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3.
J Cancer Sci Ther ; 2009: 8-18, 2010 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-20824118

RESUMO

The aim of the present study is to identify an effective and efficient expression system for purification of recombinant antiangiogenic protein tumstatin. The sequence encoding carboxy-terminal non-collagenous domain of α3 chain Type IV collagen, α3(IV)NC1 (tumstatin) was isolated from human placental tissue and cloned in three different expression vectors pET22b, pcBFT and pAcHLT-A to express it in bacteria, mammalian and Sf-9 insect cells respectively. Expression and purification profiles of tumstatin were evaluated by coomassie staining and immunoblotting, and the efficiency was determined based on the yields of soluble protein. Our results indicate that, baculovirus expression system was efficient for scalable yields of soluble protein that could be purified in its biologically active form. This baculovirus expressed tumstatin was used to evaluate its anti-angiogenic and anti-tumarogenic functions such as inhibition of endothelial cell proliferation, cell viability, migration, tube formation, cap dependent protein translation and the associated signaling mechanism including in-vivo tumor study. Our evaluated approaches using a modified baculovirus expression system shows high expression and high yield of biologically active tumstatin, as compared to two expression systems, indicating baculovirus expression system to be an ideal method for bulk production of soluble tumstatin that needed for pre-clinical and clinical trails.

4.
PLoS One ; 5(8): e12029, 2010 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-20808434

RESUMO

BACKGROUND: Weak intermolecular interactions such as hydrogen bonding and hydrophobic interactions are key players in stabilizing energetically-favored ligands, in an open conformational environment of protein structures. However, it is still poorly understood how the binding parameters associated with these interactions facilitate a drug-lead to recognize a specific target and improve drugs efficacy. To understand this, comprehensive analysis of hydrophobic interactions, hydrogen bonding and binding affinity have been analyzed at the interface of c-Src and c-Abl kinases and 4-amino substituted 1H-pyrazolo [3, 4-d] pyrimidine compounds. METHODOLOGY: In-silico docking studies were performed, using Discovery Studio software modules LigandFit, CDOCKER and ZDOCK, to investigate the role of ligand binding affinity at the hydrophobic pocket of c-Src and c-Abl kinase. Hydrophobic and hydrogen bonding interactions of docked molecules were compared using LigPlot program. Furthermore, 3D-QSAR and MFA calculations were scrutinized to quantify the role of weak interactions in binding affinity and drug efficacy. CONCLUSIONS: The in-silico method has enabled us to reveal that a multi-targeted small molecule binds with low affinity to its respective targets. But its binding affinity can be altered by integrating the conformationally favored functional groups at the active site of the ligand-target interface. Docking studies of 4-amino-substituted molecules at the bioactive cascade of the c-Src and c-Abl have concluded that 3D structural folding at the protein-ligand groove is also a hallmark for molecular recognition of multi-targeted compounds and for predicting their biological activity. The results presented here demonstrate that hydrogen bonding and optimized hydrophobic interactions both stabilize the ligands at the target site, and help alter binding affinity and drug efficacy.


Assuntos
Desenho de Fármacos , Interações Hidrofóbicas e Hidrofílicas , Sequência de Aminoácidos , Proteína Tirosina Quinase CSK , Biologia Computacional , Humanos , Ligação de Hidrogênio , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-abl/química , Proteínas Proto-Oncogênicas c-abl/metabolismo , Pirimidinas/química , Pirimidinas/metabolismo , Quinases da Família src
5.
J Cancer Sci Ther ; 2(4): 89-94, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20808724

RESUMO

Prostate cancer (PC) is the second leading cause of cancer deaths in men in America and Western Europe. Epidemiological studies suggest that prostate cancer incidences increased in last few years in Asian. The causes or consequences of increasing trend of prostate cancer incidence are not completely known. Emerging evidences suggest that among the many risk factors, inflammation is the major risk factor for developing prostate cancer and its progression to metastasis. It is proposed that exposure to environmental factors such as infectious agents, dietary agents and saturated lipids leads to injury of the prostate due to chronic inflammation and regenerative risk factor lesions referred to as proliferative inflammatory atrophy (PIA). These phenomena predominantly control by a number of proinflammatory macro molecules such as chemokines, and their receptors. Some recent studies suggest that many of these pro-inflammatory chemokines and their receptors are the products of protooncogenes in many cancers including that of the prostate. This review will focus on the current biology of chemokines and chemokine receptors in prostate cancer. An understanding of this axis may enable researchers to develop targeted strategies for prostate cancer.

