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Ilinskaya O. - One of the best experts on this subject based on the ideXlab platform.

  • Zeolites as carriers of antitumor ribonuclease binase
    2020
    Co-Authors: Lopati O., Zelenikhi P., Ilinskaya O.
    Abstract:

    © 2007 - 2019 Frontiers Media S.A. All Rights Reserved. Natural and synthetic zeolites have many applications in biomedicine and nutrition. Due to its properties, zeolites can absorb therapeutically active proteins and release them under physiological conditions. In this study we tested the clinoptilolite, chabazite, and natrolite ability to be loaded by antitumor ribonuclease binase and the cytotoxicity of the obtained complexes. We found the optimal conditions for binase loading into zeolites and established the dynamic of its release. Cytotoxic effects of zeolite-binase complexes toward colorectal cancer Caco2 cells were characterized after 24 and 48 h of incubation with cells using MTT-test. Zeolites were toxic by itselfs and reduced cells viability by 30% (clinoptilolite), 40% (chabazite), and 70% (natrolite) after 48 h of incubation. Binase complexes with clinoptilolite as well as chabazite always demonstrated enhanced toxicity (up to 57 and 60% for clinoptilolite and chabazite, respectively) in comparison with binase and zeolites separately. Our results contribute to the perspective development of binase-based complexes for therapy of colorectal cancer for or the treatment of malignant skin neoplasms where the complexes can be used in pasty form

  • Barnase and binase: Twins with distinct fates
    2020
    Co-Authors: Ilinskaya O.
    Abstract:

    RNases are enzymes that cleave RNAs, resulting in remarkably diverse biological consequences. Many RNases are cytotoxic. In some cases, they attack selectively malignant cells triggering an apoptotic response. A number of eukaryotic and bacterial RNase-based strategies are being developed for use in anticancer and antiviral therapy. However, the physiological functions of these RNases are often poorly understood. This review focuses on the properties of the extracellular RNases from Bacillus amyloliquefaciens (barnase) and Bacillus intermedius (binase), the characteristics of their biosynthesis regulation and their physiological role, with an emphasis on the similarities and differences. Barnase and binase can be regarded as molecular twins according to their highly similar structure, physical-chemical and catalytic properties. Nevertheless, the 'life paths' of these enzymes are not the same, as their expression in bacteria is controlled by diverse signals. Binase is predominantly synthesized under phosphate starvation, whereas barnase production is strictly dependent on the multifunctional Spo0A regulator controlling sporulation, biofilm formation and cannibalism. Barnase and binase also have some distinctions in practical applications. Barnase was initially suggested to be useful in research and biotechnology as a tool for studying protein-protein interactions, for RNA elimination from biological samples, for affinity purification of RNase fusion proteins, for the development of cloning vectors and for sterility acquisition by transgenic plants. Binase, as later barnase, was tested for antiviral, antitumour and immunogenic effects. Both RNases have found their own niche in cancer research as a result of success in targeted delivery and selectivity towards tumour cells. A number of eukaryotic and bacterial RNase-based strategies are being developed for use in anticancer and antiviral therapy. However, the physiological functions of these RNases are often poorly understood. Barnase and binase can be regarded as molecular twins according to their highly similar structural, physical-chemical and catalytic properties. Nevertheless, their expression in bacteria is controlled by diverse signals. © 2011 FEBS

  • Anti-influenza activity of the ribonuclease binase: Cellular targets detected by quantitative proteomics
    2020
    Co-Authors: Mahmud R.s., Mostafa A., Pleschka S., Dudkina E., Laikov A., Markelova M., Ilinskaya O.
    Abstract:

