RESUMO
Neuron-glial antigen 2 (NG2, also known as CSPG4) and hyaluronic acid receptor CD44 are chondroitin sulphate proteoglycans actively involved in brain development and its malignant transformation. Here, we aimed to compare prognostic significances of NG2, CD44 and Ki-67 expression in glioblastoma multiforme patients. Totally, 45 tissue samples and 83 paraffin-embedded tissues for 75 patients were analysed. The prognostic values of the genes were analysed using Kaplan-Meier survival curves. Grade III gliomas showed 2-fold difference in NG2 expression between anaplastic astrocytoma and oligoastrocytoma (10.1 ± 3.5 and 25.5 ± 14.5, respectively). For grade IV gliomas, upregulated NG2 expression (21.0 ± 6.8) was associated with poor glioblastoma multiforme prognosis (overall survival < 12 months) compared with glioblastoma multiforme patients with good prognosis (4.4 ± 3.2; overall survival > 12 months). Multivariate survival analysis using Cox proportional hazards model confirmed that high NG2 expression was associated with low survival of the patients (hazard ratio: 3.43; 95% confidence interval: 1.18-9.93; p = 0.02), whereas age (hazard ratio: 1.02; 95% confidence interval: 0.96-1.09; p = 0.42), tumour resection (hazard ratio: 1.03; 95% confidence interval: 0.98-1.08; p = 0.25) and sex (hazard ratio: 0.62; 95% confidence interval: 0.21-1.86; p = 0.40) did not show significant association with prognosis. Although the positive correlation was shown for NG2 and CD44 expression in the glioblastomas (Pearson coefficient = 0.954), Kaplan-Meier and multivariate survival analyses did not revealed a significant association of the increased CD44 expression (hazard ratio: 2.18; 95% confidence interval: 0.50-9.43; p = 0.30) or high Ki-67 proliferation index (hazard ratio: 1.10; 95% confidence interval: 1.02-1.20; p = 0.02) with the disease prognosis. The results suggest that upregulation of NG2/CSPG4 rather than changes in CD44 or Ki-67 expression is associated with low overall survival in glioblastoma multiforme patients, supporting NG2/CSPG4 as a potential prognostic marker in glioblastoma.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias Encefálicas/patologia , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Glioblastoma/patologia , Receptores de Hialuronatos/biossíntese , Antígeno Ki-67/biossíntese , Proteínas de Membrana/biossíntese , Adolescente , Adulto , Idoso , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidade , Proteoglicanas de Sulfatos de Condroitina/análise , Feminino , Glioblastoma/metabolismo , Glioblastoma/mortalidade , Humanos , Receptores de Hialuronatos/análise , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Antígeno Ki-67/análise , Masculino , Proteínas de Membrana/análise , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Prognóstico , Modelos de Riscos Proporcionais , Regulação para Cima , Adulto JovemRESUMO
Background: C4ST-1 catalyzes the transfer of sulfate groups in the sulfonation of chondroitin during chondroitin sulfate synthesis. Chondroitin sulfate consists of numerous copies of negatively charged sulfonic acid groups that participate in the nucleation process of biomineralization. In the present study, we obtained two CHST11 genes (PmCHST11a and PmCHST11b) which encoded the C4ST-1 and explored the functions of these genes in the synthesis of chondroitin sulfate and in the formation of the nacreous layer of shells. Results: Both PmCHST11a and PmCHST11b had a sulfotransferase-2 domain, a signal peptide and a transmembrane domain. These properties indicated that these genes localize in the Golgi apparatus. Real-time PCR revealed that both PmCHST11a and PmCHST11b were highly expressed in the central zone of the mantle tissue. Inhibiting PmCHST11a and PmCHST11b via RNA interference significantly decreased the expression levels of these genes in the central zone of the mantle tissue and the concentration of chondroitin sulfate in extrapallial fluid. Moreover, shell nacre crystallized irregularly with a rough surface after RNA interference. Conclusions: This study indicated that PmCHST11a and PmCHST11b are involved in the nacre formation of Pinctada fucata martensii through participating in the synthesis of chondroitin sulfate.
Assuntos
Sulfotransferases/metabolismo , Pinctada , Nácar/biossíntese , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Sulfotransferases/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo Real , BiomineralizaçãoRESUMO
Cornelia de Lange syndrome (CdLS) is a genetically heterogeneous disorder that presents with extensive phenotypic variability, including facial dysmorphism, developmental delay/intellectual disability (DD/ID), abnormal extremities, and hirsutism. About 65% of patients harbor mutations in genes that encode subunits or regulators of the cohesin complex, including NIPBL, SMC1A, SMC3, RAD21, and HDAC8. Wiedemann-Steiner syndrome (WDSTS), which shares CdLS phenotypic features, is caused by mutations in lysine-specific methyltransferase 2A (KMT2A). Here, we performed whole-exome sequencing (WES) of 2 male siblings clinically diagnosed with WDSTS; this revealed a hemizygous, missense mutation in SMC1A that was predicted to be deleterious. Extensive clinical evaluation and WES of 32 Turkish patients clinically diagnosed with CdLS revealed the presence of a de novo heterozygous nonsense KMT2A mutation in 1 patient without characteristic WDSTS features. We also identified de novo heterozygous mutations in SMC3 or SMC1A that affected RNA splicing in 2 independent patients with combined CdLS and WDSTS features. Furthermore, in families from 2 separate world populations segregating an autosomal-recessive disorder with CdLS-like features, we identified homozygous mutations in TAF6, which encodes a core transcriptional regulatory pathway component. Together, our data, along with recent transcriptome studies, suggest that CdLS and related phenotypes may be "transcriptomopathies" rather than cohesinopathies.
