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

Significance of iodine symporter for prognosis of the disease course and efficacy of neoadjuvant chemotherapy in patients with breast cancer of luminal and basal subtypes

The aim of the research was to study the relation between expression of Na⁺/I⁻symporter (NIS) in breast cancer (BC) of different molecular subtypes and sensitivity of BC cells to neoadjuvant chemotherapy (NACT) and to assess whether NIS expression may be used as a predictive marker of treatment efficacy. Materials and Methods: The study included 148 women with BC of stage II–III who were treated at the Precarpathian Clinical Oncology Center during 2012–2017. All patients were treated with NACT that included 2–6 cycles of chemotherapy by FAC, AC scheme with 21 day intervals. NACT efficacy was evaluated every 2 cycles by mammography according to RECIST criteria. Morphological and immunohistochemical study of NIS expression was performed by the standard methods on paraffin sections of surgically resected tumors. Results: The heterogeneity of different molecular BC subtypes regarding response to the NACT has been found. Her2/neu-positive and basal BC subtypes were the least susceptible to the NACT (p < 0.05). It was shown that NIS expression is related to the sensitivity of luminal B and basal BC subtypes to the NACT. The highest expression of NIS and impairment of its functional activity was registered in the group of patients with tumors resistant to NACT (stabilization of the disease or its progression) of luminal B (220 ± 8.6 points) and basal subtypes (290 ± 11.3 points) (p < 0.05). It was revealed that the disease-free survival of patients with BC of luminal B and basal subtypes was higher in the absence of NIS expression in tumor cells (p < 0.05). Conclusions: The results indicate that NIS can be used as an objective criterion for predicting the sensitivity of luminal B and basal BC subtypes to NACT, which will provide improved treatment outcomes in this group of patients

Correlation of iodine symporter expression in highly and low malignant cell lines of human breast cancer differed in their sensitivity to doxorubicin

Aim: To investigate the relationship of sodium/iodide symporter (NIS) expression with molecular phenotype of highly and low malignant cell lines of human breast cancer (BC) with different sensitivity to doxorubicin (Dox). Materials and Methods: The cell lines used in the analysis included T47D, MCF-7, MDA-MB-231, MDA-MB-468, and MCF/Dox. NIS expression was studied by immunocytochemical method. Results: The strongest iodine symporter expression (248 ± 1.9; 272 ± 3.2 and 289 ± 2.8 points, respectively) were found in cells of highly malignant cell lines — MDA-MB-231, MDA-MB-468, and MCF-7/Dox. NIS expression was significantly weaker (< 120 points) in two BC cell lines of low malignancy (MCF-7 and T47D). In addition, the reduced sensitivity to Dox is associated with elevation of NIS expression in both high and low malignant cells. We have demonstrated correlations between NIS levels and certain indices of BC malignancy, namely proliferative activity (r = 0.51), receptor status (estrogen receptor; r = –0.47; and progesteron receptor; r = –0.47) and invasiveness (r = 0.46). Conclusions: Our data evidence that NIS expression level correlates with BC cells indices of malignancy and their sensitivity to Dox. The results obtained suggest the necessity for further studies of NIS expression in BC patients aimed at prognosing disease course and monitoring treatment efficacy with anthracyclines

In vitro modification of cisplatin cytotoxicity with magnetic fluid

Aim: To study cytotoxicity of cisplatin conjugated with magnetic fluid (nanocomposite) upon exposure to magnetic field on sensitive and resistant to cisplatin MCF-7 human breast cancer cells. Methods: Cytotoxic activity was evaluated by MTT-test, intracellular iron accumulation was analyzed cytochemically, genotoxicity was studied by micronucleus test and DNA comet assay, ultrastructure was studied by electron microscopy techniques. Results: Nanocomposite of cisplatin was more toxic to MCF-7/S and MCF-7/CP cells compared to cisplatin in conventional pharmaceutical form. In nanocomposite-treated cells we observed more expressed signs of dystrophy (especially following application of magnetic field) and drastic alterations of nuclei ultrastructure with significant accumulation of iron nanoparticle clusters. The potent toxic action of nanocomposite is confirmed by electron microscopy and by marked genotoxicity, especially against MCF-7/CP cells. Conclusion: The enhancement of cyto- and genotoxicity of cisplatin nanocomposite combined with magnetic field in comparison with effect of convetntional cisplatin alone was demonstrated

Redox-regulation of gelatinases during growth of cisplatin-sensitive and resistant Guerin carcinoma

