Mitochondrial dysfunction significantly contributes to the sensitivity of tumor cells to anoikis and their metastatic potential.

It is well-known that the survival of metastatic cells during their dissemination plays an important role in metastasis. However, does this mean that the final result of the metastatic cascade (the volume of metastatic damage to distant organs and tissues) depends on, or at least correlates with, the degree of resistance to anoikis (distinctive hallmarks of metastatic cells)? This question remains open.The aim of the work was to study in vitro the changes in the survival rates, proliferative activity, oxidative stress, and glycolysis intensity during three days of anchorage-dependent and anchorage-independent growth of two Lewis lung carcinoma cell lines (LLC and LLC/R9) and compare these changes with the status of mitochondria and metastatic potential of the cells in vivo. Methods: The number and volume of lung metastases were estimated for each cell line after intramuscular inoculation of the cells in C57Bl/6 mice. For the in vitro study, the cells were seeded on Petri dishes pretreated with poly-HEMA or untreated dishes and then allowed to grow for 3 days. Cell viability, cell cycle progression, the level of reactive oxygen species (ROS), glucose consumption and lactate production rates were investigated daily in both growth conditions. An electron microscopy study of intracellular structures was carried out. Results: The study showed (as far as we know for the first time) a correlation between the metastatic potential of cells (determined in vivo) and their sensitivity to anoikis (assessed in vitro). The transition of LLC/R9 cells with an inherently defective mitochondrial system to the conditions of anchorage-independent growth was characterized by a decrease in survival, a slowdown in growth rates, an increase in both glucose consumption rate and intracellular ROS levels and manyfold lower metastatic potential, compared to highly metastatic LLC cells with the normal mitochondrial system.

Metformin enhances antitumor action of sodium dichloroacetate against glioma C6.

It is known that the arsenal of chemotherapeutic agents for the treatment of malignant brain tumors is quite limited, which causes the high relevance of research aimed at finding new effective antitumor regimens, including the use of energy metabolism modifiers. AIM: To investigate the anti-glioma activity of sodium dichloroacetate (DCA) and metformin (MTF) used in combination in vitro and in vivo. MATERIALS AND METHODS: Cell survival, cell cycle, apoptosis, mitochondrial membrane potential (Δψm), ATP level, the glucose consumption rate, and lactate production rate were determined in vitro in cultured glioma C6 cells. The antitumor action of agents in vivo was evaluated routinely by the prolongation of the life span of rats with transplanted intracerebral glioma C6 and was confirmed by histological examination of tumor tissue. RESULTS: The half maximal inhibitory concentration (IC50) for DCA and MTF used separately was 79.2 ± 2.1 mM and 78.4 ± 4.0 mM, respectively, whereas IC50 for DCA used in combination with 7.8 mM MTF was 3.3 fold lower (24.0 ± 1.2 mM, p < 0.05). The 1-day incubation of cells with DCA at a concentration close to IC50 (25 mM), in combination with MTF at a concentration by order lower than IC50 (7.8 mM), in contrast to their separate use, resulted in a decrease in the number of viable cells by 40% (p < 0.05); redistribution of the cells by the cell cycle phases toward decreased proportion of cells in the S-phase by 46% (p < 0.05) and an increased percentage of cells in the G0/G1 phase by 24% (p < 0.05) compared to similar indices in the control. High proapoptotic activity of DCA in combination with MTF was supported by a significantly higher percentage of apoptotic cells in vitro than in the control (18.9 ± 4.4% vs 5.7 ± 1.3%, p < 0.05) and a high number of tumor cells with signs of apoptosis revealed during the histological examination of tumor pathomorphosis. The combined effect of DCA and MTF resulted in almost 4-fold decrease of the glucose consumption rate by glioma C6 cells (0.23 ± 0.05 µmol/106 cells/h vs 0.91 ± 0.12 µmol/106 cells/h, p < 0.05) compared to the corresponding parameters in the control, and 2-fold increased rate of lactate production (1.06 ± 0.03 µmol/106 cells/h vs 0.53 ± 0.03 µmol/106 cells/h, p < 0.05). At the same time, both Δψm and the level of intracellular ATP in the glioma C6 cells treated with DCA and MTF, both separately and in combination, did not differ significantly from those indices in the control. In in vivo studies, the average life span of rats with intracranial transplanted glioma C6, treated with DCA in combination with MTF in a total dose of 1.1 and 2.6 g/kg body weight, respectively, was 50% higher (p < 0.001) than in the control group. In contrast, in the case of single-use (at a dose of 2.6 g/kg), MTF increased the life span of tumor-bearing animals just by 19% (p < 0.01), whereas DCA alone (at a dose of 1.1 g/kg) did not significantly change the survival time of rats. CONCLUSIONS: The obtained data indicate synergism of anti-glioma action of DCA and MTF in a case of their combined use both in vitro and in vivo and may be considered a starting point for the development of effective treatment regimens for malignant brain tumors based on the combined use of DCA and MTF.

