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The resistance of glioblastoma to existing therapies puts limits on quality-of-life improvements and patient survival with a glioblastoma diagnosis. The development of new effective glioblastoma therapies is based on knowledge about the mechanisms governing tumor resistance to therapeutic agents. Virotherapy is one of the most actively developing approaches to the treatment of malignant neoplasms: glioblastoma in particular. Previously, we demonstrated that the recombinant vaccinia virus VV-GMCSF-Lact exhibits in vitro cytotoxic activity and in vivo antitumor efficacy against human glioblastoma. However, the studied glioblastoma cell cultures had different sensitivities to the oncotoxic effect of the virus. In this study, we investigated cancer stem cell (CSC) surface markers in glioblastoma cells with different sensitivities to VV-GMCSFLact using flow cytometry and we assessed the levels of proteins affecting viral entry into cells and virus infection efficiency by western blotting. We showed that cell cultures more sensitive to VV-GMCSF-Lact are characterized by a greater number of cells with CSC markers and a lower level of activated Akt kinase. Akt probably inhibits lactaptin-induced apoptosis in virus-resistant cells. Hence, we suggest that the sensitivity of glioblastoma cells to the oncotoxic effect of VV-GMCSF-Lact is determined by the nature and extent of the disturbances in cell death regulation in various cultures. Further investigation of the factors affecting glioblastoma resistance to virotherapy will test this hypothesis and identify targets for antitumor therapy, combined with VV-GMCSF-Lact., (Copyright ® 2022 National Research University Higher School of Economics.)

In this study we obtained and characterized the recombinant analogue of multifunctional nucleolar phosphoprotein nucleophosmin 1 (NPM1) involved in crucial cellular processes such as transcription, reparation and mitosis. The influence ofnucleophosmin 1 on extrcellular RNAs accumulation in human adenocarcinoma cells MCF-7 was analyzed. It was found that incubation of AluY RNA (n > 300 nt), U24 snoRNA analogues (n ~ 80 nt) with Npm1-His6 resulted in RNA-protein non-covalent complexes formation, but not in case of the short oligoribonucleotide (n = 22 nt). It was shown that interaction of AluY RNA analogue with Npm1-His6 significantly increases transfection efficacy of the RNA into MCF-7 human cells. Altogether, these data allow us to conclude, that nucleophosmin 1 not only binds RNA with complex secondary structure, but also promotes uptake and internalization of such RNA by human cells.

Earlier we isolated and characterized human milk pro-apoptotic peptide - lactaptin and generated its engineered analog - RL2. It was shown that both lactaptin and RL2 are capable to induce apoptotic death of MCF-7 cells. In this report we have analyzed biochemical markers of RL2 induced MCF-7 apoptosis. The activation of initiator and effector caspases as well as mitochondrial membrane potential and cytoplasm membrane changes were analyzed using flow cytometry and Western-blot methods. We have found that RL2 induced apoptotic death of MCF-7 cells was accompanied by PS exposure on the plasma membrane surface. It also was shown that RL2 has induced dissipation of mitochondrial membrane potential and resulted in activation of initiator caspases 8, 9 and effector caspase 7.

It was found that milk of clinical healthy women contains sIgA possessing high affinity for the mammalian thymus DNA and DNA-hydrolyzing activity (sIgA-abzymes). Here we present data that such sIgA-abzymes, purified by sequential chromatography on DEAE-fractogel, heparin-sepharose, DNA-cellulose and followed by gel-filtration, are also able to hydrolyse total RNA from E. coli better than plasmid DNA. Besides, such sIgA-abzymes effectively cleaved 18S and 28S ribosomal RNA isolated from human A549 cells. It is noteworthy that the nuclease activity of sIgA-abzymes was significantly inhibited by ATP, while dATP had no effect on it. A potential role of the ribonuclease activity of sIgA-abzymes present in human milk is discussed.

Interaction of secretory immunoglobulins A of a varying degree of purity with oligonucleotides and ATP has been studied by the method of affinity modification. For this aim we used reactive derivatives of 32P-labeled deoxyoligonucleotide (ClRCH2NHp(T)14) and [gamma-32P]ATP (ClR-32PppA) or ATP (ClR-pppA) bearing a 4-[(N-2-chloroethyl-N-methyl)amino]benzylamine residue. Preparations of sIgA were obtained from human milk by sequential chromatography on protein A-sepharose (P1), DEAE-fractogel (P2) and by gel-filtration in 50 mM NaOH (P3). It was revealed, that the H- and L-chains of sIgA P1; H-, L-chains and secretory component (SC) in sIgA P2 and only SC in sIgA P3 were exposed to modification after incubation with ClRCH2NHp(T)14. LPS, DNA, tRNA, heparin, sufficiently inhibited the modification of chains of sIgA P1. These competitors did not influence the modification of H- and L-chains of sIgA P2, but DNA, tRNA, heparin, inhibited binding of SC with the modifier. Suppressing affect of binding of ClRCH2NHp(T)14 with secretory component of sIgA P3 by d(T)14 has been observed as well. The research of ClR-32PppA interaction with sIgA P3 has shown that H- and L-chains of sIgA are exposed to modification. ATP inhibited the reaction. Study of the influence of modification on the protein kinase activity of sIgA P3 has revealed, that the preliminary incubation of sIgA P3 with ClR-pppA leads to inhibition of protein kinase activity. We suggest that sIgA, possessing the protein kinase activity (sIgA-abzymes) has an ATP-binding center (catalytic center) and has an oligonucleotide-binding center as well.

