Molecular Biology Reports

, Volume 43, Issue 10, pp 1069–1078 | Cite as

Phenotype transformation of immortalized NCM460 colon epithelial cell line by TGF-β1 is associated with chromosome instability

  • Chao Huang
  • Bin Wen
Original Article


Transforming growth factor-β1 (TGF-β1) within tumor microenvironment has a pivotal function in cancer initiation and tumorigenesis, and hence this study was to observe the malignant transformation induced by TGF-β1 in an immortalized colon epithelial cell line NCM460 for better understanding the mechanisms of colon carcinogenesis. Immortalized colon epithelial cell line NCM460 was used as the model of this study, and was treated with different concentrations of TGF-β1 for different time. Then, immunofluorescence was performed to observe the change of phenotype hallmarks including adherent junction protein E-cadherin, cytoskeleton protein vimentin, and tight junction marker ZO-1, western blotting analysis was performed to detect the expression of the above three markers and two transcription factors (Snail and Slug) involved in the transformation by TGF-β1. In addition, chromosome instability (CHI) including analysis of DNA-ploid was detected by flow cytometry. Our results revealed significant loss or reduction of ZO-1 and E-cadherin, and robust emergence of vimentin in the cell line NCM460 after a 15-, 20-, and 25-day treatment with 10 ng/ml TGF-β1. Interestingly, 20 and 25 days after stimulation with 5 ng/ml TGF-β1, expression of E-cadherin and ZO-1 revealed a pattern roughly similar to that of 10 ng/ml TGF-β1, especially, both expressions was vanished and vimentin expression was dramatically increased at days 25 after TGF-β1 stimulation. After a stimulation with 10 ng/ml TGF-β1 for 15, 20, and 25 days, the levels of Snail and Slug expression in the cells were significantly up-regulated, compared with the cells treated with TGF-β1 inhibitor LY364947, PBS or balnk control (P < 0.01). Our results found that many abnormal mitotic patterns including lagging chromosomes and anaphase bridges in NCM460 cells were induced by TGF-β1 after its stimulation for 15, 20, and 25 days. Very few mitotic cells with treatment of PBS for 15, 20 and 25 days were non-diploid whose DNA content was greater or less than 4 N, but these cells were significantly increased after exposure to TGF-β1 for 15, 20, and 25 days, which was associated with the induction of hypo-diploid, hyper-diploid, and poly-diploid (P < 0.05).These data indicate that TGF-β1 induces a phenotypic transformation of normal colon epithelium similar to its pro-tumoral behaviors in TME, involving in alteration of chromosome stability.


Colon cancer Tumor microenvironment Transforming growth factor-β1 Cancer initiation Chromosome instability 



This study was supported by the National Science Foundation of China (No. 81173257).

Authors’ Contributions

Conception and design of this study was by Bin WEN, writing, acquisition and analysis of data was by Chao HUANG.


