Design, synthesis and biological activities of new brassinosteroid analogues with a phenyl group in the side chain.

We have prepared and studied a series of new brassinosteroid derivatives with a p-substituted phenyl group in the side chain. To obtain the best comparison between molecular docking and biological activities both types of brassinosteroids were synthesized; 6-ketones, 10 examples, and B-lactones, 8 examples. The phenyl group was introduced into the steroid skeleton by Horner-Wadsworth-Emmons. The docking studies were carried out using AutoDock Vina 1.05. Plant biological activities were established using different brassinosteroid bioassays in comparison with natural brassinosteroids. Differences in the production of the plant hormone ethylene were also observed in etiolated pea seedlings after treatment with new brassinosteroids. The most active compounds were lactone 8f and 6-oxo derivatives 8c and 9c, their biological activities were comparable or even better than naturally occurring brassinolide. Finally the cytotoxicity of the new derivatives was studied using human normal and cancer cell lines.

Frequency of BCR-ABL fusion transcripts in Serbian patients with chronic myeloid leukemia.

PURPOSE: The aim of this study was to analyze the occurrence of the most frequent BCR-ABL transcript variants (b3a2, b2a2 and e1a2) in Serbian patients with chronic myeloid leukemia (CML) and compare it with the occurrence reported in other populations. METHODS: We analyzed peripheral blood and bone marrow samples of 136 Serbian patients with CML by RT-PCR and cytogenetic methods. RESULTS: In 100 patients (73.5%) the b3a2 and in 34 (25%) the b2a2 forms of BCR-ABL were detected. One (0.75%) patient was BCR-ABL negative, but in lymphoblastic transformation he expressed the e1a2 [corrected] transcript of BCR-ABL. One (0.75%) patient displayed both b2a2 and b3a2 forms of BCR-ABL. Analysis of this group according to karyotype showed b3a2 predominance (79%) in patients with classic t(9;22); b2a2 was found in 20% and both b2a2 and b3a2 forms in 1%. In variant translocations b3a2 in 65% and b2a2 in 35% of the patients were detected. In contrast, the subgroup with normal karyotype expressed slight predominance of the b2a2 form (50%); b3a2 was found in 43% of the patients and one patient (7%) displayed e1a2. CONCLUSION: Predominance of the b3a2 form in Serbian patients with CML is in concordance with other relevant investigations, conducted mostly on Caucasian ethnic groups, but in contrast to the study performed on the Mestizo ethnic group in Ecuador. Slight predominance of the b2a2 form was also noticed among the patients with normal karyotype.

Endogenous cytokinins in Cocos nucifera L. in vitro cultures obtained from plumular explants.

Auxin induces in vitro somatic embryogenesis in coconut plumular explants through callus formation. Embryogenic calli and non-embryogenic calli can be formed from the initial calli. Analysis of endogenous cytokinins showed the occurrence of cytokinins with aromatic and aliphatic side chains. Fourteen aliphatic cytokinins and four aromatic cytokinins were analysed in the three types of calli and all the cytokinins were found in each type, although some in larger proportions than others. The most abundant cytokinins in each type of callus were isopentenyladenine-9-glucoside, zeatin-9-glucoside, zeatin riboside, isopentenyladenine riboside, dihydrozeatin and dihydrozeatin riboside in decreasing order. Total cytokinin content was compared between the three types of calli, and it was found to be lower in embryogenic calli compared to non-embryogenic calli or initial calli. The same pattern was observed for individual cytokinins. When explants were cultured in media containing exogenously added cytokinins, the formation of embryogenic calli in the explants was reduced. When 8-azaadenine (an anticytokinin) was added the formation of embryogenic calli and somatic embryos was increased. These results suggest that the difference in somatic embryo formation capacity observed between embryogenic calli and non-embryogenic calli is related to their endogenous cytokinin contents.

Binding of AR to SMRT/N-CoR complex and its co-operation with PSA promoter in prostate cancer cells treated with natural histone deacetylase inhibitor NaB.

