BAY 12-9566, a novel inhibitor of matrix metalloproteinases with antiangiogenic activity.

Matrix metalloproteinases (MMPs) have been implicated in tumor cell invasion, metastasis, and angiogenesis. BAY 12-9566, a novel, non-peptidic biphenyl MMP inhibitor, has shown preclinical activity on a broad range of tumor models and is currently in clinical development. The purpose of this study was to investigate the antiangiogenic activity of BAY 12-9566. In vitro, BAY 12-9566 prevented matrix invasion by endothelial cells in a concentration-dependent manner (IC50 = 8.4x10(-7) M), without affecting cell proliferation. In vivo, oral daily administration of BAY 12-9566 (50-200 mg/kg) inhibited angiogenesis induced by basic fibroblast growth factor in the Matrigel plug assay, reducing the hemoglobin content of the pellets. Histological analysis showed a reduction in the amount of functional vessels within the Matrigel. We conclude that the MMP inhibitor BAY 12-9566 inhibits angiogenesis, a property that further supports its clinical development as an antimetastatic agent.

Characterization and properties of dominant-negative mutants of the ras-specific guanine nucleotide exchange factor CDC25(Mm).

Ras proteins are small GTPases playing a pivotal role in cell proliferation and differentiation. Their activation depends on the competing action of GTPase activating proteins and guanine nucleotide exchange factors (GEF). The properties of two dominant-negative mutants within the catalytic domains of the ras-specific GEF, CDC25(Mm), are described. In vitro, the mutant GEF(W1056E) and GEF(T1184E) proteins are catalytically inactive, are able to efficiently displace wild-type GEF from p21(ras), and strongly reduce affinity of the nucleotide-free ras x GEF complex for the incoming nucleotide, thus resulting in the formation of a stable ras.GEF binary complex. Consistent with their in vitro properties, the two mutant GEFs bring about a dramatic reduction in ras-dependent fos-luciferase activity in mouse fibroblasts. The stable ectopic expression of the GEF(W1056E) mutant in smooth muscle cells effectively reduced growth rate and DNA synthesis with no detectable morphological changes.

Mesothelial cells induce the motility of human ovarian carcinoma cells.

The dissemination of ovarian carcinoma cells within the abdominal cavity involves interaction of tumor cells with the peritoneal mesothelium. The aim of our study was to investigate whether mesothelial cells might directly affect the spreading of this tumor by inducing motility and invasiveness of human ovarian carcinoma cells. Serum-free supernatants of cultured human mesothelial cells [conditioned medium (CM)] induced chemotaxis and invasiveness of the human ovarian carcinoma cell lines SK-OV-3, OVCAR-5 and A2780 in a Boyden chamber. Checkerboard analysis indicated that the stimulated motility was prevalently directional. Most of the chemotactic activity was retained by a heparin affinity column, indicating that the motility factor(s) is a heparin-binding protein. Using different monoclonal antibodies (MAbs) directed against chemotactic factors that are secreted by mesothelial cells, we found that chemotaxis was partially prevented (64.8% inhibition) by antibodies against fibronectin (FN). CM also induced haptotactic migration of ovarian carcinoma cells, and anti-FN antibodies significantly inhibited haptotaxis. The presence of FN in the CM was confirmed by Western blot analysis. Our findings suggest that mesothelium plays an active role in inducing the intraperitoneal spread of ovarian carcinoma cells, and point to FN as being one of the main mediators of mesothelium-induced ovarian carcinoma cell motility.

Paclitaxel (Taxol(R)) inhibits motility of paclitaxel-resistant human ovarian carcinoma cells.

The effect of paclitaxel on the adhesive and motility properties of human ovarian carcinoma cell lines was investigated. Paclitaxel significantly inhibited the motility of OVCAR 5, SK-OV-3, and HOC-1OTC ovarian carcinoma cell lines (IC50 = 2.1 x 10(-8), 2 x 10(-9), and 1.9 x 10(-8) m, respectively) but did not affect the adhesion of these cells to the subendothelial matrix. The association between inhibition of motility and cytotoxic activity was investigated using an A2780 subclone (1A9) and three paclitaxel-resistant variants (designated 1A9/PTX22, 1A9/PTX10, and 1A9/PTX18). Although paclitaxel did not significantly affect the adhesion to subendothelial matrix of the sublines, it completely inhibited their migration. Inhibition of migration was similar in 1A9 cells and the resistant sublines, with an IC50 of 1 x 10(-8) for 1A9 cells and 5.4 x 10(-9), 1.1 x 10(-8), and 5.2 x 10(-9) m for 1A9/PTX22, 1A9/PTX10, and 1A9/PTX18, respectively. Paclitaxel inhibited motility induced by soluble attractant (chemotaxis) and immobilized attractant (haptotaxis). Inhibition of cell motility occurred in the absence of an antiproliferative effect, because higher concentrations of paclitaxel were required to inhibit tumor cell proliferation (IC50 = 1.9 x 10(-7) and 4.6 x 10(-6), 1 x 10(-5), and 3.1 x 10(-6) m for 1A9 and 1A9/PTX22, 1A9/PTX10, and 1A9/PTX18, respectively). These data show that paclitaxel is a potent inhibitor of ovarian carcinoma cell motility and that this activity is independent of its cytotoxic activity.

Receptors, signal transduction, and spectrum of action of monocyte chemotactic protein-1 and related chemokines.

