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1.
J Exp Clin Cancer Res ; 37(1): 93, 2018 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-29712569

RESUMO

BACKGROUND: The application of VEGF signaling inhibitors have been associated with more invasive or metastatic behavior of cancers including hepatocellular carcinoma (HCC). We explored the contribution of MET pathway to the enhanced HCC invasion and metastasis by VEGF signaling inhibition, and investigated the antitumor effects of NZ001, a novel dual inhibitor of MET and VEGFR2, in HCC. METHODS: Immunocompetent orthotopic mice model of hepal-6 was established to investigate the effects of either VEGF antibody alone or in combination with the selective MET inhibitor on tumor aggressiveness. The antitumor effects of NZ001 were examined in cultured HCC cells as well as in vivo models. MET gene amplification was determined by SNP 6.0 assay. MET/P-MET expression was detected by IHC. RESULTS: Selective VEGF signaling inhibition by VEGF antibody significantly reduced in vivo tumor growth of the orthotopic mice models, simultaneously also enhanced tumor invasion and metastasis, but inhibiting MET signaling attenuated this side-effect. Further study revealed that hypoxia caused by VEGF signaling inhibition induced HIF-1α nuclear accumulation, subsequently leading to elevated total-MET expression, and synergized with HGF in inducing invasion. NZ001, a novel dual inhibitor of MET and VEGFR2, markedly inhibited both tumor growth and metastasis of HCC, which showed obvious advantages over sorafenib in not inducing more invasive and metastatic behaviors. This effect is more pronounced in HCC with MET amplification and overexpression. CONCLUSIONS: The activation of MET is responsible for the metastasis-promoting effects induced by VEGF inhibition. MET and VEGFR2 dual blockade, NZ001, has advantages over sorafenib in not inducing more invasive and metastatic behaviors; MET amplification and overexpression can be used to identify the subgroup of patients most likely to get the optimal benefit from NZ001 treatment.

2.
Cancer Res ; 73(13): 4086-97, 2013 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-23536557

RESUMO

Neuroblastoma, the most common extracranial pediatric solid tumor, is responsible for 15% of all childhood cancer deaths. Patients frequently present at diagnosis with metastatic disease, particularly to the bone marrow. Advances in therapy and understanding of the metastatic process have been limited due, in part, to the lack of animal models harboring bone marrow disease. The widely used transgenic model, the Th-MYCN mouse, exhibits limited metastasis to this site. Here, we establish the first genetic immunocompetent mouse model for metastatic neuroblastoma with enhanced secondary tumors in the bone marrow. This model recapitulates 2 frequent alterations in metastatic neuroblastoma, overexpression of MYCN and loss of caspase-8 expression. Mouse caspase-8 gene was deleted in neural crest lineage cells by crossing a Th-Cre transgenic mouse with a caspase-8 conditional knockout mouse. This mouse was then crossed with the neuroblastoma prone Th-MYCN mouse. Although overexpression of MYCN by itself rarely caused bone marrow metastasis, combining MYCN overexpression and caspase-8 deletion significantly enhanced bone marrow metastasis (37% incidence). Microarray expression studies of the primary tumors mRNAs and microRNAs revealed extracellular matrix structural changes, increased expression of genes involved in epithelial to mesenchymal transition, inflammation, and downregulation of miR-7a and miR-29b. These molecular changes have been shown to be associated with tumor progression and activation of the cytokine TGF-ß pathway in various tumor models. Cytokine TGF-ß can preferentially promote single cell motility and blood-borne metastasis and therefore activation of this pathway may explain the enhanced bone marrow metastasis observed in this animal model.


Assuntos
Neoplasias da Medula Óssea/enzimologia , Caspase 8/genética , Ganglioneuroblastoma/enzimologia , Neoplasias do Sistema Nervoso Periférico/enzimologia , Proteínas Proto-Oncogênicas/genética , Animais , Neoplasias da Medula Óssea/genética , Neoplasias da Medula Óssea/secundário , Caspase 8/metabolismo , Modelos Animais de Doenças , Ganglioneuroblastoma/genética , Ganglioneuroblastoma/secundário , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MicroRNAs/genética , MicroRNAs/metabolismo , Proteína Proto-Oncogênica N-Myc , Neoplasias do Sistema Nervoso Periférico/genética , Neoplasias do Sistema Nervoso Periférico/patologia , Transcriptoma
3.
Zhongguo Zhong Yao Za Zhi ; 36(8): 1015-8, 2011 Apr.
Artigo em Chinês | MEDLINE | ID: mdl-21809575

