gastrin-releasing-peptide and Carcinoma--Non-Small-Cell-Lung

Lung cancer is one of the most common cancers in the world. While historically, more men than women have died from lung cancer as a result of higher numbers of male smokers, the sex mortality ratio is now showing signs of narrowing. Tumors in women with lung cancer may be slightly different to those in men with lung cancer. This review focuses on biomarkers differentially expressed between female and male patients with lung cancer. There is variation in gene expression between men and women in some genes that encode carcinogen-metabolizing enzymes (CYP1A1, GSTM). Gastrin-releasing peptide (GRP), a bombesin-like peptide, is present in two actively transcribed alleles in women compared with men. Higher prevalence of infection with oncogenic variants human papilloma viruses (HPVs) HPV16 and HPV18 has been suggested in women. A higher frequency of G to T transversion was found in the p53 gene in lung tumors of women. KRAS mutation was found to be more frequent in women with resected non-small cell lung cancer (NSCLC) than in men with resected NSCLC. Epidermal growth factor receptor (EGFR) mutation is more frequently found in lung tumors from women, but the confounding effect of tobacco exposure may explain this difference. Lower levels of ERCC1 and BRCA1 have been reported in women with NSCLC. Lung tumors from women are more likely to express estrogen receptors than those from men. An in silico analysis of transcriptome datasets from lung cancer patients demonstrated that only seven genes (in at least two studies) had significantly different expression patterns in male versus female patients. All of these genes are localized on the sex chromosomes: one on chromosome X and six on chromosome Y. Many areas remain under debate and there are still significant gaps in our understanding, particularly how sex-linked factors relate to lung cancer risk, and to biological and clinical behaviors. Future research into lung cancer needs to address these gender differences more specifically.

Topics: Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Cytochrome P-450 CYP1A1; Female; Gastrin-Releasing Peptide; Gene Expression; Glutathione Transferase; Humans; Lung Neoplasms; Male; Mutation; Polymorphism, Single Nucleotide; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); ras Proteins; Receptors, Estrogen; Sex Factors; Smoking; Women's Health

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.

Topics: Adrenomedullin; Amino Acid Sequence; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Gastrin-Releasing Peptide; Humans; Lung Neoplasms; Molecular Sequence Data; Neurotensin; Peptide Hormones; Peptides

Topics: Amino Acid Sequence; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Epidermal Growth Factor; Gastrin-Releasing Peptide; Growth Substances; Humans; Insulin-Like Growth Factor I; Lung Neoplasms; Molecular Sequence Data; Peptides

As we expand our knowledge of the initiators and promoters of lung cancer, early detection and intervention strategies show great potential in individuals at high risk, especially smokers and exsmokers. Documented mutations of dominant oncogenes and tumor suppressor genes in human lung cancer cells may represent important steps in the pathogenesis of invasive cancer. The precise molecular events and their sequence that lead to tumor promotion in lung cancer, however, are less well understood. Chemointervention with agents like the retinoids may halt proliferation of cancer cells prior to the development of metastatic competence. Use of anti-growth-factor therapy and peptide hormone antagonists may also have a role in intervention approaches. This paper reviews present understanding of the initiation and promotion of lung cancer, as well as preventive strategies currently proposed for patients at risk.

Topics: Carcinogens; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Gastrin-Releasing Peptide; Gastrins; Genes, ras; Genes, Tumor Suppressor; Humans; Lung Neoplasms; Oncogenes; Peptides; Protein Processing, Post-Translational; Retinoids; Smoking; Tumor Cells, Cultured

Topics: Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Gastrin-Releasing Peptide; Growth Inhibitors; Growth Substances; Humans; Lung Neoplasms; Peptides

Small cell lung cancer (SCLC) cells express and secrete bombesin-like peptides (BLP) that can activate specific receptors that stimulate the growth of these cells. A murine monoclonal antibody, 2A11, which binds to the BLP, gastrin-releasing peptide with high affinity, has been reported to decrease the growth of SCLC cells in vitro and in athymic nude mice. A Phase I trial in lung cancer patients was performed using multiple doses of 2A11. Thirteen patients with lung cancer received 12 doses of 2A11 antibody three times a week for 4 weeks at one of four dose levels. Serum samples were obtained prior to initiation and before each dose of 2A11 antibody therapy for measurement of 2A11 antibody levels and determination of serum human anti-mouse antibody levels. A pilot imaging evaluation using 111In conjugated 2A11 monoclonal antibody was also performed in the same patients to aid in the study of pharmacokinetics and biodistribution. No toxic reactions were observed, and none of the patients developed detectable human antimouse antibody; however, no objective antitumor responses were observed. The mean trough serum 2A11 levels in patients increased with increasing dose level: 0.26+/-0.2 microg/ml, 6.7+/-6 microg/ml, 71.5+/-60 microg/ml, 248+/-184 microg/ml for dose levels 1 mg/m2, 10 mg/m2, 100 mg/m2, and 250 mg/m2, respectively. At each dose level, sustained detectable serum levels of the monoclonal antibody were achieved. Tumor uptake was noted in 11 of 12 patients who were injected with 111In conjugated 2A11. Because no dose-limiting clinical toxicity was observed, a mathematical model was used to define the recommended Phase II dose of 250 mg/m2. This trial established that repeated doses of monoclonal antibody 2A11 could be given safely to patients, and sustained levels could be achieved for a 4-week schedule. Further evaluation of the antitumor effects of 2A11 is warranted.

