OPTIMIZATION OF EXPERIMENTAL MODEL SYSTEMS FOR EVALUATING RECIPROCAL INFLUENCE OF BIFIDOBACTERIUM ANIMALIS AND HUMAN BREAST CANCER CELLS IN VITRO.

BACKGROUND: The development of human breast cancer (BC) is known to be closely related to disturbances in the mammary gland microbiota. Bacteria of the genus Bifidobacterium are an important component of normal breast microbiota and exert antitumor activity. The molecular-biological mechanisms of interaction between BC cells and microbiota members remain poorly studied yet. The aim of this study was to develop and optimize an experimental model system for the co-cultivation of BC cells with Bifidobacterium animalis in vitro. MATERIALS AND METHODS: Human ВС cells of the MCF-7, T47D, and MDA-MB-231 lines, as well as live and heat-inactivated bacteria of Bifidobacterium animalis subsp. lactis (B. animalis) were used as research objects. The growth kinetics and viability of B. animalis in the presence of different ВС cell lines and without them were determined by both the turbidimetry method and seeding on an elective nutrient medium. Glucose consumption and lactate production by bifidobacteria were assessed by biochemical methods. The viability of BC cells was determined by a standard colorimetric method. RESULTS: The growth kinetics of B. animalis in the complete DMEM nutrient medium showed standard patterns. The indicators of glucose consumption and lactate production of B. animalis confirm its physiological metabolic activity under the growth conditions. The presence of BC cells in the model system did not affect the duration of the growth phases of the B. animalis cells' population but contributed to the increase in their counts. A significant decrease in the number of live BC cells of all studied lines was observed only after 48 h of co-cultivation with live B. animalis. To achieve similar suppression of the BC cell viability, 10-30-fold higher counts of heatinactivated bacteria were required compared to live ones. CONCLUSIONS: The optimal conditions for co-cultivation of human BC cells and living B. animalis cells in vitro have been identified.

Assessment of biosafety and toxicity of hydrophilic gel for implantation in experimental in vitro and in vivo models.

BACKGROUND: The assessment of biosafety of pharmacologically active substances is crucial for determining the feasibility of their medical use. There are controversial issues regarding the use of substances of different origins as implants. METHODS: We have conducted the comprehensive studies to determine the in vivo toxicity and in vitro genotoxicity of new generation of hydrophilic gel for implantation (production name of the substance "Activegel") to detail its characteristics and assess its biosafety. RESULTS: In vivo studies have shown the absence of clinical manifestations of intoxication in animals and no abnormalities in their physiological condition, general and biochemical blood tests. Evaluation of the site of the gel application showed no inflammatory reaction and evidenced on normal state of tissues of animal skin. The results of the genotoxicity test indicated that the gel did not affect the parameters of DNA comets and the formation of micronuclei, accordingly, had no genotoxic effect on human peripheral blood lymphocytes. When studying the effect of the gel on malignantly transformed cells in vitro, it was found that the gel for implantation did not change the proliferative activity and viability of human breast cancer cells. CONCLUSIONS: Comprehensive in vitro and in vivo study using various experimental model systems showed that the hydrophilic gel for implantation "Activegel" is non-toxic.

Scientific-practical and legal problems of implementation of the personalized medicine.

The article is devoted to the comprehensive analysis of scientific, practical and legal issues of personalized medicine that is a rapidly developing science-driven approach to healthcare. It is concluded that there is lack of general legal framework for the encouragement of scientific researches and practical implementation in this field. The article shows foreign experience and prospects for the introduction of personalized medicine as a key concept of healthcare system, which is based on a selection of diagnostic, therapeutic and preventive measures that would be the most effective for a particular person in view of individual characteristics. The conclusions and proposals to improve the current legislation and development of personalized medicine in Ukraine are suggested.

Association of CD44+CD24-/low with markers of aggressiveness and plasticity of cell lines and tumors of patients with breast cancer.

