RESUMO
Chondrosarcoma is a malignancy of soft tissue and bone that has a high propensity to metastasize to distant organs. Nerve growth factor (NGF) is critical for neuronal cell growth, apoptosis, and differentiation, and also appears to promote the progression and metastasis of several different types of tumors, although the effects of NGF upon chondrosarcoma mechanisms are not very clear. We report that NGF facilitates lysyl oxidase (LOX)-dependent cellular migration and invasion in human chondrosarcoma cells, and that NGF overexpression enhances lung metastasis in a mouse model of chondrosarcoma. NGF-induced stimulation of LOX production and cell motility occurs through the inhibition of miR-149-5p expression, which was reversed by PI3K, Akt, and mTOR inhibitors and their respective short interfering RNAs. Notably, levels of NGF and LOX expression correlated with tumor stage in human chondrosarcoma samples. Thus, NGF appears to be a worthwhile therapeutic target for metastatic chondrosarcoma.
Assuntos
Condrossarcoma/genética , Proteínas da Matriz Extracelular/metabolismo , MicroRNAs/metabolismo , Fator de Crescimento Neural/metabolismo , Proteína-Lisina 6-Oxidase/metabolismo , Animais , Movimento Celular , Condrossarcoma/patologia , Humanos , Camundongos , Invasividade Neoplásica , Metástase NeoplásicaRESUMO
AIMS: Insulin-like growth factor (IGF) signaling has been documented in several human malignancies and is thought to contribute to cellular differentiation and migration, as well as malignant progression. A major binding molecule of IGF, IGF-binding protein 3 (IGFBP-3), regulates multiple IGF effects. Here, we focused on the effect of IGFBP-3 in the motility of osteosarcoma cells and examined signaling regulation. MATERIALS AND METHODS: Using a human osteosarcoma tissue array, immunohistochemical staining determined levels of IGFBP-3 expression in osteosarcoma tissue and in normal tissue. The wound healing migration assay, Transwell migration assay, luciferase reporter assay, immunofluorescence staining, Western blot and real-time quantitative PCR were performed to examine whether IGFBP-3 facilitates VCAM-1-dependent migration of osteosarcoma cells. KEY FINDINGS: In this study, we found significantly higher IGFBP-3 levels in osteosarcoma tissue compared with normal healthy tissue. IGFBP-3 treatment of two human osteosarcoma cell lines promoted cell migration and upregulated levels of VCAM-1 expression via PI3K/Akt and AP-1 signaling. SIGNIFICANCE: IGFBP-3 appears to be a novel therapeutic target in metastatic osteosarcoma.
Assuntos
Neoplasias Ósseas/metabolismo , Movimento Celular , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Osteossarcoma/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células , Imunofluorescência , Regulação Neoplásica da Expressão Gênica , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/fisiologia , Regulação para CimaRESUMO
Oral squamous cell carcinoma (OSCC) accounts for over 90% of malignant neoplasms of the mouth. In Taiwan, OSCC is the fourth most common male cancer and the fourth leading cause of male cancer death. Resistin (RETN) is an adipokine that is associated with obesity, inflammation, and various cancers. Here, we examine the association between four single nucleotide polymorphisms (SNPs) of the RETN gene (rs3745367, rs7408174, rs1862513, and rs3219175) and OSCC susceptibility as well as clinical outcomes in 935 patients with OSCC and in 1200 cancer-free healthy controls. We found that, in 1465 smokers, RETN polymorphisms carriers with the betel-nut chewing habit had a 6.708-10.882-fold greater risk of having OSCC compared to RETN wild-type carriers without the betel-nut chewing habit. Patients with OSCC who had A/A homozygous of RETN rs3219175 polymorphism showed a high risk for an advanced tumor size (> T2), compared to those patients with G/G homozygotes. In addition, A/T/G/G haplotype significantly increased the risks for OSCC by 1.376-fold. This study is the first to examine the risk factors associated with RETN SNPs in OSCC progression and development in Taiwan.
