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Tumor Biology

, Volume 35, Issue 6, pp 5189–5198 | Cite as

Bone morphogenetic protein 2 inhibits hepatocellular carcinoma growth and migration through downregulation of the PI3K/AKT pathway

  • Ying Zheng
  • Xuemei Wang
  • Haidong Wang
  • Wei Yan
  • Quan Zhang
  • Xin Chang
Research Article

Abstract

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. Previous studies have suggested that abnormal expression of BMP-4, BMP-7, and BMP-9 is correlated with tumor progression in HCC, but the role played by BMP-2 in HCC has not yet been reported. To determine the role of BMP-2 in HCC, we first investigated the effect of exogenous BMP-2 on the growth of the cell lines HCC SK-Hep-1, Hep G2, and Hep 3B. Next, we studied the function of BMP-2 in SK-Hep-1 HCC cell line using a recombinant lentivirus vector to deliver BMP-2. We also used siRNA to silence endogenous BMP-2 expression in the HCC Hep 3B cell line. Then, cell growth and migration were assayed in vitro using WST-8, wound-healing, and transwell invasion assays. Cellular apoptosis and cell-cycle distribution were assessed using flow cytometry. We also investigated the effects of BMP-2 overexpression and knockdown on the expression of proliferating cell nuclear antigen (PCNA), matrix metallopeptidase-2 (MMP-2), phosphorylated AKT (p-AKT), phosphoinositide 3-kinase p85α (PI3Kp85α), Bax, Bcl-2, caspase-3, cleaved caspase-3, p21, and cyclin E. As a result, we observed that BMP-2 inhibited the proliferation of HCC cells. Furthermore, HCC cell proliferation and migration were significantly diminished by BMP-2 overexpression, as was indicated by WST-8, would healing, and transwell assays, while knockdown of BMP-2 led to an increase in proliferation and migration of Hep 3B cells. BMP-2 overexpression significantly increased the susceptibility of SK-Hep-1 cells to low-serum-induced apoptosis, while BMP-2 knockdown reduced the susceptibility of Hep 3B cells. Overexpression of BMP-2 induced G1 phase arrest through upregulation of p21. When BMP-2 expression was elevated in SK-Hep-1 cells, the expression of PI3Kp85α, p-AKT, PCNA, and MMP-2 declined. These results suggest that BMP-2 exerts an inhibitory effect on the growth and migration of HCC cells, possibly via a blockade of PI3K/AKT signaling.

