vestibИзвестия Национальной академии наук Беларуси. Серия биологических наукProceedings of the National Academy of Sciences of Belarus, Biological Series1029-89402524-230XThe Republican Unitary Enterprise Publishing House "Belaruskaya Navuka"10.29235/1029-8940-2026-71-2-132-143vestib-1012Research ArticleСтатьиСравнительный анализ протеома в культуре клеток HeLa и HepG2 до и после обработки феназинамиComparative analysis of proteome in HeLa and HepG2 cell cultures before and after treatment with phenazinesВеремеенкоЕ. Г.VerameyenkaK. G.

Веремеенко Екатерина Геннадьевна – канд. биол. наук, доцент

пр. Независимости, 4, 220030, г. Минск

Katsiaryna G. Verameyenka – Ph. D. (Biol.), Associate Professor

4, Nezavisimosty Ave., 220030, Minsk

veremeenkoKatya@yandex.ruЛиС.LiX.

Сянпу Ли– аспирант

пр. Независимости, 4, 220030, г. Минск

Xiangpu Li – Postgraduate Student

4, Nezavisimosty Ave., 220030, Minsk

lixiangpu3@gmail.comЖизневскаяА. А.ZhiznevskayaA. A.

Жизневская Анастасия Анатольевна – выпускник кафедры генетики

пр. Независимости, 4, 220030, г. Минск

Anastasia A. Zhiznevskaya – Graduate of the Department of Genetics

4, Nezavisimosty Ave., 220030, Minsk

anastasia.zhyzneyskaya@gmail.comПокроваЕ. С.PokrovaE. S.

Покрова Екатерина Сергеевна – выпускник кафедры генетики

пр. Независимости, 4, 220030, г. Минск

Ekaterina S. Pokrova – Graduate of the Department of Genetics

4, Nezavisimosty Ave., 220030, Minsk

ekaterinapokrova01@gmail.comЗинкевичА. Г.ZinkevichA. G.

Зинкевич Алина Геннадьевна – выпускник кафедры генетики

пр. Независимости, 4, 220030, г. Минск

Alina G. Zinkevich – Graduate of the Department of Genetics

4, Nezavisimosty Ave., 220030, Minsk

zinkevicalina02@gmail.comШапироМ. А.ShapiroM. A.

Шапиро Михаил Анатольевич – науч. сотрудник

ул. Академика Купревича, 5/2, 220141, г. Минск

Mikhail A. Shapiro – Researcher

5/2, Academician Kuprevich Str., 220141, Minsk

shapiromihailanatolevich@gmail.comМаксимоваН. П.MaximovaN. P.

Максимова Наталья Павловна – д-р биол. наук, профессор, профессор кафедры генетики

пр. Независимости, 4, 220030, г. Минск

Natalia P. Maximova – D. Sc. (Biol.), Professor, Professor of the Department of Genetics

4, Nezavisimosty Ave., 220030, Minsk

nataliamaximova@yahoo.comБелорусский государственный университетBelarusian State UniversityБелорусский государственный университетBelarusИнститут биоорганической химии Национальной академии наук БеларусиInstitute of Bioorganic Chemistry of the National Academy of Sciences of Belarus202630042026712132143Copyright © Веремеенко Е.Г., Ли С., Жизневская А.А., Покрова Е.С., Зинкевич А.Г., Шапиро М.А., Максимова Н.П., 20262026Веремеенко Е.Г., Ли С., Жизневская А.А., Покрова Е.С., Зинкевич А.Г., Шапиро М.А., Максимова Н.П.Verameyenka K.G., Li X., Zhiznevskaya A.A., Pokrova E.S., Zinkevich A.G., Shapiro M.A., Maximova N.P.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://vestibio.belnauka.by/jour/article/view/1012

Действие феназиновых соединений в отношении прокариотических организмов изучено достаточно неплохо. Однако в отношении эукариотических клеток существует значительный пробел в этом вопросе.

Целью исследования является протеомный анализ культур клеток HeLa и HepG2 до и после обработки очищенными комплексами феназиновых соединений бактерий Pseudomonas chlororaphis subsp. aurantiaca и идентификация мишеней для феназинов в эукариотических клетках.

