PKA at a Cross-Road of Signaling Pathways Involved in the Regulation of Glioblastoma Migration and Invasion by the Neuropeptides VIP and PACAP

Glioblastoma (GBM) remains an incurable disease, mainly due to the high migration and invasion potency of GBM cells inside the brain. PI3K/Akt, Sonic Hedgehog (SHH), and PKA pathways play major regulatory roles in the progression of GBM. The vasoactive intestinal peptide (VIP) family of neuropeptides and their receptors, referred in this article as the “VIP-receptor system”, has been reported to regulate proliferation, differentiation, and migration in a number of tumor cell types and more particularly in GBM cells. These neuropeptides are potent activators of the cAMP/PKA pathway. The present study aimed to investigate the cross-talks between the above cited signaling cascades. Regulation by VIP-related neuropeptides of GBM migration and invasion was evaluated ex vivo in rat brain slices explanted in culture. Effects of different combinations of VIP-related neuropeptides and of pharmacological and siRNA inhibitors of PKA, Akt, and of the SHH/GLI1 pathways were tested on GBM migration rat C6 and human U87 GBM cell lines using the wound-healing technique. Quantification of nuclear GLI1, phospho-Akt, and phospho-PTEN was assessed by western-immunoblotting. The VIP-receptor system agonists VIP and PACAP-38 significantly reduced C6 cells invasion in the rat brain parenchyma ex vivo, and C6 and U87 migration in vitro. A VIP-receptor system antagonist, VIP10-28 increased C6 cell invasion in the rat brain parenchyma ex vivo, and C6 and migration in vitro. These effects on cell migration were abolished by selective inhibitors of the PI3K/Akt and of the SHH pathways. Furthermore, VIP and PACAP-38 reduced the expression of nuclear GLI1 while VIP10-28 increased this expression. Selective inhibitors of Akt and PKA abolished VIP, PACAP-38, and VIP10-28 effects on nuclear GLI1 expression in C6 cells. PACAP-38 induced a time-dependent inhibition of phospho-Akt expression and an increased phosphorylation of PTEN in C6 cells. All together, these data indicate that triggering the VIP-receptor system reduces migration and invasion in GBM cells through a PKA-dependent blockade of the PI3K/Akt and of the SHH/GLI1 pathways. Therefore, the VIP-receptor system displays anti-oncogenic properties in GBM cells and PKA is a central core in this process.

Differences in control of parasympathetic vasodilation between submandibular and sublingual glands in the rat

We examined blood flow in the submandibular gland (SMGBF) and sublingual gland (SLGBF) during electrical stimulation of the central cut end of the lingual nerve (LN) in the urethane-anesthetized rats using a laser speckle imaging flow meter. LN stimulation elicited intensity- and frequency-dependent SMGBF and SLGBF increases, and the magnitude of the SMGBF increase was higher than that of the SLGBF increase. The increase in both glands was significantly inhibited by intravenous administration of the autonomic cholinergic ganglion blocker hexamethonium. The antimuscarinic agent atropine markedly inhibited the SMGBF increase and partly inhibited the SLGBF increase. The atropine-resistant SLGBF increase was significantly inhibited by infusion of vasoactive intestinal peptide (VIP) receptor antagonist, although administration of VIP receptor antagonist alone had no effect. The recovery time to the basal blood flow level was shorter after LN stimulation than after administration of VIP. However, the recovery time after LN stimulation was significantly delayed by administration of atropine in a dose-dependent manner to the same level as after administration of VIP. Our results indicate that 1) LN stimulation elicits both a parasympathetic SMGBF increase mainly evoked by cholinergic fibers and a parasympathetic SLGBF increase evoked by cholinergic and noncholinergic fibers, and 2) VIP-ergic mechanisms are involved in the noncholinergic SLGBF increase and are activated when muscarinic mechanisms are deactivated.

