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DOI: https://doi.org/10.17650/2313-805X-2023-10-4-149-156
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Keywords: rapamycin, tamoxifen, drug resistance, MCF-7 cells, protein kinase AKT, DNA methyltransferase, LINE repeats
For citation: Andreeva O.E., Sorokin D.V., Vinokurova S.V. et al. The effect of DNA methyltransferase 3A suppression in progression of the resistance phenotype in breast cancer cells. Uspehi Molekularnoj Onkologii = Advances in Molecular Oncology 2023;10(4):149-56. DOI: https://doi.org/10.17650/2313-805X-2023-10-4-149-156
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The effect of DNA methyltransferase 3A suppression in progression of the resistance phenotype in breast cancer cells
O.E. Andreeva1, D.V. Sorokin1, 2, S.V. Vinokurova1, Yu.Yu. Shchegolev1, N.V. Elkina1, A.N. Katargin1, R.S. Faskhutdinov1, D.I. Salnikova1, 3, A.M. Scherbakov1, 2, M.A. Krasil'nikov1, 2
1N.N.. Blokhin National Medical Research Center of Oncology, Ministry of Health of Russia; 24 Kashirskoe Shosse, Moscow 115522, Russia;
2National Research Lobachevsky State University of Nizhny Novgorod; 23 Gagarin Prospekt, Nizhny Novgorod 603950, Russia; Z
3Gause Institute of New Antibiotics; Bld. 1, 11 Bol'shaya Pirogovskaya St., Moscow 119021, Russia ^
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Contacts: Olga Evgenievna Andreeva o.andreeva@ronc.ru, <
Mikhail Alexandrovich Krasil'nikov krasilnikovm1@ya.ru o
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Introduction. Rearrangement of molecular pathways and activation of bypass signaling determine the progression ^ of tumor cell resistance to various drugs. Study of the common features of resistant formation mechanisms is essential s for breast and other cancer beneficial treatments. ^
Materials and methods. The present work was performed on estrogen receptor a ERa-positive (ERa - estrogen receptor a) © MCF-7 breast cancer cells, established sublines resistant to the mTOR inhibitor rapamycin or antiestrogen tamoxifen, and ERa-negative MDA-MB-231 breast cancer cells. Methods used include MTT test, transient transfection, immunoblotting, real-time polymerase chain reaction and methylation analysis by bisulfite pyrosequencing.
Results. We have shown that the resistance of breast cancer cells to targeted and hormonal drugs is associated with the suppression of DNA methyltransferase 3A (DNMT3A) and respective changes in DNA methylation; DNMT3A knockdown results in the partial resistance to both drugs demonstrating the pivotal role of DNMT3A suppression in the progression a. of cell resistance.
Conclusion. Totally, the results obtained highlight the possible mechanism of tumor cell resistance to targeting/hormonal drugs based on the deregulation of DNMTs expression and demonstrate direct connection between DNMT3A suppression and resistance progression.
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Феномен подавления ДНК-метилтрансферазы ЗА при формировании резистентного фенотипа в клетках рака молочной железы
О.Е. Андреева1, Д.В. Сорокин12, С.В. Винокурова1, Ю.Ю. Щеголев1, Н.В. Елкина1, А.Н. Катаргин1, Р.С. Фасхутдинов1, Д.И. Сальникова1,3, А.М. Щербаков1,2, М.А. Красильников1, 2
ФГБУ«Национальный медицинский исследовательский центр онкологии им. Н.Н. Блохина» Минздрава России; Россия, 115522 Москва, Каширское шоссе, 24;
2ФГАОУВО «Национальный исследовательский Нижегородский государственный университет им. Н.И. Лобачевского»; Россия, 603022 Нижний Новгород, проспект Гагарина, 23;
3ФГБНУ«Научно-исследовательский институт новых антибиотиков им. Г.Ф. Гаузе»; Россия, Москва 119021, ул. Большая Пироговская, 11, стр. 1
Контакты: Ольга Евгеньевна Андреева o.andreeva@ronc.ru,
Михаил Александрович Красильников krasilnikovm1@ya.ru
Введение. Переключение сигнальных путей и активация параллельных сигнальных каскадов относятся к ключевым факторам, определяющим развитие резистентности опухолевых клеток, и изучение механизмов подобной реаранжировки является одной из актуальных задач современной онкологии.
