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

BACKGROUND: The 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. METHODS: mRNA 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. RESULTS: Binding 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). CONCLUSION: Targeting the GRPR and SSTR2 for nuclear imaging and/or treatment has the potential to improve BC care in primary as well as metastatic disease.

Clinical Relevance of Targeting the Gastrin-Releasing Peptide Receptor, Somatostatin Receptor 2, or Chemokine C-X-C Motif Receptor 4 in Breast Cancer for Imaging and Therapy.

UNLABELLED: Imaging and therapy using radioligands targeting receptors overexpressed on tumor cells is successfully applied in neuroendocrine tumor patients. Because expression of the gastrin-releasing peptide receptor (GRPR), somatostatin receptor 2 (SSTR2), and chemokine C-X-C motif receptor 4 (CXCR4) has been demonstrated in breast cancer, targeting these receptors using radioligands might offer new imaging and therapeutic opportunities for breast cancer patients. The aim of this study was to correlate messenger RNA (mRNA) expression of GRPR, SSTR2, and CXCR4 with clinicopathologic and biologic factors, and with prognosis and prediction to therapy response, in order to identify specific breast cancer patient groups suited for the application of radioligands targeting these receptors. METHODS: First, we studied GRPR and SSTR2 expression in 13 clinical breast cancer specimens by in vitro autoradiography and correlated this with corresponding mRNA levels to investigate whether mRNA levels reliably represent cell surface expression. Next, GRPR, SSTR2, and CXCR4 mRNA levels were measured by quantitative reverse transcriptase polymerase chain reaction in 915 primary breast cancer tissues and correlated with known clinicopathologic and biologic factors, disease-free survival, distant metastasis-free survival, and overall survival (DFS, MFS, and OS, respectively). In 224 adjuvant hormonal treatment-naïve estrogen receptor (ER, ESR1)-positive patients who received tamoxifen as first-line therapy for recurrent or metastatic disease, the expression levels of the receptors were correlated with progression-free survival. RESULTS: Our results showed a significant positive correlation between GRPR and SSTR2 expression analyzed by in vitro autoradiography and by quantitative reverse transcriptase polymerase chain reaction (Spearman's rank correlation coefficient [Rs]=0.94, P<0.001, and Rs=0.73, P=0.0042, respectively). Furthermore, high GRPR and SSTR2 mRNA levels were observed more frequently in ESR1-positive specimens, whereas high CXCR4 expression was associated with ESR1-negative specimens. Also, high mRNA expression of CXCR4 was associated with a prolonged DFS, MFS, and OS (multivariate hazard ratio MFS=0.76 [95% confidence interval, 0.64-0.90], P=0.001), whereas high mRNA levels of GRPR were associated with a prolonged progression-free survival after the start of first-line tamoxifen treatment (multivariate hazard ratio=0.68 [95% confidence interval, 0.48-0.97], P=0.031). CONCLUSION: Our data indicate that imaging and therapy using GRPR or SSTR2 radioligands might especially be beneficial for ESR1-positive breast cancer and CXCR4 radioligands for ESR1-negative breast cancer.

Synthesis and characterization of a high-affinity NOTA-conjugated bombesin antagonist for GRPR-targeted tumor imaging.

