Redox status and protein glutathionylation in binase-treated HPV16-positive SiHa Carcinoma cells

Cover Page

Cite item

Full Text

Open Access Open Access
Restricted Access Access granted
Restricted Access Subscription Access

Abstract

Human papillomavirus type 16 (HPV16) belongs to the high-risk type viruses and is associated by overexpression of E6 and E7 oncoproteins, which determine the oncogenic properties of the virus such as immortalization and malignant transformation of proliferating epithelial cells. The biogenesis of redox-sensitive proteins E6 and E7 at the early stages of viral infection leads to blocking of cell antioxidant defense system and ubiquintin-dependent degradation of p53 and Rb tumor suppressors. Maintaining high rates of tumor cell proliferation contributes to an increase in the reactive oxygen species (ROS) production level and a shift in the redox balance towards oxidative processes. Reduced glutathione (GSH) provides antioxidant protection to tumor cells through S-glutathionylation of thiol groups of redox-sensitive proteins, which leads to the appearance of multidrug-resistant forms of cancer. In this regard, drugs restoring redox balance and increasing susceptibility to antitumor therapy are of particular importance. We have established that in HPV-16-positive SiHa cells of cervical squamous cell carcinoma, Bacillus pumilus RNase (binase) modulates the redox-dependent regulatory mechanisms that ensure tumor cell resistance to apoptosis. Binase in nontoxic concentrations initiates a number of pre-apoptogenic changes, i.g., decreases ROS and GSH levels, suppresses the expression of E6 oncoprotein, activates the expression of p53 tumor suppressor, and reduces the mitochondrial potential of tumor cells. Binase-induced disruption of the mitochondrial membrane integrity is a signal for the mitochondrial apoptosis pathway activation.

Full Text

Restricted Access

About the authors

A. I. Nadyrova

Kazan Federal University

Author for correspondence.
Email: alsu.nadyrova@yandex.ru

Institute of Fundamental Medicine and Biology

Russian Federation, Kazan, 420008

I. Y. Petrushanko

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: alsu.nadyrova@yandex.ru
Russian Federation, Moscow, 119991

V. A. Mitkevich

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: alsu.nadyrova@yandex.ru
Russian Federation, Moscow, 119991

