gastrins and Glioblastoma

The effects of gastrin (G17) on the growth and migration factors of four human melanoma cell lines (HT-144, C32, G-361, and SKMEL-28) were investigated. The expression patterns of cholecystokinin (CCK)(A), CCK(B), and CCK(C) gastrin receptors were investigated in these cells and in seven clinical samples by means of reverse transcription polymerase chain reaction. Melanoma cells appear to express mRNA for CCK(C) receptors, but not for CCK(A) or CCK(B) receptors. Although gastrin does not significantly modify the growth characteristics of the cell lines under study, it significantly modifies their cell migration characteristics. These modifications occur at adhesion level by modifying the expression levels of alpha(v) and beta3 integrins, at motility level by modifying the organization of the actin cytoskeleton, and at invasion level by modifying the expression levels of matrix metalloproteinase 14. We recently demonstrated the presence of CCK(B) receptors in mouse endothelial cells involved in glioblastoma neoangiogenesis. Chronic in vivo administration of a selective CCK(B) receptor antagonist to mice bearing xenografts of human C32 melanoma cells significantly decreased levels of neoangiogenesis, resulting in considerable delays in the growth of these C32 xenografts. In conclusion, our study identifies the pleiotropic effects of gastrin on melanoma cell biology.

Topics: Actins; Animals; Antineoplastic Agents; Apoptosis; Blotting, Western; Cell Line, Tumor; Cell Movement; Cholecystokinin; Cisplatin; Cytoskeleton; Dacarbazine; DNA Primers; DNA, Complementary; Flow Cytometry; Gastrins; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Immunohistochemistry; Melanoma; Mice; Neoplasm Invasiveness; Neoplasm Transplantation; Neovascularization, Pathologic; Oligonucleotide Array Sequence Analysis; Poly(ADP-ribose) Polymerases; Receptor, Cholecystokinin B; Receptors, Cholecystokinin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Time Factors; Wound Healing

This study aims to investigate the role of gastrin-17 (G17) on angiogenesis features in gliomas both in vitro and in vivo.. The influences of G17 and G17 receptor antagonists were characterized in vitro in terms of angiogenesis on human umbilical vein endothelial cell (HUVEC) tubulogenesis processes on Matrigel and in vivo with respect to U373 orthotopic glioma xenografts. The influence of phosphatidylinositol 3'-kinase, protein kinase C, and nuclear factor-kappaB inhibitors was characterized in vitro on G17-mediated HUVEC tubulogenesis. G17-mediated release of interleukin (IL)-8 from HUVECs and G17-induced modifications in nuclear factor-kappaB DNA binding activity were characterized by means of specific enzyme-linked immunosorbent assays. The influence of G17 on E- and P-selectin expression was determined by means of computer-assisted microscopy, whereas the influence of E- and P-selectin on HUVEC migration was approached by means of antisense oligonucleotides. The chemotactic influence of G17 and IL-8 on HUVEC migration was characterized by means of computer-assisted videomicroscopy with Dunn chambers.. Messenger RNAs for cholecystokinin (CCK)A, CCKB, and CCKC receptors were present in HUVECs and microvessels dissected from a human glioblastoma. Whereas G17 significantly increased the levels of angiogenesis in vivo in the U373 experimental glioma model and in vitro in the HUVECs, the CCKB receptor antagonist L365,260 significantly counteracted the G17-mediated proangiogenic effects. G17 chemoattracted HUVECs, whereas IL-8 failed to do so. IL-8 receptor alpha (CXCR1) and IL-8 receptor beta (CXCR2) mRNAs were not detected in these endothelial cells. Gastrin significantly (but only transiently) decreased the level of expression of E-selectin, but not P-selectin, whereas IL-8 increased the expression of E-selectin. Specific antisense oligonucleotides against E- and P-selectin significantly decreased HUVEC tubulogenesis processes in vitro on Matrigel.. The present study shows that gastrin has marked proangiogenic effects in vivo on experimental gliomas and in vitro on HUVECs. This effect depends in part on the level of E-selectin activation, but not on IL-8 expression/release by HUVECs.

