Introduction. E-cadherin aberrant expression or complete loss is common for a number of human malignant neoplasms, and can be a launching mechanism of an epithelial-mesenchymal transition. Passing through epithelial-mesenchymal transition could in turn promote to the acquisition of so called cancer stem cell phenotype by the transformed cells. The objective of the present study is to reveal the influence of E-cadherin expression level on the amount of cancer stem cells in human colon cancer cell line HCT116. Materials and methods. We have created cell sublines with E-cadherin up- and downregulation and assessed the percentage of cancer stem cells using tumor formation assay, clonogenic assay; we also evaluated profile of cell pluripotency markers. Results and conclusion. We have shown that the proportion of cancer stem cells in human colon adenocarcinoma cell line HCT116 depends on the E-cadherin expression level. E-cadherin expression downregulation results in elevated expression of pluripotency genes and in the increase of proportion of cancer stem cells via activation of Wnt/ß-signalling pathway. E-cadherin upregulation has a reverse effect and decreases the amount of HCT116 cancer stem cells. Thus, E-cadherin expression restoration seems prospective in colorectal anticancer therapy.

Objective. The aim of the study was to research effect of new cyclic hydroxamic acids (CHA) I-VI on activity of Ca²+-ATPase of sarcoplasmic reticulum and cyclic guanosine monophosphodiesterase. Materials and methods. Activity of Ca²+-ATPase of sarcoplasmic reticulum and cyclic guanosine monophosphodiesterase has been evaluated. Results. It has been shown that at studied concentration (0.1 mM; 0.01 and 1 mM), CHA I-VI separate hydrolytic and transporting functions of Ca²+-ATPase of sarcoplasmic reticulum. Therefore, the antimetastatic effect of these compounds is assumed. Thus, at concentration of 0.1 mM, CHA inhibit active transport of calcium through the sarcoplasmic reticulum membrane by 40 ± 4 % (CHA-I), 50 ± 5% (CHA-II), 53 ± 5% (CHA-III), 70 ± 8% (CHA-V) and 75 ± 8% (CHA-VI) and inhibit hydrolysis of ATP by 20 ± 2%, 0 %, 0 %, 45 ± 5 % and 47 ± 3 % respectively. CHA-III, CHA-V and CHA-VI inhibit active transport of Са²+ by 46 ± 5 %, 47 ± 5 % and 60 ± 6 %, and not inhibit or inhibit by 23 ± 2 % and 27 ± 3 % respectively, hydrolysis of ATP at concentration of 0.01 mM. CHA-V inhibits reversibly and non-competitively the hydrolytic function of Ca²+-ATPase of sarcoplasmic reticulum with K = 4 x 10^-5 M. All studied CHA inhibit activity of cyclic guanosine monophosphodiesterase at concentration of 0.1 mM, by less than 20 %. Conclusion. The data obtained predicts the antimetastatic activity of compounds CHA-II, CHA-III, CHA-V, CHA-VI. We recommend to study of CHA-II, CHA-III, CHA-V, CHA-VI on animal models as promising antimetastatic drugs.

Objective. To study of known interferons (IFN) type I and IFN-inductors drugs different chemical structure on cell differentiation and expression group of genes of TLR/RLRs, referring to group of pattern-recognition receptors. Materials and methods. Cellular lines ТНР-1 (acute monocytic leukemia) и НСТ-116 (colon adenocarcinoma) were treated by interferons drugs Reaferon, Altevir, Genfakson, Infibeta 10⁵-10⁶ МЕ, IFN-inductors Ridostin 10²-10³ ßg/ml, Cycloferon 625 ßg/ml and Imunomax 2 МЕ during 24 h or 96 h at 37 °С. Gene expression were estimated by method qRT-PCR (CFX-96, Bio-Rad). CD-immuno-phenotypes of ТНР-1 cells were detected by flow cytometric fluorescence method with FITC and PE monoclonal antibodies (FACSCanto II, Becton Dickinson). Results. It was shown that tumor cells THP-1 and HCT-116 have low and variable constitutive levels gene expression of TLR-receptors 2, 3, 4, 7, 8, 9 during passages. This gene status may be connected with incorrect processing of mature mRNA forms. Genes TLR4, TLR8 and factor NFkB are stimulated by interferons and IFN-inductors more high in THP-cells and genes TLR7, TLR8, TLR9 and NFkB - in HCT-cells. Ridostin (mix ssRNA and dsRNA S. cerevisiae) is the best activator of TLR/RLRs receptors. Ridostin increases macrophage marker CD11b, Cycloferon, Imunomax stimulate levels of early T-cell antigen CD7, interferons results in growth of HLA DR. The drugs Cycloferon, Imunomax and Reaferon decrease levels of myeloid suppressor CD38. Conclusion. Group of recombinant IFNs and IFN-inductor drugs various chemical structures regulate TLR/RLRs genes expression differently in THP-1 and HCT-116. TLR/RLRs genes activation are accompanied by CD-phenotypes changes in THP-1 monocytic cell line.

