- Email: [email protected]
PO-20 Organic metallic complexes with antiplatelet and antineoplasmatic effects
5th ICTHIC Abstracts: Poster Sessions / Thrombosis Research 125 (2010) S166–S191
Results: Surgery resulted in a significant increase in metastases and anticoagulation with LMWH completely abrogated this effect. The significant difference in metastatic foci was seen at 12 hours and 3 days post surgery but not at earlier time points (10 minutes and 4 hours). This indicates that surgical stress facilitates metastases by enhancing the sustained adherence and survival of individual TCE in the vasculature while anticoagulation with LMWH prevents this effect. Fibrin clots were associated with TCE significantly more frequently in mice that underwent partial hepatectomy, as compared to mice had no surgery or that were pretreated with LMWH. Conclusion: Surgery promotes the formation of microthrombi around TCE and this appears to be the mechanism for the increase in metastases seen following surgery. Anticoagulation with LMWH appears to completely abrogate this prometastatic effect. PO-18 Tissue factor expression is associated with MMP and uPA/PAI secretion in adherent small cell lung cancer cells N. Hahn, R. Seitz, M. Heiden, U. Salge-Bartels *. Paul-Ehrlich-Institut, Langen, Germany Background: Small cell lung cancer (SCLC) clinically presents as a most aggressively growing tumor which metastasizes early. Even if chemotherapy leads to initial regression, relapses are almost inevitable due to evolving chemoresistance. Thromboembolic complications are frequent in late-stage SCLC, however, in vitro SCLC cells show only marginal activation of coagulation and negligible expression of secreted proteinases. Objective: In contrast to the parental suspension cell line NCI-H69, an adherent SCLC derivative cell line was found to highly express TF. TFassociated cellular features were searched for by use of RNA interference knock-down of TF expression. Material and Methods: From the classic SCLC cell line NCI-H69, growing in aggregates in suspension, a cell line with adherent phenotype, H69adh, was selected. TF and plasminogen activator inhibitor (PAI-1) were determined by ELISA and western blotting. Zymography was used to determine matrix metalloproteinases (MMP) 2, 9, urokinase and tissue-type plasminogen activator. TF down-regulation was performed with adherent cells using LipofectamineTM (Invitrogen) and different TF-specific Oligonucleotides (Eurogentec). Results: Fundamental differences between parental and derivative cells were found: Only H69adh cells were found to secrete PAI-1, and to express MMP9; in addition, MMP2 secretion was up-regulated. Down-regulation of TF by siRNA resulted in a clear reduction of MMP2 and MMP9 as well as of the plasminogen activator system. Discussion: The H69adh cells display characteristics essential for aggressive tumor growth. TF and TF/FVII, known as important players for the interaction of tumor cells with the coagulation system, were found to modulate also the pericellular proteolytic potential of the cells essential for the degradation of matrix proteins and for migration. PO-19 On the role of protease-activated receptors-1 and -2 (PAR-1 and PAR-2) in human glioblastoma cell lines A. Dutra-Oliveira1 *, T.C. Carneiro-Lobo1 , I. Bakker-Abreu2 , J. Scharfstein J, R.Q. Monteiro1 , A. Mariano-Oliveira1 . 1 Instituto de Bioqu´ımica M´edica, 2 Instituto de Biof´ısica Carlos Chagas Filho; UFRJ, Brazil Malignant gliomas are the most common primary brain tumor in adults. These tumors are characterized by rapid cell proliferation and a marked propensity to invade and damage surrounding tissues. Several lines of evidence indicate that blood clotting proteins and PAR receptors play an important role in tumor microenviroment. Furthermore, PAR activation may elicit several signal transduction pathways that are involved in migration, invasion, proliferation, metastasis and inhibition of apoptosis. In this context, we aimed to characterize the cellular responses upon activation of PAR-1 and PAR-2 in human glioblastoma cell lines, A172 and U87-MG. A172 cells constitutively express tissue factor and expose phosphatidylserine. Thus, we observed that these cells accelerate human plasma clotting in a number-dependent fashion. To further determine which signaling pathways are activated by PAR-1 and PAR-2, tumor cells were treated with specific agonist peptides (Trap-1 or Trap-2). Trap-1 but not Trap-2 increased the intracellular Ca+2 levels in both cell lines. Furthermore, both agonist peptides enhanced Erk-2 and Akt phosphorylation. Finally it was observed that either PAR-1 or PAR-2 activation increased VEGF production by A172 and U87-MG cells. Our findings demonstrate that U87-MG and A172 express functional PAR-1 and PAR-2 receptors, which are possibly involved in glioblastoma progression. Supported by: CNPq, FAPERJ, FAF-Onco.
