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.

Evaluation of response to tyrosine kinase inhibitors in renal cell carcinoma patients based on expression of miR-99b, -144, -210, -222, -302а and -377 in tumor tissue.

BACKGROUND: Renal cell carcinoma (RCC) is one of the most common solid tumors in adults highly resistant to conventional therapies. The expression profile of a number of miRNAs correlates with RCC response to chemotherapeutic agents. AIM: To identify the association of tumor miRNAs expression with neoadjuvant treatment response in patients with RCC. MATERIALS AND METHODS: We analyzed the expression levels of tumor miR-99b, -144, -155, -210, -222, -302а, -377 in 93 RCC patients who received pazopanib or sunitinib in neoadjuvant regimen using RT-PCR. RNU48 was used as a reference miRNA. RESULTS: The levels of expression of miR-99b and -377 are associated with the RCC response to pazopanib, and microRNA-210 and -377 to sunitinib. The characteristic expression profile of miR-99b, -144, -222, -377, and miR-302a determined in 90% of cases was delineated in pazopanib responders as opposed to nonresponders. Similarly, the characteristic expression profile of miR-210, -222, -302a and -377 was suggested for sunitinib responders. CONCLUSIONS: Levels of miR-99b, -210 and -377 expression in RCC tumor tissue might be used as a basis for future predictive panel intended for the assessment of the sensitivity to the regimens of neoadjuvant RCC treatment.

Significance of expression of tumor-associated microRNA-21 and -375 for predicting the course of cancer of oral cavity.

AIM: To investigate the features of expression of miRNA-21 and miRNA-375 in tumor cells of patients with cancer of oral cavity (COC) and to determine the possibility of their use to predict the aggressiveness of COC course. MATERIALS AND METHODS: The work is based on the results of examination and treatment of 50 patients with stage II-IV COC. miRNA expression in tumor cells was analyzed by real time reverse transcription polymerase chain reaction. RESULTS: High levels of miRNA-21 expression (> 0.26 a.u.) and miRNA-375 (> 0.36 a.u.) were determined in 72.0% and 63.0% of cases. We revealed a tendency to decreased miRNA-21 expression and increased miRNA-375 expression in tumors of patients with recurrence-free survival less than 12 months and the presence of metastatic lesions in regional lymph nodes. In patients with COC of low differentiation grade, the level of miRNA-21 was 2.0 times lower compared with tumors of moderate differentiation grade (p < 0.05), while the expression of miRNA-375, on the contrary, was higher in tumors of low differentiation grade. A decreased expression of miRNA-21 (< 0.26 a.u.) against the background of decreased levels of miRNA-375 (< 0.36 a.u.) in tumor cells was associated with worse recurrence-free survival. CONCLUSIONS: The obtained results indicate the relation between the main clinical and pathological characteristics of patients with COC and the levels of miRNA-21 and -375 expression in tumor tissue, which indicates the involvement of these miRNAs in the formation of COC malignancy evidencing on their potential usefulness as additional prognostic markers.

Prognostic significance of microRNA-200b and ERCC1 expression in tumor cells of patients with esophageal cancer.

BACKGROUND: The development of malignant tumors, including esophageal cancer (EC), could be associated with impaired expression of oncosuppressive miRNA-200b and еxcision repair cross-complementing group-1 (ERCC1), a protein involved in DNA repair and alternative splicing. AIM: To investigate the features of expression of miRNA-200b and ERCC1 in tumor cells of patients with EC and to determine the possibility of their use for prediction of EC aggressiveness. MATERIALS AND METHODS: 52 patients with EC of stages II-IV were enrolled. Expression of ERCC1 in tumor cells was assessed by immunohistochemical method, expression of miRNA-200b was evaluated by the real time reverse transcription-polymerase chain reaction. RESULTS: The expression of miRNA-200b and ERCC1 in tumor cells of EC patients is associated with their life expectancy. The characteristic features of neoplasms in patients who died within 12 months are low expression of miRNA-200b (2.87 ± 1.65 a.u.) (ρ = -0.42; p < 0.05) and high expression of ERCC1 (191.0 ± 18.6 H-Score points) (ρ = -0.48; p < 0.05). The inverse correlations between the level of miRNA-200b expression and the tumor size were found both in the group of patients with survival < 12 months (ρ = -0.42; p < 0.05) and in the group of patients with survival > 12 months after surgery (ρ = -0.53; p < 0.05). CONCLUSIONS: The obtained data indicate the feasibility of using the expression of miRNA-200b and ERCC1 in EC cells to predict the aggressiveness of esophageal cancer.

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.

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.

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.

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.

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.

Influence of exogenous lactoferrin on the oxidant/antioxidant balance and molecular profile of hormone receptor-positive and -negative human breast cancer cells in vitro.

AIM: To investigate the mechanisms of cytotoxic activity and pro-/antioxidant effect of lactoferrin on hormone receptor-positive and receptor-negative breast cancer cells in vitro. MATERIALS AND METHODS: The study was performed on receptor-positive (MCF-7, T47D) and receptor-negative (MDA-MB-231, MDA-MB-468) human breast cancer cell lines. Immunocytochemical staining, flow cytometry, low-temperature electron paramagnetic resonance, and the Comet assay were used. RESULTS: Upon treatment with lactoferrin, the increased levels of reactive oxygen species (ROS) (p < 0.05), NO generation rate by inducible NO-synthase (p < 0.05) and the level of "free" iron (p < 0.05) were observed. Moreover, the effects of lactoferrin were more pronounced in receptor-negative MDA-MB-231 and MDA-MB-468 cells. These changes resulted in increased expression of proapoptotic Bax protein (p < 0.05), reduced expression of the antiapoptotic Bcl-2 protein (p < 0.05) and level of not-oxidized mitochondrial cardiolipin (1.4-1.7-fold, p < 0.05). This, in turn, caused an increase in the percentage of apoptotic cells (by 14-24%, p < 0.05). Cytotoxic effects of lactoferrin were accompanied by an increase in the percentage of DNA in the comet tail and blocking cell cycle at G2/M phase, especially in receptor-negative cell lines. CONCLUSION: The study showed that exogenous lactoferrin causes a violation of an antioxidant balance by increasing the level of ROS, "free" iron and NO generation rate, resalting in the blocking of cell cycle at G2/M-phase and apoptosis of malignant cells.

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.