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Query: UMLS:C0025202 (melanoma)
 69,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The cytokine melanoma growth-stimulating activity (MGSA) is a growth factor for melanoma cells and a chemotaxin for neutrophils. Known purification procedures of MGSA from human sources or expression systems give a low yield and require multiple chromatography steps. Here, a fast and high-yield method for the purification of recombinant MGSA is described. Approximately 500 micrograms MGSA were recovered from the bacterial lysate of a 10 liter culture within a day. For this purpose, total mRNA of Hs294T melanoma cells was isolated and cDNA of MGSA was obtained by reverse transcription and polymerase chain reaction. The cDNA of MGSA was subcloned into the expression vector pGEX-2T, generating a fusion with the Schistosoma japonicum glutathione S-transferase gene. The fusion protein was expressed in E. coli DH5a and purified from the bacterial lysate using glutathione-sepharose beads. MGSA was cleaved from the complex of fusion protein and glutathione-sepharose beads with thrombin and purified to homogeneity by anion-exchange high-performance liquid chromatography with a Mono-S-column. The bioactivity of the recombinant MGSA was assessed by chemotactic migration and triggered [Ca2+]i-transients in human neutrophils. In addition, [125I]MGSA bound specifically to undifferentiated human leukemia cells HL-60 transfected with the cDNA of the interleukin-8 (IL-8) receptor beta with similar properties as [125I]IL-8. Thus, this described method might be a powerful tool to generate large amounts of cytokines in a short time.
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PMID:Single-step purification of recombinant melanoma growth-stimulating activity by anion-exchange high-performance liquid chromatography. 792 55

In this study, we examined the effect of triflavin, an Arg-Gly-Asp (RGD)-containing snake venom peptide, on human cervical carcinoma (HeLa) cell- and B16-F10 mouse melanoma cell-induced platelet aggregation (TCIPA) in heparinized platelet-rich plasma. TCIPA appears to play an important role in the development of certain experimental tumor metastases. Two ADP-scavenging agents, apyrase (10 U/ml) and creatine phosphate (CP) (5 mM)/creatine phosphokinase (CPK) (5 U/ml) completely inhibited B16-F10 TCIPA, but hirudin (5 U/ml) had no effect. In contrast, apyrase and CP/CPK did not inhibit HeLa TCIPA while hirudin completely inhibited it. Furthermore, HeLa cells initially induced platelet aggregation and then blood coagulation at a later stage. In addition, HeLa cells shortened, in a concentration-dependent manner, the recalcification time of normal as well as factor VIII- and IX-deficient human plasma, but did not affect the recalcification time of factor VII-deficient plasma. This suggests that HeLa TCIPA occurs via activation of the extrinsic pathway, probably owing to tumor cell expression of tissue factor-like activity. HeLa cell-induced thrombin generation was confirmed by detection of amidolytic activity towards a chromogenic substrate, S-2238 (H-D-Phe-Pip-Arg-p-NA). Triflavin and GRGDS inhibited, in a dose-dependent manner, TCIPA caused by either cell line. On a molar basis, triflavin was 10,000-30,000 times more potent than GRGDS in this regard. Moreover, monoclonal antibodies raised against glycoprotein (GP) IIb/IIIa complex (i.e., 7E3 and AP2) and against GP Ib (i.e., AP1) completely inhibited HeLa TCIPA. 7E3 and AP2 inhibited B16-F10 TCIPA by up to 80% whereas AP1 showed only 30% inhibition of B16-F10 TCIPA. In conclusion, the inhibitory effect of triflavin on HeLa and B16-F10 TCIPA may be mediated principally by the binding of triflavin to the fibrinogen receptor associated with GP IIb/IIIa complex on the platelet surface. However, GP Ib is also involved in HeLa TCIPA as thrombin formation is the key factor in triggering platelet aggregation caused by HeLa cells.
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PMID:Triflavin, an Arg-Gly-Asp-containing peptide, inhibits tumor cell-induced platelet aggregation. 822 81

