Cellular Oncology

, Volume 41, Issue 5, pp 569–580 | Cite as

β-Catenin gene promoter hypermethylation by reactive oxygen species correlates with the migratory and invasive potentials of colon cancer cells

  • Suhrid Banskota
  • Sadan Dahal
  • Eunju Kwon
  • Dong Young Kim
  • Jung-Ae KimEmail author
Original Paper



Over half of the colon cancer patients suffer from cancer-related events, mainly metastasis. Loss of β-catenin activity has previously been found to facilitate cancer cell dissociation and migration. Here, we aimed to investigate whether epigenetic silencing of β-catenin induces human colon cancer cell migration and/or invasion.


HCT-116, Caco-2, HT-29 and SW620 cell migration and invasion capacities were assessed using scratch wound healing and Matrigel invasion assays, respectively. Confocal microscopy, qRT-PCR and Western blotting were performed to determine gene expression levels, whereas methylation-specific quantitative real-time PCR was used to assess the extent of β-catenin gene (CTNNB1) promoter methylation after treatment of the cells with TPA, hydrogen peroxide, 5-aza-2′-deoxycytidine and/or VAS2870.


We found that treatment of HT-29 and Caco-2 cells (differentiated and low metastatic) with 12-O-tetradecanoyl phorbol-13-acetate (TPA; a tumor promoter) suppressed E-cadherin and β-catenin expression at both the mRNA and protein levels and, in addition, enhanced cell migration. Furthermore, we found that the CTNNB1 gene promoter methylation levels were higher in the more invasive HCT-116 and SW620 colon cancer cells than in HT-29 and CCD-841 (normal colon epithelial) cells. We also found that TPA or hydrogen peroxide induced CTNNB1 gene promoter methylation to a higher extent in HT-29 and CCD-841 cells than in HCT-116 and SW620 cells, and that the degree of CTNNB1 gene promoter methylation positively correlated with cell dissociation and migration. In addition, we found that co-treatment with 5-aza-2′-deoxycytidine (decitabine, a DNA methyl transferase inhibitor) and VAS2870 (a NADPH oxidase inhibitor) almost completely blocked the invasion of TPA-treated HT-29 and TPA-untreated HCT-116 and SW620 cells, and that these inhibitions surpassed those of the cells treated with decitabine or VAS2870 alone.


From our data we conclude that the extent of CTNNB1 gene promoter methylation by reactive oxygen species correlates with the migratory and invasive abilities of colon cancer cells. Our results suggest that epigenetic regulation of CTNNB1 may serve as a novel avenue to block colon cancer cell migration and invasion.


Colon cancer CTNNB1 gene promoter hypermethylation Reactive oxygen species NADPH oxidase (NOX) 2 Migration Invasion 



This work was supported by the National Research Foundation of Korea (NRF) funded by the Korean Ministry of Science and ICT (grant no. NRF-2017R1E1A1A01073590), and by a Yeungnam University research grant (2017).

Compliance with ethical standards

Conflict of interest

The authors have no conflict of interest to declare.


