RESUMO
Dendritic cells recognize pathogens through pattern recognition receptors such as Toll-like receptors and phagocytose and digest them by phagocytic receptors for antigen presentation. This study was designed to clarify the cross-talk between recognition and phagocytosis of microbes in dendritic cells. The murine dendritic cell line XS106 cells were stimulated with the murine C-type lectin SIGNR1 ligand lipoarabinomannan and the Toll-like receptor 2 ligand FSL-1. The co-stimulation significantly suppressed FSL-1-mediated activation of NF-κB as well as production of TNF-α, IL-6 and IL-12p40 in a dose-dependent manner. The suppression was significantly but not completely recovered by knock-down of SIGNR1. SIGNR1 was associated with Toll-like receptor 2 in XS106 cells. The co-stimulation upregulated the expression of suppressor of cytokine signalling-1 in XS106 cells, the knock-down of which almost completely recovered the suppression of the FSL-1-mediated cytokine production by lipoarabinomannan. In addition, it was found that the MyD88-adaptor-like protein in XS106 cells was degraded by co-stimulation with FSL-1 and lipoarabinomannan in the absence, but not the presence, of the proteasome inhibitor MG132 and the degradation was inhibited by knock-down of suppressor of cytokine signalling-1. This study suggests that Toll-like receptor 2-mediated signalling is negatively regulated by SIGNR1-mediated signalling in dendritic cells, possibly through suppressor of cytokine signalling-1-mediated degradation of the MyD88-adaptor-like protein.
Assuntos
Moléculas de Adesão Celular/metabolismo , Lectinas Tipo C/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Receptores de Superfície Celular/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Receptor 2 Toll-Like/metabolismo , Animais , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/imunologia , Linhagem Celular , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Diglicerídeos/farmacologia , Células HEK293 , Humanos , Subunidade p40 da Interleucina-12/imunologia , Subunidade p40 da Interleucina-12/metabolismo , Interleucina-6/imunologia , Interleucina-6/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Leupeptinas/farmacologia , Lipopolissacarídeos/farmacologia , Camundongos , NF-kappa B/imunologia , NF-kappa B/metabolismo , Oligopeptídeos/farmacologia , Fagocitose/imunologia , Fagocitose/fisiologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Transdução de Sinais/imunologia , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina/genética , Receptor 2 Toll-Like/imunologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Tumor angiogenesis is necessary for solid tumor progression and metastasis. Cyclooxygenase (COX)-2 is known to play an important role in cancer growth and invasion, and it activates the signaling pathways controlling cell proliferation, migration, apoptosis, and angiogenesis. COX-2 is reported to be expressed in many cancer cells. Several studies have reported successful treatment of cancer cells with COX-2 inhibitors (COX-2is). However, the effect of COX-2 inhibition on the tumor endothelium remains to be elucidated. Our study shows that COX-2 is expressed in the vasculature of surgically resected human tumors. To investigate the effects of COX-2 inhibition on the tumor endothelium in vitro, we isolated tumor endothelial cells (TECs) from human melanoma and oral carcinoma xenografts in mice, in which we confirmed that tumor growth was suppressed by inhibiting angiogenesis with the COX-2is NS398. COX-2 mRNA was upregulated in TECs compared to normal endothelial cells (NECs). Cell migration and proliferation were suppressed by NS398 in TECs but not in NECs. The effects of NS398 in vivo were consistent with the in vitro results. The number of CD133+ /vascular endothelial growth factor receptor-2+ cells in circulation was significantly suppressed by COX-2 inhibition. In addition, the number of progenitor marker-positive cells decreased in the tumor blood vessels after COX-2i treatment, which suggests that the homing of progenitor cells into the tumor was also blocked. We conclude that NS398 specifically targets both TECs and vascular progenitor cells without affecting NECs.
