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
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)
- et al.
Inhibition of human carcinoma cell growth and DNA synthesis by silibinin, an active constituent of milk thistle: comparison with silymarin
Cancer Lett.
(1999) - et al.
How tumors become angiogenic
Adv. Cancer Res.
(1996) - et al.
Cholesterol inhibits MMP-9 expression in human epidermal keratinocytes and HaCaT cells
FEBS Lett.
(2007) - et al.
Berberine inhibits TPA-induced MMP-9 and IL-6 expression in normal human keratinocytes
Phytomedicine
(2008) - et al.
Tumor growth of FGF or VEGF transfected MCF-7 breast carcinoma cells correlates with density of specific microvessels independent of the transfected angiogenic factor
Am. J. Pathol.
(1998) - et al.
Inhibitory effect of berberine on the invasion of human lung cancer cells via decreased productions of urokinase-plasminogen activator and matrix metalloproteinase-2
Toxicol. Appl. Pharmacol.
(2006) - et al.
Oxidative stress regulates vascular endothelial growth factor-A gene transcription through Sp1- and Sp3-dependent activation of two proximal GC-rich promoter elements
J. Biol. Chem.
(2003) - et al.
The p38 SAPK pathway regulates the expression of the MMP-9 collagenase via AP-1-dependent promoter activation
Exp. Cell Res.
(2001) - et al.
UVA-mediated downregulation of MMP-2 and MMP-9 in human epidermal keratinocytes
Biochem. Biophys. Res. Commun.
(2003) - et al.
MAP kinases and hypoxia in the control of VEGF expression
Cancer Metastasis Rev.
(2000)