Isolation, identification and biological activity of gastrin-releasing peptide 1-46 (oGRP 1-46), the primary GRP gene-derived peptide product of the pregnant ovine endometrium.

We have previously demonstrated that pregnant ovine endometrium expresses the gastrin-releasing peptide (GRP) gene at a high level following conceptus implantation. Here we report the isolation, characterization and biological activity of ovine GRP 1-46, the primary product of this gene in the pregnant endometrium. Full thickness 125-140-day pregnant sheep uterus (term is 145 day) was homogenized in 80% acetonitrile/2% trifluoroacetic acid (1:7 ACN/TFA), concentrated on reverse-phase C18 cartridges and chromatographed successively on gel filtration (Sephadex G-50) and reverse-phase HPLC (C18 muBondapak). Purification was monitored by RIA. Purified GRP peptide was analysed by mass spectrometry giving a major mass ion at 4963 which corresponds exactly to GRP 1-46. Other mass ions from pro-GRP did not contain a biologically active N-terminus or antigenic determinant. Proteolytic cleavage of pro-GRP to give rise to GRP(1-46) would require preferential cleavage at the Glu-Glu bond by a Glu-C2-like enzyme, rather than the trypsin-like and C-terminal amidation enzymes (PAM) that produce GRP(18-27) and GRP(1-27) in other tissues. GRP 1-46 was synthesized and receptor binding and biological activity tested on a range of rodent and human cell lines that express GRP-related receptors GRPR, NMBR and BRS3. GRP 1-46 bound GRPR and NMBR with low affinity, and mobilized inositol phosphate in cell lines expressing the GRPR and NMBR, but not BRS-3. This study describes a new processed product of the GRP gene, GRP 1-46, which is highly expressed in the pregnant sheep endometrium and which acts as a weak agonist at the GRPR and NMBR.

Dithiolethione compounds inhibit Akt signaling in human breast and lung cancer cells by increasing PP2A activity.

The chemopreventative effects of dithiolethione compounds are attributed to their activation of antioxidant response elements (AREs) by reacting with the Nrf2/Keap1 protein complex. In this study, we show antiproliferative effects of the dithiolethione compound ACS-1 in human cancer cell lines (A549 and MDA-MB-231) by increasing the activity of the tumor suppressor protein phoshatase 2A (PP2A). ACS-1 inhibited epidermal growth factor (EGF)-induced cellular proliferation in a concentration- and time-dependent manner. Akt activation, as determined by serine-473 phosphorylation, was inhibited by ACS-1 in cells stimulated with either EGF or fibronectin. Furthermore, ACS-1 inhibited mammalian target of rapamycin signaling and decreased c-myc protein levels. ACS-1 did not proximally alter EGF receptor or integrin signaling, but caused a concentration-dependent increase in PP2A activity. The effect of ACS-1 on Akt activation was not observed in the presence of the PP2A inhibitor okadaic acid. ACS-1 effects on PP2A activity were independent of ARE activation and cAMP formation. In addition to ACS-1, other dithiolethione compounds showed similar effects in reducing Akt activation, suggesting that this class of compounds may have other effects beyond chemoprevention.

Modulation of angiogenesis by dithiolethione-modified NSAIDs and valproic acid.

