gastrin-releasing-peptide and Colonic-Neoplasms

We aimed to explore the potential molecular mechanism and independent prognostic genes for colon cancer (CC).. Microarray datasets GSE17536 and GSE39582 were downloaded from Gene Expression Omnibus. Meanwhile, the whole CC-related dataset were downloaded from The Cancer Genome Atlas (TCGA) database. Differentially expressed mRNA (DEMs) were identified between cancer tissue samples and para-carcinoma tissue samples in TCGA dataset, followed by the KEGG pathway and GO function analyses. Furthermore, the clinical prognostic analysis including overall survival (OS) and disease-free survival (DFS) were performed in all three datasets.. A total of 633 up- and 321 down-regulated mRNAs were revealed in TCGA dataset. The up-regulated mRNAs were mainly assembled in functions including extracellular matrix and pathways including Wnt signaling. The down-regulated mRNAs were mainly assembled in functions like Digestion and pathways like Drug metabolism. Furthermore, up-regulation of UL16-binding protein 2 (ULBP2) was associated with OS in CC patients. A total of 12 DEMs including Surfactant Associated 2 (SFTA2) were potential DFS prognostic genes in CC patients. Meanwhile, the GRP and Transmembrane Protein 37 (TMEM37) were two outstanding independent DFS prognostic genes in CC.. ULBP2 might be a potential novel OS prognostic biomarker in CC, while GRP and TMEM37 could be served as the independent DFS prognostic genes in CC. Furthermore, functions including extracellular matrix and digestion, as well as pathways including Wnt signaling and drug metabolism might play important roles in the process of CC.

Topics: Animals; Biomarkers, Tumor; Colonic Neoplasms; Disease-Free Survival; Down-Regulation; Gastrin-Releasing Peptide; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genetic Markers; GPI-Linked Proteins; Humans; Intercellular Signaling Peptides and Proteins; Kaplan-Meier Estimate; Microarray Analysis; Murinae; Pulmonary Surfactant-Associated Protein A; Risk Factors; RNA, Messenger; Up-Regulation

We investigated the efficacy of a powerful antagonist of bombesin/gastrin-releasing peptide (BN/GRP) RC-3940-II administered as a single agent or in combination with cytotoxic agents on the growth of HT-29, HCT-116 and HCT-15 human colon cancer in vitro and in vivo. GRP-receptor mRNA and protein were found in all three cell lines tested. Exposure of HT-29 cells to 10 μM RC-3940-II led to an increase in the number of cells blocked in S phase and G 2/M and cells with lower G(0)/G(1) DNA content. Similar changes on the cell cycle traverse of HT-29 cells could also be seen at lower concentrations of RC-3940-II (1 μM) after pretreatment with 100 nM GRP (14-27), indicating a dose-dependent mechanism of action based on the blockage of BN/GRP induced proliferation of tumor cells at lower concentrations. Daily in vivo treatment with BN/GRP antagonist RC-3940-II decreased the volume of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic nude mice by 25 to 67% (p < 0.005). Combined treatment with RC-3940-II and chemotherapeutic agents 5-FU and irinotecan resulted in a synergistic tumor growth suppression of HT-29, HCT-116 and HCT-15 xenografts by 43% to 78%. In HT-29 and HCT-116 xenografts the inhibition for the combinations of RC-3940-II and irinotecan vs. single substances (p < 0.05) was significantly greater. These findings support the use of RC-3940-II as an anticancer agent and may help to design clinical trials using RC-3940-II in combinations with cytotoxic agents.

