Gastrin-releasing Peptide Receptor mRNA Research Articles

A comparative PET imaging study of 44gSc- and 68Ga-labeled bombesin antagonist BBN2 derivatives in breast and prostate cancer models

IntroductionRadiolabeled peptides play a central role in nuclear medicine as radiotheranostics for targeted imaging and therapy of cancer. We have recently proposed the use of metabolically stabilized GRPR antagonist BBN2 for radiolabeling with 18F and 68Ga and subsequent PET imaging of GRPRs in prostate cancer. The present work studied the impact of 44gSc- and 68Ga-labeled DOTA complexes attached to GRPR antagonist BBN2 on the in vitro GRPR binding affinity, and their biodistribution and tumor uptake profiles in MCF7 breast and PC3 prostate cancer models. MethodsDOTA-Ava-BBN2 was radiolabeled with radiometals 68Ga and 44gSc. Gastrin-releasing peptide receptor (GRPR) affinities of peptides were assessed in PC3 prostate cancer cells. GRPR expression profiles were studied in human breast cancer tissue samples and MCF7 breast cancer cells. PET imaging of 68Ga- and 44gSc-labeled peptides was performed in MCF7 and PC3 xenografts as breast and prostate cancer models. ResultsRadiopeptides [68Ga]Ga-DOTA-Ava-BBN2 and [44gSc]Sc-DOTA-Ava BBN2 were prepared in radiochemical yields of 70–80% (decay-corrected), respectively. High binding affinities were found for both peptides (IC50 = 15 nM (natGa) and 5 nM (natSc)). Gene expression microarray analysis revealed high GRPR mRNA expression levels in estrogen receptor (ER)-positive breast cancer, which was further confirmed with Western blot and immunohistochemistry. However, PET imaging showed only low tumor uptake of both radiotracers in MCF7 xenografts ([68Ga]Ga-DOTA-BBN2 (SUV60min 0.27 ± 0.06); [44gSc]Sc-DOTA-BBN2 (SUV60min 0.20 ± 0.03)). In contrast, high tumor uptake and retention were found for both radiopeptides in PC3 tumors ([68Ga]Ga-DOTA-BBN2 (SUV60min 0.46 ± 0.07); [44gSc]Sc-DOTA-BBN2 (SUV60min 0.51 ± 0.11)). ConclusionsComparison of 68Ga- and 44gSc-labeled DOTA-Ava-BBN2 peptides revealed slight but noticeable differences of the radiometal with an impact on the in vitro GRPR receptor binding properties in PC3 cells. No differences were found in their in vivo biodistribution profiles in MCF7 and PC3 xenografts. Radiopeptides [68Ga]Ga-DOTA-Ava-BBN2 and [44gSc]Sc-DOTA-Ava-BBN2 displayed comparable tumor uptake and retention profiles with rapid blood and renal clearance profiles in both tumor models. Advances in knowledge and implications for patient careThe favorable PET imaging performance of [44gSc]Sc-DOTA-Ava-BBN2 in prostate cancer should warrant the development of an [43Sc]Sc-DOTA-Ava-BBN2 analog for clinical translation which comes with a main γ-line of much lower energy and intensity compared to 44gSc.

