Bombesin-like peptide recruits disinhibitory cortical circuits and enhances fear memories.

Disinhibitory neurons throughout the mammalian cortex are powerful enhancers of circuit excitability and plasticity. The differential expression of neuropeptide receptors in disinhibitory, inhibitory, and excitatory neurons suggests that each circuit motif may be controlled by distinct neuropeptidergic systems. Here, we reveal that a bombesin-like neuropeptide, gastrin-releasing peptide (GRP), recruits disinhibitory cortical microcircuits through selective targeting and activation of vasoactive intestinal peptide (VIP)-expressing cells. Using a genetically encoded GRP sensor, optogenetic anterograde stimulation, and trans-synaptic tracing, we reveal that GRP regulates VIP cells most likely via extrasynaptic diffusion from several local and long-range sources. In vivo photometry and CRISPR-Cas9-mediated knockout of the GRP receptor (GRPR) in auditory cortex indicate that VIP cells are strongly recruited by novel sounds and aversive shocks, and GRP-GRPR signaling enhances auditory fear memories. Our data establish peptidergic recruitment of selective disinhibitory cortical microcircuits as a mechanism to regulate fear memories.

Characterization of GRP as a functional neuropeptide in basal chordate amphioxus.

Amphioxus belongs to the subphylum cephalochordata, an extant representative of the most basal chordates, whose regulation of endocrine system remains ambiguous. Here we clearly demonstrated the existence of a functional GRP neuropeptide in amphioxus, which was able to interact with GRP receptor, activate both PKC and PKA pathways, increase gh, igf, and vegf expression. We also showed that the transcription level of amphioxus grp was affected by temperature and light, indicating the role of this gene in the regulation of energy balance and circadian rhythms. In addition, the expression of the amphioxus grp was detected in cerebral vesicle that has been proposed to be the homologous organ of vertebrate brain. These data collectively suggested that a functional GRP neuropeptide had already emerged in amphioxus, which provided insights into the evolutionary origin of GRP in chordate and the functional homology between the cerebral vesicle and vertebrate brain.

Recent insights into biological functions of mammalian bombesin-like peptides and their receptors.

PURPOSE OF REVIEW: The current review highlights recent advances in physiological and pharmacological researches in biology of mammalian bombesin-like peptides (BLPs). RECENT FINDINGS: BLPs and their receptors were found to have regulatory roles in many biological processes in central nervous system. Two BLPs, neuromedin B and gastrin-releasing peptide (GRP), and their receptors are required for regulation of basal and induced sighing activity in rodents. This is the first study demonstrating central pathways involved in regulation of sighing activity. GRP receptor (GRPR) expressing neurons are excitatory glutamatergic interneurons located in the dorsal lamina without projections outside the spinal cord and mediate itch signals via vesicular glutamate transporter 2. Those neurons receive itch signals and make synapses with the parabrachial nucleus projecting spinal neurons to transmit itch signals to parabrachial nucleus. GRP expressing interneurons function in a proposed 'leaky gate model' to interpret the mechanism of both pain and itch transmission. In addition to recent advances of biology in nervous system, BLPs and their receptors were found to play potential regulatory roles in innate and adaptive immune responses and tissue development. SUMMARY: Several important biological roles of BLPs and their receptors in nervous system were identified. Together with researches regarding central roles of BLPs, studies revealing the regulatory roles of BLPs and their receptors in immunology and tissue development provide us with novel insights into understanding of the biology of BLPs and their receptors.

Gastrin-releasing peptide promotes the migration of vascular smooth muscle cells through upregulation of matrix metalloproteinase-2 and -9.

Gastrin-releasing peptide (GRP) has been reported to be implicated in the pathogenesis of inflammatory disorders. The migration and proliferation of vascular smooth muscle cells (VSMCs) are key components of vascular inflammation that leads to the development of atherosclerosis. The present study aimed to investigate the molecular effect of GRP on VSMC proliferation and migration. We report that GRP significantly enhanced the proliferation and migration of rat VSMCs. GRP increased mRNA and protein expression of matrix metalloproteinase- 2 and -9 (MMP-2/9) in VSMCs. The induction of MMP-2/9 by GRP was regulated by the activation of the signal transducer and activator of transcription-3 (STAT3). In addition, STAT3-knockdown of VSMCs by siRNA or blockade of the GRP receptor inhibited GRP-induced migration of VSMCs. Taken together, our findings indicate that GRP promotes the migration of VSMCs through upregulation of MMP-2/9 via STAT3 activation. [BMB Reports 2017; 50(12): 628-633].

