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.

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.

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.