    © 2020 by the authors. Licensee MDPI, Basel, Switzerland. Unpredictable influenza pandemics, annual epidemics, and sporadic poultry-to-human avian influenza virus infections with high morbidity and mortality rates dictate a need to develop new antiviral approaches. Targeting cellular pathways and processes is a promising antiviral strategy shown to be effective regardless of viral subtypes or viral evolution of drug-resistant variants. Proteomics-based searches provide a tool to reveal the druggable stages of the virus life cycle and to understand the putative antiviral mode of action of the drug(s). Ribonucleases (RNases) of different origins not only demonstrate antiviral effects that are mediated by the direct RNase action on viral and cellular RNAs but can also exert their impact by signal transduction modulation. To our knowledge, studies of the RNase-affected cell proteome have not yet been performed. To reveal cellular targets and explain the mechanisms underlying the antiviral effect employed by the small extra-cellular ribonuclease of Bacillus pumilus (binase) both in vitro and in vivo, qualitative shotgun and quantitative targeted proteomic analyses of the influenza A virus (IAV) H1N1pdm09-infected A549 cells upon binase treatment were performed. We compared proteomes of mock-treated, binase-treated, virus-infected, and virus-infected binase-treated cells to determine the proteins affected by IAV and/or binase. In general, IAV demonstrated a downregulating strategy towards cellular proteins, while binase had an upregulating effect. With the help of bioinformatics approaches, coregulated cellular protein sets were defined and assigned to their biological function; a possible interconnection with the progression of viral infection was conferred. Most of the proteins downregulated by IAV (e.g., AKR1B1, AKR1C1, CCL5, PFN1, RAN, S100A4, etc.) belong to the processes of cellular metabolism, response to stimulus, biological regulation, and cellular localization. Upregulated proteins upon the binase treatment (e.g., AKR1B10, CAP1, HNRNPA2B1, PFN1, PPIA, YWHAB, etc.) are united by the processes of biological regulation, cellular localization, and immune and metabolic processes. The antiviral activity of binase against IAV was expressed by the inversion of virus-induced proteomic changes, resulting in the inhibition of virus-associated processes, including nuclear ribonucleoprotein export (NCL, NPM1, Nup205, and Bax proteins involved) and cytoskeleton remodeling (RDX, PFN1, and TUBB) induced by IAV at the middle stage of single-cycle infection in A549 cells. Modulation of the immune response could be involved as well. Overall, it seems possible that binase exerts its antiviral effects in multiple ways

  • Extracellular ribonuclease from bacillus licheniformis (Balifase), a New member of the N1/T1 RNase superfamily
    2020
    Co-Authors: Sokurenko Y., Nadyrova A., Ilinskaya O.
    Abstract:

    © 2016 Yulia Sokurenko et al.The N1/T1 RNase superfamily comprises enzymes with well-established antitumor effects, such as ribotoxins secreted by fungi, primarily by Aspergillus and Penicillium species, and bacterial RNase secreted by B. pumilus (binase) and B. amyloliquefaciens (barnase). RNase is regarded as an alternative to classical chemotherapeutic agents due to its selective cytotoxicity towards tumor cells. New RNase with a high degree of structural similarity with binase (73%) and barnase (74%) was isolated and purified from Bacillus licheniformis (balifase, calculated molecular weight 12421.9 Da, pI 8.91). The protein sample with enzymatic activity of 1.5 × 106 units/A280was obtained. The physicochemical properties of balifase are similar to those of barnase. However, in terms of its gene organization and promoter activity, balifase is closer to binase. The unique feature of balifase gene organization consists in the fact that genes of RNase and its inhibitor are located in one operon. Similarly to biosynthesis of binase, balifase synthesis is induced under phosphate starvation; however, in contrast to binase, balifase does not form dimers under natural conditions. We propose that the highest stability of balifase among analyzed RNase types allows the protein to retain its structure without oligomerization

  • Binase penetration into alveolar epithelial cells does not induce cell death
    2020
    Co-Authors: Cabrera-fuentes H., Kalacheva N., Zelenikhin P., Preissner K., Kolpakov A., Barreto G., Mukhametshina R., Ilinskaya O.
    Abstract:

    Microbial ribonucleases possess a broad spectrum of biological activities, which demonstrate stimulating properties at low concentrations and cytotoxicity and genotoxicity at high concentrations. Mechanisms of their penetration into the cells still remain unclear. In this study penetration of Bacillus intermedius RNase (binase) in alveolar lung epithelial cells, type II (ATII) pneumocytes, has been investigated. Using immunofluorescence analysis we have shown for the first time internalization of binase by primary non-differentiated pneumocytes ATII. The enzyme did not penetrate in MLE-12 (Murine Lung Epithelial-12 cells). However, binase was cytotoxic towards tumor MLE-12 cells, but not ATII cells. These results clearly indicate higher sensitivity of tumor cells to binase compared to normal cells; they also demonstrate that penetration of the enzyme into alveolar epithelial cells is not directly associated with their death. © 2012 Pleiades Publishing, Ltd

Vera Ulyanova - One of the best experts on this subject based on the ideXlab platform.

