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Biomedical subjects

J Sedlák

Publications and source records attributed to J Sedlák.

At least 19 recordsLinked to original sources
PubMed2006

Garlic-derived organosulfides induce cytotoxicity, apoptosis, cell cycle arrest and oxidative stress in human colon carcinoma cell lines.

Organosulfur compounds (OSC) from garlic, especially allicin (ALI), diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS) are recognized as a group of potential chemopreventive agents. In this study, we examined the effects of OSC on human Caco-2 and HT-29 colon carcinoma cell lines. Apoptosis induction (Annexin-V-FITC/PI, fluorescein diacetate/PI, sub-G1 fraction), modulation of DNA cell cycle (G2/M arrest, phospho-H3 mitotic marker), transmembrane mitochondrial potential (JC-1) and intracellular GSH amount (monochlorobimane assay) were measured by flow cytometry and fluorimetry. Our results showed that order of OSC-induced cell death in Caco-2 and HT-29 cells increased in the range as follows: ALI < DAS = DADS < DATS and ALI = DAS < DADS < DATS, respectively. Both cell lines used are relatively resistant to OSC induced cytotoxicity, because compound concentrations required to obtain significant effect are in high micromolar range. ALI was less toxic than equimolar doses of other OSC tested with the exception of GSH modulation and G2/M arrest in Caco-2 cells. DADS-treated HT-29 cells and both DATS-treated cell lines exhibit inverse correlation between p-H3 positivity and compound concentration due to higher apoptotic rate. These results show the correlation of sulfur atoms number in OSC with their capacity in apoptosis induction and support the role of redox-sensitive "sulfhydryl switches" in maintaining intracellular redox milieu.

Anticarcinogenic Agents↗
PubMed2001

Non-steroidal anti-inflammatory agent ibuprofen-induced apoptosis, cell necrosis and cell cycle alterations in human leukemic cells in vitro.

The cytotoxic activity of the non-steroidal anti-inflammatory agent ibuprofen to human promyelocytic leukemia cell line HL-60, its multidrug-resistant subline HL-60/VCR (MDR-1 gene coded P-glycoprotein), as well as myeloma U266 and B-lymphoblastoid ARH-77 cell lines was demonstrated with the aid of flow cytometric analysis. Ibuprofen inhibited proliferation and induced apoptosis (detected as sub-G0 nuclei, fluorescein diacetate staining, Annexin-V binding cells and agarose electrophoretic detection of nucleosomal DNA fragmentation) in promyelocytic cells and, to a lesser extent, in U266 and ARH-77 cells.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2001

Conditioned medium from HeLa cells enhances motility of human monocyte-derived dendritic cells but abrogates their maturation and endocytic activity.

Progressive tumor proliferation may be associated with suppression of the immune response. Several different mechanisms can contribute to immune evasion. It is generally proposed that inhibition of dendritic cell functions would be a key mechanism by which tumors could escape immune surveillance. Therefore, the purpose of this study was to evaluate the capacity of HeLa cells conditioned medium (HeLa-CM) to modulate phenotypic and functional parameters of human peripheral blood monocyte-derived dendritic cells (DCs). Two types of reference DCs population were generated in vitro, the first cultured in the presence of IL-4 and GM-CSF which represented immature DCs (iDCs) and the second, representing mature DCs (mDCs), was raised from the iDCs by additional stimulation with a maturation cocktail - TNF-alpha, IL-1beta, IL-6, PGE2. In parallel, the iDCs were treated with HeLa-CM collected from the tumor cells. The analysis of DC populations demonstrated that the HeLa-CM prevented maturation of these cells and also impaired their capacity to uptake an antigen and stimulate proliferation of allogeneic T cells. In contrast, HeLa-CM modulated DCs exhibited a 3-fold increase mobility over iDCs. The latter functional capacity did not correlate with the levels of matrix metalloproteinase expression in the analysed cells. Altogether, our results provide evidence that HeLa cells produce soluble factors that might dramatically alter basic phenotypic and functional characteristics of DCs.

Cell Movement↗
PubMed2000

Radiation-induced apoptosis and cell cycle alterations in human carcinoma cell lines with different radiosensitivities.

