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1.
Cancer Invest ; 25(7): 632-46, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18027153

RESUMO

Integrins are heterodimeric cell adhesion receptors that mediate intercellular communication through cell-extracellular matrix interactions and cell-cell interactions. Integrins have been demonstrated to play a direct role in cancer progression, specifically in tumor cell survival, tumor angiogenesis, and metastasis. Therefore, agents targeted against integrin function have potential as effective anticancer therapies. Numerous anti-integrin agents, including monoclonal antibodies and small-molecule inhibitors, are in clinical development for the treatment of solid and hematologic tumors. This review focuses on the role of alpha(v) integrins in cancer progression, the current status of integrin-targeted agents in development, and strategies for the clinical development of anti-integrin therapies.


Assuntos
Integrina alfaV/fisiologia , Neoplasias/tratamento farmacológico , Inibidores da Angiogênese/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Remodelação Óssea/efeitos dos fármacos , Ensaios Clínicos Fase II como Assunto , Humanos , Integrina alfaV/química , Metástase Neoplásica , Neoplasias/etiologia , Transdução de Sinais
2.
Cancer Res ; 54(18): 4993-8, 1994 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-7520832

RESUMO

Disintegrins are Arg-Gly-Asp-containing proteins that inhibit integrin-mediated cell-cell and cell-matrix interactions. We have purified a disintegrin, contortrostatin, from Agkistrodon contortrix contortrix snake venom that is a potent inhibitor of human metastatic melanoma (M24 met) cell adhesion to extracellular matrix proteins. Contortrostatin inhibits M24 met cell adhesion to type I collagen, vitronectin, and fibronectin with 50% inhibitory concentration values of 20, 75, and 220 nM, respectively. Contortrostatin does not significantly inhibit adhesion of M24 met cells to laminin. 125I-labeled contortrostatin binds to M24 met cells in a saturable and displaceable manner. Scatchard analysis indicates that there are two binding sites for 125I-labeled contortrostatin on the surface of these cells. High affinity binding has a Kd of 3 nM with 165,000 sites/cells low affinity binding has a Kd of 60 nM with 500,000 sites/cell. Immobilized contortrostatin can support adhesion of M24 met cells; this binding is blocked by a monoclonal antibody to the beta 1 integrin subunit and by an antibody to the fibronectin receptor alpha 5 beta 1. The anti-vitronectin receptor (alpha v beta 5) monoclonal antibody which blocks adhesion of M24 met cells to immobilized vitronectin does not block binding of M24 met cells to immobilized contortrostatin. In an in vivo experimental metastasis model system, contortrostatin at 20 micrograms and 100 micrograms inhibits lung colonization of M24 met cells (5 x 10(5)), injected in the tail vein of scid mice, by 51 and 73%, respectively. We conclude that contortrostatin is a potent inhibitor of beta 1 integrin-mediated M24 met cell adhesion in vitro and that it also inhibits lung colonization in vivo.


Assuntos
Adesão Celular/efeitos dos fármacos , Integrinas/antagonistas & inibidores , Melanoma/secundário , Peptídeos/farmacologia , Venenos de Serpentes/farmacologia , Animais , Desintegrinas , Humanos , Integrina beta1 , Neoplasias Pulmonares/secundário , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Camundongos SCID , Peptídeos/isolamento & purificação , Peptídeos/metabolismo , Venenos de Serpentes/isolamento & purificação , Venenos de Serpentes/metabolismo
3.
Cancer Res ; 58(21): 4771-5, 1998 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-9809974

RESUMO

Integrin alphaIIb beta3 requires its cytoplasmic tails to participate in tumor cell adhesion, spreading, and migration. Using 3' rapid amplification of cDNA ends, we have amplified two alphaIIb cDNAs from human leukemia, prostate adenocarcinoma, and melanoma cells. One of these is the predicted wild-type alphaIIb cDNA, and the other is a novel truncated alphaIIb variant. This variant is unique in that it lacks the transmembrane and cytoplasmic portions of the alphaIIb light chain. The truncated alphaIIb integrin protein is expressed by human leukemia, prostate adenocarcinoma, and melanoma cells but not by platelets or normal prostate epithelial or normal breast epithelial cells. Tumor cells secrete this protein and deposit it on the extracellular matrix. To our knowledge, this is the first report of a naturally occurring variant of an alpha integrin that lacks the transmembrane and cytoplasmic tail.