  • Bacterial ribonuclease binase exerts an intra-cellular anti-viral mode of action targeting viral RNAs in influenza a virus-infected MDCK-II cells
    Virology Journal, 2018
    Co-Authors: Raihan Shah Mahmud, Christin Müller, Ahmed Mostafa, Vera Ulyanova, O N Ilinskaya, Pumaree Kanrai, Yulia Sokurenko, Julia Dzieciolowski, Irina Kuznetsova, Stephan Pleschka
    Abstract:

    Background Influenza is a severe contagious disease especially in children, elderly and immunocompromised patients. Beside vaccination, the discovery of new anti-viral agents represents an important strategy to encounter seasonal and pandemic influenza A virus (IAV) strains. The bacterial extra-cellular ribonuclease binase is a well-studied RNase from Bacillus pumilus . Treatment with binase was shown to improve survival of laboratory animals infected with different RNA viruses. Although binase reduced IAV titer in vitro and in vivo, the mode of action (MOA) of binase against IAV at the molecular level has yet not been studied in depth and remains elusive. Methods To analyze whether binase impairs virus replication by direct interaction with the viral particle we applied a hemagglutination inhibition assay and monitored the integrity of the viral RNA within the virus particle by RT-PCR. Furthermore, we used Western blot and confocal microscopy analysis to study whether binase can internalize into MDCK-II cells. By primer extension we examined the effect of binase on the integrity of viral RNAs within the cells and using a mini-genome system we explored the effect of binase on the viral expression. Results We show that (i) binase does not to attack IAV particle-protected viral RNA, (ii) internalized binase could be detected within the cytosol of MDCK-II cells and that (iii) binase impairs IAV replication by specifically degrading viral RNA species within the infected MDCK-II cells without obvious effect on cellular mRNAs. Conclusion Our data provide novel evidence suggesting that binase is a potential anti-viral agent with specific intra-cellular MOA.

  • bacterial ribonuclease binase exerts an intra cellular anti viral mode of action targeting viral rnas in influenza a virus infected mdck ii cells
    Virology Journal, 2018
    Co-Authors: Raihan Shah Mahmud, Christin Müller, Ahmed Mostafa, Vera Ulyanova, Pumaree Kanrai, Yulia Sokurenko, Julia Dzieciolowski, Irina Kuznetsova, O N Ilinskaya
    Abstract:

    Influenza is a severe contagious disease especially in children, elderly and immunocompromised patients. Beside vaccination, the discovery of new anti-viral agents represents an important strategy to encounter seasonal and pandemic influenza A virus (IAV) strains. The bacterial extra-cellular ribonuclease binase is a well-studied RNase from Bacillus pumilus. Treatment with binase was shown to improve survival of laboratory animals infected with different RNA viruses. Although binase reduced IAV titer in vitro and in vivo, the mode of action (MOA) of binase against IAV at the molecular level has yet not been studied in depth and remains elusive. To analyze whether binase impairs virus replication by direct interaction with the viral particle we applied a hemagglutination inhibition assay and monitored the integrity of the viral RNA within the virus particle by RT-PCR. Furthermore, we used Western blot and confocal microscopy analysis to study whether binase can internalize into MDCK-II cells. By primer extension we examined the effect of binase on the integrity of viral RNAs within the cells and using a mini-genome system we explored the effect of binase on the viral expression. We show that (i) binase does not to attack IAV particle-protected viral RNA, (ii) internalized binase could be detected within the cytosol of MDCK-II cells and that (iii) binase impairs IAV replication by specifically degrading viral RNA species within the infected MDCK-II cells without obvious effect on cellular mRNAs. Our data provide novel evidence suggesting that binase is a potential anti-viral agent with specific intra-cellular MOA.

  • Ribonuclease from Bacillus Acts as an Antiviral Agent against Negative- and Positive-Sense Single Stranded Human Respiratory RNA Viruses
    BioMed research international, 2017
    Co-Authors: Raihan Shah Mahmud, Christin Müller, Yulia Romanova, Ahmed Mostafa, Vera Ulyanova, Stephan Pleschka, Olga N. Ilinskaya
    Abstract:

    Bacillus pumilus ribonuclease (binase) was shown to be a promising antiviral agent in animal models and cell cultures. However, the mode of its antiviral action remains unknown. To assess the binase effect on intracellular viral RNA we have selected single stranded negative- and positive-sense RNA viruses, influenza virus, and rhinovirus, respectively, which annually cause respiratory illnesses and are characterized by high contagious nature, mutation rate, and antigen variability. We have shown that binase exerts an antiviral effect on both viruses at the same concentration, which does not alter the spectrum of A549 cellular proteins and expression of housekeeping genes. The titers of influenza A (H1N1pdm) virus and human rhinovirus serotype 1A were reduced by 40% and 65%, respectively. A preincubation of influenza virus with binase before infection significantly reduced viral titer after single-cycle replication of the virus. Using influenza A virus mini genome system we showed that binase reduced GFP reporter signaling indicating a binase action on the expression of viral mRNA. Binase reduced the level of H1N1pdm viral NP mRNA accumulation in A549 cells by 20%. Since the viral mRNA is a possible target for binase this agent could be potentially applied in the antiviral therapy against both negative- and positive-sense RNA viruses.

