Biomedical subjects

M Strnad

Publications and source records attributed to M Strnad.

At least 19 recordsLinked to original sources

PubMed2006

In vitro photodynamic therapy on melanoma cell lines with phthalocyanine.

Photodynamic therapy (PDT) is a new treatment modality of tumours. The photochemical interactions of sensitizer, light, and molecular oxygen produce singlet oxygen and other forms of active oxygen, such as peroxide, hydroxyl radical and superoxid anion. Phthalocyanine ClAlPcS(2), belonging among the promising second generation of sensitizers, was tested as an inducer of photodamage. We report the production of reactive oxygen species (ROS) and the phototoxicity of ClAlPcS(2) assessed using G361 melanoma cells. A semiconductor laser (lambda=675nm, output power 21mW) was used as a source for evocation of the photodynamic effect. ROS generation and H(2)O(2) release after PDT on G361 cells were detected using probe CM-H(2)DCFDA and recorded by luminescence spectrometer. Viability studies show, that the optimum phototoxic effect tested on G361 melanoma cells was determined in the combination of laser dose of 25Jcm(-2) and phthalocyanine ClAlPcS(2) concentration of 5microg/ml. This combination of phthalocyanine concentration and corresponding radiation dose was lethal for melanoma cells.

Cell Line, Tumor↗

PubMed2005

Photodynamic therapy with zinc-tetra(p-sulfophenyl)porphyrin bound to cyclodextrin induces single strand breaks of cellular DNA in G361 melanoma cells.

The basis of photodynamic therapy (PDT) is the phototoxicity resulting from co-action of light, sensitizer and oxygen. In this study we demonstrate in vitro phototoxicity measurement on G361 cell lines using ZnTPPS(4) sensitizer bound to cyclodextrin hpbetaCD. We have proved its photodamage effect on cancer cell lines in the visible region of spectrum. We used the halogen lamp (24V/250W) as a source of radiation. After 24h incubation of cell cultures with 10 microM ZnTPPS(4) and 1mM cyclodextrine hpbetaCD, the cells were irradiated for 7.5 min at the total irradiation dose of 12.5 Jcm(-2). Analysis of DNA damage in the cell line after PDT was proved by comet assay and using inversion fluorescent microscope with image analysis. This treatment method gave rise to DNA damage. The used radiation dose of visible light in the absence of sensitizers does not induce DNA breaks in tumour cells. In conclusion, binding of ZnTPPS(4) sensitizer to cyclodextrin hpbetaCD may improve the efficacy of PDT for the treatment of malign melanoma.

Cell Line, Tumor↗

PubMed2005

Antiproliferative activity of olomoucine II, a novel 2,6,9-trisubstituted purine cyclin-dependent kinase inhibitor.

The study describes the protein kinase selectivity profile, as well as the binding mode of olomoucine II in the catalytic cleft of CDK2, as determined from cocrystal analysis. Apart from the main cell cycle-regulating kinase CDK2, olomoucine II exerts specificity for CDK7 and CDK9, with important functions in the regulation of RNA transcription. In vitro anticancer activity of the inhibitor in a panel of tumor cell lines shows a wide potency range with a slight preference for cells harboring a wild-type p53 gene. Cell-based assays confirmed activation of p53 protein levels and events leading to accumulation of p21(WAF1). Additionally, in olomoucine II-treated cells, Mdm2 was found to form a complex with the ribosomal protein L11, which inhibits Mdm2 ubiquitin ligase function. We conclude that perturbations in RNA synthesis may lead to activation of p53 and that this contributes to the antiproliferative potency of cyclindependent kinase inhibitors.

Antineoplastic Agents↗

PubMed2005

[Erythema nodosum].

The authors describe a case of 41-year-old patient with erythema nodosum as a manifestation of colitis ulcerosa. They stress the importance of complex look at this skin disease, which can be caused by many factors. Non-specific intestinal inflammation can be a cause of the erythema nodosum in 1-2%.