  1. 1.
    M. Yilmaz, G. Christofori, Mechanisms of motility in metastasizing cells. Mol Can Res 8, 629–642 (2010)CrossRefGoogle Scholar
  2. 2.
    R. Paduch, The role of lymphangiogenesis and angiogenesis in tumor metastasis. Cell Oncol 39, 397–410 (2016)CrossRefGoogle Scholar
  3. 3.
    S. Schmidt, P. Friedl, Interstitial cell migration: Integrin-dependent and alternative adhesion mechanisms. Cell Tissue Res 339, 83–92 (2010)CrossRefPubMedGoogle Scholar
  4. 4.
    P. Devreotes, A.R. Horwitz, Signaling networks that regulate cell migration. Cold Spring Harb Perspect Biol 7, a005959 (2015)CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    L. Lobastova, D. Kraus, A. Glassmann, D. Khan, C. Steinhäuser, C. Wolff, N. Veit, J. Winter, R. Probstmeier, Collective cell migration of thyroid carcinoma cells: A beneficial ability to override unfavourable substrates. Cell Oncol 40, 63–76 (2017)CrossRefGoogle Scholar
  6. 6.
    W. Meng, M. Takeichi, Adherens junction: Molecular architecture and regulation. Cold Spring Harb Perspect Biol 1, a002899 (2009)CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    W. Birchmeier, J. Hülsken, J. Behrens, Adherens junction proteins in tumour progression. Cancer Surv 24, 129–140 (1995)PubMedGoogle Scholar
  8. 8.
    T.T. Onder, P.B. Gupta, S.A. Mani, J. Yang, E.S. Lander, R.A. Weinberg, Loss of E-cadherin promotes metastasis via multiple downstream transcriptional pathways. Cancer Res 68, 3645–3654 (2008)CrossRefPubMedGoogle Scholar
  9. 9.
    P.J. Morin, A.B. Sparks, V. Korinek, N. Barker, H. Clevers, B. Vogelstein, K.W. Kinzler, Activation of β-catenin-Tcf signaling in colon cancer by mutations in β-catenin or APC. Science 275, 1787–1790 (1997)CrossRefPubMedGoogle Scholar
  10. 10.
    V. Korinek, N. Barker, P.J. Morin, D. Van Wichen, R. De Weger, K.W. Kinzler, B. Vogelstein, H. Clevers, Constitutive transcriptional activation by a β-catenin-Tcf complex in APC−/− colon carcinoma. Science 275, 1784–1787 (1997)CrossRefPubMedGoogle Scholar
  11. 11.
    T. Li, Q. Lai, S. Wang, J. Cai, Z. Xiao, D. Deng, L. He, H. Jiao, Y. Ye, L. Liang, MicroRNA-224 sustains Wnt/β-catenin signaling and promotes aggressive phenotype of colorectal cancer. J Exp Clin Cancer Res 35, 21 (2016)CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    X. Tian, Z. Liu, B. Niu, J. Zhang, T.K. Tan, S.R. Lee, Y. Zhao, D.C.H. Harris and G. Zheng, E-cadherin/β-catenin complex and the epithelial barrier. Biomed Res Int 2011, 567305 (2011)Google Scholar
  13. 13.
    D. Kimelman, W. Xu, β-Catenin destruction complex: Insights and questions from a structural perspective. Oncogene 25, 7482–7491 (2006)CrossRefPubMedGoogle Scholar
  14. 14.
    M.P.A. Ebert, J. Yu, J. Hoffmann, A. Rocco, C. Röcken, S. Kahmann, O. Müller, M. Korc, J.J. Sung, P. Malfertheiner, Loss of beta-catenin expression in metastatic gastric cancer. J Clin Oncol 21, 1708–1714 (2003)CrossRefPubMedGoogle Scholar
  15. 15.
    Y. Miao, L. Wang, X. Zhang, X. Xu, G. Jiang, C. Fan, Y. Liu, X. Lin, J. Yu, Y. Zhang, Promoter methylation-mediated silencing of β-catenin enhances invasiveness of non-small cell lung cancer and predicts adverse prognosis. PLoS One 9, e112258 (2014)CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    F. Coppedè, Epigenetic biomarkers of colorectal cancer: Focus on DNA methylation. Cancer Lett 342, 238–247 (2014)CrossRefPubMedGoogle Scholar
  17. 17.