Topics: Animals; Benzodiazepinones; Brain Neoplasms; Cell Movement; Collagen; Drug Combinations; E-Selectin; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Female; Gastrins; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; In Vitro Techniques; Interleukin-8; Laminin; Mice; Mice, Nude; Neovascularization, Pathologic; NF-kappa B; P-Selectin; Phenylurea Compounds; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase C; Proteoglycans; Rats; Rats, Nude; Receptors, Cholecystokinin; Receptors, Interleukin-8A; Receptors, Interleukin-8B; Transplantation, Heterologous; Tumor Cells, Cultured; Umbilical Veins

Astrocytic tumors are the most common and the most malignant primary tumors of the central nervous system. We had previously observed that gastrin could significantly modulate both cell proliferation and migration of astrocytoma cells. We have investigated in the present study which genes could be targeted by gastrin in tumor astrocyte migration. Using a subtractive hybridization PCR technique we have cloned genes differentially over-expressed in human astrocytoma U373 cells treated or not with gastrin. We found about 70 genes over-expressed by gastrin. Among the genes overexpressed by gastrin, we paid particular attention to tenascin-C, S100A6 and MLCK genes because their direct involvement in cell migration features. Their gastrin-induced overexpression was quantitatively determined by competitive RT-PCR technique. We also showed by means of a reporter gene system that S100A6 and tenascin-C respective promoters were upregulated after gastrin treatment. These data show that gastrin-mediated effects in glioblastoma cells occur through activation of a number of genes involved in cell migration and suggest that gastrin could be a target in new therapeutic strategies against malignant gliomas.

Topics: Actins; Amino Acid Sequence; Biopolymers; Brain Neoplasms; Cell Cycle Proteins; Cell Movement; DNA, Complementary; Gastrins; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, Reporter; Glioblastoma; Humans; Molecular Sequence Data; Myosin-Light-Chain Kinase; Neoplasm Invasiveness; Neoplasm Proteins; Promoter Regions, Genetic; Protein Biosynthesis; Proteins; Reverse Transcriptase Polymerase Chain Reaction; rhoA GTP-Binding Protein; RNA, Messenger; RNA, Neoplasm; S100 Calcium Binding Protein A6; S100 Proteins; Stress Fibers; Subtraction Technique; Tenascin; Transfection; Tumor Cells, Cultured; Wiskott-Aldrich Syndrome Protein Family

We have investigated the potential role of gastrin in the regulation of cell growth in human astrocytic tumors. To this end we have used synthetic analogs of gastrin and cholecystokinin (CCK) which behave as gastrin and/or CCK antagonists, e.g. compounds JMV-97, JMV-209 and JMV-179. Their effects on astrocytic tumor cell proliferation was investigated by the use of the colorimetric MTT assay. The in vitro biological models used in the present study included the SW1088, U87 and U373 astrocytic tumor cell lines. The results demonstrated marked influence of gastrin and CCK antagonists in the regulation of astrocytic tumor growth. This suggests that gastrin and/or CCK antagonists might be tested in experimental glioblastoma.

Topics: Astrocytes; Astrocytoma; Brain Neoplasms; Cholecystokinin; Gastrins; Glioblastoma; Humans; Peptide Fragments; Sincalide; Tumor Cells, Cultured

