Euphol from Tapinanthus sp. Induces Apoptosis and Affects Signaling Proteins in Glioblastoma and Prostate Cancer Cells

BACKGROUND: Plants play an important role in cancer therapy. They are source of natural molecules which can induce apoptosis in cancer cells by affecting molecular mechanisms implicated in cancer progression. The MAP Kinase/ERK1/2 and PI3K/AKT signaling pathways are two classical signaling pathways implicated in cancer progression and constitute therapeutic targets against cancer. This study aimed to evaluate the effect of euphol on MAP Kinase/ERK1/2 and PI3K/AKT signaling pathways in glioblastoma and prostate cancer cells. Euphol is a tetracyclique triterpene alcohol isolated from Tapinanthus sp. which is a hemi parasitic plant belonging to Loranthaceae family. METHODS: Plant powder was extracted by maceration and euphol was isolated and described using respectively column chromatography separation on silica gel and spectroscopic data. Cytotoxic effect of euphol was evaluated using XTT assay and its effect on MAP Kinase/ERK1/2 and PI3K/AKT protein expression was investigated by Western immunoblot analysis. Apotosis was analyzed by evaluating caspase-3/7 activity. RESULTS: Our investigations demonstrated that this compound has an important cytotoxic effect on C6 and U87 MG glioblastoma (GBM) cells and PC-3 prostate cancer cells. Furthermore, euphol-induced apoptosis revealed by elevated caspase 3/7 activity, was correlated with a significant inhibition of MAP kinase/Erk 1/2 and PI3K/Akt signaling pathway in glioblastoma U87 MG cells. The reverse effect was observed in C6 glioblastoma cells, where apoptosis was correlated with a long-lasting activation of Erk 1/2.  In PC-3 cells, euphol had no or limited effect on Erk 1/2 and Akt activity. CONCLUSION: These results indicate that euphol induces cell death in glioblastoma and prostate cancer cells and regulates significantly Erk1/2 and Akt activity in glioblastoma cells.

A new flavonoid glycoside from Tapinanthus sp. (Loranthaceae) and evaluation of anticancer activity of extract and some isolated compounds.

The present work describes the isolation and anticancer activity of Tapinanthus sp. which is a hemi parasitic plant harvested on Combretum glutinosum, the host plant. Phytochemical study afforded a new flavonoid glycoside, tapinantoside (1) isolated for the first time from natural source, alongside six known compounds (2-7). Structure of compounds were elucidated by extensive spectroscopic analyses including 1 D and 2 D NMR, mass spectrometry and by comparison with literature data. The anticancer activity of extract and some isolated compounds were evaluated on glioblastoma (U87MG, C6) and prostate (PC-3) cancer cells. The methanol leaves extract showed good anticancer activity against U87 (IC50 = 21.40 µg/mL) and PC-3 cells (IC50 = 10.26 µg/mL). Compound 3 powerfully inhibits the proliferation of C6 (IC50 = 38.84 µM) and PC-3 cells (IC50 = 21.33 µM), while its effect was moderated on U87MG cells. Compound 1 and 7 were not active on all tested cancer cell lines.

Anticancer Activity of Combretum fragrans F. Hoffm on Glioblastoma and Prostate Cancer Cell Lines.

BACKGROUND: Cancer incidence has been growing in an alarming rate worldwide and new therapeutics are needed, particularly for intractable and chemoresistant cases. We evaluated the cytotoxic effects of Combretum fragrans F. Hoffm (Combretaceae) on glioblastoma (U87MG and C6) and prostate (PC-3) cancer cell lines. METHODS: The cytotoxic effect of the methanolic extract of the stem bark of Combretum fragrans was assessed using XTT (2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide) test. Expressions of Akt and ERK1/2 were determined using Western blot technique, while Caspase-3/7 kits were used to evaluate caspase-3/7 activity. RESULTS: C. fragrans extract inhibited the proliferation of U87 (IC50 = 20.13 µg/mL), C6 (IC50 = 12.17 µg/mL), and PC-3 (IC50 = 11.50 µg/mL) cells. Treatment with the extract resulted in lower levels (p < 0.001) of phospho-ERK1/2 and phospho-Akt in U87 cells, and instead, higher levels of phospho-ERK1/2 (p < 0.001) in C6 and PC-3 cells. An increase in caspase-3/7 activity was observed, mainly after 24 hours of treatment, indicating the activation of apoptotic processes. CONCLUSION: Altogether, these results suggest that C. fragrans have potent anticancer properties. This plant should be further investigated for developing new anticancer drugs.

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.

Developmental pathways associated with cancer metastasis: Notch, Wnt, and Hedgehog.