Assuntos
Inibidores de Ciclo-Oxigenase 2/uso terapêutico , Endotélio Vascular/efeitos dos fármacos , Melanoma/irrigação sanguínea , Melanoma/tratamento farmacológico , Neoplasias Bucais/irrigação sanguínea , Neoplasias Bucais/tratamento farmacológico , Neovascularização Patológica/prevenção & controle , Células-Tronco/efeitos dos fármacos , Animais , Western Blotting , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ciclo-Oxigenase 2/química , Ciclo-Oxigenase 2/metabolismo , Feminino , Humanos , Melanoma/patologia , Camundongos , Camundongos Nus , Neoplasias Bucais/patologia , Nitrobenzenos/uso terapêutico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Sulfonamidas/uso terapêuticoRESUMO
Recent findings have focused attention on the molecular consequences of the microenvironment in tumor progression, but events occurring in cancer cells themselves in response to their ambient conditions remain obscure. Here, we identify receptor activator of nuclear factor κB ligand (RANKL) as a microenvironment-specific factor essential for tumorigenesis in vivo, using head and neck squamous cell carcinoma (HNSCC) as a model. In human HNSCC tissues, RANKL is abundantly expressed, and its expression level correlates with the histological grade of differentiation. RANKL levels are significantly higher in poorly differentiated SCCs than in well or moderately differentiated SCCs. In contrast, all HNSCC cell lines tested displayed extremely low RANKL expression; however, RANKL is efficiently up-regulated when these cell lines are inoculated in the head and neck region of mice. RANKL expression is restored in a microenvironment-specific manner, and cannot be observed when the cells are inoculated in the hindlimbs. Forced expression of RANKL compensates for tumor growth in the hindlimb milieu, promotes epithelial mesenchymal transition, and induces tumor angiogenesis, in a manner independent of vascular endothelial growth factor (VEGF). These results implicate RANKL expression causatively in tumor growth and progression in HNSCC in vivo. RANKL may provide a novel functional marker for biological malignancy and a therapeutic target based on the specific nature of the microenvironment.
Assuntos
Progressão da Doença , Transição Epitelial-Mesenquimal , Neoplasias de Cabeça e Pescoço/patologia , Ligante RANK/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/irrigação sanguínea , Neoplasias de Cabeça e Pescoço/genética , Membro Posterior/patologia , Humanos , Camundongos , Neovascularização Patológica/complicações , Fenótipo , Lesões Pré-Cancerosas/patologia , Ligante RANK/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Microambiente Tumoral/genéticaRESUMO
PURPOSE: The present study aimed to measure postsurgical swallowing function in patients 5 years after the surgical treatment of tongue carcinoma. PATIENTS AND METHODS: Using a retrospective cohort study design, the investigators enrolled postsurgical patients treated for tongue carcinomas in Hokkaido University Hospital. The primary outcome variable was oropharyngeal swallow efficiency (OPSE) determined by videofluoroscopic evaluation, and OPSE at follow-up was compared with that at discharge. Other variables included current nutritional status (body mass index, serum albumin), dietary intake, self-rating of current swallowing function, and occurrence of pneumonia. Statistical analysis used the paired t test and the Spearman rank correlation. RESULTS: Swallowing function was assessed in 20 patients (11 men and 9 women) who underwent the surgical treatment of tongue carcinomas; the median age was 70 years (range, 56 to 90 yrs). The mean OPSE values for liquid and paste at follow-up were 26.6 ± 21.2 and 21.9 ± 22.5, respectively. The mean values for the body mass index and serum albumin at presentation were 22.2 ± 3.4 kg/m(2) and 4.5 ± 0.3 g/dL, respectively. All patients had a full oral intake of foods, with a mean self-rated value of 6.4 ± 2.5, a value acceptable to the patients. Pneumonia requiring hospitalization did not occur in these patients. CONCLUSIONS: The long-term follow-up of patients after the surgical treatment of tongue carcinomas showed acceptable levels of oral function and nutritional status despite objective measurements of poor swallowing efficiency assessed using videofluoroscopy.
Assuntos
Transtornos de Deglutição/etiologia , Transtornos de Deglutição/fisiopatologia , Glossectomia/efeitos adversos , Neoplasias da Língua/cirurgia , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Dieta , Feminino , Seguimentos , Glossectomia/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Estado Nutricional , Fotofluorografia/métodos , Recuperação de Função Fisiológica , Estudos Retrospectivos , Estatísticas não Paramétricas , Gravação em VídeoRESUMO
Little is known of how Toll-like receptor (TLR) ligands are processed after recognition by TLRs. This study was therefore designed to investigate how the TLR2 ligand FSL-1 is processed in macrophages after recognition by TLR2. FSL-1 was internalized into the murine macrophage cell line, RAW264.7. Both chlorpromazine and methyl-beta-cyclodextrin, which inhibit clathrin-dependent endocytosis, reduced FSL-1 uptake by RAW264.7 cells in a dose-dependent manner but nystatin, which inhibits caveolae- and lipid raft-dependent endocytosis, did not. FSL-1 was co-localized with clathrin but not with TLR2 in the cytosol of RAW264.7 cells. These results suggest that internalization of FSL-1 is clathrin dependent. In addition, FSL-1 was internalized by peritoneal macrophages from TLR2-deficient mice. FSL-1 was internalized by human embryonic kidney 293 cells transfected with CD14 or CD36 but not by the non-transfected cells. Also, knockdown of CD14 or CD36 in the transfectants reduced FSL-1 uptake. In this study, we suggest that (i) FSL-1 is internalized into macrophages via a clathrin-dependent endocytic pathway, (ii) the FSL-1 uptake by macrophages occurs irrespective of the presence of TLR2, and (iii) CD14 and CD36 are responsible for the internalization of FSL-1.