NFAT5 is involved in GRP‐enhanced secretion of GLP‐1 by high sodium

Organ to organ communication is important in the maintenance of normal fluid and electrolyte balance and blood pressure (BP). Neural mechanisms and gastrointestinal (GIT) hormones mediate the natriuresis of an oral sodium load. The GIT is the first organ exposed to ingested nutrients and most likely to react initially to a sodium load. Sensing the amount of ingested sodium by the GIT may be an important mechanism by which sodium balance is regulated. We have reported that gastrin secreted by G‐cells in the stomach and duodenum is involved in the regulation of sodium balance and BP. Besides gastrin, glucagon‐like peptide‐1 (GLP‐1) is another gut hormone, which may also participate in regulation of sodium balance and BP. It is well known that glucose‐induced GLP‐1 secretion is sodium‐dependent. The increase in intracellular calcium needed for GLP‐1 secretion is also due to sodium‐dependent cell excitability. However, the specific effect of varying sodium concentration on GLP‐1 secretion and its potential mechanism remain to be elucidated. This study aimed to determine the effect of varying sodium concentrations on GLP‐1 secretion and its potential mechanism. Here, we show that sodium can stimulate GLP‐1 secretion and that gastrin‐releasing peptide (GRP) enhances the high sodium‐induced increase in GLP‐1 secretion in a concentration and time‐dependent manner in L‐cells. The exposure of L‐cells to high (170 mM) Na+ increases GLP‐1 protein (produced by degradation of glucagon) or proglucagon mRNA expression, while Low (90 mM) and high (170 mM) Na+ increase NFAT5 and GRP receptor (GRPR) mRNA and protein expression. Silencing NFAT5 abrogates the GRP‐mediated enhancement of high sodium‐induced increase in GLP‐1 secretion and blocks the sodium‐induced increase in GRPR mRNA and protein expressions in L‐cells. Furthermore, NFAT5 silencing reduces the GLP‐1 protein or proglucagon expression in L‐cells exposed to high sodium concentration. NFAT5 silencing also reduces GRPR expression, using the promoter reporter assay; n=3/group for all studies. These results suggest that the high sodium‐induced NFAT5 expression increases expression of GRPR; GRP acting on the increased GRPR expression increases GLP‐1 secretion from L‐cells. These data give a new perspective on the mechanisms of GLP‐1 secretion, and the direct relationship between sodium and GLP‐1. These may contribute to better utilization of existing GLP‐1 based drugs and further GLP‐1‐based drug development.Effect of NaCl or GRP on GLP‐1 secretion in CCL‐251 L cells (human colorectal adenocarcinoma cells=GLP‐1 secreting cells).The same amount of cells were exposed to 90 mM, 145 mM, or 170 mM NaCl medium, and A, treated with varying concentrations of GRP (10, 100, 500, or 1000 nmol) for 4 h, or B, treated with 1000 nmol GRP at different times (1 h, 2 h, 3 h, or 4 h). GLP‐1 in the cell culture supernatant was measured by ELISA. The results are expressed as ng/ml. *P<0.05 vs. 90 mM with or without GRP. #P<0.05 vs. others, one‐way ANOVA, Holm‐Sidak test, n=3/treatment.Figure 1

Prospects of Targeting the Gastrin Releasing Peptide Receptor and Somatostatin Receptor 2 for Nuclear Imaging and Therapy in Metastatic Breast Cancer.

BackgroundThe gastrin releasing peptide receptor (GRPR) and the somatostatin receptor 2 (SSTR2) are overexpressed on primary breast cancer (BC), making them ideal candidates for receptor-mediated nuclear imaging and therapy. The aim of this study was to determine whether these receptors are also suitable targets for metastatic BC.MethodsmRNA expression of human BC samples were studied by in vitro autoradiography and associated with radioligand binding. Next, GRPR and SSTR2 mRNA levels of 60 paired primary BCs and metastases from different sites were measured by quantitative reverse transcriptase polymerase chain reaction. Receptor mRNA expression levels were associated with clinico-pathological factors and expression levels of primary tumors and corresponding metastases were compared.ResultsBinding of GRPR and SSTR radioligands to tumor tissue correlated significantly with receptor mRNA expression. High GRPR and SSTR2 mRNA levels were associated with estrogen receptor (ESR1)-positive tumors (p<0.001 for both receptors). There was no significant difference in GRPR mRNA expression of primary tumors versus paired metastases. Regarding SSTR2 mRNA expression, there was also no significant difference in the majority of cases, apart from liver and ovarian metastases which showed a significantly lower expression compared to the corresponding primary tumors (p = 0.02 and p = 0.03, respectively).ConclusionTargeting the GRPR and SSTR2 for nuclear imaging and/or treatment has the potential to improve BC care in primary as well as metastatic disease.

Gastrin-releasing peptide receptor (GRPr) promotes EMT, growth, and invasion in canine prostate cancer.