Topics: Animals; Antineoplastic Agents; Bombesin; Camptothecin; Cell Cycle Checkpoints; Cell Line, Tumor; Colonic Neoplasms; Drug Synergism; Drug Therapy, Combination; Fluorouracil; Gastrin-Releasing Peptide; HCT116 Cells; HT29 Cells; Humans; Irinotecan; Male; Mice; Mice, Nude; Peptide Fragments; Receptors, Bombesin; Transplantation, Heterologous

Epithelial cells lining the adult colon do not normally express gastrin-releasing peptide (GRP) or its receptor (GRPR). In contrast, GRP/GRPR can be aberrantly expressed in colon cancer where they are associated with improved patient survival rates. However, the mechanism of action whereby these proteins mediate their beneficial effects is not known. Heterochromatin protein 1 is an epigenetic modifier of gene transcription for which three different isoforms exist in humans: HP1(Hsα), HP1(Hsβ), and HP1(Hsγ). In breast cancer and melanoma, respectively, HP1(Hsα) and HP1(Hsβ) have been shown to modulate the aggressiveness of tumor cells in vivo. In contrast, the role of HP1 in colon cancer has not been elucidated, and a mechanism of regulating the expression of any HP1 isoform in any context has not yet been identified. In this article we demonstrate that abrogating GRP/GRPR signaling specifically down-regulates HP1(Hsβ) expression and that inhibiting GRPR signaling, or ablating HP1(Hsβ) expression, increases colon cancer cell invasiveness in vitro. These findings identify for the first time a signaling pathway regulating heterochromatin protein expression and suggest a mechanism whereby aberrantly expressed GRPR might alter the outcome of patients with colorectal cancer.

Topics: Adult; Cell Line, Tumor; Cell Nucleus; Chromobox Protein Homolog 5; Chromosomal Proteins, Non-Histone; Collagen; Colonic Neoplasms; Gastrin-Releasing Peptide; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Invasiveness; Receptors, Bombesin; RNA, Small Interfering; Signal Transduction; Time Factors

Epithelial cells lining the adult human colon do not normally express gastrin releasing peptide (GRP) or its receptor (GRPR), but both can be up regulated post malignant transformation. However, controversy exists as to the contribution these proteins make to tumor cell behavior once present. Since GRPR activation promotes proliferation, it has been assumed that their aberrant expression promotes colon cancer (CC) growth and progression. Yet we have contended that when expressed, GRP/GRPR benefits the host since in vitro studies demonstrate they enhance tumor cell attachment to the extracellular matrix and promote CC cytolysis by natural killer lymphocytes. Thus the aim of this study was to ascertain the effect of aberrant GRP/GRPR expression on patient survival. To do this we identified all CC diagnosed at a single institution from 1998 to 2002 that were classified as AJCC stage II or III (n = 88); of these 50 (57%) had sufficient tissues remaining for study. GRP/GRPR expression and natural killer cell density were determined immunohistochemically at the leading edge of each CC, and survival assessed by Kaplan Meier analysis. Expression of high levels of GRPR alone, or both GRP and GRPR, was associated with delayed CC recurrence (14.1-17.0 months, respectfully; P = 0.005) and increased survival (10.1-13.1 months, respectfully; P = 0.0124). CC expressing GRP/GRPR were associated with significantly fewer lymph node metastases than tumors not expressing these proteins, and contained significantly more CD16 + natural killer cells, than tumors not expressing these proteins. These findings demonstrate that patients whose CC express GRPR are associated with a survival advantage as compared to those whose CC do not express these proteins.

Topics: Amino Acid Sequence; Colonic Neoplasms; Gastrin-Releasing Peptide; Humans; Immunohistochemistry; Lymphatic Metastasis; Molecular Sequence Data; Receptors, Bombesin; Survival Analysis

Gastrin-releasing peptide (GRP) and its receptor (GRPR) are not normally expressed by epithelial cells lining the adult human colon. However post malignant transformation both GRP and its receptor are aberrantly expressed in the colon where we have previously shown they act to retard metastasis by enhancing tumor cell attachment to the extracellular matrix. In the present study, we show that GRP signaling via its cognate receptor when both are aberrantly expressed in human colon cancer cells causes heat shock protein 72 (Hsp72) to be expressed. We show that GRP/GRPR induces expression of Hsp72 by signaling via focal adhesion kinase. When expressed, Hsp72 promotes the binding of CD16+ and CD94+ natural killer cells, resulting in tumor cell cytolysis. These findings demonstrate the presence of a novel mechanism whereby aberrantly expressed GRP/GRPR in human colorectal cancer attenuates tumor progression and may promote a favorable outcome.

