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.

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.

[Evaluation of cyto- and genotoxic action of ferronanomagnetic and constant magnetic field in in vivo system].

Cyto- and genotoxic effects of nanoparticles on the basis of FM, CMF or their combination have been studied in AKE cells, BM cells of erythroid line, and peripheral blood lymphocytes with the use of MN test and "DNA-comet" assay. It has been shown that expression of mentioned effects is related to FM concentration and duration of tested agent action. It has been also demonstrated that action of CMF alone in the studied cells did not cause any changes in cell architectonics or affect MN counts which are associated with DNA damage. When FM and CMF were used in combination there has been observed the phenomenon of induction of CMF action with FM nanoparticles. The obtained results allow recommend MN test and "DNA-comet" assay as the markers of genome stability in the tests of genotoxic effects of nanomaterials for development of vector nanosystems.

In vitro modification of cisplatin cytotoxicity with magnetic fluid.

AIM: To study cytotoxicity of cisplatin conjugated with magnetic fluid (nanocomposite) upon exposure to magnetic field on sensitive and resistant to cisplatin MCF-7 human breast cancer cells. METHODS: Cytotoxic activity was evaluated by MTT-test, intracellular iron accumulation was analyzed cytochemically, genotoxicity was studied by micronucleus test and DNA comet assay, ultrastructure was studied by electron microscopy techniques. RESULTS: Nanocomposite of cisplatin was more toxic to MCF-7/S and MCF-7/CP cells compared to cisplatin in conventional pharmaceutical form. In nanocomposite-treated cells we observed more expressed signs of dystrophy (especially following application of magnetic field) and drastic alterations of nuclei ultrastructure with significant accumulation of iron nanoparticle clusters. The potent toxic action of nanocomposite is confirmed by electron microscopy and by marked genotoxicity, especially against MCF-7/CP cells. CONCLUSION: The enhancement of cyto- and genotoxicity of cisplatin nanocomposite combined with magnetic field in comparison with effect of convetntional cisplatin alone was demonstrated.

[Evaluation of biological effects and possible mechanisms of action of static magnetic field].

Modern views on mechanisms of interaction between static magnetic field and cells or cellular structures are reviewed. An analysis of the data about possible biotropic effects of this factor was performed. The emphasis was put on the analysis of the studies in which moderate (0.1-1 T) static magnetic fields were used, because such fields are used for targeted delivery ofmagnetosensitive nanocomposites in development of new strategies in target therapy of patients with malignant neoplasms. Based on available data it was concluded that the primary cause of changes in cells after incubation in external static magnetic field is disruption of free radical metabolism and elevation of their concentration. Such disruption causes oxidative stress, and, as a result, damages ion channels, leading to changes in cell morphology and expression of different genes and proteins, and also changes in apoptosis and proliferation.

[Visualization of iron nanoparticles accumulation and distribution features in sensitive and resistant to antitumor drugs human breast cancer cells after different time intervals of cultivation with liposomal ferromagnetic].

It was shown in vitro that visualization of iron nanoparticles by histochemical Lilly method, modified by authors for cytomorphological studies, could be a certain control of ferromagnetic income to human breast cancer MCF-7 cells with different sensitivity to antitumor drugs (doxorubicin and cisplatin). The form of the visualized iron nanoparticles, their localization and distribution towards intracellular structures was studied. It was shown that localization, dynamics of accumulation and release of ferromagnetic from cells were connected to their sensitivity to antitumor drugs and time of incubation with iron nanoparticles.

Relation between cell-to-cell adhesion and angiogenesis and clinico-morphological prognostic factors in patients with gastric cancer.

AIM: To study the relation between the expression of the molecules of cell-to-cell adhesion (E-cadherin, alpha- and beta-catenins) and vascular endothelial growth factor (VEGF) and traditional clinico-morphological characteristics of tumors to evaluate their prognostic value in the patients with gastric cancer. METHODS: To analyze the expression of E-cadherin, alpha- and beta-catenins, and VEGF the paraffin embedded tumor samples were studied by immunohistochemical analysis with the use of respective monoclonal antibodies. RESULTS: The presence of E-cadherin in tumors correlated with the absence of metastases in regional lymph nodes and was observed, as a rule, in the patients at the early stages of the disease. The presence of beta-catenin expression has been detected in gastric tumors of the patients without distant metastases, while the level of VEGF expression correlated with the degree of gastric wall injury. It has been demonstrated that the expression of E-cadherin and alpha-catenin is associated with favourable disease course and is a characteristic pattern for early stages of gastric cancer of intestinal type. However, VEGF expression is typical for the late stages of gastric cancer of diffuse type and is associated with poor prognosis. CONCLUSION: At the base of combined clinical, histological and immunohistochemical analysis of gastric tumors it has been shown that E-cadherin, alpha-catenin and VEGF could be used as informative markers of the disease course.