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.

Metabolic changes during development of Walker-256 carcinosarcoma resistance to doxorubicin.

AIM: To study indices of energy metabolism, content of K(+) and Mg(++) both in peripheral blood and in Walker-256 carcinosarcoma during development of resistance to doxorubicin. METHODS: Resistance of Walker-256 carcinosarcoma to doxorubicin has been developed through 12 subsequent transplantations of tumor after the chemotherapy. Parental strain was inhibited by drug by 65%, while transitional resistant substrains - by 30% and 2%, respectively. Determination of biochemical indices in blood serum and homogenates of tumor tissue, level of potassium, magnesium, lactate, glucose, activities of lactate dehydrogenase and glucose-6-phosphate dehydrogenase was performed with the help of biochemical and immune-enzyme analyzer GBG ChemWell 2990 (USA) using standard kits. Polarography was used to determine indices of mitochondrial oxidative phosphorylation. Study of mitochondrial membrane potential was carried out on flow cytometer Beckman Coulter Epics XL using dye JC-1. RESULTS: It has been determined that development of drug resistance causes the decrease of K(+), Mg(++), glucose content in blood serum and increase of these indices in tumor tissue. At the same time, gradual tumor's loss of sensitivity is characterized by decrease of glycolysis activity in it and activation of mitochondrial oxidative phosphorylation and pentose phosphate pathway of glucose degradation, which causes more intensive formation of NADPH. CONCLUSION: Development of drug resistance of tumor causes certain metabolic changes in organism and tumor. Further study of such changes will make possible to determine tumor and extratumor markers of resistance.

Redox-regulation of gelatinases during growth of cisplatin-sensitive and resistant Guerin carcinoma.

UNLABELLED: Study was aimed to analyze the dynamics of changes and study interrelations between content of ferritin, transferrin, active gelatinases (MMP-2 and -9) in blood serum and tumor tissue, free iron, rate of superoxide radicals generation in tumor, activity of NADPH-oxidase and iNOS in neutrophils rats with sensitive and resistant strains of Guerin carcinoma (GC). MATERIALS AND METHODS: In order to obtain resistant tumor, 12 courses of cisplatin chemotherapy have been carried out on rats bearing GC. Levels of transferrin and free iron were determined by analysis of EPR spectra from computerized radiospectrometer EPR -RE-1307 at temperature of liquid nitrogen. Rate of superoxide radicals and nitric oxide generation in tumor and neutrophils of blood was determined by EPR using spin traps at room temperature. Content of ferritin in tumor homogenate and blood serum of rats with GC was determined by ELISA method using corresponding kits. Concentration of active forms of MMP-2 and -9 in obtained samples was determined using method of zymography. RESULTS: Unregulated generation of superoxide radicals and NO by mitochondria of tumor cells and NADPH-oxidase and iNOS neutrophils via oxidation of iron-containing proteins causes the accumulation of "free iron" complexes in blood and tumor tissue of rats able to evoke oxide-induced damages of macromolecules. It has been shown that for resistant strain of carcinoma, as compared with sensitive one, significantly higher concentrations of active forms of MMP-2 and -9 in blood serum of rats are typical. Dynamics of gelatinases activity changes in tumor tissue corresponds in general with dynamics of changes in serum. In tumor tissue of rats the indices of gelatinases activity positively correlate with rate of superoxide radicals generation, content of "free iron" complexes, ferritin and activity of transferrin. Cytostatic agent increased levels of reactive oxygen species (ROS) and self-amplify rate of superoxide radicals generation. In turn, activation of MMPs via superoxide-depending regulation allows tumor cells to facilitate migration, invasion and finally - formation of metastatic centers. Mentioned above tumor "oxide phenotype" determines high level of its aggressiveness and forms corresponding level of drug resistance. CONCLUSIONS: Thus, high levels of superoxide radicals oxidize transport proteins and form free iron pool. Iron ions, via Haber - Weiss mechanism, initiate generation of the hydroxyl radicals, which also enhance oxidation processes.

