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Human colon adenocarcinoma LS180 parental cell line and selected variants, characterized by different metastatic capacity were used to examine, whether a correlation exists between β-actin expression, its subcellular distribution and metastatic potential of these cells. Cytosolic fraction (supernatant 105 000 × g), isolated from the tumor cells was used as a source for actin quantification. The higher level of β-actin was observed in the cytosol of three selected sublines to compare with LS180 parental line. Statistically significant increase of β-actin level in highly motile EB3 cells variant should be underlined to compare with the other sublines. Distinct differences in the phenotype of adenocarcinoma cell variants were found, such as the changes in cells shape, cells spreading and ability to attach to the surface of culture dish. Actin cytoskeleton was visualized with fluorescence microscopy application and microfilaments rhodamine-conjugated phalloidin staining. β-actin subcellular localization was done by immunofluorescence staining with monoclonal anti-β actin antibodies. In the elongated cells (LS180, 3LNLN), this isoactin is dispersed in the whole cell body and concentrates in pseudopods and at the leading edges, when in the rounded variant (EB3) β-actin dominates mainly in cortical ring under cellular membrane and it is also seen in the subtle protrusions. Summary of our former (Nowak et al., 2002, Acta Biochim. Polon., 49: 823) and current data lead to the conclusion that there is a distinct correlation between metastatic capacity of examined human colon adenocarcinoma cells, the state of actin polymerization, actin cytoskeleton organization and β-actin expression.
Monomeric (G), total (T) and filamentous (F) actin and the state of actin polymerisa­tion (F:G) were determined and actin filaments were visualized in hepatoma Morris 5123 cells cultured in the presence of methotrexate (MTX) at various concentration. The exposure of the cells to this drug resulted in a decrease of total and polymerised actin in cytoplasm and in some changes in actin filament organization. This coincided with a decrease of the cells' ability to migrate through Matrigel coated filters and with inhibition of tumour formation after reimplantation of the methotrexate treated cells to experimental rats.
 In this study we evaluated efficiency of DNAzymes to modulate motility of cancer cells, an important factor in the progression and metastasis of cancers. For this purpose we targeted β1 integrins that are predominant adhesive receptors in various carcinoma cell lines (CX1.1, HT29, LOVO, LS180, PC-3). To evaluate invasiveness of cancer cells, we used a transwell migration assay that allowed analyzing chemotactic migration of colon carcinoma cell lines across an ECM-coated membrane. Their adhesive properties were also characterized by the analysis of adhesion to fibronectin, laminin and collagen. In addition, the expression of major integrin subunits, selected intact β1 integrins, and other adhesive receptors (ICAM, E-selectin, uPAR) was analyzed by flow cytometry. Inhibition of β1 integrin expression by DNAzyme to β1 mRNA almost abolished the invasiveness of the CX1.1, HT29, LS180, LOVO and PC-3 cells in vitro. These data show that DNAzymes to β1 integrin subunit can be used to inhibit invasiveness of carcinoma cells.
Our studies were focused on the isolation and characterization of highly motile fraction of cells from hepatoma Morris 5123 population. Cells that underwent several migration cycles through Matrigel - coated filters were successfully cultured. The invasion index was determined by means of Matrigel invasion assay. Statically significant increase in the value of invasion factor for selected cells variant in comparison to the parental population was observed. The considerable changes in the cell shape were followed by the reorganization of the actin cytoskeleton structure including a dense subcortical congestion in the distribution of ß-actin isoform. The visualization of this protein in tumor cells was performed by immunostaining and scanning fluorescent confocal microscopy. The results were confirmed by densitometry analysis of Western blots. In addition, the increased state of actin polymerization in the cytoplasmic fraction of selected cells was determined as measured by filamentous to monomeric (F:G) actin ratio. Concluding, the selected fraction of hepatoma Morris 5123 cells with higher invasion capacity was characterized by rounded shape, remarkable increase of ß-actin level, its submembrane concentration as well as with the increased state of actin polymerization with respect to parental cells population.
Triterpene saponosides are widely distributed plant secondary metabolites characterized by relatively low systemic cytotoxicity and a range of biological activities. These include anti-inflammatory, antimicrobial, vasoprotective and antitumor properties. In particular, the ability of saponins to enhance the cytotoxicity of chemotherapeutic drugs opened perspectives for their application in combined cancer chemotherapy. Here, we used human prostate cancer DU-145 cells as an in vitro model to elucidate the synergy of the interactions between biological activities of an oleanane type 13β,28-epoxy triterpene saponoside (Lclet 4) and mitoxantrone, which is a cytostatic drug commonly used in prostate cancer therapy. No cytotoxic or pro-apoptotic effect of Lclet 4 and mitoxantrone administered at the concentrations between 0.05 and 0.1 µg/ml could be seen. In contrast, cocktails of these agents exerted synergistic pro-apoptotic effects, accompanied by the activation of the caspase 3/7 system. This effect was paralleled by attenuating effects of Lclet 4/mitoxantrone cocktails on the invasive potential, metalloproteinase expression and motility of DU-145 cells. Multifaceted and additive effects of Lclet 4 and mitoxantrone on basic cellular traits crucial for prostate cancer progression indicate that the combined application of both agents at systemically neutral concentrations may provide the basis for new promising strategies of prostate cancer chemotherapy.
The paper presents the results of several years investigations (2003-2007) on two invaders: the Himalayans Impatiens glandulifera and Asiatic Reynoutria japonica. The Sudety Mountains and their national parks are under strong pressure of both species, threatening the local vegetation. The four-year investigations have been carried out in field, glasshouse and in laboratory. Invasive species have their peculiar life histories which help them to occupy new areas. Those are above all the specific generative reproduction strategies (Impatiens) or vegetative reproduction strategies (Reynoutria). Both strategies secure the reproductive success and to capture more and more highly situated areas of the mountains. Very significant characteristics connected with the expansion of invaders is the excessively over and above the average production of seeds (Impatiens) and a huge annual increment aboveground biomass (Reynoutria). The investigated invasive species are probably not equipped with influence of allelopatic type as of greater importance is their competitive strength. The invaders can eliminate a part of the early spring flora belonging to the geophyte group and impoverish the regional biological diversity. Both the invasive plants enter also into some moist mountain forest communities.
Z tusz kurcząt izolowano termotolerancyjne szczepy Campylobacter, określano ich przynależność gatunkową, a następnie badano w nich obecność sekwencji DNA kojarzonej z inwazyjnymi właściwościami tych szczepów.
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