Radiosensitivity of examined human neoplastic cell lines was assessed with the aid of MTT assay. Differences between radiosensitive and radioresistant human neoplastic cell lines were as follow: a) radiation-induced apoptosis detected by flow cytometry was apparent in the most radiosensitive (i.e. CH-1 ovarian carcinoma cell line), but not in the radioresistant (i.e. SKOV-3 ovarian carcinoma) cell lines, b) radiation-induced G2/M arrest appeared early after irradiation (6 hours) in both the radioresistant SKOV-3 cells and in the radiosensitive CH-1 human ovarian carcinoma cell line, but a different pattern was observed 24 hours after irradiation with 2 Gy dose with G2/M arrest only in radiosensitive cell line. The radiosensitivity and resistance to radiation-induced apoptosis in the radioresistant human breast carcinoma MDA-MB-231 cell line were similar to those observed in SKOV-3 cells. These data suggest that radiation-induced apoptosis and cell cycle alterations can predict radiosensitivity at least in some examined human malignant cells in vitro.

Apoptosis↗
PubMed2000

Differential sensitivity of ovarian carcinoma cell lines to apoptosis induced by the IMPDH inhibitor benzamide riboside.

The differential sensitivity of examined human ovarian carcinoma cell lines (CH1, A-2780 and SKOV-3) to the IMPDH inhibitor, benzamide riboside (BR), was demonstrated with the aid of MTT assay. Present data show that all three examined ovarian carcinoma cell lines were sensitive to the cytotoxic effects of BR in the order of sensitivity CH1, SKOV-3, A-2780, (IC50 = 2.8, 4.0 and 7.4 microM, respectively). Although the IC50 of SKOV-3 cells was similar to that previously determined by others, more than 20% of SKOV-3 cells remained viable in a plateau up to 40 microM BR concentration. This relative resistance of SKOV-3 cells to BR corresponded to the absence ofBR-induced apoptosis in SKOV-3 cells, which together with clearly demonstrated sensitivity of CH1 cells to BR-induced apoptosis, established by flow cytometry (presence of nuclei with sub-G0 DNA content, Annexin V binding) and western blotting (poly-ADP-ribosyl-polymerase (PARP) cleavage), further stressed the role of drug-induced apoptosis in the over-all drug-induced cytotoxicity.

Apoptosis↗
PubMed2000

Radiation-induced DNA damage and repair evaluated with 'comet assay' in human ovarian carcinoma cell lines with different radiosensitivities.

Radiation-induced DNA damage and kinetics of DNA repair was evaluated in three human ovarian carcinoma cell lines (i.e. CH-1, A-2780 and SKOV-3) with different sensitivities to ionizing radiation and radiation-induced apoptosis with the aid of single cell gel electrophoresis (SCGE, the comet assay). A good correlation was found between the initial level of DNA breaks and radiation induced apoptosis in CH-1 and SKOV-3 cell lines. While the radiation-sensitive CH-1 cell line manifested the highest level of initial DNA breakage and a significant delay in DNA break rejoining, the inverse correlation was found in the radiation-resistant cell line SKOV-3. Intermediate initial level of breaks was induced in the A-2780 cell line characterized by the intermediate sensitivity to X-ray radiation in comparison to CH-1 and SKOV-3 cells, however, the kinetics of DNA repair was comparable with radiation-resistant cell line SKOV-3. Our data suggest that the comet assay could be a promising tool for prediction of intrinsic cell radiosensitivity. This method might be considered as a supplementary technique to the more reliable but time consuming clonogenic assay.

Apoptosis↗
PubMed1999

Cell surface immunophenotype and gelatinase activity of the human breast carcinoma cell line (MCF-7/6) with functionally defective E-cadherin.

Expression of differentiation and adhesion cell surface antigens (LewisX - CD15, CD44, syndecan 1 - CD138 and basigin/EMMPRIN - CD147) were determined on the cell surface of human breast carcinoma MCF7 cells in vitro with the aid of flow cytometry and compared with that of MCF-7/6 cells, with functionally defective E-cadherin system and increased biological aggressiveness. The major cell surface alterations in MCF-7/6 cells compared with the parental MCF-7 cell line were a markedly increased CD15 (LewisX) and CD44 antigen cell surface expression on MCF-7/6 cells. There were no major differences between parental MCF-7 and MCF-7/6 cells in cell surface syndecan 1, basigin/EMMPRIN, E-cadherin and high affinity non-integrin laminin receptor expression. The constitutive cell surface gelatinase A and B activities were absent on MCF-7 and faint in MCF-7/6 cells. Both phorbol ester TPA and tumor necrosis factor TNF-alpha induced a marked up-regulation of gelatinase B only in MCF-7/6 cells. No marked differences in penetration of MCF-7 vs. MCF-7/6 cells into collagen/fibroblast matrix in vitro were observed. The increased expression of CD15 (LewisX), CD44 antigen and TNF-alpha-inducible gelatinase B on MCF-7/6 cells may represent auxiliary factors contributing to the increased biological aggressiveness of MCF-7/6 cells.