The lipid content of cisplatin- and doxorubicin-resistant MCF-7 human breast cancer cells.

AIM: To perform the comparative study both of qualitative and quantitative content of lipids in parental and drug resistant breast cancer cells. MATERIALS AND METHODS: Parental (MCF-7/S) and resistant to cisplatin (MCF-7/CP) and doxorubicin (MCF-7/Dox) human breast cancer cells were used in the study. Cholesterol, total lipids and phospholipids content were determined by means of thin-layer chromatography. RESULTS: It was found that cholesterol as well as cholesterol ethers content are significantly higher but diacylglycerols, triacyl-glycerols content are significantly lower in resistant cell strains than in parental (sensitive) cells. Moreover the analysis of individual phospholipids showed the increase of sphingomyelin, phosphatidylserine, cardiolipin, phosphatidic acid and the decrease of phosphatidy-lethanolamine, phosphatidylcholine in MCF-7/CP and MCF-7/Dox cells. CONCLUSION: Obtained results allow to suggest that the lipid profile changes can mediate the modulation of membrane fluidity in drug resistant MCF-7 breast cancer cells.

Gold nanoparticles synthesis and biological activity estimation in vitro and in vivo.

UNLABELLED: The aim of the work was the synthesis of gold nanoparticles (GNP) of different sizes and the estimation of their biological activity in vitro and in vivo. MATERIALS AND METHODS: Water dispersions of gold nanoparticles of different sizes have been synthesized by Davis method and characterized by laser-correlation spectroscopy and transmission electron microscopy methods. The GNP interaction with tumor cells has been visualized by confocal microscopy method. The enzyme activity was determined by standard biochemical methods. GNP distribution and content in organs and tissues have been determined via atomic-absorption spectrometry method; genotoxic influence has been estimated by "Comet-assay" method. RESULTS: The GNP size-dependent accumulation in cultured U937 tumor cells and their ability to modulate U937 cell membrane Na(+),K(+)-АТР-ase activity value has been revealed in vitro. Using in vivo model of Guerin carcinoma it has been shown that GNP possess high affinity to tumor cells. CONCLUSIONS: Our results indicate the perspectives of use of the synthesized GNP water dispersions for cancer diagnostics and treatment. It's necessary to take into account a size-dependent biosafety level of nanoparticles.

Characteristics of homocysteine-induced multidrug resistance of human MCF-7 breast cancer cells and human A2780 ovarian cancer cells.

AIM: To study the influence of homocysteine on the mechanisms of drug resistance formation. METHODS: In current study human MCF-7 breast cancer cells and A2780 ovarian cancer cells sensitive to anticancer drugs were used. To access the viability of cells, we applied 3-[4,5-dimethylthiazol-2-1]-2,5-diphenyltetrazolium bromide colorimetric assay (MTT-test). Expression of Bcl-2, p-glycoprotein (P-gp), glutathione S-transferase (GST) and E-cadherin was studied by immunocytochemistry. RESULTS: A2780 and MCF-7 cells were treated by homocysteine. It was shown that every next treatment with homocysteine (up to 5th) decreased the sensitivity of A2780 and MCF-7 cells to cytotoxic drugs. Immunocytochemical study of molecular profile of A2780 and MCF-7 cells after long-term cultivation with homocysteine has been carried out and has revealed that such treatment resulted in the induction of Bcl-2, P-gp, GST and E-cadherin expression. This indicates that incubation of studied cells with homocysteine leads to simultaneous induction of expression of drug resistance markers to cisplatin and doxorubicin. CONCLUSION: Cultivation of MCF-7 and A2780 cells with homocysteine leads to simultaneous development of resistance to doxorubicine and cisplatin. The development of drug resistance is diverse for different drugs and varies among cell lines.

Postoperative autovaccinotherapy for patients with gastric cancer and expression of some proteins in tumor tissue.