BACKGROUND AND PURPOSE: Angiogenesis involves multiple signaling pathways that must be considered when developing agents to modulate pathological angiogenesis. Because both cyclooxygenase inhibitors and dithioles have demonstrated anti-angiogenic properties, we investigated the activities of a new class of anti-inflammatory drugs containing dithiolethione moieties (S-NSAIDs) and S-valproate. EXPERIMENTAL APPROACH: Anti-angiogenic activities of S-NSAIDS, S-valproate, and the respective parent compounds were assessed using umbilical vein endothelial cells, muscle and tumor tissue explant angiogenesis assays, and developmental angiogenesis in Fli:EGFP transgenic zebrafish embryos. KEY RESULTS: Dithiolethione derivatives of diclofenac, valproate, and sulindac inhibited endothelial cell proliferation and induced Ser(78) phosphorylation of hsp27, a known molecular target of anti-angiogenic signaling. The parent drugs lacked this activity, but dithiolethiones were active at comparable concentrations. Although dithiolethiones can potentially release hydrogen sulphide, NaSH did not reproduce some activities of the S-NSAIDs, indicating that the dithioles regulate angiogenesis through mechanisms other than release of H(2)S. In contrast to the parent drugs, S-NSAIDs, S-valproate, NaSH, and dithiolethiones were potent inhibitors of angiogenic responses in muscle and HT29 tumor explants assessed by 3-dimensional collagen matrix assays. Dithiolethiones and valproic acid were also potent inhibitors of developmental angiogenesis in zebrafish embryos, but the S-NSAIDs, remarkably, lacked this activity. CONCLUSIONS AND IMPLICATION: S-NSAIDs and S-valproate have potent anti-angiogenic activities mediated by their dithiole moieties. The novel properties of S-NSAIDs and S-valproate to inhibit pathological versus developmental angiogenesis suggest that these agents may have a role in cancer treatment.

Peptide hormones and lung cancer.

Several peptide hormones have been identified which alter the proliferation of lung cancer. Small cell lung cancer (SCLC), which is a neuroendocrine cancer, produces and secretes gastrin releasing peptide (GRP), neurotensin (NT) and adrenomedullin (AM) as autocrine growth factors. GRP, NT and AM bind to G-protein coupled receptors causing phosphatidylinositol turnover or elevated cAMP in SCLC cells. Addition of GRP, NT or AM to SCLC cells causes altered expression of nuclear oncogenes, such as c-fos, and stimulation of growth. Antagonists have been developed for GRP, NT and AM receptors which function as cytostatic agents and inhibit SCLC growth. Growth factor antagonists, such as the NT1 receptor antagonist SR48692, facilitate the ability of chemotherapeutic drugs to kill lung cancer cells. It remains to be determined if GRP, NT and AM receptors will served as molecular targets, for development of new therapies for the treatment of SCLC patients. Non-small cell lung cancer (NSCLC) cells also have a high density of GRP, NT, AM and epidermal growth factor (EGF) receptors. Several NSCLC patients with EGF receptor mutations respond to gefitinib, a tyrosine kinase inhibitor. Gefitinib relieves NSCLC symptoms, maintaining stable disease in patients who are not eligible for systemic chemotherapy. It is important to develop new therapeutic approaches using translational research techniques for the treatment of lung cancer patients.

VIP-ellipticine derivatives inhibit the growth of breast cancer cells.

The effects of vasoactive intestinal peptide (VIP)-ellipticine (E) derivatives were investigated on breast cancer cells. VIP-ALALA-E and VIP-LALA-E inhibited 125I-VIP binding to MCF-7 cells with an IC(50) values of 1 and 0.2 microM respectively. VIP-ALALA-E and VIP-LALA-E caused elevation of cAMP in MCF-7 cells with ED(50) values of 1 and 0.1 microM. VIP-LALA-E caused increased c-fos mRNA in MCF-7 cells. Radiolabeled VIP-LALA-E was internalized at 37 degrees C and delivered the cytotoxic E into MCF-7 cells. VIP-LALA-E inhibited the clonal growth of MCF-7 cells, decreased cell viability based on trypan blue exclusion and reduced 35S-methionine uptake. These results indicate that VIP-E derivatives function as breast cancer VPAC(1) receptor agonists which inhibit MCF-7 cellular viability.

Neuropeptides: brain messengers of many faces.

Neuropeptides 2001, 2nd Joint Meeting of the European Neuropeptide Club and the American Summer Neuropeptide Conference (11th Annual Meeting). 6-11 May 2001 with Satellite Symposium, Israeli-French Symposium, Israel Ministry of Science, Culture and Sport, 6 May 2001, held at Maale Hachmicha and Tel Aviv University, Israel.

CI-988 inhibits growth of small cell lung cancer cells.