The gastrin-releasing peptide receptor (GRPr) is upregulated in early and late-stage human prostate cancer (PCa) and other solid tumors of the mammary gland, lung, head and neck, colon, uterus, ovary, and kidney. However, little is known about its role in prostate cancer. This study examined the effects of a heterologous GRPr agonist, bombesin (BBN), on growth, motility, morphology, gene expression, and tumor phenotype of an osteoblastic canine prostate cancer cell line (Ace-1) in vitro and in vivo. The Ace-1 cells were stably transfected with the human GRPr and tumor cells were grown in vitro and as subcutaneous and intratibial tumors in nude mice. The effect of BBN was measured on cell proliferation, cell migration, tumor growth (using bioluminescence), tumor cell morphology, bone tumor phenotype, and epithelial-mesenchymal transition (EMT) and metastasis gene expression (quantitative RT-PCR). GRPr mRNA expression was measured in primary canine prostate cancers and normal prostate glands. Bombesin (BBN) increased tumor cell proliferation and migration in vitro and tumor growth and invasion in vivo. BBN upregulated epithelial-to-mesenchymal transition (EMT) markers (TWIST, SNAIL, and SLUG mRNA) and downregulated epithelial markers (E-cadherin and β-catenin mRNA), and modified tumor cell morphology to a spindle cell phenotype. Blockade of GRPr upregulated E-cadherin and downregulated VIMENTIN and SNAIL mRNA. BBN altered the in vivo tumor phenotype in bone from an osteoblastic to osteolytic phenotype. Primary canine prostate cancers had increased GRPr mRNA expression compared to normal prostates. These data demonstrated that the GRPr is important in prostate cancer growth and progression and targeting GRPr may be a promising strategy for treatment of prostate cancer. Prostate 76:796-809, 2016. © 2016 Wiley Periodicals, Inc.

Comparative Anatomy of Gastrin-releasing Peptide Pathways in the Trigeminal Sensory System of Mouse and the Asian House Musk Shrew Suncus murinus.

Gastrin-releasing peptide (GRP) has recently been identified as an itch-signaling molecule in the primary afferents and spinal cord of rodents. However, little information exists on the expression and localization of GRP in the trigeminal somatosensory system other than in rats. We examined the generality of the trigeminal GRP system in mammals using two distinct species, suncus as a model of specialized placental mammals known to have a well-developed trigeminal sensory system and mice as a representative small laboratory animal. We first analyzed the gross morphology of the trigeminal somatosensory system in suncus to provide a brainstem atlas on which to map GRP distribution. Immunohistochemical analyses showed that 8% of trigeminal ganglion neurons in suncus and 6% in mice expressed GRP. Expression was restricted to cells with smaller somata. The GRP-containing fibers were densely distributed in the superficial layers of the caudal part of the trigeminal spinal nucleus (Vc) but rare in the rostral parts, both in suncus and mice. Expression of GRP receptor mRNA and protein was also detected in the Vc of suncus. Taken together, these results suggest that the trigeminal GRP system mediating itch sensation is conserved in mammals.

Self administration of oxycodone by adolescent and adult mice affects striatal neurotransmitter receptor gene expression

Illicit use of prescription opioid analgesics (e.g., oxycodone) in adolescence is a pressing public health issue. Our goal was to determine whether oxycodone self administration differentially affects striatal neurotransmitter receptor gene expression in the dorsal striatum of adolescent compared to adult C57BL/6J mice. Groups of adolescent mice (4weeks old, n=12) and of adult mice (11weeks old, n=11) underwent surgery during which a catheter was implanted into their jugular veins. After recovering from surgery, mice self administered oxycodone (0.25mg/kg/infusion) 2h/day for 14 consecutive days or served as yoked saline controls. Mice were sacrificed within 1h after the last self-administration session and the dorsal striatum was isolated for mRNA analysis. Gene expression was analyzed with real time PCR using a commercially available neurotransmitter receptor PCR array containing 84 genes. We found that adolescent mice self administered less oxycodone than adult mice over the 14days. Monoamine oxidase A (Maoa) and neuropeptide Y receptor 5 mRNA levels were lower in adolescent mice than in adult mice without oxycodone exposure. Oxycodone self administration increased Maoa mRNA levels compared to controls in both age groups. There was a positive correlation of the amount of oxycodone self administered in the last session or across 14 sessions with Maoa mRNA levels. Gastrin-releasing peptide receptor mRNA showed a significant Drug×Age interaction, with point-wise significance. More genes in the dorsal striatum of adolescents (19) changed in response to oxycodone self administration compared to controls than in adult (4) mice. Overall, this study demonstrates that repeated oxycodone self administration alters neurotransmitter receptors gene expression in the dorsal striatum of adolescent and adult mice.

