vasoactive-intestinal-peptide and Cell-Transformation--Neoplastic

Glucagon-like peptide-2 (GLP-2) is a peptide hormone with multiple beneficial effects on the intestine, including expansion of the mucosal surface area through stimulation of crypt cell proliferation, as well as enhancement of nutrient digestion and absorption. Recent advances in clinical trials involving GLP-2 necessitate elucidation of the exact signaling pathways by which GLP-2 acts. In particular, the GLP-2 receptor has been localized to several intestinal cell types that do not include the proliferating crypt cells, and the actions of GLP-2 have thus been linked to a complex network of indirect mediators that induce diverse signaling pathways. The intestinotropic actions of GLP-2 on the colon have been shown to be mediated through the actions of keratinocyte growth factor and insulin-like growth factor (IGF)-2, whereas small intestinal growth has been linked to IGF-1, IGF-2, and ErbB ligands, as well as the IGF-1 receptor and ErbB. The cellular source of these mediators remains unclear, but it likely includes the intestinal subepithelial myofibroblasts. Conversely, the anti-inflammatory and blood flow effects of GLP-2 are dependent on vasoactive intestinal polypeptide released from submucosal enteric neurons and nitric oxide, respectively. Finally, recent studies have suggested that GLP-2 not only modulates intestinal stem cell behavior but may also promote carcinogenesis in models of sporadic colon cancer. Further consideration of the molecular cross-talk and downstream signaling pathways mediating the intestinotropic effects of GLP-2 is clearly warranted.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Cell Proliferation; Cell Transformation, Neoplastic; Colonic Neoplasms; Fibroblast Growth Factor 7; Glucagon-Like Peptide 2; Glucagon-Like Peptide-2 Receptor; Humans; Intestinal Mucosa; Intestines; Mice; Nitric Oxide; Rats; Receptors, Glucagon; Signal Transduction; Somatomedins; Stem Cells; Vasoactive Intestinal Peptide

There is no question that gut peptides are trophic for normal gut mucosa. Gut peptides can function in an endocrine, paracrine or autocrine fashion. We examined the effects of gut peptides on the growth of animal and human cancers of the gastrointestinal (GI) tract and pancreas in vivo and in vitro. We also examined the role of growth factors and bioamines in the regulation of growth of human endocrine tumors. Our studies have shown that gut peptides (gastrin, VIP, neurotensin, and bombesin) regulate growth of some cancers of the GI tract and pancreas. We have found that peptide action is mediated through specific receptors and that cell-specific differences in receptor expression occur. We have also begun to examine the intracellular signal-transduction pathways involved in endocrine and autocrine actions of these peptides on growth of GI cancers. We have found that cell-type-specific differences exist among the various signal-transduction pathways (cyclic AMP, phosphatidylinositol hydrolysis (PI), intracellular calcium ([Ca2+]i) mobilization, and tyrosine phosphorylation) and that different receptors for the same hormone may be linked to different signal-transduction pathways depending upon cell type. We have also found that autocrine growth regulation of human pancreatic carcinoid occurs through specific receptor-mediated signal-transduction pathways. We will discuss the mechanisms of action and potential therapeutic uses of manipulation of gut hormone levels or hormone antagonists to inhibit the growth of GI tract cancers.

Topics: Animals; Bombesin; Cell Division; Cell Line, Transformed; Cell Transformation, Neoplastic; Gastrins; Gastrointestinal Hormones; Gastrointestinal Neoplasms; Humans; Neurotensin; Pancreatic Neoplasms; Receptors, Gastrointestinal Hormone; Signal Transduction; Vasoactive Intestinal Peptide