Targeting prostate cancer cells with genetically engineered polypeptide-based micelles displaying gastrin-releasing peptide.

In recent years G protein-coupled receptors (GPCRs) have emerged as crucial tumorigenic factors that drive aberrant cancer growth, metastasis and angiogenesis. Consequently, a number of GPCRs are strongly expressed in cancer derived cell lines and tissue samples. Therefore a rational anti-cancer strategy is the design of nano-medicines that specifically target GPCRs to bind and internalise cytotoxic drugs into cancer cells. Herein, we report the genetic engineering of a self-assembling nanoparticle based on elastin-like polypeptide (ELP), which has been fused with gastrin releasing peptide (GRP). These nanoparticles increased intracellular calcium concentrations when added to GRP receptor positive PC-3 prostate cancer cells, demonstrating specific receptor activation. Moreover, GRP-displaying fluorescent labelled nanoparticles showed specific cell-surface interaction with PC-3 prostate cancer cells and increased endocytic uptake. These nanoparticles therefore provide a targeted molecular carrier system for evaluating the delivery of cytotoxic drugs into cancer cells.

The BB2 receptor antagonist BW2258U89 attenuates the feeding responses evoked by exogenous gastrin releasing peptide-29.

This confirmatory work is aimed to test that the hypothesis that the gastrin releasing peptide (GRP) receptor - the BB2 receptor - is necessary for reduction of meal size (MS) and prolongation of the intermeal interval (IMI) by the small and the large forms of GRP in the rat, GRP-10 and GRP-29, and to confirm the sites of action regulating such responses - the vascular bed of the celiac artery (CA, supplying stomach and upper duodenum). To pursue these aims we measured first MS and IMI length in response to GRP-10 and GRP-29 (0, 0.5nmol/kg) infused in the CA (n=8 rats) and the cranial mesenteric artery (CMA, supplying the small and part of the large intestine, n=8 rats) in near spontaneously free feeding rats pretreated with the BB2 receptor antagonist BW2258U89 (0.1mg/kg) in the same arteries prior to the onset of the dark cycle. We found that GRP-29, but not GRP-10, infused by the CA reduced MS and prolonged the IMI by decreasing meal latency and meal duration and the BB2 receptor antagonist BW2258U89 infused in the same artery attenuated these responses. These results suggest that the BB2 receptor is necessary for reduction of MS and prolongation of the IMI by exogenous GRP-29, and the vascular bed of the CA, stomach and upper duodenum, contains sites of action regulating these feeding responses.

Critical evaluation of the expression of gastrin-releasing peptide in dorsal root ganglia and spinal cord.

There are substantial disagreements about the expression of gastrin-releasing peptide (GRP) in sensory neurons and whether GRP antibody cross-reacts with substance P (SP). These concerns necessitate a critical revaluation of GRP expression using additional approaches. Here, we show that a widely used GRP antibody specifically recognizes GRP but not SP. In the spinal cord of mice lacking SP (Tac1KO), the expression of not only GRP but also other peptides, notably neuropeptide Y (NPY), is significantly diminished. We detectedGrpmRNA in dorsal root ganglias using reverse transcription polymerase chain reaction, in situ hybridization and RNA-seq. We demonstrated thatGrpmRNA and protein are upregulated in dorsal root ganglias, but not in the spinal cord, of mice with chronic itch. Few GRP(+)immunostaining signals were detected in spinal sections following dorsal rhizotomy and GRP(+)cell bodies were not detected in dissociated dorsal horn neurons. Ultrastructural analysis further shows that substantially more GRPergic fibers form synaptic contacts with gastrin releasing peptide receptor-positive (GRPR(+)) neurons than SPergic fibers. Our comprehensive study demonstrates that a majority of GRPergic fibers are of primary afferent origin. A number of factors such as low copy number ofGrptranscripts, small percentage of cells expressingGrp, and the use of an eGFP GENSAT transgenic as a surrogate for GRP protein have contributed to the controversy. Optimization of experimental procedures facilitates the specific detection of GRP expression in dorsal root ganglia neurons.

Peptide Synthesis through Cell-Free Expression of Fusion Proteins Incorporating Modified Amino Acids as Latent Cleavage Sites for Peptide Release.