Assuntos
Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Progressão da Doença , Estudos de Associação Genética , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Polimorfismo de Nucleotídeo Único/genética , Resistina/genética , Areca , Estudos de Casos e Controles , Frequência do Gene/genética , Predisposição Genética para Doença , Haplótipos/genética , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Chondrosarcoma is the second most prevalent general primary tumor of bone following osteosarcoma. Chondrosarcoma development may be linked to angiogenesis, which is principally elicited by vascular endothelial growth factor-A (VEGF-A). VEGF-A level has been recognized as a prognostic marker in angiogenesis. WNT1-inducible signaling pathway protein-3 (WISP)-3/CCN6 belongs to the CCN family and is involved in regulating several cellular functions, including cell proliferation, differentiation, and migration. Nevertheless, the effect of WISP-3 on VEGF-A production and angiogenesis in human chondrosarcoma remains largely unknown. This current study shows that WISP-3 promoted VEGF-A production and induced angiogenesis of human endothelial progenitor cells. Moreover, WISP-3-enhanced VEGF-A expression and angiogenesis involved the c-Src and p38 signaling pathways, while miR-452 expression was negatively affected by WISP-3 via the c-Src and p38 pathways. Our results illustrate the clinical significance of WISP-3, VEGF-A and miR-452 in human chondrosarcoma patients. WISP-3 may illustrate a novel therapeutic target in the metastasis and angiogenesis of chondrosarcoma.
Assuntos
Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Proteínas de Sinalização Intercelular CCN/metabolismo , Condrossarcoma/genética , Condrossarcoma/metabolismo , Células Progenitoras Endoteliais/metabolismo , MicroRNAs/genética , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Neoplasias Ósseas/patologia , Proteínas de Sinalização Intercelular CCN/genética , Proteína Tirosina Quinase CSK , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Condrossarcoma/patologia , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Camundongos , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Quinases da Família src/metabolismoRESUMO
BACKGROUND: Osteoarthritis (OA) is manifested by synovial inflammation and cartilage destruction that is directly linked to synovitis, joint swelling and pain. In the light of the role of synovium in the pathogenesis and the symptoms of OA, synovium-targeted therapy is a promising strategy to mitigate the symptoms and progression of OA. Transforming growth factor beta 1 (TGF-ß1), a secreted homodimeric protein, possesses unique and potent anti-inflammatory and immune-regulatory properties in many cell types. Heme oxygenase 1 (HO-1) is an inducible anti-inflammatory and stress responsive enzyme that has been proven to prevent injuries caused by many diseases. Despite the similar anti-inflammatory profile and their involvement in the pathogenesis of arthritic diseases, no studies have as yet explored the possibility of any association between the expression of TGF-ß1 and HO-1. METHODOLOGY/PRINCIPAL FINDINGS: TGF-ß1-induced HO-1 expression was examined by HO-1 promoter assay, qPCR, and Western blotting. The siRNAs and enzyme inhibitors were utilized to determine the intermediate involved in the signal transduction pathway. We showed that TGF-ß1 stimulated the synthesis of HO-1 in a concentration- and time-dependent manner, which can be mitigated by blockade of the phospholipase (PLC)γ/protein kinase C alpha (PKC)α pathway. We also showed that the expression of miRNA-519b, which blocks HO-1 transcription, is inhibited by TGF-ß1, and the suppression of miRNA 519b could be reversed via blockade of the PLCγ/PKCα pathway. CONCLUSIONS/SIGNIFICANCE: TGF-ß1 stimulated the expression of HO-1 via activating the PLCγ/PKCα pathway and suppressing the downstream expression of miRNA-519b. These results may shed light on the pathogenesis and treatment of OA.
