RESUMO
Current diagnostic markers for gastric cancer are not sufficiently specific or sensitive for use in clinical practice. The aims of this study are to compare the proteomes of serum samples from patients with gastric cancers and normal controls, and to develop useful tumor markers of gastric cancer by quantitative proteomic analysis. We identified a total of 388 proteins with a ≤1% FDR and with at least two unique peptides from the sera of each group. Among them, 215, 251, and 260 proteins were identified in serum samples of patients in an advanced cancer group, early cancer group, and normal control group, respectively. We selected differentially expressed proteins in cancer patients compared with those of normal controls via semiquantitative analyses comparing the spectral counts of identified proteins. These differentially expressed proteins were successfully verified using an MS-based quantitative assay, multiple reactions monitoring analysis. Four proteins (vitronectin, clusterin isoform 1, thrombospondin 1, and tyrosine-protein kinase SRMS) were shown to have significant changes between the cancer groups and the normal control group. These four serum proteins were able to discriminate gastric cancer patients from normal controls with sufficient specificity and selectivity.
Assuntos
Biomarcadores Tumorais/sangue , Proteômica/métodos , Neoplasias Gástricas/sangue , Neoplasias Gástricas/diagnóstico , Adulto , Área Sob a Curva , Estudos de Casos e Controles , Feminino , Ontologia Genética , Humanos , Masculino , Pessoa de Meia-Idade , Curva ROC , Reprodutibilidade dos Testes , Estatística como AssuntoRESUMO
Spinocerebellar ataxia type 1 (SCA1), an autosomal-dominant neurodegenerative disorder, is caused by expansion of the polyglutamine tract within ataxin-1 (ATXN1). The AXH domain of ATXN1 can mediate neurodegeneration through its interaction with other proteins. We have previously showed that the ubiquitin-conjugating enzyme UbcH6 modulates the transcriptional repression activity of ATXN1 through ubiquitylation. In the present study, we sought to identify sites in the AXH domain that are ubiquitylated by UbcH6. Systematic replacement of each lysine residue in the AXH domain revealed that the lysine at 589 (K589) of ATXN1 is essential for its ubiquitylation by UbcH6. Mass spectrometry studies further confirmed the ubiquitylation site. Interestingly, protein aggregation was significantly enhanced in mutant AXH K589R, implying that the aggregation is strongly associated with the level of ATXN1 expression. Our study may suggest a therapeutic potential of UbcH6 in the treatment of SCA1.
Assuntos
Lisina , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Ubiquitinação , Sequência de Aminoácidos , Ataxina-1 , Ataxinas , Sítios de Ligação/genética , Células HEK293 , Humanos , Lisina/química , Lisina/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Agregados Proteicos , Ligação Proteica , Estrutura Terciária de Proteína/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação/genéticaRESUMO
Protein tyrosine nitration (PTN) is a PTM that regulates signal transduction and inflammatory responses, and is related to neurodegenerative and cardiovascular diseases. The cellular function of PTN remains unclear because the low stoichiometry of PTN limits the identification and quantification of nitrated peptides. Effective enrichment is an important aspect of PTN analysis. In this study, we analyzed the in vivo nitroproteome elicited by mating signal transduction in Saccharomyces cerevisiae using a novel chemical enrichment method followed by LC-MS/MS. Nitroproteome profiling successfully identified changes in the nitration states of 14 proteins during mating signal transduction in S. cerevisiae, making this the first reported in vivo nitroproteome in yeast. We investigated the biological functions of these nitroproteins and their relationships to mating signal transduction in S. cerevisiae using a protein-protein interaction network. Our results suggest that PTN and denitration may be involved in nonreactive nitrogen species-mediated signal transduction and can provide clues for understanding the functional roles of PTN in vivo.