Chlorinated analogues of Leu and Ile are incorporated during cell-free expression of peptides fused to protein, by exploiting the promiscuity of the natural biosynthetic machinery. They then act as sites for clean and efficient release of the peptides simply by brief heat treatment. Dehydro analogues of Leu and Ile are similarly incorporated as latent sites for peptide release through treatment with iodine under cold conditions. These protocols complement enzyme-catalyzed methods and have been used to prepare calcitonin, gastrin-releasing peptide, cholecystokinin-7, and prolactin-releasing peptide prohormones, as well as analogues substituted with unusual amino acids, thus illustrating their practical utility as alternatives to more traditional chemical peptide synthesis.

Dosimetry and first clinical evaluation of the new 18F-radiolabeled bombesin analogue BAY 864367 in patients with prostate cancer.

UNLABELLED: The aim of this first-in-man study was to demonstrate the feasibility, safety, and tolerability, as well as provide dosimetric data and evaluate the imaging properties, of the bombesin analogue BAY 864367 for PET/CT in a small group of patients with primary and recurrent prostate cancer (PCa). METHODS: Ten patients with biopsy-proven PCa (5 with primary PCa and 5 with prostate-specific antigen recurrence after radical prostatectomy) were prospectively selected for this exploratory clinical trial with BAY 864367, a new (18)F-labeled bombesin analogue. PET scans were assessed at 6 time points, up to 110 min after intravenous administration of 302 ± 11 MBq of BAY 864367. Imaging results were compared with (18)F-fluorocholine PET/CT scans. Dosimetry was calculated using the OLINDA/EXM software. RESULTS: Three of 5 patients with primary disease showed positive tumor delineation in the prostate, and 2 of 5 patients with biochemical relapse showed a lesion suggestive of recurrence on the BAY 864367 scan. Tumor-to-background ratio averaged 12.9 ± 7.0. The ratio of malignant prostate tissue to normal prostate tissue was 4.4 ± 0.6 in 3 patients with tracer uptake in the primary PCa. Mean effective dose was 4.3 ± 0.3 mSv/patient (range, 3.7-4.9 mSv). CONCLUSION: BAY 864367, a novel (18)F-labeled bombesin tracer, was successfully investigated in a first-in-man clinical trial of PCa and showed favorable dosimetric values. Additionally, the application was safe and well tolerated. The tracer delineated tumors in a subset of patients, demonstrating the potential of gastrin-releasing-peptide receptor imaging.

Sequence analysis and feeding responses evoked by the large molecular form of gastrin releasing peptide (GRP) in the rat GRP-29.

Microisolation techniques utilizing several reverse phase high performance liquid chromatography (HPLC) steps have resulted in the purification of two rat gastrin releasing peptide (GRP) forms suitable for microsequence and mass spectral analysis. The sequence of the larger form is APVSTGAGGGTVLAKMYPRGSHWAVGHLM-amide and the smaller form is GSHWAVGHLM-amide which is the carboxyl terminal decapeptide of the larger peptide. The peptides were synthesized and their feeding patterns e.g. first meal size (MS), intermeal interval (IMI) and satiety ratio (SR, IMI/MS) were determined in overnight food-, but not water deprived, male Sprague Dawley rats. The peptides were administered in the femoral vein (0, 0.21, 0.41 and 1.03 nmol/kg) immediately before presenting the rats with a 10% sucrose solution. We found that (1) GRP-10 (all doses) and GRP-29 (0.41 nmol/kg) reduced first MS, (2) both peptides prolonged IMI length and (3) both peptides increased the SR to similar extents. In conclusion, GRP-10 and GRP-29 are the two endogenous forms of GRP in the rat intestine and they reduce short term feeding to similar extents when administered intravenously.

Selection of DNA aptamers with affinity for pro-gastrin-releasing peptide (proGRP), a tumor marker for small cell lung cancer.

Aptamers are single-strand oligonucleotides that are generated by the systemic evolution of ligands by exponential enrichment (SELEX) technique and that can bind to target molecules specifically. However, only a few aptamers have been developed to date against tumor markers. To utilize aptamers for tumor diagnosis, a variety of aptamers are required. Here, a single-stranded DNA aptamer specific for pro-gastrin-releasing peptide (proGRP), a marker for small cell lung cancer, was selected using SELEX. After selection, identical sequences were found in the DNA library. This sequence was selected and its binding affinity to proGRP was evaluated using surface plasmon resonance.

Selective killing of cancer cells by peptide-targeted delivery of an anti-microbial peptide.