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.

Enterosorption as a method to decrease the systemic toxicity of cisplatin.

UNLABELLED: A perspective adsorptive method to minimize systemic toxic effects of chemotherapy is enterosorption (ES). However, the capabilities of this method are far from being completely studied. The question remains opened - should ES be initiated in the first hours on completing cytostatic infusion without the risk of their anticancer activity to be decreased. AIM: to analyze ES influence on anticancer activity and toxic reactions of cisplatin (CP) upon the use of carbon enterosorbent in 1 h after intravenous administration of cytostatic. METHODS: CP at the dose of 1 mg/kg body weigh (BW) was administered to Guerin carcinoma-bearing rats each second day for two weeks. Enterosorbents on the basis of highly activated carbon fibers were administered by per os daily 1 h after CP injection. 3 days after the last CP administration the rats were weighted and blood under ether narcosis has been taken for biochemical examination. Tumors and innate organs were isolated, weighted, and fixed in 4% buffered formalin for morphologic examination. RESULTS: In rats administered with CP at the background of ES, BW loss was in 1.6 times lower than in animals after CP session. Relative kidney weight in CP-treated rats was 33.9% higher than in normal ones (p ≤ 0.05). No significant differences were detected between relative kidney weights in the CP + ES-treated and intact animals. Introduction of ES allowed prevent an 30% increase of creatinin content observed in blood plasma after CP treatment (р ≤ 0.05). Urea content was 1.7 times lower in blood plasma of CP + ES-treated rats than after CP treatment. CP caused significant toxic injuries in kidneys, liver, and spleen tissues. Morphologic structure of organs in rats treated with CP at the background of ES was affected at much lower degree. In tumors, large areas of newly formed connective tissue and blood vessels have been fixed after the CP+ES action instead of large necrotic area observed after CP treatment. ES caused insignificant suppression of Guerin carcinoma growth and had additional impact to inhibitory action of CP. CONCLUSION: Active carbon enterosorbents which are administrated just 1 h after CP administration possesses detoxicating potential sufficient for significant elimination of toxic effect of the cytostatic at the background of complete preservation of its antitumor activity.

Distribution and accumulation of liposomal form of doxorubicin in breast cancer cells of MCF-7 line.

AIM: To study distribution and accumulation of liposomal form of doxorubicin in human breast cancer cells of MCF-7 line and Dox-resistant subline MCF-7/Dox. METHODS: High performance liquid chromatography and laser confocal microscopy were used. RESULTS: It has been shown that conventional form of doxorubicin was more efficiently delivered to the MCF-7 cells already after 30 min of incubation amounting to its maximum concentration after 4 h. MCF-7/Dox cells are characterized by lower doxorubicin accumulation rate compared with parental cells. The quantity of accumulated liposomal form of doxorubicin is high in MCF-7 cells, and, what is important, Dox-resistant cells accumulated higher levels of liposomal form of doxorubicin than its conventional form. CONCLUSION: It has been shown that intracellular distribution and accumulation of liposomal forms of doxorubicin in parental and Dox-resistant MCF-7 cells differs from that of conventional doxorubicin.

Ultrastructural changes in tumor cells treated with liposomal forms of anticancer drugs.

AIM: To determine the main ultrastructural changes in MCF-7 sublines sensitive and resistant to cytotoxic action of anticancer drugs, resulting from the treatment with conventional and liposomal forms of cisplatin and doxorubicin. METHODS: Electron microscopy, light microscopy, MTT-test. RESULTS: It has been shown that the phenomenon of drug resistance is associated with complication of ultrastructural organization of cells and more high differentiation by the main cytomorphologic characteristics which promote their resistance to cytotoxic action of anticancer preparations. Cytoarchitectonics of all resistant cells possesses common patterns and doesn't depend on the particular drugs toward which the resistance has been developed. It has been shown that the cells of the parental form MCF-7 line are more sensitive to cytotoxic action of doxorubicin than to cisplatin. Liposomal forms of anticancer drugs used at the same concentrations that the conventional ones, especially that of doxorubicin, caused more expressed alterations in ultrastructural organization of cells of all studied sublines with dominance of apoptotic processes. CONCLUSION: Evaluating an effect of equal concentrations of cisplatin and doxorubicin in conventional and liposomal forms, one may conclude on higher cytotoxic action of doxorubicin vs. cisplatin that is expressed in a wider spectrum of ultrastructural changes of cell architectonics in different sublines of MCF-7 cells and higher rate of apoptosis.