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Материалы и методы. Настоящая работа выполнена на ERa-положительных (ERa - эстрогеновый рецептор а) клетках рака молочной железы MCF-7 и полученных из них сублиниях, устойчивых к ингибитору mTOR рапамицину или антиэстрогену тамоксифену, а также на ERa-отрицательных клетках рака молочной железы MDA-MB-231. Используемые методы включают тест МТТ, транзиторную трансфекцию, иммуноблоттинг, полимеразную цепную реакцию в реальном времени и анализ метилирования с помощью бисульфитного пиросеквенирования. Результаты. Мы показали, что резистентность клеток рака молочной железы к таргетным и гормональным препаратам связана с подавлением ДНК-метилтрансферазы ЗА (DNMT3A) и соответствующими изменениями в метилировании ДНК. Нокдаун DNMT3A приводит к частичной резистентности к обоим препаратам, что демонстрирует ключевую роль подавления DNMT3A в развитии резистентности опухолевых клеток.
Заключение. В целом, полученные результаты свидетельствуют о возможном механизме формирования устойчивости опухолевых клеток к таргетным/гормональным препаратам, основанном на дерегуляции экспрессии DNMT, и демонстрируют прямую связь между подавлением DNMT3A и развитием резистентности.
Ключевые слова: рапамицин, тамоксифен, лекарственная устойчивость, клетки MCF-7, протеинкиназа AKT, ДНК-метилтрансфераза, повторы LINE
Для цитирования: Андреева O.E., Сорокин Д.В., Винокурова С.В. и др. Феномен подавления ДНК-метилтрансферазы ЗА при формировании резистентного фенотипа в клетках рака молочной железы. Успехи молекулярной онкологии 2023;10(4):149-56. (На англ.). DOI: https://doi.org/10.17650/2313-805X-2023-10-4-149-156
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INTRODUCTION
The development of acquired drug resistance of tumor cells is among the key factors limiting the efficiency of antitumor therapy [1—5]. There are various mechanisms which are respondent for the formation of the resistant phe-notype of cancer cells, including the activation of ABC (ATP-binding cassette) transporters, mutations of targeted genes, rearrangement of signaling pathways, etc. [6—8]. Among them, the reconstruction of the epigenetic machinery belongs to the main events involved in the progression and maintenance of the low drug sensitivity of tumor cells [9—12]. DNA (de)methylation involved in the different regulation of genes is associated with the progression of tumor cells resistance, including genes encoding ABC transporters, growth signaling proteins, DNA repair enzymes, growth factors and receptors, etc. [13, 14].
DNA methyltransferases (DNMTs) belong to the key enzymes, which are responsible for DNA methylation and epigenetic regulation of gene expression [15, 16]. Several studies demonstrate various changes in the expression or activity of DNMTs in resistant tumors [17—19], however, the mechanism of the precise regulation of DNMTs is still unclear.
Earlier, we have shown that prolonged treatment of MCF-7 breast cancer cells with mammalian target of rapa-mycin (mTOR) pathway inhibitors, rapamycin or metformin, resulted in the development of resistant clones characterized by constitutive activation of growth-related pathways [20, 21]. Because the activation ofbypass growth signaling is among the key features of the acquired hormonal resistance, we proposed the existence of common mechanism respondent for the formation of cell resistance to both mTOR-targeting and hormonal agents.
Here we have shown that the progression of the resistance of breast cancer cells to targeted and hormonal drugs involved the suppression of DNMT3A axis and the respective changes in DNA methylation, and demonstrated the pivotal role of DNMT3A suppression in the progression of the cell resistance. Following studies will delineate the
mechanism of DNMT3A suppression in resistant tumors, and highlight the perspectives of the applying of these parameters as an additional criterion for the prognosis of tumor resistance.
MATERIALS AND METHODS
Cell lines and evaluation of antiproliferative activity. The
MCF-7 cells (ATCC HTB-22) and MDA-MB-231 cells (ATCC HTB-26™) were cultured at 37 °C and 5 % CO2 in DMEM medium (PanEco, Russia) containing 4.5 g/l glucose and 10 % fetal bovine serum (HyClone, USA). Prolonged treatment of the parent MCF-7 cells with tamoxifen and rapamycin was used to obtain resistant sublines MCF-7/T and MCF-7/Rap respectively [20, 22]. The MTT assay [23] with modifications as described [24] was used to determine the cell response to the drugs after the treatment of the cells with tamoxifen or rapamycin.
