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.

EFFECTS OF DEXTRAN-GRAFT-POLYACRYLAMIDE/ZnO NANOPARTICLES ON PROSTATE CANCER CELL LINES IN VITRO.

BACKGROUND: The combination of zinc oxide (ZnO) nanoparticles (NPs) with carriers enhances the anticancer effect of nanocomposites. AIM: To explore the mechanisms of cytotoxic action of dextran-graft-polyacrylamide (D-g-PAA/ZnO) NPs against prostate cancers cell lines in vitro. MATERIALS AND METHODS: Dextran-polyacrylamide was used as a matrix for the synthesis of ZnO NPs. Prostate cancer cells LNCaP, DU-145 and PC-3 were treated with D-g-PAA/ZnO NPs. The expression of Bax, Bcl-2, p53 and Ki-67 was studied using immunocytochemical analysis. Cytomorphological changes in cells were detected after their incubation with nanocomposites for 24 h. RESULTS: The treatment with D-g-PAA/ZnO NPs caused the increase in the Bax and p53 and the decrease in Ki-67 and Bcl-2 expression. Morphological changes associated with apoptosis were registered: decrease in cell size, appearance of cytoplasmic vacuolation, condensation of chromatin, blebbing. CONCLUSIONS: Treatment with D-g-PAA/ZnO nanocomposite led to the initiation of apoptotic cell death in prostate cancer cells in vitro.

EXPRESSION OF OSTEOPONTIN AND OSTEONECTIN IN BREAST AND PROSTATE CANCER CELLS WITH DIFFERENT SENSITIVITY TO DOXORUBICIN.

BACKGROUND: According to modern literature, osteopontin (OPN) and osteonectin (ON) are involved not only in the formation of the aggressive phenotype of malignantly transformed cells, but also in the realization of cytotoxic effects of some antitumor drugs. AIM: To study the changes of the expression of OPN and ON and their mRNAs (SPP1 and SPARC) upon exposure to doxorubicin (Dox) in breast cancer (BCa) and prostate cancer (PCa) cell lines with different sensitivity to Dox. MATERIALS AND METHODS: Cell lines of BCa (MCF-7 and MDA-MB-231) and PCa (LNCaP and DU-145) were cultured in the presence of Dox at IC30 concentrations for 24 h. OPN and ON levels were assessed by immunocytochemical (ICH) and Western blot analysis. SPP1 and SPARC mRNA levels were assessed by quantitative PCR. RESULTS: Dox treatment resulted in the significant decrease in the expression of both OPN and ON in MCF-7 and LNCaP cells. Similarly, Dox treatment downregulated both SPP1 and SPARC in MDA-MB-231 and DU-145 cells. Dox did not affect ON expression in MDA-MB-231 and DU-145 cells although the significant decrease in the level of SPARC mRNA has been evident. In contrast, no significant differences in SPP1 and SPARC mRNA levels were detected in LNCaP cells. CONCLUSION: The changes in the expression of OPN and ON proteins and their corresponding genes in BCa and PCa cells may be related to the intrinsic mechanisms of Dox effects in cells differing by malignant phenotype and Dox sensitivity.

HUMAN MICROBIOTA AND BREAST CANCER.

Breast cancer is the leading malignancy in women worldwide. To date, much is known about the molecular subtypes of these malignant neoplasms and the mechanisms of drug resistance. Significant success has been achieved in approaches to early diagnosis, which allows identifying the tumor process in the early stages of development. Recently, the study of the influence of the human body microbiota on cancer development and the effectiveness of treatment has become an actively developing field of research. This review presents an analysis of the literature data on this issue.

SLAMF1/CD150 expression and topology in prostate and breast cancer cell lines.

