Advertisement

Tumor Biology

, Volume 35, Issue 2, pp 1075–1082 | Cite as

Bufalin inhibits the differentiation and proliferation of human osteosarcoma cell line hMG63-derived cancer stem cells

  • Yuewen ChangEmail author
  • Yongfang Zhao
  • Hongsheng Zhan
  • Xiaoen Wei
  • Tianjin Liu
  • Bo Zheng
Research Article

Abstract

Cancer stem cells (CSCs) play an important role in drug resistance of tumor and are responsible for high recurrence rates. Agents that can suppress the proliferation and differentiation of CSCs would provide new opportunity to fight against tumor recurrence. In this study, we developed a new strategy to enrich CSCs in human osteosarcoma cell line hMG63. Using these CSCs as model, we tested the effect of bufalin, a traditional Chinese medicine, on the proliferation and differentiation of CSCs. hMG63 cells were cultured in poly-HEMA-treated dish and cancer stem cell-specific medium. In this nonadhesive culture system, hMG63 formed spheres, which were then collected and injected into the immunodeficient mice. Cisplatin was administered every 3 days for five times. The enriched xenograft tumors were cultured in cancer stem cell-specific medium again to form tumor spheres. Expression of cancer stem cell markers of these cells was measured by flow cytometry. These cells were then treated with bufalin, and the proliferation and differentiation ability were indicated by the expression level of molecular markers and the formation of sphere again in vitro. We obtained a low CD133+/CD44 cell population with high-level stem cell marker. When treated with bufalin, the sphere could not get attached to the flask and failed to differentiate, which was indicated by the stable expression of stem cell marker CD133 and OCT-4 in the condition permissive to differentiation. Treatment of bufalin also suppressed the single cells isolated from the sphere to form sphere again in the nonadhesive culture system, and a decreased expression of proliferation marker Ki67 was also detected in these cells. Sphere-formed and chemoresistant colon xenograft tumors in immunodeficient mice could enrich cancer stem cell population. Bufalin could inhibit proliferation and differentiation of CSCs.

Keywords

Sphere culture Cancer stem cells Osteosarcoma Bufalin 

Notes

Acknowledgments

This work is supported by grant from Natural Science Foundation of Shanghai (13ZR1442500).