Assuntos
Fibroblastos/metabolismo , Heme Oxigenase-1/genética , MicroRNAs/genética , Osteoartrite/genética , Fator de Crescimento Transformador beta1/genética , Anticorpos/farmacologia , Artroplastia do Joelho , Carbazóis/farmacologia , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Inibidores Enzimáticos/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Regulação da Expressão Gênica , Heme Oxigenase-1/metabolismo , Humanos , Indóis/farmacologia , Maleimidas/farmacologia , MicroRNAs/metabolismo , Osteoartrite/metabolismo , Osteoartrite/patologia , Osteoartrite/cirurgia , Fosfolipases/antagonistas & inibidores , Fosfolipases/genética , Fosfolipases/metabolismo , Cultura Primária de Células , Proteína Quinase C-alfa/antagonistas & inibidores , Proteína Quinase C-alfa/genética , Proteína Quinase C-alfa/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Chondrosarcoma is the second most frequently occurring type of bone malignancy that is characterized by the distant metastasis propensity. Vascular endothelial growth factor-C (VEGF-C) is the chief lymphangiogenic mediator, and makes crucial contributions to tumor lymphangiogenesis. Leptin is an adipocytokine and has been indicated to facilitate tumorigenesis, angiogenesis and metastasis. However, the effect of leptin on VEGF-C regulation and lymphangiogenesis in human chondrosarcoma has hugely remained a mystery. Our results showed a clinical correlation between leptin and VEGF-C as well as tumor stage in human chondrosarcoma tissues. We further demonstrated that leptin promoted VEGF-C production and secretion in human chondrosarcoma cells. The conditioned medium from leptin-treated chondrosarcoma cells induced lymphangiogenesis of human lymphatic endothelial cells. We also found that leptin-induced VEGF-C is mediated by the FAK, PI3K and Akt signaling pathway. Furthermore, the expression of microRNA-27b was negatively regulated by leptin via the FAK, PI3K and Akt cascade. Our study is the first to describe the mechanism of leptin-promoted lymphangiogenesis by upregulating VEGF-C expression in chondrosarcomas. Thus, leptin could serve as a therapeutic target in chondrosarcoma metastasis and lymphangiogenesis.
Assuntos
Neoplasias Ósseas/metabolismo , Condrossarcoma/metabolismo , Leptina/metabolismo , Linfangiogênese , MicroRNAs/biossíntese , Proteínas de Neoplasias/metabolismo , RNA Neoplásico/biossíntese , Fator C de Crescimento do Endotélio Vascular/biossíntese , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Condrossarcoma/patologia , HumanosRESUMO
Chondrosarcoma is the second most common primary malignant bone cancer, with potential for local invasion and distant metastasis. Chemokine CCL5 (formerly RANTES) of the CC-chemokine family plays a crucial role in metastasis. Angiogenesis is essential for the cancer metastasis. However, correlation of CCL5 with vascular endothelial growth factor (VEGF) expression and angiogenesis in human chondrosarcoma is still unknown. CCL5-mediated VEGF expression was assessed by qPCR, ELISA, and Western blotting. CCL5-induced angiogenesis was examined by migration and tube formation in endothelial progenitor cells in vitro. CCL5 increased VEGF expression and also promoted chondrosarcoma conditional medium-mediated angiogenesis in vitro and in vivo. Stimulation of chondrosarcoma with CCL5 augmented PI3K and Akt phosphorylation, while PI3K and Akt inhibitor or siRNA abolished CCL5-induced VEGF expression and angiogenesis. We also demonstrated CCL5 inhibiting miR-200b expression and miR-200b mimic reversing the CCL5-enhanced VEGF expression and angiogenesis. Moreover, in chondrosarcoma patients showed the positive correlation between CCL5 and VEGF; negative correlation between CCL5 and miR-200b. Taken together, results demonstrate CCL5 promoting VEGF-dependent angiogenesis in human chondrosarcoma cells by down-regulating miR-200b through PI3K/Akt signaling pathway.