Adult↗

PubMed2004

Haematological malignancies in childhood in Croatia: investigating the theories of depleted uranium, chemical plant damage and 'population mixing'.

Some of potential causes proposed to explain the reported increase of haematological malignancies in childhood during or after the war period in several countries include depleted uranium, chemical pollution and population mixing theory. The aim of this study was to define the population of Croatian children aged 0-14 years who were potentially exposed to each of those risks during the war and to investigate any possible association between the exposure and the incidence of haematological malignancies. The authors analyzed the data reported by the Cancer Registry of Croatia during the pre-war period (1986-1990), war period (1991-1995) and post-war period (1996-1999). In the group of 10 counties potentially exposed to depleted uranium and two counties where chemical war damage occurred, no significant difference in incidence of the studied haematological malignancies was noted in comparison to pre-war period. The incidence of lymphatic leukaemia significantly increased in four counties where population mixing had occurred during the war period, supporting the 'mixing theory'. In those counties, the incidence of Hodgkin's lymphoma decreased during and after the war. In Croatia as a whole, decreases in incidence of myeloid leukaemias during war and non-Hodgkin lymphoma after the war were noted.

Adult↗

PubMed2004

Synergic effects of the cyclin-dependent kinase (CDK) inhibitor olomoucine and androgen-antagonist bicalutamide on prostatic cancer cell lines.

Currently, mechanisms leading to both apoptosis induction and the development of hormone-independence of prostate carcinoma cells are intensively studied. Attention is also given to the possibility of restoring cell sensitivity to hormone-antagonists. The present study focuses on the effect of the combined synthetic cyclin-dependent kinase [CDK] inhibitor, olomoucine and the antiandrogen bicalutamide on hormone-insensitive (DU-145) and hormone-sensitive (LNCaP) prostate cancer cell lines. In both cell lines reduction in cell viability was significantly higher when olomoucine and bicalutamide were applied in combination when compared to separate application of both these drugs. The setting of optimal concentrations for both substances was important for the final effect on both cell lines. The proliferation arrest was accompanied by a decrease in cyclin D1 expression and the activation of p21Waf1/Cip1 and p27Kip1 pathways in both cell lines. Contrary to the previously described effect of 200 microM olomoucine, weak AR induction after treatment with effective concentrations of olomoucine was not seen in the hormone- insensitive cell line DU-145. The related reaction of DU-145 and LNCaP cell lines to treatment with combined olomoucine and bicalutamide likely provides evidence that the inhibitory effect of bicalutamide may not only be associated with its antiandrogenic properties. The tested substances probably influence different regulatory pathways and these have co-operative impact on the cell cycle outcome. Understanding antitumor and antihormone actions of both agents is essential for the development of novel therapeutic schemes integrating substances with different action. Our results show that the combination of synthetic CDK inhibitors and hormone- antagonists may be one of a number of possible alternatives.

Androgen Antagonists↗

PubMed2002

Antiproliferative effect of plant cytokinin analogues with an inhibitory activity on cyclin-dependent kinases.

In this study, analogues of olomoucine, a previously described plant cytokinin analogue with cyclin-dependent kinase (CDK) inhibitory activity, were investigated for effect on CDK1 and CDK2 and for effect on cell proliferation. Eight new compounds exhibit stronger inhibitory activity on CDK1 and CDK2 and on cell proliferation than olomoucine. Some active compounds showed low inhibition of proliferation of normal myeloid growth. Improvement of inhibitory activity of known compounds with a C6-benzylamino group was brought about by substitution with one hydroxyl. Also, new C2 substituents associated with inhibitory activity on CDK and on cell proliferation are described. There was a significant correlation between effect on CDK and antiproliferative effect on the KG1 and Molt3 cell lines and on primary human lymphocytes, strongly suggesting that at least part of the antiproliferative effect of cytokinin analogues was due to inhibition of CDK activity. Cytokinin analogues induced apoptosis in a time- and concentration-dependent manner and changes in cell cycle distribution. The antiproliferative and pro-apoptotic effects of plant cytokinin analogues suggest that they are a new class of cytostatic agents and that they may find an application in the chemotherapy of cancer.