The effects of cholecystokinin (CCK) antagonists on small cell lung cancer (SCLC) cells were investigated. CI-988, L-365,260, and L-364,718 inhibited specific (125)I-CCK-8 binding to NCI-H209 cells with IC(50) values of 5, 2, and 200 nM. ([R-(R*,R*)]-4[[2-[[3-(1H-Indole-3-yl)-2-methyl-1-oxo-2-[[tricyclo[3.3.1.1(3,7)]- dec-2-yloxy)carbonyl[amino]propyl]amino]-1-phenylethyl]amino]-4-oxobutanoic acid) (CI-988; 100 nM) inhibited the ability of 10 nM CCK-8 to elevate cytosolic Ca(2+) in 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2'-amino-5'-methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester-loaded NCI-H209 cells. By Western blot, CI-988 inhibited tyrosine phosphorylation of focal adhesion kinase and paxillin stimulated by CCK-8. Also, CI-988 inhibited tyrosine phosphorylation of mitogen-activated protein kinase stimulated by CCK-8. By Northern blot, CI-988 antagonized the ability of 10 nM CCK-8 to increase c-fos mRNA in NCI-H209 cells. Also, CI-988 inhibited the ability of CCK-8 to increase vascular endothelial cell growth factor mRNA. Using a [3-(4,5 dimethylthiazol-2-yl)-2.5-diphenyl-2H-tetrazolium bromide] and clonogenic assay, CI-988 inhibited the proliferation of NCI-H209 cells in vitro. Using nude mice, CI-988 inhibited the proliferation of NCI-H209 xenografts. These results suggest that CI-988 is a CCK(2) receptor antagonist that inhibits the proliferation of SCLC cells.

VIP receptor antagonists and chemotherapeutic drugs inhibit the growth of breast cancer cells.

The effects of vasoactive intestinal peptide (VIP) antagonists on breast cancer cells were investigated. (N-stearyl, norleucine17)VIP hybrid ((SN)VIPhyb) inhibited specific 125I-VIP binding to MCF7, SKBR3, T47D ZR75-1 and MDA-MB231 cells with high affinity (IC50 values of 0.03-0.06 microM). (SN)VIPhyb, 1 microM, inhibited the ability of 10 nM VIP to cause elevation of cAMP and to increase c-fos mRNA. Micromolar concentrations of (SN)VIPhyb inhibited the proliferation of MDA-MB231 or MCF7 cells using a MTT and clonogenic assay. Using a MTT assay, (SN)VIPhyb enhanced the ability of taxol and doxorubicin to inhibit breast cancer growth. Using nude mice bearing MDA-MB231 xenografts, VIPhyb potentiated the ability of taxol to inhibit proliferation. The results indicate that VIP receptor antagonists increase the ability of chemotherapeutic drugs to kill breast cancer cells.

A lipophilic vasoactive intestinal peptide analog enhances the antiproliferative effect of chemotherapeutic agents on cancer cell lines.

BACKGROUND: Vasoactive intestinal peptide (VIP) is one of several small neuropeptides that affect cancer growth. A lipophilic VIP analog, stearyl-Nle(17)-neuroten-sin(6-11)VIP(7-28) (SNH) that inhibited lung carcinoma growth has been described previously. The experiments performed were clonogenic assays in vitro and tumor xenografts in nude mice in vivo. These studies were now extended to colon carcinoma and to combination therapy with chemotherapeutic agents. METHODS: Assays were performed with cell lines, and tumor proliferation was assessed using the (3-[4,5-dimethylthiazol-2-yl-5]-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H tetrazolium) (MTS) colorimetric assay for mitochondrial function of living cells. RESULTS: The lipophilic analog (SNH) enhanced the antiproliferative activity of diverse chemotherapeutic agents: doxorubicine (antibiotic); vinorelbine (vinca alkaloid, antimicrotubule formation); paclitaxel (antimicrotubule agent); gemcitabine (antimetabolite); irinotecan (topoisomerase I inhibitor); and cisplatin (platinum compound acting as an alkylating agent). In all cases, the antiproliferative effect of SNH and the chemotheraputic agent was at least additive and for some combinations and concentrations even synergistic. For example, 2 microM of the antagonist that produced a 15-20% growth inhibition in the nonsmall cell lung carcinoma cell line reduced the IC(50) by 2-4-fold for most of the chemotherapeutic agents tested. Higher analog concentrations were even more efficacious. Similar results were obtained with colon carcinoma cell lines. CONCLUSIONS: Chemotherapeutic treatment of advanced solid tumors, such as nonsmall cell lung carcinoma, colon carcinoma, or prostate carcinoma, achieves a response rate of between 10% and 30% with significant toxicity. Combination therapy with the lipophilic VIP analog SNH and the preferred chemotherapeutic agent may greatly enhance the response rate, and by permitting a dose reduction, should significantly reduce side effects.