Background. Aranose is alkylating antineoplastic agent with cytotoxic and cytostatic properties. Liposomal form of aranose has advantages compared with traditional dosage form. Another alkylating agent, cisplatin, promotes mRNA increase expression level of genes, which control external apoptosis way. Objective. We assume that liposomal form of aranose can change the CD95/Fas, TNFR1, TNFR2, TRAIL, DR3 and DR4/5 expression level in melanoma cell lines mel Kor, mel Z, mel Mtp, mel Mtp-X, mel Ibr, mel Ch, mel Is and mel R. Materials and methods. There are two forms of aranose were used in this work: liposomal form and traditional diluted form for injection. Melanoma cell lines were incubated with both forms. We used RQ PCR to evaluate mRNA expression mRNA expression of DR3, DR4/5, CD95/Fas, TNFR1, TNFR2 and TRAIL relatively gene ABL. Results. CD95/FAS mRNA expression level was most changeable. Liposomal aranose increase CD95/FAS expression level in mel Ibr and mel Mtp-X cell lines and reduced CD95/FAS expression level in mel Ch and mel Is. Conclusion. Thus, liposomal aranose can change mRNA CD95/FAS expression level in melanoma cell lines. Using this process, liposomal form can affect the apoptotic cell death of melanoma.

Background. Development of new models of a human disseminative melanoma of skin with existence of a molecular and genetic target for specific therapy increases productivity of the preclinical researches in vitro and in vivo new the anti-melanoma drugs or their combinations. Such opportunity is realized by adaptation to in vivo of the original human pigment-free skin melanoma cell line mel Rac and receiving s. c. xenograft under monitoring, transplant, morphological, molecular and genetic (NRAS mutation) and chemotherapeutic (sensitivity to an inhibitor of NRAS a trametinib) characteristics. Objective. Receiving from the cell line mel Rac s. c. xenograft of a human pigmented melanoma of skin with a mutation of NRAS and sensitive to specific target therapy. Materials and methods. Human pigment-free skin melanoma cell line mel Rac from the Collection of Russian Cancer Research Center and immunodeficient female of Balb/c nude mice cultivation of Russian Cancer Research Center was used. Required characteristics are defined at a multiple s. c. transplanting in vivo by methods of transplant biology, a light microscopy, molecular genetics and the experimental chemotherapy. Sensitivity to a NRAS inhibitor to a trametinib was estimated under monitoring of rate of the tumor growth (V_t/V₀) on indexes, adequate for patients: existence of the complete remission and possibility of recurrence. Results. At s. c. transplantation of 10⁷ cell of mel Rac line are received cytological identical intertwined s. c. xenografts with a stable kinetics of growth on 4-9 passages (a latent phase 8 days, exponential - to 14 days, stationary - to 24 days) and existence of a mutation of NRAS. Trametinib in a single dose of 0,3 mg/kg caused the complete remission during a 14-day course and within 7 days after its cancellation without recurrence. Conclusion. Receiving with cell line mel Rac s. c. xenograft of a human pigment-free melanoma of skin with a mutation of NRAS and sensitive to specific target therapy is suitable for preclinical studying of the specific for this target new anti-melanoma drugs.