Antimicrobial peptides selectively kill bacteria while maintaining low mammalian cell cytotoxicity. However, they become cytotoxic subsequent to internalization. Here we have conjugated the lytic peptide (KLAKLAK)(2) to either a cancer-cell binding peptide (LTVSPWY) selected from peptide libraries or to a gastrin-releasing peptide (GNHWAVGHLM) in order to direct the lytic peptide to cancer cells. Peptide cytotoxicity was tested in breast MCF-7 and MDA-MB-231 cancer cells. The fusion peptides were internalized by cancer cells, disintegrated the cell membrane and induced rapid killing of the cells with IC50 values as low as 4-7 µM. Peptide cytotoxicity was dependent on the targeting receptor. Indeed, addition of free targeting peptide reduced cell killing. Blood lymphocytes and normal human mammary epithelial cells were less sensitive to the fusion peptides. Although most of the cells were killed by necrosis, fusion peptides branched with DNA oligonucleotides induced apoptosis as assayed by annexin V staining and activation of caspase 3. Therefore, the new designed drug peptides might provide a potent and selective anticancer therapy.

Bombesin-like peptides stimulate growth hormone secretion mediated by the gastrin-releasing peptide receptor in cattle.

This study was designed to determine the effects of bombesin-like peptides (BLPs) on the secretion of growth hormone (GH) and to characterize the receptor subtypes mediating these effects in cattle. Four experiments were conducted: (1) six steers were randomly assigned to receive intravenous (IV) bolus injections of 0, 0.2, 1.0, 12.5 and 50.0 µg/kg neuromedin C (NMC); (2) seven pre-weaned calves were IV injected with 1.0 µg/kg NMC; (3) six steers were IV injected with 2.5µg/kg bovine gastrin-releasing peptide (GRP), 1.0 µg/kg NMC combined with 20.0 µg/kg [d-Lys(3)]-GHRP-6 (an antagonist for the GH secretagogue receptor type 1a [GHS-R1a]), 1.0 µg/kg NMC combined with 20.0 µg/kg N-acetyl-GRP(20-26)-OCH(2)CH(3) (N-GRP-EE, an antagonist for the GRP receptor), 20.0 µg/kg N-GRP-EE alone, 1.0 µg/kg neuromedin B (NMB); and (4) four rats were IV injected 1.0 µg/kg NMC. A serial blood sample was collected before and after injection. Plasma GH levels dose-dependently increased at 5 min after NMC injection and the minimal effective dose was 1.0 µg/kg. Plasma GH level was elevated by GRP, but not by NMB. The NMC-induced elevation of GH was completely blocked by N-GRP-EE. The administration of NMC elevated GH level in pre-weaned calves but not in rats. Ghrelin level was unaffected by any treatments; and [d-Lys(3)]-GHRP-6 did not block the NMC-induced elevation of GH. The results indicate BLP-induced elevation of GH levels is mediated by the GRP receptor but not through a ghrelin/GHS-R1a pathway in cattle.

Efficient siRNA targeted delivery into cancer cells by gastrin-releasing peptides.

Small interfering RNAs (siRNAs) have displayed considerable promise for the treatment of cancer. However, their delivery to the desired cell population remains a challenging task. Here we have covalently conjugated a siRNA against survivin to gastrin-releasing peptides (GRPs) to direct siRNA molecules to cancer cells that express the GRP receptor. The cellular uptake of the peptide-siRNA conjugates was tested in breast MDA-MB 231 cancer cells, which express the GRP receptor. Fluorescein-tagged GRP-siRNA conjugates were taken up by cancer cells but not normal mammary epithelial cells or human blood monocytes. By 120 min of incubation, most of the cells have taken up the conjugates. Excess free peptide inhibited uptake, implying dependence of uptake on GRP receptor. Moreover, bitargeting of siRNA molecules by GR and luteinizing hormone-releasing peptides accelerated the uptake kinetics by MDA-MB 231 cells when compared to monotargeted siRNAs. Peptide-siRNA conjugates, but not free siRNAs, inhibited the expression of survivin, an endogenous gene involved in cancer cell survival. None of the peptide-siRNA conjugates induced the expression of inflammatory cytokines or interferon α in human blood leukocytes. Overall, the data demonstrate the feasibility of GRP receptor-mediated targeted delivery of siRNAs to cancer cells, an important step for RNA interference therapy in humans.

Pro-GRP-derived peptides are expressed in colorectal cancer cells and tumors and are biologically active in vivo.

