Anti-Tumor Active Isopropylated Fused Azaisocytosine-Containing Congeners Are Safe for Developing Danio rerio as Well as Red Blood Cells and Activate Apoptotic Caspases in Human Breast Carcinoma Cells.

New isopropylated fused azaisocytosine-containing congeners (I-VI) have previously been reported as promising anticancer drug candidates, so further research on these molecules in the preclinical development phase is fully justified and necessary. For this reason, in the present paper, we assess the toxicity/safety profiles of all the compounds using Danio rerio and red blood cell models, and examine the effect of the most selective congeners on the activation of apoptotic caspases in cancer and normal cells. In order to evaluate the effect of each molecule on the development of zebrafish embryos/larvae and to select the safest compounds for further study, various phenotypic parameters (i.e., mortality, hatchability, heart rate, heart oedema, yolk sac utilization, swim bladder development and body shape) were observed, and the half maximal lethal concentration, the maximal non-lethal concentration and no observed adverse effect concentration for each compound were established. The effect of all the isopropylated molecules was compared to that of an anticancer agent pemetrexed. The lipophilicity-dependent structure-toxicity correlations were also determined. To establish the possible interaction of the compounds with red blood cells, an ex vivo hemolysis test was performed. It was shown that almost all of the investigated isopropylated congeners have no adverse phenotypic effect on zebrafish development during five-day exposure at concentrations up to 50 µM (I-III) or up to 20 µM (IV-V), and that they are less toxic for embryos/larvae than pemetrexed, demonstrating their safety. At the same time, all the molecules did not adversely affect the red blood cells, which confirms their very good hemocompatibility. Moreover, they proved to be activators of apoptotic caspases, as they increased caspase-3, -7 and -9 levels in human breast carcinoma cells. The conducted research allows us to select-from among the anticancer active drug candidates-compounds that are safe for developing zebrafish and red blood cells, suitable for further in vivo pharmacological tests.

Anticancer active trifluoromethylated fused triazinones are safe for early-life stages of zebrafish (Danio rerio) and reveal a proapoptotic action.

The main purpose of this investigation was to evaluate the effect of anticancer active compounds (I-VIII) on zebrafish development in order to select the safest molecules. Larval mortality, embryo hatchability and malformations were end-points used to assess the acute toxicity among embryos and larvae from compounds-/pemetrexed-treated and control groups. LC50 and MNLC (maximal non-lethal concentration) were determined. Lipophilicity-dependent structure-toxicity relationships were established. The results clearly indicated that the majority of test molecules are safe for zebrafish individuals and simultaneously are less toxic than an anticancer agent - pemetrexed. The subsequent aim of this study was to elucidate the molecular mechanism of antiproliferative activity of the most selective compounds. Substantially increased activation of caspase-6 and -8 in cancerous cell lines confirmed the proapoptotic action of molecules examined. Considering the safety for zebrafish individuals, the title compounds as inducers of apoptosis are promising drug candidates in the preclinical phase of drug development.

The Role of Mitochondria in Carcinogenesis.

The mitochondria are essential for normal cell functioning. Changes in mitochondrial DNA (mtDNA) may affect the occurrence of some chronic diseases and cancer. This process is complex and not entirely understood. The assignment to a particular mitochondrial haplogroup may be a factor that either contributes to cancer development or reduces its likelihood. Mutations in mtDNA occurring via an increase in reactive oxygen species may favour the occurrence of further changes both in mitochondrial and nuclear DNA. Mitochondrial DNA mutations in postmitotic cells are not inherited, but may play a role both in initiation and progression of cancer. One of the first discovered polymorphisms associated with cancer was in the gene NADH-ubiquinone oxidoreductase chain 3 (mt-ND3) and it was typical of haplogroup N. In prostate cancer, these mutations and polymorphisms involve a gene encoding subunit I of respiratory complex IV cytochrome c oxidase subunit 1 gene (COI). At present, a growing number of studies also address the impact of mtDNA polymorphisms on prognosis in cancer patients. Some of the mitochondrial DNA polymorphisms occur in both chronic disease and cancer, for instance polymorphism G5913A characteristic of prostate cancer and hypertension.

