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1.
Int J Cancer ; 135(6): 1487-96, 2014 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-24347491

RESUMO

Regorafenib, a novel multikinase inhibitor, has recently demonstrated overall survival benefits in metastatic colorectal cancer (CRC) patients. Our study aimed to gain further insight into the molecular mechanisms of regorafenib and to assess its potential in combination therapy. Regorafenib was tested alone and in combination with irinotecan in patient-derived (PD) CRC models and a murine CRC liver metastasis model. Mechanism of action was investigated using in vitro functional assays, immunohistochemistry and correlation with CRC-related oncogenes. Regorafenib demonstrated significant inhibition of growth-factor-mediated vascular endothelial growth factor receptor (VEGFR) 2 and VEGFR3 autophosphorylation, and intracellular VEGFR3 signaling in human umbilical vascular endothelial cells (HuVECs) and lymphatic endothelial cells (LECs), and also blocked migration of LECs. Furthermore, regorafenib inhibited proliferation in 19 of 25 human CRC cell lines and markedly slowed tumor growth in five of seven PD xenograft models. Combination of regorafenib with irinotecan significantly delayed tumor growth after extended treatment in four xenograft models. Reduced CD31 staining indicates that the antiangiogenic effects of regorafenib contribute to its antitumor activity. Finally, regorafenib significantly delayed disease progression in a murine CRC liver metastasis model by inhibiting the growth of established liver metastases and preventing the formation of new metastases in other organs. In addition, our results suggest that regorafenib displays antimetastatic activity, which may contribute to its efficacy in patients with metastatic CRC. Combination of regorafenib and irinotecan demonstrated an increased antitumor effect and could provide a future treatment option for CRC patients.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Compostos de Fenilureia/farmacologia , Piridinas/farmacologia , Animais , Camptotecina/administração & dosagem , Camptotecina/análogos & derivados , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Irinotecano , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/secundário , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Metástase Neoplásica , Compostos Organoplatínicos/farmacologia , Oxaliplatina , Compostos de Fenilureia/administração & dosagem , Piridinas/administração & dosagem , Distribuição Aleatória , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
2.
FASEB J ; 25(10): 3325-35, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21685330

RESUMO

Lymphatic metastasis constitutes a critical route of disease dissemination, which limits the prognosis of patients with pancreatic ductal adenocarcinoma (PDAC). As lymphangiogenesis has been implicated in stimulation of lymphatic metastasis by vascular endothelial growth factor-C (VEGF-C) and VEGF-D, we studied the effect of the angioregulatory growth factor angiopoietin-2 (Ang-2) on PDAC progression. Ang-2 was found to be expressed in transformed cells of human PDAC specimens, with corresponding Tie-2 receptors present on blood and lymphatic endothelium. In vitro in PDAC cells, Ang-2 was subject to autocrine/paracrine TGF-ß stimulation (2-fold induction, P=0.0106) acting on the -61- to +476-bp element of the human Ang-2 promoter. In turn, Ang-2 regulated the expression of genes involved in cell motility and tumor suppression. Orthotopic PDAC xenografts with forced expression of Ang-2, but not Ang-1, displayed increased blood and lymphatic vessel density, and an enhanced rate of lymphatic metastasis (6.7- to 9.1-fold, P<0.01), which was prevented by sequestration of Ang-2 via coexpression of soluble Tie-2. Notably, elevated circulating Ang-2 in patients with PDAC correlated with the extent of lymphatic metastasis. Furthermore, median survival was reduced from 28.4 to 7.7 mo in patients with circulating Ang-2 ≥ 75th percentile (P=0.0005). These findings indicate that Ang-2 participates in the control of lymphatic metastasis, constitutes a noninvasive prognostic biomarker, and may provide an accessible therapeutic target in PDAC.