Topics: Animals; Antibodies, Monoclonal; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Female; Gastrin-Releasing Peptide; Humans; Immunoglobulin G; Indium Radioisotopes; Lung Neoplasms; Male; Mice; Radioimmunodetection

To evaluate a newly developed Hybiome ProGRP chemiluminescent assay.. Analytical sensitivity, precision, recovery, and equivalency of serum and plasma, serum stability, and complement interference of the Hybiome ProGRP assay were evaluated. Serum specimens from 318 individuals including 38 small cell lung cancer (SCLC), 65 non-small cell lung cancer (NSCLC), 53 benign lung diseases, and 162 healthy controls were assessed using the Hybiome ProGRP assay and Roche Elecsys ProGRP assay, and the results were compared.. The Hybiome ProGRP assay showed good analytical sensitivity, precision, and accuracy, and it showed equivalence between serum and plasma and serum stability. The methodological comparison results showed good correlation between the Hybiome and Roche assays (slope, 0.9889; intercept, 1.28). Both the Hybiome and Roche assays showed good ability to distinguish between SCLC and NSCLC. Based on 95% specificity in the NSCLC cohort, a clinical differentiation cut-off for separating SCLC from NSCLC patients was 114 pg/mL for the Hybiome assay and 117 pg/mL for the Roche assay; the AUC was 0.9166 and the sensitivity was 71.05% for Hybiome and 0.9045 and 76.32% for Roche, respectively.. The Hybiome ProGRP chemiluminescent assay shows good analytical performance and good correlation with the Roche Elecsys ProGRP assay.

Topics: Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Gastrin-Releasing Peptide; Humans; Immunoassay; Lung Neoplasms; Peptide Fragments; Recombinant Proteins; Sensitivity and Specificity

Testing for circulating biomarkers in lung cancer is hampered by the insufficient specificity. We aimed to assess the relative diagnostic accuracy of pro-gastrin-releasing peptide (ProGRP) for the differential diagnosis of small cell lung cancer and compare it with more conventional biomarkers.. We enrolled a cohort of 390 patients with a clinical suspicion of lung cancer and for whom a histologic assessment was available. Serum or plasma samples were assessed for ProGRP, carcinoembryonic antigen, CYFRA 21-2, and neuron-specific enolase. The performance of each biomarker in discriminating the small cell lung cancer and squamous cell carcinoma/adenocarcinoma from non-malignant lung disease, and small cell lung cancer from squamous cell carcinoma/adenocarcinoma, was assayed by receiver operating characteristic curve analysis.. At the cut-off levels suggested by the manufacturers, ProGRP and neuron-specific enolase showed an almost identical sensitivity of 55.2% and 55.6%, respectively, in discriminating small cell lung cancer with respect to non-malignant lung disease. In order to quantify the added value of ProGRP to other conventional markers, we ran a multivariable logistic regression analysis, but the results showed that no markers improve the performance of ProGRP.. ProGRP and neuron-specific enolase individually appear more accurate than other conventional biomarkers for small cell lung cancer, but the union of two markers does not increase the accuracy. The very small target group of patients with small cell lung cancer is a limitation of this study, which can explain why ProGRP alone does not show a sensitivity higher than neuron-specific enolase, as reported by other authors.

Topics: Adenocarcinoma; Aged; Antigens, Neoplasm; Biomarkers, Tumor; Carcinoembryonic Antigen; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Diagnosis, Differential; Female; Follow-Up Studies; Gastrin-Releasing Peptide; Humans; Keratin-19; Lung Neoplasms; Male; Peptide Fragments; Prognosis; Recombinant Proteins; ROC Curve; Small Cell Lung Carcinoma

Gastrin-releasing peptide (GRP) is a mitogen for lung epithelial cells and initiates signaling through a G-protein-coupled receptor, gastrin-releasing peptide receptor (GRPR). Because GRPR transactivates the epidermal growth factor receptor (EGFR), we investigated induction by GRP of Akt, an EGFR-activated signaling pathway, and examined effects of GRP on viability of non-small cell lung carcinoma (NSCLC) cells exposed to the EGFR tyrosine kinase inhibitor gefitinib. GRP induced Akt activation primarily through c-Src-mediated transactivation of EGFR. Transfection of dominant-negative c-Src abolished GRP-induced EGFR and Akt activation. GRP induced release of amphiregulin, and pre-incubation with human amphiregulin neutralizing antibody eliminated GRP-induced Akt phosphorylation. Pretreatment with phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 completely blocked GRP-initiated Akt phosphorylation. These results suggest that GRP stimulates Akt activation primarily via c-Src activation, followed by extracellular release of the EGFR ligand amphiregulin, leading to the activation of EGFR and PI3K. Pretreatment of NSCLC cells with GRP resulted in an increase in the IC(50) of gefitinib of up to 9-fold; this protective effect was mimicked by the pretreatment of cells with amphiregulin and reversed by Akt or PI3K inhibition. GRP appears to rescue NSCLC cells exposed to gefitinib through release of amphiregulin and activation of the Akt pathway, suggesting GRPR and/or EGFR autocrine pathways in NSCLC cells may modulate therapeutic response to EGFR inhibitors.

