Functional properties of individual sub-domains of the fibrin(ogen) αC-domains.

Background: Fibrinogen is a large polyfunctional plasma protein consisting of a number of structural and functional domains. Among them, two αC-domains, each formed by the amino acid residues Аα392-610, are involved in fibrin polymerization, activation of fibrinolysis, platelet aggregation, and interaction with different cell types. Previous study revealed that each fibrinogen αC-domain consists of the N-terminal and C-terminal sub-domains. The major objections of the present study were to test functional role of these sub-domains in the above mentioned processes. Methods: To achieve these objections, we used specific proteases to prepare two truncated forms of fibrinogen, fibrinogen desAα505-610 and fibrinogen desAα414-610, missing their N-terminal and both N- and C-terminal sub-domains, respectively. Results: Our study with these truncated forms using turbidity measurements and electron microscopy revealed that the N- and C-terminal subdomains both contribute to protofibril formation and their lateral aggregation into fibers during fibrin polymerization process. These two sub-domains also contributed to platelet aggregation with the N-terminal sub-domains playing a more significant role in this process. At the same time, the C-terminal sub-domains make the major contribution to the plasminogen activation process. Further, our experiments revealed that the C-terminal sub-domains are involved in endothelial cell viability and migration of cancer cells. Conclusions: Thus, the results obtained establish the functional role of individual sub-domains of the αC-domains in fibrin polymerization, activation of fibrinolytic system, platelet aggregation, and cellular interactions. General significance: The present study expands our understanding of the functional role of individual fibrinogen domains and their specific portions in various fibrin(ogen)-dependent processes.

Synthesis, spectral characterization of novel Pd(II), Pt(II) π-coordination compounds based on N-allylthioureas. Cytotoxic properties and DNA binding ability.

Four novel Pd2+ and Pt2+ mononuclear π-coordination compounds with general formula [M(HL)1,2Cl2], M=Pd2+, Pt2+ have been synthesized by reaction of [PdCl4]2-, [PtCl4]2- anions with N-allyl-4-morpholinethiocarboxamide (HL1) and N-Allyl-N'-tert-butylthiourea (HL2). All complexes have been characterized by single-crystal X-ray diffraction study and 1H, 13C NMR spectroscopy. Cytotoxic, cytostatic and proapoptotic activities of compounds have been determined in vitro on HeLa cell line and compared with cisplatin as etalon drug. All complex compounds possessed pronounced cytotoxic activity with IC50 indexes in range of 2·10-6-1.5·10-4М (IC50 of cisplatin is 5.7∙10-5М) and showed proapoptotic, cytostatic and antisyntetic influence higher or comparable with cisplatin. The comparative influence of cisplatin and synthesized metal complexes on pTZ19R* plasmid DNA was monitored by agarose gel electrophoresis. All compounds showed high affinity to DNA that correlates with observed cytostatic and proapoptotic levels. In general Pd(II) compounds showed higher activity than Pt(II) ones.

The mechanism of VEGF-mediated endothelial cells survival and proliferation in conditions of unfed-culture.

The mechanisms of VEGF-mediated effects on endothelial cells during cancer development and progression is not clear. In present study the biological effects of VEGF, VEGF-rich culture medium of peritoneal macrophages from mice with Lewis lung carcinoma were studied on MAEC cell line under conditions of unfed culture. We have shown that VEGF increased cell proliferation by the 5th day of culturing vs control and anti-VEGF-treated cells. This effect was associated with increased consumption of glucose and NO production by the 2nd day while decreased ­ on the 5th day of cell culturing. VEGF-mediated NO production was dependent on Ca2+ ions. Block of Ca2+-channels (LaCl3) had more pronounced inhibitory effect vs chelator of Ca2+ ions (EDTA). It was shown that peritoneal macrophages are the main suppliers of VEGF at tumor angiogenesis, as evidenced by the data obtained on model system of endothelial cells synchronized in G0/G1 phase.

Effect of Pd(II) and Ni(II) coordination compounds with 4-amino-3-mercapto-5-methyl-1,2,4-triazole on the mitochondrial dehydrogenases activity.

Pd(I) and Ni(II) complex compounds: [Pd(AMMT)2]Cl2 (1), [Pd(AMMT)4]Cl2 (2) and [Ni(AMMT)2(H2O)](NO3)2 (3) with 4-amino-3-mercapto-5-methyl-1,2,4-triazole (AMMT) have been synthesized. The spectral characteristics of 1, 2 were studied by 1H (13C) NMR and UV-Vis spectroscopy. X-ray diffraction studies established that all complexes contain the AMMT molecule, which are coordinated to the central metal ion in the thione tautomeric form. At the ratio M: L = 1:2 ligand is coordinated in bidentate chelate manner by the nitrogen of amino- and sulfur of mercapto group (compounds 1, 3). But the molar ratio M: L = 1:4 leads to monodentate coordination of AMMT molecules only by sulfur of mercaptogroup (complex 2). Vacant coordination sites of the metal ion are occupied by water molecules (complex 3). The screening of complexes 1-3 and starting compounds [AMMT, K2PdCl4 (4), Ni(NO3)2 · 6H2O (5)] by their mitochondrial dehydrogenase activity have been performed by us for the first time, resulting in established that the Pd(II) complexes (1, 2), Pd(II) salt (4) and AMMT normalize the activity of mitochondrial dehydrogenases of cancer HeLa cells, identified by MTT-test. In contrast, the Ni(II) complex (3) and Ni(II) salt (5) do not stimulate the activity of mitochondrial dehydrogenases. It has been found, that all investigated compounds do not affect on the cell cycle and the level of apoptotic cells as well as do not show a toxic effect. Thus, these results indicate that AMMT and Pd(II) complexes may be used as modifiers of mitochondrial respiration, which dysfunction is particularly evident in the tumor cells.