Topics: Cell Line, Tumor; Colonic Neoplasms; Cytotoxicity, Immunologic; Focal Adhesion Protein-Tyrosine Kinases; Gastrin-Releasing Peptide; HSP72 Heat-Shock Proteins; Humans; Killer Cells, Natural; NK Cell Lectin-Like Receptor Subfamily D; Receptors, Bombesin; Receptors, IgG; Signal Transduction; Up-Regulation

In this report, we present the synthesis and evaluation of the (99m)Tc-labeled beta-Ala-BN(7-14)NH2 (ABN = beta-Ala-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2) as a new radiotracer for tumor imaging in the BALB/c nude mice bearing HT-29 human colon cancer xenografts. The gastrin releasing peptide receptor binding affinity of ABN and HYNIC-ABN (6-hydrazinonicotinamide) was assessed via a competitive displacement of (125)I-[Tyr4]BBN bound to the PC-3 human prostate carcinoma cells. The IC 50 values were calculated to be 24 +/- 2 nM and 38 +/- 1 nM for ABN and HYNIC-ABN, respectively. HYNIC is the bifunctional coupling agent for (99m)Tc-labeling, while tricine and TPPTS (trisodium triphenylphosphine-3,3',3''-trisulfonate) are used as coligands to prepare the ternary ligand complex [(99m)Tc(HYNIC-ABN)(tricine)(TPPTS)] in very high yield and high specific activity. Because of its high hydrophilicity (log P = -2.39 +/- 0.06), [(99m)Tc(HYNIC-ABN)(tricine)(TPPS)] was excreted mainly through the renal route with little radioactivity accumulation in the liver, lungs, stomach, and gastrointestinal tract. The tumor uptake at 30 min postinjection (p.i.) was 1.59 +/- 0.23%ID/g with a steady tumor washout over the 4 h study period. As a result, it had the best T/ B ratios in the blood (2.37 +/- 0.68), liver (1.69 +/- 0.41), and muscle (11.17 +/- 3.32) at 1 h p.i. Most of the injected radioactivity was found in the urine sample at 1 h p.i., and there was no intact [(99m)Tc(HYNIC-ABN)(tricine)(TPPTS)] detectable in the urine, kidney, and liver samples. Its metabolic instability may contribute to its rapid clearance from the liver, lungs, and stomach. Despite the steady radioactivity washout, the tumors could be clearly visualized in planar images of the BALB/c nude mice bearing the HT-29 human colon xenografts at 1 and 4 h p.i. The favorable excretion kinetics from the liver, lungs, stomach, and gastrointestinal tract makes [(99m)Tc(HYNIC-ABN)(tricine)(TPPTS)] a promising SPECT radiotracer for imaging colon cancer.

Topics: Amino Acid Sequence; Animals; Bombesin; Colonic Neoplasms; Cross-Linking Reagents; Gastrin-Releasing Peptide; HT29 Cells; Humans; Kinetics; Ligands; Mice; Mice, Inbred BALB C; Niacin; Organotechnetium Compounds; Radiochemistry; Tissue Distribution; Tomography, Emission-Computed, Single-Photon; Transplantation, Heterologous

Gastrin-releasing peptide (GRP) and its receptor (GRPR) act as morphogens when expressed in colorectal cancer (CRC), promoting the assumption of a better differentiated phenotype by regulating cell motility in the context of remodeling and retarding tumor cell metastasis by enhancing cell-matrix attachment. Although we have shown that these processes are mediated by focal adhesion kinase (FAK), the downstream target(s) of GRP-induced FAK activation are not known. Since osteoblast differentiation is mediated by FAK-initiated upregulation of ICAM-1 (Nakayamada S, Okada Y, Saito K, Tamura M, Tanaka Y. J Biol Chem 278: 45368-45374, 2003), we determined whether GRP-induced activation of FAK alters ICAM-1 expression in CRC and, if so, determined the contribution of ICAM-1 to mediating GRP's morphogenic properties. Caco-2 and HT-29 cells variably express GRP/GRPR. These cells only express ICAM-1 when GRPR are present. In human CRC, GRPR and ICAM-1 are only expressed by better differentiated tumor cells, with ICAM-1 located at the basolateral membrane. ICAM-1 expression was only observed subsequent to GRPR signaling via FAK. To study the effect of ICAM-1 expression on tumor cell motility, CRC cells expressing GRP, GRPR, and ICAM-1 were cultured in the presence and absence of GRPR antagonist or monoclonal antibody to ICAM-1. CRC cells engaged in directed motility in the context of remodeling and were highly adherent to the extracellular matrix, only in the absence of antagonist or ICAM-1 antibody. These data indicate that GRP upregulation of ICAM-1 via FAK promotes tumor cell motility and attachment to the extracellular matrix.