6.
Curr Eye Res ; 35(1): 45-55, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20021254

RESUMO

PURPOSE: The potential role of arresten (alpha1(IV)NC1) as an endogenous angiogenesis inhibitor in the prevention of bFGF mediated retinal angiogenesis and regulation of matrix metalloproteinase-2 activation has not been explored. METHODS: Mouse retinal endothelial cells (MREC) were cultured on type IV collagen and treated with basic fibroblast growth factor (bFGF) alone or in the presence of arresten at concentrations ranging from 1 to 10 microg/ml. The proliferation of MRECs were evaluated using 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay, and bFGF stimulated endothelial cell migration was assessed using Boyden chamber. Expression of matrix metalloproteinase-2 (MMP-2) was assessed by reverse transcription polymerase chain reaction (RT-PCR) analysis using RNA isolated from MRECs. Secretion and activation of MMP-2 in arresten-treated conditioned MREC growth medium was determined by gelatin zymography and Western blotting. RESULTS: Different doses of bFGF induced MREC proliferation was significantly inhibited upon arresten treatment (P < 0.005). The bFGF-induced migration was significantly inhibited by arresten at 1 and 10 microg/ml concentrations (P < 0.01). The bFGF stimulated expression of MMP-2 mRNA and secretion of MMP-2 in MREC was not affected and interestingly activation of MMP-2 was suppressed by arresten in a dose and time dependent manner. CONCLUSIONS: Inhibitory effects of arresten on proliferation, migration and MMP-2 activation but not on expression and secretion of MMP-2 in MREC; this early work with arresten supports potential therapeutic action in retinal neovascularization dependent disorders.


Assuntos
Arrestina/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/antagonistas & inibidores , Metaloproteinase 2 da Matriz/metabolismo , Vasos Retinianos/citologia , Animais , Western Blotting , Células Cultivadas , Colorimetria , Endotélio Vascular/citologia , Endotélio Vascular/enzimologia , Ativação Enzimática , Fator 2 de Crescimento de Fibroblastos/farmacologia , Metaloproteinase 2 da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , Proteínas Recombinantes/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
7.
Invest Ophthalmol Vis Sci ; 50(10): 4567-75, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19443723

RESUMO

PURPOSE: To determine the impact of the antiangiogenic factor alpha1(IV)NC1 on vascular endothelial growth factor-mediated proangiogenic activity in mouse retinal endothelial cells (MRECs). METHODS: Primary culture of MRECs was established as previously described and was used to determine the effects of alpha1(IV)NC1 on the proangiogenic activity of VEGF. Cell proliferation was evaluated using [(3)H]-thymidine incorporation and 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide colorimetric assays. Cell migration was determined using modified Boyden chamber and scratch wound assays and tube formation was assessed on basement membrane matrix (BMM). Intracellular signaling events Bcl-2/Bcl-x(L) and caspase-3/poly (ADP-ribose) polymerase (PARP) activities were evaluated in cells stimulated with VEGF and plated on type IV collagen-coated dishes. Apoptosis was assessed by measuring caspase activity and by performing quantitative fluorescence analysis using fluorescence-activated cell sorting assay. Subcutaneously injected VEGF induced in vivo neovascularization was studied with the BMM plug assay. RESULTS: VEGF-induced subconfluent MREC proliferation, migration, and tube formation were significantly inhibited by alpha1(IV)NC1 at 1 muM (P < 0.001). alpha1(IV)NC1 induced MREC apoptosis is mediated by inhibition of Bcl-2 and Bcl-x(L) expression and activation of caspase-3/PARP through FAK/p38-MAPK signaling. In addition, alpha1(IV)NC1 dose dependently inhibited VEGF-mediated neovascularization in vivo. CONCLUSIONS: alpha1(IV)NC1 inhibited VEGF-mediated angiogenesis by promoting apoptosis and caspase-3/PARP activation and by negatively impacting FAK/p38-MAPK phosphorylation, Bcl-2, and Bcl-x(L) expression leading to MREC death. The endothelial-specific inhibitory actions of recombinant alpha1(IV)NC1 may be of benefit in the treatment of a variety of eye diseases with a neovascular component.