Assuntos
Neoplasias Ósseas/irrigação sanguínea , Quimiocina CCL5/metabolismo , Condrossarcoma/irrigação sanguínea , MicroRNAs/genética , Neovascularização Patológica , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Apoptose , Western Blotting , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Movimento Celular , Proliferação de Células , Células Cultivadas , Quimiocina CCL5/genética , Embrião de Galinha , Condrossarcoma/metabolismo , Condrossarcoma/patologia , Membrana Corioalantoide , Regulação para Baixo , Células Progenitoras Endoteliais/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Masculino , Camundongos , Camundongos Nus , Fosfatidilinositol 3-Quinases/genética , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Inflammatory response and articular destruction are common symptoms of osteoarthritis (OA) and rheumatoid arthritis (RA). Leptin, an adipocyte-secreted hormone that centrally regulates weight control, may exert proinflammatory effects in the joint, depending on the immune response. Yet, the mechanism of leptin interacting with the arthritic inflammatory response is unclear. This study finds that leptin increased expression of oncostatin M (OSM) in human osteoblasts in a concentration- and time-dependent manner. In addition, OBRl, but not OBRs receptor antisense oligonucleotide, abolished the leptin-mediated increase of OSM expression. On the other hand, leptin inhibited miR-93 expression; an miR-93 mimic reversed leptin-increased OSM expression. Stimulation of osteoblasts with leptin promoted Akt phosphorylation, while pretreatment of cells with Akt inhibitor or siRNA reversed leptin-inhibited miR-93 expression. Our results showed that leptin heightened OSM expression by downregulating miR-93 through the Akt signaling pathway in osteoblasts, suggesting leptin as a novel target in arthritis treatment.
Assuntos
Regulação para Baixo , Leptina/farmacologia , MicroRNAs/metabolismo , Oncostatina M/metabolismo , Osteoblastos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células Cultivadas , Humanos , MicroRNAs/genética , Oncostatina M/genética , Osteoblastos/metabolismo , Receptores para Leptina/genética , Receptores para Leptina/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: Leptin, 16kDa product of obese gene, is adipocytokine playing critical role in regulation of body weight. In recent years, leptin is also defined as potent angiogenic factor involving in tumorigenesis, angiogenesis, and metastasis. However, it is unknown whether leptin regulates VEGF production in human chondrosarcoma and contributing the tumor-associated angiogenesis. METHODS: We analyzed protein level of leptin and VEGF in human chondrosarcoma tissues. Effects of leptin on chondrosarcoma cells were examined by in vitro and in vivo assays. In addition, intracellular signal pathways were investigated by pharmacological and genetic approaches. RESULTS: We found that both leptin and VEGF are highly expressed in human chondrosarcoma tissues, and positively correlated with tumor stage. Leptin increases VEGF production by activating OBRl receptor and MAPKs (p38, ERK, and JNK), which in turn enhances binding of AP-1 transcription factor to VEGF promoter, resulting in the transactivation of VEGF expression and subsequently promoting migration and tube formation in endothelial progenitor cells (EPCs). In vivo, knockdown leptin significantly reduces angiogenesis and tumor growth. CONCLUSION: Leptin may be a therapeutic target of angiogenesis and metastasis in chondrosarcoma. GENERAL SIGNIFICANCE: These findings provide better understanding of pathogenesis of chondrosarcoma and can utilize this knowledge to design new therapeutic strategy.
RESUMO
Chondrosarcoma, a primary malignant bone cancer, has potential for local invasion and distant metastasis, especially to the lungs. Patients diagnosed with it show poor prognosis. Paeonol (2'-hydroxy-4'-methoxyacetophenone), the main active compound of traditional Chinese remedy Paeonia lactiflora Pallas, exhibits anti-inflammatory and anti-tumor activity; whether paeonol regulates metastatic chondrosarcoma is largely unknown. Here, we find paeonol do not increase apoptosis. By contrast, at non-cytotoxic concentrations, paeonol suppresses migration and invasion of chondrosarcoma cells. We also demonstrate paeonol enhancing miR-141 expression and miR-141 inhibitor reversing paeonol-inhibited cell motility; paeonol also reduces protein kinase C (PKC)d and c-Src kinase activity. Since paeonol inhibits migration and invasion of human chondrosarcoma via up-regulation of miR-141 via PKCd and c-Src pathways, it thus might be a novel anti-metastasis agent for treatment of metastatic chondrosarcoma.