AIM: To search for additional molecular-biological markers of cancer stem cell (CSC) involved in the development of intra-tumor heterogeneity for the detection of features of the breast cancer (BC) pathogenesis. MATERIALS AND METHODS: Expression of estrogen receptors (ER), progesterone receptors (PR), Her2/neu, E- and N-cadherin, CD24, CD44, Bcl-2, Bax, Slug, P-gp, glutathione-S-transferase (GST) and metallothionein in cell lines was determined by the immunocytochemical method. Expression of ER, PR, Her2/neu, CD24 and CD44 in the surgical material of BC patients were determined by the immunohistochemical method. The levels of the miRNA were determined using real-time polymerase chain reaction. RESULTS: Cells of high-grade malignancy (HGM), MDA-MB-231 and MDA-MB-468 are characterized by high expression of stem cell markers compared to the cells of low-grade malignancy (LGM), T47D and MCF-7: CD44 levels in T47D and MCF-7 cells were in range of 72-79 points, which is significantly lower than in HGM cells (p < 0.05). Also, HGM cells with the properties of CSC were characterized by high expression of antiapoptotic proteins, the transcription factor Slug, and low levels of proapoptotic protein Bax (p < 0.05) compared to LGM cells. In cells with CSC characteristics an increased expression of transferrin and its receptor, ferritin, fentorin and hepcidin was revealed indicating activation of the endogenous iron metabolism. The characteristic feature of HGM cells with CSC phenotype were the increased levels of oncogenic miR-221, -155 and -10b by 60%, 92% and 78%, respectively, and decreased levels of oncosuppressive miR-29b, -34a and -200b by 8.4 ± 0.3, 4.6 ± 0.2, and 3.4 ± 0.6 times compared to MCF-7 line cells. It has been established that the development of resistance to cytostatics is accompanied by increased aggressiveness of tumor cells, loss of expression of hormonal receptors and acquiring of stem phenotype. In particular, increased expression of P-gp was observed in BC cells during the development of resistance to doxorubicin, of GST during the development of resistance to cisplatin along with increased CD44 expression (p < 0.05). We have revealed the relation between the presence of cells with the CSC phenotype (CD44+CD24-/low) and clinical and pathological characteristics of BC patients, their survival and BC sensitivity to neoadjuvant therapy (p > 0.05). CONCLUSIONS: The dependence between the expression of CSC markers and the degree of malignancy of tumor cells, development of resistance to cytostatics in vitro was established as well as the predictive value of the detection of the CSC for the individual prognosis of the BC course and sensitivity of the tumors to the treatment.

Immunophenotypic characteristics and karyotype analysis of bone marrow-derived mesenchymal stem cells of rabbits during in vitro cultivation.

The aim of this study was to establish the immunophenotypic profile and karyotypic stability of bone marrow mesenchymal stem cells (MSCs) of rabbits at the early passages in vitro following the application of different methods of dissociation of cellular material. MSCs were obtained from the femur bone marrow of three clinically healthy rabbits under general anaesthesia. Bone marrow aspirate was seeded in Petri dishes and cultured in a CO2 incubator with 5% CO2 at 37.0oC using a standard procedure. Immunohistochemical detection of nuclear proteins, cytoskeletal proteins and cell adhesion were performed by immunohistochemical analysis and karyotype analysis of MSCs following the enzyme and chelating methods of dissociation of the cell monolayer. The results of the immunophenotypic analysis of rabbit bone marrow MSCs showed that at the first, seventh, twelfth, and eighteenth passages these cells express markers of mesenchymal, muscle, epithelial and nerve cells. The choice of the enzyme or chelating method of dissociation of a culture of rabbit mesenchymal stem cells affects their cytogenetic variability. Dissociation of the MSCs monolayer with ethylenediaminetetraacetic acid produces a cell culture with fewer quantitative and qualitative changes in the chromosome apparatus as compared to the enzyme method. Rabbit MSCs express markers of mesenchymal (vimentin, actin), muscle, epithelial and nerve (E-cadherin, N-cadherin) cells that are essential for differentiation of these cells. The chelating method of dissociation of a culture of rabbit mesenchymal stem cells, using ethylenediaminetetraacetic acid during cultivation, is more advantageous than the enzyme method of dissociation because it leads to less cytogenetic variability.

Establishment and characterization of new breast and ovarian cancer cell lines as a model for studying cellular plasticity in vitro.

AIM: To compare biological properties of primary tumor cells isolated from malignant effusion of cancer patients with the same cells of permanent lines established during their long-term cultivation in vitro and to assess the impact of phenotypic conversion that was caused by changes in their microenvironment on their behavioral characteristics. MATERIALS AND METHODS: The study was performed on primary cell cultures from pleural effusion or ascites of breast and ovarian cancer and permanent cell lines derived from them, namely permanent ovarian cancer cell line I, permanent ovarian cancer cell line II and permanent breast cancer cell line I. Biological characteristics were studied using standard cell culture methods and immunocytochemical assays. RESULTS: Three new cell lines were established from breast and ovarian cancer and cell morphology, migration activity, the kinetics of growth, colony forming activity in semisolid agar and sensitivity to anticancer drug were examined. These characteristics were compared with those of the primary tumor cells. It has been shown that among the primary tumor cells from malignant effusion, cells with mese-nchymal characteristics were the most prevalent. Cultivation of primary cancer cells in vitro leads to a phenotypic change of their population: it becomes more homogeneous in morphology with predominantly epithelial-like cells. Also, later after a number of cell doublings in vitro, the cell population changes to include cells primarily with immunophenotypic properties characteristic of epithelial cells. These changes include increase in number of E-cadherin-positive cells and a decrease in number of vimentin and α- smooth muscle actin-positive cells. It was found that significant changes in expression of epithelial-mesenchymal transition associated proteins in cells during their cultivation in vitro in new microenvironment are accompanied by a rapid change in their sensiti-vity to anticancer drugs. CONCLUSIONS: The new breast and ovarian cancer cell lines were established and characterized. The induction of phenotypic transdifferentiation in malignant cells from pleural effusion and ascites can be an important approach for suppressing the progression of neoplastic process.