Rhodamine-marked bombesin: a novel means for prostate cancer fluorescence imaging

The gastrin releasing peptide receptor (GRPR) has been found to be strongly expressed in various types of cancers such as prostate and breast carcinomas. The GRPR ligands gastrin releasing peptide and bombesin can play a very significant role in cancer therapy and diagnostics. In this study we synthesized unlabeled bombesin BBN along with two conjugates in which the correct bombesin (BBN-Rhd) and a mutant bombesin (mBBN-Rhd) sequence was coupled to rhodamine, a fluorescent dye. These novel rhodamine fluorescent conjugates were used to study the targeting and uptake of bombesin on a cellular level. Nine different human cell lines including both tumor and healthy cells were examined using flow cytometry and confocal laser scanning microscopy. GRPR mRNA expression analysis was performed and it was found that the receptor is highly expressed in LNCaP and PC3 cells compared to the rest of other cell lines. Competition experiments were performed to verify the receptor dependence of the labeled conjugates using unmarked bombesin. The present study is a first attempt at direct fluorescence imaging of living cells using bombesin and its target, the GRPR. A rhodamine bombesin conjugate can be used as marker to differentiate between healthy cells and malignant cells such as prostate hyperplasia and prostate carcinoma in the early detection of cancer.

Combination of gastrin-releasing peptide antagonist with cytotoxic agents produces synergistic inhibition of growth of human experimental colon cancers

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 G2/M and cells with lower G0/G1 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.

Elevated gastrin‐releasing peptide receptor mRNA expression in buccal mucosa: Association with head and neck squamous cell carcinoma

Expression of gastrin-releasing peptide receptor (GRPR) is elevated in mucosa adjacent to head and neck squamous cell carcinoma (HNSCC) compared with mucosa from cancer-free controls, suggesting elevated GRPR expression may indicate presence of HNSCC. We measured GRPR mRNA levels in histologically normal buccal mucosa from 65 surgical patients with HNSCC and 75 cancer-free control subjects using quantitative polymerase chain reaction (PCR). We tested for association between GRPR expression and HNSCC and evaluated differences in patient progression-free survival (PFS). Buccal GRPR expression was higher in cases but not controls who were active smokers (p = .04). High GRPR expression was associated with HNSCC (odds ratio [OR] = 3.55; 95% confidence interval [CI] = 1.15-10.93), even after adjustment for age, sex, tobacco use, and sample storage time. PFS did not differ between patients with HNSCC with high versus low GRPR expression (p = .22). Elevated buccal GRPR expression was significantly associated with HNSCC independent of known risk factors but was not an indicator of disease prognosis.

Immunohistochemical analysis of gastrin-releasing peptide receptor (GRPR) and possible regulation by estrogen receptor βcx in human prostate carcinoma