Assuntos
Adenocarcinoma/patologia , Angiopoietina-2/metabolismo , Metástase Linfática/fisiopatologia , Neoplasias Pancreáticas/patologia , Adenocarcinoma/metabolismo , Adenocarcinoma/secundário , Idoso , Angiopoietina-2/sangue , Angiopoietina-2/genética , Animais , Biomarcadores , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Linfangiogênese/fisiologia , Masculino , Camundongos , Camundongos SCID , Neoplasias Experimentais , Neoplasias Pancreáticas/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor TIE-2/genética , Receptor TIE-2/metabolismo
3.
Int J Cancer ; 129(1): 245-55, 2011 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-21170960

RESUMO

Angiogenesis, a critical driver of tumor development, is controlled by interconnected signaling pathways. Vascular endothelial growth factor receptor (VEGFR) 2 and tyrosine kinase with immunoglobulin and epidermal growth factor homology domain 2 play crucial roles in the biology of normal and tumor vasculature. Regorafenib (BAY 73-4506), a novel oral multikinase inhibitor, potently inhibits these endothelial cell kinases in biochemical and cellular kinase phosphorylation assays. Furthermore, regorafenib inhibits additional angiogenic kinases (VEGFR1/3, platelet-derived growth factor receptor-ß and fibroblast growth factor receptor 1) and the mutant oncogenic kinases KIT, RET and B-RAF. The antiangiogenic effect of regorafenib was demonstrated in vivo by dynamic contrast-enhanced magnetic resonance imaging. Regorafenib administered once orally at 10 mg/kg significantly decreased the extravasation of Gadomer in the vasculature of rat GS9L glioblastoma tumor xenografts. In a daily (qd)×4 dosing study, the pharmacodynamic effects persisted for 48 hr after the last dosing and correlated with tumor growth inhibition (TGI). A significant reduction in tumor microvessel area was observed in a human colorectal xenograft after qd×5 dosing at 10 and 30 mg/kg. Regorafenib exhibited potent dose-dependent TGI in various preclinical human xenograft models in mice, with tumor shrinkages observed in breast MDA-MB-231 and renal 786-O carcinoma models. Pharmacodynamic analyses of the breast model revealed strong reduction in staining of proliferation marker Ki-67 and phosphorylated extracellular regulated kinases 1/2. These data demonstrate that regorafenib is a well-tolerated, orally active multikinase inhibitor with a distinct target profile that may have therapeutic benefit in human malignancies.


Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Compostos de Fenilureia/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Feminino , Imageamento por Ressonância Magnética , Camundongos , Camundongos Nus , Fosforilação , Ratos , Ratos Endogâmicos F344
4.
Invest Ophthalmol Vis Sci ; 49(5): 1836-42, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18436817

RESUMO

PURPOSE: To analyze whether tyrosine kinase inhibitors blocking VEGF receptors (PTK787/ZK222584 [PTK/ZK] and ZK261991 [ZK991]) can inhibit not only hemangiogenesis but also lymphangiogenesis and whether treatment with tyrosine kinase inhibitors after corneal transplantation can improve graft survival. METHODS: Inflammatory corneal neovascularization was induced by corneal suture placement. One treatment group received PTK/ZK, and the other treatment group received ZK991. Corneas were analyzed histomorphometrically for pathologic corneal hemangiogenesis and lymphangiogenesis. The inhibitory effect of tyrosine kinase inhibitors on lymphatic endothelial cells (LECs) in vitro was analyzed with a colorimetric (BrdU) proliferation ELISA. Low-risk allogeneic (C57Bl/6 to BALB/c) corneal transplantations were performed; the treatment group received ZK991, and grafts were graded for rejection (for 8 weeks). RESULTS: Treatment with tyrosine kinase inhibitors resulted in a significant reduction of hemangiogenesis (PTK/ZK by 30%, P < 0.001; ZK991 by 53%, P < 0.001) and lymphangiogenesis (PTK/ZK by 70%, P < 0.001; ZK991 by 71%, P < 0.001) in vivo. Inhibition of proliferation of LECs in vitro was also significant and dose dependent (PTK/ZK, P < 0.001; ZK991, P < 0.001). Comparing the survival proportions after corneal transplantation, treatment with ZK991 significantly improved graft survival (68% vs. 33%; P < 0.02). CONCLUSIONS: Tyrosine kinase inhibitors blocking VEGF receptors are potent inhibitors not only of inflammatory corneal hemangiogenesis but also lymphangiogenesis in vivo. Tyrosine kinase inhibitors seem to have the ability to restrain the formation of the afferent and efferent arm of the immune reflex arc and are therefore able to promote graft survival after corneal transplantation.


