Top

BMC Cancer

Published in:

Open Access 01-12-2024 | Breast Cancer | Research

Methyl CpG binding protein MBD2 has a regulatory role on the BRCA1 gene expression and its modulation by resveratrol in ER+, PR+ & triple-negative breast cancer cells

Authors: Ram Krishna Sahu, Simran Tandon, Shalini Singh, Bhudev Chandra Das, Suresh T Hedau

Published in: BMC Cancer | Issue 1/2024

Abstract

Background

Resveratrol has demonstrated its ability to regulate BRCA1 gene expression in breast cancer cells, and previous studies have established the binding of MBD proteins to BRCA1 gene promoter regions. However, the molecular mechanism underlying these interactions remains to be elucidated. The aimed to evaluate the impact of MBD proteins on the regulation of BRCA1, BRCA2, and p16 genes and their consequential effects on breast cancer cells.

Methods

Efficacy of resveratrol was assessed using the MTT assay. Binding interactions were investigated through EMSA, ChIP, & MeIP assay. Expression analyses of MBD genes and proteins were conducted using qRT-PCR and western blotting, respectively. Functional assays, including clonogenic, migratory, and sphere formation assays were used to assess cancer cells’ colony-forming, metastatic, and tumor-forming abilities. The cytotoxicity of resveratrol on cancer cells was also tested using an apoptosis assay.

Results

The study determined an IC50 of 30µM for resveratrol. MBD proteins were found to bind to the BRCA1 gene promoter. Resveratrol exhibited regulatory effects on MBD gene expression, subsequently impacting BRCA1 gene expression and protein levels. Higher concentrations of resveratrol resulted in reduced colony and sphere formation, decreases migration of cancer cells, and an increases number of apoptotic cells in breast cancer cells.

Impact

Identification of MBD2-BRCA1 axis indicates their significant role in the induction of apoptosis and reduction of metastasis and proliferation in breast cancer cells. Further therapy can be designed to target these MBD proteins and resveratrol could be used along with other anticancer drugs to target breast cancer.

Conclusions

In conclusion MBD2 protein interact to the BRCA1 gene promoter, and resveratrol modulates MBD2 gene expression, which in turn regulates BRCA1 gene expression, and inhibits cell proliferation, migration, and induces apoptosis in ER+, PR+ & Triple negative breast cancer cells.

Available only for authorised users

GLOBOCAN 2020: New Global Cancer Data.

Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021;71(3):209–49. https://doi.org/10.3322/caac.21660.CrossRefPubMed

Sharma S, Kelly TK, Jones PA. Epigenetics in cancer. Carcinogenesis. 2010;31(1):27–36. https://doi.org/10.1093/carcin/bgp220.CrossRefPubMed

Handy DE, Castro R, Loscalzo J. Epigenetic modifications: basic mechanisms and role in cardiovascular disease. Circulation. 2011;123(19):2145–56. https://doi.org/10.1161/circulationaha.110.956839.CrossRefPubMedPubMedCentral

Dawson MA, Kouzarides T. Cancer epigenetics: from mechanism to therapy. Cell. 2012;150(1):12–27. https://doi.org/10.1016/j.cell.2012.06.013.CrossRefPubMed

Li L, Chen BF, Chan WY. An epigenetic regulator: methyl-CpG-binding domain protein 1 (MBD1). Int J Mol Sci. 2015;16(3):5125–40. https://doi.org/10.3390/ijms16035125.CrossRefPubMedPubMedCentral

Parry L, Clarke AR. The Roles of the Methyl-CpG Binding Proteins in Cancer. Genes Cancer. 2011;2(6):618–30. https://doi.org/10.1177/1947601911418499.CrossRefPubMedPubMedCentral

Wood KH, Zhou Z. Emerging Molecular and Biological Functions of MBD2, a Reader of DNA Methylation. Front Genet. 2016;7:93. https://doi.org/10.3389/fgene.2016.00093.CrossRefPubMedPubMedCentral

Lin X, Nelson WG. Methyl-CpG-binding domain protein-2 mediates transcriptional repression associated with hypermethylated GSTP1 CpG islands in MCF-7 breast cancer cells. Cancer Res. 2003;63(2):498–504.PubMed

10.

Roy R, Chun J, Powell SN. BRCA1 and BRCA2: different roles in a common pathway of genome protection. Nat Rev Cancer. 2011;12(1):68–78. https://doi.org/10.1038/nrc3181.CrossRefPubMedPubMedCentral

11.

Petrucelli N, Daly MB, Pal T. BRCA1- and BRCA2 -Associated Hereditary Breast and Ovarian Cancer Summary Genetic counseling Suggestive Findings. GeneReviews: University of Washington, Seattle; 1998. p. 1–37.

12.

Gilkes DM, Sidney T, Comprehensive K, Johns T. HHS Public Access. 2017;4:1–17.