Gastrin-releasing peptide (GRP) belongs to the family of bombesin-like peptides. GRP was demonstrated to stimulate the proliferation and invasiveness of androgen-independent prostate carcinoma. GRP mediates its action through the membrane-bound receptor, GRP receptor (GRPR), which is characterized by a high-affinity binding for both GRP and bombesin. In human prostate cancer tissue, GRPR mRNA was reported to be detectable in more than 90% but its immunolocalizaition has not been reported. Therefore, in this study we immunolocalized GRPR in 51 human prostate cancer cases and correlated the findings with several clinicopathological parameters in order to better understand the function and regulation of GRPR in human prostate cancer. GRPR was immnolocalized in carcinoma cells and their values were significantly associated with Gleason score and immunoreactivity of estrogen receptor βcx (ERβcx) that is one of splicing variants of ligand dependent transcription factor, ERβ, and considered to be prognostic factor of prostate cancer patients. The amounts of GRPR and ERβcx mRNA in three prostate cancer cell lines PC-3, DU-145 and LNCaP evaluated by quantitative RT-PCR (qPCR) analysis were also significantly correlated. In addition, we established stable transformants of prostate carcinoma cell line PC-3 introduced with ERβcx, and confirmed that GRPR mRNA was induced in ERβcx over-expressing PC-3 cells by qPCR analysis. These results also suggest that ERβcx contributes to prostate cancer development possibly through mediating GRPR expression in carcinoma cells.

Gastrin-Releasing Peptide Receptors Regulate Proliferation of C6 Glioma Cells through a Phosphatidylinositol 3-Kinase-Dependent Mechanism

Gastrin-releasing peptide (GRP) has been proposed as a major growth factor in brain tumors, and GRP receptor (GRPR) antagonists show antiproliferative effects in experimental gliomas. However, the underlying molecular events downstream of GRPR activation remain poorly understood. In the present study, we examined the role of the GRPR in regulating proliferation of glioma cells in vitro and its possible interaction with the phosphatidylinositol 3-kinase (PI3K) signaling pathway. Expression of GRPR mRNA and protein in C6, U-87MG, and U-373MG glioma cells was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. Proliferation of C6 and U-87MG, but not U-373MG cells was significantly inhibited by the GRPR antagonist RC-3095, whereas the GRPR agonist bombesin (BB) significantly enhanced proliferation of C6 cells. The BB-induced stimulatory effect on cell proliferation was prevented by either RC-3095 or the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. Our results provide the first evidence that the GRPR regulates proliferation of C6 glioma cells and suggest that PI3K is required for GRPR-mediated stimulation of glioma growth.

Gender Differences: Implications for Clinical Trials and Practice

Gender Differences: Implications for Clinical Trials and Practice

Progastrin-releasing peptide and gastrin-releasing peptide receptor mRNA expression in non-tumor tissues of the human gastrointestinal tract

To investigate the expression of gastrin-releasing peptide (GRP) and GRP-receptor mRNA in non-tumor tissues of the human esophagus, gastrointestinal tract, pancreas and gallbladder using molecular biology techniques. Poly A(+) mRNA was isolated from total RNA extracts using an automated nucleic acid extractor and, subsequently, converted into single-stranded cDNA (ss-cDNA). PCR amplifications were carried out using gene-specific GRP and GRP-receptor primers. The specificity of the PCR amplicons was further confirmed by Southern blot analyses using gene-specific GRP and GRP-receptor hybridization probes. Expression of GRP and GRP-receptor mRNA was detected at various levels in nearly all segments of the non-tumor specimens analysed, except the gallbladder. In most of the biopsy specimens, co-expression of both GRP and GRP-receptor mRNA appeared to take place. However, expression of GRP mRNA was more prominent than was GRP-receptor mRNA. GRP and GRP-receptor mRNAs are expressed throughout the gastrointestinal tract and provides information for the future mapping and determination of its physiological importance in normal and tumor cells.

Stimulation of proliferation and migration of a colorectal cancer cell line by amidated and glycine-extended gastrin-releasing peptide via the same receptor