В ходе исследования было установлено, что основная масса белков, обнаруженных нами в протеоме культур клеток HeLa и HepG2, задействована в регуляции мембранного потенциала клеток, сигнальных каскадах, вовлеченных в процессы онкогенеза, регуляции кальция в сердечной мышце, функционировании нервной системы и ответе на ксенобиотики. Также было зарегистрировано накопление гистоновых белков и белков-модификаторов хроматина, способствующих его переводу в гетерохроматин в культурах HeLa и HepG2 после обработки феназинами. В то же время содержание ферментов декомпактизации хроматина снижается в обеих культурах. Концентрация некоторых рибосомных белков существенно меняется в культуре клеток HeLa, тогда как в культуре клеток HepG2 регистрируется массовое увеличение их содержания.

The effect of phenazine compounds on prokaryotic organisms has been well studied. However, there is a large gap with regard to eukaryotic cells.

The aim of this study is the proteomic analysis of HeLa and HepG2 cell cultures before and after treatment with purified complexes of phenazine compounds from the bacterium Pseudomonas chlororaphis subsp. aurantiaca and the identification of targets for phenazines in eukaryotic cells.

In the course of this study, most proteins found in the proteome of HeLa and HepG2 cell cultures were found to be involved in the regulation of cell membrane potential, signalling cascades involved in oncogenesis, calcium regulation in cardiac muscle, nervous system function and response to xenobiotics. In addition, an accumulation of histone proteins and chromatin modifiers that promote conversion to heterochromatin was detected in HeLa and HepG2 cultures after phenazine treatment. At the same time, the content of chromatin decompaction enzymes decreases in both cultures. The concentration of some ribosomal proteins changes significantly in the HeLa cell culture, while in the HepG2 cell culture there is a massive increase in their content.

