Biomedical subjects

Janko Kos

Publications and source records attributed to Janko Kos.

24 records · Page 2Linked to original sources

PubMed2002

Genotoxicity of trivalent chromium in bacterial cells. Possible effects on DNA topology.

Trivalent chromium is a metal required for proper sugar and fat metabolism. However, it has been suggested that it causes DNA damage in in vitro test systems, although in vivo toxicity has not yet been proved. In the present study, the effect of Cr3+ on bacterial cells was tested with the Pro-Tox (C) assay, and its cellular uptake was measured with flame atomic absorption spectroscopy. The potential genotoxicity of Cr3+ was further examined by the study of its influence on a bacterial type II topoisomerase. Cr3+ was shown to cause DNA damage and inhibit topoisomerase DNA relaxation activity, probably by preventing the formation of the covalent link between enzyme and double helix. In addition, Cr3+ decreases the viability and/or proliferation rate of eukaryotic cells such as murine B16 melanoma cells and human MCF-10A neoT ras-transformed human epithelial cells. The possible implication for Cr3+ intake by humans is discussed.

Animals↗

PubMed2002

Lysosomal enzymes, cathepsins in brain tumour invasion.

The expression patterns of different classes of peptidases in central nervous system (CNS) tumours have been most extensively studied in astrocytomas and meningiomas. Although the two types of tumours are very different in most respects, both may invade locally into normal brain. This process of invasion includes increased synthesis and secretion of lysosomal proteolytic enzymes - cathepsins. Aspartic endopeptidase cathepsin (Cat) D levels were found to be elevated in high-grade astrocytoma and partial inhibition of glioblastoma cell invasion by anti-Cat D antibody suggests that the enzyme activity is involved in the invasion process. Several studies on cysteine endopeptidase (CP) Cat B in gliomas agreed that transcript abundance, protein level and activity of Cat B increased in high-grade astrocytoma cultures compared with low-grade astrocytoma cultures and normal brain. Moreover, in glioma biopsies Cat B levels correlated with evidence of clinical invasion and it has been demonstrated that Cat B both in tumour cells and in endothelial cells can serve as a new biological marker for prognosis in glioblastoma patients. A high level of Cat B protein was also a diagnostic marker for invasive types of meningioma, distinguishing between histomorphologically benign, but invasive meningiomas and noninvasive, so-called clear-benign meningiomas. Cat L was also significantly increased in high-grade astrocytoma compared with low-grade astrocytoma and normal brain. Specific Cat L antibodies and antisense Cat L RNA transfection significantly lowered glioblastoma cell invasion. In meningioma, Cat L was a less-significant marker of invasion than Cat B. In contrast to cathepsins, the activities of endogenous cysteine peptidase inhibitors (CPIs), including stefins, cystatins and kininogens, were significantly higher in benign and atypical meningioma cell extracts than in malignant meningioma, and low-grade compared to high-grade astrocytoma. However, very low levels of stefins A and B were found in meningioma and glioblastoma tissues. Further studies on the expression levels and balance between cysteine endopeptidases (CPs) and CPIs would improve the clinical application of cathepsins in prognosis, which would lead to more-informed therapeutic strategies.

Animals↗

PubMed2002

Molecular cloning and chimerisation of an inhibitory anti-cathepsin B antibody and its expression in Chinese hamster ovary cells.

The lysosomal cysteine protease cathepsin B is one of several proteases that have been linked to tumour progression. Its increased expression and secretion in tumour cells may facilitate the degradation of extracellular matrix proteins, leading to tumour cell invasion and metastasis. Specific inhibitory monoclonal antibodies are a possible alternative to synthetic inhibitors as a therapeutic tool for cancer treatment. An inhibitory monoclonal antibody, which binds to an epitope near the active site of cathepsin B and inhibits its proteolytic activity, was prepared and its effect on invasion of ras-transformed MCF-10A neoT cells was tested in vitro. Here we present the nucleotide sequences of the heavy and light chains of the inhibitory antibody and compare them to the murine immunoglobulin germline sequences for possible somatic hypermutations. Since no harmful mutations were found, a mouse/human chimeric antibody was constructed by fusing murine V(H) and V(L) variable regions of the inhibitory antibody with human gamma 1 and K constant regions, respectively. Chinese hamster ovary K1 cells were co-transfected with expression vectors pcD-NA3L and pcDNA3H and the reactivity of the isolated chimeric antibody was tested by ELISA and Western blotting. We could demonstrate an inhibitory effect of the chimeric antibody.