Two novel classes of fused azaisocytosine-containing congeners as promising drug candidates: Design, synthesis as well as in vitro, ex vivo and in silico studies.

Searching for new less toxic anticancer drug candidates is a big challenge from a medical point of view. The present investigation was aimed at describing two independent synthetic approaches based on isosteric replacements, spectroscopic characteristics, in vitro anticancer and ex vivo antihaemolytic activities of novel molecules (9-22) and correlations between their standardised lipophilicity indices, computed log Paverage values and pharmacokinetic descriptors. Two novel protocols for annelation of the triazinone template on hydrazinylideneimidazolidines (1-8) (showing a high reactivity towards electrophilic reagents, such as ethyl trifluoropyruvate and ethyl 3-methyl-2-oxobutyrate) were developed for the first time, giving rise to two original classes of highly conjugated azaisocytosine-containing molecules (9-16 and 17-22). Both syntheses proceeded under basic conditions to yield the most probable intermediates (e.g. hemiaminals and imines), which in refluxing two-component solvent mixtures or a suitable solvent cyclised through closing the triazinone ring on functionalised imidazolidines in both cases. All fused azaisocytosine-containing congeners were investigated with the purpose of preselecting possible drug candidates with a better selectivity that could be suitable for further more detailed drug development studies. The majority of test molecules revealed strong antiproliferative effects in most tumour cell cultures and they were more cytotoxic against tumour cells than anticancer drug - pemetrexed. These cytotoxicities may be associated with the activation of initiator and executioner caspases (confirmed for compound 12) which are inducers of apoptosis. Simultaneously, three bioisosteres bearing the trifluoromethyl moiety at the C-3 and the ortho substitution at the phenyl ring (10, 12 and 13) proved to be the most promising in terms of selectivity as they were less or equally toxic to normal cells as pemetrexed. It was shown that isosteric replacement of the ethyl group in antitumour active congeners by the trifluoromethyl or isopropyl group was favourable for the selectivity of the designed drug-like molecules. Almost all new compounds revealed the protective effects in an ex vivo model of oxidatively stressed rat erythrocytes (better or comparable than that of ascorbic acid/Trolox), proving that they are safe to red blood cells. The statistically significant and predictive QSAR equations were derived that describe relationships between some pharmacokinetic descriptors (such as log Ka, HSA, fu, brain, Caco-2, log Kp) and lipophilicity parameters of test molecules. Among all molecules with anticancer profile, the possible drug candidates seem to be 10, 12, 13, 19 and 21 which are the least toxic for normal cells, deprived of haemolytic effects on oxidatively-stressed red blood cells and have the optimum pharmacokinetic descriptors in terms of their lipophilicity parameters. Because of a high development potential they should be utilised in further more extended in vivo investigations aimed at developing novel less toxic anticancer agents.

In vitro effects of a new fused azaisocytosine-like congener on relative cell proliferation, necrosis and cell cycle in cancer and normal cell cultures.

The study was aimed at describing the mode of action of an innovative drug-like congener of fused azaisocytosine-EIMTC (ethyl 8-(4-methoxyphenyl)-4-oxo-6,7-dihydroimidazo[2,1-c][1,2,4]triazine-3-carboxylate)-on cancer cells in early in vitro oncology-related bioassays. Micromolar concentrations of EIMTC were effective at inhibiting the growth of two types of malignant multiple myeloma cells (including cells resistant to thalidomide) while having less cytotoxic effect on normal HSF cells. Furthermore, EIMTC was disclosed as capable of producing the statistically significant decrease in the number of cells in the S phase (in HeLa, TOV112D, T47D and Vero cells) and in the G2/M phase (in TOV112D cells) as well as evoking the distinctly higher necrosis rates in malignant than normal cells of the same epithelial origin. These results are promising in the sense that the bicyclic nucleobase-like structure related to azaisocytosine may target epithelial cancer cells and inhibit their growth while having less effect on normal cells. This may be due to induction of necrosis.