Apoptosis↗

PubMed2002

Synthetic inhibitors of CDKs induce different responses in androgen sensitive and androgen insensitive prostatic cancer cell lines.

AIMS: Because of the high prevalence of prostatic cancer and the limitations of its treatment, enormous effort has been put into the development of new therapeutic modalities. One potential tool is the use of cyclin dependent kinase (CDK) inhibitors, which are based on the trisubstituted derivatives of purine. The aim of this study was to analyse alterations of the regulatory pathways in both androgen sensitive and androgen insensitive prostatic cancer cell lines (LNCaP and DU-145, respectively) after blockage of the cell cycle by the synthetic CDK inhibitors, olomoucine and bohemine. METHODS: The effects of olomoucine and bohemine were studied on the following parameters: (1) cell proliferation, by measurement of DNA content; (2) viability, by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and/or XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) test; and (3) the expression of p53, pRB, Bcl-2, Bax, p16, p21, p27, cyclins A, B, D1, E, p34(cdc2), and the androgen receptor (AR), by western blot analysis. RESULTS: Both olomoucine and bohemine were potent inhibitors of growth and viability; however, bohemine was two to three times more effective than olomoucine. The sensitivity of LNCaP cells to both agents was significantly higher. After treatment, both cell lines revealed quite different spectra of protein expression. CONCLUSIONS: These results indicate the existence of specific cell cycle regulating pathways in both cell lines, which may be associated with both p53 and AR status. CDK inhibitors exhibited valuable secondary effects on the expression of numerous regulators and thus may modulate the responsiveness of tumour cells to treatment, including treatment with hormone antagonists.

Cell Division↗

PubMed2002

Nuclear receptors in early hormone refractory prostate cancer and their relationship to apoptosis-related proteins.

The expression of several genes involved in apoptosis and cell cycle control can be regulated by steroid hormones and related agents via their nuclear receptors. Members of the bcl-2 gene family participate in the regulation of apoptosis in a diverse range of cell types and are implicated in the development of hormone refractory prostate cancer and resistance to anti-cancer therapy. The aim of this study, therefore, was to examine the expression of several nuclear receptors in relation to the expression of apoptosis and cell cycle related proteins in a series of patients with early hormone refractory prostate cancer. Analysis of protein expression revealed only a weak association between Bcl-2 and AR. Bax positivity and p27Kip1 expression were significantly more frequent in the AR-positive tumors, whereas RXRbeta expression was more frequently observed in the AR-negative group. The expression of AR, Bax and p27Kip1 was inversely related, and the expression of RXRbeta directly related, to Gleason pattern status. These results suggest that the immunophenotype of early hormone refractory prostate cancer may be different to that seen in more advanced stage disease. Androgen withdrawal therapy employing anti-androgens may elicit different signalling pathways that may be dependent on ARstatus and ARsensitivity.

Apoptosis↗

PubMed2001

Regulation of plant growth by cytokinin.

Cytokinins are a class of plant-specific hormones that play a central role during the cell cycle and influence numerous developmental programs. Because of the lack of biosynthetic and signaling mutants, the regulatory roles of cytokinins are not well understood. We genetically engineered cytokinin oxidase expression in transgenic tobacco plants to reduce their endogenous cytokinin content. Cytokinin-deficient plants developed stunted shoots with smaller apical meristems. The plastochrone was prolonged, and leaf cell production was only 3-4% that of wild type, indicating an absolute requirement of cytokinins for leaf growth. In contrast, root meristems of transgenic plants were enlarged and gave rise to faster growing and more branched roots. These results suggest that cytokinins are an important regulatory factor of plant meristem activity and morphogenesis, with opposing roles in shoots and roots.

