Evgeniy Alekseevich Beloborodov

Colorectal cancer is the third most common cancer and the second most fatal cancer worldwide. One of the strategies for colorectal cancer treatment is targeted peptide therapy that targets certain types of receptors overexpressed on the cell surface. A promising target is a bombesin receptor GRPR, which is abnormally expressed in colorectal cancer. In this paper, we suggest to use BBN/C1-C2 molecule, created on the basis of bombesin, a peptide tropic to GRPR, and knottin, acting as a scaffold to stabilize a molecule, for colorectal cancer treatment. BBN/C1-C2 acts as a GRPR receptor antagonist, which determines BBN/C1-C2 role in the survival of cancer cells. Objective: The aim of the paper is to study the effect of BBN/C1-C2 peptide on colorectal cancer cell survival. Materials and Methods. The effect of BBN/C1-C2 peptide obtained by solid-phase synthesis on cancer cell survival was assessed in HCT-116 culture using fluorescence microscopy (apoptosis, necrosis) and a cell analyzer (cell adhesion dynamics) 3 and 24 hours after exposure. Results: The BBN/C1-C2 peptide (concentrations from 0.2 to 20 μM) was able not only to inhibit cell proliferation, but also to cause cell death (apoptosis) three hours after incubation. Conclusion. BBN/C12-C2 molecule, created on the basis of a GRPR agonist built into a knottin molecule, can be considered as a prototype for a radiopharmaceutical to treat colon tumors.

Myocardial infarction is one of the most common cardiovascular diseases worldwide. Interruption of coronary blood flow subjects cardiomyocytes to stress, while reperfusion reintroduces substrate and oxygen, providing a sharp normalization of the intracellular milieu. All this paradoxically increases cell death, resulting in ischemia-reperfusion injury. It has long been known that sodium overload in the myocardium is one of the most important mechanisms underlying the ischemia-reperfusion injury. In our work, we used mu-agatoxin-Aa1a to reduce the influx of Na+ at the reperfusion stage and, thereby, to reduce damage. Objective. The aim of the study is to examine the effect of mu-agatoxin-Aa1a on cardiomyocyte cell death in an in vitro ischemia-reperfusion injury model. Materials and Methods. The study was performed on H9C2 cardiomyocyte culture. Fluorescence microscopy was used to assess the apoptosis and necrosis levels, and sodium ion concentration. Spectrophotometric method for determining lactate dehydrogenase concentration was used to confirm the necrosis level. The ischemia-reperfusion injury model was based on 18-hour culture incubation in the medium with partial glucose, serum, and oxygen deprivation, followed by transfer to a nutrient medium for 2 hours. The toxin was added at the beginning of the reperfusion stage Results: In case of increased cell death caused by apoptosis and necrosis in the ischemia-reperfusion injury model, the addition of toxin at a concentration of 50 nM causes an increase in cell survival due to a decrease in the sodium ion concentration. Conclusion. The peptide toxin mu-agatoxin-Aa1a, which targets sodium voltage-gated channels and can change their conductance, at a concentration of 50 nM significantly reduces cardiomyocyte death in an in vitro model of ischemia-reperfusion injury.

The study objective. This paper reviews the express of C/D box snoRNAs (small nucleolar RNAs) and possibility of their use as biomarkers of radioresistance in chromosomal abnormalities. Material and Methods. The study compared the values of log2FC express of snoRNA C/Dbox in radiosensitive (HL-60) and radioresistant (K562) cell lines with different levels of chromosomal abnormalities. The cells were irradiated with X-ray radiation once at a dose of 4 Gy. The expression of snoRNA C/D was evaluated 1, 4 and 24 hours after irradiation, using new generation sequencing (NGS) MiSeq. Results. Different log2FC values were obtained in HL-60 and K562 cell lines 1 hour, 4 and 24 hours after irradiation. Positive expression of C/D snoRNA prevails in HL-60 throughout the experiment. In K562, the predominance of positive values of C/D snoRNA expression was observed 4 hours after irradiation, and negative values of log2FC were observed 24 hours later. the more anomalies there were in the chromosome, the greater the difference in expression we observed. at the same time, the number of C/D snoRNA changed maximally 24 hours after irradiation in the studied cell lines. We noted a greater number of C/D snoRNAs in the HL-60 cell line, and only 3 expressed C/D snoRNAs in the 15th marker chromosome in K562 out of 16 in HL-60 in the same chromosome. Conclusion. Our study showed a low informative value of using C/D snoRNAs family as markers of radiosensitivity in the presence of chromosomal abnormalities in cancer cells.