Assuntos
Códon sem Sentido , Síndrome de Cornélia de Lange , Exoma , Regulação da Expressão Gênica , Fenótipo , Transcriptoma , Adolescente , Adulto , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Criança , Pré-Escolar , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Proteoglicanas de Sulfatos de Condroitina/genética , Proteínas Cromossômicas não Histona/biossíntese , Proteínas Cromossômicas não Histona/genética , Síndrome de Cornélia de Lange/genética , Síndrome de Cornélia de Lange/metabolismo , Síndrome de Cornélia de Lange/patologia , Exonucleases , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Heterozigoto , Histona Desacetilases/biossíntese , Histona Desacetilases/genética , Histona-Lisina N-Metiltransferase , Humanos , Lactente , Masculino , Proteína de Leucina Linfoide-Mieloide/biossíntese , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas/genética , Proteínas/metabolismo , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genéticaRESUMO
Giant cell tumor of bone (GCTB) displays worrisome clinical features such as local recurrence and occasionally metastatic disease which are unpredictable by morphology. Additional routinely usable biomarkers do not exist. Gene expression profiles of six clinically defined groups of GCTB and one group of aneurysmal bone cyst (ABC) were determined by microarray (n = 33). The most promising differentially expressed genes were validated by Q-PCR as potential biomarkers in a larger patient group (n = 41). Corresponding protein expression was confirmed by immunohistochemistry. Unsupervised hierarchical clustering reveals a metastatic GCTB cluster, a heterogeneous, non-metastatic GCTB cluster, and a primary ABC cluster. Balanced score testing indicates that lumican (LUM) and decorin (DCN) are the most promising biomarkers as they have lower level of expression in the metastatic group. Expression of dermatopontin (DPT) was significantly lower in recurrent tumors. Validation of the results was performed by paired and unpaired t test in primary GCTB and corresponding metastases, which proved that the differential expression of LUM and DCN is tumor specific rather than location specific. Our findings show that several genes related to extracellular matrix integrity (LUM, DCN, and DPT) are differentially expressed and may serve as biomarkers for metastatic and recurrent GCTB.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias Ósseas/genética , Decorina/biossíntese , Tumor de Células Gigantes do Osso/genética , Neoplasias Pulmonares/secundário , Adolescente , Adulto , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Criança , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Proteoglicanas de Sulfatos de Condroitina/genética , Análise por Conglomerados , Decorina/genética , Regulação para Baixo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Proteínas da Matriz Extracelular/biossíntese , Proteínas da Matriz Extracelular/genética , Feminino , Perfilação da Expressão Gênica , Tumor de Células Gigantes do Osso/metabolismo , Tumor de Células Gigantes do Osso/patologia , Humanos , Imuno-Histoquímica , Sulfato de Queratano/biossíntese , Sulfato de Queratano/genética , Lumicana , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo Real , Transcriptoma , Adulto JovemRESUMO
Old astrocyte specifically induced substance (OASIS), a basic leucine zipper transcription factor of the cAMP response element binding/Activating transcription factor family, is induced in reactive astrocytes in vivo and has important roles in quality control of protein synthesis at the endoplasmic reticulum. Reactive astrocytes produce a non-permissive environment for regenerating axons by up-regulating chondroitin sulfate proteoglycans (CSPGs). In this study, we focus on the potential role of OASIS in CSPG production in the adult mouse cerebral cortex. CS-C immunoreactivity, which represents chondroitin sulfate moieties, was significantly attenuated in the stab-injured cortices of OASIS knockout mice compared to those of wild-type mice. We next examined expression of the CSPG-synthesizing enzymes and core proteins of CSPGs in the stab-injured cortices of OASIS knockout and wild-type mice. The levels of chondroitin 6-O-sulfotransferase 1 (C6ST1, one of the major enzymes involved in sulfation of CSPGs) mRNA and protein increased after cortical stab injury of wild-type, but not of OASIS knockout, mice. A C-terminal deletion mutant OASIS over-expressed in rat C6 glioma cells increased C6ST1 transcription by interacting with the first intron region. Neurite outgrowth of cultured hippocampal neurons was inhibited on culture dishes coated with membrane fractions of epidermal growth factor-treated astrocytes derived from wild type but not from OASIS knockout mice. These results suggest that OASIS regulates the transcription of C6ST1 and thereby promotes CSPG sulfation in astrocytes. Through these mechanisms, OASIS may modulate axonal regeneration in the injured cerebral cortex. OASIS, an ER stress-responsive CREB/ATF family member, is up-regulated in the reactive astrocytes of the injured brain. We found that the up-regulated OASIS is involved in the transcriptional regulation of C6ST1 gene, which promotes chondroitin sulfate proteoglycan (CSPG) sulfation. We conclude that OASIS functions as an anti-regenerative transcription factor by establishing a non-permissive microenvironment to regenerating axons.