13.

Yoshida K, Miki Y. Role of BRCA1 and BRCA2 as regulators of DNA repair, transcription, and cell cycle in response to DNA damage. Cancer Sci. 2004;95(11):866–71. https://doi.org/10.1111/j.1349-7006.2004.tb02195.x.CrossRefPubMed

14.

Liggett WH Jr, Sidransky D. Role of the p16 tumor suppressor gene in cancer. J Clin Oncol. 1998;16(3):1197–206. https://doi.org/10.1200/JCO.1998.16.3.1197.CrossRefPubMed

15.

Li Y, Barbash O, Diehl JA. Regulation of the cell cycle. In book: The Molecular basis of cancer. 2015. p 165–178. e 2. https://doi.org/10.1016/B978-1-4557-4066-6.00011-1.

16.

Indovina P, Pentimalli F, Casini N, Vocca I, Giordano A. RB1 dual role in proliferation and apoptosis: cell fate control and implications for cancer therapy. Oncotarget. 2015;6(20):17873–90. https://doi.org/10.18632/oncotarget.4286.CrossRefPubMedPubMedCentral

17.

Fustier P, Le Corre L, Chalabi N, Vissac-Sabatier C, Communal Y, Bignon YJ, Bernard-Gallon DJ. Resveratrol increases BRCA1 and BRCA2 mRNA expression in breast tumour cell lines. Br J Cancer. 2003;89(1):168–72. https://doi.org/10.1038/sj.bjc.6600983.CrossRefPubMedPubMedCentral

18.

Bishayee A. Cancer prevention and treatment with resveratrol: from rodent studies to clinical trials. Cancer Prev Res (Phila). 2009;2(5):409–18. https://doi.org/10.1158/1940-6207.CAPR-08-0160.CrossRefPubMed

19.

Koushki M, Amiri-Dashatan N, Ahmadi N, Abbaszadeh HA, Rezaei-Tavirani M. Resveratrol: A miraculous natural compound for diseases treatment. Food Sci Nutr. 2018;6(8):2473–90. https://doi.org/10.1002/fsn3.855.CrossRefPubMedPubMedCentral

20.

Ko JH, Sethi G, Um JY, Shanmugam MK, Arfuso F, Kumar AP, Bishayee A, Ahn KS. The Role of Resveratrol in Cancer Therapy. Int J Mol Sci. 2017;18(12):2589. https://doi.org/10.3390/ijms18122589.CrossRefPubMedPubMedCentral

21.

Carter LG, D’Orazio JA, Pearson KJ. Resveratrol and cancer: focus on in vivo evidence. Endocr Relat Cancer. 2014;21(3):R209-25. https://doi.org/10.1530/ERC-13-0171.CrossRefPubMedPubMedCentral

22.

Auriol E, Billard LM, Magdinier F, Dante R. Specific binding of the methyl binding domain protein 2 at the BRCA1-NBR2 locus. Nucleic Acids Res. 2005;33(13):4243–54. https://doi.org/10.1093/nar/gki729.CrossRefPubMedPubMedCentral

23.

Altenburg JD, Bieberich AA, Terry C, Harvey KA, Vanhorn JF, Xu Z, Jo Davisson V, Siddiqui RA. A synergistic antiproliferation effect of curcumin and docosahexaenoic acid in SK-BR-3 breast cancer cells: unique signaling not explained by the effects of either compound alone. BMC Cancer. 2011;11:149. https://doi.org/10.1186/1471-2407-11-149.CrossRefPubMedPubMedCentral

24.

Yang X, Xu Y, Wang T, Shu D, Guo P, Miskimins K, Qian SY. Inhibition of cancer migration and invasion by knocking down delta-5-desaturase in COX-2 overexpressed cancer cells. Redox Biol. 2017;11:653–62. https://doi.org/10.1016/j.redox.2017.01.016.CrossRefPubMedPubMedCentral

25.

Rice P, Longden I, Bleasby A. EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet. 2000;16(6):276–7. https://doi.org/10.1016/s0168-9525(00)02024-2.CrossRefPubMed

26.

Ye J. Gel Shift / EMSA Protocol References : Pennington Biomedical Research Center. 1996;269:3–10.

27.

Cornelussen RN, Gupta S, Knowlton AA. Regulation of prostaglandin A1-induced heat shock protein expression in isolated cardiomyocytes. J Mol Cell Cardiol. 2001;33(8):1447–54. https://doi.org/10.1006/jmcc.2001.1406.CrossRefPubMed

28.

Ishida A, Fujita N, Kitazawa R, Tsuruo T. Transforming growth factor-beta induces expression of receptor activator of NF-kappa B ligand in vascular endothelial cells derived from bone. J Biol Chem. 2002;277(29):26217–24. https://doi.org/10.1074/jbc.M111093200.CrossRefPubMed

29.

