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.

BIOMAGNETISM OF DRUG-SENSITIVE AND DRUG-RESISTANT MALIGNANT TUMORS AFTER INJECTION OF FERROMAGNETIC NANOCOMPOSITE.

Magnetic signals emitted by living organisms, regardless of a biological species, are important biophysical indicators. The study of these indicators is very relevant and promising for the visualization of the tumor process and the development of technologies using artificial intelligence when it comes to malignant neoplasms, particularly resistant to chemotherapy. AIM: To measure magnetic signals from transplantable rat tumors and their counterparts resistant to cytostatics for evaluating the features of the accumulation of iron-containing nanocomposite Ferroplat. MATERIALS AND METHODS: Doxorubicin (Dox)-sensitive and Dox-resistant Walker-256 carcinosarcoma and cisplatin-sensitive and cisplatin-resistant Guerin's carcinoma transplanted in female Wistar rats were studied. The magnetism of tumors, liver and heart was determined using Superconductive Quantum Interference Device (SQUID) - magnetometry in a non-contact (13 mm over the tumor) way using specially designed computer programs. In a group of the experimental animals, a ferromagnetic nanocomposite (Ferroplat) was administered as a single intravenous injection and biomagnetism was assessed in 1 h. RESULTS: The magnetic signals coming from Dox-resistant Walker-256 carcinosarcoma in the exponential growth phase were significantly higher in comparison with sensitive tumor. Intravenous administration of Ferroplat increased biomagnetism by at least an order of magnitude, especially in resistant tumors. At the same time, the magnetic signals of the liver and heart were within the magnetic noise. CONCLUSION: The use of SQUID-magnetometry with ferromagnetic nanoparticles as a contrast agent is a promising approach for visualization of malignant neoplasms with varying sensitivity to chemotherapy.

Biomagnetism of tumor in rats with Guerin's carcinoma after injection of ferromagnetic nanocomposite (Ferroplat): contactless measurement.

AIM: In order to develop fundamentally new technologies for non-invasive and safer diagnosis of cancer, we aimed to detect non-contact magnetic signals from a malignant tumor in animals treated or not-treated with the ferromagnetic nanocomposite Ferroplat. MATERIALS AND METHODS: Guerin's carcinoma was used as a model of tumor growth. The biomagnetism of the tumor was evaluated in the dynamics of its growth. Ten days after tumor transplantation, Ferroplat was administered intravenously to half of the animals with the tumor and to half of the control animals. The magnitude of the magnetic signals was determined 1 h and every two days after administration of the nanocomposite using a Superconducting Quantum Interference Device magnetometer of the original design. RESULTS: We have found that the magnetic signals coming from the tumor are significantly higher compared to control tumor-free animals. Intravenous administration of a ferromagnetic nanocomposite (Ferroplat: Fe3O4 + cisplatinum) led to a significant increase of the magnetic signal, especially in the tumor tissue, and inhibition of Guerin's carcinoma growth. Ferromagnetic nanoparticles (32.7 nm) are retained in malignant cells for a longer time than in normal ones. CONCLUSION: Tumor cells accumulate iron nanoparticles more intensively than normal ones. Nanocomposite Ferroplat can be used for a targeted delivery of cisplatin to malignant cells.

Association of circulating miR-21, -205, and -182 with response of luminal breast cancers to neoadjuvant FAC and AC treatment.

AIM: To identify the association of serum miRNAs with neoadjuvant polychemotherapy response in patients with breast cancer of luminal A and B subtypes. MATERIALS AND METHODS: We analyzed the expression levels of circulating miR-21, -155, -182, -373, -199a, -205, -375 in serum of 182 breast cancer patients using real-time polymerase chain reaction. Each case was characterized by TMN criteria using morphological and immunohistochemical analyses. RESULTS: Serum levels of miR-205 and -375 are associated with the response of luminal A tumors and miR-205 and -21 with the response of luminal B tumors to neoadjuvant polychemotherapy in fluorouracil + doxorubicin + cyclophosphamide and doxorubicin + cyclophosphamide regimens. In addition, we found correlation of miR-155, -182, -199a, -375 with 3-year relapse-free survival of patients. Based on the obtained data, we developed innovative prognostic and predictive panels to assess the drug sensitivity of tumors and lower the risk of breast cancer recurrence, which would significantly improve the treatment outcomes and the quality of life of patients. CONCLUSIONS: Serum levels of miR-155, -182, -199a, -205, -375 can be used as predictive and prognostic panel for monitoring BC course in Ukrainian population.

