BIOMAGNETISM OF DRUG-SENSITIVE AND DRUG-RESISTANT MALIGNANT TUMORS AFTER INJECTION OF FERROMAGNETIC NANOCOMPOSITE.

Magnetic signals emitted by living organisms, regardless of a biological species, are important biophysical indicators. The study of these indicators is very relevant and promising for the visualization of the tumor process and the development of technologies using artificial intelligence when it comes to malignant neoplasms, particularly resistant to chemotherapy. AIM: To measure magnetic signals from transplantable rat tumors and their counterparts resistant to cytostatics for evaluating the features of the accumulation of iron-containing nanocomposite Ferroplat. MATERIALS AND METHODS: Doxorubicin (Dox)-sensitive and Dox-resistant Walker-256 carcinosarcoma and cisplatin-sensitive and cisplatin-resistant Guerin's carcinoma transplanted in female Wistar rats were studied. The magnetism of tumors, liver and heart was determined using Superconductive Quantum Interference Device (SQUID) - magnetometry in a non-contact (13 mm over the tumor) way using specially designed computer programs. In a group of the experimental animals, a ferromagnetic nanocomposite (Ferroplat) was administered as a single intravenous injection and biomagnetism was assessed in 1 h. RESULTS: The magnetic signals coming from Dox-resistant Walker-256 carcinosarcoma in the exponential growth phase were significantly higher in comparison with sensitive tumor. Intravenous administration of Ferroplat increased biomagnetism by at least an order of magnitude, especially in resistant tumors. At the same time, the magnetic signals of the liver and heart were within the magnetic noise. CONCLUSION: The use of SQUID-magnetometry with ferromagnetic nanoparticles as a contrast agent is a promising approach for visualization of malignant neoplasms with varying sensitivity to chemotherapy.

Biomagnetism of tumor in rats with Guerin's carcinoma after injection of ferromagnetic nanocomposite (Ferroplat): contactless measurement.

AIM: In order to develop fundamentally new technologies for non-invasive and safer diagnosis of cancer, we aimed to detect non-contact magnetic signals from a malignant tumor in animals treated or not-treated with the ferromagnetic nanocomposite Ferroplat. MATERIALS AND METHODS: Guerin's carcinoma was used as a model of tumor growth. The biomagnetism of the tumor was evaluated in the dynamics of its growth. Ten days after tumor transplantation, Ferroplat was administered intravenously to half of the animals with the tumor and to half of the control animals. The magnitude of the magnetic signals was determined 1 h and every two days after administration of the nanocomposite using a Superconducting Quantum Interference Device magnetometer of the original design. RESULTS: We have found that the magnetic signals coming from the tumor are significantly higher compared to control tumor-free animals. Intravenous administration of a ferromagnetic nanocomposite (Ferroplat: Fe3O4 + cisplatinum) led to a significant increase of the magnetic signal, especially in the tumor tissue, and inhibition of Guerin's carcinoma growth. Ferromagnetic nanoparticles (32.7 nm) are retained in malignant cells for a longer time than in normal ones. CONCLUSION: Tumor cells accumulate iron nanoparticles more intensively than normal ones. Nanocomposite Ferroplat can be used for a targeted delivery of cisplatin to malignant cells.

The influence of lactoferrin on elemental homeostasis and activity of metal-containing enzymes in rats with Walker-256 carcinosarcoma.

AIM: To investigate the content of essential elements (EE): copper, zinc, magnesium, iron and calcium and the evaluation of the activity of metal-containing enzymes - ceruloplasmin (CP), myeloperoxidase (MPO) and the content of transferrin (TF) in blood plasma (BP) and tumor tissue (TT) of animals with Walker-256 carcinosarcoma treated with lactoferrin (LF). MATERIALS AND METHODS: The study of the EE content and the activity of the abovementioned enzymes was carried out on rats with Walker-256 carcinosarcoma treated with LF at the doses of 1 and 10 mg/kg of body weight. The quantitative content of EE in BP and TT of animals was determined using the inductively coupled plasma atomic emission spectroscopy (ICP-AES). Determination of CP activity, content of TF and hemochromes was performed using the method of electron paramagnetic resonance (EPR), and MPO - by unified biochemical method. RESULTS: The introduction of LF at the doses of 1 and 10 mg/kg resulted in a decrease in the ratio of Cu/Zn in BP and even more expressed decrease of Ca/Mg ratio in TT. Administration of LF, especially at a dose of 10 mg/kg, affected the increase in CP and MPO activity in BP. It has been shown that administration of LF at a dose of 10 mg/kg led to an increase in oxidative products of destruction of the hemoglobin-hemochrom system in the TT, against the background of lowering the TF content. CONCLUSIONS: The administration of LF, especially at a dose of 10 mg/kg, led to metabolic alterations associated with inhibition of the tumor process. The detected modulating effect of LF on the content of the EE and the activity of the CP and MPO may be a basis for correction of the elemental balance in carcinogenesis.