Keywords

Bone morphogenetic protein 2 Hepatocellular carcinoma Migration PI3K AKT 

Notes

Conflicts of interest

None

References

  1. 1.
    El-Serag HB. Epidemiology of viral hepatitis and hepatocellular carcinoma. Gastroenterology. 2012;142(6):1264–73 e1. doi: 10.1053/j.gastro.2011.12.061.PubMedCentralCrossRefPubMedGoogle Scholar
  2. 2.
    Shin JW, Chung YH. Molecular targeted therapy for hepatocellular carcinoma: current and future. World J Gastroenterol. 2013;19(37):6144–55. doi: 10.3748/wjg.v19.i37.6144.PubMedCentralCrossRefPubMedGoogle Scholar
  3. 3.
    Wozney JM, Rosen V, Celeste AJ, Mitsock LM, Whitters MJ, Kriz RW, et al. Novel regulators of bone formation: molecular clones and activities. Science. 1988;242(4885):1528–34.CrossRefPubMedGoogle Scholar
  4. 4.
    Liu H, Bao D, Xia X, Chau JF, Li B. An unconventional role of BMP-Smad1 signaling in DNA damage response: a mechanism for tumor suppression. J Cell Biochem. 2013. doi: 10.1002/jcb.24698.PubMedCentralGoogle Scholar
  5. 5.
    Kim M, Choe S. BMPs and their clinical potentials. BMB Rep. 2011;44(10):619–34.PubMedCentralCrossRefPubMedGoogle Scholar
  6. 6.
    Fei ZH, Yao CY, Yang XL, Huang XE, Ma SL. Serum BMP-2 up-regulation as an indicator of poor survival in advanced non-small cell lung cancer patients. Asian Pac J Cancer Prev. 2013;14(9):5293–9.CrossRefPubMedGoogle Scholar
  7. 7.
    Raida M, Clement JH, Ameri K, Han C, Leek RD, Harris AL. Expression of bone morphogenetic protein 2 in breast cancer cells inhibits hypoxic cell death. Int J Oncol. 2005;26(6):1465–70.PubMedGoogle Scholar
  8. 8.
    Raida M, Clement JH, Leek RD, Ameri K, Bicknell R, Niederwieser D, et al. Bone morphogenetic protein 2 (BMP-2) and induction of tumor angiogenesis. J Cancer Res Clin Oncol. 2005;131(11):741–50. doi: 10.1007/s00432-005-0024-1.CrossRefPubMedGoogle Scholar
  9. 9.
    Lai TH, Fong YC, Fu WM, Yang RS, Tang CH. Osteoblasts-derived BMP-2 enhances the motility of prostate cancer cells via activation of integrins. Prostate. 2008;68(12):1341–53. doi: 10.1002/pros.20799.CrossRefPubMedGoogle Scholar
  10. 10.
    Hsu YL, Huang MS, Yang CJ, Hung JY, Wu LY, Kuo PL. Lung tumor-associated osteoblast-derived bone morphogenetic protein-2 increased epithelial-to-mesenchymal transition of cancer by Runx2/Snail signaling pathway. J Biol Chem. 2011;286(43):37335–46. doi: 10.1074/jbc.M111.256156.PubMedCentralCrossRefPubMedGoogle Scholar
  11. 11.
    Shirai YT, Ehata S, Yashiro M, Yanagihara K, Hirakawa K, Miyazono K. Bone morphogenetic protein-2 and −4 play tumor suppressive roles in human diffuse-type gastric carcinoma. Am J Pathol. 2011;179(6):2920–30. doi: 10.1016/j.ajpath.2011.08.022.PubMedCentralCrossRefPubMedGoogle Scholar
  12. 12.
    Wang L, Park P, Zhang H, La Marca F, Claeson A, Than K, et al. BMP-2 inhibits tumor growth of human renal cell carcinoma and induces bone formation. Int J Cancer. 2012;131(8):1941–50. doi: 10.1002/ijc.27444.CrossRefPubMedGoogle Scholar
  13. 13.
    Wang L, Park P, Zhang H, La Marca F, Claeson A, Valdivia J, et al. BMP-2 inhibits the tumorigenicity of cancer stem cells in human osteosarcoma OS99–1 cell line. Cancer Biol Ther. 2011;11(5):457–63.PubMedCentralCrossRefPubMedGoogle Scholar
  14. 14.
    Xu CP, Ji WM, van den Brink GR, Peppelenbosch MP. Bone morphogenetic protein-2 is a negative regulator of hepatocyte proliferation downregulated in the regenerating liver. World J Gastroenterol. 2006;12(47):7621–5.PubMedCentralPubMedGoogle Scholar
