Epigallocatechin Gallate (EGCG), an Active Phenolic Compound of Green Tea, Inhibits Tumor Growth of Head and Neck Cancer Cells by Targeting DNA Hypermethylation
Abstract
:1. Introduction
2. Material and Methods
2.1. Chemicals and Antibodies
2.2. HNSCC Cell Lines and Cell Culture Conditions
2.3. Assay for Global DNA Methylation and DNMT Activity
2.4. Analysis of 5mC in DNA following Dot-Blot Assay
2.5. Analysis of Protein Levels Using Western Blotting
2.6. Tumor Xenograft Study in Balb/C Nude Mice
2.7. Immunohistochemical Detection and Analysis
2.8. Statistical Analysis
3. Results
3.1. The Basal Levels of Global DNA Methylation in HNSCC Cell Lines
3.2. EGCG Treatment Inhibits DNA Hypermethylation in HNSCC Cell Lines
3.3. EGCG Inhibits 5-mC Expression in SCC-1 and FaDu Cells
3.4. EGCG Treatment Inhibits DNMT Activity, DNMT (DNMT1, DNMT3A, and DNMT3B) Protein Expression in SCC-1 and FaDu Cells
3.5. Effects of 5Aza-dc, a Potent Inhibitor of DNA Methylation, Alone or in Combination of EGCG, on DNA Hypermethylation and DNMT Activity
3.6. Effect of EGCG on DNA Demethylation in HNSCC Cells
3.7. EGCG Reactivates Tumor Suppressor Genes in HNSCC Cells
3.8. Growth Inhibitory Potential of EGCG on FaDu Xenograft in Nude Mice
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
References
- Arantes, L.M.; de Carvalho, A.C.; Melendez, M.E.; Carvalho, A.L.; Goloni-Bertollo, E.M. Methylation as a biomarker for head and neck cancer. Oral. Oncol. 2014, 50, 587–592. [Google Scholar] [CrossRef] [PubMed]
- Siegel, R.L.; Miller, K.D.; Wagle, N.S.; Jemal, A. Cancer statistics, 2023. CA A Cancer J. Clin. 2023, 73, 17–48. [Google Scholar] [CrossRef]
- Gormley, M.; Creaney, G.; Schache, A.; Ingarfield, K.; Conway, D.I. Reviewing the epidemiology of head and neck cancer: Definitions, trends and risk factors. Br. Dent. J. 2022, 233, 780–786. [Google Scholar] [CrossRef] [PubMed]
- Chauhan, R.; Trivedi, V.; Rani, R.; Singh, U. A Study of Head and Neck Cancer Patients with Reference to Tobacco Use, Gender, and Subsite Distribution. South Asian J. Cancer 2022, 11, 46–51. [Google Scholar] [CrossRef]
- Patterson, R.H.; Fischman, V.G.; Wasserman, I.; Siu, J.; Shrime, M.G.; Fagan, J.J.; Koch, W.; Alkire, B.C. Global Burden of Head and Neck Cancer: Economic Consequences, Health, and the Role of Surgery. Otolaryngol.–Head Neck Surg. 2020, 162, 296–303. [Google Scholar] [CrossRef]
- Kim, H.S.; Quon, M.J.; Kim, J.A. New insights into the mechanisms of polyphenols beyond antioxidant properties; lessons from the green tea polyphenol, epigallocatechin 3-gallate. Redox Biol. 2014, 2, 187–195. [Google Scholar] [CrossRef] [Green Version]
- Khan, M.A.; Hussain, A.; Sundaram, M.K.; Alalami, U.; Gunasekera, D.; Ramesh, L.; Hamza, A.; Quraishi, U. (-)-Epigallocatechin-3-gallate reverses the expression of various tumor-suppressor genes by inhibiting DNA methyltransferases and histone deacetylases in human cervical cancer cells. Oncol. Rep. 2015, 33, 1976–1984. [Google Scholar] [CrossRef] [Green Version]
- Fang, M.Z.; Wang, Y.; Ai, N.; Hou, Z.; Sun, Y.; Lu, H.; Welsh, W.; Yang, C.S. Tea polyphenol (-)-epigallocatechin-3-gallate inhibits DNA methyltransferase and reactivates methylation-silenced genes in cancer cell lines. Cancer Res. 2003, 63, 7563–7570. [Google Scholar] [PubMed]
- Fang, M.Z.; Chen, D.; Sun, Y.; Jin, Z.; Christman, J.K.; Yang, C.S. Reversal of hypermethylation and reactivation of p16INK4a, RARbeta, and MGMT genes by genistein and other isoflavones from soy. Clin. Cancer Res. 2005, 11, 7033–7041. [Google Scholar] [CrossRef] [Green Version]
- Rhind, N.; Russell, P. Signaling pathways that regulate cell division. Cold Spring Harb. Perspect. Biol. 2012, 4, a005942. [Google Scholar] [CrossRef] [Green Version]
- Vandiver, A.R.; Idrizi, A.; Rizzardi, L.; Feinberg, A.P.; Hansen, K.D. DNA methylation is stable during replication and cell cycle arrest. Sci. Rep. 2015, 5, 17911. [Google Scholar] [CrossRef] [Green Version]