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.

Immunohistochemical studies of protein kinase D (PKD) 2 expression in malignant human lymphomas.

AIM: To study the PKD2 expression, autophosphorylation and localization in reactive lymph nodes and tumors of lymphoid tissues. MATERIALS AND METHODS: Specific antibodies, which recognize PKD1/2 or PKD2 and autophosphorylated PKD1/2, were used for immunohistochemical and biochemical studies of tonsils, reactive lymph nodes, tumor samples of non-Hodgkin's lymphoma (NHL) and Hodgkin's lymphoma (HL). RESULTS: Immunohistochemical and biochemical analysis of PKD1 and PKD2 expression showed PKD2 expression in tonsils, reactive lymph nodes and tumor tissues from patients with NHL and HL. Furthermore, we were not able to reveal PKD1 expression in studied lymphoid tissues. In tonsils and reactive lymph nodes the PKD2 expression was detected in T and B cell zones with highest level in germinal centers of lymphoid follicles and the maximum level of autophosphorylation in the light zones of the germinal centers. We found that low level of PKD2 expression and autophosphorylation was characteristic feature for mantle cell lymphomas, Burkitt's lymphomas, and in 50% of CLL/small lymphocytic lymphomas. Lymphoma cells of germinal center origin and with activated B cell phenotype (diffuse large B cell lymphomas, HL) and anaplastic large cells lymphoma demonstrated the high level of PKD2 expression and autophosphorylation. CONCLUSIONS: The level of PKD2 expression and autophosphorylation in neoplastic cells corresponds to the expression pattern of this kinase in their normal analogs, and to the level of cell differentiation and activation.

An ultrastructural study of phagocytosis and shrinkage in nutritive phagocytes of the sea urchin Anthocidaris crassispina.

The ultrastructural mechanisms of waste-sperm phagocytosis and postspawning shrinkage were studied for accessory cells (nutritive phagocytes; NPs) of the sea urchin Anthocidaris crassispina. Sperm cells were phagocytosed by NPs; they penetrated into the cytoplasm of the NPs inside heterophagosomes formed by an invagination of the cell membrane. Single-sperm-containing heterophagosomes aggregated to form large multisperm heterophagosomes that were accompanied by cytoplasmic vesicles and lipids. Two types of vesicle, viz., Golgi-complex-derived electron-dense vesicles ("zymogen granules") and smooth-endoplasmic-reticulum-derived electron-lucent vesicles, were incorporated within multisperm heterophagosomes. Completed multisperm heterophagosomes were transformed into electron-dense remnant bodies, the content of which underwent destruction, resulting in "empty" vacuoles inside the remnant body. The "empty" vacuoles were then compressed by the surrounding cytoplasm. Shrinkage of NPs occurred upon completion of sperm degeneration in gonad tubules. This process was undertaken by structures termed cell-size-reducing autolysosomes, which performed two types of autolysis, and resulted in the formation of "cheese-hole"-like vacuoles in the cytoplasm of NPs. Subsequent cytoplasmic compression of these vacuoles was required for the reduction in size of NPs, an essential event for remodeling the cell for the next gametogenetic cycle.

Monoclonal antibodies of IPO series against B cell differentiation antigens in leukemia and lymphoma immunophenotyping.

Monoclonal antibodies (mAbs) of IPO series were developed following immunization with human B cell lines RPMI-1788, Daudi, and spleen cells from a patient with hairy cell leukemia. Reactivity of these mAbs was studied on 19 human cell lines, mononuclear cells of 50 healthy persons and 142 patients with leukemias and lymphomas. It was shown that mAbs IPO-3, IPO-10 and IPO-24 define B cell-specific antigens expressed at different stages of maturation. MAb IPO-3 reacted with activated B lymphocytes. MAb IPO-10 defined the antigen which appears on B cell progenitors following HLA-DR and proceeding CD19, CD10, CD22, CD37; cy mu and CD20 and have been lost during terminal differentiation. The antigen detected by mAb IPO-24 was expressed throughout B cell ontogeny from pre-B cell until the B-blasts. MAb IPO-4 detected an antigen of activated T and B lymphocytes. These mAbs are useful tools in the leukemia and lymphoma phenotypic characterization and classification.