Tumor Biology

, Volume 37, Issue 8, pp 11397–11407 | Cite as

Silibinin inhibits triple negative breast cancer cell motility by suppressing TGF-β2 expression

  • Sangmin Kim
  • Jeonghun Han
  • Myeongjin Jeon
  • Daeun You
  • Jeongmin Lee
  • Hee Jung Kim
  • Sarang Bae
  • Seok Jin Nam
  • Jeong Eon Lee
Original Article

Abstract

Transforming growth factor-beta (TGF-β) is a multifunctional cytokine that regulates many biological events including cell motility and angiogenesis. Here, we investigated the role of elevated TGF-β2 level in triple negative breast cancer (TNBC) cells and the inhibitory effect of silibinin on TGF-β2 action in TNBC cells. Breast cancer patients with high TGF-β2 expression have a poor prognosis. The levels of TGF-β2 expression increased significantly in TNBC cells compared with those in non-TNBC cells. In addition, cell motility-related genes such as fibronectin (FN) and matrix metalloproteinase-2 (MMP-2) expression also increased in TNBC cells. Basal FN, MMP-2, and MMP-9 expression levels decreased in response to LY2109761, a dual TGF-β receptor I/II inhibitor, in TNBC cells. TNBC cell migration also decreased in response to LY2109761. Furthermore, we observed that TGF-β2 augmented the FN, MMP-2, and MMP-9 expression levels in a time- and dose-dependent manner. In contrast, TGF-β2-induced FN, MMP-2, and MMP-9 expression levels decreased significantly in response to LY2109761. Interestingly, we found that silibinin decreased TGF-β2 mRNA expression level but not that of TGF-β1 in TNBC cells. Cell migration as well as basal FN and MMP-2 expression levels decreased in response to silibinin. Furthermore, silibinin significantly decreased TGF-β2-induced FN, MMP-2, and MMP-9 expression levels and suppressed the lung metastasis of TNBC cells. Taken together, these results suggest that silibinin suppresses metastatic potential of TNBC cells by inhibiting TGF-β2 expression in TNBC cells. Thus, silibinin may be a promising therapeutic drug to treat TNBC.

Keywords

Silibinin TGF-β2 Fibronectin MMP-2 MMP-9 Triple negative breast cancer 

Notes

Compliance with ethical standards

Human and animal rights and informed consent

The mice were kept in pathogen-free animal housing in accordance with the Institute for Laboratory Animal Research Guide for the Care and Use of Laboratory Animals and were used according to protocols approved by the appropriate Institutional Review Board of the Samsung Medical Center (Seoul, Korea).

Conflicts of interest

None

Financial support

This research was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health and Welfare, Republic of Korea (HI14C3418), and by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2015R1D1A1A01057585).

Supplementary material

13277_2016_5000_MOESM1_ESM.tif (1.5 mb)
Supplement 1 TGF-β2, FN, MMP-2, and MMP-9 expression levels and cell migration are suppressed by silibinin in TNBC cells. (A) After serum starvation for 24 h, TNBC cells were treated with 50 μM Sil for 24 h. TGF-β2 protein expression level was analyzed by confocal microscopy. (B) After serum starvation for 24 h, MDA-MB231 TNBC cells were treated with or without 25 and 50 μM Sil for 24 h. FN protein expression levels were analyzed by western blotting. MMP-2 and MMP-9 protein expression levels were analyzed by zymography. (C) The migrating ability of TNBC cells was analyzed using the wound healing assay. These results are representative of three independent experiments. Con, control; Sil, silibinin. (TIF 1565 kb)

