Vanillin Inhibits Matrix Metalloproteinase-9 Expression through Down-Regulation of Nuclear Factor- B Signaling Pathway in Human Hepatocellular Carcinoma Cells

Vanillin has been reported to exhibit anti-invasive and antimetastatic activities by suppressing the enzymatic activity of matrix metalloproteinase-9 (MMP-9). However, the underlying mechanism of anti-invasive activity remains unclear so far. Herein we demonstrate that vanillin reduced 12-O-tetradecanoylphorbol13-acetate (TPA)-induced MMP-9 gelatinolytic activity and suppressed cell invasion through the down-regulation of MMP-9 gene transcription in HepG2 cells. Vanillin significantly reduced the 6.6fold invasive capacity of HepG2 cells in noncytotoxic concentrations, and this anti-invasive effect was concentration-dependent in the Matrigel invasion assay. Moreover, vanillin significantly suppressed the TPA-induced enzymatic activity of MMP-9 and decreased the induced mRNA level of MMP-9. Analysis of the transcriptional regulation indicated that vanillin suppressed MMP-9 transcription by inhibiting nuclear factorB (NFB) activity. Western blot further confirmed that vanillin inhibited NFB activity through the inhibition of I Bphosphorylation and degradation. In conclusion, vanillin might be a potent antiinvasive agent that suppresses the MMP-9 enzymatic activity via NFB signaling pathway. Tumor invasion and metastasis require increased expressions of matrix metalloproteinases (MMPs) (Stamenkovic, 2000). MMP family is involved in the degradation of extracellular matrix, and members of MMPs have been implicated in malignancy and metastasis (Westermarck and Kahari, 1999; Stamenkovic, 2000). Among the members of MMPs, elevated serum levels of MMP-9 were reported in patients with hepatocellular carcinoma (HCC) (Hayasaka et al., 1996). MMP-9 gene (also termed gelatinase B or 92-kDa type IV collagenase) was found to highly express in HCC with invasive potential (Arii et al., 1996). Therefore, MMP-9 is suggested to serve as a new serum marker of HCC in patients with chronic liver disease (Paradis et al., 2005). MMP-9 degrades basement membrane type IV collagen and expresses during cellular invasion and metastasis (Nelson et al., 2000). The activity of MMP-9 is tightly controlled, mainly at the transcription level (Stamenkovic, 2000). The promoter of MMP-9 is highly conserved and is shown to contain multiple functional elements, including nuclear factorB (NFB) and activator protein 1 (AP-1) elements (Sato and Seiki, 1993). NFB has been shown to regulate the expression of a number of genes, such as vascular endothelial growth factor and MMP-9, whose products are involved in tumorigenesis (Garg and Aggarwal, 2002). Several evidences indicate that the suppression of MMP-9 prevents invasion and metastasis (Cha et al., 1996). MMP-9 has been considered to be an important factor in facilitating invasion and metastasis in pancreatic cancer (Nagakawa et al., 2002). Moreover, Murono et al. (2000) demonstrated that the invasiveness induced by Epstein-Barr virus latent membrane protein 1 is correlated with the induction of MMP-9. Thus, agents that inhibit the activation of NFB or MMP-9 may exhibit the therapeutic potential for the suppression of carcinogenesis and tumor metastasis (Banerjee et al., 2002; Garg and Aggarwal, 2002). This work was supported by grants from National Research Program for Genomic Medicine, National Science and Technology Program for Agricultural Biotechnology, National Science Council, Committee on Chinese Medicine and Pharmacy, Department of Health (CCMP 96-RD-201 and CCMP 97-RD-201), and China Medical University, Taiwan. C.-Y.H. and T.-Y.H. contributed equally to this work. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.108.049502. □S The online version of this article (available at http://molpharm. aspetjournals.org) contains supplemental material. ABBREVIATIONS: MMP, matrix metalloproteinase; AP-1, activator protein 1; DMEM, Dulbecco’s modified Eagle’s medium; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HCC, hepatocellular carcinoma; MMP-9, matrix metalloproteinase-9; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide; NFB, nuclear factorB; RLU, relative luciferase unit; PCR, polymerase chain reaction; RT-PCR, reverse transcription-polymerase chain reaction; TPA, 12-O-tetradecanoylphorbol-13-acetate. 