Thrombin has been shown to activate tumor-cell adhesion to platelets, fibronectin and von Willebrand factor 2- to 3-fold in vitro, and enhance metastasis 10- to 156-fold in vivo. We therefore elected to determine whether thrombin binds to tumor cells and whether thrombin-treated tumor cells enhance their adhesion to endothelial cells, the first barrier to tumor invasion and metastasis. Thrombin-treated human and hamster melanoma cells (SK-Mel-28 and HM-29) enhanced their adhesion to bovine aortic endothelial cells 2.1- to 2.3-fold, respectively. Similar results were obtained with bovine capillary endothelial cells. Thrombin activation of tumor cells was rapid, reaching its peak 15 min after thrombin activation; and transient, declining to baseline levels by 60 min. 125I-thrombin bound to both SK-Mel-28 and HM-29 cells in a saturation-dependent manner, was inhibitable by unlabelled thrombin, and could be 90% washed away with buffer following 30 min of incubation. Electron microscopy of tumor cells bound to fibronectin-coated millipore filters revealed adhesion of naive as well as thrombin-treated tumor cells to endothelial cells and subendothelial matrix between endothelial cells. Neither mode of adhesion was preferentially enhanced by thrombin-treated tumor cells. Both naive and thrombin-treated SK-Mel-28 cells had the adhesive ligand integrin receptors: alpha 3 beta l (fibronectin, laminin, collagen); alpha 5 beta l (fibronectin); alpha v beta x (vitronectin). Receptors for the beta 2 integrin family (LFA-I and Mac-I) were not found, nor were receptors of the beta 3 integrin family, GPIIIa. The receptor ligands fibronectin and vitronectin were present. None of the above receptors or ligands increased their density or appeared de novo after thrombin stimulation. Thus, 2 melanoma cell lines have thrombin receptors which, when occupied, lead to enhanced adhesion of tumor cells to endothelium and subendothelial matrix.
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PMID:Thrombin stimulates melanoma tumor-cell binding to endothelial cells and subendothelial matrix. 847 56

Specific antibodies to tissue factor pathway inhibitor (TFPI) were used in immunohistochemical procedures to determine the distribution of TFPI in normal and neoplastic human tissues. TFPI was restricted to megakaryocytes and the endothelium of the microvasculature in normal and abnormal tissues, but was not found in the endothelium of larger vessels or in hepatocytes. TFPI was also detected in macrophages in the villi of term placenta. Tumor-associated macrophages in several types of malignancy that we have shown previously to express a complete tissue factor-initiated pathway of coagulation and thrombin generation also manifested TFPI. By contrast, malignant cells in small cell carcinoma of the lung, renal cell carcinoma, and malignant melanoma that we have shown previously to express coagulation factors together with tumor cell-associated fibrin formation failed to stain for TFPI. We postulate that TFPI may be lacking from the latter malignancies because of the absence of the appropriately configured tissue factor-factor VIIa-factor Xa complex required for TFPI binding.
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PMID:Distribution of tissue factor pathway inhibitor in normal and malignant human tissues. 849 49

Osteopontin is an adhesive glycoprotein implicated in numerous diseases associated with inflammation and remodeling. There are several structural domains in osteopontin that are of particular interest. The RGD motif is a cell attachment sequence shown to be critical for cell adhesion through alphav-containing integrins. In close proximity to the RGD domain is the thrombin cleavage site. Previous observations suggest that thrombin cleavage of osteopontin occurs in vivo and may be physiologically important. To study the functional significance of osteopontin cleavage by thrombin, we made glutathione S-transferase-osteopontin fusion proteins. These proteins contain either the N- or C-terminal domains expected to be formed following thrombin cleavage at the Arg169-Ser170 peptide bond. We compared these osteopontin fragments with native osteopontin in their ability to support adhesion of several different cell lines and identified the receptors mediating these interactions. Our data show that the N-terminal osteopontin fragment, which contains the RGD domain, supports adhesion of a melanoma cell line that is unable to bind native osteopontin. This suggests that osteopontin adhesive interactions may be regulated by thrombin cleavage. We also demonstrate that osteopontin contains a cryptic binding activity, which can be recognized by a novel osteopontin receptor. This receptor has been identified as the alpha9beta1 integrin.
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PMID:Osteopontin N-terminal domain contains a cryptic adhesive sequence recognized by alpha9beta1 integrin. 891 Apr 76

