Background. Glucose is one of the main sources of energy in cells. Glucose consumption by cancer cells is markedly higher compared to normal cells and increases with malignant progression. The initial step in glucose metabolism is its transport across the plasma membrane, which is mediated by the GLUT family of glucose transporters. Although patterns of GLUT gene expression in cancer have already been identified, studying the mechanisms of their activation represents a promising approach to differentially block glucose-regulated metabolism in cancer cells. In this work, we investigated the dependence of the efficiency of adenosine triphosphate (ATP) synthesis and the regulation of the expression of the genes of classical “glucose transporters” GLUT1–GLUT4 on the amount of glucose in the culture medium.

Aim. Study of glucose-dependent ATP synthesis and expression of glucose transporter genes: GLUT1, GLUT2, GLUT3, GLUT4, involved in glucose transport in human myeloma cells RPMI8226.

Materials and methods. The human myeloma cell line RPMI8226 was used in this work. Cell viability was assessed using a colorimetric method. The effect of glucose on ATP synthesis in cells was determined using the luminescent method. The expression of mRNA in cells was studied using real-time quantitative polymerase chain reaction.

Results. The study found that ATP synthesis in RPMI8226 cells depends on glucose metabolism. Decreased viability of RPMI8226 cells strongly correlates with decreased levels of newly synthesized ATP. This cell line is characterized by relatively high initial expression of the GLUT1 gene and the GLUT3 gene, relatively moderate expression of the GLUT2 gene, and relatively weak expression of the GLUT4 gene. Studies on glucose deprivation revealed activation of the expression of all these glucose transporter genes, but the highest expression was characteristic of the GLUT2 gene and GLUT4 gene,

Conclusion. Based on the study, we conclude that for RPMI8226 myeloma cells, glucose is one of the important sources of energy metabolism. Expression of glucose transporter genes: GLUT1, GLUT2, GLUT3, GLUT4 depends on glucose concentration, but the initial level of expression does not predict the nature of its changes during glucose deprivation.

Background. Pneumolysin (Ply) – bacterial cholesterol-dependent cytolysin produced by almost all strains of Streptococcus pneumoniae. Antibodies induced to pneumolysin for some extend are able to neutralize its pathogenic effect on cells.

Aim. Investigation of the effect of monoclonal antibodies obtained to native (nPly) and recombinant (rPly) forms of Ply on the neutralization of the cytolytic activity of bacterial Ply in vitro and their preventive properties on mice challenged with a virulent strain of S. pneumoniae.

Materials and methods. mAbs to the nPly and rPly forms of Ply was obtained from the ascitic fluid of mice. Polyclonal antibodies pAbs (rPly) were obtained from the serum of rabbits immunized with rPly. The inhibition of erythrocyte hemolysis was assessed by the minimum concentration of mAbs (nPly), pAbs (rPly) and pAbs (rPly), which prevents lysis of erythrocytes induced by nPly. The neutralizing ability of antibodies was evaluated on the ovarian cells of the Chinese hamster CHO-K1 by the minimum concentration of antibodies necessary to protect cells from 4 cytopathogenic doses of nPly. The protective properties of the antibodies were studied with a single intraperitoneal injection of 200 µg of antibodies to mice, followed by challenge with a lethal dose of S. pneumoniae.

Results. It was shown that 24 hours after intraperitoneal administration of nPly to mice, the body weight of the animals decreased and degenerative forms of red blood cells appeared in the blood. In in vitro experiments, nPly caused hemolysis of erythrocytes and destruction of CHO-K1 cells by the mechanism of apoptosis. mAbs (nPly), mAbs (rPly) and pAbs (rPly) inhibited erythrocyte hemolysis and neutralized the destruction of CHO-K1 cells. The highest neutralizing activity was shown by mAbs (rPly) and pAbs (rPly). A single injection of the studied antibodies to mice caused a decrease of their death when infected with the S. pneumoniae strain. There were no differences in the protective activity of antibodies.

Conclusion. Methods based on neutralizing the toxic effect of Ply S. pneumoniae with the help of antibodies can be considered as a tool to reduce the pathogenicity of pneumococci.