Amidated gastrin-releasing peptide (GRP) is the prototypical autocrine growth factor. Nonamidated peptides derived from the C terminus of pro-GRP are also biologically active in colorectal cancer (CRC) cell lines in vitro, via a receptor distinct from the GRP receptor. The aims of this study were to measure the amounts of pro-GRP-derived peptides in human CRC cell lines and tumors, characterize the immunoreactive peptide, and investigate its effect on proliferation in vitro and in vivo. Pro-GRP-derived peptides were quantitated by region-specific ELISA in extracts of five human CRC cell lines and 20 tumors. The immunoreactive material was purified by HPLC and its mass and sequence established by mass spectrometry. The concentration of GRPamide was determined by RIA. Proliferation of DLD-1 cells and murine gastrointestinal mucosa was measured by [(3)H]-thymidine incorporation and mitotic index, respectively. In CRC cell extracts, ELISA for pro-GRP-derived peptides detected 3-152 fmol/10(6) cells. The immunoreactive peptide was purified and identified as pro-GRP42-98. Resected stage III tumors contained significantly less pro-GRP immunoreactivity than stage II tumors, and no amidated GRP was detected in cell lines or tumors. Stable transfection of DLD-1 cells with pro-GRP short hairpin RNA, or treatment with a monoclonal anti-pro-GRP antibody, significantly reduced proliferation. Pro-GRP42-98, pro-GRP47-68, and pro-GRP80-97 significantly stimulated mitosis in colonic, but not small intestinal, mucosa of 10-wk-old mice. We conclude that nonamidated peptides derived from the C terminus of pro-GRP are expressed in significant quantities in CRC cell lines and tumors and stimulate the proliferation of CRC cells and of normal colonic mucosa. Such peptides are attractive targets for novel CRC therapies.

Neuropeptide signaling differentially affects phase maintenance and rhythm generation in SCN and extra-SCN circadian oscillators.

Circadian rhythms in physiology and behavior are coordinated by the brain's dominant circadian pacemaker located in the suprachiasmatic nuclei (SCN) of the hypothalamus. Vasoactive intestinal polypeptide (VIP) and its receptor, VPAC(2), play important roles in the functioning of the SCN pacemaker. Mice lacking VPAC(2) receptors (Vipr2(-/-)) express disrupted behavioral and metabolic rhythms and show altered SCN neuronal activity and clock gene expression. Within the brain, the SCN is not the only site containing endogenous circadian oscillators, nor is it the only site of VPAC(2) receptor expression; both VPAC(2) receptors and rhythmic clock gene/protein expression have been noted in the arcuate (Arc) and dorsomedial (DMH) nuclei of the mediobasal hypothalamus, and in the pituitary gland. The functional role of VPAC(2) receptors in rhythm generation and maintenance in these tissues is, however, unknown. We used wild type (WT) and Vipr2(-/-) mice expressing a luciferase reporter (PER2::LUC) to investigate whether circadian rhythms in the clock gene protein PER2 in these extra-SCN tissues were compromised by the absence of the VPAC(2) receptor. Vipr2(-/-) SCN cultures expressed significantly lower amplitude PER2::LUC oscillations than WT SCN. Surprisingly, in Vipr2(-/-) Arc/ME/PT complex (Arc, median eminence and pars tuberalis), DMH and pituitary, the period, amplitude and rate of damping of rhythms were not significantly different to WT. Intriguingly, while we found WT SCN and Arc/ME/PT tissues to maintain a consistent circadian phase when cultured, the phase of corresponding Vipr2(-/-) cultures was reset by cull/culture procedure. These data demonstrate that while the main rhythm parameters of extra-SCN circadian oscillations are maintained in Vipr2(-/-) mice, the ability of these oscillators to resist phase shifts is compromised. These deficiencies may contribute towards the aberrant behavior and metabolism associated with Vipr2(-/-) animals. Further, our data indicate a link between circadian rhythm strength and the ability of tissues to resist circadian phase resetting.

Development of a new bombesin analog radiolabeled with lutetium-177: in vivo evaluation of the biological properties in Balb-C mice.