The gastrin-releasing peptide receptor (GRPR/BB2) is a molecular target for the visualization of prostate cancer. This work focused on the development of high-affinity, hydrophilic, antagonistic, bombesin-based imaging agents for PET and SPECT. The bombesin antagonist analog d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 ([d-Phe(6),Sta(13),Leu(14)]bombesin[6-14]) was synthesized and conjugated to 1,4,7-triazacyclononane-N,N',N″-triacetic acid (NOTA) via a diethylene glycol (PEG2) linker. The resulting conjugate, NOTA-PEG2-[d-Phe(6),Sta(13),Leu(14)]bombesin[6-14] (NOTA-P2-RM26), was labeled with (68)Ga (T1/2 = 68 min, positron emitter) and (111)In (T1/2 = 2.8 days, gamma emitter). The labeling stability, specificity, inhibition efficiency (IC50), and dissociation constant (KD) of both labeled compounds as well as their cellular retention and internalization were investigated. The pharmacokinetics of the dual isotope ((111)In/(68)Ga)-labeled peptide in both normal NMRI mice and PC-3 tumor-bearing Balb/c nu/nu mice was also studied. NOTA-P2-RM26 was labeled with (111)In and (68)Ga at a radiochemical yield of >98%. Both conjugates were shown to have high specificity and binding affinity for GRPR. The KD value was determined to be 23 ± 13 pM for the (111)In-labeled compound in a saturation binding experiment. In addition, (nat)In- and (nat)Ga-NOTA-P2-RM26 showed low nanomolar binding inhibition concentrations (IC50 = 1.24 ± 0.29 nM and 0.91 ± 0.19 nM, respectively) in a competitive binding assay. The internalization rate of the radiolabeled conjugates was slow. The radiometal-labeled tracers demonstrated rapid blood clearance via the kidney and GRPR-specific uptake in the pancreas in normal mice. Tumor targeting and biodistribution studies in mice bearing PC-3 xenografts displayed high and specific uptake in tumors (8.1 ± 0.4%ID/g for (68)Ga and 5.7 ± 0.3%ID/g for (111)In) and high tumor-to-background ratios (tumor/blood: 12 ± 1 for (68)Ga and 10 ± 1 for (111)In) after only 1 h p.i. of 45 pmol of peptide. The xenografts were visualized by gamma and microPET cameras shortly after injection. In conclusion, the antagonistic bombesin analog NOTA-PEG2-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 (NOTA-P2-RM26) is a promisindg candidate for prostate cancer imaging using PET and SPECT/CT.

Autocrine effects of neuromedin B stimulate the proliferation of rat primary osteoblasts.

Neuromedin B (NMB) is a mammalian bombesin-like peptide that regulates exocrine/endocrine secretion, smooth muscle contraction, body temperature, and the proliferation of some cell types. Here, we show that mRNA encoding Nmb and its receptor (Nmbr) are expressed in rat bone tissue. Immunohistochemical analysis demonstrated that NMB and NMBR colocalize in osteoblasts, epiphyseal chondrocytes, and proliferative chondrocytes of growth plates from mouse hind limbs. Then, we investigated the effect of NMB on the proliferation of rat primary cultured osteoblasts. Proliferation assays and 5-bromo-2'-deoxyuridine incorporation assays demonstrated that NMB augments the cell number and enhances DNA synthesis in osteoblasts. Pretreatment with the NMBR antagonist BIM23127 inhibited NMB-induced cell proliferation and DNA synthesis. Western blot analysis showed that NMB activates ERK1/2 MAPK signaling in osteoblasts. Pretreatment with the MAPK/ERK kinase inhibitor U0126 attenuated NMB-induced cell proliferation and DNA synthesis. We also investigated the effects of molecules that contribute to osteoblast proliferation and differentiation on Nmb expression in osteoblasts. Real-time PCR analysis demonstrated that 17ß-estradiol (E2) and transforming growth factor ß1 increase and decrease Nmb mRNA expression levels respectively. Finally, proliferation assays revealed that the NMBR antagonist BIM23127 suppresses E2-induced osteoblast proliferation. These results suggest that NMB/NMBR signaling plays an autocrine or paracrine role in osteoblast proliferation and contributes to the regulation of bone formation.

Anticancer activity of a peptide combination in gastrointestinal cancers targeting multiple neuropeptide receptors.