Assuntos
Lesões Encefálicas/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Sulfotransferases/biossíntese , Animais , Astrócitos/metabolismo , Western Blotting , Lesões Encefálicas/genética , Córtex Cerebral/metabolismo , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Proteoglicanas de Sulfatos de Condroitina/genética , Modelos Animais de Doenças , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulfotransferases/genética , Transcrição Gênica , Carboidrato SulfotransferasesRESUMO
Chondroitin sulfate proteoglycan 4 (CSPG4), a transmembrane proteoglycan originally identified in melanoma cells, has been reported to be expressed in breast cancer cells. This study was performed to examine the expression and significance of CSPG4 in a cohort of breast cancer patients. Immunohistochemical analysis of CSPG4 was performed on tissue microarrays constructed from tissue specimens from 240 breast cancer patients. CSPG4 staining was correlated with clinical and pathological characteristics, overall survival (OS), and disease recurrence. Contradicting to a previous report, our results showed that high CSPG4 expression was not related to triple-negative status of breast cancer patients. The Kaplan-Meier method showed that high CSPG4 expression was significantly associated with shorter time to recurrence (TTR). Patients with high CSPG4 expression had poorer OS and shorter TTR in a multivariate survival analysis after adjustment for stage, tumor grade, expression of estrogen receptor and progesterone receptor, and HER2 overexpression. This study showed that high CSPG4 expression correlates with disease recurrence and OS in breast cancers.
Assuntos
Neoplasias da Mama/mortalidade , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Proteínas de Membrana/biossíntese , Recidiva Local de Neoplasia/mortalidade , Adulto , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Feminino , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/metabolismo , Prognóstico , Fatores de Tempo , Análise Serial de Tecidos/estatística & dados numéricosRESUMO
Lumican, a member of the class II small leucine-rich proteoglycan family, regulates the assembly and diameter of collagen fibers in the extracellular matrix of various tissues. We previously reported that lumican expression in the stromal tissues of pancreatic ductal adenocarcinoma (PDAC) correlates with tumor invasion, and tends to correlate with poor prognosis. Lumican stimulates growth and inhibits the invasion of a PDAC cell line. In the present study, we performed a global shotgun proteomic analysis using lumican-overexpressing PANC1 cells and lumican downregulated PANC1 cells to identify candidate proteins that are regulated by lumican and related to cell growth and invasion in PDAC cells. A total of 448 proteins were identified from lumican-overexpressing PANC1 and control cells. Additionally, 451 proteins were identified from lumican-downregulated PANC1 cells and control cells. As a result of semi-quantification based on spectral counting, 174 differentially expressed proteins were identified by lumican upregulation, and 143 differentially expressed proteins were identified by lumican downregulation. The expression levels of 24 proteins, including apoptosis- and invasion-related proteins correlated with lumican expression levels. It is likely that the expression of these proteins is regulated by lumican, and that they are involved in apoptosis and invasion in PDAC. These findings suggest that lumican may be involved in cell growth and invasion through the regulation of these 24 proteins expressed in PDAC.
Assuntos
Apoptose/genética , Carcinoma Ductal Pancreático/patologia , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Sulfato de Queratano/metabolismo , Invasividade Neoplásica/genética , Neoplasias Pancreáticas/patologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Regulação para Baixo , Expressão Gênica , Humanos , Sulfato de Queratano/biossíntese , Lumicana , Metalotioneína/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Prognóstico , Isoformas de Proteínas/metabolismo , Proteômica , Regulação para CimaRESUMO
The stromal reaction surrounding tumors leads to the formation of a tumor-specific microenvironment, which may play either a restrictive role or a supportive role in the growth and progression of the tumors. Lumican, a small leucine-rich proteoglycan (SLRP) of the extracellular matrix (ECM), regulates collagen fibrillogenesis. Recently, lumican has also been shown to regulate cell behavior during embryonic development, tissue repair and tumor progression. The role of lumican in cancer varies according to the type of tumor. In this study we analyze the role of lumican in the pathogenesis of prostate cancer both in vivo and in vitro. Overall lumican up-regulation was observed in the primary tumors analyzed through both real-time PCR and immunostaining. The increase in lumican expression was observed in the reactive stroma surrounding prostate primary tumors with fibrotic deposition surrounding the acinar glands. In vitro analysis demonstrated that lumican inhibited both the migration and invasion of metastatic prostate cancer cells isolated from lymph node, bone and brain. Moreover, prostate cancer cells seeded on lumican presented a decrease in the formation of cellular projections, lamellipodia detected by a decreased rearrangement in ZO-1, keratin 8/18, integrin ß1 and MT1-MMP, and invadopodia detected by disruption of α-smooth muscle actin, cortactin and N-WASP. Moreover, a significant increase in prostate cancer cell invasion was observed through the peritoneum of lumican knockout mice, further demonstrating the restrictive role lumican present in the ECM has on prostate cancer invasion. In conclusion, lumican present in the reactive stroma surrounding prostate primary tumors plays a restrictive role on cancer progression, and we therefore postulate that lumican could be a valuable marker in prostate cancer staging.