Topics: Antibodies; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Dose-Response Relationship, Drug; Enzyme Activation; ErbB Receptors; Gastrin-Releasing Peptide; Gastrointestinal Agents; Gefitinib; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinazolines; Receptors, Bombesin; Signal Transduction; src-Family Kinases; Transfection

Bombesin (BN) or gastrin-releasing peptide (GRP) can stimulate the growth of neoplasms such as breast cancer and small-cell lung carcinoma (SCLC). Antagonists of BN/GRP have been shown to inhibit these cancers. We evaluated whether antagonists of BN/GRP can suppress the growth of human non-SCLC (NSCLC) xenografted into nude mice. The effect of the administration of BN/GRP antagonist RC-3940-II on the growth of H460 and A549 NSCLC cell lines orthotopically xenografted into the intrapulmonary interstitium was examined. Protein levels of K-Ras, COX-2, Akt/pAkt, WT p53, Erk1/2, and lung resistance-related protein (LRP) in tumors were analyzed by Western blot analaysis, and receptors for BN/GRP were investigated by radioligand-binding studies. The effect of RC-3940-II on the proliferation of H460 and A549 cells in vitro was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays. High-affinity receptors for BN/GRP were found on tumors. Treatment with RC-3940-II significantly (P < 0.001) inhibited growth of H460 and A549 NSCLC xenografts by 30-50% and led to an improved performance status, compared with controls. In H460 NSCLC, the antitumor effect was associated with a significant (P < 0.001) reduction in protein levels of K-Ras, COX-2, pAkt, and pERK1/2 and with a major augmentation in the expression of WT p53, compared with controls. In A549 NSCLC, pAkt and LRP were significantly down-regulated. Our findings demonstrate the efficacy of BN/GRP antagonist RC-3940-II for the treatment of NSCLC. The suppression of K-Ras, COX-2, pAkt, and LRP, as well as the up-regulation of WT p53 might contribute to the antitumor action of BN/GRP antagonists.

Topics: Animals; Bombesin; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cyclooxygenase 2; Disease Progression; Gastrin-Releasing Peptide; Humans; Lung Neoplasms; Mice; Mice, Nude; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Peptide Fragments; Phosphorylation; Proto-Oncogene Proteins c-akt; ras Proteins; Receptors, Bombesin; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

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.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Bombesin; Carcinoma, Non-Small-Cell Lung; Cell Division; Cytoskeletal Proteins; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Gastrin-Releasing Peptide; Genistein; Humans; Lung Neoplasms; Paxillin; Phosphoproteins; Phosphorylation; Protein-Tyrosine Kinases; Tumor Cells, Cultured; Tyrosine

The effects of bombesin (BB) on mitogen activated protein (MAP) kinase were investigated using non-small cell lung cancer (NSCLC) cells. By Western blot, both 42 and 44 kDalton forms of MAP kinase were present in NCI-H1299 and NCI-H838 cells. Addition of BB to NCI-H1299 cells resulted in phosphorylation of the MAP kinase substrate myelin basic protein (MBP). Phosphorylation of MBP was maximal 6 min after the addition of 10 nM BB to NCI-H1299 cells. Addition of gastrin releasing peptide (GRP) or GRP14-27 but not GRP1-16 to NCI-H 1299 cells caused MBP phosphorylation. The effects of BB were inhibited by BW2258U89, a BB receptor antagonist, and PD98059, a MAP kinase kinase inhibitor. Also, PD98059 inhibited the clonal growth of NCI-H1299 cells. These data suggest that MAP kinase may be an important regulatory enzyme in NSCLC.