Assuntos
Nitratos/análise , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Tirosina/análise , Sequência de Aminoácidos , Modelos Moleculares , Dados de Sequência Molecular , Nitratos/metabolismo , Mapas de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Proteômica , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/química , Transdução de Sinais , Espectrometria de Massas em Tandem , Tirosina/metabolismoRESUMO
Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) such as gefitinib are one of gold standard treatment options for nonsmall-cell lung cancer (NSCLC) patients, which eventually fail due to the acquired resistance and relapse because of the development of secondary activating mutations such as T790M in EGFR. Predicting chemo-responsiveness of cancer patients provides a major challenge in chemotherapy. The goal of the present study is to determine whether phospholipid signatures of tumor extracellular vesicles (EV) are associated with gefitinib-resistance of NSCLC. A sophisticated MS-based shotgun lipidomic assays were performed for in-depth analysis of the lipidomes of gefitinib-resistant (PC9R) and responsive (PC9) NSCLC cells and their shed EV from these cell lines (PC9EV or PC9REV). Lipid MALDI-MS analysis showed that EV phospholipid composition was significantly distinct in PC9R, compared to PC9 cells. Following statistical analyses has identified 35 (20 positive and 15 negative ion mode) differentially regulated lipids, which are significantly over- or underexpressed in PC9R EV, compared to PC9 EV (p value < 0.01, fold change > 1.5). Our phospholipid signatures suggest that EV associates with drug sensitivity, which is worthy of additional investigation to assess chemoresistance in patients with NSCLC treated with anti-EGFR TKIs.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas , Extratos Celulares , Resistencia a Medicamentos Antineoplásicos , Espaço Extracelular , Fosfolipídeos , Quinazolinas/farmacologia , Extratos Celulares/química , Extratos Celulares/farmacologia , Linhagem Celular Tumoral , Vesículas Citoplasmáticas/química , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/fisiologia , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/fisiologia , Gefitinibe , Humanos , Fosfolipídeos/farmacologia , Fosfolipídeos/fisiologiaRESUMO
Polycaprolactone (PCL) nanofibers (PCL-NF) with uniform fibrous structure were fabricated by electrospinning. However, PCL-NF has hydrophobic surface, lacks functional groups and hence it is not a good substrate for cell adhesion. To improve the cell adhesion, PCL-NF surfaces were modified by low pressure RF discharge plasma treatment using monomer such as acrylic acid or oxygen gas. The plasma treated PCL-NFs improved the wettability and cell proliferation.
Assuntos
Substitutos Ósseos/química , Nanofibras/química , Poliésteres/química , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Camundongos , Osteoblastos/efeitos dos fármacos , Gases em Plasma , Poliésteres/farmacologiaRESUMO
Polycaprolactone (PCL)/TiO2 composite films (PTCFs) were prepared by a solvent casting method at various concentrations of TiO2 (1, 3, 5, and 10 wt%) and then treated using oxygen plasma. The hydrophilicity of the oxygen plasma treated PTCFs increased as the treatment time was increased, due to the oxygen induced production of polar species at the surface of the PTCFs. In vitro bioactivities of the composite films were examined by immersion in simulated body fluid for up to 7 days. It was found that the oxygen plasma treatment significantly influenced the in vitro bioactivity of the PTCFs.
Assuntos
Fosfatos de Cálcio/síntese química , Materiais Revestidos Biocompatíveis/síntese química , Nanocompostos/química , Oxigênio/química , Poliésteres/química , Titânio/química , Materiais Biomiméticos/química , Líquidos Corporais/química , Teste de Materiais , Nanocompostos/ultraestrutura , Tamanho da Partícula , Gases em Plasma/química , Propriedades de SuperfícieRESUMO
Jasin-hwan-gagambang (BHH10), a modified prescription of Jasin-hwan, contains Astragalus membranaceus, Cinnamomum cassia, and Phellodendron amurense, and it has been traditionally used to treat osteoporosis and other inflammatory diseases. In this study, we systematically investigated the protective effects of BHH10 in ovariectomy (OVX)-induced rats. Sprague-Dawley rats were randomly divided into sham and OVX subgroups. The rats in the OVX group were treated with vehicle, BHH10, alendronate (ALN), and 17ß-estradiol (E2). BHH10 treatment significantly inhibited OVX-induced increases in body weight and uterus atrophy. In addition, it significantly increased the bone mineral density (BMD) and prevented a decrease in trabecular bone volume, connectivity density, trabecular number, thickness, and separation at the total femur and femur neck. The OVX rats showed significant decreases in the serum levels of calcium and phosphorous and significant increases in the serum levels of cholesterol, low-density lipoprotein cholesterol, alkaline phosphatase, osteocalcin, C-telopeptide type 1 collagen, and bone morphogenetic protein-2. These changes were significantly reduced to near sham levels by administration of BHH10 to OVX rats. BHH10-treated rats had a greater bone mass, a better structural architecture of the bone, and higher levels of biochemical markers of the bone than did the ALN-treated or E2-treated rats. These results suggest that BHH10 reverses osteoporosis in OVX rats by stimulating bone formation or regulating bone resorption and is not associated with toxicity.
