Modern views on the role of main components of stroma and tumor microinvironment in invasion, migration and metastasis.

The review presents modern ideas about tumor microenvironment, which most researchers recognize as the main "player" in tumor cell invasion, cell migration and metastasis. The current data on the main components of the stroma and the microenvironment, which play the role of the driving force in tumor progression, are analyzed. In particular, the review highlights the issues of origin, biological traits, phenotypic plasticity, functional heterogeneity of activated fibroblasts - myofibroblasts and tumor-associated fibroblasts, which in recent years have received much attention. Such components of the extracellular matrix proteome as collagen and matrix metalloproteinases are discussed in detail. They are mostly produced by activated fibroblasts and, on the one hand, initiate the development of desmoplasia due to type I collagen and, on the other hand, promote degradation of extracellular matrix proteins due to metalloproteinases, which generally leads to tissue remodeling that promotes tumor progression. Possibilities of using the most important indicators of extracellular matrix remodeling as potential markers and targets of clinical strategy are discussed.

Morphological characteristics and expression of adhesion markers in cells of low differentiated endometrial carcinoma.

The aim of the study was to evaluate the morphological features of endometrioid carcinoma of the endometrium (ECE) of low differentiation grade with different invasive potential and to characterize their molecular phenotype by the expression of a number of adhesion markers. MATERIALS AND METHODS: We have studied the samples of operation material of 37 patients with ECE of low differentiation grade with deep invasion (> ½ myometrium), n = 26, and with invasion < ½ myometrium, n = 11, with the use of morphological and immunohistochemical methods, and flow cytometry. RESULTS: In the morphological study of tumors with deep invasion in the myometrium, we have detected pronounced structural heterogeneity, which became the basis for the discretion of two groups of tumors with different characteristics of morphological phenotypes. In the majority of cases, solid layers and glandular-like structures are detected, and the similarity of the tumor epithelium with the elements of the endometrium is completely lost. In such tumors high expression of adhesion molecules - E-cadherin, CD44, CD24, and ß-catenin and low expression of the marker of mesenchymal tissues - vimentin were determined. Other tumors were characterized by morphological features of the epithelial-mesenchymal transition (EMT), with the decrease of the expression of E-cadherin, ß-catenin, CD24, CD44, and a significant increase in vimentin expression in comparison with these indices in tumors without signs of EMT. In ECEs that invade < ½ myometrium, the morphological indices of malignancy were less pronounced, which was associated by the changes in the expression of the molecular markers. CONCLUSION: This comprehensive study has established associations between the morphological heterogeneity of ECE and the expression of adhesion markers and vimentin, which is important for understanding the mechanisms of tumor cell migration.

The influence of lactoferrin on elemental homeostasis and activity of metal-containing enzymes in rats with Walker-256 carcinosarcoma.

AIM: To investigate the content of essential elements (EE): copper, zinc, magnesium, iron and calcium and the evaluation of the activity of metal-containing enzymes - ceruloplasmin (CP), myeloperoxidase (MPO) and the content of transferrin (TF) in blood plasma (BP) and tumor tissue (TT) of animals with Walker-256 carcinosarcoma treated with lactoferrin (LF). MATERIALS AND METHODS: The study of the EE content and the activity of the abovementioned enzymes was carried out on rats with Walker-256 carcinosarcoma treated with LF at the doses of 1 and 10 mg/kg of body weight. The quantitative content of EE in BP and TT of animals was determined using the inductively coupled plasma atomic emission spectroscopy (ICP-AES). Determination of CP activity, content of TF and hemochromes was performed using the method of electron paramagnetic resonance (EPR), and MPO - by unified biochemical method. RESULTS: The introduction of LF at the doses of 1 and 10 mg/kg resulted in a decrease in the ratio of Cu/Zn in BP and even more expressed decrease of Ca/Mg ratio in TT. Administration of LF, especially at a dose of 10 mg/kg, affected the increase in CP and MPO activity in BP. It has been shown that administration of LF at a dose of 10 mg/kg led to an increase in oxidative products of destruction of the hemoglobin-hemochrom system in the TT, against the background of lowering the TF content. CONCLUSIONS: The administration of LF, especially at a dose of 10 mg/kg, led to metabolic alterations associated with inhibition of the tumor process. The detected modulating effect of LF on the content of the EE and the activity of the CP and MPO may be a basis for correction of the elemental balance in carcinogenesis.