Assuntos
Proteoglicanas de Sulfatos de Condroitina/biossíntese , Sulfato de Queratano/biossíntese , Neoplasias da Próstata/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular , Proteoglicanas de Sulfatos de Condroitina/deficiência , Humanos , Integrina beta1/metabolismo , Sulfato de Queratano/deficiência , Lumicana , Masculino , Camundongos , Camundongos Knockout , Neoplasias da Próstata/patologia , Regulação para CimaRESUMO
NG2/CSPG4 is a complex surface-associated proteoglycan (PG) recognized to be a widely expressed membrane component of glioblastoma (WHO grade IV) cells and angiogenic pericytes. To determine the precise expression pattern of NG2/CSPG4 on glioblastoma cells and pericytes, we generated a panel of >60 mouse monoclonal antibodies (mAbs) directed against the ectodomain of human NG2/CSPG4, partially characterized the mAbs, and performed a high-resolution distributional mapping of the PG in human foetal, adult and glioblastoma-affected brains. The reactivity pattern initially observed on reference tumour cell lines indicated that the mAbs recognized 48 immunologically distinct NG2/CSPG4 isoforms, and a total of 14 mAbs was found to identify NG2/CSPG4 isoforms in foetal and neoplastic cerebral sections. These were consistently absent in the adult brain, but exhibited a complementary expression pattern in angiogenic vessels of both tumour and foetal tissues. Considering the extreme pleomorphism of tumour areas, and with the aim of subsequently analysing the distributional pattern of the NG2/CSPG4 isoforms on similar histological vessel typologies, a preliminary study was carried out with endothelial cell and pericyte markers, and with selected vascular basement membrane (VBM) components. On both tumour areas characterized by 'glomeruloid' and 'garland vessels', which showed a remarkably similar cellular and molecular organization, and on developing brain vessels, spatially separated, phenotypically diversified pericyte subsets with a polarized expression of key surface components, including NG2/CSPG4, were disclosed. Interestingly, the majority of the immunolocalized NG2/CSPG4 isoforms present in glioblastoma tissue were present in foetal brain, except for one isoform that seemed to be exclusive of tumour cells, being absent in foetal brain. The results highlight an unprecedented, complex pattern of NG2/CSPG4 isoform expression in foetal and neoplastic CNS, discriminating between phenotype-specific and neoplastic versus non-neoplastic variants of the PG, thus opening up vistas for more selective immunotherapeutic targeting of brain tumours.
Assuntos
Neoplasias Encefálicas/metabolismo , Encéfalo/metabolismo , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Feto/metabolismo , Glioblastoma/metabolismo , Proteínas de Membrana/biossíntese , Proteínas de Neoplasias/biossíntese , Pericitos/metabolismo , Adulto , Animais , Anticorpos Monoclonais Murinos/química , Anticorpos Antineoplásicos/química , Encéfalo/patologia , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Feminino , Feto/patologia , Regulação Neoplásica da Expressão Gênica , Glioblastoma/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pericitos/patologia , Isoformas de Proteínas/biossínteseRESUMO
BACKGROUND: Molecular events responsible for the onset and progression of peripheral occlusive arterial disease (POAD) are incompletely understood. Gene expression profiling may point out relevant features of the disease. METHODS: Tissue samples were collected as operatory waste from a total of 36 patients with (n = 18) and without (n = 18) POAD. The tissues were histologically evaluated, and the patients with POAD were classified according to Leriche-Fontaine (LF) classification: 11% with stage IIB, 22% with stage III, and 67% with stage IV. Total RNA was isolated from all samples and hybridized onto Agilent 4×44K Oligo microarray slides. The bioinformatic analysis identified genes differentially expressed between control and pathologic tissues. Ten genes with a fold change ≥ 2 (1 with a fold change ≥ 1.8) were selected for quantitative polymerase chain reaction validation (GPC3, CFD, GDF10, ITLN1, TSPAN8, MMP28, NNMT, SERPINA5, LUM, and FDXR). C-reactive protein (CRP) was assessed with a specific assay, while nicotinamide N-methyltransferase (NNMT) was evaluated in the patient serum by enzyme-linked immunosorbent assay. RESULTS: A multiple regression analysis showed that the level of CRP in the serum is correlated with the POAD LF stages (r(2) = 0.22, P = 0.046) and that serum NNMT is higher in IV LF POAD patients (P = 0.005). The mRNA gene expression of LUM is correlated with the LF stage (r(2) = 0.45, P = 0.009), and the mRNA level of ITLN1 is correlated with the ankle-brachial index (r(2) = 0.42, P = 0.008). CONCLUSIONS: Our analysis shows that NNMT, ITLN1, LUM, CFD, and TSPAN8 in combination with other known markers, such as CRP, could be evaluated as a panel of biomarkers of POAD.
