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The effect of the cytostatic antitumor drug ONC201 on the number of mitochondrial nucleoids and the average size of mitochondria in the BT474 human breast cancer cells was studied. It was shown that incubation of BT474 cells with ONC201 (10 μM) for 24 h reduces the number of nucleoids from 262 ± 46 to 140 ± 27 per cell, and the number of mitochondrial nucleoids was further reduced to 67 ± 22 per cell in cells incubated with the drug for 48 h. It was found that the ONC201-induced change in the number of nucleoids per cell depended on the duration of the presence of the agent in the incubation medium. Short-term (24 h) and long-term (48 h) exposure of cells to a single dose of ONC201 led to a reversible and irreversible change in the number of nucleoids per cell, respectively. Thus, after 24 h of exposure and subsequent 120 h washout with ONC201-free medium, the number of nucleoids was restored to 211 ± 47 per cell, while 120-h washout of the drug after 48 h of treatment did not lead to the restoration of the number of nucleoids, and their number remained at the level of 70 ± 59 per cell. Changes in the number of mitochondrial nucleoids, regardless of the duration of cell treatment with ONC201, positively correlated with changes in the average size of mitochondria as an indicator of their morphology. It is concluded that ONC201 has a cytostatic effect on BT474 cells in culture, suppresses cell proliferation, and induces a reversible or irreversible decrease in the number of mitochondrial nucleoids and fragmentation of mitochondria, depending on the duration of treatment.

Previously, we found a significant increase in TRAIL (TNFα-related apoptosis-inducing ligand) resistance of acute myeloid leukemia (AML) cells in multicellular aggregates in vitro that simulate the location of leukemia cells in the bone marrow. In this study, we tried to assess whether the increased resistance of the AML cells to the cytotoxic effect of TRAIL in multicellular aggregates in vitro can be associated with the launch of mechanisms mediated by cell differentiation or the acquisition of a pro-inflammatory phenotype. The phenotype of THP-1 cells in aggregated and disaggregated cultures was studied in comparison with the phenotype of a disaggregated THP-1 cell culture exposed to factors that induce monocyte-like differentiation or pro-inflammatory phenotype. It was found that an increase in the TRAIL resistance of the THP-1 cells in the aggregated culture was accompanied by the appearance of signs characteristic of monocyte-like differentiation and a pro-inflammatory phenotype. The results are of interest for developing approaches for suppressing TRAIL resistance of AML cells in human bone marrow.

Antibiotic resistance of bacteria is a topical problem on a global scale. Sometimes vigorous human activity leads to an increase in the number of bacteria carrying resistance genes in the environment. Antimicrobial peptides (AMPs) and similar compounds are potential candidates for combating antibiotic-resistant bacteria. Previously, we proposed and successfully tested on Thermus thermophilus a new mechanism of AMP action. This mechanism of directed coaggregation is based on the interaction of a peptide capable of forming fibrils with a target protein. In this work, we discuss the criteria for choosing a target for the targeted action of AMP, describe the features of the "parental" S1 ribosomal proteins T. thermophilus and Escherichia coli and the studied peptides using bioinformatic analysis methods, assess the antimicrobial effect of the synthesized peptides on a model organism of E. coli and cytotoxicity on cells of human fibroblasts. The obtained results will be important for the creation of new AMPs for pathogenic organisms.Antibiotikorezistentnost' bakteriĭ iavliaetsia zlobodnevnoĭ problemoĭ mirovogo masshtaba. Podchas aktivnaia chelovecheskaia deiatel'nost' privodit k uvelicheniiu predstavlennosti v okruzhaiushcheĭ srede bakteriĭ, ustoĭchivykh k deĭstviiu antibiotikov. Antimikrobnye peptidy (AMP) iavliaiutsia potentsial'nymi lekarstvennymi kandidatami dlia bor'by s takimi mikroorganizmami. Ranee nami byl predlozhen i uspeshno aprobirovan na primere Thermus thermophilus novyĭ mekhanizm deĭstviia AMP, osnovannyĭ na napravlennoĭ koagregatsii (vzaimodeĭstvii peptida, sposobnogo formirovat' fibrilly s belkom-mishen'iu). V nastoiashcheĭ rabote rassmotreny kriterii vybora misheni dlia napravlennogo deĭstviia AMP, opisany osobennosti “roditel'skikh” S1 ribosomnykh belkov Thermus thermophilus i Escherichia coli i issleduemykh peptidov s primeneniem metodov bioinformaticheskogo analiza, provedena otsenka antimikrobnogo deĭstviia sintezirovannykh peptidov na model'nom organizme Escherichia coli i tsitotoksichnosti na kletkakh fibroblastov cheloveka.

