Expression of tissue factor signaling pathway elements correlates with the production of vascular endothelial growth factor and interleukin-8 in human astrocytoma patients.

The expression levels of tissue factor (TF), the clotting initiator protein, have been correlated with angiogenesis and the histological grade of malignancy in glioma patients. The pro-tumor function of TF is linked to a family of G protein-coupled receptors known as protease-activated receptors (PARs), which may be activated by blood coagulation proteases. Activation of PARs elicits a number of responses, including the expression of vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8). In the present study, we analyzed the expression of TF signaling pathway elements (TF, PAR1 and PAR2) and evaluated their correlation with the expression of downstream products (VEGF and IL-8) in human astrocytoma patients. Quantitative PCR (qPCR) showed a significant increase in TF expression in grade IV (glioblastoma) tumors, which was inversely correlated with the expression of the tumor-suppressor PTEN. Immunohistochemistry and qPCR analyses demonstrated a highly significant elevation in the expression of PAR1, but not PAR2, in tumor samples from high-grade astrocytoma patients. The elevated VEGF expression levels detected in the high-grade astrocytoma samples were positively correlated with TF, PAR1 and PAR2 expression. In addition, IL-8 was significantly increased in glioblastoma patients and positively correlated with TF and PAR2 expression. Further in vitro assays employing the human glioma cell lines U87-MG and HOG demonstrated that a synthetic peptide PAR2 agonist stimulated VEGF and IL-8 production. Our findings suggest a role for TF signaling pathway elements in astrocytoma progression, particularly in glioblastoma. Therefore, TF/PAR signaling elements may be suitable targets for the development of new therapies for the treatment of aggressive glioma.

Inhibition of tissue factor by ixolaris reduces primary tumor growth and experimental metastasis in a murine model of melanoma.

Melanoma is a highly metastatic cancer and there is strong evidence that the clotting initiator protein, tissue factor (TF), contributes to its aggressive pattern. TF inhibitors may attenuate primary tumor growth and metastasis. In this study, we evaluated the effect of ixolaris, a TF inhibitor, on a murine model of melanoma B16F10 cells. Enzymatic assays performed with B16F10 and human U87-MG tumor cells as the TF source showed that ixolaris inhibits the generation of FX in either murine, human or hybrid FVIIa/TF complexes. The effect of ixolaris on the metastatic potential was further estimated by intravenous injection of B16F10 cells in C57BL/6 mice. Ixolaris (250 µg/kg) dramatically decreased the number of pulmonary tumor nodules (4 ± 1 compared to 47 ± 10 in the control group). Furthermore, a significant decrease in tumor weights was observed in primary tumor growth assays in animals treated with ixolaris (250 µg/kg) from days 3 to 18 after a subcutaneous inoculation of melanoma cells. Remarkably, immunohistochemical analyses showed that inhibition of melanoma growth by ixolaris is accompanied by a significant downregulation of both vascular endothelial growth factor (VEGF) expression and microvascular density in the tumor mass. Our data demonstrate that ixolaris targets B16F10 cell-derived TF, resulting in the reduction of both the primary tumor growth and the metastatic potential of melanoma, as well as the inhibition of tumor angiogenesis. Therefore TF may be a potential target for the treatment of this aggressive malignancy.

Protease-activated receptor-2 (PAR2) mediates VEGF production through the ERK1/2 pathway in human glioblastoma cell lines.

Glioblastoma (GBM) is a highly aggressive cancer type characterized by intense neovascularization. Several lines of evidence indicate that blood clotting enzymes play an important role in the tumor microenvironment, mainly through the activation of protease-activated receptors (PAR). In particular, PAR1 and PAR2 isoforms may activate signal transduction pathways that promote a number of pro-tumoral responses. However, little is known concerning the role of PAR1/PAR2 in GBM progression. In this study, we investigated the expression and function of PAR1 and PAR2 in the human GBM cell lines A172 and U87-MG. We also evaluated the effect of agonist peptides for PAR1 (PAR1-AP) and PAR2 (PAR2-AP) on signaling pathways and the expression of vascular endothelial growth factor (VEGF). Immunoblotting assays showed that A172 and U87-MG constitutively express PAR1 and PAR2. Treatment of GBM cells with PAR1-AP or PAR2-AP enhanced Akt (protein kinase B) and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in a time-dependent manner. LY29042 and PD98059, inhibitors of the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways, decreased PAR-mediated activation of Akt and ERK1/2, respectively. In addition, we observed that PAR2, but not PAR1, activation increased VEGF secretion in U87-MG and A172 cells. Notably, only PD98059 reduced PAR2-mediated VEGF production by GBM cells. Our results suggest that PAR2 modulates VEGF production through the MAPK/ERK1/2 pathway, and not the PI3K/Akt pathway, in human GBM cell lines. Therefore, the PAR2/MAPK signaling axis might be regarded as a relevant target for adjuvant treatment of GBM with a possible impact on tumor angiogenesis.

alpha(9)beta(1) integrin engagement inhibits neutrophil spontaneous apoptosis: involvement of Bcl-2 family members.

