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PubMed2025

Proteomic hub proteins CDKN2B, TRAPPC2L, WFS1, and ARPP19 drive biochemical recurrence and metastatic progression in prostate cancer: Protein macromolecule action.

The biological characteristics and metastasis mechanism of prostate cancer are complex, involving the important role of many proteins in cell transcriptional regulation. This study focused on the role of the proteomic hub proteins CDKN2B, TRAPPC2L, WFS1 and ARPP19 in the biochemical recurrence and metastasis progression of prostate cancer. Cross-platform transcriptome integration and differential expression analysis were used to evaluate transcriptome characteristics in a prostate cancer cohort. Functional enrichment analysis was performed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation, and weighted gene co-expression network analysis (WGCNA) was used to investigate cancer progression subtypes. It was found that prostate cancer progression showed significant transcriptome heterogeneity, and low-expression genes dominated. We reveal the important role of epithelial-immune interactions and inflammatory signaling in transcriptional remodeling in prostate cancer. The co-expression network topology analysis showed that the immune-metabolic center module plays a central role in cancer progression. CDKN2B was identified as a key transcriptional determinant in prostate cancer typing, while TRAPPC2L and WFS1 acted as core transcriptional regulators, driving metastatic heterogeneity. ARPP19 and LOC650152 also show important transcriptional driving effects in advanced prostate cancer.

Humans↗
PubMedDate not supplied

In search of secreted protein biomarkers for the anti-inflammatory effect of beta2-adrenergic receptor agonists: application of DIGE technology in combination with multivariate and univariate data analysis tools.

Two-dimensional difference gel electrophoresis (DIGE) in combination with univariate (Student's t-test) and multivariate data analysis, principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were used to study the anti-inflammatory effects of the beta(2)-adrenergic receptor (beta(2)-AR) agonist zilpaterol. U937 macrophages were exposed to the endotoxin lipopolysaccharide (LPS) to induce an inflammatory reaction, which was inhibited by the addition of zilpaterol (LZ). This inhibition was counteracted by addition of the beta(2)-AR antagonist propranolol (LZP). The extracellular proteome of the U937 cells induced by the three treatments were examined by DIGE. PCA was used as an explorative tool to investigate the clustering of the proteome dataset. Using this tool, the dataset obtained from cells treated with LPS and LZP were separated from those obtained from LZ treated cells. PLS-DA, a multivariate data analysis tool that also takes correlations between protein spots and class assignment into account, correctly classified the different extracellular proteomes and showed that many proteins were differentially expressed between the proteome of inflamed cells (LPS and LZP) and cells in which the inflammatory response was inhibited (LZ). The Student's t-test revealed 8 potential protein biomarkers, each of which was expressed at a similar level in the LPS and LZP treated cells, but differently expressed in the LZ treated cells. Two of the identified proteins, macrophage inflammatory protein-1beta (MIP-1beta) and macrophage inflammatory protein-1alpha (MIP-1alpha) are known secreted proteins. The inhibition of MIP-1beta by zilpaterol and the involvement of the beta(2)-AR and cAMP were confirmed using a specific immunoassay.

Adrenergic Agonists↗
PubMed2003

In vivo proteomic analysis of cytokine expression in laser capture-microdissected urothelial cells of obstructed ureteropelvic junction procured by laparoscopic dismembered pyeloplasty.