  • extracellular ribonuclease from bacillus licheniformis balifase a new member of the n1 t1 rnase superfamily
    BioMed Research International, 2016
    Co-Authors: Yulia Sokurenko, Vera Ulyanova, Alsu I Nadyrova, O N Ilinskaya
    Abstract:

    The N1/T1 RNase superfamily comprises enzymes with well-established antitumor effects, such as ribotoxins secreted by fungi, primarily by Aspergillus and Penicillium species, and bacterial RNase secreted by B. pumilus (binase) and B. amyloliquefaciens (barnase). RNase is regarded as an alternative to classical chemotherapeutic agents due to its selective cytotoxicity towards tumor cells. New RNase with a high degree of structural similarity with binase (73%) and barnase (74%) was isolated and purified from Bacillus licheniformis (balifase, calculated molecular weight 12421.9 Da, pI 8.91). The protein sample with enzymatic activity of 1.5 × 106 units/A280 was obtained. The physicochemical properties of balifase are similar to those of barnase. However, in terms of its gene organization and promoter activity, balifase is closer to binase. The unique feature of balifase gene organization consists in the fact that genes of RNase and its inhibitor are located in one operon. Similarly to biosynthesis of binase, balifase synthesis is induced under phosphate starvation; however, in contrast to binase, balifase does not form dimers under natural conditions. We propose that the highest stability of balifase among analyzed RNase types allows the protein to retain its structure without oligomerization.

  • direct inhibition of oncogenic kras by bacillus pumilus ribonuclease binase
    Biochimica et Biophysica Acta, 2016
    Co-Authors: O N Ilinskaya, Vera Ulyanova, Indrabahadur Singh, Elena Dudkina, Airat R Kayumov, Guillermo Barreto
    Abstract:

    RAS proteins function as molecular switches that transmit signals from cell surface receptors into specific cellular responses via activation of defined signaling pathways (Fang, 2015). Aberrant constitutive RAS activation occurs with high incidence in different types of cancer (Bos, 1989). Thus, inhibition of RAS-mediated signaling is extremely important for therapeutic approaches against cancer. Here we showed that the ribonuclease (RNase) binase, directly interacts with endogenous KRAS. Further, molecular structure models suggested an inhibitory nature of binase-RAS interaction involving regions of RAS that are important for different aspects of its function. Consistent with these models, phosphorylation analysis of effectors of RAS-mediated signaling revealed that binase inhibits the MAPK/ERK signaling pathway. Interestingly, RAS activation assays using a non-hydrolysable GTP analog (GTPγS) demonstrated that binase interferes with the exchange of GDP by GTP. Furthermore, we showed that binase reduced the interaction of RAS with the guanine nucleotide exchange factor (GEF), SOS1. Our data support a model in which binase-KRAS interaction interferes with the function of GEFs and stabilizes the inactive GDP-bound conformation of RAS thereby inhibiting MAPK/ERK signaling. This model plausibly explains the previously reported, antitumor-effect of binase specific towards RAS-transformed cells and suggests the development of anticancer therapies based on this ribonuclease.

O N Ilinskaya - One of the best experts on this subject based on the ideXlab platform.

  • Zeolites as Carriers of Antitumor Ribonuclease Binase
    Frontiers Media S.A., 2019
    Co-Authors: Vera Khojaewa, Oleg Lopatin, Pavel Zelenikhin, O N Ilinskaya
    Abstract:

    Natural and synthetic zeolites have many applications in biomedicine and nutrition. Due to its properties, zeolites can absorb therapeutically active proteins and release them under physiological conditions. In this study we tested the clinoptilolite, chabazite, and natrolite ability to be loaded by antitumor ribonuclease binase and the cytotoxicity of the obtained complexes. We found the optimal conditions for binase loading into zeolites and established the dynamic of its release. Cytotoxic effects of zeolite-binase complexes toward colorectal cancer Caco2 cells were characterized after 24 and 48 h of incubation with cells using MTT-test. Zeolites were toxic by itselfs and reduced cells viability by 30% (clinoptilolite), 40% (chabazite), and 70% (natrolite) after 48 h of incubation. Binase complexes with clinoptilolite as well as chabazite always demonstrated enhanced toxicity (up to 57 and 60% for clinoptilolite and chabazite, respectively) in comparison with binase and zeolites separately. Our results contribute to the perspective development of binase-based complexes for therapy of colorectal cancer for or the treatment of malignant skin neoplasms where the complexes can be used in pasty form