Assuntos
Arteriopatias Oclusivas/genética , Proteoglicanas de Sulfatos de Condroitina/genética , Citocinas/genética , Regulação da Expressão Gênica , Sulfato de Queratano/genética , Lectinas/genética , RNA Mensageiro/genética , Índice Tornozelo-Braço , Arteriopatias Oclusivas/diagnóstico , Arteriopatias Oclusivas/metabolismo , Proteína C-Reativa/metabolismo , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Citocinas/biossíntese , Ensaio de Imunoadsorção Enzimática , Feminino , Artéria Femoral/metabolismo , Artéria Femoral/patologia , Seguimentos , Proteínas Ligadas por GPI/biossíntese , Proteínas Ligadas por GPI/genética , Humanos , Sulfato de Queratano/biossíntese , Lectinas/biossíntese , Lumicana , Masculino , Pessoa de Meia-Idade , Nicotinamida N-Metiltransferase/sangue , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Lumican expression in the stromal tissues of pancreatic ductal adenocarcinoma (PDAC) correlates with tumor invasion, and tends to correlate with poor prognosis. We used gene transfection techniques to examine the biological roles of lumican secreted from PDAC cells. Lumican-transfected PANC-1 cells secreted a 70-kDa lumican protein and had an active ERK pathway. Transfection stimulated PANC-1 cell growth, increased cell adhesion to laminin, inhibited cell invasion, and decreased active matrix metalloproteinase-9. Down-regulation of lumican using siRNA resulted in opposite cell behavior. Thus, the 70-kDa lumican secreted by PDAC cells plays important roles in cell growth and invasion.
Assuntos
Carcinoma Ductal Pancreático/metabolismo , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Sulfato de Queratano/metabolismo , Neoplasias Pancreáticas/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Proteoglicanas de Sulfatos de Condroitina/genética , Regulação para Baixo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Glicosilação , Humanos , Integrina alfa3/biossíntese , Sulfato de Queratano/biossíntese , Sulfato de Queratano/genética , Lumicana , Sistema de Sinalização das MAP Quinases , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Invasividade Neoplásica , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , TransfecçãoRESUMO
Dermatopontin (DPT), a component of the extracellular matrix (ECM), is involved in promotion of cellular adhesion and ECM assembly activities. However, the role of DPT in the pathogenesis of carcinoma is unclear. We evaluated DPT expression in human oral cancer and its possible roles including cellular adhesion and invasiveness. We first investigated the DPT mRNA and protein expression status in human oral squamous cell carcinoma (OSCC)-derived cells. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and immunoblotting analysis detected frequent downregulation of DPT in OSCC-derived cells compared to human normal oral keratinocytes. To assess the epigenetic regulation of DPT, OSCC-derived cells were treated with a histone deacetylase inhibitor, sodium butyrate (NaB). NaB restored the DPT expression in OSCC-derived cells. DPT-overexpressed cells were examined whether DPT could contribute to cellular adhesion and invasiveness. Markedly, increased adhesion and decreased invasiveness in DPT-overexpressed cells were found compared to mock-transfected cells. Adhesion of DPT-overexpressed cells was inhibited by α3ß1 integrin functional blocking antibody. OSCC-derived cells treated with NaB also decreased invasiveness. The expression status of DPT in primary OSCCs (n = 97) was analyzed and compared to clinicopathological behavior. DPT expression in primary OSCCs was significantly lower (p < 0.05) than in the normal counterparts and was correlated significantly (p < 0.05) with regional lymph node metastasis. Our data provided strong evidence that downregulation of DPT is a characteristic event in OSCCs and that DPT was correlated with cellular adhesion and invasiveness. Therefore, DPT might play an important role in regulating tumor invasion and metastasis.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Metástase Linfática , Neoplasias Bucais/patologia , Idoso , Idoso de 80 Anos ou mais , Anticorpos Bloqueadores , Butiratos/farmacologia , Ácido Butírico/farmacologia , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/secundário , Adesão Celular , Linhagem Celular Tumoral , Proliferação de Células , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Proteínas da Matriz Extracelular/biossíntese , Feminino , Regulação Neoplásica da Expressão Gênica , Inibidores de Histona Desacetilases/farmacologia , Humanos , Integrina alfa3beta1/imunologia , Queratinócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/metabolismo , Invasividade Neoplásica , RNA Mensageiro/biossínteseRESUMO
Endocan is a dermatan sulfate proteoglycan (DSPG) that has been observed in the cytoplasm of endothelial cells of small and large vessels in lung, kidney, liver, colon, ovary and brain tumors. This DSPG has been implicated in the regulation of cellular activities such as adhesion, migration, and proliferation. Given the important roles played by endocan in such processes, we sought to determine whether this DSPG is present in the chicken embryo aortic wall in embryonic days 12 and 14, when intimal thickening and endothelial transformation are notorious. Immunolabeling of serial paraffin cross-sections revealed endocan immunoreactivity at the endothelium and some mesenchymal cells constituting the intimal thickening but not in the cells arranged in lamellar layers. We also investigated whether endocan was present in monolayers of primary embryonic aortic endothelial cells attached to fibronectin when they were deprived of serum and stimulated with epidermal growth factor. Immunofluorescence determined that in the epidermal growth factor (EGF) condition where separating, detaching, and migrating cells were observed, endocan appeared organized in arrays typical of focal complexes in the leading edge of these cells. In serum-free medium condition in which the endothelial cells displayed a cobblestone appearance, endocan appeared mainly delineating the margin of many cells. This study demonstrates for the first time the presence of endocan during the aortic wall remodeling, and provides evidence that suggests a possible contribution of this DSPG in the endothelial-mesenchymal transition (EndoMT) process.
