Proliferative and Differentiation Potential of Multipotent Mesenchymal Stem Cells Cultured on Biocompatible Polymer Scaffolds with Various Physicochemical Characteristics.

Biocompatibility of film and fibrous scaffolds from polylactide-based polymers and the relationship between their architecture and the functional characteristics of mesenchymal stem cells were studied. Cell culturing on polylactide-based film and fibrous matrixes did not deteriorate cell morphology and their proliferation and differentiation capacities. The rate of cell proliferation and penetration in microporous 3D matrices with the same porosity parameters and pore size depended on their spatial organization. The above materials can be used as scaffolds for mesenchymal stem cells for creation of tissue engineering implants. The scaffold size and structure should be determined by the defects in the organs in which the regeneration processes have to be stimulated.

[Molecular mechanisms of niclosamide antitumor activity].

In this review the recent data regarding the antitumor activity of niclosamide and the molecular mechanisms of its antitumor activity are presented. Niclosamide has been used in the clinic for the treatment of intestinal parasite infections. In recent years in several screening investigations of various drugs and chemical compounds niclosamide was identified as a potential anticancer agent. Niclosamide not only inhibits the Wnt/ß-catenin, mTORC1, STAT3, NF-κB and Notch signaling pathways, but also targets mitochondria in cancer cells to induce growth inhibition and apoptosis. A number of studies have established the anticancer activity of niclosamide in both in vitro and in vivo in xenotransplantation models using human tumors and immunodeficient mice. It is important that niclosamide is active not only against tumor cells but also cancer stem cells. Normal cells are resistant to niclosamide. The accumulated experimental data suggest niclosamide is a promising drug for the treatment of various types of cancer.

[Biodistribution of Rifabutin Polymeric Transport Form].

BACKGROUND: One way to increase drug efficacy is to provide a drug delivery transport system to the target organ. A widely used method is to incorporate the drug in a biodegradable polymer composition with forming nanosized drug's transport forms. Objective: Our aim was to investigate the tissue biodistribution of antibiotic rifabutin transport system based on lactic and glycolic acids copolymer, and to compare it with the pure substance of rifabutin. METHODS: These substances were administered to two groups of rats intragastrically in the doses of 10 mg/kg. After a certain period of time, the animals were sacrificed by cervical dislocation. Samples preparation for analysis was carried out of the liquid-liquid extraction. Active substance's concentrations were measured by high performance liquid chromatography method. RESULTS: The study included 8-week-aged Wistar rats of both sexes weighing 0.22 ± 0.02 kg. Animals were divided into 2 groups. The study group received polymer form of antibiotic, and the comparison group received substance of rifabutin. In intervals of 10 min, 30 min, 1 h, 2 h, 4 h, 7h, 15 h, 24 h after drug administration liver, lung, spleen, kidney, intestines, stomach, heart and brain were resected respectively. Organs were measured by their weight. The drug was not detected in the brain. Rifabutin was determined in other examined tissues within 10 minutes and the maximum drug concentration in organs was fixed in 1.5-3.5 hours after administration. The rifabutin concentrations defined in the lungs were significantly higher in polymerform (p < 0.05). The polymer form's distribution coefficient was higher in the liver and lungs (15.83 and 10.14 µg/g respectively) in comparison with the substance one. The minimum amount of the active ingredient was observed in the heart (0.02 µg/g). CONCLUSION: It is shown that the inclusion of the drug in a polymeric form substantially alters its localization in organs and tissues. Extensive biodistribution nanorifabutin in lung tissue, liver and spleen is established.

Telomerase as a tumor marker in diagnosis of prostatic intraepithelial neoplasia and prostate cancer.

BACKGROUND: Early diagnosis of prostate cancer (CaP) can be addressed by studying prostatic intraepithelial neoplasia (PIN) as precancer (high-grade PIN or HGPIN). This article attempts to analyze the diagnostic role of telomerase as an early marker of carcinogenesis. METHODS: Complex urological patient evaluation and assessment of telomerase activity. RESULTS: Out of 92 patients 44% were diagnosed with CaP, 49% with low-grade PIN (LGPIN) in association with benign prostatic hyperplasia (BPH), and 7% with HGPIN in association with BPH. Active telomerase (AT) in prostate biopsy specimens was detected in 98% of patients with CaP, in 33% of patients with HGPIN, and in 20% of patients with LGPIN. In the event of simultaneous detection of AT and PIN in initial prostate biopsy specimens, further monitoring for 0.5-4.0 years revealed CaP development in 50-56% of cases. Further follow-up of patients with PIN and absent telomerase activity in initial biopsy specimens did not demonstrate the development of CaP. The PSA level was significantly higher in patients with active telomerase in the prostate tissue than in telomerase negative patients. CONCLUSIONS: Telomerase activity in the prostate tissue increases the risk of CaP development in patients with PIN. Detection of telomerase activity in prostate biopsy specimens from patients with PIN enables selection of a group of patients with high risk of CaP development and reduction of the number of prostate biopsies performed in other patients.

