, Volume 8, Issue 1, pp 319–322 | Cite as

Nitric Oxide Production Correlates with Cell Death of Fibroblasts Treated by Bacillus pumilus Ribonuclease

  • Pavel Zelenikhin
  • Andreas Koschinski
  • Olga Ilinskaya


NO is a crucial messenger in tumor cell signaling. High levels of nitric oxide synthase expression may be cytostatic or cytotoxic for tumor cells, whereas low levels can have the opposite effect and promote tumor growth. Bioimaging is a major technique to visualize the nitric oxide level in living cells and to compare it with physiological outcomes. In this report, we used two fluorescent probes, DAA and DAF-FM diacetate, in order to visualize NO levels in normal and ras-transformed fibroblasts treated by the bacterial ribonuclease binase (Bacillus pumilus RNase). To assess selective toxicity of binase towards cells expressing the ras oncogene, a fluorescent live/dead dye was applied. Here we compared the NO levels in normal and ras-transformed fibroblasts to elucidate the role of NO in the apoptotic signaling cascade induced by binase.


Nitric oxide Binase Toxicity Ras 



The study was performed within the Russian Government Program of Competitive Growth of Kazan Federal University and was supported by the Russian Science Foundation (project no. 14-14-00522).


  1. 1.
    Fukumura, D., Kashiwagi, S., & Jain, R. K. (2006). The role of nitric oxide in tumor progression. Nature Reviews Cancer, 6, 521–534. Scholar
  2. 2.
    Scicinski, J., Oronsky, B., Ning, S., Knox, S., Peehl, D., Kim, M., Langecker, P., & Fangera, G. (2015). NO to cancer: the complex and multifaceted role of nitric oxide and the epigenetic nitric oxide donor, RRx-001. Redox Biology, 6, 1–8. Scholar
  3. 3.
    Eroglu, E., Gottschalk, B., Charoensin, S., Blass, S., Bischof, H., Rost, R., Madreiter-Sokolowski, C. T., Pelzmann, B., Bernhart, E., Sattler, W., Hallstrom, S., Malinski, T., Waldeck-Weiermair, M., Graier, W. F., & Malli, R. (2016). Development of novel FP-based probes for live-cell imaging of nitric oxide dynamics. Nature Communications, 7, 10623. Scholar
  4. 4.
    Francis, S. H., Busch, J. L., Corbin, J. D., & Sibley, D. (2010). cGMP-dependent protein kinases and cGMP phosphodiesterases in nitric oxide and cGMP action. Pharmacological Reviews, 62(3), 525–563. Scholar
  5. 5.
    Kyle, B. D., Hurst, S., Swayze, R. D., Sheng, J., & Braun, A. P. (2013). Specific phosphorylation sites underlie the stimulation of a large conductance, Ca(2+)-activated K(+) channel by cGMP-dependent protein kinase. FASEB Journal, 27(5), 2027–2038. Scholar
  6. 6.
    Zelenikhin, P., Pukhovskaya, V., Garipov, A., Makeeva, A., Sokolova, E., & Ilinskaya, O. (2016). Obvious and hidden reasons of breast cancer cell sensitivity to antitumor RNase. BioNanoScience., 6(4), 528–533.CrossRefGoogle Scholar
  7. 7.
    Mitkevich, V. A., Kretova, O. V., Petrushanko, I. Y., Burnysheva, K. M., Sosin, D. V., Simonenko, O. V., Ilinskaya, O. N., Tchurikov, N. A., & Makarov, A. A. (2013). Ribonuclease binase apoptotic signature in leukemic Kasumi-1 cells. Biochimie, 95(6), 1344–1349.CrossRefGoogle Scholar
  8. 8.
    Cabrera-Fuentes, H. A., Aslam, M., Saffarzadeh, M., Kolpakov, A., Zelenikhin, P., Preissner, K. T., & Ilinskaya, O. N. (2013). Internalization of Bacillus intermedius ribonuclease (BINASE) induces human alveolar adenocarcinoma cell death. Toxicon, 69, 219–226.CrossRefGoogle Scholar
  9. 9.