Assuntos
Densidade Óssea/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Osteoporose/tratamento farmacológico , Extratos Vegetais/farmacologia , Alendronato/farmacologia , Animais , Astragalus propinquus/química , Peso Corporal , Reabsorção Óssea/prevenção & controle , Cinnamomum aromaticum/química , Modelos Animais de Doenças , Estradiol/farmacologia , Feminino , Fêmur/efeitos dos fármacos , Tamanho do Órgão , Osteocalcina/sangue , Ovariectomia , Phellodendron/química , Ratos , Ratos Sprague-Dawley , Testes de Toxicidade Aguda , Testes de Toxicidade CrônicaRESUMO
Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs), including gefitinib, are the first-line treatment of choice for nonsmall cell lung cancer patients who harbor activating EGFR mutations, however, acquired resistance to EGFR-TKIs is inevitable. The main objective of this study was to identify informative protein signatures of extracellular vesicles (EV) derived from gefitinib-resistant nonsmall cell lung cancer cells using proteomics analysis. Nano-LC-MS/MS analysis identified with high confidence (false discovery rate < 0.05, fold change ≥2) 664 EV proteins enriched in PC9R cells, which are resistant to gefitinib due to EGFR T790M mutation. Computational analyses suggested components of several signal transduction mechanisms including the AKT (also PKB, protein kinase B)/mTOR (mechanistic target of rapamycin) pathway are overrepresented in EV from PC9R cells. Treatment of recipient cells with EV harvested from PC9R cells increased phosphorylation of signaling molecules, and enhanced proliferation, invasion, and drug resistance to gefitinib-induced apoptosis. Dose- and time-dependent pharmaceutical inhibition of AKT/mTOR pathway overcame drug resistance of PC9R cells and those of H1975 exhibiting EGFR T790M mutation. Our findings provide new insight into an oncogenic EV protein signature regulating tumor microenvironment, and will aid in the development of novel diagnostic strategies for prediction and assessment of gefitinib resistance.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares/tratamento farmacológico , Quinazolinas/farmacologia , Microambiente Tumoral , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Gefitinibe , Humanos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Mutação Puntual , Inibidores de Proteínas Quinases/farmacologia , Proteômica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Microambiente Tumoral/efeitos dos fármacosRESUMO
Hepatitis B virus (HBV) X protein (HBx) is a key player in HBV replication as well as HBV-induced hepatocellular carcinoma (HCC). However, the pathogenesis of HBV infection and the mechanisms of host-virus interactions are still elusive. In this study, a combination of affinity purification and mass spectrometry was applied to identify the host factors interacting with HBx in hepatoma cells. Thirteen proteins were identified as HBx binding partners. Among them, we first focused on determining the functional significance of the interaction between HBx and hepatocystin. A physical interaction between HBx and hepatocystin was confirmed by co-immunoprecipitation and Western blotting. Immunocytochemistry demonstrated that HBx and hepatocystin colocalized in the hepatoma cells. Domain mapping of both proteins revealed that the HBx C-terminus (amino acids 110-154) was responsible for binding to the mannose 6-phosphate receptor homology domain (amino acids, 419-525) of hepatocystin. Using translation and proteasome inhibitors, we found that hepatocystin overexpression accelerated HBx degradation via a ubiquitin-independent proteasome pathway. We demonstrated that this effect was mediated by an interaction between both proteins using a HBx deletion mutant. Hepatocystin overexpression significantly inhibited HBV DNA replication and expression of HBs antigen concomitant with HBx degradation. Using the hepatocystin mutant constructs that bind HBx, we also confirmed that hepatocystin inhibited HBx-dependent HBV replication. In conclusion, we demonstrated for the first time that hepatocystin functions as a chaperon-like molecule by accelerating HBx degradation, and thereby inhibits HBV replication. Our results suggest that inducing hepatocystin may provide a novel therapeutic approach to control HBV infection.