[The role of adiponectin in the pathogenesis of the metabolic syndrome and approach to therapy].

In this adiponectin-focused review, the pathophysiological role and the potential therapeutic benefits of adiponectin in metabolic syndrome (MetS) are analysed. MetS is recognized as clusters several metabolic abnormalities and the leading cause of cardiovascular diseases. Insulin resistance (IR) is a key factor in the pathogenesis MetS. Adiponectin is the most abundant and adipose-specific adipokine. Adiponectin acts through the activation of AMP-activated protein kinase and peroxisome proliferator-activated receptor-alpha (PPARalpha) pathways. The wide distribution of adiponectin receptors in various organs and tissues suggests that adiponectin has pleiotropic effects on numerous physiological processes. Its well-known insulin-sensitizing, anti-inflammatory and antiatherosclerotic properties, accumulating evidence suggests that adiponectin may have cardioprotective properties. There is an evidence that adiponectin decreases systematic IR and generally predicts cardiovascular diseases. Recent therapeutic strategies have focused on the indirect upregulation of adiponectin through the administration of various therapeutic agents and/or lifestyle modifications. Weight loss, diet, lifestyle changes and/or medications including orlistat, sibutramine, rimonabant, increase level of adiponectin. Also insulin sensitizers, including thiazolidinediones, and lipid-lowering agents, including statins and fibrates, upregulate adiponectin and may improve IR. The wider use of new treatment approaches appears to signal of a new era in the management of cardiovascular diseases, diabetes mellitus and MetS.

Effect of anthralin on cell viability in human prostate adenocarcinoma.

The study revealed the key role of serine protease hepsin activity in transition of in situ prostate adenocarcinoma into the metastasizing form. Inhibition of hepsin activity suppresses the invasive growth of the tumor. Hepsin is an convenient target for pharmacological agents, so the study of its inhibitory mechanisms is a promising avenue in drug development. Assay of proteolytic activity in various tumor cell lines in vitro showed that this activity in prostate adenocarcinoma cells significantly surpasses proteolytic activity in other examined tumor cell lines. Selective cytotoxic action of anthralin, an inhibitor of hepsin activity, on human adenocarcinoma cells was demonstrated in comparison with other tumor cell lines.

[The distribution of iodine-125 labeled alpha-fetoprotein in the animal organism and its accumulation in the tumor].

The distribution of iodine-125 labeled human alpha-fetoprotein in mice was studied after its intravenous injection. The maximal accumulation of alpha-fetoprotein in different tissues and organs of animals was observed mainly 5 hours after injection. Then the protein was gradually eliminated from the body. In the liver, intestine and blood of intact animals 125I-alpha-fetoprotein persists for at least three days. Accumulation of alpha-fetoprotein in various tissues and organs may determine the different biological effects of this protein. In the mice with transplanted lymphatic leukemia cells P388 the high level of alpha-fetoprotein accumulation was detected in the tumor tissue, reaching 6% of the injected amount per 1 g of tissue. This allows considering the radionuclide-labeled alpha-fetoprotein as a promising medical radionuclide marker for the radiological detection of malignant tumors.

[Analysis of complex formation of human recombinant HSP70 with tumor-associated peptides].

Molecular chaperones of HSP70 family assists presentation of exogenous antigenic peptides by antigen-presenting cells (APC). HSP70-peptide complexes are powerful immunotherapeutic agents, which enhance cross-presentation of captured antigen in dendritic cells and macrophages. Several clinical trials have shown that HSP-based cancer vaccines possess good efficacy and safety. However, sometime it is impossible to isolate sufficient amount of vaccine. These make us to pay attention for recombinant HSP70-based vaccines and to optimize in vitro complex formation mechanism. Here we have investigated two human recombinant proteins HSP70(HYB) and HSC70. Optimal values of ADP concentration, pH, temperature and peptides excess are determined in this work. We have also shown that proposed complex formation method enriches eluted from HSP70-complexes peptide repertoire compared to in vivo assembled ones.