Amino Acid Sequence↗

PubMed1997

The Expression of Lysosomal Proteinases and Their Inhibitors in Breast Cancer: Possible Relationship to Prognosis of the Disease.

Proteolytic enzymes have been proposed as new biological prognostic indicators to facilitate decisions about treatment of breast cancer patients following surgery. We reported earlier that the activities of cysteine proteinases (CP), cathepsin (Cat) B and cathepsin (Cat) L and the expression of stefin A might be associated with breast tumor progression and prognosis. Here, the protein concentrations of Cats D, B and L and stefin A have been measured in a series of 60 matched pairs of breast tumours and control adjacent tissues, using ELISAs developed in our laboratory. Median tumor concentrations of Cat D (47 pm/mg), Cat B (222 ng/mg) and Cat L (88 ng/mg) were significantly (p<0.0005) increased by 7 fold, 27 fold and 6 fold, respectively. Much greater increases in the activities of Cat B (63 fold) and of Cat L (274 fold) were found, indicating activation of proCat B and proCat L and/or to a decrease in specific endogenous cystatins. However, the 1.6-fold decreased (p<0.0001) levels of inhibition by cystatins could not be entirely responsible for more than 100-fold increased ratio of CP:cystatins activity. Moreover, stefin A was either increased or decreased in tumor samples, resulting in a 1.4-fold median increase in tumors. Comparing the biological parameters with the established histo-pathological prognosticators, we found that the increased protein concentration of Cat B was associated with lymph node involvement (p<0.009) and higher stage (p<0.003), and both Cat B and Cat L activities were more increased in high grade tumours (p<0.05). Survival analysis revealed that stefin A was the most significant prognostic factor for disease-free (p<0.008) and overall survival (p<0.02), followed by increased Cat B activity and protein concentration. Cat L was of borderline significance while Cat D was not significant for prognosis. We conclude that enhanced activation of CP, due partially to an imbalance between cysteine proteinases and inhibitors is linked to the progression of breast cancer. Larger sample size is needed to confirm the prognostic significance of stefin A, Cat B and Cat L.

Journal Article↗

PubMedDate not supplied

Intracellular delivery of cysteine protease inhibitor cystatin by polymeric nanoparticles.

Two polymers chitosan and poly(lactide-co-glycolide) copolymer (PLGA) were investigated to develop nanoparticles (NPs) for delivery of protein drug substance into tumour cells. Cystatin was selected as a model protein drug due to its high potential to inhibit cysteine proteases, known to trigger the invasive process. Ionotropic gelation of chitosan with tripolyposphate and precipitation of PLGA polymer from a double emulsion system by a solvent diffusion process were used to produce 250-300 nm in diameter NPs. The cellular uptake of NPs was tested on a transformed human breast epithelial cell line, MCF-10A neoT, characterized by an increased expression of cysteine proteases and highly invasive cell phenotype. The influence of NPs on cell viability was evaluated by MTT test showing IC50 400 microg/ml for PLGA and 5 mg/ml for chitosan NPs. As determined by fluorescence microscopy chitosan NPs did not enter the cells during 1-hour incubation whereas the same amount of PLGA NPs were in the cells already after 5 min of incubation. Cystatin delivered into MCF-10A neoT cells by PLGA NPs effectively inhibited intracellular proteolytic activity of cathepsin B, as detected by specific fluorogenic substrate Z-Arg2 cresyl violet. On the contrary, free cystatin in solution did not internalise into the cells and inhibit cathepsin B. To conclude, PLGA NPs with cystatin but not chitosan NPs were targeted through endocytosis to the lysosomal compartments that are rich of proteases enzymes. Our results suggest new strategy to inactivate intracellular tumour-associated proteases, and consequently the invasion behaviour of tumour cells, which could contribute to cancer therapy.

Cell Line, Tumor↗

PubMedDate not supplied

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.

Carcinoma, Non-Small-Cell Lung↗