BACKGROUND AND PURPOSE: Ureteropelvic junction obstruction (UPJO) is defined as an impediment to urinary flow from the renal pelvis into the ureter. The exact cause remains an enigma despite investigations along embryologic, anatomic, and histologic lines. Our goal was to investigate in vivo the expression profile of cytokines in hyperplastic urothelial cells as a means of determining the source of UPJO. MATERIALS AND METHODS: Cellular proteomes of matched normal and hyperplastic urothelial cells were analyzed by laser capture microdissection (LCM) and tissue microdissection and human cytokine proteomic chips. All specimens (N = 9) were surgically obtained from patients undergoing laparoscopic dismembered pyeloplasty and were immediately embedded in O.C.T. solution and flash-frozen in liquid nitrogen. Tissue sections (6 microm) were mounted on uncoated glass slides using a cryostat, fixed in 70% ethanol, stained with hematoxylin and eosin, and sequentially dehydrated in ethanol and xylene. Typically, paired samples of normal and hyperplastic urothelial cells were procured on separate caps from serial sections of each specimen by the Arcturus PixCell II LCM system using 3000 laser pulses and a spot diameter of 30 microm. Total proteins were harvested and quantitated. Differential expression profile analysis of 43 cytokines in normal and hyperplastic cells were performed using human protein chips. Briefly, the membranes were initially probed with protein (150 ng) from normal or hyperplastic cells and sequentially reacted with a cocktail of biotinylated cytokine antibodies and horseradish peroxidase-conjugated streptavidin. The membranes were developed using enhanced chemiluminescence and analyzed by densitometry. RESULTS: Comparative densitometric analysis revealed twofold to fourfold upregulation of growth-related oncogene alpha (GRO-alpha), interleukin (IL)-1alpha, interferon (INF)-gamma, IL-8, tumor necrosis factor (TNF)-alpha, RANTES, and macrophage inflammatory protein-1beta (MIP-1beta) and twofold to fourfold downregulation of transforming growth factor (TGF)-beta and IL-10 in hyperplastic urothelial cells compared with paired control cells. CONCLUSIONS: We here report the efficient application of LCM and proteomic array chips for expression profile analysis of cytokines in vivo. Because the etiology and pathogenesis of UPJO are still fragmentary, the marked heterogeneity of the observed cytokine alterations reported here may be of significance. Further studies are required to elucidate the functional significance of the differentially expressed cytokines in the pathogenesis of the disease.

Adult↗
PubMed2005

Effects of diet-induced hypercholesterolemia on testosterone-regulated protein expression in mice liver.

We report here testosterone deficiency in diet-induced hypercholesterolemic mice. We attempted to compare the liver protein profiles between hypercholesterolemic and normal mice by using the technology of proteomics. Protein extractions from mice livers were separated by two-dimensional electrophoresis and the gels were analysed with image analysis software 2D Elite 4.0. The differentially expressed proteins were identified primarily by comparing with reference gel images in the SWISS 2DPAGE database and then confirmed by peptide mass fingerprinting. Sixteen differentially expressed protein spots (>2-fold) were detected and 8 of which were identified as major urinary proteins, carbonic anhydrase III, and glutathione S-transferase P2, which are known to be regulated by androgens. The expression of these three proteins was statistically lower in the livers of hypercholesterolemic mice. Meanwhile, the testosterone levels in serum, testis, and liver were lower in hypercholesterolemic mice than those in normal mice, when human chorionic gonadotrophin was injected intramuscularly. These results suggest a testosterone deficiency in diet-induced hypercholesterolemic mice.

Animals↗
PubMed2006

Proteome analysis of rat liver mitochondria reveals a possible compensatory response to endotoxic shock.

Organ failure induced by endotoxic shock has recently been associated with affected mitochondrial function. In this study, effects of in vivo lipopolysaccharide-challenge on protein patterns of rat liver mitochondria in treated animals versus controls were studied by two-dimensional electrophoresis (differential image gel electrophoresis). Significant upregulation was found for ATP-synthase alpha chain and superoxide dismutase [Mn]. Our data suggest that endotoxic shock mediated changes in the mitochondrial proteome contribute to a compensatory reaction (adaptation to endotoxic shock) rather than to a mechanism of cell damage.

Animals↗
PubMed2026

Ascites reprograms innate lymphoid immune cells in ovarian cancer by promoting ILC2 enrichment and dysfunctional NK-cell states.