Assuntos
Carcinoma Hepatocelular/metabolismo , Glucosidases/fisiologia , Vírus da Hepatite B/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Neoplasias Hepáticas/metabolismo , Transativadores/metabolismo , Replicação Viral/fisiologia , Sequência de Aminoácidos , Proteínas de Ligação ao Cálcio , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Linhagem Celular Tumoral , Glucosidases/química , Glucosidases/metabolismo , Humanos , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Espectrometria de Massas , Dados de Sequência Molecular , Ligação Proteica , Proteínas Virais Reguladoras e AcessóriasRESUMO
Formononetin (1), a plant-derived phytoestrogen, possesses bone protective properties. To address the potential therapeutic efficacy and mechanism of action of 1, we investigated its antiosteoclastogenic activity and its effect on nuclear factor-kappaB ligand (RANKL)-induced bone-marrow-derived macrophages (BMMs). Compound 1 markedly inhibited RANKL-induced osteoclast differentiation in the absence of cytotoxicity, by regulating the expression of osteoprotegerin (OPG) and RANKL in BMMs and in cocultured osteoblasts. Compound 1 significantly inhibited RANKL-induced tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, monocyte chemoattractant protein-1 (MCP-1), regulated on activation normal T cell expressed and secreted (RANTES), and macrophage inflammatory protein-1α (MIP-1α) in a concentration-dependent manner. These effects were accompanied by a decrease in RANKL-induced activation of the NF-κB p65 subunit, degradation of inhibitor κBα (IκBα), induction of NF-κB, and phosphorylation of AKT, extracellular-signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK). NF-κB siRNA suppressed AKT, ERK, JNK, and p38 MAPK phosphorylation. Furthermore, 1 significantly suppressed c-Fos and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), key transcription factors during osteoclastogenesis. SP600125, a specific inhibitor of JNK, reduced RANKL-induced expression of phospho-c-Jun, c-Fos, and NFATc1 and inhibited osteoclast formation. These results suggested that 1 acted as an antiresorption agent by blocking osteoclast activation.
Assuntos
Isoflavonas/farmacologia , NF-kappa B/antagonistas & inibidores , Fitoestrógenos/farmacologia , Quimiocina CCL2 , Interleucina-6/metabolismo , Isoflavonas/química , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Macrófagos/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Estrutura Molecular , Osteoblastos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Fitoestrógenos/química , Proteínas Proto-Oncogênicas c-fos/efeitos dos fármacos , Ligante RANK/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Arginine, an α-amino acid, has been reported to exert beneficial effects that ameliorate health problems and prevent excessive fat deposition. In this study, we investigated whether the activation of cell signaling by arginine can induce osteogenic differentiation and modulate excessive adipogenic differentiation in human mesenchymal stem cells (MSCs). Arginine potently induced the expression of type Iα1 collagen, osteocalcin, and ALP in a dose-dependent manner without causing cytotoxicity. Arginine significantly increased the mRNA expression of the osteogenic transcription factors runt-related transcription factor 2 (Runx2), DIx5, and osterix. Furthermore, arginine demonstrated its antiadipogenicity by decreasing adipocyte formation and triglyceride (TG) content in MSCs and inhibiting the mRNA expression of the adipogenic transcription factors peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), and fatty acid binding protein 4 (Fabp4). This effect was associated with increased expression of Wnt5a, and nuclear factor of activated T-cells (NFATc), and was abrogated by antagonists of Wnt and NFATc, which indicated a role of Wnt and NFATc signaling in the switch from adipogenesis to osteoblastogenesis induced by arginine. In conclusion, this is the first report of the dual action of arginine in promoting osteogenesis and inhibiting adipocyte formation through involving Wnt5a and NFATc signaling pathway.