A novel peptide combination consisting of four synthetic neuropeptide analogs of Vasoactive Intestinal Peptide (VIP), Bombesin, Substance P and Somatostatin has been found to have potent anticancer activity in vitro and in vivo. The receptors of these four neuropeptides are known to be over expressed in various cancers. We have found the presence of native neuropeptides in the culture supernatant of the primary tumor cells of human colon adenocarcinomas. It was further demonstrated by receptor-ligand assays that not only do these tumor cells synthesize and secrete four peptide hormones but also possess specific high affinity receptors on their surface. Screening a large panel of analogs to the four peptide hormones on tumor cell proliferation led to the identification of four cytotoxic analogs, the combination of which was code-named DRF7295. The design and synthesis of the peptide analogs have been described in this paper. In vitro anticancer activity of DRF7295 was studied in a large panel of human tumor cells. Gastrointestinal tumor cells of the colon, pancreas and duodenum were found to be most sensitive to DRF7295 with moderate activity seen in glioblastoma, prostate, leukemia and those of oral cancer cells. Efficacy studies in xenograft models of colon and duodenum resulted in T/C% of less than 40%, which is indicative of strong tumor regressing potential of DRF7295 in gastrointestinal cancers. Acute and long-term toxicity studies as well as safety pharmacology studies conducted indicate the safety of the drug upon systemic administration with no significant adverse pharmacological effects.

In vitro and in vivo antitumor effects of cytotoxic camptothecin-bombesin conjugates are mediated by specific interaction with cellular bombesin receptors.

Most human tumors overexpress or ectopically express peptide hormone/neurotransmitter receptors, which are being increasingly studied as a means to selectively deliver cytotoxic agents. Although a number of peptide ligand-constructs demonstrate tumor cytotoxicity, the role of specific tumoral receptor interaction in its mediation is unclear. To address this question, we synthesized camptothecin (CPT) bombesin (Bn) analogs, in which CPT was coupled via a novel carbamate linker, L2 [N-(N-methyl-amino-ethyl)-glycine carbamate], that were chemically similar but differed markedly in their potency/affinity for human Bn receptors. We then examined their ability to interact with Bn receptors and cause in vitro and in vivo tumor cytotoxicity. CPT-L2-[D-Tyr(6),beta-Ala(11),D-Phe(13),Nle(14)] Bn (6-14) (BA3) bound with high affinity and had high potency for all three human Bn receptor subtypes, whereas CPT-L2-[D-Tyr(6),beta-Ala(11), D-Phe(13),Nle(14)] Bn (6-14) [D-Phe-CPT-L2-BA3] had >1400-fold lower affinity/potency. (125)I-CPT-L2-BA3 but not (125)I-D-Phe-CPT-L2-BA3 was internalized by Bn receptor subtype-containing cells. CPT-L2-BA3 displayed significantly more cytotoxicity than D-Phe-CPT-L2-BA3 toward NCI-H1299 lung cancer cells in both 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide and clonogenic assays and more potently inhibited H1299 xenograft growth in nude mice. CPT-L2-BA3 was also metabolically more stable than its parent peptide and inhibited growth of a number of other tumor cell lines in vitro and in vivo. These results demonstrate that specific tumoral receptor interaction is important in mediating the ability of peptide ligand-cytotoxic constructs to cause cytotoxicity. Because many tumors overexpress Bn receptors, these results also demonstrate that CPT-L2-BA3 will be a useful agent for delivering receptor-mediated cytotoxicity to many different human tumors.

Targeting gastrin-releasing peptide receptors on small cell lung cancer cells with a bispecific molecule that activates polyclonal T lymphocytes.