sIgA possessing ability to hydrolyse plasmid DNA to linear forms was purified from human milk by sequential chromatography on protein A-sepharose, DEAE-Fractogel and DNA-cellulose. It was discovered that incubation of sIgA with nuclei of porcine embryo kidney cells permeabilized by Triton X-100 causes formation of electrophoretically mobile forms of nuclear nucleic acids and inhibition of phosphorylation of nuclear proteins. We suppose that sIgA possessing affinity to DNA and endonuclease activity can cause degradation of cell nuclear chromatin.

The influence of nucleic acids (DNA, tRNA), synthetic oligonucleotides, and polysaccharides (lipopolysaccharides from Escherichia coli, heparin) on protein kinase and lipid kinase activities of preparations of human secretory immunoglobulin A (sIgA) has been studied. The preparations of sIgA were isolated from human milk by chromatography on the column with Protein A-Sepharose and DEAE-sorbent (sIgA1), by affinity chromatography of sIgA1 on DNA-cellulose (sIgA2), and by gel-filtration of sIgA1 in buffer containing 5% dioxane (sIgA3). Two 32P-labeled products with high and low electrophoretic mobility in polyacrylamide gel containing SDS were found after incubation of sIgA1 and sIgA2 with [gamma-32P]ATP. The product with low electrophoretic mobility was degraded in 10% trichloroacetic acid giving a radioactive background in lanes of the polyacrylamide gel. 32P-Labeled phospholipids were found among the phosphorylation products. Soluble and immobilized DNA increase lipid kinase activity of preparations of sIgA. In this case the secretory component and H-chains of sIgA were degraded. Fractions possessing lipid kinase activity were precipitated in the presence of heparin (1 mg/ml), and lipid kinase activity was separated from sIgA by gel-filtration in buffer containing 5% dioxane. 32P-Labeled products were formed in the presence of [gamma-32P]ATP as well as [32P]ortho-phosphoric acid. The influence of heparin and synthetic deoxy- and ribooligonucleotides on casein kinase activity of sIgA3 was studied. It was observed that deoxyribooligonucleotides in micromolar concentrations increased the rate of casein phosphorylation in the presence of sIgA3 and [gamma-32P]ATP. It has been proposed that catalytically active sIgA have an affinity to DNA (anti-DNA sIgA) and can be present in human milk as a part of lipoprotein complexes.

Oligonucleotides (ON) 4 to 60 nucleotides in length were isolated by ion-exchange chromatography on a column with Fractogel TSK DEAE-650 (M) from human milk which was hydrolyzed with proteinase K. ON from 60 to 16 nucleotides were degraded by RNase A but were resistant to DNase I, and, thus, they were ribooligonucleotides. In the presence of [gamma-32P]ATP, ON and heparin inhibited the phosphorylation of 38- and 20-kD milk proteins and failed to affect the phosphorylation of a 76-kD protein. Human milk is believed to contain polyanion-dependent and polyanion-independent protein kinases. The influence of the ON on the activity of the cytotoxic fraction of human milk alpha-lactalbumin towards human mammary gland carcinoma MCF-7 cells was studied. The ON inhibited the cytostatic and cytotoxic effects of alpha-lactalbumin. Synthetic oligonucleotides and heparin had similar effects. The endogenous ON are suggested to be involved in the regulation of cytotoxic activity of human milk.

The antibody (AB) fraction containing sIgA and IgG was isolated from human milk by Protein A-Sepharose chromatography and was shown to possess affinity to DNA-cellulose. Ion-exchange HPLC of these AB on a TSK DEAE-5PW column resulted in the isolation of a fraction containing sIgA and oligonucleotides (ON). Gel-filtration of the AB fraction revealed the presence of ON with length 4-8 nucleotides co-isolating with sIgA. sIgA Preparations purified on DEAE-Fractogel and DNA-cellulose contained lipids which were phosphorylated in the presence of [gamma-32P]ATP. The affinity of HPLC-purified IgG and sIgA to calf thymus DNA, Escherichia coli DNA and total tRNA, and plasmid DNA was demonstrated. IgG was shown to bind to thymus DNA and E. coli DNA, and sIgA was shown to bind to E. coli DNA and tRNA. Nucleic acids of intestinal microflora are supposed to participate in induction of the secretory immune response.