  1. 1.
    Tchekmedyian A, Messuti A, Richelli R, Stein S, Silveira A, Iade B, Cohen H (2009) Colorectal cancer prevention. Am J Gastroenterol 104:S551–S576CrossRefGoogle Scholar
  2. 2.
    Walther A, Johnstone E, Swanton C, Midgley R, Tomlinson I, Kerr D (2009) Genetic prognostic and predictive markers in colorectal cancer. Nat Rev Cancer 9:489–499CrossRefPubMedGoogle Scholar
  3. 3.
    Schwitalla S, Ziegler PK, Horst D, Becker V, Kerle I, Begus-Nahrmann Y, Lechel A, Rudolph KL, Langer R, Slotta-Huspenina J, Bader FG, Prazeres da Costa O, Neurath MF, Meining A, Kirchner T, Greten FR (2013) Loss of p53 in enterocytes generates an inflammatory microenvironment enabling invasion and lymph node metastasis of carcinogen-induced colorectal tumors. Cancer Cell 23:93–106CrossRefPubMedGoogle Scholar
  4. 4.
    Chang C-C, Lin B-R, Wu T-S, Jeng Y-M, Kuo M-L (2014) Input of microenvironmental regulation on colorectal cancer: role of the CCN family. World J Gastroenterol 20(22):6826–6831CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Quante M, Varga J, Wang TC, Greten FR (2013) The gastrointestinal tumor microenvironment. Gastroenterology 145(1):63–78CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Su X, Ye J, Hsueh EC, Zhang Y, Hoft DF, Peng G (2010) Tumor microenvironments direct the recruitment and expansion of human Th17 cells. J Immunol 184:1630–1641CrossRefPubMedGoogle Scholar
  7. 7.
    Labelle M, Begum S, Hynes RO (2011) Direct signaling between platelets and cancer cells induces an epithelial-mesenchymal-like transition and promotes metastasis. Cancer Cell 20:576–590CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Lee JH, Kang MJ, Han HY, Lee MG, Jeong SI, Ryu BK, Ha TK, Her NG, Han J, Park SJ, Lee KY, Kim HJ, Chi SG (2011) Epigenetic alteration of PRKCDBP in colorectal cancers and its implication in tumor cell resistance to TNFα-induced apoptosis. Clin Cancer Res 17:7551–7562CrossRefPubMedGoogle Scholar
  9. 9.
    Jallepalli PV, Lengauer C (2001) Chromosome segregation and cancer: cutting through the mystery. Nat Rev Cancer 1:109–117CrossRefPubMedGoogle Scholar
  10. 10.
    Shih I-M, Zhou W, Goodman SN, Lengauer C, Kinzler KW, Vogelstein B (2001) Evidence that genetic instability occurs at an early stage of colorectal tumorigenesis. Cancer Res 61:818–822PubMedGoogle Scholar
  11. 11.
    Beckman RA, Loeb LA (2005) Genetic instability in cancer: theory and experiment. Semin Cancer Biol 15:423–435CrossRefPubMedGoogle Scholar
  12. 12.
    Deng W, Tsao SW, Kwok YK, Wong E, Huang XR, Liu S, Tsang CM, Ngan HY, Cheung AN, Lan HY, Guan XY, Cheung AL (2008) Transforming growth factor beta1 promotes chromosomal instability in human papillomavirus 16 E6E7-infected cervical epithelial cells. Cancer Res 68(17):7200–7209CrossRefPubMedGoogle Scholar
  13. 13.
    Ying X, Sun Y, He P (2015) Bone morphogenetic protein-7 inhibits EMT-associated genes in breast cancer. Cell Physiol Biochem 37:1271–1278CrossRefPubMedGoogle Scholar
  14. 14.
    Neuzillet C, Tijeras-Raballand A, Cohenb R, Cros J, Faivre S, Raymond E, de Gramont A (2015) Targeting the TGFβ pathway for cancer therapy. Pharmacol Ther 147:22–31CrossRefPubMedGoogle Scholar
  15. 15.
    Neuzillet C, de Gramont A, Tijeras-Raballand A, de Mestier L, Cros J, Faivre S, Raymond E (2014) Perspectives of TGF-beta inhibition in pancreatic and hepatocellular carcinomas. Oncotarget 5:78–94PubMedGoogle Scholar
  16. 16.
    Van De Water L, Varney S, Tomasek JJ (2013) Mechanoregulation of the myofibroblast in wound contraction, scarring, and fibrosis: opportunities for new therapeutic intervention. Adv Wound Care 2:122–141CrossRefGoogle Scholar
  17. 17.
    Pohlers D, Brenmoehl J, Löffler I, Müller CK, Leipner C, Schultze-Mosgau S, Stallmach A, Kinne RW, Wolf G (2009) TGF-beta and fibrosis in different organs-molecular pathway imprints. Biochim Biophys Acta 1792:746–756CrossRefPubMedGoogle Scholar
  18. 18.