Transfection of small interfering RNA. Scrambled nonspecific siRNA and DNMT3A specific siRNA were purchased from Syntol. Oligonucleotides were dissolved in annealing buffer (50 mM NaCl, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA), annealed at 95 °C and used for transfection with Lipofectamine 2000 (Thermo Fisher Scientific, USA). The following sequences of siRNA were used in the study: scrambled siRNA 5'-UUCUCCGAACGUGUCAC-GUTT-3', DNMT3A siRNA 5'-GCCAAGGUCAUUG-CAGGAATT-3', with corresponding antisense sequences.
Total RNA isolation and quantitative real-time polymerase chain reaction. Total RNA was extracted from cells using TRIzol reagent (Invitrogen, USA) following the manufacturer's instructions. cDNA was synthesized from 1 ^g of total RNA by reverse transcription using the iScript™ Advanced cDNA Synthesis Kit (Bio-Rad, USA). Quantitative real-time polymerase chain reaction (RT-PCR) was performed using 5X qPCRmix-HS SYBR (Evrogen, Russia) with the following conditions: initial denaturation for 3 min at 95 °C, followed by 40 cycles at 95 °C for 15 s, at Ta 60 °C for 15 s (Ta — temperature annealing), and at 72 °C for 30 s. All PCR reactions were performed in triplicate,
and gene expression was normalized relative to that of human p-Actin (ACTB), which was used as internal control. Primers used for RT-PCR are listed in table 1. Amplification and data analysis were performed on CFX96 touch RT-PCR Detection System (Bio-Rad, USA); Bio-Rad CFX Manager software v. 3.1 was used for threshold cycle (Ct) value calculations and data analysis. Relative mRNA expression was determined using the AACt method [25].
Table 1. Sequence of primers used in real-time polymerase chain reaction
Gene Forward primer (5'-3') Reverse primer (5'-3')
DNMT1 CGACTACATCAA AGGCAGCAACCTG TGGAGTGGACTTG TGGGTGTTCTC
DNMT3a TGGCAGGATAG CCAAGTTCAG GCTGGTCTTT GCCCTGCTTTATG
ACTB ATGTGGCCGAGGA CTTTGATT AGTGGGGTGG CTTTTAGGATG
Immunoblotting. Preparation of the cell lysates for im-munoblotting was conducted as described previously in [26]. The lysates were separated by 10 % SDS-PAGE, transferred to a nitrocellulose membrane (PerkinElmer, USA), and processed as described earlier [27]. After the treatment with 5 % nonfat milk (Applichem, Germany) the membranes were incubated with primary antibodies (Cell Signaling Technology) overnight at +4 °C. For the standardization of loading, the antibodies against a-tubulin (Cell Signaling Technology, USA) were used; the secondary antibodies corresponding IgGs conjugated with horseradish peroxidase were provided by Jackson ImmunoResearch (USA). The detection was performed using Mruk and Cheng's protocol [28] and an ImageQuant LAS4000 system for chemiluminescence (GE HealthCare, USA). Densi-tometry for immunoblotting data was performed using Im-ageJ software (Wayne Rasband). The protocol for densitometry was provided by The University of Queensland with the recommendations from the work [29].
Methylation analysis by bisulfite pyrosequencing. Methylation analysis was carried out using a bisulfite pyrosequencing method. Briefly, genomic DNA was isolated using the ExtractDNA Blood & Cells kit (Evrogen, Russia) and modified with sodium bisulfite using the EZ DNA Meth-ylation-Gold™ Kit (Zymo Research, USA) according to the manufacturer's protocol. Bisulfite treated DNA was amplified with a pair of gene-specific primers using 5х MaSTaqDD PCR master mix (Dialat Ltd., Russia). One of these primers was biotinylated for subsequent immobilisation of a specific DNA-fragment onto a solid support (by biotin/streptavidin coupling). Primers and assays used for pyrosequencing are listed in table 2. The PCR products were sequenced by pyrosequencing PyroMark Q24 (Qiagen, Germany) using a specific sequencing primers (table 2). The obtained sequences were analyzed using PyroMark Q24 Advanced Software, which allows analysis of methylation levels of CpG sites.
Statistical analysis. Each experiment was repeated three times with three technical replicates. Statistical analysis was performed using Microsoft Excel. Results were expressed as mean + SD (standard deviation value) if not stated explicitly. A p-value of <0.05 was considered statistically significant.