Assuntos
Neovascularização da Córnea/tratamento farmacológico , Sobrevivência de Enxerto/efeitos dos fármacos , Ceratoplastia Penetrante , Linfangiogênese/efeitos dos fármacos , Inibidores de Proteínas Quinases/uso terapêutico , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Feminino , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fosforilação , Ftalazinas/uso terapêutico , Piridinas/uso terapêutico , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo
5.
Cancer Med ; 5(11): 3176-3185, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27734608

RESUMO

Regorafenib is an orally administered inhibitor of protein kinases involved in tumor angiogenesis, oncogenesis, and maintenance of the tumor microenvironment. Phase III studies showed that regorafenib has efficacy in patients with advanced gastrointestinal stromal tumors or treatment-refractory metastatic colorectal cancer. In clinical studies, steady-state exposure to the M-2 and M-5 metabolites of regorafenib was similar to that of the parent drug; however, the contribution of these metabolites to the overall observed clinical activity of regorafenib cannot be investigated in clinical trials. Therefore, we assessed the pharmacokinetics and pharmacodynamics of regorafenib, M-2, and M-5 in vitro and in murine xenograft models. M-2 and M-5 showed similar kinase inhibition profiles and comparable potency to regorafenib in a competitive binding assay. Inhibition of key target kinases by all three compounds was confirmed in cell-based assays. In murine xenograft models, oral regorafenib, M-2, and M-5 significantly inhibited tumor growth versus controls. Total peak plasma drug concentrations and exposure to M-2 and M-5 in mice after repeated oral dosing with regorafenib 10 mg/kg/day were comparable to those in humans. In vitro studies showed high binding of regorafenib, M-2, and M-5 to plasma proteins, with unbound fractions of ~0.6%, ~0.9%, and ~0.4%, respectively, in murine plasma and ~0.5%, ~0.2%, and ~0.05%, respectively, in human plasma. Estimated free plasma concentrations of regorafenib and M-2, but not M-5, exceeded the IC50 at human and murine VEGFR2, suggesting that regorafenib and M-2 are the primary contributors to the pharmacologic activity of regorafenib in vivo.


Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos/farmacologia , Compostos de Fenilureia/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Inibidores da Angiogênese/farmacocinética , Animais , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , Metaboloma , Metabolômica/métodos , Camundongos , Compostos de Fenilureia/farmacocinética , Ligação Proteica , Mapeamento de Interação de Proteínas , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Quinases/metabolismo , Piridinas/farmacocinética , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Mech Dev ; 119(2): 165-75, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12464430

RESUMO

In endothelial cells that form capillary-like structures in vitro a variety of genes is upregulated as we have demonstrated previously. In addition to well known genes, we also identified genes never described in endothelial cells before. Here, we report the further characterization of one selected gene called cysteine-rich motor neuron 1 (CRIM1). CRIM1 is strongly upregulated in endothelial cells during tube formation and is expressed by a variety of adherent growing cell lines whereas cell lines grown in suspension do not express CRIM1. By using antisense technology we were able to inhibit CRIM1 expression and demonstrate impaired formation of capillary-like structures in vitro in transfected endothelial cells. Furthermore, we show that CRIM1 is a glycosylated type I transmembrane protein, that accumulates at sites of close cell-to-cell contact upon stimulation. Finally, we found CRIM1 protein to be expressed by endothelial cells of the inner lining of blood vessels in vivo. Taken together our results imply a possible role of CRIM1 in capillary formation and maintainance during angiogenesis.


Assuntos
Capilares/metabolismo , Endotélio Vascular/citologia , Proteínas de Membrana , Proteínas Nucleares/fisiologia , Proteínas , Sequência de Aminoácidos , Animais , Northern Blotting , Western Blotting , Receptores de Proteínas Morfogenéticas Ósseas , Membrana Celular/metabolismo , Células Cultivadas , Colágeno/metabolismo , DNA Complementar/metabolismo , Regulação para Baixo , Combinação de Medicamentos , Regulação da Expressão Gênica no Desenvolvimento , Glicosilação , Humanos , Imuno-Histoquímica , Laminina/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Modelos Genéticos , Dados de Sequência Molecular , Neovascularização Fisiológica , Proteínas Nucleares/biossíntese , Oligonucleotídeos Antissenso/farmacologia , Estrutura Terciária de Proteína , Proteoglicanas/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Distribuição Tecidual , Transfecção , Regulação para Cima
7.
Int J Oncol ; 27(3): 669-79, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16077915

