EFFECT OF LACTATE DEHYDROGENASE INHIBITION BY OXAMATE ON LEWIS LUNG CARCINOMA CELLS WITH DIFFERENT METASTATIC POTENTIAL.

BACKGROUND: Today, the ability for metabolic reprogramming is considered one of the distinguishing features of metastatically active tumor cells, a classic example of which is aerobic glycolysis. Despite a large number of studies in this direction, the question of the relationship between the intensity of aerobic glycolysis and the metastatic potential of tumor cells remains almost completely open. The work aimed to investigate the effect of the lactate dehydrogenase (LDH) inhibitor on the viability and several characteristics of Lewis lung carcinoma cells with different metastatic potential. MATERIALS AND METHODS: High-metastatic (LLC) and low-metastatic (LLC/R9) variants of Lewis lung carcinoma cells were used. After 24 h of tumor cells incubation with or without 40 mM sodium oxamate, cell viability, the concentration of glucose and lactate in the incubation medium, distribution of cells by the cell cycle phases, and intracellular ROS production were estimated. RESULTS: It was revealed that regardless of the metastatic potential, LLC cells are heterogeneous in terms of both the involvement of aerobic glycolysis in their growth and survival processes and the sensitivity to the cytotoxic/cytostatic action of an LDH inhibitor. 35% of cells of either LLC variant form an oxamate-resistant subpopulation while 65% are oxamate-sensitive. The rate of glucose consumption of LLC/R9 cells in the absence of oxamate is almost twice higher compared to LLC and, as a result, the sensitivity of these cells to the cytotoxic/cytostatic effect of oxamate also is significantly higher (the IC50 for LLC/R9 cells is by 35.8% lower than that for LLC cells, p < 0.05). Approximately one-third of the cells of both LLC and LLC/R9 variants can survive and proliferate when aerobic glycolysis is completely inhibited by oxamate. This indicates metabolic reprogramming (either pre-existing or dynamically arising in response to inhibition of glycolysis) of this subpopulation of cells, within which not only the survival of cells but also their proliferative activity is most likely based on glutamine metabolism. CONCLUSIONS: Such metabolic heterogeneity of metastatically active cells indicates that inhibition of glycolysis as monotherapy is insufficient for effective antimetastatic therapy. Presumably, more effective would be to involve various inhibitors of metabolic processes that ensure the metabolic plasticity of metastatic cells.

ROS production by circulating phagocytes and Guerin carcinoma resistance to cisplatin.

BACKGROUND: Tumor drug resistance remains a primary cause of unsuccessful cancer therapy. The search for biological markers of the sensitivity/resistance of malignant neoplasms to drug therapy is an urgent and important task, the solution of which will increase the effectiveness of anticancer chemotherapy. AIM: To study the relationship between the functional activity (parameters of the phagocytosis and reactive oxygen species (ROS) production) of neutrophils and monocytes in the peripheral blood of rats with transplanted Guerin carcinoma and the degree of its sensitivity to cisplatin (Cpt). MATERIALS AND METHODS: The original and Cpt-resistant variants of Guerin carcinoma were transplanted to female Wistar rats 2.5 months old. The parameters of the phagocytic activity of circulating neutrophils and monocytes were determined by the degree of ingestion of inactivated and FITC-labeled staphylococci using flow cytometry. The number of ROS-generating cells and the intensity of ROS production by phagocytes were determined by flow cytometry using 2',7'-dichlorodihydrofluorescein diacetate. RESULTS: The growth of both variants of Guerin carcinoma caused a statistically significant decrease in the intensity of neutrophil phagocytosis by more than 47% with a tendency to the reduction of the intensity of phagocytosis by monocytes. The phagocytic activity of circulating neutrophils and monocytes did not differ significantly between the groups of animals with the original and Cpt-resistant variant of Guerin carcinoma. In contrast, the intensity of ROS generation by both monocytes and neutrophils in the peripheral blood of animals with Cpt-resistant tumor increased by more than 86% as compared to original carcinoma-bearing rats. CONCLUSION: This study provides evidence that the intensity of ROS production by circulating monocytes and neutrophils may reflect the degree of tumor sensitivity to Cpt. Increased intensity of ROS production could serve as a pretreatment predictor of the formation of tumor drug resistance.

Tumor microenvironment changes tumor cell sensitivity to action of energy metabolism modifiers.