    J. Yuan, N.L. Luceño, B. Sander, M.M. Golas, Synergistic anti-cancer effects of epigenetic drugs on medulloblastoma cells. Cell Oncol 40, 263–279 (2017)CrossRefGoogle Scholar
  18. 18.
    M.S.G. Montani, M. Granato, C. Santoni, P. Del Porto, N. Merendino, G. D’Orazi, A. Faggioni, M. Cirone, Histone deacetylase inhibitors VPA and TSA induce apoptosis and autophagy in pancreatic cancer cells. Cell Oncol 40, 167–180 (2017)CrossRefGoogle Scholar
  19. 19.
    M. Staberg, S.R. Michaelsen, R.D. Rasmussen, M. Villingshøj, H.S. Poulsen, P. Hamerlik, Inhibition of histone deacetylases sensitizes glioblastoma cells to lomustine. Cell Oncol 40, 21–32 (2017)CrossRefGoogle Scholar
  20. 20.
    K. GrØnbÆk, C. Hother, P.A. Jones, Epigenetic changes in cancer. APMIS 115, 1039–1059 (2007)CrossRefPubMedGoogle Scholar
  21. 21.
    P.M. Das, R. Singal, DNA methylation and cancer. J Clin Oncol 22, 4632–4642 (2004)CrossRefPubMedGoogle Scholar
  22. 22.
    P.A. Jones, S.B. Baylin, The epigenomics of cancer. Cell 128, 683–692 (2007)CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    S.H. Lin, J. Wang, P. Saintigny, C.-C. Wu, U. Giri, J. Zhang, T. Menju, L. Diao, L. Byers and J.N. Weinstein, Genes suppressed by DNA methylation in non-small cell lung cancer reveal the epigenetics of epithelial–mesenchymal transition. BMC Genomics 15, 1079 (2014)CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    M. Esteller, Epigenetics in cancer. N Engl J Med 2008, 1148–1159 (2008)CrossRefGoogle Scholar
  25. 25.
    D.C. Radisky, D.D. Levy, L.E. Littlepage, H. Liu, C.M. Nelson, J.E. Fata, D. Leake, E.L. Godden, D.G. Albertson, M.A. Nieto, Rac1b and reactive oxygen species mediate MMP-3-induced EMT and genomic instability. Nature 436, 123–127 (2005)CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    S. Banskota, S.C. Regmi, J.A. Kim, NOX1 to NOX2 switch deactivates AMPK and induces invasive phenotype in colon cancer cells through overexpression of MMP-7. Mol Cancer 14, 123 (2015)CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    S. Banskota, S.C. Regmi, J. Gautam, P. Gurung, Y.J. Lee, S.K. Ku, J.H. Lee, J. Lee, H.W. Chang, S.J. Park, J.A. Kim, Serotonin disturbs colon epithelial tolerance of commensal E. Coli by increasing NOX2-derived superoxide. Free Radic Biol Med 106, 196–207 (2017)CrossRefPubMedGoogle Scholar
  28. 28.
    K. Mori, M. Shibanuma, K. Nose, Invasive potential induced under long-term oxidative stress in mammary epithelial cells. Cancer Res 64, 7464–7472 (2004)CrossRefPubMedGoogle Scholar
  29. 29.
    Q. Wu, X. Ni, ROS-mediated DNA methylation pattern alterations in carcinogenesis. Curr Drug Targets 16, 13–19 (2015)CrossRefPubMedGoogle Scholar
  30. 30.
    M. Esteller, CpG island hypermethylation and tumor suppressor genes: A booming present, a brighter future. Oncogene 21, 5427–5440 (2002)CrossRefPubMedGoogle Scholar
  31. 31.
    R. Franco, O. Schoneveld, A.G. Georgakilas, M.I. Panayiotidis, Oxidative stress, DNA methylation and carcinogenesis. Cancer Lett 266, 6–11 (2008)CrossRefPubMedGoogle Scholar
  32. 32.
    S.O. Lim, J.M. Gu, M.S. Kim, H.S. Kim, Y.N. Park, C.K. Park, J.W. Cho, Y.M. Park and G. Jung, Epigenetic changes induced by reactive oxygen species in hepatocellular carcinoma: Methylation of the E-cadherin promoter. Gastroenterology 135, 2128–2140 (2008)CrossRefGoogle Scholar
  33. 33.