The combined effect of melatonin and cytarabin at low concentration on the cells of acute promyelocytic leukemia HL-60 was studied. It has been shown that the combined effect of these substances led to a decrease in the number of cells by 70% and the mitotic activity index by 75%. It was found that the combined effect of melatonin and cytarabin also contributed to a decrease in the expression of mitochondrial proteins. It has been demonstrated that the combined use of melatonin with low concentration of cytarabin (2 nM) reduced the expression of the voltage-dependent anion channel (VDAC1). Melatonin in combination with cytarabin similarly affected the expression of translocator protein (TSPO) and antiapoptotic protein Bcl-2. The synergistic effect of melatonin in the combined action with cytarabin has been revealed, which provides an increase in the effectiveness of its antitumor action.

—The resistance of leukemic cells to the action of chemotherapeutic drugs is the main reason for insufficient efficacy in the treatment of acute myeloid leukemia. Microenvironmental factors play one of the key roles in the formation of the phenotype of drug resistance of leukemic cells. The elucidation of the nature of drug resistance mediated by the conditions of the microenvironment is important for the identification of new pharmacological targets for conservative directed therapy. We conducted a study of the role of differentiation in the macrophage direction in increasing the drug resistance of acute myeloid leukemia cells in multicellular aggregates. It was shown that maturation does not occur in the macrophage direction in multicellular aggregates. These data indicate that other mechanisms contribute to an increased drug resistance of acute myeloid leukemia cells in multicellular aggregates.

Octacalcium phosphate (OCP, Ca8H2(PO4)6·5H2O) is known to be a possible precursor of biological hydroxyapatite formation of organic bone tissue. OCP has higher biocompatibility and osseointegration rate compared to other calcium phosphates. In this work, the synthesis of low-temperature calcium phosphate compounds and substituted forms of those at physiological temperatures is shown. Strontium is used to improve bioactive properties of the material. Strontium was inserted into the OCP structure by ionic substitution in solutions. The processes of phase formation of low-temperature OCP with theoretical substitution of strontium for calcium up to 50 at.% in conditions close to physiological, i.e., temperature 35–37 °C and normal pressure, were described. The effect of strontium substitution range on changes in the crystal lattice of materials, the microstructural features, surface morphology and biological properties in vitro has been established. The results of the study indicate the effectiveness of using strontium in OCP for improving biocompatibility of OCP based composite materials intended for bone repair.

Various amyloid aggregates, in particular, aggregates of amyloid β-proteins, demonstrate in vitro and in vivo cytotoxic effects associated with impairment of cell adhesion. We investigated the effect of amyloid aggregates of smooth-muscle titin on smooth-muscle-cell cultures. The aggregates were shown to impair cell adhesion, which was accompanied by disorganization of the actin cytoskeleton, formation of filopodia, lamellipodia, and stress fibers. Cells died after a 72-h contact with the amyloid aggregates. To understand the causes of impairment, we studied the effect of the microtopology of a titin-amyloid-aggregate-coated surface on fibroblast adhesion by atomic force microscopy. The calculated surface roughness values varied from 2.7 to 4.9 nm, which can be a cause of highly antiadhesive properties of this surface. As all amyloids have the similar structure and properties, it is quite likely that the antiadhesive effect is also intrinsic to amyloid aggregates of other proteins. These results are important for understanding the mechanisms of the negative effect of amyloids on cell adhesion.

Melatonin (N-acetyl-5-methoxytryptamine MEL) is an indolamine that has antioxidant, anti-inflammatory and anti-tumor properties. Moreover, MEL is capable of exhibiting both anti-apoptotic and pro-apoptotic effects. In the normal cells, MEL possesses antioxidant property and has an anti-apoptotic effect, while in the cancer cells it has pro-apoptotic action. We investigated the combined effect of MEL and navitoclax (ABT-737), which promotes cell death, on the activation of proliferation in acute promyelocytic leukemia on a cell model HL-60. The combined effect of these compounds leads to a reduction of the index of mitotic activity. The alterations in the level of anti- and pro-apoptotic proteins such as BclxL, Bclw, Mcl-1, and BAX, membrane potential, Ca2+ retention capacity, and ROS production under the combined action of MEL and ABT-737 were performed. We obtained that MEL in combination with ABT-737 decreased Ca2+ capacity, dropped membrane potential, increased ROS production, suppressed the expression of anti-apoptotic proteins such as BclxL, Bclw, and Mcl-1, and enhanced the expression of pro-apoptotic BAX. Since, MEL modulates autophagy and endoplasmic reticulum (ER) stress in cancer cells, the combined effect of MEL and ABT-737 on the expression of ER stress and autophagy markers was checked. The combined effect of MEL and ABT-737 (0.2 &mu, M) increased the expression of protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), leading to a decrease in the level of binding immunoglobulin protein (BIP) followed by an increase in the level of C/EBP homologous protein (CHOP). In this condition, the expression of ERO1 decreased, which could lead to a decrease in the level of protein disulfide isomerase (PDI). The obtained data suggested that melatonin has potential usefulness in the treatment of cancer, where it is able to modulate ER stress, autophagy and apoptosis.