Assuntos
Aorta/embriologia , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Dermatan Sulfato/biossíntese , Células Endoteliais/metabolismo , Endotélio/embriologia , Mesoderma/embriologia , Animais , Aorta/citologia , Linhagem Celular , Embrião de Galinha , Galinhas , Células Endoteliais/citologia , Endotélio/citologia , Mesoderma/citologiaRESUMO
BACKGROUND: Previously we have shown that a fraction of the matrix metalloproteinase-9 (MMP-9) synthesized by the macrophage cell line THP-1 was bound to a chondroitin sulphate proteoglycan (CSPG) core protein as a reduction sensitive heteromer. Several biochemical properties of the enzyme were changed when it was bound to the CSPG. METHODOLOGY/PRINCIPAL FINDINGS: By use of affinity chromatography, zymography, and radioactive labelling, various macrophage stimulators were tested for their effect on the synthesis of the proMMP-9/CSPG heteromer and its components by THP-1 cells. Of the stimulators, only PMA largely increased the biosynthesis of the heteromer. As PMA is an activator of PKC, we determined which PKC isoenzymes were expressed by performing RT-PCR and Western Blotting. Subsequently specific inhibitors were used to investigate their involvement in the biosynthesis of the heteromer. Of the inhibitors, only Rottlerin repressed the biosynthesis of proMMP-9/CSPG and its two components. Much lower concentrations of Rottlerin were needed to reduce the amount of CSPG than what was needed to repress the synthesis of the heteromer and MMP-9. Furthermore, Rottlerin caused a minor reduction in the activation of the PKC isoenzymes δ, ε, θ and υ (PKD3) in both control and PMA exposed cells. CONCLUSIONS/SIGNIFICANCE: The biosynthesis of the proMMP-9/CSPG heteromer and proMMP-9 in THP-1 cells involves a Rottlerin-sensitive pathway that is different from the Rottlerin sensitive pathway involved in the CSPG biosynthesis. MMP-9 and CSPGs are known to be involved in various physiological and pathological processes. Formation of complexes may influence both the specificity and localization of the enzyme. Therefore, knowledge about biosynthetic pathways and factors involved in the formation of the MMP-9/CSPG heteromer may contribute to insight in the heteromers biological function as well as pointing to future targets for therapeutic agents.
Assuntos
Acetofenonas/farmacologia , Benzopiranos/farmacologia , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Proteoglicanas de Sulfatos de Condroitina/química , Metaloproteinase 9 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/química , Multimerização Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Macrófagos/metabolismo , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Estrutura Quaternária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacologia , Fatores de TempoRESUMO
BACKGROUND: We have previously demonstrated that four members of the family of small leucine-rich-proteoglycans (SLRPs) of the extracellular matrix (ECM), named decorin, biglycan, lumican and fibromodulin, are deeply remodeled in mouse uterine tissues along the estrous cycle and early pregnancy. It is known that the combined action of estrogen (E2) and progesterone (P4) orchestrates the estrous cycle and prepares the endometrium for pregnancy, modulating synthesis, deposition and degradation of various molecules. Indeed, we showed that versican, another proteoglycan of the ECM, is under hormonal control in the uterine tissues. METHODS: E2 and/or medroxiprogesterone acetate (MPA) were used to demonstrate, by real time PCR and immunoperoxidase staining, respectively, their effects on mRNA expression and protein deposition of these SLRPs, in the uterine tissues. RESULTS: Decorin and lumican were constitutively expressed and deposited in the ECM in the absence of the ovarian hormones, whereas deposition of biglycan and fibromodulin were abolished from the uterine ECM in the non-treated group. Interestingly, ovariectomy promoted an increase in decorin, lumican and fibromodulin mRNA levels, while biglycan mRNA conspicuously decreased. Hormone replacement with E2 and/or MPA differentially modulates their expression and deposition. CONCLUSIONS: The patterns of expression of these SLRPs in the uterine tissues were found to be hormone-dependent and uterine compartment-related. These results reinforce the existence of subpopulations of endometrial fibroblasts, localized into distinct functional uterine compartments, resembling the organization into basal and functional layers of the human endometrium.