Assuntos
Neoplasias/química , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/análise , Processamento Alternativo , Sequência de Aminoácidos , Sequência de Bases , DNA Complementar/análise , Humanos , Imuno-Histoquímica , Leucemia/metabolismo , Masculino , Melanoma/metabolismo , Dados de Sequência Molecular , Peso Molecular , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Neoplasias da Próstata/química , Biossíntese de Proteínas , Células Tumorais Cultivadas
4.
Cancer Res ; 56(8): 1902-8, 1996 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-8620512

RESUMO

The binding of autocrine motility factor (AMF) to its cell surface receptor, gp78, stimulates tumor cell motility. In this report, we provide evidence that stimulation of gp78 by either AMF or a monoclonal antibody to gp78 (3F3A) increases adhesion and spreading of metastatic murine melanoma (B16a) cells on fibronectin. This gp78-regulated increase is mediated by up-regulation of surface alphaIIbbeta3++ and alpha5beta1 integrin receptors. In addition, AMF treatment of B16a cells increased translocation of alphaIIbbeta3 and alpha5beta1 from the cytoplasm to the cell surface. However, alphaIIbbeta3 and alpha5beta1 demonstrate separate and unique staining patterns at the surface of B16a cells in response to stimulation of gp78. Furthermore, stimulation of B16a cells with AMF increased their invasion through Matrigel. This stimulated invasion was inhibited by antibodies to alphaIIbbeta3 but not by antibodies to alpha5beta1. The increased integrin surface expression and function in response to AMF was blocked by N-benzyl-N-hydroxy-5-phenylpentanamide, an inhibitor of 12-lipoxygenase, and calphostin C, an inhibitor of protein kinase C. The results demonstrate that AMF stimulates integrin-mediated B16a cell adhesion, spreading, and invasion, and these events are regulated by a signaling pathway involving 12-lipoxygenases and protein kinase C.


Assuntos
Adesão Celular , Glucose-6-Fosfato Isomerase/farmacologia , Melanoma Experimental/patologia , Melanoma Experimental/fisiopatologia , Metástase Neoplásica , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Membrana Celular/fisiologia , Colágeno , Meios de Cultivo Condicionados , Inibidores de Ciclo-Oxigenase/farmacologia , Combinação de Medicamentos , Fibronectinas , Fibrossarcoma , Citometria de Fluxo , Glucose-6-Fosfato Isomerase/isolamento & purificação , Humanos , Laminina , Inibidores de Lipoxigenase/farmacologia , Camundongos , Modelos Biológicos , Invasividade Neoplásica , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/biossíntese , Proteoglicanas , Receptores de Fibronectina/biossíntese , Transdução de Sinais , Células Tumorais Cultivadas
5.
Cancer Res ; 57(12): 2522-8, 1997 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-9192835

RESUMO

Integrins play an important role in mediating tumor cell-extracellular matrix (ECM) and tumor cell-endothelial cell interactions. The integrin alphaIIb beta3 (GPIIb-IIIa) is expressed on the surface of platelets in an inactive state and requires a conformational change to recognize extracellular matrix proteins such as fibrinogen, fibronectin, vitronectin, and others. In this study, we questioned whether human melanoma cells express the alphaIIb beta3 integrin. Reverse transcription-PCR/Southern blotting, Northern blotting, and dot blotting demonstrated the presence of the platelet-type alphaIIb beta3 integrin in human melanoma WM 983B, WM 983A, and WM 35 cells. AP-2, a monoclonal antibody (mAb) to alphaIIb beta3, positively stained two human melanoma specimens, indicating expression of this integrin in vivo. Phorbol 12-myristate 13-acetate and 12(S)-hydroxyeicosatetraenoic acid, two activators of protein kinase C, stimulated adhesion of melanoma cells to immobilized fibronectin and PAC-1, a mAb to alphaIIb beta3. PAC-1 specifically recognizes the conformationally active form of platelet alphaIIb beta3. Phorbol 12-myristate 13-acetate-stimulated adhesion of WM 983B cells to PAC-1 was completely blocked by an RGD peptide, thus providing evidence that tumor cell adhesion to PAC-1 is mediated via the alphaIIb beta3 integrin but not the Fc receptor. Confocal immunofluorescent studies demonstrated that fibronectin-adherent melanoma cells possess an intracellularly localized pool of high-affinity alphaIIb beta3. Invasion of WM 983B cells through fibronectin was stimulated by 12(S)-hydroxyeicosatetraenoic acid, and this stimulated invasion was blocked by the mAb PAC-1. The data suggest that melanoma cells express the high-affinity alphaIIb beta3 integrin, which is involved in tumor invasion.


