The lipid content of cisplatin- and doxorubicin-resistant MCF-7 human breast cancer cells

Aim. To perform the comparative study both of qualitative and quantitative content of lipids in parental and drug resistant breast cancer cells. Materials and methods. Parental (MCF-7/S) and resistant to cisplatin (MCF-7/CP) and doxorubicin (MCF-7/Dox) human breast cancer cells were used in the study. Cholesterol, total lipids and phospholipids content were determined by means of thin-layer chromatography. Results. It was found that cholesterol as well as cholesterol ethers content are significantly higher but diacylglycerols, triacyl­glycerols content are significantly lower in resistant cell strains than in parental (sensitive) cells. Moreover the analysis of individual phospholipids showed the increase of sphingomyelin, phosphatidylserine, cardiolipin, phosphatidic acid and the decrease of phosphatidy­lethanolamine, phosphatidylcholine in MCF-7/CP and MCF-7/Dox cells. Conclusion. Obtained results allow to suggest that the lipid profile changes can mediate the modulation of membrane fluidity in drug resistant MCF-7 breast cancer cells

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

Gold nanoparticles synthesis and biological activity estimation in vitro and in vivo

The aim of the work was the synthesis of gold nanoparticles (GNP) of different sizes and the estimation of their biological activity in vitro and in vivo. Materials and Methods: Water dispersions of gold nanoparticles of different sizes have been synthesized by Davis method and characterized by laser-correlation spectroscopy and transmission electron microscopy methods. The GNP interaction with tumor cells has been visualized by confocal microscopy method. The enzyme activity was determined by standard biochemical methods. GNP distribution and content in organs and tissues have been determined via atomic-absorption spectrometry method; genotoxic influence has been estimated by “Comet-assay” method. Results: The GNP size-dependent accumulation in cultured U937 tumor cells and their ability to modulate U937 cell membrane Na+,K+-АТР-ase activity value has been revealed in vitro. Using in vivo model of Guerin carcinoma it has been shown that GNP possess high affinity to tumor cells. Conclusions: Our results indicate the perspectives of use of the synthesized GNP water dispersions for cancer diagnostics and treatment. It’s necessary to take into account a size-dependent biosafety level of nanoparticles

Antitumor and genotoxic effects of lactoferrin in Walker-256 tumor-bearing rats

Aim: To investigate the influence of exogenous lactoferrin (LF) on tumor growth, energy and lipid metabolism of Walker-256 carcinosarcoma and to assess genotoxic effects of LF. Materials and Methods: The study was performed on Walker-256 tumor-bearing rats. Total lipids and phospholipids were determined by thin-layer chromatography. Comet assay was used to investigate the genotoxic effects of LF. Results: Daily i.p. administrations of exogenous LF at concentrations of 1 mg/kg and 10 mg/kg starting from the 4th day after tumor transplantation suppressed growth of Walker-256 carcinosarcoma by almost 44%. After treatment with recombinant LF in both doses, the phospholipid composition of Walker-256 carcinosarcoma cells was changed (3-fold increase of phosphatidylethanolamine, 3.4-fold increase of phosphatidylcholine, and 1.8-fold increase of sphingomyelin, while the cardiolipin content decreased by 67%. Exogenous LF was not genotoxic for bone marrow cells (as assessed by the ratio of PCE/NCE, number of micronuclei) and peripheral blood lymphocytes (percentage of DNA in the tail of a comet) in Walker-256 carcinosarcoma-bearing rats. Conclusion: Exogenous LF caused the inhibition of Walker-256 carcinosarcoma growth and a decrease in the microviscosity of plasma cell membranes, and exerted no genotoxicity toward bone marrow cells and peripheral blood of experimental animals. Key Words: lactoferrin, breast cancer, Walker-256 carcinosarcoma, phospholipid content, genotoxicity

Metabolic changes during development of Walker-256 carcinosarcoma resistance to doxorubicin