RESUMO

The presence of lymphatic metastases is a strong indicator for poor prognosis in patients with ductal pancreatic cancer. In order to better understand the mechanisms controlling lymphatic growth and lymph node metastasis in human ductal pancreatic cancer, we analyzed the expression pattern of the vascular endothelial growth factor-D (VEGF-D), its receptor VEGF-receptor-3 (VEGFR-3) and the lymphatic endothelium-specific hyaluronan receptor LYVE-1 in a panel of 19 primary human ductal pancreatic tumors and 10 normal pancreas specimens. We further addressed the biological function of VEGF-D for induction of lymphatic metastasis in a nude mouse xenograft model using two human ductal pancreatic cancer cell lines with overexpression of VEGF-D. Compared to normal human pancreas, pancreatic cancer tissue showed overexpression of VEGF-D and VEGFR-3 in conjunction with a high lymphatic vascularization as determined by immunohistochemistry and in situ hybridization. Tumors derived from VEGF-D-overexpressing cells had a higher microvessel density compared to their mock-controls, as determined based on CD31 immunohistochemistry. Importantly, these tumors also revealed a significant induction of intra- and peritumoral lymphatics, as judged from immunohistochemical detection of LYVE-1 expression. This was associated with a significant increase in lymphatic vessel invasion by tumor cells and an increased rate of lymphatic metastases, as indicated by pan-cytokeratin reactive cells in lymph nodes. Our results suggest that VEGF-D plays a pivotal role in stimulating lymphangiogenesis and lymphatic metastasis in human ductal pancreatic cancer, and therefore represents a novel therapeutic target for this devastating disease.


Assuntos
Carcinoma Ductal Pancreático/patologia , Neoplasias Pancreáticas/patologia , Fator D de Crescimento do Endotélio Vascular/genética , Animais , Western Blotting , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Imuno-Histoquímica , Hibridização In Situ , Linfangiogênese , Metástase Linfática , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Pâncreas/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante Heterólogo , Fator D de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas de Transporte Vesicular
8.
J Med Chem ; 45(26): 5687-93, 2002 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-12477352

RESUMO

Two readily synthesized anthranilamide, VEGF receptor tyrosine kinase inhibitors have been prepared and evaluated as angiogenesis inhibitors. 2-[(4-Pyridyl)methyl]amino-N-[3-(trifluoromethyl)phenyl]benzamide (5) and N-3-isoquinolinyl-2-[(4-pyridinylmethyl)amino]benzamide (7) potently and selectively inhibit recombinant VEGFR-2 and VEGFR-3 kinases. As a consequence of their physicochemical properties, these anthranilamides readily penetrate cells and are absorbed following once daily oral administration to mice. Both 5 and 7 potently inhibit VEGF-induced angiogenesis in an implant model, with ED(50) values of 7 mg/kg. In a mouse orthotopic model of melanoma, 5 and 7 potently inhibited both the growth of the primary tumor as well as the formation of spontaneous peripheral metastases. The anthranilamides 5 and 7 represent a new structural class of VEGFR kinase inhibitors, which possess potent antiangiogenic and antitumor properties.


Assuntos
Antineoplásicos/síntese química , Benzamidas/síntese química , Inibidores Enzimáticos/síntese química , Isoquinolinas/síntese química , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , ortoaminobenzoatos/síntese química , Administração Oral , Amidas/síntese química , Amidas/química , Amidas/farmacologia , Inibidores da Angiogênese/síntese química , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Benzamidas/química , Benzamidas/farmacologia , Células CHO , Cricetinae , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Isoquinolinas/química , Isoquinolinas/farmacologia , Metástase Linfática , Melanoma/tratamento farmacológico , Melanoma/patologia , Camundongos , Fosforilação , ortoaminobenzoatos/química , ortoaminobenzoatos/farmacologia
9.
Thromb Haemost ; 90(3): 501-10, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12958620