Assuntos
Antígenos CD36/imunologia , Clatrina/imunologia , Diglicerídeos/farmacologia , Endocitose/imunologia , Receptores de Lipopolissacarídeos/imunologia , Macrófagos Peritoneais/imunologia , Oligopeptídeos/farmacologia , Receptor 2 Toll-Like/imunologia , Animais , Antígenos CD36/genética , Linhagem Celular , Clorpromazina/farmacologia , Clatrina/genética , Clatrina/metabolismo , Diglicerídeos/imunologia , Diglicerídeos/metabolismo , Antagonistas de Dopamina/farmacologia , Relação Dose-Resposta a Droga , Endocitose/efeitos dos fármacos , Endocitose/genética , Humanos , Ionóforos/farmacologia , Receptores de Lipopolissacarídeos/genética , Macrófagos Peritoneais/metabolismo , Microdomínios da Membrana/genética , Microdomínios da Membrana/imunologia , Microdomínios da Membrana/metabolismo , Camundongos , Camundongos Knockout , Nistatina/farmacologia , Oligopeptídeos/imunologia , Oligopeptídeos/metabolismo , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismoRESUMO
Tumor angiogenesis is necessary for solid tumor progression and metastasis. Increasing evidence indicates that tumor endothelial cells (TECs) are more relevant to the study of tumor angiogenesis than normal endothelial cells (NECs) because their morphologies and gene expression are different from NECs. However, it is challenging to isolate and culture large numbers of pure ECs from tumor tissue since the percentage of ECs is only about 1-2% and tumor cells and fibroblasts easily overgrow them. In addition, there has been concern that isolated TECs may lose their special phenotype once they are dissociated from tumor cells. In this study, we have successfully purified murine TECs from four different human tumor xenografts and NECs from murine dermal tissue. Isolated ECs expressed endothelial markers, such as CD31, VE-cadherin (CD144), and endoglin (CD105), for more than 3 months after isolation. TECs maintained tumor endothelial-specific markers, such as tumor endothelial marker 8 (TEM8) and aminopeptidase N (APN), as in tumor blood vessels in vivo. In addition, TECs were more proliferative and motile than NECs. TECs showed a higher response to VEGF and higher expression of VEGF receptors-1 and -2 than NECs did. Stem cell antigen-1 was up-regulated in all four TECs, suggesting that they have a kind of stemness. Cultured TECs maintain distinct biological differences from NECs as in vivo. In conclusion, it was suggested that TECs are relevant material for tumor angiogenesis research.
Assuntos
Linhagem Celular Tumoral , Células Endoteliais/patologia , Neovascularização Patológica/patologia , Inibidores da Angiogênese/isolamento & purificação , Inibidores da Angiogênese/farmacologia , Animais , Antígenos CD/biossíntese , Antígenos CD1/biossíntese , Biomarcadores Tumorais/biossíntese , Caderinas/biossíntese , Ensaios de Seleção de Medicamentos Antitumorais , Endoglina , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Proteínas dos Microfilamentos , Neovascularização Patológica/metabolismo , Receptores de Superfície Celular , Receptores de Peptídeos/biossíntese , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Modulation of the physiologically influential Na(+),K(+)-ATPase is a complex process involving a wide variety of factors. To determine the possible effects of the protein tyrosine phosphatase (PTP) inhibitors dephostatin and Et-3,4-dephostatin on human and pig, renal cells and enzymatic extracts, we treated our samples (15 min-24 h) with those PTP inhibitors (0-100 microM). PTP inhibitors were found to possess a concentration-dependent inhibition of Na(+),K(+)-ATPase activity in both human and pig samples. The inhibition was similarly demonstrated on all cellular, microsomal fraction and purified Na(+),K(+)-ATPase levels. Despite rigorous activity recovery attempts, the PTP inhibitors' effects were sustained on Na(+),K(+)-ATPase activity. Western blotting experiments revealed the expression of both alpha(1)- and beta(1)-subunits in both human and pig tissues. alpha(1)-Subunits possessed higher tyrosine phosphorylation levels with higher concentrations of PTP inhibitors. Meanwhile, serine/threonine residues of both alpha(1)- and beta(1)-subunits demonstrated diminished phosphorylation levels upon dephostatin treatment. Accordingly, we provide evidence that Na(+),K(+)-ATPase can be regulated through tyrosine phosphorylation of primarily their alpha(1)-subunits, using PTP inhibitors.