Fan J, Chen YC, Watkins T, Dang CV, Gorospe M, Cheadle C. Array-based nuclear run-on analysis. Methods Mol Biol. 2012;809:505–17. https://doi.org/10.1007/978-1-61779-376-9_33.CrossRefPubMedPubMedCentral

30.

Zhou P, Wan X, Zou Y, Chen Z, Zhong A. Transforming growth factor beta (TGF-β) is activated by the CtBP2-p300-AP1 transcriptional complex in chronic renal failure. Int J Biol Sci. 2020;16(2):204–15. https://doi.org/10.7150/ijbs.38841.CrossRefPubMedPubMedCentral

31.

Gao L, Yang M, Wei Z, Gu M, Yang L, Bai C, Wu Y, Li G. MSTN Mutant Promotes Myogenic Differentiation by Increasing Demethylase TET1 Expression via the SMAD2/SMAD3 Pathway. Int J Biol Sci. 2020;16(8):1324–34. https://doi.org/10.7150/ijbs.40551.CrossRefPubMedPubMedCentral

32.

Vaher K, Anier K, Jürgenson M, Harro J, Kalda A. Cocaine-induced changes in behaviour and DNA methylation in rats are influenced by inter-individual differences in spontaneous exploratory activity. J Psychopharmacol. 2020;34(6):680–92. https://doi.org/10.1177/0269881120916137.CrossRefPubMed

33.

Wang J, Bian C, Li J, Couch FJ, Wu K, Zhao RC. Poly(ADP-ribose) polymerase-1 down-regulates BRCA2 expression through the BRCA2 promoter. J Biol Chem. 2008;283(52):36249–56. https://doi.org/10.1074/jbc.M803693200.CrossRefPubMedPubMedCentral

34.

Mirza S, Sharma G, Parshad R, Gupta SD, Pandya P, Ralhan R. Expression of DNA methyltransferases in breast cancer patients and to analyze the effect of natural compounds on DNA methyltransferases and associated proteins. J Breast Cancer. 2013;16(1):23–31. https://doi.org/10.4048/jbc.2013.16.1.23.CrossRefPubMedPubMedCentral

35.

Rafehi H, Orlowski C, Georgiadis GT, Ververis K, El-Osta A, Karagiannis TC. Clonogenic assay: adherent cells. J Vis Exp. 2011;49:2573. https://doi.org/10.3791/2573.CrossRef

36.

Jonkman JE, Cathcart JA, Xu F, Bartolini ME, Amon JE, Stevens KM, Colarusso P. An introduction to the wound healing assay using live-cell microscopy. Cell Adh Migr. 2014;8(5):440–51. https://doi.org/10.4161/cam.36224.CrossRefPubMedPubMedCentral

37.

Liu K, Liu PC, Liu R, Wu X. Dual AO/EB staining to detect apoptosis in osteosarcoma cells compared with flow cytometry. Med Sci Monit Basic Res. 2015;21:15–20. https://doi.org/10.12659/MSMBR.893327.CrossRefPubMedPubMedCentral

38.

Bartosh TJ, Ylostalo JH. Preparation of anti-inflammatory mesenchymal stem/precursor cells (MSCs) through sphere formation using hanging-drop culture technique. Curr Protoc Stem Cell Biol. 2014 ;28:Unit 2B.6. https://doi.org/10.1002/9780470151808.sc02b06s28.

39.

Johnson S, Chen H, Lo PK. In vitro Tumorsphere Formation Assays. Bio Protoc. 2013;3(3): e325. https://doi.org/10.21769/bioprotoc.325.CrossRefPubMed

40.

Ribble D, Goldstein NB, Norris DA, Shellman YG. A simple technique for quantifying apoptosis in 96-well plates. BMC Biotechnology. 2005;5:12. https://doi.org/10.1186/1472-6750-5-12.CrossRefPubMedPubMedCentral

41.

Baylin SB, Jones PA. Epigenetic Determinants of Cancer. Cold Spring Harb Perspect Biol. 2016;8(9):a019505. https://doi.org/10.1101/cshperspect.a019505.CrossRefPubMedPubMedCentral

42.

Phillips T. The role of methylation in gene expression. Nature Education. 2008;1:116.

43.

Bannister AJ, Kouzarides T. Regulation of chromatin by histone modifications. Cell Res. 2011;21(3):381–95. https://doi.org/10.1038/cr.2011.22.CrossRefPubMedPubMedCentral

44.

Papoutsis AJ, Lamore SD, Wondrak GT, Selmin OI, Romagnolo DF. Resveratrol prevents epigenetic silencing of BRCA-1 by the aromatic hydrocarbon receptor in human breast cancer cells. J Nutr. 2010;140(9):1607–14. https://doi.org/10.3945/jn.110.123422.CrossRefPubMedPubMedCentral

45.

