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.

Oxamate, an inhibitor of lactate dehydrogenase, can stimulate M2 polarization of peritoneal macrophages in mice with Lewis lung carcinoma.

BACKGROUND: Inhibition of aerobic glycolysis of cancer cells is considered a promising therapeutic strategy for the treatment of neoplasms. Some inhibitors of energy metabolism can affect not only tumor cells but also the functional polarization of tumor-associated macrophages, which may either enhance the antitumor effect of such agents or impair their antitumor efficacy. AIM: To investigate the effect of oxamate, a lactate dehydrogenase (LDH) inhibitor, on the polarization of peritoneal macrophages (PMP) in both intact mice and mice with transplanted Lewis lung carcinoma (LLC). MATERIALS AND METHODS: The low-metastatic LLC variant, LLC/R9, was transplanted to female C57Bl/6 mice. Sodium oxamate was used as the test agent at concentrations of 0.02, 0.2, and 2 mg/ml. Macrophage polarization in tumor-bearing mice was estimated on day 23 after tumor transplantation by assessing nitric oxide (NO) production and arginase activity as functional indices of PMPs polarization. RESULTS: Oxamate can affect the functional polarization of PMPs in both intact mice and animals with transplanted LLC/R9. Oxamate in all studied concentrations changed the markers of PMPs polarization in intact mice (decreasing NO levels and activating arginase activity) that indicated the stimulation of M2 polarization. In tumor-bearing animals, stimulation of M2 polarization is observed at low concentrations of oxamate (0.02 mg/ml), but its high concentrations (2.0 mg/ml) causes M1 polarization, which is characterized by three-fold increase in the level of NO and a decrease in the level of arginase activity. CONCLUSION: Oxamate, an inhibitor of LDH, can stimulate M2 polarization of peritoneal macrophages of mice bearing LLC in a dose-dependent manner.

Role of tumor/endothelial cell interactions in tumor growth and metastasis.

BACKGROUND: It is known that interactions between tumor and endothelial cells have a significant influence on the growth and metastasis of malignant tumors. AIM: To study the reciprocal effect of Lewis lung carcinoma (LLC) and endothelial cells on the growth rate of each other upon their co-cultivation in vitro and to assess the contribution of such tumor/endothelial cell crosstalk to in vivo LLC growth and metastasis. MATERIALS AND METHODS: Two variants of Lewis lung carcinoma cells, high-metastatic (LLC) and low-metastatic (LLC/R9), and murine aorta endothelial cell line (MAEC) were used. Kinetics of tumor cell growth in vitro and in vivo, electrokinetic properties of tumor cells and their adhesion to endothelial monolayer, and the number of tumor and endothelial viable cells after 1-day contact or non-contact co-cultivation were estimated. RESULTS: LLC/R9 had significantly higher growth rate in vivo (as opposed to in vitro) than LLC. However, the number and volume of lung metastatic lesions in LLC/R9-bearing mice were 4.5-fold (p < 0.05) and 3.6-fold lower (p < 0.05), respectively, compared to those in LLC-bearing mice. Non-contact co-cultivation of LLC/R9 + MAEC caused more than a 34% (p < 0.05) LLC/R9-induced increase in the number of MAEC and a 60% (p < 0.05) MAEC-induced increase in the number of LLC/R9 cells as compared to those of corresponding controls (cells cultured alone). In contrast, in the case of LLC + MAEC, both the number of LLC and MAEC cells after their non-contact co-cultivation and cultivation alone did not differ significantly. Contact co-cultivation LLC+MAEC (in contrast to LLC/R9+MAEC) caused more than a 50% (p < 0.01) LLC-induced decrease in the number of MAEC and a 50% decrease (p < 0.05) MAEC-induced in the number of LLC cells as compared to the corresponding controls. Both tumor cell variants showed a bimodal distribution of cells by ζ-potential, but in the case of LLC there was observed a shift towards high values due to 52% of cells with a surface charge density > 10 C/m2, while in the case of LLC/R9 such a subpopulation was absent and 19% of cells had a surface charge < 5 C/m2. The number of LLC cells that adhered to the monolayer of endothelial cells was by 65% (p < 0.05) higher than that of LLC/R9 cells. CONCLUSION: Obtained data demonstrated that the tumor/endothelial cell relationships might reflect the features of tumor growth and metastasis of a malignant tumor.