RESULTS
DNMT3A expression and cell resistance. The experiments were performed on MCF-7 breast cancer cells and rapamycin-resistant (MCF-7/Rap) and tamoxifen-resist-ant (MCF-7/T) sublines developed under prolonged treatment of the parent cells with mTOR inhibitor rapamycin or selective estrogen ERa modulator tamoxifen respectively [20, 22]. In a parallel, the study of the tamoxifen-resistant ERa-negative MDA-MB-231 breast cancer cells were performed.
Study of the DNA methylation enzymes showed the significant suppression of DNMT3A protein level in both MCF-7 resistant sublines, whereas the expression of DN-MT1 was not changed significantly (fig. 1a). Similarly,
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Table 2. Primers and pyrosequencing assays
Gene Forward primer (5'-3') Reverse primer (5'-3') Sequencing primer (5'-3') Assay
YGTGGTGYG
LINE-1 TGAGTTAGGTG TGGGATATAGT bio~AAAATCAAAA AATTCCCTTTC GTTAGGTGTG GGATATAGTTT TYGTTTTTTAA GTYGGTTT GAAAAGYGTAATA TTYGGGTGGGA
DNMT3A cg03463641 TAGTATTGGGGT TGGGGATAGTAG bio~ACCTTAACCCTAT AAAACAAAATAACCTC TAGTATTGGGGT TGGGGATAGTAG YGTTGGTTTA ATYGYGTYGTA ATTTTTAG
DNMT3A cg21629895 TGGAAGATTTTGT GTGTGTTTATATAT biotin-AAATCAAAAA CCTAAAACCCTAAAC TGGAAGATTTTGT GTGTGTTTATATAT YGTTTTTATTTT TTTATYGTGGGGG TTGTTTTTTTTTTTT ATGGAGYGTTT
Note. Symbol Y represents C or T according to the IUPAC codes.
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RT-PCR analysis revealed the marked decrease in the level of mRNA DNMT3A in the resistant cells when the level of mRNA DNMT1 was decreased slightly (fig. 1b). Similarly to the MCF-7 resistant sublines, the ERa-negative MDA-MB-231 cells were characterized with the marked decrease in the protein and mRNA level of DN-MT3A when the DNMT1 level was even slightly increased (fig. 1a, b).
Cell resistance and DNA methylation. To investigate the effect of DNMT3A suppression on the global change in DNA methylation, we examined the methylation level of long interspersed nucleotide element 1 (LINE-1). LINE-1 is a major genetic element, making up ~17 % of the entire genome [30]. CpG sites located within LINE-1 and their methylation levels correlate with the global methylation status of genomic DNA and therefore often used as a surrogate marker for assessing global DNA methylation alterations [31].
The analysis of LINE-1 methylation in the resistant sublines revealed a slight decrease in that in tamoxifen- and rapamycin-resistant cells as well as in the MDA-MB-231 cell line (not significant)) (fig. 2) supporting the repression of DNMT3A activity in the resistant cells.
In addition, we analyzed the methylation of regulatory sequences, a CpG-island (CGI) in the promoter of the DNMT3A gene and in transitional areas, termed shores, to elucidate potential mechanisms of suppression of DN-MT3A expression in resistant cells. There were no significant changes in the DNA sequence methylation levels in the CGI and S-shore regions in the MCF-7/T and MCF-7/Rap resistant cells compared to the parental cell line. According to The Cancer Genome Atlas (TCGA) (breast cancer data collection, BRCA), increased methylation of S-shore region (genomic coordinates chr2:25565794—
25565842, hg19/Human) is associated with reduced DN-MT3A expression. Thus, DNA methylation, at least of the S-shore region of DNMT3A gene, does not seem to be responsible for suppression of DNMT3A expression in resistant cells. In contrast, we found increased methylation in the CpG-island in DNMT3A promoter in MDA-MB-231 cells, which may be one of the reasons for the decreased DNMT3A activity in these cells (fig. 2).
Influence of DNMT3a knockdown on the cell resistance. As can be seen in fig. 3, knockdown of DNMT3A by siRNA resulted in the progression of partial resistance of MCF-7 cells to both tamoxifen and rapamycin underlining the direct association between DNMT3A suppression and formation of the resistant phenotype.
Totally, we have revealed the pivotal role of suppression of DNMT3A in the formation of the cell resistant phenotype. Namely, we have described the suppression of DN-MT3A axis in the rapamycin- ¡and tamoxifen-resistant cells, and demonstrated the similar progression of partial cell resistance under DNMT3A ¡knockdown; we have shown that suppression of DNMT3A axis was associated with the decrease in the DNA methylation. The following studies will delineate the prevalence of DNMT3A suppression among the resistant tumors, and reveal the possible applying of these parameters as the criteria of the cell resistance to targeting drugs.