Assuntos
Adipogenia/efeitos dos fármacos , Arginina/farmacologia , Células-Tronco Mesenquimais/citologia , Fatores de Transcrição NFATC/metabolismo , Osteogênese/efeitos dos fármacos , Proteínas Wnt/metabolismo , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Diferenciação Celular/efeitos dos fármacos , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , RNA Mensageiro/metabolismo , Transdução de SinaisRESUMO
The effective removal of (137) Cs(+) â ions from contaminated groundwater and seawater and from radioactive nuclear waste solutions is crucial for public health and for the continuous operation of nuclear power plants. Various (137) Cs(+) â removers have been developed, but more effective (137) Cs(+) â removers are still needed. A novel microporous vanadosilicate with mixed-valence vanadium (V(4+) and V(5+) ) ions is now reported, which shows an excellent ability for Cs(+) â capture and immobilization from groundwater, seawater, and nuclear waste solutions. This material is superior to other known materials in terms of selectivity, capacity, and kinetics, and at very low Cs(+) â concentrations, it was found to be the most effective material for the removal of radioactive Cs(+) â ions under the test conditions. This novel vanadosilicate also contains hexadeca-coordinated Cs(+) â ions, which corresponds to the highest coordination number ever described.
RESUMO
Microvesicles (MVs, also known as exosomes, ectosomes, microparticles) are released by various cancer cells, including lung, colorectal, and prostate carcinoma cells. MVs released from tumor cells and other sources accumulate in the circulation and in pleural effusion. Although recent studies have shown that MVs play multiple roles in tumor progression, the potential pathological roles of MV in pleural effusion, and their protein composition, are still unknown. In this study, we report the first global proteomic analysis of highly purified MVs derived from human nonsmall cell lung cancer (NSCLC) pleural effusion. Using nano-LC-MS/MS following 1D SDS-PAGE separation, we identified a total of 912 MV proteins with high confidence. Three independent experiments on three patients showed that MV proteins from PE were distinct from MV obtained from other malignancies. Bioinformatics analyses of the MS data identified pathologically relevant proteins and potential diagnostic makers for NSCLC, including lung-enriched surface antigens and proteins related to epidermal growth factor receptor signaling. These findings provide new insight into the diverse functions of MVs in cancer progression and will aid in the development of novel diagnostic tools for NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/química , Exossomos/química , Neoplasias Pulmonares/química , Proteínas de Neoplasias/análise , Derrame Pleural/metabolismo , Adulto , Idoso , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Cromatografia Líquida , Bases de Dados de Proteínas , Receptores ErbB/metabolismo , Exossomos/metabolismo , Feminino , Humanos , Pulmão/química , Neoplasias Pulmonares/metabolismo , Masculino , Microscopia Eletrônica , Proteínas de Neoplasias/química , Proteínas de Neoplasias/classificação , Proteoma/análise , Proteoma/química , Proteômica , Reprodutibilidade dos Testes , Transdução de Sinais , Espectrometria de Massas em TandemRESUMO
RATIONALE: Protein post-translational modifications (PTMs) are directly involved in protein function and cellular activities. Among them, glycosylation and phosphorylation are particularly important modifications on proteins located at extracellular and intracellular domains, respectively. However, the combined detection using phospho- and glycoproteomics is limited mainly due to protocol differences. METHODS: In this study, we developed a novel method for both phospho- and glycoproteome detection from a single sample batch, in which a titanium dioxide cartridge was used to capture the phosphoproteome, and the flow-through solution was processed for capturing N-linked glycopeptides using hydrazide resin. RESULTS: By using 1 mg of protein from kidney tissue lysates from normal and diseased rats, we concurrently identified 437 glycosites/358 phosphosites and 468 glycosites/369 phosphosites in normal and disease kidneys, respectively, by liquid chromatography/tandem mass spectrometric analysis. CONCLUSIONS: Compared with individual PTM analyses, the combined PTM analysis clearly provides more broad implications for PTMs related to the pathological status and discovery of biomarker candidates. Furthermore, the combined protocol thoroughly showed its advantages in enrichment efficiency and biological interpretation compared with current methods.
