Clinical Orthopaedics and Related Research®

, Volume 472, Issue 3, pp 865–873 | Cite as

Chick Embryo Extract Demethylates Tumor Suppressor Genes in Osteosarcoma Cells

  • Xiaodong Mu
  • Bolat Sultankulov
  • Riddhima Agarwal
  • Adel Mahjoub
  • Trevor Schott
  • Nicholas Greco
  • Johnny Huard
  • Kurt Weiss
Symposium: Musculoskeletal Tumor Society 2012 Symposium



Epigenetics is the study of changes in gene expression or cellular phenotype caused by mechanisms other than changes in the underlying DNA sequence. It is widely accepted that cancer has genetic and epigenetic origins. The idea of epigenetic reprogramming of cancer cells by an embryonic microenvironment possesses potential interest from the prospect of both basic science and potential therapeutic strategies. Chick embryo extract (CEE) has been used for the successful expansion of many specific stem cells and has demonstrated the ability to facilitate DNA demethylation.


The current study was conducted to compare the status of DNA methylation in highly metastatic and less metastatic osteosarcoma cells and to investigate whether CEE may affect the epigenetic regulation of tumor suppressor genes and thus change the metastatic phenotypes of highly metastatic osteosarcoma cells.


K7M2 murine OS cells were treated with CEE to determine its potential effect on DNA methylation, cell apoptosis, and invasion capacity.


Our current results suggest that the methylation status of tumor suppressor genes (p16, p53, and E-cadherin) is significantly greater in highly metastatic mouse ostoesarcoma K7M2 cells in comparison with less metastatic mouse osteosarcoma K12 cells. CEE treatment of K7M2 cells caused demethylation of p16, p53, and E-cadherin genes, upregulated their expression, and resulted in the reversion of metastatic phenotypes in highly metastatic osteosarcoma cells.


CEE may promote the reversion of metastatic phenotypes of osteosarcoma cells and can be a helpful tool to study osteosarcoma tumor reversion by epigenetic reprogramming.

Clinical Relevance

Demethylation of tumor suppressor genes in osteosarcoma may represent a novel strategy to diminish the metastatic potential of this neoplasm. Further studies, both in vitro and in vivo, are warranted to evaluate the clinical feasibility of this approach as an adjuvant to current therapy.


Osteosarcoma Tumor Suppressor Gene Cancer Stem Cell Osteosarcoma Cell Metastatic Phenotype 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



We acknowledge the support of The Pittsburgh Foundation and the Houy family in loving memory of Jon Houy.