O. N. Ilinskaya

Kazan Federal University

Email: alsu.nadyrova@yandex.ru

Institute of Fundamental Medicine and Biology

Russian Federation, Kazan, 420008

References

  1. Green R.M., Graham M., O’Donovan M.R., Chipman J.K., Hodges N.J. (2006) Subcellular compartmentalization of glutathione: correlations with parameters of oxidative stress related to genotoxicity. Mutagenesis. 21, 383–390. doi: 10.1093/mutage/gel043
  2. Kennedy L., Sandhu J.K., Harper M.E., Cuperlovic-Culf M. (2020) Role of glutathione in cancer: from mechanisms to therapies. Biomolecules. 10, 1429. doi: 10.3390/biom10101429.
  3. Schafer F.Q., Buettner G.R. (2001) Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Radic. Biol. Med. 30, 1191–1212. doi: 10.1016/s0891-5849(01)00480-4
  4. Buettner G.R., Wagner B.A., Rodgers V.G. (2013) Quantitative redox biology: an approach to understand the role of reactive species in defining the cellular redox environment. Cell Biochem. Biophys. 67, 477–483. doi: 10.1007/s12013-011-9320-3
  5. Townsend D.M., Tew K.D., Tapiero H. (2003) The importance of glutathione in human disease. Biomed. Pharmacother. 57, 145–155. doi: 10.1016/S0753-3322(03)00043-X
  6. Vafa O., Wade M., Kern S., Beeche M., Pandita T.K., Hampton G.M., Wahl G.M. (2002) c-Myc can induce DNA damage, increase reactive oxygen species, and mitigate p53 function: a mechanism for oncogene-induced genetic instability. Mol. Cell. 9, 1031–1044. doi: 10.1016/S1097-2765(02)00520-8
  7. Weinberg F., Hamanaka R., Wheaton W.W., Weinberg S., Joseph J., Lopez M., Kalyanaraman B., Mutlu G., Budinger S., Chandel N.S. (2010) Mitochondrial metabolism and ROS generation are essential for Kras-mediated tumorigenicity. Proc. Natl. Acad. Sci. USA. 107, 8788–8793. doi: 10.1073/pnas.1003428107
  8. Ballatori N., Krance S.M., Marchan R., Hammond C.L. (2009) Plasma membrane glutathione transporters and their roles in cell physiology and pathophysiology. Mol. Aspects Med. 30, 13–28. doi: 10.1016/j.mam.2008.08.004
  9. Mieyal J.J., Gallogly M.M., Qanungo S., Sabens E.A., Shelton M.D. (2008) Molecular mechanisms and clinical implications of reversible protein S-glutathionylation. Antioxid. Redox Signal. 10, 1941–1988. doi: 10.1089/ars.2008.2089
  10. Miller O.G., Mieyal J.J. (2015) Sulfhydryl-mediated redox signaling in inflammation: role in neurodegenerative diseases. Arch. Toxicol. 89, 1439–1467. doi: 10.1007/s00204-015-1496-7
  11. Xue X., Wang B., Du W., Zhang C., Song Y., Cai Y., Cen D., Wang L., Xiong Y., Jiang P., Zhu S., Zhao K.N., Zhang L. (2016) Generation of affibody molecules specific for HPV16 E7 recognition. Oncotarget. 7, 73995–74005. doi: 10.18632/oncotarget.12174
  12. Wondrak G.T. (2009) Redox-directed cancer therapeutics: molecular mechanisms and opportunities. Antioxid. Redox Signal. 11, 3013–3069. doi: 10.1089/ars.2009.2541
  13. Tew K.D., Townsend D.M. (2011) Redox platforms in cancer drug discovery and development. Curr. Opin. Chem. Biol. 15, 156–161. doi: 10.1016/j.cbpa.2010.10.016
  14. Mironova N.L., Petrushanko I.Y., Patutina O.A., Sen’kova A.V., Simonenko O.V., Mitkevich V.A., Markov O.V., Zenkova M.A., Makarov A.A. (2013) Ribonuclease binase inhibits primary tumor growth and metastases via apoptosis induction in tumor cells. Cell Cycle. 12, 2120–2131. doi: 10.4161/cc.25164
  15. 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, 1344–1349. doi: 10.1016/j.biochi.2013.02.016
  16. Бурнышева К.М., Петрушанко И.Ю., Спирин П.В., Прасолов В.С., Макаров А.А., Митькевич В.А. (2016) Рибонуклеаза биназа вызывает гибель клеток острого Т-лимфобластного лейкоза, индуцируя в них апоптоз. Молекуляр. биология. 50, 347–352. doi: 10.7868/S0026898416020038
  17. Шульга А.А., Окороков А.Л., Панов К.И., Курбанов Ф.Т., Чернов Б.К., Скрябин К.Г., Кирпичников М.П. (1994) Суперпродукция рибонуклеазы Bacillus intermedius 7P (биназы) в E. coli. Молекулярная биология. 28(2), 453–463.
  18. Mitkevich V.A., Burnysheva K.M., Petrushanko I.Y., Adzhubei A.A., Schulga A.A., Chumakov P.M., Makarov A.A. (2017) Binase treatment increases interferon sensitivity and apoptosis in SiHa cervical carcinoma cells by downregulating E6 and E7 human papilloma virus oncoproteins. Oncotarget. 8, 72666–72675. doi: 10.18632/oncotarget.20199