Topics: Amino Acid Sequence; Cell Line; Cell Movement; Colonic Neoplasms; Colorectal Neoplasms; Extracellular Matrix; Focal Adhesion Protein-Tyrosine Kinases; Gastrin-Releasing Peptide; Humans; Intercellular Adhesion Molecule-1; Molecular Sequence Data; Morphogenesis; Peptide Fragments; Receptors, Bombesin

Although there is abundant evidence that gastrin-releasing peptide acts as a mitogen in various carcinoma cell lines, the effect of administration of gastrin-releasing peptide on the colorectal mucosa in vivo has not been reported. The aims of this study were to determine whether continuous infusion of gastrin-releasing peptide stimulated proliferation or accelerated carcinogenesis in the rat gastrointestinal tract and other organs. The possible requirement for C-terminal amidation for mitogenic activity in vivo was also investigated. Proliferation was measured in the colon by metaphase index and by immunostaining for the proliferation marker Ki-67, and in other tissues by immunostaining alone. Acceleration of colorectal carcinogenesis was assessed by counting aberrant crypt foci after treatment with the carcinogen azoxymethane. Defunctioning of the rectum reduced both the proliferative index and the crypt height of the rectal mucosa of untreated rats. Treatment with amidated or glycine-extended gastrin-releasing peptide for 4 weeks using implanted mini-osmotic pumps resulted in a two- to three-fold increase in proliferation, and an increase in crypt height, in the defunctioned rectal mucosa (p<0.001), with smaller but significant increases in the caecum and distal colon. No changes in proliferation were detected in lung, pancreas or gastric mucosa. The numbers of aberrant crypt foci in the mid-colon, distal colon and rectum following treatment with azoxymethane were also significantly increased by infusion with amidated or glycine-extended gastrin-releasing peptide. We conclude that administration of gastrin-releasing peptide to mature rats stimulates proliferation and accelerates carcinogenesis in the colorectal mucosa, and that C-terminal amidation is not essential for either effect. Gastrin-releasing peptides could thus potentially act as promoters of colorectal carcinogenesis.

Topics: Animals; Azoxymethane; Cell Proliferation; Colon; Colonic Neoplasms; Gastrin-Releasing Peptide; Glycine; Male; Mitosis; Precancerous Conditions; Rats; Rats, Sprague-Dawley; Rectum; Species Specificity; Time Factors

Gastrin-releasing peptide (GRP) and its receptor (GRPR) are aberrantly up-regulated in colon cancer. When expressed, they act as morphogens, retaining tumor cells in a better differentiated state and retarding metastasis. To identify targets activated in response to GRPR signaling we studied Caco-2 and HT-29 cells, colon cancer cell lines that expresses GRPR as a function of confluence. Total cell protein was extracted from pre-confluent cells (expressing GRP/GRPR) cultured in serum-free media in the presence or absence of GRPR-specific antagonist; as well as from confluent cells that do not express GRPR. Overall, we identified 5 proteins that are specifically down-regulated after GRP/GRPR expression: Bach2, creatine kinase B, p47, and two that could not be identified; and 6 proteins that are up-regulated: gephyrin, HSP70, HP1, ICAM-1, ACAT, and one that could not be identified. These findings suggest that the mechanism(s) by which GRP/GRPR mediate its morphogenic effects in colon cancer involve the actions of a number of hitherto unappreciated proteins.