Indomethacin reduces lung adenoma number in A/J mice.

The effects of indomethacin on A/J mice were investigated. The non-steroidal antiinflammatory drug (NSAID) indomethacin reduced significantly the number of lung adenomas 3, 4 or 8 months after urethane injection by 28, 30 and 29% respectively. The density of apoptotic cell bodies increased 2.9-fold in the lung adenomas of A/J mice treated with indomethacin. By immunocytochemistry, COX-2 immunoreactivity was present in the cytosol of lung adenomas, and in epithelial cells lining the bronchioli and bronchus as well as type 2 alveolar cells. COX-1 immunostaining was similar to that of COX-2 in the lungs of urethane-injected mice treated with or without indomethacin. By RT-PCR, COX-1 and COX-2 PCR products were present in mouse lung adenomas, alveoli and bronchioli. These results suggest that indomethacin may inhibit COX-1 and COX-2 in the A/J mouse lung resulting in reduced adenoma formation.

SR48692 is a neurotensin receptor antagonist which inhibits the growth of small cell lung cancer cells.

Neurotensin (NT) is an autocrine growth factor for some small cell lung cancer (SCLC) cells. In this communication, the effects of a non-peptide NT receptor antagonist, SR48692, were investigated using SCLC cells. (3)H-SR48692 bound with high affinity (IC(50) = 20 nM) to NCI-H209 cells. Also, NT and SR48692 inhibited specific (125)I-NT binding with high affinity (IC(50) values of 2 and 200 nM). In contrast, the NT(2) receptor agonist, levocabastine, had little effect on specific (125)I-NT binding, second messenger production and proliferation using NCI-H209 cells. SR48692 (5 microM) antagonized the ability of NT (10 nM) to cause elevated cytosolic Ca2+ in Fura-2 AM loaded NCI-H209 cells. SR48692 antagonized the ability of NT to cause elevation of c-fos mRNA in these cells. Using a MTT proliferation assay, SR48692 inhibited NCI-H209 and H345 proliferation in a concentration-dependent manner. Using a clonogenic assay, 1 microM SR48692, reduced NCI-H209 colony number. Also, SR48692 (0.4 mg/kg per day) inhibited NCI-H209 xenograft proliferation in nude mice. These results suggest that SR48692 is a NT(1) receptor antagonist which inhibits SCLC growth.

Prostaglandin E2 and vasoactive intestinal peptide increase vascular endothelial cell growth factor mRNAs in lung cancer cells.

The effects of prostaglandin E2 (PGE2) and vasoactive intestinal peptide (VIP) on vascular endothelial cell growth factor (VEGF) mRNAs were investigated using lung cancer cells. By RT-PCR, VEGF(121), VEGF(165), and VEGF(189), but not VEGF(206) isoforms were detected in all lung cancer cell lines and biopsy specimens examined. By Northern blot, VEGF mRNA was detected in all small cell lung cancer (SCLC) and non-SCLC (NSCLC) cell lines examined. PGE2, VIP and forskolin caused increased VEGF expression in a time- and concentration-dependent manner using NSCLC cell line NCI-H157. Approximately 1 microM PGE2, 0.1 microM VIP and 50 microM forskolin caused cAMP elevation, 64-, 33- and 128-fold, respectively, using NCI-H157 cells after 5 min. The increase in cAMP caused by PGE(2) and VIP was reversed by somatostatin (SST). Also 1 microM PGE2, 0.1 microM VIP and 50 microM forskolin increased the VEGF mRNA 2.0-, 1.5- and 2.3-fold, respectively, after 4 h. The increase in VEGF mRNA caused by PGE2, VIP and forskolin was inhibited by H-89, a protein kinase A inhibitor. A VIP receptor antagonist, VIPhybrid, inhibited the increase in cAMP and VEGF mRNA caused by VIP. By ELISA, VEGF was detected in the conditioned media exposed to the lung cancer cell lines. These results suggest that VEGF synthesis in and secretion from lung cancer cells can be regulated by agents, which cause adenylyl cyclase 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.