RESUMO

OBJECTIVE: To investigate pharmacokinetic parameters of peoniflorin, albiflorin and amygdaloside after administration of Guizhi Fuling capsule in beagle dogs. METHOD: Plasma was collected from forelimb vein of Beagle dogs after oral administration of Guizhi Fuling capsule. HPLC-MS/MS method was used to determine the concentrations of constituents in plasma. The pharmacokinetic parameters were analyzed by program DAS 2.0. RESULT: The limit of quantitation of peoniflorin, albiflorin and amygdaloside were 0.25, 2.64, 0.04 microg x L(-1), respectively. After administrated with different doses, half-life of peoniflorin in dogs were 4.33, 3.62 h, albiflorin were 6.16, 5.91 h, amygdaloside were 2.43, 1.32 h. The AUC(0-t) of all components were related to dose. CONCLUSION: The pharmacokinetic course of peoniflorin, albiflorin and amygdaloside can be described by two-compartment model, and these components have high expose.


Assuntos
Amigdalina/sangue , Benzoatos/sangue , Hidrocarbonetos Aromáticos com Pontes/sangue , Medicamentos de Ervas Chinesas/administração & dosagem , Medicamentos de Ervas Chinesas/farmacocinética , Glucosídeos/sangue , Administração Oral , Animais , Área Sob a Curva , Cápsulas/administração & dosagem , Cápsulas/farmacocinética , Cromatografia Líquida de Alta Pressão/métodos , Cães , Meia-Vida , Masculino , Monoterpenos , Espectrometria de Massas em Tandem/métodos
4.
Pediatr Blood Cancer ; 55(4): 629-38, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20589651

RESUMO

BACKGROUND: Demethylating agents may alter the expression of genes involved in chemotherapy resistance. We conducted a phase I trial to determine the toxicity and molecular effects of the demethylating agent, decitabine, followed by doxorubicin and cyclophosphamide in children with refractory solid tumors. PROCEDURE: Stratum A included children with any solid tumor; Stratum B included neuroblastoma patients only. Patients received a 1-hr decitabine infusion for 7 days, followed by doxorubicin (45 mg/m(2)) and cyclophosphamide (1 g/m(2)) on day 7. Pharmacokinetic studies were performed after the first dose of decitabine. Biological studies included methylation and gene expression analyses of caspase-8, MAGE-1 and fetal hemoglobin (HbF), and expression profiling of pre- and post-treatment peripheral blood and bone marrow cells. RESULTS: The maximum-tolerated dose of decitabine was 5 mg/m(2)/day for 7 days. Dose-limiting toxicities at 10 mg/m(2)/day were neutropenia and thrombocytopenia. Decitabine exhibited rapid clearance from plasma. Three of 9 patients in Stratum A and 4/12 patients in Stratum B had stable disease for > or = 4 months. Sustained MAGE-1 demethylation and increased HbF expression were observed in the majority of patients post-treatment (12/20 and 14/16, respectively). Caspase-8 promoter demethylation and gene expression were seen in 2/7 bone marrow samples. Differentially expressed genes were identified by microarray analysis. CONCLUSION: Low-dose decitabine when combined with doxorubicin/cyclophosphamide has tolerable toxicity in children. However, doses of decitabine capable of producing clinically relevant biologic effects were not well tolerated with this combination. Alternative strategies of combining demethylating agents with non-cytotoxic, biologically targeted agents such as histone deacetylase inhibitors should be explored.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias/tratamento farmacológico , Neuroblastoma/tratamento farmacológico , Adolescente , Adulto , Antígenos de Neoplasias/genética , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Azacitidina/administração & dosagem , Azacitidina/efeitos adversos , Azacitidina/análogos & derivados , Azacitidina/farmacocinética , Caspase 8/genética , Criança , Pré-Escolar , Ciclofosfamida/administração & dosagem , Ciclofosfamida/efeitos adversos , Ciclofosfamida/farmacocinética , Metilação de DNA , Decitabina , Doxorrubicina/administração & dosagem , Doxorrubicina/efeitos adversos , Doxorrubicina/farmacocinética , Feminino , Hemoglobina Fetal/genética , Perfilação da Expressão Gênica , Humanos , Lactente , Masculino , Antígenos Específicos de Melanoma , Proteínas de Neoplasias/genética
5.
Biochim Biophys Acta ; 1783(6): 1055-67, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18342014