Aim: To study indices of energy metabolism, content of K+ and Mg++ both in peripheral blood and in Walker-256 carcinosarcoma during development of resistance to doxorubicin. Methods: Resistance of Walker-256 carcinosarcoma to doxorubicin has been developed through 12 subsequent transplantations of tumor after the chemotherapy. Parental strain was inhibited by drug by 65%, while transitional resistant substrains — by 30% and 2%, respectively. Determination of biochemical indices in blood serum and homogenates of tumor tissue, level of potassium, magnesium, lactate, glucose, activities of lactate dehydrogenase and glucose-6-phosphate dehydrogenase was performed with the help of biochemical and immune-enzyme analyzer GBG ChemWell 2990 (USA) using standard kits. Polarography was used to determine indices of mitochondrial oxidative phosphorylation. Study of mitochondrial membrane potential was carried out on flow cytometer Beckman Coulter Epics XL using dye JC-1. Results: It has been determined that development of drug resistance causes the decrease of K+, Mg++, glucose content in blood serum and increase of these indices in tumor tissue. At the same time, gradual tumor’s loss of sensitivity is characterized by decrease of glycolysis activity in it and activation of mitochondrial oxidative phosphorylation and pentose phosphate pathway of glucose degradation, which causes more intensive formation of NADPH. Conclusion: Development of drug resistance of tumor causes certain metabolic changes in organism and tumor. Further study of such changes will make possible to determine tumor and extratumor markers of resistance. Key Words: tumor, drug resistance, potassium, magnesium, glycolysis, pentose phosphate cycle, mitochondrial membrane potential

Biodistribution analysis of cisplatin in liposomal form in animals with cisplatinresistant and cisplatin-sensitive carcinoma

Aim: To analyze the relation between pharmacokinetics of cisplatin in liposomal form and antitumor efficacy toward cisplatinresistant and cisplatin-sensitive variants of Guerin carcinoma. Concentration of platinum was measured by atomic absorption spectrophotometry (С115М1 “Selmi”, Ukraine). Elimination constant was calculated based on the dynamics of cisplatin concentration in time period between 1 h to 24 h using nonlinear regression analysis. Area under curve (AUC24) was calculated by the trapezium method. Results: It was shown that for liposomal form of cisplatin (LCp) AUC24 in tumor practically didn’t depend on the level of the tumor sensitivity, while in animals with the resistant variant (CpRGC), AUC24 for free cisplatin (FCp) decreased by 70% less (p < 0.001) as compared to the sensitive tumor strain (CpSGC). Significant decrease of elimination constant of LCp compared to FCp in blood serum of rats bearing either CpRGC or CpSGC tumors favors cisplatin accumulation in tumor tissues with low vascularization level. The dynamics of cisplatin concentration in CpRGC variant was characterized by 90% higher level in 24 h after administration of LCp as compared to FCp (p < 0.05). This fact may explain increased antitumor efficacy of LCp compared to FCp toward CpRGC variant. In the study of kidney function, AUC24 index for LCp was by 68.6% (p < 0.01) and 50.7% (p < 0.05) lower than AUC24 index for FCp in rats with CpRGC and CpSGC variants, respectively. No significant differences have been found in biodistribution of cisplatin in both pharmaceutical forms in liver and lung in CpRGC-r CpSGC-bearing rats. Conclusion: The results suggest that cisplatin in liposomal form possesses higher specificity of antitumor action than free cisplatin

Ultrastructural and some functional changes in tumor cells treated with stabilized iron oxide nanoparticles

The aim of this paper is to study the ultrastructure and some functional indexes of tumor cells treated with stabilized iron nanoparticles in vitro

Peculiarities of antioxidant system and iron metabolism in organism during development of tumor resistance to cisplatin

