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Biomedical subjects

Janko Kos

Publications and source records attributed to Janko Kos.

At least 19 recordsLinked to original sources
PubMed2006

Cystatin C as a potential marker for relapse in patients with non-Hodgkin B-cell lymphoma.

The concentration of cysteine protease inhibitor cystatin C was determined in sera from 59 patients with non-Hodgkin B-cell lymphoma using ELISA. The sera from 43 age and sex matched healthy blood donors served as controls. Cystatin C was significantly increased in sera of patients without therapy (mean 1136+/-SE 105.7ng/ml, p=0.00001) and with therapy (mean 1073+/-52ng/ml, p=0.001) compared to controls (mean 819+/-28ng/ml). The highest levels were determined in sera of patients with a relapse (mean 1680+/-196ng/ml). By using immunofluorescence staining and confocal microscopy we determined immature dendritic cells as a major population of cystatin C positive cells in affected lymph nodes. Our study reports for the first time that cystatin C is a potential marker for relapse in patients with non-Hodgkin B-cell lymphoma.

Adolescent↗
PubMed2006

Carboxypeptidase cathepsin X mediates beta2-integrin-dependent adhesion of differentiated U-937 cells.

Cathepsin X is a lysosomal carboxypeptidase with a potential role in processes of inflammation and immune response. The integrin-binding motifs RGD and ECD, present in the pro- and in mature forms of cathepsin X, respectively, suggest that this enzyme might have a function in cell signaling and adhesion. In this study, we report that cysteine protease inhibitors E-64 and CA-074 and 2F12 monoclonal antibody, all of which inhibit cathepsin X activity, significantly reduced adhesion of differentiated U-937 cells to polystyrene- and fibrinogen-coated surfaces via Mac-1 integrin receptor, whereas their binding to vitronectin, fibronectin or Matrigel was not affected. On the other hand, cathepsin X, added to differentiating U-937 cells, stimulated their adhesion. Using confocal microscopy, we demonstrated that the pro-form of cathepsin X was co-localized with beta(2) and beta(3) integrin subunits and its mature form solely with the beta(2) integrin subunit with the most intense signal in cell-cell junctions in differentiated U-937 cells and in co-cultures with endothelial cells. Our results indicate that active cathepsin X mediates the function of beta(2) integrin receptors during cell adhesion and that it could also be involved in other processes associated with beta(2) integrin receptors such as phagocytosis and T cell activation.

Antibodies, Monoclonal↗
PubMed2006

Intracellular proteolytic activity of cathepsin B is associated with capillary-like tube formation by endothelial cells in vitro.

The lysosomal cysteine protease cathepsin B is implicated in degradation of extracellular matrix (ECM), a crucial step in a variety of physiological and pathological processes, including tumor dissemination and angiogenesis. In this study, we analyzed the contribution of extracellular and intracellular cathepsin B activity on the formation of capillary-like tubular structures by human umbilical vein endothelial cells (HUVECs) grown on Matrigel matrix, using general and specific cysteine protease inhibitors. We demonstrated, by confocal assay using quenched fluorescent protein substrate DQ-collagen IV, that endothelial cells degrade ECM both intracellularly and pericellularly. Intracellular cathepsin B activity detected by degradation of Z-Arg-Arg cresyl violet substrate was co-localized with the products of DQ-collagen IV degradation in the perinuclear region and in the capillary-like tubular structures. Treatment of cells with membrane-permeable CA-074 Me effectively abolished intracellular cathepsin B activity, and resulted in reduced tube length (32.3+/-9.4% at 10 microM), total tubule area (49.6+/-12.4% at 10 microM), and the number of branch points of tubules (47.5+/-7.7% at 10 microM) in a dose-dependent manner. In contrast, CA-074 (0.1-10 microM), a membrane-impermeable cathepsin B specific inhibitor, general cysteine protease inhibitors chicken cystatin (5 microM) and E-64 (10 microM), and the metalloprotease inhibitor Minocycline (10 microM) showed no significant inhibitory effect in our angiogenesis model. These results show that, besides multiple regulatory molecules, intracellular cathepsin B also contributes to the neovascularization process and should be considered as a potential therapeutic target.