Assuntos
Rim/enzimologia , Subunidades Proteicas/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Tirosina/metabolismo , Animais , Western Blotting , Catecolaminas/farmacologia , Linhagem Celular Tumoral , Citometria de Fluxo , Humanos , Hidroquinonas/farmacologia , Compostos Nitrosos/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Tirosina Fosfatases/antagonistas & inibidores , SuínosRESUMO
E4orf6 plays an important role in the transportation of cellular and viral mRNAs and is known as an oncogene product of adenovirus. Here, we show that E4orf6 interacts with pp32/leucine-rich acidic nuclear protein (LANP). E4orf6 exports pp32/LANP from the nucleus to the cytoplasm with its binding partner, HuR, which binds to an AU-rich element (ARE) present within many protooncogene and cytokine mRNAs. We found that ARE-mRNAs, such as c-fos, c-myc, and cyclooxygenase-2, were also exported to and stabilized in the cytoplasm of E4orf6-expressing cells. The oncodomain of E4orf6 was necessary for both binding to pp32/LANP and effect for ARE-mRNA. C-fos mRNA was exported together with E4orf6, E1B-55kD, pp32/LANP, and HuR proteins. Moreover, inhibition of the CRM1-dependent export pathway failed to block the export of ARE-mRNAs mediated by E4orf6. Thus, E4orf6 interacts with pp32/LANP to modulate the fate of ARE-mRNAs by altering the CRM1-dependent export pathway.
Assuntos
Adenoviridae/metabolismo , Proteínas E4 de Adenovirus/metabolismo , Carioferinas/metabolismo , Neuropeptídeos/metabolismo , Proteínas Nucleares/metabolismo , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Adenoviridae/genética , Proteínas E1B de Adenovirus/genética , Proteínas E1B de Adenovirus/metabolismo , Proteínas E4 de Adenovirus/genética , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Humanos , Carioferinas/genética , Neuropeptídeos/genética , Proteínas Nucleares/genética , Fases de Leitura Aberta/genética , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico/fisiologia , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , RNA Mensageiro/genética , RNA Viral/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Proteína Exportina 1RESUMO
Tenascin-X (TNX), which has a molecular mass of roughly 450 kDa, is the largest member of the tenascin family. Complete deficiency of TNX in humans leads to a recessive form of Ehlers-Danlos syndrome (EDS). TNX is expressed abundantly in a variety of tissues, especially in cardiac muscle and in perivascular stroma. Human TNX is also present in serum with an apparent molecular size of 140 kDa. In the present study, we investigated the expression levels of TNX protein in thoracic and abdominal aortic aneurysm tissues. The level of TNX was significantly increased in both aortic aneurysm tissues compared with that in adjacent normal tissues. Next, to compare TNX levels in serum from both patients with thoracic aortic aneurysm and patients with abdominal aortic aneurysm with levels in serum from healthy individuals, we developed a sandwich enzyme-linked immunosorbent assay (ELISA) using TNX-specific antibodies. Measurement of TNX serum concentrations in both aortic aneurysm patients and controls showed that the levels were almost the same. These results indicate that TNX expression is significantly elevated in both thoracic and abdominal aortic aneurysm tissues but that the increase in TNX levels in both tissues does not result in an increase in TNX serum concentration in patients with TAA or AAA.
Assuntos
Aorta/metabolismo , Aneurisma da Aorta Abdominal/metabolismo , Aneurisma da Aorta Torácica/metabolismo , Tecido Conjuntivo/metabolismo , Tenascina/metabolismo , Anticorpos/sangue , Estudos de Casos e Controles , Síndrome de Ehlers-Danlos/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Tenascina/imunologiaRESUMO
We investigated the possible roles of estrogen on plasma membrane Ca(2+)-ATPase (PMCA) in human fibroblast-like synovial cells (HFLS) and mouse macrophage-like cells (RAW 264.7). Western blots revealed the expression of PMCA 2 and 4 in both cells. In vitro treatments with 17beta-estradiol for 24 hours resulted in a concentration dependent decrease in PMCA expression. Moreover, Ca(2+)-ATPase specific activity was similarly decreased with estrogen treatments. However, treatments for 1 hour in the presence or absence of cycloheximide demonstrated non-significant effects. These results suggest that estrogen has a modulatory role on Ca(2+) homeostasis through decreasing PMCA expression and abating their activity.