Topics: Amino Acid Sequence; Bombesin; Calcium-Calmodulin-Dependent Protein Kinases; Carcinoma, Non-Small-Cell Lung; Gastrin-Releasing Peptide; Humans; Lung Neoplasms; Molecular Sequence Data; Phosphorylation; Tumor Cells, Cultured

Gastrin-releasing peptide (GRP), a member of the bombesin family of peptides, has been shown to have mitogenic activity in small cell lung carcinoma (SCLC), and to be produced by SCLC in an autocrine fashion. In this report, we demonstrate that both GRP and another member of the bombesin family of peptides, neuromedin B (NMB), are also autocrine growth factors for non-small cell lung carcinoma (NSCLC). Using the reverse transcription-polymerase chain reaction (RT-PCR), we have detected mRNA for the neuromedin B receptor (NMBR) in all 14 of the NSCLC cell lines examined. GRP receptor (GRPR) mRNA was also expressed in the majority of NSCLC cell lines (nine of 14). By immunoblotting using SDS-PAGE gradient gels fixed in trichloroacetic acid, GRP and NMB were found in fractions of culture medium that had been purified by high pressure liquid chromatography (HPLC) from NSCLC cell lines. NMB was detected in the conditioned medium of seven of nine cell lines and GRP in seven of nine cell lines; both peptides were produced in six cell lines. In four of the cell lines where both peptides were produced, the relative amount of NMB secreted into the medium was 7-15 times that of GRP; in the other two cases, the relative amounts of GRP and NMB were equivalent. Cultured human bronchial epithelial (HBE) cells expressed the GRPR and NMBR but did not produce either peptide. A subline of A549 cells that was adapted to grow in serum-free and growth factor-free conditions, termed A549-R(0), secreted both bombesin-like peptides (BLPs) into the culture medium. Using either a colony-forming assay or a BrDU incorporation assay, both NMB and GRP were found to be mitogens for three NSCLC cell lines that express mRNA for BLP receptors and secrete BLPs, regardless of which peptide and/or receptor subtype was detected. The monoclonal antibody 2A11, which preferentially recognizes GRP, was able to block the in vitro proliferative response to GRP in the BrDU incorporation assay, and partially blocked the response to NMB. The 2A11 antibody could only partially block the in vivo growth of cell lines that showed proliferative responses to BLPs. 2A11 antibody was more effective against the 239T cell line, which secreted a low amount of GRP into the medium (0.6 nM), compared to the 201T cell line, which secreted a higher amount of both GRP and NMB (4.2 nM and 36.6 nM, respectively). These results suggest that both NMB and GRP are autocrine growth factors for NSCLC, but that the production o

Topics: Animals; Antibodies, Blocking; Antibodies, Monoclonal; Autocrine Communication; Carcinoma, Non-Small-Cell Lung; Cell Division; Culture Media, Conditioned; Female; Gastrin-Releasing Peptide; Humans; Immunoblotting; Lung Neoplasms; Mice; Mice, Nude; Mitogens; Neurokinin B; Receptors, Bombesin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Neutral endopeptidase (NEP; CALLA, CD10, EC 3.4.24.11) is a cell surface endopeptidase that hydrolyses bioactive peptides, including the bombesin-like peptides, as well as other neuropeptides. Bombesin-like peptides and other neuropeptides are autocrine growth factors for both small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Low expression of NEP has been reported in SCLC and NSCLC cell lines. NEP inhibition has been shown to increase proliferation in one cell line. To date, NEP expression has not been quantitatively evaluated in normal adult lung, SCLC or NSCLC tumors, paired uninvolved lung from the same patient, or in other pulmonary neoplasms such as mesotheliomas and carcinoids. We examined the expression of NEP in these tissues and human cell lines using immunohistochemistry, flow cytometry, enzyme activity, ELISA, Western blot, and reverse transcription (RT)-PCR. Uninvolved lung tissue from different individuals displayed considerable variation in NEP activity and protein. By immunohistochemistry, NEP expression was detectable in alveolar and airway epithelium, fibroblasts of normal lung, and in mesotheliomas, whereas it was undetectable in most SCLC, adenocarcinoma, squamous cell carcinoma, and carcinoid tumors of the lung. NEP activity and protein levels were lower in all SCLC and adenocarcinoma tumors when compared to adjacent uninvolved lung, often at levels consistent with expression derived from contaminating stroma. NEP expression and activity were reduced or undetectable in most SCLC and lung adenocarcinoma cell lines. NEP mRNA by RT-PCR was not expressed or was in low abundance in the majority of lung cancer cell lines. The majority of lung tumors did not express NEP by RT-PCR as compared with normal adjacent lung. In addition, recombinant NEP abolished, whereas an NEP inhibitor potentiated, the calcium flux generated by neuropeptides in some lung cancer cell lines, demonstrating potential physiological significance for low NEP expression. NEP, therefore, is a signal transduction and possibly a growth modulator for both SCLC and NSCLC, emphasizing the role of neuropeptides in the pathogenesis of the major histological forms of lung cancer.