Assuntos
Acetofenonas/farmacologia , Condrossarcoma/metabolismo , MicroRNAs/metabolismo , Proteína Quinase C-delta/metabolismo , Regulação para Cima , Quinases da Família src/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Humanos , MicroRNAs/genética , Transdução de SinaisRESUMO
Chondrosarcoma, a primary malignant bone cancer, has a potent capacity to invade locally and cause distant metastasis, especially to the lungs. Patients diagnosed with it have poor prognosis. Naringin, polymethoxylated flavonoid commonly found in citrus fruits, has anti-oxidant, anti-inflammatory and anti-tumor activity; whether naringin regulates migration of chondrosarcoma is largely unknown. Here we report that naringin does not expedite apoptosis in human chondrosarcoma. By contrast, at noncytotoxic concentrations, naringin suppressed migration and invasion of chondrosarcoma cells. Vascular cell adhesion molecule-1 (VCAM-1) of the immunoglobulin superfamily is linked with metastasis; we found incubation of chondrosarcoma cells with naringin reducing mRNA transcription for, and cell surface expression of, VCAM-1. We also observed that naringin enhancing miR-126 expression, and miR-126 inhibitor reversed the naringin-inhibited cell motility and VCAM-1 expression. Therefore, naringin inhibits migration and invasion of human chondrosarcoma via down-regulation of VCAM-1 by increasing miR-126. Thus, naringin may be a novel anti-migration agent for the treatment of migration in chondrosarcoma.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Condrossarcoma/tratamento farmacológico , Flavanonas/farmacologia , Molécula 1 de Adesão de Célula Vascular/metabolismo , Apoptose/efeitos dos fármacos , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Condrossarcoma/patologia , Citrus , Frutas , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Terapia de Alvo Molecular , Invasividade Neoplásica/prevenção & controle , Molécula 1 de Adesão de Célula Vascular/genéticaRESUMO
BACKGROUND: Leptin, an adipocyte-secreted hormone that centrally regulates weight control, may exert proinflammatory effects in the joint, depending on the immune response. Leptin is abundantly expressed in osteoarthritis (OA) cartilage and synovium. However, the relationship between leptin and interleukin-6 (IL-6) in OA synovial fibroblasts (OASFs) remains obscure. METHODOLOGY/PRINCIPAL FINDINGS: Stimulation of OASFs with leptin induced IL-6 expression in a concentration- and time-dependent manner. OASFs expressed the long (OBRl) and short (OBRs) isoforms of the leptin receptor. However, OBRl, but not OBRs, antisense oligonucleotide (AS-ODN) abolished the leptin-mediated increase of IL-6 expression. Transfection with insulin receptor substrate (IRS)-1 siRNA decreased leptin-induced IL-6 production. In addition, pretreatment of cells with PI3K, Akt, or AP-1 inhibitor also inhibited the potentiating action of leptin. Leptin-induced AP-1 activation was inhibited by OBRl, IRS-1, PI3K, or Akt inhibitors and siRNAs. CONCLUSIONS/SIGNIFICANCE: Our results showed that leptin activates the OBRl receptor, which in turn activates IRS-1, PI3K, Akt, and AP-1 pathway, leading to up-regulation of IL-6 expression.
Assuntos
Fibroblastos/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Leptina/metabolismo , Receptores para Leptina/metabolismo , Transdução de Sinais/genética , Membrana Sinovial/metabolismo , Células Cultivadas , Humanos , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Leptina/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , Receptores para Leptina/genética , TransfecçãoRESUMO