Clear renal cell carcinoma (cRCC) is an aggressive and fatal neoplasm. The present work was undertaken to investigate the antiproliferative potential of vasoactive intestinal peptide (VIP) exposure on non-tumoral (HK2) and tumoral (A498, cRCC) human proximal tubular epithelial cell lines. Reverse transcription and semiquantitative PCR was used at the VIP mRNA level whereas enzyme immunoanalysis was performed at the protein level. Both renal cell lines expressed VIP as well as VIP/pituitary adenylate cyclase-activating peptide (VPAC) receptors whereas only HK2 cells expressed formyl peptide receptor-like 1 (FPRL-1). Receptors were functional, as shown by VIP stimulation of adenylyl cyclase activity. Treatment with 0.1μM VIP (24h) inhibited proliferation of A498 but not HK2 cells as based on a reduction in the incorporation of [(3)H]-thymidine and BrdU (5'-Br-2'-deoxyuridine), PCNA (proliferating-cell nuclear antigen) expression and STAT3 (signal transducer and activator of transcription 3) expression and activation. VPAC(1)-receptor participation was established using JV-1-53 antagonist and siRNA transfection. Growth-inhibitory response to VIP was related to the cyclic adenosine monophosphate (cAMP)/exchange protein directly activated by cAMP (EPAC)/phosphoinositide 3-kinase (PI3-K) signaling systems as shown by studies on adenylate cyclase stimulation, and using the EPAC-specific compound 8CPT-2Me-cAMP and specific kinase inhibitors such as H89, wortmannin and PD98059. The efficacy of VIP on the prevention of tumor progression was confirmed in vivo using xenografted athymic mouse. These actions support a potential role of this peptide and its agonists in new therapies for cRCC.

Topics: Animals; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Cyclic AMP; Gene Expression Regulation, Neoplastic; Humans; Intracellular Space; Kidney Neoplasms; Mice; Mice, Nude; Receptors, Vasoactive Intestinal Peptide; Receptors, Vasoactive Intestinal Polypeptide, Type I; RNA, Messenger; Signal Transduction; STAT3 Transcription Factor; Vasoactive Intestinal Peptide; Xenograft Model Antitumor Assays

The carcinogenic potential of vasoactive intestinal peptide (VIP) was analyzed in non-tumor human prostate epithelial cells (RWPE-1) and in vivo xenografts. VIP induced morphological changes and a migratory phenotype consistent with stimulation of expression/activity of metalloproteinases MMP-2 and MMP-9, decreased E-cadherin-mediated cell-cell adhesion, and increased cell motility. VIP increased cyclin D1 expression and cell proliferation that was blocked after VPAC(1)-receptor siRNA transfection. Similar effects were seen in RWPE-1 tumors developed by subcutaneous injection of VIP-treated cells in athymic nude mice. VIP acts as a cytokine in RWPE-1 cell transformation conceivably through epithelial-mesenchymal transition (EMT), reinforcing VIP role in prostate tumorigenesis.

Topics: Animals; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Humans; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Prostatic Neoplasms; Receptors, Vasoactive Intestinal Polypeptide, Type I; 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

A non-transformed small-intestinal cell line from the rat (IEC-6) and a human colon cancer cell line (HT 29) were examined for their trophic response to sensory neuropeptides. Substance P, neurokinin A (NKA), calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP), and peptide YY (PYY) were tested. Epidermal growth factor (EGF), insulin, and somatostatin-14 were also used. Interaction studies were performed on IEC-6 cells by combining EGF or insulin with somatostatin-14. The sensory neuropeptides had no effect either on IEC-6 cell growth and DNA synthesis or on HT29 cell growth. EGF and insulin stimulated cell growth and DNA synthesis in IEC-6 cells and cell growth in HT 29 cells in a dose-dependent fashion. Somatostatin-14 had no effect either alone or in combination with EGF or insulin on IEC-6 cell growth and DNA synthesis. HT 29 cell growth was inhibited by somatostatin-14 only in the presence of serum with a maximal and significant response at 10(-7) M. Our observations suggest that the sensory neuropeptides do not exert a direct growth-regulatory effect either on IEC-6 cells or on HT 29 cells. Somatostatin, however, inhibits serum-induced HT 29 cell growth but does not interfere directly with the proliferative effect of serum, EGF, or insulin on IEC-6 cells in this model.