BACKGROUND: High-grade serous ovarian cancer (HGSOC) is commonly accompanied by malignant ascites, a clinically relevant tumor niche that promotes immune evasion, metastasis, and treatment resistance. Although natural killer (NK)-cell dysfunction has been described in ovarian cancer, the broader innate lymphoid landscape of ascites and the mechanisms linking ascites-derived signals to innate immune suppression remain insufficiently resolved. METHODS: We performed single-cell RNA sequencing of NK/innate lymphoid cells from ovarian cancer ascites to define cellular heterogeneity and differentiation states. Functional assays assessed NK-cell cytotoxicity, degranulation, and receptor expression following exposure to patient-derived ascites, with or without transforming growth factor-β (TGF-β) receptor inhibition. Proteomic profiling was used to characterize the soluble ascites milieu, and clinical associations were examined for innate lymphoid subsets. RESULTS: Single-cell analysis identified eight transcriptionally distinct NK/innate lymphoid states, including cytotoxic, precursor, early-like, tolerant/immunoregulatory, regulatory, proinflammatory, and innate lymphoid populations. Ovarian cancer ascites was characterized by depletion of cytotoxic and precursor NK-cell states together with enrichment of early-like, tolerant, regulatory, pro-inflammatory, and innate lymphoid cell (ILC) populations. Trajectory analysis indicated impaired maturation toward terminally differentiated cytotoxic NK cells. Notably, ascites contained an expanded population of programmed cell death protein 1 (PD-1)+ ILC2s, which were more abundant in patients with shorter progression-free survival. In functional assays, short-term exposure of healthy donor NK cells to ascites suppressed degranulation and tumor-cell killing, reduced expression of activating receptors including NKp30 and DNAM-1, increased inhibitory receptor expression, and shifted NK cells toward a CD56highCD16low phenotype. Proteomic profiling supported a soluble milieu consistent with type 2 immune skewing and NK-cell suppression. Importantly, TGF-β receptor inhibition partially restored NK-cell activation and function in the presence of ascites. CONCLUSIONS: HGSOC ascites establishes a type 2-skewed immunoregulatory niche that coordinately drives NK cell dysfunction and PD-1+ ILC2 accumulation. The findings identify TGF-β-linked suppression and ascites-associated immune regulators as candidate immunotherapeutic vulnerabilities for restoring antitumor immunity in ovarian cancer.

Humans↗
PubMed2006

Proteomic analysis for the identification of proteins related to methotrexate resistance.

Methotrexate(MTX) is one of the most important and frequently used drugs in cancer therapy, but the efficacy of this drug is often compromised by the development of resistance in cancer cells. To seek and identify differentially expressed proteins related to MTX resistance and provide clues for the mechanism of MTX resistance, proteins from cell line MTX300 (resistant to 300 micromol/L MTX) and its control cell line 3T3R500 were separated by two-dimensional electrophoresis (2-DE). The colloidal Coomassie brilliant blue-stained 2-DE gels were subjected to image analysis, which revealed several spots with high levels of differential expression between MTX300 and 3T3R500. The protein spot with highest differential expression was submitted for tryptic peptide mass fingerprinting(PMF) for identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). MS analysis and database searches revealed it to be dihydrofolate reductase (DHFR), which was subsequently confirmed by Western blot. The result suggested that DHFR might play an important role in the MTX resistance.

Animals↗
PubMed2006

Proteomic analysis of tobacco mosaic virus-infected tomato (Lycopersicon esculentum M.) fruits and detection of viral coat protein.