In this study, we investigated the anticancer effects of a new benzimidazole derivative, 1-benzyl-2-phenyl -benzimidazole (BPB), in human chondrosarcoma cells. BPB-mediated apoptosis was assessed by the MTT assay and flow cytometry analysis. The in vivo efficacy was examined in a JJ012 xenograft model. Here we found that BPB induced apoptosis in human chondrosarcoma cell lines (JJ012 and SW1353) but not in primary chondrocytes. BPB induced upregulation of Bax, Bad and Bak, downregulation of Bcl-2, Bid and Bcl-XL and dysfunction of mitochondria in chondrosarcoma. In addition, BPB also promoted cytosolic releases AIF and Endo G. Furthermore, it triggered extrinsic death receptor-dependent pathway, which was characterized by activating Fas, FADD and caspase-8. Most importantly, animal studies revealed a dramatic 40% reduction in tumor volume after 21 days of treatment. Thus, BPB may be a novel anticancer agent for the treatment of chondrosarcoma.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Benzimidazóis/farmacologia , Neoplasias Ósseas/patologia , Condrossarcoma/patologia , Animais , Benzimidazóis/química , Células Cultivadas , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Camundongos SCID , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
INTRODUCTION: Thrombin is a key factor in the stimulation of fibrin deposition, angiogenesis, and proinflammatory processes. Abnormalities in these processes are primary features of osteoarthritis (OA). Heme oxygenase (HO)-1 is a stress-inducible rate-limiting enzyme in heme degradation that confers cytoprotection against oxidative injury. Here, we investigated the intracellular signaling pathways involved in thrombin-induced HO-1 expression in human synovial fibroblasts (SFs). METHODS: Thrombin-mediated HO-1 expression was assessed with quantitative real-time (q)PCR. The mechanisms of action of thrombin in different signaling pathways were studied by using Western blotting. Knockdown of protease-activated receptor (PAR) proteins was achieved by transfection with siRNA. Chromatin immunoprecipitation assays were used to study in vivo binding of Nrf2 to the HO-1 promoter. Transient transfection was used to examine HO-1 activity. RESULTS: Osteoarthritis synovial fibroblasts (OASFs) showed significant expression of thrombin, and expression was higher than in normal SFs. OASFs stimulation with thrombin induced concentration- and time-dependent increases in HO-1 expression. Pharmacologic inhibitors or activators and genetic inhibition by siRNA of protease-activated receptors (PARs) revealed that the PAR1 and PAR3 receptors, but not the PAR4 receptor, are involved in thrombin-mediated upregulation of HO-1. Thrombin-mediated HO-1 expression was attenuated by thrombin inhibitor (PPACK), PKCδ inhibitor (rottlerin), or c-Src inhibitor (PP2). Stimulation of cells with thrombin increased PKCδ, c-Src, and Nrf2 activation. CONCLUSION: Our results suggest that the interaction between thrombin and PAR1/PAR3 increases HO-1 expression in human synovial fibroblasts through the PKCδ, c-Src, and Nrf2 signaling pathways.
Assuntos
Fibroblastos/enzimologia , Heme Oxigenase-1/fisiologia , Receptor PAR-1/fisiologia , Receptores de Trombina/fisiologia , Transdução de Sinais/fisiologia , Líquido Sinovial/enzimologia , Trombina/fisiologia , Células Cultivadas , Fibroblastos/fisiologia , Humanos , Receptores Ativados por Proteinase/fisiologia , Líquido Sinovial/fisiologiaRESUMO
Hepatocyte growth factor (HGF) has been demonstrated to stimulate osteoblast proliferation and participated bone remodeling. Bone morphogenetic protein-2 (BMP-2) is a crucial mediator in bone formation during fracture healing. However, the effects of HGF in BMP-2 expression in human osteoblasts are large unknown. Here we found that HGF induced BMP-2 expression in human osteoblasts dose-dependently. HGF-mediated BMP-2 production was attenuated by c-Met inhibitor or siRNA. Pretreatment with FAK inhibitor or JNK inhibitor (SP600125) also blocked the potentiating action of HGF. Stimulation of osteoblasts with HGF enhanced FAK phosphorylation, JNK phosphorylation, and RunX2 translocation from cytosol to the nucleus. HGF-mediated Runx2 binding to BMP-2 promoter was inhibited by c-Met inhibitor, FAK inhibitor, and SP600125. The binding of Runx2 to the BMP-2 promoter, as well as the recruitment of p300 and the enhancement of histones H3 and H4 acetylation on the BMP-2 promoter was enhanced by HGF. Our results suggest that HGF increased BMP-2 production in human osteoblasts via the c-Met receptor/FAK/JNK/Runx2 and p300 signaling pathways.