A vasoactive intestinal peptide antagonist inhibits the growth of glioblastoma cells.

The effects of a vasoactive intestinal peptide (VIP) receptor antagonist (VIPhyb) on human glioblastoma cells were characterized. Pituitary adenylate cyclase activating polypeptide (125I-PACAP-27) bound with high affinity to U87, U118, and U373 cells. Specific 125I-PACAP-27 binding to U87 cells was inhibited, with high affinity, by PACAP but not VIP or VIPhyb (IC50 = 10, 1500, and 500 nM, respectively). By reverse transcriptase-polymerase chain reaction (RT-PCR), a major 305 bp band was observed indicative of PAC1 receptors. PACAP-27 caused cAMP elevation and the increase in cAMP caused by PACAP-27, was inhibited by the VIPhyb. Also, PACAP-27 caused cytosolic Ca2+ elevation in Fura-2AM loaded U87 cells and the VIPhyb inhibited this increase. Using the MTT growth assay, the VIPhyb was shown to inhibit glioblastoma growth in a concentration-dependent manner. Using a clonogenic assay in vitro, 10 microM VIPhyb significantly inhibited proliferation of U87, U118, and U373 cells. In vivo, 0.4 microg/kg VIPhyb inhibited U87 xenograft proliferation in nude mice. These results suggest that the VIPhyb antagonizes PAC1 receptors on glioblastoma cells and inhibits their proliferation.

Bombesin and gastrin releasing peptide increase tyrosine phosphorylation of focal adhesion kinase and paxillin in non-small cell lung cancer cells.

The effects of some oncogenes, growth factors and neuropeptides are mediated by tyrosine phosphorylation of focal adhesion kinase (p125(FAK)) and paxillin cytoskeletal proteins. In this study the ability of bombesin/gastrin releasing peptide (BB/GRP) to stimulate tyrosine phosphorylation of p125(FAK) and paxillin in non-small cell lung cancer (NSCLC) H1299 cells was investigated. BB, 100 nM caused increased p125(FAK) and paxillin tyrosine phosphorylation maximally after 1 min. The effect of BB on p125(FAK) and paxillin tyrosine phosphorylation was concentration-dependent, being half maximal at 4-8 nM. Also, 100 nM GRP, GRP(14-27) but not GRP(1-16) increased p125(FAK) and paxillin tyrosine phosphorylation indicating that the C-terminal of GRP is essential. BW2258U89, a GRP receptor antagonist, caused a dose-dependent inhibition of BB-stimulated p125(FAK) and paxillin tyrosine phosphorylation with an IC50 value of 3 microM. Cytochalasin D (0.3 microM), which inhibits actin polymerization, reduced the ability of BB to stimulate tyrosine phosphorylation of p125(FAK) and paxillin. Genistein (50 microM) and H-7 (50 microM), which are kinase inhibitors, reduced the tyrosine phosphorylation of p125(FAK) and paxillin stimulated by BB. Also, treatment of NCI-H1299 cells with FAK antisense resulted in decreased FAK tyrosine kinase activity and proliferation. These results suggest that p125(FAK) is an important enzyme for NSCLC proliferation.

Nonpeptide neuromedin B receptor antagonists inhibit the proliferation of C6 cells.