Capillaries↗
PubMed2006

Cysteine cathepsins: regulators of antitumour immune response.

Cysteine cathepsins are lysosomal cysteine proteases that are involved in a number of important biological processes, including intracellular protein turnover, propeptide and hormone processing, apoptosis, bone remodelling and reproduction. In cancer, the cathepsins have been linked to extracellular matrix remodelling and to the promotion of tumour cell motility, invasion, angiogenesis and metastasis, resulting in poor outcome of cancer patients; however, cysteine cathepsins are also involved at different levels of the innate and adaptive immune responses. Their best known role in this aspect is their contribution to major histocompatibility complex class II antigen presentation, the processing of progranzymes into proteolytically active forms, cytotoxic lymphocyte self-protection, cytokine and growth factor degradation and, finally, the induction of cytokine expression and modulation of integrin function. This review is focused on the role of cysteine cathepsins in the antitumour immune response and the evaluation of their pro- and anticancer behaviours during the regulation of these processes.

Animals↗
PubMed2006

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.

Adolescent↗
PubMed2005

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.

Antibodies, Monoclonal↗
PubMed2005

Differentiation- and maturation-dependent content, localization, and secretion of cystatin C in human dendritic cells.

Antigen-presenting cells (APC) play a pivotal role in the initiation of the T cell-mediated and antigen-specific immune response. The suggested role of endogenous inhibitor cystatin C (CyC) is to modulate cysteine proteases (cathepsins) present in human APC. To test this hypothesis, dendritic cells (DC) were generated in vitro from isolated monocytes, and changes in content, localization, and secretion of CyC and cathepsins S, L, and H (CatS, -L, and -H, repsectively) were followed in response to interleukin-4, enabling monocyte differentiation, and to tumor necrosis factor alpha (TNF-alpha), enabling DC maturation. A large increase in intracellular CyC accompanied the differentiation of monocytes to immature DC, also shown by strong immunolabeling of Golgi in immature DC. On DC maturation, intracellular CyC levels decreased, and CyC was mostly absent from the Golgi. On prolonged incubation of mature DC with TNF-alpha, CyC was found located in the proximity of the plasma membrane, indicating that the transport of CyC from Golgi was not blocked as the result of the arrested exocytosis in mature DC. The secretion of CyC ceased, consistent with the peak of the surface expression of phenotypic markers (CD40, CD54, CD80, CD83, CD86, and major histocompatibility complex class II), characteristic for the mature DC stage, whereas the secretion of cathepsins did not correlate with the maturation stage. The difference in localization of CyC and of CatS, -L, and -H in immature and mature DC shows that the regulatory potential of CyC toward CatS, -L, and -H inside DC is limited. However, these interactions may occur extracellularly in lymph, as suggested by the large excess of CyC over secreted CatS, -L, and -H, and they may facilitate DC migration to lymph nodes.

Antigen Presentation↗
PubMed2005

Inhibitory properties of cystatin F and its localization in U937 promonocyte cells.

Cystatin F is a recently discovered type II cystatin expressed almost exclusively in immune cells. It is present intracellularly in lysosome-like vesicles, which suggests a potential role in regulating papain-like cathepsins involved in antigen presentation. Therefore, interactions of cystatin F with several of its potential targets, cathepsins F, K, V, S, H, X and C, were studied in vitro. Cystatin F tightly inhibited cathepsins F, K and V with Ki values ranging from 0.17 nM to 0.35 nM, whereas cathepsins S and H were inhibited with 100-fold lower affinities (Ki approximately 30 nM). The exopeptidases, cathepsins C and X were not inhibited by cystatin F. In order to investigate the biological significance of the inhibition data, the intracellular localization of cystatin F and its potential targets, cathepsins B, H, L, S, C and K, were studied by confocal microscopy in U937 promonocyte cells. Although vesicular staining was observed for all the enzymes, only cathepsins H and X were found to be colocalized with the inhibitor. This suggests that cystatin F in U937 cells may function as a regulatory inhibitor of proteolytic activity of cathepsin H or, more likely, as a protection against cathepsins misdirected to specific cystatin F containing endosomal/lysosomal vesicles. The finding that cystatin F was not colocalized with cystatin C suggests distinct functions for these two cysteine protease inhibitors in U937 cells.

