hirudin and Lung-Neoplasms

Inhibition of coagulation greatly limits cancer metastasis in many experimental models. Cancer cells trigger coagulation, through expression of tissue factor or P-selectin ligands that have correlated with worse prognosis in human clinical studies. Cancer cells also affect coagulation through expression of thrombin and release of microparticles that augment coagulation. In the cancer-bearing host, coagulation facilitates tumour progression through release of platelet granule contents, inhibition of Natural Killer cells and recruitment of macrophages. We are revisiting this literature in the light of recent studies in which treatment of clinical cohorts with anticoagulant drugs led to diminished metastasis.

Topics: Animals; Anticoagulants; Blood Coagulation; Blood Platelets; Cysteine Endopeptidases; Cytoplasmic Granules; Hirudins; Humans; Killer Cells, Natural; Lung Neoplasms; Macrophages; Mice; Neoplasm Metastasis; Neoplasm Proteins; Neoplasms, Experimental; Neoplastic Cells, Circulating; Neuraminidase; P-Selectin; Platelet Activation; Platelet Aggregation Inhibitors; Rats; Thrombin; Thrombophilia; Thromboplastin

Cancer cachexia and cancer-associated thrombosis are potentially fatal outcomes of advanced cancer. Nevertheless, thrombin expression in non-small cell lung cancer (NSCLC) primary tumour tissues and the association between prognosis of NSCLC patients remain largely unknown.. Clinical pathological analysis was performed to determine the relationship between thrombin and tumour progression. Effects of r-hirudin and direct thrombin inhibitor peptide (DTIP) on cancer progression were evaluated. Western blotting, immunohistochemistry, and immunofluorescence were used to explore the inhibition mechanism of r-hirudin and DTIP. The therapeutic effect of the combination of DTIP and chemotherapy was determined.. Thrombin expression in NSCLC tissues was closely related to clinicopathological features and the prognosis of patients. Thrombin deficiency inhibited tumour progression. The novel thrombin inhibitors, r-hirudin and DTIP, inhibited cell invasion and metastasis in vitro. They inhibited tumour growth and metastasis in orthotopic lung cancer model, inhibited cell invasion, and prolonged survival after injection of tumour cells via the tail vein. They also inhibited angiogenesis and spontaneous metastases from subcutaneously inoculated tumours. The promotion by thrombin of invasion and metastasis was abolished in PAR-1-deficient NSCLC cells. r-hirudin and DTIP inhibited tumour progression through the thrombin-PAR-1-mediated RhoA and NF-κB signalling cascades via inhibiting MMP9 and IL6 expression. DTIP potentiated chemotherapy-induced growth and metastatic inhibition and inhibited chemotherapy-induced resistance in mice.. Thrombin makes a substantial contribution, together with PAR-1, to NSCLC malignancy. The anti-coagulants, r-hirudin and DTIP, could be used in anti-tumour therapy and a combination of DTIP and chemotherapy might improve therapeutic effects.

Topics: Animals; Antineoplastic Agents; Antithrombins; Carcinoma, Non-Small-Cell Lung; Fibrinolytic Agents; Hirudins; Interleukin-6; Lung Neoplasms; Matrix Metalloproteinase 9; Mice; Neoplasm Metastasis; NF-kappa B; Thrombin

Topics: Cell Line, Tumor; Escherichia coli; Gene Expression; Hirudins; Humans; Lung Neoplasms; Neoplasm Invasiveness; Neoplasm Metastasis; Recombinant Fusion Proteins; Superoxide Dismutase