Tobacco mosaic virus (TMV) is a widespread plant virus from the genus Tobamovirus that affects tobacco and tomato plants causing a pathology characterised by cell breakage and disorganisation in plant leaves and fruits. In this study we undertook a proteomic approach to investigate the molecular and biochemical mechanisms potentially involved in tomato fruit defence against the viral infection. The comparison of 2-D gels from control and TMV-infected but asymptomatic tomato fruits revealed changes in several proteins. The differential expression of peptidases, endoglucanase, chitinase and proteins participating in the ascorbate-glutathione cycle in infected fruits suggests that pathogenesis-related proteins and antioxidant enzymes may play a role in the protection against TMV infection. TMV coat protein appeared as a prominent spot in 2-D gels from TMV-infected asymptomatic fruits. A Triton X-114 phase-partitioning step of tomato protein extracts favoured the solubilisation of TMV coat protein and the enrichment of two aminopeptidases not present in control fruits. PMF and MS/MS data of the 2-D gel-isolated TMV coat protein is proposed as a powerful analysis method for the simultaneous tobamovirus detection, species determination and strain differentiation in virus-infected fruit commodities.

Capsid Proteins↗
PubMed2026

Proteomic insights into azoospermia: protein differences in testicular tissue between non-obstructive and obstructive azoospermia patients.

Non-obstructive azoospermia (NOA) and obstructive azoospermia (OA) are the main classifications of severe male infertility, but the molecular mechanism of NOA remains poorly understood. This study aimed to identify potential biomarkers and pathological mechanisms by comparing the proteomic differences in testicular tissues of NOA and OA patients. Through proteomic analysis based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) of testicular samples from 5 NOA patients and 5 OA patients, we identified 5264 proteins, among which 717 differentially expressed proteins (DEPs) were found between the two groups (242 upregulated and 475 downregulated in NOA). Bioinformatics analysis indicated that these DEPs were significantly associated with reproductive development, gametogenesis, and cell structural stability. On the basis of this, six candidate proteins, including dysferlin (DYSF), myoferlin (MYOF), mitsugumin 53 (MG53), cluster of differentiation 63 (CD63), caveolin-3 (CAV3), and calpain-3 (CAPN3), were selected from the DEPs and verified in an expanded sample set (37 NOA cases and 28 OA cases) through quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, confirming their dysregulation in NOA. These findings provide new proteomic insights into NOA, highlighting the disruption of membrane repair and structural pathways, and offer potential biomarkers for understanding its pathogenesis.

Humans↗
PubMed2005

Chemoprevention of breast cancer, proteomic discovery of genistein action in the rat mammary gland.

Genistein, the primary isoflavone component of soy, consumed in the diet during the prepubertal period only, and the combined prepubertal and adult periods, suppresses chemically induced mammary cancer in rats. Gestational or adult-only exposures do not provide protection. An inverse relation exists between cancer susceptibility and mammary gland differentiation. The current study used proteomic technology to investigate genistein mechanisms of action as related to programming against chemically induced mammary cancer. Rats were injected subcutaneously with 500 microg genistein/g body weight on d 16, 18, and 20 postpartum. At d 21, mammary glands were subjected to 2-dimensional polyacrylamide gel electrophoresis. After gel scanning, image analysis, and MS, 6 proteins were determined to be differentially regulated and identified. One protein, GTP-cyclohydrolase 1 (GTP-CH1), was confirmed as being significantly upregulated at d 21 by immunoblot analysis. Investigation of downstream signaling from GTP-CH1 showed that tyrosine hydroxylase was upregulated and vascular endothelial growth factor receptor 2 (VEGFR2) was downregulated in the mammary glands of 50-d-old rats treated with genistein in the prepubertal period. This and previous work suggest that early prepubertal exposure to genistein enhances cell proliferation by upregulating GTP-CH1 and the epidermal growth factor (EGF)-signaling pathway, and hence cell differentiation and gland maturation. This unique developmental maturation leads to a new biochemical blueprint, whereby the cells have reduced EGF signaling and VEGFR2, which renders the mature mammary gland less proliferative and less susceptible to cancer. This study demonstrated the usefulness of proteomics for the discovery of novel pathways that may be involved in cancer prevention.

Animals↗
PubMed2005

Response of the anaerobe Desulfovibrio vulgaris Hildenborough to oxidative conditions: proteome and transcript analysis.