Biomarkers, Tumor↗
PubMed2005

Nanoscale polymer carriers to deliver chemotherapeutic agents to tumours.

Nanoscale polymer carriers have the potential to enhance the therapeutic efficacy of antitumour drugs as they can regulate their release, improve their stability and prolong circulation time by protecting the drug from elimination by phagocytic cells or premature degradation. Moreover, nanoscale polymeric carriers are capable of accumulating in tumour cells and tissues due to enhanced permeability and retention effect or by active targeting bearing ligands designed to recognise overexpressed tumour-associated antigens. The diversity in the polymer structures being studied as drug carriers in cancer therapy allows an optimal solution for a particular drug to be provided regarding its delivery and efficacy, and thus the patient's quality of life. This review is focused on the different types of nanoscale polymer carriers used for the delivery of chemotherapeutic agents and on the factors that affect their cellular uptake and trafficking.

Animals↗
PubMed2004

Poly(lactide-co-glycolide) nanoparticles as a carrier system for delivering cysteine protease inhibitor cystatin into tumor cells.

Cystatins are able to inhibit the tumor-associated activity of intracellular cysteine proteases cathepsins B and L and have been suggested as potential anticancer drugs. We have incorporated chicken cystatin, a model protein inhibitor of cysteine proteases, in poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) to improve its bioavailability and delivery into tumor cells. Cystatin-loaded NPs, 300-350 nm in diameter, were prepared by the double emulsion solvent diffusion method using low energy emulsification to preserve the biological activity of the protein. PLGA NPs and cystatin-loaded PLGA NPs at concentrations higher than 80 microg/ml were cytotoxic towards MCF-10A neoT cells, but not free cystatin at concentrations up to 5 microM. To visualize the uptake of cystatin into living MCF-10A neoT cells, NPs loaded with Alexa Fluor 488-labeled cystatin were added to the culture medium. They rapidly internalized into the cells, whereas the uptake of free-labeled cystatin was very slow. Cystatin, released from the NPs, effectively inhibited cathepsin B activity, as detected by degradation of specific Z-Arg-Arg cresyl violet substrate. In contrast, the same amount of free cystatin showed no inhibition of intracellular cathepsin B. Our results show that PLGA NPs are a useful carrier system for rapid delivery of protein inhibitors into tumor cells, enabling effective inhibition of intracellular proteolysis. The approach can be applied to other protein drugs active against intracellular targets.

Animals↗
PubMed2004

Serum cystatin C in patients with head and neck carcinoma.

BACKGROUND: The balance between proteinases of various classes and their inhibitors was found to be of critical importance for local invasion and metastasizing of tumor cells. The aim of the present study was to determine the changes in the serum cystatin C concentration in patients with squamous cell carcinoma of the head and neck. METHODS: In the sera of 34 patients with squamous cell carcinoma of the head and neck, the concentration of cysteine proteinase inhibitor cystatin C was determined using ELISA. The serum samples were collected at diagnosis (group A) and 7 to 407 days (median, 59 days) after the therapy (group B). The sera of 30 healthy blood donors served as controls (group C). RESULTS: A significant increase in the median concentration of cystatin C was found in the patients' sera (group A: 573 ng/ml, P<0.0001; group B: 551 ng/ml, P<0.0001) compared to control group C (320 ng/ml), whereas no difference was observed between groups A and B (P>0.05). Cystatin C concentrations in the sera of group A correlated with the site of primary tumor (P=0.035), being higher in the patients with non-laryngeal tumors (658 ng/ml) than in those with larynx primaries (529 ng/ml). There was a significant trend (RS=-0.535, P=0.049) towards lower cystatin C concentrations with an increasing time delay in post-treatment serum sampling (group B), which was observed in the patients with no relapse of the disease and a sampling later than 45 days after the completion of therapy. CONCLUSIONS: These results add to the knowledge of the role of cystatin C in invasive behavior of squamous cell carcinoma of the head and neck, and suggest its potential role as a tumor marker in this particular type of cancer.

Adult↗
PubMed2004

Cystatin incorporated in poly(lactide-co-glycolide) nanoparticles: development and fundamental studies on preservation of its activity.

