V.A. Sergeeva - Research Scientist, Research Centre for Medical Genetics (Moscow) Е-mail: tracytheplane@gmail.com E.S. Ershova - Leading Research Scientist, Research Centre for Medical Genetics (Moscow) Е-mail: es-ershova@rambler.ru E.M. Malinovskaya - Senior Research Scientist, Research Centre for Medical Genetics (Moscow) Е-mail: tigerilina@mail.ru L.V. Kameneva - Senior Research Scientist, Research Centre for Medical Genetics (Moscow) Е-mail: al4004@mail.ru P.E. Umriukhin - Dr.Sc. (Med.), Professor, I.M. Sechenov First Moscow State medical university; Senior Research Scientist, P.K. Anokhin Institute of Normal Physiology (Moscow) Е-mail: pavelum@mail.ru A.V. Zhilenkov - Engineer-researcher, Functional Materials for Electronics and Medicine Laboratory, Institute for Problems of Chemical Physics of Russian Academy of Sciences (Chernogolovka, Moscow region) Е-mail: zhilenkow91@inbox.ru P.A. Troshin - Ph.D (Chem.), Head of the Laboratory, Functional Materials for Electronics and Medicine Laboratory, Skolkovo Institute of Science and Technology (Moscow) Е-mail: troshin2003@inbox.ru N.N. Veiko - Dr.Sc. (Biol.), Chief Researcher, Research Centre for Medical Genetics (Moscow) Е-mail: satelit32006@yandex.ru S.V. Kostyuk - Dr.Sc. (Biol.), Head of Molecular Biology Laboratory, Research Centre for Medical Genetics (Moscow) Е-mail: svet-vk@yandex.ru

It has been shown that fullerene [C60] nanoparticles have potential anti-viral, antioxidant and anti-cancer activity. Moreover, they can potentially be used as vectors for drug delivery across the biological barriers. Various studies have shown that well-known drugs increase their anti-cancer activity in conjugates with [C60 fullerenes]. The present work describes the effect of a novel water-soluble fullerene [C60] derivative F-243 on the cells of MCF-7 breast cancer cell line. The derivative is fluorescent both in aqueous solutions and in the cells allowing its detection within the cytoplasm without attachment of fluorescent tags. F-243 penetrates into the cytoplasm within 3-24 hours after start of incubation. Despite the fact that the derivative is an acceptor of electrons due to its chemical structure, in the cytoplasm it causes elevation of reactive oxygen species (ROS). We have already observed a similar effect in case of a different fullerene [C60] derivative. We have shown that the elevation in ROS levels is connected to the increase in the amounts of NOX4-oxidase. It is widely known that active ROS production leads to oxidative stress which can result in DNA damage and cell cycle arrest. In this study we show that the fullerene derivative F-243 causes oxidative modifications of DNA (8-oxodG) and double-strand breaks (H2AX), which brings the cell cycle progression to a halt. DNA breaks activate kinases ATM and ATR that initiate the arrest of the cell-cycle. However, the reparation system remains inactive and pro-apoptotic tp53 levels are increased, whereas anti-apoptotic bcl2 is decreased. Apart from that, the investigated derivative descreases mitochondrial membrane potential (mitotracker). All these events lead to apoptosis. Thus, the investigated [C60] fullerene derivative is toxic to the breast cancer cell line MCF-7. It could have potential anti-cancer effect if delivered straight into the tumour. However, if the cells remain alive after F-243 treatment they might develop more mutations due to DNA breaks reparation and become more malignant. Thus, it is necessary to choose a correct dose and this requires further investigation.

 

