hirudin and Adenocarcinoma

An in vitro cultured monolayer system of alveolar epithelial cells was used as a model to investigate the transport pathway of the peptides and proteins, salmon calcitonin (sCT), insulin (INS), recombinant hirudin (rHAV2), and recombinant human growth hormone (rhGH), in pulmonary epithelium. Human lung adenocarcinoma A549 cells formed continuous monolayers when grown on the polycarbonate filters of Transwell plates. The transport of the peptides and proteins having MW of 3400-22,000 Da was studied under different conditions. The results showed that the apparent permeability coefficients (P(app)) of these macromolecules across A549 cell monolayers ranged from 2x10(-6) to 5x10(-6) cm s(-1) and exhibited a good inverse correlation with molecular weight. No concentration, direction, or temperature dependence was observed in the permeation of sCT, INS, and rHAV2. While the P(app) of rhGH in the BA direction (2.25x10(-6) cm s(-1)) was less than that in the AB direction at both concentrations (3.20x10(-6) and 3.29x10(-6) cm s(-1)). The P(app) values of rhGH were concentration and temperature independent in the AB direction. These findings suggest that the hydrophilic peptides and proteins used in this study, sCT, INS, rHAV2, and rhGH, appear to cross the A549 cell monolayers via a paracellular pathway by a passive diffusion mechanism.

Topics: Adenocarcinoma; Biological Transport; Calcitonin; Hirudins; Humans; Insulin; Molecular Weight; Peptides; Permeability; Proteins; Pulmonary Alveoli; Tumor Cells, Cultured

Using a chemoinvasion assay, we show that platelets promote invasiveness of 5 pancreatic adenocarcinoma cell lines.. Gelatin zymography and Western blot analysis were performed to detect metalloproteinase-9 (MMP-9) secreted from tumor cells in the presence or absence of platelets. The effects of antiplatelet agents on the invasiveness of tumor cells and the secretion level of MMP-9 were evaluated.. The number of traversed tumor cells significantly increased when incubated with platelets compared without platelets in all cell lines. The MMP-9 band was detected in all tumor cell lines, and the intensity was obviously greater in conditions of incubation with platelets than without. In the experiment of antiplatelet agents effects, it was confirmed that invasiveness of tumor cells significantly decreased following incubation with cilostazol depending on the concentration in spite of the presence of platelets. The level of MMP-9 also significantly decreased in the ELISA analysis.. These data mean platelets activate invasiveness of tumor cells because of enhanced MMP-9 secretion. Furthermore, anti-platelet drugs may inhibit invasiveness of tumor cells due to decreased MMP-9 secretion, and this inhibition may lead to the suppression of tumor cell invasion. We propose that antiplatelet agents are applicable in clinical treatment to inhibit metastasis of malignant tumor cells.

Topics: Adenocarcinoma; Adenosine Diphosphate; Adult; Blood Platelets; Cell Line, Tumor; Cilostazol; Collagen; Drug Combinations; Drug Screening Assays, Antitumor; Eicosapentaenoic Acid; Epoprostenol; Hirudins; Humans; Laminin; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Neoplasm Proteins; Pancreatic Neoplasms; Platelet Activation; Platelet Aggregation Inhibitors; Proteoglycans; Tetrazoles; Thrombin

An in vitro cultured monolayer system of alveolar epithelial cells was used as a model to investigate the transport pathway peptides or proteins, salmon calcitonin (sCT), insulin (INS), recombinant hirudin (rHAV2), and recombinant human growth hormone (rhGH), in pulmonary epithelium in vivo.. Human lung adenocarcinoma A549 cells formed continuous monolayers with growing polycarbonate filters of Transwell plate. Transport studies of macromolecules in the monolayer system were carried out after 6 days in culture. The transport of peptides or proteins with MW 3,400 - 22,000 was studied in cultured human lung adenocarcinoma A549 cell monolayers at different conditions.. The results showed that the apparent permeability coefficients (Papp) of these macromolecules across A549 cell monolayers ranged from 2 x 10(-6) to 5 x 10(-6) cm x s(-1) and exhibited good inverse correlation with molecule weight. No concentration, direction and temperature dependence were observed in the permeation of sCT, INS and rHAV2. While the Papp of rhGH in the BA direction (2.25 x 10(-6) cm x s(-1)) was significantly less than that in the reverse direction. The Papp values of rhGH were concentration and temperature independent in the AB direction.. These findings suggest that the hydrophilic peptides and proteins, salmon calcitonin, insulin, recombinant hirudin, and recombinant human growth hormone used in this study, appeared to penetrate the A549 cell monolayers via a paracellular pathway by passive diffusion mechanism.