PURPOSE: Gastrin-releasing peptide (GRP) is a growth factor for small cell lung cancer (SCLC). GRP belongs to the bombesin peptide family and has significant homology to bombesin. We constructed a bispecific molecule, OKT3xAntag2, by conjugating a monoclonal antibody OKT3 (anti-CD3) with a bombesin/GRP antagonist (Antag2) and evaluated cytotoxicity against SCLC cells. EXPERIMENTAL DESIGN: We tested binding of the bispecific molecule to SCLC cell lines and T cells by flow cytometry, antibody-dependent cellular cytotoxicity (ADCC) of SCLC cells in vitro and in a murine SCLC xenograft model. We studied SCLC apoptosis and necrosis during ADCC and the activity and cleavage of caspase-3, caspase-9, and poly(ADP-ribose) polymerase (PARP). RESULTS: The bispecific molecule functions as a cross-linker between T cells and SCLC cells, induces T cell activation, and mediates ADCC of SCLC cells; 40% to 80% growth inhibition of SCLC cells mediated by the bispecific molecule at low effector to target cell ratios was achieved. Activation of T cells by the bispecific molecule resulted in significant increases in IFNgamma production and apoptosis and necrosis of SCLC cells associated with cleavage of PARP and caspase-3. Targeted immunotherapy with the bispecific molecule-armed human T cells significantly reduced SCLC tumor burdens in a mouse model. CONCLUSION: The bispecific molecule OKT3xAntag2 mediates growth inhibition and apoptosis of SCLC cells by activated T cells through activation and cleavage of caspase-3 and PARP in vitro and in vivo. Clinical trials of this bispecific molecule through adoptive transfer of ex vivo activated T cells in GRP receptor-positive tumors, such as SCLC, are warranted.

Gastrin-releasing peptide receptor as a molecular target for psychiatric and neurological disorders.

The mammalian bombesin (BB)-like peptide gastrin-releasing peptide (GRP) stimulates cell proliferation, displays a range of neuroendocrine activities, and acts as a growth factor in the pathogenesis of several types of human cancer. Several lines of evidence have indicated that GRP and its receptor (GRPR) might also be involved in the neurochemical alterations associated with psychiatric and neurological disorders. GRP and GRPR are distributed throughout the mammalian central nervous system (CNS). Altered levels of BB-like peptides have been found in the CNS of patients with schizophrenia and Parkinson's disease. Dysfunctions in GRPR-induced cellular calcium signaling have been reported in fibroblasts from patients with Alzheimer's disease. A translocation in the GRPR gene has been associated with autism. Pharmacological and genetic studies in rodents have shown that GRPRs in brain areas such as the dorsal hippocampus and amygdala are importantly involved in regulating synaptic plasticity and aspects of behavior that might be altered in disorders such as anxiety, schizophrenia, depression, autism and dementia. Behaviors modulated by the GRPR in rodents include grooming, food intake, stereotypy, social behavior, and emotionally-motivated learning and memory. Together, these findings support the view that the GRPR should be considered a therapeutic target for a subset of CNS diseases.

Targeted chemotherapy with cytotoxic bombesin analogue AN-215 inhibits growth of experimental human prostate cancers.

We developed a powerful cytotoxic analogue of bombesin AN-215, in which the bombesin (BN)-like carrier peptide is conjugated to 2-pyrrolino doxorubicin (AN-201). Human prostate cancers express high levels of receptors for BN/gastrin releasing peptide (GRP) that can be used for targeted chemotherapy. The effects of targeted chemotherapy with cytotoxic BN analogue AN-215 were evaluated in nude mice bearing subcutaneous xenografts of DU-145, LuCaP-35, MDA-PCa-2b and intraosseous implants of C4-2 human prostate cancers. Intraosseous growth of C4-2 tumors was monitored by serum PSA. BN/GRP receptors were evaluated by 125I-[Tyr4]BN binding assays and RT-PCR. The effects of AN-215 on apoptosis and cell proliferation were followed by histology, and the expression of Bcl-2 and Bax protein was determined by Western blot analysis. Targeted analog AN-215 significantly inhibited growth of subcutaneously implanted DU-145, LuCaP-35 and MDA-PCa-2b prostate cancers by 81% to 91% compared to controls, while cytotoxic radical AN-201 was less effective and more toxic. Serum PSA levels of mice bearing intraosseous C4-2 prostate tumors were significantly reduced. In LuCaP-35 tumors administration of BN antagonist RC-3095 prior to AN-215 blocked the receptors for BN/GRP and inhibited the effects of AN-215. High affinity receptors for BN/GRP and their m-RNA were detected on membranes of all 4 tumor models. Therapy with AN-215, but not with AN-201, decreased the ratio of Bcl-2/Bax in DU-145 and the expression of antiapoptotic Bcl-2 in LuCaP-35 tumors. The presence of BN/GRP receptors on primary and metastatic prostate cancers makes possible targeted chemotherapy with AN-215 for the treatment of this malignancy.