The influence of five anti-hormone and/or anti-growth factor neutralizing antibodies on the in vitro proliferation of four human astrocytic tumor cell lines (U87, U138, U373, H4) is quantitatively described by means of a new tool which makes it possible to evaluate cell growth and cell clone architecture concomitantly. This tool relies upon the combined use of the digital cell image analyses of Feulgen-stained nuclei and the Delaunay and Voronoi mathematical triangulation and paving techniques. Of the five anti-hormone and/or anti-growth factors tested here, the anti-luteinizing hormone-releasing hormone (LHRH) antibody induced the most marked perturbation in the U138 and U373 cell lines, whereas this role was played by the anti-epidermal growth factor (EGF) antibody in the U87 and H4 cell lines. The anti-gastrin (G) antibody significantly modified the growth and/or cell clone architecture of the U138, U87 and H4 cell lines, as did the anti-transforming growth factor alpha (TGFalpha) antibody. The anti-transforming growth factor beta (TGFbeta) antibody modified the growth and/or cell clone architecture of the four cell lines under study. If the five antibodies are taken into consideration, the results strongly suggest that four (the anti-G, the anti-EGF, the anti-LHRH and the anti-TGFalpha) act as inhibitory agents on some glioma cell line proliferation, while the fifth one, i.e. the anti-TGFbeta, act as a stimulator of cell proliferation, perhaps by abrogating the inhibitory effects of TGFbeta on proliferation. A comparison of cell growth data with cell clone architecture characteristics provided further evidence of some specific influence exercised by a given hormone and/or growth factor on glioma cell proliferation. Indeed, the anti-LHRH antibody caused the most pronounced perturbations in the U138 and U373 cell clone architecture; this feature was observed in the H4 cell line and, to a lesser extent in the U87 one after the anti-EGF antibody had been used.

Topics: Antibodies, Monoclonal; Cell Division; Epidermal Growth Factor; Gastrins; Glioblastoma; Gonadotropin-Releasing Hormone; Humans; Neuroblastoma; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

We investigated the effects of somatostatin analogues and a synthetic bombesin/gastrin-releasing peptide (GRP) antagonist on the growth of the human malignant glioma cell lines U-87MG and U-373MG transplanted to nude mice or cultured in vitro. Nude mice bearing s.c. implanted U-87MG or U-373MG tumors were treated for 4 and 6 weeks, respectively, with various somatostatin analogues or bombesin/GRP antagonist RC-3095. Somatostatin analogues RC-160, RC-160-II, and RC-101-I, given s.c. in doses of 100 micrograms/animal/day, inhibited the growth of U-87MG xenografts as shown by more than 60% reduction in tumor volumes and 45% reduction in tumor weights compared with the control group. Bombesin/GRP antagonist RC-3095, given s.c. at a dose of 20 micrograms/animal twice daily, had the greatest inhibitory effect and decreased tumor volumes and weights by approximately 79% and 72%, respectively. The growth of U-373MG xenografts was also significantly inhibited by treatment with analogue RC-160 or antagonist RC-3095. The mean survival time of nude mice, inoculated orthotopically with U-87MG cells into the brain, was significantly prolonged by 4.9 days by treatment with antagonist RC-3095. Serum gastrin levels in animals bearing U-87MG tumors, treated with antagonist RC-3095 or somatostatin analogues, were decreased compared with controls. All three somatostatin analogues also reduced serum growth hormone levels. Receptor analyses demonstrated high-affinity binding sites for bombesin, somatostatin, and epidermal growth factor on membranes of U-87MG and U-373MG tumors. The concentration of binding sites for epidermal growth factor on both tumors was significantly decreased after in vivo treatment with antagonist RC-3095 or the somatostatin analogues. In studies in vitro, RC-3095, added to the culture medium, significantly inhibited the proliferation of U-87MG and U-373MG cells in the presence of GRP(14-27), as measured by cell number, but only a moderate suppression of growth of both cell lines was observed with somatostatin analogue RC-160. These results demonstrate that bombesin/GRP antagonist RC-3095 and somatostatin analogues such as RC-160 can inhibit the growth of human glioblastoma cell lines U-87MG and U-373MG in vitro as well as in vivo. Our work suggests the merit of further investigations of these analogues for the possible development of new approaches for treatments of patients with malignant gliomas.

Topics: Amino Acid Sequence; Animals; Bombesin; Cell Division; Drug Screening Assays, Antitumor; ErbB Receptors; Gastrins; Glioblastoma; Male; Mice; Mice, Nude; Molecular Sequence Data; Peptide Fragments; Receptors, Bombesin; Somatostatin; Tumor Cells, Cultured