Topics: Adenocarcinoma; Biological Transport; Calcitonin; Cell Line, Tumor; Epithelial Cells; Hirudins; Human Growth Hormone; Humans; Insulin; Lung Neoplasms; Peptides; Permeability; Proteins; Pulmonary Alveoli

Coagulation has long been known to facilitate metastasis. To pinpoint the steps where coagulation might play a role in the metastasis, we used three-dimensional visualization of direct infusion of fluorescence labeled antibody to observe the interaction of tumor cells with platelets and fibrinogen in isolated lung preparations. Tumor cells arrested in the pulmonary vasculature were associated with a clot composed of both platelets and fibrin(ogen). Initially, the cells attached to the pulmonary vessels were rounded. Over the next 2 to 6 hours, they spread on the vessel surface. The associated clot was lysed coincident with tumor cell spreading. To assess the importance of clot formation, we inhibited coagulation with hirudin, a potent inhibitor of thrombin. The number of tumor cells initially arrested in the lung of hirudin-treated mice was essentially the same as in control mice. However, tumor cell spreading and subsequent retention of the tumor cells in the lung was markedly inhibited in the anticoagulated mice. These associations of the tumor cells with platelets were independent of tumor cell expression of P-selectin ligands. This work identifies tumor cell spreading onto the vascular surface as an important component of the metastatic cascade and implicates coagulation in this process.

Topics: Adenocarcinoma; Animals; Blood Coagulation; Blood Platelets; Cell Communication; Cell Line, Tumor; Colonic Neoplasms; Fibrinogen; Fibrosarcoma; Hirudins; Humans; Lung; Lung Neoplasms; Melanoma; Melanoma, Experimental; Mice; Mice, Nude; Neoplasm Metastasis; Neoplasms; Neoplastic Cells, Circulating; Rats; Thrombin

Topics: Adenocarcinoma; Adult; Anticoagulants; Arterial Occlusive Diseases; Autoimmune Diseases; Drug Resistance; Female; Heparin; Heparin, Low-Molecular-Weight; Hepatitis C, Chronic; Hirudins; Humans; Inflammatory Bowel Diseases; Male; Middle Aged; Neoplasms, Unknown Primary; Platelet Aggregation Inhibitors; Portal Vein; Pulmonary Embolism; Recombinant Proteins; Recurrence; Thrombocytopenia; Vena Cava, Inferior; Venous Thrombosis; Warfarin

PC-3 cells, a metastatic human prostate adenocarcinoma line, caused dose-dependent platelet aggregation in heparinised human platelet-rich plasma (PRP). PC-3 tumour cell-induced platelet aggregation (TCIPA) was completely inhibited by hirudin (5 U/ml) and limited by increasing concentrations of apyrase. This TCIPA was unaffected by cysteine proteinase inhibition with E-64 (10 microM), but was limited by cell pretreatment with phospholipase A2. PC-3 cell suspension caused marked, dose-dependent decreases in plasma recalcification times using normal, Factor VIII-deficient and Factor IX-deficient, but not Factor VII-deficient, human plasma. This effect was potentiated in cell lysates, but was inhibited in intact cells preincubated with sphingosine. Overall, these data suggest that PC-3 TCIPA arises from PC-3 tissue factor activity expression. Trigramin and rhodostomin, RGD-containing snake venom peptides which antagonise the binding of fibrinogen to platelet membrane glycoprotein IIb-IIIa, prevented PC-3 TCIPA. Similarly, synthetic peptide GRGDS as well as monoclonal antibodies against platelet membrane glycoproteins IIb-IIIa and Ib prevented PC-3 TCIPA, which was unaffected by control peptide GRGDS. On a molar basis, trigramin (IC50, 0.11 microM) and rhodostomin (IC50, 0.03 microM) were approximately 5000 and 18000 times, respectively, more potent than GRGDS (IC50, 0.56 mM).

Topics: Adenocarcinoma; Antibodies, Monoclonal; Apyrase; Cysteine Proteinase Inhibitors; Fibrinogen; Glycoproteins; Hirudins; Humans; Intercellular Signaling Peptides and Proteins; Male; Peptides; Phospholipases A; Phospholipases A2; Platelet Aggregation; Platelet Aggregation Inhibitors; Prostatic Neoplasms; Thrombin; Tumor Cells, Cultured