Assuntos
Melanoma/metabolismo , Invasividade Neoplásica , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacologia , Adulto , Anticorpos Monoclonais/farmacologia , Northern Blotting , Southern Blotting , Adesão Celular/efeitos dos fármacos , Epitopos/metabolismo , Feminino , Fibronectinas/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Imuno-Histoquímica , Melanoma/patologia , Microscopia Confocal , Oligopeptídeos/farmacologia , RNA Mensageiro/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacos
6.
Cancer Res ; 56(21): 5071-8, 1996 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-8895766

RESUMO

The integrin alphaIIb(beta)3 was initially believed to be expressed only in cells from the megakaryocytic lineage, such as platelets or HEL cells. In this study, we report for the first time that human prostate carcinoma PC-3 and DU-145 cells express alphaIIb(beta)3. Reverse transcription-PCR from HEL (positive control), PC-3, and DU-145 cells amplified a predicted alphaIIb fragment that hybridized to the full-length alphaIIb cDNA probe. DNA sequencing of the PCR fragments revealed 100% sequence homology to the corresponding extracellular domain of platelet alphaIIb but minimal sequence homology to integrins (alpha)v or a5. An RNase protection assay was used to confirm the results from reverse transcription-PCR. An antisense riboprobe to alphaIIb mRNA hybridized to total RNA from HEL, PC-3, and DU-145 cells, suggesting that alphaIIb mRNA is transcribed in these tumor cells. In situ hybridization on surgical specimens from human prostate tumor tissue stained positive with an antisense riboprobe to alphaIIb mRNA. The expression of alphaIIb(beta)3 protein in PC-3 and DU-145 cells was demonstrated by Western and dot blotting and flow cytometry with monoclonal antibodies (mAbs) to alphaIIb (MAB 1990), beta3, and alphaIIb(beta)3 (AP-2). A protein kinase C activator, phorbol 12-myristate 13-acetate, increased the adhesion of PC-3 cells to PAC-1, a mAb specific to the high-affinity state of alphaIIb(beta)3, by more than 80-fold. The invasion of DU-145 cells through a reconstituted basement membrane was blocked 40-50% by mAbs AP-2 or PAC-1. These data collectively suggest that: (a) prostate tumor cells express alphaIIb(beta)3; (b) surface expression of alphaIIb(beta)3 integrin is regulated by protein kinase C; and (c) mAbs to this receptor inhibit invasion of prostate cancer cells through a reconstituted basement membrane.


Assuntos
Complexo Glicoproteico GPIIb-IIIa de Plaquetas/análise , Neoplasias da Próstata/química , Humanos , Masculino , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/fisiologia , Reação em Cadeia da Polimerase , Neoplasias da Próstata/patologia , RNA Mensageiro/análise , Células Tumorais Cultivadas
7.
Clin Exp Metastasis ; 16(5): 437-45, 1998 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10091939

RESUMO

In resting platelets integrin alphaIIbbeta3 is constitutively expressed in an inactive state and it does not recognize soluble proteins. Platelet activation results in a conformational change of the low-affinity alphaIIbbeta3 to a high-affinity state which then recognizes plasma fibrinogen. The ectopic expression of alphaIIbbeta3 integrin in rodent and human cells derived from solid tumors is well documented, although little is known about its affinity state in these tumor cells. In this study we analysed expression and function of high-affinity alphaIIbbeta3 in murine metastatic melanoma B16a cells by using a mAb that specifically recognizes high-affinity alphaIIbbeta3 (PAC-1). These tumor cells while in suspension bound PAC-1 and fibrinogen. Immunofluorescent studies of B16a cells indicated that high-affinity alphaIIbbeta3 is associated with the Golgi complex and the cell surface. Stimulation of B16a cells with a PKC-activator, 12(S)-HETE, induced translocation of the high-affinity integrin from an intracellular pool to the plasma membrane, which resulted in increased tumor cell adhesion to fibronectin. In addition to participating in 12(S)-HETE-stimulated adhesion of B16a cells, the high-affinity alphaIIbbeta3 integrin is also involved in tumor cell invasion through a reconstituted basement membrane. In conclusion, results from this study suggest that in non-megakaryocytic lineage B16a cells alphaIIbbeta3 is constitutively expressed in a high-affinity state, and that this conformation participates in tumor cell adhesion and invasion.