NANOG as prognostic factor of prostate cancer course.

The variability of the clinical course of prostate cancer (PC) indicates the need to find factors that could predict the aggressive potential of neoplasms accounting the biological characteristics of tumor cells. In this context, the role of NANOG, a transcription factor involved in maintaining pluripotency and one of the markers of cancer stem cells (CSCs), is being actively studied today. AIM: To investigate the level of NANOG mRNA in tumor tissue of patients with PC and to analyze the possibility of its use as a marker of the disease course. MATERIALS AND METHODS: The study involved 85 patients with PC of stages II-IV. Morphological and immunohistochemical studies were performed on serial paraffin sections of resected PC using monoclonal antibodies to Ki-67 and androgen receptor. NANOG and miR-214 mRNA expression in tumor cells was analyzed by real-time reverse transcription polymerase chain reaction. The identification of CSCs was performed by double-labeled immunohistochemical method using primary antibodies to CD24 and CD44. RESULTS: We have revealed notable variability of NANOG mRNA levels in tumor tissue of patients with PC (mean 4.18 ± 0.65 a.u. with individual deviations from 0.11 ± 0.03 a.u. to 15.24 ± 0.36 a.u.). According to NANOG mRNA levels, two groups of the PC patients were delineated: group 1 and group 2, with the average NANOG mRNA levels of 2.12 ± 0.16 a.u., and 8.68 ± 1.24 a.u., respectively. The NANOG mRNA levels in tumor tissue of PC patients of groups 1 and 2 correlated with preoperative serum prostate-specific antigen level (r = 0.58; p < 0.05 and r = 0.64; p < 0.05, respectively), tumor volume (r = 0.42; p < 0.05 and r = 0.72; p < 0.05, respectively), regional lymph node metastases (r = 0.70; p < 0.05 and r = 0.75; p < 0.05, respectively). High NANOG mRNA levels in tumor cells were associated with such molecular and biological features of PC as androgen receptor expression (r = 0.52; p < 0.05), high proliferative activity (r = 0.60; p < 0.05) and the presence of CSC markers (r = 0.75; p < 0.05). CONCLUSIONS: The findings indicate that NANOG is involved in the formation of the PC malignancy and should be further studied as a potential marker for the prediction of the disease course.

Human microbiota and effectiveness of cancer chemotherapy.

This review presents up-to-date information on the effects of microbiota on the individual chemotherapy sensitivity in cancer treatment. Recent studies have shown that a fine balance between the intestinal microbiota and the immune system is crucial for maintaining an efficacy of cancer chemotherapy. A number of antitumor drugs have complex mechanisms of action involving not only direct effects but also the activity of the intestinal microbiota and the immune system. A unique combination of these factors contributes to the individual chemotherapy sensitivity.

Tumor microenvironment-derived miRNAs as prognostic markers of breast cancer.

AIM: To study expression of miRNA derived from tumor microenvironment in patients with breast cancer (BC) as the aspect of tumor-host interaction. MATERIALS AND METHODS: The expression levels of estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2 (HER2/neu) were analyzed in tissue of BC using immunohistochemical method. Relative expression levels of the miR-155, -320a, and -205 were examined in tissue and sera from BC patients using quantitative reverse transcription polymerase chain reaction. RESULTS: Serum and tissue miR-155, -320a, and -205 levels in patients with BC are of low diagnostic value as such for differentiation of malignant and non-malignant breast neoplasms. Nevertheless, we established the relation of circulating and tissue miR-155, -320a, and -205 to lymph node metastases and basal breast cancer subtype. CONCLUSIONS: Changes of miR-155, -320a, and -205 expression in tumor tissue and sera of BC patients provide information about major clinical-pathological characteristics of BC.

The influence of lactoferrin on elemental homeostasis and activity of metal-containing enzymes in rats with Walker-256 carcinosarcoma.