Synthesis, structure elucidation and identification of antiproliferative activities of a novel class of thiophene bioisosteres bearing the privileged 7,8-dihydroimidazo[2,1-c][1,2,4]triazin-4(6H)-one scaffold.

The straightforward and practical synthesis route and remarkable antitumour activities in vitro of a novel class of thiophene bioisosteres (10-18) are disclosed. These molecules were obtained with good overall yields via the reaction of 1-aryl-2-hydrazonoimidazolidine hydroiodides with ethyl 2-oxo-2-(2-thienyl)acetate in the presence of triethylamine in refluxing DMF/methanol mixture. All the synthesized compounds proved to be markedly effective against human tumour cells: A549, HeLa, T47D and TOV112D and more cytotoxic than pemetrexed against A549, HeLa and T47D cells. Among these strongly antiproliferative active molecules, the disclosed three thiophene bioisosteres (11, 17 and 18) are proposed as the most promising anticancer lead structures for the rational design of more selective antitumour agents because they proved to be markedly lower cytotoxic towards normal than tumour cells. Results from the bioassay based on a double fluorochrome staining were worthy to be described because they provide a clue to the mode of action of one (18) of the most promising anticancer lead structures of the series.

Synthesis, antiproliferative and antimicrobial activity of new Mannich bases bearing 1,2,4-triazole moiety.

Abstract This study presents the synthesis, antiproliferative and antimicrobial evaluation of a new series of Mannich base derivatives containing 1,2,4-triazole system. New compounds were prepared by the reaction of 4,5-disubstituted 1,2,4-triazole-3-thiones with formaldehyde and various amines. The structures of the prepared compounds were confirmed by means of (1)H NMR, (13)C NMR and elemental analyses. Twelve compounds were evaluated for their in vitro antiproliferative activities against six chosen cancer cell lines. All synthesized compounds were screened for their in vitro antimicrobial activity by using the agar dilution technique. For 17 potentially active compounds, their antibacterial activity was confirmed on the basis of MIC (minimal inhibitory concentration) by broth microdilution method using the reference Gram-positive and Gram-negative bacterial strains.

Cytotoxic, antioxidant, antimicrobial properties and chemical composition of rose petals.

BACKGROUND: Rosa rugosa petals are used for production of teas, jams, wines and juices. Despite the wide availability of rose cultivars, comprehensive information on petal chemical composition and healthful properties is still lacking. Therefore, the aim of this study was analysis of cytotoxic, antioxidant and antimicrobial activity and chemical composition of rugosa rose petals. RESULTS: Petals of R. rugosa were evaluated for their cytotoxic effect against cervical (HeLa) and breast cancer (T47D) cell lines and for antiradical activity (with DPPH•). As a result, significant cytotoxic (up to 100% of dead cells) and antiradical properties (IC50 1.33-0.08 mg mg⁻¹ DPPH•) were demonstrated. Moreover, notable antimicrobial activity against eight bacterial (i.e. Staphylococcus. epidermidis, S. aureus, Bacillus subtilis, Micrococcus luteus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis) and two yeast strains (Candida. albicans, C. parapsilosis) was shown. Total phenolic, flavonoid, phenolic acid, tannin, carotenoid and polysaccharide content in petals was determined using spectrophotometric methods. Liquid chromatography-electrospray ionization-tandem mass spectrometry was used to thoroughly analyze phenolic acids and flavonoid glycosides in the methanolic extract and fractions obtained after its separation. Five phenolic acids and six flavonoids previously not reported in the plant material were identified. CONCLUSION: This is the first such detailed report on chemical composition and biological activity of R. rugosa petals.

