№ 5 / 2017 / 5-10

GLUTAREDOXIN AND GLUTATHIONE AS THE MOLECULES REGULATING BREAST EPITHELIAL CELL PROLIFERATION UNDER ROSCOVITINE-INDUCED OXIDATIVE STRESS

Author: Shakhristova E.V.¹, Stepovaya E.A.², Nosareva О.L.³, Rudikov E.V.⁴, Novitsky V.V.⁵

Abstract

The objective: to determine the role of glutathione and glutaredoxin in regulation of proliferation of HBL-100 human breast epithelial cells under roscovitine-induced oxidative stress. Material and methods. The HBL-100 cell line (human breast epithelial cells) was used in the study. The cell culture was incubated in the presence and absence of roscovitine in the final concentration of 20 mcmol for 18 h. The production of reactive oxygen intermediate, the cells distribution among cycling state and the amount of Annexin V positive cells were determined using flow cytometry. The concentrations of reduced and oxidized glutathione and protein SH groups were measured by spectrophotometry. The levels of glutaredoxin, cyclin E and cyclin-dependent protein kinases were estimated by Western blotting with monoclonal antibodies. Results and discussion. The effects of roscovitine in the HBL-100 cells resulted in cell cycle block in G₂/М phases with the decreased level of cyclin-dependent protein kinase 2 and activation of oxidative stress. It was accompanied by the elevated generation of reactive oxygen intermediate and the decrease in the concentrations of reduced glutathione and protein SH groups. We found out that roscovitine has anti-proliferative effects that are determined not only by the impact on the cyclin level, cyclin conformations and cyclin-dependent protein kinase activity, but also by the shift in the intracellular oxidant/antioxidant ratio. The model of free radical oxidation proposed in our work enabled to establish the role of the glutathione/glutaredoxin system in impairing cell cycle phase progression. It indicated a possibility to regulate cellular proliferation through modulating functional properties of redox-dependent proteins in different pathologies accompanied by oxidative stress.

Key words

proliferation, glutaredoxin, glutathione, cyclin-dependent kinase, oxidative stress

References

Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding // Anal. Biochem. 1976. 7. (1, 2). 248–254.

Cappellini A., Chiarini F., Ognibene A. et al. The cyclin-dependent kinase inhibitor roscovitine and the nucleoside analog sangivamycininduce apoptosis in caspase-3 deficient breast cancer cells independent of caspase mediated P-glycoprotein cleavage: implications for therapy of drug resistant breast cancers // Cell Cycle. 2009. 8. (9). 1421–1425.

Dubininа E.E. Products of metabolism of oxygen in the functional activity of cells (life and death, creation and destruction). Physiological and clinical-biochemical aspects. Saint-Petersburg, 2006. 400 р. [In Rusian].

Halliwell B., Whiteman M. Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean? // Br. J. Pharmacol. 2004. 142. (2). 231–255.

Kalinina E.V., Chernov N.N., Aleid R. et al. Current views of antioxidative activity of glutathione and glutathione-depending enzymes // Vestnik Rossiyskoy akademii meditsinskikh nauk = Annals of the Russian academy of medical sciences. 2010. (3). 46–54. [In Rusian].

Lu J., Holmgren A. The thioredoxin antioxidant system // Free Radic. Biol. Med. 2014. 66. 75–87.

Menshchikova E.B., Zenkov N.K., Lankin V.Z. et al. Oxidative stress: Pathological conditions and diseases. Novosibirsk, 2008. 284 р. [In Rusian].

Murphy M.P., Holmgren A., Larsson N.G. et al. Unraveling the biological roles of reactive oxygen species // Cell Metab. 2011. 13. (4). 361–366.

Rahman I., Kode A., Biswas S.K. Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method // Nat. Protoc. 2006. 1. (6). 3159–3165.

Rajnai Z., Méhn D., Beéry E., et al. ATP-binding cassette B1 transports seliciclib (R-roscovitine), a cyclin-dependent kinase inhibitor // Drug Metab. Dispos. 2010. 38. (11). 2000–2006.

Ray P.D., Huang B.W., Tsuji Y. Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling // Cell Signal. 2012. 24. (5). 981–990.

Shakhristova E.V., Stepovaya E.A., Ryazantseva N.V. et al. Role of glutathione system redox potential in apoptosis dysregulation in MCF-7 breast adenocarcinoma // Bull. Exp. Biol. Med. 2016. 160(3). 364–367.

Shan W., Zhong W., Zhao R. et al. Thioredoxin 1 as a subcellular biomarker of redox imbalance in human prostate cancer progression // Free Radic. Biol. Med. 2010. 49. (12). 2078–2087.

Stepovaya E.A., Shakhristova E.V., Ryazantseva N.V. et al. The role of oxidative protein modification and the glutathione system in modulation of the redox status of breast epithelial cells // Biochemistry (Mosc.). Suppl Series B. Biomed. Chem. 2016. 10. (3). 235–239.

Wang J., Boja E.S., Tan W. et al. Reversible glutathionylation regulates actin polymerization in A431 cells // J. Biol. Chem. 2001. 276. (51). 47763–47766.

About Authors (Correspondence):

Shakhristova E.V. – candidate of medical sciences, assistant professor of the chair for biochemistry and molecular biology with the course of clinical laboratory diagnostics, e-mail: shaxristova@yandex.ru

Full Text

snmzh_5-2017_shahristova_i_dr.pdf

Received: 30/10/2017

Accepted: 30/10/2017