Assuntos
Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Colágeno Tipo IV/farmacologia , Endotélio Vascular/efeitos dos fármacos , Quinase 1 de Adesão Focal/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Inibidores da Angiogênese/farmacologia , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Ativação Enzimática , Epitopos , Citometria de Fluxo , Camundongos , Neovascularização Patológica/etiologia , Neovascularização Patológica/prevenção & controle , Fosforilação , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Recombinantes/farmacologia , Vasos Retinianos/citologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Proteína bcl-X/metabolismo
8.
J Cancer Sci Ther ; 1(2): 1-4, 2009 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-20740081
9.
Exp Cell Res ; 314(18): 3292-305, 2008 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-18775695

RESUMO

Physiological and pathological turnover of basement membranes liberates biologically active cryptic molecules. Several collagen-derived fragments possess anti-angiogenic activity. Arresten is the 26-kDa non-collagenous domain of type IV collagen alpha1 chain. It functions as an efficient inhibitor of angiogenesis and tumor growth in mouse models, but its anti-angiogenic mechanism is not completely known. Here we show that arresten significantly increases apoptosis of endothelial cells in vitro by decreasing the amount of anti-apoptotic molecules of the Bcl-family; Bcl-2 and Bcl-xL. Although the pro-apoptotic effect of arresten is endothelial cell specific in vitro, in mouse tumors arresten induced apoptosis both in endothelial and tumor cells. The tumor cell apoptosis is likely an indirect effect due to the inhibition of blood vessel growth into the tumor. The active site of arresten was localized by deletion mutagenesis within the C-terminal half of the molecule. We have previously shown that arresten binds to alpha1beta1 integrin on human umbilical vein endothelial cells. However, the microvascular endothelial cells (MLECs) are more important in the context of tumor vasculature. We show here that arresten binds also to the microvascular endothelial cells via alpha1beta1 integrin. Furthermore, it has no effect on Matrigel neovascularization or the viability of integrin alpha1 null MLECs. Tumors implanted on integrin alpha1 deficient mice show no integrin alpha1 expression in the host-derived vascular endothelium, and thus arresten does not inhibit the tumor growth. Collectively, this data sheds more light into the anti-angiogenic mechanism of arresten.


Assuntos
Inibidores da Angiogênese/farmacologia , Colágeno Tipo IV/química , Células Endoteliais/efeitos dos fármacos , Integrina alfa1beta1/metabolismo , Proteínas Recombinantes/farmacologia , Proteínas Supressoras de Tumor/farmacologia , Inibidores da Angiogênese/genética , Inibidores da Angiogênese/metabolismo , Animais , Aorta/citologia , Apoptose/efeitos dos fármacos , Western Blotting , Bovinos , Linhagem Celular Tumoral , Proliferação de Células , Células Cultivadas , Colágeno Tipo IV/genética , Células Endoteliais/citologia , Humanos , Integrina alfa1beta1/genética , Camundongos , Camundongos Knockout , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Artéria Pulmonar/citologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteína bcl-X/efeitos dos fármacos , Proteína bcl-X/metabolismo
10.
Pharm Res ; 25(12): 2731-9, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18551250

RESUMO

Growing tumors develop additional new blood vessels to meet the demand for adequate nutrients and oxygen, a process called angiogenesis. Cancer is a highly complex disease promoted by excess angiogenesis; interfering with this process poses for an attractive approach for controlling tumor growth. This hypothesis led to the identification of endogenous angiogenesis inhibitors generated from type IV collagen, a major component of vascular basement membrane (VBM). Type IV collagen and the angiogenesis inhibitors derived from it are involved in complex roles, than just the molecular construction of basement membranes. Protease degradation of collagens in VBM occurs in various physiological and pathological conditions and produces several peptides. Some of these peptides are occupied in the regulation of functions conflicting from those of their original integral molecules. Tumstatin (alpha3(IV)NC1), a proteolytic C-terminal non-collagenous (NC1) domain from type IV collagen alpha3 chain has been highlighted recently because of its potential role in anti-angiogenesis, however its biological actions are not limited to these processes. alpha3(IV)NC1 inhibits proliferation by promoting endothelial cell apoptosis and suppresses diverse tumor angiogenesis, thus making it a potential candidate for future cancer therapy. The present review surveys the physiological functions of type IV collagen and discovery of alpha3(IV)NC1 as an antiangiogenic protein with a comprehensive overview of the knowledge gained by us towards understanding its signaling mechanisms.