The effect of 2D tungsten disulfide nanoparticles on Lewis lung carcinoma cells in vitro.

The unique physicochemical properties of modern two-dimensional (2D) nanomaterials with graphene-like structures make them promising candidates for biology and medicine purposes. In this article, we investigate the influence of the two-dimensional tungsten disulfide (2D WS2) water suspension nanoparticles obtained by an improved mechanochemical method from powdered WS2 on morphological and structural characteristics of Lewis lung carcinoma cells using FT-IR, Raman spectroscopy, and confocal microscopy. The characterization of the 2D WS2 nanoparticles by different physical methods is given also. We have highlighted that 2D WS2 does not exert cytotoxic activity in the case of 1 day incubation with tumor cells. Prolongation of the incubation period up to 2 days has caused a statistically significant (p < 0.05) concentration-dependent decrease of the number of viable cells by more than 30% with the maximum cytotoxic effect at concentrations of 2D WS2 close to 2 µg ml-1. In the Raman spectra of 2D WS2 treated cells the bands centered at 354 cm-1 and 419 cm-1, which are assigned to characteristics and modes of WS2 nanoparticles were observed. The obtained data indicate, that the cytotoxic effect of 2D WS2 on tumor cells in the case of long-term incubation is realized particularly through the ability of 2D WS2 to enter tumor cells and/or accumulate on their surface, which gives a rationale to conduct further studies of their antitumor efficacy in vitro and in vivo when combined with chemotherapeutic drugs.

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.

Metformin enhances cytotoxic action of dichloroacetate against Lewis lung carcinoma cells in vitro.

Tumor cell metabolism is considered one of the hallmarks of cancer. This concept is exploited in the development of new ways of anticancer therapy based on the use of substances capable of changing drastically bioenergetic metabolism of tumor cells. Among them, sodium dichloroace-tate (DCA), an inhibitor of pyruvate dehydrogenase kinase, and metformin (MTF), an antidiabetic hypoglycemic drug, an inhibitor of the mitochondrial respiratory chain (complex I), both have been long used in clinical non-oncological practice, and presently are considered promising candidates in oncology. AIM: To study the capability of MTF to enhance the antitumor action of DCA against Lewis lung carcinoma cells in vitro. MATERIALS AND METHODS: LLC/R9, a low metastatic variant of Lewis lung carcinoma cells, was used. Effects of 30 mM DCA in combination with 2 mM MTF on cell survival, cell cycle distribution, apoptosis, mitochondrial potential, intracellular ATP level, glucose consumption, and lactate production rates were determined in vitro. RESULTS: MTF was shown to enhance the cytotoxic/cytostatic action of DCA against LLC/R9 cells in vitro. Treatment of LLC/R9 cells with 30 mM DCA in combination with 2 mM MTF resulted in a 39% decrease in the number of viable cells (p < 0.05), a 2.8-fold increase of the number of dead cells (p < 0.05), a near 2-fold decrease in the proportion of cells at the S-phase (p < 0.05), a 4-fold increase in the apoptosis (p < 0.05) and significant reduction (p < 0.05) of the mitochondrial membrane potential of tumor cells as compared to corresponding values in control. DCA alone reduced glucose consumption and lactate production rates by more than 26% (p < 0.05) and 34% (p < 0.05), respectively, whereas MTF counteracted these effects. Nevertheless, in the cells treated with both DCA and DCA in combination with MTF, the intracellular adenosine triphosphate increased by 33-35% compared with that in the control (p < 0.05). CONCLUSION: MTF enhanced the cytotoxic/cytostatic action of DCA against LLC/R9 cells in vitro, which points on their possible synergistic antitumor action in vivo.

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.

Time-dependent cytotoxicity of dichloroacetate and metformin against Lewis lung carcinoma.