New chelation strategy allows for quick and clean 99mTc-labeling of synthetic peptides.

Analogues of bombesin have been synthesized in which a N2S2 (bis-mercaptoacetyl functionalized diaminopropionic acid) or a N3S (mercaptoacetyl-Gly-Gly-Gly) radiometal-chelating center has been incorporated that allows radiolabeling of these peptides with 99mTc without the need for conjugation or harsh reaction conditions. A mild radiolabeling is possible by using an acetyl-moiety as sulfur protecting group, which can be removed by mild hydroxylamine-treatment at room temperature before radiolabeling. Retained receptor binding is demonstrated in competitive binding experiments with 99mTc-radiolabeled peptides and PC-3 cells with bombesin receptors.

Preclinical evaluation of therapeutic effects of targeted cytotoxic analogs of somatostatin and bombesin on human gastric carcinomas.

BACKGROUND: New modalities are necessary for the treatment of patients with unresectable gastric carcinoma. The authors investigated whether receptors for somatostatin and bombesin were present in human gastric carcinoma lines and tested the antitumor effects of cytotoxic somatostatin analog AN-238 and cytotoxic bombesin conjugate AN-215. METHODS: Nude mice bearing AGS, Hs 746T, and NCI-N87 human gastric carcinomas were treated with AN-238, AN-215, or their cytotoxic moiety 2-pyrrolinodoxorubicin (AN-201). Tumor growth reduction and tumor doubling times were calculated, and histologic characteristics of cell proliferation and apoptosis were examined. The expression of mRNA for somatostatin and bombesin receptors in tumors was investigated by reverse transcriptase-polymerase chain reaction. Subtypes 2 and 5 of somatostatin receptor proteins (sst2 and sst5, respectively) were analyzed using immunohistochemistry and immunoblotting. Binding assays were performed with radiolabeled somatostatin and bombesin analogs. RESULTS: Cytotoxic bombesin analog AN-215 powerfully inhibited the growth of AGS carcinomas that expressed high-affinity subtype 1 bombesin receptors. All three carcinomas expressed high-affinity sst2 and sst5 receptors. Cytotoxic somatostatin analog AN-238 exerted a strong inhibitory effect on NCI-N87 and Hs 746T carcinomas, which exhibited high concentrations of somatostatin receptors, but had a weaker effect on AGS tumors, which expressed the lowest receptor levels. AN-201 had only nonsignificant effects. CONCLUSIONS: Experimental human gastric carcinomas that expressed high-affinity subtype 1 bombesin receptors were inhibited by cytotoxic bombesin analog AN-215, and tumors with high concentrations of sst2 or sst5 somatostatin receptors were suppressed by cytotoxic somatostatin analog AN-238. These findings suggest that this class of targeted compounds should be considered for the therapy of patients with advanced gastric carcinoma.

Inhibition of proliferation of human small cell lung cancer cells expressing an autocrine system for gastrin releasing peptide by antisense oligodeoxynucleotides to gastrin releasing peptide receptor.

The objectives of this study were to investigate the effect of antisense (AS) oligodeoxynucleotides (ODNs) directed against gastrin releasing peptide (GRP) receptor mRNA on proliferation of human small cell lung cancer (SCLC) NCI-H345 cells which express the autocrine system for GRP. The methods used were to expose human SCLC cell lines to antisense ODNs or sense ODNs and to measure their proliferation by spectrophotometric assay or viable cell counts. Our results demonstrated that the single or combined AS ODNs against GRP receptor inhibited proliferation of human SCLC NCI-H345 cells significantly by 37% (P<0.01), but did not inhibit proliferation of either human bronchial epithelial BEAS 2B cells or human SCLC NCI-N417 cells, neither of which express the GRP autocrine system. The sense controls did not significantly inhibit proliferation compared with no treatment controls. Specificity was also demonstrated by the observation that cells exposed to AS ODNs had a decrease in GRP receptor expression as measured by specific binding of 34% (P<0.01), and when all three AS ODNs were used, binding was decreased by 60% (P<0.03). Furthermore, AS ODNs decreased by 75% the maximum percentage of cells responding to GRP in an intracellular calcium release assay. Our conclusions are that antisense ODNs directed against a GRP receptor which is involved in an autocrine loop in human SCLC cells inhibited proliferation of these cells by their impact on reducing GRP receptor expression. Further development of means of increasing AS ODN specificity and effectiveness in human SCLC cell is warranted.