Assuntos
Melanoma Experimental/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/biossíntese , Animais , Adesão Celular/efeitos dos fármacos , Fosfatase 2 de Especificidade Dupla , Fibrinogênio/metabolismo , Humanos , Melanoma Experimental/patologia , Melanoma Experimental/secundário , Camundongos , Invasividade Neoplásica , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/fisiologia , Proteína Fosfatase 2 , Proteínas Tirosina Fosfatases/metabolismo
8.
Thromb Haemost ; 85(6): 1037-42, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11434681

RESUMO

Megakaryocytic genes such as alphaIIbbeta3 can be expressed by malignant cells as part of the disturbances in their gene regulation. However, the function of the gene product greatly depends on the interaction of the ectopic protein with the new environment. The outside-in signaling of the ectopically expressed alphaIIbbeta3 integrin was studied in B16a murine melanoma cells using a monoclonal antibody, specifically directed to the activated conformation of alphaIIbbeta3, PAC-1 and the physiological ligand, fibrinogen. Ligation of alphaIIbbeta3 induced down-regulation of FAK but serine phosphorylation of three protein bands, 20/21, 85 and 140 kDa within 1-15 min. Flow cytometry indicated that the ligation of the receptor in B16a cells induces approximately 50% increase in phosphoserine positive cells within 5-15 min. 12-lipoxygenase is placed downstream in the signaling pathway, since ligation of alphaIIbbeta3 induces 12-HETE production within 5 min and pretreatment of tumor cells with select lipoxygenase inhibitior, Baicalein, prevents the increase in serine phosphorylation. Confocal microscopy of adherent tumor cells demonstrated rearrangement of actin filaments upon alphaIIbbeta3 ligation paralleled by downregulation of p125FAK and phoshotyrosine+ adhesion plaques and translocation of PKCalpha to stress fibers and cortical actin. PKC appears to be the major effector serine kinase of the alphaIIbbeta3-coupled signaling pathway, since pretreatment of tumor cells with a select PKC inhibitor, Calphostin C, prevents the ligation-induced serine phosphorylation. Previous studies have indicated a role for the 12-lipoxygenase-PKC signaling pathway in platelet aggregation as well as tumor invasion, therefore the involvement of this cascade in the signaling of the ectopic alphaIIbbeta3 integrin may partially explain its role in tumor progression.


Assuntos
Araquidonato 12-Lipoxigenase/farmacologia , Melanoma/patologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/fisiologia , Proteína Quinase C/farmacologia , Transdução de Sinais/fisiologia , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/análise , Animais , Anticorpos Monoclonais/farmacologia , Adesão Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fibrinogênio/metabolismo , Citometria de Fluxo , Camundongos , Microscopia Confocal , Fosforilação/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologia , Proteína Quinase C/antagonistas & inibidores , Serina/metabolismo , Células Tumorais Cultivadas
9.
Thromb Haemost ; 85(5): 896-902, 2001 May.
Artigo em Inglês | MEDLINE | ID: mdl-11372685

RESUMO

Abciximab (c7E3 Fab, ReoPro) blocks GPIIb/IIIa and alphavbeta3 and inhibits thrombotic and proliferative events only in humans and non-human primates. The bivalent F(ab')2 fragment is an effective anti-thrombotic agent in canine models. In the present study, 7E3 F(ab')2 was also found to bind to rat GPIIb/IIIa (KD = 27 +/- 4 microg/mL) and alphavbeta3 (KD = 9 +/- 8 microg/mL), to block in vitro rat platelet aggregation (IC50 = 16 +/- 6 microg/mL), and to inhibit alphavbeta3-mediated microvessel sprout formation in a rat aortic ring assay. Following administration of 7E3 F(ab')2 (4 mg/kg) to rats, platelet aggregation was completely blocked for up to 6 h and thrombus formation in response to a rat abdominal aorta double crush injury was prevented. Effective chronic dosing was achieved with 6 mg/kg daily I.P. injections. In vitro mixing experiments indicated that 7E3 F(ab')2 redistributed to unlabeled platelets in 2 h. Ex vivo, 7E3 F(ab')2 was detected on platelets for up to 4 days after a single 4-mg/kg injection. These data suggest that 7E3 F(ab')2 may be a useful agent to study the effects of GPIIb/IIIa and alphavbeta3 blockade in rat models of thrombosis and vascular disease.