The use of inhibitors of energy metabolism of malignant cells is a new promising trend in the treatment of cancer patients, based on one of the unique features of the malignant cell, namely the dominance of glycolysis over oxidative phosphorylation, even in the presence of oxygen, the so-called Warburg effect. AIM: To study time-dependent cytotoxicity of sodium dichloroacetate (DCA) and metformin (MTF) against metastatic tumor cells and action of these agents on tumor cell migration. MATERIALS AND METHODS: In the study low metastatic LLC/R9 variant of Lewis lung carcinoma was used. The number of living cells in the cytotoxic test was evaluated using sulforhodamine B after 1, 2 and 3 days of cell incubation in vitro. The parameters of the sensitivity of tumor cells to the action of DCA and MTF in vitro were calculated using nonlinear and linear regression of experimental data. The effect of DCA and MTF on cellular motility in vitro was evaluated using a Boyden chamber by calculation of the number of cells that migrated to the bottom side of the filter within 3 days of incubation. The statistical analysis of the data was carried out with the use of descriptive methods, Student's t-criterion, nonlinear, and linear regression analysis. RESULTS: IC50 of DCA was found to be equal to 50.8 ± 7.6 mM at the first day of incubation with LLC/R9 cells and decreased by 1.9 (p < 0.05) and 2.1 (p < 0.05) times at the 2nd and 3rd days, respectively. Despite the almost identical ІC50 at the 2nd and 3rd days, an increase in the incubation period of cells with DCA for up to 3 days increased the C0 parameter, which reflects the maximum concentration of the agent that does not exhibit cytotoxic effects, by 93% (p < 0.05) compared to this at the 2nd day (16.2 ± 1.4 mM vs 8.4 ± 1.0 mM, correspondently). Unlike DCA, the LLC/R9 cell population was not homogeneous by the sensitivity to the action of MTF; at least at the 3rd day, an appearance of MTF-resistant subpopulation was observed, accounting for 35% of all cells. IC50 of MTF was equal to 12.1 ± 0.6 mM, and unlike DCA, this index progressively decreased at the 2nd and 3rd days by 1.4 (p < 0.05) and 9.3 times (p < 0.05) respectively. Action of DCA at a concentration of 25 mM alone and in combination with MTF at the concentrations of 0.1 mM and 0.7 mM resulted in an increase in cell migration by 65% (p < 0.05), 63% (p < 0.05) and 78.5% (p < 0.05), respectively. There was no significant effect of MTF on the tumor cell migration. CONCLUSIONS: The sensitivity of the metastatic Lewis lung carcinoma cells to the action of the modifiers of the energy metabolism increased significantly with an increase in the incubation period, apparently, primarily due to the shortage of nutrient substrates and, in particular, glucose, indicating the relevance of their combined use as well as with other agents, which promote the deficiency of glucose in the tumor microenvironment.

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.

Cytotoxic activity of metformin in vitro does not correlate with its antitumor action in vivo.