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

UNLABELLED: 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.

Modifying effects of 5-azacytidine on metal-containing proteins profile in Guerin carcinoma with different sensitivity to cytostatics.

AIM: To assess the influence of the treatment with 5-azacytidine (5-aza) on the profile of metal-containing proteins and factors of their regulation in Guerin carcinoma cells in vivo. MATERIALS AND METHODS: The study was conducted on Wistar rats transplanted with wild-type Guerin carcinoma (Guerin/WT) and its strains resistant to cisplatin (Guerin/CP) or doxorubicin (Guerin/Dox). Animals were distributed in 6 groups treated with 5-aza and control animals without treatment. 5-Aza was injected by i.v. route (1 injection in 4 days at a dose of 2 mg/kg starting from the 4th day after tumor transplantation, 4 injections in total). Ferritin levels in blood serum and tumor tissue were measured by ELISA, transferrin and free iron complexes - by low-temperature EPR, miRNA-200b, -133a and -320a levels and promoter methylation - by real-time quantitative reverse transcription polymerase chain reaction. RESULTS: The study has shown that 5-aza treatment caused demethylation of promoter regions of fth1 and tfr1 genes in all studied Guerin carcinoma strains. 5-Aza treatment resulted in a significant decrease of ferritin levels in tumor tissue (by 32.1% in Guerin/WT strain, by 29.8% in Guerin/Dox and by 69.1% in Guerin/CP). These events were accompanied by 3.5-fold and 2-fold increase of free iron complexes levels in tumor tissue of doxorubicin and cisplatin resistant strains, respectively. Also, 5-aza treatment resulted in significantly elevated levels of miR-200b, -133a, 320a expression in tumor tissue. After 5-aza treatment, ferritin levels in blood serum of animals with Guerin/Dox were increased by 23.9%, while in Guerin/Wt and Guerin/CP they were decreased by 17 and 16%, respectively. CONCLUSION: Alterations of epigenetic regulation upon in vivo treatment with 5-aza change the levels of metal-containing proteins due to DNA demethylation and altered miRNA expression profiles in Guerin carcinoma cells.

Remodulating effect of doxorubicin on the state of iron-containing proteins, and redox characteristics of tumor with allowance for its sensitivity to cytostatic agents.

The study was aimed at determining the changes of metal-containing proteins in blood serum and tumor tissue of animals with parental and doxorubicin-resistant strains of Walker-256 carcinosarcoma before and after the cytostatic administration. It has been shown that upon doxorubicin action the levels of total iron and transferrin in the tissues from the both groups of animals decreased while that of ferritine simultaneously increased with more pronounced pattern in the group of animals with resistant tumor strain. It has been shown that upon the action of doxorubicin in tumor tissue of animals with different sensitivity to the cytostatic there could be observed oppositely directed changes in the redox state of these cells that in turn determined the content of " free iron" complexes, RO S generation and concentration of active forms of matrix metaloproteinase- 2 and matrix metaloproteinase-9, namely, the increase of these indexes in animals with parental strain and their decrease in animals with the resistant one. So, our study has demonstrated the remodulating effect of doxorubicin on the state of metal-containing proteins and redox characteristics of tumor dependent on its sensitivity to cytostatic, at the levels of the tumor and an organism. These data may serve as a criterion for the development of programs for the correction of malfunction of iron metabolism aimed at elevating tumor sensitivity to cytostatic agents.

Modifying effects of lactoferrin in vitro on molecular phenotype of human breast cancer cells.

AIM: To assess the role of endogenous lactoferrin (LF) in the formation of the molecular phenotype of human breast cancer (BC) cell lines with varying degrees of malignancy, including cisplatin/doxorubicin resistant cell lines, and identify possible impact of exogenous LF. MATERIALS AND METHODS: 5 breast cell lines of different origin - MCF-10 A, MCF-7, including doxorubicin/cisplatin resistant ones, T47D, MDA-MB-231, and MDA-MB-468. Immunocytochemistry: expression of LF, Ki-67, adhesion molecules E- and N-cadherin, CD44, CD24 rating the invasive potential of cells. RESULTS: Expression of LF in human BC cell lines varies. It is associated with the heterogeneity of molecular profiles of cell lines in terms of adhesion. A link has been established between the level of LF expression in the resistant cell line MCF-7/CP and MCF-7/Dox, features of their molecular profile and invasive properties. Exogenous LF was shown to be capable of modifying the molecular profile and invasive properties of all the studied cell lines including resistant ones (MCF-7/CP and MCF-7/Dox). CONCLUSIONS: The sensitivity of cytostatic-resistant cell lines (MCF-7/CP and MCF-7/Dox) tends to increase under the influence of exogenous LF. It is likely that this effect is due to LF-mediated inhibition of the expression of proteins associated with drug resistance.

