Induction of oxidative stress by hydrogen peroxide in vitro in breast epithelial cells for studying MCF-7 tumor cell line apoptosis
Author Affiliations1Siberian State Medical University, 634050, Tomsk, Moscowski Trakt, 2
2Siberian State Medical University of Minzdrav of Russia, 634050, Tomsk, Moskovsky Tract, 2; National Research Tomsk State University, 634050, Tomsk, Lenin av., 36
3Siberian State Medical University, 634050, Tomsk, Moscowski Trakt, 2
4Siberian State Medical University, 634050, Tomsk, Moscowski Trakt, 2
Abstract
The research objective. Selection of the hydrogen peroxide concentration that is able to induce oxidative stress and trigger breast epithelial cell apoptosis without eliminating cells via cell death. Material and methods. The cell culture of human breast epithelium (HBL-100) and MCF-7 tumor cell line (epithelial-like eadenocarcinoma of human breast) have been chosen as the objects of the research. Modeling of oxidative stress in HBL-100 cell line has been achieved by adding hydrogen peroxide in the final concentrations of 0.2; 0.3; 0.5 and 1.0 mMole. The level of reactive oxygen species, the concentration of total, reduced and oxidized glutathione as well as their ratio in HBL-100 epithelial cell line have been evaluated in order to carry out comparative analysis of the degree of oxidative stress with MCF-7 tumor cells line. Results and discussion. The optimal concentration of hydrogen peroxide has been established – 0.3 mMole, at which the increase in the concentration of reactive oxygen species in the cell is registered that may be compared to the one in tumor cells and also may be compared to the value of the reduced glutathione-oxidized glutathione ratio; more over, formation of the maximum amount of annexin V-positive cells and the minimum amount of propidium iodide- positive cells takes place.
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References
- Arhipov S.A., Shkurupij V.A., Zajkovskaja M.V. et al. Mixed effects on Н2О2 macrophages, fibroblasts in the conditions of modeling of oxidative stress // Sovremennye naukoemkie tekhnologii. 2010. (8). 76–77. (in Russian)
- Vladimirov Yu.A. Free radicals in biological systems // Sorosovskij obrazovatel'nyi zhurnal. 2000. 6. (12). 13–19. (in Russian)
- .Davydov M.I., Aksel' E.M. Cancer morbidity of the population of Russia and CIS countries in 2008 // Vestnik Rossiyskogo onkologicheskogo tsentra imeni Nikolaya Nikolaevicha Blokhina. 2010. 2. (21). 52–86. (in Russian)
- 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 Russian)
- Kalinina E.V., Chernov N.N., Aleid R. et al. Modern representations about antioxidant role of glutathione and glutathione-dependent enzymes // Vestnik Rossijskoy Akademii meditsinskikh nauk. 2010. (3). 46–54. (in Russian)
- Kondakova I.V., Kakurina G.V., Smirnova L.P., Borunov E.V. Regulation of tumor cell proliferation and apoptosis by free radicals // Sibirskiy onkologicheskiy zhurnal. 2005. (1). 58–61. (in Russian)
- Kulinskij V.I., Kolesnichenko L.S. Glutathione system. I. Synthesis, transport, glutathione transferase, glutathione peroxidase // Biomeditsinskaya Khimiya. 2009. 55. (3). 255–277. (in Russian)
- Menshchikova E.B., Zenkov N.K., Lankin V.Z. et al. Oxidative stress: Pathological conditions and diseases. Novosibirsk, 2008. 284р. (in Russian)
- Oktyabrsky O.N., Smirnova C.V. Redox regulation of cellular functions // Biochemistry. 2007. 72. (2). 132–145. (in Russian)
- Ryazanceva N.V., Novickiy V.V., Zhukova O.B. et al. Role of reactive oxygen species and Bcl-2 family proteins in TNF-α-induced apoptosis of lymphocytes // Bull. Exp. Bio.l Med. 2010. 149. (2). 180–183. (in Russian)
- Starikova Ye.G., Tashireva L.A., Vasilieva O.A. et al. Participation of redox signalization in nitric oxide-, carbon monoxide- and hudrogen sulfide-mediated regulation of apoptosis and cell cycle // Bulleten' sibirskoy meditsiny. 2013. 12. (1). 49–54. (in Russian)
- Stepovaya E.A., Petina G.V., Zhavoronok T.V. et al. Role of thiol-disulfide system in mechanisms of functional changes in neutrophils under conditions of oxidative stress // Bull. Exp. Biol. Med. 2010. 150. (2). 198–202. (in Russian)
- Chissova V.I., Starinskij V.V., Petrova G.V. Malignant neoplasms in Russia in 2011 (morbidity and mortality). Moscow, 2013. 289 р. (in Russian)
- Anderson M.E. Determination of glutathione and glutathione sulfide in biological samples // Methods Enzymol. 1985. (113). 548–555.
- Brigelius-Flohé R., Flohé L. Basic principles and emerging concepts in the redox control of transcription factors // Antioxid Redox Signal. 2011. 15. (8). 2335–2381.
- Droge W. Free radicals in the physiological control of cell function // Physiol. Rev. 2002. 82. (1). 476–485.
- Halliwell B. Biochemistry of oxidative stress // Biochem. Soc. Trans. 2007. (5). 1147–1150.
- Halliwell B. Free radicals and antioxidants: updating a personal view // Nutr Rev. 2012. 70. (5). 257–265.
- 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. 231–255.
- Khamaisi M., Kavel O., Rosenstock M. et al. Effect of inhibition of glutathione synthesis on insulin action: in vivo and in vitro studies using buthionine sulfoximine // Biochem. J. 2000. 349. (2). 579–586.
- 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.
- Oberley T.D. Oxidative damage and cancer // Am. J. Pathol. 2002. 160. (2). 403–408.
- 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.
24. 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.
About Authors (Correspondence):
Shakhristova E.V. – candidate of medical sciences, head of the laboratory of molecular medicine
Ryazantseva N.V. – doctor of medical sciences, professor, head of the chair for molecular medicine and clinical laboratory diagnostics, e-mail: office@ssmu.tomsk.ru
Stepovaya E.A. – doctor of medical sciences, professor of the chair for biochemistry and molecular biology, e-mail: muir@mail.ru
Nosareva О.L. – candidate of medical sciences, assistant professor of the chair for biochemistry and molecular biology, e-mail: olnosareva@yandex.ru
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