  1. 1.
    Allegrucci C, Rushton MD, Dixon JE, Sottile V, Shah M, Kumari R, Watson S, Alberio R, Johnson AD. Epigenetic reprogramming of breast cancer cells with oocyte extracts. Mol Cancer. 2011;10:7.PubMedCentralPubMedGoogle Scholar
  2. 2.
    Baylin SB, Ohm JE. Epigenetic gene silencing in cancer—a mechanism for early oncogenic pathway addiction? Nat Rev Cancer. 2006;6:107–116.PubMedCrossRefGoogle Scholar
  3. 3.
    Beavon IR. The E-cadherin-catenin complex in tumour metastasis: structure, function and regulation. Eur J Cancer. 2000;36:1607–1620.PubMedCrossRefGoogle Scholar
  4. 4.
    Benassi MS, Molendini L, Gamberi G, Ragazzini P, Sollazzo MR, Merli M, Asp J, Magagnoli G, Balladelli A, Bertoni F, Picci P. Alteration of pRb/p16/cdk4 regulation in human osteosarcoma. Int J Cancer. 1999;84:489–493.PubMedCrossRefGoogle Scholar
  5. 5.
    Bielack SS, Kempf-Bielack B, Delling G, Exner GU, Flege S, Helmke K, Kotz R, Salzer-Kuntschik M, Werner M, Winkelmann W, Zoubek A, Jurgens H, Winkler K. Prognostic factors in high-grade osteosarcoma of the extremities or trunk: an analysis of 1,702 patients treated on neoadjuvant cooperative osteosarcoma study group protocols. J Clin Oncol. 2002;20:776–790.PubMedCrossRefGoogle Scholar
  6. 6.
    Bui HT, Kwon DN, Kang MH, Oh MH, Park MR, Park WJ, Paik SS, Van Thuan N, Kim JH. Epigenetic reprogramming in somatic cells induced by extract from germinal vesicle stage pig oocytes. Development. 2012;139:4330–4340.PubMedCrossRefGoogle Scholar
  7. 7.
    Carrle D, Bielack SS. Current strategies of chemotherapy in osteosarcoma. Int Orthop. 2006;30:445–451.PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Cucina A, Biava PM, D’Anselmi F, Coluccia P, Conti F, di Clemente R, Miccheli A, Frati L, Gulino A, Bizzarri M. Zebrafish embryo proteins induce apoptosis in human colon cancer cells (Caco2). Apoptosis. 2006;11:1617–1628.PubMedCrossRefGoogle Scholar
  9. 9.
    Das PM, Singal R. DNA methylation and cancer. J Clin Oncol. 2004;22:4632–4642.PubMedCrossRefGoogle Scholar
  10. 10.
    Demircan B, Dyer LM, Gerace M, Lobenhofer EK, Robertson KD, Brown KD. Comparative epigenomics of human and mouse mammary tumors. Genes Chromosomes Cancer. 2009;48:83–97.PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Diez-Torre A, Andrade R, Eguizabal C, Lopez E, Arluzea J, Silio M, Arechaga J. Reprogramming of melanoma cells by embryonic microenvironments. Int J Dev Biol. 2009;53:1563–1568.PubMedCrossRefGoogle Scholar
  12. 12.
    Diller L, Kassel J, Nelson CE, Gryka MA, Litwak G, Gebhardt M, Bressac B, Ozturk M, Baker SJ, Vogelstein B, et al. p53 functions as a cell cycle control protein in osteosarcomas. Mol Cell Biol. 1990;10:5772–5781.PubMedCentralPubMedGoogle Scholar
  13. 13.
    Esteller M. CpG island hypermethylation and tumor suppressor genes: a booming present, a brighter future. Oncogene. 2002;21:5427–5440.PubMedCrossRefGoogle Scholar
  14. 14.
    Esteller M. Epigenetic gene silencing in cancer: the DNA hypermethylome. Hum Mol Genet. 2007;16(Spec No 1):R50–59.PubMedCrossRefGoogle Scholar
  15. 15.
    Ferrari S, Smeland S, Mercuri M, Bertoni F, Longhi A, Ruggieri P, Alvegard TA, Picci P, Capanna R, Bernini G, Muller C, Tienghi A, Wiebe T, Comandone A, Bohling T, Del Prever AB, Brosjo O, Bacci G, Saeter G. Neoadjuvant chemotherapy with high-dose Ifosfamide, high-dose methotrexate, cisplatin, and doxorubicin for patients with localized osteosarcoma of the extremity: a joint study by the Italian and Scandinavian Sarcoma Groups. J Clin Oncol. 2005;23:8845–8852.PubMedCrossRefGoogle Scholar
  16. 16.
    Gharaibeh B, Lu A, Tebbets J, Zheng B, Feduska J, Crisan M, Peault B, Cummins J, Huard J. Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique. Nat Protoc. 2008;3:1501–1509.PubMedCrossRefGoogle Scholar
  17. 17.
    Greger V, Passarge E, Hopping W, Messmer E, Horsthemke B. Epigenetic changes may contribute to the formation and spontaneous regression of retinoblastoma. Hum Genet. 1989;83:155–158.PubMedCrossRefGoogle Scholar
  18. 18.
    Hansen MF. Genetic and molecular aspects of osteosarcoma. J Musculoskelet Neuronal Interact. 2002;2:554–560.PubMedGoogle Scholar
  19. 19.
    Hayden JB, Hoang BH. Osteosarcoma: basic science and clinical implications. Orthop Clin North Am. 2006;37:1–7.PubMedCrossRefGoogle Scholar
  20. 20.
    Hendrix MJ, Seftor EA, Seftor RE, Kasemeier-Kulesa J, Kulesa PM, Postovit LM. Reprogramming metastatic tumour cells with embryonic microenvironments. Nat Rev Cancer. 2007;7:246–255.PubMedCrossRefGoogle Scholar