In this work we describe the first results of radiolabeling with lutetium-177 ((177)Lu) and in vivo biodistribution and pharmacokinetics studies in normal Balb-c mice of a new bombesin analog (BEFG2)--DOTA-Phe-X-BBN(6-14), where X is a spacer of two aminoacids. Bombesin (BBN) is an amphibian analog of human gastrin releasing peptide (GRP). Development of radiolabeled BBN derivatives as agents for diagnostic imaging and systemic radiotherapy has increased considerable because of the observation that GRP receptors (GRPr) are over-expressed in a variety of human tumor cells, such as prostate tumor cells. (177)Lu-labeled peptides are attractive due to the excellent radiophysical properties and commercial availability of the radiometal. BEFG2 was successfully labeled with high yield and kept stable for more than 96 hours at 2-8 degrees C and 1 hour in human plasma. Data analysis obtained from the in vivo studies showed that the amount of BEFG2 present in plasma decreased rapidly and became almost undetectable at 60 min p.i., indicating rapid peptide excretion, which is performed mainly by renal pathway. In addition, biodistribution and single photon emission tomography showed low abdominal accumulation of (177)Lu-DOTA- Phe-X-BBN(6-14), indicating that this analog is a potential candidate for tumors target therapy.

Discovery and optimization of a novel Neuromedin B receptor antagonist.

The discovery and parallel synthesis of potent, small molecule antagonists of Neuromedin B receptor based on the ary-hexahydro-dibenzodiazepin-1-one core is described.

Molecular characterization of ghrelin and gastrin-releasing peptide in Atlantic cod (Gadus morhua): cloning, localization, developmental profile and role in food intake regulation.

Full-length complementary deoxyribonucleic acid as well as genomic sequences encoding for two gastrointestinal appetite-related peptides, ghrelin and for gastrin-releasing peptide (GRP) were cloned from Atlantic cod (Gadus morhua) stomach using reverse transcription and rapid amplification of complementary deoxyribonucleic acid ends. Semi-quantitative reverse transcriptase polymerase chain reaction shows that both ghrelin and GRP are widely distributed in several peripheral tissues and throughout cod brain, although expression levels are very low. During development, ghrelin was detected at the cleavage stage, with low expression levels persisting until the first-feeding stage, while GRP was detected at the blastula stage, showing increased expression from the pre-hatching stage on. Juvenile cod fed medium rations displayed periprandial changes in gut ghrelin, but not GRP, expression, with higher expression levels at meal time compared to 2h before feeding time. Ghrelin gut mRNA expression was not affected by rations, whereas GRP gut mRNA expression was higher in fish fed high rations as compared to fish fed low rations. Neither ghrelin nor GRP gut mRNA expressions were affected by 30 days starvation or 5 days re-feeding.

Oncogenic activation of androgen receptor.

BACKGROUND: There is considerable evidence implicating the aberrant activation or "reactivation" of androgen receptor in the course of androgen-ablation therapy as a potential cause for the development of castration-resistant prostate cancer. Several non-mutually exclusive mechanisms including the inappropriate activation of androgen receptor (AR) by non-steroids have been postulated. The present work is aimed to understand the role of neuropeptides released by neuroendocrine transdifferentiated prostate cancer cells in the aberrant activation of AR. OBJECTIVES: The study was designed to study how neuropeptides such as gastrin-releasing peptide activate AR and to define the crucial signal pathways involved, in the hope to identify therapeutic targets. METHODS AND MATERIALS: Androgen-dependent LNCaP cell line was used to study the effects of bombesin/gastrin-releasing peptide on the growth of the cell line and the transactivation of AR. The neuropeptide was either added to the media or introduced as a transgene in LNCaP cells to study its paracrine or autocrine effect on LNCaP growth under androgen-deprived conditions. The activation of AR was monitored by reporter assay, chromatin immunoprecipitation (ChIP) of AR, translocation into the nucleus and cDNA microarray of the AR response genes. RESULTS: Bombesin/gastrin releasing peptides induce androgen-independent growth of LNCaP in vitro and in vivo. It does so by activating AR, which is accompanied by the activation of Src tyrosine kinase and its target c-myc oncogene. The bombesin or Src-activated AR induces an overlapping set of AR response genes as androgen, but they also a unique set of genes. Intriguingly, the Src-activated and androgen-bound ARs differ in their binding specificity toward AR response elements, indicating the receptors activated by these 2 mechanisms are not conformationally identical. Finally, Src inhibitor was shown to effectively block the activation of AR and the growth effects induced by bombesin. CONCLUSION: The results showed that AR can be activated by neuropeptide, a ligand for G-protein coupled receptor, in the absence of androgen. The activation goes through Src-tyrosine kinase pathway, and tyrosine kinase inhibitor is a potentially useful adjunctive therapy during androgen ablation.