Assuntos
Inibidores da Angiogênese/farmacologia , Autoantígenos/farmacologia , Colágeno Tipo IV/farmacologia , Neoplasias/tratamento farmacológico , Neovascularização Patológica/prevenção & controle , Transdução de Sinais/fisiologia , Animais , Autoantígenos/uso terapêutico , Colágeno Tipo IV/fisiologia , Colágeno Tipo IV/uso terapêutico , Humanos , Integrina alfa3beta1/fisiologia , Integrina alfaVbeta3/fisiologia , Neoplasias/irrigação sanguínea
11.
Clin Med Oncol ; 2: 73-81, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-21892268

RESUMO

Non-collagenous α3 chain of type IV collagen or α3(IV)NC1, a 28 kDa C-terminal domain of collagen type IV is a specific inhibitor of endothelial cell translation and angiogenesis. In the present study we have cloned and expressed mouse α3(IV)NC1 in baculovirus system. The recombinant protein was expressed in soluble form and tested for several of its biological functions. We identified that this recombinant mouse α3(IV)NC1 specifically inhibited proliferation, translation and tube formation of endothelial cells. Also, we show that α3(IV)NC1 treatment results in apoptosis specifically in proliferating endothelial cells. In addition we report for the first time that mouse α3(IV)NC1 inhibits migration and p38 MAPK phosphorylation in addition to inhibition of FAK/Akt/mTOR/4E-BP1 signaling. In mice α3(IV)NC1 treatment reduced tumor growth and CD-31 positive endothelial vasculature in tumors. Collectively, our data demonstrate the expression of biologically active form of mouse α3(IV)NC1 in Sf-9 cells and provide important mechanistic insights on α3(IV)NC1 antiangiogenic actions in endothelial cells.

12.
Blood ; 110(4): 1168-77, 2007 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-17426256

RESUMO

Human alpha3 chain, a noncollagenous domain of type IV collagen [alpha3(IV)NC1], inhibits angiogenesis and tumor growth. These biologic functions are partly attributed to the binding of alpha3(IV)NC1 to alphaVbeta3 and alpha3beta1 integrins. alpha3(IV)NC1 binds alphaVbeta3 integrin, leading to translation inhibition by inhibiting focal adhesion kinase/phosphatidylinositol 3-kinase/Akt/mTOR/4E-BP1 pathways. In the present study, we evaluated the role of alpha3beta1 and alphaVbeta3 integrins in tube formation and regulation of cyclooxygenase-2 (COX-2) on alpha3(IV)NC1 stimulation. We found that although both integrins were required for the inhibition of tube formation by alpha3(IV)NC1 in endothelial cells, only alpha3beta1 integrin was sufficient to regulate COX-2 in hypoxic endothelial cells. We show that binding of alpha3(IV)NC1 to alpha3beta1 integrin leads to inhibition of COX-2-mediated pro-angiogenic factors, vascular endothelial growth factor, and basic fibroblast growth factor by regulating IkappaBalpha/NFkappaB axis, and is independent of alphaVbeta3 integrin. Furthermore, beta3 integrin-null endothelial cells, when treated with alpha3(IV)NC1, inhibited hypoxia-mediated COX-2 expression, whereas COX-2 inhibition was not observed in alpha3 integrin-null endothelial cells, indicating that regulation of COX-2 by alpha3(IV)NC1 is mediated by integrin alpha3beta1. Our in vitro and in vivo findings demonstrate that alpha3beta1 integrin is critical for alpha3(IV)NC1-mediated inhibition of COX-2-dependent angiogenic signaling and inhibition of tumor progression.