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.

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.

Pharmacological effect of aminoferrocene in mice with L1210 leukemia.

AIM: To study the cytostatic and some biological effects of aminoferrocene using mice with L1210 lymphoid leukemia. MATERIALS AND METHODS: Experiments were performed on BDF1 male mice (DBA/2, female × C57Bl/6, male) with transplantable L1210 lymphoid leukemia. Determination of antitumor activity of Benzyl-Fc Boron (Bn), it was injected intraperitoneally 6 times daily, starting on day 2 after L1210 leukemia cell transplantation. Doses of Bn such as 26; 260 and 2600 µg/kg were used. The determination of intracellular content of cardiolipin, thiols, reactive oxygen species (ROS) and also analysis of Annexin V positivity and mitochondrial transmembrane potential (JC-1 staining) were performed with use of flow cytometry. The levels of "free iron" complexes, transferrin active forms and the rate of NO generation were measured by EPR-specroscopy. RESULTS: Six daily injections of Bn at a dose of 26 µg/kg resulted in an increased survival of mice with L1210 leukemia by 28% (p < 0.05). Bn led to an increase of apoptotic cells number and ROS amount in leukemia cells. Besides, Bn caused a decrease of cardiolipin and nonprotein thiol compounds content. The membrane electrochemical potential of cell mitochondria was decreased also after Bn administration. Studies using EPR-spectroscopy revealed a significant increase in a level of "free iron", content of transferrin active species and generation rate of NO by inducible NO-synthase in L1210 cells after aminoferrocene administration. CONCLUSION: Our data indicate that Benzyl-Fc Boron can be promising candidate for realizing a new strategy of anticancer therapy with the use of ROS-inducing agents.

Prevention of myelosuppression by combined treatment with enterosorbent and granulocyte colony-stimulating factor.

UNLABELLED: Hematotoxicity and its complication are the prominent limiting factors for rational treatment of malignancies. Granulocyte colony-stimulating factor (G-CSF) is used to increase granulocyte production. It has been shown previously that enterosorption causes prominent myeloprotective activity also. Still, no trial was performed to combine both of them. AIM: To study the influence of combination of enterosorption and pharmaceutical analogue of naturally occurring G-CSF (filgrastim) on bone marrow protection and the growth of grafted tumor in a case of injection of melphalan (Mel). MATERIALS AND METHODS: Mel injections were used for promotion of bone marrow suppression in rats. Carbon granulated enterosorbent C2 (IEPOR) was used for providing of enteral sorption detoxifying therapy. Filgrastim was used to increase white blood cells (WBC) count. RESULTS: The simultaneous usage of enterosorption and filgrastim had maximum effectiveness for restoring of all types of blood cells. WBC count was higher by 138.3% compared with the Mel group. The increase of platelets count by 98.5% was also observed. In the group (Mel + C2 + filgrastim) the absolute neutrophils count was twofold higher, in comparison with rats of Mel group. CONCLUSION: Simultaneous administration of G-CSF-analogue and carbonic enterosorbent C2 is a perspective approach for bone marrow protection, when the cytostatic drug melphalan is used. Such combination demonstrates prominent positive impact on restoring of all types of blood cells and had no influence on the antitumor efficacy.

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.

Increase of antitumor activity of cisplatin using agonist of gonadotropin-realising hormone and inhibitor of aromatase on the model of ascites ovarian tumor.