Signaling through the androgen receptor (AR) plays a critical role in prostate cancer progression. The AR is a classical nuclear receptor (NR) providing a link between signaling molecule and transcription response. Histone deacetylase inhibitors- (HDACI) have antiproliferative and proapoptotic effects on prostate cancer cells and their implication in silence AR signaling may have potential therapeutic use. We aimed to study the inhibitory effects of the corepressor SMRT (Silencing Mediator for Retinoid and Thyroid -hormone receptors) which forms a complex together with nuclear receptor corepressor (N-CoR) and with histone deacetylase 3 (HDAC3) on AR activity.The androgen-sensitive prostate cancer cell line LNCaP and androgen-insensitive prostate cancer cell line C4-2 both AR-positive, and androgen-insensitive DU145 and PC3 prostate cancer cell lines were treated with two HDACIs, sodium butyrate (NaB) and/or trichostatin A (TSA). We amplified immunoprecipitated DNA by conventional PCR and in the -following step we used the chromatin immunoprecipitation (ChIP) analysis coupled with quantitative PCR for monitoring NaB induced formation of AR-SMRT/N-CoR complex binding on the PSA promoter. The co-immunoprecipitation assay revealed increase in AR-SMRT formation in NaB treated cells. Simultaneously, the Western blot analysis showed a significant decrease in AR protein expression. In conclusion, the inhibitory effect of NaB on AR gene expression seems to be specific and unique for prostate cancer AR-positive cell lines and corresponds with its ability to stimulate AR-SMRT complex formation. We suggest that AR and SMRT/N-CoR corepressors may form a stable complex in vitro and NaB may facilitate the interaction between AR nuclear steroid receptor and SMRT corepressor prote.

In vitro and in silico studies of interaction of synthetic 2,6,9-trisubstituted purine kinase inhibitors BPA-302, BP-21 and BP-117 with liver drug-metabolizing cytochromes P450

An evaluation of possible interactions with enzymes of drug metabolism (cytochromes P450, CYP) is an important part of studies on safety and, in general, on the properties of any drug or biologically active compound. The article is focused on the preliminary metabolic study of selected 2,6,9-trisubstituted purine kinase inhibitors with significant anticancer activities which we have developed. The compounds BP-21 and BP-117 represent strong CDK inhibitors and the compound BPA-302 was developed as selective FLT3-ITD kinase inhibitor. Here, emphasis is placed on interactions of these compounds with the nine most important forms of CYP to evaluate the possibility of inhibition of these enzymes. The possibility of their inhibitory effect was studied in vitro on selected human liver microsomal CYP enzymes. The most affected enzyme was CYP2C19. Its activity dropped to 22 % of its original value by BPA 302, to 13 % by BP-21 and to 6 % by BP-117 at the highest concentration tested (250 µmol·l(-1)). The results suggest that the metabolism of concomitantly administered drugs should not be significantly affected at lower doses. Molecular docking of BPA-302 indicated that it can bind to active site of both CYP2C19 and CYP2D6 enzymes above the heme cofactor corroborating the experimental data.

Expression of programmed cell death proteins in patients with chronic myeloid leukemia.

PURPOSE: Chronic myelogenous leukemia (CML) is a malignant myeloproliferative disease developing out of pluripotent hematopoietic stem cells that contain the fusion Bcr-Abl gene. The mechanisms that lead to these changes at molecular level are still unknown as are the mechanisms that increase the proliferative capacity of these cells. Disorders that occur in the process of apoptosis represent one of the possible molecular mechanisms that bring about disease progress. In our study we analyzed the presence of mutated (mut) p53 gene and the amplification of Bax proteins in patients with CML. PATIENTS AND METHODS: This study included 30 patients with CML (23 in chronic phase, 7 in blast transformation). Using immunohistochemistry with alkaline phosphatase / anti-alkaline phosphatase (APAAP) method we analyzed the expression of cell death proteins p53 and Bax in mononuclear bone marrow cells. Polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) method was used to analyze the presence of mut p53 gene in mononuclear peripheral blood cells. Reverse transcription-polymerase chain reaction (RT-PCR) method was used to analyze the presence of Bcr-Abl in peripheral blood cells. RESULTS: High expression of Bax protein was detected in all analyzed patients, but no significant differences were noticed among them. No mut p53 gene was detected in any of the analyzed samples. Bcr-Abl b3a2 protein form was detected in all patients with variant translocations. CONCLUSION: Lack of mut p53 product in the peripheral blood and bone marrow cells in patients with CML suggests that this gene plays no important role in disease pathology. Increased level of Bax protein expression is an essential characteristic of CML cells but it is not related with the clinical stage of disease.

Sensitivity of different cell lines to phototoxic effect of disulfonated chloroaluminium phthalocyanine.