Assuntos
Colágeno Tipo IV/metabolismo , Ciclo-Oxigenase 2/metabolismo , Regulação Neoplásica da Expressão Gênica , Integrina alfa3beta1/fisiologia , Integrina alfaVbeta3/antagonistas & inibidores , Neovascularização Patológica/metabolismo , Teratocarcinoma/metabolismo , Inibidores da Angiogênese/genética , Inibidores da Angiogênese/farmacologia , Animais , Adesão Celular , Hipóxia Celular , Proliferação de Células , Células Cultivadas , Colágeno Tipo IV/genética , Ciclo-Oxigenase 2/química , Inibidores de Ciclo-Oxigenase 2/farmacologia , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Immunoblotting , Imunoprecipitação , Masculino , Camundongos , NF-kappa B/genética , NF-kappa B/metabolismo , Neoplasias , Neovascularização Patológica/tratamento farmacológico , Artéria Pulmonar/citologia , Artéria Pulmonar/metabolismo , Teratocarcinoma/irrigação sanguínea , Teratocarcinoma/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/metabolismo
13.
Gene Regul Syst Bio ; 1: 217-26, 2007 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-19936090

RESUMO

Vascular basement membrane (VBM) derived molecules are regulators of certain biological activities such as cell growth, differentiation and angiogenesis. Angiogenesis is regulated by a systematic controlled balance between VBM derived antiangiogenic factors and proangiogenic growth factors. In the normal physiological state, equilibrium is maintained between the antiangiogenic and proangiogenic factors. The antiangiogenic factors (molecules), which are generated by the proteolytic cleavage of the VBM, include; alpha1 chain non-collagenous (NC1) domain of type XVIII collagen (endostatin) and the NC1 domains from the alpha chains of Type IV collagen considered as endogenous angiogenesis inhibitors. These collagen derived NC1 domains have a pivotal role in the regulation of tumor angiogenesis, thus making them attractive alternate candidates for cancer therapies. In this review we illustrate a comprehensive overview of the knowledge gained from the signaling mechanisms of Type IV collagen derived endogenous inhibitors in angiogenesis.

14.
Protein Expr Purif ; 49(2): 211-8, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16631378

RESUMO

alpha1(IV)NC1, a cleavage fragment of the carboxy terminal non-collagenous human alpha1 chain of type IV collagen, is derived from the extracellular matrix specifically by MMP-2. Recently we determined the in vitro and in vivo anti-angiogenic activity of alpha1(IV)NC1 and presently, its role in cancer therapy is under evaluation. To characterize alpha1(IV)NC1 as a potential candidate for drug development and to test its efficacy in animal models, an effective method to produce a purified active form of alpha1(IV)NC1 is needed. In the present study, expression of alpha1(IV)NC1 in Sf9 cells using baculovirus expression system was discussed, this method was found to be effective in the production of a functionally active soluble form of the recombinant protein. The purified protein showed its characteristic activities such as inhibiting cell proliferation, migration, and tube formation in endothelial cells.


Assuntos
Inibidores da Angiogênese/genética , Inibidores da Angiogênese/isolamento & purificação , Colágeno Tipo IV/genética , Colágeno Tipo IV/isolamento & purificação , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Clonagem Molecular , Colágeno Tipo IV/farmacologia , Colágeno Tipo IV/uso terapêutico , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Expressão Gênica , Humanos , Neoplasias/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Estrutura Terciária de Proteína/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Spodoptera/citologia
15.
J Clin Invest ; 115(10): 2801-10, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16151532

RESUMO

Human noncollagenous domain 1 of the alpha1 chain of type IV collagen [alpha1(IV)NC1], or arresten, is derived from the carboxy terminal of type IV collagen. It was shown to inhibit angiogenesis and tumor growth in vivo; however, the mechanisms involved are not known. In the present study we demonstrate that human alpha1(IV)NC1 binds to alpha1beta1 integrin, competes with type IV collagen binding to alpha1beta1 integrin, and inhibits migration, proliferation, and tube formation by ECs. Also, alpha1(IV)NC1 pretreatment inhibited FAK/c-Raf/MEK/ERK1/2/p38 MAPK activation in ECs but had no effect on the PI3K/Akt pathway. In contrast, alpha1(IV)NC1 did not affect proliferation, migration, or the activation of FAK/c-Raf/MEK1/2/p38/ERK1 MAPK pathway in alpha1 integrin receptor knockout ECs. Consistent with this, alpha1(IV)NC1 elicited significant antiangiogenic effects and tumor growth inhibition in vivo but failed to do the same in alpha1 integrin receptor knockout mice. This suggests a highly specific, alpha1beta1 integrin-dependent antiangiogenic activity of alpha1(IV)NC1. In addition, alpha1(IV)NC1 inhibited hypoxia-induced expression of hypoxia-inducible factor 1alpha and VEGF in ECs cultured on type IV collagen by inhibiting ERK1/2 and p38 activation. This unravels a hitherto unknown function of human alpha1(IV)NC1 and suggests a critical role for integrins in hypoxia and hypoxia-induced angiogenesis. Collectively, the above data indicate that alpha1(IV)NC1 is a potential therapeutic candidate for targeting tumor angiogenesis.