S171
PO-20 Organic metallic complexes with antiplatelet and antineoplasmatic effects S. Karkabounas, P. Veltsistas, R. Liasko, A. Metsios, M.P. Makris, A. Evangelou, P.E. Makris *. Laboratory of Physiology of University of Ioannina and Aristotle University of Thessaloniki, Greece The new complexes of Vanadium (V) and Stannum (Sn) with organic ligants were synthesized and tested in vitro for their cytotoxic effects on liomyosarcoma cells and ex vivo for possible inhibition of platelet aggregation. The inhibition of platelet function facilitates the inhibition of angiogenesis that triggers the inhibition of tumor growth, while it disables the formation of thrombus from platelets and cancer cells thus impeding the bloodstream metastasis. Material: We used platelet rich plasma (PRP) from 10 healthy males (25±10 years old). Method: The organic complexes of V (with cys, mercaptopropionyl-glycine, putrescine) and Sn (with mercaptonicotinic acid and cys) were diluted in DMSO. The platelet aggregation was tested with ADP and PAF stimulators and also with liosarcomatic cells in the Ca-500 aggregometer of Chronolog co. The final concentrations of the complexes were from 10−3 to 10−9 M before the addition of the reagent. For control we used the platelet aggregation (with ADP, PAF and liomyosarcoma cells as stimulators) without the organic complexes. Results: All the complexes showed a significant inhibition of platelet aggregation. The IC50 of the V and Sn complexes were the following: (Table 1. Inhibition results). Conclusion: In V complexes, both V and the ligants were responsible for the inhibition of aggregation while in Sn complexes only the ligants. Both metals (V and Sn) have antineoplasmatic effects though the organic complexes were stronger and with fewer side effects. Table 1. Inhibition of platelet function by organic metal complexes and liomyosarcoma cells Complexes V–cvs V–V–MPG Vputr Sn–MNA Sn–Cys
ADP IC50 −3
2×10 M 3×10−3 M 8×10−3 M 3×10−5 M 2×10−4 M
PAF IC50 −4
3×10 M 8×10−4 M 5×10−4 M 2×10−5 M 2×10−3 M
LMC IV50 5×10−3 M 2×10−3 M 3×10−4 4×10−5 M 4×10−3 M
PO-21 Characterization of tumor cells thrombin generation (TG) capacity by the calibrated automated assay (CAT) M. Marchetti1 *, E. Diani1 , E. Cantalino1 , H. ten Cate2 , A. Falanga1 . 1 Division of Immunohematology and Transfusion Medicine & Hemostasis and Thrombosis Center, Ospedali Riuniti, Bergamo, Italy, 2 Department of Internal Medicine, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, Netherlands Introduction: Tumor cells can induce TG and fibrin formation by expressing procoagulant proteins. Thrombin is a multifunctional serine protease, which can be involved in tumor progression. Aim and Methods: CAT assay was used to characterize the TG activity of different tumor cell lines: i.e. MDA.MB231 and MCF7 (breast), H69 (small cell lung), and NB4, HEL, and K562 (all myelogenous leukemia). The endothelial HMEC-1 and embryonic kidney HEK-293 cell lines were used as non-tumor cells. CAT was performed in normal pool plasma (NPP), FVII-, FXII- and FIX-deficient plasmas, and in the presence or absence of anti-TF antibody. Results: In NPP the highest TG was produced by MDA.MB231, while the lowest by HEL and K562 cells. MDA.MB231-induced TG was not significantly affected by FXII and FVII depletion. In addition, the high TG levels by MDA.MB231 in FIX-def plasma plus anti-TF antibody suggested an important contribution of FVII-independent procoagulants in their TG capacity. The NB4-induced TG was not FXII-dependent, but was significantly reduced in FVII-def plasma, indicating a major role of TF for this cell line. HEL and K562 TG capacity was very low in both NPP and in FVII-def plasma, and became undetectable in FXII-def plasma; this indicated a main role of contact activation in the TG capacity of these cells. MCF7 H69, HMEC-1 and HEK-239 cells, all showed a slight decrease of TG in either FVII or FXII-def plasma, suggesting a role for both TF and contact activation. The TG parameters of all cell lines strongly correlated to tissue factor (TF) and cancer procoagulant (CP) levels.