METALLOPROTEINS DURING DEVELOPMENT OF WALKER-256 CARCINOSARCOMA RESISTANT PHENOTYPE.

The study was focused on the detection of changes in serum and tumor metal-containing proteins in animals during development ofdoxorubicin-resistant phenotype in malignant cells after 12 courses of chemotherapy. We found that on every stage of resistance development there was a significant increase in content of ferritin and transferrin proteins (which take part in iron traffick and storage) in Walker-256 carc'inosarcoma tissue. We observed decreased serumferritin levels at the beginning stage of the resistance development and significant elevation of this protein levels in the cases withfully developed resistance phenotype. Transferrin content showed changes opposite to that offerritin. During the development of resistance phenotype the tumor tissue also exhibited increased 'free iron' concentration that putatively correlate with elevation of ROS generation and levels of MMP-2 and MMP-9 active forms. The tumor non-protein thiol content increases gradually as well. The serum of animals with early stages of resistance phenotype development showed high ceruloplasmin activity and its significant reduction after loss of tumor sensitivity to doxorubicin. Therefore, the development of resistance phenotype in Walker-256 carcinosarcoma is accompanied by both the deregulation of metal-containing proteins in serum and tumor tissue and by the changes in activity of antioxidant defense system. Thus, the results of this study allow us to determine the spectrum of metal-containing proteins that are involved in the development of resistant tumor phenotype and that may be targeted for methods for doxorubicin sensitivity correction therapy.

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 acti-vity of NADPH-oxidase and iNOS activity in neutrophils, blood serum and tumor homogenates was measured by EPR-spectro-scopy. 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.

Alteration in lipid composition of plasma membranes of sensitive and resistant Guerin carcinoma cells due to the action of free and liposomal form of cisplatin.

AIM: To study in vivo changes of lipid composition of plasma membranes of sensitive and resistant to cisplatin Guerin carcinoma cells under influence of free and liposomal cisplatin forms. MATERIALS AND METHODS: The isolation of plasma membranes from parental (sensitive) and resistant to cisplatin Guerin carcinoma cells was by differential ultracentrifugation in sucrose density gradient. Lipids were detected by method of thin-layer chromatography. RESULTS: It was determined that more effective action of cisplatin liposomal form on resistant cells is associated with essential abnormalities of conformation of plasma membrane due to change of lipid components and architectonics of rafts. It results in the increase of membrane fluidity. CONCLUSION: Reconstructions in lipid composition of plasma membranes of cisplatin-resistant Guerin carcinoma cells provide more intensive delivery of drug into the cells, increase of its concentration and more effective interaction with cellular structural elements.

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.

Ultrastructural changes in tumor cells treated with liposomal forms of anticancer drugs.

AIM: To determine the main ultrastructural changes in MCF-7 sublines sensitive and resistant to cytotoxic action of anticancer drugs, resulting from the treatment with conventional and liposomal forms of cisplatin and doxorubicin. METHODS: Electron microscopy, light microscopy, MTT-test. RESULTS: It has been shown that the phenomenon of drug resistance is associated with complication of ultrastructural organization of cells and more high differentiation by the main cytomorphologic characteristics which promote their resistance to cytotoxic action of anticancer preparations. Cytoarchitectonics of all resistant cells possesses common patterns and doesn't depend on the particular drugs toward which the resistance has been developed. It has been shown that the cells of the parental form MCF-7 line are more sensitive to cytotoxic action of doxorubicin than to cisplatin. Liposomal forms of anticancer drugs used at the same concentrations that the conventional ones, especially that of doxorubicin, caused more expressed alterations in ultrastructural organization of cells of all studied sublines with dominance of apoptotic processes. CONCLUSION: Evaluating an effect of equal concentrations of cisplatin and doxorubicin in conventional and liposomal forms, one may conclude on higher cytotoxic action of doxorubicin vs. cisplatin that is expressed in a wider spectrum of ultrastructural changes of cell architectonics in different sublines of MCF-7 cells and higher rate of apoptosis.