BACKGROUND: SLAMF1/CD150 receptor is aberrantly expressed in malignant hematopoietic cells compared to ubiquitous expression in their normal analogues. However, the data about CD150 expression and function outside the hematopoietic system are limited. The aim of this pilot study was to examine the profile of mRNA expression of CD150 isoforms and the protein topology in highly and low malignant breast (BC) and prostate cancer (PC) cell lines. MATERIALS AND METHODS: The study was performed on BC T47D, MDA-MB-231, ВСС/Р and BC/ML cell lines and PC LNCap, Du-145 and PC-3 cell lines. The quantitative polymerase chain reaction was applied for study of CD150 isoforms mRNA expression and flow cytometry was used for determination of protein localization. RESULTS: Analyzed BC cell lines did not express CD150 on the cell surface membrane (csCD150-), but more than 45% of cells were positive for CD150 cytoplasmic reaction (cyCD150+). The cyCD150 expression level in T47D cells of luminal BC subtype was higher than in basal BC cell lines MDA-MB-231, ВСС/Р and BC/ML. The PC cell lines expressed CD150 both on the cell surface and in cytoplasm. The highest number of csCD150+ and cyCD150+ cells was revealed in less aggressive androgen responsive, non-metastatic LNCap cell line. All studied BC and PC cell lines expressed mRNA of canonical transmembrane mCD150 and novel nCD150 isoforms but with different pattern of prevalence. Soluble CD150 isoform was revealed at the low level only in BCC/P BC cell line and LNCap, PC-3 PC cell lines. CONCLUSIONS: We have shown that BC and PC cell lines differentially expressed multifunctional receptor CD150 at the mRNA and protein levels that may indicate its association with the degree of their malignancy.

Effect of antitumor drugs in low concentrationson the biological, immunophenotypic and cytogenetic characteristics of human colon cancer cells in vitro.

OBJECTIVE: To estimate the impact of the low-dose anticancer drugs (ACD) with the different mechanisms of action and human interferon (IFN) alpha 2b on the biological properties, immunophenotypic and cytogenetic characteristics of colon cancer cells in vitro. MATERIALS AND METHODS: The study was performed on human colon cancer cell lines COLO 205, HT-29 and 3C-P treated with ACD and IFN in subtoxic concentrations. Expression of CD44, N-cadherin, vimentin, ß-catenin, ERCC1 and Slug was assessed by immunocytochemical method. Using cytogenetic analysis, the numbers of mitoses, cells with micronuclei, apoptotic cells and cells with nuclear protrusions were studied. RESULTS: The prolonged exposure (up to 30 days) of colon cancer cells to low-dose ACD (0.2-0.5 µg/ml cisplatin and 0.1-0.2 µg/ml irinotecan) in combination with IFN (500-1000 IU/ml) led to 37-fold decreased colony-forming activity of these cell and 10-fold reduction of the number of cells expressing mesenchymal protein markers (N-cadherin, vimentin). Also, in COLO 205 cells treated with ACD and IFN the number of SLUG- and CD44-positive cells decreased by 92 and by 85%, respectively. Long-term cultivation of HT-29 cells in the presence of cisplatin and IFN resulted in 5-fold suppression of ERCC1 expression. The cytogenetic analysis has shown that the ACD, IFN and their combinations in subtoxic concentrations caused significant genotoxic effect, suppression of cell proliferation and accumulation of cells with micronuclei. The sensitivity of colon cancer cells to ACD in standard cytotoxic concentrations did not change after prolonged low-dose exposure. CONCLUSION: The data showed that the prolonged action of the low doses of ACD on human colon cancer cells resulted in the suppression of cell proliferation, colony-forming activity in soft agar, expression of epithelial-mesenchymal transition-associated markers and significant cytogenetic changes.

Inhibition of malignant potential and expression of proteins associated with epithelial-mesenchymal transition in Lewis lung carcinoma cells transduced with murine ifn-ß gene in recombinant baculovirus.

AIM: To analyze biological characteristics, malignant potential and expression of proteins associated with epithelial-mesenchymal transition in murine lung carcinoma cells transduced with interferon-beta (ifn-ß) gene in baculovirus vector. MATERIALS AND METHODS: The study was performed on Lewis lung carcinoma (LL) cells transduced with ifn-ß gene in recombinant baculovirus vector. Biological characteristics of the LL cells were studied with the use of standard cell culture methods, cytogenetic and immunocytochemical assays. RESULTS: Recombinant baculovirus-mediated transduction of LL cells with ifn-ß gene resulted in significant decrease of cell growth rate and density both in complete and serum-free medium. Also, LL cells transduction with ifn-ß gene significantly inhibited cell migration in vitro. Transduction of LL cells by baculovirus vector with or without ifn-ß gene caused significant genotoxic effect in these cells. Furthermore, ifn-ß gene transfer to lung carcinoma cells resulted in significant increase of nuclear expression of p19(ARF) (p < 0.01), p21(WAF1) (p < 0.01), cytoplasmic expression of E-cadherin (p < 0.005) and inhibition of transcription factors of epithelial-mesenchymal transition (EMT) Twist (p < 0.005) and Slug (p < 0.01) expression. CONCLUSIONS: Transduction with ifn-ß gene of LL cells in recombinant baculovirus resulted in acquirement of less malignant phenotype in vitro and suppressed expression of proteins associated with EMT.