Antigens, Surface↗
PubMed1999

Angiogenesis inhibitor TNP-470: cytotoxic effects on human neoplastic cell lines.

Angiostatic substance TNP-470 displayed moderate cytotoxicity towards human leukemia HL-60, HL-60/ADR, HL-60/VCR and myeloma ARH77 cell lines with IC50 in the range 5-10 microM of concentrations and slightly higher IC50 for myeloma cell line U266. IC50 for ovarian CH-1, A2780 and A2780/ADR cell lines was in the range 10-15 microM with the exception of platinum-resistant SKOV3 cell line (more than 40 microM ). The IC50 values for MDA-MB-231 and MCF-7 breast carcinoma cell lines were 15 and 25 microM, respectively. In human hemopoietic neoplastic cell lines examined, TNP-470 induced the appearance of subpopulation with sub-G0 DNA content, suggesting the apoptosis-inducing potential of TNP-470 in these cells. No TNP-470-induced drug uptake modulation in drug-resistant leukemia cell line HL-60/VCR was observed. TNP-470 induced accumulation of cells in G0/G1 phase of cell cycle. There was no TNP-470-induced inhibition of MMP collagenase activity or MMP (MMP2 and MMP9) production in the human fibrosarcoma cells HT 1080 in vitro.

Angiogenesis Inhibitors↗
PubMed1999

Detection of apoptotic changes in HeLa cells after treatment with paracetamol and sodium fluoride.

Apoptosis is genetically programmed cell death, an irreversible process of cell senescence with characteristic features (cell shrinkage, chromatin condensation, DNA fragmentation, apoptotic bodies) different from necrosis. Several effective in vitro methods for qualitative and quantitative detection of apoptotic events have been developed. Chromatin degradation, reductions in cell volume and other apoptosis-associated changes in cell morphology and physiology can be quickly analysed by multiparametric flow cytometry (FC) using small numbers of intact cells. One further method used for morphological determination of apoptotic changes is the fluorescent microscopic technique (FM) based on labeling of cells with fluorochromes acridine orange and ethidium bromide. In our experiments FC was used for determination of DNA changes in HeLa cells based on staining of DNA by fluorochrome propidium iodide (PI). Among the tested chemicals (paracetamol and sodium fluoride) apoptotic process could only be detected in paracetamol-treated cells. Apoptosis was induced mainly in cells treated with paracetamol (concentration range 4-5 mg/ml) for 8 h and following incubation for 18 h in fresh medium without paracetamol. The results obtained by the FM method correlated with the results obtained by FC.

Acetaminophen↗
PubMed1999

Detection of apoptosis in a heterogenous cell population using flow cytometry.

Apoptosis induced in human leukemic cells (promyelocytic human leukemic cells HL-60, multidrug-resistant subline HL-60/VCR) and human ovarian carcinoma cells (A2780 and multidrug-resistant subline A2780/ADR) in vitro was detected by flow cytometric analysis or DNA electrophoresis. The cytofluorometric techniques utilized, i. e. detection of phosphatidylserine exposed at the outer surface of the plasma membrane, identification of cells with "sub-G0" DNA content or increased light side scatter (cell internal structure) correlated with the electrophoretic determination of DNA fragmentation ("DNA ladder"). Detection of the 34 kDa mitochondrial protein recognized by the monoclonal antibody Apo2.7 yielded elevated percentages of apoptotic cells, suggesting that this technique detecting both early and late apoptosis in digitonin-fixed cells might not be restricted to the specific detection of programmed cell death.

Apoptosis↗
PubMed1998

Staurosporine enhanced benzamide riboside-induced apoptosis in human multidrug-resistant promyelocytic leukemia cells (HL-60/VCR) in vitro.