Cytokinins↗

PubMed2001

Potent induction of wild-type p53-dependent transcription in tumour cells by a synthetic inhibitor of cyclin-dependent kinases.

Activation of the p53 tumour suppressor protein by distinct forms of stress leads to inhibition of cellular proliferation by inducing cell cycle arrest or apoptosis. The cyclin-dependent kinase inhibitor roscovitine has been shown to induce nuclear accumulation of wild-type p53 in human untransformed and tumour-derived cells. We analyzed the response of different human tumour cell lines to roscovitine treatment with respect to their p53 status. Striking induction of wild-type p53 protein and dramatic enhancement of p53-dependent transcription, coinciding with p21WAF1 induction, was observed in wildtype, but not mutant, p53-bearing tumour cells after treatment with roscovitine. The transcriptional activity of p53 was substantially higher in roscovitine-treated cells than in cells irradiated with ultraviolet C or ionizing radiation, even though all these agents induced a similar amount of p53 accumulation. These results highlight the therapeutic potential of roscovitine as an anticancer drug, especially in tumours retaining a functional wild-type p53 pathway.

Animals↗

PubMed2001

Preparation, physicochemical properties and biological activity of copper(II) complexes with 6-(2-chlorobenzylamino)purine (HL1) or 6-(3-chlorobenzylamino)purine (HL2). The single-crystal X-ray structure of.

Copper(II) complexes of 6-(2-chlorobenzylamino)purine (HL1) and 6-(3-chlorobenzylamino)purine (HL2), respectively, were prepared. Depending on the pH of the medium and the molar ratio of reactants the following mononuclear (trigonal-bipyramidal) and dinuclear (octahedral, trigonal-bipyramidal or tetrahedral) complexes were isolated: [Cu2(mu-HL1)2(mu-Cl2)2(HL1)2Cl2] (1a,b), [Cu2(mu-Cl)2(mu-L1)2(H2O)2] (2a), [Cu2(mu-Cl)2(mu-L2)2(H2O)2] (2b), [Cu(H+L2)2Cl3]Cl.H2O (3a,b), [Cu2(mu-Cl)2(HL1)2Cl2] (4a), and [Cu2(mu-Cl)2(HL2)2Cl2] (4b). The compounds were characterized by elemental analyses, electronic, infrared and mass (FAB+, ES+) spectral data, magnetic susceptibility temperature dependence measurements and molar conductivity data. An X-ray single-crystal structural analysis of [Cu(H+L2)2Cl3]Cl.2H2O (3b) showed that the Cu2+ ion is penta-coordinated by three chloride ions and by two H+L2 ligands. Thus, the Cu2+ ion adopts a distorted trigonal bipyramidal coordination geometry with the protonated H+L2 ligands coordinated in trans apical positions, while the three chloride ions are situated in an equatorial plane. The cytotoxic activity of the complexes was determined by a calcein AM assay. Mouse melanoma cell line B16-FO, human malignant melanoma cell line G361, human osteogenic sarcoma cell line HOS and human breast adenocarcinoma cell line MCF7 were used. IC50 values, the drug concentrations lethal to 50% of the tumor cells, were estimated. One of the important mechanisms responsible for the cytotoxicity of cytokinin-derived compounds, the inhibition of cyclin-dependent kinases by the studied complexes, was also determined.

Animals↗

PubMed2001

Epidemiological investigation of school-related injuries in Koprivnica County, Croatia.