Although amidated forms of gastrin-releasing peptide (GRP) have been identified as autocrine growth factors in small cell lung cancer, their role in the development and progression of colorectal carcinoma is less clear. In addition, the biological activity of non-amidated gastrin-releasing peptide has not been investigated in colorectal carcinoma cells. We therefore investigated the effect of bombesin (a homologue of gastrin-releasing peptide) on proliferation, migration and inositol phosphate production in the human colorectal carcinoma cell line DLD-1, and determined the ability of gastrin-releasing peptide receptor antagonists to inhibit these effects. We also compared the biological activities of amidated and non-amidated GRP in the same assays. Treatment with either bombesin, or amidated or non-amidated GRP resulted in significant increase in proliferation, and in migration in a wound-healing assay. Both the mitogenic and migratory effects of amidated and non-amidated forms were inhibited by the GRP receptor antagonist [ d-Phe 6, Leu-NHet 13, des-Met 14]–bombesin(6–13). The presence of GRP receptor mRNA and GRP binding sites in three colorectal carcinoma cell lines was demonstrated by RT-PCR and by binding of radiolabelled bombesin, respectively. Transfection of DLD-1 cells with a dominant negative phosphatidylinositol 3-kinase did not affect bombesin-stimulated cell proliferation, but inhibited bombesin-stimulated cell migration. Bombesin and GRPgly activated phospholipase C, mitogen-activated protein kinase and focal adhesion kinase. We conclude that both amidated and non-amidated forms of gastrin-releasing peptide accelerate proliferation and migration of DLD-1 human colorectal carcinoma cells via the gastrin-releasing peptide receptor, but that phosphatidylinositol 3-kinase is only involved in the cell migration signalling pathway. Our results suggest a potential role for gastrin-releasing peptide receptor antagonists in the management of colorectal carcinoma.

Dexamethasone regulation of gastrin-releasing peptide receptor in human lung cells

We investigated the effects of the glucocorticoid, dexamethasone (Dex), on expression of the gastrin-releasing peptide (GRP) receptor by human small cell lung carcinoma (SCLC) SHP77 cells. After 12 h of 10 nM Dex exposure, a six-fold increase in the peak of GRP receptor mRNA compared with untreated controls (10.5±4 versus 1.65±0.15 attomols/μg total RNA, respectively, P<0.05) occurred. GRP receptor mRNA levels fell to less than 0.5 attomols/μg total RNA after 24 h; in Dex-treated cells, these levels rose to 1.2 compared with 0.12 attomols/μg total RNA in the absence of Dex after 7 days. A significant increase ( P<0.05) in the GRP receptor-specific binding was also found. Stimulation of SHP77 cell proliferation (25–35% in the presence of 10–100 nM Dex; P<0.0001) was observed after 4–8 days of exposure; this stimulation was inhibited by GRP receptor antagonists. SHP77 cell content and concentration of bombesin-like peptides (BLP) in conditioned medium (approximately 4 nM) was unchanged by Dex. Stimulation of human SCLC SHP77 cell proliferation by Dex may, in part, occur via effects on the GRP autocrine system in these cells.

Expression of progastrin-releasing peptide and gastrin-releasing peptide receptor mRNA transcripts in tumor cells of patients with small cell lung cancer.

Small cell lung cancer (SCLC) is a rapidly growing neoplasm accounting for approximately 20% of patients with lung cancer. Progastrin-releasing peptide (proGRP) is produced in about two-thirds of SCLC tumors and is used as a specific marker for SCLC. Although GRP is known to have a variety of biological functions, only limited information is available concerning expression of proGRP mRNA and protein, and that of the receptor for GRP (GRPR) in SCLC tumors. In individuals with SCLC, the levels of serum proGRP(31-98) were measured by enzyme-linked immunosorbent assay. Expression of proGRP as well as GRPR mRNA in SCLC tumor tissues was investigated by reverse transcription-nested polymerase chain reaction (PCR) amplification. The proportions of alternatively spliced proGRP mRNA transcripts were analyzed in proGRP-producing tumors by nested and competitive PCR amplification. Finally, production of proGRP protein in SCLC tumor was evaluated by using immunohistochemical staining with a polyclonal human anti-proGRP antibody. ProGRP mRNA transcripts could be detected only in tumor tissues recovered from individuals with high serum proGRP levels. The proportions of mRNA subtypes in each case were nearly the same, revealing type I of 55.4+/-7.6%, type II with 21-b deletion of 1.8+/-3.6%, and type III with 19-b deletion of 42.8+/-4.3%, respectively. ProGRP protein production was demonstrated in tumor tissues exclusively from individuals exhibiting high serum proGRP levels. In contrast, GRPR mRNA transcripts were detectable in cancer cells from two of five proGRP-expressing tumor tissues. ProGRP mRNA expression is closely related with the synthesis of proGRP protein which is eventually released into the blood. It is suggested GRP may function as an autocrine growth factor for cancer cells in a subgroup of SCLC patients through, at least in part, upregulation of GRPR expression.

