phosphoramidon and Lung-Neoplasms

This study was designed to characterize the endothelin pathway in an immortalized human adenocarcinoma-derived alveolar epithelial cell line (A549) and human umbilical vein endothelial cell line (HUVEC).. The release of ET-1 and big-ET-1 was measured in the incubation medium of both cell lines. The expression of mRNAs coding for the endothelin isoforms (hppET-1, -2, -3), the endothelin converting enzymes (hECE-1a, b, c, and d) and the hETA and hETB receptors was investigated using RT-PCR. The expression of ECE-1 mRNA in various human tissues and in A549 cells was investigated by Northern blot analysis and the subcellular localization of ECE-1 in A549 cells was investigated by immunoblotting using a polyclonal antibody.. Under control conditions, HUVEC release both ET-1 and big-ET-1 (ratio 5 to 1) while in A549 cells the big-ET-1 levels were below the threshold of detection. The release of these two peptides was minimally affected by various inhibitors of peptidases. However, in both cell lines phosphoramidon produced a concentration-dependent inhibition of ET-1 release and an enhanced accumulation of big-ET-1. Both HUVEC and A549 cells express the mRNAs for ppET-1, ET-A, and ET-B receptor subtypes and ECE-1 (isoforms ECE-1b, c and/or d). In addition, in HUVEC the mRNAs for ppET-2 and for the isoform ECE-1a were also detected. In A549 cells, ECE-1 had a preferential subcellular localization in the membrane fraction but was not detected in the cytosol.. Both A549 and HUVEC produce and release endothelin-1 through a specific enzymatic pathway, whether or not ECE-1 is the only enzyme involved remains to be determined. A549 might be used as a screening assay for drug discovery such as for inhibitors of endothelin-1 release.

Topics: Adenocarcinoma; Aspartic Acid Endopeptidases; Cells, Cultured; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Endothelium, Vascular; Glycopeptides; Humans; Lung Neoplasms; Metalloendopeptidases; Protein Precursors; RNA, Messenger; Tumor Cells, Cultured; Umbilical Veins

Endothelin-1 may be involved in the pathogenesis of asthma by causing bronchial smooth muscle constriction and airway remodelling. Bronchial epithelial cells represent an important source of endothelin-1 in this disease, and increased release of epithelial cell-derived endothelin-1 may contribute to the genesis of subepithelial fibrosis by promoting fibroblast proliferation and collagen production. In this study, we demonstrate that endothelin-1 upregulates fibronectin gene expression and fibronectin release in bronchial epithelial cells via an ETA receptor. Fibronectin is an important component of the extracellular matrix which is deposited in excess in the subepithelial area of asthmatic bronchial mucosa, and it represents a potent chemotactic factor for fibroblasts. Thus, endothelin-1 may induce subepithelial fibrosis both directly and by the autocrine mechanism reported here.

Topics: Blotting, Northern; Bronchi; Cells, Cultured; Dose-Response Relationship, Drug; Endothelin Receptor Antagonists; Endothelins; Epithelium; Fibronectins; Gene Expression; Glycopeptides; Humans; Kinetics; Lung Neoplasms; Peptides, Cyclic; Receptors, Endothelin; RNA, Messenger; Time Factors; Vasoconstrictor Agents; Viper Venoms

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

The ligand-binding properties of the gastrin-releasing peptide (GRP) receptor and the cellular processing of GRP have been studied in the small-cell lung cancer (SCLC) cell line COR-L42. Scatchard analysis of GRP receptor expression indicated a single class of high-affinity receptors (Kd 1.5 nM) and approx. 6700 receptors/cell. GRP bound to its receptor with a Ki of 2.4 nM. The bombesin-related peptides neuromedin B (NMB) and phyllolitorin also bound to GRP receptors with Ki values of 22.7 and 59.1 nM respectively. Binding of 125I-GRP to COR-L42 cells increased rapidly at 37 degrees, achieved a maximum at 10 min and declined rapidly thereafter. At 4 degrees C, maximum binding was achieved at 30 min and the subsequent decline in cell-associated radioactivity was slower than that seen at 37 degrees C. Acid/salt extraction, to separate surface-bound ligand from internalized GRP, indicated that after receptor binding 125I-GRP was rapidly internalized. To determine the pathway of 125I-GRP degradation, binding studies were carried out with the lysosomotropic agent chloroquine (5 mM), and with phosphoramidon (10 microM), an inhibitor of the membrane-bound enzyme (EC 3.4.24.11). Both agents markedly inhibited the degradation of GRP, indicating that this process involves a lysosomal pathway and a phosphoramidon-sensitive pathway, possibly involving the EC 3.4.24.11 enzyme. GRP receptor down-regulation was observed following a 10 min exposure to 100 nM-GRP. With longer pretreatment times the number of binding sites recovered to 80% of control values. Treatment with 5 mM-chloroquine plus GRP or cycloheximide (10 micrograms/ml) plus GRP demonstrated that the majority of GRP receptors are recycled. NMB and phyllolitorin pretreatment did not influence the subsequent binding of 125I-GRP, suggesting that these peptides do not down-regulate GRP receptors.

Topics: Anti-Bacterial Agents; Binding, Competitive; Bombesin; Carcinoma, Small Cell; Cell Line; Chloroquine; Gastrin-Releasing Peptide; Glycopeptides; Humans; Kinetics; Ligands; Lung Neoplasms; Peptides; Receptors, Bombesin; Receptors, Neurotransmitter

Bombesin-like peptides are essential autocrine growth factors for many small cell carcinomas (SCCas) of the lung. Herein, we demonstrate that these malignant pulmonary neuroendocrine cells express low levels of the cell surface metalloendopeptidase CD10/neutral endopeptidase 24.11 (CD10/NEP, common acute lymphoblastic leukemia antigen) and that this enzyme hydrolyzes bombesin-like peptides. The growth of bombesin-like peptide-dependent SCC as is inhibited by CD10/NEP and potentiated by CD10/NEP inhibition. The results provide evidence that CD10/NEP is involved in the regulation of tumor cell proliferation. Since SCCa of the lung occurs almost exclusively in cigarette smokers and cigarette smoke inactivates CD10/NEP, decreased cell surface CD10/NEP enzymatic activity may be causally related to the development of SCCa of the lung.

Topics: Amino Acid Sequence; Antigens, Differentiation; Antigens, Neoplasm; Bombesin; Carcinoma, Small Cell; Cell Division; Cell Line; DNA Replication; Fetus; Glycopeptides; Humans; Immunohistochemistry; Kinetics; Lung; Lung Neoplasms; Molecular Sequence Data; Neprilysin; Receptors, Bombesin; Receptors, Neurotransmitter; Substrate Specificity