The method of two-dimensional protein gel electrophoresis was used to evaluate the changes at the proteins level following oxygen exposure of the anaerobic sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough. Fifty-seven proteins showed significant differential expression. The cellular concentration of 35 proteins decreased while that of nineteen increased as a specific consequence of oxidative conditions. The proteins that were less abundant belonged to various functional categories such as nucleic acid and protein biosynthesis, detoxification mechanisms, or cell division. Interestingly, quantitative real-time PCR revealed that the genes encoding detoxification enzymes (rubrerythrins, superoxide reductase) are down regulated. The loss of viability of D. vulgaris Hildenborough under these oxidative conditions (Fournier et al., J. Biol. Chem. 279 (2004) 1785) can be directly related to the decrease in the cellular concentrations of these proteins, thereby specifying the toxicity of oxygen for the cells. Among the proteins that were more abundant under oxygen exposure, several thiol-specific peroxidases (thiol-peroxidase, BCP-like protein, and putative glutaredoxin) were identified. Using RT-PCR, the up-regulation of the genes encoding the thiol-peroxidase and the BCP was demonstrated. That is the first time that these proteins have been shown to be involved in the defense of D. vulgaris toward an oxidative stress. Several hypothetical proteins were also shown to be differentially expressed. A function in the defense mechanism against an oxidative stress is proposed for these uncharacterized proteins.

Anaerobiosis↗
PubMed2006

Subcellular and functional proteomic analysis of the cellular responses induced by Helicobacter pylori.

Helicobacter pylori infection is a crucial factor in the pathogenesis of several digestive disorders, including peptic ulcers, chronic gastritis, and gastric cancer. Moreover H. pylori induces disease-specific protein expression in gastric epithelial cells. The aim of the present study was to characterize proteins differentially expressed in H. pylori-infected gastric epithelial AGS cells. An in vitro model was established using a multiplicity of infection of 100 and evaluating the effectiveness of H. pylori infection by functional analyses. Changes in protein patterns were identified using a proteomic approach consisting of two-dimensional fluorescence difference gel electrophoresis and mass spectrometry. The expression of many proteins was found to be altered, and 28 of these were identified and classified as protein synthesis- and folding-related proteins, cytoskeleton proteins, metabolic enzymes, transcription- and translation-related proteins, angiogenesis/metastasis-related proteins, cell communication/signal transduction-related proteins, or others (oxygen-regulated protein and oncoprotein). The expression profiles of eight of these proteins, laminin gamma-1 chain precursor, valosin-containing protein, heat shock 70-kDa protein, mitochondrial matrix protein P1, FK506-binding protein 4, T-complex protein 1, enolase alpha, and 14-3-3 beta were further examined in cancerous and paired surrounding normal tissues by immunoblot assay and immunohistochemical staining to identify molecular targets that may be involved in the pathogenesis of H. pylori-induced gastric diseases. On the basis of our results, valosin-containing protein, mitochondrial matrix protein P1, T-complex protein 1, enolase alpha, and 14-3-3 beta may play a crucial role in H. pylori-induced gastric carcinogenesis by mediating antiapoptotic and proliferative responses.

Blotting, Western↗
PubMed2006

Meta-analysis of differentiating mouse embryonic stem cell gene expression kinetics reveals early change of a small gene set.