References

  1. 1.
    Clarke MF, Dick JE, Dirks PB, Eaves CJ, Jamieson CHM, Jones DL, et al. Cancer stem cells—perspectives on current status and future directions: AACR Workshop on cancer stem cells. Cancer Res. 2006;66(19):9339–44.PubMedCrossRefGoogle Scholar
  2. 2.
    Clarke MF, Dick JE, Dirks PB, Eaves CJ, Jamieson CH, Jones DL, et al. Cancer stem cells—perspectives on current status and future directions: AACR Workshop on cancer stem cells. Cancer Res. 2006;66(19):9339–44.PubMedCrossRefGoogle Scholar
  3. 3.
    Jordan CT, Guzman ML, Noble M. Cancer stem cells. N Engl J Med. 2006;355(12):1253–61.PubMedCrossRefGoogle Scholar
  4. 4.
    Dalerba P, Cho RW, Clarke MF. Cancer stem cells: models and concepts. Annu Rev Med. 2007;58:267–84.PubMedCrossRefGoogle Scholar
  5. 5.
    Costea DE, Tsinkalovsky O, Vintermyr OK, Johannessen AC, Mackenzie IC. Cancer stem cells—new and potentially important targets for the therapy of oral squamous cell carcinoma. Oral Dis. 2006;12(5):443–54.PubMedCrossRefGoogle Scholar
  6. 6.
    Locke M, Heywood M, Fawell S, Mackenzie IC. Retention of intrinsic stem cell hierarchies in carcinoma-derived cell lines. Cancer Res. 2005;65(19):8944–50.PubMedCrossRefGoogle Scholar
  7. 7.
    Pérez-Caro M, Cobaleda C, González-Herrero I, Vicente-Dueñas C, Bermejo-Rodríguez C, Sánchez-Beato M, et al. Cancer induction by restriction of oncogene expression to the stem cell compartment. EMBO J. 2009;28(1):8–20.PubMedCrossRefGoogle Scholar
  8. 8.
    Vermeulen L, Todaro M, Mello FS, Sprick MR, Kemper K, Alea MP, et al. Single-cell cloning of colon cancer stem cells reveals a multi-lineage differentiation capacity. Proc Natl Acad Sci. 2008;105(36):13427–32.PubMedCrossRefGoogle Scholar
  9. 9.
    Yang YM, Chang JW. Current status and issues in cancer stem cell study. Cancer Investig. 2008;26(7):741–55.CrossRefGoogle Scholar
  10. 10.
    Wright MH, Calcagno A, Salcido CD, Carlson MD, Ambudkar SV, Varticovski L. Brca1 breast tumors contain distinct CD44+/CD24− and CD133+ cells with cancer stem cell characteristics. Breast Cancer Res. 2008;10(1):R10-R.CrossRefGoogle Scholar
  11. 11.
    Hadnagy A, Gaboury L, Beaulieu R, Balicki D. SP analysis may be used to identify cancer stem cell populations. Exp Cell Res. 2006;312(19):3701–10.PubMedCrossRefGoogle Scholar
  12. 12.
    Fuller J. Stem cell aging and cancer. Sci Aging Knowl Environ. 2006;2006(9):pe12.CrossRefGoogle Scholar
  13. 13.
    Lahad JP. Stem cell-ness: a “magic marker” for cancer. J Clin Investig. 2005;115(6):1463–7.PubMedCrossRefGoogle Scholar
  14. 14.
    Shen S, Yang W, Wang Z, Lei X, Xu L, Wang Y, et al. Tumor-initiating cells are enriched in CD44(hi) population in murine salivary gland tumor. PloS One. 2011;6(8):e23282.PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Savage SA, Mirabello L. Using epidemiology and genomics to understand osteosarcoma etiology. Sarcoma. 2011;2011:548151.PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Jaffe N, Carrasco H, Raymond K, Ayala A, Eftekhari F. Can cure in patients with osteosarcoma be achieved exclusively with chemotherapy and abrogation of surgery? Cancer. 2002;95(10):2202–10.PubMedCrossRefGoogle Scholar
  17. 17.
    Meyers PA, Schwartz CL, Krailo M, Kleinerman ES, Betcher D, Bernstein ML, et al. Osteosarcoma: a randomized, prospective trial of the addition of ifosfamide and/or muramyl tripeptide to cisplatin, doxorubicin, and high-dose methotrexate. J Clin Oncol. 2005;23(9):2004–11.PubMedCrossRefGoogle Scholar
  18. 18.
    Bruland OS, Pihl A. On the current management of osteosarcoma. A critical evaluation and a proposal for a modified treatment strategy. Eur J Cancer. 1997;33(11):1725–31.PubMedCrossRefGoogle Scholar
  19. 19.
    Gibbs CP, Kukekov VG, Reith JD, Tchigrinova O, Suslov ON, Scott EW, et al. Stem-like cells in bone sarcomas: implications for tumorigenesis. Neoplasia. 2005;7(11):967–76.PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Levings PP, McGarry SV, Currie TP, Nickerson DM, McClellan S, Ghivizzani SC, et al. Expression of an exogenous human Oct-4 promoter identifies tumor-initiating cells in osteosarcoma. Cancer Res. 2009;69(14):5648–55.PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Han KQ, Huang G, Gu W, Su YH, Huang XQ, Ling CQ. Anti-tumor activities and apoptosis-regulated mechanisms of bufalin on the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice. World J Gastroenter. 2007;13(24):3374–9.Google Scholar