Thrombomodulin (TM) is a predominantly endothelial transmembrane glycoprotein that modulates hemostatic function through a domain that controls thrombin-mediated proteolysis and an N-terminal lectin-like domain that controls inflammatory processes. To test the hypothesis that TM is a determinant of malignancy and dissect the importance of these functional domains in cancer biology, metastatic potential was evaluated in TM(Pro) mice expressing a mutant form of TM with reduced thrombin affinity and TM(LeD) mice lacking the N-terminal lectin-like domain. Studies of TM(Pro) mice revealed that TM is a powerful determinant of hematogenous metastasis. TM(Pro) mice exhibited a strongly prometastatic phenotype relative to control mice that was found to result from increased survival of tumor cells newly localized to the lung rather than any alteration in tumor growth. The impact of the TM(Pro) mutation on metastasis was dependent on both tumor cell-associated tissue factor and thrombin procoagulant function. In contrast, expression of a mutant form of TM lacking the lectin-like domain had no significant impact on metastasis. These studies directly demonstrate for the first time that TM-mediated regulation of tumor cell-driven procoagulant function strongly influences metastatic potential and suggest that endothelial cell-associated modulators of hemostasis may represent novel therapeutic targets in limiting tumor dissemination.

Topics: Animals; Carcinoma, Lewis Lung; Female; Hirudins; Humans; Lectins; Liver Neoplasms; Lung Neoplasms; Lymphatic Metastasis; Male; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Neoplastic Cells, Circulating; Oligonucleotides, Antisense; Platelet Count; Prothrombin; Recombinant Proteins; Sarcoma, Experimental; Thrombin; Thrombomodulin

Cancer progression is facilitated by blood coagulation. Anticoagulants, such as Hirudin and low molecular weight heparins (LMWHs), reduce metastasis mainly by inhibition of thrombin formation and L- and P-selectin-mediated cell-cell adhesion. It is unknown whether the effects are dependent on cancer cell type. The effects of anticoagulants on tumor development of K1735 and B16 melanoma cells and CT26 colon cancer cells were investigated in mouse lung. Tumor load was determined noninvasively each week up to day 21 in all experiments using bioluminescence imaging. Effects of anticoagulants on tumor development of the three cell lines were correlated with the fibrin/fibrinogen content in the tumors, expression of tissue factor (TF), protease activated receptor (PAR)-1 and -4 and CD24, a ligand of L- and P-selectins. Hirudin inhibited tumor development of B16 cells in lungs completely but did not affect tumor growth of K1735 and CT26 cells. Low molecular weight heparin did not have an effect on K1735 melanoma tumor growth either. TF and PAR-4 expression was similar in the three cell lines. PAR-1 and CD24 were hardly expressed by K1735, whereas CT26 cells expressed low levels and B16 high levels of PAR-1 and CD24. Fibrin content of the tumors was not affected by LMWH. It is concluded that effects of anticoagulants are dependent on cancer cell type and are correlated with their CD24 and PAR-1 expression.

Topics: Animals; Anticoagulants; Blood Coagulation; CD24 Antigen; Cell Line, Tumor; Fibrin; Fibrinogen; Heparin, Low-Molecular-Weight; Hirudins; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; P-Selectin; Receptor, PAR-1; Thromboplastin; Transplantation, Heterologous

Tumor/host-generated thrombin (endogenous thrombin) was investigated with tumor growth and metastasis experiments in mice by the use of hirudin, a highly potent specific inhibitor of thrombin. Pretreatment with hirudin inhibited tumor implantation in nude or syngeneic mice, following subcutaneous injection of 2 human and 2 murine tumors. Hirudin induced a considerable lag period in the appearance of tumor growth, compared with phosphate-buffered saline (PBS) treatment, but had no effect on established tumor nodule growth in vivo or on tumor growth in vitro. Hirudin treatment induced central necrosis of the tumor nodule compared with no effect with PBS treatment. Greater protection was noted with longer duration of treatment. Tumor seeding into blood was examined with green fluorescent protein (GFP)-labeled tumor cells. Hirudin inhibited seeding into the blood as well as systemic organs which varied from complete protection to 15- to 32-fold in the blood and 17- to 395-fold in the lung. Hirudin inhibited spontaneous metastases from subcutaneously implanted tumor by reducing the number of tumor nodules in the lungs. Mouse survival in animals injected subcutaneously with highly aggressive 4T1 cells revealed 5 of 5 deaths of PBS-treated animals on day 40 compared with no deaths with hirudin treatment, with prolongation of survival with hirudin treatment of 16 days to more than 31 days. Thus, endogenous thrombin contributes to tumor implantation, seeding, and spontaneous metastasis. A potent antithrombin agent should be of clinical benefit to patients with cancer.

