hirudin and Glioma

Glioma is the most common primary malignant brain tumour, and survival is poor. Hirudin has anticancer pharmacological effects through suppression of glioma cell progression, but the molecular target and mechanism are poorly understood. In this study, we observed that hirudin dose- and time-dependently inhibited glioma invasion, migration and proliferation. Mechanistically, hirudin activated LC3-II but not Caspase-3 to induce the autophagic death of glioma cells by decreasing the phosphorylation of mTOR and its downstream substrates ULK1, P70S6K and 4EBP1. Furthermore, hirudin inhibited glioma growth and induced changes in autophagy in cell-derived xenograft (CDX) nude mice, with a decrease in mTOR activity and activation of LC3-II. Collectively, our results highlight a new anticancer mechanism of hirudin in which hirudin-induced inhibition of glioma progression through autophagy activation is likely achieved by inhibition of the mTOR signalling pathway, thus providing a molecular basis for hirudin as a potential and effective clinical drug for glioma therapy.

Topics: Animals; Apoptosis; Autophagy; Cell Line, Tumor; Cell Proliferation; Glioma; Hirudins; Humans; Mice; Mice, Nude; TOR Serine-Threonine Kinases

Patients with central nervous system (CNS) malignancies have a substantial risk for developing both thrombotic and bleeding disorders. The risk of venous thromboembolism (VTE) is substantially higher in these patients, both in the perioperative period and throughout their disease course. Patients with CNS malignancy harbor a latent hypercoagulability, which predisposes to VTE, as do postoperative immobility, hemiparesis, and other factors. The management of VTE in these patients is complex, given the significant morbidity and mortality associated with intratumoral hemorrhage. In the past, the perceived risk of intracranial hemorrhage limited the use of anticoagulation for the management of VTE with many favoring nonpharmacologic methods for prophylaxis and treatment. Inferior vena cava (IVC) filters have since lost favor at many centers given significant complications, which appear to be more frequent in patients with CNS malignancy. Recent studies have demonstrated safe and efficacious use of anticoagulation in these patients with a low incidence of intracranial hemorrhage. Treatment of established VTE is now recommended in this population with many centers favoring low-molecular-weight heparin (LMWH) versus oral warfarin for short- or long-term treatment. We advocate a multimodality approach utilizing compression stockings, intermittent compression devices, and heparin in the perioperative setting as the best proven method to reduce the risk of VTE. In the absence of a strict contraindication to systemic anticoagulation, such as previous intracranial hemorrhage or profound thrombocytopenia, we recommend LMWH in patients with newly diagnosed VTE and a CNS malignancy.

Topics: Antibodies, Monoclonal, Humanized; Anticoagulants; Arginine; Bevacizumab; Central Nervous System Neoplasms; Fondaparinux; Glioblastoma; Glioma; Hemorrhage; Heparin, Low-Molecular-Weight; Hirudins; Humans; Pipecolic Acids; Polysaccharides; Postoperative Complications; Pulmonary Embolism; Recombinant Proteins; Sulfonamides; Thrombocytopenia; Vena Cava Filters; Venous Thromboembolism; Venous Thrombosis; Warfarin

In addition to a recognized role in the coagulation cascade, thrombin is known to have other functions via G protein-coupled receptors, including protease-activated receptor-1 (PAR-1). To investigate the relationship between PAR-1 activation and proinflammatory cytokine expression, we studied the responsiveness of C6 cells to thrombin and to the agonist PAP-1-activating peptide (PAR-1-AP).. Cultured C6 rat glioma cells were stimulated with human alpha-thrombin or PAR-1-AP. To study mRNA expression changes, total RNA was isolated from the C6 cells, reverse transcribed, and amplified by real-time polymerase chain reaction. Three proinflammatory cytokines were studied: interleukin-6 (IL-6), interleukin-1beta (IL-1beta), and tumor necrosis factor-alpha (TNF-alpha). To measure cytokine release, cell-free supernatants were assayed using enzyme-linked immunosorbent assay (ELISA).. By quantitative real time reverse transcriptase polymerase chain reaction, thrombin (5 U/mL) exposure significantly increased mRNA expression of the proinflammatory cytokines: IL-6 (2.8 +/- 0.4, multiple of control), IL-1beta (4.8 +/- 1.6), and TNF-alpha (16.5 +/- 4.2). Effects on IL-6 mRNA expression were dose-dependent and matched by increments in IL-6 protein secretion. Effects of thrombin on IL-6 mRNA expression could be inhibited by hirudin. PAR-1-AP exposure also significantly increased mRNA expression of IL-6, IL-1beta and TNF-alpha. PAR-1 mRNA is expressed in C6 cells.. Both thrombin and its agonist, PAR-1-AP, significantly increased mRNA expression of pro-inflammatory cytokines in C6 glioma cells via PAR-1 activation.

Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Cytokines; Fibrinolytic Agents; Gene Expression; Glioma; Hemostatics; Hirudins; Interleukin-1; Interleukin-6; Oligopeptides; Pancreatitis-Associated Proteins; Rats; RNA, Messenger; Thrombin; Tumor Necrosis Factor-alpha

Thrombin is a key enzyme in the blood coagulation system where it converts fibrinogen to fibrin. It participates in a variety of biological processes such as the induction of mitogenesis and of morphological changes, the production of cytokines and growth factors, and apoptosis. To clarify the role of thrombin in the proliferation of human malignant gliomas, we investigated its effect on the expression of vascular endothelial growth factor (VEGF) in vitro and determined its intrinsic expression in human glioma tissues. In 3 human glioma cell lines tested, U-87 MG, U-251 MG, and U-105 MG, thrombin induced the VEGF mRNA expression and protein in a dose- and time-dependent manner. The thrombin receptor expression was detectable by RT-PCR and immunoblot. The secretion of VEGF protein in glioma cells was stimulated by the thrombin receptor agonist peptide and the induction of VEGF was significantly blocked by the thrombin inhibitor hirudin, indicating that the up-regulation of VEGF was mediated by the thrombin/thrombin receptor pathway. Immunoblot analysis demonstrated that prothrombin, the precursor of thrombin, was distributed in all 10 glioma tissues examined. In situ hybridization and immunohistochemical analysis revealed the co-localization of prothrombin mRNA-positive and GFAP-positive cells in the glioma tissues. Although various factors may be involved in the up-regulation of VEGF, our results suggest that human gliomas per se express prothrombin, and that thrombin, converted from prothrombin in glioma tissues, substantially stimulates angiogenesis in an autocrine fashion.

Topics: Blotting, Northern; DNA, Complementary; Dose-Response Relationship, Drug; Endothelial Growth Factors; Enzyme-Linked Immunosorbent Assay; Glioma; Hirudins; Humans; Immunoblotting; Immunohistochemistry; In Situ Hybridization; Lymphokines; Neovascularization, Pathologic; Plasmids; Protease Inhibitors; Prothrombin; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Messenger; Thrombin; Time Factors; Tumor Cells, Cultured; Up-Regulation; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Thrombin at nanomolar concentrations induces rapid changes in the second messenger Ca2+ in a glial astrocyte-type cell line. Continuous application of the protease thrombin causes regular [Ca2+]i oscillations (amplitude 109 nM, spike length 48 s) which are suppressed by hirudin. Reduction of [Ca2+]ex (from 1.8 mM to 50 microM) reversibly abolishes the oscillations indicating the contribution of Ca2+ influx to generation of the oscillations. Thrombin receptor-activating peptide (TRAP, 1-10 microM) causes similar Ca2+ oscillations which depend, like the oscillations induced by thrombin, on the continuous presence of agonist. Thus, we can deduce that cell surface receptors are responsible for the effect of thrombin on glioma cells.

Topics: Animals; Calcium; Cell Line; Endopeptidases; Fura-2; Glioma; Hirudins; Rats; Receptors, Thrombin; Thrombin; Time Factors

Treatment of rat glioma C6 cells with a beta-adrenergic agonist leads to a rise in cAMP level and a subsequent change in cell morphology from an epithelial to an astrocyte type of appearance. This morphological change is reverted by the addition of thrombin. In 10-15 min the cells acquire their normal epithelial morphology. The reversion by thrombin is inhibited by hirudin, but not by antithrombin III (an inhibitor of the proteolytic action of thrombin). Using the intracellular Ca2(+)-indicator fura-2, we observed that the addition of thrombin to the glioma cells generated a Ca2(+)-signal which was inhibited by pretreatment of the cells with hirudin or with 1 mM neomycin. These results suggest that thrombin uses the phospholipid-inositol pathway to counteract the morphological response, which was induced by activation of the cAMP pathway.

Topics: Animals; Antithrombin III; Astrocytes; Blood; Calcium; Culture Media; Epithelial Cells; Glioma; Hirudins; Ionomycin; Isoproterenol; Microscopy, Electron, Scanning; Neomycin; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Signal Transduction; Tetradecanoylphorbol Acetate; Thrombin; Tumor Cells, Cultured

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 different mechanisms of aggregation of heparinized human platelet-rich plasma have been identified with two tumor cell lines: In neither case are these mechanisms dependent on platelet-derived ADP. U87MG cells from a glioblastoma line of human origin caused a single irreversible wave of aggregation simultaneously with the onset of platelet secretion, and this was inhibited by heparin and hirudin but not by apyrase or phospholipase D. In contrast, Hut 20 cells from an undifferentiated tumor cell line of murine origin gave an initial reversible wave followed by a second irreversible wave, which then led to secretion. The first wave of platelet aggregation was unaffected by heparin or hirudin but was inhibited by apyrase, and the second wave was inhibited by phospholipase D. Citrate caused irreversible inhibition with either cell line, and aggregation did not occur with gel filtered platelets. These results suggest that platelet aggregation by the Hut 20 line is initially dependent on ADP released from the tumor cells, whereas aggregation induced by the U87MG line is dependent on a procoagulant activity of the tumor cell surface.

Topics: Adenosine Diphosphate; Animals; Apyrase; Blood Platelets; Cell Line; Cell Membrane; Glioma; Heparin; Hirudins; Humans; Membrane Proteins; Mice; Neoplasms, Experimental; Phospholipase D; Platelet Aggregation