Cancer is a leading cause of death worldwide. A promising modality for cancer treatment is peptide receptor radionuclide therapy. Therapeutic radionuclide is delivered using peptide-based vectors, which can bind to specific receptors on the cancer cell surface. Bombesin receptors are one of the receptors peculiar to many types of cancer, which can be targeted by peptide vectors. Peptides have a number of advantages, but they also have one serious drawback: low stability in the internal environment. To solve the problem, it is possible to the include a therapeutic peptide in the structure of a highly stable knottin peptide. Objective. The aim of the study is to examine the stability of BBN/C1-C2 structure, created on the basis of U5-scytotoxinSth1a knottin and bombesin tropic to bombesin receptor, and the ability of this structure to bind to target receptors on the cancer cell surface. Materials and Methods. BBN/C1-C2 peptide was obtained by solid-phase peptide synthesis. Then, is underwent chromatography purification under analytical chromatography and mass spectrometry control. Stability was studied by analytical chromatography. Competitive inhibition analysis was carried out using a fluorescently labeled GRP peptide with excess BBN/C1-C2 and fluorescently labeled BBN/C1-C2 with GRP bombesin receptor inhibitor. Cancer cell line PC-3 expressing bombesin receptors and normal cell line CHO-K1 not expressing bombesin receptors were used in the work. Results. The conducted studies have shown that hybrid BBN/C1-C2 peptide based on bombesin peptide inserted into the U5-scytotoxinSth1a knottin framework between the first and second cysteine residues has a greater stability compared to the commercial radiopharmaceutical PSMA-617. BBN/C1-C2 peptide is specific to bombesin receptor: it binds to PC-3 cancer cell line with a target bombesin receptor on its surface, and does not bind to the healthy CHO-K1 cell line, without a target receptor. BBN/C1-C2 peptide shows high affinity for the bombesin receptor, since GRP prevents its binding to the PC-3 cell line.

Ischemia contributes to many pathological conditions encountered in clinical practice. Besides, subsequent reperfusion may worsen tissue damage, exacerbating injuries caused by ischemia. Shifts in the balance of calcium and sodium ions play a major role in the development of ischemia-reperfusion injury. Inhibitors of calcium and sodium ion channels located on the membrane surface can help to avoid a sharp disturbance in the ion balance. Although such inhibitors reduce cell death, their mechanisms of action differ. The aim of the study is to conduct a comparative analysis of the mechanisms of action of peptide inhibitors of calcium and sodium channels on ischemia-reperfusion damage to epithelial cells. Materials and Methods. Peptide synthesizer was used for toxin synthesis. Chromatography and mass spectrometry were used for quality control. Analysis of cell death, changes in calcium and sodium ion concentrations, and pH levels were performed using fluorescent dyes and a multimodal reader. Results. It was found that peptide inhibitors of calcium and sodium channels reduce apoptosis and necrosis levels in CHO-K1 culture under simulated ischemia/reperfusion. The calcium channel inhibitor reduces cell death by lowering calcium and sodium ion concentrations and maintaining physiological pH levels throughout the reperfusion phase. The sodium channel inhibitor reduces death by lowering calcium and increasing sodium concentrations, and by maintaining an elevated pH throughout the reperfusion phase. Conclusion. Although both calcium and sodium concentrations as well as their mutual influence play an important role in the development of ischemia-reperfusion injury, inhibition of certain channels has different effects on intracellular processes with the same result, namely reduced cell death.

Prostate cancer is one of the most commonly diagnosed cancers worldwide. Targeted therapy is an anticancer strategy using short peptides targeting prostate-specific membrane antigen (PSMA). However, short peptides have a number of disadvantages, including low stability in vivo. This problem can be solved by using toxins with an inhibitory cystine knot with a short built in peptide. The aim of the study is to examine the stability and ability of the PSMA/C1-C2 peptide, created on the base of knottin U5-scytotoxin-Sth1a and a short peptide tropic to PSMA, to bind to receptors on the surface of prostate cancer cells and to compare the results with a market image drug PSMA I&T and a specific PSMA inhibitor. Materials and Methods. A solid-phase peptide synthesizer was used for peptide synthesis, chromatography and mass spectrometry were applied for analysis and purification. A competitive inhibition analysis was performed with radiolabeled commercial PSMA I&T in excess of PSMA/C1-C2 and FAM-labeled PSMA/C1-C2 with a specific PSMA inhibitor 2-PMPA. PSMA-positive culture 22Rv1 and PSMA-negative culture PC-3 were used as cell cultures. Chromatography was used to access peptide stability. Results. As a result, it was observed that the synthesized PSMA/C1-C2 structure, created on the basis of knottin and a short peptide, prevents binding of PSMA I&T to receptors on the surface of 22Rv1 cells. The decrease in PSMA/C1-C2 binding in the presence of 2-PMPA suggests that PSMA/C1-C2 is targeted for prostate-specific membrane antigen. In addition, PSMA/C1-C2 peptide has increased stability compared to PSMA I&T.