Topics: Cell Line, Tumor; Colonic Neoplasms; Culture Media, Serum-Free; Electrophoresis, Gel, Two-Dimensional; Gastrin-Releasing Peptide; Humans; Protein Binding; Proteome; Receptors, Bombesin; Signal Transduction; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Gastrin-releasing peptide (GRP) is typically viewed as a growth factor in cancer. However, we have suggested that in colon cancer, GRP acts primarily as a morphogen when it and its receptor (GRP-R) are aberrantly upregulated. As such, GRP/GRP-R act(s) primarily to modulate processes contributing to the assumption or maintenance of tumor differentiation. One of the most important such processes is the ability of tumor cells to achieve directed motility in the context of tissue remodeling. Yet the cellular conditions affecting GRP/GRP-R expression, and the biochemical pathways involved in mediating its morphogenic properties, remain to be established. To study this, we evaluated the human colon cancer cell lines Caco-2 and HT-29 cells. We found that confluent cells do not express GRP/GRP-R. In contrast, disaggreation and plating at subconfluent densities results in rapid GRP/GRP-R upregulation followed by their progressive decrease as confluence is achieved. GRP/GRP-R coexpression correlated with that of focal adhesion kinase (FAK) phosphorylation of Tyr(397), Tyr(407), Tyr(861), and Tyr(925) but not Tyr(576) or Tyr(577). To more specifically evaluate the kinetics of GRP/GRP-R upregulation, we wounded confluent cell monolayers. At t = 0 h GRP/GRP-R were not expressed, yet cells immediately began migrating into the gap created by the wound. GRP/GRP-R were first detected at approximately 2 h, and maximal levels were observed at approximately 6 h postwounding. The GRP-specific antagonist [d-Phe(6)]-labeled bombesin methyl ester had no effect on cell motility before GRP-R expression. In contrast, this agent increasingly attenuated cell motility with increasing GRP-R expression such that from t = 6 h onward no further cell migration into the gap was observed. Overall, these findings indicate the existence of GRP-independent and -dependent phases of tumor cell remodeling with the latter mediating colon cancer cell motility during remodeling via FAK.

Topics: Caco-2 Cells; Cell Movement; Colonic Neoplasms; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Focal Adhesions; Gastrin-Releasing Peptide; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Metastasis; Phosphorylation; Protein-Tyrosine Kinases; Receptors, Bombesin; Tumor Cells, Cultured; Up-Regulation

To establish whether gastrin releasing peptide (GRP) and the GRP receptor (GRPR) are expressed together in gastrointestinal carcinoid tumours.. Twenty six carcinoid tumours from the stomach, small intestine, appendix, and colorectum were investigated by immunohistochemistry for GRP and GRPR.. GRP was detected in nine of 19 tumours and GRPR in 22 of 26. Coexpression of both the ligand and receptor was seen in six of 19 cases. GRPR but not GRP was more strongly expressed in appendix and colonic tumours.. GRP and GRPR are produced by a large number of gastrointestinal carcinoid tumours. An autocrine/paracrine pathway may exist for GRP stimulated cell proliferation in some of these neoplasms, analogous to that seen in small cell anaplastic carcinoma of the lung.

Topics: Appendiceal Neoplasms; Carcinoid Tumor; Colonic Neoplasms; Gastrin-Releasing Peptide; Gastrointestinal Neoplasms; Humans; Neoplasm Proteins; Receptors, Bombesin; Stomach Neoplasms

Gastrin-releasing peptide (GRP) and its receptor (GRP-R) are not normally expressed by epithelial cells lining the colon but are aberrantly expressed in cancer, where they act as morphogens and regulate tumor cell differentiation. Studies of colon cancer formation in mice genetically incapable of synthesizing GRP-R suggested that this receptor's morphogenic properties were mediated via focal adhesion kinase (FAK). We therefore set out to determine the presence of both total and phosphorylated forms of FAK in human colon cancer specimens as a function of tumor cell differentiation and GRP/GRP-R co-expression. Ten colon cancers containing 25 regions of distinct differentiation were randomly selected from our GI Cancer Tumor Bank. All specimens were immunohistochemically probed using antibodies recognizing GRP, GRP-R, total FAK, and FAK specifically phosphorylated at tyrosine (Y) 397, 407, 576, 577, 861, and 925. Antibody-specific chromogen was determined by quantitative immunohistochemistry (IHC) for each region of defined differentiation. Here we confirm that GRP/GRP-R co-expression is a function of differentiation, with highest levels observed in well-differentiated tumor cells. We also show that the amount of total FAK and of FAK phosphorylated at Y397 and Y407 tightly correlates with differentiation and with the amount of GRP/GRP-R co-expression. These findings are consistent with GRP/GRP-R acting as a morphogen by activating FAK, and suggest that this occurs via phosphorylation of this enzyme at two specific tyrosine residues.