BACKGROUND: Taking into account differences in the bioenergetics between malignant and normal cells a search of antitumor drugs among the modifiers of tumor metabolism has a reasonable excuse. Earlier it was found that the cytotoxic/cytostatic action of sodium dichloroacetate (DCA) against Lewis lung carcinoma (LLC) cells in vitro was enhanced in the case of its combination with metformin (MTF). AIM: To study the antitumor action of DCA in combination with MTF against LLC in vivo. MATERIALS AND METHODS: LLC/R9, a low metastatic variant of LLC cells, was used. LLC/R9 bearing mice were treated with MTF (at a total dose 0.15 g/kg b.w.) alone or in combination with DCA (at a total dose of 0.75 g/kg b.w.). LLC/R9 growth kinetics and the primary tumor growth and metastasis indices on the 23rd day after tumor cell inoculation were evaluated by routine procedures. The state of the electron transport chain of mitochondria in tumor cells was studied using electron paramagnetic resonance. The content of lactate and glucose in blood plasma from mice was measured by enzymatic methods using biochemical analyzer. The number of tumor-associated macrophages (TAMs) and their distribution by M1/M2 phenotype were estimated by flow cytometry using antibodies against CD68 and CD206. RESULTS: In LLC/R9-bearing mice treated with DCA in combination with MTF, tumor growth and metastasis indices, as well as circulating glucose and lactate levels were not significantly different from those in the control group. The level of nitrosylation of non-heme and heme proteins and the content of iron-sulfur centers in the mitochondria of tumor cells in LLC/R9-bearing mice administered with DCA in combination with MTF did not also differ from the corresponding indices in control. Instead, in tumors treated with MTF alone and in combination with DCA the total CD68+ TAMs count was almost 27% (p < 0.05) and 43% lower (p < 0.05) correspondingly than that in control, but this decrease was not accompanied by redistribution of CD68+/CD206+ and CD68+/D206- subsets. CONCLUSION: DCA in combination with MTF, at least in doses applied, did not affect LLC/R9 growth and metastasis in vivo. The complete absence of an antitumor effect of DCA in combination with MTF was simultaneously associated with the absence of significant changes in the functional state of electron transport chain of mitochondria in tumor cells, circulating glucose and lactate levels, and the decrease of the TAMs amount in tumors. It suggests that the antitumor activity of DCA and MTF could be determined by both their local effects within a tumor and their multiple systemic impacts.

Influence of metformin, sodium dichloroacetate and their combination on the hematological and biochemical blood parameters of rats with gliomas C6.

BACKGROUND: The efficacy of antimetabolic therapy of malignant neoplasms could not be explained solely by the direct mechanisms of action of such energy metabolism inhibitors as sodium dichloroacetate (DCA) and metformin (MTF). The indirect effects of DCA and MTF on the organs and tissues, which could play significant role in the antitumor activity of these agents, have not been thoroughly explored. AIM: To investigate the effect of MTF, DCA and their combination on the survival of rats with C6 glioma and major haematological and biochemical blood parameters. MATERIALS AND METHODS: DCA and MTF were administered orally to inbred female rats for 11 days starting from the second day after tumor cell transplantation at a total dose of 1.1 and 2.6 g/kg, respectively. When combined treatment was used, MTF was administered 3 hours after the administration of DCA. The content of lactate and pyruvate in blood plasma was determined on the ChemWell® 2910 (Combi) automatic analyzer. Blood parameters were determined using the Particle Counter PCE-210 automatic hematology analyzer. RESULTS: The administration of DCA did not significantly affect the life span of rats with C6 glioma. Duration of life of rats, which were administered with MTF only, was significantly higher (by 19.1%, p < 0.01). Combined administration of DCA + MTF prolonged life span of animals with glioma by 50% (p < 0.001). The positive result of antitumor activity of MTF alone and in combination with DCA correlated with a decrease in the mean platelet volume/platelet count (MPV/PLT) ratio by 75.0% (p < 0.05) compared with tumor control. In addition, the expressed antitumor effect of combination therapy with DCA and MTF was associated with a decrease (p < 0.05) in glucose and lactate levels in blood plasma of rats with C6 glioma by 10% and 41.4%, respectively, compared to tumor control. Analysis of blood parameters showed that the growth of C6 glioma was accompanied by the development of leukopenia, anemia and thrombocytopenia. The introduction of DCA caused the correction of manifestations of anemia and leukopenia, but did not affect the level of platelets in the blood of animals with glioma. MTF alone and in combination with DCA positively influenced the number of white blood cells and caused complete thrombocytopenia correction, increasing platelet count by more than 200% (p < 0.001). CONCLUSION: The ability of MTF either used alone or in combination with DCA to influence the development of C6 glioma which is manifested in an increase in the lifespan of rats has been revealed. The most pronounced antitumor effect was recorded against the background of the combined use of these agents, which may be due to their ability to lower the levels of lactate and glucose in the blood of tumor-bearing rats. It is proved that MTF both in monotherapy and in combination with DCA provides correction of anemia and thrombocytopenia, which arise at the background of glioma C6 growth.

2-Deoxy-D-glucose enhances dichloroacetate antitumor action against Lewis lung carcinoma.