AIM: To investigate the content of essential elements (EE): copper, zinc, magnesium, iron and calcium and the evaluation of the activity of metal-containing enzymes - ceruloplasmin (CP), myeloperoxidase (MPO) and the content of transferrin (TF) in blood plasma (BP) and tumor tissue (TT) of animals with Walker-256 carcinosarcoma treated with lactoferrin (LF). MATERIALS AND METHODS: The study of the EE content and the activity of the abovementioned enzymes was carried out on rats with Walker-256 carcinosarcoma treated with LF at the doses of 1 and 10 mg/kg of body weight. The quantitative content of EE in BP and TT of animals was determined using the inductively coupled plasma atomic emission spectroscopy (ICP-AES). Determination of CP activity, content of TF and hemochromes was performed using the method of electron paramagnetic resonance (EPR), and MPO - by unified biochemical method. RESULTS: The introduction of LF at the doses of 1 and 10 mg/kg resulted in a decrease in the ratio of Cu/Zn in BP and even more expressed decrease of Ca/Mg ratio in TT. Administration of LF, especially at a dose of 10 mg/kg, affected the increase in CP and MPO activity in BP. It has been shown that administration of LF at a dose of 10 mg/kg led to an increase in oxidative products of destruction of the hemoglobin-hemochrom system in the TT, against the background of lowering the TF content. CONCLUSIONS: The administration of LF, especially at a dose of 10 mg/kg, led to metabolic alterations associated with inhibition of the tumor process. The detected modulating effect of LF on the content of the EE and the activity of the CP and MPO may be a basis for correction of the elemental balance in carcinogenesis.

Overexpression of the mitochondrial ribosomal protein S18-2 in the invasive breast carcinomas.

BACKGROUND: Recent studies allow to consider the mitochondrial ribosomal protein S18-2 (MRPS18-2, S18-2) as a potential oncoprotein, which suggests the need for further characterization of its expression in tumors of different genesis including breast cancer (BC). The aim of the study was to analyze the expression of the S18-2 protein in BC of luminal A and basal subtypes. MATERIALS AND METHODS: Operational material of BC patients stage І-ІІ (luminal A subtype, n = 30, and basal subtype, n = 10) was studied with the use of morphological, immunohistochemical, statistical and bioinformatic methods. RESULTS: Using the immunohistochemical analysis, we found that the S18-2 protein showed the nuclear signal in 66.7% of luminal A subtype BC samples and 80.0% of basal subtype BC samples. The variability of the S18-2 expression in both the luminal A and basal subtypes of BC was revealed. Noteworthy, the number of cells expressing S18-2 in high-proliferating tumors of luminal A and basal subtype is significantly higher than in tumors with a low proliferative potential (p < 0.05). In 10 samples of luminal A subtype, the nuclear S18-2 signal was higher than median value. Moreover, the S18-2 protein was overexpressed in 4 out of such 10 samples. Metastases in the lymph nodes were found in 3 out of 4 patients with the stage II BC, low differentiation grade of the tumor and high proliferative activity. The bioinformatic analysis confirms our preliminary findings that the trend for increasing expression of the S18-2 protein in tumors correlates with the aggressiveness of malignant BC. CONCLUSION: The S18-2 protein may be a marker of cancer aggressiveness in BC patients.

Influence of ferromagnetic nanocomposite (Ferroplat) on human breast cancer cells of different malignancy degrees: pro/antioxidant balance and energy metabolism.