It is known that metformin is a hypoglycemic drug used to treat type II diabetes mellitus. Recently active studies of its antitumor activity in relation to different types of malignant cells are conducted. AIM: To determine the relationship between cytotoxic activity of metformin in vitro and its antitumor activity in vivo. MATERIALS AND METHODS: The rat C6 glioma cell line and mouse Lewis lung carcinoma cells (LLC) were used in this work. The number of living cells in the cytotoxic test was evaluated using sulforhodamine B. Parameters of tumor cell susceptibility to metformin activity in vitro were calculated using nonlinear and linear regression of experimental data. The antitumor action of metformin in vivo was evaluated routinely by the extension of survival time (ST) (in rats with intracerebral C6 glioma) and its effect on the volume of the primary tumor, the number and volume of metastases (in mice with LLC). RESULTS: In cultured LLC cells in vitro, the proportions of metformin-resistant (A1, %) and metformin-sensitive (A2, %) subpopulations were 10.0 ± 2.2% and 92.0 ± 3.5%, respectively, in terms of the total number of living cells. Parameter t, which characterizes the sensitivity of cancer cells to metformin action (the lower is the value of this parameter the higher is sensitivity of cells to metformin cytotoxicity), for metformin-resistant and metformin-sensitive subpopulations was: t1(mM) = ∞ and t2(mM) = 2.9 ± 0.3, correspondingly. For metformin-sensitive subpopulation of LLC cells IC50 (mM) = 2.42 ± 0.34. The volume of the primary tumor, the amount and volume of metastases in mice receiving metformin at a dose of Dmin (0.15 g/kg) and Dmax (0.3 g/kg) values did not significantly differ from those in the control. However, in the case of Dmin, there was a tendency to increased volume of the primary tumor, in the case of Dmax, there was a tendency to increased volume of metastases. The analogical parameters (A1, A2, b1, b2, IC50 (1), IC50 (2)) characterizing cell sensitivity to the action of metformin in vitro were obtained in relation to C6 glioma cells. In metformin-resistant subpopulation, these parameters were: A1 (%) = 72.3 ± 1.4; b1 (%/mM) = 0.43 ± 0.005; IC50 (1) (mM) = 84.1 ± 2.4. For metformin-sensitive subpopulation, these parameters were: A2 (%) = 30.8 ± 2.3; b2 (%/mM) = 2.87 ± 0.4; IC50 (2) (mM) = 5.37 ± 0.45. In vivo, a statistically significant anti-glioma effect of metformin was observed: at a dose of Dmax (5.2 g/kg) administration of this preparation resulted in a prolongation of the mean ST of tumor-bearing rats by 23% (p < 0.05) compared with that in the control. CONCLUSIONS: We found no correlation between the cytotoxic/cytostatic action of metformin in vitro and its antitumor activity in vivo on the two types of tumor cells; these results indicate a significant contribution of the tumor microenvironment to the implementation of the antitumor activity of the drug.

Impact of lactic acidosis on the survival of Lewis lung carcinoma cells.

AIM: To investigate the effect of lactic acidosis on the survival of Lewis lung carcinoma cells under glucose-deprived conditions. MATERIALS AND METHODS: LLC/R9 variant of Lewis lung carcinoma cells was cultured in glucose deficit or complete culture medium. Conditions of lactic acidosis, lactosis, and acidosis were generated in glucose deficit medium. Cell survival, cell cycle, apoptosis, autophagy, and the content of glucose, lactate, vascular endothelial growth factor in the culture medium were determined. Light and fluorescent microscopy, flow cytometry, spectrophotometry, and ELISA were used. RESULTS: It has been found that 24 h incubation of tumor cells under lactic acidosis caused (i) the reduction of the number of living cells by 33% (p < 0.05) and 56% (p < 0.05); (ii) the inhibition of apoptosis by 4.3-fold (p < 0.05) and 3.3-fold (p < 0.05); (iii) the reduction of the rate of glucose consumption by 2-fold (p < 0.05) and 2.5-fold (p < 0.05); (iv) an increase of lactate production more than twice (p < 0.05) and 1.6-fold (p < 0.05) compared with these indexes under conditions of glucose deficiency or complete glucose-containing medium, respectively. However, on the second day of culture under lactic acidosis, the number of viable cells reached a maximum, in contrast to culture in the complete medium. The number of live cells on the seventh day of culture under lactic acidosis exceeded almost 2-3 times (p < 0.05) that in the culture under conditions of the glucose deprivation or in complete medium. On the third day under lactic acidosis the autophagolysosomes count was 54% (p < 0.05) lower that that under glucose deficit. CONCLUSIONS: Lactic acidosis promoted the survival and proliferation of Lewis lung carcinoma cells by energy system reprogramming directed on inhibition of apoptosis and autophagy, a significant decrease in the rate of glucose utilization and activation of glutaminolysis and, consequently, increase of the lactate production rate. Inhibition of lactate production by tumor cells may be considered as a promising approach for more efficient antiangiogenic treatment of cancer.

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.

Effect of enterosorption on paraneoplastic syndrome manifestations in mice with highly angiogenic variant of Lewis lung carcinoma.