  19. Pal D., Rai A., Checker R., Patwardhan R.S., Singh B., Sharma D., Sandur S.K. (2021) Role of protein S-glutathionylation in cancer progression and development of resistance to anti-cancer drugs. Arch. Biochem. Biophys. 704, 108890. doi: 10.1016/j.abb.2021.108890
  20. Ilinskaya O.N., Singh I., Dudkina E., Ulyanova V., Kayumov A., Barreto G. (2016) Direct inhibition of oncogenic KRAS by Bacillus pumilus ribonuclease (binase). Biochim. Biophys. Acta. 1863, 1559–1567. doi: 10.1016/j.bbamcr.2016.04.005
  21. Mitkevich V.A., Petrushanko I.Y., Spirin P.V., Fedorova T.V., Kretova O.V., Tchurikov N.A., Prassolov V.S., Ilinskaya O.N., Makarov A.A. (2011) Sensitivity of acute myeloid leukemia Kasumi-1 cells to binase toxic action depends on the expression of KIT and АML1-ETO oncogenes. Cell Cycle. 10, 4090–4097. doi: 10.4161/cc.10.23.18210
  22. Митькевич В.А., Орлова Н.Н., Петрушанко И.Ю., Симоненко О.В., Спирин П.В., Прокофьева М.М., Горностаева А.С., Stocking C., Макаров А.А., Прасолов В.С. (2013) Экспрессия онкогена FLT3-ITD сообщает предшественникам B-клеток мыши линии BAF3 чувствительность к цитотоксическому действию биназы. Молекуляр. биология. 47, 282–282. https://doi.org/10.7868/s0026898413020092
  23. Zur Hausen H. (2002) Papillomaviruses and cancer: from basic studies to clinical application. Nat. Rev. Cancer. 2, 342–350. doi: 10.1038/Nrc798
  24. Velu C.S., Niture S.K., Doneanu C.E., Pattabiraman N., Srivenugopal K.S. (2007) Human p53 is inhibited by glutathionylation of cysteines present in the proximal DNA-binding domain during oxidative stress. Biochemistry. 46, 7765–7780. doi: 10.1021/bi700425y
  25. Petrushanko I.Y., Yakushev S., Mitkevich V.A., Kamanina Y.V., Ziganshin R.H., Meng X., Anashkina A.A., Makhro A., Lopina O.D., Gassmann M., Makarov A.A., Bogdanova A. (2012) S-glutathionylation of the Na, K-ATPase catalytic α subunit is a determinant of the enzyme redox sensitivity. J. Biol. Chem. 287, 32195–32205. doi: 10.1074/jbc.M112.391094
  26. Alevizopoulos K., Calogeropoulou T., Lang F., Stournaras C. (2014) Na+/K+ ATPase inhibitors in cancer. Curr. Drug Targets. 15, 988–1000. doi: 10.2174/1389450115666140908125025
  27. Bejček J., Spiwok V., Kmoníčková E., Rimpelová S. (2021) Na+/K+-ATPase revisited: on its mechanism of action, role in cancer, and activity modulation. Molecules. 26, 1905. doi: 10.3390/molecules26071905
  28. Lushchak V.I. (2012) Glutathione homeostasis and functions: potential targets for medical interventions. J. Amino Acids. 2012, 736837. doi: 10.1155/2012/736837
  29. Zou J., Shang X., Li C., Ouyang J., Li B., Liu X. (2019) Effects of cadmium on mineral metabolism and antioxidant enzyme activities in Salix matsudana Koidz. Pol. J. Environ. Stud. 28, 989–999. doi: 10.15244/pjoes/81697
  30. Allocati N., Masulli M., Di Ilio C., Federici L. (2018) Glutathione transferases: substrates, inihibitors and pro-drugs in cancer and neurodegenerative diseases. Oncogenesis. 7, 8. doi: 10.103/s41389-017-0025-3
  31. Ardelt B., Juan G., Burfeind P., Salomon T., Wu J.M., Hsieh T.C., Li X., Sperry R., Pozarowski P., Shogen K., Ardelt W., Darzynkiewicz Z. (2007) Onconase, an anti-tumor ribonuclease suppresses intracellular oxidative stress. Int. J. Oncol. 31, 663–669. doi: 10.3892/ijo.31.3.663
  32. Tsai S.Y., Ardelt B., Hsieh T.C., Darzynkiewicz Z., Shogen K., Wu J.M. (2004) Treatment of Jurkat acute T-lymphocytic leukemia cells by onconase (Ranpirnase) is accompanied by an altered nucleocytoplasmic distribution and reduced expression of transcription factor NF-B. Int. J. Oncol. 25, 1745–1752. doi: 10.3892/ijo.25.6.1745
  33. Fratelli M., Gianazza E., Ghezzi P. (2004) Redox proteomics: identification and functional role of glutathionylated proteins. Expert Rev. Proteomics. 1, 365–376. doi: 10.1586/14789450.1.3.365
  34. Dalle-Donne I., Rossi R., Giustarini D., Colombo R., Milzani A. (2007) S-glutathionylation in protein redox regulation. Free Radic. Biol. Med. 43, 883–898. doi: 10.1016/j.freeradbiomed.2007.06.014