феназиныпротеоммалигнизированные культуры клетокMetascape-анализphenazinesproteomemalignant cell culturesMetascape analysisИсследование выполнено в рамках Государственной программы «Биотехнологии-2» подпрограммы «Геномика, эпигеномика и биоинформационный анализ» (№ гранта 20241244) при финансовой поддержке Министерства образования Республики Беларусь. Выражаем благодарность В. Э. Сяховичу, канд. биол. наук, доценту, заведующему научно-исследовательской лабораторией УЗ «Национальная антидопинговая лаборатория», Ю. С. Бабакиной, канд. биол. наук, вед. науч. сотруднику этой же лаборатории, за возможность проведения протеомного анализа на базе лаборатории.The study was carried out within the framework of the State Program “Biotechnology-2”, subprogram “Genomics, epigenomics and bioinformation analysis” (grant No. 20241244) with the financial support of the Ministry of Education of the Republic of Belarus. We would like to express our gratitude to V. E. Syakhovich, Ph. D. (Biol.), Associate Professor, Head of the Research Laboratory of the National Anti-Doping Laboratory, and Yu. S. Babakina, Ph. D. (Biol.), Leading Researcher of the same Laboratory, for the opportunity to conduct proteomic analysis at the laboratory.ReferencesА Pan-cancer molecular subtypes revealed by mass-spectrometry-based proteomic characterization of more than 500 human cancers / F. Chen, D. S. Chandrashekar, S. Varambally, C. J. Creighton // Nature Communications. – 2019. – Vol. 10. – Art. 5679. https://doi.org/10.1038/s41467-019-13528-0Chen F., Chandrashekar D. S., Varambally S., Creighton C. J. A Pan-cancer molecular subtypes revealed by massspectro metry-based proteomic characterization of more than 500 human cancers. Nature Communications, 2019, vol. 10, art. 5679. https://doi.org/10.1038/s41467-019-13528-0High throughput proteomics identifies a high-accuracy 11 plasma protein biomarker signature for ovarian cancer / S. Enroth, M. Berggrund, M. Lycke [et al.] // Communications Biology. – 2019. – Vol. 2. – Art. 221. https://doi.org/10.1038/s42003-019-0464-9Enroth S., Berggrund M., Lycke M., Broberg J., Lundberg M., Assarsson E., Olovsson M., Stålberg K., Sundfeldt K., Gyllensten U. High throughput proteomics identifies a high-accuracy 11 plasma protein biomarker signature for ovarian cancer. Communications Biology, 2019, vol. 2, art. 221. https://doi.org/10.1038/s42003-019-0464-9Application of Proteomics in Cancer: Recent Trends and Approaches for Biomarkers Discovery / Y. W. Kwon, H. S. Jo, S. Bae [et al.] // Frontiers in Medicine. – 2021. – Vol. 8. – P. 747–760. https://doi.org/10.3389/fmed.2021.747333Kwon Y. W., Jo H. S., Bae S., Seo Y., Song P., Song M., Yoon J. H. Application of Proteomics in Cancer: Recent Trends and Approaches for Biomarkers Discovery. Frontiers in Medicine, 2021, vol. 8, pp. 747–760. https://doi.org/10.3389/fmed.2021.747333Proteomics in decoding cancer: A review / E. Gheybi, P. Hosseinzadeh, V. Tayebi-Khorrami [et al.] // Clinica Chimica Acta. – 2025. – Vol. 574. – Art. 120132. https://doi.org/10.1016/j.cca.2025.120302Gheybi E., Hosseinzadeh P., Tayebi-Khorrami V., Rostami M., Soukhtanloo M. Proteomics in decoding cancer: A review. Clinica Chimica Acta, 2025, vol. 574, art. 120132. https://doi.org/10.1016/j.cca.2025.120302Proteomic approaches in the study of cancers / K. U. Nisa, N. Tarfeen, S. Wani [et al.] // Proteomics. A Promising Approach for Cancer Research / eds.: S. Ali, S. Majid, M. U. Rehman. – San Diego, 2023. – Ch. 8. – Р. 205–217. https://doi.org/10.1016/b978-0-323-95072-5.00002-xNisa K. U., Tarfeen N., Wani S., Nisa Q., Ali Sh., Wali A. F. Proteomic approaches in the study of cancers. Proteomics. A Promising Approach for Cancer Research. San Diego, 2023, сh. 8, pp. 205–217. https://doi.org/10.1016/b978-0-323-950725.00002-xCancerProteome: a resource to functionally decipher the proteome landscape in cancer / D. Lv, D. Li, Y. Cai [et al.] // Nucleic Acids Research. – 2024. – Vol. 52, N D1. – P. D1155–D1162. https://doi.org/10.1093/nar/gkad824Lv D., Li D., Cai Y., Guo J., Chu S., Yu J., Liu K., Jiang T., Ding N., Jin X., Li Y., Xu J. CancerProteome: a resource to functionally decipher the proteome landscape in cancer. Nucleic Acids Research, 2024, vol. 52, no. D1. pp. D1155–D1162. https://doi.org/10.1093/nar/gkad824Giurini, E. F. Redefining bioactive small molecules from microbial metabolites as revolutionary anticancer agents / E. F. Giurini, A. Godla, K. H. Gupta // Cancer Gene Therapy. – 2024. – Vol. 31, N 2. – P. 187–206. https://doi.org/10.1038/s41417-023-00715-xGiurini E. F., Godla A., Gupta K. H. Redefining bioactive small molecules from microbial metabolites as revolutionary anticancer agents. Cancer Gene Therapy, 2024, vol. 31, no. 2, pp. 187–206. https://doi.org/10.1038/s41417-023-00715-xAntimicrobial, anti-inflammatory, anticancer and antiviral activity of bioactive compounds from Pseudomonas aeruginosa isolated from Mediterranean Sea, Alexandria, Egypt / A. N. Mohamed, A. K. S. H. Mohamed, A. M. Zahran [et al.] // Microbial Biosystems. – 2025. – Vol. 10, N 1. – P. 123–134. https://doi.org/10.21608/mb.2025.322359.1173Mohamed A. N., Mohamed A. K. S. H., Zahran A. M., Gad A. M., Mekky A. E. Antimicrobial, anti-inflammatory, anticancer and antiviral activity of bioactive compounds from Pseudomonas aeruginosa isolated from Mediterranean Sea, Alexandria, Egypt. Microbial Biosystems, 2025, vol. 10, no. 1, pp. 123–134. https://doi.org/10.21608/mb.2025.322359.1173Novel approach of phenazine derivatives isolation from Pseudomonas culture medium / M. A. Shapira, K. G. Verameyenka, K. V. Liavonchyk [et al.] // Process Biochemistry. – 2021. – Vol. 111, N 2. – P. 325–331. https://doi.org/10.1016/j.procbio.2021.11.004Shapira M. A., Verameyenka K. G., Liavonchyk K. V., Dobysh A. A., Yantsevich A. V., Maksimova N. P. Novel approach of phenazine derivatives isolation from Pseudomonas culture medium. Process Biochemistry, 2021, vol. 111, no. 2, pp. 325–331. https://doi.org/10.1016/j.procbio.2021.11.004The Cytotoxic Activity of Phenazine Compounds from Pseudomonas chlororaphis subsp. aurantiaca against the HeLa Cell Line / A. A. Zhyzneyskaya, A. A. Lukashevich, N. P. Maksimova, E. G. Veremeenko // Molecular Genetics, Microbiology and Virology. – 2023. – Vol. 38, N 4. – P. 215–221. https://doi.org/10.3103/S0891416823040079Zhyzneyskaya A. A., Lukashevich A. A., Maksimova N. P., Veremeenko E. G. The Cytotoxic Activity of Phenazine Compounds from Pseudomonas chlororaphis subsp. aurantiaca against the HeLa Cell Line. Molecular Genetics, Microbiology and Virology, 2023, vol. 38, no. 4, pp. 215–221. https://doi.org/10.3103/S0891416823040079Statistical Approach to Protein Quantification / S. Gerster, T. Kwon, C. Ludwig [et al.] // Molecular and Cellular Proteomics. – 2014. – Vol. 13, N 2. – P. 666–677. https://doi.org/10.1074/mcp.m112.025445Gerster S., Kwon T., Ludwig C., Matondo M., Vogel C., Marcotte E. M., Aebersold R., Bühlmann P. Statistical Approach to Protein Quantification. Molecular and Cellular Proteomics, 2014, vol. 13, no. 2, pp. 666–677. https://doi.org/10.1074/mcp.m112.025445Protein measurement with the folin phenol reagent / O. H. Lowry, N. J. Rosebrough, A. L. Farr, R. J. Randall // Journal of Biological Chemistry. – 1951. – Vol. 193, N 1. – P. 265–275. https://doi.org/10.1016/s0021-9258(19)52451-6Lowry O. H., Rosebrough N. J., Farr A. L., Randall R. J. Protein measurement with the folin phenol reagent. Journal of Biological Chemistry, 1951, vol. 193, no. 1, pp. 265–275, https://doi.org/10.1016/s0021-9258(19)52451-6Expression stability of 13 housekeeping genes during carbon starvation of Pseudomonas aeruginosa / B. Alqarni, B. Colley, J. Klebensberger [et al.] // Journal of Microbiological Methods. – 2016. – Vol. 127. – P. 