Synthesis, structure elucidation and in vitro anticancer activities of novel derivatives of diethyl (2E)-2-[(2E)-(1-arylimidazolidin-2-ylidene)hydrazono]succinate and ethyl (4-oxo-8-aryl-4,6,7,8-tetrahydroimidazo[2,1-c][1,2,4]triazin-3-yl)acetate.

The worked out and optimized synthesis routes and remarkable antitumour activities in vitro of novel polynitrogenated derivatives of diethyl (2E)-2-[(2E)-(1-arylimidazolidin-2-ylidene)hydrazono]succinate (7-10) and ethyl (4-oxo-8-aryl-4,6,7,8-tetrahydroimidazo[2,1-c][1,2,4]triazin-3-yl)acetate (11-16) are presented. Small molecules based on the privileged 7,8-dihydroimidazo[2,1-c][1,2,4]triazin-4(6H)-one scaffold (11-16) were obtained with fairly modest to good overall yields by very facile addition reactions of the nucleophilic centred 1-aryl-2-hydrazonoimidazolidine hydroiodides to diethyl acetylenedicarboxylate (DEAD) in the presence of triethylamine (TEA) and a subsequent cyclocondensation of the putative intermediate chain hydrazones. Heterobicyclic products 12 and 14-16 could also be prepared in high overall yields by an effective intramolecular cyclocondensation of the isolated stable and antiproliferative active heterocyclic hydrazones, namely, diethyl (2E)-2-[(2E)-(1-arylimidazolidin-2-ylidene)hydrazono]succinates (7-10), performed in refluxing DMF. These intermediates are the first products to be formed in the result of an addition of the nucleophilic reactants, namely, 1-aryl-2-hydrazonoimidazolidines of the 1-6 type, bearing the basic nitrogen atom of the hydrazono moiety (N-NH2), to the carbon-carbon triple bond of the highly electrophilic alkyne, that is, DEAD. Molecular structures of the synthesized compounds (7-16) in the DMSO-d6 solutions were verified by (1)H NMR and (13)C NMR spectral data. These were finally confirmed based on the advanced 2D HMBC and HMQC NMR experiments, which were performed for the two representatives (8 and 11) of the two synthesized sets of the bioactive substances. Among the majority of antiproliferative active molecules, the disclosed herein ethyl [4-oxo-8-(3-chlorophenyl)-4,6,7,8-tetrahydroimidazo[2,1-c][1,2,4]triazin-3-yl]acetate (14) is proposed as a promising lead structure for the design of novel highly selective antitumour agents because of the distinctly marked lower cytotoxicity towards the primary cell line of normal HSF cells and several-fold higher against cancer cells used. A double fluorochrome mix-staining was performed in order to find out about the possible mode of action by which this novel small heterobicycle reveals remarkable antiproliferative effects in vitro. Taking into account the obtained double staining results, this small molecule was identified as capable of inducing significantly higher levels of necrotic cells in human cancer cell lines (T47D and HeLa) than in normal HSF cells. Furthermore, its cytotoxicity against cells was found to be connected to the predominant induction of necrosis over apoptosis.

Thermal Decomposition Path-Studied by the Simultaneous Thermogravimetry Coupled with Fourier Transform Infrared Spectroscopy and Quadrupole Mass Spectrometry-Of Imidazoline/Dimethyl Succinate Hybrids and Their Biological Characterization.