Topics: Algorithms; Cell Differentiation; Colonic Neoplasms; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Focal Adhesions; Gastrin-Releasing Peptide; Humans; Immunohistochemistry; Phosphorylation; Protein-Tyrosine Kinases; Receptors, Bombesin; Tyrosine

Little is known about the factors involved in regulating the appearance, or differentiation, of solid tumors including those arising from the colon. We herein demonstrate that the mitogen gastrin-releasing peptide (GRP) is a morphogen, critically important in regulating the differentiation of murine colon cancer. Although epithelial cells lining the mouse colon do not normally express GRP and its receptor (GRP-R), both are aberrantly expressed by all better differentiated cancers in wild-type C57BL/6J mice treated with the carcinogen azoxymethane. Whereas small tumors in both wild-type and GRP-R-deficient (i.e., GRP-R-/-) mice are histologically similar, larger tumors become better differentiated in the former but degenerate into more poorly differentiated mucinous adenocarcinomas in the latter. This alteration in phenotype is attributable to GRP increasing focal adhesion kinase expression in GRP-R-expressing tumors. Consistent with GRP acting as a mitogen, GRP/GRP-R coexpressing tumors in wild-type animals also contain more proliferating cells than those occurring in GRP-R-/- mice. Yet tumors are similarly sized in animals of either genotype receiving azoxymethane for identical times, a finding attributable to the significantly higher number of apoptotic cells detected in GRP/GRP-R coexpressing cancers. Thus, these findings indicate that although GRP is a mitogen, aberrant expression does not result in increased tumor growth. Rather, the mitogenic properties of GRP are subordinate to it acting as a morphogen, where it and its receptor are critically involved in regulating colon cancer histological progression by promoting a well-differentiated phenotype.

Topics: Adenocarcinoma; Animals; Apoptosis; Azoxymethane; Carcinogens; Cell Differentiation; Cell Division; Colonic Neoplasms; Epithelial Cells; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Gastrin-Releasing Peptide; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitogens; Protein-Tyrosine Kinases; Proto-Oncogene Proteins pp60(c-src); Receptors, Bombesin

Gastrin-releasing peptide (GRP) is a mitogen and morphogen important in the development of human colon cancers. Although epithelial cells lining the colon do not normally express GRP or its receptor (GRP-R), most human tumors express GRP-R mRNA. Yet functional protein has only been detected in 24 to 40% of colon cancers. To elucidate the reason for the difference between the expression of GRP/GRP-R mRNA and protein, we studied nine human colon cancer cell lines. Quantitative polymerase chain reaction revealed that all colon cancer cell lines expressed similar amounts of mRNA for both GRP as well as GRP-R. Yet binding studies using (125)I-Tyr(4)-bombesin detected functional receptors on only five of the nine cell lines studied. Conformational fragment-length polymorphism analysis indicated that although mRNA for the ligand GRP was never mutated, mRNA for the GRP-R was always mutated. Sequencing revealed that the message for GRP-R contained between two and seven separate mutations at the nucleotide level. This resulted in 14 separate coding mutations, 2 of which were observed in more than one cell line. Each mutation was individually recreated by site-directed mutagenesis and studied in transiently transfected Chinese hamster ovary-K1 cells. Alteration of Pro(145) into a tyrosine, of Val(317) into a glutamic acid, and insertion of a 32-nucleotide segment resulting in a frameshift distal to Asp(137) all resulted in GRP receptors incapable of binding ligand. Thus, these data indicate that human colon cancers commonly express GRP and GRP-R mRNA but that receptor mutations account for the failure of functional protein to be generated.