Assuntos
Inibidores da Angiogênese/metabolismo , Colágeno Tipo IV/metabolismo , Células Endoteliais/metabolismo , Sangue Fetal/metabolismo , Integrina alfa1beta1/metabolismo , Sistema de Sinalização das MAP Quinases , Neovascularização Patológica/metabolismo , Inibidores da Angiogênese/genética , Inibidores da Angiogênese/farmacologia , Hipóxia Celular/efeitos dos fármacos , Colágeno Tipo IV/genética , Colágeno Tipo IV/farmacologia , Células Endoteliais/citologia , Ativação Enzimática , Sangue Fetal/citologia , Deleção de Genes , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Integrina alfa1beta1/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neovascularização Patológica/tratamento farmacológico , Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína/genética , Fator A de Crescimento do Endotélio Vascular/biossíntese
16.
Proc Natl Acad Sci U S A ; 102(8): 2934-9, 2005 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-15710885

RESUMO

Disruption of the systemic angiogenesis balance to favor enhanced angiogenesis is speculated to represent a key step in the growth of tumors. Although a major emphasis has been placed on the increase of angiogenesis stimulators, such as VEGF, on the disruption of the angiogenic balance, the potential role of the physiological levels of endogenous inhibitors of angiogenesis on tumor growth is poorly understood. Here, we use three independent lines of mice deficient in tumstatin, endostatin, or thrombospondin-1 (TSP-1), to address the role that these endogenous angiogenesis inhibitors play in tumor growth. Our experiments demonstrate that normal physiological levels of these inhibitors serve to retard the growth of tumors, and that their absence leads to enhanced angiogenesis and a 2- to 3-fold increase in tumor growth. The tumor-suppressive action of TSP-1, endostatin, and tumstatin correlates with expression of CD36 receptor, alpha5beta1 integrin, and alphavbeta3 integrin on proliferating endothelial cells, respectively. Moreover, tumors grow 2-fold faster in the tumstatin/TSP-1 double-knockout mice, compared with either the tumstatin- or the TSP-1-deficient mice, strongly suggesting that ceiling rate of cancer growth is not completely dependent on the genetic defects of cancer cells but also depends on the host-derived tumor microenvironment. Additionally, tumor growth in transgenic mice overproducing endostatin specifically in the endothelial cells (a 1.6-fold increase in the circulating levels; mimicking Down's syndrome condition) is 3-fold slower than the tumor growth in wild-type mice. Collectively, our data suggest that physiological levels of endogenous inhibitors of angiogenesis can serve as endothelium-specific tumor suppressors.


Assuntos
Inibidores da Angiogênese/fisiologia , Autoantígenos/fisiologia , Colágeno Tipo IV/fisiologia , Endostatinas/fisiologia , Trombospondina 1/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Linhagem Celular Tumoral , Colágeno Tipo IV/biossíntese , Colágeno Tipo XVIII/biossíntese , Integrina beta3/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/irrigação sanguínea , Neoplasias Experimentais/patologia , Neoplasias Experimentais/prevenção & controle
17.
Cancer Res ; 64(5): 1570-4, 2004 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-14996710