AIM: To study the efficacy of autovaccine in the treatment of gastric cancer and significance of molecular factors having prognostic values for disease outcome to evaluate its efficacy in clinical setting. PATIENTS AND METHODS: 150 patients with histologically proven adenocarcinoma of the stomach of stages II, III or IV were enrolled into study. 86 patients have been treated with autovaccine (AV) after operation. Expression of p53, Bcl-2, receptors of tyrosine kinase, vascular endothelial growth factor (VEGF), capital IE, Cyrillic-cadherin, alpha-catenin and beta-catenin was determined in paraffin embedded tumor samples by means of immunohistochemical method with the use of respective monoclonal antibodies. RESULTS: It was shown that application of AV has resulted in the increase of 3-year overall survival of patients having stage III of disease by more than 30%, but those having stage IV - only around 14%. The increase of 3-year overall survival of patients with metastases in lymph nodes (N1-2) was observed also in more than 30%. It has been suggested the optimal phenotype for vaccine application: small er, Cyrillic53(+), EGFR(+), HER-2 neu (+), beta-catenin (+), VEGF(+) and Bcl-2(+) with no dependence on E-cadherin and alpha-catenin presence. CONCLUSION: It was determined that the best effect of AV application is observed in patients with category capital TE, Cyrillic3-4, poorly-differentiated tumors, metastases in lymph nodes (N1-2), but without distant metastases (capital EM, Cyrillic0). Gastric cancer patients with p53, EGFR, HER-2/neu, beta-catenin, VEGF and Bcl-2-positive tumors are the favorable group for the treatment with AV in the adjuvant regime.

The use of nanoferromagnetics to increase the cytotoxic effect of antitumor drugs.

AIM: To investigate the influence of ferromagnetic nanoparticles on antitumor effect of doxorubicin and mitochondria oxidative phosphorylation. METHODS: The study was carried out on the mice-hybrids (C57Bl/6xDBA/2) with intraperitoneally (i/p) transplantated Ehrlich ascitic carcinoma. Single i/p injection of doxorubicin (Dox), stabilized ferromagnetic nanoparticles (Fe3O4; 20-40 nm; FM) or their combination were performed 7 days after tumor transplantation. The cytotoxic effect of agents, morphology and cell cycle of tumor cells were studied 24, 48 and 72 h after Dox administration. RESULTS: The investigations showed that ferromagnetic nanoparticles increased the cytotoxic effect of doxorubicin on Ehrlich ascsmall i, Ukrainiantic carcinoma mainly 48 h after agents' administration. The largest number of apoptotic cells was observed in group of animals in which doxorubicin was administered before ferromagnetic nanoparticles. Moreover, the ferromagnetic nanoparticles at concentration 1.45 microg Fe/ml and, particularly, 7.25 microg Fe/ml decreased mitochondria oxygen consumption in phosphorylation state that may negatively influence their living capability. CONCLUSIONS: Obtained data point out the perspective of use of certain sized FM nanoparticles to increase the cytotoixc effect of antitumor drugs.

Molecular profile and cell cycle in MCF-7 and MCF-7/Dox cells exposed to conventional and liposomal forms of doxorubicin.