The ability of nonpeptide antagonists to interact with neuromedin B receptors on C6 cells was investigated. 2-[3-(2, 6-Diisopropyl-phenyl)-ureido]3-(1H-indol-3-yl)-2-methyl-N-(1-pyridin- 2-yl-cyclohexylmethyl)-proprionate (PD165929), 3-(1H-indol-3-yl)-2-methyl-2-[3(4-nitro-phenyl)-ureido]-N-(1-pyridin- 2-yl-cyclohexylmethyl)-propionamide (PD168368) and 3-(1H-indol-3-yl)-N-[1-(5-methoxy-pyridin-2-yl)-cyclohexylmethyl]- 2-m ethyl-2-[3-(4-nitro-phenyl)-ureido]-propionamide (PD176252) inhibited (125I-Tyr0)neuromedin B binding with IC50 values of 2000, 40 and 50 nM, respectively. Because neuromedin B is a G-protein coupled serpentine receptor, the effects of neuromedin B antagonists on second messenger production and proliferation were investigated. PD168368 inhibited the ability of 10 nM neuromedin B to cause elevation of cytosolic Ca2+, whereas it had no effect on basal cytosolic Ca2+. PD168368 inhibited the ability of 100 nM neuromedin B to cause elevation of c-fos mRNA. Also, PD168368 in a dose-dependent manner inhibited the ability of 100 nM neuromedin B to cause phosphorylation of focal adhesion kinase. Using a [3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay, the order of antagonist potency to inhibit C6 proliferation was PD168368=PD176252>PD165929. Also, 1 microM PD168368 and PD176252 significantly inhibited colony number using a proliferation assay in vitro. PD168368 significantly inhibited C6 xenograft growth in nude mice in vivo. These results indicate that PD168368 is a C6 cell neuromedin B receptor antagonist, which inhibits proliferation.

Enhanced tumorigenesis and reduced transforming growth factor-beta type II receptor in lung tumors from mice with reduced gene dosage of transforming growth factor-beta1.

To elucidate the role of transforming growth factor-beta1 (TGF-beta1) and the TGF-beta type II receptor (TGF-beta RII) as tumor-suppressor genes in lung carcinogenesis, we mated C57BL/6 mice heterozygous (HT) for deletion of the TGF-beta1 gene with A/J mice to produce AJBL6 TGF-beta1 HT progeny and their wild-type (WT) littermates. Immunohistochemical staining, in situ hybridization, and northern blot analyses showed lower staining and hybridization for TGF-beta1 protein and mRNA, respectively, in the lungs of normal HT mice versus WT mice. Competitive reverse transcription-polymerase chain reaction (CRT-PCR) amplification showed the level of TGF-beta1 mRNA in the lungs of HT mice to be fourfold lower than the level in WT lung. When challenged with ethyl carbamate, lung adenomas were detected in 55% of HT mice by 4 mo but only in 25% of WT littermates at this time. Whereas all HT mice had adenomas by 6 mo, it was not until 10 mo before all WT mice had adenomas. After 12 mo, the average number of adenomas was fivefold higher in HT lungs than in WT lungs. Most dramatic was the appearance of lung carcinomas in HT mice 8 mo before they were visible in WT mice. Thus, the AJBL6 TGF-beta1 HT mouse provides an excellent model system to examine carcinogen-induced lung tumorigenesis by increasing progressive lesion incidence and multiplicity relative to their WT littermates. Immunohistochemical staining showed expression of the TGF-beta type I receptor (TGF-beta RI) at moderate to strong levels in lung adenomas and carcinomas in HT and WT mice. In contrast, whereas weak immunostaining for TGF-beta RII was detected in 67% of HT carcinomas at 12 mo, only 22% of WT carcinomas showed weak staining for this protein. Individual lung carcinomas showing reduced TGF-beta RII expression and adjacent normal bronchioles were excised from HT lungs using laser capture microdissection, and CRT-PCR amplification of the extracted RNA showed 12-fold less TGF-beta RII mRNA in these carcinomas compared with bronchioles. Decreasing TGF-beta RII mRNA levels occurred with increasing tumorigenesis in lung hyperplasias, adenomas, and carcinomas, with carcinomas having fourfold and sevenfold lower levels of TGF-beta RII mRNA than adenomas and hyperplasias, respectively. These data show enhanced ethyl carbamate-induced lung tumorigenesis in AJBL6 HT mice compared with WT mice, suggesting that both TGF-beta1 alleles are necessary for tumor-suppressor activity. Reduction of TGF-beta RII mRNA expression in progressive stages of lung tumorigenesis in HT mice suggests that loss of TGF-beta RII may play an important role in the promotion of lung carcinogenesis in mice with reduced TGF-beta1 gene dosage when challenged with carcinogen.

