Corinne Chadéneau

Study of a cyclopamine glucuronide prodrug for the selective chemotherapy of glioblastoma.

The first glucuronide prodrug of the hedgehog signaling inhibitor cyclopamine was synthesized. The carbamoyl derivatisation of cyclopamine significantly decreased its toxicity towards the U87 human glioblastoma cell line. However, when the prodrug was incubated with beta-glucuronidase in the culture media, the active drug was efficiently released thereby restoring its anti-proliferative activity.

The VIP-receptor system in neuroblastoma cells.

Neuroblastoma (NB), the most common extracranial tumor during childhood arises from the embryonic sympathetic nervous system. Remarkably, NB can spontaneously regress, even after metastasis, leading to complete remission. Subpopulations of neuroblastic (N-type) and nonneuronal cells coexist in NB. Expression of the high-affinity nerve growth factor (NGF) TrkA receptor in NB is correlated with good prognosis, while MYCN amplification is associated with advanced stages of disease. N-type cells undergo differentiation when treated with different compounds, such as retinoids, phorbol esters, growth and neurotrophic NGF and neuropeptides, especially vasoactive intestinal peptide (VIP). These substances stabilize proliferation, leading to a more mature neuronal phenotype, neurite outgrowth and induction of expression of sympathetic neuronal markers. Therefore, receptors for these substances and their associated signalling pathways, appear like promising targets for the development of novel NB therapeutics. The aim of the present review is to summarize the quite considerable array of data, concerning production of VIP and related peptides, expression of their receptors in NB and the key regulation exerted by the VIP-receptor system in the control of NB cell behaviour.

Atypical nuclear localization of VIP receptors in glioma cell lines and patients.

An increasing number of G protein-coupled receptors, like receptors for vasoactive intestinal peptide (VIP), are found in cell nucleus. As VIP receptors are involved in the regulation of glioma cell proliferation and migration, we investigated the expression and the nuclear localization of the VIP receptors VPAC1 and VPAC2 in this cancer. First, by applying Western blot and immunofluorescence detection in three human glioblastoma (GBM) cell lines, we observed a strong nuclear staining for the VPAC1 receptor and a weak nuclear VPAC2 receptor staining. Second, immunohistochemical staining of VPAC1 and VPAC2 on tissue microarrays (TMA) showed that the two receptors were expressed in normal brain and glioma tissues. Expression in the non-nuclear compartment of the two receptors significantly increased with the grade of the tumors. Analysis of nuclear staining revealed a significant increase of VPAC1 staining with glioma grade, with up to 50% of GBM displaying strong VPAC1 nuclear staining, whereas nuclear VPAC2 staining remained marginal. The increase in VPAC receptor expression with glioma grades and the enhanced nuclear localization of the VPAC1 receptors in GBM might be of importance for glioma progression.

The vasoactive intestinal peptide-receptor system is involved in human glioblastoma cell migration

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumor in adults. This cancer has an infiltrative nature and the median survival of patients is about one year. Vasoactive intestinal peptide (VIP) belongs to a structurally related family of polypeptides and is a major regulatory factor in the central and peripheral nervous systems. VIP regulates proliferation of astrocytes and of numerous cancer cell lines and modulates migration in prostatic and colonic cancer cell lines. Little is known about the involvement of VIP and its receptors (VIP-receptor system) in proliferation or migration of GBM cells. The effects of VIP, PACAP and of synthetic VIP antagonists were tested in two human GBM cell lines, M059K and M059J, established from two different parts of a single tumor. In these cells, the data revealed that the VIP-receptor system did not affect proliferation but controlled cell migration. Indeed, in M059K cells which express components of the VIP receptor system, the VIP receptor antagonists and a PACAP antibody enhanced migration. The VIP receptor antagonists increased generation of typical migration-associated processes: filopodia and lamellipodia, and activation of Rac1 and Cdc42 GTPases. Reciprocally, in M059J cells which poorly express the VIP-receptor system, treatments with the agonists VIP and PACAP resulted in decreased cell migration. Furthermore, the peptides appeared to act through a subclass of binding sites displaying an uncommon very high affinity for these ligands. Taken together, these observations suggest that components of the VIP-receptor system negatively regulate cell migration, thus showing potential anti-oncogenic properties.

Neuropeptides of the VIP family inhibit glioblastoma cell invasion

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.