DISCUSSION
The high level of tumor cell resistance to drug treatment is among the key factors determining the relatively low efficiency of anticancer therapy [2]. As known, long-term drug treatment is accompanied with the progression of acquired drug resistance of tumors, and, in the case of the specific targeting drugs, may be based on the rearrangement
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Fig. 1. DNMTs protein and RNA expression levels in the cell lines: a — Western blotting of the protein samples of MCF-7, MCF-7/T, MCF-7/Rap and MDA-MB-231 cells. Densitometry was used to quantify changes in protein expression; b — RT-PCR analysis of the expression of DNMT3A and DNMT1 in MCF-7, MCF-7/T, MCF-7/Rap and MDA-MB-231 cells
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Fig. 2. Methylation levels in the studied cell lines: a — average methylation level of the six CpG dinucleotides in the LINE-1 region in MCF-7, MCF-7/Rap, MCF-7/T and MDA-MB-231 cells; b — average methylation of the four CpG dinucleotides within CpG-island (CGI) in the promoter of the DNMT3A gene and of the three CpGs in the adjacent N-shore sequence
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signaling pathways respondent for the regulation of cell proliferation [3].
Similar to targeted drug resistance, progression of cell resistance to hormonal drugs possesses the common features, i.e. the rearrangement of signaling pathways and activation of bypass estrogen-independent pathways resulting in the restoration of cell proliferation [6]. A number of works, including our studies, described the changes in the profile of proteins and nucleic acids in the resistant cells [5, 20, 32—34], revealed microRNAs involved in the development of the resistance of tumor cells [35], and demonstrated the participation of exosomes in the transfer of hormonal resistance [36].
It should be noted that the mechanism of maintaining of cell signaling rearrangement and, in particular, the significance of epigenomic changes, i.e. DNA methylation, in the development of tumor cells resistance are currently
being actively studied using various experimental models [37]. ERa was found to regulate gene expression in breast cancer cells in part via DNA methylation whereas the loss of ERa results in the progression of estrogen resistance associated with the hypomethylation and overexpression of mitogen signaling genes [13]. Totally, the comparative analysis of the DNA methylation profile of the estrogen-sensitive and resistant breast cancer cells revealed number of genes hyper- or hypomethylated in the resistant cells [13, 14]. Among the recently studied genes some of histone variants overexpressed in the resistant cells [38], hypomethyl-ation of Lactate dehydrogenase genes in the MCF-7 resistant clones [39] and some others. Earlier, using the panel of various MCF-7 resistant derivates we have identified six coding genes, PRKCZ, TRAPPC9, ASIC2, C2CD4a, ZNF787 and CRTAC1, differentially methylated in the resistant cells [40].
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In the present work, the expression and activity of the key DNA methyltransferases in the MCF-7 resistant sublines was analyzed. As revealed, both tamoxifen-resistant and rapamycin-resistant cells were characterized by common features — constitutive suppression of DNMT3A associated with hypomethylation of numerous DNA regulatory sequences. Knockdown of DNMT3A in parental MCF-7 cells was accompanied with the development of partial cell resistance to rapamycin and tamoxifen, indicating the involvement of DNMT3A in the formation of cell response to the tested drugs. The association of DNMT3A suppression with cell resistance was confirmed in the experiments on the
ERa-negative MDA-MB-231 breast cancer cells showing the low level of DNMT3A expression in MDA-MB-231 cells compared with the estrogen-dependent MCF-7 cells.
CONCLUSION
Totally, the results obtained highlight the possible mechanism of tumor cell resistance to targeting/hormonal drugs based on the deregulation of DNMTs expression and demonstrate direct connection between DNMT3A suppression and resistance progression; further studies are required to identify the factors involved in DNMT3 deregulation in the resistant cells.
