vasoactive-intestinal-peptide and Glioblastoma

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are neuropeptides acting through VPAC1, VPAC2 and PAC1 receptors (referred here as the VIP-receptor system). In the central nervous system, VIP and PACAP are involved in neurogenesis, cell differentiation and migration, suggesting that they could be implicated in the development of glioblastoma (GBM). The infiltrative nature of GBM remains a major problem for the therapy of these tumors. We previously demonstrated that the VIP-receptor system regulated cell migration of the human cell lines M059J and M059K, derived from a single human GBM. Here, we evaluated the involvement of the VIP-receptor system in GBM cell invasion. In Matrigel invasion assays, M059K cells that express more the VIP-receptor system than M059J cells were less invasive. Invasion assays performed in the presence of agonists, antagonists or anti-PACAP antibodies as well as experiments with transfected M059J cells overexpressing the VPAC1 receptor indicated that the more the VIP-receptor system was expressed and activated, the less the cells were able to invade. Western immunoblotting experiments revealed that the VIP-receptor system inactivated the signaling protein AKT. Invasion assays carried out in the presence of an AKT inhibitor demonstrated the involvement of this signaling kinase in the regulation of cell invasion by the VIP-receptor system in M059K cells. The inhibition by VIP of invasion and AKT was also observed in U87 cells. In conclusion, VIP and PACAP act as anti-invasive factors in different GBM cell lines, a function mediated by VPAC1 inhibition of AKT signaling in M059K cells.

Topics: Apoptosis; Blotting, Western; Cell Adhesion; Cell Movement; Cell Proliferation; Cyclic AMP; Glioblastoma; Humans; Neoplasm Invasiveness; Neuroprotective Agents; Proto-Oncogene Proteins c-akt; Receptors, Vasoactive Intestinal Peptide; Signal Transduction; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

The growth rate of numerous cancer cell lines is regulated in part by actions of neuropeptides of the vasoactive intestinal peptide (VIP) family, which also includes pituitary adenylate cyclase-activating peptide (PACAP), glucagon, and peptide histidine/isoleucine (PHI). The aim of this work was to investigate the effect of these peptides on the growth of the rat glioblastoma cell line C6 in vitro. We also sought to determine which binding sites were correlated with the effects observed. Proliferation studies performed by means of a CyQuant trade mark assay showed that VIP and PACAP strongly stimulated C6 cell proliferation at most of the concentrations tested, whereas PHI increased cell proliferation only when associated with VIP. Two growth hormone-releasing factor (GRF) derivatives and the VIP antagonist hybrid peptide neurotensin-VIP were able to inhibit VIP-induced cell growth stimulation, even at very low concentrations. Binding experiments carried out on intact cultured C6 cells, using 125I-labeled VIP and PACAP as tracers, revealed that the effects of the peptides on cell growth were correlated with the expression on C6 cells of polyvalent high-affinity VIP-PACAP binding sites and of a second subtype corresponding to very high-affinity VIP-selective binding species. The latter subtype, which interacted poorly with PACAP with a 10,000-fold lower affinity than VIP, might mediate the antagonist effects of neurotensin- VIP and of both GRF derivatives on VIP-induced cell growth stimulation.

Topics: Animals; Antineoplastic Agents; Binding Sites; Binding, Competitive; Brain Neoplasms; Cell Division; Cell Line, Tumor; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Drug Synergism; Glioblastoma; Growth Hormone-Releasing Hormone; Neuropeptides; Neurotensin; Peptide PHI; Pituitary Adenylate Cyclase-Activating Polypeptide; Rats; Receptors, Cell Surface; Vasoactive Intestinal Peptide

The effects of a vasoactive intestinal peptide (VIP) receptor antagonist (VIPhyb) on human glioblastoma cells were characterized. Pituitary adenylate cyclase activating polypeptide (125I-PACAP-27) bound with high affinity to U87, U118, and U373 cells. Specific 125I-PACAP-27 binding to U87 cells was inhibited, with high affinity, by PACAP but not VIP or VIPhyb (IC50 = 10, 1500, and 500 nM, respectively). By reverse transcriptase-polymerase chain reaction (RT-PCR), a major 305 bp band was observed indicative of PAC1 receptors. PACAP-27 caused cAMP elevation and the increase in cAMP caused by PACAP-27, was inhibited by the VIPhyb. Also, PACAP-27 caused cytosolic Ca2+ elevation in Fura-2AM loaded U87 cells and the VIPhyb inhibited this increase. Using the MTT growth assay, the VIPhyb was shown to inhibit glioblastoma growth in a concentration-dependent manner. Using a clonogenic assay in vitro, 10 microM VIPhyb significantly inhibited proliferation of U87, U118, and U373 cells. In vivo, 0.4 microg/kg VIPhyb inhibited U87 xenograft proliferation in nude mice. These results suggest that the VIPhyb antagonizes PAC1 receptors on glioblastoma cells and inhibits their proliferation.

Topics: Animals; Calcium; Cyclic AMP; Cytosol; Dose-Response Relationship, Drug; Glioblastoma; Humans; Mice; Mice, Nude; Neuropeptides; Neurotensin; Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Vasoactive Intestinal Peptide; Recombinant Fusion Proteins; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Transplantation, Heterologous; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Functional VIP/PACAP receptors were identified in the human glioblastoma cell line T98G, based on the relative potency of VIP, PACAP and PACAP-38 to stimulate adenylate cyclase activity. Analysis of the T98G cells mRNA by reverse transcription followed by a polymerase chain reaction (RT-PCR) demonstrated the expression of the mRNA coding for the VIP2 receptor subclass only. VIP, PACAP-27 and PACAP-38 were potent and efficIent inhibitors of cell proliferation, assessed by the colorimetric MTT assay. VIP, PACAP-27 and PACAP-38 also reduced the incorporation of 3H-thymidine in T98G cells, but did not significantly alter the percentage of cells present at each stage of the cell cycle. Thus, VIP and PACAP, probably acting through a VIP2 receptor subtype, decreased cell proliferation.

Topics: Adenylyl Cyclases; Brain Neoplasms; Cell Cycle; Cell Division; DNA Replication; DNA, Neoplasm; Enzyme Activation; Glioblastoma; Growth Inhibitors; Humans; Neoplasm Proteins; Neuropeptides; Pituitary Adenylate Cyclase-Activating Polypeptide; Polymerase Chain Reaction; Receptors, Vasoactive Intestinal Peptide; Receptors, Vasoactive Intestinal Peptide, Type II; RNA, Messenger; Tumor Cells, Cultured; Vasoactive Intestinal Peptide