  22. 22.
    Watabe M, Ito K, Masuda Y, Nakajo S, Nakaya K. Activation of AP-1 is required for bufalin-induced apoptosis in human leukemia U937 cells. Oncogene. 1998;16(6):779–87.PubMedCrossRefGoogle Scholar
  23. 23.
    Yeh JY, Huang WJ, Kan SF, Wang PS. Effects of bufalin and cinobufagin on the proliferation of androgen dependent and independent prostate cancer cells. Prostate. 2003;54(2):112–24.PubMedCrossRefGoogle Scholar
  24. 24.
    Li D, Qu X, Hou K, Zhang Y, Dong Q, Teng Y, et al. PI3K/Akt is involved in bufalin-induced apoptosis in gastric cancer cells. Anti Cancer Drugs. 2009;20(1):59–64.PubMedCrossRefGoogle Scholar
  25. 25.
    Wang J, Yin JQ, Jia Q, Shen JN, Huang G, Xie XB, et al. Bufalin induces apoptosis in osteosarcoma U-2OS and U-2OS methotrexate 300-resistant cell lines in vitro. Zhonghua Zhong Liu Za Zhi [Chin J Oncol]. 2010;32(10):734–8.Google Scholar
  26. 26.
    Chiou SH, Yu CC, Huang CY, Lin SC, Liu CJ, Tsai TH, et al. Positive correlations of Oct-4 and Nanog in oral cancer stem-like cells and high-grade oral squamous cell carcinoma. Clin Cancer Res. 2008;14(13):4085–95.PubMedCrossRefGoogle Scholar
  27. 27.
    Jordan CT. Cancer stem cell biology: from leukemia to solid tumors. Curr Opin Cell Biol. 2004;16(6):708–12.PubMedCrossRefGoogle Scholar
  28. 28.
    Mohseny AB, Machado I, Cai Y, Schaefer KL, Serra M, Hogendoorn PC, et al. Functional characterization of osteosarcoma cell lines provides representative models to study the human disease. Lab Investig. 2011;91(8):1195–205.PubMedCrossRefGoogle Scholar
  29. 29.
    Yu F, Yao H, Zhu P, Zhang X, Pan Q, Gong C, et al. let-7 Regulates self renewal and tumorigenicity of breast cancer cells. Cell. 2007;131(6):1109–23.PubMedCrossRefGoogle Scholar
  30. 30.
    Eyler CE, Rich JN. Survival of the fittest: cancer stem cells in therapeutic resistance and angiogenesis. J Clin Oncol. 2008;26(17):2839–45.PubMedCentralPubMedCrossRefGoogle Scholar
  31. 31.
    Uchida S, Yokoo S, Yanagi Y, Usui T, Yokota C, Mimura T, et al. Sphere formation and expression of neural proteins by human corneal stromal cells in vitro. Invest Ophthalmol Vis Sci. 2005;46(5):1620–5.PubMedCrossRefGoogle Scholar
  32. 32.
    Bertolini G, Roz L, Perego P, Tortoreto M, Fontanella E, Gatti L, et al. Highly tumorigenic lung cancer CD133+ cells display stem-like features and are spared by cisplatin treatment. Proc Natl Acad Sci. 2009;106(38):16281–6.PubMedCrossRefGoogle Scholar
  33. 33.
    Yu CH, Kan SF, Pu HF, Jea Chien E, Wang PS. Apoptotic signaling in bufalin- and cinobufagin-treated androgen-dependent and -independent human prostate cancer cells. Cancer Sci. 2008;99(12):2467–76.PubMedCrossRefGoogle Scholar
  34. 34.
    Xie CM, Chan WY, Yu S, Zhao J, Cheng CH. Bufalin induces autophagy-mediated cell death in human colon cancer cells through reactive oxygen species generation and JNK activation. Free Radic Biol Med. 2011;51(7):1365–75.PubMedCrossRefGoogle Scholar
  35. 35.
    Chen Y, Guo Q, Zhang B, Kang M, Xie Q, Wu Y. Bufalin enhances the antitumor effect of gemcitabine in pancreatic cancer. Oncol Lett. 2012;4(4):792–8.PubMedCentralPubMedGoogle Scholar
  36. 36.
    Tsai SC, Yang JS, Peng SF, Lu CC, Chiang JH, Chung JG, et al. Bufalin increases sensitivity to AKT/mTOR-induced autophagic cell death in SK-HEP-1 human hepatocellular carcinoma cells. Int J Oncol. 2012.Google Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2013

Authors and Affiliations

  • Yuewen Chang
    • 1
    Email author
  • Yongfang Zhao
    • 1
  • Hongsheng Zhan
    • 1
  • Xiaoen Wei
    • 1
  • Tianjin Liu
    • 2
  • Bo Zheng
    • 2
  1. 1.Department of Orthopedics and TraumatologyShuguang Hospital Affiliated to Shanghai University of Traditional Chinese MedicineShanghaiChina
  2. 2.Shanghai GeneCell Biological Technology Co., LTDShanghaiChina

Personalised recommendations