AIM: To study the correcting effects of microgranulated HSGD enterosorbent on hematological, morphological and biochemical indices of paraneoplastic syndrome in mice with highly angiogenic variant of Lewis lung carcinoma LLC/R9. METHODS: The study was performed on male С57/ВL6 mice with transplanted LLC/R9. Enterosorbent HSGD was administered daily at a dose of 0.625 g/kg for 2 weeks starting from 7(th) day after tumor cell transplantation. When enterosorption was completed, an analysis of peripheral blood, biochemical indices and morphological structure of tumor, lung, liver, spleen and thymus was carried out by standard methods. RESULTS: It has been shown that administration of enterosorbent did not affect LLC/R9 growth but resulted in nearly two fold decrease of the volume of lung metastases (p < 0.05). Erythrocyte number and hemoglobin level were higher by 30.0% (p < 0.05) and 23.3% (p < 0.05), respectively, in mice treated with enterosorbents as compared to untreated animals. In addition sorbent treatment completely normalized the thrombocyte index resulting in elevation of platelet number by 54.5% (p < 0.01) up to their level in intact mice. The morphological examination of liver and biochemical analysis of peripheral blood evidenced on significant positive correcting effect of enterosorption on histological structure of this organ and its functional activity. Normalization of total proteins and serum albumin level as well as significant decrease of total lipid concentration by 29% (p < 0.01) in blood of treated mice were observed. CONCLUSION: Positive influence of microgranulated carbon sorbent on some hematological, morphological and biochemical indices of tumor associated symptoms in LLC/R9-bearing mice denotes that enterosorption-based therapy can be considered as a prospective treatment for correction of some paraneoplastic syndrome signs in cancer patients.

Aconitine-containing agent enhances antitumor activity of dichloroacetate against Ehrlich carcinoma.

UNLABELLED: Significant variability of anticancer efficacy of dichloroacetate (DCA) stimulated an active search for the agents capable to enhance it antitumor action. Therefore, the aim of this work is the study of capability of aconitine-containing antiangiogenic agent BC1 to enhance anticancer activity of DCA against Ehrlich carcinoma. MATERIALS AND METHODS: DCA (total dose was 1.3 g/kg of b.w.) and BC1 (total dose was 0.9 mg/kg of b.w.) were administered per os starting from the 2(nd) and 3(rd) days, respectively (8 admini-strations for each agent). Antitumor efficacy of agents was estimated. Lactate level, LDH activity and the state of mitochondrial electron transport chain in tumor cells as well as phagocytic activity and reactive oxygen species (ROS) production of tumor-associated macrophages (TAM) were studied. RESULTS: Combined administration of DCA and ВС1 resulted in 89.8% tumor growth inhibition (p < 0.001), what is by 22.5% (p < 0.05) higher that that of DCA alone. This combined treatment was accompanied with a decrease of lactate level in tumor tissue by 30% (p < 0.05) and significant elevation of LDH activity by 70% (p < 0.01). Increased level of NO-Fe-S clusters and 2-fold reduction of Fe-S cluster content were revealed in tumor tissue of mice after DCA and BC1 administration. It was shown that combined therapy did not effect TAM quantity and their phagocytic activity but stimulated ROS production by TAMs by 78% (p < 0.05) compared to this index in control animals. CONCLUSION: Antiangiogenic agent ВС1 in combination with DCA considerably enhances antitumor activity of DCA via significant decrease of Fe-S-containing protein level resulted from substantial elevation of nitrosylation of these proteins.

Effect of dichloroacetate on Lewis lung carcinoma growth and metastasis.