AIM: To study antitumor activity of triptorelin - agonist of gonadotropin-releasing hormone and exemestane - inhibitor of aromatase in monotherapy and in combination with cisplatin on the model of receptor-positive for estrogens and progesterone malignant ascites transplantable ovarian tumor (TOT), to assess therapeutic pathomorphosis and level of VEGF expression in tumor cells using diffe-rent combinations of cytostatics and hormonal drugs. MATERIALS AND METHODS: 72 female Wistar rats, which underwent intraperitoneal transplantation of ascitic TOT, by 5·10(6) cells per animal, have been involved in the study. Rats were divided into 8 groups, 9 rats in each group. Histological study with assessment of therapeutic pathomorphosis in TOT and immunohistochemical study has been carried out. Survival of animals in the studied groups has been evaluated. RESULTS: Among animals treated in regimen of monotherapy, the most pronounced antiangiogenic activity in TOT has been observed on application of hormonal drugs (triptorelin - 39.4 ± 1.9 and exemestane - 33.9 ± 1.4%; р = 0.003), the highest grade of treatment pathomorphosis in TOT has been observed at treatment with cisplatin (11.7%; р = 0.001). Combination of triptorelin and exemestane has amplified antiangiogenic activity in TOT (12.2 ± 0.9%; р = 0.001), but has not significantly changed rates of pathomorphosis (22.1 ± 0.4%; р=0.005) and survival of animals (32.2%; р = 0.007) as compared with the same rates in rats treated with hormonal drugs in monotherapy. Significant correlation between VEGF expression and pathomorphosis has been established (relative part of viable tumor tissue (RPVTT)) in TOT (r = 0.712; р = 0.001), as well as between RPVTT and life-span of animals (r = -0.320; р = 0.007). However, lack of correlation between VEGF expression in cells of TOT and survival of rats has been determined (r = -0.194; р = 0.11). Combination of cytostatic agent with triptorelin or exemestane has demonstrated significantly high rates of therapeutic pathomorphosis (10.1 ± 0.1% and 16.2 ± 0.3%, respectively) and antiangiogenic activity in TOT (21.4 ± 1.4% and 15.0 ± 1.3%, respectively) as well as the highest survival of animals (100.0%, increase of life-span (ILS) = 231.9% and 85.7%, ILS = 205.8%, respectively) as compared with the same one in rats treated in regimen of monotherapy with cisplatin, triptorelin, exemestane or by combination of hormonal drugs. Among animals treated by combination of cytostatic drug with triptorelin, two were cured, and among rats, which received cisplatin and exemestane, one animal was cured. CONCLUSIONS: Triptorelin and exemestane increase antitumor activity of cisplatin in respect to the malignant ascitic TOT and significantly increase survival of animals, especially when triptorelin and cisplatin are used in combination.

Peculiarities of antioxidant system and iron metabolism in organism during development of tumor resistance to cisplatin.

AIM: To study in vivo the peculiarities of changes of iron metabolism and antioxidant system in dynamics of growth of Guerin carcinoma with different sensitivity to cisplatin. MATERIALS AND METHODS: In order to evaluate the content of metallothionein-1 (MT-1) in tumor homogenates and blood serum of rats with cisplatin-sensitive and cisplatin-resistant Guerin carcinoma the immunoenzyme method was used. The evaluation of ceruloplasmin activity, content of "free iron" complexes, superoxide and NO-generating acti-vity of NADPH-oxidase and iNOS activity in neutrophils, blood serum and tumor homogenates was measured by EPR-spectro-scopy. RESULTS: Maximal accumulation of MT-1 in blood serum and tumor, more pronounced in resistant strain, at the border of latent and exponential phase of growth has been shown that is the evidence of protective role of this protein in the respect to the generation of free radical compounds. It has been determined that in animals with cisplatin-resistant strain of Guerin carcinoma, increase of "free iron" complexes is more apparent both on the level of tumor and organism on the background on increase of CP/TR ratio that is the consequence of organism antioxidant protection system disorder. CONCLUTIONS: Mentioned changes in metabolism of iron with its accumulation in tumor and further reprogramming of mitochondria metabolism and activity of NADPH-oxidase for non-transformed cells are favorable conditions for the formation of oxidative phenotype of tumor.

Alteration in lipid composition of plasma membranes of sensitive and resistant Guerin carcinoma cells due to the action of free and liposomal form of cisplatin.