Assuntos
Estradiol/farmacologia , Macrófagos/efeitos dos fármacos , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Animais , Western Blotting , Cálcio/metabolismo , Linhagem Celular , Cicloeximida/farmacologia , Humanos , Macrófagos/citologia , Macrófagos/enzimologia , Camundongos , Inibidores da Síntese de Proteínas/farmacologia , Líquido Sinovial/citologia , Líquido Sinovial/efeitos dos fármacos , Líquido Sinovial/enzimologiaRESUMO
OBJECTIVE: To assess and develop a monocortical mandibular bone grafting procedure for reconstruction of alveolar cleft. DESIGN: Prospective study. SETTING: Hokkaido University Hospital. PATIENTS: Forty-two consecutive Japanese patients who had been treated for a total of 48 clefts according to a strict clinical protocol. Mean age at bone grafting was 6 years 11 months. INTERVENTIONS: Bone grafting was performed by harvesting lateral cortical bone plates from the symphysis and/or body and then placing them on the labial and palatal openings of the alveolar process defect. No particulate bone grafts were packed into the bony cavity. Mean follow-up after bone grafting was 37 months. MAIN OUTCOME MEASURES: Status of the grafted area and eruption of cleft-adjacent teeth were assessed prospectively using computed tomography and periapical radiography. RESULTS: At 6 months postoperatively, computed tomography showed sufficient bone bridge formation at the cleft site in 85.4% of clefts. Periapical radiography showed ≥75% of the root surfaces of cleft-adjacent teeth were covered with spanning bone in 83.3% of clefts. In 92.6% of clefts in which the cleft-adjacent canine was uncovered with bone during follow-up, the canines erupted spontaneously. CONCLUSIONS: Monocortical mandibular bone grafting appears extremely effective for sufficient bone bridge formation and facilitation of cleft-adjacent teeth eruption. The procedure is advantageous in that the quantity of bone required per unit volume of cleft defect is relatively reduced, and larger clefts can thus be treated.
Assuntos
Enxerto de Osso Alveolar/métodos , Transplante Ósseo/métodos , Fissura Palatina/cirurgia , Mandíbula/cirurgia , Coleta de Tecidos e Órgãos/métodos , Autoenxertos/transplante , Criança , Pré-Escolar , Fenda Labial/cirurgia , Dente Canino/fisiologia , Feminino , Seguimentos , Humanos , Masculino , Osteogênese/fisiologia , Estudos Prospectivos , Radiografia Interproximal/métodos , Procedimentos de Cirurgia Plástica/métodos , Deiscência da Ferida Operatória/etiologia , Tomografia Computadorizada por Raios X/métodos , Erupção Dentária/fisiologia , Dente Impactado/etiologia , Sítio Doador de Transplante/cirurgia , Resultado do TratamentoRESUMO
The polyphenol epigallocatechin-3 gallate (EGCG) in green tea suppresses tumor growth by direct action on tumor cells and by inhibition of angiogenesis, but it is not known whether it specifically inhibits tumor angiogenesis. We examined the anti-angiogenic effect of EGCG on tumor-associated endothelial cells (TEC), endothelial progenitor cells (EPC), and normal endothelial cells (NEC). EGCG suppressed the migration of TEC and EPC but not NEC. EGCG also inhibited the phosphorylation of Akt in TEC but not in NEC. Furthermore, vascular endothelial growth factor-induced mobilization of EPC into circulation was inhibited by EGCG. MMP-9 in the bone marrow plasma plays key roles in EPC mobilization into circulation. We observed that expression of MMP-9 mRNA was downregulated by EGCG in mouse bone marrow stromal cells. In an in vivo model, EGCG suppressed growth of melanoma and reduced microvessel density. Our study showed that EGCG has selective anti-angiogenic effects on TEC and EPC. It is suggested that EGCG could be a promising angiogenesis inhibitor for cancer therapy.