  • Bacterial ribonuclease binase exerts an intra-cellular anti-viral mode of action targeting viral RNAs in influenza a virus-infected MDCK-II cells
    Virology Journal, 2018
    Co-Authors: Raihan Shah Mahmud, Christin Müller, Ahmed Mostafa, Vera Ulyanova, O N Ilinskaya, Pumaree Kanrai, Yulia Sokurenko, Julia Dzieciolowski, Irina Kuznetsova, Stephan Pleschka
    Abstract:

    Background Influenza is a severe contagious disease especially in children, elderly and immunocompromised patients. Beside vaccination, the discovery of new anti-viral agents represents an important strategy to encounter seasonal and pandemic influenza A virus (IAV) strains. The bacterial extra-cellular ribonuclease binase is a well-studied RNase from Bacillus pumilus . Treatment with binase was shown to improve survival of laboratory animals infected with different RNA viruses. Although binase reduced IAV titer in vitro and in vivo, the mode of action (MOA) of binase against IAV at the molecular level has yet not been studied in depth and remains elusive. Methods To analyze whether binase impairs virus replication by direct interaction with the viral particle we applied a hemagglutination inhibition assay and monitored the integrity of the viral RNA within the virus particle by RT-PCR. Furthermore, we used Western blot and confocal microscopy analysis to study whether binase can internalize into MDCK-II cells. By primer extension we examined the effect of binase on the integrity of viral RNAs within the cells and using a mini-genome system we explored the effect of binase on the viral expression. Results We show that (i) binase does not to attack IAV particle-protected viral RNA, (ii) internalized binase could be detected within the cytosol of MDCK-II cells and that (iii) binase impairs IAV replication by specifically degrading viral RNA species within the infected MDCK-II cells without obvious effect on cellular mRNAs. Conclusion Our data provide novel evidence suggesting that binase is a potential anti-viral agent with specific intra-cellular MOA.

  • bacterial ribonuclease binase exerts an intra cellular anti viral mode of action targeting viral rnas in influenza a virus infected mdck ii cells
    Virology Journal, 2018
    Co-Authors: Raihan Shah Mahmud, Christin Müller, Ahmed Mostafa, Vera Ulyanova, Pumaree Kanrai, Yulia Sokurenko, Julia Dzieciolowski, Irina Kuznetsova, O N Ilinskaya
    Abstract:

    Influenza is a severe contagious disease especially in children, elderly and immunocompromised patients. Beside vaccination, the discovery of new anti-viral agents represents an important strategy to encounter seasonal and pandemic influenza A virus (IAV) strains. The bacterial extra-cellular ribonuclease binase is a well-studied RNase from Bacillus pumilus. Treatment with binase was shown to improve survival of laboratory animals infected with different RNA viruses. Although binase reduced IAV titer in vitro and in vivo, the mode of action (MOA) of binase against IAV at the molecular level has yet not been studied in depth and remains elusive. To analyze whether binase impairs virus replication by direct interaction with the viral particle we applied a hemagglutination inhibition assay and monitored the integrity of the viral RNA within the virus particle by RT-PCR. Furthermore, we used Western blot and confocal microscopy analysis to study whether binase can internalize into MDCK-II cells. By primer extension we examined the effect of binase on the integrity of viral RNAs within the cells and using a mini-genome system we explored the effect of binase on the viral expression. We show that (i) binase does not to attack IAV particle-protected viral RNA, (ii) internalized binase could be detected within the cytosol of MDCK-II cells and that (iii) binase impairs IAV replication by specifically degrading viral RNA species within the infected MDCK-II cells without obvious effect on cellular mRNAs. Our data provide novel evidence suggesting that binase is a potential anti-viral agent with specific intra-cellular MOA.