References

  1. 1.
    Dent R, Trudeau M, Pritchard KI, Hanna WM, Kahn HK, Sawka CA, et al. Triple-negative breast cancer: clinical features and patterns of recurrence. Clin Cancer Res. 2007;13:4429–34. doi: 10.1158/1078-0432.CCR-06-3045.CrossRefPubMedGoogle Scholar
  2. 2.
    Lin NU, Claus E, Sohl J, Razzak AR, Arnaout A, Winer EP. Sites of distant recurrence and clinical outcomes in patients with metastatic triple-negative breast cancer: high incidence of central nervous system metastases. Cancer. 2008;113:2638–45. doi: 10.1002/cncr.23930.CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Khokher S, Qureshi MU, Mahmood S, Nagi AH. Association of immunohistochemically defined molecular subtypes with clinical response to presurgical chemotherapy in patients with advanced breast cancer. Asian Pac J Cancer Prev. 2013;14:3223–8.CrossRefPubMedGoogle Scholar
  4. 4.
    Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial-mesenchymal transitions in development and disease. Cell. 2009;139:871–90. doi: 10.1016/j.cell.2009.11.007.CrossRefPubMedGoogle Scholar
  5. 5.
    Feng XH, Derynck R. Specificity and versatility in tgf-beta signaling through Smads. Annu Rev Cell Dev Biol. 2005;21:659–93. doi: 10.1146/annurev.cellbio.21.022404.142018.CrossRefPubMedGoogle Scholar
  6. 6.
    Ikushima H, Miyazono K. TGFbeta signalling: a complex web in cancer progression. Nat Rev Cancer. 2010;10:415–24. doi: 10.1038/nrc2853.CrossRefPubMedGoogle Scholar
  7. 7.
    Xu J, Lamouille S, Derynck R. TGF-beta-induced epithelial to mesenchymal transition. Cell Res. 2009;19:156–72. doi: 10.1038/cr.2009.5.CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Giampieri S, Manning C, Hooper S, Jones L, Hill CS, Sahai E. Localized and reversible TGFbeta signalling switches breast cancer cells from cohesive to single cell motility. Nat Cell Biol. 2009;11:1287–96. doi: 10.1038/ncb1973.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Kim S, Lee J, Jeon M, Nam SJ, Lee JE. Elevated TGF-beta1 and -beta2 expression accelerates the epithelial to mesenchymal transition in triple-negative breast cancer cells. Cytokine. 2015;75:151–8. doi: 10.1016/j.cyto.2015.05.020.CrossRefPubMedGoogle Scholar
  10. 10.
    Padua D, Zhang XH, Wang Q, Nadal C, Gerald WL, Gomis RR, et al. TGFbeta primes breast tumors for lung metastasis seeding through angiopoietin-like 4. Cell. 2008;133:66–77. doi: 10.1016/j.cell.2008.01.046.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Ehata S, Hanyu A, Fujime M, Katsuno Y, Fukunaga E, Goto K, et al. Ki26894, a novel transforming growth factor-beta type i receptor kinase inhibitor, inhibits in vitro invasion and in vivo bone metastasis of a human breast cancer cell line. Cancer Sci. 2007;98:127–33. doi: 10.1111/j.1349-7006.2006.00357.x.CrossRefPubMedGoogle Scholar
  12. 12.
    Kim S, Lee Y, Seo JE, Cho KH, Chung JH. Caveolin-1 increases basal and TGF-beta1-induced expression of type I procollagen through PI-3 kinase/Akt/mTOR pathway in human dermal fibroblasts. Cell Signal. 2008;20:1313–9. doi: 10.1016/j.cellsig.2008.02.020.CrossRefPubMedGoogle Scholar
  13. 13.
    Lv ZD, Na D, Liu FN, Du ZM, Sun Z, Li Z, et al. Induction of gastric cancer cell adhesion through transforming growth factor-beta1-mediated peritoneal fibrosis. J Exp Clin Cancer Res. 2010;29:139. doi: 10.1186/1756-9966-29-139.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Pankov R, Yamada KM. Fibronectin at a glance. J Cell Sci. 2002;115:3861–3.CrossRefPubMedGoogle Scholar