RESUMO

Caspase-8 is frequently deleted or silenced in neuroblastoma and other solid tumor such as medulloblastoma and small cell lung carcinoma. Caspase-8 expression can be re-established in neuroblastoma cell lines by treatment with demethylating agents or with IFN-gamma. Here we show that four different retinoic acid (RA) derivatives also increase caspase-8 protein expression in neuroblastoma, medulloblastoma and small cell lung carcinoma cell lines. This increase in protein expression is mirrored by an increase in RNA expression in NB cells. However, the promoter region of the caspase-8 gene was not responsible for the induction of caspase-8 expression. Rather, we identified another intronic region containing a CREB binding site that was required for maximal induction of caspase-8 via RA. DNA-protein interaction assays revealed increased phospho-CREB binding to this response element in RA-treated NB cells. Furthermore, mutations of the CREB binding site completely blocked caspase-8 induction in the luciferase reporter system assay and transfection of dominant-negative form of CREB repressed the up-regulation of caspase-8 by RA. Importantly, RA-released cells maintained caspase-8 expression for at least 2-5 days and were more sensitive to doxorubicin and TNFalpha. Thus, RA treatment in conjunction with TNFalpha and/or subsets of cytotoxic agents may have therapeutic benefits.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Caspase 8/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Neuroblastoma/patologia , Transcrição Genética , Tretinoína/farmacologia , Antibióticos Antineoplásicos/farmacologia , Apoptose/fisiologia , Western Blotting , Carcinoma de Células Pequenas/tratamento farmacológico , Carcinoma de Células Pequenas/genética , Carcinoma de Células Pequenas/patologia , Caspase 8/metabolismo , Proliferação de Células/efeitos dos fármacos , Imunoprecipitação da Cromatina , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Metilação de DNA , Doxorrubicina/farmacologia , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Íntrons/genética , Luciferases/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Meduloblastoma/tratamento farmacológico , Meduloblastoma/genética , Meduloblastoma/patologia , Mutação/genética , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Elementos de Resposta/genética , Transdução de Sinais , Ativação Transcricional , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/farmacologia
6.
Biochim Biophys Acta ; 1763(10): 1000-10, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16979248

RESUMO

Loss of caspase-8 expression and resistance to cytotoxic agents occurs frequently in late stage neuroblastoma (NB). Interferon-gamma (IFN-gamma) induces caspase-8 in NB cells, sensitizing them to death receptor mediated apoptosis. This study characterizes the kinetics of this phenomenon and examines the effects of IFN-gamma on global gene expression to determine whether IFN-gamma responses are achievable at physiologically relevant doses and to define the biological effects of this cytokine. Here we examine the IFN-gamma responses of 16 NB cell lines. A single <5-min exposure to IFN-gamma (0.5 ng/ml) induced caspase-8 expression in all non-expressing cell lines and in 3/6 cell lines which already expressed high caspase-8. This increase in caspase-8 proteins was observed within 16 h and persisted for up to 9 days. Furthermore, IFN-gamma pretreatment of NB cells increased doxorubicin-induced apoptosis nearly 3-fold. Microarray analysis was used to identify additional genes involved in proliferation, signaling and apoptosis whose expression was modulated via IFN-gamma. Altered expression of these genes should further enhance the responsiveness of NB cells to chemotherapeutics. Thus, the use of IFN-gamma to sensitize NB cells to cytotoxic agents represents an attractive therapeutic strategy and warrants further investigation.


Assuntos
Regulação Neoplásica da Expressão Gênica , Interferon gama/farmacologia , Neuroblastoma/metabolismo , Apoptose , Caspase 8/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Doxorrubicina/farmacologia , Sinergismo Farmacológico , Humanos , Metilação , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/metabolismo , Transdução de Sinais , Fatores de Tempo
7.
Biochemistry ; 43(46): 14584-93, 2004 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-15544329

RESUMO

Activation of many Rho family GTPase pathways involves the signaling module consisting of the Dbl-like guanine nucleotide exchange factors (GEFs), the Rho GTPases, and the Rho GTPase specific effectors. The current biochemical model postulates that the GEF-stimulated GDP/GTP exchange of Rho GTPases leads to the active Rho-GTP species, and subsequently the active Rho GTPases interact with and activate the effectors. Here we report an unexpected finding that the Dbl oncoprotein, Cdc42 GTPase, and PAK1 can form a complex through their minimum functional motifs, i.e., the Dbl-homolgy (DH) and Pleckstrin-homology domains of Dbl, Cdc42, and the PBD domain of PAK1. The Dbl-Cdc42-PAK1 complex is sensitive to the nucleotide-binding state of Cdc42 since either dominant negative or constitutively active Cdc42 readily disrupts the ternary binding interaction. The complex formation depends on the interactions between the DH domain of Dbl and Cdc42 and between Cdc42 and the PBD domain of PAK1 and can be reconstituted in vitro by using the purified components. Furthermore, the Dbl-Cdc42-PAK1 ternary complex is active in generating signaling output through the activated PAK1 kinase in the complex. The GEF-Rho-effector ternary intermediate is also found in other Dbl-like GEF, Rho GTPase, and effector interactions. Finally, PAK1, through the PDB domain, is able to accelerate the GEF-induced GTP loading onto Cdc42. These results suggest that signal transduction through Cdc42 and possibly other Rho family GTPases could involve tightly coupled guanine nucleotide exchange and effector activation mechanisms and that Rho GTPase effector may have a feedback regulatory role in the Rho GTPase activation.