AIM: To compare the molecular profile and cell cycle of sensitive and resistant to doxorubicin MCF-7 breast cancer cells upon exposition to conventional or liposome-encapsulated forms of doxorubicin. METHODS: capital EM, Cyrilliccapital TE, Cyrilliccapital TE, Cyrillic-test, immunocytochemistry, flow cytometry. RESULTS: In sensitive MCF-7 cells the exposure to conventional but not liposomal form of doxorubicin decreased metallothionein (MT) expression demonstrating activation of MT-detoxification system. In doxorubicin-resistant cells (MCF-7/Dsmall o, Cyrillicsmall ha, Cyrillic) MT expression drastically decreased. Conventional but not liposomal form of doxorubicin reduced the levels of expression of steroid hormones receptors on MCF-7 sensitive cells. The exposure of MCF-7 cells to conventional form of doxorubicin resulted in the decrease of p53 expression and the increase of Bcl-2 expression. In MCF-7/Dsmall o, Cyrillicsmall ha, Cyrillic cells neither conventional nor liposomal form of doxorubicin altered Bcl-2 expression. The exposure of MCF-7 but not MCF-7/Dsmall o, Cyrillicsmall ha, Cyrillic to doxorubicin in conventional form caused cell cycle arrest in G0/G1. Upon exposure to doxorubicin in liposomal form, cell cycle blockage in G0/G1 phase was observed in both sensitive and resistant sublines. CONCLUSION: The differential effects of the conventional and liposomal forms of doxorubicin in sensitive and resistant cells have been demonstrated. Exposure of MCF-7 and MCF-7/Dsmall o, Cyrillicsmall ha, Cyrillic cells to doxorubicin in liposomal form alters the molecular profile and cell distribution over cell cycle phases.

Molecular profile and cell cycle in MCF-7 cells resistant to cisplatin and doxorubicin.

AIM: To compare ultrastructure, phenotypic profile and cell cycle progression of MCF-7 human breast cancer cells and MCF7 sublines resistant to cisplatin (MCF-7/DDP) and doxorubicin (MCF-7/DOX). METHODS: MTT-test, immunocytochemistry, flow cytometry, electron microscopy. RESULTS: The development of drug resistance to cisplatin and doxorubicin in MCF-7 cells upon the culturing of the initial cells with the raising concentrations of cytostatics was accompanied by the increase in cells adhesion, the increasing differentiation grade and the loss of steroid hormone receptors. Besides, it was shown that antiapoptotic mechanisms (decrease of Bcl-2 expression) and intracellular glutathione detoxifying system are involved in the process of cisplatin resistance development in MCF-7 cells. At the same time, P-glycoprotein overexpression in cells resistant to doxorubicin suggests MDR-dependent mechanism. Both doxorubicin- and cisplatin-resistant cells are characterized by the changes in the expression of several cell cycle regulators -- Ki-67, cyclin D1, pRb and p21). CONCLUSION: The long-time culture of MCF-7 cells with cytostatic drugs results in the decreased cyclin D1, pRb, and Ki-67 expression and increased p21 expression with the increasing differentiation grade of the resistant cells. The underlying mechanisms of resistance to cisplatin and doxorubicin in MCF-7 cells may be different.

Expression of drug resistance proteins in triple-receptor-negative tumors as the basis of individualized therapy of the breast cancer patients.

AIM: To evaluate the efficacy of the application of various chemotherapy schemes based on the immunohistochemical study of expression patterns of proteins associated with the drug resistance P-glycoprotein (P-gp), glutathione-S-transferase (GST), metallothioneins (MT) of breast cancer (BC) patients with the triple-receptor-negative (RE(-), RP(-), HER-2/neu(-)) cancer. METHODS AND RESULTS: P-gp, GST and MT expression in BC-biopsy samples from 60 BC patients was evaluated by immunohistochemical analysis. The results of the clinical observations showed that 3-years relapse-free survival rate of the patients of with P-gp, GST and MT-positive tumors treated with taxoter + adriablastin / taxoter + cyclophosphamide (TA/TC), gemcitabine + carboplatin, or TC + bevacizumab was 61.5%, 78.6% and 81.2% respectively, vs 41.2% in the control group with P-gp, GST and MT-negative tumors treated with adriablastin + cyclophosphamide (p<0.05), while overrall suvival rates were 84.4%, 92.6% and 93.8% respectively vs 70.6% in the control group (p<0.05). CONCLUSION: The study points on the possibility to elevate the efficiency of polychemotherapy by its individualization based on the expression patterns of P-gp, GST and MT on tumor cells of the patients with the triple-receptor-negative BC.

Modifying influence of prolonged action of interferon on phenotypic characteristics of human lung cancer cells in vitro.