The thermal decomposition path of synthetically and pharmacologically useful hybrid materials was analyzed in inert and oxidizing conditions for the first time and presented in this article. All the imidazoline/dimethyl succinate hybrids (1-5) were studied using the simultaneous thermogravimetry (TG) coupled with Fourier transform infrared spectroscopy (FTIR) and quadrupole mass spectrometry (QMS). It was found that the tested compounds were thermally stable up to 200-208 °C (inert conditions) and up to 191-197 °C (oxidizing conditions). In both furnace atmospheres, their decomposition paths were multi-step processes. At least two major stages (inert conditions) and three major stages (oxidizing conditions) of their decomposition were observed. The first decomposition stage occurred between T5% and 230-237 °C. It was connected with the breaking of one ester bond. This led to the emission of one methanol molecule and the formation of radicals capable of further radical reactions in both used atmospheres. At the second decomposition stage (Tmax2) between 230-237 °C and 370 °C (inert conditions), or at about 360 °C (oxidizing conditions), the cleavage of the second ester bond and N-N and C-C bonds led to the emission of CH3OH, HCN, N2, and CO2 and other radical fragments that reacted with each other to form clusters and large clusters. Heating the tested compounds to a temperature of about 490 °C resulted in the emission of NH3, HCN, HNCO, aromatic amines, carbonyl fragments, and the residue (Tmax2a) in both atmospheres. In oxidizing conditions, the oxidation of the formed residues (Tmax3) was related to the production of CO2, CO, and H2O. These studies confirmed the same radical decomposition mechanism of the tested compounds both in inert and oxidizing conditions. The antitumor activities and toxicities to normal cells of the imidazoline/dimethyl succinate hybrids were also evaluated. As a result, the two hybrid materials (3 and 5) proved to be the most selective in biological studies, and therefore, they should be utilized in further, more extended in vivo investigations.

The Expression of Signaling Genes in Breast Cancer Cells.

The aim of the study was to investigate the effect of paclitaxel on the expression of genes encoding signaling factors in breast cancer cells in in vitro conditions after incubation with the said chemotherapeutic. The tested cells were harvested from the mammary glands of 36 patients with early breast cancer. The microarray technology was employed for the identification of gene expression. For this purpose, mRNA isolated from tumor cells was used. A significant effect of paclitaxel on the genome of breast cancer cells was confirmed. Paclitaxel changed the functions of cancer cells by increasing the expression of most genes encoding signaling proteins and receptors. The analysis of the results suggested that this cytostatic agent produces a beneficial therapeutic effect at a lower dose (60 ng/mL). In contrast, a high dose of paclitaxel (300 ng/mL) was associated with a high cytotoxicity.

Novel Venetin-1 nanoparticle from earthworm coelomic fluid as a promising agent for the treatment of non-small cell lung cancer.

The present research shows the antitumor activity of a protein-polysaccharide complex Venetin-1 obtained from the coelomic fluid of Dendrobaena veneta earthworms against A549 cancer cells. The investigations are a continuation of experiments on the antitumor activity of coelomic fluid obtained from this species. The Venetin-1 nanoparticle was obtained after thermal treatment of the coelomic fluid, separation from coelomocytes, filtration, and lyophilization. The preparation showed a selective effect on cancer cells, whereas normal cells were unaffected. Venetin-1 was effective against the lung cancer cells at doses of 31.3 and 62.5 µg/ml, and the results were imaged using light microscopy and scanning electron microscopy (SEM). The cells died mainly via the apoptosis pathway. Necrotic cells appeared sporadically in the microscopic view. SEM imaging revealed complete destruction of the A549 cells after the incubation with Venetin-1. The atomic force microscopy (AFM) analyses showed changes in the topography, peak force error images, and Young's modulus (elasticity) of the A549 cells after the incubation with Venetin-1. The transmission electron cryomicroscopy (Cryo-TEM) analysis indicated a polymeric nature of the analyzed preparation. The samples of Venetin-1 showed a very homogeneous size profile with the microparticle size of approximately 58.23 nm. A significant decrease in Venetin-1 binding to sphingomyelin was observed. Venetin-1 lost its pore-forming activity or deactivation of the pore-forming activity occurred. This confirms the absence of hemolytic capacity of Venetin-1 towards red blood cells. The conducted analyses show the suitability of the obtained complex for biomedical research. The next step will consist in analyses of the effect of Venetin-1 on the immune system in mice.