1. Panchuk R.R., Prylutska S.V., Chumak V.V., Skorokhyd N.R., Lehka L.V., Evstigneev M.P., Prylutskyy Yu. I., Berger W., Heffeter P., Scharff P., Ritter U., Stoika R.S. Application of C60 Fullerene-Doxorubicin Complex for Tumor Cell Treatment In Vitro and In Vivo // Journal of Biomedical Nanotechnology. 2015. № 11. R. 1139-1152.
2. Raza K., Thotakura N., Kumar P., Joshi M., Bhushan S., Bhatia A., Kumar V., Malik R., Sharma G., Guru S.K., Katare O.P. C60-fullerenes for delivery of docetaxel to breast cancer cells: A promising approach for enhanced efficacy and better pharmacokinetic profile // Int J Pharm. 2015. № 10. V. 495(1) R. 551-559.
3. Prylutska S.V., Burlaka A.P., Prylutskyy Y.I., Ritter U., Scharff P. Pristine C(60) fullerenes inhibit the rate of tumor growth and metastasis // Exp Oncol. 2011 № 33. R. 162-164.
4. Weyemi, Dupuy U.C. The emerging role of ROS-generating NADPH oxidase NOX4 in DNA-damage responses // Mutat Res. 2012. V. 751. R. 77-81.
5. Sedelnikova O.A., Redon C.E., Dickey J.S., Nakamura A.J., Georgakilas A.G., Bonner W.M. Role of oxidatively induced DNA lesions in human pathogenesis // Mutat.Res. 2010. № 704. R. 152-159.
6. Lambeth, J.D. Nox enzymes, ROS, and chronic disease: an example of antagonistic pleiotropy // Free Radic Biol Med. 2007. № 43. R. 332‒347.
7. LeBel C.P., Ischiropoulos H., Bondy S.C. Evaluation of the probe 2_,7_-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress // Chem. Res. Toxicol. 1992. № 5. P. 227-231.
8. Cossarizza A., Ferraresi R., Troiano L., Roat E., Gibellini L., Bertoncelli L., Nasi M., Pinti M. Simultaneous analysis of reactive oxygen species and reduced glutathione content in living cells by polychromatic flow cytometry // Nat Protoc. 2009. № 4. P. 1790-1797.
9. Royall J.A., Ischiropoulos H. Evaluation of 2_,7_-dichlorofluorescin and dihydrorhodamine 123 as fluorescent probes for intracellular H2O2 in cultured endothelial cells // Arch. Biochem. Biophys. 1993. № 302. P. 348-355.
10. Zhu H., Bannenberg G.L., Moldeus P., Shertzer H.G. Oxidation pathways for the intracellular probe 2_,7_-dichlorofluorescein // Arch. Toxicol. 1994. № 68. P. 582-587.
11. Ershova E.S., Sergeeva V.A., Chausheva A.I., Zheglo D.G., Nikitina V.A., Smirnova T.D., Kameneva L.V., Porokhovnik L.N., Kut-sev S.I., Troshin P.A., Voronov I.I., Khakina E.A., Veiko N.N., Kostyuk S.V. Toxic and DNA damaging effects of a functionalized fullerene in human embryonic lung fibroblasts // Mutat Res Genet Toxicol Environ Mutagen. 2016 № 805 P. 46-57.
12. Chen F., Haigh S., Barman S., Fulton D.J. From form to function: the role of Nox4 in the cardiovascular system // Front Physiol. 2012. № 1. P. 412.
13. Scully R., Xie A. Double strand break repair functions of histone H2AX // Mutat Res. 2013. № 750. P. 5-14.
14. Lobrich M., Shibata A., Beucher A., Fisher A., Ensminger M., Goodarzi A.A., Barton O., Jeggo P.A. H2AX foci analysis for monitoring DNA double-strand break repair. Strengths, limitations and oprtimization // Cell Cycle. 2010. № 9. P. 662-669.
15. Ermakov A.V., Konkova M.S., Kostyuk S.V., Smirnova T.D., Malinovskaya E.M., Efremova L.V., Veiko N.N. An extracellular DNA mediated bystander effect produced from low dose irradiated endothelial cells // Mutat Res. 2011. № 712. P. 1-10.
16. Goodarzi A.A., Jeggo P.A. The repair and signaling responses to DNA double-strand breaks. Adv Genet. 2013. № 82. P. 1-45.
17. Mermershtain I., Glover J.N. Structural mechanisms underlying signaling in the cellular response to DNA double strand breaks // Mutat Res. 2013. № 750. P. 15-22.
18. Wu J., Sun L., Chen X., Du F., Shi H., Chen C. et al. Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA // Science. 2013. № 339. P. 826-830.
19. Abdelwahid E., Rolland S., Teng X., Conradt B., Hardwick J.M., White K. Mitochondrial involvement in cell death of non-mammalian eukaryotes // Biochim. Biophys. Acta. 2011. № 1813. P. 597-607.