Russian Journal of Biological Physics and Chemisrty Russian Journal of Biological Physics and Chemisrty АКТУАЛЬНЫЕ ВОПРОСЫ БИОЛОГИЧЕСКОЙ ФИЗИКИ И ХИМИИ 2499-9962 54350 Общая биофизика General biophysics Общая биофизика CHANGE IN THE LEVEL OF ROS IN HELA KYOTO CELLS AT DIFFERENT STAGES OF CISPLATIN-INDUCED CELL DEATH ИЗМЕНЕНИЕ УРОВНЯ АФК В КЛЕТКАХ HELA KYOTO НА РАЗНЫХ СТАДИЯХ ЦИСПЛАТИН-ИНДУЦИРОВАННОЙ КЛЕТОЧНОЙ ГИБЕЛИ Щукина К М Shchukina K M ksushaschukina1995@yandex.ru Неруш А С Nerush A S Орлова А Г Orlova A G Национальный исследовательский Нижегородский государственный университет им. Н.И. Лобачевского ru Lobachevsky State University of Nizhny Novgorod ru Федеральный исследовательский центр Институт прикладной физики РАН ru Institute of Applied Physics RAS ru Федеральный исследовательский центр Институт прикладной физики РАН ru Institute of Applied Physics RAS ru 25 12 2018 25 12 2018 3 4 699 705 20 12 2018 20 12 2018 https://rusjbpc.ru/en/nauka/article/54350/view

Цисплатин - химиотерапевтический препарат, успешно применяемый в лечении широкого спектра опухолей. Данный препарат может вызвать митохондриальную дисфункцию и повышенное производство активных форм кислорода (АФК), что приводит к повреждению мембраны, ДНК и других важных элементов клетки. Целью работы было исследование изменений уровня АФК на разных стадиях клеточной гибели, вызванной цисплатином. В работе использовалась линия HeLa Kyoto, флуоресцентный краситель DCFH-DA, маркер апоптоза ФЭ-Аннексин V и витальный краситель 7-актиноаминомицин D. С использованием метода проточной цитофлуориметрии было показано, что большинство жизнеспособных клеток после обработки препаратом интенсивно окрашиваются DCFH-DA, что свидетельствует об активно идущих в них окислительных процессах и высоком уровне АФК. На стадии раннего апоптоза количество АФК уменьшается по сравнению с необработанным препаратом клетками.

Cisplatin is a chemotherapeutic drug, successfully used in the treatment of a wide range of tumors. This drug can cause mitochondrial dysfunction and increased production of reactive oxygen spices (ROS), which leads to the damage of membrane, DNA and other important cell elements. The aim of the study was to investigate the changes in the level of ROS at different stages of cell death caused by cisplatin. We used the HeLa Kyoto cell line, the fluorescent dye DCFH-DA, the marker of apoptosis PE-Annexin V, and the vital dye 7-actinaminomycin D. Using flow cytometry, it was shown that after treatment most of viable cells are intensely stained with DCFH-DA, which indicates active oxidation processes and high level of ROS. At the stage of early apoptosis, the amount of ROS decreases as compared to the untreated cells.