RESUMO

Vascular endothelial growth factor (VEGF) is a potent mitogen for endothelial cells and plays a central role in angiogenesis and vasculogenesis. Therefore, VEGF and its receptors VEGFR-1 and VEGFR-2 are prime targets for anti-angiogenic intervention which is thought to be one of the most promising approaches in cancer therapy. Recently, we have discovered a VEGFR-2-derived peptide ((247)RTELNVGIDFNWEYP(261)) representing a potential binding site to VEGF. Using the spot synthesis technique, systematic D-amino acid substitutional analyses of this peptide were conducted and the resulting D,L-peptides inhibit VEGF binding to VEGFR-2 at half maximal concentration of 30 nM. The serum-stable D,L-peptides further inhibited autophosphorylation of the VEGFR-2 at nanomolar concentrations. Testing of the peptides in a spheroid-based angiogenesis assay demonstrated a potent anti-angiogenic effect in vitro. The rational design of potent and stable anti-angiogenic peptide inhibitors from their parent receptors provides a feasible route to develop novel leads for anti-angiogenic medicines.


Assuntos
Inibidores da Angiogênese/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Peptídeos/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Sequência de Aminoácidos , Inibidores da Angiogênese/síntese química , Sítios de Ligação , Relação Dose-Resposta a Droga , Estabilidade de Medicamentos , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Humanos , Peptídeos/síntese química , Fosforilação/efeitos dos fármacos , Relação Estrutura-Atividade , Veias Umbilicais/citologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
10.
ChemMedChem ; 9(1): 61-6, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24285584

RESUMO

The transcription factors hypoxia-inducible factor-1 and -2 (HIF-1 and HIF-2) orchestrate a multitude of processes that allow tumor cells to survive under conditions of low oxygen and nutrients, and that lead to resistance to some apoptotic pathways and facilitate invasion and metastasis. Therefore, inhibition of transactivation by HIF has become an attractive target in cancer research. Herein we present the results of a cell-based screening approach that led to the discovery of substituted 1H-pyrazole-3-carboxamides. Chemical optimization of the hit class with respect to potency and metabolic stability is described; it resulted in novel 5-(1H-pyrazol-3-yl)-1,2,4-oxadiazoles that inhibit the hypoxia-induced accumulation of HIF-1α and HIF-2α. The HIF inhibitory potency in the screening cell system was improved from IC50 190 to 0.7 nM, and significant parts of the SAR are disclosed. For a key compound, the ability to suppress the hypoxia-induced expression of HIF target genes was studied in A549 human lung adenocarcinoma cells. The same compound shows a favorable pharmacokinetic profile in rats after i.v. and p.o. administration.


Assuntos
Amidas/química , Hipóxia Celular , Oxidiazóis/química , Pirazóis/química , Administração Oral , Amidas/farmacocinética , Amidas/toxicidade , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Meia-Vida , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Injeções Intravenosas , Ratos , Relação Estrutura-Atividade , Transcrição Gênica/efeitos dos fármacos
11.
Target Oncol ; 6(3): 155-62, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21660559

RESUMO

Serum lactate dehydrogenase (LDH) is a well-known clinical surrogate parameter. A high activity of LDH is associated with a poor prognosis in different tumor types. Here we demonstrate by a gene silencing approach that LDH-A is critical for in vivo but not in vitro growth of HT29 colon carcinoma cells. We provide evidence that the suppression of the LDH-A gene leads to an increased level of hypoxia inducible factor 1α (HIF1α) but in consequence not to an increase of HIF1 regulated proteins such as carbonic anhydrase IX (CAIX), vascular endothelial growth factor (VEGF), prolyl-hydroxylase 2 (PHD2), and factor-inhibiting HIF (FIH) in cell cultures and tumor lysates. This effect is independent of LDH activity in vivo. We conclude that LDH-A has an influence on the activity of HIF1α and thus on the adaptation of cells to a hypoxic tumor microenvironment in HT29 colon cells. We suggest the use of LDH-M as a potential therapeutic target for anticancer treatment.