Assuntos
Proteína Morfogenética Óssea 2/biossíntese , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Proteína p300 Associada a E1A/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Fator de Crescimento de Hepatócito/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Sequência de Bases , Proteína Morfogenética Óssea 2/genética , Linhagem Celular , Subunidade alfa 1 de Fator de Ligação ao Core/antagonistas & inibidores , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Quinase 1 de Adesão Focal/antagonistas & inibidores , Quinase 1 de Adesão Focal/genética , Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Proteínas Recombinantes/farmacologiaRESUMO
The maturation of mastoparan B, the major toxin peptide in the venom of Vespa basalis, requires enzymatic cleavage of its prosequence presumably via sequential liberation of dipeptides. The putative processing enzyme, dipeptidyl peptidase IV, was expressed as a glycosylated His-tag fusion protein (rDPP-IV) via the baculovirus expression system. rDPP-IV purified by one-step nickel-affinity chromatography was verified by Western blot and LC-MS/MS analysis. The k(cat)/K(m) of rDPP-IV was determined to be in the range of 10-500 mM(-1)·S(-1) for five synthetic substrates. The optimal temperature and pH for rDPP-IV were determined to be 50 °C and pH 9. Enzymatic activity of rDPP-IV was significantly reduced by 80 and 60% in the presence of sitagliptin and phenylmethylsulfonyl fluoride respectively.
Assuntos
Dipeptidil Peptidase 4/genética , Dipeptidil Peptidase 4/metabolismo , Vespas/enzimologia , Vespas/genética , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Linhagem Celular , Cromatografia de Afinidade , Dipeptidil Peptidase 4/química , Dipeptidil Peptidase 4/isolamento & purificação , Expressão Gênica , Dados de Sequência MolecularRESUMO
Chondrosarcoma is a malignant primary bone tumor that responds poorly to both chemotherapy and radiation therapy. (-)-Epigallocatechin-3-gallate (EGCG), the major polyphenol in green tea, has been shown to inhibit tumorigenesis and cancer cell growth in animal models. The aim of this study was to elucidate the mechanism of EGCG-induced apoptosis of human chondrosarcoma cells. EGCG induced cell apoptosis in human chondrosarcoma cell lines but not primary chondrocytes. EGCG induced upregulation of Bax and Bak, downregulation of Bcl-2 and Bcl-XL, and dysfunction of mitochondria in chondrosarcoma. We also found that the accumulation of reactive oxygen species (ROS) is a critical mediator in EGCG-induced cell death. EGCG induced apoptosis signal-regulating kinase 1 (ASK1) dephosphorylation and its dissociation from 14-3-3. Treatment of chondrosarcoma cells with EGCG induced p38 and c-jun-NH2-kinase (JNK) phosphorylation. Transfection with ASK1 siRNA or p38 and JNK mutant antagonized the EGCG-induced cell apoptosis. Therefore, EGCG triggered ROS and activated the ASK1-p38/JNK pathway, resulting chondrosarcoma cell death. Importantly, animal studies revealed a dramatic reduction in tumor volume after 24 days of treatment. Thus, EGCG may be a novel anti-cancer agent for the treatment of chondrosarcoma.
Assuntos
Apoptose/efeitos dos fármacos , Catequina/análogos & derivados , Condrossarcoma/tratamento farmacológico , Condrossarcoma/metabolismo , MAP Quinase Quinase Quinase 5/metabolismo , Animais , Anticarcinógenos/farmacologia , Anticarcinógenos/uso terapêutico , Western Blotting , Catequina/farmacologia , Catequina/uso terapêutico , Linhagem Celular Tumoral , Citometria de Fluxo , Humanos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos SCID , RNA Interferente Pequeno , Espécies Reativas de Oxigênio/metabolismo , Rodaminas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The positive inotropic effect of cardiac glycosides lies in their reversible inhibition on the membrane-bound Na(+)/K(+)-ATPase in human myocardium. Steroid-like compounds containing a core structure similar to cardiac glycosides are found in many Chinese medicines conventionally used for promoting blood circulation. Some of them are demonstrated to be Na(+)/K(+)-ATPase inhibitors and thus putatively responsible for their therapeutic effects via the same molecular mechanism as cardiac glycosides. On the other hand, magnesium lithospermate B of danshen is also proposed to exert its cardiac therapeutic effect by effectively inhibiting Na(+)/K(+)-ATPase. Theoretical modeling suggests that the number of hydrogen bonds and the strength of hydrophobic interaction between the effective ingredients of various medicines and residues around the binding pocket of Na(+)/K(+)-ATPase are crucial for the inhibitory potency of these active ingredients. Ginsenosides, the active ingredients in ginseng and sanqi, substantially inhibit Na(+)/K(+)-ATPase when sugar moieties are attached only to the C-3 position of their steroid-like structure, equivalent to the sugar position in cardiac glycosides. Their inhibitory potency is abolished, however, when sugar moieties are linked to C-6 or C-20 position of the steroid nucleus; presumably, these sugar attachments lead to steric hindrance for the entrance of ginsenosides into the binding pocket of Na(+)/K(+)-ATPase. Neuroprotective effects of cardiac glycosides, several steroid-like compounds, and magnesium lithospermate B against ischemic stroke have been accordingly observed in a cortical brain slice-based assay model, and cumulative data support that effective inhibitors of Na(+)/K(+)-ATPase in the brain could be potential drugs for the treatment of ischemic stroke.