Aim : To study in vivo the peculiarities of changes of iron metabolism and antioxidant system in dynamics of growth of Guerin carcinoma with different sensitivity to cisplatin. Materials and Methods: In order to evaluate the content of metallothionein-1 (MT-1) in tumor homogenates and blood serum of rats with cisplatin-sensitive and cisplatin-resistant Guerin carcinoma the immunoenzyme method was used. The evaluation of ceruloplasmin activity, content of “free iron” complexes, superoxide and NO-generating acti­vity of NADPH-oxidase and iNOS activity in neutrophils, blood serum and tumor homogenates was measured by EPR-spectro­scopy. Results: Maximal accumulation of MT-1 in blood serum and tumor, more pronounced in resistant strain, at the border of latent and exponential phase of growth has been shown that is the evidence of protective role of this protein in the respect to the generation of free radical compounds. It has been determined that in animals with cisplatin-resistant strain of Guerin carcinoma, increase of “free iron” complexes is more apparent both on the level of tumor and organism on the background on increase of CP/TR ratio that is the consequence of organism antioxidant protection system disorder. Conclutions: Mentioned changes in metabolism of iron with its accumulation in tumor and further reprogramming of mitochondria metabolism and activity of NADPH-oxidase for non-transformed cells are favorable conditions for the formation of oxidative phenotype of tumor. Key Words: Guerin carcinoma, metallothionein-1, antioxidant system, transferrin, ceruloplasmin, “free iron” complexes, activity of NADPH-oxidase and iNOS neutrophils

Prevention of myelosuppression by combined treatment with enterosorbent and granulocyte colony-stimulating factor

Hematotoxicity and its complication are the prominent limiting factors for rational treatment of malignancies. Granulocyte colony-stimulating factor (G-CSF) is used to increase granulocyte production. It has been shown previously that enterosorption causes prominent myeloprotective activity also. Still, no trial was performed to combine both of them. Aim: To study the influence of combination of enterosorption and pharmaceutical analogue of naturally occurring G-CSF (filgrastim) on bone marrow protection and the growth of grafted tumor in a case of injection of melphalan (Mel). Materials and Methods: Mel injections were used for promotion of bone marrow suppression in rats. Carbon granulated enterosorbent C2 (IEPOR) was used for providing of enteral sorption detoxifying therapy. Filgrastim was used to increase white blood cells (WBC) count. Results: The simultaneous usage of enterosorption and filgrastim had maximum effectiveness for restoring of all types of blood cells. WBC count was higher by 138.3% compared with the Mel group. The increase of platelets count by 98.5% was also observed. In the group (Mel + C2 + filgrastim) the absolute neutrophils count was twofold higher, in comparison with rats of Mel group. Conclusion: Simultaneous admi­nistration of G-CSF-analogue and carbonic enterosorbent C2 is a perspective approach for bone marrow protection, when the cytostatic drug melphalan is used. Such combination demonstrates prominent positive impact on restoring of all types of blood cells and had no influence on the antitumor efficacy. Key Words: myelotoxicity, melphalan, enterosorption, granulocyte colony-stimulating factor

The use of nanoferromagnetics to increase the cytotoxic effect of antitumor drugs

Aim: To investigate the influence of ferromagnetic nanoparticles on antitumor effect of doxorubicin and mitochondria oxidative phosphorylation. Methods: The study was carried out on the mice-hybrids (C57Bl/6xDBA/2) with intraperitoneally (i/p) transplantated Ehrlich ascitic carcinoma. Single i/p injection of doxorubicin (Dox), stabilized ferromagnetic nanoparticles (Fe3O4; 20–40 nm; FM) or their combination were performed 7 days after tumor transplantation. The cytotoxic effect of agents, morphology and cell cycle of tumor cells were studied 24, 48 and 72 h after Dox administration. Results: The investigations showed that ferromagnetic nanoparticles increased the cytotoxic effect of doxorubicin on Ehrlich ascіtic carcinoma mainly 48 h after agents’ administration. The largest number of apoptotic cells was observed in group of animals in which doxorubicin was administered before ferromagnetic nanoparticles. Moreover, the ferromagnetic nanoparticles at concentration 1.45 μg Fe/ml and, particularly, 7.25 μg Fe/ml decreased mitochondria oxygen consumption in phosphorylation state that may negatively influence their living capability. Conclusions: Obtained data point out the perspective of use of certain sized FM nanoparticles to increase the cytotoixc effect of antitumor drugs