Topics: Amino Acid Sequence; Animals; Binding Sites; Caco-2 Cells; CHO Cells; Colonic Neoplasms; Cricetinae; Gastrin-Releasing Peptide; Humans; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Conformation; Receptors, Bombesin; RNA, Messenger; Transfection; Tumor Cells, Cultured

Epithelial cells lining the adult human colon do not normally express gastrin-releasing peptide (GRP) or its receptor (GRPR). In contrast, approximately one-third of human colon cancers and cancer cell lines have been shown to express GRP-binding sites. Because GRPR activation causes the proliferation of many cancer cell lines, GRP has been presumed to act as a clinically significant growth factor. Yet GRP has not been shown to be expressed by colon cancers in humans nor has the effect of GRP and/or GRPR coexpression on tumor behavior been investigated. We therefore determined GRP and GRPR expression by immunohistochemistry in 50 randomly selected colon cancers resected between 1980 and 1997, all 37 associated lymph node and liver metastases, and 20 polyps. Tumor sections studied were those that contained the margin and adjacent nonmalignant epithelium. Overall, 84% of cancers aberrantly expressed GRP or GRPR, with 62% expressing both ligand and receptor, whereas expression was not observed in adjacent normal epithelium. Consistent with the previously established mitogenic capabilities of GRP, tissues coexpressing GRP and GRPR were more likely to express proliferating cell nuclear antigen than tissues not expressing both ligand and receptor. Yet GRP/GRPR coexpression was seen with equal frequency in stage A as in stage D cancers and was only detected in 1 in 37 metastases. Furthermore, Kaplan-Meier analysis did not reveal any difference in patient survival between those whose tumors did or did not express GRP/GRPR. In contrast, GRP/GRPR coexpression was found in all well-differentiated tumor regions, whereas poorly differentiated tissues never coexpressed GRP/GRPR. Overall, these data indicate that, although GRP is a mitogen, it is not a clinically significant growth factor in human colon cancers. Rather, the strong association of GRP/GRPR coexpression with tumor differentiation raises the possibility that these proteins primarily act in vivo as morphogens.

Topics: Adult; Aged; Aged, 80 and over; Colonic Neoplasms; Female; Gastrin-Releasing Peptide; Humans; Immunohistochemistry; Ligands; Male; Middle Aged; Mitogens; Neoplasm Staging; Receptors, Bombesin; Survival Analysis

The neuropeptide bombesin stimulates tumour cell proliferation in vitro. Through pharmacological testing, 20-40% of human colorectal tumours have been shown to be equipped with bombesin/gastrin releasing peptide receptor (GRP-R). The aim of the present study was to test whether GRP-R expression is correlated with tumour characteristics and usual prognostic factors in colorectal adenocarcinomas. A sensitive reverse transcription (RT)-competitive polymerase chain reaction (PCR) method was validated by studying GRP-R mRNA in separated layers of normal colonic wall, and GRP-R mRNA levels (in parallel with binding studies) in colon cancer cell lines LoVo and Caco-2. GRP-R mRNA levels were then determined in 29 surgical tumour specimens and the results compared with tumour histology and, using histochemistry, with the accumulation of p53 protein and a Ki-67 cell proliferation index. The mRNA was not detected in normal colonic epithelium, whereas a distinct signal was observed after amplification in 27/29 (93%) tumour specimens. Estimates of mRNA levels in the 27 positive tumours ranged from 52 to 8000 amol/0.25 microgram total RNA, and were significantly higher in poorly/moderately differentiated tumours (P < 0.05) and in tumours with lymphatic vessel invasion (P < 0.01). There was no relationship with p53 accumulation or to the proliferation index. Our results show that GRP-R mRNA can be detected in most colorectal tumour specimens, and suggest a link between high mRNA levels and both tumour dedifferentiation and lymph vessel invasion, but not proliferation.