Topics: Adenocarcinoma; Adult; Base Sequence; Blotting, Western; Bradykinin; Calcium; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Cell Line; DNA Primers; Enzyme Inhibitors; Gastrin-Releasing Peptide; Gene Expression; Glycopeptides; Humans; Immunohistochemistry; Lung; Lung Neoplasms; Mesothelioma; Molecular Sequence Data; Neoplasm Metastasis; Neprilysin; Peptides; Polymerase Chain Reaction; Pulmonary Alveoli; Recombinant Proteins; RNA, Messenger; Signal Transduction; Tumor Cells, Cultured

Previously, GRP receptors were characterized in small cell lung cancer cells and here non-small cell lung cancer (NSCLC) cells were investigated: (125I-Tyr4) bombesin (BN) or 125I-GRP bound with high affinity to NCI-H720 (lung carcinoid) and NCI-H1299 (large cell carcinoma) cells. Binding was specific, time dependent, and saturable. Specific (125I-Tyr4)BN binding to NCI-H1299 cells was inhibited with high affinity by GRP, BN, GRP14-27, (D-Phe6)BN6-13methyl ester, moderate affinity by NMB, and low affinity by GRP1-16. BN (10 nM) transiently elevated cytosolic calcium in a dose dependent manner. BN caused translocation of protein kinase C from the cytosol to the membrane and the translocation caused by BN was reversed by (D-Phe6)BN6-13methylester. BN stimulated arachidonic acid release and the increase caused by BN was reversed by (D-Phe6)BN6-13methylester. Using a clonogenic assay, BN stimulated the growth of NCI-H720 cells, and the number of colonies was reduced using (D-Phe6)BN6-13methylester. These data suggest that GRP receptors that are present in lung carcinoid and NSCLC cells may regulate proliferation.

Topics: Arachidonic Acid; Bombesin; Calcium; Carcinoid Tumor; Carcinoma, Large Cell; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Gastrin-Releasing Peptide; Humans; Lung Neoplasms; Neoplasm Proteins; Peptides; Receptors, Bombesin

Small cell lung cancers (SCLC) and some non-small cell lung cancers (NSCLC) have neuroendocrine features which include production of a variety of neuropeptides, cell surface expression of the receptors for these peptides, and autocrine stimulation by the peptides. Previous studies showed that some peptide antagonists and anti-peptide antibodies inhibited the growth of SCLC cell lines which expressed receptors for the specific peptide. We and others showed that the heterogeneity of peptide receptor expression and responsiveness was a major potential obstacle for developing therapeutic uses of peptide antagonists. In this manuscript we evaluated the effects of 11 peptide antagonists (3 bombesin-specific, 2 cholecystokinin-specific, 1 arginine vasopressin (AVP)-specific, and 5 substance P derivatives with broad specificity) on peptide-induced calcium mobilization and growth of SCLC and NSCLC cell lines. For each antagonist, we determined the dose-response effects, specificity of peptide antagonism, and biological stability in serum using Indo-1AM-based flow cytometric assays. We found that the three bombesin antagonists, S30, SC196, and L336,175, varied in potency from 10 nM to 10 microM, varied in serum stability from 6 h to more than 24 h, and had no effect on the calcium response elicited by other peptides. None of these compounds effectively inhibited the growth of SCLC cell lines in [3H]dThd and cell growth assays in vitro. Similarly, the three cholecystokinin and AVP antagonists were highly specific for cholecystokinin and AVP, respectively, had widely varying potency, but had little inhibitory effect on SCLC growth in vitro. In contrast, the five substance P derivatives inhibited the calcium response to bombesin, AVP, bradykinin, and fetal bovine serum. None of these five antagonists were as potent as the six specific antagonists described above, but they were more effective in inhibiting the growth of SCLC cell lines in vitro. These substance P derivatives inhibited the growth of peptide-sensitive SCLC cell lines more efficiently than their inhibition of peptide-insensitive NSCLC or breast cancer cell lines. Relatively high concentrations of these substance P derivatives were required to inhibit in vitro growth, even in the absence of added peptide. It is likely that more potent broad spectrum antagonists, toxins, or radiolabeled stable antagonists will need to be developed for maximal clinical development of this type of anti-growth factor therapy.

Topics: Amino Acid Sequence; Bombesin; Bradykinin; Calcium; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Cholecystokinin; Gastrin-Releasing Peptide; Humans; Lung Neoplasms; Molecular Sequence Data; Neuropeptides; Peptides; Substance P; Tumor Cells, Cultured

We investigated the effects of our synthetic bombesin/gastrin-releasing peptide (GRP) antagonists and somatostatin analogue RC-160 on the growth of human small-cell lung carcinoma (SCLC) and non-small-cell lung carcinoma (non-SCLC) lines in nude mice. Athymic nude mice bearing xenografts of the SCLC NCl-H69 line or non-SCLC NCl-H157 line were treated for 5 and 4 weeks, respectively, with somatostatin analogue RC-160 or various bombesin/GRP antagonists. RC-160, administered s.c. peritumorally at a dose of 100 micrograms per animal per day, inhibited the growth of H69 SCLC xenografts as shown by more than 70% reduction in tumour volumes and weights, as compared with the control group. Bombesin/GRP antagonists, RC-3440, RC-3095 and RC-3950-II, given s.c. peritumorally at a dose of 20 micrograms per animal per day, also inhibited the growth of H69 SCLC tumours. RC-3950-II had the greatest inhibitory effect and decreased tumour volume and weights by more than 80%. The growth of H-157 non-SCLC xenografts was significantly reduced by treatment with RC-160, but not with bombesin/GRP antagonist RC-3095. In mice bearing either tumour model, administration of RC-160 significantly decreased serum growth hormone and gastrin levels. Specific high-affinity receptors for bombesin and somatostatin were found on membranes of SCLC H69 tumours, but not on non-SCLC H157 tumours. Receptor analyses demonstrated high-affinity binding sites for epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on the membranes of H69 and H157 tumours. EGF receptors were down-regulated on H69 tumours after treatment with RC-160 and bombesin/GRP antagonists. The concentration of binding sites for EGF and IGF-I on the H157 tumours was decreased after treatment with RC-160, but bombesin/GRP antagonist RC-3095 had no effect. These results demonstrate that bombesin/GRP antagonists inhibit the growth of H-69 SCLC, but not of H-157 non-SCLC xenografts in nude mice, whereas somatostatin analogue RC-160 is effective in both tumour models. This raises the possibility that these peptide analogues could be used selectively in the treatment of various subclasses of lung cancer.

