ecallantide and Lung-Neoplasms

Interleukin-like epithelial-to-mesenchymal transition inducer (ILEI) is an essential cytokine in tumor progression that is upregulated in several cancers, and its altered subcellular localization is a predictor of poor survival in human breast cancer. However, the regulation of ILEI activity and the molecular meaning of its altered localization remain elusive.. The influence of serum withdrawal, broad-specificity protease inhibitors, different serine proteases and plasminogen depletion on the size and amount of the secreted ILEI protein was investigated by Western blot analysis of EpRas cells. Proteases with ILEI-processing capacity were identified by carrying out an in vitro cleavage assay. Murine mammary tumor and metastasis models of EpC40 and 4T1 cells overexpressing different mutant forms of ILEI were used-extended with in vivo aprotinin treatment for the inhibition of ILEI-processing proteases-to test the in vivo relevance of proteolytic cleavage. Stable knockdown of urokinase plasminogen activator receptor (uPAR) in EpRas cells was performed to investigate the involvement of uPAR in ILEI secretion. The subcellular localization of the ILEI protein in tumor cell lines was analyzed by immunofluorescence. Immunohistochemistry for ILEI localization and uPAR expression was performed on two human breast cancer arrays, and ILEI and uPAR scores were correlated with the metastasis-free survival of patients.. We demonstrate that secreted ILEI requires site-specific proteolytic maturation into its short form for its tumor-promoting function, which is executed by serine proteases, most efficiently by plasmin. Noncleaved ILEI is tethered to fibronectin-containing fibers of the extracellular matrix through a propeptide-dependent interaction. In addition to ILEI processing, plasmin rapidly increases ILEI secretion by mobilizing its intracellular protein pool in a uPAR-dependent manner. Elevated ILEI secretion correlates with an altered subcellular localization of the protein, most likely representing a shift into secretory vesicles. Moreover, altered subcellular ILEI localization strongly correlates with high tumor cell-associated uPAR protein expression, as well as with poor survival, in human breast cancer.. Our findings point out extracellular serine proteases, in particular plasmin, and uPAR as valuable therapeutic targets against ILEI-driven tumor progression and emphasize the prognostic relevance of ILEI localization and a combined ILEI-uPAR marker analysis in human breast cancer.

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Cytokines; Disease Progression; Epithelial-Mesenchymal Transition; Female; Fibrinolysin; Humans; Kaplan-Meier Estimate; Leukocyte Elastase; Lung Neoplasms; Mice, Nude; Neoplasm Proteins; Neoplasm Transplantation; Plasma Kallikrein; Protein Processing, Post-Translational; Protein Transport; Proteolysis; Receptors, Urokinase Plasminogen Activator

Tissue kallikrein (hK1) and plasma kallikrein (PK, hKB1) are serine proteases that produce biologically active kinin peptides from endogenous kininogen substrates. There is evidence linking the kallikreins and the mitogenic kinin peptides to carcinogenesis. The aim of this study was to investigate the expression of tissue prokallikrein (pro-hK1), plasma prekallikrein (PPK, pre-hKB1) and kinin B1 and B2 receptor proteins in different subtypes of lung cancer. Immunohistochemistry, using specific antibodies, was performed on archived normal lung sections and sections from adenocarcinomas, squamous cell carcinomas, large cell carcinomas, small cell carcinomas and carcinoid tumours of the lung. Immunoperoxidase labelling was visualised by brightfield microscopy and immunofluorescence labelling by confocal microscopy. Extensive cytoplasmic expression of pro-hK1 and PPK was observed, which was similar in small cell and non-small cell tumours. However, nuclear labelling for the kallikreins was absent or limited. The kinin B1 and B2 receptors were highly expressed in the cytoplasm of all tumour types and in the nuclei of non-small cell tumours. Further studies are required to assess the functional significance of the expression of hK1, PK and kinin receptors in lung tumours, and whether any of these proteins may be potential biomarkers for specific subtypes of lung carcinoma.

Topics: Carcinoma; Humans; Lung; Lung Neoplasms; Plasma Kallikrein; Receptor, Bradykinin B1; Receptor, Bradykinin B2; Tissue Kallikreins

Glycoproteins in human serum play fundamental roles in many biological processes, and also have clinical value as biomarkers for disease progression and treatment. In this study, we isolated glycoproteins from the sera of three healthy individuals and three lung adenocarcinoma patients using multilectin affinity chromatography. The recovered glycoproteins were subjected to treatment with peptide-N-glycosidase F (PNGase F) and in-gel digestion by trypsin. Tryptic peptides were analyzed by nano-LC coupled to ESI-MS/MS and the MS/MS spectra were processed by Bioworks 3.2 and an in-house bioinformatics tool, ProtAn. Approximately 90% of the proteins identified contained more than one potential glycosylation site. Comparison of the serum glycoproteome of healthy and adenocarcinoma individuals revealed 38 cancer-selective proteins. Among them, 60% have previously been reported as low abundance proteins in human sera. We identified several cancer-selective proteins that have been previously characterized as potential indicators of lung cancer in serum or plasma, including haptoglobin (HP), inter-alpha-trypsin inhibitor heavy chain 4 (ITI-H4), complement C3 precursor, and leucine-rich alpha-2-glycoprotein. In addition, plasma kallikrein (KLKB1) and inter-alpha-trypsin inhibitor heavy chain 3 (ITI-H3) were identified as being potentially elevated in the lung cancer group, and were validated by Western blot analysis. Furthermore, approximately 18 kDa plasma kallirein protein fragment was detected at high levels in 25 out of 28 adenocarcinoma patients, while one of the eight normal individuals showed moderate positive. The results suggest that KLKB1 represents a potential candidate serum biomarker of lung cancer.

Topics: Adenocarcinoma; Aged; Alpha-Globulins; Biomarkers, Tumor; Blood Proteins; Blotting, Western; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Gene Expression; Glycoproteins; Humans; Lectins; Lung Neoplasms; Male; Middle Aged; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; Plasma Kallikrein; Reproducibility of Results; Tandem Mass Spectrometry; Up-Regulation

To investigate the kinin generation pathways in acute and chronic airway inflammation.. BALF from patients with acute, chronic airway inflammation and healthy controls were collected. Kinins, Plasma kallikrein, alpha 2-macroglobulin and toluenesulphonyl-arginine methyl ester esterase activity (TAME-ea) in BALF were studied.. Kinins and TAME-ea values were significantly higher in the BALF of patients with acute and chronic airway inflammation than those in the controls, but there was no significant difference between acute and chronic groups; PK and alpha 2-M values were significantly higher in the acute group than the in chronic one. Gel filtration revealed the highest TAME-ea peak at about 800,000 in the acute group, corresponding with the first alpha 2-M peak, whereas at about 40,000 in chronic bronchitis. The inhibition test of the TAME-ea showed that the TAME-ea peak at 800,000 was mainly due to PK and the TAME-ea peak at 40,000 was mainly due to TK.. The results indicated that in acute airway inflammation kinins seem to be mainly generated by PK, whereas in chronic inflammation kininogenases other than PK--such as TK--seem to be more important.

Topics: Acute Disease; Aged; alpha-Macroglobulins; Bronchitis; Bronchitis, Chronic; Bronchoalveolar Lavage Fluid; Female; Humans; Kinins; Lung Neoplasms; Male; Middle Aged; Peptide Hydrolases; Plasma Kallikrein; Protease Inhibitors