- Costello, J.F.; Plass, C. Methylation matters. J. Med. Genet. 2001, 38, 285–303. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Li, E.; Beard, C.; Jaenisch, R. Role for DNA methylation in genomic imprinting. Nature 1993, 366, 362–365. [Google Scholar] [CrossRef]
- Pfeifer, G.P.; Tanguay, R.L.; Steigerwald, S.D.; Riggs, A.D. In vivo footprint and methylation analysis by PCR-aided genomic sequencing: Comparison of active and inactive X chromosomal DNA at the CpG island and promoter of human PGK-1. Genes. Dev. 1990, 4, 1277–1287. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Prasad, R.; Katiyar, S.K. Prostaglandin E2 Promotes UV radiation-induced immune suppression through DNA hypermethylation. Neoplasia 2013, 15, 795–804. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Costello, J.F.; Fruhwald, M.C.; Smiraglia, D.J.; Rush, L.J.; Robertson, G.P.; Gao, X.; Wright, F.A.; Feramisco, J.D.; Peltomaki, P.; Lang, J.C.; et al. Aberrant CpG-island methylation has non-random and tumour-type-specific patterns. Nat. Genet. 2000, 24, 132–138. [Google Scholar] [CrossRef]
- Issa, J.P.; Vertino, P.M.; Wu, J.; Sazawal, S.; Celano, P.; Nelkin, B.D.; Hamilton, S.R.; Baylin, S.B. Increased cytosine DNA-methyltransferase activity during colon cancer progression. J. Natl. Cancer Inst. 1993, 85, 1235–1240. [Google Scholar] [CrossRef]
- Lin, S.Y.; Yeh, K.T.; Chen, W.T.; Chen, H.C.; Chen, S.T.; Chang, J.G. Promoter CpG methylation of caveolin-1 in sporadic colorectal cancer. Anticancer Res. 2004, 24, 1645–1650. [Google Scholar]
- Baylin, S.B.; Jones, P.A. Epigenetic Determinants of Cancer. Cold Spring Harb. Perspect. Biol. 2016, 8. [Google Scholar] [CrossRef] [Green Version]
- Prasad, R.; Singh, T.; Katiyar, S.K. Honokiol inhibits ultraviolet radiation-induced immunosuppression through inhibition of ultraviolet-induced inflammation and DNA hypermethylation in mouse skin. Sci. Rep. 2017, 7, 1657. [Google Scholar] [CrossRef] [Green Version]
- Kansal, V.; Agarwal, A.; Harbour, A.; Farooqi, H.; Singh, V.K.; Prasad, R. Regular Intake of Green Tea Polyphenols Suppresses the Development of Nonmelanoma Skin Cancer through miR-29-Mediated Epigenetic Modifications. J. Clin. Med. 2022, 11, 398. [Google Scholar] [CrossRef]
- Jensen, M.M.; Jorgensen, J.T.; Binderup, T.; Kjaer, A. Tumor volume in subcutaneous mouse xenografts measured by microCT is more accurate and reproducible than determined by 18F-FDG-microPET or external caliper. BMC Med. Imaging 2008, 8, 16. [Google Scholar] [CrossRef] [Green Version]
- Bachmanov, A.A.; Reed, D.R.; Beauchamp, G.K.; Tordoff, M.G. Food intake, water intake, and drinking spout side preference of 28 mouse strains. Behav. Genet. 2002, 32, 435–443. [Google Scholar] [CrossRef]
- Nair, A.B.; Jacob, S. A simple practice guide for dose conversion between animals and human. J. Basic. Clin. Pharm. 2016, 7, 27–31. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Zhang, W.; Xu, J. DNA methyltransferases and their roles in tumorigenesis. Biomark. Res. 2017, 5, 1. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Uysal, F.; Ozturk, S.; Akkoyunlu, G. DNMT1, DNMT3A and DNMT3B proteins are differently expressed in mouse oocytes and early embryos. J. Mol. Histol. 2017, 48, 417–426. [Google Scholar] [CrossRef]
- Robert, M.F.; Morin, S.; Beaulieu, N.; Gauthier, F.; Chute, I.C.; Barsalou, A.; MacLeod, A.R. DNMT1 is required to maintain CpG methylation and aberrant gene silencing in human cancer cells. Nat. Genet. 2003, 33, 61–65. [Google Scholar] [CrossRef] [PubMed]
- Zhao, R.; Choi, B.Y.; Lee, M.H.; Bode, A.M.; Dong, Z. Implications of Genetic and Epigenetic Alterations of CDKN2A (p16(INK4a)) in Cancer. eBioMedicine 2016, 8, 30–39. [Google Scholar] [CrossRef] [Green Version]