AIM: To study in vivo changes of lipid composition of plasma membranes of sensitive and resistant to cisplatin Guerin carcinoma cells under influence of free and liposomal cisplatin forms. MATERIALS AND METHODS: The isolation of plasma membranes from parental (sensitive) and resistant to cisplatin Guerin carcinoma cells was by differential ultracentrifugation in sucrose density gradient. Lipids were detected by method of thin-layer chromatography. RESULTS: It was determined that more effective action of cisplatin liposomal form on resistant cells is associated with essential abnormalities of conformation of plasma membrane due to change of lipid components and architectonics of rafts. It results in the increase of membrane fluidity. CONCLUSION: Reconstructions in lipid composition of plasma membranes of cisplatin-resistant Guerin carcinoma cells provide more intensive delivery of drug into the cells, increase of its concentration and more effective interaction with cellular structural elements.

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.

Biodistribution analysis of cisplatin in liposomal form in animals with cisplatin-resistant and cisplatin-sensitive carcinoma.

AIM: To analyze the relation between pharmacokinetics of cisplatin in liposomal form and antitumor efficacy toward cisplatin-resistant and cisplatin-sensitive variants of Guerin carcinoma. METHODS: Concentration of platinum was measured by atomic absorption spectrophotometry (C115M1 "Selmi", Ukraine). Elimination constant was calculated based on the dynamics of cisplatin concentration in time period between 1 h to 24 h using nonlinear regression analysis. Area under curve (AUC24) was calculated by the trapezium method. RESULTS: It was shown that for liposomal form of cisplatin (LCp) AUC24 in tumor practically didn't depend on the level of the tumor sensitivity, while in animals with the resistant variant (CpRGC), AUC24 for free cisplatin (FCp) decreased by 70% less (p < 0.001) as compared to the sensitive tumor strain (CpSGC). Significant decrease of elimination constant of LCp compared to FCp in blood serum of rats bearing either CpRGC or CpSGC tumors favors cisplatin accumulation in tumor tissues with low vascularization level. The dynamics of cisplatin concentration in CpRGC variant was characterized by 90% higher level in 24 h after administration of LCp as compared to FCp (p < 0.05). This fact may explain increased antitumor efficacy of LCp compared to FCp toward CpRGC variant. In the study of kidney function, AUC24 index for LCp was by 68.6% (p < 0.01) and 50.7% (p < 0.05) lower than AUC24 index for FCp in rats with CpRGC and CpSGC variants, respectively. No significant differences have been found in biodistribution of cisplatin in both pharmaceutical forms in liver and lung in CpRGC- or CpSGC-bearing rats. CONCLUSION: The results suggest that cisplatin in liposomal form possesses higher specificity of antitumor action than free cisplatin.

Ultrastructural and some functional changes in tumor cells treated with stabilized iron oxide nanoparticles.

AIM: To study the ultrastructure and some functional indexes of tumor cells treated with stabilized iron nanoparticles in vitro. METHODS: 3-[4,5dimethylthiazol-2-1]-2,5-diphenyltetrazolium bromide (MTT)-test, electron microscopy, polarography with applying of closed Clark's electrode. RESULTS: It was shown that cultivation of cells with stabilized Fe(3)O(4) leads to intracellular accumulation of ferromagnetic nanoparticles. The most active ferromagnetic uptake by cells has been observed after 24 and 48 h of incubation. The presence of ferromagnetic in cells led to altered mitochondrial structure that caused the decrease of oxygen uptake rate in the cells of all studied lines. Ferromagnetic released from the majority of cells via exocytosis or clasmacytosis after a certain period of time. The number of dead cells or cells with severe damage was moderate, so cytotoxic action of stabilized iron oxide nanoparticles was minimal toward the studied cell lines. CONCLUSION: the presence of ferromagnetic nanoparticles in culture medium led to alterations in mitochondria ultrastructural organization and decrease of oxygen uptake by mitochondria in sensitive and anticancer-drugs resistant cells.

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.

Changes in VEGF level and tumor growth characteristics during lewis lung carcinoma progression towards cis-DDP resistance.