Results: Surgery resulted in a significant increase in metastases and anticoagulation with LMWH completely abrogated this effect. The significant difference in metastatic foci was seen at 12 hours and 3 days post surgery but not at earlier time points (10 minutes and 4 hours). This indicates that surgical stress facilitates metastases by enhancing the sustained adherence and survival of individual TCE in the vasculature while anticoagulation with LMWH prevents this effect. Fibrin clots were associated with TCE significantly more frequently in mice that underwent partial hepatectomy, as compared to mice had no surgery or that were pretreated with LMWH. Conclusion: Surgery promotes the formation of microthrombi around TCE and this appears to be the mechanism for the increase in metastases seen following surgery. Anticoagulation with LMWH appears to completely abrogate this prometastatic effect. PO-18 Tissue factor expression is associated with MMP and uPA/PAI secretion in adherent small cell lung cancer cells N. Hahn, R. Seitz, M. Heiden, U. Salge-Bartels *. Paul-Ehrlich-Institut, Langen, Germany Background: Small cell lung cancer (SCLC) clinically presents as a most aggressively growing tumor which metastasizes early. Even if chemotherapy leads to initial regression, relapses are almost inevitable due to evolving chemoresistance. Thromboembolic complications are frequent in late-stage SCLC, however, in vitro SCLC cells show only marginal activation of coagulation and negligible expression of secreted proteinases. Objective: In contrast to the parental suspension cell line NCI-H69, an adherent SCLC derivative cell line was found to highly express TF. TFassociated cellular features were searched for by use of RNA interference knock-down of TF expression. Material and Methods: From the classic SCLC cell line NCI-H69, growing in aggregates in suspension, a cell line with adherent phenotype, H69adh, was selected. TF and plasminogen activator inhibitor (PAI-1) were determined by ELISA and western blotting. Zymography was used to determine matrix metalloproteinases (MMP) 2, 9, urokinase and tissue-type plasminogen activator. TF down-regulation was performed with adherent cells using LipofectamineTM (Invitrogen) and different TF-specific Oligonucleotides (Eurogentec). Results: Fundamental differences between parental and derivative cells were found: Only H69adh cells were found to secrete PAI-1, and to express MMP9; in addition, MMP2 secretion was up-regulated. Down-regulation of TF by siRNA resulted in a clear reduction of MMP2 and MMP9 as well as of the plasminogen activator system. Discussion: The H69adh cells display characteristics essential for aggressive tumor growth. TF and TF/FVII, known as important players for the interaction of tumor cells with the coagulation system, were found to modulate also the pericellular proteolytic potential of the cells essential for the degradation of matrix proteins and for migration. PO-19 On the role of protease-activated receptors-1 and -2 (PAR-1 and PAR-2) in human glioblastoma cell lines A. Dutra-Oliveira1 *, T.C. Carneiro-Lobo1 , I. Bakker-Abreu2 , J. Scharfstein J, R.Q. Monteiro1 , A. Mariano-Oliveira1 . 1 Instituto de Bioqu´ımica M´edica, 2 Instituto de Biof´ısica Carlos Chagas Filho; UFRJ, Brazil Malignant gliomas are the most common primary brain tumor in adults. These tumors are characterized by rapid cell proliferation and a marked propensity to invade and damage surrounding tissues. Several lines of evidence indicate that blood clotting proteins and PAR receptors play an important role in tumor microenviroment. Furthermore, PAR activation may elicit several signal transduction pathways that are involved in migration, invasion, proliferation, metastasis and inhibition of apoptosis. In this context, we aimed to characterize the cellular responses upon activation of PAR-1 and PAR-2 in human glioblastoma cell lines, A172 and U87-MG. A172 cells constitutively express tissue factor and expose phosphatidylserine. Thus, we observed that these cells accelerate human plasma clotting in a number-dependent fashion. To further determine which signaling pathways are activated by PAR-1 and PAR-2, tumor cells were treated with specific agonist peptides (Trap-1 or Trap-2). Trap-1 but not Trap-2 increased the intracellular Ca+2 levels in both cell lines. Furthermore, both agonist peptides enhanced Erk-2 and Akt phosphorylation. Finally it was observed that either PAR-1 or PAR-2 activation increased VEGF production by A172 and U87-MG cells. Our findings demonstrate that U87-MG and A172 express functional PAR-1 and PAR-2 receptors, which are possibly involved in glioblastoma progression. Supported by: CNPq, FAPERJ, FAF-Onco.