- Auerkari, E.I. Methylation of tumor suppressor genes p16(INK4a), p27(Kip1) and E-cadherin in carcinogenesis. Oral Oncol. 2006, 42, 5–13. [Google Scholar] [CrossRef] [PubMed]
- Witcher, M.; Emerson, B.M. Epigenetic silencing of the p16(INK4a) tumor suppressor is associated with loss of CTCF binding and a chromatin boundary. Mol. Cell. 2009, 34, 271–284. [Google Scholar] [CrossRef] [Green Version]
- Majid, S.; Kikuno, N.; Nelles, J.; Noonan, E.; Tanaka, Y.; Kawamoto, K.; Hirata, H.; Li, L.C.; Zhao, H.; Okino, S.T.; et al. Genistein induces the p21WAF1/CIP1 and p16INK4a tumor suppressor genes in prostate cancer cells by epigenetic mechanisms involving active chromatin modification. Cancer Res. 2008, 68, 2736–2744. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Go, J.H. Methylation analysis of cyclin-dependent kinase inhibitor genes in primary gastrointestinal lymphomas. Mod. Pathol. 2003, 16, 752–755. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Johnson, D.E.; Burtness, B.; Leemans, C.R.; Lui, V.W.Y.; Bauman, J.E.; Grandis, J.R. Head and neck squamous cell carcinoma. Nat. Rev. Dis. Primers 2020, 6, 92. [Google Scholar] [CrossRef]
- Ferlay, J.; Colombet, M.; Soerjomataram, I.; Mathers, C.; Parkin, D.M.; Pineros, M.; Znaor, A.; Bray, F. Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods. Int. J. Cancer 2019, 144, 1941–1953. [Google Scholar] [CrossRef] [Green Version]
- Takeshima, H.; Ushijima, T. Accumulation of genetic and epigenetic alterations in normal cells and cancer risk. NPJ Precis. Oncol. 2019, 3, 7. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Ilango, S.; Paital, B.; Jayachandran, P.; Padma, P.R.; Nirmaladevi, R. Epigenetic alterations in cancer. Front. Biosci. (Landmark Ed.) 2020, 25, 1058–1109. [Google Scholar] [CrossRef]
- Sharma, S.; Kelly, T.K.; Jones, P.A. Epigenetics in cancer. Carcinogenesis 2010, 31, 27–36. [Google Scholar] [CrossRef] [Green Version]
- Issa, J.P.; Kantarjian, H.M. Targeting DNA methylation. Clin. Cancer Res. 2009, 15, 3938–3946. [Google Scholar] [CrossRef] [Green Version]
- Collado, M.; Blasco, M.A.; Serrano, M. Cellular senescence in cancer and aging. Cell 2007, 130, 223–233. [Google Scholar] [CrossRef] [Green Version]
- Nabel, E.G. CDKs and CKIs: Molecular targets for tissue remodelling. Nat. Rev. Drug. Discov. 2002, 1, 587–598. [Google Scholar] [CrossRef]
- Katiyar, S.K.; Singh, T.; Prasad, R.; Sun, Q.; Vaid, M. Epigenetic alterations in ultraviolet radiation-induced skin carcinogenesis: Interaction of bioactive dietary components on epigenetic targets. Photochem. Photobiol. 2012, 88, 1066–1074. [Google Scholar] [CrossRef] [PubMed]
- Berletch, J.B.; Liu, C.; Love, W.K.; Andrews, L.G.; Katiyar, S.K.; Tollefsbol, T.O. Epigenetic and genetic mechanisms contribute to telomerase inhibition by EGCG. J. Cell. Biochem. 2008, 103, 509–519. [Google Scholar] [CrossRef] [PubMed] [Green Version]
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Agarwal, A.; Kansal, V.; Farooqi, H.; Prasad, R.; Singh, V.K. Epigallocatechin Gallate (EGCG), an Active Phenolic Compound of Green Tea, Inhibits Tumor Growth of Head and Neck Cancer Cells by Targeting DNA Hypermethylation. Biomedicines 2023, 11, 789. https://doi.org/10.3390/biomedicines11030789
Agarwal A, Kansal V, Farooqi H, Prasad R, Singh VK. Epigallocatechin Gallate (EGCG), an Active Phenolic Compound of Green Tea, Inhibits Tumor Growth of Head and Neck Cancer Cells by Targeting DNA Hypermethylation. Biomedicines. 2023; 11(3):789. https://doi.org/10.3390/biomedicines11030789
Chicago/Turabian StyleAgarwal, Anshu, Vikash Kansal, Humaira Farooqi, Ram Prasad, and Vijay Kumar Singh. 2023. "Epigallocatechin Gallate (EGCG), an Active Phenolic Compound of Green Tea, Inhibits Tumor Growth of Head and Neck Cancer Cells by Targeting DNA Hypermethylation" Biomedicines 11, no. 3: 789. https://doi.org/10.3390/biomedicines11030789