Assuntos
Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Fator 1 Induzível por Hipóxia/metabolismo , L-Lactato Desidrogenase/metabolismo , Animais , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Neoplasias do Colo/enzimologia , Neoplasias do Colo/genética , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Células HT29 , Humanos , Fator 1 Induzível por Hipóxia/genética , Isoenzimas/deficiência , Isoenzimas/genética , Isoenzimas/metabolismo , L-Lactato Desidrogenase/deficiência , L-Lactato Desidrogenase/genética , Lactato Desidrogenase 5 , Camundongos , Camundongos Nus , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Transfecção , Transplante Heterólogo , Regulação para Cima
12.
Chembiochem ; 6(3): 550-7, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15742376

RESUMO

The angiogenesis inhibitor PTK 787/ZK 222584 (PTK/ZK) blocks all known VEGF receptor (VEGFR) tyrosine kinases, including the lymphangiogenic VEGFR3, in the lower nanomolar range. From a panel of 100 kinases only PDGFR, c-kit, and c-fms are inhibited beyond those in the nanomolar range. PTK/ZK functions as a competitive inhibitor at the ATP-binding site of the receptor kinase as shown here in kinetic experiments. The VEGF signal blockade in microvascular endothelial cells (MVEC) results in a blockade of MVEC proliferation (IC50=30 nM), without affecting the proliferation of normal tissue cells and tumor cells. The efficacy of PTK/ZK depends on its continuous presence within the endothelial target cells. Early removal attenuates its antiproliferative activity in vitro. Growth inhibition of endothelial cells is fully reversible as demonstrated by "washout" experiments. Without inhibiting tumor cell proliferation directly, PTK/ZK results in a significant retardation of tumor growth in a number of experimental tumor models of different tissue origin. Combination of PTK/ZK with an antiandrogen revealed additive effects on tumor-growth inhibition. Treatment efficacy was monitored both by tumor weight and by the determination of serum concentrations of the surrogate marker PSA. PTK/ZK is currently being investigated in patients with different solid tumor types for its therapeutic utility. Preliminary data from phase I/II clinical trials of PTK/ZK as a monotherapy suggested a positive safety and tolerability profile, which we interpret to be a consequence of the high selectivity of the drug for a limited number of kinases. Preliminary response, time to progression, and overall survival data were promising.1 Based on these encouraging results, PTK/ZK is currently in Phase III clinical trials for metastatic colorectal cancer.


Assuntos
Neoplasias/tratamento farmacológico , Neoplasias/patologia , Ftalazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Antineoplásicos/química , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Neoplasias/irrigação sanguínea , Neoplasias/metabolismo , Fosforilação/efeitos dos fármacos , Ftalazinas/química , Inibidores de Proteínas Quinases/química , Piridinas/química , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo
13.
Biochem Biophys Res Commun ; 313(1): 80-8, 2004 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-14672701

RESUMO

Signaling between the ligand ephrinB2 and the respective receptors of the EphB class is known to play a vital role during vascular morphogenesis and angiogenesis. The relative contribution of each EphB receptor type present on endothelial cells to these processes remains to be determined. It has been shown that ephrinB2-EphB receptor signal transduction leads to a repulsive migratory behavior of endothelial cells. It remained unclear whether this anti-migratory effect can be mediated by EphB4 signaling alone or whether other EphB receptors are necessary. It also remained unclear whether the kinase activity of EphB4 is pivotal to its action. To answer these questions, we developed a cellular migration system solely dependent on ephrinB2-EphB4 signaling. Using this system, we could show that EphB4 activation leads to the inhibition of cell migration. Furthermore we identified PP2, a known inhibitor of kinases of the Src family, and PD 153035, a known inhibitor of EGF receptor kinase, as inhibitors of EphB4 kinase activity. Using PP2, the inhibition of cell migration by ephrinB2 could be relieved, demonstrating that the kinase function of EphB4 is of prominent importance in this process. These results show that EphB4 activation is not only accompanying ephrinB2 induced repulsive behavior of cells, but is capable of directly mediating this effect.


Assuntos
Movimento Celular/efeitos dos fármacos , Efrina-B2/metabolismo , Efrina-B2/farmacologia , Receptor EphB4/metabolismo , Animais , Células CHO , Células Cultivadas , Cricetinae , Endotélio Vascular/citologia , Inibidores Enzimáticos/farmacologia , Efrina-B2/antagonistas & inibidores , Receptores ErbB/antagonistas & inibidores , Humanos , Microcirculação , Fosforilação , Pirimidinas/farmacologia , Quinazolinas/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Fatores de Crescimento do Endotélio Vascular/farmacologia , Quinases da Família src/antagonistas & inibidores
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