The metabolism of BW2258U89, a GRP receptor antagonist.

BW2258U89 is a gastrin releasing peptide (GRP) receptor antagonist which inhibits the proliferation of the neuroendocrine tumor small cell lung cancer (SCLC). Here the biological activity of BW2258U89 and its metabolite were investigated. Using mass spectroscopy (LC-ESI/MS) techniques, three major peaks for BW2258U89 were observed with mass/charge (m/z) ratios of 1081.6, 541.4 and 361.4. After metabolism by mouse plasma enzymes, the major product had a m/z ratio of 1082.5, 541.9 and 361.8 suggesting that BW2258U89 was deamidated. Deamidated (Da) BW2258U89 was synthesized and it inhibited ((125)I-Tyr(4)) BB binding to NCI-H345 SCLC cells with an IC(50)value of 450 nM; BW2258U89 had an IC(50)value of 17 nM. BW2258U89 (1 microM) antagonized the ability of 50 nM BB to elevate cytosolic Ca(2+)in NCI-H345 cells, whereas 1 microM (Da) BW2258U89 did not. One micromolar BW2258U89 antagonized the increase in NCI-H345 c-fos mRNA caused by 10 nM BB, whereas 1 microM (Da) BW2258U89 had little effect. One microM BW2258U89 inhibited NCI-H345 clonal growth significantly whereas 1 microM (Da) BW2258U89 did not. These data suggest that an amidated C-terminal is important for antagonism of SCLC GRP receptors by BW2258U89.

(Tyr(0),Bpa(4))bombesin is a GRP receptor agonist.

(Tyr(0),Bpa(4))bombesin, (YB)BB was synthesized and its biologic activity evaluated using T47D breast cancer cells. ((125)I-Tyr(0), Bpa(4))BB bound with high affinity (K(d) = 5 nM) to T47D cells. Specific ((125)I-Tyr(0),Bpa(4))BB binding was inhibited with high affinity by BB, BW2258U89, GRP, GRP(14-27) and NMB (IC(50) values of 10, 2, 15, 20, and 150 nM)but not GRP(1-16) (IC(50) value of > 1000 nM). ((125)I-Tyr(0),Bpa(4))BB bound to the surface of T47D cells at 4 degrees C but was internalized at 37 degrees C. After binding at 4 degrees C followed by irradiation using ultraviolet light, ((125)I-Tyr(0),Bpa(4))BB labeled a 75 kDa protein using T47D cells. (Tyr(0),Bpa(4))BB, 10 nM, elevated cytosolic calcium using T47D cells within 10 s. Also (Tyr(0),Bpa(4))BB, 10 nM, elevated c-fos mRNA after 45 min. These results indicate that (Tyr(0),Bpa(4))BB is an agonist for GRP receptors.

Sigma ligands inhibit the growth of small cell lung cancer cells.

The effects of sigma ligands on small cell lung cancer (SCLC) cells were investigated. 125I-N-(2-(piperidino)ethyl)-2-iodobenazmide (2-IBP) bound with high affinity to SCLC cell line NCI-H209 and NCI-N417. Specific 125I-2-IBP binding was inhibited with high affinity by ifendipine, haloperidol, (2-piperidinyl-aminoethyl)-4-iodobenzamide (IPAB) and 1,3-ditolylguanidine (DTG) with IC50 values of 3, 10, 15 and 90 nM respectively. In vitro, 10 microM 2-IBP, haloperidol or IPAB inhibited NCI-N417 proliferation using a MTT or clonogenic assay. In vivo, 4 mg/kg IPAB or 2-IBP inhibited NCI-N417 xenograft proliferation. 125I-2-IBP localized to the SCLC tumors after subcutaneous injection. These results suggest that sigma ligands may be utilized to localize and inhibit the proliferation of SCLC tumors.