Topics: Amino Acid Sequence; Animals; Antineoplastic Agents; Binding Sites; Body Weight; Bombesin; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Cell Division; Gastrin-Releasing Peptide; Gastrins; Gonadotropin-Releasing Hormone; Growth Hormone; Humans; Lung Neoplasms; Male; Mice; Mice, Nude; Molecular Sequence Data; Neoplasm Transplantation; Peptide Fragments; Peptides; Receptors, Somatotropin; Somatostatin; Substrate Specificity; Transplantation, Heterologous

Non-small cell lung cancers with neuroendocrine differentiation (NSCLC-NE) may demonstrate biologic behavior intermediate between non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) with impact on prognosis. We studied the expression of four well-defined neuroendocrine (NE) markers: neuron-specific enolase (NSE), chromogranin A, Leu-7, gastrin-releasing peptide, and a panel of three non-NE markers, including vimentin, and the epithelial markers carcino-embryonic antigen (CEA) by immunohistochemistry, and mucin by histochemistry in 237 resected NSCLCs from patients on six LCSG protocols. Twenty-nine (12%) tumors were positive for 2 or more NE markers. An NE differentiation score was calculated but failed to correlate with recurrence as did other combinations of markers. However, the presence of tissue staining for CEA was strongly associated with improved survival (p = 0.011), whereas the presence of mucin was associated with a worse outcome (p < 0.001). Individually, CEA and mucin remained prognostic even when corrected for stage, histologic features, and performance status. We conclude that NE differentiation is not predictive of recurrence in patients with resected NSCLC but data on patterns of CEA and mucin expression may improve prognostication and permit rational design of new therapeutic approaches.

Topics: Antigens, Differentiation; Biomarkers, Tumor; Carcinoembryonic Antigen; Carcinoma, Non-Small-Cell Lung; Chromogranin A; Chromogranins; Gastrin-Releasing Peptide; Humans; Immunohistochemistry; Lung Neoplasms; Neoplasm Recurrence, Local; Neuroendocrine Tumors; Peptides; Phosphopyruvate Hydratase; Prognosis; Proportional Hazards Models; Vimentin

Gastrin-releasing peptide is an important growth-modulating factor in developing lung epithelium. It is known to be produced by small cell carcinomas of the lung, and an autocrine loop involving gastrin-releasing peptide and its receptor has been demonstrated in many small cell lung tumors. We investigated whether such an autocrine loop could also be demonstrated in non-small cell lung carcinoma, since gastrin-releasing peptide is known to stimulate human bronchial epithelial cells, from which non-small cell tumors should emerge. We report here that gastrin-releasing peptide is produced by a bronchiolo-alveolar carcinoma cell line (A549) adapted to serum-free and growth factor-free conditions. A549 cells adapted to these conditions, termed A549-R0 cells, display extensive membrane interdigitations, Golgi apparatus, and secretory-like granules, and grow as a mixture of attached colonies and floating cells. Gastrin-releasing peptide is present in the conditioned medium produced by A549-R0 cells. Colony formation of cells derived from a squamous cell carcinoma of the lung, 239T, was stimulated 9-fold by A549-R0 conditioned medium or by authentic gastrin-releasing peptide, measured in serum-free conditions. The growth stimulatory activity was inhibited by a monoclonal antibody to gastrin-releasing peptide. Transcripts for receptors for the bombesin family of peptides were also demonstrated in A549-R0 cells and 239T cells. These results demonstrate that non-small cell lung carcinomas can secrete gastrin-releasing peptide and can also respond to the peptide.