Assuntos
Glicopeptídeos/análise , Nefrocalcinose/induzido quimicamente , Fosfopeptídeos/análise , Ácido Fítico/toxicidade , Proteoma/efeitos dos fármacos , Proteômica/métodos , Sequência de Aminoácidos , Animais , Cromatografia Líquida , Feminino , Rim/química , Rim/efeitos dos fármacos , Dados de Sequência Molecular , Nefrocalcinose/metabolismo , Ácido Fítico/administração & dosagem , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas em TandemRESUMO
Mesenchymal stem cells (MSCs) have emerged as a promising means for treating degenerative or incurable diseases. Recent studies have shown that microvesicles (MVs) from MSCs (MSC-MVs) contribute to recovery of damaged tissues in animal disease models. Here, we profiled the MSC-MV proteome to investigate their therapeutic effects. LC-MS/MS analysis of MSC-MVs identified 730 MV proteins. The MSC-MV proteome included five positive and two variable known markers of MSCs, but no negative marker, as well as 43 surface receptors and signaling molecules controlling self-renewal and differentiation of MSCs. Functional enrichment analysis showed that cellular processes represented by the MSC-MV proteins include cell proliferation, adhesion, migration, and morphogenesis. Integration of MSC's self-renewal and differentiation-related genes and the proteome of MSC-conditioned media (MSC-CM) with the MSC-MV proteome revealed potential MV protein candidates that can be associated with the therapeutic effects of MSC-MVs: (1) surface receptors (PDGFRB, EGFR, and PLAUR); (2) signaling molecules (RRAS/NRAS, MAPK1, GNA13/GNG12, CDC42, and VAV2); (3) cell adhesion (FN1, EZR, IQGAP1, CD47, integrins, and LGALS1/LGALS3); and (4) MSC-associated antigens (CD9, CD63, CD81, CD109, CD151, CD248, and CD276). Therefore, the MSC-MV proteome provides a comprehensive basis for understanding the potential of MSC-MVs to affect tissue repair and regeneration.
Assuntos
Vesículas Citoplasmáticas/química , Células-Tronco Mesenquimais/citologia , Proteínas/análise , Proteômica/métodos , Diferenciação Celular/fisiologia , Processos de Crescimento Celular/fisiologia , Células Cultivadas , Vesículas Citoplasmáticas/metabolismo , Descoberta de Drogas , Humanos , Células-Tronco Mesenquimais/química , Células-Tronco Mesenquimais/metabolismo , Proteínas/química , Proteínas/classificação , Proteoma/análise , Transdução de SinaisRESUMO
To evaluate melatonin's ability to enhance ovarian cancer cells to cisplatin treatment for ovarian cancer, this study was performed. Melatonin by itself had no significant cytotoxicity against SK-OV-3 cells, while cisplatin suppressed the cell viability in a dose-dependent manner. Combined treatment with cisplatin and melatonin synergistically inhibited the viability of SK-OV-3 cells with the synergism between two drugs (1 > combination index). In contrast, melatonin revealed the protective effect against cisplatin-induced cytotoxicity in OSEN normal ovarian epithelial cells. Cotreatment with cisplatin and melatonin increased the sub-G1 DNA contents and TdT-mediated dUTP nick end-labeling (TUNEL)-positive cells compared with cisplatin control in SK-OV-3 cells, suggesting that melatonin augments cisplatin-induced apoptosis. Consistently, combined treatment of cisplatin and melatonin increased the cleavage of caspase-3 and poly-(ADP-ribose) polymerase (PARP). Importantly, melatonin synergistically inhibited the phosphorylation of extracellular signal-regulated kinase (ERK) along with dephosphorylation of 90-kDa ribosomal S6 kinase (p90RSK) and heat shock protein 27 (HSP27) induced by cisplatin. Furthermore, melatonin remarkably blocked the expression and colocalization of p90RSK and HSP27 by combination treatment with cisplatin. Taken together, our findings demonstrate that melatonin enhances cisplatin-induced apoptosis via the inactivation of ERK/p90RSK/HSP27 cascade in SK-OV-3 cells as a potent synergist to cisplatin treatment.