Objective. The aim of the study was to research the toxicity of L-asparaginase Was79 in rabbits. Materials and methods. Chronic toxicity of L-asparaginase Was79, obtained by modification of native enzyme Wolinella succinogenes in Research Institute of Genetics and Selection, was performed in male and female Russian chinchilla rabbits. L-asparaginase was injected intravenously at the 1 and 5 therapeutic dose (15 x 100 IU/kg or 15 x 500 IU/kg with 24-h interval). The following parameters were tested: body mass, clinical and biochemical blood tests, urinalysis, electrocardiography, pathomorphological evaluation of internal organs. Resutls. The results of the study suggest that the treatment with L-asparaginase Was79 does not influence on the function of heart and kidneys, but damages their structure. Loss in body mass, diarrhea and alteration of stomach and intestine mucosa could be interpreted as evidence of gastrointestinal toxicity. Hematological toxicity was exhibited as a decrease of total leukocyte count, lymphocyte and neutrophils count level in peripheral blood and atrophy of lymphoid tissue of the spleen, thymus and lymph nodes. Elevation of total and direct bilirubin in serum and histopathological findings in liver were found in groups treated with both high and low doses of Was79. Conclusion. Most of these abnormalities were reversible and dose-dependent.

Background. In N.N. Blokhin Russian Cancer Research Center have been synthesized a number of hypothalamic hormone somatostatin analogues, including pentapeptide cyphetrylin, and drug Cyphetrylin tablets 6 mg has been created. One of the drug standardization step is development of assay technique for acting substance in the final preparation. Objective. Development and validation of cyphetrylin assay in tablets. Materials and methods. The study used cyphetrylin, tablets 6 mg, сyphetrylin powder substance (FSBI “N.N. Blokhin Russian Cancer Research Center”, branch “Naukaprofi”), lactose monohydrate, cellulose microcrystalline, potato starch, povidone, talc, magnesium stearate (European Pharmacopoeia current edition), 95 % ethyl alcohol. Method of assay - spectrophotometry. Results. The technique for assay of сyphetrylin in drug “Cyphetrylin, tablets 6 mg” has been developed. Validation was performed to confirm test validity and accuracy. The technique was estimated by validation characteristics: specificity, linearity, accuracy, precision (repeatability and intermediate precision). Conclusion. The developed technique has the required characteristics: specificity, accuracy, repeatability, intermediate precision, linearity and can be used in the range of 70-130 % of nominal сyphetrylin content in preparation.

Background. In N.N. Blokhin Russian Cancer Research Center has been developed injectable dosage form based on the substance of indolocarbazole derivative LCS-1208, studied its antitumor activity and mechanism of antitumor effect, which was the basis for the pre-clinical study of the drug toxicity. Objective. The study of subchronic toxicity of formulation of glycoside derivative of indolocarbazole - LCS-1208 after intraperitoneal application to rats. Materials and methods. Dosage form of study drug: lyophilisate for preparation of solution for injection of 9,0 mg in vial. The study was conducted on 40 healthy non inbred male rats, which were obtained from the breeding of the N.N. Blokhin Russian Cancer Research Center. It was studied the action of the drug on the peripheral blood, the functional state of the liver, kidney and gastrointestinal tract of animals. Results. The drug LCS-1208 by daily intraperitoneal administration to rats for 15 days in the 3 studied doses (total dose 200 mg/kg, 100 mg/kg and 50 mg/kg) did not cause death of animals, had no effect on the general condition of the animals, did not cause external manifestations of toxicity, did not change behavioral responses of animals and had no effect on the functioning of the studied organs. Conclusion. The results obtained by the study of the subchronic toxicity in rats were the basis for further preclinical toxicology study of the drug LCS-1208.