Rapid protein kinase D translocation in response to G protein-coupled receptor activation. Dependence on protein kinase C.

Protein kinase D (PKD)/protein kinase C (PKC) mu is a serine/threonine protein kinase that can be activated by physiological stimuli like growth factors, antigen-receptor engagement and G protein-coupled receptor (GPCR) agonists via a phosphorylation-dependent mechanism that requires PKC activity. In order to investigate the dynamic mechanisms associated with GPCR signaling, the intracellular translocation of a green fluorescent protein-tagged PKD was analyzed by real-time visualization in fibroblasts and epithelial cells stimulated with bombesin, a GPCR agonist. We found that bombesin induced a rapidly reversible plasma membrane translocation of green fluorescent protein-tagged PKD, an event that can be divided into two distinct mechanistic steps. The first step, which is exclusively mediated by the cysteine-rich domain in the N terminus of PKD, involved its translocation from the cytosol to the plasma membrane. The second step, i.e. the rapid reverse translocation of PKD from the plasma membrane to the cytosol, required its catalytic domain and surprisingly PKC activity. These findings provide evidence for a novel mechanism by which PKC coordinates the translocation and activation of PKD in response to bombesin-induced GPCR activation.

A bombesin receptor subtype-3 peptide increases nuclear oncogene expression in a MEK-1 dependent manner in human lung cancer cells.

A synthetic peptide, (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) was used to investigate the signal transduction mechanisms of bombesin receptor subtype-3. Using NCI-1299#5 human lung cancer cells stably transfected with bombesin receptor subtype-3, 100 nM (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) elevated the cytosolic Ca2+ from 150 to 250 nM within 10 s. Addition of (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) caused phosphorylation of mitogen activated protein kinase in a time- and concentration-dependent manner. The mitogen activated protein kinase phosphorylation caused by (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) was inhibited by 2'-amino-3'-methyoxyflavone (PD98059), a mitogen activated protein kinase kinase (MEK-1) inhibitor. Using a luciferase reporter gene construct, (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) caused Elk-1 activation after 10 min and the increase in Elk-1 activation caused by (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) was inhibited by PD98059 as well as a dominant-negative MEK-1. (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) caused increased c-fos as well as c-jun mRNAs 1 h after addition to NCI-H1299#5 cells. The 47-fold increase in c-fos mRNA caused by 100 nM (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) was inhibited by PD98059, a dominant-negative MEK-1 and a substance P antagonist but not (3-phenylpropanoyl-D-Ala(24), Pro(26), Psi(26,27), Phe(27))GRP-(20-27) (BW2258U89), a GRP receptor antagonist. These results indicate that (D-Phe(6), beta-Ala(11), Phe(13), Nle(14))bombesin-(6-14) caused increased nuclear oncogene expression and upstream events include mitogen activated protein kinase phosphorylation and Elk-1 activation.

Inhibition of growth of MDA-MB-468 estrogen-independent human breast carcinoma by bombesin/gastrin-releasing peptide antagonists RC-3095 and RC-3940-II.