    Garipov, A. R., Nesmelov, A. A., Cabrera-Fuentes, H. A., & Ilinskaya, O. N. (2014). Bacillus intermedius ribonuclease (BINASE) induces apoptosis in human ovarian cancer cells. Toxicon, 15(92), 54–59. Scholar
  10. 10.
    Ilinskaya, O., Decker, K., Koschinski, A., Dreyer, F., & Repp, H. (2001). Bacillus intermedius ribonuclease as inhibitor of cell proliferation and membrane current. Toxicology, 156, 101–107.CrossRefGoogle Scholar
  11. 11.
    Ilinskaya, O., Singh, I., Dudkina, E., Ulyanova, V., Kayumov, A., & Barreto, G. (2016). Direct inhibition of oncogenic KRAS by Bacillus pumilus ribonuclease (binase). Biochimica et Biophysica Acta - Molecular Cell Research, 1863(7), 1559–1567.CrossRefGoogle Scholar
  12. 12.
    Ilinskaya, O. N., Koschinski, A., Repp, H., Mitkevich, V., Dreyer, F., Scholtz, J. M., Pace, C. N., & Makarov, A. (2008). RNase induced apoptosis: fate of calcium–activated potassium channels. Biochemie, 90, 717–725.CrossRefGoogle Scholar
  13. 13.
    Sokurenko, J. V., Zelenikhin, P. V., Ulyanova, V. V., Kolpakov, A. I., Muller, D., & Ilinskaya, O. N. (2015). Identification of 2',3'-cGMP as an intermediate of RNA catalytic cleavage by binase and evaluation of its biological action. Bioorganicheskaia Khimiia, 41(1), 37–43.Google Scholar
  14. 14.
    Denninger, J. W., & Marletta, M. A. (1999). Guanylate cyclase and the NO/cGMP signaling pathway. Biochimica et Biophysica Acta, 1411, 334–350.CrossRefGoogle Scholar
  15. 15.
    Marin, M. J., Thomas, P., Fabregat, V., Luis, S. V., Russell, D. A., & Galindo, F. (2011). Fluorescence of 1,2-diaminoanthraquinone and its nitric oxide reaction product within macrophage cells. Chembiochem, 12(16), 2471–2477.CrossRefGoogle Scholar
  16. 16.
    Xu, W., Liu, L. Z., Loizidou, M., Ahmed, M., & Charles, I. G. (2002). The role of nitric oxide in cancer. Cell Research, 12, 311–320.CrossRefGoogle Scholar
  17. 17.
    Wang, Y., Chen, C., Loake, G. J., & Chu, C. (2010). Nitric oxide: promoter or suppressor of programmed cell death? Protein & Cell, 1, 133–142. Scholar
  18. 18.
    Rukoyatkina, N., Walter, U., Friebe, A., & Gambaryan, S. (2011). Differentiation of cGMP-dependent and -independent nitric oxide effects on platelet apoptosis and reactive oxygen species production using platelets lacking soluble guanylyl cyclase. Thrombosis and Haemostasis, 106(5), 922–933. Scholar
  19. 19.
    Brüne, B. (2003). Nitric oxide: NO apoptosis or turning it ON? Cell Death and Differentiation, 10, 864–869. Scholar
  20. 20.
    Kook, H., Itoh, H., Choi, B. S., Sawada, N., Doi, K., Hwang, T. J., Kim, K. K., Arai, H., Baik, Y. H., & Nakao, K. (2003). Physiological concentration of atrial natriuretic peptide induces endothelial regeneration in vitro. American Journal of Physiology - Heart and Circulatory Physiology, 284(4), H1388–H1397. Scholar
  21. 21.
    Isshiki, K., Matsuda, S., Tsuji, A., & Yuasa, K. (2012). cGMP-dependent protein kinase I promotes cell apoptosis through hyperactivation of death-associated protein kinase 2. Biochemical and Biophysical Research Communications, 422(2), 280–284. Scholar
  22. 22.
    Mitkevich, V. A., Tchurikov, N. A., Zelenikhin, P. V., Petrushanko, I. Y., Makarov, A. A., & Ilinskaya, O. N. (2010). Binase cleaves cellular noncoding RNAs and affects coding mRNAs. The FEBS Journal, 277(1), 186–196.CrossRefGoogle Scholar

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Authors and Affiliations

  1. 1.Kazan (Volga region) Federal UniversityKazanRussia
  2. 2.Department of physiology, Anatomy and GeneticsUniversity of OxfordOxfordUK

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