Background. Ferroptosis is  a  type of  non-apoptotic iron-dependent form of  regulated cell death. Ferroportin is the only exporter of iron from cells, however, the involvement of ferroportin in cell ferroptosisis poorly understood. Activation or blocking of  ferroptosis pathways may provide a  therapeutic strategy for the  treatment of a  number of diseases. Control of ferroportin expression will make it possible to select a more effective tumor therapy. Obtaining monoclonal antibodies to human ferroportin will help to further study its role in ferroptosis of tumor cells.

Aim. Obtaining and characterization of monoclonal antibodies to human ferroportin.

Materials and methods. Mouse monoclonal antibodies against a synthetic peptide corresponding to the ferroportin protein region were obtained using hybridomic technology. The peptide was synthesized by the method of solid-phase automatic synthesis.

Results. A 15-membered peptide with the sequence ELKQLNLHKDTEPKP (ELK) was selected and synthesized. Conjugates of the synthetic peptide with carrier proteins were obtained. 4 stable hybridomic clones were obtained, and samples of monoclonal antibodies were obtained. The samples were successfully tested using methods of flow cytometry, enzyme immunoassay, and polyacrylamide gel electrophoresis.

Conclusion. Four antibodies against ferroportin binding the ELK peptide were obtained and characterized. The antibodies can be used in further studies of iron transport in tumor cells.

Background. Long-wave fluorescent proteins (FP) exhibit significant potential for in vivo investigations involving fluorescent tumor models in laboratory animals. If tumors expressing FP exhibit enhanced immunogenicity in a specific strain of immunocompetent mice as compared with the original tumors, it becomes challenging to delineate the immune component contributing to the antitumor effects of cytotoxic therapy.

Aim. To establish mouse tumor cell lines 4T1 that consistently express the original protein TagRFP and the mutant protein TagRFP-L228A, to develop tumor models based on these cell lines, and to assess the humoral immune response of Balb / c mice to such tumors.

Materials and methods. Eukaryotic plasmids pcDNA3 containing genes encoding the expression of the original protein TagRFP (TagRFP-WT) and the L228A mutant protein (TagRFP-L228A) were obtained from prokaryotic plasmids by genetic engineering methods. Individual 4T1 tumor cell clones expressing TagRFP-WT and TagRFP-L228A were obtained by sequential liposomal transfection of cells with eukaryotic plasmids, selection, and cloning. Tumor models were obtained by subcutaneous inoculation of suspensions of 4T1, 4T1-TagRFP-WT, and 4T1-TagRFP-L228A cells into female Balb / c mice. After 4 weeks, the immune response to TagRFP protein was evaluated by enzyme-linked immunosorbent assay (ELISA) by binding of mouse venous blood sera to the parent TagRFP-WT protein.

Results. Plasmids for the expression of TagRFP-WT and TagRFP-L228A proteins in eukaryotic cells and 4T1 cell clones stably expressing these proteins have been obtained. After inoculation of 4T1-TagRFP-WT, 4T1-TagRFP-L228A and 4T1 cells, tumors developed in all mice of the corresponding groups. Using an ELISA on microplates with adsorbed TagRFP-WT protein, the presence of a humoral immune response to the original fluorescent protein was shown in Balb / c mice with fluorescent tumors, in contrast to mice with 4T1 tumors. Compared to the sera of mice with the 4T1-TagRFP-WT tumors, the binding to the original protein TagRFP-WT of the sera of mice with the 4T1-TagRFP-L228A tumors decreases by more than 4 times.

Conclusion. The single amino acid substitution L228A in the TagRFP protein leads to a significant reduction in the humoral immune response to the TagRFP-L228A protein expressed by tumors compared to the original TagRFP-WT. This will allow using TagRFP-L228A expressing cell lines to create optimized tumor models for Balb / c mice.

Background. The study of new representatives of indolo[2,3-a]carbazole N-glycosides with a biological activity broad spectrum remains a relevant direction in the highly effective antitumor drugs creation.

Aim. To evaluate the efficacy of the lyophilized dosage form of a new derivative of indolocarbazole N-glycoside 6-picolinamido-12-(β-D-xylopyranosyl)indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-dione (LCS-1269-lyo) on transplantable mice colon adenocarcinoma (CAC) and on SW620 human colon cancer xenografts.