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Cisplatino/farmacologia , Proteínas de Choque Térmico HSP27/metabolismo , Melatonina/farmacologia , Neoplasias Ovarianas/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , MAP Quinases Reguladas por Sinal Extracelular , Feminino , Imunofluorescência , Humanos , Marcação In Situ das Extremidades Cortadas , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Melittin (1) is a major polypeptide in honey bee venom that has been used traditionally against chronic inflammation and cancer. However, its molecular mechanism has not been determined. In this study, the antitumor effect of 1 was compared with that of NS398, a cyclooxygenase-2 (COX-2) inhibitor, in vivo and in vitro. Subcutaneous injection of 1 at 0.5 and 5 mg/kg suppressed significantly vascular endothelial growth factor (VEGF)-A-transfected highly metastatic Lewis lung cancer (VEGF-A-hm LLC) tumor growth by 25% and 57%, respectively. Also, 1 inhibited significantly the number of vessels around VEGF-A-hm LLC cells. The results were superior to those obtained in the mice treated with NS398. Compound 1 dose-dependently inhibited proliferation and tube formation in human umbilical vein endothelial cells (VEGF-A-HUVECs), without affecting cell viability in native HUVECs. In addition, 1 decreased the expression of VEGF receptor-2 (VEGFR-2), COX-2, and prostaglandin E2 (PGE2) in VEGF-A-transfected HUVECs. These effects were accompanied by a reduction of the phosphorylation of extracellular signal-regulated kinase 1/2 and c-jun N-terminal kinase, whereas it increased the phosphorylation of p38 mitogen-activated protein kinase (MAPK). SB203580 abolished the downregulation of COX-2 and VEGFR-2 and the inhibition of cell proliferation by 1. The antitumor activity of 1 may be associated with antiangiogenic actions via inhibiting VEGFR-2 and inflammatory mediators involved in the MAPK signaling pathway.
Assuntos
Neoplasias do Colo/tratamento farmacológico , Inibidores de Ciclo-Oxigenase 2/farmacologia , Meliteno/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Dinoprostona/antagonistas & inibidores , Relação Dose-Resposta a Droga , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Camundongos , Nitrobenzenos/farmacologia , Sulfonamidas/farmacologiaRESUMO
BACKGROUND: WIN-34B is a novel Oriental medicine, which represents the n-butanol fraction prepared from dried flowers of Lonicera japonica Thunb and dried roots of Anemarrhena asphodeloides BUNGE. The component herb of WIN-34B is used for arthritis treatment in East Asian countries. The aim of this study was to determine the cartilage-protective effects and mechanisms of WIN-34B and its major phenolic compounds, chlorogenic acid and mangiferin, in osteoarthritis (OA) human cartilage explants culture and chondrocytes. METHODS: The investigation focused on whether WIN-34B and its standard compounds protected cartilage in interleukin (IL)-1ß-stimulated cartilage explants culture and chondrocytes derived from OA patients. Also, the mechanisms of WIN-34B on matrix metalloproteinases (MMPs), tissue inhibitor of matrix metalloproteinases (TIMPs), inflammatory mediators, and mitogen-activated protein kinases (MAPKs) pathways were assessed. RESULTS: WIN-34B was not cytotoxic to cultured cartilage explants or chondrocytes. WIN-34B dose-dependently inhibited the release of glycosaminoglycan and type II collagen, increased the mRNA expression of aggrecan and type II collagen, and recovered the intensity of proteoglycan and collagen by histological analysis in IL-1ß-stimulated human cartilage explants culture. The cartilage protective effect of WIN-34B was similar to or better than that of chlorogenic acid and mangiferin. Compared to chlorogenic acid and mangiferin, WIN-34B displayed equal or greater decreases in the levels of MMP-1, MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5, and markedly up-regulated TIMP-1 and TIMP-3. WIN-34B inhibited inflammatory mediators involved in cartilage destruction, such as prostaglandin E2, nitric oxide, tumor necrosis factor-alpha, and IL-1ß. The phosphorylation of extracellular signal-regulated kinase, c-Jun N-terminal kinase (JNK), and p38 was significantly reduced by WIN-34B treatment, while phosphorylation of JNK was only inhibited by chlorogenic acid or mangiferin in IL-1ß-stimulated chondrocytes. CONCLUSIONS: WIN-34B is potentially valuable as a treatment for OA by virtue of its suppression of MMPs, ADAMTSs, and inflammatory mediators, and it's up-regulation of TIMP-1 and TIMP-3 involved in the MAPK pathway.