Preservation of biological activity is still a major challenge for successful formulation and delivery of protein drugs. Cystatin, a potential protein drug in cancer therapy, was incorporated in poly(lactide-co-glycolide) nanoparticles by the water-in-oil-in-water emulsion solvent diffusion technique. In order to preserve the biological activity of cystatin, a specific modification of the method of producing nanoparticles was introduced. The activity of cystatin was strongly influenced by the stirring rate during preparation and, to a lesser extent, by selected organic solvents. A synergistic effect of mechanical stirring and sonication, both at low energy levels, enabled nanoparticles to be formed without denaturing the cystatin. Nanoparticles produced by the optimised method ranged from 300 to 350 nm in diameter with 85% of the starting cystatin activity. The loading efficiency of cystatin depends on polymer type and ranged from 12 to 57%, representing an actual loading of 0.6-2.6% (w/w). Among various cryo-/lyoprotectants bovine serum albumin was identified as the most successful. The use of a protein protectant prior to nanoparticle formation was essential to maintaining the biologically active three-dimensional structure of cystatin. In addition, a specific type of poly(lactide-co-glycolide) polymer, particularly in terms of its functional groups, was identified to be important in retaining cystatin activity. Cystatin incorporated into nanoparticles in this way maintains its structural integrity, making it suitable for effective drug delivery.

Animals↗
PubMed2004

Cysteine cathepsins (proteases)--on the main stage of cancer?

Cysteine cathepsins are involved in degradation of extracellular matrix, facilitating growth, invasion, and metastasis of tumor cells, in tumor angiogenesis, in apoptosis, and in events of inflammatory and immune responses. In this issue of Cancer Cell, demonstrate association of increased cathepsins activity with angiogenic vasculature and invasive fronts of carcinomas during tumorigenesis in transgenic mouse models using activity-based chemical probes and in vivo imaging. Moreover, this study shows that a broad-spectrum cysteine cathepsin inhibitor effectively blocks several stages of tumorigenesis in the RIP1-Tag2 transgenic mouse model, offering new therapeutic opportunities in cancer treatment.

Animals↗
PubMed2004

Inhibitory p41 isoform of invariant chain and its potential target enzymes cathepsins L and H in distinct populations of macrophages in human lymph nodes.

Activation of the CD4(+) T-cell mediated immune response relies on the proteolytic capacity of enzymes involved in modulating major histocompatibility complex (MHC) II-associated antigen presentation in antigen-presenting cells (APC). The MHC II-associated chaperone molecule p41 isoform of invariant chain (inhibitory p41 Ii) has been suggested to regulate stability and activity of cathepsin L in these APC. In the present study the human lymph node distribution of non-inhibitory p31 Ii and inhibitory p41 Ii have been compared by differential labelling, using two specific monoclonal antibodies. The distribution of p41 Ii, but not p31 Ii, matched the distribution of cathepsins L and H in subcapsular and cortical sinuses and germinal centres. Co-localization of p41 Ii with cathepsin H was confirmed in strongly CD68(+) sinus-lining macrophages, acting as APC. Furthermore, p41 Ii was determined together with cathepsins L and H in tingible body macrophages, highly phagocytic, but not antigen-presenting cells inside germinal centres. With respect to the physiological function that these two populations of macrophages have in human lymph nodes, our results support a regulatory function of p41 Ii towards cathepsins L and H in human macrophages, associated with the processes of phagocytosis rather than antigen presentation.

Animals↗
PubMed2003

Intracellular and extracellular cathepsin B facilitate invasion of MCF-10A neoT cells through reconstituted extracellular matrix in vitro.