Master developmental pathways, such as Notch, Wnt, and Hedgehog, are signaling systems that control proliferation, cell death, motility, migration, and stemness. These systems are not only commonly activated in many solid tumors, where they drive or contribute to cancer initiation, but also in primary and metastatic tumor development. The reactivation of developmental pathways in cancer stroma favors the development of cancer stem cells and allows their maintenance, indicating these signaling pathways as particularly attractive targets for efficient anticancer therapies, especially in advanced primary tumors and metastatic cancers. Metastasis is the worst feature of cancer development. This feature results from a cascade of events emerging from the hijacking of epithelial-mesenchymal transition, angiogenesis, migration, and invasion by transforming cells and is associated with poor survival, drug resistance, and tumor relapse. In the present review, we summarize and discuss experimental data suggesting pivotal roles for developmental pathways in cancer development and metastasis, considering the therapeutic potential. Emerging targeted antimetastatic therapies based on Notch, Wnt, and Hedgehog pathways are also discussed.

Mesenchymal stromal cells' role in tumor microenvironment: involvement of signaling pathways.

Mesenchymal stromal cells (MSCs) are adult multipotent stem cells residing as pericytes in various tissues and organs where they can differentiate into specialized cells to replace dying cells and damaged tissues. These cells are commonly found at injury sites and in tumors that are known to behave like " wounds that do not heal." In this article, we discuss the mechanisms of MSCs in migrating, homing, and repairing injured tissues. We also review a number of reports showing that tumor microenvironment triggers plasticity mechanisms in MSCs to induce malignant neoplastic tissue formation, maintenance, and chemoresistance, as well as tumor growth. The antitumor properties and therapeutic potential of MSCs are also discussed.

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.

A Novel Platinum-Maurocalcine Conjugate Induces Apoptosis of Human Glioblastoma Cells by Acting through the ROS-ERK/AKT-p53 Pathway.

Glioblastoma multiforme (GBM) is a highly malignant and aggressive primary brain tumor. In spite of an arsenal of therapeutic interventions, the prognosis of glioblastoma remains very poor. Cisplatin-based therapy is one of the most important chemotherapy treatments for GBM, although its efficacy is limited by drug resistance and undesirable side effects. In the present study, we designed a chimera molecule containing the platinum binding moiety MBL-III-7 (1) attached N-terminal to the sequence of d-maurocalcine (D-MCa), a protease-resistant and highly efficient cell-penetrating peptide (CPP) derived from the Tunisian chactid scorpion toxin, L-MCa. The concept behind this design is that MCa, through its cell retention properties, should reduce cell expulsion of the platinum complex and increase its efficiency. The anti-cancer properties of the synthesized platinum analogue Pt-MBL-III_7-D_MCa (Pt-1-DMCa) were assessed in human glioblastoma cells (U87) by assaying cell viability and apoptosis. The new molecule exhibited enhanced anti-cancer efficacy compared to cisplatin, especially at low doses. By inducing intracellular oxidative stress, Pt-1-DMCa potentiated platinum-induced DNA damage and led to enhanced p53 phosphorylation, followed by increased activation of both mitochondrial and death receptor pathways. Decreased phosphorylated AKT and ERK levels were associated with the apoptosis induced by the novel synthesized cisplatin analogue. Our results suggested that a chimera between platinum and a maurocalcine-derived CPP is a highly successful anti-cancer compound that works by targeting the intracellular redox system. Pt-1-DMCa is an interesting candidate for a preclinical assessment of platinum-based therapy in GBM treatments and possibly other cancer types.

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 ß-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 ß-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 ß-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.

New targeted therapies for breast cancer: A focus on tumor microenvironmental signals and chemoresistant breast cancers.

Breast cancer is the most frequent female malignancy worldwide. Current strategies in breast cancer therapy, including classical chemotherapy, hormone therapy, and targeted therapies, are usually associated with chemoresistance and serious adverse effects. Advances in our understanding of changes affecting the interactome in advanced and chemoresistant breast tumors have provided novel therapeutic targets, including, cyclin dependent kinases, mammalian target of rapamycin, Notch, Wnt and Shh. Inhibitors of these molecules recently entered clinical trials in mono- and combination therapy in metastatic and chemo-resistant breast cancers. Anticancer epigenetic drugs, mainly histone deacetylase inhibitors and DNA methyltransferase inhibitors, also entered clinical trials. Because of the complexity and heterogeneity of breast cancer, the future in therapy lies in the application of individualized tailored regimens. Emerging therapeutic targets and the implications for personalized-based therapy development in breast cancer are herein discussed.

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.

Signaling pathways in breast cancer: therapeutic targeting of the microenvironment.

Breast cancer is the most common cancer in women worldwide. Understanding the biology of this malignant disease is a prerequisite for selecting an appropriate treatment. Cell cycle alterations are seen in many cancers, including breast cancer. Newly popular targeted agents in breast cancer include cyclin dependent kinase inhibitors (CDKIs) which are agents inhibiting the function of cyclin dependent kinases (CDKs) and agents targeting proto-oncogenic signaling pathways like Notch, Wnt, and SHH (Sonic hedgehog). CDKIs are categorized as selective and non-selective inhibitors of CDK. CDKIs have been tried as monotherapy and combination therapy. The CDKI Palbocyclib is now a promising therapeutic in breast cancer. This drug recently entered phase III trial for estrogen receptor (ER) positive breast cancer after showing encouraging results in progression free survival in a phase II trials. The tumor microenvironment is now recognized as a significant factor in cancer treatment response. The tumor microenvironment is increasingly considered as a target for combination therapy of breast cancer. Recent findings in the signaling pathways in breast cancer are herein summarized and discussed. Furthermore, the therapeutic targeting of the microenvironment in breast cancer is also considered.