Electroacupuncture Attenuates Collagen-Induced Arthritis in Rats through Vasoactive Intestinal Peptide Signalling-Dependent Re-Establishment of the Regulatory T Cell/T-Helper 17 Cell Balance

Objective Imbalance between T-helper 17 (Th17) cells and regulatory T (Treg) cells is causally linked to the development of rheumatoid arthritis (RA). In this study, we tested the hypothesis that electroacupuncture (EA) confers therapeutic benefits in RA through activation of vasoactive intestinal peptide (VIP)-dependent signalling and restoration of the Th17/Treg cell balance. Materials and Methods A collagen-induced arthritis (CIA) model was induced in Sprague–Dawley rats by injection of bovine type II collagen in incomplete Freund's adjuvant on day 0 and day 7. Three days after the second injection, EA was given at acupuncture points GB39 and ST36 three times per week for 4 weeks. To block VIP signalling, [D-P-Cl-Phe(6)-Leu(17)]-VIP, a VIP receptor antagonist, was administered intraperitoneally 30 min before EA. Inflammatory and pathological responses in the joint were assessed. Synovial VIP receptor mRNA levels and Treg and Th17 cell frequencies in the spleen were determined. Results EA significantly reduced the severity of CIA, as evidenced by reduced paw volumes, arthritis scores and inflammation scores. EA significantly increased mRNA expression of the VIP receptor VPAC1 and led to an elevation in CD4+FOXP3+ Treg cell frequency and a reduction in CD4+IL17+ Th17 cell frequency. Pre-injection of a VIP receptor antagonist significantly reversed EA-induced expansion of Treg cells, but did not alter the frequencies of Th17 cells. Conclusions EA exerts anti-inflammatory effects in a collagen-induced rat model of arthritis. These effects appear to be mediated through activation of VIP signalling and re-establishment of the Th17/Treg cell balance.

VIP-Receptor Antagonist Enhanced Antileukemic Activity In Mice

Background and Objective We have previously published that antagonizing vasoactive intestinal peptide (VIP) receptors dramatically decreases PD-1 expression on activated CD8+ T-cells and increases antiviral immunity (Blood 2013, 121:2347-51; PLoS One 2013, 8: e63381). Herein we tested whether short-term pharmacological antagonism of VIP signaling could induce anti-tumor immune responses in mice. Methods B6 mice were inoculated with 0.5 - 2 x 106 luciferase+ murine acute myeloid leukemia cells (Luc+ C1498) and B10BR mice were injected with 3 x 106 luciferase+ murine acute T-cell lymphoma cells (Luc+ LBRM) through tail vein. Mice were treated with one or more daily s.c. injections of 10 μg VIPhyb. Survival of groups that received 1, 3 or 7 doses of VIPhyb were compared with PBS treated controls. Tumor growth was monitored weekly by bioluminescence imaging (BLI). Cytokine expression and expression of immune markers PD-1, PD-1H (VISTA), and PD-L1 on blood and spleen leukocytes were analyzed by flow cytometry. Results Long-term survival (day 80) of tumor-bearing B6 and B10BR mice that received a single-dose of VIPhyb one day before tumor inoculation was 80% for both mouse strains harboring both leukemia cells (p<0.01 vs. control, B6 n=10 and B10BR n=5, Figure 1). A single injection of VIPhyb was more effective than multiple doses in achieving long-term tumor-free survival, with 60% survival among C1498-tumor bearing mice (p<0.01 vs. controls, n=5) treated with 3 doses, and 46% survival in mice (p<0.01 vs. control, n=13) with C1498 and 40% survival in mice (p=0.06 vs. control, n=5,) with LBRM treated with 7 doses. None of the control mice inoculated with C1498 (n=21) or LBRM (n=10) that received PBS injections survived to day 55. To explore the therapeutic effect of VIPhyb on established tumors, B6 mice and B10BR were treated with 7 daily doses of VIPhyb starting 8 days or 15 days after inoculation with Luc+ C1498 or Luc+ LBRM, respectively. Survival of B6 mice bearing C1498 and B10BR mice bearing LBRM that received delayed administration of VIPhyb was 60% (p<0.001 vs. control, n=10) and 20% (p=0.039 vs. control, n=5), respectively, compared with 0 % survival (and faster tumor growth) among control mice (B6 n=10; B10BR n=5) that received PBS injections. Tumor burdens in VIPhyb treated mice measured by BLI showed slower tumor growth, and regression of established tumors compared with mice that received PBS (Figure 1). To elucidate the mechanisms whereby VIPhyb induced anti-tumor activities, expression of serum cytokines (IFN-γ, TNF-α, IL-10 and IL-13), expression of co-inhibitory molecules PD-1, PD-1H, PD-L1, and effector molecules Fas-L and granzyme B were measured in T-cells from VIPhyb- and PBS-treated tumor-bearing B10BR mice. Blood and splenic activated (CD62L-CD25+CD69+) and memory (CD62L+/-CD44+) CD8+ T-cells from VIPhyb-treated tumor–bearing mice expressed higher levels of IFN-γ, FAS-L and granzyme B, and lower levels of PD-1 (but not VISTA/PD-1H) in activated CD8+ T-cells compared with those from PBS-treated mice (Figure 2). Expression levels of TNF-α, IL-10, IL-13, and PD-L1 in blood and splenic dendritic cells were not different comparing with tumor-bearing VIPhyb-treated with PBS-treated control mice. Conclusion Treatment with a small molecule antagonist of VIP-receptor, VIPhyb, dramatically increased immune/T-cell specific anti-leukemic activity. The mechanism by which administration of a VIP receptor antagonist enhanced anti-tumor immunity includes increasing productions of IFN-γ, and expression of FAS-L and granzyme B in and decreasing expression of PD-1 in activated CD8+ T-cells, leading to enhance anti-tumor cytotoxicity. Disclosures: No relevant conflicts of interest to declare.