  35. Fiaschi T., Cozzi G., Raugei G., Formigli L., Ramponi G., Chiarugi P. (2006) Redox regulation of β-actin during integrin-mediated cell adhesion. J. Biol. Chem. 281, 22983–22991. doi: 10.1074/jbc.M603040200
  36. Lu G.D., Shen H.M., Chung M.C., Ong C.N. (2007) Critical role of oxidative stress and sustained JNK activation in aloe-emodin-mediated apoptotic cell death in human hepatoma cells. Carcinogenesis. 28, 1937–1945. doi: 10.1093/carcin/bgm143
  37. Cuadrado A., Garcia-Fernandez L.F., Gonzalez L., Suarez Y., Losada A., Alcaide V., Martinez T., Fernandez-Sousa J.M., Sanchez Puelles J.M., Munoz A. (2003) Aplidin induces apoptosis in human cancer cells via glutathione depletion and sustained activation of the epidermal growth factor receptor, Src, JNK, and p38 MAPK. J. Biol. Chem. 278, 241–250. doi: 10.1074/jbc.M201010200
  38. Ji L., Shen K., Jiang P., Morahan G., Wang Z. (2011) Critical roles of cellular glutathione homeostasis and jnk activation in andrographolide-mediated apoptotic cell death in human hepatoma cells. Mol. Carcinog. 50, 580–591. doi: 10.1002/mc.20741
  39. Velu C.S., Niture S.K., Doneanu C.E., Pattabiraman N., Srivenugopal K.S. (2007) Human p53 is inhibited by glutathionylation of cysteines present in the proximal DNA-binding domain during oxidative stress. Biochemistry. 46, 7765–7780. doi: 10.1021/bi700425y
  40. Mitkevich V.A., Petrushanko I.Y., Kretova O.V., Zelenikhin P.V., Prassolov V.S., Tchurikov N.A., Ilinskaya O.N., Makarov A.A. (2010) Oncogenic c-kit transcript is a target for binase. Cell Cycle. 9, 2674–2678. doi: 10.4161/cc.9.13.12150
  41. Mitkevich V.A., Petrushanko I.Y., Makarov A.A. (2019) RNases disrupt the adaptive potential of malignant cells: perspectives for therapy. Front. Pharmacol. 10, 922. doi: 10.3389/fphar.2019.00922
  42. Mijatovic T., Dufrasne F., Kiss R. (2012) Na+/K+-ATPase and cancer. Pharm. Pat. Anal. 1, 91–106. doi: 10.4155/ppa.12.3
  43. Eskiocak U., Ramesh V., Gill J.G., Zhao Z., Yuan S.W., Wang M., Vandergriff T., Shackleton M., Quintana E., Frankel A., Johnson T., DeBerardinis R., Morrison S.J. (2016) Synergistic effects of ion transporter and MAP kinase pathway inhibitors in melanoma. Nat. Commun. 7, 12336. doi: 10.1038/ncomms12336
  44. Ren J., Gao X., Guo X., Wang N., Wang X. (2022) Research progress in pharmacological activities and applications of cardiotonic steroids. Front. Pharmacol. 13, 902459. doi: 10.3389/fphar.2022.902459
  45. Ayogu J.I., Odoh A.S. (2020) Prospects and therapeutic applications of cardiac glycosides in cancer remediation. ACS Comb. Sci. 22, 543–553. doi: 10.1021/acscombsci.0c00082
  46. Chang Y.M., Shih Y.L., Chen C.P., Liu K.L., Lee M.H., Lee M.Z., Hou H.T., Huang H.C., Lu H.F., Peng S.F.., Chen K.W., Yeh M.Y., Chung J.G. (2019) Ouabain induces apoptotic cell death in human prostate DU 145 cancer cells through DNA damage and TRAIL pathways. Environ. Toxicol. 34, 1329–1339. doi: 10.1002/tox.22834
  47. Osman M.H., Farrag E., Selim M., Osman M.S., Hasanine A., Selim A. (2017) Cardiac glycosides use and the risk and mortality of cancer; systematic review and meta-analysis of observational studies. PloS One. 12, e0178611. doi: 10.1371/journal.pone.0178611
  48. Ortega A.L., Mena S., Estrela J.M. (2011) Glutathione in cancer cell death. Cancers. 3, 1285–1310. doi: 10.3390/cancers3011285
  49. Ghosh S., Pulinilkunnil T., Yuen G., Kewalramani G., An D., Qi D., Abrahani A., Rodrigues B. (2005) Cardiomyocyte apoptosis induced by short-term diabetes requires mitochondrial GSH depletion. Am. J. Physiol. Heart Circ. Physiol. 289, H768–H776. doi: 10.1152/ajpheart.00038.2005
  50. Armstrong J.S., Steinauer K.K., Hornung B., Irish J.M., Lecane P., Birrell G.W., Peehl D.M., Knox S.J. (2002) Role of glutathione depletion and reactive oxygen species generation in apoptotic signaling in a human B lymphoma cell line. Cell Death Differ. 9, 252–263. doi: 10.1038/sj.cdd.4400959
  51. Makarov A.A., Kolchinsky A., Ilinskaya O.N. (2008) Binase and other microbial RNases as potential anticancer agents. BioEssays. 30, 781–790. doi: 10.1002/bies.20789