AIM: To study the relationship between tumor angiogenic potential and its growth and metastasis using Lewis lung carcinoma (LLC) models with different degree of resistance to cis-diamminedichloroplatinum (cis-DDP). METHODS: LLC and its two cis-DDP-resistant variants (LLC-9 LLC-19), were used. For determination of angiogenic potential of LLC, LLC-9 and LLC-19, the level of VEGF production by these tumor cells in vitro and the level of circulating VEGF during tumor growth in vivo was measured by enzyme-linked immunosorbent assay. RESULTS: Progressive decrease of LLC-9 and LLC-19 sensitivity to action of cis-DDP evidenced in vitro (IC50=0.0077+/-0.0005 mg/ml and 0.0156+/-0.0008 mg/ml respectively vs. 0.004+/-0.0003 mg/ml for LLC, p<0.05) and in vivo (index of primary tumor growth inhibition by cis-DDP was 26% and 3% respectively vs. 46%; index of metastasis inhibition--46% and 11% vs. 65%, p<0.05) was accompanied by the significant changes of tumor angiogenic potential. The level of VEGF production by primary culture of LLC-9 in vitro was 1.5 fold higher (p<0.05) than that by primary culture of LLC, whereas there were no differences in the level of VEGF production between LLC-19 and LLC. The level of circulating VEGF drastically increased in the initial phase of LLC-9 and LLC-19 growth in vivo, whereas in LLC bearing mice the dynamic changes of VEGF level are characterized by the presence of long-term latent period (t(lag)=17.0+/-0.3 days). In LLC bearing mice the character of changes of circulating VEGF level significantly correlated with the number of metastases (p<0.001) but not with tumor volume; while in LLC-9 bearing mice - with tumor volume (p<0.01) and the number of metastases (p<0.05). Although maximum level of circulating VEGF was significantly (p<0.05) higher in LLC-9 bearing mice than that in LLC bearing mice, maximum number of lung metastases was significantly (p<0.05) lower in LLC-9 bearing mice vs LLC. In contrast to LLC-9, in LLC-19 bearing mice the level of metastatic injury was significantly elevated (p<0.05) and the level of circulating VEGF considerably correlated with both tumor volume (p<0.01) and metastatic index (p<0.01). CONCLUSION: There is revealed a direct correlation between the level of circulating VEGF and all parameters of tumor progression observed only in the cases of highly resistant tumors, whilst elevation of circulating VEGF level during tumor growth in vivo could be considered as a marker of metastasis not dependent on a drug resistance of tumor.

Antitumor resistance formation in animals in early neonatal period.

Immunization of new-born mice during the first three days after birth enhances immunity in adult life, and it remains high throughout the entire life course. The antitumor resistance is raised so high in order to produce cancerogenesis blockade caused by 20-methylcholanthrene injection to adult animals even one year after vaccination. The dynamics of growth of new-borns shows that immediately after birth the animal growth proceeds very quickly, following an exponential law. The same occurs in rats and even in human beings. Vaccination of animals during fast growth phase ensures an antitumor effect, while vaccination after fast growth phase leads to antitumor immunity breakdown. It was assumed fetal lymphocytes do not attain full sexual maturity and their cytotoxic properties start developing only after birth. This hypothesis is in full agreement with our results. Vaccination of new-borns during fast growth phase leads to a significant increase of immature lymphocytes of those clones which are sensitive to an injected antigen. Upon vaccination termination, these lymphocytes attain complete maturation and remain in tissues, thereby ensuring a prolonged and high immunity. Our earlier experiments clearly demonstrated the increase in lymphocytes number in animals which had been exposed to early postnatal immunization. The kinetic analysis showed that full tumor destruction would be only possible if the rate of tumor cells destruction by cytotoxic lymphocytes is greater than the rate of tumor cells reproduction, as the tumor is incapable to make up for loss of its own cells. Immunization during fast growth phase does allow maximum increase in the number of cytotoxic lymphocytes and, thereby, enhance an entire cell immunity - unattainable after full lymphocyte maturation.

[Ultrastructural organization of carcinoma Lewis (3LL) cells during cisplatin resistance formation].

The electron-microscopic analysis of the morphological status of 3LL (Lewis) carcinoma tumour cells in the process of cisplatin resistant phenotype formation has been performed. It was shown that selection of tumour cells forming cell clones characterized by more complicated nuclear and cytoplasm organization took place. The tumour cells had the diffused nuclear chromatin; nuclear envelope had the numerous pores with expanded diaphragms. The prominent nucleoli consisted of the active centres surrounded by considerable areas of the condensed nucleolar chromatin. Cell cytoplasm contained the well-developed Goldgi complex and the numerous well-structured myelinoid formations in the form of dense-wrapped concentric membrane structures. The obtained data can morphologically confirm the hypothesis of Gately D.P. and Howel S.B., 1993, thain the process of resistant phenotype formation the tumour cells can create the cellular mechanisms to remove the drug from the cell and to correct the damages of the cellular nucleus and cytoplasm.