  • extracellular ribonuclease from bacillus licheniformis balifase a new member of the n1 t1 rnase superfamily
    BioMed Research International, 2016
    Co-Authors: Yulia Sokurenko, Vera Ulyanova, Alsu I Nadyrova, O N Ilinskaya
    Abstract:

    The N1/T1 RNase superfamily comprises enzymes with well-established antitumor effects, such as ribotoxins secreted by fungi, primarily by Aspergillus and Penicillium species, and bacterial RNase secreted by B. pumilus (binase) and B. amyloliquefaciens (barnase). RNase is regarded as an alternative to classical chemotherapeutic agents due to its selective cytotoxicity towards tumor cells. New RNase with a high degree of structural similarity with binase (73%) and barnase (74%) was isolated and purified from Bacillus licheniformis (balifase, calculated molecular weight 12421.9 Da, pI 8.91). The protein sample with enzymatic activity of 1.5 × 106 units/A280 was obtained. The physicochemical properties of balifase are similar to those of barnase. However, in terms of its gene organization and promoter activity, balifase is closer to binase. The unique feature of balifase gene organization consists in the fact that genes of RNase and its inhibitor are located in one operon. Similarly to biosynthesis of binase, balifase synthesis is induced under phosphate starvation; however, in contrast to binase, balifase does not form dimers under natural conditions. We propose that the highest stability of balifase among analyzed RNase types allows the protein to retain its structure without oligomerization.

  • direct inhibition of oncogenic kras by bacillus pumilus ribonuclease binase
    Biochimica et Biophysica Acta, 2016
    Co-Authors: O N Ilinskaya, Vera Ulyanova, Indrabahadur Singh, Elena Dudkina, Airat R Kayumov, Guillermo Barreto
    Abstract:

    RAS proteins function as molecular switches that transmit signals from cell surface receptors into specific cellular responses via activation of defined signaling pathways (Fang, 2015). Aberrant constitutive RAS activation occurs with high incidence in different types of cancer (Bos, 1989). Thus, inhibition of RAS-mediated signaling is extremely important for therapeutic approaches against cancer. Here we showed that the ribonuclease (RNase) binase, directly interacts with endogenous KRAS. Further, molecular structure models suggested an inhibitory nature of binase-RAS interaction involving regions of RAS that are important for different aspects of its function. Consistent with these models, phosphorylation analysis of effectors of RAS-mediated signaling revealed that binase inhibits the MAPK/ERK signaling pathway. Interestingly, RAS activation assays using a non-hydrolysable GTP analog (GTPγS) demonstrated that binase interferes with the exchange of GDP by GTP. Furthermore, we showed that binase reduced the interaction of RAS with the guanine nucleotide exchange factor (GEF), SOS1. Our data support a model in which binase-KRAS interaction interferes with the function of GEFs and stabilizes the inactive GDP-bound conformation of RAS thereby inhibiting MAPK/ERK signaling. This model plausibly explains the previously reported, antitumor-effect of binase specific towards RAS-transformed cells and suggests the development of anticancer therapies based on this ribonuclease.

Olga N. Ilinskaya - One of the best experts on this subject based on the ideXlab platform.

  • Ribonuclease from Bacillus Acts as an Antiviral Agent against Negative- and Positive-Sense Single Stranded Human Respiratory RNA Viruses
    BioMed research international, 2017
    Co-Authors: Raihan Shah Mahmud, Christin Müller, Yulia Romanova, Ahmed Mostafa, Vera Ulyanova, Stephan Pleschka, Olga N. Ilinskaya
    Abstract:

    Bacillus pumilus ribonuclease (binase) was shown to be a promising antiviral agent in animal models and cell cultures. However, the mode of its antiviral action remains unknown. To assess the binase effect on intracellular viral RNA we have selected single stranded negative- and positive-sense RNA viruses, influenza virus, and rhinovirus, respectively, which annually cause respiratory illnesses and are characterized by high contagious nature, mutation rate, and antigen variability. We have shown that binase exerts an antiviral effect on both viruses at the same concentration, which does not alter the spectrum of A549 cellular proteins and expression of housekeeping genes. The titers of influenza A (H1N1pdm) virus and human rhinovirus serotype 1A were reduced by 40% and 65%, respectively. A preincubation of influenza virus with binase before infection significantly reduced viral titer after single-cycle replication of the virus. Using influenza A virus mini genome system we showed that binase reduced GFP reporter signaling indicating a binase action on the expression of viral mRNA. Binase reduced the level of H1N1pdm viral NP mRNA accumulation in A549 cells by 20%. Since the viral mRNA is a possible target for binase this agent could be potentially applied in the antiviral therapy against both negative- and positive-sense RNA viruses.