Assuntos
Biglicano/biossíntese , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Decorina/biossíntese , Estradiol/farmacologia , Proteínas da Matriz Extracelular/biossíntese , Sulfato de Queratano/biossíntese , Acetato de Medroxiprogesterona/farmacologia , Proteoglicanas/biossíntese , Útero/metabolismo , Animais , Matriz Extracelular/metabolismo , Feminino , Fibromodulina , Lumicana , Camundongos , Útero/efeitos dos fármacosRESUMO
The AT-hook transcription factor HMGA2 is an oncogene involved in the tumorigenesis of many malignant neoplasms. HMGA2 overexpression is common in both early and late-stage high-grade ovarian serous papillary carcinoma. To test whether HMGA2 participates in the initiation of ovarian cancer and promotion of aggressive tumor growth, we examined the oncogenic properties of HMGA2 in ovarian surface epithelial (OSE) cell lines. We found that introduction of HMGA2 overexpression was sufficient to induce OSE transformation in vitro. HMGA2-mediated OSE transformation resulted in tumor formation in the xenografts of nude mice. By silencing HMGA2 in HMGA2-overexpressing OSE and ovarian cancer cell lines, the aggressiveness of tumor cell growth behaviors was partially suppressed. Global gene profiling analyses revealed that HMGA2-mediated tumorigenesis was associated with expression changes of target genes and microRNAs that are involved in epithelial-to-mesenchymal transition (EMT). Lumican, a tumor suppressor that inhibits EMT, was found to be transcriptionally repressed by HMGA2 and was frequently lost in human high-grade serous papillary carcinoma. Our findings show that HMGA2 overexpression confers a powerful oncogenic signal in ovarian cancers through the modulation of EMT genes.
Assuntos
Transformação Celular Neoplásica/genética , Cistadenocarcinoma Papilar/genética , Cistadenocarcinoma Seroso/genética , Transição Epitelial-Mesenquimal/genética , Proteína HMGA2/genética , Neoplasias Ovarianas/genética , Animais , Estudos de Casos e Controles , Linhagem Celular Transformada , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Proteoglicanas de Sulfatos de Condroitina/genética , Cistadenocarcinoma Papilar/metabolismo , Cistadenocarcinoma Papilar/patologia , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patologia , Feminino , Perfilação da Expressão Gênica , Proteína HMGA2/biossíntese , Humanos , Sulfato de Queratano/biossíntese , Sulfato de Queratano/genética , Lumicana , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Transfecção , Transplante HeterólogoRESUMO
Our previous study has reported that electroacupuncture (EA) promotes survival, differentiation of bone marrow mesenchymal stem cells (MSCs), and functional improvement in spinal cord-transected rats. In this study, we further investigated the structural bases of this functional improvement and the potential mechanisms of axonal regeneration in injured spinal cord after MSCs and EA treatment. Five experimental groups, 1) sham control (Sham-control); 2) operated control (Op-control); 3) electroacupuncture treatment (EA); 4) MSCs transplantation (MSCs), and 5) MSCs transplantation combined with electroacupuncture (MSCs + EA), were designed for this study. Western blots and immunohistochemical staining were used to assess the fibrillary acidic protein (GFAP) and chondroitin sulfate proteoglycans (CSPGs) proteins expression. Basso, Beattie, Bresnahan (BBB) locomotion test, cortical motor evoked potentials (MEPs), and anterograde and retrograde tracing were utilized to assess cortical-spinal neuronal projection regeneration and functional recovery. In the MSCs + EA group, increased labeling descending corticospinal tract (CST) projections into the lesion site showed significantly improved BBB scales and enhanced motor evoked potentials after 10 weeks of MSCs transplant and EA treatment. The structural and functional recovery after MSCs + EA treatment may be due to downregulated GFAP and CSPGs protein expression, which prevented axonal degeneration as well as improved axonal regeneration.