Assuntos
Anticorpos Monoclonais/farmacologia , Fragmentos Fab das Imunoglobulinas/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Receptores de Vitronectina/antagonistas & inibidores , Trombose/tratamento farmacológico , Abciximab , Animais , Anticorpos Monoclonais/química , Aorta/lesões , Aorta/patologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Fragmentos Fab das Imunoglobulinas/química , Cinética , Masculino , Camundongos , Microcirculação , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Ratos , Ratos Endogâmicos , Receptores de Vitronectina/imunologia , Receptores de Vitronectina/metabolismo , Trombose/prevenção & controle
10.
Thromb Res ; 73(1): 39-52, 1994 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-8178312

RESUMO

Proteins that inhibit glycoprotein (GP) IIb/IIIa mediated platelet aggregation have been purified from the venom of two snake species. A small platelet aggregation inhibitor (p1.AI), multisquamatin (Mr = 5,700), was purified from Echis multisquamatus venom by hydrophobic interaction HPLC and two steps on C18 reverse phase HPLC. A larger p1.AI, contortrostatin (Mr = 15,000), was purified by a similar HPLC procedure from the venom of Agkistrodon contortrix contortrix. Both p1.AIs inhibit ADP-induced human, canine and rabbit platelet aggregation using platelet rich plasma (PRP). Multisquamatin has an IC50 of 97 nM, 281 nM and 333 nM for human, canine and rabbit PRP, respectively. Contortrostatin has an IC50 of 49 nM, 120 nM and 1,150 nM for human, canine and rabbit PRP, respectively. In a competitive binding assay using 125I-7E3 (a monoclonal antibody to GPIIb/IIIa that inhibits platelet aggregation) both contortrostatin and multisquamatin demonstrated GPIIb/IIIa specific binding to human and canine platelets. The IC50 for contortrostatin displacement of 7E3 binding to human and canine GPIIb-/IIIa is 27 nM and 16 nM, respectively and for multisquamatin it is 3 nM and 63 nM, respectively. Our results indicate that both p1.AIs inhibit platelet aggregation by binding with high affinity to GPIIb/IIIa.


Assuntos
Venenos de Crotalídeos/química , Inibidores da Agregação Plaquetária/isolamento & purificação , Venenos de Víboras/química , Animais , Anticorpos Monoclonais , Sítios de Ligação/fisiologia , Cães , Eletroforese em Gel de Poliacrilamida , Humanos , Inibidores da Agregação Plaquetária/sangue , Glicoproteínas da Membrana de Plaquetas/imunologia , Glicoproteínas da Membrana de Plaquetas/metabolismo , Coelhos
11.
Toxicon ; 32(12): 1521-31, 1994 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-7725320

RESUMO

Fibrolase, a zinc metalloproteinase possessing direct-acting fibrinolytic activity, has been previously purified from southern copperhead (Agkistrodon contortrix contortix) snake venom. We recently reported that a pool of southern copperhead venom from different geographical locations possesses two isoforms of fibrolase (fib1 and fib2) [Loayza, S. L. et al. (1994) J. Chromat. B, in press]. We now report that venom from individual southern copperhead snakes contains the two isoforms which can be separated by a three-step high performance liquid chromatography (HPLC) procedure consisting of hydrophobic interaction chromatography, hydroxylapatite chromatography and weak cation exchange chromatography. Utilizing mass spectrometry we determined that fib1 has a molecular mass of 22,879 atomic mass units (amu) compared to 22,753 amu for fib2. These results support earlier observations during amino acid sequence analysis that a truncated version of the enzyme is produced which is missing the amino-terminal amino acid (< Glu-Arg-Phe-Pro vs. the intact enzyme < Glu-Gln-Arg-Phe-Pro, where < Glu is cyclized glutamine). The truncated version of fibrolase (fib2) has full fibrinolytic activity compared to fib1. EC50 values (concentration of enzyme required to degrade 50% of fibrin in a micro-fibrin plate assay) are 6.4 (+/- 1.0) microM and 5.2 (+/- 0.8) microM for fib 1 and fib2, respectively. Therefore, loss of the amino-terminal amino acid does not appear to influence enzymatic activity. We conclude that the two isoforms of fibrolase arise from variations in the molecular processing of the enzyme by the snake venom gland rather than being caused by the pooling of southern copperhead venoms from different geographical locations.


