卷 66, 编号 3 (2024)

This mini-review systematizes information on methods for quantitative assessment of intracellular hydrogen peroxide concentration based on the use of a genetically encoded peroxide sensor HyPer. Two approaches are being considered: 1) calibration of the biosensor using exogenous hydrogen peroxide, based on assessing the rate of peroxide penetration into cells and intracellular peroxidase activity; 2) direct determination of the intracellular peroxide content, based on measuring the level of oxidation of the biosensor, the oxidation reaction constant and the reduction reaction constant of HyPer in the cells. The use of these methods makes it possible to solve a wide range of tasks in cellular redox biology — to determine the range of physiological and damaging concentrations of hydrogen peroxide in cells, to evaluate the effectiveness of the antioxidant defense system in various cellular compartments under conditions of oxidative stress, to determine the contribution of various enzymatic systems to the peroxidase activity of cells, and to characterize antioxidant defense systems in various biological contexts (in the process of cellular senescence, differentiation, reprogramming, during the development of pathologies). The described methods can be adapted for other genetically encoded hydrogen peroxide biosensors.

Over the past decades, gene therapy based on the adenoviral E1A has proven its benefit against a number of tumor diseases, both in animal models and in clinical studies. It has been shown that in addition to its own antiproliferative activity, E1A also has the ability to enhance the cytotoxic effect of some anticancer drugs. The use of E1A in combination therapy can solve a number of problems in clinical oncology, among which the most pressing is the problem of drug-resistance of tumor cells. This work describes the establishment of a cell model based on human colorectal cancer cells HCT116 and cisplatin-resistant HCT116/C cells with doxycycline-inducible expression of adenoviral E1A. We have shown the concentration-dependent and time-dependent dynamics of E1A expression upon doxycycline treatment, and shown the antiproliferative effect of adenoviral E1A in the HCT116-E1A and HCT116/C-E1A cells in vitro in experiments assessing viability in MTT and clonogenic activity tests and in vivo in xenograft mouse models. Thus, as a result of our work, a model was created to explore the antiproliferative and sensitizing properties of E1A in platinum-sensitive and platinum-resistant colorectal cancer cells and to search for new approaches to anticancer therapy both in vitro and in vivo. The resulting cell line is a convenient model for selecting the most promising combinations of cytostatic drugs with E1A-based gene therapy.

The effect of N-acyl derivatives of 2-amino-4,6-di-tert-butylphenolon the functions of neutrophils was studied. It has been established that these derivatives with a free hydroxyl group in the benzene ring, in contrast to O-methylated ones, modify the properties of cells, which is expressed in a decrease in hypochlorous acid generation during the “respiratory burst” formation. These compounds are scavengers of HOCl/OCl^– generated by activated neutrophils and reduce the secretion of myeloperoxidase (MPO) from cells. N-(3,5-di-tert-butyl-2-hydroxyphenyl)acetamide has been shown to be the most effective hypochlorous acid scavenger. This substance significantly suppresses the secretory degranulation of neutrophils and has a cytoprotective effect under conditions of halogenating stress.

An important task of topical application of medicines in the treatment of eyes is to achieve a compromise between their effectiveness and safety. The development of new multifunctional local ophthalmic drug delivery systems and in vitro screening of potential medicinal eye products are key areas in solving this problem. In this study, primary in vitro screening of the effect of echinochrome (Ech), the carrageenan complex of echinochrome (CRG/Ech) and its liposomal form (CRG/Ech-Lip) was performed on cultured epithelial cells of the outer shell of the eyeball: conjunctival epithelial cells (Chang Conjunctiva, Clone 1-5c-4) and corneal epithelium human (HCE). The cell viability was assessed by their morphology and metabolic activity using light microscopy and MTT test methods. The direct dependence of the intensity of the cytotoxic effect of Ech on its concentration in the nutrient medium, the form of use, the cellular test system and the incubation time of cells was revealed. Ech in the form of an alcoholic solution in its final concentration of 0.1 mg/ml of the nutrient medium exhibits pronounced cytoxicity against both cellular test systems. The same final concentration of Ech in the nutrient medium, but already as part of the carrageenan complex of echinochrome (CRG/Ech), turned out to be critical only for the viability of corneal epithelial cells, the survival rate of conjunctival cells under these conditions was about 50 %. A high biocompatibility of the liposomal form of the carrageenan complex of echinochrome (CRG/Ech-Lip) with cells of both test systems and a stimulating cytoprotective effect against the cells of the conjunctiva epithelium was revealed.

A cell is a complex three-dimensional system, which possesses a number of highly dynamic structures with extended, rugged, and uneven morphology. The actin cytoskeleton consists of fibrillar and globular actin, as well as auxiliary proteins that regulate organization. The shape and the rearrangements of actin cytoskeleton are closely related to functioning of the cell. The ability to characterize these changes allows scientists to confirm or refute any hypotheses in the research. Obtaining a numerical equivalent of the actin cytoskeleton organization could help compare actin structures in biological experiments (example: exposure to biologically active substances). The review summarizes methods for analyzing images of intracellular actin structures labeled with phalloidin using ImageJ. The methods considered make it possible to obtain a quantitative characteristic of the organization of actin structures for further evaluation and comparison of experimental results.