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

Аim: 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.Цель: провести анализ кинетики роста и пролиферативной гетерогенности клеток карциномы легкого Льюис (LLC) при их росте в монослое на протяжении 5 сут без замены культуральной среды (unfed culture). Методы: иммуноферментный метод определения уровня продукции VEGF опухолевыми клетками; глюкозооксидазный метод определения уровня глюкозы в среде инкубации; математическое моделирование. Результаты: предложенная математическая модель хорошо описывает экспериментальные данные, что позволяет определить кинетические параметры роста клеток LLC и предсказать изменения количества пролиферирующих и покоящихся клеток (пролиферативная гетерогенность) в процессе их роста. Кинетика роста клеток LLC носит немонотонный характер — в течение первых 3 сут их роста количество клеток экспоненциально увеличивается и далее, по достижении максимальной плотности, снижается. В то же время уменьшение количества живых клеток/увеличение количества мертвых клеток тесно связано с истощением глюкозы в среде инкубации. Зависимость скорости перехода клеток из состояния пролиферации в состояние покоя от содержания глюкозы в среде инкубации имеет выраженный двухфазный характер. В широком диапазоне относительно высоких концентраций глюкозы (> 1,0 мг/ мл) скорость перехода близка к нулю. При концентрациях < 0,7 мг/мл скорость перехода стремительно возрастает, что приводит к резким изменениям клеточного состава клеточной популяции. В интервале от 70 до 90 ч практически все пролиферирующие клетки переходят в состояние покоя. Скорость гибели покоящихся клеток относительно низкая, что, в частности, связано с низким уровнем потребления глюкозы этими клетками по сравнению с таковым пролиферирующими клетками. Установлено, что в процессе роста скорость продукции VEGF клетками LLC монотонно снижается, несмотря на то, что в среде инкубации его уровень монотонно нарастает. Выводы: низкая зависимость скорости перехода клеток LLC из состояния пролиферации в состояние покоя от уровня глюкозы (> 0,7 мг/мл) в среде инкубации, а также низкий уровень их гибели обусловливает медленное снижение пула покоящихся клеток в популяции, что значительно повышает их шансы выжить в условиях дефицита питательных субстратов

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

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 и 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 (tlag = 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.Цель: изучить взаимосвязь между ангиогенным потенциалом опухоли и распространенностью опухолевого процесса на модели карциномы Льюис разной степени резистентности к действию cis-DDP. Методы: в работе использовали карциному легкого Льюис (LLC), а также два ее варианта (LLC-9 и LLC-19), полученные на 9-м и 19-м этапе экспериментальной прогрессии LLC в направлении формирования резистентности к cis-DDP in vivo. Ангиогенный потенциал LLC, LLC-9 и LLC-19 оценивали иммуноферментным методом по уровню продукции VEGF первичными культурами in vitro, а также по динамике изменений уровня циркулирующего VEGF в процессе роста LLC in vivo. Результаты: прогрессивное снижение чувствительности LLC-9 и LLC-19 к действию cis-DDP, подтверждавееся in vitro ( 50 = 0,0077 ± 0,0005 мг/мл для LLC-9 и 0,0156 ± 0,0008 мг/мл для LLC-19 против 0,004 ± 0,0003 мг/мл для LLC, p < 0,05) и in vivo (индекс торможения цитостатиком роста LLC-9 и LLC-19 cоставлял 26 и 3% соответственно против 46% для LLC; метастазирования — 46 и 11% против 65% для LLC, p < 0,05), сопровождалось значительными изменениями ее ангиогенного потенциала. Уровень продукции in vitro n vitro первичной культурой LLC-9 был в 1,5 раза выше (p < 0,05) по сравнению с таковым первичной культурой LLC, тогда как уровень продукции VEGF первичной культурой LLC-19 практически не отличался от такового для LLC. У ровень циркулирующего VEGF резко повышался уже на ранних этапах роста LLC-9 и LLC-19 in vivo, тогда как у мышей с LLC динамика изменения его уровня характеризовалась длительным латентным периодом (tlag = 17,0 ± 0,3 сут). Характер изменений уровня циркулирующего VEGF в процессе роста LLC (p < 0,001) коррелировал с количеством метастазов; у животных с LLC-9 — с объемом опухоли (p < 0,01) и количеством (p < 0,05) метастазов. Несмотря на то, что максимальный уровень циркулирующего VEGF у мышей с LLC-9 значительно (p < 0,05) выше, чем у мышей с LLC, максимальное количество метастазов в легких у мышей с LLC-9 достоверно (p < 0,05) меньше, чем у мышей с LLC. В отличие от LLC-9, в процессе роста LLC-19, сопровождавшегося повышением уровня метастатического поражения легких p < 0,05), уровень циркулирующего VEGF коррелировал как с объемом опухоли (p < 0.01), так и с исследованными показателями метастазирования (p 0.01). Выводы: прямую корреляционную взаимосвязь между уровнем циркулирующего VEGF и изученными показателями распространенности опухолевого процесса выявляют лишь для опухолей с высокой степенью лекарственной резистентности, тогда как повышение уровня циркулирующего VEGF в процессе роста опухоли можно рассматривать в качестве маркера имеющихся метастазов независимо от степени ее резистентности

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. Key Words: sodium dichloroacetate, metformin, C6 glioma, hematological indices, lactate, glucose

Hypoxia enhances antitumor activity of dichloroacetate

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 corre­sponding 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. Key Words: dichloroacetate, glioma C6, hypoxia, hyperoxia, reactive oxygen species, apoptosis

2-deoxy-D-glucose enhances dichloroacetate antitumor action against lewis lung carcinoma

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 СD14⁺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 (р < 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 СD14⁺ 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 СD14⁺cells infiltrating tumor tissue. Taking into account significant antimetastatic activity of DCA this substance could be considered as a promising antimetastatic agent

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 14th and 17th 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 14th day of growth it sharply increased by 147% (17th day, LLC) and 217% (20th day, LLC/R9), and then decreased to the level registered at 14th 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. Key Words: Lewis lung carcinoma variants, reactive oxygen species, lipid peroxidation, antioxidant enzymes, glutathione dependent enzymes

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

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 2nd and 3rd days, respectively (8 admini­strations 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. Key Words: Ehrlich carcinoma, dichloroacetate, aconitine-containing agent, tumor-associated macrophages, mitochondrial electron transport chain

Effectiveness of sodium dichloroacetate against glioma с6 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 7th 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; р < 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