Stem cell differentiation involves critical changes in gene expression. Identification of these should provide endpoints useful for optimizing stem cell propagation as well as potential clues about mechanisms governing stem cell maintenance. Here we describe the results of a new meta-analysis methodology applied to multiple gene expression datasets from three mouse embryonic stem cell (ESC) lines obtained at specific time points during the course of their differentiation into various lineages. We developed methods to identify genes with expression changes that correlated with the altered frequency of functionally defined, undifferentiated ESC in culture. In each dataset, we computed a novel statistical confidence measure for every gene which captured the certainty that a particular gene exhibited an expression pattern of interest within that dataset. This permitted a joint analysis of the datasets, despite the different experimental designs. Using a ranking scheme that favored genes exhibiting patterns of interest, we focused on the top 88 genes whose expression was consistently changed when ESC were induced to differentiate. Seven of these (103728_at, 8430410A17Rik, Klf2, Nr0b1, Sox2, Tcl1, and Zfp42) showed a rapid decrease in expression concurrent with a decrease in frequency of undifferentiated cells and remained predictive when evaluated in additional maintenance and differentiating protocols. Through a novel meta-analysis, this study identifies a small set of genes whose expression is useful for identifying changes in stem cell frequencies in cultures of mouse ESC. The methods and findings have broader applicability to understanding the regulation of self-renewal of other stem cell types.

Adaptation, Physiological↗
PubMed2005

Rapid detection of fetal aneuploidy using proteomics approaches on amniotic fluid supernatant.

OBJECTIVE: Conventional chromosomal studies or fluorescent in situ hybridization takes days to diagnose fetal aneuploidies during amniocentesis. Here, we evaluated the value of mass spectrometry-based clinical proteomics analysis on amniotic fluid supernatant (AFS) as a rapid detection of fetal aneuploidies. METHODS: Proteomics profiles generated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) after fractionating samples with functionalized magnetic beads were used for differentiating 60 normal karyotypic from 20 aneuploid AFS. After the discriminating models were generated using genetic algorithm, we evaluated the clinical efficacy of the models in detecting aneuploidies in two batches (each n=30) of AFS prior to the release of chromosomal diagnoses. RESULTS: Within hours, the two-step proteomics analysis of AFS with the C18 model, followed by the weak cation exchange model, was able to detect aneuploid AFS at 3.3% disease prevalence rate with 100% sensitivity, 72 to 96% specificity, 11 to 50% positive predictive value, and 100% negative predictive value. CONCLUSION: Clinical proteomics analysis of AFS using magnetic beads-based sample preparation and MALDI-TOF-MS can be used as a rapid detection for fetal aneuploidies. With perfect sensitivity and negative predictive value of the two-step proteomics method, it may be used for rapid detection of aneuploid AFS immediately after amniocentesis. Further large-scale examinations are apparently needed to verify the clinical value of this rapid detection.

Amniotic Fluid↗
PubMed2003

Early drug safety evaluation: biomarkers, signatures, and fingerprints.

When target organ toxicity arises in animal models during routine drug safety evaluation, it raises several key questions: Is this target organ toxicity related to the pharmacology? What is the mode of action (MOA)? Is the target organ toxicity relevant to humans? Pathology or prior knowledge of the compound class may provide clues on a possible MOA for toxicity. However, if this deductive approach yields no results, the inductive approach offered by new technologies can generate novel research leads. For example, toxicogenomics can generate a gene expression profile of the toxicity that can be compared with reference compounds or with other candidate drugs. Similarly, proteomic analysis of the protein profile at the toxic vs. the efficacious dose can provide clues on MOA for the toxicity and may allow differentiation of the pathways of the toxic response from those required for pharmacological activity.

Animals↗
PubMed2006

Comparative proteomic analysis between the invasive and commensal strains of Staphylococcus epidermidis.

Staphylococcus epidermidis is one of the major causative agents for nosocomial infections. To reveal the pathogenesis factors, we performed the comparative proteomic analysis of invasive ATCC35984 and commensal ATCC12228 strains by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry. The differentially expressed proteins were involved in carbohydrate metabolism, sugar binding, lipid degradation and amino acid binding. In addition, we demonstrated that the trap gene was transcribed by 3.657+/-0.156 (P<0.01) -fold higher in ATCC35984 than in ATCC12228. Levels of accumulation-associated protein (AAP) were found to be low by the immuno-dot blotting assay in ATCC12228, which is unable to form biofilm. Our results suggest that the target of RNAIII activating protein and AAP may contribute to S. epidermidis virulence and biofilm formation.

Amino Acids↗