Topics: Animals; Cell Line, Tumor; Hirudins; Humans; Lung Neoplasms; Mice; Mice, Nude; Necrosis; Neoplasm Metastasis; Neoplasm Transplantation; Neoplastic Cells, Circulating; Survival Rate; Thrombin; Transplantation, Heterologous; Transplantation, Isogeneic

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

Detailed studies of tumor cell-associated procoagulants and fibrinolytic factors have implied that local thrombin generation and fibrin deposition and dissolution may be important in tumor growth and dissemination. To directly determine whether fibrin(ogen) or plasmin(ogen) are determinants of the metastatic potential of circulating tumor cells, this study examined the impact of genetic deficits in each of these key hemostatic factors on the hematogenous pulmonary metastasis of 2 established murine tumors, Lewis lung carcinoma and the B16-BL6 melanoma. In both tumor models, fibrinogen deficiency strongly diminished, but did not prevent, the development of lung metastasis. The quantitative reduction in metastasis in fibrinogen-deficient mice was not due to any appreciable difference in tumor stroma formation or tumor growth. Rather, tumor cell fate studies indicated an important role for fibrin(ogen) in sustained adhesion and survival of tumor cells within the lung. The specific thrombin inhibitor, hirudin, further diminished the metastatic potential of circulating tumor cells in fibrinogen-deficient mice, although the inhibitor had no apparent effect on tumor cell proliferation in vitro. The absence of plasminogen and plasmin-mediated fibrinolysis had no significant impact on hematogenous metastasis. The authors concluded that fibrin(ogen) is a critical determinant of the metastatic potential of circulating tumor cells. Furthermore, thrombin appears to facilitate tumor dissemination through at least one fibrin(ogen)-independent mechanism. These findings suggest that therapeutic strategies focusing on multiple distinct hemostatic factors might be beneficial in the containment of tumor metastasis.

Topics: Animals; Carcinoma, Lewis Lung; Cell Adhesion; Disease Models, Animal; Fibrinogen; Fibrinolysin; Fibrinolysis; Fibrinolytic Agents; Hemostasis; Hirudins; Histocytochemistry; Lung Neoplasms; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neoplasm Metastasis; Neoplastic Cells, Circulating; Neovascularization, Pathologic; Thrombin

Thrombomodulin (TM) is an anticoagulant molecule expressed on the endothelial cell surface and soluble TM antigen, which is present in human plasma and urine, represents the products of limited proteolytic cleavage of cell-surface TM. Recently, it was demonstrated that TM is also expressed on the surface of several tumor cells and the expression level of TM negatively correlated with malignancy in cancer. We investigated the effect of soluble TM isolated from human urine (uTM) on the invasion and metastasis of murine melanoma cells (B16F10 cells) through a reconstituted basement membrane (Matrigel) and in a murine model of experimental lung metastasis. Matrigel reconstituted with uTM inhibited the invasion of B16F10 cells in a dose-dependent manner in a range from 10 to 1000 ng/ml uTM as compared with the control Matrigel without uTM. The inhibitory action of uTM was not altered in the presence of an excess amount of hirudin, an inhibitor of thrombin proteolytic activity, but abolished in the presence of anti-human TM IgG. Matrigel reconstituted with thrombin (1 NIH unit/ml) enhanced the invasion level of cells by 1.5-fold relative to the control Matrigel without thrombin. The thrombin-enhanced invasion of B16F10 cells was repressed by addition of hirudin (10 units/ml) or uTM (100 ng/ml) into the Matrigel. Matrigel reconstituted with hirudin (10 units/ml) and uTM (100 ng/ml) additionally accelerated the inhibitory activity of hirudin or uTM on the thrombin-enhanced invasion of B16F10 cells. Moreover, metastatic colonies formed in the lungs of mice injected intravenously with B16F10 cells were significantly reduced by injection of uTM once a day up to 2 days after co-injection of uTM with the cells. These results suggested that Matrigel reconstituted with uTM inhibited the invasion of B16F10 cells in vitro through a thrombin-independent mechanism and the injection of uTM suppressed experimental lung metastasis of the cells in mice.