AIM: To assess the prevalence of injuries in elementary schools and determine specific risk groups of school-age children. METHODS: According to the 1991 census, there were 6,398 children between 7 and 14 years of age in the study area of the former Koprivnica district. During the 1992-1997 period, 354 children were injured in school. The registration of injured children was performed via structured questionnaires filled out at the emergency clinic and outpatient surgical clinic of the General Hospital in Koprivnica. The mechanism of accident and activities preceding it were categorized according to the Nordic Medico-Statistical Committee classification. Chi-square test was used to determine groups of school children at specific risk and a classification tree was made on the basis of minimum entropy values for age, sex, activity, and mechanism of injury. RESULTS: The highest injury rate of was recorded in 12-year-olds (21.7%). Upper extremities were most common site of injury (52.8%), whereas the most common type of injury was contusion (45.2%). The rate of head injuries was 3.2 times higher in younger (aged 7-10) children, whereas the rate of sports injuries was 3.5-fold higher in older (aged 11-14) children (p=0.001). Entropy classification revealed younger school-age children to be at the highest risk of contusion due to a blow from a ball, an object, or contact during sports activities. CONCLUSION: In Koprivnica County, most school-related injuries occurred during sport activities (42%) and play during recess (55%), with specific differences in age and sex.

Adolescent↗

PubMed2000

Docking-based development of purine-like inhibitors of cyclin-dependent kinase-2.

The cell division cycle is controlled by cyclin-dependent kinases (cdk), which consist of a catalytic subunit (cdk1-cdk8) and a regulatory subunit (cyclin A-H). Purine-like inhibitors of cyclin-dependent kinases have recently been found to be of potential use as anticancer drugs. Rigid and flexible docking techniques were used for analysis of binding mode and design of new inhibitors. X-ray structures of three (ATP, olomoucine, roscovitine) cdk2 complexes were available at the beginning of the study and were used to optimize the docking parameters. The new potential inhibitors were then docked into the cdk2 enzyme, and the enzyme/inhibitor interaction energies were calculated and tested against the assayed activities of cdk1 (37 compounds) and cdk2 (9 compounds). A significant rank correlation between the activity and the rigid docking interaction energy has been found. This implies that (i) the rigid docking can be used as a tool for qualitative prediction of activity and (ii) values obtained by the rigid docking technique into the cdk2 active site can also be used for the prediction of cdk1 activity. While the resulting geometries obtained by the rigid docking are in good agreement with the X-ray data, the flexible docking did not always produce the same inhibitor conformation as that found in the crystal.

CDC2 Protein Kinase↗

PubMed2000

Proteomics approach in classifying the biochemical basis of the anticancer activity of the new olomoucine-derived synthetic cyclin-dependent kinase inhibitor, bohemine.

The aim of this study was to use two-dimensional electrophoresis (2-DE) coupled with multivariate principal component analysis (PCA) to characterize the quantitative changes in the protein composition of the CEM T-lymphoblastic leukemia cell line after treatment with bohemine (BOH), a synthetic olomoucin-derived cyclin-dependent kinase inhibitor (CDKI). Cell classification, reflecting protein patterns, clearly distinguished two main groups: one group consists of 9, 12 and 24 h treated BOH cells while the second is represented by the 0 and 24 h control untreated cells and the 6 h BOH-exposed CEM lymphoblasts. Discriminant protein spots differentially expressed in the BOH-treated CEM cells were selected for identification by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) or electrospray ionization-tandem MS (ESI-MS/MS). Five of the selected protein spots were unequivocally identified as alpha-enolase, triosephosphate isomerase, eukaryotic initiation factor 5A, and alpha- and beta-subunits of Rho GDP-dissociation inhibitor 1. These proteins, all significantly downregulated in CEM T-lymphoblast leukemia in the course of BOH treatment, are known to play an important role in cellular functions such as glycolysis, protein biosynthesis, and cytoskeleton rearrangement. These results indicate that the cellular effects of olomoucine-derived CDKIs are not dependent on their ability to inhibit CDKs and could be mediated by several factors such as a decrease in protein synthesis and/or glycolysis which in turn diminishes the ability of cancer cells to function.

Antineoplastic Agents↗

PubMed1999

Increased steady state mRNA levels of the STM and KNAT1 homeobox genes in cytokinin overproducing Arabidopsis thaliana indicate a role for cytokinins in the shoot apical meristem.