Topics: Base Sequence; Bombesin; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cell Line; Culture Media, Conditioned; Culture Media, Serum-Free; Culture Techniques; DNA Primers; Gastrin-Releasing Peptide; Gastrointestinal Hormones; Gene Expression; Growth Substances; Humans; Immunohistochemistry; Lung Neoplasms; Microscopy, Electron; Molecular Sequence Data; Neuropeptides; Peptide Biosynthesis; Peptides; Protein Precursors; Radioimmunoassay; Receptors, Bombesin; Tumor Cells, Cultured; Tumor Stem Cell Assay

Gastrin-releasing peptide (GRP; mammalian bombesin) is present in the neuroendocrine cells of human fetal lung and in small cell lung carcinomas (SCLCs), where it may act as a growth factor. Considering the potential importance of GRP as a tumor marker, we have conducted a retrospective immunohistochemical analysis of 176 lung tumors for markers of GRP gene expression, as well as several other markers of neuroendocrine cell differentiation: chromogranin A, neuron-specific enolase, and calcitonin. The majority of carcinoids contained mature GRP, in contrast to only a minority of SCLCs and large cell lung carcinomas (LCLCs). However, a majority of SCLCs and LCLCs contained proGRP immunoreactivity. In situ hybridization did not add any information beyond what was obtained using proGRP antisera. In spite of sharing these neuroendocrine cell markers, SCLCs are associated with a graver prognosis than LCLCs. No prognostic significance was associated with immunostaining for GRP or several other markers of neuroendocrine cell differentiation.

Topics: Adult; Aged; Biomarkers; Carcinoid Tumor; Carcinoma; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Female; Gastrin-Releasing Peptide; Gene Expression; Humans; Immunoenzyme Techniques; In Situ Hybridization; Lung Neoplasms; Male; Middle Aged; Peptides; Retrospective Studies; RNA, Messenger; RNA, Neoplasm; Survival Analysis

Gastrin-releasing peptide (GRP), a mammalian bombesin-like peptide, has been shown to be an important autocrine growth factor for small cell lung cancer (SCLC). However, not all SCLC cell lines express the GRP gene or respond mitogenically to GRP stimulation, suggesting the existence of other autocrine pathways in this tumor. Neuromedin B (NMB), the mammalian counterpart of amphibian ranatensin, has been shown to be a mitogen for SCLC cell lines in vitro. To determine whether NMB is a potential autocrine growth factor for lung tumors, NMB gene expression, peptide synthesis, and secretion have been investigated in a panel of SCLC and non-SCLC (NSCLC) cell lines. Northern blot analysis and enzymatic amplification from mRNA by polymerase chain reaction showed that the NMB gene was expressed in all SCLC and NSCLC cell lines examined. In contrast, the GRP gene was expressed in four of six classic SCLC cell lines but not in variant SCLC or NSCLC cell lines. Immunoreactive NMB was detected by radioimmunoassay in the majority of classic SCLC, in one of three variant SCLC and in one of three NSCLC cell lines, and secreted NMB was detected in medium conditioned by a SCLC and a NSCLC cell line. The present study also demonstrated the presence of immunoreactive GRP in the absence of detectable GRP gene expression. The antiserum used in the GRP radioimmunoassay failed to cross-react with NMB but showed some cross-reactivity with amphibian phyllolitorin raising the possibility that certain SCLC cell lines may produce a phyllolitorin-like peptide.

Topics: Base Sequence; Blotting, Northern; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Gastrin-Releasing Peptide; Gene Expression; Humans; Lung Neoplasms; Molecular Sequence Data; Neurokinin B; Peptide Biosynthesis; Polymerase Chain Reaction; RNA; Tumor Cells, Cultured

Small cell lung cancer (SCLC) tumor progression can involve partial or complete conversion to a more treatment-resistant non-small cell (NSCLC) phenotype. In a cell culture model of this phenomenon, we have previously demonstrated that insertion of the viral Harvey ras gene (v-Ha-ras) into SCLC cell lines with amplification and overexpression of the c-myc gene induced many NSCLC phenotypic features. We now report that the v-Ha-ras gene can also induce morphologic, biochemical, and growth characteristics consistent with the NSCLC phenotype in an N-myc amplified SCLC cell line, NCI-H249. We show that v-Ha-ras has novel effects on these cells, abrogating an SCLC-specific growth requirement for gastrin-releasing peptide, and inducing mRNA expression of three NSCLC-associated growth factors and receptors, platelet-derived growth factor B chain, transforming growth factor-alpha (TGF-alpha), and epidermal growth factor receptor (EGF-R). TGF-alpha secretion and EGF-R also appear, consistent with the induction of an autocrine loop previously shown to be growth stimulatory for NSCLC in culture. These data suggest that N-myc and v-Ha-ras represent functional classes of genes that may complement each other in bringing about the phenotypic alterations seen during SCLC tumor progression, and suggest that such alterations might include the appearance of growth factors and receptors of potential importance for the growth of the tumor and its surrounding stroma.