AIM: To study the effect of Ferroplat (FrP) on the indexes of pro/antioxidant balance and energy metabolism in breast cancer cells of different malignancy degree and different sensitivity to drug therapy. MATERIALS AND METHODS: The study was carried out on breast cancer cells of low (T47D, MCF-7) and high malignancy degree (MCF-7/DDP (cisplatin-resistant), MDA-MB-231, MDA-MB-468) using cell culture techniques, immunocytochemical, biochemical, biophysical methods, flow cytometry and polarography. RESULTS: We established that the addition of FrP to the culture medium reduces the activity of glucose-6-phosphate dehydrogenase (G6PDH), superoxide dismutase (SOD) and the level of non-protein thiols by 32-41% (p < 0.05). At the same time, there was an increase of the total level of ROS and the rate of NO generation by inducible NO synthase by 1.7-2.5 times (p < 0.05). This testifies that FrP disturbs the antioxidant balance in cells, resulting in their death. Also, the use of FrP led to a decrease in the rate of oxygen absorption in MCF-7 and T47D cells by 26% and 25%, respectively (p < 0.05). In cells of high malignancy degree this index decreased by 38-40% under the influence of FrP. Incubation of MCF-7 and T47D cells with the indicated agent also reduced the content of phospholipid cardiolipin by 15-16% (p < 0.05), and in MDA-MB-231, MCF-7/DDP, MDA-MB-468 cells - by 29%, 30% and 32%, respectively. In addition, the effect of FrP caused a decrease in the levels of Mg2+ and lactate in MCF-7 and T47D cells by 21-29% and 14-24%, respectively, whereas in MDA-MB-231, MDA-MB-468, MCF-7/DDP cells - by 34-38% and 32-35%, respectively. In this case, the agent raised the level of glucose in the cells of low malignancy degree by 20-23% (p < 0.05), and in the cells of high malignancy degree and with the phenotype of drug resistance - by 31-36%. However, the nanocomposite did not affect the activity of lactate dehydrogenase in all studied breast cancer cells. CONCLUSION: The study has shown that FrP has an effect on the pro/antioxidant balance and energy metabolism of cancer cells. In addition, the denoted effect of FrP was more pronounced in the breast cancer cells with a high malignancy degree and the phenotype of drug resistance.

Antitumor and genotoxic effects of lactoferrin in Walker-256 tumor-bearing rats.

AIM: To investigate the influence of exogenous lactoferrin (LF) on tumor growth, energy and lipid metabolism of Walker-256 carcinosarcoma and to assess genotoxic effects of LF. MATERIALS AND METHODS: The study was performed on Walker-256 tumor-bearing rats. Total lipids and phospholipids were determined by thin-layer chromatography. Comet assay was used to investigate the genotoxic effects of LF. RESULTS: Daily i.p. administrations of exogenous LF at concentrations of 1 mg/kg and 10 mg/kg starting from the 4th day after tumor transplantation suppressed growth of Walker-256 carcinosarcoma by almost 44%. After treatment with recombinant LF in both doses, the phospholipid composition of Walker-256 carcinosarcoma cells was changed (3-fold increase of phosphatidylethanolamine, 3.4-fold increase of phosphatidylcholine, and 1.8-fold increase of sphingomyelin, while the cardiolipin content decreased by 67%. Exogenous LF was not genotoxic for bone marrow cells (as assessed by the ratio of PCE/NCE, number of micronuclei) and peripheral blood lymphocytes (percentage of DNA in the tail of a comet) in Walker-256 carcinosarcoma-bearing rats. CONCLUSION: Exogenous LF caused the inhibition of Walker-256 carcinosarcoma growth and a decrease in the microviscosity of plasma cell membranes, and exerted no genotoxicity toward bone marrow cells and peripheral blood of experimental animals.

Effects of exogenous lactoferrin on phenotypic profile and invasiveness of human prostate cancer cells (DU145 and LNCaP) in vitro.

AIM: To investigate the biological effects of exogenous lactoferrin (LF) on phenotypic profile and invasiveness of human prostate cancer (PC) cells in vitro. MATERIALS AND METHODS: Human PC cell lines (LNCaP, DU-145) were cultured with an exogenous LF at a dose corresponding to IC30. The expression levels of steroid hormone receptors (androgen receptor, estrogen receptor, progesterone receptor), Her2/neu, Ki-67, E- and N-cadherin, were monitored by immunohistochemical analysis. The levels of miRNAs were assessed using q-PCR. The invasive activity of the cells was examined in a standard invasion test. RESULTS: Exogenous LF reduced expression of steroid hormone receptors (ERα and PR) and Ki-67 in both PC cell lines. The expression of E-cadherin increased significantly in LF-treated DU-145 cells. Also, we established the decrease in invasive activity upon LF treatment by 40% and 30% in DU-145 and LNCaP cells, respectively. In DU-145 cells, incubation with exogenous LF resulted in an increase in the expression of oncosuppressive (miR-133a and miR-200b) miRNAs. CONCLUSIONS: Exogenous LF causes the changes in phenotypic characteristics of PC cells and levels of oncogenic and oncosuppressive miRNAs involved in the regulation of key cellular processes.

Significance of iodine symporter for prognosis of the disease course and efficacy of neoadjuvant chemotherapy in patients with breast cancer of luminal and basal subtypes.