RESUMO

Low-dose cyclophosphamide (LDC) induces selective apoptosis of endothelial cells within the vascular bed of tumors. Here, we investigated a hypothesis that the effect of LDC is mediated by the pro-apoptotic action of endogenous inhibitors of angiogenesis. Tumors treated with LDC demonstrate similar expression of matrix metalloproteinases and also basement membrane-derived angiogenesis inhibitors when compared with wild-type tumors, whereas the expression of thrombospondin-1 (TSP-1) is significantly elevated in LDC-treated tumors. We used mice with an absence of type XVIII collagen (endostatin) or type IV collagen alpha3 chain (tumstatin) or TSP-1 to assess the contribution of these endogenous inhibitors of angiogenesis on LDC-mediated tumor suppression. Lack of TSP-1 in the host in addition to tumor cells leads to diminished capacity of LDC to suppress tumor growth, whereas the absence of endostatin and tumstatin did not alter the effect of LDC. LDC treatment predominantly induces selective expression of TSP-1 in tumor cells and peri-vascular cells and facilitates apoptosis of proliferating endothelial cells, with minimal direct effect on tumor cells and peri-vascular cells. These studies indicate that TSP-1 contributes to tumor growth suppression induced by LDC and suggest that tumors that express high basal level of TSP-1 may be more susceptible to tumor suppression by such a regimen. This study also makes a strong case for TSP-1 expression levels as a potential predictive marker for the successful use of LDC in cancer patients.


Assuntos
Inibidores da Angiogênese/farmacologia , Apoptose/efeitos dos fármacos , Ciclofosfamida/farmacologia , Células Endoteliais/efeitos dos fármacos , Neoplasias Experimentais/tratamento farmacológico , Trombospondina 1/fisiologia , Animais , Autoantígenos/fisiologia , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Colágeno Tipo IV/fisiologia , Ciclofosfamida/uso terapêutico , Endostatinas/fisiologia , Células Endoteliais/patologia , Metaloproteinases da Matriz/análise , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Trombospondina 1/análise
18.
Cancer Cell ; 3(6): 589-601, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12842087

RESUMO

We demonstrate a physiological role for tumstatin, a cleavage fragment of the alpha3 chain of type IV collagen (Col IValpha3), which is present in the circulation. Mice with a genetic deletion of Col IValpha3 show accelerated tumor growth associated with enhanced pathological angiogenesis, while angiogenesis associated with development and tissue repair are unaffected. Supplementing Col IValpha3-deficient mice with recombinant tumstatin to a normal physiological concentration abolishes the increased rate of tumor growth. The suppressive effects of tumstatin require alphaVbeta3 integrin expressed on pathological, but not on physiological, angiogenic blood vessels. Mice deficient in matrix metalloproteinase-9, which cleaves tumstatin efficiently from Col IValpha3, have decreased circulating tumstatin and accelerated growth of tumor. These results indicate that MMP-generated fragments of basement membrane collagen can have endogenous function as integrin-mediated suppressors of pathologic angiogenesis and tumor growth.


Assuntos
Autoantígenos/fisiologia , Carcinoma Pulmonar de Lewis/prevenção & controle , Colágeno Tipo IV/fisiologia , Integrina alfaVbeta3/metabolismo , Neovascularização Patológica , Animais , Autoantígenos/farmacologia , Membrana Basal/química , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patologia , Divisão Celular , Colágeno/metabolismo , Colágeno Tipo IV/farmacologia , Combinação de Medicamentos , Endotélio Vascular/metabolismo , Epitopos , Feminino , Heterozigoto , Homozigoto , Humanos , Laminina/metabolismo , Regeneração Hepática , Pulmão/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout/embriologia , Camundongos Knockout/crescimento & desenvolvimento , Neovascularização Fisiológica , Gravidez , Prenhez , Proteoglicanas/metabolismo , Proteínas Recombinantes/farmacologia , Taxa de Sobrevida , Células Tumorais Cultivadas , Cicatrização
19.
Proc Natl Acad Sci U S A ; 100(8): 4766-71, 2003 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-12682293