A new cyclopamine glucuronide prodrug with improved kinetics of drug release.

We prepared a new glucuronide prodrug of cyclopamine designed to target selectively the Hedgehog signalling pathway of cancer cells. This prodrug includes a novel self-immolative linker bearing a hydrophilic side chain that can be easily introduced via"click chemistry". With this design, the prodrug exhibits reduced toxicity compared to the free drug on U87 glioblastoma cells. However, in the presence of ß-glucuronidase, the prodrug conducts to the quick release of cyclopamine thereby restoring its antiproliferative activity.

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.

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.

Vasoactive intestinal peptide decreases MYCN expression and synergizes with retinoic acid in a human MYCN-amplified neuroblastoma cell line.

Neuroblastoma is a pediatric tumor which can spontaneously regress or differentiate into a benign tumor. MYCN oncogene amplification occurs in 22% of neuroblastomas and is associated with poor prognosis. Retinoic acid (RA), a molecule able to induce differentiation and to decrease MYCN expression, is used in the therapy of neuroblastomas. The neuropeptide vasoactive intestinal peptide (VIP) is known to control proliferation or differentiation of numerous cancer cells. In vitro, VIP induces differentiation of neuroblastoma cells. To determine whether VIP could modulate MYCN expression, we carried out real-time quantitative RT-PCR and Western immunoblot analyses in human neuroblastoma SH-SY5Y and IMR-32 cells. The results indicated that VIP reduced MYCN mRNA and protein expression, especially in the MYCN-amplified IMR-32 cells, with a maximal and transient decrease by approximately 50% after few hours of treatment with VIP at 10(-6) M. This effect was compared to that of RA at 10(-5) M, which induced a diminution of MYCN mRNA expression by approximately 25% after few days of treatment. This indicated that VIP and RA display complementary kinetics. Cotreatments showed that VIP and RA had synergistic effects on regulation of expression of MYCN proteins. VIP and RA cotreatments regulated also expression of two MYCN target genes, SKP2 and TP53INP1. These results suggest that VIP, in combination with RA may have a potential therapeutic benefit in neuroblastomas with MYCN amplification, a genetic abnormality associated with poor prognosis.

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.

Lack of vasoactive intestinal peptide reduces testosterone levels and reproductive aging in mouse testis.

The neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide have long been considered as putative regulators of testicular functions. In vitro evidence suggests that VIP could play an important role in testosterone biosynthesis. However, the endogenous role of VIP on testicular functions remained to be demonstrated. In C57BL/6 mice exhibiting a complete disruption of the VIP gene, we first observed here that serum testosterone levels were lower than those of WT littermates. At the age of 4 months, this phenotype was accompanied with a reduction of expression of StAR and 3-beta-hydroxysteroid dehydrogenase (3beta-HSD) in the testis. In addition, serum levels of FSH but not LH were reduced in young knock-out (KO) males. Testicular anatomy also revealed a higher percentage of degenerated seminiferous tubules in the 4-month-old VIP KO animals when compared with WT. In 15-month-old animals, control males showed typical testicular aging, including a severe degeneration of seminiferous tubules, a dramatic decrease in serum levels of testosterone, and a reduction in StAR and 3beta-HSD gene expression. In age-matching VIP KO males, the levels of serum testosterone and steroidogenic enzymes were still very low. Interestingly, in contrast to that observed in young mice, testicular degeneration at 15 months was significantly less severe in VIP KO than WT mice. All together, these results suggest that 1) VIP is an important factor for regulating testosterone biosynthesis and FSH secretion and 2) VIP may influence testicular aging.

Unconventional binding sites and receptors for VIP and related peptides PACAP and PHI/PHM: an update.

The 28-amino-acid neuropeptide VIP and related peptides PACAP and PHI/PHM modulate virtually all of the vital functions in the body. These peptides are also commonly recognized as major regulators of cell growth and differentiation. Through their trophic and cytoprotective functions, they appear to play major roles in embryonic development, neurogenesis and the progression of a number of cancer types. These peptides bind to three well-characterized subtypes of G-protein coupled receptors: VPAC1 and VPAC2 share a common high affinity in the nanomolar range for VIP and PACAP; a third receptor type, PAC1, has been characterized for its high affinity for PACAP but its low affinity for VIP. Complex effects and pharmacological behaviors of these peptides suggest that multiple subtypes of binding sites may cooperate to mediate their function in target cells and tissues. In this complex response, some of these binding sites correspond to the definition of the conventional receptors cited above, while others display unexpected pharmacological and functional properties. Here we present potential clues that may lead investigators to further characterize the molecular nature and functions of these atypical binding species.