Assuntos
Anticarcinógenos/farmacologia , Catequina/análogos & derivados , Células Endoteliais/efeitos dos fármacos , Melanoma/metabolismo , Neovascularização Patológica/metabolismo , Células-Tronco/efeitos dos fármacos , Animais , Western Blotting , Catequina/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Regulação para Baixo , Flavonoides/farmacologia , Citometria de Fluxo , Expressão Gênica/efeitos dos fármacos , Humanos , Metaloproteinase 9 da Matriz/efeitos dos fármacos , Metaloproteinase 9 da Matriz/metabolismo , Melanoma/genética , Melanoma/patologia , Camundongos , Microvasos/efeitos dos fármacos , Microvasos/metabolismo , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Fenóis/farmacologia , Polifenóis , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Chá , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Silicone rubbers are widely used as tissue implants because of their flexibility and chemical stability. However, they have limited cellular adhesiveness and may cause problems in the long term. In this study, a coating of carbon nanotubes (CNTs) was applied to silicone rubber to improve its cellular adhesiveness. Scanning electron micrograph of this coating revealed that CNTs had formed a densely packed meshwork; the Ra values and protein adsorption capacity were enhanced. Although the contact angle did not change after coating, it decreased after immersion into a culture medium. After cultivation for 6 d, while Saos-2 cells were hardly observed on untreated silicone, the cells proliferated on CNT-coated silicone. Thus, CNT coating might be a simple and effective solution to problems associated with silicone implants.
Assuntos
Materiais Revestidos Biocompatíveis/química , Nanotecnologia/métodos , Nanotubos de Carbono/química , Osteoblastos/citologia , Osteoblastos/fisiologia , Elastômeros de Silicone/química , Engenharia Tecidual/métodos , Absorção , Adesão Celular , Técnicas de Cultura de Células/métodos , Linhagem Celular , Movimento Celular , Proliferação de Células , Cristalização/métodos , Matriz Extracelular/química , Humanos , Teste de Materiais , Tamanho da Partícula , Porosidade , Propriedades de SuperfícieRESUMO
Parathyroid hormone-related protein (PTHrP) is detected in many aggressive tumors and involved in malignant conversion; however, the underlying mechanism remains obscure. Here, we identified PTHrP as a mediator of epidermal growth factor receptor (EGFR) signaling to promote the malignancies of oral cancers. PTHrP mRNA was abundantly expressed in most of the quiescent oral cancer cells, and was significantly upregulated by EGF stimulation via ERK and p38 MAPK. PTHrP silencing by RNA interference, as well as EGFR inhibitor AG1478 treatment, significantly suppressed cell proliferation, migration, and invasiveness. Furthermore, combined treatment of AG1478 and PTHrP knockdown achieved synergistic inhibition of malignant phenotypes. Recombinant PTHrP substantially promoted cell motility, and rescued the inhibition by PTHrP knockdown, suggesting the paracrine/autocrine function of PTHrP. These data indicate that PTHrP contributes to the malignancy of oral cancers downstream of EGFR signaling, and may thus provide a therapeutic target for oral cancer.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Receptores ErbB/metabolismo , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Transdução de Sinais , Proliferação de Células , Humanos , Células Tumorais CultivadasRESUMO
HEK293 cells stably expressing DC-SIGN (293/DC-SIGN) were examined for phagocytosis of Escherichia coli. 293/DC-SIGN stable transfectants were able to mediate phagocytosis of E. coli. The phagocytosis was inhibited by EDTA or several inhibitors specific for Syk kinase, Raf kinase and the transcription factor NF-kappaB. DC-SIGN consists of characteristic domains and motifs such as CRD, neck, incomplete ITAM, dileucine and tri-acidic cluster. HEK293 cells expressing mutants of DC-SIGN were also examined for the phagocytosis. It was found that Ca(2+) binding sites in the CRD of DC-SIGN were involved in phagocytosis of bacteria as well as multimerization of DC-SIGN, and the neck region played a role in efficiency of binding to microbes as well as multimerization of the protein.
Assuntos
Cálcio/metabolismo , Moléculas de Adesão Celular/metabolismo , Escherichia coli/imunologia , Lectinas Tipo C/metabolismo , Fagocitose , Receptores de Superfície Celular/metabolismo , Sítios de Ligação , Moléculas de Adesão Celular/genética , Linhagem Celular , Ácido Edético/farmacologia , Inibidores de Histona Desacetilases , Histona Desacetilases/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lectinas Tipo C/genética , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Fagocitose/efeitos dos fármacos , Fagocitose/genética , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Receptores de Superfície Celular/genética , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/metabolismo , Quinase SykRESUMO
Matrix metalloproteinase (MMP) is closely involved in the degradation of extracellular matrix and confers invasive and metastatic potential to malignant tumors. MMP-2 is a type-IV collagenase secreted as a proenzyme that is activated on the surface of the tumor cell by membrane-type 1-MMP (MT1-MMP). MT1-MMP plays a critical role during tumor progression and metastasis. We investigated the expression levels of E1AF and MT1-MMP in malignant melanoma cell lines and specimens from patients in order to clarify the mechanisms responsible for the invasion and metastasis of malignant melanoma. High levels of E1AF and MT1-MMP mRNA expression were observed in melanoma cells by Northern blotting and real-time PCR. The expression level was highly correlated with an invasive potential determined by an in vitro invasion assay. The down-regulation of MT1-MMP was identified when E1AF was knocked down by RNA interference. These results suggest that E1AF plays a crucial role in the invasion and metastasis of malignant melanoma through up-regulating the MT1-MMP expression.