  21. 21.
    Honoki K, Tsujiuchi T, Mori T, Yoshitani K, Tsutsumi M, Takakura Y, Mii Y. Expression of the p16INK4a gene and methylation pattern of CpG sites in the promoter region in rat tumor cell lines. Mol Carcinog. 2004;39:10–14.PubMedCrossRefGoogle Scholar
  22. 22.
    Jost JP. Nuclear extracts of chicken embryos promote an active demethylation of DNA by excision repair of 5-methyldeoxycytidine. Proc Natl Acad Sci U S A. 1993;90:4684–4688.PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Kalyani A, Hobson K, Rao MS. Neuroepithelial stem cells from the embryonic spinal cord: isolation, characterization, and clonal analysis. Dev Biol. 1997;186:202–223.PubMedCrossRefGoogle Scholar
  24. 24.
    Kashima T, Nakamura K, Kawaguchi J, Takanashi M, Ishida T, Aburatani H, Kudo A, Fukayama M, Grigoriadis AE. Overexpression of cadherins suppresses pulmonary metastasis of osteosarcoma in vivo. Int J Cancer. 2003;104:147–154.PubMedCrossRefGoogle Scholar
  25. 25.
    Khanna C, Prehn J, Yeung C, Caylor J, Tsokos M, Helman L. An orthotopic model of murine osteosarcoma with clonally related variants differing in pulmonary metastatic potential. Clin Exp Metastasis. 2000;18:261–271.PubMedCrossRefGoogle Scholar
  26. 26.
    Kiss NB, Geli J, Lundberg F, Avci C, Velazquez-Fernandez D, Hashemi J, Weber G, Hoog A, Ekstrom TJ, Backdahl M, Larsson C. Methylation of the p16INK4A promoter is associated with malignant behavior in abdominal extra-adrenal paragangliomas but not pheochromocytomas. Endocr Relat Cancer. 2008;15:609–621.PubMedCrossRefGoogle Scholar
  27. 27.
    Kulesa PM, Kasemeier-Kulesa JC, Teddy JM, Margaryan NV, Seftor EA, Seftor RE, Hendrix MJ. Reprogramming metastatic melanoma cells to assume a neural crest cell-like phenotype in an embryonic microenvironment. Proc Natl Acad Sci U S A. 2006;103:3752–3757.PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    Lunyak VV, Rosenfeld MG. Epigenetic regulation of stem cell fate. Hum Mol Genet. 2008;17:R28–36.PubMedCrossRefGoogle Scholar
  29. 29.
    Messerschmitt PJ, Garcia RM, Abdul-Karim FW, Greenfield EM, Getty PJ. Osteosarcoma. J Am Acad Orthop Surg. 2009;17:515–527.PubMedGoogle Scholar
  30. 30.
    Meyers PA, Schwartz CL, Krailo MD, Healey JH, Bernstein ML, Betcher D, Ferguson WS, Gebhardt MC, Goorin AM, Harris M, Kleinerman E, Link MP, Nadel H, Nieder M, Siegal GP, Weiner MA, Wells RJ, Womer RB, Grier HE. Osteosarcoma: the addition of muramyl tripeptide to chemotherapy improves overall survival—a report from the Children’s Oncology Group. J Clin Oncol. 2008;26:633–638.PubMedCrossRefGoogle Scholar
  31. 31.
    Mu X, Isaac C, Schott T, Huard J, Weiss K. Rapamycin Inhibits ALDH Activity, Resistance to Oxidative Stress, and Metastatic Potential in Murine Osteosarcoma Cells. Sarcoma. 2013;2013:480713.PubMedCentralPubMedCrossRefGoogle Scholar
  32. 32.
    Onder TT, Gupta PB, Mani SA, Yang J, Lander ES, Weinberg RA. Loss of E-cadherin promotes metastasis via multiple downstream transcriptional pathways. Cancer Res. 2008;68:3645–3654.PubMedCrossRefGoogle Scholar
  33. 33.
    Pajtler K, Bohrer A, Maurer J, Schorle H, Schramm A, Eggert A, Schulte JH. Production of chick embryo extract for the cultivation of murine neural crest stem cells. J Vis Exp. 2010.Google Scholar
  34. 34.
    Pollina EA, Brunet A. Epigenetic regulation of aging stem cells. Oncogene. 2011;30:3105–3126.PubMedCrossRefGoogle Scholar
  35. 35.
    Rajasingh J, Lambers E, Hamada H, Bord E, Thorne T, Goukassian I, Krishnamurthy P, Rosen KM, Ahluwalia D, Zhu Y, Qin G, Losordo DW, Kishore R. Cell-free embryonic stem cell extract-mediated derivation of multipotent stem cells from NIH3T3 fibroblasts for functional and anatomical ischemic tissue repair. 2008;102:e107–117.Google Scholar
  36. 36.
    Rao-Bindal K, Kleinerman ES. Epigenetic regulation of apoptosis and cell cycle in osteosarcoma. Sarcoma. 2011;2011:679457.PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© The Association of Bone and Joint Surgeons® 2013

Authors and Affiliations

  • Xiaodong Mu
    • 1
  • Bolat Sultankulov
    • 2
  • Riddhima Agarwal
    • 1
  • Adel Mahjoub
    • 1
  • Trevor Schott
    • 1
  • Nicholas Greco
    • 1
  • Johnny Huard
    • 1
  • Kurt Weiss
    • 1
  1. 1.Cancer Stem Cell Laboratory, Stem Cell Research Center, Department of Orthopaedic SurgeryUniversity of PittsburghPittsburghUSA
  2. 2.Department of BiophysicsNazarbayev University Research and Innovation SystemAstanaKazakhstan

Personalised recommendations