Topics: Animals; Calcitonin Gene-Related Peptide; Cell Division; Cell Line, Transformed; Cell Transformation, Neoplastic; Cells, Cultured; Colonic Neoplasms; DNA, Neoplasm; Dose-Response Relationship, Drug; Epidermal Growth Factor; Epithelium; Gastrointestinal Hormones; Humans; Insulin; Intestinal Mucosa; Intestines; Neurokinin A; Neurons, Afferent; Neuropeptides; Peptide YY; Peptides; Rats; Somatostatin; Substance P; Transforming Growth Factor beta; Vasoactive Intestinal Peptide

The activation of the cAMP signaling pathway by vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP) and related peptides was studied (i) in normal peripheral human monocytes and THP-1 leukemic human monocytes, (ii) in their derived macrophage counterparts respectively obtained after spontaneous differentiation or retinoic acid (RA) treatment, and (iii) in human bronchoalveolar macrophages. In THP-1 monocytes, PACAP increased basal adenylate cyclase activity 5.3-fold, with an affinity 50-times greater than that of VIP or helodermin (Ka = 3.2 x 10(-11) M VIP), whereas in normal peripheral monocytes, PACAP and VIP exhibited similar affinities and only increased cAMP generation 2-fold (EC50 = 10(-9) M). Spontaneous and RA-induced differentiation into normal and leukemic macrophages induced a progressive loss of cAMP production and regulation of superoxide anion production by VIP and related peptides. The neoplastic transformation in THP-1 monocytes and the deficiencies in the cAMP cascade observed during the terminal differentiation of normal and leukemic human macrophages may relate to a differential genetic expression of the VIP/PACAP receptor subtypes, and alterations in the functional activity of the stimulatory and inhibitory Gs/Gi subunits of adenylate cyclase.

Topics: Adenylyl Cyclases; Cell Differentiation; Cell Transformation, Neoplastic; Colforsin; Cyclic AMP; Enzyme Activation; Humans; Hydrogen Peroxide; Isoproterenol; Leukemia; Macrophages; Monocytes; Neuropeptides; Pituitary Adenylate Cyclase-Activating Polypeptide; Pulmonary Alveoli; Signal Transduction; Superoxides; Tretinoin; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Elevated serum levels of vasoactive intestinal peptide (VIP) are associated with some cases of neuroblastoma and correlate with a favorable prognosis. VIP has previously been shown in our laboratory to cause the in vitro growth inhibition and morphological differentiation of the human neuroblastoma cell line, LA-N-5. It is now shown that LA-N-5 cells express immunoreactive VIP and bear specific VIP receptors. Antagonism of endogenous VIP, either by competitive inhibition or receptor blockade, increased cell proliferation, suggesting that VIP is operative in normal growth regulation. Intracellular and extracellular levels of VIP were also shown to increase significantly during the retinoic acid-induced differentiation of these cells. Furthermore, a concomitant marked increase in VIP receptor expression was demonstrated with cellular differentiation. These receptors remain functional as evidenced by a matching increase in the level of detectable cAMP generated in response to exogenous VIP. It is concluded that VIP is a normal autoregulator of neuroblastoma cell growth and differentiation, and that retinoic acid-mediated differentiation may be, in part, due to endogenous VIP.

Topics: Cell Transformation, Neoplastic; Humans; Neuroblastoma; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Tretinoin; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

