral or antiviral with respect to VSV replication. Identification of host proteins-virus interactions beneficial for virus would be particularly exciting as they can provide new ways to combat viral infections via control of host components.

Altered Proteome of Extracellular Vesicles Derived from Bladder Cancer Patients Urine.

Proteomic analysis of extracellular vesicles (EVs) from biological fluid is a powerful approach to discover potential biomarkers for human diseases including cancers, as EV secreted to biological fluids are originated from the affected tissue. In order to investigate significant molecules related to the pathogenesis of bladder cancer, EVs were isolated from patient urine which was analyzed by mass spectrometry based proteomics. Comparison of the EV proteome to the whole urine proteome demonstrated an increased number of protein identification in EV. Comparative MS analyses of urinary EV from control subjects and bladder cancer patients identified a total of 1,222 proteins. Statistical analyses provided 56 proteins significantly increased in bladder cancer urine, including proteins for which expression levels varied by cancer stage (P-value < 0.05). While urine represents a valuable, noninvasive specimen for biomarker discovery in urologic cancers, there is a high degree of intra- and inter-individual variability in urine samples. The enrichment of urinary EV demonstrated its capability and applicability of providing a focused identification of biologically relevant proteins in urological diseases.

A novel method to quantify sphingosine 1-phosphate by immobilized metal affinity chromatography (IMAC).

Sphingosine 1-phosphate (S1P), a lysophospholipid mediator that signals through G protein-coupled receptors, regulates a wide plethora of biological responses such as angiogenesis and immune cell trafficking. Detection and quantification of S1P in biological samples is challenging due to its unique physicochemical nature and occurrence in trace quantities. In this report, we describe a new method to selectively enrich S1P and dihydro-S1P from biological samples by the Fe(3+) gel immobilized metal affinity chromatography (IMAC). The eluted S1P from IMAC was dephosphorylated, derivatized with o-phthalaldehyde (OPA), and detected by high-performance liquid chromatography (HPLC) coupled to a fluorescence detector. IMAC purification of S1P was linear for a wide range of S1P concentration. Using this assay, secretion of endogenous S1P from endothelial cells, fibroblasts and colon cancer cells was demonstrated. We also show that dihydro-S1P was the major sphingoid base phosphate secreted from HUVEC over expressed with Sphk1 cDNA. Pharmcological antagonists of ABC transporters, glyburide and MK-571 attenuated endogenous S1P release. This assay was also used to demonstrate that plasma S1P levels were not altered in mice deficient for ABC transporters, Abca1, Abca7 and Abcc1/Mrp1. IMAC-based affinity-enrichment coupled with a HPLC-based separation and detection system is a rapid and sensitive method to accurately quantify S1P.

Exosomal proteome analysis of cerebrospinal fluid detects biosignatures of neuromyelitis optica and multiple sclerosis.

Quantitative proteomic analysis of exosomes isolated from cerebrospinal fluid (CSF) of neuromyelitis optica (NMO) patients detected signature proteins differentiating NMO from multiple sclerosis (MS) and idiopathic longitudinally extensive transverse myelitis. Exosomes with good yields were obtained using ultracentrifugation from pooled CSF assisted by chemokine-based clustering strategy, which improved target molecule identification by providing amplified fold change values. 442 significant proteins generated a list of signature molecules of diseases validated primarily by the identification of known markers such as glial fibrillary acidic protein (GFAP) and fibronectin specific to NMO and MS respectively. MetaCore pathway analysis of significant proteins supported the involvement of these proteins in disease progression via neurological pathway. Expression levels of target molecules from orthogonal label-free quantification employing quadrupole-Orbitrap hybrid mass spectrometry were in good agreement with those from Western blotting. Additional investigation of GFAP and fibronectin as representative disease molecules revealed their presence in intact exosomes as detected by flow cytometry. This comprehensive study suggests that the exosomal proteomic analysis of CSF can be applied to the identification and characterization of inflammatory disorders of the central nervous system.

Identification of anti-metastatic drug and natural compound targets in isogenic colorectal cancer cells.

Therapeutic strategies for cancer treatment often remain challenging due to the cumulative risk derived from metastasis, which has been described as an aggressive state of cancer cell proliferation often resulting in failure of clinical therapy. In the current study, anti-metastatic properties of three chemotherapeutic drugs and three compounds from natural sources were investigated by comparative proteomic analysis. Proteomic profile comparison of the isogenic primary and metastatic colon cancer cell lines SW480 and SW620 identified two potential metastasis related molecular targets: fatty acid synthase and histone H4. To demonstrate their biological roles in cancer metastasis, the expression of these target genes was suppressed by siRNA transfection. Subsequent cell migration assays demonstrated reduced migratory effects. SW620 cells were treated with six anti-cancerous components. Through comprehensive proteomic analysis, three of the tested compounds, oxaliplatin, ginsenoside 20(S)-Rg3 and curcumin, were revealed to have a suppressive effect on FASN and histone H4 expression. SW620 cells treated with these drugs showed significantly reduced migratory activity, which suggests that drug-induced targeted suppression of these genes may affect cell migration. The validity of the proteomic datasets was verified by knowledgebase pathway analysis and immunoblotting assays. The anti-metastatic components revealed by the current proteomic analysis represent promising chemotherapeutic candidates for the treatment of colorectal adenocarcinoma. BIOLOGICAL SIGNIFICANCE: The current study demonstrates anti-metastatic activity of chemotherapeutics and natural components by the suppression of target molecules, fatty acid synthase and histone H4 identified by a comparative proteomic analysis employing the isogenic primary and metastatic colon cancer cell lines, SW480 and SW620. Three tested drugs, namely, oxaliplatin, ginsenoside 20(S)-Rg3 and curcumin were revealed to possess suppressive effects on fatty acid synthase and histone H4 and reduce metastasis as determined by cell migration assay. Data were confirmed by the correlation between spectral counts from proteomic data and Western blot analysis, which were in good agreement with immunohistochemistry.

A draft map of rhesus monkey tissue proteome for biomedical research.

Though the rhesus monkey is one of the most valuable non-human primate animal models for various human diseases because of its manageable size and genetic and proteomic similarities with humans, proteomic research using rhesus monkeys still remains challenging due to the lack of a complete protein sequence database and effective strategy. To investigate the most effective and high-throughput proteomic strategy, comparative data analysis was performed employing various protein databases and search engines. The UniProt databases of monkey, human, bovine, rat and mouse were used for the comparative analysis and also a universal database with all protein sequences from all available species was tested. At the same time, de novo sequencing was compared to the SEQUEST search algorithm to identify an optimal work flow for monkey proteomics. Employing the most effective strategy, proteomic profiling of monkey organs identified 3,481 proteins at 0.5% FDR from 9 male and 10 female tissues in an automated, high-throughput manner. Data are available via ProteomeXchange with identifier PXD001972. Based on the success of this alternative interpretation of MS data, the list of proteins identified from 12 organs of male and female subjects will benefit future rhesus monkey proteome research.