Islet function phenotype in gastrin-releasing peptide receptor gene-deficient mice.

Gastrin-releasing peptide (GRP) is an islet neuropeptide that stimulates insulin secretion. To explore whether islet GRP contributes to neurally mediated insulin secretion, we studied GRP receptor (GRPR)-deleted mice. By using RT-PCR we showed that GRPR mRNA is expressed in islets of wild-type mice, but is lost in GRPR-deleted mice. Functional studies revealed that GRP potentiates glucose-stimulated insulin secretion in wild-type animals, but not in GRPR-deleted mice. This shows that GRPR is the receptor subtype mediating GRP-induced insulin secretion and that GRPR-deleted mice are tools for studying the physiological role of islet GRP. We found that GRPR-deleted mice display 1) augmentation of the insulin response to glucose by a mechanism inhibited by ganglionic blockade; 2) increased insulin responsiveness also to the cholinergic agonist carbachol, but not to arginine; 3) impaired insulin and glucagon responses to autonomic nerve activation by 2-deoxyglucose; 4) normal islet adaptation to high fat-induced insulin resistance and fasting; and 5) normal islet cytoarchitecture, as revealed by immunocytochemistry of insulin and glucagon. In conclusion, 1) GRPR is the receptor subtype mediating the islet effects of GRP; 2) GRP contributes to insulin secretion induced by activation of the autonomic nerves; and 3) deletion of GRPR is compensated by increased cholinergic sensitivity.

Gastrin-releasing peptide is a growth factor for human neuroblastomas.

To evaluate whether gastrin-releasing peptide (GRP) and GRP receptor (GRP-R) expression correlate with tumor behavior and to examine the mitogenic actions of GRP on neuroblastomas. Neuroblastoma is the most common solid tumor of infants and children. Despite recent advances in multimodality treatment regimens, the survival for advanced-stage tumors remains dismal. Neuroblastomas are known to produce GRP; however, the proliferative effects of GRP on neuroblastomas have not been elucidated. Sections of paraffin-embedded neuroblastomas from 33 patients were analyzed for GRP and GRP-R protein expression by immunohistochemistry. Functional binding of GRP-R to the Ca2+ signaling pathway was examined. In addition, the proliferative effect of GRP on neuroblastoma cells (SK-N-SH, IMR-32, SH-SY5Y, LAN-1) was determined. Immunohistochemical analysis showed GRP and GRP-R protein expression in neuroblastomas; an increased expression of GRP-R was noted in a higher percentage of undifferentiated tumors compared with tumors that were benign. GRP-R mRNA was confirmed in neuroblastoma cell lines. GRP treatment resulted in intracellular calcium [Ca2+]i mobilization in two cell lines (SK-N-SH, LAN-1). GRP treatment stimulated growth of all four neuroblastoma cell lines; this effect was inhibited in SK-N-SH cells by pretreatment with GRP antibody. These findings show increased GRP-R expression in the more aggressive and undifferentiated neuroblastomas. The synchronous expression of GRP and its receptor, GRP-R, suggests a role for these proteins in tumor growth. Moreover, these findings show enhanced proliferation of neuroblastoma cells in vitro after GRP treatment, suggesting that GRP may act as an autocrine and/or paracrine growth factor for neuroblastomas. Treatment with specific GRP-R antagonists may provide novel adjuvant therapy for neuroblastomas in children.

Gastrin-releasing peptide receptor-mediated autocrine growth in squamous cell carcinoma of the head and neck.

