Comparative study of anticancer efficacy of aсonitinecontaining agent BC1 against ascite and solid forms of Ehrlich’s carcinoma

Aim: To study anticancer activity of аconitine-containing agent BC1 in vivo. Methods: BC1 water solution was administered per os to mice bearing ascite or solid form of Ehrlich’s carcinoma. Anticancer effect of BC1 administered per os was evaluated by the indexes of tumor growth inhibition and average life span of experimental animals. Results: BC1 didn’t show anticancer activity in the case of ascite form of Ehrlich’s carcinoma. At the same time treatment with BC1 resulted in 77.3% growth inhibition of solid form of Ehrlich’s carcinoma (p < 0.05). Conclusion: BC1 is active in vivo against tumor with angiogenesis-dependent growth.Цель: изучить противоопухолевую активность аконитинсодержащего агента ВС1 in vivo.Методы: асцитную и солидную формы карциномы Эрлиха перевивали белым беспородным мышам внутрибрюшинно и подкожно соответственно. Противоопухолевую активность ВС1 оценивали по показателям ингибирования роста опухоли и средней продолжительности жизни животных. Результаты: ВС1 не оказывал противоопухолевого действия в отношении асцитной формы карциномы Эрлиха, но был активен против солидной формы, приводя к 77,3% ингибированию роста опухоли (p < 0,05). Выводы: ВС1 оказывает выраженное противоопухолевое действие в отношении злокачественных новообразований с ангиогенеззависимым ростом

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

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

Effect of dichloroacetate on Lewis lung carcinoma growth and metastasis

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% (р < 0.05) and apoptosis level was triply lower (р < 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% (р < 0.05) and 94% (р < 0.05) lower, respectively, than these indices in the control group. DCA treatment resulted in nearly 30% increase (р < 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 gmed = 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. Key Words: dichloroacetate, Lewis lung carcinoma, lactacidosis

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 7th 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. Key Words: paraneoplastic syndrome, Lewis lung carcinoma LLC/R9, enterosorption

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