Sahu RK, Verma VV, Kumar A, Tandon S, Chandra Das B, Hedau ST. In silico prediction and interaction of resveratrol on methyl-CpG binding proteins by molecular docking and MD simulations study. RSC Adv. 2022;12(18):11493–504. https://doi.org/10.1039/D2RA00432A.CrossRefPubMedPubMedCentral

46.

Günther K, Rust M, Leers J, Boettger T, Scharfe M, Jarek M, Bartkuhn M, Renkawitz R. Differential roles for MBD2 and MBD3 at methylated CpG islands, active promoters and binding to exon sequences. Nucleic Acids Res. 2013;41(5):3010–21. https://doi.org/10.1093/nar/gkt035.CrossRefPubMedPubMedCentral

47.

Magdinier F, Billard LM, Wittmann G, Frappart L, Benchaïb M, Lenoir GM, Guérin JF, Dante R. Regional methylation of the 5’ end CpG island of BRCA1 is associated with reduced gene expression in human somatic cells. FASEB J. 2000;14(11):1585–94. https://doi.org/10.1096/fj.14.11.1585.CrossRefPubMed

48.

Mueller CR, Roskelley CD. Regulation of BRCA1 expression and its relationship to sporadic breast cancer. Breast Cancer Res. 2003;5(1):45–52. https://doi.org/10.1186/bcr557.CrossRefPubMed

49.

Zhao Y, Tang H, Zeng X, Ye D, Liu J. Resveratrol inhibits proliferation, migration and invasion via Akt and ERK1/2 signaling pathways in renal cell carcinoma cells. Biomed Pharmacother. 2018;98:36–44. https://doi.org/10.1016/j.biopha.2017.12.029.CrossRefPubMed

50.

Xiong W, Yin A, Mao X, Zhang W, Huang H, Zhang X. Resveratrol suppresses human glioblastoma cell migration and invasion via activation of RhoA/ROCK signaling pathway. Oncol Lett. 2016;11(1):484–90. https://doi.org/10.3892/ol.2015.3888.CrossRefPubMed

51.

Wu H, Chen L, Zhu F, Han X, Sun L, Chen K. The Cytotoxicity Effect of Resveratrol: Cell Cycle Arrest and Induced Apoptosis of Breast Cancer 4T1 Cells. Toxins (Basel). 2019;11(12):731. https://doi.org/10.3390/toxins11120731.CrossRefPubMedPubMedCentral

52.

Lang F, Qin Z, Li F, Zhang H, Fang Z, Hao E. Apoptotic Cell Death Induced by Resveratrol Is Partially Mediated by the Autophagy Pathway in Human Ovarian Cancer Cells. PLoS One. 2015;10(6):e0129196. https://doi.org/10.1371/journal.pone.0129196.CrossRefPubMedPubMedCentral

Title: Methyl CpG binding protein MBD2 has a regulatory role on the BRCA1 gene expression and its modulation by resveratrol in ER+, PR+ & triple-negative breast cancer cells
Authors: Ram Krishna Sahu
Simran Tandon
Shalini Singh
Bhudev Chandra Das
Suresh T Hedau
Publication date: 01-12-2024
Publisher: BioMed Central
Keywords: Breast Cancer
Breast Cancer
Epigenetics
Published in: BMC Cancer / Issue 1/2024
Electronic ISSN: 1471-2407
DOI: https://doi.org/10.1186/s12885-024-12274-x

Webinar | 19-02-2024 | 17:30 (CET)

Keynote webinar | Spotlight on antibody–drug conjugates in cancer

Watch now

Antibody–drug conjugates (ADCs) are novel agents that have shown promise across multiple tumor types. Explore the current landscape of ADCs in breast and lung cancer with our experts, and gain insights into the mechanism of action, key clinical trials data, existing challenges, and future directions.

Dr. Véronique Diéras

Prof. Fabrice Barlesi

Developed by: Springer Medicine

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Abstract

Background

Methods

Results

Conclusions

Please log in to get access to this content

Other articles of this Issue 1/2024

Interaction of immune cells with renal cancer development: Mendelian randomization (MR) study

Glycosylation-related genes mediated prognostic signature contribute to prognostic prediction and treatment options in ovarian cancer: based on bulk and single‑cell RNA sequencing data

Dual effect of vitamin D3 on breast cancer-associated fibroblasts

Identification of PANoptosis-related signature reveals immune infiltration characteristics and immunotherapy responses for renal cell carcinoma

Tretinoin improves the anti-cancer response to cyclophosphamide, in a model-selective manner

Monitoring trends in the absolute lymphocyte count and the neutrophil-to-lymphocyte ratio in patients with breast cancer receiving eribulin

Keynote webinar | Spotlight on antibody–drug conjugates in cancer