This study investigates the consequences of endogenously enhanced biosynthesis of the plant hormone cytokinin in Arabidopsis thaliana (L.) Heynh. Transcriptional control of the bacterial ipt gene by the Drosophila melanogaster hsp70 promoter enabled temperature-dependent increased cytokinin production in transgenic plants. Heat-treated plants accumulated higher levels of unbound and bound zeatin-type cyto-kinins, the latter being preferentially N-conjugated glucosides. Cytokinin overproduction significantly increased the biomass of seedlings. Ipt transgenics had higher steady state mRNA levels of the shoot meristem specifying homeobox genes KNAT1 and STM, similar to the cytokinin-overproducing shoot meristem mutant amp1 (hpt, cop2, pt) This finding, together with previously described phenotypic similarities between transgenic cytokinin-overproducing plants and plants overexpressing the KNAT1 or KN1 genes, suggests that these factors act on the same pathway. We hypothesize that cytokinins act upstream of KNAT1 and STM. The influence of cytokinins on homeobox genes provides a link between the hormone and the developmental genes and indicates a role for cytokinins in the shoot apical meristem.

Alkyl and Aryl Transferases↗

PubMed1999

Dynamics of cytokinins in apical shoot meristems of a day-neutral tobacco during floral transition and flower formation

This study considered cytokinin distribution in tobacco (Nicotiana tabacum L.) shoot apices in distinct phases of development using immunocytochemistry and quantitative tandem mass spectrometry. In contrast to vegetative apices and flower buds, we detected no free cytokinin bases (zeatin, dihydrozeatin, or isopentenyladenine) in prefloral transition apices. We also observed a 3-fold decrease in the content of cytokinin ribosides (zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine) during this transition phase. The group concluded that organ formation (e.g. leaves and flowers) is characterized by enhanced cytokinin content, in contrast to the very low endogenous cytokinin levels found in prefloral transition apices, which showed no organogenesis. The immunocytochemical analyses revealed a differing intracellular localization of the cytokinin bases. Dihydrozeatin and isopentenyladenine were mainly cytoplasmic and perinuclear, whereas zeatin showed a clear-cut nuclear labeling. To our knowledge, this is the first time that this phenomenon has been reported. Cytokinins do not seem to act as positive effectors in the prefloral transition phase in tobacco shoot apices. Furthermore, the differences in distribution at the cellular level may be indicative of a specific physiological role of zeatin in nuclear processes.

Journal Article↗

PubMed1999

Synthetic cyclin dependent kinase inhibitors. New generation of potent anti-cancer drugs.

The unsatisfactory results of current anti-cancer therapies require the active search for new drugs, new treatment strategies and a deeper understanding of the host-tumour relationship. From this point of view, the drugs with a capacity to substitute the functions of altered tumour suppressor genes are of prominent interest. Since one of the main functions of oncosuppressors is to mediate cell cycle arrest via modification of cyclin dependent kinases (CDKs) activity, the compounds with ability to substitute altered functions of these genes in neoplastic cells are of prominent interest. Synthetic inhibitors of cyclin dependent kinases (CDKIs) are typical representatives of such drugs. Olomoucine (OC), flavopiridol (FP), butyrolactone I (BL) and their derivatives selectively inhibit CDKs and thus constrain tumor cell proliferation under in vitro and/or in vivo conditions. We originally discovered OC and its inhibitory activity toward CDK1 family of CDKs, and recently reported the induction of apoptosis and tumor regression following OC application. Moreover, the OC family of synthetic CDKIs has the capacity of directly inhibit CDK7, the principal enzyme required for activating other CDKs, and thus these compounds are the first known CDK7 inhibitors. Its unique mechanism of action and potent anti-cancer activity under both in vitro and in vivo conditions provide a unique tool to inhibit tumour cell proliferation, and to selectively induce apoptosis in neoplastic tissues. The mechanisms of anti-cancer activities of FP, BL, OC and related synthetic CDKIs are compared and discussed in this paper.

Antineoplastic Agents↗