[Characterization of recombinant human HSP70's domains functions and interdomain interactions].

We investigated ATP-ase and peptide-binding activity of recombinant human heat shock protein HSP70(A1B), HSC70, and two hybrid proteins derived from those. The UV-spectral recorded data was used to characterize conformational rearrangements, which were induced by domain replacement or HSP70-peptide interaction. We have shown that N-terminal domain dramatically affect substrate specificity of C-terminal peptide-binding domain. This proposes new hypothesis for HSP70 chaperone machinery. The linear dependence between ATP-ase activity and peptide complex ratio was found. This relationship could be used for unlabeled peptide-HSP70 complex quantification.

[High-efficient renaturation of immobilized recombinant C-terminal fragment of human alpha-fetoprotein].

C-terminal fragment of a human oncofetal alpha-fetoprotein (AFP) may be used in targeted cytostatics delivery to malignant cells of many tumors. AFP fragment (from 404 to 595 amino acids residues of a full-sized protein) was cloned and produced in Escherichia coli cells, BL21 strain (DE3) in the form of inclusion bodies. To obtain a functionally active protein, is it necessary to renature the protein. The renaturation procedure of the AFP third domain (rAFP3D) is considerably complicated by the fact that the protein is hydrophobic and contains a large number of S-S bonds. A renaturation technique of rAFP3D immobilized on silicic metal chelate resin has been developed. The yield of renatured C-terminal fragment was no less than 60% with purity on the order of 98%. The developed technique has been applied for the first time for hydrophobic protein with a large number of S-S bonds. The approach can be applied for efficient renaturation of other hydrophobic proteins with a large number of disulfide bonds for scientific and practical purposes.

Isolation, purification, and study of properties of recombinant hepsin from Escherichia coli.

A recombinant hepsin-producing strain of Escherichia coli was obtained and the conditions for hepsin expression in a bacterial system were optimized. To study the physicochemical properties of the enzyme, a procedure for purification of active recombinant hepsin using metal-chelate affinity chromatography and ion-exchange chromatography was developed. The interaction of recombinant hepsin with various peptide substrates is characterized. The dose-dependent inhibition of the recombinant hepsin enzyme activity by anthralin in vitro and an increase in the hepsin enzymatic activity in the presence of resveratrol were revealed.

[Urokinase-type plasminogen activator and its inhibitor type 1 in the diagnosis of evolving complications in coronary heart disease].

Fibrinolytic system components are important in the regulation of thrombogenesis therefore the aim of the investigation was to compare the level of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor 1 (PAI-1) in the monocytes of patients with stable coronary heart disease (CHD) and acute coronary syndrome (ACS) to reveal an association of the content of these proteins with the severity of disease, by applying two different techniques: immunocytochemistry and flow cytofluorimetry. The counts of uPA- and PAI-1-expressing monocytes were equal in each case and accounted for 81.9-99.9% in all groups. At the same time, the level of PAI-1 was higher than that of uPA and significantly higher in patients with ACS than in those without ACS and in the controls. No significant differences were found in uPA levels between the ACS and stable CHD groups; however, it was significantly higher in the patient groups than in the control one. The detection of the higher expression of PAI-1 in the peripheral blood monocytes of patients with ACS suggests that it can be used as a marker of disease severity.

Targeted delivery of anti-cancer growth inhibitory peptides derived from human alpha-fetoprotein: review of an International Multi-Center Collaborative Study.

The alpha-fetoprotein derived growth inhibitory peptide (GIP) is a 34-amino acid peptide composed of three biologically active subfragments. GIP-34 and its three constituent segments have been synthesized, purified, and studied for biological activity. The GIP-34 and GIP-8 have been characterized as anticancer therapeutic peptides. An multicenter study was initiated to elucidate the means by which these peptide drugs could be targeted to tumor cells. The study first established which cancer types were specifically targeted by the GIP peptides in both in vitro and in vivo investigations. It was next demonstrated that radiolabeled peptide ((125)I GIP-34) is specifically localized to rodent breast tumors at 24 h post-injection. The radionuclide studies also provided evidence for a proposed cell surface receptor; this was confirmed in a further study using fluorescent-labeled GIP-nanobeads which localized at the plasma membrane of MCF-7 breast cancer cells. Finally, it was readily demonstrated that GIP conjugated to either fluorescein or doxorubicin (DOX) underwent tumor cell uptake; subsequently, DOX-GIP conjugates induced cytotoxic cell destruction indicating the utility of GIP segments as cancer therapeutic agents. Following a discussion of the preceding results, a candidate cell surface receptor family was proposed which correlated with previous published reports for a putative AFP/GIP receptor.