Assuntos
Axônios/fisiologia , Eletroacupuntura/métodos , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/fisiologia , Regeneração Nervosa/fisiologia , Traumatismos da Medula Espinal/terapia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Proteína Glial Fibrilar Ácida/biossíntese , Imuno-Histoquímica , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Ratos , Ratos Sprague-Dawley , Traumatismos da Medula Espinal/metabolismo , Análise de SobrevidaRESUMO
Melanoma chondroitin sulfate proteoglycan (MCSP) is a plasma membrane-associated proteoglycan that facilitates the growth, motility, and invasion of tumor cells. MCSP expression in melanoma cells enhances integrin function and constitutive activation of Erk1,2. The current studies were performed to determine the mechanism by which MCSP expression promotes tumor growth and motility. The results show that MCSP expression in radial growth phase, vertical growth phase, or metastatic cell lines causes sustained activation of Erk1,2, enhanced growth, and motility which all require the cytoplasmic domain of the MCSP core protein. MCSP expression in a radial growth phase cell line also promotes an epithelial-to-mesenchymal transition based on changes in cell morphology and the expression of several epithelial-to-mesenchymal transition markers. Finally, MCSP enhances the expression of c-Met and hepatocyte growth factor, and inhibiting c-Met expression or activation limits the increased growth and motility of multiple melanoma cell lines. The studies collectively show the importance of MCSP in promoting progression by an epigenetic mechanism and they indicate that MCSP could be targeted to delay or inhibit tumor progression in patients.
Assuntos
Movimento Celular/fisiologia , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Regulação Neoplásica da Expressão Gênica , Melanoma/metabolismo , Melanoma/patologia , Animais , Processos de Crescimento Celular , Linhagem Celular Tumoral , Proteoglicanas de Sulfatos de Condroitina/genética , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Ativação Enzimática , Células Epiteliais/patologia , Feminino , Humanos , Sistema de Sinalização das MAP Quinases , Melanoma/genética , Mesoderma/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismoRESUMO
BACKGROUND: ErbB receptors and their cognate ligands are implicated in cancer progression. Their expression in gastrointestinal cancer, however, has not been systemically studied. MATERIALS AND METHODS: The expression of four ErbB receptors and a panel of ErbB ligands were determined by reverse transcription-PCR in two gastric (TMK1, MKN-45) and two colon (SW1116, HT-29) cancer cell lines. Cell proliferation was measured by MTT assay while gene knockdown was achieved by RNA interference. RESULTS: ErbB1, ErbB2 and ErbB3 receptors and five known or putative ErbB ligands, namely, epiregulin, epidermal growth factor (EGF), heparin-binding EGF, transforming growth factor alpha (TGFalpha) and neuroglycan-C were expressed in all four cell lines. Knockdown of neuroglycan-C, however, did not affect cell proliferation. CONCLUSION: This study profiles the expression of ErbB receptors and their cognate ligands in gastric and colon cancer cells. These findings might lay the basis for the development of ErbB pathway-directed therapeutics for gastrointestinal cancer.
Assuntos
Adenocarcinoma/metabolismo , Neoplasias do Colo/metabolismo , Proteínas Oncogênicas v-erbB/biossíntese , Neoplasias Gástricas/metabolismo , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Neoplasias do Colo/patologia , Fator de Crescimento Epidérmico/biossíntese , Epirregulina , Células HT29 , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Ligantes , Neurregulinas/biossíntese , Neoplasias Gástricas/patologiaRESUMO
BACKGROUND: Chondroitin sulfate proteoglycan (CSPG) is a major component of the glial scar. It is considered to be a major obstacle for central nervous system (CNS) recovery after injury, especially in light of its well-known activity in limiting axonal growth. Therefore, its degradation has become a key therapeutic goal in the field of CNS regeneration. Yet, the abundant de novo synthesis of CSPG in response to CNS injury is puzzling. This apparent dichotomy led us to hypothesize that CSPG plays a beneficial role in the repair process, which might have been previously overlooked because of nonoptimal regulation of its levels. This hypothesis is tested in the present study. METHODS AND FINDINGS: We inflicted spinal cord injury in adult mice and examined the effects of CSPG on the recovery process. We used xyloside to inhibit CSPG formation at different time points after the injury and analyzed the phenotype acquired by the microglia/macrophages in the lesion site. To distinguish between the resident microglia and infiltrating monocytes, we used chimeric mice whose bone marrow-derived myeloid cells expressed GFP. We found that CSPG plays a key role during the acute recovery stage after spinal cord injury in mice. Inhibition of CSPG synthesis immediately after injury impaired functional motor recovery and increased tissue loss. Using the chimeric mice we found that the immediate inhibition of CSPG production caused a dramatic effect on the spatial organization of the infiltrating myeloid cells around the lesion site, decreased insulin-like growth factor 1 (IGF-1) production by microglia/macrophages, and increased tumor necrosis factor alpha (TNF-alpha) levels. In contrast, delayed inhibition, allowing CSPG synthesis during the first 2 d following injury, with subsequent inhibition, improved recovery. Using in vitro studies, we showed that CSPG directly activated microglia/macrophages via the CD44 receptor and modulated neurotrophic factor secretion by these cells. CONCLUSIONS: Our results show that CSPG plays a pivotal role in the repair of injured spinal cord and in the recovery of motor function during the acute phase after the injury; CSPG spatially and temporally controls activity of infiltrating blood-borne monocytes and resident microglia. The distinction made in this study between the beneficial role of CSPG during the acute stage and its deleterious effect at later stages emphasizes the need to retain the endogenous potential of this molecule in repair by controlling its levels at different stages of post-injury repair.