—A comparative study of the expression of pro- and anti-apoptotic receptors to cytokine TRAIL (TNF-related apoptosis-inducing ligand) on immature blast cells of acute myeloid leukemia, on the cells of acute myeloid leukemia differentiated into monocyte-like and macrophage-like cells, and on normal monocytes and macrophages has been performed. It has been shown that a decrease in the expression of proapoptotic receptors to TRAIL and the acquisition of resistance to TRAIL-induced apoptosis occurs not only during the differentiation of acute myeloid leukemia cells into monocyte-like and macrophage-like cells, but also during the maturation of normal monocytes into macrophages. Thus, the differentiation of both acute myeloid leukemia cells and normal myeloid cells in the direction of monocyte-macrophages mediates a decrease in the expression of proapoptotic receptors to TRAIL, which in turn can determine the resistance of normal myeloid cells to TRAIL-induced apoptosis.

The development and testing of new antimicrobial peptides (AMPs) represent an important milestone toward the development of new antimicrobial drugs that can inhibit the growth of pathogens and multidrug-resistant microorganisms such as Pseudomonas aeruginosa, Gram-negative bacteria. Most AMPs achieve these goals through mechanisms that disrupt the normal permeability of the cell membrane, which ultimately leads to the death of the pathogenic cell. Here, we developed a unique combination of a membrane penetrating peptide and peptides prone to amyloidogenesis to create hybrid peptide: “cell penetrating peptide + linker + amyloidogenic peptide”. We evaluated the antimicrobial effects of two peptides that were developed from sequences with different propensities for amyloid formation. Among the two hybrid peptides, one was found with antibacterial activity comparable to antibiotic gentamicin sulfate. Our peptides showed no toxicity to eukaryotic cells. In addition, we evaluated the effect on the antimicrobial properties of amino acid substitutions in the non-amyloidogenic region of peptides. We compared the results with data on the predicted secondary structure, hydrophobicity, and antimicrobial properties of the original and modified peptides. In conclusion, our study demonstrates the promise of hybrid peptides based on amyloidogenic regions of the ribosomal S1 protein for the development of new antimicrobial drugs against P. aeruginosa.

We have shown that hydroxycobalamin (vitamin B12b) increases the toxicity of diethyldithiocarbamate (DDC) to tumor cells by catalyzing the formation of disulfiram (DSF) oxi-derivatives. The purpose of this study was to elucidate the mechanism of tumor cell death induced by the combination DDC + B12b. It was found that cell death induced by DDC + B12b differed from apoptosis, autophagy, and necrosis. During the initiation of cell death, numerous vacuoles formed from ER cisterns in the cytoplasm, and cell death was partially suppressed by the inhibitors of protein synthesis and folding, the IP3 receptor inhibitor as well as by thiols. At this time, a short-term rise in the expression of ER-stress markers BiP and PERK with a steady increase in the expression of CHOP were detected. After the vacuolization of the cytoplasm, functional disorders of mitochondria and an increase in the generation of superoxide anion in them occurred. Taken together, the results obtained indicate that DDC and B12b used in combination exert a synergistic toxic effect on tumor cells by causing severe ER stress, extensive ER vacuolization, and inhibition of apoptosis, which ultimately leads to the induction of paraptosis-like cell death.

Recent demonstration that mechanism of action of small molecule imipridone ONC201 could be mediated through mitochondrial targeting (Graves et al., 2019; Greer et al., Ishizawa et al., 2019), was confirmed by identification of mitochondrial caseinolythic protease (ClpP), as the only target of ONC201 and its chemical analog TR57 (Graves et al., 2019). We further studied direct effect of these drugs on mitochondrial functions and mtDNA distribution in human breast cancer cells. Using respirometry and confocal microscopy, we demonstrated dose- and time dependent suppression of mitochondria oxygen consumption and degradation of mtDNA in cultured human BT-474 cell lines, treated with ONC201 and TR57. Both drugs, used in this study, induced significant and reversible decrease in the rate of proliferation as well as the content of mtDNA within the mitochondria (> by 50%), which was released into the cytosol. Semi-quantitative analysis of confocal images of mitochondrial matrix (MTDR, red fluorescence) and mtDNA (SYBR Green-1, green fluorescence) demonstrated 30-35% release of mtDNA from mitochondria/mitochondrial fragments. Exposure to ONC201 (10µM) or TR57 (50nM) for 48 h decreased mtDNA content by 60% and 85%, respectively. Effect of these drugs was reversible and washout of drugs restored the rate of proliferation as well as mtDNA content in time-dependent manner. Within 24h of washout, the total cellular mtDNA reached almost 90% of initial level, demonstrating fast activation of mtDNA synthesis upon removal of toxins. RT-PCR approach to monitor mtDNA in the cytosol and mitochondria confirmed release of mtDNA into the cytosol and associated decrease of mtDNA within mitochondria/mitochondrial fragments, following ONC201 or TR57 treatment of BT-474 cells. Taken together, our data demonstrate that these drugs reversibly modulate and regulate intracellular mtDNA content, through ONC201 and TR57 dependent regulation of mitochondrial biogenesis in human breast cancer cells. In conclusion, ONC201 & TR57 dependent reversible inhibition of cell proliferation and decrease in mtDNA content allow suggesting drug-dependent regulation of mitochondrial biogenesis in these BCC. Our observation warrants new direction in the elucidation of the mechanism of action of these drugs and search for novel efficient anti-cancer drugs. Support of Funding Information: Russian Science Foundation № 19-75-20145 Citation Format: Margarita I. Kobyakova, Serazhutdin Abdullaev, Yana V. Evstratova, Artem Mishukov, Irina Odinokova, Lee M. Graves, Ekhson Holmuhamedov. ONC201 & TR57 reversibly depletes mtDNA content & regulates mitochondrial biogenesis in BT-474, human breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 991.