[Purification and characterization of recombinant human alpha-fetoprotein fragment, corresponding to the C-terminal structural domain].

Human alpha-fetoprotein (hAFP) is the main human oncofetal protein. Receptor of hAFP is expressed on the surface of different types of cancer cells, but not produced by normal cells of the adult organism. The hAFP interacts with the receptor via its third domain. The conjugates of native hAFP with a variety of natural cytostatic agents inhibit growth of cancer cells in vivo and in vitro. The C-terminal hAFP fragment comprising amino acids from 404 to 595 of the native hAFP was expressed in E. coli BL21 (DE3) strain. The level of the protein expression was no less than 150 mg/l. Highly efficient purification and refolding procedures were developed. The final protein yield was no less than 50% with purity of about 95%. Refolded rAFP3D bound specifically with cancer cells carrying hAFP receptor and was accumulated by them. This rAFP3D can be used as a carrier for the targeted drug delivery to cancer cells.

[Regulation of human dendrite cells' physiological functions with the recombinant heat shock protein Hsp70].

Reference literature review. The dendrite cells (DC) are the most important antigen-representing cells of the organism. They are the target for various vaccines, and on the basis of the DC cellular anti-tumour and anti-viral vaccines (DC vaccines) have been developed. At the same time, the DC can be a convenient model for studying activity and action mechanisms of different immune-therapeutic preparations. One of the aspects of the DC use optimization for induction of antigen-specific immune response might involve use of the heat shock proteins (Hsp), the Hsp70 in particular. This protein can be used for administration of protein antigens into the DC and for regulation of the DC activity. Knowledge of the DC physiology and specifics of interrelationship among the Hsp70 and its complexes with the DC antigens of different differentiation degree is an important aspect for implementation of these ideas. Human Hsp70 has been shown both to bring antigens to the DC and to regulate activity of the DC as well as to optimize induction of antigen-specific cellular immune response.

[Prognostic, diagnostic, and therapeutic prospects of using adiponectine as a biomarker in cardiobascular diseases].

The role of adipose tissue as an important endocrine organ is today beyond doubt. The adipose tissue is known to be a source of many biologically active substances, adipocitokines, one of them being the adiponectine possessing anti-inflammatory, antiatherogenic and cardioprotective properties; it is believed to be one of prospective biomarkers for risk assessment, for diagnostics and, possibly, for therapy of cardiovascular diseases.

[Live xenogenic vaccine in the prevention of metastases of uveal melanoma].

By the results of experimental studies and by being guided by Protocol No. 22 dated January 22, 2004, by the Ministry of Health of the Russian Federation, the authors have conducted clinical trials of biotherapy used in oncological care. They describe the first experience in using xenovaccination in 35 patients with uveal melanoma to prevent hematogenic metastases. The follow-ups of the patients lasted within 14-49 months after initiation of the vaccination. No recurrences were observed in 30 patients during follow-ups whose durations averaged 41.3 months. The authors consider that this is reason to regard xenovaccination as an available method for the prevention of metastases of uveal melanoma.

Localization of Mullerian inhibiting substance receptors in various human cancer cell lines.

Recombinant human MIS (rhMIS) produced in transfected Chinese hamster ovary cells has been purified by immunoaffinity chromatography. In the absence of reducing agents, 140 kD homodimer and several oligomers with molecular masses from 280 to 1000 kD are present. Homodimer, tetramer, and higher-molecular-weight rhMIS fractions reduced survival of tumor cells. For these experiments, FITC-labeled rhMIS was used for binding and endocytosis studies by flow cytometry. Flow cytometry performed on MIS-sensitive cancer cell lines demonstrated specific binding of rhMIS. The majority of rhMIS receptors have cytosolic localization. Thus, the level of MIS receptors on the cell membrane was proportional to the content of MIS-binding proteins in the whole cell and defines a level of receptor-mediated endocytosis. The immunopurified rhMIS caused significant growth inhibition of ovarian and prostate adenocarcinoma and melanoma human cell lines in inhibition assays.