  15. 15.
    Yang YH, Chen K, Li B, Chen JW, Zheng XF, Wang YR, et al. Estradiol inhibits osteoblast apoptosis via promotion of autophagy through the ER-ERK-mTOR pathway. Apoptosis. 2013;18(11):1363–75. doi: 10.1007/s10495-013-0867-x.CrossRefPubMedGoogle Scholar
  16. 16.
    Herrera B, Inman GJA. Rapid and sensitive bioassay for the simultaneous measurement of multiple bone morphogenetic proteins. Identification and quantification of BMP4, BMP6 and BMP9 in bovine and human serum. BMC Cell Biol. 2009;10:20. doi: 10.1186/1471-2121-10-20.PubMedCentralCrossRefPubMedGoogle Scholar
  17. 17.
    Chen A, Wang D, Liu X, He S, Yu Z, Wang J. Inhibitory effect of BMP-2 on the proliferation of breast cancer cells. Mol Med Rep. 2012;6(3):615–20. doi: 10.3892/mmr.2012.962.PubMedGoogle Scholar
  18. 18.
    Wang G, Cao X, Lai S, Luo X, Feng Y, Xia X, et al. PI3K stimulates DNA synthesis and cell-cycle progression via its p55PIK regulatory subunit interaction with PCNA. Mol Cancer Ther. 2013;12(10):2100–9. doi: 10.1158/1535-7163.MCT-12-0920.CrossRefPubMedGoogle Scholar
  19. 19.
    Strzalka W, Ziemienowicz A. Proliferating cell nuclear antigen (PCNA): a key factor in DNA replication and cell cycle regulation. Ann Bot. 2011;107(7):1127–40. doi: 10.1093/aob/mcq243.PubMedCentralCrossRefPubMedGoogle Scholar
  20. 20.
    Kang S, Song J, Kang H, Kim S, Lee Y, Park D. Insulin can block apoptosis by decreasing oxidative stress via phosphatidylinositol 3-kinase- and extracellular signal-regulated protein kinase-dependent signaling pathways in HepG2 cells. Eur J Endocrinol. 2003;148(1):147–55.CrossRefPubMedGoogle Scholar
  21. 21.
    Wang YD, Yang F, Chen WD, Huang X, Lai L, Forman BM, et al. Farnesoid X receptor protects liver cells from apoptosis induced by serum deprivation in vitro and fasting in vivo. Mol Endocrinol. 2008;22(7):1622–32. doi: 10.1210/me.2007-0527.CrossRefPubMedGoogle Scholar
  22. 22.
    Harper JW, Adami GR, Wei N, Keyomarsi K, Elledge SJ. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell. 1993;75(4):805–16.CrossRefPubMedGoogle Scholar
  23. 23.
    Ghosh-Choudhury N, Ghosh-Choudhury G, Celeste A, Ghosh PM, Moyer M, Abboud SL, et al. Bone morphogenetic protein-2 induces cyclin kinase inhibitor p21 and hypophosphorylation of retinoblastoma protein in estradiol-treated MCF-7 human breast cancer cells. Biochim Biophys Acta. 2000;1497(2):186–96.CrossRefPubMedGoogle Scholar
  24. 24.
    Ghosh-Choudhury N, Woodruff K, Qi W, Celeste A, Abboud SL, Ghosh Choudhury G. Bone morphogenetic protein-2 blocks MDA MB 231 human breast cancer cell proliferation by inhibiting cyclin-dependent kinase-mediated retinoblastoma protein phosphorylation. Biochem Biophys Res Commun. 2000;272(3):705–11. doi: 10.1006/bbrc.2000.2844.CrossRefPubMedGoogle Scholar
  25. 25.
    Yang LJ, Jin Y. Immunohistochemical observations on bone morphogenetic protein in normal and abnormal conditions. Clin Orthop Relat Res. 1990;257:249–56.PubMedGoogle Scholar
  26. 26.
    Laitinen M, Jortikka L, Halttunen T, Nevalainen J, Aho AJ, Marttinen A, et al. Measurement of total and local bone morphogenetic protein concentration in bone tumours. Int Orthop. 1997;21(3):188–93.PubMedCentralCrossRefPubMedGoogle Scholar
  27. 27.