0026-895X/09/7501-151–157$20.00 MOLECULAR PHARMACOLOGY Vol. 75, No. 1 Copyright © 2009 The American Society for Pharmacology and Experimental Therapeutics 49502/3416676 Mol Pharmacol 75:151–157, 2009 Printed in U.S.A. 151 http://molpharm.aspetjournals.org/content/suppl/2008/10/03/mol.108.049502.DC1 Supplemental material to this article can be found at: at A PE T Jornals on O cber 3, 2017 m oharm .aspeurnals.org D ow nladed from Vanillin (4-hydroxy-3-methoxybenzaldehyde) is the major component of natural vanilla, which is one of the most widely used flavor component in food and personal products, with an estimated annual worldwide consumption of more than 2000 tons (Rao and Ravishankar, 2000). Besides its flavor qualities, vanillin exhibits the antimicrobial potential and has been used as a natural food preservative (Cerrutti et al., 1997). Moreover, vanillin displays the antioxidant activity through the protection of membranes against photosensitization-induced oxidative damage in rat liver mitochondria (Aruoma, 1999; Kamat et al., 2000). Vanillin inhibits chemical and physical mutagens-induced mutagenesis in both bacteria and mammalian cells (Keshava et al., 1998; Shaughnessy et al., 2006). It also displays chemopreventive effects in multiorgan carcinogenesis and hepatocarcinogenesis models in rats (Akagi et al., 1995). Vanillin has been known to inhibit cell invasion and migration, suppress enzymatic activity of MMP-9, and reduce the numbers of lung-metastasized colonies in mice (Lirdprapamongkol et al., 2005). However, how the MMP-9 activity is regulated by vanillin is still unclear. Herein we demonstrate that the suppression of MMP-9 activity by vanillin is via the NFB signaling pathway in HepG2 cells. Materials and Methods Cell Culture and Transfection. HepG2 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT). Vanillin, purchased from Sigma (St. Louis, MO), was dissolved in dimethyl sulfoxide and stocked at 30°C. 12-O-Tetradecanoylphorbol-13-acetate (TPA), purchased from Sigma, was dissolved in ethanol at 0.5 mg/ml. HepG2 cells were transiently transfected with 5 g of plasmid DNAs by SuperFect transfection reagent (QIAGEN, Valencia, CA) and then kept in a humidified incubator at 37°C with 5% CO2. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide Assay. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma and dissolved in phosphate-buffered saline (137 mM NaCl, 1.4 mM KH2PO4, 4.3 mM Na2HPO4, and 2.7 mM KCl, pH 7.2). Cell viability was monitored by MTT colorimetric assay as described previously (Hsiang et al., 2005). In brief, various amounts of vanillin were added to subconfluent cell monolayers for 24 h, and one-tenth volume of 5 mg/ml MTT was added to the culture medium. After a 4-h incubation at 37°C, equal volume of 0.04 N HCl in isopropanol was added to dissolve the MTT formazan, and the absorbance value was measured at 570 nm using a microplate reader. Cell viability (measured as a percentage) was calculated by (optical density of vanillin-treated cells/optical density of solvent-treated cells). Cell Invasion Assay. Cell invasion was measured using the Matrigel-coated film insert (8 m pore size) fitting into 24-well invasion chambers (BD Biosciences, San Jose, CA) as described previously (Hsiang et al., 2007). In brief, HepG2 cells (5 10), which were resuspended in 200 l of DMEM, were added to the top compartment of the invasion chamber and treated with various amounts of vanillin in DMEM. Thirty minutes later, 100 ng/ml TPA was added to the medium, and the Matrigel invasion chambers were incubated at 37°C in 5% CO2. After a 24-h incubation, the filter inserts were removed from the wells, and the cells on the top side of the filter were removed using cotton swabs. The cells in the bottom surface of the filter were stained with crystal violet, and the cell number was counted with a microscope. Invasive -fold was calculated