Elsevier

Phytomedicine

Volume 16, Issues 6–7, June 2009, Pages 573-580
Phytomedicine

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

https://doi.org/10.1016/j.phymed.2008.11.006Get rights and content

Abstract

Matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) expression are pivotal steps in cancer metastasis. Herein, we investigated the effect of silibinin, a major constituent (flavanolignan) of the fruits of Silybum marianum, on 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced MMP-9 and VEGF expression in MCF-7 human breast cancer cells. The expression of MMP-9 and VEGF in response to TPA was increased, whereas TPA-induced MMP-9 and VEGF expression was decreased by silibinin. To investigate the regulatory mechanism of silibinin on TPA-induced MMP-9 and VEGF expression, we pretreated cells with various inhibitors, such as UO126 (MEK1/2 inhibitor), SP600125 (JNK inhibitor), and SB203580 (p38 inhibitor). Interestingly, TPA-induced MMP-9 expression was significantly inhibited by UO126, but not by SP600125 and SB203580. In addition, we pretreated cells with 100 μM silibinin prior to TPA treatment. TPA-induced MEK and ERK phosphorylation was significantly decreased by silibinin in MCF7 cells. TPA-induced VEGF expression was also suppressed by UO126. On the other hand, we found that adenoviral constitutive active-MEK (Ad-CA-MEK) significantly increased MMP-9 and VEGF expression. Taken together, we suggest that the inhibition of TPA-induced MMP-9 and VEGF expression by silibinin is mediated by the suppression of the Raf/MEK/ERK pathway in MCF-7 breast cancer cells.

Introduction

Silibinin is a major bioactive flavanolignan that has been isolated from milk thistle seeds, and has been used as a traditional medicine (Singh and Agarwal 2002). Silibinin was reported to be a chemopreventive agent against skin cancer in a murine model (Singh and Agarwal 2002), and known to trigger cell cycle arrest and apoptosis in lung cell carcinoma cells, such as SHP-77 and A-549 cells (Sharma et al. 2003). However, the role of silibinin has not been fully elucidated for breast cancer. Herein, we investigated the effect of silibinin on TPA-induced MMP-9 and VEGF expression in the MCF-7 breast cancer cell line.

The matrix metalloproteinases (MMPs) are a major group of enzymes that regulate cell-matrix composition (Deryugina and Quigley 2006). MMPs are zinc-binding endopeptidases that can degrade virtually all extracellular matrix (ECM) components (Overall and Dean 2006). Abnormally severe expression of MMPs contributes to various pathologic processes, including rheumatoid arthritis, osteoarthritis, angiogenesis, invasion, and metastasis in carcinoma (Chambers and Matrisian 1997; Egeblad and Werb 2002; Murphy and Docherty 1992). MMP-9 belongs to the gelatinase subgroup of the MMP family and the expression and activity of MMP-9 have been associated with different stages of carcinoma progression through breakdown of the basement membrane (Stetler-Stevenson et al. 1993; Zeng and Guillem 1996). Zucker et al. (1993) reported that MMP-9 is increased in the plasma of patients with colon and breast cancers and these inductions do not only regulate tumor invasiveness, but also tumor growth and angiogenesis (Sang 1998).

VEGF is a one of the most important pro-angiogenic growth factors (Leung et al. 1989) and suggested to be a potential therapeutic target in solid tumors, including breast cancer (Jensen et al. 1982). VEGF is highly expressed by malignant and non-malignant cells in response to extracellular stimuli, such as inflammation, hypoxia, and cytokines (Bouck et al. 1996; Ferrara 1999). VEGF-overexpressed breast cancer cells have both greater cell growth and greater metastatic potential (McLeskey et al. 1998), and the role of VEGF has been investigated both in vitro and in vivo with breast cancer models.

The aim of the current study was to evaluate the effect of silibinin on TPA-induced MMP-9 and VEGF expression. The results suggest that silibinin inhibits TPA-induced ERK phosphorylation and then prevents TPA-induced MMP-9 and VEGF expression in MCF-7 human breast cancer cells. Silibinin may be an effective ingredient for anti-cancer therapy by preventing cancer metastasis through the down-regulation of MMP-9 and VEGF in breast cancer.

Section snippets

Reagents and cell cultures

Cell culture media, antibiotics, and 10% Zymogram gel were purchased from Life Technologies (Rockville, MD, USA). Rabbit polyclonal anti-phospho-ERK and anti-JNK antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). MMP-9 antibody was obtained from Lab Vision (Neomarker; Fremont, CA, USA). TPA was purchased from R&D Systems (Minneapolis, MN, USA). Silibinin [2,3-Dihydro-3-(4-hydroxy-3-methoxyphenyl)-2-(hydroxymethyl)-6-(3,5,7-trihydroxy-4-oxobenzopyran-2-yl)benzodioxin] (

The basal levels of expression of MMP-9 and VEGF are increased by TPA treatment of MCF-7 cells

The chemical structure of silibinin is shown in Fig. 1A. To verify the effect of silibinin on cell viability, we treated cells with silibinin at the concentrations indicated for 24 h. MCF-7 cell viabilities were not affected by silibinin at various concentrations (Fig. 1B). Thus, in additional experiments, we demonstrated that the reduction in TPA-induced MMP-9 and VEGF expression by silibinin was irrelevant to cell viability.

To examine the time response of TPA on MMP-9 expression, we treated

Discussion

Silibinin, one of the major flavanones, has a wide range of pharmacologic effects, such as inhibition of DNA synthesis, cell proliferation, cell cycle progression, and apoptosis in various cancer cell lines, including breast cancer (Bhatia et al. 1999; Sharma et al. 2003). However, the mechanism underlying the inhibitory action of silibinin on tumor metastasis and angiogenesis in breast cancer has not been completely elucidated. Therefore, we investigated whether silibinin prevents TPA-induced

Acknowledgements

This work was supported in part by Grant C-A5-822-1 from In-SUNG Foundation for Medical Research and by the Samsung Biomedical Research Institute Grant #SBRI C-A9-313-1.

References (28)

  • A.F. Chambers et al.

    Changing views of the role of matrix metalloproteinases in metastasis

    J. Natl. Cancer Inst.

    (1997)
  • E.I. Deryugina et al.

    Matrix metalloproteinases and tumor metastasis

    Cancer Metastasis Rev.

    (2006)
  • M. Egeblad et al.

    New functions for the matrix metalloproteinases in cancer progression

    Nat. Rev. Cancer

    (2002)
  • N. Ferrara

    Molecular and biological properties of vascular endothelial growth factor

    J. Mol. Med.

    (1999)
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