  15. 15.
    Ritzenthaler JD, Han S, Roman J. Stimulation of lung carcinoma cell growth by fibronectin-integrin signalling. Mol Biosyst. 2008;4:1160–9. doi: 10.1039/b800533h.CrossRefPubMedGoogle Scholar
  16. 16.
    Fernandez-Garcia B, Eiro N, Marin L, Gonzalez-Reyes S, Gonzalez LO, Lamelas ML, et al. Expression and prognostic significance of fibronectin and matrix metalloproteases in breast cancer metastasis. Histopathology. 2014;64:512–22. doi: 10.1111/his.12300.CrossRefPubMedGoogle Scholar
  17. 17.
    Bae YK, Kim A, Kim MK, Choi JE, Kang SH, Lee SJ. Fibronectin expression in carcinoma cells correlates with tumor aggressiveness and poor clinical outcome in patients with invasive breast cancer. Hum Pathol. 2013;44:2028–37. doi: 10.1016/j.humpath.2013.03.006.CrossRefPubMedGoogle Scholar
  18. 18.
    Rintoul RC, Sethi T. Extracellular matrix regulation of drug resistance in small-cell lung cancer. Clin Sci (Lond). 2002;102:417–24.CrossRefGoogle Scholar
  19. 19.
    Hayashida T, Poncelet AC, Hubchak SC, Schnaper HW. TGF-beta1 activates MAP kinase in human mesangial cells: a possible role in collagen expression. Kidney Int. 1999;56:1710–20. doi: 10.1046/j.1523-1755.1999.00733.x.CrossRefPubMedGoogle Scholar
  20. 20.
    Owens LV, Xu L, Craven RJ, Dent GA, Weiner TM, Kornberg L, et al. Overexpression of the focal adhesion kinase (p125FAK) in invasive human tumors. Cancer Res. 1995;55:2752–5.PubMedGoogle Scholar
  21. 21.
    Jeon M, Lee J, Nam SJ, Shin I, Lee JE, Kim S. Induction of fibronectin by HER2 overexpression triggers adhesion and invasion of breast cancer cells. Exp Cell Res. 2015;333:116–26. doi: 10.1016/j.yexcr.2015.02.019.CrossRefPubMedGoogle Scholar
  22. 22.
    Sharma G, Singh RP, Chan DC, Agarwal R. Silibinin induces growth inhibition and apoptotic cell death in human lung carcinoma cells. Anticancer Res. 2003;23:2649–55.PubMedGoogle Scholar
  23. 23.
    Kim S, Choi JH, Lim HI, Lee SK, Kim WW, Kim JS, et al. Silibinin prevents TPA-induced MMP-9 expression and VEGF secretion by inactivation of the Raf/MEK/ERK pathway in MCF-7 human breast cancer cells. Phytomedicine. 2009;16:573–80. doi: 10.1016/j.phymed.2008.11.006.CrossRefPubMedGoogle Scholar
  24. 24.
    Kim S, Kim SH, Hur SM, Lee SK, Kim WW, Kim JS, et al. Silibinin prevents TPA-induced MMP-9 expression by down-regulation of COX-2 in human breast cancer cells. J Ethnopharmacol. 2009;126:252–7. doi: 10.1016/j.jep.2009.08.032.CrossRefPubMedGoogle Scholar
  25. 25.
    Kim S, Han J, Kim JS, Kim JH, Choe JH, Yang JH, et al. Silibinin suppresses EGFR ligand-induced CD44 expression through inhibition of EGFR activity in breast cancer cells. Anticancer Res. 2011;31:3767–73.PubMedGoogle Scholar
  26. 26.
    Kim S, Lee HS, Lee SK, Kim SH, Hur SM, Kim JS, et al. 12-O-Tetradecanoyl phorbol-13-acetate (TPA)-induced growth arrest is increased by silibinin by the down-regulation of cyclin B1 and cdc2 and the up-regulation of p21 expression in MDA-MB231 human breast cancer cells. Phytomedicine. 2010;17:1127–32. doi: 10.1016/j.phymed.2010.03.013.CrossRefPubMedGoogle Scholar
  27. 27.
    Deep G, Singh RP, Agarwal C, Kroll DJ, Agarwal R. Silymarin and silibinin cause G1 and G2-M cell cycle arrest via distinct circuitries in human prostate cancer PC3 cells: a comparison of flavanone silibinin with flavanolignan mixture silymarin. Oncogene. 2006;25:1053–69. doi: 10.1038/sj.onc.1209146.CrossRefPubMedGoogle Scholar