Photodynamic therapy (PDT) is a treatment for cancer involving three key components: sensitizer, light and tissue oxygen. A sensitizer is a chemical compound that can be excited by light of a specific wavelength. Phthalocyanine ClAlPcS(2), belonging among the promising second generation of sensitizers, was evaluated as an inducer of photodamage on NIH3T3 (mouse fibroblasts), B16 (mouse melanoma), MCF7 (human breast adenocarcinoma) and G361 (human melanoma) cell lines. A semiconductor laser was used as a source for evocation of the photodynamic effect. We report the influence of various concentrations of the sensitizer in combination with laser irradiation on the photodamage of cells. Viability of cells was determined by means of molecular probes (Calcein AM and ethidium homodimer) for fluorescence microscopy. The quantitative changes of cell viability in relation to sensitizer concentrations and laser irradiation were proved by fluorometric measurement. We detected phototoxicity evoked by laser irradiated sensitizer in all studied cell lines. In addition, the viability studies showed that G361 melanoma cells and MCF7 breast adenocarcinoma cells were more sensitive than NIH3T3 mouse fibroblasts and B16 mouse melanoma to photodynamic damage induced by ClAlPcS(2).

Breast, colon, and prostate screening in the adult population of Croatia: does rural origin matter?

INTRODUCTION: The aim of this study was to investigate the utilization of breast, colon and prostate cancer screening in the adult Croatian population in a period without national cancer screening programs, with a special interest in respondents' rural versus urban origin. METHODS: Self-reported screening utilization was investigated in the Croatian Adult Health Survey, which collected health-related information from a representative sample of the adult Croatian population. Breast cancer screening was investigated in women aged over 40 years, while colon and prostate screening was investigated in respondents aged over 50 years. The data were analysed using binary logistic regression. RESULTS: One in five women reported breast cancer screening uptake in the year preceding the survey (22.5%), while only 4.5% reported a colon screening. A total of 6.1% men reported colon screening, while 13.7% of men reported having a prostate cancer screening. Respondents with rural origin reported all sites screening utilization less frequently than those of urban origin (breast: 14.5% vs 27.4%; prostate: 9.6% vs 16.3%; colon-men: 5.7% vs 6.3%; colon-women: 3.6% vs 5.1%; respectively). Multivariable models indicated that people with higher socio-economic status more commonly reported breast and prostate cancer screening uptake. Access to health care was the only independent variable associated with colon cancer screening in men, and the strongest variable associated with colon cancer screening in women. Rural origin was associated only with lower odds of breast screening (adjusted odds ratio 0.60 [95% confidence interval 0.48-0.74]), while in the remaining models, rural origin was not a significant predictor for cancer screening uptake. CONCLUSIONS: Opportunistic cancer screening uptake is low in the Croatian adult population, with existing socio-economic differences in breast and prostate screening, and their absence in colon cancer screening. Rural origin was significantly associated with breast screening, even after adjustment to socioeconomic status and problems in access to health care. Lack of rural origin significance in the other screening sites could be related to small sample sizes of people who reported opportunistic utilization. Overall, access to health care is the strongest cancer screening predictor, and this should have a prominent role in the development of a systematic cancer screening program on a national level.

Immunogold-EM analysis reveal brefeldin a-sensitive clusters of auxin in Arabidopsis root apex cells.

Immunogold electron microscopy (EM) study of Arabidopsis root apices analyzed using specific IAA antibody and high-pressure freeze fixation technique allowed, for the first time, vizualization of subcellular localization of IAA in cells assembled intactly within plant tissues. Our quantitative analysis reveals that there is considerable portion of IAA gold particles that clusters within vesicles and membraneous compartments in all root apex cells. There are clear tissue-specific and developmental differences of clustered IAA in root apices. These findings have significant consequences for our understanding of this small molecule which is controlling plant growth, development and behavior.

Anti-angiogenic effects of novel cyclin-dependent kinase inhibitors with a pyrazolo[4,3-d]pyrimidine scaffold.

BACKGROUND AND PURPOSE: Cyclin-dependent kinase 5 (CDK5) has recently emerged as an attractive target in several tumour entities. Inhibition of CDK5 has been shown to have anti-angiogenic effects in vitro and in vivo. However, potent inhibitors of CDK5, which can be applied in vivo, are still scarce. We have recently developed a new series of 5-substituted 3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyrimidines that show a preference for inhibiting CDK5 and tested them in vitro and in vivo in a murine model of hepatocellular carcinoma. EXPERIMENTAL APPROACH: All compounds were initially examined for effects on proliferation of HUVECs. The most potent compounds were then tested on migration, and one of them, LGR2674, was selected for assessing effects on nuclear fragmentation, cell cycle, cell viability and metabolic activity. Furthermore, LGR2674 was tested in a tube formation assay and in vivo in a murine model of hepatocellular carcinoma, induced by s.c. injection of HUH7 cells (measurement of in vivo toxicity, tumour vascularization, tumour cell proliferation and tumour size). KEY RESULTS: LGR2674 showed an EC50 in the low nanomolar range in the proliferation and migration assays. Cytotoxic effects started at 50 nM, a concentration that did not influence the cell cycle. In vivo, LGR2674 was well tolerated and caused a clear reduction in vessel density in the tumours; also tumour cell proliferation was inhibited and tumour growth retarded. CONCLUSIONS AND IMPLICATIONS: Pyrazolo[4,3-d]pyrimidine is a novel scaffold for the development of potent CDK inhibitors with in vivo potential. Such structures are good candidates for broadening our pharmacological arsenal against various tumours.