The inosine monophosphate (IMP) dehydrogenase inhibitor benzamide riboside (BR) induced apoptosis (detected with the aid of flow cytometric identification of cells with sub-G0 DNA content and increased side angle light scatter) equally or slightly more intensively in the multidrug-resistant human promyelocytic leukemia cell line (HL-60/VCR: MDR-1 gene, Pgp positive) in comparison with the parental drug sensitive HL-60 cells. Staurosporine alone induced relatively low level of apoptosis in parental HL-60 cells but higher level (approximately 35%) of apoptosis in multidrug-resistant HL-60/VCR cells after 24 hour induction. The combination of benzamide riboside and staurosporine induced in both drug-sensitive and drug-resistant HL-60 cells a marked proportion of apoptotic cells already after short (6 hour) induction (more than 30% of apoptotic cells).

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Hematopoietic cell differentiation antigens (CD system 1997). Cancer research relevance.

The 6th International Workshop on Leukocyte Antigens (white cell differentiation antigens) continued the international cooperative effort aimed at characterization of all leukocyte cell surface antigens with respect to their biochemical properties, cell- and cell line expression, molecular and cellular function(s) and eventual disease relevance. Significantly, among 36 newly defined CD clusters identified with the aid of numerous monoclonal antibodies submitted to the workshop [17], 8 new clusters belonged to the endothelial section, 6 new CD clusters were identified within cytokine receptor section and 5 such clusters were characterized in the adhesion structure section, i.e. as antigens with the pattern of expression also on cells, tissues and cell lines outside the hematopoietic system (Tables 1-3). Minority of newly defined clusters appurtened to lineage-specific or non-lineage hematopoietic differentiation antigens (Table 4), i.e. 5 new CD clusters within myeloid section, 3 new clusters within both non-lineage and NK antigens, 2 T-cell antigens and one new CD cluster defined within both B-cell or platelet sections.

Antigens, CD↗
PubMed1998

Pentoxifylline stimulates drug-induced apoptosis in leukemic cells.

Camptothecin (CAM) and cisplatin (cis-diamminedichloroplatinum(II), cis-Pt) were used as inducers of apoptosis in the mouse leukemic L1210 cells. Relatively high concentrations of 50 micromol cis-Pt and 50 micromol CAM, respectively, were used to induce the apoptotic DNA ladder. The simultaneous treatment of L1210 cells by the drug and pentoxifylline (PTX) resulted in a decrease of drug concentrations necessary for the induction of apoptosis. This study revealed that a cell cycle G2 checkpoint inhibitor PTX reduces time intervals necessary for the onset of drug-induced apoptosis in these cells. This fact might be important as the earlier onset of programmed cell death may decrease a risk of tumor cells to become resistant to drug therapy.

Animals↗
PubMed1997

Protein kinase inhibitor-induced alterations of drug uptake, cell cycle and surface antigen expression in human multidrug-resistant (Pgp and MRP) promyelocytic leukemia HL-60 cells.

Protein kinase inhibitors staurosporine and CGP 41251, a benzoylated derivative of staurosporine with selective PKC inhibitory activity, reversed the decreased rhodamine 123 uptake in HL-60/VCR (with Pgp-mediated drug resistance) but not in HL-60/ADR (MRP-mediated drug resistance) cells. CGP 41251 reversed the decreased rhodamine 123 uptake in HL-60/VCR cells more efficiently (when compared on a equimolar basis) than staurosporine. However, the protein tyrosine kinase inhibitor genistein unexpectedly modulated the decreased rhodamine 123 uptake in Pgp positive (HL-60/VCR) cells, but not in HL-60/ADR (MRP positive) cells. Cell surface phenotype of both HL-60 drug-resistant cell sublines was compared with that of the parental, drug-sensitive HL-60 cells. Both drug-resistant cell lines expressed markedly decreased levels of cell surface HLA class I antigen in comparison with the parental HL-60 cells. A similar decreased cell surface expression of HLA class II/DR on both drug-resistant, as well as of CD59 (protectin) on HL-60/ADR cells was found. Both protein kinase C inhibitors studied (staurosporine and CGP 41251) exhibited variable effects on cell surface antigen (HLA, ICAM-1, CD59) expression, suggesting complex interactions between PKC-dependent and -independent mechanisms in the regulation of surface antigen expression in these cell lines. Staurosporine differed from CGP 41251 in the cell cycle alterations induced in the HL-60 cell lines examined. Staurosporine induced the accumulation of cells in the G2/M phase of the cell cycle and the appearance of pre-G0 (apoptotic) cells in both examined drug-resistant cell lines. Staurosporine induced the appearance of cells with high DNA content in HL-60/ADR, but not in HL-60/VCR cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Taxol-enhanced cytotoxic effect of radiation in human promyelocytic leukemia cells: relative resistance of multidrug-resistant HL-60 cells in vitro.