Lysosomal cysteine proteinase cathepsin B is implicated in remodeling the extracellular matrix, a crucial step in the process of tumor cell invasion. In this study the contributions of intracellular and extracellular cathepsin B activities in the invasion of ras-transformed human breast epithelial cells, MCF-10A neoT, were assessed using specific cathepsin B neutralizing monoclonal antibody (Mab) 2A2, together with other general and specific cysteine proteinase inhibitors. We showed that the degradation of extracellular matrix by living MCF-10A neoT cells was predominantly intracellular, as imaged by confocal assays using quenched fluorescent substrate DQ-collagen IV. CA-074, a membrane-impermeable cathepsin B-selective inhibitor and its membrane-permeable analogue CA-074Me showed similar inhibition of invasion at 10 microM, i.e., 24.9 and 27.0%, respectively. Neutralizing monoclonal antibody exhibited a significantly higher inhibitory effect, decreasing invasion at 0.5 microM by 42.7%. Tumor cells may internalize monoclonal antibody; therefore, 2A2 Mab could impair both the intracellular and the extracellular fractions of cathepsin B activity. However, both 2A2 Mab and cathepsin B-selective inhibitors were less potent than the general cysteine proteinase inhibitors chicken cystatin and E-64, indicating that other cysteine proteinases, presumably cathepsin L, are involved in invasion. Our results show that intracellular and extracellular cathepsin B activity contribute to in vitro invasion of MCF-10A neoT cells and suggest that inhibitors capable of impairing both fractions have a potential as new anticancer drugs.

Antibodies, Monoclonal↗
PubMed2003

Selective suppression of cathepsin L by antisense cDNA impairs human brain tumor cell invasion in vitro and promotes apoptosis.

Invasion and metastasis of certain tumors are accompanied by increased mRNA protein levels and enzymatic activity of cathepsin L. Cathepsin L has also been suggested to play a role in the proteolytic cascades associated with apoptosis. To investigate the role of cathepsin L in brain tumor invasion and apoptosis, the human glioma cell line, IPTP, was stably transfected with full-length antisense and sense cDNA of cathepsin L. Down-regulation of cathepsin L by antisense cDNA significantly impaired (up to 70%) glioma cell invasion in vitro and markedly increased glioma cell apoptosis induced by staurosporine. Compared to control and parental cell lines, antisense down-regulation of cathepsin L was associated with an earlier induction of caspase-3 activity. Up-regulation of cathepsin L activity by sense cDNA was associated with reduced apoptosis and later induction of caspase-3 activity. Moreover, down-regulation of cathepsin L lowered the expression of antiapoptotic protein Bcl-2, whereas up-regulation increased the expression of Bcl-2, indicating that cathepsin L acts upstream of caspase-3. These data show that cathepsin L is an important protein mediating the malignancy of gliomas and its inhibition may diminish their invasion and lead to increased tumor cell apoptosis by reducing apoptotic threshold.

Apoptosis↗
PubMed2002

Comparison of potential biological markers cathepsin B, cathepsin L, stefin A and stefin B with urokinase and plasminogen activator inhibitor-1 and clinicopathological data of breast carcinoma patients.

Cysteine, serine and metalloproteinases and their respective inhibitors are involved in tumor cell invasion and may have prognostic value for the outcome of malignant disease. The aim of the study was to compare the expression of new potential biological tumor markers, the lysosomal cysteine proteinases and their endogenous inhibitors, with that of the serine proteinases and their inhibitors in breast cancinoma and to relate their levels to the clinicopathological factors of the disease. Enzyme-linked immunosorbent assays (ELISAs) were used to measure cysteine cathepsin B (CatB) and cathepsin L (CatL) and their inhibitors, stefin A (StA) and stefin B (StB), together with urokinase (u-PA) and plasminogen activator inhibitor-1 (PAI-1), in 150 cytosols of primary invasive breast carcinoma. A good correlation was found between the levels of the two cysteine proteinases but only a moderate one between those of the cysteine and serine proteinases. u-PA and PAI-1 levels correlated positively with histological grade and negatively with estrogen receptor (ER) status. PAI-1 correlated with most clinicopathological factors that indicate the progression of the disease, while cathepsins and stefins were independent of these factors. In the total group of patients, high u-PA and PAI-1 and low StB levels correlated significantly with shorter disease-free survival (DFS), while CatB, CatL and StA did not. In lymph node negative patients, high CatB and CatL were also associated with shorter DFS, while u-PA remained the most significant of all these biological markers. In conclusion, this retrospective study showed u-PA to be of better prognostic relevance than the cysteine proteinases, though CatB and CatL were relevant for prognosis in lymph node negative breast cancer patients.

Adult↗