Ischemic and reperfusion injury is a critical condition, as it is necessary to control cell death and maintain tissue function. Restoration of nutrient and oxygen flow causes secondary damage to ischemic cells and is called reperfusion injury. Reperfusion injury causes, on the one hand, fluctuations in ion concentration inside cells, in particular sodium ions, due to changes in the conductivity of voltage-dependent ion channels, and, on the other hand, activation of the antioxidant system as a response to oxidative stress, in which the key role is given to reactive oxygen species and nitric oxide. Thus, the effect of ion channel inhibitors on the progression of oxidative stress, apoptosis and necrosis during reperfusion is of particular interest. The aim of the study is to examine the impact of sodium channels on oxidative stress under ischemic and reperfusion injury and sodium channel blockers action. Materials and Methods. The authors studied the influence of the synthesized peptide toxin, an inhibitor of voltage-gated sodium channels, under modelled ischemia/reperfusion in CHO-K1 culture on the level of apoptosis, necrosis, and oxidative stress (concentration of reactive oxygen species, nitric oxide, and glutathione) using fluorescent dyes and fluorescence microplate reader. Results. Data obtained indicate a decreased level of apoptosis and necrosis, and a control level of nitric oxide under toxin at a nanomolar concentration. At the same time, the concentrations of reactive oxygen species and glutathione did not change. Thus, the inhibitor toxin acted as a protective agent by preventing a decrease in the nitric oxide concentration, which favorably affected the survival of the cell culture during reperfusion after ischemia.

The H/ACA snoRNA family is involved in pseudouridine biogenesis. It prevents genetic changes in cells and makes them more stable due to ribosomal RNA characteristics. Therefore, the study of H/ACA snoRNA expression in cell lines with chromosomal disorders after irradiation is of particular interest. The purpose of the study is to analyze the effect of chromosomal disorders on H/ACA snoRNA expression in radioresistant K562 and radiosensitive HL-60 cell lines after radiation exposure. Materials and Methods. K562 and HL-60 cell lines were exposed to radiation (4 Gy). H/ACA snoRNA expression was analyzed by NGS sequencing (1, 4, and 24 hours after irradiation). Results. The authors revealed differences in H/ACA snoRNA expression by chromosomes in the studied cell lines, as well as the impact of chromosomal abnormalities on H/ACA snoRNA expression after radiation exposure. Changes in the copy number of normal chromosomes lead to minor changes in H/ACA snoRNA expression. Marker chromosomes disrupt H/ACA snoRNA expression. Thus, is becomes impossible to use H/ACA snoRNAs located in abnormal chromosomes as radioresistance markers. Moreover, marker chromosomes decrease the number of H/ACA snoRNAs expressed in K562, despite the greater amount of genetic material.

Despite its efficacy, radiation therapy faces the challenges connected with accelerated reproduction of tumor cells and radioresistance of malignant neoplasms. The aim of the study was to analyze the impact of the gene expression level and intermolecular interaction networks on the development of tumor cell radioresistance. Materials and Methods. The authors used 4 tumor cell lines: (K562, HCT-116p53 (+/+), HCT-116p53 (–/–), and Me45. To study the cell line transcriptome. Affymetrix high-density hybridization DNA chips (HGU133A series) were used. Bioinformatic analysis of gene expression dynamics was performed using the original Gene Selector program. Intermolecular interaction networks were studied using the STRING online system. Results. After exposure to ionizing radiation at a dose of 4 Gy, the expression level of DAAM1, IFNAR2, PALLD, and STK17A genes increases in K562 cell line and decreases in HCT-116p53 (+/+), HCT-116p53 (–/–) and Me45. Numerous protein complexes of the studied genes were found with STRING online system. Thus, DAAM1, IFNAR2, PALLD, and STK17A genes influence the activity of some particles in the network of intermolecular interactions. Selected DAAM1, IFNAR2, PALLD and STK17A genes and protein-protein complexes encoded by DAAM1, TNK2, PTBP2 and DVL2; IFNAR2, STAT2, IRF9, JAK1, GNB2L1 and IFNAR1; PALLD, LPP and ACTN2 genes can be used as potential targets. Their modulation can increase the response of malignant neoplasm cells to ionizing radiation.

Prostate cancer is the most common type of cancer in males. Approximately 1.3 million cases of prostate cancer and over 400,000 deaths from the disease are diagnosed annually. The number of deaths is expected to double by 2040. Common methods of prostate cancer treatment have many disadvantages; one of them is the relapse risk. The shortcomings of traditional therapy have led to peptide-receptor radionuclide therapy. The aim of the study is to examine binding efficiency of Lu177 labeled knottins containing PSMA-tropic peptide in different domains in vitro and their biodistribution in vivo. Materials and Methods. We used prostate cancer cell (LNCaP, PC3) and ovarian fibroblast cell (CHO-K1) cultures. The peptides were synthesized using a peptide synthesizer (ResPepSL, Intavis). We studied peptide stability, their toxicity, binding to cell cultures, and biodistribution on the example of breast adenocarcinoma-bearing BALB/c mice. Chromatographic methods and radiometric techniques were used. Results. The synthesized peptides with GTIQPYPFSWGY sequence inserted into U5-cytotoxin-Sth1a node are more stable in blood plasma and saline than 177Lu-PSMA-617a, but have a similar degree of binding. Biodistribution studies in BALB/c mice show a higher binding index of the synthesized peptide if compared to 177Lu-PSMA-617. Conclusion. Modified peptides with a PSMA-tropic peptide inserted into the structure of U5-Sth1a toxin demonstrate high stability both in saline and in blood plasma, as well as good binding to cell cultures and tumors.