Integrin signaling is comprised of well-characterized pathways generally involved in cell survival. alpha(9)beta(1) integrin has recently become a target of study and has been shown to present pro-survival effects on neutrophils. However, there are no detailed studies on how alpha(9)beta(1) integrin-coupled signaling pathways interact and how they converge to finally modulate spontaneous apoptosis in neutrophils. In this regard we sought to investigate the main signaling events triggered by alpha(9)beta(1) integrin engagement and how these signaling pathways modulate the apoptotic program of human neutrophils. Using VLO5, a snake venom disintegrin shown to bind to alpha(9)beta(1) integrin in neutrophils, we demonstrate that alpha(9)beta(1) integrin engagement leads to the activation of integrin signaling pathways and potently reduces neutrophil spontaneous apoptosis. These effects are dependent on the activation of PI3K and MAPK pathways, since both LY294002 (PI3K inhibitor) or PD95059 (MEK inhibitor) reverted the effects of VLO5/alpha(9)beta(1) interaction. Moreover we show that VLO5/alpha(9)beta(1) engagement induces NF-kappaB nuclear translocation and increases the ratio between anti- and pro-apoptotic proteins by inducing the degradation of pro-apoptotic protein Bad and increasing the expression of anti-apoptotic protein Bcl-x(L). VLO5 also inhibited the early steps of neutrophil spontaneous apoptosis by preventing Bax translocation to the outer mitochondrial membrane and consequent cytochrome c release. In conclusion, as the mechanistic details of alpha(9)beta(1) integrin signaling pathways in human neutrophils becomes clearer, it should become possible to develop new therapeutic agents for human diseases where neutrophils play a prominent role.

Prothrombin fragments containing kringle domains induce migration and activation of human neutrophils.

The cross-talk between inflammatory and coagulation cascades has been demonstrated. Prothrombin processing releases the protease domain (thrombin) along with two catalytically inactive kringle-containing derivatives: prothrombin fragments 1 (F1) and 2 (F2). It is well established that thrombin is able to trigger an inflammatory response but the possible effects of prothrombin fragments on leukocyte functions are still unknown. In this report, we demonstrate for the first time that both F1 and F2 prothrombin fragments, interfere with intracellular functional signaling pathways to modulate human neutrophil migration. In addition, we show that thrombin, fragment 1 and fragment 2 induce human neutrophil chemotaxis. The effect of fragment 2, but not fragment 1, was partially inhibited by pertussis toxin, an inhibitor of G(alphai)-signaling. The pre-treatment of cells with fragment 2 inhibited thrombin-induced chemotaxis, while both fragments impaired neutrophil migration induced by interleukin-8. F1 and F2 increased the expression and activation of G-protein-coupled receptor kinase-2, which has emerged as a key effector in the desensitization of chemokine receptors. In parallel, prothrombin fragments activated extracellular signal-regulated kinase 1/2, stimulating its phosphorylation and nuclear translocation, and induced inhibitor of kappa-B phosphorylation and degradation followed by nuclear factor-kappa B translocation to nucleus. Furthermore, both prothrombin fragments induced interleukin-8 gene expression in human neutrophils. These findings suggest that the interference with neutrophil signaling and function, caused by kringle-containing prothrombin fragments may desensitize these cells to respond to further activation by thrombin and interleukin-8 during inflammatory and coagulation responses.

Alternagin-C, a disintegrin-like protein, induces vascular endothelial cell growth factor (VEGF) expression and endothelial cell proliferation in vitro.