The aim of the research was to study the relation between expression of Na.

Artemisinin modulating effect on human breast cancer cell lines with different sensitivity to cytostatics.

AIM: To explore effects of Artemisinin on a series of breast cancer cells with different sensitivity to typical cytotoxic drugs (doxorubicin - Dox; cisplatin - DDP) and to investigate possible artemisinin-induced modification of the mechanisms of drug resistance. MATERIALS AND METHODS: The study was performed on wild-type breast cancer MCF-7 cell line (MCF-7/S) and its two sublines MCF-7/Dox and MCF-7/DDP resistant to Dox and DDP, respectively. The cells were treated with artemisinin and iron-containing magnetic fluid. The latter was added to modulate iron levels in the cells and explore its role in artemisinin-induced effects. The MTT assay was used to monitor cell viability, whereas changes of expression of selected proteins participating in regulation of cellular iron homeostasis were estimated using immunocytochemical methods. Finally, relative expression levels of miRNA-200b, -320a, and -34a were examined by using qRT-PCR. RESULTS: Artemisinin affects mechanisms of the resistance of breast cancer cells towards both Dox and DDP at sub-toxic doses. The former drug induces changes of expression of iron-regulating proteins via different mechanisms, including epigenetic regulation. Particularly, the disturbances in ferritin heavy chain 1, lactoferrin, hepcidin (decrease) and ferroportin (increase) expression (р ≤ 0.05) were established. The most enhanced increase of miRNA expression under artemisinin influence were found for miRNA-200b in MCF-7/DDP cells (7.1 ± 0.98 fold change), miRNA-320a in MCF-7/Dox cells (2.9 ± 0.45 fold change) and miRNA-34a (1.7 ± 0.15 fold change) in MCF-7/S cells. It was observed that the sensitivity to artemisinin can be influenced by changing iron levels in cells. CONCLUSIONS: Artemisinin can modify iron metabolism of breast cancer cells by its cytotoxic effect, but also by inducing changes in expression of iron-regulating proteins and microRNAs (miRNAs), involved in their regulation. This modification affects the mechanisms that are implicated in drug-resistance, that makes artemisinin a perspective modulator of cell sensitivity towards chemotherapeutic agents in cancer treatment.

Correlation of iodine symporter expression in highly and low malignant cell lines of human breast cancer differed in their sensitivity to doxorubicin.

UNLABELLED: To investigate the relationship of sodium/iodide symporter (NIS) expression with molecular phenotype of highly and low malignant cell lines of human breast cancer (BC) with different sensitivity to doxorubicin (Dox). MATERIALS AND METHODS: The cell lines used in the analysis included T47D, MCF-7, MDA-MB-231, MDA-MB-468, and MCF/Dox. NIS expression was studied by immunocytochemical method. RESULTS: The strongest iodine symporter expression (248 ± 1.9; 272 ± 3.2 and 289 ± 2.8 points, respectively) were found in cells of highly malignant cell lines - MDA-MB-231, MDA-MB-468, and MCF-7/Dox. NIS expression was significantly weaker (< 120 points) in two BC cell lines of low malignancy (MCF-7 and T47D). In addition, the reduced sensitivity to Dox is associated with elevation of NIS expression in both high and low malignant cells. We have demonstrated correlations between NIS levels and certain indices of BC malignancy, namely proliferative activity (r = 0.51), receptor status (estrogen receptor; r = -0.47; and progesteron receptor; r = -0.47) and invasiveness (r = 0.46). CONCLUSIONS: Our data evidence that NIS expression level correlates with BC cells indices of malignancy and their sensitivity to Dox. The results obtained suggest the necessity for further studies of NIS expression in BC patients aimed at prognosing disease course and monitoring treatment efficacy with anthracyclines.

Lactoferrin expression in breast cancer in relation to biologic properties of tumors and clinical features of disease.