 цисплатин активные формы кислорода апоптоз cisplatin reactive oxygen spices apoptosis

Szatrowski T.P., Nathan C.F. Production of large amounts of hydrogen peroxide by human tumor cells. Cancer Res., 1991, vol. 51, pp. 794-798. Szatrowski T.P., Nathan C.F. Production of large amounts of hydrogen peroxide by human tumor cells. Cancer Res., 1991, vol. 51, pp. 794-798. Toyokuni S., Okamoto K., Yodoi J., Hiai H. Pers is tent oxidative s tres s in cancer. FEBS Lett., 1995, vol. 358, pp. 1-3. Toyokuni S., Okamoto K., Yodoi J., Hiai H. Pers is tent oxidative s tres s in cancer. FEBS Lett., 1995, vol. 358, pp. 1-3. Liou G.-Y., Storz P. Reactive oxygen species in cancer. Free Radic Res., 2010, vol. 44, no. 5, pp. 479-496. Liou G.-Y., Storz P. Reactive oxygen species in cancer. Free Radic Res., 2010, vol. 44, no. 5, pp. 479-496. Trachootham D., Alexandre J., Huang P. Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach? Nat. Rev. Drug Discov., 2009, vol. 8, no. 7, pp. 579-591. Trachootham D., Alexandre J., Huang P. Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach? Nat. Rev. Drug Discov., 2009, vol. 8, no. 7, pp. 579-591. Conklin K.A. Chemotherapy-associated oxidative stress: impact on chemotherapeutic effectiveness. Integrative Cancer Therapies, 2004, vol. 3, no. 4, pp. 294-300. Conklin K.A. Chemotherapy-associated oxidative stress: impact on chemotherapeutic effectiveness. Integrative Cancer Therapies, 2004, vol. 3, no. 4, pp. 294-300. Florea A.-M., Bus selberg D. Cisplatin as an anti-tumor drug: cellular mechanisms of activity, drug resistance and induced side effects. Cancers, 2011, vol. 3, no. 1, pp. 1351-1371. Florea A.-M., Bus selberg D. Cisplatin as an anti-tumor drug: cellular mechanisms of activity, drug resistance and induced side effects. Cancers, 2011, vol. 3, no. 1, pp. 1351-1371. Loskotova H., Brabec V. DNA interactions of cisplatin tethered to the DNA minor groove binder distamycin. Eur. J. Biochem., 1999, vol. 266, pp. 392-402. Loskotova H., Brabec V. DNA interactions of cisplatin tethered to the DNA minor groove binder distamycin. Eur. J. Biochem., 1999, vol. 266, pp. 392-402. Choi Y.-M., Kim H.-K., Shim W., Anwar M.A., Kwon J.-W., Kwon H.-K., Kim H.J., Jeong H., Kim H.M., Hwang D., Kim H.S., Choi S. Mechanism of Cisplatin-Induced Cytotoxicity Is Correlated to Impaired Metabolism Due to Mitochondrial ROS Generation. PLoS One, 2015, vol. 10, no. 8, p. 21. Choi Y.-M., Kim H.-K., Shim W., Anwar M.A., Kwon J.-W., Kwon H.-K., Kim H.J., Jeong H., Kim H.M., Hwang D., Kim H.S., Choi S. Mechanism of Cisplatin-Induced Cytotoxicity Is Correlated to Impaired Metabolism Due to Mitochondrial ROS Generation. PLoS One, 2015, vol. 10, no. 8, p. 21. Kim H.-J., Lee J-H., Kim S.-J., Oh G.S., Moon H.-D., Kwon K.-B., Park C., Park B.H., Lee H.-K, Chung S.-Y., Park R., So H.-S. Roles of NADPH Oxidases in Cisplatin-Induced Reactive Oxygen Species Generation and Ototoxicity. JNeurosci, 2010, vol. 30, no. 11, рр. 3933-3946. Kim H.-J., Lee J-H., Kim S.-J., Oh G.S., Moon H.-D., Kwon K.-B., Park C., Park B.H., Lee H.-K, Chung S.-Y., Park R., So H.