Assuntos
Venenos de Crotalídeos/enzimologia , Isoenzimas/isolamento & purificação , Metaloendopeptidases/isolamento & purificação , Agkistrodon , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Fibrina/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Espectrometria de Massas , Metaloendopeptidases/química , Metaloendopeptidases/metabolismo , Dados de Sequência Molecular
12.
Pathol Oncol Res ; 6(3): 163-74, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-11033455

RESUMO

Abciximab (ReoPro) is a mouse-human chimeric monoclonal antibody Fab fragment of the parent murine monoclonal antibody 7E3, and was the first of these agents approved for use as adjunct therapy for the prevention of cardiac ischemic complications in patients undergoing percutaneous coronary intervention (PCI). Abciximab binds with high avidity to both the non-activated and activated form of the GPIIb/IIIa receptor of platelets, the major adhesion receptor involved in aggregation. Additional cardiovascular indications for abciximab are unstable angina, carotid stenting, ischemic stroke and peripheral vascular diseases. Abciximab also interacts with two other integrin receptors; the a av b b3 receptor, which is present in low numbers on platelets but in high density on activated endothelial and smooth muscle cells, and a aMb b2 integrin which is present on activated leukocytes. Cell types that express integrins GPIIb/IIIa and a av b b3 such as platelets, endothelial and tumor cells have been implicated in angiogenesis, tumor growth and metastasis. Since abciximab interacts with high avidity to integrins GPIIb/IIIa and a av b b3, it is reasonable to assume that it may possess anti-angiogenic properties in angiogenesis-related diseases, as well as anti-metastastatic properties in case of disseminating tumors expressing the target integrin receptors.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Fragmentos Fab das Imunoglobulinas/uso terapêutico , Isquemia/terapia , Metástase Neoplásica/tratamento farmacológico , Inibidores da Agregação Plaquetária/uso terapêutico , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Trombose/terapia , Abciximab , Ensaios Clínicos como Assunto , Humanos , Isquemia/fisiopatologia , Metástase Neoplásica/fisiopatologia , Trombose/fisiopatologia
15.
Biochem Biophys Res Commun ; 275(2): 690-5, 2000 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-10964724

RESUMO

The arachidonic acid metabolite of 12 lipoxygenase, 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) promotes metastatic behavior of tumor cells. In this study we set out to identify 12(S)-HETE signaling pathways, and their contribution to cellular functions in A431 epidermoid carcinoma. (1) 12(S)-HETE stimulated phosphotyrosine associated PI3 kinase activity. (2) 12(S)-HETE stimulated ERK1/2 in a PI3 kinase dependent manner. (3) PI3 kinase affected the 12(S)-HETE stimulated Raf/MEK/ERK cascade at the level of MEK. (4) 12(S)-HETE stimulated ERK1/2 via PKCzeta. (5) 12(S)-HETE stimulated cell migration on laminin, which was eliminated by PI3 kinase and cPKC inhibitors, but it was unaffected by inhibition of ERK1/2.


Assuntos
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Ativação Enzimática , Humanos , Isoenzimas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Quinase C/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas
16.
J Biol Chem ; 275(49): 38831-41, 2000 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-10952974

RESUMO

12(S)-Hydroxyeicosatetraenoic acid (12(S)-HETE), a 12-lipoxygenase metabolite of arachidonic acid, has multiple effects on tumor and endothelial cells, including stimulation of invasion and angiogenesis. However, the signaling mechanisms controlling these physiological processes are poorly understood. In a human epidermoid carcinoma cell line (i.e. A431), 12(S)-HETE activates extracellular signal-regulated kinases 1/2 (ERK1/2), which is mediated by upstream kinases MEK and Raf. 12(S)-HETE stimulates phosphorylation of phospholipase Cgamma1 and activity of protein kinase Calpha (PKCalpha). In addition, independent of PKC 12(S)-HETE increases tyrosine phosphorylation of Shc, and Grb2, stimulates association between Shc and Src, and increases the activity of Ras, via Src family kinases. Furthermore, at low (10-100 nm) concentrations 12(S)-HETE counteracts epidermal growth factor-stimulated activation of ERK1/2 via stimulating protein tyrosine phosphatases. We also present evidence that 12(S)-HETE stimulates ERK1/2 via G proteins and that A431 cells have multiple binding sites for 12(S)-HETE. Finally, inhibition of 12-lipoxygenase induced apoptosis of A431 cells, which was reversed by addition of exogenous 12(S)-HETE. Collectively we demonstrate that the activation of ERK1/2 by 12(S)-HETE may be regulated by multiple receptors triggering PKC-dependent and PKC-independent pathways in A431 cells.