The cytotoxicity and cytostatic effects of imipridone family drugs ONC201 and its analog TR57 (Madera, USA) in variety of cultured breast cancer cell line (estrogen dependent BT474) was manifested through induction of ISR (Integrated Stress Response), UPRER and UPRMT. The major consequences of drug treatment are inhibition of proliferation and mitochondrial structural and functional damage(s). Our preliminary data demonstrated that 24 h exposure of BT474 leads to dysregulation and suppression in mitochondrial Ca2+ uptake and Ca2+ capacity (Odinokova et al., 2021). In the present work, we extend our study to include the effect of these drugs on the rate of migration intact and treated cells in culture. Treatment of BT474 cells in culture for 24h resulted in decreased motility of cells from 17 ± 2 µm/h in the absence of the drugs to 5 ± 1 µm/h and to 3±1 µm/h in cells treated with 10µM of ONC201 or 50nM of TR57 (n=5). This suppression was fully reversible and removal of drugs for 120h resulted in time dependent restoration of the initial motility of BT474 cells, and TR57 caused larger inhibitory effect on motility as compared with ONC201. Buffering of extracellular Ca2+ with 5 mM EGTA or intracellular Ca2+ with 10 µM BAPTA, 30 min suppressed motility of cells (19 ± 2 µm/h vs 6 ± 1 µm/h). Gd3+, selective blocker of store operated Ca2+ entrance (SOCE) pathway at concentration of 10 µM suppressed the motility of BT474 cells, and rate of migration changed for from 16 ± 2 µm/h in control to 7± 2 µm/h, in Gd3+ treated cells. In line with our preliminary data, taken together these data indicate that ONC201 and/or TR57 induced dysfunction in mitochondrial Ca2+ homeostasis, which translates into the suppression of the migration of human breast cancer cells in culture. Support of Funding Information: Russian Science Foundation № 19-75-20145 Citation Format: Yana Evstratova, Margarita Kobyakova, Irina Odinokova, Alexander Stolyarov, Artyom Mishukov, Lee M. Graves, Ekhson Holmuhamedov. Reversible and dose-dependent suppression of motility of human breast cancer cells with ONC201 and/or TR57 in culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2888.

Melatonin is produced by the pineal gland. It can be regarded as an anticancer agent and used for combined therapy, owing to its oncostatic, antioxidant, and immunoregulatory activities. Retinoic acid is widely used for the treatment of acute promyelocytic leukemia, however, it has adverse effects on the human organism. We investigated the effect of melatonin and reduced concentrations of retinoic acid on the activation of proliferation in acute promyelocytic leukemiaon a cell model HL-60. The combined effect of these compounds leads to a reduction in the number of cells by 70% and the index of mitotic activity by 64%. Combined treatment with melatonin and retinoic acid decreased the expression of the Bcl-2. The mitochondrial isoform VDAC1 can be a target in the treatment of different tumors. The combined effect of and retinoic acid at a low concentration (10 nM) decreased VDAC1 expression. Melatonin in combination with retinoic acid produced a similar effect on the expression of the translocator protein. The coprecipitation of VDAC with 2&prime, 3&prime, cyclonucleotide-3&prime, phosphodiesterase implies a possible role of its in cancer development. The combined effect of retinoic acid and melatonin decreased the activity of the electron transport chain complexes. The changes in the activation of proliferation in HL-60 cells, the mitotic index, and Bcl-2 expression under combined effect of retinoic acid (10 nM) with melatonin (1 mM) are similar to changes that are induced by 1 &mu, M retinoic acid. Our results suggest that MEL is able to improve the action the other chemotherapeutic agent.