Chemokines are a bipartite family of chemotactic proteins that bear the structural hallmark of four cysteine residues, the first two of which are in tandem. The spectrum of action of C-C chemokines, monocyte chemotactic protein-1 (MCP-1), MCP-2, and MCP-3, in particular, encompasses, in addition to monocytes, other leukocyte populations. Evidence is presented that MCP-1, MCP-2, and MCP-3 are active on natural killer cells. Available information on receptor usage by MCP-1 and related chemokines and signal transduction pathways is reviewed. A better understanding of signaling mechanisms will provide a new basis for therapeutic strategies.

Activation of phospholipase D by interleukin-8 in human neutrophils.

Interleukin 8 (IL-8), a member of the C-X-C branch of the chemokine superfamily, stimulated the breakdown of 1-O-[3H]alkyl-2-acyl-sn-glycero-3-phosphocholine ([3H]EAPC) and the formation of 1-O-[3H]alkyl-2-acyl-phosphatidic acid ([3H]-EAPA) in human polymorphonuclear leukocytes (PMN) in the presence of cytochalasin B. In addition, the mass of diradyl-PA was increased with similar kinetics. In the presence of ethanol, 1-O-[3H]alkyl-2-acyl-phosphatidylethanol ([3H]EAPEt) was formed at the expense of [3H]EAPA formation, indicating the activation of phospholipase D by the cytokine. The effect was time- and concentration-dependent, reaching a plateau at 30 seconds with the maximally activating concentration of 120 nmol/L IL-8. Preincubation of cells with 1 microgram/mL Bordetella pertussis toxin inhibited the breakdown of [3H]EAPC and [3H]EAPA formation, indicating a role for a pertussis toxin-sensitive guanosine triphosphate-binding protein. Formation of phosphatidic acid (PA) correlated with activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, the oxidative burst enzyme, with both events occurring in the same concentration range. Inhibition of PA formation, by the presence of ethanol, also inhibited the oxidative burst stimulation by IL-8. Pretreatment of PMN with 10 nmol/L platelet-activating factor potentiated both [3H]EAPA accumulation and activation of NADPD oxidase by IL-8. Collectively, these data show that IL-8 stimulates the metabolism of choline-containing phosphoglycerides in human PMN and support a role for PA in the signaling mechanisms used by IL-8 to stimulate PMN function.

Receptors and transduction pathways for monocyte chemotactic protein-2 and monocyte chemotactic protein-3. Similarities and differences with MCP-1.

MCP-2 and MCP-3 are recently identified members of the Cys-Cys chemokine family with high sequence similarity with MCP-1 (62% and 71%, respectively). The present study was aimed at defining receptor usage and signal transduction pathways of MCP-2 and MCP-3 in human monocytes in comparison with MCP-1. MCP-2 and MCP-3 induced migration of monocytes with a typical bell-shaped curve and maximal response at 10 and 50 ng/ml, respectively. The maximal response elicited by MCP-2 and MCP-3 was lower (approximately 60%) than that of MCP-1. Pertussis toxin (PTox) inhibited the chemotactic activity of MCP-3 and MCP-1 (IC50 = 6.2 and 4.4 ng/ml, respectively), whereas cholera toxin (CTox) had little effect on these two chemokines (IC50 > 1000 ng/ml). In contrast, MCP-2-induced chemotaxis was blocked by CTox (IC50 = 75 ng/ml) and relatively unaffected by PTox. MCP-3 and MCP-1 induced a rapid increase in intracellular Ca2+ concentration, whereas MCP-2, in the range of concentrations active on chemotaxis, did not. MCP-1-, MCP-2-, and MCP-3-induced chemotactic responses were blocked by C-I, a serine/threonine kinase inhibitor, and by genistein, a tyrosine kinase inhibitor, with the MCP-2 response being more sensitive than those induced by MCP-1 and MCP-3. MCP-1 and MCP-3 rapidly induced arachidonic acid release whereas MCP-2 was ineffective. MCP-1 and MCP-3 cross-desensitized with each other in terms of Ca2+ transients and displaced with a comparable efficiency labeled MCP-1 from human monocytes. On the other hand, MCP-2 did not cross-desensitize with MCP-1 and MCP-3 and only partially (20%) displaced labeled MCP-1. Thus, in spite of high sequence similarity, MCP-2 differed considerably from MCP-1 and MCP-3 in terms of sensitivity to CTox and PTox, arachidonate and calcium mobilization, and capacity to compete for labeled MCP-1.

Synergism between platelet activating factor and C-C chemokines for arachidonate release in human monocytes.

Monocyte Chemotactic Protein(MCP)-1 and other members of the C-C branch of the chemokine superfamily (RANTES, MIP1 alpha/LD78, and MCP-3) induced, at chemotactic concentrations, the release of [3H]arachidonic acid in prelabeled human monocytes. Arachidonate release was potentiated (2 to 4 fold) in the presence of platelet activating factor (PAF). PAF effect was blocked by a specific receptor antagonist, WEB 2187, and was not present when inactive analogs, PAF inactive enantiomer, or lysoPAF were used. Thus, the synergistic action of PAF with C-C chemokines, in terms of arachidonate release and chemotaxis, is specific for this alkylphospholipid and is receptor mediated. Reciprocal potentiation of PAF and C-C chemokines may be important at sites of inflammation.