MCF-7 cells, a metastatic human breast carcinoma line, caused dose-dependent platelet aggregation in heparinized human platelet-rich plasma (PRP). MCF-7 tumor cell-induced platelet aggregation (TCIPA) was almost blocked by apyrase (0.5 U/ml) and completely inhibited by hirudin (5 U/ml). This TCIPA was unaffected by cysteine proteinase inhibition with E-64 (10 microM), but was limited by cell pretreatment with phospholipase A2. MCF-7 cell suspension caused marked, dose-dependent decrease in plasma recalcification times using normal, Factor VIII-deficient, and Factor IX-deficient human plasma. This effect was potentiated in cell lysates but was inhibited in intact cells preincubated with sphingosine. MCF-7 cell suspension did not affect the recalcification time of Factor VII-deficient plasma. Taken together, these data suggest that MCF-7 TCIPA arises from MCF-7 tissue factor activity expression. Trigramim and rhodostomin, RGD-containing snake venom peptides which antagonized the binding of fibrinogen to platelet membrane glycoprotein IIb/IIIa, prevented MCF-7 TCIPA. Likewise, synthetic peptide GRGDS as well as monoclonal antibodies against human tissue factor, platelet membrane glycoprotein IIb/IIIa and Ib prevented MCF-7 TCIPA, which was unaffected by control peptide GRGES. On a molar basis, trigramin (IC50, 0.1 microM) and rhodostomin (IC50, 0.03 microM), were about 5,000 and 18,000 times, respectively, more potent than GRGDS (IC50, 0.54 mM).

Topics: Adenocarcinoma; Animals; Antibodies, Monoclonal; Apyrase; Breast Neoplasms; Crotalid Venoms; Cysteine Proteinase Inhibitors; Female; Hirudins; Humans; Intercellular Signaling Peptides and Proteins; Oligopeptides; Peptides; Phospholipases A; Phospholipases A2; Platelet Aggregation; Platelet Aggregation Inhibitors; Sphingosine; Tumor Cells, Cultured

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

Involvement of platelet membrane glycoproteins (GP) in interactions between platelets and tumor cells was studied by using two human tumor cell lines and two monoclonal antibodies against platelet membrane GP. HMV-I cells derived from vaginal melanoma induced platelet aggregation in heparinized plasma, which was not followed by coagulation. M7609 cells derived from colon adenocarcinoma also induced platelet aggregation in heparinized plasma, which, on the contrary, was followed by coagulation. Aggregating activities of the HMV-I cells were abolished by pretreatment with neuraminidase or trypsin, but M7609 activity was not labile to these enzymes. Aggregations induced by M7609 were inhibited by hirudin or MD805, while those by HMV-I were not. M7609 cells dose dependently shortened the recalcification time of normal as well as Factor IX-deficient plasmas, while they were not effective in shortening the time of Factor II- or Factor VII-deficient plasmas. The procoagulant activity of HMV-I cells was 1000 times less than M7609 on the basis of cell numbers. When human platelets were preincubated with monoclonal anti-GPIb or anti-GPIIb/IIIa complex antibodies, neither cell line could cause aggregations. These findings suggest that both GPIb and the GPIIb/IIIa complex on the platelet surface are involved in the thrombin-dependent and -independent platelet aggregations induced by tumor cells.

Topics: Adenocarcinoma; Antibodies, Monoclonal; Cell Communication; Cell Line; Colonic Neoplasms; Female; Hirudins; Humans; Melanoma; Neuraminidase; Platelet Aggregation; Platelet Membrane Glycoproteins; Thrombin; Trypsin; Tumor Cells, Cultured; Vaginal Neoplasms

Topics: Adenocarcinoma; Animals; Apyrase; Cell Line; Colonic Neoplasms; Glioma; Hirudins; Humans; Kinetics; Lung Neoplasms; Melanoma; Mesothelioma; Mice; Neoplasms; Neoplasms, Experimental; Neuroblastoma; Phospholipases; Phosphoric Monoester Hydrolases; Platelet Aggregation

Two lines of mouse tumor cells were shown to be capable of aggregating mouse and rabbit platelets in vitro. This process required higher Mg2+ concentrations than were needed by other commonly used platelet-aggregating agents. Platelet-aggregating activity was also found in tumor cell membrane fragments. This membrane-bound platelet-aggregating material contained protein, lipid, and carbohydrate moieties. The presence of all three appeared to be essential for stimulating platelet aggregation. Destruction of any component abolished its activity: protein by trypsin; lipid by phospholipase A2 and non-ionic detergents; and sialic acid by neuraminidase. Platelet aggregation induced by tumor cell membrane fragments was associated with a secretory release reaction. In this process, growth-promoting activity for tumor cells was also released from platelets. These results underline the importance of platelets in establishing tumor metastases.

Topics: Adenocarcinoma; Adenosine Diphosphate; Animals; Aspirin; Cell Line; Cell Membrane; Growth Substances; Hirudins; In Vitro Techniques; Kidney Neoplasms; Magnesium; Mice; Neoplasms, Experimental; Neuroblastoma; Platelet Aggregation; Rabbits