Synthesis, structure elucidation, determination of the lipophilicity and identification of antitumour activities in vitro of novel 3-(2-furanyl)-8-aryl-7,8-dihydroimidazo[2,1-c][1,2,4]triazin-4(6H)-ones with a low cytotoxicity towards normal human skin fibroblast cells.

Eleven novel 3-(2-furanyl)-8-aryl-7,8-dihydroimidazo[2,1-c][1,2,4]triazin-4(6H)-ones (12-22) were designed and obtained from appropriate 1-aryl-2-hydrazonoimidazolidines (1-11) by condensation reaction with 2-oxo-2-furanacetic acid and subsequent cyclocondensation of intermediate chain derivatives. IR, (1)H NMR and (13)C NMR spectra and elemental analyses confirmed the chemical structure of all the synthesized compounds. The reversed-phase HPLC method was optimized and proved to be applicable and reliable for the analysis of these unknown small molecules (12-22). These compounds were chromatographed on octadecyl silica (ODS) stationary phase and their hydrophobic parameters expressed as the log k(w) values were determined by RP-HPLC, using mixtures of methanol and water as mobile phases with different methanol concentrations. Octane-1-sulfonic acid sodium salt (OSA-Na) and 20% acetate buffer (pH 3.5) was added to the mobile phase (eluent containing 0.01 M/L OSA-Na in organic modifier (MeOH)-buffered mobile phase). The high values of regression coefficients (r >0.9841) for all the compounds investigated proved the excellent fit between experimental data and the Snyder-Soczewinski equation. Results obtained from the reversed-phase HPLC were compared both with those theoretically calculated and with those obtained from an ALOGPS 2.1. software by the use of nine different computational methods for estimation of log P. The predicted values of log P by use of AB log P algorithm revealed the best correlation with the experimental log k(w) values for the investigated solutes, since a good correlation (r=0.7760) between these quantities was found. The majority of novel imidazotriazinones were found to be evidently effective in vitro against human cancerous cells (HeLa and T47D) in an effective concentration of 50 µg/mL. Five compounds (13, 15, 16, 18 and 22) revealed remarkable antiproliferative activities and selective cytotoxicities for cancer cells over normal HSF cells. Therefore these ones may be considered as a basis for the design of novel useful non-toxic (13, 15 and 16) and low toxic (18 and 22) anticancer agents.

The determination of changes in the expression of genes for selected specific transcriptional factors in in vitro ductal breast cancer cells under the influence of paclitaxel.

This study aimed to determine the changes in the expression of genes for selected specific transcriptional factors that have both activating and repressing functions in in vitro ductal breast cancer cells, under the influence of paclitaxel, applying the microarray technique. The cells are treated with 60 ng/ml and 300 ng/ml doses of paclitaxel that correspond to those applied in breast cancer therapy. About 60 ng/ml doses of paclitaxel cause a statistically significant increase in expression of all the 16 analysed genes coding transcriptional factors, ranging from 1.84-fold (for PO4F2) to 4.65-fold (for LMO4) (p < 0.05) in comparison with the control cells, and enhanced the taxane mechanism of action. The 300 ng/ml doses of paclitaxel cause a cytotoxic effect in the cells. In this article, we argue that these changes in gene expression values may constitute prognostic and predictive factors in ductal breast cancer therapy.

Anti-oxidant activity of superoxide dismutase and glutathione peroxidase enzymes in skeletal muscles from slaughter cattle infected with Taenia saginata.