Assuntos
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacologia , Carcinoma de Células Escamosas/enzimologia , MAP Quinase Quinase Quinase 1 , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Quinase C/metabolismo , Transdução de Sinais/fisiologia , Araquidonato 12-Lipoxigenase/metabolismo , Ativação Enzimática , Fator de Crescimento Epidérmico/antagonistas & inibidores , Fator de Crescimento Epidérmico/farmacologia , Humanos , Isoenzimas/metabolismo , Cinética , Inibidores de Lipoxigenase , Proteína Quinase 3 Ativada por Mitógeno , Fosfolipase C gama , Fosforilação , Proteína Quinase C-alfa , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Fosfolipases Tipo C/metabolismo
17.
J Chromatogr B Biomed Appl ; 662(2): 227-43, 1994 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-7719479

RESUMO

Fibrolase, the fibrinolytic enzyme from Agkistrodon contortrix contortrix snake venom, is a zinc metalloproteinase with a molecular mass of 23 kDa. We report a method to isolate two isoforms of natural fibrolase (fib1 and fib2) and three isoforms of recombinant fibrolase (r-fib1, r-fib2 and r-fib3) using CM 300 cation-exchange high-performance liquid chromatography. Utilizing mass spectrometry we characterized differences in molecular masses of the isoforms of r-fibrolase. These findings suggest that the isoforms differ by minor sequence variations at their amino-termini. Since the stability of fibrolase is exquisitely sensitive to the removal of zinc, we examined the EDTA sensitivity of the isoforms of fibrolase and r-fibrolase to determine if their different chromatographic behavior is related to differences in their zinc affinities. All of the isoforms examined appear to have similar zinc binding affinities. Thus, the IC50 (concentration of EDTA to produce 50% inhibition of enzymatic activity) for fib1 is 160 microM. For the closely related r-fib1, the IC50 is 180 microM. Similarly, r-fib3 has an IC50 of 140 microM.


Assuntos
Venenos de Crotalídeos/enzimologia , Isoenzimas/isolamento & purificação , Metaloendopeptidases/isolamento & purificação , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Venenos de Crotalídeos/química , Ácido Edético/química , Eletroforese em Gel de Poliacrilamida , Isoenzimas/química , Espectrometria de Massas , Metaloendopeptidases/química , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Zinco/metabolismo
18.
J Biol Chem ; 269(35): 21940-3, 1994 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-7520909

RESUMO

Tyrosine phosphorylation of multiple platelet proteins is regulated by the integrin alpha IIb beta 3. In order to further examine integrin-regulated tyrosine phosphorylation, we have used small Arg-Gly-Asp-containing snake venom proteins (termed disintegrins) that inhibit platelet aggregation to competitively block the agonist-induced binding of fibrinogen to alpha IIb beta 3. One structurally unique disintegrin, contortrostatin (which appears to be a disulfide-linked dimer of 13.5 kDa with two Arg-Gly-Asp sites), was found to trigger signaling events typically mediated by fibrinogen cross-linking of alpha IIb beta 3, as demonstrated by tyrosine phosphorylation of the tyrosine kinase pp72syk and a 140-kDa protein. Contortrostatin and another disintegrin, multisquamatin (a monomer of 5.7 kDa with a single Arg-Gly-Asp site), did not affect thrombin-induced platelet shape change, secretion, or integrin-independent tyrosine phosphorylation; however, they inhibited aggregation and aggregation-dependent tyrosine phosphorylation of numerous proteins, including the focal adhesion kinase pp125FAK. Our results suggest that structurally distinct disintegrins have varying effects on tyrosine phosphorylation; while monomeric multisquamatin and dimeric contortrostatin both inhibit aggregation-dependent tyrosine phosphorylation, contortrostatin also possesses a unique functional activity that allows it to activate an intracellular signaling pathway leading to tyrosine phosphorylation. This activity may be involved in the function of this snake venom protein on hemostasis.


