rhodostomin and Colonic-Neoplasms

The interactions between tumour cells and the microvasculature, including the adhesion of tumour cells to endothelium and extracellular matrix (ECM) as well as their migratory ability, are prerequisites for metastasis to occur. In this study we showed that thrombin is capable of enhancing in vitro tumour cell metastatic potential in terms of adhesive properties and migratory response. Following exposure to subclotting concentrations of thrombin, SW-480 human colon adenocarcinoma cells exhibited increased adhesion to both the endothelium and ECM component (i.e. fibronectin). Likewise, the pretreatment of thrombin enhanced the migratory ability of SW-480 cells. The enhanced adhesion was significantly inhibited by complexing of thrombin with its inhibitor hirudin, or by serine proteinase inhibition with 3,4-DCI, but was unaffected by pretreatment of tumour cells with actinomycin D or cycloheximide. The effect of thrombin resulted in an upregulated cell-surface expression of beta 3 integrins, a group of receptors mediating interactions between tumour cells and endothelial cells, and between tumour cells and ECM. Antibodies against beta 3 integrins effectively blocked both the enhanced adhesion and migration. This thrombin-mediated up-regulation of beta 3 integrins involved the activation of protein kinase C (PKC) as thrombin-enhanced adhesion was diminished by PKC inhibition. Rhodostomin, an Arg-Gly-Asp-containing antiplatelet snake venom peptide that antagonises the binding of ECM toward beta 3 integrins on SW-480 cells, was about 600 and 500 times, more potent that RGDS in inhibiting thrombin-enhanced adhesion and migration respectively. Our data suggest that PKC inhibitors as well as rhodostomin may serve as inhibitory agents in the prevention of thrombin-enhanced metastasis.

Topics: Adenocarcinoma; Amino Acid Sequence; Antibodies, Monoclonal; Antigens, CD; Cell Adhesion; Cell Movement; Colonic Neoplasms; Endothelium, Vascular; Enzyme Inhibitors; Fibronectins; Humans; Integrin beta3; Molecular Sequence Data; Oligopeptides; Peptides; Platelet Aggregation Inhibitors; Platelet Membrane Glycoproteins; Protein Kinase C; Thrombin; Tumor Cells, Cultured

Integrins are a superfamily of cell surface glycoproteins that mediate cell-extracellular matrix (ECM) and cell-cell adhesion. Immunofluorescence microscopy and flow cytometric analysis using anti-integrin mAbs as the primary binding ligands demonstrated that the platelet integrin receptor alpha IIb beta 3, as well as alpha v beta 3, alpha 5 beta 1 and alpha 6 beta 1, are present on the surface of SW-480 human colon adenocarcinoma cells. Monoclonal antibodies (mAbs) against alpha IIb beta 3 and alpha 5 beta 1 inhibited unstimulated basal adhesion to fibronectin by approximately 30% and 40%, respectively. The surface immunoreactivity of tumor cells for alpha IIb beta 3 was enhanced by pretreatment (5 min) with a phorbol ester (12-O-tetradecanoylphorbol-13-acetate (TPA)) or a lipoxygenase metabolite of arachidonic acid, 12-hydroxyeicosatetraenoic acid (12-HETE) in a dose- and time-dependent manner. SW-480 cells possess a large intracellular pool of alpha IIb beta 3, from which the receptor complex translocates to the cell surface following pretreatment with TPA or 12(S)-HETE. This pretreatment enhances adhesion to fibronectin, which is mediated exclusively by alpha IIb beta 3 integrins. Staurosporine was found to block alpha IIb beta 3 up-regulation and enhanced-adhesion. TPA and 12(S)-HETE also facilitated the redistribution of alpha IIb beta 3 during the enhanced-spreading process. Rhodostomin, an Arg-Gly-Asp- (RGD) containing antiplatelet snake venom peptide, was about 400-times more potent than RGDS at inhibiting control, TPA- or 12(S)-HETE-enhanced adhesion of SW-480 cells to fibronectin. The binding of mAbs against alpha IIb beta 3, alpha v beta 3 and alpha 5 beta 1 was inhibited by pretreatment with rhodostomin, suggesting that rhodostomin binds via its RGD sequence to multiple integrin receptors (i.e., alpha IIb beta 3, alpha v beta 3, alpha 5 beta 1) expressed on the SW-480 cell surface, inhibiting cell adhesion to ECM.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adenocarcinoma; Antibodies, Monoclonal; Binding Sites, Antibody; Cell Adhesion; Colonic Neoplasms; Extracellular Matrix; Fibronectins; Fluorescent Antibody Technique; Humans; Hydroxyeicosatetraenoic Acids; Integrins; Peptides; Platelet Aggregation Inhibitors; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Up-Regulation

SW-480 cells, derived from a primary human colon adenocarcinoma, caused dose-dependent platelet aggregation in heparinized human platelet-rich plasma. SW-480 tumour cell-induced platelet aggregation (TCIPA) was completely inhibited by hirudin (5 U/ml) but unaffected by apyrase (10 U/ml). This TCIPA was also unaffected by cysteine proteinase inhibition with E-64 (10 microM) but was limited by cell pretreatment with phospholipase A2. SW-480 cell suspension caused marked dose-dependent decreases in plasma recalcification times using normal, factor VIII-deficient and factor IX-deficient human plasma. This effect was potentiated with cell lysates but inhibited in intact cells pretreated with sphingosine. SW-480 cell suspension did not affect the recalcification time of factor VII-deficient plasma. Moreover, monoclonal antibody against human tissue factor completely abolished SW-480 TCIPA. Taken together, these data suggest that SW-480 TCIPA arises from SW-480 tissue factor activity expression. Trigramin and rhodostomin, RGD-containing snake venom peptides, which antagonize the binding of fibrinogen to platelet membrane glycogen IIb/IIIa, prevented SW-480 TCIPA. Likewise, synthetic peptide GRGDS as well as monoclonal antibodies against platelet membrane glycoprotein IIb/IIIa and Ib prevent SW-480 TCIPA, which was unaffected by control peptide GRGES. On a molar basis, trigramin (IC50 0.09 microM) and rhodostomin (IC50 0.03 microM) were about 6000 and 18,000 times, respectively, more potent than GRGDS (IC50 0.56 mM).

Topics: Adenocarcinoma; Apyrase; Calcium; Colonic Neoplasms; Cysteine Proteinase Inhibitors; Fibrinogen; Hirudins; Humans; Intercellular Signaling Peptides and Proteins; Kinetics; Peptides; Phospholipases A; Phospholipases A2; Platelet Aggregation; Platelet Aggregation Inhibitors; Tumor Cells, Cultured