UNLABELLED: Aerobic glycolysis that supports high proliferation rate and survival of tumor cells in unfavorable conditions is among fundamental features of tumor metabolism. The search for active modulators of energetic metabolism capable of suppressing tumor growth and metastasis could result in higher effectiveness of anticancer therapy. AIM: To study antitumor and antimetastatic activity of the modulators of energetic metabolism dichloroacetate (DCA) and 2-deoxy-D-glucose (2DG) used in combination treatment of Lewis lung carcinoma (LLC). MATERIALS AND METHODS: As experimental tumor model, LLC/R9 variant was used. DCA and 2DG were administered per os to С57Bl/6 mice 5 times per week for 3 weeks at a total dose of 1.5 and 0.98 g/kg, respectively, as single agents or in combination starting from the following day after tumor cell transplantation. Growth of primary tumor and number and volume of lung metastases were registered. Lactate and pyruvate content was determined by enzymatic methods using lactate dehydrogenase. Electron paramagnetic resonance was used for analyzing the functional state of the components of mitochondrial respiratory chain. Engulfing activity and reactive oxygen species (ROS) production in tumor-associated CD14(+) cells was analyzed by flow cytometer with the use of FITC-labeled staphylococcus, and by spectrofluorometry with the use of 2.7-dichlorofluorescein diacetate, respectively. RESULTS: DCA administered as a single agent did not affect primary tumor growth but decreased the number and volume of lung metastases by 60% (p < 0.05) and 90% (p < 0.05), respectively. In mice treated with 2DG only, primary tumor volume as well as the number and volume of lung metastases were not affected. Combination treatment with DCA and 2DG resulted in the decrease of primary tumor volume, the number and volumes of lung metastases by 70; 46, and 90%, respectively (p < 0.05). High antitumor activity of DCA + 2DG was associated with 31% decrease (p < 0.05) of lactate content in tumor tissue and 120% increase (p < 0.01) of ROS production in CD14(+) cells recruited to the region of tumor growth. CONCLUSION: 2DG that possesses neither antitumor nor antimetastatic activity against LLC/R9 significantly enhanced antitumor activity of DCA with accompanying inhibition of glycolysis and increase of cytotoxic activity of CD14(+) cells infiltrating tumor tissue. Taking into account significant antimetastatic activity of DCA this substance could be considered as a promising antimetastatic agent.

Effectiveness of sodium dichloroacetate against glioma C6 depends on administration schedule and dosage.

BACKGROUND: Anticancer action of sodium dichloroacetate (DCA) could be related to its ability to activate oxidative phosphorylation leading to enhanced generation of reactive oxygen species and induction of apoptosis. On the other hand, activation of oxidative phosphorylation could promote tumor cell survival, in particular, via increased ATP synthesis. Such ambiguous effects of DCA could influence its anticancer effectiveness, depending on biological properties of a tumor, schedule of DCA administration and its dosage. The aim of the study was to analyze anticancer effect of DCA against glioma С6 in rats under conditions of different schedules of its administration and various dosages. MATERIALS AND METHODS: The study was carried out in Wistar rats with intracerebrally transplanted glioma С6 cells. Therapy with DCA was performed as follows: daily for 6 days starting from the second day after tumor cell transplantation (schedule І) or 7(th) day (schedule ІІ) at a dose of 1.0 g/kg, or daily for 13 days starting from the second day at doses of 1.0; 1.5 or 4.5 g/kg (schedule ІІІ). An influence of hypoxia on anticancer effect of DCA was studied using hypoxic chambers where oxygen content was maintained at a level of 12.5-13% for 3 h after DCA administration to glioma С6 bearing rats. The state of mitochondrial electron transport chain components in tumor cells was studied using electron paramagnetic resonance. RESULTS: It has been shown that therapy with DCA using schedule I resulted in 15% decrease of animals life span (LS; < 0.05), while the use of schedule II had no effect on this index. Prolonged administration of DCA (schedule ІІІ) resulted in significant antitumor effect and increased LS of rats by 25.5% (p < 0.05). Under hypoxic conditions, treatment with DCA resulted in a significant increase of animal LS by 15-22%. Dosage of DCA had a moderate effect of its anticancer action. Maximal effect, an increase of LS by 34.5% (p < 0.05) was detected at a dose of 1.5 g/kg. It has been shown that anticancer activity of DCA under all studied conditions is not related to its influence on a functional state of tumor cell mitochondria. CONCLUSION: Anticancer effect of DCA significantly depends on a schedule of its administration; being administered at equal total dose, but dependent on the schedule DCA could cause ambiguous effects varying from tumor growth stimulation to significant anticancer activity. Under hypoxic conditions, anticancer efficacy of DCA against glioma С6 is significantly enhanced.

[Systems biology approach to the functional role of enzymatic modification of bacterial ribosome].

In this work we describe methodology for studying the role of bacterial ribosome modification in the regulation of gene expression. Ribosomal components modification influences translation efficiencies of certain mRNAs. Proteome analysis allows us to identify cellular protein composition change caused by ribosome modification gene knockout. Particular stage of gene expression responsible for certain protein concentration change could be found using reporter constructs. After identification of mRNA species, whose translation is influenced by ribosome modification we can determine exact mRNA region responsible for the observed changes. The developed methodology can be applied for studying other translational control mechanisms.