The purpose of this study was to evaluate whether or not, using sensitive analytical methods for the measurement of coagulation and fibrinolysis enzyme activity, there was a hypercoagulable state in patients with melanoma, and whether differences existed between those with or without metastases. Seventy-one patients were studied, 45 with localized tumors (stages Ia and Ib) and 26 with metastases (stages II-IV). Plasma level of activated factor VII, prothrombin fragment 1 + 2, thrombin-antithrombin complex, fibrinopeptide A, plasmin-antiplasmin complex and D-dimer were much higher in the whole group of 71 patients than in 45 controls with benign nevi. However, when melanoma patients with or without metastases were compared, there were smaller differences, with only thrombin-antithrombin complex, plasmin-antiplasmin and D-dimer significantly higher in metastatic melanoma. These results indicate that in patients carrying a tumor endowed with high procoagulant activity in vitro, there is a laboratory picture of hypercoagulability with secondary hyperfibrinolysis in vivo. However, differences between patients with localized and metastatic tumors for markers of hypercoagulability are not striking, in spite of the fact that metastatic cells support greater coagulant activity than primary cells in vitro.
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PMID:Hypercoagulability and hyperfibrinolysis in patients with melanoma. 892 92

Studies have been carried out to investigate aspects of the structure of thrombomodulin, an endothelial cell glycoprotein that binds thrombin and accelerates both the thrombin-dependent activation of protein C and the inhibition of antithrombin III. We have determined the shape of SolulinTM, a soluble recombinant form of human thrombomodulin missing the transmembrane and cytoplasmic domains, by electron microscopy of preparations rotary-shadowed with tungsten. Solulin appears to be an elongated molecule about 20 nm long that has a large nodule at one end and a smaller nodule near the other end from which extends a thin strand. About half of the molecules form bipolar dimers apparently via interactions between these thin strands. Electron microscopy of complexes formed between Solulin and human alpha-thrombin revealed that a single thrombin molecule appears to bind to the smaller nodule of Solulin, suggesting that this region contains the epidermal growth factor-like domains 5 and 6. Epidermal growth factor-like domains 1-4 comprise the connector between the small and large nodule, which is the lectin-like domain; the thin strand at the other end of the molecule is the carbohydrate-rich region. With chondroitin sulfate-containing soluble thrombomodulin produced from either human melanoma cells Bowes or Chinese hamster ovary cells, a higher percentage of molecules bound thrombin and, in some cases, two thrombin molecules were attached to one soluble thrombomodulin in approximately the same region. These structural studies provide insight into the structure of thrombomodulin and its interactions with thrombin as well as aspects of the mechanisms of its actions.
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PMID:The shape of thrombomodulin and interactions with thrombin as determined by electron microscopy. 894 Jan 62