UNLABELLED: A hallmark of malignancy is excessive tumor glycolysis, even in the presence of oxygen, which causes lactacidosis in the tumor microenvironment and favors tumor cell proliferation and survival. For this reason antimetabolic agents which target tumor cell metabolism are being researched extensively as promising anticancer drugs. AIM: To study the effect of lactacidosis on survival of Lewis lung carcinoma (LLC) cells at the conditions of nutritional substrate deficiency in vitro and evaluate antitumor and antimetastatic activity against LLC/R9 in vivo. MATERIALS AND METHODS: LLC variant LLC/R9 was used as experimental tumor model. Tumor cell viability was determined using trypan blue staining. Apoptosis level was counted with the use of Hoechst 33258 dye. Lactate content in the tumor tissue was evaluated by enzyme method with the use of lactate dehydrogenase. Reactive oxygen species was determined using 2.7-dichlorofluorescein diacetate. Effects of dichloroacetate (DCA) on the growth and metastasis of LLC/R9 were analyzed by routine procedures. Evaluation of DCA effect toward electron-transport chain (ETC) components was performed using EPR. RESULTS: It has been shown that at the conditions of lactacidosis and glucose deficiency, LLC/R9 cell viability in vitro was higher by 30% (p < 0.05) and apoptosis level was triply lower (p < 0.05) than these indices at the conditions of glucose deficiency only. In mice with transplanted LLC/R9 tumors treated for 3 weeks per os with DCA at the total dose of 1.5 g/kg of body weight starting from the next day after tumor transplantation, the primary tumor volume was just by 30% lower than that in control group. At the same time, the number and volume of lung metastases in animals treated with DCA were by 59% (p < 0.05) and 94% (p < 0.05) lower, respectively, than these indices in the control group. DCA treatment resulted in nearly 30% increase (p < 0.05) of lactate content in tumor tissue compared to that in the control, but did not affect significantly the levels of heme iron complexes with NO (at g med = 2.007) in mitochondrial ETC proteins and Fe-S cluster proteins (at g = 1.94) in tumor cells. CONCLUSIONS: It has been shown that lactacidosis significantly promoted LLC/R9 cell survival at the conditions of glucose deficiency in vitro. If LLC/R9 developed in vivo, DCA as the compound with antilactacidosis activity did not suppress significantly the primary tumor growth but exerted significant antimetastatic activity.

Hypoxia enhances antitumor activity of dichloroacetate.

UNLABELLED: It is known that glycolysis contributes to the survival of tumor cells by providing them with energetic and plastic substrates. Dichloroacetate (DCA) as inhibitor of kinase pyruvate dehydrogenase shifts balance of energy metabolism of tumor cells from aerobic glycolysis towards oxidative phosphorylation. The aim of the study was to investigate cytostatic/cytotoxic effect of DCA on glioma C6 cells at the conditions of different oxygenation of the cell incubation medium. MATERIALS AND METHODS: DCA action on glioma C6 cells was investigated upon the conditions of normoxia, hypoxia (1% of oxygen) and hyperoxia (30% and 95% of oxygen) in vitro. The number and viability of tumor cells were assessed using trypan blue dye-exclusion test. Apoptosis was determined using dye Hoechst 33258. Lactate production by tumor cells was determined by enzymatic method using lactate dehydrogenase. Cell cycle distribution was studied using flow cytometry. Reactive oxygen species (ROS) content was evaluated using 2´,7´-dichlorofluorescein diacetate. RESULTS: By the data of in vitro cytotoxicity, upon hypoxia IC50 value of DCA was three times lower (p < 0.05) than that upon normoxic conditions (18.2 ± 3.9 mM vs. 51.2 ± 8.1 mM). Hypoxia itself enhanced the ROS production in glioma cells by 113.5% (p < 0.05) that correlated with increase of apoptosis by 292% (p < 0.05). In hypoxic glioma C6 cells DCA did not significantly influence the ROS production, but decreased hypoxia-induced apoptosis by 3.5-6.5 times (p < 0.05) and significantly increased cell death rates via necrosis (p < 0.05). In contrast to hypoxia, upon the conditions of hyperoxia IC50 values for DCA did not differ from the corre-sponding values upon the normoxia conditions and at 30% and 95%oxygen content were equal to 35.8 ± 7.2 mM and 42.3 ± 5.1 mM respectively. CONCLUSION: According to the obtained results, hypoxia enhances cytostatic/cytotoxic effects of DCA in glioma C6 cells via high level of DCA-induced necrosis of tumor cells and hypoxia-induced ROS hyperproduction.

Dynamics of changes of antioxidant system indexes during the growth of two Lewis lung carcinoma variants.