    Braig S, Bosserhoff AK. Death inducer-obliterator 1 (Dido1) is a BMP target gene and promotes BMP-induced melanoma progression. Oncogene. 2013;32(7):837–48. doi: 10.1038/onc.2012.115.CrossRefPubMedGoogle Scholar
  28. 28.
    Maegdefrau U, Amann T, Winklmeier A, Braig S, Schubert T, Weiss TS, et al. Bone morphogenetic protein 4 is induced in hepatocellular carcinoma by hypoxia and promotes tumour progression. J Pathol. 2009;218(4):520–9. doi: 10.1002/path.2563.CrossRefPubMedGoogle Scholar
  29. 29.
    Li W, Cai HX, Ge XM, Li K, Xu WD, Shi WH. Prognostic significance of BMP7 as an oncogene in hepatocellular carcinoma. Tumour Biol. 2013;34(2):669–74. doi: 10.1007/s13277-012-0594-x.CrossRefPubMedGoogle Scholar
  30. 30.
    Herrera B, Garcia-Alvaro M, Cruz S, Walsh P, Fernandez M, Roncero C, et al. MP9 is a proliferative and survival factor for human hepatocellular carcinoma cells. PLoS One. 2013;8(7):e69535. doi: 10.1371/journal.pone.0069535.PubMedCentralCrossRefPubMedGoogle Scholar
  31. 31.
    Eliseev RA, Dong YF, Sampson E, Zuscik MJ, Schwarz EM, O'Keefe RJ, et al. Runx2-mediated activation of the Bax gene increases osteosarcoma cell sensitivity to apoptosis. Oncogene. 2008;27(25):3605–14. doi: 10.1038/sj.onc.1211020.CrossRefPubMedGoogle Scholar
  32. 32.
    Rici RE, Alcantara D, Fratini P, Wenceslau CV, Ambrosio CE, Miglino MA, et al. Mesenchymal stem cells with rhBMP-2 inhibits the growth of canine osteosarcoma cells. BMC Vet Res. 2012;8:17. doi: 10.1186/1746-6148-8-17.CrossRefPubMedGoogle Scholar
  33. 33.
    Zhou JD, Shen F, Ji JS, Zheng K, Huang M, Wu JC. FAM9C plays an anti-apoptotic role through activation of the PI3K/Akt pathway in human hepatocellular carcinoma. Oncol Rep. 2013;30(3):1275–84. doi: 10.3892/or.2013.2592.PubMedGoogle Scholar
  34. 34.
    Fan L, Song B, Sun G, Ma T, Zhong F, Wei W. Endoplasmic reticulum stress-induced resistance to doxorubicin is reversed by paeonol treatment in human hepatocellular carcinoma cells. PLoS One. 2013;8(5):e62627. doi: 10.1371/journal.pone.0062627.PubMedCentralCrossRefPubMedGoogle Scholar
  35. 35.
    Fu Z, Ren L, Wei H, Lv J, Che X, Zhu Z, et al. Effects of tyroserleutide on phosphatidylinositol 3'-kinase/AKT pathway in human hepatocellular carcinoma cell. J Drug Target. 2013. doi: 10.3109/1061186X.2013.844820.PubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2014

Authors and Affiliations

  • Ying Zheng
    • 1
  • Xuemei Wang
    • 2
  • Haidong Wang
    • 3
  • Wei Yan
    • 4
  • Quan Zhang
    • 5
  • Xin Chang
    • 6
  1. 1.Department of AnesthesiaHuai’an First People’s Hospital, Nanjing Medical UniversityHuai’anPeople’s Republic of China
  2. 2.Department of UltrasoundObstetrics and Gynecology Hospital of Fudan UniversityShanghaiPeople’s Republic of China
  3. 3.Department of Obstetrics and GynecologyHuai’an First People’s Hospital, Nanjing Medical UniversityHuai’anPeople’s Republic of China
  4. 4.Department of GastroenterologyHuai’an First People’s Hospital, Nanjing Medical UniversityHuai’anPeople’s Republic of China
  5. 5.Department of OncologyHuai’an First People’s Hospital, Nanjing Medical UniversityHuai’anPeople’s Republic of China
  6. 6.Department of Imaging Medicine CenterHuai’an First People’s Hospital, Nanjing Medical UniversityHuai’anPeople’s Republic of China

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