Materials and methods. In studies on female Balb / c mice with subcutaneously (s.c.) transplanted CAC, a single intravenous (i.v.) administration of LCS-1269-lyo was used at doses of 80, 90, 100, 120 mg / kg and five-time administrataion daily at doses of 80 and 90 mg / kg. The LCS-1269-lyo study on SW620 effectiveness was carried out at doses of 60 and 90 mg / kg with five-time daily intraperitoneal (i.p.) administration to male Balb / c nude mice with subcutaneously transplanted SW620 xenografts. The antitumor effect was assessed by tumor growth inhibition and an increase in the lifespan of animals receiving LCS-1269-lyo, compared to the controls.

Results. A dose-dependent reliable antitumor effect of LCS-1269-lyo was observed on CAС after a single exposure at doses of 80, 90, 100 and 120 mg / kg compared to the control group of mice. LCS-1269-lyo, when administered intravenously five times at doses of 80 and 90 mg / kg (total doses of 400 and 450 mg / kg), significantly inhibited CAC growth by 89–88 and 86–84 % (p <0,05) over 30 days of observation and increased the life span of Balb / c mice by 36 and 54 %, respectively. The LCS-1269-lyo effectiveness in a total dose of 450 mg / kg on CAC has been confirmed by the activity of LCS-1269-lyo on SW620 xenografts in Balb / c nude mice with tumor growth inhibition 72,9–71,0 % (p <0,05) during 8 days of observation.

Conclusion. The results of the antitumor activity evaluation of LCS-1269 in a lyophilized dosage form on the model of CAC mice transplantable colon adenocarcinoma and on human colon cancer SW620 subcutaneous xenografts allow to continue preclinical studies of the drug.

Background. A key component of pharmaceutical development is the optimization and justification of technological processes, since the influence of critical process parameters (KPP) on critical quality indicators (CPC) is one of the dimensions of the design field within which a medicinal product (LP) is created. This study shows the relationship between the conditions of wet granulation and the characteristics of GSB-106 tablets.

Aim. To study the effect of wet granulation PPC on the main parameters of GSB-106 tablets in the development of an optimal technological regime using the Box–Behnken plan.

Materials and methods. Equipment used: high-speed mixer granulator GSL-12 (Etorch, China), granulator with screening function WG-30 (Pharmag, Germany); manual hydraulic press PRG-50 (Russia); strength analyzer TBF 1000 (Copley Scientific, UK); disintegration analyzer SVM 221 (Erweka, Germany); PTF 3DR abrasion analyzer

(Pharma Test, Germany). The mathematical planning of the experiment was carried out using the surface response method with a three-level Box–Behnken plan, regression and variance analysis were applied, and multi-criteria optimization using generalized desirability by Derringer–Suich.

Results. To develop the technological process, 15 experiments were conducted in the entire range of varying factors, such as the duration of mixing, the speed of rotation of the blades, and the amount of granulating solution. The following pharmaceutical and technological characteristics were studied: crushing strength and disintegration of tablets at a pressing pressure of 5 kN / m², 10 kN / m², as well as abrasion resistance. Based on the data obtained, regression analysis equations were developed for each studied characteristic, the obtained models were compared by determination coefficients, the statistical significance of the equation terms was evaluated, and regression equations were optimized by getting rid of statistically insignificant equation terms. After constructing a mathematical model with adequate predictive ability, multi-criteria optimization was performed using generalized Derringer–Suich desirability.

Conclusion. Multi-criteria optimization allowed us to determine the most optimal combination of control factors in accordance with the prioritization of the desirability of pharmaceutical and technological characteristics. The identified optimal technological regime consists in adding 68.69 ml of purified water and stirring the tablet mixture for 9.92 minutes at a speed of a paddle mixer for 200 rpm, which provide the most optimal pharmaceutical and technological characteristics of the tablets.

Background. It is known that anemic syndrome (AS) is often detected in cancer patients during disease diagnosis. AS, along with tumor size and disease stage, is considered as an independent prognostic factor affecting survival.

Aim. To analyze hematological parameters of peripheral blood and ferrokinetic parameters to clarify the role of hepcidin (GP-25), interleukin 6 (IL-6) in early and differential diagnostics of AS variants in patients with rectal cancer (RC).

Materials and methods. The study was conducted in 32 patients with RC before treatment. Hematological parameters of peripheral blood were studied on a Sysmex XE-2100 analyzer (Japan). Determination of the concentration of ferritin, GP-25, IL-6, erythropoietin was performed in blood plasma using ELISA.