Assuntos
Proteínas E1A de Adenovirus/biossíntese , Proteínas E1A de Adenovirus/fisiologia , Regulação Enzimológica da Expressão Gênica , Metaloproteinase 14 da Matriz/biossíntese , Melanoma/metabolismo , Melanoma/patologia , Proteínas Proto-Oncogênicas c-ets/biossíntese , Proteínas Proto-Oncogênicas c-ets/fisiologia , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/fisiologia , Linhagem Celular Tumoral , Colágeno/química , Combinação de Medicamentos , Ativação Enzimática , Humanos , Laminina/química , Modelos Biológicos , Invasividade Neoplásica , Metástase Neoplásica , Fenótipo , Proteoglicanas/química , Interferência de RNA , Regulação para CimaRESUMO
Matrix metalloproteinases (MMPs) play important roles in the invasion and metastasis to soft tissues of carcinomas including, oral squamous cell carcinomas (SCCs). Although, osteoclastic bone resorption is an important step in bone involvement in a variety of malignancies, the mechanism of bone involvement of oral SCC remains unclear. Once cancer cells arrest in bone, the bone is a storehouse of a variety of cytokines and growth factors and thus provides an extremely fertile environment for cell growth. The bone-invasive oral cancer cell line, BHY, transcriptionally expressed detectable levels of TGF-beta, IL-1beta, IL-8, parathyroid hormone-related protein (PTHrP) and vascular endothelial growth factor (VEGF) mRNAs and failed to express GM-CSF, IL-6, and TNF-alpha. Furthermore, the BHY-conditioned medium greatly upregulated IL-6 and RANKL/ODF mRNA expression in osteoblasts, suggesting a potential indirect stimulation of osteoclastogenesis via the osteogenic lineage. Seven out of eleven patients with carcinomas of the lower alveolus and gingiva showing infiltrative bone involvement expressed PTHrP mRNA. These data suggest that the occurrence of PTHrP may be an indication of developing oral malignant carcinomas.
Assuntos
Carcinoma de Células Escamosas/patologia , Diferenciação Celular , Neoplasias Bucais/patologia , Osteoblastos/citologia , Osteoclastos/citologia , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Animais , Células da Medula Óssea , Carcinoma de Células Escamosas/metabolismo , Células Cultivadas , Técnicas de Cocultura , Meios de Cultivo Condicionados/farmacologia , Citocinas/metabolismo , Expressão Gênica , Neoplasias Gengivais/genética , Neoplasias Gengivais/metabolismo , Neoplasias Gengivais/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Bucais/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Proteína Relacionada ao Hormônio Paratireóideo/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Several epidemiological studies have reported that temporomandibular disorder is more prevalent in women, which suggests the involvement of sex hormones, such as estrogen, in the pathogenesis of this disease. PCR amplification and Western blotting were employed to target the expression of estrogen receptors (ERs) in human fibroblast-like synovial and ATDC5 cells. The effect of estrogen was investigated through the expression of RANKL, osteoprotegerin (OPG), M-CSF/CSF-1 and c-fms. We showed expression of M-CSF/ CSF-1 and c-fms, with time-dependent increase in both after the addition of estrogen. Based on previous studies reporting that M-CSF/CSF-1 regulates the proliferation and differentiation of hemopoietic progenitor cells into mature macrophages, we put forward a new hypothesis based on the increased inflammation and tendency of females to suffer more from temporomandibular disorder (TMD) in the presence of external exacerbating factors. Detection of RANKL and OPG in ATDC5 and expression of both in HFLS was confirmed with complete disappearance of the RANKL band, and marked increase in the expression of OPG after 1 h from the addition of estrogen.
