Neuropeptide Y and its Y2 receptor: potential targets in neuroblastoma therapy.

Neuroblastomas are pediatric tumors that develop from sympathetic precursors and express neuronal proteins, such as neuropeptide Y (NPY). NPY is a sympathetic neurotransmitter acting via multiple receptors (Y1-Y5R). Both NPY and Y2Rs are commonly expressed in neuroblastoma cell lines and tissues. The peptide secreted from neuroblastomas stimulates tumor cell proliferation and angiogenesis. As both processes are Y2R-mediated, the aim of this study was to assess Y2R as a potential therapeutic target for neuroblastoma. In vitro, Y2R antagonist (BIIE0246) prevented activation of p44/42 mitogen-activated protein kinase (MAPK) induced by endogenous NPY, which resulted in decreased proliferation and induction of Bim-mediated apoptosis. Similar growth-inhibitory effects were achieved with NPY small interfering RNA (siRNA) and Y2R siRNA. In vivo, Y2R antagonist significantly inhibited growth of SK-N-BE(2) and SK-N-AS xenografts, which was associated with decreased activation of p44/42 MAPK, as well as reduced proliferation (Ki67) and increased apoptosis (TdT-mediated dUTP nick end labeling; TUNEL). The Y2R antagonist also exerted an antiangiogenic effect. In vitro, it reduced the proliferation of endothelial cells induced by neuroblastoma-conditioned media. Consequently, the Y2R antagonist-treated xenografts had decreased vascularization and a high degree of focal fibrosis. In human neuroblastoma tissues, the expression of Y2R was observed in both tumor and endothelial cells, while NPY was predominantly expressed in neuroblastoma cells. In summary, Y2R is a promising new target for neuroblastoma therapy affecting both cancer cells and tumor vasculature.

Critical role of dipeptidyl peptidase IV in neuropeptide Y-mediated endothelial cell migration in response to wounding.

Recently, we have discovered that neuropeptide Y (NPY), a sympathetic neurotransmitter, is also present in human umbilical endothelial cells (HUVECs), and is potently chemotactic and angiogenic by acting on one or several of Y1-Y5 receptors. In HUVECs, NPY is co-localized with dipeptidyl peptidase IV (DPPIV) which cleaves Tyr(1)-Pro(2) from NPY(1-36) to form NPY(3-36) resulting in the formation of a non-Y1 receptor agonist, which remains angiogenic. Presently we studied the effects of DPPIV's blockade using monoclonal antibodies (mAbs) on migration of HUVECs in response to NPY(1-36) or NPY(3-36) following cell wounding. Both peptides caused similar dose-dependent increases in cell migration (+80% at 0.1 nM) 12 h after wounding. DPPIV mAbs, E19 and E26, significantly reduced HUVEC's migration below that of the untreated cells, and blocked responses to NPY(1-36) but not NPY(3-36). Enhanced expression of DPPIV was found in the migrating cells and in cells with their protrusions at the edge of the wound (immunostaining and Western blot). Thus, DPPIV's expression is stimulated by endothelial wounding and its enzymatic activity is required for NPY-mediated chemotaxis. Furthermore, this suggests that non-Y1 receptors activated by NPY(3-36) (Y2, Y3 and/or Y5) mediate angiogenic effects of NPY.

Revascularization of ischemic tissues with SIKVAV and neuropeptide Y (NPY).

Angiogenesis, the process of new vessel growth, is necessary for many normal physiological and pathological processes such as tumor growth, wound healing and ischemia. We have recently examined in vitro and in vivo the ability of two potent angiogenic compounds, SIKVAV (a peptide derived from the alpha chain of laminin-1) and Neuropeptide Y (NPY) to revascularize ischemic tissue. These compounds were tested in an ex vivo capillary sprouting angiogenesis assay that uses rat aortic rings. Both NPY and SIKVAV in the presence of VEGF, stimulated the formation of long sprouts at concentrations of 1 ng NPY (0.2 pmol/L) and 100 micrograms SIKVAV. In comparison very little sprouting occurred in the control rings and 50 ng of VEGF alone was required to induce equivalent number of sprouts as NPY. SIKVAV and NPY were further tested in vivo in a rat hindlimb ischemic model. Both compounds (500 micrograms SIKVAV and 10 ng of NPY) were embedded in the rat hind limb following unilateral ligation of the femoral artery 1 cm proximal to the adductor hiatus. After two weeks control peptides show little or no revascularization of the hindlimb distal to the ligation; however, both SIKVAV and NPY demonstrated a two-fold increase in new vessels in the region proximal to the ligation. Histological sections of latex perfused hindlimb demonstrated that ligated limbs had very few latex-filled dermal capillaries. Limbs treated with SIKVAV and NPY, however, demonstrated normal distribution in the dermal capillary beds. These data indicate that both SIKVAV and NPY are potent angiogenic factors that show promising potential clinical application to the revascularization of ischemic tissue.

Effect of oxafun on the antibiosis between Bacillus subtilis and Phoma betae.

The fungicide oxafun decreased the production of antibiotic substances active against Phoma betae, by Bacillus subtillis. This was confirmed by reduced effectiveness of antibiotic substances against Phoma infection in micropot experiment. Besides, the bacterial metabolites effected cell membrane permeability of mycelium and resulted in some relationships of fungicide and antibiotic substances.