Background. Concentration of a prostate specific antigen (PSA) in blood serum considerably increases in case of development of a prostate adenocarcinoma, in tens and hundreds times exceeding extreme values of the range of the determined concentration for all existing diagnostic test systems that limits possibilities of their application for the purpose of systematic monitoring of a condition of patients and efficiency of the treatment. At the same time, change of standard algorithm of accomplishment of the laboratory analysis stated in instructions to the relevant test systems allows to expand the range of the determined concentration of the PSA significantly. Objective. The present research is executed for the purpose of creation of algorithm of quantitative detection of the total PSA concentration in blood serum specimens by enzyme immunoassay (EIA) suitable at the same time for primary laboratory diagnostics, screening and systematic monitoring of a condition of patients with the verified diagnosis of a prostate adenocarcinoma. Materials and methods. In present investigation serially made EIA-kits for quantitative determination of level of a total PSA blood serum specimens certified by the Russian Ministry of Health for clinical laboratory diagnostics are used. In research samples of blood serum of almost healthy donors, and also patients with not tumoral diseases of a prostate, and also patients with the verified diagnosis of a prostate adenocarcinoma, including in a stage of a dissemination of tumoral process were applied. Results. It is shown that linearity of the defined concentration of the total PSA at consecutive two-fold dilutions of blood serum samples by the “zero” standard remains dilution up to 1024 times that allows to execute correctly measurement of total PSA level in the range up to 30 000 ng/ml. Conclusion. The simple algorithm of performance of quantitative detection of PSA by EIA at patients with a prostate adenocarcinoma is developed and approved.

Introduction. Prognostical indexes for follicular lymphoma (FLIPI, FLIPI-2 (Follicular Lymphoma International Prognostic Index)) not completely reflect the variability of disease and its prognosis. There appeared new data concerning the prognostic factors which can predict the disease behaviour more thoroughly. Search for new prognostic factors which can predict both immediate and late progression risk in follicular lymphoma is one of the most important tasks in oncohaematology. Objective. To compare the frequeneies of clinical and laboratory characteristics at diagnosis which negatively influenced the prognosis of follicular lymphoma was the purpose of this research. Matherials and methods._From 222 patients with follicular lymphoma we have identified 79 (36 %) aggressive course of the disease (1^st group): some patients died from follicular lymphoma progression during less than 3 years from diagnosis, in others - early relapses/progression took place in less than 24 months from start of treatment. We compared the frequences of clinical and laboratory characteristics at diagnosis which negatively influenced the prognosis of follicular lymphoma in patients of 1^st group with 143 patients of control (standard prognosis) group. 2 groups of follicular lymphoma patients were compared: 1^st group - 79 patients with unfavourable prognosis and 2^nd group - 143 patients with favourable prognosis. Results. Patients from the 1^st group compared to the 2^nd group had worse estimation according to ECOG, more frequent diminution of serum protein, more frequent Hb lower than 120 g/l, more frequent intoxication symptoms at diagnostics, more frequent involvement of more than 1 extranodal organ, more frequent involvement of spleen, worse treatment response, higher proportion of high risk according to FLIPI. New prognostic factors not included into existing prognostic models were identified. Negative prognostic factors for overall survival (OS) and progression-free survival (PFS) were diminution of serum protein level (OS p = 0,03; PFS p = 0,021), involvement of spleen (OS p = 0,000, PFS p = 0,002) and bad response (stabilization, progression) to 1^st line therapy (OS p = 0,000, PFS p = 0,000). Symptoms of intoxication negatively influenced OS (p = 0,000), but not PFS (p = 0,0127). Conclusion. Except of standard prognostic factors, several other facors can negatively influence the prognosis of follicular lymphoma: diminution of total serum protein, involvement of spleen, unsatisfactory response to 1^st line treatment (stabilization, progression), intoxication symptoms. These parameters are necessary to estimate and use for more thorough estimation of progression risk in follicular lymphoma.

Objective of the research was determination level of RRM1 expression in ovarian cancer tissue. Materials and methods. To avoid disadvantages of those evaluations, the present study of the RRM1 expression level in ovarian cancer tissue determination was held using strictly quantitative immunofluorescence flow cytometry-associated method developed by the authors. Results. RRM1 expression was detected in 100 % of samples of ovarian serous adenocarcinoma, with significant differences in the indicator level in variable patients. The high level of RRM1 (≥ 40 % of cells expressing the marker) was detected in 65 % of the tumor samples studied, low (< 40 %) - in 35 % of cases. Data on the lower expression of RRM1 in one-third of the test samples from primary serous ovarian cancer corresponds with clinical observations of gemcitabine monotherapy efficiency in 25-30 % of patients. Conclusion. The authors believe that a strictly quantitative determination of the marker level in tumor tissue can help to overcome the ambiguity of RRM1 clinical significance estimation in gemcitabine treatment effectiveness prediction.