Assuntos
Estrogênios/farmacologia , Fibroblastos/efeitos dos fármacos , Membrana Sinovial/efeitos dos fármacos , Idoso , Animais , Western Blotting , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Reabsorção Óssea/prevenção & controle , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica/efeitos dos fármacos , Hormônios Esteroides Gonadais/farmacologia , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/prevenção & controle , Fator Estimulador de Colônias de Macrófagos/genética , Fator Estimulador de Colônias de Macrófagos/metabolismo , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Ligante RANK/genética , Ligante RANK/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor Ativador de Fator Nuclear kappa-B/análise , Receptor Ativador de Fator Nuclear kappa-B/efeitos dos fármacos , Receptor Ativador de Fator Nuclear kappa-B/genética , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Membrana Sinovial/citologia , Membrana Sinovial/metabolismo , Articulação Temporomandibular/efeitos dos fármacos , Articulação Temporomandibular/metabolismo , Articulação Temporomandibular/patologia , Transtornos da Articulação Temporomandibular/genética , Transtornos da Articulação Temporomandibular/metabolismo , Transtornos da Articulação Temporomandibular/prevenção & controleRESUMO
OBJECTIVE: To evaluate the diagnostic accuracy of fluorodeoxyglucose positron emission tomography (FDG-PET) relative to computed tomography (CT) for detecting metastatic cervical lymph nodes in patients with squamous cell carcinoma of the head and neck (HNSCC), and to ascertain the factors that affect this accuracy. METHODS: A total of 1076 lymph nodes obtained from 35 neck dissections in 26 HNSCC patients who preoperatively underwent both FDG-PET and CT were retrospectively analyzed. For pathological metastatic lymph nodes, the lymph node size (short-axis diameter), the ratio of intranodal tumor deposits, and the size of intranodal tumor deposits (maximum diameter of metastatic foci in each lymph node) were histologically recorded. RESULTS: Forty-six lymph nodes from 23 neck sides were pathologically diagnosed metastases. The sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of FDG-PET evaluated individually per neck side were 74%, 92%, 80%, 94%, and 65%, respectively, whereas those of CT were 78%, 58%, 71%, 78%, and 58%, respectively. FDG-PET detected 100% of metastatic lymph nodes > or =10 mm, intranodal tumor deposits > or =9 mm, and intranodal tumor deposits with a ratio >75%, whereas no nodes or tumor deposits smaller than 5 mm were detected. The spatial resolution limitations of FDG-PET were responsible for 16 of 20 (80%) false-negative PET results in lymph nodes. CONCLUSIONS: FDG-PET is a useful tool for preoperative evaluation of the neck because it accurately detects metastatic lymph nodes > or =10 mm and has fewer false-positive cases than CT. The high specificity of FDG-PET for lymph node metastases may play an important role in avoiding unnecessary neck dissection.
Assuntos
Carcinoma de Células Escamosas/diagnóstico por imagem , Carcinoma de Células Escamosas/secundário , Fluordesoxiglucose F18/farmacocinética , Neoplasias de Cabeça e Pescoço/patologia , Tomografia por Emissão de Pósitrons/normas , Adulto , Idoso , Protocolos Clínicos/normas , Reações Falso-Negativas , Reações Falso-Positivas , Feminino , Neoplasias de Cabeça e Pescoço/cirurgia , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Esvaziamento Cervical , Estadiamento de Neoplasias , Compostos Radiofarmacêuticos/farmacocinética , Sensibilidade e Especificidade , Tomografia Computadorizada por Raios XRESUMO
A significant amount of evidence has been accumulated to show that Toll-like receptors (TLRs) function as sensors for microbial invasion. However, little is known about how signalling triggered by TLRs leads to the phagocytosis of pathogens. This study was designed to determine whether stimulation of TLR2 mainly with the lipopeptide FSL-1 plays a role in the phagocytosis of pathogens by macrophages. FSL-1 enhanced the phagocytosis of Escherichia coli to a markedly greater extent than it did that of Staphylococcus aureus, but did not enhance the phagocytosis of latex beads. FSL-1 stimulation resulted in enhanced phagocytosis of bacteria by macrophages from TLR2(+/+) mice but not by those from TLR2(-/-) mice. Chinese hamster ovary cells stably expressing TLR2 failed to phagocytose these bacteria, but the cells expressing CD14 did. FSL-1 induced upregulation of the expression of phagocytic receptors, including MSR1, CD36, DC-SIGN and Dectin-1 in THP-1 cells. Human embryonic kidney 293 cells transfected with DC-SIGN and MSR1 phagocytosed these bacteria. These results suggest that the FSL-1-induced enhancement of phagocytosis of bacteria by macrophages may be explained partly by the upregulation of scavenger receptors and the C-type lectins through TLR2-mediated signalling pathways, and that TLR2 by itself does not function as a phagocytic receptor.