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.

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.

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.

[Erythema nodosum]. / 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%.

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.

Cytokinin-derived cyclin-dependent kinase inhibitors: synthesis and cdc2 inhibitory activity of olomoucine and related compounds.

Cyclin-dependent kinases (cdk) have recently raised considerable interest in view of their essential role in the regulation of the cell division cycle. The structure-activity relationships of cdk inhibition showed that the 1, 3; and 7 positions of the purine ring must remain free, probably for a direct interaction, in which it behaves as a hydrogen bond acceptor. Olomoucine (6-(benzylamino)-2-[(2-hydroxyethyl)amino]-9-methylpurine, OC), roscovitine (6-(benzylamino)-2(R)-[[1-(hydroxymethyl)propyl]amino]-9-isopropylpur ine), and other N6,2,9-trisubstituted adenines were found to exert a strong inhibitory effect on the p34cdc2/cyclin B kinase. Removal or change of the side chain at position 2 or the hydrophobic group at position 9 dramatically decreased the inhibitory activity of olomoucine or roscovitine. Inhibition of cdk with OC and related compounds clearly arrests cell proliferation of many tumor cell lines at G1/S and G2/M transitions and also triggers apoptosis in the target tumor cells in vitro and in vivo. Thus, from a pharmacological point of view, OC may represent a model compound for a new class of antimitotic and antitumor drugs.

Helicobacter pylori and gastric cancer in Croatia.

The seroprevalence of Helicobacter pylori infection was studied in a population from two areas of Croatia with significantly different average gastric cancer (GC) cumulative incidence and mortality rates. In a random sample of 456 blood sera from both areas, which was tested with the ELISA Helicobacter pylori-antibody test, 48.8% of people in the north and 53.3% in the south of the country were found to be infected. The difference between the two areas in the seroprevalence of the infection was not statistically significant, nor did their populations differ in age, sex, educational background, smoking habit or wine consumption. Our results do not point to any association between the prevalence of Helicobacter pylori infection and the level of cumulative incidence and GC mortality levels.

Comparison of oriented and random antibody immobilization in immunoaffinity chromatography of cytokinins.

Immunosorbents for the plant hormones cytokinins prepared by random antibody immobilization (to Affi-Gel 10) and by oriented approach via oxidized carbohydrate moieties on the Fc region (to Affi-Gel Hz or hydrazide derivative of Perloza MT 200) have been compared. Both approaches yielded immunosorbents with high dynamic capacity (ca. 5-10 nmol ml gel-1). Oriented antibody immobilization did not exhibit crucial effects in the case of low-molecular-mass cytokinins. Antibodies immobilized via a spacer to Affi-Gel 10 have probably enough conformational freedom to enable good accessibility to cytokinins. The sorbents were used in analysis of endogenous cytokinins in maize seeds. In phosphatase treated samples trans-zeatin and its riboside were predominant.

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.

In vitro toxicity testing of supramolecular sensitizers for photodynamic therapy.

We report the phototoxicity of meso-tetrakis(4-sulphonatophenyl)porphine (TPPS4) and zinc metallocomplex (ZnTPPS4) sensitizers in the presence or absence of 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD) on G361human melanoma cells. Morphological changes in cell cultures have been evaluated using inversion fluorescent microscope and image analysis. Viability of cells was determined by means of molecular probes for fluorescence microscopy (LIVE/DEAD kit- double staining with Calcein AM and Ethidium Homodimer). The quantitative changes of cell viability in relation to sensitizers concentrations and irradiation doses were proved by fluorometric measurement with fluoroscan Ascent. We found that the most effective sensitizer is ZnTPPS4 bound to HP-beta-CD, since the IC50 value was 12.5 g/ml at the dose of light radiation of 10 J/cm2.