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ЛИТЕРАТУРА/REFERENCES
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1. Mansoori В., Mohammadi A., Davudian S. et al. The Different mechanisms of cancer drug resistance: a brief review. Adv Pharm Bull 2017;7(3):339-48. DOI: 10.15171/apb.2017.041
2. Aleksakhina S.N., Kashyap A., Imyanitov E.N. Mechanisms of acquired tumor drug resistance. Biochim Biophys
Acta Rev Cancer 2019;1872(2):188310. DOI: 10.1016/j.bbcan.2019.188310
3. Cree I.A., Charlton P. Molecular chess? Hallmarks of anti-cancer drug resistance. BMC Cancer 2017;17(1):10.
DOI: 10.1186/s12885-016-2999-1
4. Konjevic G., Mirjacic Martinovic K., Vuletic A. et al. Low expression of CD161 and NKG2D activating NK receptor is associated with impaired NK cell cytotoxicity in metastatic melanoma patients. Clin Exp Metastasis 2007;24(1):1-11. DOI: 10.1007/s10585-006-9043-9
5. Scherbakov A.M., Krasil'nikov M.A., Kushlinskii N.E. Molecular mechanisms of hormone resistance of breast cancer. Bull Exp Biol Med 2013;155(3):384-95. DOI: 10.1007/s10517-013-2160-y
6. Clarke R., Tyson J.J., Dixon J.M. Endocrine resistance in breast cancer — an overview and update. Mol Cell Endocrinol 2015;418 Pt. 3(03):220-34.
DOI: 10.1016/j.mce.2015.09.035
7. Araki K., Miyoshi Y. Mechanism of resistance to endocrine therapy in breast cancer: the important role of PI3K/Akt/mTOR in estrogen receptor-positive, HER2-negative breast cancer. Breast Cancer 2018;25(4)392-401. DOI: 10.1007/s12282-017-0812-x
8. Citi V., Del Re M., Martelli A. et al. Phosphorylation of AKT and ERK1/2 and mutations of PIK3CA and PTEN are predictive
of breast cancer cell sensitivity to everolimus in vitro. Cancer Chem Pharmacol 2018;81(4):745-54. DOI: 10.1007/s00280-018-3543-6
9. Xie W., Sun H., Li X. et al. Ovarian cancer: epigenetics, drug resistance, and progression. Cancer Cell Int 2021;21(1):1—16. DOI: 10.1186/s12935-021-02136-y
10. Hazra A., Bose P., Sunita P. et al. Molecular epigenetic dynamics in breast carcinogenesis. Arch Pharm Res 2021;44(8):741—63. DOI: 10.1007/s12272-021-01348-0
11. Andreeva O.E., Shchegolev Y.Y., Scherbakov A.M. et al. The phenomenon of the cross-resistance of breast cancer to target and hormonal drugs: the role of epigenetic reconstruction. Med Sci Forum 2023;20(1):5. DOI: 10.3390/IECC2023-14220
12. Vinokurova S. Epigenetics of virus-induced tumors: perspectives for therapeutic targeting. Curr Pharm Des 2017;23(32):4842-61. DOI: 10.2174/1381612823666170822100627
13. Ariazi E.A., Taylor J.C., Black M.A. et al. A new role for ERalpha: silencing via DNA methylation of basal, stem cell, and emt genes. Mol Cancer Res 2017;15(2):152-64.
DOI: 10.1158/1541-7786.MCR-16-0283
14. Lin X., Li J., Yin G. et al. Integrative analyses of gene expression and DNA methylation profiles in breast cancer cell line models
of tamoxifen-resistance indicate a potential role of cells with stemlike properties. Breast Cancer Res 2013;15(6):R119. DOI: 10.1186/bcr3588
15. Jin B., Robertson K.D. DNA methyltransferases, DNA damage repair, and cancer. Adv Exp Med Biol 2013;754:3-29.
DOI: 10.1007/978-1-4419-9967-2_1
16. Moore L.D., Le T., Fan G. DNA methylation and its basic function. Neuropsychopharmacology 2013;38(1):23-38.
DOI: 10.1038/npp.2012.112
17. Jahangiri R., Mosaffa F., Emami Razavi A. et al. Altered DNA methyltransferases promoter methylation and mRNA expression are associated with tamoxifen response in breast tumors. J Cell Physiol 2018;233(9):7305-19. DOI: 10.1002/jcp.26562
18. He D., Wang X., Zhang Y. et al. DNMT3A/3B overexpression might be correlated with poor patient survival, hypermethylation and low expression of ESR1/PGR in endometrioid carcinoma: an analysis of The Cancer Genome Atlas. Chin Med J (Engl) 2019;132(2): 161-70. DOI: 10.1097/CM9.0000000000000054
19. Chen B.F., Chan W.Y. The de novo DNA methyltransferase DNMT3A in development and cancer. Epigenetics 2014;9(5):669-77. DOI: 10.4161/epi.28324
20. Shchegolev Y.Y., Sorokin D.V., Scherbakov A.M. et al. Exosomes are involved in the intercellular transfer of rapamycin resistance in the breast cancer cells. Bioimpacts 2023;13(4):313-21.