UNLABELLED: To determine the patterns of lactoferrin (LF) expression in breast cancer (BC) in relation to biologic properties of the neoplasms and clinical features of the disease course. MATERIALS AND METHODS: Clinical specimens of 266 BC patients (115 patients with BC of stages I-II - retrospective study, and 151 BC patients - prospective study) were analyzed. Morphological, immunohistochemical and statistical methods were used. RESULTS: The number of LF-positive tumors in retrospective and prospective groups was similar (52.1 and 52.8%, respectively). Among common clinical criteria for prognosis of the disease outcome in BC patients (patient's age; stage of the disease; histological type, differentiation grade, receptor status; presence of metastases), a strong correlation was found only between expression indexes of LF and estrogen receptors (ER). In ER-positive tumors expression of LF was significantly higher than in ER-negative tumors (35 vs 18%). 5-Year survival rate of BC patients was higher in LF-positive group (70 vs 52% in LF-negative group). The presence of regional metastasis tended to correlate with an increased number of LF-positive tumors. In the patients with invasive ductal carcinoma, expression level of LF moderately correlated with occurrence of luminal A subtype (r = 0.43), while in the patients with invasive lobular carcinoma this index strongly correlated with occurrence of luminal B subtype (r = 0.71). LF expression correlated positively with low and moderate differentiation grade of luminal B or basal tumors, and negatively with luminal B or basal tumors of high differentiation grade (r = -0.57 and -0.63, respectively). CONCLUSION: It has been shown that LF expression in breast tumors correlated with life expectancy of BC patients and important physiologic and clinical features of the disease, while the character of such relation strongly depended on molecular phenotype of tumor, i.e. luminal A, luminal B or basal.

Effect of 5-azacytidine on miRNA expression in human breast cancer cells with different sensitivity to cytostatics.

AIM: To analyze expression of miRNA in human breast cancer cells, sensitive and resistant to cisplatin and doxorubicin, and to explore possible modification of drug sensitivity via treatment of cells with 5-azacytidine (5-aza), a demethylating agent. MATERIALS AND METHODS: The study was performed on wild-type MCF-7 cell line (MCF-7/S) and its two sublines MCF-7/Dox and MCF-7/DDP resistant to doxorubicin and cisplatin, respectively. Cells were treated with 5-aza, cisplatin, doxorubicin and their combinations. Relative expression levels of miRNA-221, -200b, -320a, -10b, -34a, -122 and -29b were examined, using qRT-PCR. The MTT assay was used to monitor cell viability. RESULTS: We compared miRNA expression profiles in MCF-7/S and drug resistant MCF-7/Dox and MCF-7/DDP cells. Changes of miRNA-221, -200b, -320a, -10b, -34a, -122 and -29b were observed in both resistant cell lines. The most significant differences were found for miRNA-200b (decreased in 50.0 ± 2.6 and 63.0 ± 3.1 times for MCF-7/Dox and MCF-7/DDP cells, respectively) and for oncogenic miRNA-221 levels (increase in 62.0 ± 5.7 times for MCF-7/Dox and 83.8 ± 7.2 times for MCF-7/DDP cells). 5-aza treatment caused an increase of miRNA-10b, -122, -200b levels in MCF-7/S cells, miRNA-34a, -10b, -122, -200b and -320a levels in MCF-7/Dox cells and miRNA-34a, -10b, -200b and -320a levels in MCF-7/DDP cells. Pretreatment of all studied lines with 5-aza resulted in the increase of their sensitivity to studied cytostatics. In particular, the IC50 of doxorubicin decreased by 2-, 4- and 3-fold for cell lines MCF-7/S, MCF-7/Dox and MCF-7/DDP cells, respectively, and IC50 of cisplatin in studied cultures decreased by 3-, 2- and 1.5-fold, respectively. CONCLUSIONS: It was shown that use of 5-aza can modify sensitivity of breast cancer cells to cytotoxic drugs not only by it's demetylation effect, but also by changes in expression of miRNAs, involved in cell proliferation, migration and drug resistance development.

Modifying effects of 5-azacytidine on metal-containing proteins profile in Guerin carcinoma with different sensitivity to cytostatics.