BACKGROUND: The growth of breast carcinoma is promoted by autocrine growth factors such as the bombesin (BN)-like peptides and epidermal growth factor (EGF). The stimulatory action of BN-like peptides can be blocked by the use of BN/gastrin-releasing peptide (GRP) antagonists. METHODS: The authors investigated the effects of synthetic BN/GRP antagonists RC-3095 and RC-3940-II on tumor growth and the expression of mRNA for EGF receptors and three BN receptor subtypes in MDA-MB-468 human breast carcinoma. Athymic nude mice with xenografts of MDA-MB-468 human breast carcinoma were injected subcutaneously for 6 weeks with RC-3940-II at doses of 20 or 40 microg/day. In another study, the effects of RC-3940-II and RC-3095 were compared. RESULTS: RC-3940-II caused a significant and dose-dependent growth inhibition of MDA-MB-468 tumors in nude mice; therapy with either dose of RC-3940-II significantly (P<0.01) reduced the mean final tumor volume and weight compared with controls. RC-3940-II induced a persistent regression of > 50% of all tumors. One of 3 tumors treated with 20 microg of RC-3940-II and 3 of 5 tumors treated with 40 microg were found to have regressed completely by the end of the study. When RC-3940-II and RC-3095 were compared at the dose of 20 microg/day, both powerfully suppressed growth of MDA-MB-468 tumors, with RC-3940-II causing a complete regression of 2 tumors and RC-3095 a complete regression of 1 tumor. Receptor analyses of untreated MDA-MB-468 tumors revealed an overexpression of EGF receptors and two classes of binding sites for BN/GRP. mRNAs for receptors of GRP, neuromedin B, and BN receptor subtype-3 were detected by reverse transcriptase-polymerase chain reaction. CONCLUSIONS: A virtual arrest of growth or regression of MDA-MB-468 human breast carcinoma after therapy with RC-3940-II and RC-3095 indicates that these BN/GRP antagonists could provide a new treatment modality for breast tumors expressing BN and EGF receptors.

Role of adenosine and N-methyl-D-aspartate receptors in mediating haloperidol-induced gene expression and catalepsy.

Acute blockade of dopamine D(2) receptors by the typical antipsychotic drug haloperidol leads to alterations in neuronal gene expression and behavior. In the dorsolateral striatum, the levels of mRNA for the immediate-early gene c-fos and the neuropeptide gene neurotensin/neuromedin N (NT/N) are significantly increased by haloperidol. An acute behavioral response to haloperidol is catalepsy, considered to be a rodent correlate of some of the immediate extrapyramidal motor side effects seen in humans. Several lines of evidence suggest a link between neurotensin induction in the dorsolateral striatum and catalepsy. We hypothesize that both striatal gene induction and catalepsy elicited by haloperidol arise from the combined effect of excitatory adenosinergic and glutamatergic inputs acting at adenosine A(2A) and N-methyl-D-aspartate (NMDA) receptors, respectively. In agreement with our previous reports, adenosine antagonists reduced haloperidol-induced c-fos and neurotensin gene expression as well as catalepsy. In agreement with other reports, the noncompetitive NMDA receptor antagonist MK-801 also reduced gene expression and catalepsy in response to haloperidol. The competitive NMDA receptor antagonist LY235959 decreased haloperidol-induced catalepsy. We show here that blocking both A(2A) and NMDA receptors simultaneously in conjunction with haloperidol resulted in a combined effect on gene expression and behavior that was greater than that for block of either receptor alone. Both c-fos and NT/N mRNA levels were reduced, and catalepsy was completely abolished. These results indicate that the haloperidol-induced increases in c-fos and NT gene expression in the dorsolateral striatum and catalepsy are driven largely by adenosine and glutamatergic inputs acting at A(2A) and NMDA receptors.

Bombesin-like peptides depolarize rat hippocampal interneurones through interaction with subtype 2 bombesin receptors.

