neuromedin-b and Carcinoma--Non-Small-Cell-Lung

Neuromedin B (NMB), a member of the bombesin family of peptides, is an autocrine growth factor for many lung cancer cells. The present study investigated the ability of NMB to cause transactivation of the epidermal growth factor (EGF) receptor in lung cancer cells. By Western blot, addition of NMB or related peptides to NCI-H1299 human non-small cell lung cancer (NSCLC) cells, caused phosphorylation of Tyr(1068) of the EGF receptor. The signal was amplified using NCI-H1299 cells stably transected with NMB receptors. The transactivation of the EGF receptor or the tyrosine phosphorylation of ERK caused by NMB-like peptides was inhibited by AG1478 or gefitinib (tyrosine kinase inhibitors) and NMB receptor antagonist PD168368 but not the GRP receptor antagonist, BW2258U89. The transactivation of the EGF receptor caused by NMB-like peptides was inhibited by GM6001 (matrix metalloprotease inhibitor), PP2 (Src inhibitor), or transforming growth factor (TGF)alpha antibody. The transactivation of the EGF receptor and the increase in reactive oxygen species caused by NMB-like peptides was inhibited by N-acetylcysteine (NAC) or Tiron. Gefitinib inhibited the proliferation of NCI-H1299 cells and its sensitivity was increased by the addition of PD168368. The results indicate that the NMB receptor regulates EGF receptor transactivation by a mechanism dependent on Src as well as metalloprotease activation and generation of reactive oxygen species.

Topics: Acetylcysteine; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Enzyme Inhibitors; ErbB Receptors; Gefitinib; Humans; Neurokinin B; Phosphorylation; Phosphotyrosine; Quinazolines; Reactive Oxygen Species; Receptors, Bombesin; Transcriptional Activation; 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

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