Assuntos
Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Oncogênicas de Retroviridae/fisiologia , Transdução de Sinais/fisiologia , Proteína cdc42 de Ligação ao GTP/fisiologia , Animais , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Células COS , Cercopithecus aethiops , Sinergismo Farmacológico , Ativação Enzimática , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Modelos Moleculares , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Oncogênicas de Retroviridae/química , Proteínas Oncogênicas de Retroviridae/metabolismo , Homologia Estrutural de Proteína , Frações Subcelulares/química , Frações Subcelulares/metabolismo , Proteína cdc42 de Ligação ao GTP/química , Proteína cdc42 de Ligação ao GTP/metabolismo , Quinases Ativadas por p21 , Proteínas rho de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/fisiologia
8.
J Biol Chem ; 279(5): 3777-86, 2004 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-14597635

RESUMO

Rho family GTPases play important roles in a variety of cellular processes, including actin cytoskeleton reorganization, transcription activation, and DNA synthesis. Dominant negative mutants of Rho GTPases, such as T17NRac1, that block the endogenous Rho protein activation by sequestering upstream guanine nucleotide exchange factors (GEFs) have been widely used to implicate specific members of the Rho family in various signaling pathways. We show here that such an approach could produce potentially misleading results since many Rho GEFs can interact with multiple Rho proteins promiscuously, and overexpression of one dominant negative Rho protein mutant may affect the activity of other members of the Rho family. Based on the available structural information, we have identified the highly conserved amino acid pairing of Asn(1406)Trio-Asp(65)Rac1 of the GEF-Rho GTPase interaction as the critical catalytic machinery required for the Rac1 GDP/GTP exchange reaction. The N1406A/D1407A mutant of Trio acted dominant negatively in vitro by retaining Rac1 binding activity but losing GEF catalytic activity and competitively inhibited Rac1 activation by wild type Trio. It readily blocked the platelet-derived growth factor (PDGF)-induced lamellipodia formation and inhibited the wild type Trio-induced serum response factor activation. Moreover the mutant was able to selectively inhibit Dbl-induced Rac1 activation without affecting RhoA activity in cells. In contrast to the non-discriminative inhibitory effect displayed by T17NRac1, the Trio mutant was ineffective in inhibiting PDGF-stimulated DNA synthesis and Dbl-induced transformation, revealing the Rac-independent functions of PDGF and Dbl. These studies identify a conserved pair of amino acid residues of the Trio-Rac interaction that is likely to be essential to the GEF catalysis of Rho family GTPases and demonstrate that a dominant negative mutant derived from a Rho GTPase regulator constitutes a new generation of specific inhibitors of Rho GTPase signaling pathways.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Mutação , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Proteínas rac de Ligação ao GTP/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Asparagina/química , Ácido Aspártico/química , Catálise , Sequência Conservada , DNA/metabolismo , DNA Complementar/metabolismo , Ativação Enzimática , Escherichia coli/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Genes Dominantes , Genes Reporter , Glutationa Transferase/metabolismo , Humanos , Luciferases/metabolismo , Camundongos , Microscopia de Fluorescência , Modelos Moleculares , Dados de Sequência Molecular , Células NIH 3T3 , Fator de Crescimento Derivado de Plaquetas/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Retroviridae/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/genética
9.
Mol Cell ; 9(1): 109-19, 2002 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11804590

RESUMO

We have identified ARAP1 and ARAP2 and examined ARAP1 as a possible link between phosphoinositide-, Arf-, and Rho-mediated cell signaling. ARAP1 contains Arf GAP, Rho GAP, Ankyrin repeat, Ras-associating, and five PH domains. In vitro, ARAP1 had Rho GAP and phosphatidylinositol (3,4,5) trisphosphate (PIP3)-dependent Arf GAP activity. ARAP1 associated with the Golgi. The Rho GAP activity mediated cell rounding and loss of stress fibers when ARAP1 was overexpressed. The Arf GAP activity mediated changes in the Golgi apparatus and the formation of filopodia, the latter a consequence of increased cellular activity of Cdc42. The Arf GAP and Rho GAP activities both contributed to inhibiting cell spreading. Thus, ARAP1 is a PIP3-dependent Arf GAP that regulates Arf-, Rho-, and Cdc42-dependent cell activities.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Fosfatidilinositóis/metabolismo , Proteínas/genética , Proteínas/metabolismo , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Células COS , Adesão Celular , Movimento Celular , Clonagem Molecular , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Alinhamento de Sequência
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