A number of studies have demonstrated the pivotal role of collagen molecules in modulating cell growth and differentiation. In order to analyze the direct effects of collagen type I on the osteoblastic phenotype, we have devised an in vitro culture system for studying the interactions between bovine collagen type I and Saos-2 cells, a human osteoblastic cell line. Saos-2 cells were cultured both on top of collagen-coated culture dishes as well as inside a three-dimensional collagen network. Plating on dishes treated with collagen induced maximal adhesion of Saos-2 cells after 24-hour incubation. Cells cultured on collagen gel matrix expressed about 2.5-fold more alkaline phosphatase when compared with untreated plastic dishes. On collagen-coated dishes the responsiveness of Saos-2 cells to parathyroid hormone was decreased, whereas no modifications were observed in the effect of vasoactive intestinal peptide on these cells. Using a microfluorimetric measurement of DNA, an increase of proliferation was observed in Saos-2 cells cultured on collagen gel. Saos-2 cells were also able to colonize collagen sponges and in this three-dimensional network they were able to synthesize osteocalcin, as assessed both by immunocytochemistry and radioimmunoassay. In this study we have demonstrated that bovine collagen type I exhibits favorable effects on attachment and functional and growth activities of a human osteoblastic cell line, encouraging its use as a bone graft material.

Topics: Alkaline Phosphatase; Bone Neoplasms; Cell Adhesion; Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Collagen; Cyclic AMP; Cytophotometry; DNA, Neoplasm; Extracellular Matrix; Fluorescent Antibody Technique; Gels; Humans; Immunohistochemistry; Microscopy, Electron; Osteoblasts; Osteocalcin; Osteosarcoma; Parathyroid Hormone; Phenotype; Radioimmunoassay; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Cells of the F9 murine embryonal carcinoma (F9 EC) line have an activity that stimulates transcription from early adenovirus gene promoters. One such promoter, that of the E4 gene, is transcribed efficiently in F9 EC cells and is activated further as these cells differentiate to parietal endoderm-like cells (F9 PE). The sequences required for this in vivo regulation include the activating transcription factor (ATF)-binding site, and consistent with this we show that complexes formed on this site are regulated as F9 EC cells differentiate. Another ATF site-containing promoter is that of the human vasoactive intestinal polypeptide (VIP). In contrast to the E4 promoter, the VIP promoter is transcriptionally inactive throughout differentiation, a feature that correlates with distinct binding activities on its ATF site. We define five ATF site-binding activities in F9 cells that can be distinguished from each other by their precise sequence requirements and their regulation during differentiation. From these activities, we define those that bind in a promoter-specific or promoter-common fashion to the E4 and VIP promoters. These data indicate that members of the ATF family of transcription factors are differentiation-regulated and support the idea that they provide diverse transcriptional stimuli.

Topics: Activating Transcription Factors; Base Sequence; Binding Sites; Blood Proteins; Cell Transformation, Neoplastic; Cyclic AMP; Humans; In Vitro Techniques; Molecular Sequence Data; Neoplasm Proteins; Promoter Regions, Genetic; Teratoma; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Intestinal epithelial cells from 19-day-old rat fetuses underwent electropermeabilization and were successfully transfected by three recombinant plasmids containing the cloned oncogenes from the human adenovirus type 2 early region E1A (SLC-11 cells) and polyoma virus and simian virus 40 large T tumor antigens (SLC-21 and SLC-41 cells). SLC-11 cells were propagated for 21 months in culture (current passage, 76; doubling time, 17 hr) and were immortalized by E1A, as shown by RNA transfer blot (Northern blot) analysis and indirect immunofluorescence of the nuclear oncoproteins. These cells were not tumorigenic in either athymic nude mice or syngeneic Wistar rats and showed a nearly normal karyotype with minimal chromosomal changes. The immortalized epithelial cell line SLC-11 retained several of the phenotypes observed in the parent cells of the intestinal mucosa, including cytoplasmic villin, cytokeratins, enkephalinase, and cell surface receptors sensitive to vasoactive intestinal peptide. It is concluded that immortal SLC-11 cells are a suitable model for studying the proliferation and differentiation of epithelial intestinal cells and analyzing cancer progression in the gastrointestinal tract.

Topics: Adenoviridae; Animals; Antigens, Polyomavirus Transforming; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cyclic AMP; DNA, Recombinant; Epithelium; Fetus; Fluorescent Antibody Technique; Intestines; Karyotyping; Microscopy, Electron; Nucleic Acid Hybridization; Oncogenes; Plasmids; Rats; RNA; Simian virus 40; Transfection; Vasoactive Intestinal Peptide