AIM: To study an intensity of prooxidant processes and activity of antioxidant enzymes in tumor tissue of two Lewis lung carcinoma variants (LLC and LLC/R9) differing by their proliferative, metastatic, and angiogenic potential (LLC/R9 as compared to LLC is characterized by lower metastatic activity, higher proliferative and angiogenic potential). MATERIALS AND METHODS: The in vitro study was carried out using cultured LLC and LLC/R9 cell lines, and in vivo on 50 female С57/BL6 mice. The indexes of prooxidant processes and activity of antioxidant enzymes have been studied using the methods of experimental oncology, optical spectroscopy, fluorescent spectroscopy, electron paramagnetic resonance, statistical analysis. RESULTS: There has been determined the coherence of results on 1.5 fold higher (p < 0.01) level of spontaneous generation of reactive oxygen species (ROS) by LLC cells in vitro compared to LLC/R9 cells, and twice (p < 0.05) higher content of secondary products of lipid peroxidation at 14(th) and 17(th) day of tumor growth in LLC compared to that in LLC/R9. It has been shown that deficiency of nutrient substrates determines an increase (p < 0.01) of ROS production in LLC/R9 cells what is in congruence with the data on accumulation of nitrosyl complexes of heme iron in mitochondria of LLC/R9 during tumor growth. Activity of superoxide dismutase during tumor growth has altered in unmonotonous way: starting from 14(th) day of growth it sharply increased by 147% (17(th) day, LLC) and 217% (20(th) day, LLC/R9), and then decreased to the level registered at 14(th) day. Progressive decrease of activity of glutathione peroxidase (GP) and glutathione-S-transferase (GST) during LLC growth has been accompanied with the decrease of the level of reduced glutathione (GSH) by 70% (p < 0.05). In the case of LLC/R9 the decrease of GP activity at initial stages of tumor growth correlated with significant increase of GSH level in the tumor--by 250% (p < 0.01). It has been shown that in LLC/R9 tumors (unlike to LLC), GSH utilization is mostly provided by GST, its significantly higher activity has been detected in LLC/R9 tumors compared to LLC. CONCLUSION: We have revealed a number of peculiarities of antioxidant system functioning in LLC and LLC/R9 tumors and have shown a relation between an activity of antioxidant system and some biological properties of studied tumor variants.

Analysis of growth kinetics and proliferative heterogeneity of Lewis lung carcinoma cells growing as unfed culture.

AIM: To analyze the growth kinetics and proliferative heterogeneity of Lewis lung carcinoma (LLC) cells during their growth in monolayer for 5 days without replacement of culture medium (unfed culture). METHODS: Cell biology methods, sandwich enzyme-linked immunosorbent assay for vascular endothelial growth factor (VEGF) detection (ELISA), enzymatic glucose-oxidase method for glucose measurements, mathematical modeling. RESULTS: Created mathematical model showed good fit to experimental data; that allowed to determine kinetic (model) parameters of LLC cells and predict the changes in number of proliferating and quiescent cells (proliferative heterogeneity) during their growth. It was shown that growth kinetics of viable LLC cells possesses non-monotonous character - during first three days of growth the number of cells raised exponentially, with following decrease after the maximal level was achieved. At the same time the decrease of number of viable cells/increase of number of dead cells has been observed upon complete depletion of culture medium by glucose content. Glucose dependence of cell transition rate from proliferation to resting state predicted by mathematical model possessed a pronounced two-phase character. At a wide range of relatively high glucose concentrations (> 1.0 mg/ml) the transition rate was close to zero. At concentrations lower than 0.7 mg/ml, the rate of transition swiftly increased resulting in sharp change in cellular composition. At an interval from 70 to 90 h, practically all proliferating cells transited to a resting state. The rate of quiescent cell death was relatively low, and this was in part caused by too low level of glucose consumption compared to proliferating cells. It was shown that during LLC cells growth VEGF production rate decreased monotonously in spite of the fact that the level of VEGF in incubation medium increased monotonously. Observed monotonous decrease of VEGF production rate could not be explained by VEGF degradation in incubation medium (our results displayed the stability of VEGF molecule during investigations). CONCLUSIONS: Weak dependence of cell transition rate from proliferating to resting state from glucose level (> 0.7 mg/ml) and low rate of cell death provided slow decrease of the pool of quiescent cells in the population, thus significantly increasing their chance to survive upon nutritional deficiency.

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.