Cytotoxic effects of sequential taxol (paclitaxel) and X-irradiation on drug-sensitive human cultured promyelocytic leukemia (HL-60) cell line and its multidrug-resistant sublines were examined using photometric MTT test and flow cytometry. Paclitaxel (at concentrations 1-10 nmol) stimulated the cytotoxic effect of irradiation in HL-60 and to a lesser extent also in HL-60/ADR, but not in HL-60/VCR cells. The stimulation of radiation-induced cytotoxic effect by paclitaxel correlated with its potential to induce cell cycle and viability alterations identified with flow cytometric analysis (i.e. increased propidium iodide staining, increased side scatter, decreased forward angle scatter, accumulation of necrotic cell detritus, apoptotic pre-G0 cells and cells in the G2/M phase of the cell cycle).

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

The leukocyte common antigen (CD45) on human pre-B leukemia cells: variant glycoprotein form expression during the cell exposure to phorbol ester is blocked by a nonselective protein kinase inhibitor H7.

The human pre-B acute lymphoblastic leukemia cell line REH6 was utilized for characterization of CD45 glycoprotein by monoclonal antibodies (mAb) recognizing four distinct CD45 antigen specificities, i.e. nonrestricted CD45, restricted CD45RA, CD45RB and CD45R0. Immunoprecipitation revealed two antigen specificities on REH6 cells of m.w. 220 kDa and 190 kDa, both presenting wide range of isoelectric point pI approximately 6.0-7.5. Nonrestricted CD45 epitopes were not affected by the sialyl acid cleavage with sodium metaperiodate or neuraminidase, but were sensitive to both, tunicamycin, the N-glycosylation inhibitor and monensin, an inhibitor of protein transport through the Golgi compartment. O-sialoglycoprotein endopeptidase from Pasteurella haemolytica A1 partially cleaved CD45RA and CD45RB epitopes, while nonrestricted CD45 determinants were not affected by this enzyme. Limited proteolysis of this antigen resulted in the appearance of 160-180 kDa peptide domains which retained CD45 epitopes. Further, the treatment of cells with phorbol myristate acetate (PMA) induced marked down-regulation of 220 and 190 kDa isoforms and the appearance of new 210, 180 and 170 kDa variant glycoprotein forms which were not found on parental cells. This PMA effect was not accompanied by the programmed cell death and was markedly blocked by a nonselective protein kinase (PK) inhibitor isoquinoline sulfonamide H7. Modulation of CD45 by phorbol esters might serve as an in vitro model for an additional insight into the function of CD45 in hematopoietic cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1997

Human multidrug-resistant (MRP,p190) myeloid leukemia HL-60/ADR cells in vitro: resistance to the mevalonate pathway inhibitor lovastatin.

Mevalonate pathway inhibitor lovastatin inhibited proliferation of human multidrug-resistant promyelocytic leukemia HL-60/ADR cells in vitro, with MRP-gene coded p190 mediated drug resistance, to a markedly lesser extent than that of the parental drug sensitive HL-60 cells and also that of the other human multidrug resistant (MDR-1, P-glycoprotein) myeloid leukemia cell line HL-60/VCR. The sensitivity of the examined human leukemia cell lines to the cytostatic activity of lovastatin correlated approximately with the potential of lovastatin to induce the characteristic cell cycle alteration (i.e. the accumulation of lovastatin-treated cells in the G0/G1 phase of the cell cycle). The P-glycoprotein positive HL-60/VCR cells and the parental drug sensitive HL-60 cells were more sensitive to this cell cycle alteration than the HL-60/ADR multidrug resistant leukemia cells with MRP drug resistance. Lovastatin (72 hours, 20 micromol) induced apoptosis and cell necrosis in HL-60 cells, apoptosis but not cell necrosis in HL-60/VCR cells and neither apoptosis nor necrosis in HL-60/ADR cells.

Antineoplastic Agents↗