  28. 28.
    Kim S, Choi JH, Lim HI, Lee SK, Kim WW, Cho S, et al. EGF-induced MMP-9 expression is mediated by the JAK3/ERK pathway, but not by the JAK3/STAT-3 pathway in a SKBR3 breast cancer cell line. Cell Signal. 2009;21:892–8.CrossRefPubMedGoogle Scholar
  29. 29.
    Cho JW, Il KJ, Lee KS. Downregulation of type i collagen expression in silibinin-treated human skin fibroblasts by blocking the activation of Smad2/3-dependent signaling pathways: potential therapeutic use in the chemoprevention of keloids. Int J Mol Med. 2013;31:1148–52. doi: 10.3892/ijmm.2013.1303.PubMedGoogle Scholar
  30. 30.
    Rhee J, Han SW, Oh DY, Kim JH, Im SA, Han W, et al. The clinicopathologic characteristics and prognostic significance of triple-negativity in node-negative breast cancer. BMC Cancer. 2008;8:307. doi: 10.1186/1471-2407-8-307.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Oft M, Heider KH, Beug H. TGFbeta signaling is necessary for carcinoma cell invasiveness and metastasis. Curr Biol. 1998;8:1243–52.CrossRefPubMedGoogle Scholar
  32. 32.
    Thomas DA, Massague J. TGF-beta directly targets cytotoxic t cell functions during tumor evasion of immune surveillance. Cancer Cell. 2005;8:369–80. doi: 10.1016/j.ccr.2005.10.012.CrossRefPubMedGoogle Scholar
  33. 33.
    Kopp A, Jonat W, Schmahl M, Knabbe C. Transforming growth factor beta 2 (TGF-beta 2) levels in plasma of patients with metastatic breast cancer treated with tamoxifen. Cancer Res. 1995;55:4512–5.PubMedGoogle Scholar
  34. 34.
    Eldred JA, Hodgkinson LM, Dawes LJ, Reddan JR, Edwards DR, Wormstone IM. MMP2 activity is critical for TGFbeta2-induced matrix contraction—implications for fibrosis. Invest Ophthalmol Vis Sci. 2012;53:4085–98. doi: 10.1167/iovs.12-9457.CrossRefPubMedGoogle Scholar
  35. 35.
    Cheng S, Pollock AS, Mahimkar R, Olson JL, Lovett DH. Matrix metalloproteinase 2 and basement membrane integrity: a unifying mechanism for progressive renal injury. FASEB J. 2006;20:1898–900. doi: 10.1096/fj.06-5898fje.CrossRefPubMedGoogle Scholar
  36. 36.
    Kaur M, Velmurugan B, Tyagi A, Deep G, Katiyar S, Agarwal C, et al. Silibinin suppresses growth and induces apoptotic death of human colorectal carcinoma LoVo cells in culture and tumor xenograft. Mol Cancer Ther. 2009;8:2366–74. doi: 10.1158/1535-7163.MCT-09-0304.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Kim S, Jeon M, Lee J, Han J, Oh SJ, Jung T, et al. Induction of fibronectin in response to epidermal growth factor is suppressed by silibinin through the inhibition of STAT3 in triple negative breast cancer cells. Oncol Rep. 2014;32:2230–6. doi: 10.3892/or.2014.3450.PubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2016

Authors and Affiliations

  • Sangmin Kim
    • 1
  • Jeonghun Han
    • 1
  • Myeongjin Jeon
    • 1
    • 2
  • Daeun You
    • 1
    • 2
  • Jeongmin Lee
    • 1
  • Hee Jung Kim
    • 1
  • Sarang Bae
    • 1
  • Seok Jin Nam
    • 1
  • Jeong Eon Lee
    • 1
    • 2
  1. 1.Department of Sugery, Samsung Medical CenterSungkyunkwan University School of MedicineSeoulSouth Korea
  2. 2.Department of Health Sciences and TechnologySamsung Advanced Institute for Health Sciences and TechnologySeoulSouth Korea

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