Combined influence of teichoic acids from Staphylococcus aureus and heterometallik Cu/Cd ethylenediamine complex on peritoneal macrophages and tumor cells.

We investigated the effects of teichoic acid (TA) from Staphylococcus aureus Wood 46 on tumor growth and metastasis of the experimental Lewis lung carcinoma (LLC) in mice. Intranasal administration of TA alone aggravated both tumor growth and metastasis, whereas combined administration of TA with a synthetic bimetallic (copper : cadmium) ethylene diamine complex PO244 resulted in pronounced antitumor and antimetastatic effects. The group of animals subjected to the combined treatment with TA and PO244 manifested the highest degree of lymphocyte infiltration into the tumor tissue, compared to the control group and those exposed to TA or PO244 alone. Moreover, the combined treatment negatively affected the adhesive properties of peritoneal macrophages in the LLC bearing mice. Co-cultivation of the isolated macrophages with primary LLC cultures revealed significant (p < 0.05) cytotoxic and cytostatic effects, detected as an increased level of apoptosis and a reduced fraction of replicating cells.

[Potential cytostatic effect of the maleimide derivative 1-(4-Cl-benzyl)-3-Cl-4-(CF3-phenylamino)-1-H-pyrrol-2,5-dione].

The cytostatic/cytotoxic effects of the maleimide derivative 1-(4-Cl-Benzyl)-3-Cl-4-(CF3-phenylamino)-1H-pyrrol-2,5-dione (MI-1) have been estimated on epithelial derived human cell cultures (Colo 205, MCF-7, and Hela). The anticancer and toxic effects of MI-1 have been investigated on DMH-induced cancer development and normal colon morphology in rats. The results showed that the compound studied has low cytotoxicity but produces a strong antiproliferative effect on cell cultures and partially suppresses colon cancer development in DMH-induced model. The MI-1 effect on normal colon mucosa is insignificant, and no destructive changes have been detected in the intestine of rats. This maleimide derivate can be considered as a promising anticancer drug.

Immunological markers of anti-tumor dendritic cells vaccine efficiency in patients with non-small cell lung cancer.

AIM: To investigate the quantitative and functional status of peripheral blood lymphocytes in patients with non-small cell lung cancer during DC-vaccine therapy and identify the most informative immunological parameters which are associated with clinical outcome. MATERIALS AND METHODS: The study was conducted within the framework of randomized phase III clinical trial of DC-vaccine efficacy in patients with non-small cell lung cancer. Quantitative composition of peripheral blood lymphocytes was determined by flow cytometry. Cytokines mRNA expression level was estimated using real-time RT-PCR. RESULTS: In our study the most pronounced changes in the immune system have been defined after fourth DC-vaccine injection. Immunologic features such as reduction the MIP-1α mRNA expression level, increasing the RANTES mRNA expression level and NK-cells count, retention CD4/CD8 ratio at physiological level were associated with favorable clinical outcome after DC-immunotherapy. CONCLUSIONS: Immunological markers established in our investigation can be used for estimation of DC-immunotherapy efficiency. The results of our research are very promising, but these data should be confirmed in further studies with a large cohort of patients.

[Mitokorrektine stimulates angiogenesis in vitro].

The effect of mitokorrectine (complex native oligopeptides isolated from neonatal pig brain) on endothelial cells in culture was investigated. It was revealed that the drug concentration-dependently induces angiogenesis in vitro. Mitogen effect of Mitokorrectine was shown by MTT-test and routine cell count in concentration diapason (0.1-1 mg/ml) which means an increased number of cells by 25 +/- 5% and cell subpopulation of proliferative pool (G2/M+S) 1,8 times in concentration diapason mitokorrectine (0.01-0.05 mg/ml) in comparison with control. In 3-D culture and in stationary phase we detected induction of differentiation of endothelial cells, a decrease the level of NO production and enhancement of glucose metabolism and stimulation of formation of capillary-like tubes.

[Effect of dendritic cell autovaccine on the treatment outcome in patients with ovarian cancer].

During I/II phase clinical trial in ovarian cancer (OC) patients two types of autologous anticancer vaccines based on dendritic cells have been tested, and a comparative analysis of their effectiveness have been performed. It was shown that the anticancer vaccines based on DC, "loaded" with autologous tumor cell lysate obtained by treatment of tumor cells by cytotoxic lectins B. subtilis had higher efficiency, compared with the standard DC--autovaccine. The presence of antigen-specific immune response observed after at least four vaccinations. Obtained results open the prospects to improve the basic treatment of OC patients by this method. The results of immunological examinations create preconditions for individual optimization of the DC-vaccine therapy.