182–187. https://doi.org/10.1016/j.mimet.2016.06.008Alqarni B., Colley B., Klebensberger J., McDougald D., Rice S. A. Expression stability of 13 housekeeping genes du ring carbon starvation of Pseudomonas aeruginosa. Journal of Microbiological Methods, 2016, vol. 127, pp. 182–187. https://doi.org/10.1016/j.mimet.2016.06.008Highly expressed RPLP2 inhibits ferroptosis to promote hepatocellular carcinoma progression and predicts poor prognosis / J. Guo, M. Huang, S. Deng [et al.] // Cancer Cell International. – 2023. – Vol. 23, N 1. – Art. 278. https://doi.org/10.1186/s12935-023-03140-0Guo J., Huang M., Deng S., Wang H., Wang Z., Yan B. Highly expressed RPLP2 inhibits ferroptosis to promote hepato cellular carcinoma progression and predicts poor prognosis. Cancer Cell International, 2023, vol. 23, no. 1, art. 278. https://doi.org/10.1186/s12935-023-03140-0RPLP2 activates TLR4 in an autocrine manner and promotes HIF-1alpha-induced metabolic reprogramming in hepatocellular carcinoma / Q. Yang, X. Meng, J. Chen [et al.] // Cell Death Discovery. – 2023. – Vol. 9, N 1. – Art. 440. https:// doi.org/10.1038/s41420-023-01719-0Yang Q., Meng X., Chen J., Li X., Huang Y., Xiao X., Li R., Wu X. RPLP2 activates TLR4 in an autocrine manner and promotes HIF-1alpha-induced metabolic reprogramming in hepatocellular carcinoma. Cell Death Discovery, 2023, vol. 9, no. 1, art. 440. https://doi.org/10.1038/s41420-023-01719-0Expression of the ribosomal proteins Rplp0, Rplp1, and Rplp2 in gynecologic tumors / A. Artero-Castro, J. Castellvi, A. García [et al.] // Human Pathology. – 2011. – Vol. 42, N 2. – P. 194–203. https://doi.org/10.1016/j.humpath.2010.04.020Artero-Castro A., Castellvi J., García A., Hernández J., Ramón y Cajal S., LLeonart M. E. Expression of the ribosomal proteins Rplp0, Rplp1, and Rplp2 in gynecologic tumors. Human Pathology, 2011, vol. 42, no. 2, pp. 194–203. https:// doi.org/10.1016/j.humpath.2010.04.020Ribosomal protein RPL5 regulates colon cancer cell proliferation and migration through MAPK/ERK signaling pathway / H. Zhang, J. Liu, Q. Dang [et al.]. // BMC Molecular and Cell Biology. – 2022. – Vol. 23, N 1. – Art. 48. https://doi.org/10.1186/s12860-022-00448-zZhang H., Liu J., Dang Q., Wang X., Chen J., Lin X., Yang N., Du J., Shi H., Liu Y., Han J. Ribosomal protein RPL5 regulates colon cancer cell proliferation and migration through MAPK/ERK signaling pathway. BMC Molecular and Cell Biology, 2022, vol. 23, no. 1, art. 48. https://doi.org/10.1186/s12860-022-00448-zRibosomal proteins in hepatocellular carcinoma: mysterious but promising / Q. Su, H. Sun, L. Mei [et al.] // Cell and Bioscience. – 2024. – Vol. 14. – Art. 133. https://doi.org/10.1186/s13578-024-01316-3Su Q., Sun H., Mei L., Yan Y., Ji H., Chang L., Wang L. Ribosomal proteins in hepatocellular carcinoma: mysterious but promising. Cell and Bioscience, 2024, vol. 14, art. 133. https://doi.org/10.1186/s13578-024-01316-3Targeting RPL23 restores chemosensitivity of cisplatin-resistant ovarian carcinoma by inhibiting EMT / Y. Liu, S. Lai, J. He [et al.] // Cytotechnology. – 2022. – Vol. 74, N 3. – P. 421–432. https://doi.org/10.1007/s10616-022-00535-1Liu Y., Lai S., He J., Wan J., Fu F., Jinlong Y. Targeting RPL23 restores chemosensitivity of cisplatin-resistant ovarian carcinoma by inhibiting EMT. Cytotechnology, 2022, vol. 74, no. 3, pp. 421–432. https://doi.org/10.1007/s10616-022-00535-1Price-Whelan, A. Rethinking ‘secondary’ metabolism: physiological roles for phenazine antibiotics / A. PriceWhelan, L. E. Dietrich, D. K. Newman // Nature Chemical Biology. – 2006. – Vol. 2. – P. 71–78. https://doi.org/10.1038/nchembio764Price-Whelan A., Dietrich L. E., Newman D. K. Rethinking ‘secondary’ metabolism: physiological roles for phenazine antibiotics. Nature Chemical Biology, 2006, vol. 2, pp. 71–78. https://doi.org/10.1038/nchembio764

The authors declare that there are no conflicts of interest present.