Cystatins in non-small cell lung cancer: tissue levels, localization and relation to prognosis.

Cystatins regulate tumour-associated cysteine proteases, however, their role in tumour progression is not clear yet. To assess their relevance in the progression of non-small cell lung cancer (NSCLC) the protein level, cysteine protease activity (CPI) and localization of type I (stefins A and B) and type II (C, E/M and F) cystatins were defined in tumours and control lung counterparts from 165 patients. The medians of CPI activity, stefins A and B were significantly greater in tumour than in lung tissue (2.1-fold, 1.7-fold, 1.2-fold, respectively, all p<0.001). The median levels of cystatin C and cystatin E/M were lower in tumour tissue (0.9-fold, p=0.06; 0.6-fold, p<0.01). In all the samples the levels of cystatin F were below the detection limit. Immunohistochemical analysis revealed the presence of all cystatins in tumour cells and infiltrated inflammatory cells such as macrophages and neutrophils. In univariate survival analysis patients with high levels of stefin A, stefin B and CPI activity exhibited a better survival probability (p=0.05, p=0.05, p<0.01, respectively). In contrast, cystatins C and E/M provided no prognostic information. In multivariate analysis the most powerful predictor of survival was the pTNM stage (p<0.0001; RR 3.5), followed by stefin A, stefin B and CPI activity (all p=0.03; RR 1.5). Our results suggest that only stefins A and B, i.e. type I cystatins, are up-regulated in lung tumours and thus able to counteract harmful tumour-associated proteolytic activity. As biological markers they may add independent prognostic information for better assessment of low- and high-risk patients with NSCLC.

Carboxypeptidases cathepsins X and B display distinct protein profile in human cells and tissues.

Cathepsin X, a recently discovered lysosomal cysteine protease, shares common structural features and activity properties with cysteine protease cathepsin B. Based on its widespread mRNA distribution in primary tumors and tumor cell lines, a redundant function in tumor progression has been proposed. In this study, we have shown that these two related proteases exhibit different profiles with respect to their protein distribution in cells and tissues and to their possible roles in malignancy. Protein level of cathepsin X did not differ significantly between matched pairs of lung tumor and adjacent lung tissue obtained from patients with lung cancer whereas that of cathepsin B was 9.6-fold higher in tumor compared to adjacent lung tissue. Immunohistochemical analysis of lung tumor cathepsin X revealed very faint staining in tumor cells but positive staining in infiltrated histiocytes, alveolar macrophages, bronchial epithelial cells, and alveolar type II cells. Cathepsin X stained positive also in CD68+ cells in germinal centers of secondary follicles in lymph nodes, corresponding to tingible body macrophages. Two cell lines with proven invasive behavior, MCF-10A neoT and MDA-MB 231, showed positive staining for cathepsin B, but negative for cathepsin X. We showed that the invasive potential of MCF-10A neoT cells can be impaired by specific inhibitor of cathepsin B but not by that of cathepsin X. Cathepsin X was found in large amounts in the pro-monocytic U-937 cell line, in monocytes and in dendritic cells, generated from monocytes in vitro. Our results show that cathepsin X is not involved in degradation of extracellular matrix, a proteolytic event leading to tumor cell invasion and metastasis. Its expression, restricted to immune cells suggests a role in phagocytosis and the regulation of immune response.

Expression, proliferation activity and clinical significance of cathepsin B and cathepsin L in operated lung cancer.

AIMS: To immunohistochemically investigate the expression of cathepsin B and cathepsin L in various lung cancer cell types in association with the patients' prognosis. MATERIALS AND METHODS: Histological slides obtained from formalin-fixed, paraffin-embedded tissue of 120 potentially curative resected lung cancer specimens (20 cases of each of the following cell types: squamous cell, adeno, large cell anaplastic, small cell anaplastic, intrapulmonary metastases, mesotheliomas) were quantitatively immunohistochemically analysed with an automated image analysing system and correlated with the patients' prognosis and the tumour proliferation rate measured by the expression of Ki-67. RESULTS: The expression of cathepsin B was most frequently present in large cell anaplastic carcinomas and missing in small cell carcinomas. That of cathepsin L was less frequently seen, and mainly expressed in macrophages and adenocarcinoma tumour cells. The structural heterogeneity of cathepsin B expression measured by syntactic structure analysis was greater than that of cathepsin L. The expression of cathepsin B is of prognostic significance in non-small cell lung cancer, and ranges directly after the pN and pT stages in multivariate statistical analysis. CONCLUSION: In concordance with the literature, the expression of cathepsin B in non-small cell lung cancer should be considered as a significant prognostic parameter in contrast to that of cathepsin L, which is not related to the patients' outcome at a statistically significant level.