Supplementary files

Supplementary Files
Action
1. JATS XML
Download
2. Fig. 1. Cytofluorimetric analysis of ROS (a) and reduced glutathione (б) content in SiHa cells incubated with binase (0.8 μM) for 24, 48, and 72 h. Results are presented as the fluorescence signal of DHR123 (ROS) and ThiolTrackerTM Violet (GSH) dyes obtained from cells incubated with binase relative to untreated cells. *p ≤ 0.05.

Download (246KB)
Indexing metadata
3. Fig. 2. Cytofluorimetric analysis of the content of cells with reduced mitochondrial potential (a) and dead cells (б) after treatment with binase (0.8 μM) for 24, 48 and 72 h. *p ≤ 0.05.

Download (255KB)
Indexing metadata
4. Fig. 3. Effect of binase on the levels of HPV-16 E6 and p53 proteins in SiHa cells. a – Electrophoretic analysis of proteins in SiHa cell lysates. Here and below: M – molecular weight markers of proteins PageRuler Prestained Protein Ladder (“Thermo Fisher Scientific”, USA), control – untreated cells; binase – cells were incubated with 0.8 μM binase for 48 h. б – Relative content of p53 and E6 proteins in lysates of SiHa cells treated with binase. The level of the corresponding proteins in the control is taken as 100%. в – Change in the degree of glutathionylation of the p53 protein (GSS-p53) upon treatment of SiHa cells with binase. The fold change was calculated as GSS-p53/p53; the value of GSS-p53/p53 in the control is taken as one. *p ≤ 0.05.

Download (154KB)
Indexing metadata
5. Fig. 4. Effect of binase on the level of α1-subunit of Na,K-ATPase in SiHa cells. a – Electrophoretic analysis of the content of α1-subunit of Na,K-ATPase in SiHa cells. For designations, see the legend to Fig. 3. Change in the degree of glutathionylation (б) and content (в) of α1-subunit of Na,K-ATPase in lysates of SiHa cells treated with binase. The values ​​in the control are taken as one. *p ≤ 0.05.

Download (85KB)
Indexing metadata

Copyright (c) 2024 Russian Academy of Sciences