Topics: Aged; Cell Transformation, Neoplastic; Colonic Neoplasms; Female; Gastrin-Releasing Peptide; Humans; Immunohistochemistry; Lymphatic Diseases; Neoplasm Invasiveness; Neoplasm Proteins; RNA, Messenger

Nude mice bearing xenografts of HT-29 human colon cancer cell line were treated for 4 weeks with somatostatin analog (RC-160), bombesin/gastrin releasing peptide (GRP) antagonists (RC-3095 and RC-3440). In three separate experiments somatostatin analog RC-160 (50 micrograms/day) released from microgranules significantly reduced tumor growth. Bombesin/GRP antagonists, RC-3095 and RC-3440 injected subcutaneously (s.c.) twice daily at a dose of 10 micrograms had the greatest and consistently significant inhibitory effect on tumor growth. RC-3095 given once daily s.c. at a dose of 20 micrograms was less effective. RC-3095 also inhibited metastatic tumor growth after intrasplenic injection of HT-29 cells in nude mice. Specific binding sites of somatostatin, bombesin and epidermal growth factor (EGF) were detected on intact HT-29 cells or on the membranes from HT-29 tumor xenografts. The inhibitory effects of bombesin antagonists on tumor growth were consistently linked with a significant down-regulation of EGF receptors. Bombesin/GRP antagonists and somatostatin analogs could be considered for the development of new hormonal therapies for colon cancer.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Bombesin; Colonic Neoplasms; Drug Screening Assays, Antitumor; ErbB Receptors; Gastrin-Releasing Peptide; Gastrins; Growth Hormone; Humans; Insulin-Like Growth Factor I; Liver Neoplasms; Male; Mice; Mice, Nude; Neoplasm Transplantation; Peptide Fragments; Peptides; Radioligand Assay; Somatostatin

In the present study, we characterized specific binding of bombesin (BBS)/gastrin-releasing peptide (GRP) to mouse colon cancer (MC-26) cells. MC-26 cells were inoculated into male BALB/c mice subdermally, and tumors were harvested from mice 21-28 days postinoculation. Tumor membranes were analyzed for binding to GRP-related peptides, using either 125I-GRP or 125I-tyrosine4-BBS. Under optimal binding assay conditions, BBS displaced specific binding of both 125I-GRP and 125I-tyrosine4-BBS in a dose-dependent manner, and a curvilinear displacement resulted. Specific binding data, analyzed by either a Scatchard or a Lineweaver-Burk plot, demonstrated presence of 2 classes of specific binding sites, arbitrarily named type I and type II sites. Type I sites had a high binding affinity [Kd 0.45 +/- 0.05 nM (SE)] and a relatively low capacity (226 +/- 27 fmol/mg membrane protein), whereas type II sites had a 10-20-fold lower binding affinity and approximately 6-7-fold higher capacity. BBS/GRP binding sites were specific for GRP-related peptides and demonstrated no significant binding affinity for all other unrelated peptides tested. Relative binding affinity of GRP analogues was in the order of GRP (14-27) greater than neuromedin C greater than or equal to BBS greater than or equal to GRP (1-27) greater than neuromedin B (for the later, P greater than 0.05 versus other peptides). Two BBS receptor antagonists, [D-Arg1,D-trp7,9,Leu11]-substance P (spantide) and [Leu13-psi-(CH2NH)Leu14]BBS also inhibited specific binding of 125I-GRP in a dose-dependent manner. Molecular weight of GRP/BBS binding proteins on tumor membranes was determined by cross-linking methods. A major molecular form (greater than 80-90%) (Mr approximately 75,000) and a minor Mr approximately 180,000 band were evident, both under reducing and nonreducing conditions. BBS (0.5-50 nM) demonstrated a significant dose-dependent growth effect on MC-26 cells in vitro, in terms of [3H]thymidine and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide uptake; these studies indicate that the BBS/GRP binding sites on MC-26 cells may serve as functional receptors and mediate the growth effects of BBS on MC-26 cells.

Topics: Animals; Bombesin; Cell Division; Colonic Neoplasms; Gastrin-Releasing Peptide; Iodine Radioisotopes; Male; Membranes; Mice; Mice, Inbred BALB C; Molecular Weight; Peptides; Tumor Cells, Cultured