Cystatins C, E/M and F in human pleural fluids of patients with neoplastic and inflammatory lung disorders.

Secretory type 2 cystatins, like cystatins C, E/M and F, are thought to be involved in many pathobiological processes, including vascular amyloidosis, rheumatoid arthritis, Alzheimer's disease, osteoporosis, viral and bacterial infections, inflammatory disorders and tumour invasion and metastasis. In order to define the levels of cystatins C, E/M, and F in pleural effusions and to investigate whether these cystatins correlate with diagnostic parameters of pleural and lung diseases, we determined their concentrations in 160 pleural effusions. The median concentration of cystatin C in pleural effusions was 1437 microg/l (95.8 nM), ranging between 18-3967 microg/l. Cystatin C did neither correlate with malignant nor with benign diseases. The concentration of cystatin E/M was significantly higher in effusions of primary pleural tumours (mesotheliomas) compared to secondary pleural tumours and benign diseases. Furthermore, there was a significant correlation between the concentration of cystatin E/M of mesotheliomas and the pleural fluid tumour cell count and of cystatin C. The median values of cystatin F were significantly increased in parapneumonic/empyema thoracis pleural effusions and tuberculous pleurisy compared to malignant pleural effusions, respectively. The concentration of cystatin F in benign effusions correlated significantly with diagnostic parameters and inflammation (total protein; lactate dehydrogenase; C-reactive protein). Finally, only in the group of parapneumonic/empyema thotatin F and the neutrophil count. In conclusion, pleural effusions of different origin contain high levels of cystatin C, perhaps constituting the major part of an inhibitor reservoir. The level of cystatin E/M appears to be significantly associated with primary pleural tumours and cystatin F correlates with inflammatory processes of lung disorders.

Presentation of NO-metabolites (nitrate/nitrite) in blood serum and pleural effusions from cancer patients with pleurisy.

To assess whether cancer-induced pleurisy is associated with an alteration of nitric oxide (NO)-synthase activity, the levels of nitrate/nitrite (NOx) were measured in blood serum (BS) and pleural effusion (PE) of 35 cancer patients (secondary pleural metastases and mesotheliomas), eight patients with benign lung diseases, and in BS of nine healthy donors. It was found that (1) BS of patients with secondary pleural metastases had an elevated level (P<0.015) of NOx (59.7+/-24.4 microM, n=28) in comparison with control level of BS for healthy donors (43.4+/-13.5 microM, n=9); (2) BS of mesotheliomas (32.1+/-12.2 microM, n=4) had significantly (P<0.05) lower level of NOx compared to BS of benign patients (61.2+/-28.8, n=6); (3) differences in mean levels of NOx in BS and same PE of examined patient groups did not reach statistical significance, excepting sub-group of patients with primary mammary carcinoma; (4) significant interindividual differences of NOx in all groups of patients were revealed; (5) fluids from about 11% of cancer patients contained extremely high levels of NOx over 100 microM; (6) a significant elevation of apparent NOx level in BS and PE of patients with secondary pleural metastases in comparison with those in BS of healthy donors was revealed when the native, i.e. protein-contained, samples were managed with Griess reagent. The results described here, point up the diverse role of NOx in cancer patients. Its role is far from being clear but it seems that NOx acts as a signaling mediator during the formation of pleural metastases and might be considered as a non-specific marker in the corresponding PE. Furthermore, NOx could be used to give rationale of proper application of anticancer drugs affecting diversely NO-synthase activity in cells. Besides, a casual effectiveness of NOx measurements in native samples from cancer patients using Griess reagent needs additional elaboration.