    T. Ikenoue, H. Ijichi, N. Kato, F. Kanai, T. Masaki, W. Rengifo, M. Okamoto, M. Matsumura, T. Kawabe, Y. Shiratori, Analysis of the β-catenin/T cell factor signaling pathway in 36 gastrointestinal and liver Cancer cells. Cancer Sci 93, 1213–1220 (2002)Google Scholar
  34. 34.
    M. Ilyas, I. Tomlinson, A. Rowan, M. Pignatelli, W. Bodmer, β-Catenin mutations in cell lines established from human colorectal cancers. Proc Natl Acad Sci U S A 94, 10330–10334 (1997)CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    D. Ahmed, P. Eide, I. Eilertsen, S. Danielsen, M. Eknaes, M. Hektoen, G. Lind, R. Lothe, Epigenetic and genetic features of 24 colon cancer cell lines. Oncogenesis 2, e71 (2013)CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    B.C. Park, D. Thapa, Y.S. Lee, M.K. Kwak, E.S. Lee, H.G. Choi, C.S. Yong, J.A. Kim, 1-furan-2-yl-3-pyridin-2-yl-propenone inhibits the invasion and migration of HT1080 human fibrosarcoma cells through the inhibition of proMMP-2 activation and down regulation of MMP-9 and MT1-MMP. Eur J Pharmacol 567, 193–197 (2007)CrossRefPubMedGoogle Scholar
  37. 37.
    S.C. Regmi, S.Y. Park, S.K. Ku, J.A. Kim, Serotonin regulates innate immune responses of colon epithelial cells through Nox2-derived reactive oxygen species. Free Radic Biol Med 69, 377–389 (2014)CrossRefPubMedGoogle Scholar
  38. 38.
    O. Tetsu, F. McCormick, Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells. Nature 398, 422–426 (1999)CrossRefPubMedGoogle Scholar
  39. 39.
    T. Brabletz, A. Jung, S. Dag, F. Hlubek, T. Kirchner, β-Catenin regulates the expression of the matrix metalloproteinase-7 in human colorectal cancer. Am J Pathol 155, 1033–1038 (1999)CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    B.P.L. Wijnhoven, W.N.M. Dinjens, M. Pignatelli, E-cadherin-catenin cell-cell adhesion complex and human cancer. Br J Surg 87, 992–1005 (2000)CrossRefPubMedGoogle Scholar
  41. 41.
    J. Theys, B. Jutten, R. Habets, K. Paesmans, A.J. Groot, P. Lambin, B.G. Wouters, G. Lammering, M. Vooijs, E-cadherin loss associated with EMT promotes radioresistance in human tumor cells. Radiother Oncol 99, 392–397 (2011)CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Y. Wang, J. Shi, K. Chai, X. Ying, B.P. Zhou, The role of Snail in EMT and tumorigenesis. Curr Cancer Drug Targets 13, 963–972 (2013)CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    J.R. Graff, J.G. Herman, R.G. Lapidus, H. Chopra, R. Xu, D.F. Jarrard, W.B. Isaacs, P.M. Pitha, N.E. Davidson, S.B. Baylin, E-cadherin expression is silenced by DNA hypermethylation in human breast and prostate carcinomas. Cancer Res 55, 5195–5199 (1995)PubMedGoogle Scholar
  44. 44.
    A.O. Chan, S.K. Lam, B.C. Wong, W.M. Wong, M.F. Yuen, Y.H. Yeung, W.M. Hui, A. Rashid, Y.L. Kwong, Promoter methylation of E-cadherin gene in gastric mucosa associated with helicobacter pylori infection and in gastric cancer. Gut 52, 502–506 (2003)CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    S.Y. Shin, C.G. Kim, E.H. Jho, M.S. Rho, Y.S. Kim, Y.H. Kim, Y.H. Lee, Hydrogen peroxide negatively modulates Wnt signaling through downregulation of β-catenin. Cancer Lett 212, 225–231 (2004)CrossRefPubMedGoogle Scholar

Copyright information

© International Society for Cellular Oncology 2018
corrected publication June 2018

Authors and Affiliations

  1. 1.College of PharmacyYeungnam UniversityGyeongsanRepublic of Korea

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