-S. Roles of NADPH Oxidases in Cisplatin-Induced Reactive Oxygen Species Generation and Ototoxicity. JNeurosci, 2010, vol. 30, no. 11, rr. 3933-3946. Guo J, Xu B., Han Q., Zhou H., Xia Y., Gong C., Dai X., Li Z., Wu G. Ferroptosis: A Novel Anti-tumor Action for Cisplatin. Cancer Res. Treat., 2018, vol. 50, no. 2, pp. 445-460. Guo J, Xu B., Han Q., Zhou H., Xia Y., Gong C., Dai X., Li Z., Wu G. Ferroptosis: A Novel Anti-tumor Action for Cisplatin. Cancer Res. Treat., 2018, vol. 50, no. 2, pp. 445-460. Marullo R., Werner E., Degtyareva N., Moore B., Altavilla G., Ramalingam S., Doetsch P. Cisplatin Induces a Mitochondrial-ROS Response That Contributes to Cytotoxicity Depending on Mitochondrial Redox Status and Bioenergetic Functions. PLoS One, 2013, vol. 8, no. 11, p. 15. Marullo R., Werner E., Degtyareva N., Moore B., Altavilla G., Ramalingam S., Doetsch P. Cisplatin Induces a Mitochondrial-ROS Response That Contributes to Cytotoxicity Depending on Mitochondrial Redox Status and Bioenergetic Functions. PLoS One, 2013, vol. 8, no. 11, p. 15. Pavelescu L.A. On reactive oxygen species measurement in living systems? J. Med. Life, 2015, vol. 8, pp. 38-42. Pavelescu L.A. On reactive oxygen species measurement in living systems? J. Med. Life, 2015, vol. 8, pp. 38-42. Belova А.S., Orlova A.G., Balalaeva I.V., Antonova N.O., Maslennikova A.V., Mishina N.M., Zagaynova E.V. Hydrogen peroxide detection in viable and apoptotic tumor cells under action of cisplatin and bleomycin. Photonics & Lasers in Medicine, 2016, vol. 5, no. 2, pp. 113-121. Belova A.S., Orlova A.G., Balalaeva I.V., Antonova N.O., Maslennikova A.V., Mishina N.M., Zagaynova E.V. Hydrogen peroxide detection in viable and apoptotic tumor cells under action of cisplatin and bleomycin. Photonics & Lasers in Medicine, 2016, vol. 5, no. 2, pp. 113-121. Louis K.S., Siegel A.C. Cell viability analysis using trypan blue: manual and automated methods. Methods Mol. Biol., 2011, vol. 740, pp. 7-12. Louis K.S., Siegel A.C. Cell viability analysis using trypan blue: manual and automated methods. Methods Mol. Biol., 2011, vol. 740, pp. 7-12. Burow S., Valet G. Flow-cytometric characterization of stimulation, free radical formation, peroxidase activity and phagocytosis of human granulocytes with 2.7-dichlorofluorescein (DCF). Eur. J. Cell. Biol., 1987, vol. 43, pp. 128-133. Burow S., Valet G. Flow-cytometric characterization of stimulation, free radical formation, peroxidase activity and phagocytosis of human granulocytes with 2.7-dichlorofluorescein (DCF). Eur. J. Cell. Biol., 1987, vol. 43, pp. 128-133. Rothe G., Valet G. Flow cytometric analysis of respiratory burst activity in phagocytes with hydroethidine and 2, 7-dichlorofl uorescin. J. Leukoc. Biol., 1990, vol. 47, pp. 440-448. Rothe G., Valet G. Flow cytometric analysis of respiratory burst activity in phagocytes with hydroethidine and 2, 7-dichlorofl uorescin. J. Leukoc. Biol., 1990, vol. 47, pp. 440-448. Белова А.С., Орлова А.Г., Брилкина А.А., Масленникова А.В. Чувствительность клеток линии Hela Kyoto трансфицированных сенсором HyPer2 к действию цисплатина. СТМ, 2014, т. 6, № 4, c. 7-13. [Belova А.S., Orlova A.G., Brilkina А.