Alternagin-C (ALT-C), a disintegrin-like protein purified from the venom of the Brazilian snake Bothrops alternatus, interacts with the major collagen I receptor, the alpha(2)beta(1) integrin, inhibiting collagen binding. Here we show that ALT-C also inhibits the adhesion of a mouse fibroblast cell line (NIH-3T3) to collagen I (IC(50) 2.2 microm). In addition, when immobilized on plate wells, ALT-C supports the adhesion of this cell line as well as of human vein endothelial cell (HUVEC). ALT-C (3 microm) does not detach cells that were previously bound to collagen I. ALT-C (5 nm) induces HUVEC proliferation in vitro, and it inhibits the positive effect of vascular endothelial growth factor (VEGF) or FGF-2 on the proliferation of these cells, thus suggesting a common mechanism for these proteins. Gene expression analysis of human fibroblasts growing on ALT-C- or collagen-coated plates showed that ALT-C and collagen I induce a very similar pattern of gene expression. When compared with cells growing on plastic only, ALT-C up-regulates the expression of 45 genes including the VEGF gene and down-regulates the expression of 30 genes. Fibroblast VEGF expression was confirmed by RT-PCR and ELISA assay. Up-regulation of the VEGF gene and other growth factors could explain the positive effect on HUVEC proliferation. ALT-C also strongly activates Akt/PKB phosphorylation, a signaling event involved in endothelial survival and angiogenesis. In conclusion, ALT-C acts as a survival factor, promoting adhesion and endothelial cell proliferation.

RGD- and MLD-disintegrins, jarastatin and EC3, activate integrin-mediated signaling modulating the human neutrophils chemotaxis, apoptosis and IL-8 gene expression.

The effects of jarastatin (JT), a monomeric RGD-disintegrin, were compared with those of the heterodimeric MLD-disintegrin, EC3, on human neutrophil activation and functions. Both disintegrins inhibited neutrophil chemotaxis induced by fMet-Leu-Phe and were also potent chemotactic agents. These effects were accompanied by an increase in actin polymerization, and both were inhibited by genistein, a tyrosine kinase inhibitor. While JT, but not other RGD-disintegrins, inhibited EC3-induced chemotaxis, EC3 was not able to inhibit JT effect. The chemotactic effect of JT was blocked by anti-alpha(M) antibody whereas anti-alpha(9)beta(1) inhibited EC3 effect. Both JT and EC3 induced focal adhesion kinase (FAK) and phosphoinositide 3-kinase (PI3K) activation. Accordingly, LY294002, a PI3K inhibitor, impaired their chemotactic effect on neutrophils. JT induced Erk-2 translocation to nucleus and a delay of the spontaneous apoptosis of neutrophils in vitro. In contrast, EC3 inhibited Erk-2 activation and had a proapoptotic effect. These effects were reverted by PD98059, an MEK 1/2 inhibitor and blocked by z-VAD-FMK, a caspase inhibitor. In addition, JT, but not EC3, increased the IL-8 mRNA levels in neutrophils. The data indicate that JT and EC3 directly activate an integrin-coupled signaling and modulate the MAPK pathway in different ways, leading the neutrophils to express different functional response.

Alternagin-C, a nonRGD-disintegrin, induces neutrophil migration via integrin signaling.

Recently, a new protein containing a disintegrin domain, alternagin-C (Alt-C), was purified from Bothrops alternatus venom. Unlike other disintegrins, in Alt-C an ECD amino acid mogif takes the place of the RGD sequence. Most disintegrins contain an RGD/KGD sequence and are very potent inhibitors of platelet aggregation, as well as other cell interactions with the extracellular matrix, including tumor cell metastasis and angiogenesis. The present study investigated the effects of Alt-C on human neutrophil chemotaxis in vitro and the activation of integrin-mediated pathways. Alt-C showed a potent chemotactic effect for human neutrophils when compared to N-formyl-methionyl-leucyl-phenylalanine peptide (fMLP), a classic chemotactic agent. Moreover, preincubation of neutrophils with Alt-C significantly inhibited chemotaxis toward fMLP and itself. In addition, a peptide containing an ECD sequence presented a chemotactic activity and significantly inhibited chemotaxis induced by Alt-C and fMLP. A significant increase of F-actin content was observed in cells treated with Alt-C, showing that the chemotactic activity of Alt-C on neutrophils is driven by actin cytoskeleton dynamic changes. Furthermore, this protein was able to induce an increase of phosphotyrosine content triggering focal adhesion kinase activation and its association with phosphatidylinositol 3-kinase. Alt-C was also able to induce a significant increase in extracellular signal-regulated kinase 2 nuclear translocation. The chemotactic activity of Alt-C was partially inhibited by LY294002, a specific phosphatidylinositol 3-kinase inhibitor, and by PD98056, a Map kinase kinase inhibitor. These findings suggest that Alt-C can trigger human neutrophil chemotaxis modulated by intracellular signals characteristic of integrin-activated pathways and that these effects could be related to the ECD mogif present in disintegrin-like domain.