AIM: To study modifying influence of interferon (IFN) on some phenotypic properties of human non-small-cell lung cancer cells (NSCLC) upon prolonged exposition of the cells with IFN. MATERIALS AND METHODS: A-549 cells were cultivated with IFN at increasing concentrations for a long period of time (up to 1 year). Cell morphology and ultrastructure were studied by light and electron microscopy. Expression of adhesion protein E-cadherin, and vimentin, cytosceleton protein associated with tumor cell migration and invasion, antigen of proliferating cells Ki-67, angiogenesis-stimulating protein VEGF were studied using the method of immunocytochemistry. Autonomity of the cell growth was studied with the use of colony formation in soft agar, platting efficiency assay, and growth in serum-free medium. RESULTS: It has been shown that prolonged action of IFN results in significant and irreversible inhibition of manifestation of malignant phenotype: decreased of proliferative potential and inhibited autonomy of the cells; in complicated cell ultrastructure; in decreased expression vimentin and in increased expression of E-cadherin. Also, an inhibiting influence of IFN on expression of EGF receptors and VEGF in tumor cells have been shown. CONCLUSIONS: The data are showing that prolonged exposition of NSCLC cells to IFN is accompanied by stable phenotypic alterations of the cells directed on significant loss of malignancy and their shift to more differentiated phenotype.

Expression of CD40 by the cells of benign and malignant breast tumors and antitumor action of autologous lymphocytes against chemoresistant and chemosensitive tumors.

AIM: To study the expression of CD40 by cells of benign and malignant tumors of mammary gland, and to compare the efficacy of lymphocytes antitumor activity against drug resistant and sensitive breast tumors in relevance to CD40 expression. METHODS: Breast tumor explants were cultured with autologous lymphocytes in double diffusion chambers. The results were evaluated by morphological criteria of explants growth. Expression level of molecules on tumor cells was analyzed using immunohistochemical method (paraffin embedded slides), and on lymphocytes - by the method of indirect immunofluorescence. RESULTS: The highest level of CD40 expression was detected on cells of chemoresistant malignant breast tumors, and the lowest one - on cells of benign breast tumors. The decreased CD40 expression on lymphocytes from patients with drug resistant breast cancer was compared with that on lymphocytes of the patients with drug sensitive breast cancer. The study of antitumor activity of autologous lymphokine activated killer cells (LAK) has shown their pronounced antitumor activity against drug resistant malignant breast tumors. CONCLUSION: Marked antitumor activity of LAK from the patients with drug resistant breast cancer is associated with high expression level of CD40 on tumor cells and with its decreased expression on lymphocytes.

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.

The role of expression of the components of proteome in the formation of molecular profile of human ovarian carcinoma A2780 cells sensitive and resistant to cisplatin.

AIM: To study the expression of proteins that characterize drug resistance, proliferation and apoptosis of human ovarian carcinoma cells. METHODS: The study was carried out on human ovarian carcinoma A2780 cells and on the A2780/DDP8 subline resistant to cisplatin. Expression of the surface and intracellular antigens (p53, Bcl-2, CD95, antigen of proliferating cells, metallothioneins, drug resistance proteins (P-glycoprotein (P-gp), glutathione-S-transferase), molecules of adhesion (E-cadherin, alpha- and beta-catenins) was studied by immunocytochemical method. RESULTS: It has been shown that the formation of the resistance to cisplatin in A2780/DDP8 cells is accompanied by the increase of expression of glutathione-S-transferase and Bcl-2, by the decrease of expression of CD95-antigene and proliferation potential of the cells, by appearance of EGF receptors and elevation of expression level of E-cadherin, alpha- and beta-catenins, proving the enhancement of adhesive properties of tumor cells. CONCLUSION: Antiapoptotic program seems to be the leading mechanism of the development of the resistance to cisplatin in A2780/DDP8 cells and is realized via high expression of Bcl-2. During the development of drug resistance of A2780/DDP cells, the program of glutathione detoxification is functionally replacing the decrease of the content of metallothioneins.