А., Maslennikova A.V., The sensitivity of Hela Kyoto cell line transfected with sensor HyPer2 to cisplatin. STM, 2014, vol. 6, no. 4, pp. 7-13. (In Russ.)] Belova A.S., Orlova A.G., Brilkina A.A., Maslennikova A.V. Chuvstvitel'nost' kletok linii Hela Kyoto transficirovannyh sensorom HyPer2 k deystviyu cisplatina. STM, 2014, t. 6, № 4, c. 7-13. [Belova A.S., Orlova A.G., Brilkina A.A., Maslennikova A.V., The sensitivity of Hela Kyoto cell line transfected with sensor HyPer2 to cisplatin. STM, 2014, vol. 6, no. 4, pp. 7-13. (In Russ.)] Crow J.P. Dichlorodihydrofluorescein and dihydrorhodamine 123 are sensitive indicators of peroxynitrite in vitro: implications for intracellular measurement of reactive nitrogen and oxygen species. Nitric Oxide, 1997, vol. 1, pp. 145-157. Crow J.P. Dichlorodihydrofluorescein and dihydrorhodamine 123 are sensitive indicators of peroxynitrite in vitro: implications for intracellular measurement of reactive nitrogen and oxygen species. Nitric Oxide, 1997, vol. 1, pp. 145-157. Wang H., Joseph J.A. Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Radic. Biol. Med., 1999, vol. 27, pp. 612-616. Wang H., Joseph J.A. Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Radic. Biol. Med., 1999, vol. 27, pp. 612-616. Franskevych D.V., Grynyuk I.I., Prylutska S.V., Matyshevska O.P. Modulation of cisplatin-induced reactive oxygen species production by fullerene C(60) in normal and transformed lymphoid cells. Ukr. Biochem. J., 2016, vol. 88, pp. 44-50. Franskevych D.V., Grynyuk I.I., Prylutska S.V., Matyshevska O.P. Modulation of cisplatin-induced reactive oxygen species production by fullerene C(60) in normal and transformed lymphoid cells. Ukr. Biochem. J., 2016, vol. 88, pp. 44-50. Itoh T., Terazawa R., Kojima K., Nakane K., Deguchi T., Ando M., Tsukamasa Y., Ito M., Nozawa Y. Cisplatin induces production of reactive oxygen species via NADPH oxidase activation in human prostate cancer cells. Free Radic. Res., 2011, vol. 45, no. 9, pp. 1033-1039. Itoh T., Terazawa R., Kojima K., Nakane K., Deguchi T., Ando M., Tsukamasa Y., Ito M., Nozawa Y. Cisplatin induces production of reactive oxygen species via NADPH oxidase activation in human prostate cancer cells. Free Radic. Res., 2011, vol. 45, no. 9, pp. 1033-1039. Kalyanaraman B., Darley-Usmar V., Davies K.J.A., Dennery P.A, Forman H.J., Grisham M.B., Mann G.E., Moore K., Jackson Roberts II L., Ischiropoulose H. Measuring reactive oxygen and nitrogen species with fluorescent probes: challenges and limitations. Free Radic. Biol. Med., 2012, vol. 52, no. 1, p. 6. Kalyanaraman B., Darley-Usmar V., Davies K.J.A., Dennery P.A, Forman H.J., Grisham M.B., Mann G.E., Moore K., Jackson Roberts II L., Ischiropoulose H. Measuring reactive oxygen and nitrogen species with fluorescent probes: challenges and limitations. Free Radic. Biol. Med., 2012, vol. 52, no. 1, p. 6. Karlsson M., Kurz T., Brunk U.T., Nilsson. S.E., Frennesson C.I. What does the commonly used DCF test for oxidative stress really show? Biochem. J., 2010, vol. 428, pp. 183-190. Karlsson M., Kurz T., Brunk U.T., Nilsson. S.E., Frennesson C.I. What does the commonly used DCF test for oxidative stress really show? Biochem. J., 2010, vol. 428, pp. 183-190. Qian S.Y., Buettner G.R. Iron and dioxygen chemistry is an important route to initiation of biological and free radical oxidations: an electron paramagnetic resonance spin trapping study. Free Radic. Biol. Med., 1999, vol. 26, pp. 1447-1456. Qian S.Y., Buettner G.R. Iron and dioxygen chemistry is an important route to initiation of biological and free radical oxidations: an electron paramagnetic resonance spin trapping study. Free Radic. Biol. Med., 1999, vol. 26, pp. 1447-1456.