Background. Human melanoma cell line mel Cher have a different cell forms and is appraised for grade of differentiation as low-diffe-rentiation melanoma. Objective. The purpose of this study was development and characterization of subclone mel Cher 5C. Materials and methods. For received subclone mel Cher 5C was applied method of colony formation in conditions with low concentration of embryo calf serum (5 %). Then was carry out selection spindle-like cells, their long cultivation and estimate morphological and immunological characteristics. Results. Subclone mel Cher 5C cells differed for morphology from primary line mel Cher. This cells had spindle-like form and had capacity to form spherical type colony on the surface of monolayer during their growth. Subclone cells has more intensive growth compare with cells of primary line. Subclone cells had differences by immunological phenotype and didn’t expressed antigene HLA-DR. Conclusions. Subclone cells had a fusiform shape, in the growth process of the formed spheroid colonies monomorphic type, did not Express antigens of histocompatibility complex II.

Background. Beta-III tubulin (TUBB3) is a tumor-specific isoform of the microtubule protein beta-tubulin. TUBB3 is considered to be a marker of adverse prognosis and tumor resistance to therapy with taxanes and Vinka alkaloids. Association between TUBB3 expression and histological type of the tumor has not been studied properly yet. Objective. The expression level of TUBB3 in non-small cell lung cancer biopsy specimens has been measured on 2 groups of patients with adenocarcinoma and squamous cell carcinoma. Materials and methods. The samples with adenocarcinoma (n = 43) and squamous cell carcinoma (n = 39) were converted to suspension, filtered, fixed with 4 % formaldehyde, stained with monoclonal antibodies for TUBB3 and DyLight 650-conjugated secondary antibodies to mouse IgG and analyzed by flow-cytometry. The method was developed in N.N. Blokhin Russian Cancer Research Center. Results. The average level of TUBB3 expression in the adenocarcinoma group is higher than in the squamous cell carcinoma group (33.1 ± 12.4 % of cells expressing the marker in adenocarcinoma vs. 26.0 ± 13.6 % in squamous cell carcinoma; differences were statistically significant). The average level of TUBB3 expression in adenocarcinoma is 29.7 ± 8.1 % in female and is 34.9 ± 13.9 % in male (differences statistically insignificant). Since the group of patients with adenocarcinoma was presented by both men and women, while the group of patients with squamous cell carcinoma was only men, from the analysis was excluded all female patients. Differences between the groups remained statistically significant. Conclusion. TUBB3 expression in tumor tissue does not depend on the gender, at least among patients with adenocarcinoma of the lung, and at the same time, the level of TUBB3 in adenocarcinoma tissue is higher in comparison with squamous cell carcinoma tissue.

Background. Bone marrow is the mostfrequent metastatic site in follicular lymphoma, 40-70 % cases. It’s unfovourable prognostic role is stated in the index FLIPI-2 (Follicular Lymphoma International Prognostic Index-2). Objective. To study both prognostic role of bone marrow involvement and it’s relation to erythropoiesis peculiarities in follicular lymphoma was the purpose of this research. Materials and methods. Histological study was performed in 269 follicular lymphoma patients. Erythropoiesis peculiarities were studied in that patients according to standard myelogram analysis. Results. Bone marrow involvement was noted according to trephine biopsy section staining in 37,9 % of follicular lymphoma case (102 from 269). Bone marrow involvement did not influenced the prognosis (overall survival) in all period of observation (p = 0,18). Longterm survival (more than 48 months) was negatively influenced by bone marrow involvement (p = 0,04). Intertrabecular pattern of follicular lymphoma growth in bone marrow was negative prognostic factor (p = 0,02). We noted negative correlation between bone marrow involvement and the elevation of orthochromic normoblasts in bone marrow of patients with follicular lymphoma. In cause of bone marrow such elevation was noted in 67 %, and in the absense of involvement - in 78 % (p = 0,043). Elevation of orthochromic normoblasts did not influenced the overall survival of follicular lymphoma patients (p = 0,89). Conclusion. Bone marrow involvement in follicular lymphoma plays prognostically unfavourable role in long-time observation periods (later than 48 months). The most unfavourable are the intertrabecular patchy lesions. Involvement of bone marrow is in opposite relations to elevation of orthochromic normoblast, but the latter sign is of no prognostic significance.