Vasoactive intestinal peptide produces long-lasting changes in neural activity in the suprachiasmatic nucleus

The neuropeptide vasoactive intestinal peptide (VIP) is expressed at high levels in the neurons of the suprachiasmatic nucleus (SCN). While VIP is known to be important to the input and output pathways from the SCN, the physiological effects of VIP on electrical activity of SCN neurons are not well known. Here the impact of VIP on firing rate of SCN neurons was investigated in mouse slice cultures recorded during the night. The application of VIP produced an increase in electrical activity in SCN slices that lasted several hours after treatment. This is a novel mechanism by which this peptide can produce long-term changes in central nervous system physiology. The increase in action potential frequency was blocked by a VIP receptor antagonist and lost in a VIP receptor knockout mouse. In addition, inhibitors of both the Epac family of cAMP binding proteins and cAMP-dependent protein kinase (PKA) blocked the induction by VIP. The persistent increase in spike rate following VIP application was not seen in SCN neurons from mice deficient in Kv3 channel proteins and was dependent on the clock protein PER1. These findings suggest that VIP regulates the long-term firing rate of SCN neurons through a VIPR2-mediated increase in the cAMP pathway and implicate the fast delayed rectifier (FDR) potassium currents as one of the targets of this regulation.

F-18 Labeled Vasoactive Intestinal Peptide Analogue in the PET Imaging of Colon Carcinoma in Nude Mice

As large amount of vasoactive intestinal peptide (VIP) receptors are expressed in various tumors and VIP-related diseases, radiolabeled VIP provides a potential PET imaging agent for VIP receptor. However, structural modification of VIP is required before being radiolabeled and used for VIP receptor imaging due to its poor in vivo stability. As a VIP analogue, [R8, 15, 21, L17]-VIP exhibited improved stability and receptor specificity in preliminary studies. In this study, F-18 labeled [R8,15,21, L17]-VIP was produced with the radiochemical yield being as high as33.6%±3%(decay-for-corrected,n=5) achieved within 100 min, a specific activity of 255 GBq/μmol, and a radiochemical purity as high as 99% as characterized by radioactive HPLC, TLC, and SDS-Page radioautography. A biodistribution study in normal mice also demonstrated fast elimination of F-18 labeled [R8,15,21, L17]-VIP in the blood, liver, and gastrointestinal tracts. A further micro-PET imaging study in C26 colon carcinoma bearing mice confirmed the high tumor specificity, with the tumor/muscle radioactivity uptake ratio being as high as 3.03 at 60 min following injection, and no apparent radioactivity concentration in the intestinal tracts. In addition, blocking experiment and Western Blot test further confirmed its potential in PET imaging of VIP receptor-positive tumor.