Evaluation of Cytotoxic Properties of a Cyclopamine Glucuronide Prodrug in Rat Glioblastoma Cells and Tumors

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. Activation of the developmental hedgehog (Hh) pathway is observed in GBM, particularly in the so-called glioma stem cells (GSCs). An inhibitor of this pathway is the steroidal alkaloid cyclopamine, an antagonist of the Hh coreceptor Smoothened (SMO). To limit the toxicity of cyclopamine toward Hh-dependent non-tumor cells, our group previously reported the synthesis of a prodrug (called 1b), designed to deliver cyclopamine in the presence of β-glucuronidase, an enzyme found in the necrotic area of GBM. Here, we aimed to analyze the in vitro, ex vivo, and in vivo cytotoxic properties of this prodrug in the C6 rat GBM cells. In the presence of β-glucuronidase, the activated prodrug 1b was toxic and downregulated expression of Gli1, a Hh target gene, in C6 cells and C6-GSCs, but not in normal rat astrocytes in which the Hh pathway is weakly activated. In the absence of β-glucuronidase, prodrug 1b displayed no obvious toxicity toward rat brain tissue explants while cyclopamine clearly affected brain tissue viability. When administered to rats bearing fluorescent C6-derived GBM, the prodrug 1b reduced the tumor density more efficiently than cyclopamine. Prodrug 1b thus appears as a promising concept to optimize confinement of cyclopamine cytotoxicity within the tumors, with more limited effects in the surrounding normal brain tissue.

Vasoactive intestinal peptide-induced neuritogenesis in neuroblastoma SH-SY5Y cells involves SNAP-25.

Vasoactive intestinal peptide (VIP) is a neuropeptide known to regulate proliferation and differentiation in normal and tumoral cells. We previously reported that VIP induced neuritogenesis in human neuroblastoma SH-SY5Y cells cultured in serum-free medium. This neuritogenesis was associated with a regulated expression of neuronal cytoskeleton markers. To further characterize the neuroblastic cell differentiation induced by VIP in human SH-SY5Y cells, we investigated expression of synaptosomal-associated protein of 25 kDa (SNAP-25), a protein implicated in exocytosis associated with different processes, including neurite outgrowth. Western immunoblotting and real-time RT-PCR analyses revealed that VIP increased expression of the SNAP-25 protein and the level of both SNAP-25a and SNAP-25b mRNA isoforms. Immunofluorescence experiments indicated that SNAP-25 was mainly located in neurites and at the plasma membrane in SH-SY5Y cells treated with VIP. RNA interference experiments demonstrated that SNAP-25 was involved in VIP-induced neuritogenesis. In conclusion, SNAP-25 is up-regulated and implicated in neuritogenesis in human neuroblastoma SH-SY5Y cells treated with the neuropeptide VIP.

The small G-proteins Rap 1 as potential targets of vasoactive intestinal peptide effects in the human colonic cancer cells HT29

We recently reported that the vasoactive intestinal peptide (VIP) potently inhibited proliferation and induced in parallel a strong cAMP rise, in the human colonic cancer cell line HT29. In this study, we investigated whether Rap 1 proteins could be potential targets of VIP effects in HT29 cells. These Ras-related proteins in which activity was demonstrated to be regulated by PKA phosphorylation, are considered as potential modulators of the Ras / Raf / MAP kinases cascade that governs cell growth control. Our data revealed that the Rap 1a isoform is highly expressed in HT29 cells and mainly localized in a late endosomal compartment. In these cells, VIP induces Rap 1 phosphorylation and a yet unidentified modification that leads to their acidification. This latter Rap 1 acidification seems to be, at least partially, cAMP-dependent. It is concluded that in HT29 cells, Rap 1 proteins may be part of a VIP-induced signaling cascade.

VIP and PACAP analogs regulate therapeutic targets in high-risk neuroblastoma cells.

Neuroblastoma (NB) is a pediatric cancer. New therapies for high-risk NB aim to induce cell differentiation and to inhibit MYCN and ALK signaling in NB. The vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating polypeptide (PACAP) are 2 related neuropeptides sharing common receptors. The level of VIP increases with NB differentiation. Here, the effects of VIP and PACAP analogs developed for therapeutic use were studied in MYCN-amplified NB SK-N-DZ and IMR-32 cells and in Kelly cells that in addition present the F1174L ALK mutation. As previously reported by our group in IMR-32 cells, VIP induced neuritogenesis in SK-N-DZ and Kelly cells and reduced MYCN expression in Kelly but not in SK-N-DZ cells. VIP decreased AKT activity in the ALK-mutated Kelly cells. These effects were PKA-dependent. IMR-32, SK-NDZ and Kelly cells expressed the genes encoding the 3 subtypes of VIP and PACAP receptors, VPAC1, VPAC2 and PAC1. In parallel to its effect on MYCN expression, VIP inhibited invasion in IMR-32 and Kelly cells. Among the 3 PACAP analogs tested, [Hyp(2)]PACAP-27 showed higher efficiency than VIP in Kelly cells. These results indicate that VIP and PACAP analogs act on molecular and cellular processes that could reduce aggressiveness of high-risk NB.