AIM: To assess the influence of the treatment with 5-azacytidine (5-aza) on the profile of metal-containing proteins and factors of their regulation in Guerin carcinoma cells in vivo. MATERIALS AND METHODS: The study was conducted on Wistar rats transplanted with wild-type Guerin carcinoma (Guerin/WT) and its strains resistant to cisplatin (Guerin/CP) or doxorubicin (Guerin/Dox). Animals were distributed in 6 groups treated with 5-aza and control animals without treatment. 5-Aza was injected by i.v. route (1 injection in 4 days at a dose of 2 mg/kg starting from the 4th day after tumor transplantation, 4 injections in total). Ferritin levels in blood serum and tumor tissue were measured by ELISA, transferrin and free iron complexes - by low-temperature EPR, miRNA-200b, -133a and -320a levels and promoter methylation - by real-time quantitative reverse transcription polymerase chain reaction. RESULTS: The study has shown that 5-aza treatment caused demethylation of promoter regions of fth1 and tfr1 genes in all studied Guerin carcinoma strains. 5-Aza treatment resulted in a significant decrease of ferritin levels in tumor tissue (by 32.1% in Guerin/WT strain, by 29.8% in Guerin/Dox and by 69.1% in Guerin/CP). These events were accompanied by 3.5-fold and 2-fold increase of free iron complexes levels in tumor tissue of doxorubicin and cisplatin resistant strains, respectively. Also, 5-aza treatment resulted in significantly elevated levels of miR-200b, -133a, 320a expression in tumor tissue. After 5-aza treatment, ferritin levels in blood serum of animals with Guerin/Dox were increased by 23.9%, while in Guerin/Wt and Guerin/CP they were decreased by 17 and 16%, respectively. CONCLUSION: Alterations of epigenetic regulation upon in vivo treatment with 5-aza change the levels of metal-containing proteins due to DNA demethylation and altered miRNA expression profiles in Guerin carcinoma cells.

Modifying effects of lactoferrin in vitro on molecular phenotype of human breast cancer cells.

AIM: To assess the role of endogenous lactoferrin (LF) in the formation of the molecular phenotype of human breast cancer (BC) cell lines with varying degrees of malignancy, including cisplatin/doxorubicin resistant cell lines, and identify possible impact of exogenous LF. MATERIALS AND METHODS: 5 breast cell lines of different origin - MCF-10 A, MCF-7, including doxorubicin/cisplatin resistant ones, T47D, MDA-MB-231, and MDA-MB-468. Immunocytochemistry: expression of LF, Ki-67, adhesion molecules E- and N-cadherin, CD44, CD24 rating the invasive potential of cells. RESULTS: Expression of LF in human BC cell lines varies. It is associated with the heterogeneity of molecular profiles of cell lines in terms of adhesion. A link has been established between the level of LF expression in the resistant cell line MCF-7/CP and MCF-7/Dox, features of their molecular profile and invasive properties. Exogenous LF was shown to be capable of modifying the molecular profile and invasive properties of all the studied cell lines including resistant ones (MCF-7/CP and MCF-7/Dox). CONCLUSIONS: The sensitivity of cytostatic-resistant cell lines (MCF-7/CP and MCF-7/Dox) tends to increase under the influence of exogenous LF. It is likely that this effect is due to LF-mediated inhibition of the expression of proteins associated with drug resistance.

METALLOPROTEINS DURING DEVELOPMENT OF WALKER-256 CARCINOSARCOMA RESISTANT PHENOTYPE.

The study was focused on the detection of changes in serum and tumor metal-containing proteins in animals during development ofdoxorubicin-resistant phenotype in malignant cells after 12 courses of chemotherapy. We found that on every stage of resistance development there was a significant increase in content of ferritin and transferrin proteins (which take part in iron traffick and storage) in Walker-256 carc'inosarcoma tissue. We observed decreased serumferritin levels at the beginning stage of the resistance development and significant elevation of this protein levels in the cases withfully developed resistance phenotype. Transferrin content showed changes opposite to that offerritin. During the development of resistance phenotype the tumor tissue also exhibited increased 'free iron' concentration that putatively correlate with elevation of ROS generation and levels of MMP-2 and MMP-9 active forms. The tumor non-protein thiol content increases gradually as well. The serum of animals with early stages of resistance phenotype development showed high ceruloplasmin activity and its significant reduction after loss of tumor sensitivity to doxorubicin. Therefore, the development of resistance phenotype in Walker-256 carcinosarcoma is accompanied by both the deregulation of metal-containing proteins in serum and tumor tissue and by the changes in activity of antioxidant defense system. Thus, the results of this study allow us to determine the spectrum of metal-containing proteins that are involved in the development of resistant tumor phenotype and that may be targeted for methods for doxorubicin sensitivity correction therapy.