Assuntos
Anemarrhena/química , Cartilagem Articular/efeitos dos fármacos , Condrócitos/metabolismo , Mediadores da Inflamação/metabolismo , Lonicera/química , Metaloproteinases da Matriz/metabolismo , Osteoartrite/enzimologia , Extratos Vegetais/farmacologia , Cartilagem Articular/enzimologia , Cartilagem Articular/metabolismo , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Condrócitos/enzimologia , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Regulação para Baixo/efeitos dos fármacos , Feminino , Flores/química , Humanos , Técnicas In Vitro , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Masculino , Inibidores de Metaloproteinases de Matriz/metabolismo , Metaloproteinases da Matriz/genética , Pessoa de Meia-Idade , Osteoartrite/tratamento farmacológico , Osteoartrite/genética , Osteoartrite/metabolismo , Extratos Vegetais/isolamento & purificação , Raízes de Plantas/química , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
The presence of malignant ascites in the peritoneal cavity is a poor prognostic indicator of low survival rate. Various cancer cells, including those of colorectal cancer (CRC), release microvesicles (exosomes) into surrounding tissues and peripheral circulation including malignant ascites. Although recent progress has revealed that microvesicles play multiple roles in tumor progression, the protein composition and the pathological function of malignant ascites-derived microvesicles are still unknown. Here, we report the first global proteomic analyses of highly purified microvesicles derived from human CRC ascites. With 1-D SDS-PAGE and nano-LC-MS/MS analyses, we identified a total of 846 microvesicular proteins from ascites of three CRC patients with high confidence; 384 proteins were identified in at least two patients. We identified proteins that might function in tumor progression via disruption of epithelial polarity, migration, invasion, tumor growth, immune modulation, and angiogenesis. Furthermore, we identified several potential diagnostic markers of CRC including colon-specific surface antigens. Our proteomic analyses will help to elucidate diverse functions of microvesicles in cancer progression and will aid in the development of novel diagnostic tools for CRC.
Assuntos
Ascite/patologia , Neoplasias Colorretais/patologia , Exossomos/química , Proteínas de Neoplasias/análise , Proteoma/análise , Eletroforese em Gel de Poliacrilamida/métodos , Humanos , Espectrometria de Massas em Tandem/métodosRESUMO
Recently, the rapid emergence of microbial pathogens which are resistant to currently available antibiotics has triggered considerable interest searching for naturally occuring antimicrobial peptides (AMPs). Because AMPs from food organisms are comparatively nontoxic, a number of them are used as sources, purified in new antibiotics. Herein, an antibacterial peptide (heat-stable KPS-1) was isolated from Korean pen shell (Atrina pectinata) by the following procedures: solvent-extraction, heating, ultrafiltration, and RP-HPLC. The molecular weight of KPS-1 (4549.1 Da) was revealed by MALDI-TOF/MS analysis. Interestingly, KPS-1 inhibited in vitro growth of Gram-negative bacteria, including Escherichia coli, E. coli O157, Pseudomonas aeruginosa, Enterobacter sakazakii, and Salmonella typhimurium, at pH 5.2, rather than at pH 7.2. Its minimal inhibitory concentrations (MICs) were ranged from 20 to 80 µg/ml; however, it was not effective against human red blood cells at a concentration of 500 µg/ml. This suggests that this peptide is useful as a clinical agent for some human organs in an acidic environment.