Gastrin-releasing peptide receptor (GRPR)-mediated autocrine growth appears to be an early marker of susceptibility to tobacco-related lung cancers. Because expression of GRPR, however, has not been reported in squamous cell carcinoma of the head and neck (SCCHN), we investigated its expression and that of its ligand GRP in normal mucosa and SCCHN tissues and the involvement of these proteins in the proliferation of SCCHN cells. We assessed GRPR messenger RNA (mRNA) expression in specimens from 25 patients with SCCHN, six control noncancer patients, and 14 SCCHN cell lines by use of quantitative reverse transcriptase-polymerase chain reaction. We used neutralizing GRP monoclonal antibody 2A11 to block the GRP-GRPR interaction in SCCHN cell lines and xenografts and assessed the antibody's effect on proliferation by counting cultured cells or measuring xenograft tumor volume in vivo. All statistical tests were two-sided. Tumor and mucosa tissues, respectively, from SCCHN patients expressed sixfold and fourfold higher levels of GRPR mRNA than normal mucosa tissue from noncancer patients (P<.001). The levels of GRPR expression in the tumor and adjacent normal epithelium of individual patients with SCCHN were correlated (r =.652; P =.001), suggesting that increased GRPR expression is an early event in SCCHN formation. SCCHN cells expressed fivefold higher levels of GRPR mRNA than did cultured normal mucosal epithelial cells (P =.005). GRP stimulated proliferation of SCCHN cells in a dose-dependent fashion (P =.006). Neutralizing GRP monoclonal antibody 2A11 inhibited SCCHN cell proliferation in vitro and in vivo. Median survival was 54 months in patients with higher levels of GRPR mRNA and was not reached in those with lower levels. GRP and GRPR appear to participate in an autocrine regulatory pathway in SCCHN. Thus, strategies that specifically target GRP and/or GRPR may be effective therapeutic approaches for this disease.

GRP-receptor-mediated signal transduction, gene expression and DNA synthesis in the human pancreatic adenocarcinoma cell line HPAF

Bombesin-like peptides have been implicated as growth factors in various human cancers. Human adenocarcinoma cell lines (Capan-1, Capan-2, MiaPaCa-2 and HPAF) were tested to determine whether they express the gastrin-releasing peptide-preferring bombesin receptor (GRPR) and neuromedin B-preferring bombesin receptor (NMBR). Using RT-PCR the highest level of GRP receptor mRNA was found in HPAF cells. NMB receptor mRNA expression moderate in all cell lines investigated. We therefore selected the HPAF cell line to investigate whether bombesin treatment affects intracellular Ca 2+ ([Ca 2+] i), cAMP level, DNA synthesis as a measure of cell proliferation, and expression of three transcription factors: c-fos, c-myc and high mobility group protein IY (HMG-I(Y)). Bombesin administration led to an immediate increase in free intracellular Ca 2+ concentration ([Ca 2+] i) but did not change cAMP levels. The peptide also enhanced [ 3H]thymidine incorporation in HPAF cells (but not in the other cell lines), an effect that was concentration dependent, reaching 36 ± 5% stimulation over control values at 24 h with an EC 50 of 2.27 × 10 −12 M. Furthermore, bombesin stimulated c-fos, c-myc and HMG-I(Y) expression in a time-dependent manner: the c-fos mRNA level increased dramatically in the first 30 min of exposure, then returned to basal level within 2 h, while the c-myc and HMG-I(Y) mRNA levels peaked at 2 h and 4h, respectively. All actions of bombesin were blocked by BME (D-Phe 6-bombesin-(6–13)-methylester), a selective GRP receptor antagonist, but not by the NMB receptor antagonist BIM-23127 (D-Nal-cyclo[Cys-Tyr-D-Trp-Orn-Val-Cys]-Nal-NH 2). We conclude that HPAF cells express mRNA for GRP receptors and that functional receptors are present in the cell membrane. The occupation of these receptors leads to a sequence of intracellular events involving rapid mobilization of intracellular Ca 2+, expression of c-fos, c-myc and HMG-I(Y) mRNA, and stimulation of cell proliferation. Conversely, although NMB receptor mRNA can be detected, its actual translation to functional receptors does not reach a detectable level.