Dmitriy Evgen'EVich Sugak

Myocardial infarction is one of the common cardiovascular diseases. Sodium overload in the myocardium is another of the most important mechanisms underlying ischemia/reperfusion injury. Na⁺ entry into the cell through ion channels can be controlled using peptide toxins targeting ion channels. In our work, we analyze the use of mu-agatoxin-Aa1a, as a sodium channel modulator, as a cytoprotective agent in ischemia/reperfusion injury. For IRI modeling in vitro, the H9C2 culture was placed under oxygen-glucose deprivation conditions followed by reperfusion. Cell death and ion dynamics were recorded using a fluorescent plate reader. Reversible myocardial ischemia of rats in vivo was created by applying an occluder under the main branch of the left coronary artery. Histological and biochemical changes were recorded. The study demonstrated the ability of mu-agatoxin-Aa1a myocardial cells to adapt to rapidly changing extracellular conditions upon restoration of normal blood flow after a period of ischemia. Thus, the study allows us to consider mu-agatoxin-Aa1a as a biologically active molecule with a targeted effect to enhance the adaptation of cardiomyocytes to OGD/reperfusion conditions.

Cutaneous melanoma is one of the most aggressive malignant neoplasms, characterized by high metastatic potential and resistance to chemotherapy and radiotherapy. Photodynamic therapy (PDT) is considered an effective local treatment for superficial melanoma due to its ability to induce selective cell death with minimal damage to surrounding tissue. Recently, riboflavin has been increasingly considered as a potential photosensitizer for PDT, due to its natural origin and low toxicity. The aim of the study is to evaluate the effect of 450 nm laser radiation, riboflavin, and their concomitant use on the intracellular concentration of sodium, potassium, and calcium ions, as well as on cell death in the B16F10 melanoma cell culture. Materials and Methods. B16F10 melanoma cells were incubated with riboflavin and exposed to laser radiation (450 nm, 4.2 J/cm2). Intracellular concentrations of sodium, potassium, and calcium ions, as well as apoptosis and necrosis levels, were assessed using fluorescence microscopy with ion-selective dyes, Yo-PRO, and propidium iodide. Quantitative analysis was performed based on corrected total cell fluorescence. Results. Laser radiation and riboflavin, when administered independenty, induced moderate changes in ion homeostasis and cell death markers. Combined photodynamic therapy resulted in a pronounced disruption of ionic balance, characterized by increased intracellular Na+ and Ca2+ concentrations and a decrease in K+ levels. Furthermore, a significant increase in fluorescent markers for apoptosis and necrosis was obsered, especially 3 hours after exposure.

The search for diagnostic tests assessing the cardiovascular disease risk is a major focus in medicine. Genetic factors together with external influence and concomitant diseases contribute to ischemic damage. Despite the active use of targeted therapy drugs, little data has been obtained on their negative impact. To solve the problem, it is proposed to use arthropod peptide toxins that have selectivity for ion channels, chemical, thermal and biological stability, and resistance to proteases. Objective: The aim of the paper is to study the effect of mu-agatoxin-Aa1a toxin on the expression profile of Scn5a, Scn4a, Cacna1c, Plce1, Elavl1, Senp, Parp1, Xiap and Orail1 genes under ischemia-reperfusion injury model. Materials and methods. H9C2 cell culture was used. The ischemia-reperfusion injury model was reproduced by incubating the cells in a low-glucose, serum and oxygen (18 h) medium, and subsequent incubation in a complete nutrient medium (2 h). The toxin was introduced at the initial stage of reperfusion. Solid-phase peptide synthesis was used for this purpose. Real-time PCR was used to measure the amount of gene expression. The primer sequence was selected in the NCBI nucleotide sequence database. Statistical processing was conducted using OriginPro 2018. Results: The mu-agatoxin-Aa1a toxin at a concentration of 50 nM in the H9C2 cell culture under ischemia-reperfusion injury contributed to the suppression of Scn4a, Cacna1c, Plce1, Orail1 and Scn5a gene expression and the enhancement of Elavl1 and Xiap gene expression. Conclusion. The obtained results provide new insights into the intracellular molecular mechanisms induced by ischemia-reperfusion injury. The mu-agatoxin-Aa1a toxin, which affects the modulation of Scn5a, Scn4a, Cacna1c, Plce1, Elavl1, Senp, Parp1, Xiap and Orail1 gene expression, can be used for targeted regulation of the main elements of the pathological reaction.

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.

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.

Ischemia–reperfusion injury (IRI) is a common phenomenon that develops both from natural causes and during major operations. Many intracellular processes mediated by calcium ions are involved in the development of IRI. Currently, chemical calcium channel blockers are used but they have a number of limitations. In this article, we study the effect of the omega-hexatoxin-Hv1a peptide toxin, an alternative to chemical calcium channel blockers, on the mechanisms of IRI development in epithelial cell culture. The toxin was produced using solid phase peptide synthesis. IRI was caused by deprivation of glucose, serum and oxygen. The data obtained demonstrate that the omega-hexatoxin-Hv1a toxin in nanomolar concentrations is able to prevent the development of apoptosis and necrosis in epithelial cells by reducing the concentration of calcium, sodium and potassium ions, as well as by delaying rapid normalization of the pH level, affecting the mitochondrial potential and oxidative stress. This toxin can be used as an alternative to chemical calcium channel blockers for preventing tissue and organ IRI due to its low-dose requirement and high bioavailability.

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.