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.

Hepcidin as a possible marker in determination of malignancy degree and sensitivity of breast cancer cells to cytostatic drugs.

AIM: To investigate the role of hepcidin (Hepc) in the formation of cells malignant phenotype in vitro and its expression in the dyna-mics of growth of Walker-256 carcinosarcoma 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, MCF/CP, and MCF/Dox. Hepc expression was studied by immunocytochemical method. "Free" iron content was determined by EPR spectroscopy. Determination of Hepc expression in homogenates of tumor tissue and in blood serum of rats with Dox-sensitive and -resistant Walker-256 carcinosarcoma was performed. RESULTS: It was found that Hepc levels in breast cancer (BC) cells with high degree of malignancy (MDA-MB-231, MDA-MB-468) and drug-resistant phenotype (MCF/CP, MCF/Dox) were by 1.5-2 times higher (p < 0.05) in comparison with sensitive and less malignant BC cells. The development of drug-resistant phenotype in Walker-256 carcinosarcoma cells was accompanied by increasing of Hepc and "free" iron content (by 2.4 and 1.2 times, respectively). CONCLUSION: The data of in vitro and in vivo research evidenced on involvement of Hepc in formation of BC cells malignant phenotype and their resistance to Dox.

Remodulating effect of doxorubicin on the state of iron-containing proteins, and redox characteristics of tumor with allowance for its sensitivity to cytostatic agents.

The study was aimed at determining the changes of metal-containing proteins in blood serum and tumor tissue of animals with parental and doxorubicin-resistant strains of Walker-256 carcinosarcoma before and after the cytostatic administration. It has been shown that upon doxorubicin action the levels of total iron and transferrin in the tissues from the both groups of animals decreased while that of ferritine simultaneously increased with more pronounced pattern in the group of animals with resistant tumor strain. It has been shown that upon the action of doxorubicin in tumor tissue of animals with different sensitivity to the cytostatic there could be observed oppositely directed changes in the redox state of these cells that in turn determined the content of " free iron" complexes, RO S generation and concentration of active forms of matrix metaloproteinase- 2 and matrix metaloproteinase-9, namely, the increase of these indexes in animals with parental strain and their decrease in animals with the resistant one. So, our study has demonstrated the remodulating effect of doxorubicin on the state of metal-containing proteins and redox characteristics of tumor dependent on its sensitivity to cytostatic, at the levels of the tumor and an organism. These data may serve as a criterion for the development of programs for the correction of malfunction of iron metabolism aimed at elevating tumor sensitivity to cytostatic agents.

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.

Antitumor and antimetastatic activities of vaccine prepared from cisplatin-resistant lewis lung carcinoma.

AIM: To study antitumor and antimetastatic activities of antitumor vaccine (ATV) prepared from cisplatin (CP) sensitive and resistant strains of Lewis lung carcinoma (LLC). METHODS: The inhibition of tumor growth, and the mean survival time of the tumor-bearing animals, the number and the volume of metastases were measured as the indices of ATV efficacy. The activity of cytotoxic T-lymphocytes and natural killer cells, peritoneal macrophages (Mph), the level of tumor necrosis factor and the total proteolytic activity of blood plasma (PA) were assessed. RESULTS: ATV from CP resistant LLC prepared using cytolectin (CL) of capital VE, Cyrillic. subtilis capital VE, Cyrillic-7025 significantly inhibited growth of CP resistant tumors (by 52%) and increased mean survival time (MST) of animals (by 44.6%). The index of metastasis inhibition for ATV prepared from CP sensitive or resistant LLC was 154.5% and 227.0%, respectively. In all vaccine-treated animals, Mph activity was shown to be significantly increased. In spite of high antitumor and antimetastatic effects of ATV prepared from CP resistant LLC, PA in plasma of animals inoculated with CP resistant LLC was increased significantly upon vaccine administration.

[N-stearoylethanolamine inhibits growth and metastasis of the Lewis carcinoma and modulates lipid composition of the lung during tumorogenesis in mice].

We investigated the influence of N-stearoylethanolamine (NSE) on tumor growth and metastasis of the lung Lewis carcinoma in mice. The effect of NSE on lipid composition of lung tissue under tumorogenesis was also studied. We demonstrated that NSE inhibited the tumor growth and decreased the volume and quantity of metastases being administered from the fourth day after injection of tumor cells to the last day of experiment and being administrated from the 21th day after injection of tumor cells to the last day of the experiment. The analysis of the lipid composition of the lung tissue showed the decrease of total phospholipid levels and change of the phospholipid spectra under tumor growth. The decreasing of the concentration of phosphatidylcholine, sphyngomyeline, phosphatidylserine and lysophosphatidylcholine in the lung tissue of tumor-bearding mice in comparison with lung of intact animals was observed. It was found that administration of NSE increased the level of lysophosphatidylcholine and decreased the concentration of phosphatidylinositol in investigated tissues. The content of sphingosine was increased in lung tissue of mice fed by NSE in comparison with tumor-bearing mice. The carcinoma development was associated by the significant decreasing of cholesterol level and by the increasing of unsaturated fatty acids in membrane phospholipids. The amount of the tiobarbituric acid (TBA) reactive substances in tumor-bearing mice was elevated. The administration of NSE inhibited the accumulation of TBA reacting compounds.