Topics: Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Cell Division; Eflornithine; Epidermal Growth Factor; ErbB Receptors; Gastrin-Releasing Peptide; Gene Expression; Genes, ras; Lung Neoplasms; Peptides; Platelet-Derived Growth Factor; Transforming Growth Factors

Pretreatment serum neuron specific enolase (NSE) and plasma bombesin/gastrin releasing peptide (BN/GRP) were measured in 92 lung cancer patients and 17 controls. The mean level of NSE (p less than 0.001) and BN/GRP (p less than 0.05) was significantly raised in patients with small cell lung cancer (SCLC, n = 62) compared to non-SCLC (n = 30) and controls. The mean concentration of NSE in extensive SCLC was significantly greater (p less than 0.005) than in limited stage but with a substantial overlap of values. Forty-seven out of 62 SCLC patients had at least one of the two markers raised (sensitivity 76%, specificity 83%), 44 had raised NSE (sensitivity 71%, specificity 89%) but only 24 had BN/GRP raised (sensitivity 42%, specificity 91%). At restaging, 16 of 19 patients with SCLC responsive to chemotherapy showed a significant fall of NSE; on the other hand, BN/GRP fell significantly in only 3 patients, remaining unchanged in the majority of responding patients. In conclusion, the combined determination of NSE and BN/GRP in SCLC, at diagnosis and during the follow-up, was not found to be superior to NSE determination alone.

Topics: Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Bombesin; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Female; Gastrin-Releasing Peptide; Humans; Lung Neoplasms; Male; Middle Aged; Peptides; Phosphopyruvate Hydratase

A selected group of 263 pulmonary neuroendocrine tumours comprised 156 small cell carcinomas, five combined cell carcinomas, nine atypical carcinoid/small cell carcinomas, 32 atypical carcinoids, ten large cell/small cell carcinomas, and 51 carcinoid tumours. These were compared with a group of 109 non-small cell carcinomas, using four markers of neuroendocrine differentiation to determine differences in reactivity between the two groups and among the variants of neuroendocrine tumour. The antibodies used were neuron-specific enolase (NSE), protein gene product (PGP) 9.5, human bombesin, and the C-terminal flanking peptide of human bombesin (CTP). Most small cell carcinomas, carcinoid tumours, and atypical carcinoid variants showed immunoreactivity for both NSE and PGP 9.5 but a significant number of non-small cell carcinomas, mainly squamous cell carcinomas, were also positive (11 and 35 per cent, respectively). Bombesin was specific for neuroendocrine tumours, being demonstrable in 35 per cent carcinoids and 24 per cent small cell carcinomas, but staining was focal and often confined to scattered cells. Diffuse strongly positive immunoreactivity for CTP was seen in the majority of malignant neuroendocrine tumours, but only 12 per cent of carcinoid tumours were positive and non-small cell carcinomas were negative. CTP is therefore of potential value as a specific marker of malignant neuroendocrine tumours, particularly if the amount of biopsy material is limited and the tumour is an unusual variant, such as atypical carcinoid or large cell-small cell carcinoma.

Topics: Biomarkers, Tumor; Bombesin; Carcinoid Tumor; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Gastrin-Releasing Peptide; Humans; Immunoenzyme Techniques; Lung Neoplasms; Neuropeptides; Peptide Fragments; Peptides; Phosphopyruvate Hydratase; Ubiquitin Thiolesterase

To demonstrate unbalanced distribution of subunits of human chorionic gonadotropin (hCG) in the lung and lung tumors and to clarify its significance in differentiation and carcinogenesis of the lung, immunohistochemistry was performed on human fetus, infant, and adult lungs, and endocrine and nonendocrine tumors of the lung. Tissues were immunostained for alpha-subunits and for beta-subunits of glycoprotein hormones (hCG, luteinizing hormone, follicle stimulating hormone, and thyroid stimulating hormone), serotonin, and gastrin-releasing peptide. Immunoreactive alpha-subunit was first identified in endocrine-like cells at the 39th gestational week, and was found in all infant lungs and two-thirds of adult lungs. The hCG beta-immunoreactive cells were extremely rare in an adult lung, and were not found in fetus or infant lungs. The alpha-subunit-containing cells were present in neuroepithelial bodies, tumorlets, carcinoid tumors, and small cell carcinomas of the lung (SCCL). There were occasionally alpha-subunit-containing cells in non-SCCL but one of the carcinomas also contained many serotonin-positive and gastrin-releasing peptide-positive cells in the same region. All alpha-subunit-immunoreactive cells lacked immunoreactivity for beta-subunits of glycoprotein hormones, except some for hCG beta in one carcinoid tumor. Immunoreactive cells for isolated hCG beta appeared much more frequently in non-SCCL than in SCCL. Most non-SCCL containing hCG beta-positive cells did not show alpha-subunit-immunoreactivity. Thus, immunohistochemical distribution of hCG-subunits was unbalanced and hCG-subunits may be expressed through an independent mechanism, commonly in the lung and lung tumors. The significance of isolated alpha-subunit is further discussed in light of multidirectional differentiation of lung neoplasms (14, 17).

Topics: Adult; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Chorionic Gonadotropin; Chorionic Gonadotropin, beta Subunit, Human; Fetus; Gastrin-Releasing Peptide; Histocytochemistry; Humans; Infant, Newborn; Lung; Lung Neoplasms; Peptide Fragments; Peptides; Serotonin