Platelet agonists are known to contribute to the regulation of cytoplasmic Ca2+ levels in tumor cells and this property could be relevant in the stimulation of cell proliferation. In the present study we investigated the ability of ADP, collagen and thrombin to increase cytoplasmic Ca2+ levels in different human tumor cell lines (mesothelioma, DND-1A melanoma, HeLa uterine carcinoma) and we analyzed the effect of the calcium channel blocker verapamil on Ca2+ fluxes and on in vitro tumor cell growth. ADP was able to induce a transient increase in the cytoplasmic Ca2+ concentration in tumor cells from all lines; collagen showed this effect in mesothelioma cells and in HeLa cells, and thrombin was effective only in mesothelioma cells. Verapamil inhibited Ca2+ fluxes induced by the effective agonists in a dose-dependent manner. Values of IC50 for inhibition of ADP-induced Ca2+ transients were 63.5 microM in mesothelioma cells, 97.3 microM in DND-1A cells and 93.5 microM in HeLa cells, while those for inhibition of collagen-induced Ca2+ movements were slightly higher (170.2 microM in mesothelioma cells and 112.3 microM in HeLa cells) and the value of IC50 for inhibition of thrombin-induced Ca2+ fluxes (evaluated only in mesothelioma cells) was lower (22.5 microM). The drug dose-dependently also inhibited the in vitro growth of tumor cells; values of IC50 for growth inhibition were 21.8 microM in mesothelioma cells, 9.1 microM in DND-1A cells and 6.4 microM in HeLa cells, suggesting that the antiproliferative activity of verapamil was partly Ca(2+)-independent. These data may be of interest to elucidate the mechanisms of the two-way interactions of tumors with the hemostatic system and may help to identify new pharmacologic strategies for their control.
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PMID:Verapamil inhibits to different extents agonist-induced Ca2+ transients in human tumor cells and in vitro tumor cell growth. 903 Feb 40

Regional limb perfusion with antineoplastic agents stresses the local vasculature in a variety of ways. However, by monitoring the perfusates from limbs treated with melphalan alone or with melphalan plus tumor necrosis factor (TNF) and interferon-gamma (IFN-gamma), we were able to distinguish the effect of the cytokines on the observed coagulant and fibrinolytic responses. We collected samples of effluent from a series of lower extremities that were perfused with the cytokines and/or melphalan as treatment for localized melanoma. Both regimens produced statistically significant evidence of coagulant and fibrinolytic activation. However, limbs receiving cytokines in addition to the melphalan responded with a sharper rise in tissue plasminogen activator (tPA) and plasmin (plasmin-antiplasmin complexes [PAP]) than limbs treated with melphalan alone. Evidence of thrombin formation (prothrombin fragment 1 + 2 [F1 + 2], thrombin-antithrombin complexes [TAT]) was also greater when the cytokines were included, although the response was delayed and less consistent than the fibrinolytic activation.
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PMID:Fibrinolytic and coagulant responses to regional limb perfusions of tumor necrosis factor, interferon-gamma, and/or melphalan. 903 49

The 9E3/CEF4 gene codes for a chemokine that is highly homologous to human interleukin-8 and melanoma growth-stimulating activity/groalpha. These chemokines belong to a family of molecular mediators that are importantly involved in inflammation, wound healing, tumor development, and viral entry into cells. On the chorioallantoic membrane the 9E3 protein is chemotactic for monocyte/macrophages and lymphocytes and is angiogenic. In cultured chicken embryo fibroblasts, which have many of the properties of wound fibroblasts, the gene is stimulated by a variety of agents including oncogenes, growth factors, phorbol esters, and thrombin. The strong stimulation of 9E3 by thrombin in culture correlates well with the observation that in young chicks this gene is stimulated to very high levels in fibroblasts upon wounding and remains high throughout wound repair. Activation of 9E3 by thrombin: (i) occurs very rapidly, one minute exposure to thrombin is sufficient to initiate the signals necessary for gene activation; (ii) is independent of mitogenesis; (iii) operates through the proteolytically activated receptor for thrombin; (iv) is mediated by tyrosine kinases, including c-src and the epidermal growth factor (EGF) receptor, rather than Ser/Thr kinases such as protein kinase C and protein kinase A. Inhibition of either c-src or the EGF receptor tyrosine kinase inhibits the stimulation of 9E3 by thrombin. We show here for the first time that activation of the EGF receptor through a cell-surface receptor that does not have tyrosine kinase activity can lead to expression of an immediate early response gene which encodes for a secreted protein, a chemokine. This rapidly activated tyrosine kinase pathway may be a general stress response by which in vivo a localized cell population reacts to emergency situations such as viral infection, wounding, or tumor growth.
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PMID:Thrombin aivation of the 9E3/CEF4 chemokine involves tyrosine kinases including c-src and the epidermal growth factor receptor. 947 78


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