  • internalization of bacillus intermedius ribonuclease binase induces human alveolar adenocarcinoma cell death
    Toxicon, 2013
    Co-Authors: Hector A Cabrerafuentes, P V Zelenikhin, Klaus T Preissner, Muhammad Aslam, Mona Saffarzadeh, Alexei Kolpakov, Olga N. Ilinskaya
    Abstract:

    Ribonuclease (RNase) treatment represents a novel mechanism based approach to anticancer therapy as an alternative to the DNA damaging drugs commonly used in clinical practice. Apart from their ribonucleolytic activity, cytotoxic effects have attracted a considerable attention to RNases because of their potential as selective agents for treatment of certain malignancies. Among these enzymes, Binase, an RNase from Bacillus intermedius, has shown promising results. Here, we have found that binase selectively attacked human A549 alveolar adenocarcinoma cells to trigger an apoptotic response, whereas normal lung epithelial cells LEK were not affected by the ribonuclease. The tumor transformation led to the modification of certain cellular characteristics causing cell sensitivity to binase. Although a general mode for RNases cytotoxicity includes their penetration into the cell, translocation to the cytosol and degradation of ribonucleic acid, many aspects of this process have not been fully elucidated. Our data revealed the following time-dependent changes induced by binase in A549 cells: (a) fast permanent internalization of the enzyme during the first hours of treatment; (b) temporary increase in cellular permeability for macromolecules during the 4–6 h of treatment; (c) apoptotic alterations in population after 24 h and (d) DNA fragmentation and cell death after 72 h of treatment with binase. Elucidation of these molecular strategies used by this promising toxin provides us essential information for the development of new anticancer drugs.

  • binase penetration into alveolar epithelial cells does not induce cell death
    Biochemistry (moscow) Supplement Series B: Biomedical Chemistry, 2012
    Co-Authors: Hector A Cabrerafuentes, N V Kalacheva, Regina T Mukhametshina, P V Zelenikhin, A I Kolpakov, Guillermo Barreto, Klaus T Preissner, Olga N. Ilinskaya
    Abstract:

    Microbial ribonucleases possess a broad spectrum of biological activities, which demonstrate stimulating properties at low concentrations and cytotoxicity and genotoxicity at high concentrations. Mechanisms of their penetration into the cells still remain unclear. In this study penetration of Bacillus intermedius RNase (binase) in alveolar lung epithelial cells, type II (ATII) pneumocytes, has been investigated. Using immunofluorescence analysis we have shown for the first time internalization of binase by primary non-differentiated pneumocytes ATII. The enzyme did not penetrate in MLE-12 (Murine Lung Epithelial-12 cells). However, binase was cytotoxic towards tumor MLE-12 cells, but not ATII cells. These results clearly indicate higher sensitivity of tumor cells to binase compared to normal cells; they also demonstrate that penetration of the enzyme into alveolar epithelial cells is not directly associated with their death.

Zelenikhin P. - One of the best experts on this subject based on the ideXlab platform.

  • Antitumor macrophage response to bacillus pumilus ribonuclease (Binase)
    2020
    Co-Authors: Makeeva A., Rodriguez-montesinos J., Zelenikhin P., Nesmelov A., Preissner K., Cabrera-fuentes H., Ilinskaya O.
    Abstract:

    © Copyright 2017 Anna Makeeva et al. Extracellular bacterial ribonucleases such as binase from Bacillus pumilus possess cytotoxic activity against tumor cells with a potential for clinical application. Moreover, they may induce activation of tumor-derived macrophages either into the M1-phenotype with well-documented functions in the regulation of the antitumor immune response or into M2-macrophages that may stimulate tumor growth, metastasis, and angiogenesis. In this study, binase or endogenous RNase1 (but not RNA or short oligonucleotides) stimulated the expression of activated NF-κB p65 subunit in macrophages. Since no changes in MyD88 and TRIF adaptor protein expression were observed, toll-like receptors may not be involved in RNase-related NF-κB pathway activation. In addition, short exposure (0.5 hr) to binase induced the release of cytokines such as IL-6, MCP-1, or TNF-α (but not IL-4 and IL-10), indicative for the polarization into antitumor M1-macrophages. Thus, we revealed increased expression of activated NF-κB p65 subunit in macrophages upon stimulation by binase and RNase1, but not RNA or short oligonucleotides

  • Binase cleaves cellular noncoding RNAs and affects coding mRNAs
    2020
    Co-Authors: Tchurikov N., Makarov A., Zelenikhin P., Ilinskaya O.
    Abstract:

    Bacterial RNases are promising tools for the development of anticancer drugs. Neoplastic transformation leads to enhanced accumulation of rRNA and tRNA, and altered expression of regulatory noncoding RNAs. Cleavage of RNA in cancer cells is the main reason for the cytotoxic effects of exogenic RNases. We have shown that binase, a cytotoxic ribonuclease from Bacillus intermedius, affects the total amount of intracellular RNA and the expression of proapoptotic and antiapoptotic mRNAs. For four cell lines, we visualized cellular RNA by fluorescence microscopy, and determined RNA levels, viability and apoptosis by flow cytometry. We found that the level of cellular RNA was decreased in cells that were sensitive to the cytotoxic effects of binase. The RNA level was lowered by 44% in HEK cells transfected with the hSK4 gene of the Ca 2+-activated potassium channels (HEKhSK4) and by 20% in kit-transformed myeloid progenitor FDC-P1iR1171 cells. The most significant decrease in RNA levels was registered in the subpopulations of apoptotic cells. However, the binase-induced RNA decrease did not correlate with apoptosis. Kit-transformed cells with binase-induced RNA decrease retained viability if the interleukin-dependent proliferation pathway was activated. Using quantitative RT-PCR with RNA samples isolated from the binase-treated HEKhSK4 cells, we found that the amount of mRNA of the antiapoptotic bcl-2 gene in vivo was reduced about two-fold. In contrast, expression of the proapoptotic genes p53 and hSK4 was increased 1.5-fold and 4.3-fold, respectively. These results show that binase is a regulator of RNA-dependent processes of cell proliferation and apoptosis. © 2009 FEBS

  • The combined action of binase and bleomycin on human lung adenocarcinoma cells
    2020
    Co-Authors: Zelenikhin P., Makeeva A., Nguen T., Siraj Y., Ilinskaya O.
    Abstract:

    © 2016, Pleiades Publishing, Ltd.Some microbial ribonucleases (RNases) demonstrate selective cytotoxic effect against a wide range of tumor cells. In this context combined use of cytotoxic RNases in complex therapy with other chemotherapeutic agents appears to be especially promising. In this study we have investigated the apoptosisinduced effect of Bacillus pumilus RNase (binase) in combination with known antitumor antibiotic bleomycin on human lung adenocarcinoma A549 cells. The combined effect of high concentrations of these agents did not have any mutual increase in their apoptosisinduced action, while a combination of nonapoptotic concentrations resulted in the increase of proportion of apoptotic cells up to 22% as compared with individual effect of bleomycin (6%) and binase (12%) used separately. These results indicate that binase and bleomycin are effective in combination of their low concentrations and ineffective in combination of their high concentrations

  • Combined action of binase and bleomycin toward human lung adenocarcinoma cells
    2020
    Co-Authors: Zelenikhin P., Makeeva A., Nguen T., Siraj Y., Ilinskaya O.
    Abstract:

    Some microbial ribonucleases (RNases) demonstrate selective cytotoxic effect against a wide range of tumor cells. In this context combined use of cytotoxic RNases in complex therapy with other chemotherapeutic agents appears to be especially promising. In this study we have investigated the apoptosis-induced effect of Bacillus pumilus RNase (binase) in combination with known anti-tumor antibiotic bleomycin on human lung adenocarcinoma A549 cells. The combined effect of high concentrations of these agents did not have any mutual increase in their apoptosis-induced action, while a combination of non-apoptotic concentrations resulted in the increase of the proportion of apoptotic cells up to 22% as compared with individual effect of bleomycin (6%) and binase (12%) used separately. These results indicate that binase and bleomycin are effective in combination of their low concentrations and ineffective in combination of their high concentrations

  • Binase Do Not Change Level of Intracellular RNA in Breast Cancer Cells
    2020
    Co-Authors: Sokolova E., Zelenikhin P.
    Abstract:

    © 2017, Springer Science+Business Media, LLC. We analyzed changes of intracellular RNA level in breast cancer cells using BT-20, BT-474, and MCF-7 cell lines. It is the continuation of our former studying the effect of bacterial ribonucleases on physiological and biochemical parameters of breast cancer cells. For the first time, we tested ability of Bacillus pumilus RNase (binase) to effect the level of intracellular RNA in these cells. Differences in total RNA level under of the binase action on the cells of triple negative, triple positive, and double positive breast cancer during 48 h of treatment were not detected. The sensitivity of cells to binase was not associated with a reduction of the intracellular RNA level