interleukin-8 and 3-(5--hydroxymethyl-2--furyl)-1-benzylindazole

Glutamate-triggered signal transduction is thought to contribute widely to cancer pathogenesis. In melanoma, overexpression of the metabotropic glutamate receptor (GRM)-1 occurs frequently and its ectopic expression in melanocytes is sufficient for neoplastic transformation. Clinical evaluation of the GRM1 signaling inhibitor riluzole in patients with advanced melanoma has demonstrated tumor regressions that are associated with a suppression of the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathways. Together, these results prompted us to investigate the downstream consequences of GRM1 signaling and its disruption in more detail. We found that melanoma cells with enhanced GRM1 expression generated larger tumors in vivo marked by more abundant blood vessels. Media conditioned by these cells in vitro contained relatively higher concentrations of interleukin-8 and VEGF due to GRM1-mediated activation of the AKT-mTOR-HIF1 pathway. In clinical specimens from patients receiving riluzole, we confirmed an inhibition of MAPK and PI3K/AKT activation in posttreatment as compared with pretreatment tumor specimens, which exhibited a decreased density of blood vessels. Together, our results demonstrate that GRM1 activation triggers proangiogenic signaling in melanoma, offering a mechanistic rationale to design treatment strategies for the most suitable combinatorial use of GRM1 inhibitors in patients.

Topics: Angiogenesis Inhibitors; Animals; Cell Movement; Heterocyclic Compounds, 3-Ring; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Indazoles; Inhibitor of Apoptosis Proteins; Interleukin-8; Melanoma; Mice; Mice, Nude; Neoplasm Transplantation; Neovascularization, Pathologic; Platelet Endothelial Cell Adhesion Molecule-1; Receptors, Metabotropic Glutamate; Riluzole; Signal Transduction; Sirolimus; Skin Neoplasms; Survivin; Tumor Burden; Vascular Endothelial Growth Factor A

In the presence of external Ca2+, pretreatment of neutrophils with 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) inhibited the cyclopiazonic acid (CPA)-induced [Ca2+](i) elevation in a concentration- but not a time-dependent manner, while YC-1 had no effect on the Ca2+ signals in a Ca2+-free medium. YC-1 failed to inhibit ATP- and interleukin-8 (IL-8)-induced [Ca2+](i) changes. Addition of YC-1 after cell activation strongly inhibited the CPA-induced [Ca2+](i) changes. In a classical Ca2+ readdition protocol, a similar extent inhibition of Ca2+ spike by YC-1 introduced either prior to or after CPA stimulation was obtained. In rat neutrophils, mRNA for endothelial differentiation gene (edg)1, edg5, edg6 and edg8, the putative targets for sphingosine 1-phosphate (S1P), could be detected. However, S1P was found to have little effect on Ca(2+) signals. YC-1 did not inhibit but enhanced the sphingosine-induced [Ca2+](i) changes. Inhibition by YC-1 of CPA-induced [Ca2+](i) changes was not prevented by 7-nitroindazole and N-(3-aminomethyl)benzylacetamidine (1400W), two nitric oxide synthase (NOS) inhibitors, by aristolochic acid, a phospholipase A(2) inhibitor, or by suspension in a Na(+)-deprived medium. YC-1 did not affect the mitochondrial membrane potential. Moreover, YC-1 did not alter [Ca2+](i) changes in response to ionomycin after CPA and formyl-Met-Leu-Phe (fMLP) stimulation in a Ca2+-free medium. YC-1 had no effect on the basal [Ca2+](i) level, the pharmacologically isolated plasma membrane Ca2+-ATPase activity, and Ba2+ entry into CPA-activated cells. YC-1 alone resulted in the accumulation of actin filaments in neutrophils, while significantly reduced the intensity of actin filament staining in the subsequent activation with CPA. These results indicate that YC-1 inhibited CPA-activated store-operated Ca2+ entry (SOCE) probably through the direct blockade of channel activation and/or the disruption of the integrity of the actin cytoskeleton necessary for supporting Ca2+ entry pathway in neutrophils.

Topics: Actins; Adenosine Triphosphate; Amidines; Animals; Aristolochic Acids; Barium; Benzylamines; Calcium; Calcium Signaling; Calcium-Transporting ATPases; Drug Interactions; Enzyme Inhibitors; Indazoles; Indoles; Interleukin-8; Membrane Potentials; Mitochondria; Neutrophils; Platelet Aggregation Inhibitors; Rats; Rats, Sprague-Dawley; Receptors, Lysosphingolipid; Sodium; Sphingosine