[Phospholipid and cholesterol levels in the tumor cell plasma membranes with different sensitivity to doxorubicin]. / Vmist fosfolipidiv ta kholesterolu u plazmatychnykh membranakh pukhlynnykh klityn iz riznoiu chutlyvistiu do doksorubitsynu.

The investigation is aimed to study qualitative and quantitative composition of phospholipids, cholesterol content and lipids unsaturation index in plasma membranes of Guerin's carcinoma cells sensitive or resistant to doxorubicin. The comparison of infrared spectra and phospholipids unsaturation index showed that the unsaturation level of fatty acids in plasma membrane from resistant cells was lower than that from sensitive carcinoma cells. 31P-NMR spectroscopy of plasma membranes phospholipids shows the increase of phosphatidylserine and sphingomyeline content in plasma membrane isolated from resistant tumor as compared with sensitive tumor. The levels of phosphatidylcholine and phosphatidylethanolamine were equal in drug-resistant and drug-sensitive carcinoma strains. Changes in plasma membrane from resistant cells result in elevation of plasma membrane microviscosity and phosphatidylserine level increase can suggest the activation of P-glycoprotein-mediated efflux of doxorubicin.

Surface enhanced IR absorption of nucleic acids from tumor cells: FTIR reflectance study.

The data on surface enhanced IR absorption (SEIRA) of nucleic acids deposited on a metal substrate were obtained using FTIR in reflectance mode. A 200-400 A thick gold film on a glass plate was the metal substrate. The approximate enhancement factors of the SEIRA for different vibrations of nucleic acids in our experimental conditions were 3-5. The roughness of the Au surface was about 50 A. Application of this method to nucleic acids isolated from tumor cells revealed some possible peculiarities of their structural organization, namely, the appearance of unusual sugar and base conformations, modification of the phosphate backbone, redistribution of the H-bond net, and so forth. This method enhanced a set of the bands, which is impossible to observe in conventional IR geometry. The SEIRA spectra of the RNA from tumor cells showed more sensitivity to the grade of tumor malignancy than the spectra of the DNA. After application of the anticancer drug doxorubicin to sensitive and resistant strains, the DNA isolated from these strains had different spectral features, especially in the region of the phosphate I and II bands. As induced by anticancer drugs, the conformational changes in the DNA from resistant and sensitive cancer strains could be characterized with different levels of structure disordering.

[Changes in energy metabolism in the brain of bemegride-kindled rabbits]. / Zminy enerhetychnoho metabolizmu v tkanynakh mozku kroliv pry bemehrydovomu kindlinhu.

Changes in the energetic metabolism in the cortex were examined in bemegride-kindled rabbits. A reliable decrease in the ATP and an increase in the AMP contents in the cortical tissues as well as low ATP/AMP and energetic charge coefficients have been found after kindling the rabbits for 14 and 30-days as compared to the control groups of animals. Considerable disorders in the oxidative phosphorilation in the mitochondria of the cortical tissues have been determined after the succinate and glutamate oxidation: a decrease in the oxygen utilization rate at V3 active metabolic state, lowering the respiratory control coefficient and the disscciation between phosphorilation and oxidation. Those disorders were more expressed after glutamate oxidation as compared to a succinate one. In addition, it has been determined that oxygen utilization at V2 state was reliably decreased; both coefficient of energetic production and ATP-ase reserve activity lowered.

[Effect of ceruloplasmin on the oxygen tension in muscular tissue of experimental animals]. / Vplyv tseruloplazminu na napruzhennia kysniu v míazevii tkanyni eksperymentalnykh tvaryn.

The polarographic method using platinum electrode has been applied to study the effect of ceruloplasmin (CP) on the oxygen tension (pO2), oxygen saturation rate and rate of oxygen utilization in the muscular tissue of high-leukemic AKR mice, C57BL/6 mice with transplanted Lewis lung carcinoma (3LL) and rats after gamma-irradiation in a dose of 7 Gr. It has been shown that CP in AKR mice improves oxygen saturation of the muscular tissue. This effect is also evident in the case of the marked pO2 decrease in the muscle and its oxygen saturation rate (animals with Lewis lung carcinoma and after gamma-irradiation).