    Papageorgis P (2015) TGF-β signaling in tumor initiation, epithelial-to-mesenchymal transition, and metastasis. J Oncol. doi: 10.1155/2015/587193 PubMedPubMedCentralGoogle Scholar
  19. 19.
    Cano A, Pérez-Moreno MA, Rodrigo I et al (2000) The transcription factor Snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression. Nat Cell Biol 2(2):76–83CrossRefPubMedGoogle Scholar
  20. 20.
    Savagner P, Yamada KM, Thiery JP (1997) The zinc-finger protein slug causes desmosome dissociation, an initial and necessary step for growth factor-induced epithelial-mesenchymal transition. J Cell Biol 137(6):1403–1419CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Yang J, Mani SA, Donaheretal JL (2004) Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell 117(7):927–939CrossRefPubMedGoogle Scholar
  22. 22.
    Lo H-W, Hsu S-C, Xia W et al (2007) Epidermal growth factor receptor cooperates with signal transducer and activator of transcription 3 to induce epithelial-mesenchymal transition in cancer cells via up-regulation of TWIST gene expression. Cancer Res 67(19):9066–9076CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Wang X, Allen TD, May RJ, Lightfoot S, Houchen CW, Huycke MM (2008) Enterococcus faecalis induces aneuploidy and tetraploidy in colonic epithelial cells through a bystander effect. Cancer Res 68(23):9909–9917CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Stewénius Y, Gorunova L, Jonson T, Larsson N, Höglund M, Mandahl N, Mertens F, Mitelman F, Gisselsson D (2005) Structural and numerical chromosome changes in colon cancer develop through telomere-mediated anaphase bridges, not through mitotic multipolarity. Proc Natl Acad Sci USA 102:5541–5546CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Malkoski SP, Haeger SM, Cleaver TG, Rodriguez KJ, Li H, Lu SL, Feser WJ, Barón AE, Merrick D, Lighthall JG, Ijichi H, Franklin W, Wang XJ (2012) Loss of transforming growth factor beta type II receptor increases aggressive tumor behavior and reduces survival in lung adenocarcinoma and squamous cell carcinoma. Clin Cancer Res 18(8):2173–2183CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Salvo E, Garasa S, Dotor J, Morales X, Peláez R, Altevogt P, Rouzaut A (2014) Combined targeting of TGF-β1 and integrin β3 impairs lymph node metastasis in a mouse model of non-small-cell lung cancer. Mol Cancer 13:112CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Tian M, Neil J, Schiemann W (2011) Transforming growth factor-ß and the hallmarks of cancer. Cell Signal 23(6):951–962CrossRefPubMedGoogle Scholar
  28. 28.
    Zamarron B, Chen W (2011) Dual roles of immune cells and their factors in cancer development and progression. Int J Biol Sci 7(5):651–658CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Halaoui R, McCaffrey L (2015) Rewiring cell polarity signaling in cancer. Oncogene 34:939–950CrossRefPubMedGoogle Scholar
  30. 30.
    Negrini S, Gorgoulis VG, Halazonetis TD (2010) Genomic instability-an evolving hallmark of cancer. Nat Rev Mol Cell Biol 11(3):220–228CrossRefPubMedGoogle Scholar
  31. 31.
    Weaver BA, Silk AD, Montagna C, Verdier-Pinard P, Cleveland DW (2007) Aneuploidy acts both oncogenically and as a tumor suppressor. Cancer Cell 11(1):25–36CrossRefPubMedGoogle Scholar
  32. 32.
    Marx J (2002) Debate surges over the origins of genomic defects in cancer. Science 297(5581):544–546CrossRefPubMedGoogle Scholar
  33. 33.
    Li R, Sonik A, Stindl R, Rasnick D, Duesberg P (2000) Aneuploidy versus gene mutation hypothesis of cancer: recent study claims mutation but is found to support aneuploidy. Proc Natl Acad Sci USA 97(7):3236–3241CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Szylberg Ł, Janiczek M, Popiel A, Marszałek A (2015) Large bowel genetic background and inflammatory processes in carcinogenesis-systematic review. Adv Clin Exp Med 24(4):555–563CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  • Chao Huang
    • 1
  • Bin Wen
    • 1
  1. 1.Institute of Pi-WeiGuangzhou University of Chinese MedicineGuangzhouChina

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