It is known that highly reactive oxygene species produced during normal cellular metabolism represent a powerful effector mechanism against parasites. Superoxide dismutase (SOD) and glutathione peroxidase (GPx) belong to the main defense anti-oxidants that prevent the formation of new free radical species. The aim of this study was to assess the activities of SOD and GPx in cattle tissues infected with Taenia saginata. We observed a statistically significant increase in the SOD and GPx activities (p=0.00003, 0.00008, respectively, Student's t-test) in skeletal muscles infected with T. saginata in spectrophotometric analysis. With the use of western blot technique, SOD synthesis stimulation has appeared in the host tissues containing cysticerci in contrast with the control samples. There was no statistically significant increase in the GPx band intensity observed in the studied samples in comparison to controls (Gene Tools Version 4.01 program). These results support the significance of anti-oxidant processes in host defense mechanism during parasitic infections.

Antitumor activity and apoptotic action of coelomic fluid from the earthworm Dendrobaena veneta against A549 human lung cancer cells.

It is known that earthworm coelomic fluid (CF) can affect not only cancer but also normal cells. The study demonstrated that the CF of the earthworm Dendrobaena veneta exhibited cytotoxicity against A549 lung cancer cells but did not toward the bronchial epithelial cell line BEAS-2B. The selective effect on the tumor cells was achieved after a short-term CF heat pre-treatment at 70 °C. The cytotoxic effect of the CF was time- and concentration-dependent. The CF noticeably decreased the viability and affected the morphology of the A549 cells. Scanning electron microscopy revealed a different degree of destruction of the nucleus and cytoplasm of A549 cells. As determined by atomic force microscopy, the cell surface roughness increased while the cell stiffness was reduced upon the CF treatment. A twofold increase in the caspase 3, 4, 5, and 10 levels was observed in the A549 cells after the incubation with the CF. The results obtained by flow cytometry using Annexin V confirmed the proapoptotic effect of the earthworm CF on A549 lung cancer cells. The D. veneta CF and active fraction obtained with cytotoxicity toward A549 lung cancer is an interesting and promising preparation for further biological, chemical, and biomedical research.

Sida hermaphrodita seeds as the source of anti - Candida albicans activity.

Sida hermaphrodita is a perennial herbaceous plant with potential economic importance; however, there is no information about its antimicrobial properties. The aim of our study was to analyze the morphology and metabolic activity of Candida albicans cells after exposure to the extract from S. hermaphrodita seeds, determine its cytotoxicity against human skin fibroblasts and carry out chemical analysis of the extract. Microscopic analysis showed that the crude seed extract (CSE) caused a significant decrease in the metabolic activity of fungal cells, clear cell deformation, and budding disturbances. The analysis of cytotoxicity showed no influence of the extract on the fibroblasts. The CSE and seed extract after dialysis (DSE) were analyzed using electrophoretic, chromatographic, and spectroscopic methods. SDS-PAGE electrophoresis showed the presence of proteins and carbohydrate compounds in the extract. The Raman spectroscopy analysis of the DSE confirmed the presence of proteins, while FTIR analyses revealed the occurrence of albumin-type proteins. The NMR and GC-MS analyses showed the presence of carbohydrates in the seed extract. The MALDI and ESI LC-MS/MS analysis of the CSE and the DSE fractions revealed the occurrence of vicilin-type and plant lipid transfer proteins. The seed extract is a promising formulation to use in C. albicans infections.

Synthesis, determination of the lipophilicity, anticancer and antimicrobial properties of some fused 1,2,4-triazole derivatives.

