hirudin and Neoplasm-Metastasis

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

The metastasis of tumor cells is one of the major obstacles to successful clinical therapy. A treatment strategy by incorporating a specific inhibitor of thrombin, recombinant hirudin with stealthy liposomal vinblastine, was used in this study for inhibiting the metastasis of tumor cells and enhancing the efficacy of anti-tumor agents. In vitro cytotoxicity, cell adhesion to extracellular matrix (ECM) proteins, and cell invasion and migration assays were performed on human A375 melanoma cell line. In vivo measurement of coagulation parameters, inhibition of tumor growth, and inhibition of metastasis were assessed in female BALB/c mice. In vitro, vinblastine or stealthy liposomal vinblastine alone was effective to inhibit the growth of A375 cells. On the contrary, hirudin had no influence on either cytotoxicity when treating with hirudin alone or hirudin plus vinblastine. In addition, in vitro results showed that hirudin had no impact on the adhesion of tumor cells to extracellular matrix proteins, and metastasis and invasion of tumor cells. In mice, hirudin significantly inhibited the activity of thrombin. Furthermore, administered at the initial implantation of murine B16 melanoma cells, hirudin evidently delayed the growth of tumor, and depressed the occurrence of experimental lung metastasis. A subsequent administration of stealthy liposomal vinblastine resulted in further inhibiting growth and metastasis of tumor, indicating that hirudin plus stealthy liposomal vinblastine exhibited a significant anti-metastasis effect and slightly potent effect against tumor growth as compared with stealthy liposomal vinblastine alone. In conclusion, administration of recombinant hirudin followed by giving stealthy liposomal vinblastine may be beneficial for inhibiting the growth and metastasis of melanoma in vivo. The likely mechanism could be associated with inhibition of thrombin after administration of hirudin.

Topics: Animals; Antineoplastic Agents, Phytogenic; Cell Adhesion; Cell Line, Tumor; Cell Movement; Drug Carriers; Drug Screening Assays, Antitumor; Drug Synergism; Extracellular Matrix Proteins; Hirudins; Homeostasis; Humans; Liposomes; Male; Melanoma; Melanoma, Experimental; Mice; Mice, Inbred BALB C; Neoplasm Invasiveness; Neoplasm Metastasis; Particle Size; Protease Inhibitors; Recombinant Proteins; Tetrazolium Salts; Thiazoles; Vinblastine

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

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

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

Saos-2 cells, derived from a primary human osteosarcoma, caused dose-dependent platelet aggregation in heparinised human platelet-rich plasma. Saos-2 tumour cell-induced platelet aggregation (TCIPA) was completely inhibited by hirudin but unaffected by apyrase. The cell suspension shortened the plasma recalcification times of normal, factor VIII-deficient and factor IX-deficient human plasmas in a dose-dependent manner. However, the cell suspension did not affect the recalcification time of factor VII-deficient plasma. Moreover, a monoclonal antibody (MAb) against human tissue factor completely abolished TCIPA. Flow cytometric analysis using anti-integrin MAbs as the primary binding ligands demonstrated that the integrin receptors alpha v beta 3, alpha 5 beta 1 and alpha 6 beta 1 were present of Saos-2 cells, which might mediate tumour cell adhesion to extracellular matrix. Rhodostomin, an Arg-Gly-Asp (RGD)-containing snake venom peptide which antagonises the binding of fibrinogen to platelet membrane glycoprotein IIb/IIIa, prevented Saos-2 TCIPA as well as tumour cell adhesion to vitronectin, fibronectin and collagen type I. Likewise, the synthetic peptide Gly-Arg-Gly-Asp-Ser (GRGDS) showed a similar effect. On a molar basis, rhodostomin was about 18,000 and 1000 times, respectively, more potent than GRGDS in inhibiting TCIPA and tumour cell adhesion.

Topics: Amino Acid Sequence; Antibodies, Monoclonal; Apyrase; Blood Coagulation Disorders; Blood Coagulation Factors; Bone Neoplasms; Cell Adhesion; Enzyme Activation; Extracellular Matrix; Hirudins; Humans; Integrins; Molecular Sequence Data; Neoplasm Metastasis; Neoplasm Proteins; Oligopeptides; Osteosarcoma; Peptides; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet Membrane Glycoproteins; Protein Binding; Thrombin; Tumor Cells, Cultured

Tumor cell-induced platelet aggregation (TCIPA) is considered to be a critical step in hematogenous metastasis.. TCIPA was studied in vitro in six human pancreatic carcinoma cell lines (PC 3, PC 44, AsPC1, BxPC3, Capan2, Panc1).. Whereas all cell lines induced aggregation of washed platelets in the presence of minimal amounts of platelet-poor plasma, five cell lines also induced aggregation of platelets in platelet-rich plasma. The thrombin-antagonist hirudin inhibited TCIPA in all cell lines indicating that TCIPA is thrombin-dependent. Since pretreatment of tumor cells with phospholipase A2 or C inhibited TCIPA, the thrombin-generating activity might be confined to the tumor cell surface. Further support for a prothrombinase activity was provided by the observation that all cell lines were able to induce the aggregation of washed platelets after addition of purified coagulation factors II and V.. Pancreatic carcinoma cells are able to induce platelet aggregation via activation of thrombin. This might support metastasis in pancreatic cancer and possibly explain the incidence of thrombosis in this tumor.

Topics: Antithrombins; Hirudins; Humans; Neoplasm Metastasis; Pancreatic Neoplasms; Platelet Aggregation; Platelet Aggregation Inhibitors; Thrombin; Tumor Cells, Cultured