Impact of stromal cell components of tumor microenvironment on epithelial-mesenchymal transition in breast cancer cells.

BACKGROUND: Cell and tissue homeostasis results from the dynamic balance of cell - cell and cell - extracellular component crosstalk that regulates proliferation, differentiation, and apoptosis of cells as well as secretion and activation of soluble factors and/or deposition of extracellular matrix (ECM) components. AIM: The aim of the work was to study the crosstalk between tumor cells and stromal cell components using noncontact co-cultivation in vitro system. MATERIALS AND METHODS: Human and rat breast cancer (BC) cell lines, normal human fibroblasts (NHF) and endothelial cells, and aspirates of bone marrow (BM) of BC patients with different clinical course of the disease (groups "Remission" (BM-R) and "Progression" (BM-P)) were used in noncontact co-cultivation system in vitro. The cell growth, expression of epithelial-mesenchymal transition (EMT) and tumor stem cell markers (E-cadherin, vimentin, CD44), Ki-67, p21 and Slug were investigated using immunocytochemical analysis. RESULTS: Analysis of expression of E- and N-cadherin, vimentin and Slug in BC cells has shown that T-47D and MRS-T5 cells possess mesenchymal phenotype, while MCF-7 and MRS cells possess mostly epithelial phenotype with a part of cells with mesenchymal patterns. Upon noncontact co-cultivation of fibroblasts with Т-47D or MRS-Т5 cells, BC cells acquired higher proliferative activity compared to the control cells (р < 0.05) or MCF-7 and MRS cells co-cultivated with fibroblasts. Upon noncontact co-cultivation of Т-47D cells with normal fibroblasts and BM cells from BC patients from group "Progression" there were observed increased quantity of CD44(+) Т-47D cells (by 26%), decreased quantity of Е-cadherin(+) Т-47D cells, and appearance of vimentin-positive cells. In co-cultivation variant Т-47D + NHF + BM-R ("Remission") the quantity of CD44(+) Т-47D cells significantly decreased (р < 0.005) and E-cadherin expression remained unaltered compared to control cells. At the same time, in NHF cell population (co-cultivation variant Т-47D + NHF + BM-P) there was detected significant increase of quantity of р21+-cells (р < 0.005), cytoplasmic localization of p21, and nuclear localization of Slug. Expression of vimentin did not alter in any variant of co-cultivation. CONCLUSION: The new integration cell system for investigation of the mechanisms of interaction between tumor cells and the tumor microenvironment in vitro was developed. The significant changes in proliferative activity of TC dependently on its -ЕМT-status were detected after their interaction with fibroblasts and endothelial cells in noncontact co-cultivation system. BM cells of BC patients had different modifying influence on TC dependent on clinical BC course. The activation of ЕМT program was revealed in TC upon noncontact co-cultivation with BM cells of BC patients with progression of the disease.

The effect of monotherapy and combined therapy with NSC-631570 (ukrain) on growth of low- and high-metastasizing B16 melanoma in mice.

BACKGROUND: NSC-631570 (ukrain) is a semisynthetic derivative of the Chelidonium majus alcaloids and the alkylans thiotepa. It exerts a selective cytotoxic effect on tumor cells in vitro and in vivo and shows the ability to modulate immunocyte functions. Purpose. The aim of our work was to carry out a comparative investigation of the effects of NSC-631570 alone or in combination with pathogen-associated molecules (PAM) on the growth of low- and high-metastasizing melanoma B16 in mice. METHODS: NSC-631570 was administered intravenously and PAM intramuscularly to tumor-bearing mice seven times every third day, starting from the second day after the transplantation of tumor cells. The effect of monotherapy and combined therapy on tumor growth was evaluated by the indices of tumor growth inhibition in experimental animals. Cell cycle distribution of cancer cells was determined by flow cytometry. TAP1 and TAP2 expression was evaluated by RT-PCR. The metabolic activity of phagocytes was determined by NBT-test, phagocytosis was tested by flow cytometry, and arginase activity was estimated by colorimetric determination of urea. RESULTS: Combined therapy and monotherapy with NSC-631570 resulted in significant inhibition of tumor growth in melanoma-bearing mice. Monotherapy with Ukrain was more effective in mice with high-metastasizing tumors. The therapeutic efficacy of NSC-631570 used in combination with PAM was more expressed in mice with low-metastasizing melanoma. CONCLUSION: The effectiveness of monotherapy and combined therapy with NSC-631570 in the treatment of melanoma B16 depends on the biological properties of the tumor and the immune state of the organism.