DOI: 10.34172/bi.2023.27490
21. Scherbakov A.M., Sorokin D.V., Tatarskiy V.V.Jr. et al. The phenomenon of acquired resistance to metformin in breast cancer cells: the interaction of growth pathways and estrogen receptor signaling. IUBMB life 2016;68(4):281-92. DOI: 10.1002/iub.1481
22. Semina S.E., Scherbakov A.M., Kovalev S.V. et al. Horizontal transfer of tamoxifen resistance in MCF-7 cell derivates: proteome study. Cancer Invest 2017;35(8):506-18.
DOI: 10.1080/07357907.2017.1368081
23. Iselt M., Holtei W., Hilgard P. The tetrazolium dye assay for rapid in vitro assessment of cytotoxicity. Arzneimittel-Forschung 1989;39(7):747-9.
24. Scherbakov A.M., Vorontsova S.K., Khamidullina A.I. et al. Novel pentacyclic derivatives and benzylidenes of the progesterone series cause anti-estrogenic and antiproliferative effects and induce apoptosis in breast cancer cells. Invest New Drugs 2023;41(1): 142-52. DOI: 10.1007/s10637-023-01332-z
25. Livak K.J., Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods (San Diego, Calif) 2001;25(4):402-8.
DOI: 10.1006/meth.2001.1262
26. Scherbakov A.M., Komkov A.V., Komendantova A.S. et al. Steroidal pyrimidines and dihydrotriazines as novel classes
of anticancer agents against hormone-dependent breast cancer cells. Front Pharmacol 2017;8:979. DOI: 10.3389/fphar.2017.00979
27. Scherbakov A.M., Lobanova Y.S., Shatskaya V.A. et al. Activation of mitogenic pathways and sensitization to estrogen-induced apoptosis: two independent characteristics of tamoxifen-resistant breast cancer cells? Breast Cancer Res Treat 2006;100(1):1 —11. DOI: 10.1007/s10549-005-9075-x
28. Mruk D.D., Cheng C.Y. Enhanced chemiluminescence (ECL) for routine immunoblotting: an inexpensive alternative to commercially available kits. Spermatogenesis 2011;1(2):121—2.
DOI: 10.4161/spmg.1.2.16606
29. Taylor S.C., Berkelman T., Yadav G. et al. A defined methodology for reliable quantification of Western blot data. Mol Biotechnol 2013;55(3):217-26. DOI: 10.1007/s12033-013-9672-6
PMID: 23709336
30. Lander E.S., Linton L.M., Birren B. et al. Initial sequencing and analysis of the human genome. Nature 2001;409(6822):860-921. DOI: 10.1038/35057062
31. Yang A.S., Estecio M.R., Doshi K. et al. A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements. Nucleic Acids Res 2004;32(3):e38.
DOI: 10.1093/nar/gnh032
32. Dong C., Wu J., Chen Y. et al. Activation of PI3K/AKT/mTOR pathway causes drug resistance in breast cancer. Front Pharmacol 2021;12:628690. DOI: 10.3389/fphar.2021
33. Liu R., Chen Y., Liu G. et al. PI3K/AKT pathway as a key link modulates the multidrug resistance of cancers. Cell Death Dis 2020;11(9):797. DOI: 10.1038/s41419-020-02998-6
34. Scherbakov A.M., Basharina A.A., Sorokin D.V. et al. Targeting hormone-resistant breast cancer cells with docetaxel: a look inside the resistance. Cancer Drug Resist 2023;6(1):103-15.
DOI: 10.20517/cdr.2022.96
35. Meng D., Li Z., Ma X. et al. MicroRNA-1280 modulates cell growth and invasion of thyroid carcinoma through targeting estrogen receptor alpha. Cell Mol Biol (Noisy-le-grand) 2016;62 (3):1—6.
36. Xu C.G., Yang M.F., Ren Y.Q. et al. Exosomes mediated transfer of lncRNA UCA1 results in increased tamoxifen resistance in breast cancer cells. Eur Rev Med Pharmacol Sci 2016;20(20):4362-8.