Assuntos
Plaquetas/metabolismo , Venenos de Crotalídeos/farmacologia , Peptídeos/farmacologia , Venenos de Víboras/farmacologia , Sequência de Aminoácidos , Sítios de Ligação , Desintegrinas , Fibrinogênio/metabolismo , Integrinas/metabolismo , Dados de Sequência Molecular , Peptídeos/química , Fosforilação/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas , Conformação Proteica , Transdução de Sinais , Trombina/farmacologia , Tirosina/efeitos dos fármacos , Tirosina/metabolismo
19.
Int J Cancer ; 72(4): 642-8, 1997 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-9259405

RESUMO

The integrin alphaIIb beta3 is a membrane receptor which was considered to be expressed only in cells of megakaryocytic lineage. We have shown that alphaIIb beta3 is expressed in mouse melanoma B16a cells, and in human prostate adenocarcinoma cells. The purpose of this study was to determine whether the megakaryocytic product alphaIIb beta3 was functionally expressed in other non-megakaryocyte lineage tumor cells. By using the reverse transcription polymerase chain reaction (RT-PCR), we have obtained data demonstrating that alphaIIb beta3 is expressed in a variety of tumor cell lines (17) derived from different species (human, rat and mouse) and of different histological origins (skin, blood, lung, liver, kidney, cervix, colon, bladder, breast and prostate). Immunostaining of tumor cells with a monoclonal antibody (MAb) to alphaIIb beta3 demonstrates that alphaIIb beta3 protein is also expressed in tumor cells. A protein kinase C activator PMA stimulates adhesion of tumor cells to fibronectin and fibrinogen, and this stimulated adhesion is blocked by a function-blocking MAb directed to alphaIIb beta3. Our results indicate that the megakaryocytic gene product alphaIIb beta3 integrin is widely expressed among tumor cells of non-megakaryocytic lineage, suggesting that ectopic expression of this integrin may play an important role in tumor progression.


Assuntos
Plaquetas/metabolismo , Neoplasias/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/biossíntese , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Adesão Celular/fisiologia , Humanos , Integrinas/biossíntese , Masculino , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Dados de Sequência Molecular , Neoplasias/patologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/fisiologia , Reação em Cadeia da Polimerase , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA Mensageiro/metabolismo , Ratos , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Transcrição Gênica , Células Tumorais Cultivadas
20.
Prostate ; 35(3): 185-92, 1998 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-9582087

RESUMO

BACKGROUND: Integrins participate in cell-cell and cell-matrix interactions. In this study we determined whether alphaII(b)beta3 integrin is involved in metastasis of human prostate adenocarcinoma cells. METHODS: Prostate adenocarcinoma PC-3 and DU-145 cell lines express alphaII(b)beta3. Northern blotting, 5'-RACE, and immunofluorescent localization confirmed expression of alphaIIb integrin in prostate adenocarcinoma cells. We used orthotopic/ectopic site of implantation and lung colonization assays in SCID mice to determine whether alphaII(b)beta3 participates in metastatasis of tumor cells. RESULTS: Immunofluorescent localization of alphaIIb integrin in fibronectin-adherent DU-145 and PC-3 cells is remarkably different. In DU-145 cells the integrin localizes to focal contact sites, whereas it is predominantly intracellular in PC-3 cells. Both tumor cell lines are tumorigenic when implanted subcutaneously or intraprostatically in SCID mice, but only DU-145 cells injected intraprostatically metastasize. Flow cytometry with a mAb directed to alphaII(b)beta3 revealed higher expression of alphaII(b)beta3 in DU-145 tumor cell suspensions isolated from the prostate when compared to DU-145 tumor cells from the subcutis. Function-blocking mAbs to alphaII(b)beta3 inhibit lung colonization of tail vein-injected DU-145 cells. CONCLUSIONS: Altogether, the data suggest that alphaII(b)beta3 integrin participates in the metastatic progression of prostatic adenocarcinoma.


Assuntos
Adenocarcinoma/metabolismo , Neoplasias Pulmonares/secundário , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Neoplasias da Próstata/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/secundário , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Imunofluorescência , Expressão Gênica , Humanos , Técnicas In Vitro , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Camundongos SCID , Dados de Sequência Molecular , Fenótipo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/fisiologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Células Tumorais Cultivadas/metabolismo
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