1. Whole-cell patch-clamp recordings were made from visually identified hippocampal interneurones in slices of rat brain tissue in vitro. Bath application of the bombesin-like neuropeptides gastrin-releasing peptide (GRP) or neuromedin B (NMB) produced a large membrane depolarization that was blocked by pre-incubation with the subtype 2 bombesin (BB2) receptor antagonist [D-Phe6, Des-Met14]bombesin-(6-14)ethyl amide. 2. The inward current elicited by NMB or GRP was unaffected by K+ channel blockade with external Ba2+ or by replacement of potassium gluconate in the electrode solution with caesium acetate. 3. Replacement of external NaCl with Tris-HCl significantly reduced the magnitude of the GRP-induced current at -60 mV. In contrast, replacement of external NaCl with LiCl had no effect on the magnitude of this current. 4. Photorelease of caged GTPgammaS inside neurones irreversibly potentiated the GRP-induced current at -60 mV. Similarly, bath application of the phospholipase C (PLC) inhibitor U-73122 significantly reduced the size of the inward current induced by GRP. 5. Reverse transcription followed by the polymerase chain reaction using cytoplasm from single hippocampal interneurones demonstrated the expression of BB2 receptor mRNA together with glutamate decarboxylase (GAD67). 6. Although bath application of GRP or NMB had little or no effect on the resting membrane properties of CA1 pyramidal cells per se, these neuropeptides produced a dramatic increase in the number and amplitude of miniature inhibitory postsynaptic currents in these cells in a TTX-sensitive manner.

Novel expression and regulation of gastrin gene in human ovarian cancer cell line, SW626.

Gastrin-secreting tumors have been identified in ectopic locations including the ovary; the mechanisms regulating gastrin gene expression, its distribution, and signaling pathways in these ectopic tissues are not known. The purpose of our present study was to determine: (1) whether the gastrin gene and peptide could be detected in ovarian cancer cell lines, (2) if functional gastrin releasing peptide receptors (GRP-R) are present, and (3) whether gastrin gene expression is altered by GRP. Five ovarian cancer cell lines (SW626, OVCA 420, OVCA 429, OVCA 432, and OVCA 433) were analyzed. We identified gastrin gene and peptide expression in the SW626 cell line but not in the OVCA lines. SW626 cells express a functional GRP-R that is correctly coupled to the Ca2+ signaling pathway. Treatment of SW626 cells with bombesin, the amphibian equivalent of GRP, inhibited expression of the gastrin gene in a time- and dose-dependent fashion. The SW626 ovarian cancer cell line will provide a useful model to further define regulation and expression of both the gastrin gene and peptide in ectopic (nongastrointestinal) tissues.

Discovery of a high affinity radioligand for the human orphan receptor, bombesin receptor subtype 3, which demonstrates that it has a unique pharmacology compared with other mammalian bombesin receptors.

An orphan receptor discovered in 1993 was called bombesin receptor subtype 3 (BRS-3) because of 47-51% amino acid identity with bombesin (Bn) receptors. Its pharmacology is unknown, because no naturally occurring tissues have sufficient receptors to allow studies. We made two cell lines stably expressing the human BRS-3 (hBRS-3). hBRS-3 was overexpressed in the human non-small cell lung cancer cells, NCI-H1299, and the other was made in Balb 3T3 cells, which lack endogenous BRS-3. [D-Phe6,beta-Ala11,Phe13, Nle14]Bn-(6-14) (where Nle represents norleucine) was discovered to have high potency for stimulating inositol phosphate formation in both cell lines. [125I-D-Tyr6,beta-Ala11,Phe13, Nle14]Bn-(6-14) bound to both cell lines with high affinity. Neither Bn nor 14 other naturally occurring Bn peptides bound to hBRS-3 with a Kd <1000 nM. Twenty-six synthetic peptides that are high affinity agonists or antagonists at other bombesin receptors had an affinity >1000 nM. Guanosine 5'-(beta,gamma-imido)triphosphate inhibited binding to both cells due to a change in receptor affinity. These results demonstrate hBRS-3 has a unique pharmacology. It does not interact with high affinity with any known natural agonist or high affinity antagonist of the Bn receptor family, suggesting the natural ligand is either an undiscovered member of the Bn peptide family or an unrelated peptide. The availability of these cell lines and the hBRS-3 ligand should facilitate identification of the natural ligand for BRS-3, its pharmacology, and cell biology.