Assuntos
Circulação Sanguínea/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Animais , Glicosídeos Cardíacos/farmacologia , Medicamentos de Ervas Chinesas/química , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Modelos TeóricosRESUMO
BACKGROUND: Long-term storage (aging) with periodic drying of fresh oolong tea gives rise to so-called old oolong tea. Alteration of aroma compounds is expected when a fresh oolong tea is converted into an old one, as the two teas smell drastically different. The aim of this study was to compare the volatile compounds in fresh and old oolong teas. RESULTS: Significant differences were observed between the volatile compounds in fresh and old oolong teas. This observation suggested that long straight chains of alcohols and acids were putatively decomposed while shorter-chain acids, their amide derivatives and many nitrogen-containing compounds were generated during the tea conversion processes. The overall patterns of volatile compounds observed in five different preparations of old oolong tea were fundamentally identical. This consensus pattern was different from that observed in oolong tea either stored for more than 10 years without drying or prepared at relatively low temperatures and short baking time. CONCLUSION: Characteristic aroma nitrogen-containing compounds, including N-ethylsuccinimide, 2-acetylpyrrole, 2-formylpyrrole and 3-pyridinol, were consistently found in the examined old oolong teas. These compounds might be regarded as typical constituents at least for a certain kind of old oolong tea.
Assuntos
Camellia sinensis/química , Dessecação , Manipulação de Alimentos/métodos , Chá/química , Compostos Orgânicos Voláteis/análise , Odorantes , Folhas de Planta/química , TemperaturaRESUMO
AIM: To examine if steroid-like compounds found in many Chinese medicinal products conventionally used for the promotion of blood circulation may act as active components via the same molecular mechanism triggered by cardiac glycosides, such as ouabain. METHODS: The inhibitory potency of ouabain and the identified steroid-like compounds on Na(+)/K(+)-ATPase activity was examined and compared. Molecular modeling was exhibited for the docking of these compounds to Na(+)/K(+)-ATPase. RESULTS: All the examined steroid-like compounds displayed more or less inhibition on Na(+)/K(+)-ATPase, with bufalin (structurally almost equivalent to ouabain) exhibiting significantly higher inhibitory potency than the others. In the pentacyclic triterpenoids examined, ursolic acid and oleanolic acid were moderate inhibitors of Na(+)/K(+)-ATPase, and their inhibitory potency was comparable to that of ginsenoside Rh2. The relatively high inhibitory potency of ursolic acid or oleanolic acid was due to the formation of a hydrogen bond between its carboxyl group and the Ile322 residue in the deep cavity close to two K(+) binding sites of Na(+)/K(+)-ATPase. Moreover, the drastic difference observed in the inhibitory potency of ouabain, bufalin, ginsenoside Rh2, and pentacyclic triterpenoids is ascribed mainly to the number of hydrogen bonds and partially to the strength of hydrophobic interaction between the compounds and residues around the deep cavity of Na(+)/K(+)-ATPase. CONCLUSION: Steroid-like compounds seem to contribute to therapeutic effects of many cardioactive Chinese medicinal products. Chinese herbs, such as Prunella vulgaris L, rich in ursolic acid, oleanolic acid and their glycoside derivatives may be adequate sources for cardiac therapy via effective inhibition on Na(+)/K(+)-ATPase.