RESUMO

Tumstatin and endostatin are two inhibitors of angiogenesis derived from precursor human collagen molecules known as alpha 3 chain of type IV collagen and alpha1 chain of type XVIII collagen, respectively. Although both these inhibitors are noncollagenous (NC1) domain fragments of collagens, they only share a 14% amino acid homology. In the present study we evaluated the functional receptors, mechanism of action, and intracellular signaling induced by these two collagen-derived inhibitors. Human tumstatin prevents angiogenesis via inhibition of endothelial cell proliferation and promotion of apoptosis with no effect on migration, whereas human endostatin prevents endothelial cell migration with no effect on proliferation. We demonstrate that human tumstatin binds to alpha v beta 3 integrin in a vitronectin/fibronectin/RGD cyclic peptide independent manner, whereas human endostatin competes with fibronectin/RGD cyclic peptide to bind alpha 5 beta 1 integrin. The activity of human tumstatin is mediated by alpha v beta 3 integrin, whereas the activity of human endostatin is mediated by alpha 5 beta 1 integrin. Additionally, although human tumstatin binding to alpha v beta 3 integrin leads to the inhibition of Cap-dependent translation (protein synthesis) mediated by focal adhesion kinase/phosphatidylinositol 3-kinase/Akt/mTOR/4E-BP1 pathway, human endostatin binding to alpha 5 beta 1 integrin leads to the inhibition of focal adhesion kinase/c-Raf/MEK1/2/p38/ERK1 mitogen-activated protein kinase pathway, with no effect on phosphatidylinositol 3-kinase/Akt/mTOR/4E-BP1 and Cap-dependent translation. Collectively, such distinct properties of human tumstatin and human endostatin provide the first insight into their diverse antiangiogenic actions and argue for combining them for targeting tumor angiogenesis.


Assuntos
Autoantígenos/fisiologia , Colágeno Tipo IV/fisiologia , Colágeno/fisiologia , Integrina alfa5beta1/fisiologia , Integrina alfaVbeta3/fisiologia , Neovascularização Patológica/prevenção & controle , Fragmentos de Peptídeos/fisiologia , Sequência de Aminoácidos , Autoantígenos/genética , Autoantígenos/farmacologia , Ligação Competitiva , Células Cultivadas , Colágeno/genética , Colágeno/farmacologia , Colágeno Tipo IV/genética , Colágeno Tipo IV/farmacologia , Colágeno Tipo XVIII , Endostatinas , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Fibronectinas/metabolismo , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Modelos Biológicos , Dados de Sequência Molecular , Neovascularização Patológica/fisiopatologia , Oligopeptídeos , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/farmacologia , Ligação Proteica , Proteínas Tirosina Quinases/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Vitronectina/metabolismo
20.
J Biol Chem ; 278(15): 12605-8, 2003 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-12538598

RESUMO

There are about 2.5 million glomeruli in the kidneys each consisting of a barrel of glomerular basement membrane surrounded by glomerular endothelial cells on the inside and glomerular epithelial cells with established foot processes (podocytes) on the outside. Defects in this filtration apparatus lead to glomerular vascular leak or proteinuria. The role of vascular endothelial growth factor (VEGF) in the regulation of glomerular vascular permeability is still unclear. Recent studies indicate that patients receiving anti-VEGF antibody therapy may have an increased incidence of proteinuria. In a different setting, pregnancies complicated by preeclampsia are associated with elevated soluble VEGF receptor 1 protein (sFlt-1), endothelial cell dysfunction and proteinuria. These studies suggest that neutralization of physiologic levels of VEGF, a key endothelial survival factor, may lead to proteinuria. In the present study, we evaluated the potential of anti-VEGF neutralizing antibodies and sFlt-1 in the induction of proteinuria. Our studies demonstrate that anti-VEGF antibodies and sFlt-1 cause rapid glomerular endothelial cell detachment and hypertrophy, in association with down-regulation of nephrin, a key epithelial protein in the glomerular filtration apparatus. These studies suggest that down-regulation or neutralization of circulating VEGF may play an important role in the induction of proteinuria in various kidney diseases, some forms of cancer therapy and also in women with preeclampsia.


Assuntos
Anticorpos , Fatores de Crescimento Endotelial/antagonistas & inibidores , Fatores de Crescimento Endotelial/sangue , Peptídeos e Proteínas de Sinalização Intercelular/sangue , Glomérulos Renais/fisiologia , Linfocinas/antagonistas & inibidores , Linfocinas/sangue , Proteinúria/etiologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Fatores de Crescimento Endotelial/imunologia , Fatores de Crescimento Endotelial/fisiologia , Endotélio Vascular/fisiologia , Endotélio Vascular/fisiopatologia , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Glomérulos Renais/fisiopatologia , Linfocinas/imunologia , Linfocinas/fisiologia , Camundongos , Fator A de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/imunologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Fatores de Crescimento do Endotélio Vascular
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