37. Romero-Garcia S., Prado-Garcia H., Carlos-Reyes A. Role
of DNA methylation in the resistance to therapy in solid tumors. Front Oncol 2020;10:1152. DOI: 10.3389/fonc.2020.01152
38. Nayak S.R., Harrington E., Boone D. et al. A role for histone H2B variants in endocrine-resistant breast cancer. Horm Cancer 2015;6(5-6):214-24. DOI: 10.1007/s12672-015-0230-5
39. Hamadneh L., Al-Lakkis L., Alhusban A.A. et al. Changes
in Lactate production, lactate dehydrogenase genes expression and DNA methylation in response to tamoxifen resistance development in MCF-7 cell line. Genes (Basel) 2021;12(5). DOI: 10.3390/genes12050777
40. Андреева O.E., Сигин В.О., Стрельников В.В. и др. Изменение профиля метилирования ДНК в тамоксифен-резистентных сублиниях клеток MCF-7. Сибирский онкологический журнал 2019;18(5):45-53. DOI: 10.21294/1814-4861-2019-18-5-45-53 Andreeva O., Sigin V., Strelnikov V. et al. Changes in DNA methylation profile in tamoxifen-resistant MCF-7 sublines. Sibirskij onkologicheskij zhurnal = Siberian Journal of Oncology 2019;18(5):45-53. (In Russ.).
DOI: 10.21294/1814-4861-2019-18-5-45-53
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Вклад авторов
О.Е. Андреева: разработка концепции исследования, редактирование, методология, транзиторная трансфекция; Д.В. Сорокин: иммуноблоттинг;
C.В. Винокурова: разработка концепции исследования, анализ полученных данных, методология, редактирование; Ю.Ю. Щеголев: получение резистентных сублиний, проведение МТТ-теста;
Н.В. Елкина: выделение РНК, проведение ПЦР, анализ полученных данных; А.Н. Катаргин: исследование метилирования, анализ полученных данных; Р.С. Фасхутдинов: исследование метилирования, анализ полученных данных; Д.И. Сальникова: проведение МТТ-теста;
А.М. Щербаков: статистическая обработка данных, анализ полученных данных, подготовка иллюстративного материала; М.А. Красильников: идея и организация исследования, разработка концепции исследования, анализ полученных данных, написание текста статьи. Authors' contributions
O.E. Andreeva: development of the research concept, editing, methodology, transient transfection;
D.V. Sorokin: immunoblotting;
S.V. Vinokurova: development of the research concept, analysis of the data obtained, methodology, editing;
Yu.Yu. Shchegolev: obtaining resistant sublines, conducting the MTT test;
N.V. Elkina: RNA isolation, conducting the PCR, analysis of the obtained data;
A.N. Katargin: methylation study, analysis of data obtained;
R.S. Faskhutdinov: methylation study, analysis of data obtained;
D.I. Salnikova: conducting the MTT test;
А.М. Scherbakov: statistical data processing, analysis of the obtained data, preparation of illustrative material;
M.A. Krasil'nikov: idea and organization of the study, development of the research concept, analysis of the data obtained, article writing.
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ORCID авторов / ORCID of authors
O.E. Andreeva: https://orcid.org/0000-0002-6015-6619 D.V. Sorokin: https://orcid.org/0000-0002-1264-7405 S.V. Vinokurova: https://orcid.org/0000-0003-1615-3928 Yu.Yu. Shchegolev: https://orcid.org/0000-0002-1490-6781 N.V. Elkina: https://orcid.org/0000-0002-0503-6016 A.N. Katargin: https://orcid.org/0000-0002-7405-0671 R.S. Faskhutdinov: https://orcid.org/0000-0002-0050-7798 D.I. Salnikova: https://orcid.org/0000-0002-0809-3710 A.M. Scherbakov: https://orcid.org/0000-0002-2974-9555 M.A. Krasil'nikov: https://orcid.org/0000-0002-5902-7633
со Conflict of interest. The authors declare no conflict of interest.
^ Конфликт интересов. Авторы заявляют об отсутствии конфликта интересов. О
Funding. The research was funded by the Russian Science Foundation (grant No. 19-15-00245; https://rscf.ru/project/22-15-35008/). Финансирование. Исследование финансировалось Российским научным фондом (грант № 19-15-00245; https://rscf.ru/project/22-15-35008).
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Article submitted: 14.11.2023. Accepted for publication: 05.12.2023. Статья поступила: 14.11.2023. Принята к публикации: 05.12.2023.