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 ascsmall i, Ukrainiantic 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 microg Fe/ml and, particularly, 7.25 microg 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.

[The prognostic significance of cytogenetic, morphological and clinical indices in patients with malignant skin melanoma]. / Prognosticheskaia znachimost' tsitogeneticheskikh, morfologicheskikh i klinicheskikh pokazatelei u bol'nykh zlokachestvennoi melanomoi kozhi.

Retrospective estimation of the prognostic importance of parameters, that are different in biological essence, for the persons that have died from malignant melanoma of the skin with use of the adequate mathematical approaches is carried out. A spectrum of important cytogenetic, morphologic and clinical factors which have influence on the prognosis of disease is determined. For group of living patients the prognosed survival time are individually calculated.

[A comparison of the morphofunctional characteristics of the peripheral blood lymphocytes and the immune status of patients with malignant melanoma of the skin]. / Sopostavlenie morfo-funktsional'nykh osobennostei limfotsitov perifericheskoi krovi i immunnogo statusa u bol'nykh zlokachestvennoi melanomoi kozhi.

As a results of the study of immune status of 75 patients with malignant skin melanoma, some signs of immunodeficiency with failure of cell and humoral reactions were revealed. The degree of quantitative and qualitative changes of immune system depended on clinical-morphological features of malignancy development.

[Segregation and genetic dispersion analysis of the pedigrees of breast cancer patients in the Kiev region]. / Segregatsionnyi i genetiko-dispersionnyi analiz rodoslovnykh bol'nykh rakom molochnoi zhelezy kievskogo regiona.

Complex clinical-genealogical and genetic study of 261 patients with breast cancer (BC) was carried out in Kiev region. It was demonstrated that the multifactorial origin of BC was of first importance in this population. The share of genetic component in the susceptibility to BC comprise 55.68 +/- 2.44%. Risk of the origin of malignant tumors in progeny was calculated.

[Pathomorphosis of metastatic malignant melanoma of the skin as effected by preoperative endolymphatic polychemotherapy]. / Patomorfoz metastazov zlokachestvennoi melanomy kozhi pod vliianiem predoperatsionnoi éndolimfaticheskoi polikhimioterapii.

In 59 patients with malignant skin melanoma, the effect of regional endolymphatic therapy on pathomorphosis of lymphogenic metastases and long-term results of treatment was studied. The essential morphologic changes at the sites of melanoma and higher indices of patients' survival in the group studied when compared to the control group have been noted.

[Proliferative activity of pigmented nevus cells and malignant melanoma of human skin]. / Osobennosti proliferativnoi aktivnosti pigmentnykh kletok nevusov i zlokachestvennykh melanom kozhi cheloveka.

The findings in the study of special features of proliferative activity in 58 pigmented neoplasms of skin have revealed a pronounced heterogeneity of tumor cell populations, malignant melanomas based on the values of the mitotic index (MI) and label index (LI). It is shown that while evaluating tumor anaplasia, these parameters provide more information as compared to the morphological characteristics.

[A karyotype and chromatin study of the peripheral blood lymphocytes of patients with pigmented skin neoplasms]. / Issledovanie kariotipa i khromatina limfotsitov perifericheskoi krovi bol'nykh pigmentnymi novoobrazovaniiami kozhi.

Rearrangements of genetic material in the peripheral blood lymphocyte nuclei have been found in patients with dysplastic pigmented nevi and malignant melanomas (15 cases), in contrast to the control (10 healthy persons). It is expressed as an increase in the number of chromosome aberrations in the karyotype and as a change of the Feulgen-positive substance correlation in the interphase nuclei of the given cells towards an increase in the active-functioning DNA structures.

[Clinico-morphologic manifestations of the regression of a primary focus of malignant melanoma of the skin]. / Kliniko-morfologicheskie proiavleniia regressii pervichnogo ochaga zlokachestvennoi melanomy kozhi.

The general laws and clinico-morphologic peculiarities of the regression of cutaneous melanoma in 4 patients, which occurred spontaneously or under the influence of the local use of non-conventional methods of therapy are presented. It is shown that resorption of primary neoplasm is not the ultimate outcome of the tumour process, and because of the development of metastases, the prognosis remains unfavourable. Regression of a primary focus of cutaneous melanoma is estimated as one of the manifestations of the progression of tumour growth.