S171
PO-20 Organic metallic complexes with antiplatelet and antineoplasmatic effects S. Karkabounas, P. Veltsistas, R. Liasko, A. Metsios, M.P. Makris, A. Evangelou, P.E. Makris *. Laboratory of Physiology of University of Ioannina and Aristotle University of Thessaloniki, Greece The new complexes of Vanadium (V) and Stannum (Sn) with organic ligants were synthesized and tested in vitro for their cytotoxic effects on liomyosarcoma cells and ex vivo for possible inhibition of platelet aggregation. The inhibition of platelet function facilitates the inhibition of angiogenesis that triggers the inhibition of tumor growth, while it disables the formation of thrombus from platelets and cancer cells thus impeding the bloodstream metastasis. Material: We used platelet rich plasma (PRP) from 10 healthy males (25±10 years old). Method: The organic complexes of V (with cys, mercaptopropionyl-glycine, putrescine) and Sn (with mercaptonicotinic acid and cys) were diluted in DMSO. The platelet aggregation was tested with ADP and PAF stimulators and also with liosarcomatic cells in the Ca-500 aggregometer of Chronolog co. The final concentrations of the complexes were from 10−3 to 10−9 M before the addition of the reagent. For control we used the platelet aggregation (with ADP, PAF and liomyosarcoma cells as stimulators) without the organic complexes. Results: All the complexes showed a significant inhibition of platelet aggregation. The IC50 of the V and Sn complexes were the following: (Table 1. Inhibition results). Conclusion: In V complexes, both V and the ligants were responsible for the inhibition of aggregation while in Sn complexes only the ligants. Both metals (V and Sn) have antineoplasmatic effects though the organic complexes were stronger and with fewer side effects. Table 1. Inhibition of platelet function by organic metal complexes and liomyosarcoma cells Complexes V–cvs V–V–MPG Vputr Sn–MNA Sn–Cys
ADP IC50 −3
2×10 M 3×10−3 M 8×10−3 M 3×10−5 M 2×10−4 M
PAF IC50 −4
3×10 M 8×10−4 M 5×10−4 M 2×10−5 M 2×10−3 M
LMC IV50 5×10−3 M 2×10−3 M 3×10−4 4×10−5 M 4×10−3 M
PO-21 Characterization of tumor cells thrombin generation (TG) capacity by the calibrated automated assay (CAT) M. Marchetti1 *, E. Diani1 , E. Cantalino1 , H. ten Cate2 , A. Falanga1 . 1 Division of Immunohematology and Transfusion Medicine & Hemostasis and Thrombosis Center, Ospedali Riuniti, Bergamo, Italy, 2 Department of Internal Medicine, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, Netherlands Introduction: Tumor cells can induce TG and fibrin formation by expressing procoagulant proteins. Thrombin is a multifunctional serine protease, which can be involved in tumor progression. Aim and Methods: CAT assay was used to characterize the TG activity of different tumor cell lines: i.e. MDA.MB231 and MCF7 (breast), H69 (small cell lung), and NB4, HEL, and K562 (all myelogenous leukemia). The endothelial HMEC-1 and embryonic kidney HEK-293 cell lines were used as non-tumor cells. CAT was performed in normal pool plasma (NPP), FVII-, FXII- and FIX-deficient plasmas, and in the presence or absence of anti-TF antibody. Results: In NPP the highest TG was produced by MDA.MB231, while the lowest by HEL and K562 cells. MDA.MB231-induced TG was not significantly affected by FXII and FVII depletion. In addition, the high TG levels by MDA.MB231 in FIX-def plasma plus anti-TF antibody suggested an important contribution of FVII-independent procoagulants in their TG capacity. The NB4-induced TG was not FXII-dependent, but was significantly reduced in FVII-def plasma, indicating a major role of TF for this cell line. HEL and K562 TG capacity was very low in both NPP and in FVII-def plasma, and became undetectable in FXII-def plasma; this indicated a main role of contact activation in the TG capacity of these cells. MCF7 H69, HMEC-1 and HEK-239 cells, all showed a slight decrease of TG in either FVII or FXII-def plasma, suggesting a role for both TF and contact activation. The TG parameters of all cell lines strongly correlated to tissue factor (TF) and cancer procoagulant (CP) levels.