Topics: Animals; Collagen; Dose-Response Relationship, Drug; Drug Combinations; Hirudins; Humans; Immunoglobulin G; Laminin; Lung Neoplasms; Melanoma; Mice; Neoplasm Transplantation; Proteoglycans; Prothrombin; Thrombin; Thrombomodulin; Tumor Cells, Cultured

Early cellular events in the lung which may lead to the development of pulmonary metastases (PM) are still poorly understood. Thrombin, a key component of the coagulation cascade, may be involved in the development of PM as it has been shown to be an enhancer of platelet-tumor interaction in vitro and metastasis in vivo, and because it has been found in high levels in lungs from patients with PM. In this study, we assessed the potential role of thrombin in promoting PM by inducing an enhancement of tumor cell adhesion to platelets and tumor cell chemoinvasion and proliferation. We used bronchoalveolar lavage fluid (BALF) from 20 patients with PM. Results were compared with those from healthy controls. We found an enhancement of adhesion of PM-BALF-treated tumor cells to untreated platelets. BALF from patients with PM significantly increased chemoinvasion and proliferation in three human tumor cell lines. These activities were attenuated significantly by a thrombin inhibitor: hirudin. These results indicate that the thrombin present in the lungs of patients with PM is, at least in part, responsible for their adhesive, invasive and mitogenic activity on three different tumor cell lines. They also suggest that thrombin may be involved in the development of PM.

Topics: Adhesiveness; Antithrombins; Bronchoalveolar Lavage Fluid; Hirudins; Humans; In Vitro Techniques; Lung; Lung Neoplasms; Neoplasm Metastasis; Neoplastic Processes; Thrombin; Tumor Cells, Cultured

Recombinant desulfatohirudin (r-hirudin), a highly specific inhibitor of thrombin, was examined to determine whether it would inhibit production of experimental lung metastasis by B16-F10 melanoma cells. In in vitro assays using mouse plasma, the high level of procoagulant activity in B16-F10 cells was significantly inhibited by r-hirudin in a dose-dependent manner. From 15 to 120 min after s.c. administration into C57BL/6 mice, r-hirudin (10 mg/kg) markedly prolonged clotting time in a time course pattern that directly correlated with that of blood distribution of 125I-labeled r-hirudin. The production of experimental lung metastasis by B16-F10 cells was significantly inhibited by r-hirudin administered s.c. at time points ranging from 120 min before to 60 min after tumor cell inoculation with the most significant effects found in mice given r-hirudin 15 or 2 min before the i.v. injection of tumor cells. The organ distribution of [125I]IdUrd-labeled tumor cells demonstrated a clear difference in the lungs of mice treated with r-hirudin and the lungs of control mice, and these differences directly correlated with the number of lung tumor colonies found 3 weeks later. The inhibition of lung metastasis was not due to direct antitumor effects of r-hirudin. These results suggest that inhibition of coagulation events by r-hirudin significantly inhibit experimental lung metastasis during a critical time of 60 min after the entry of tumor cells into the circulation.

Topics: Animals; Blood Coagulation; Fibrinolytic Agents; Hirudin Therapy; Hirudins; Humans; Lung Neoplasms; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Recombinant Proteins

Cultured SK-OS-10 cells (human osteogenic sarcoma metastatic to lung) shed microvesicles (dia. 300-1000 nm) that contained procoagulant and proaggregatory activities inhibitable by hirudin, by anti-tissue factor antibody and by phospholipase A2. These results show that SK-OS-10 cells belong to a group including U87MG human glioblastoma and HL-60 promyelocytic leukemia in which these activities are due to a thrombin-dependent mechanism arising from the presence of tissue factor on the surface of the tumor cells and their shed microvesicles.

Topics: Antibodies; Blood Platelets; Cell Line; Creatine Kinase; Hirudins; Humans; Inclusion Bodies; Lung Neoplasms; Osteosarcoma; Phosphocreatine; Phospholipases; Platelet Activating Factor; Platelet Aggregation; Thromboplastin

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