Study was aimed to analyze the dynamics of changes and study interrelations between content of ferritin, transferrin, active gelatina­ses (MMP-2 and -9) in blood serum and tumor tissue, free iron, rate of superoxide radicals generation in tumor, activity of NADPH-oxidase and iNOS in neutrophils rats with sensitive and resistant strains of Guerin carcinoma (GC). Materials and Methods: In order to obtain resistant tumor, 12 courses of cisplatin chemotherapy have been carried out on rats bearing GC. Levels of transferrin and free iron were determined by analysis of EPR spectra from computerized radiospectrometer EPR ­RE-1307 at temperature of liquid nitrogen. Rate of superoxide radicals and nitric oxide generation in tumor and neutrophils of blood was determined by EPR using spin traps at room temperature. Content of ferritin in tumor homogenate and blood serum of rats with GC was determined by ELISA method using corresponding kits. Concentration of active forms of MMP-2 and -9 in obtained samples was determined using method of zymography. Results: Unregulated generation of superoxide radicals and NO by mitochondria of tumor cells and NADPH-oxidase and iNOS neutrophils via oxidation of iron-containing proteins causes the accumulation of “free iron” complexes in blood and tumor tissue of rats able to evoke oxide-induced damages of macromolecules. It has been shown that for resistant strain of carcinoma, as compared with sensitive one, significantly higher concentrations of active forms of MMP-2 and -9 in blood serum of rats are typical. Dynamics of gelatinases activity changes in tumor tissue corresponds in general with dynamics of changes in serum. In tumor tissue of rats the indices of gelatinases activity positively correlate with rate of superoxide radicals generation, content of “free iron” complexes, ferritin and activity of transferrin. Cytostatic agent increased levels of reactive oxygen species (ROS) and self-amplify rate of superoxide radicals generation. In turn, activation of MMPs via superoxide-depending regulation allows tumor cells to facilitate migration, invasion and finally — formation of metastatic centers. Mentioned above tumor “oxide phenotype” determines high level of its aggressiveness and forms corresponding level of drug resistance. Conclusions: Thus, high levels of superoxide radicals oxidize transport proteins and form free iron pool. Iron ions, via Haber — Weiss mechanism, initiate generation of the hydroxyl radicals, which also enhance oxidation processes. Key Words: drug resistance, redox-regulation, iron-containing proteins, matrix metalloproteinases

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

Lactoferrin expression in breast cancer in relation to biologic properties of tumors and clinical features of disease

Aim: To determine the patterns of lactoferrin (LF) expression in breast cancer (BC) in relation to biologic properties of the neoplasms and clinical features of the disease course. Materials and Methods: Clinical specimens of 266 BC patients (115 patients with BC of stages I–II — retrospective study, and 151 BC patients — prospective study) were analyzed. Morphological, immunohistochemical and statistical methods were used. Results: The number of LF-positive tumors in retrospective and prospective groups was similar (52.1 and 52.8%, respectively). Among common clinical criteria for prognosis of the disease outcome in BC patients (patient’s age; stage of the disease; histological type, differentiation grade, receptor status; presence of metastases), a strong correlation was found only between expression indexes of LF and estrogen receptors (ER). In ER-positive tumors expression of LF was significantly higher than in ER-negative tumors (35 vs 18%). 5-Year survival rate of BC patients was higher in LF-positive group (70 vs 52% in LF-negative group). The presence of regional metastasis tended to correlate with an increased number of LF-positive tumors. In the patients with invasive ductal carcinoma, expression level of LF moderately correlated with occurrence of luminal A subtype (r = 0.43), while in the patients with invasive lobular carcinoma this index strongly correlated with occurrence of luminal B subtype (r = 0.71). LF expression correlated positively with low and moderate differentiation grade of luminal B or basal tumors, and negatively with luminal B or basal tumors of high differentiation grade (r = −0.57 and −0.63, respectively). Conclusion: It has been shown that LF expression in breast tumors correlated with life expectancy of BC patients and important physiologic and clinical features of the disease, while the character of such relation strongly depended on molecular phenotype of tumor, i.e. luminal A, luminal B or basal

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

Comparative study of dye-loaded liposome accumulation in sensitive and resistant human breast cancer cells

The aim of this research is to study the dynamics and efficiency of liposome accumulation in sensitive and resistant human breast cancer cells. Methods: Methods of fluorescence microscopy, fluorescence microspectroscopy and MTT-test have been used. Results: The liposome-to-cell interaction and dye cellular uptake in sensitive, cisplatin-resistant and doxorubicin-resistant MCF-7 human breast cancer cells have been analyzed using time changes in both fluorescence resonance energy transfer signal from the donor probe DiO to the acceptor one DiI preloaded in liposomes and cell image brightness. Conclusion: Obtained results show that resistant cells accumulate dye-loaded liposomes more effectively and reveal more effective dye molecule cellular uptake