Results. Almost half (47 %) of the patients with RC had AS in two variants: iron deficiency anemia and functional iron deficiency (FID). All variants were characterized by microcytosis, hypochromia of erythrocytes and reticulocytes, which indicated iron deficiency erythropoiesis. However, in patients with stages III–IV of the disease and with FID, the concentration of ferritin, GP-25, IL-6, the number of erythrocyte fragmentocytes was statistically significantly higher, and the levels of erythrocytes, hemoglobin, hematocrit were lower than in patients with iron deficiency anemia. This indicated a long-term process of AS formation or anemia of chronic disease with FID. Deviations from the reference value of GP-25 and ferritin levels were detected not only in patients with AS, but also in individual patients without hematological signs of anemia, which did not exclude the latent stage of AS. Erythropoietin deficiency relative to the severity of AS was established in all variants.

Conclusion. The study was small in number, which does not allow us to draw an unambiguous conclusion about the role of GP-25 and IL-6 in the pathogenesis of the AS variant with FJ in patients with RC and requires further study.

Aim. To assess the prognostic value of TWIST and E-cadherin expression in primary tumor samples of patients with early-stage cutaneous melanoma and locally advanced rectal cancer. To determine their association with clinicopathological features, risk of progression, and treatment response.

Materials and methods. A retrospective analysis of two patient cohorts was performed. The first cohort included

101 patients with pT1a–1b cutaneous melanoma. The second cohort comprised 75 patients with locally advanced rectal cancer (T2–4N0–2M0) who underwent neoadjuvant chemoradiotherapy. In all cases, immunohistochemical assessment of TWIST and E-cadherin expression in primary tumors was carried out. Associations with disease progression, treatment effectiveness, and histopathological features were analyzed.

Results. In melanoma patients, reduced E-cadherin expression (<80 %) and elevated TWIST expression (>20 %) were significantly associated with 5-year disease progression (p <0.01). In rectal cancer patients, similar expression profiles correlated with poor response to chemoradiotherapy (tumor regression grading: 3–4), low tumor regression grade, and reduced overall and disease-free survival (p <0.05). TWIST and E-cadherin demonstrated independent prognostic significance in both cancer types.

Conclusion. Altered TWIST and E-cadherin expression in primary tumors reflects epithelial – mesenchymal transition activation, associated with aggressive clinical behavior, therapy resistance, and higher risk of disease progression. These markers may be used to refine risk stratification and guide treatment personalization.

Background. Minimal residual disease (MRD) study is a necessary step for detailed analysis of tumor clone persistence in bone marrow of patients with multiple myeloma (MM), which is important for assessing the depth of remission during treatment. MRD assessment can be useful for building a prognosis of MM and choosing patient management tactics.

Aim. To assess the frequency of MRD and its prognostic significance in patients with MM during treatment.

Materials and methods. The study included 56 patients with MM, the average age of which was 54.9 ± 1.3 years.

All patients received induction chemotherapy, which was carried out according to the Vrd regimen. After induction therapy and 100 days after autologous hematopoietic stem cell transplantation (auto-HSCT), MRD status was assessed. MRD status was assessed by multicolor flow cytometry (FACSCanto II, Kaluza Analysis v2.1 software, USA). Monoclonal antibodies: CD45, CD19, CD27, CD56, CD28, CD38, CD117, CD19, CD81, immunoglobulin light chains kapa / lambda, 7ADD (Becton Diсkinson, USA).

Results. The frequency of MRD-negative status after induction therapy was 35.7 %, after auto-HSCT – 56.7 %.

A decrease in the number of abnormal plasma cells (PC) in the bone marrow by 1.3 times was noted compared to the induction therapy stage. Conversion of MRD-positive cases was established during treatment. In the group with the PC content in the bone marrow after induction therapy more than 0.01 %, but less than 0.1 %, the MRD status changed after auto-HSCT and became negative in 53 % of patients. In the presence of PC more than 0.01 % after induction, the progression rate increased more than 2-fold and was 50.0 % (p <0.05). The average number of PCs in patients without disease progression detected after auto-HSCT was significantly lower: 0.02 % versus 0.31 %, p <0.05.

Conclusion. The frequency of MRD-negative status at the stage of induction therapy and after auto-HSCT differs, so several control points are important. After auto-HSCT, there is a conversion of MRD-positive cases to negative ones. Progression of MM was observed more often with MRD-positive status. Monitoring of MRD can help in selecting candidates for joining maintenance treatment of MM.