3-Unsubstituted and 3-substituted-7-aryl-5H-6,7-dihydroimidazo[2,1-c][1,2,4]triazoles (1-14) were designed and obtained from biologically active 1-aryl-2-hydrazonoimidazolidines by cyclocondensation reaction with triethyl orthoformates (1-4), phenoxyacetic acid derivatives (5-13) and carbon disulfide (14), respectively. Their chemical structures were confirmed by IR, (1)H NMR, (13)C NMR, MS spectra and elemental analysis. In the high performance liquid chromatographic series of experiments, fourteen synthesized compounds (1-14) were chromatographed on octadecyl silica adsorbent and their lipophilicity parameter (logk(W)) was determined using various aqueous systems: mixture of water and organic modifiers (methanol - MeOH, acetonitrile - MeCN or dioxane - DX). Compounds 7 and 12 were evaluated for their cytotoxic activity against three cancer cell lines: human Caucasian colon adenocarcinoma cell line - LS180 (ECACC 87021202), human uterus carcinoma cell line - SiHa (ECACC 85060701) and human breast carcinoma cell line - T47D (ECACC 85102201). Compound 12 was found to be the most effective in vitro against human colon adenocarcinoma cell line (LS180). Moreover, the distinctly marked lower cytotoxicity of compounds 7 and 12 against the normal cell line - human skin fibroblasts (HSF) and almost several-fold higher against the examined cancer cell lines was ascertained. The cytotoxic effect of imidazotriazole 7 was noticed on DNA structure of breast cancer cell line (T47D) by using the comet assay. Compound 7 in concentration of 29.3 microM was found to possess efficiency for DNA strand breakage. In particular, this led to cutting of the DNA strands and formation of small fragments of DNA - two higher and one lighter in comparison with control DNA. Moreover, significant viability decreases in the human leukaemic RPMI 8226 cells treated with different concentrations of imidazotriazoles 8-12 were observed, suggesting their antiproliferative properties. Besides, three tested compounds (9, 13, 14) revealed significant antimicrobial activities with MIC values in the range of 30.9-44.0 microM. Compound 13 showed superior antibacterial activity to ampicillin and chloramphenicol in vitro, whereas 14 displayed superior antifungal activity to miconazole.

Synthesis, structure elucidation and identification of antitumoural properties of novel fused 1,2,4-triazine aryl derivatives.

Synthesis, structure elucidation and anticancer activities of novel fused 1,2,4-triazine aryl derivatives containing the ethoxycarbonyl (6-10) and carbohydrazide formations (11-15) are presented. Molecular structures of the synthesized compounds were confirmed by IR, (1)H NMR, (13)C NMR, EI-MS spectra and elemental analyses. Antitumour activities in vitro for heterobicyclic hydrazides of the type 11-14 were evaluated by BrdU method for human LS180, SiHa and T47D carcinoma cells. Amongst them, hydrazide 14 has exhibited remarkable inhibitory effect against SiHa and LS180 tumour cells, and simultaneously was found to be non-toxic towards the human normal cell line-HSF cells. Furthermore, the pulse field gel electrophoresis experiment was performed for characterizing DNA-cleaving activity of heterobicycle 14. The DNA fragments of 2500, 2000 and 500 kilobase pairs (kbp) were commonly detected in the cancer cell lines (SiHa, LS180 and T47D) treated with compound 14. DNA fragmentation pattern, since three types of fragments induced by the tested hydrazide of the type 14 were detected, suggesting a way of interaction with DNA. It is worth pointing out, that DNA strand breaks were also produced in human breast cancer (T47D) cells, a cell line where the induction of DNA fragmentation is very difficult. Moreover, the statistically significant apoptotic activity in T47D human breast cancer cells for the tested heterobicycle 14 was proved using the annexin V-binding assay. The antiproliferative properties in vitro for compounds 6-14 were evaluated by MTT method for human leukaemic Jurkat cells. Significant viability decreases in Jurkat cells treated with different concentrations of compounds 10 and 11 were observed, suggesting that these derivatives have antiproliferative activities. Their acute toxicities were established. For these compounds the influence on the central nervous system of mice in behavioural tests was examined. Molecular structure for free base of the intermediate 4 was confirmed by (1)H-(1)H COSY, HMBC and HMQC correlations.

Taxanes and gene expression in breast cancer cells.

We analyzed expression of genes participating in apoptotic and oncogenesis processes under influence of paclitaxel applied in treatment of breast carcinoma. Analysis showed, that in a group of cells where paclitaxel was administered at lower dose (60 ng/mL) it caused statistically significant increase of expression of pro and antiapoptotic genes, genes coding caspases and oncogenes in comparison to control group.