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Silibinin Inhibits Invasion of Oral Cancer Cells by Suppressing the MAPK Pathway

¹ Institute of Biochemistry,
³ School of Medical Technology, Chung Shan Medical University, No. 110, Section 1, Chien Kuo N. Road, Taichung 402, Taiwan; and
² Department of Food Science, Central Taiwan University of Science and Technology, No. 11 Pu-tzu Lane, Pu-tzu Road, Taichung 406, Taiwan

^* corresponding author, scchu{at}ctust.edu.tw

	ABSTRACT

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Oral squamous cell carcinoma (OSCC) is the most common malignancy of the oral cavity. Here, we provide molecular evidence associated with the anti-metastatic effect of silibinin by showing a marked inhibition of the invasion and motility of SCC-4 tongue cancer cells, with 89% and 66.4% of inhibition, respectively, by 100 µM of silibinin. This effect was associated with a reduced expression of MMP-2 and u-PA, together with an enhanced expression of TIMP-2 and PAI-1. Silibinin also exerted an inhibitory effect on the phosphorylation of ERK1/2. Additionally, pre-treatment of SCC-4 cancer cells with 10 and 20 µM of U0126, a specific MEK inhibitor, resulted in a reduced expression of MMP-2 (18.7 and 51.4%) and u-PA (19.2 and 48.9%) concomitantly with a marked inhibition of cell invasion (13.7 and 45.7%). Finally, silibinin was evidenced by its inhibition of the metastasis of Lewis lung carcinoma (LLC) cells in vivo. These results suggested that silibinin can reduce the invasion and metastasis of tumor cells, and such a characteristic may be of great value in the development of a potential cancer therapy.

KEY WORDS: silibinin • invasion • MMP-2 • u-PA

	INTRODUCTION

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Squamous cell carcinoma (SCC) of the tongue is a common malignancy of the oral cavity, with an annual rate of 1.51 males and 0.99 females per 100,000 persons in the United States (Canto and Devesa, 2002). Nearly 50% of OSCC patients present with pathologic or clinical evidence of nodal metastases. The five-year survival rate is less than 50% for patients with a single unilateral lymph node metastasis and less than 25% for patients with bilateral metastases (Zhang et al., 2002). Thus, metastasis has been a major challenge for a successful cancer treatment to improve patient outcome.

The metastatic process is comprised of multiple events involving cell motility, cell invasion, surface adhesion properties, and degradation of extracellular matrix (ECM) (Stetler-Stevenson et al. 1993). Several classes of proteases may be involved in the proteolytic events that occur during cell invasion, including serine proteases and matrix metalloproteinases (MMPs), such as urokinase-type plasminogen activator (u-PA), MMP-2, and membrane type 1-MMP (MT1-MMP) (Westermarck and Kahari, 1999; Rao, 2003); their expression and activity against matrix macromolecules have been linked to the development of malignant phenotypes (Vihinen et al., 2005) and the promotion of cell invasiveness and metastasis of OSCC (Shimada et al., 2000; Geletu et al., 2004).

Silibinin is a polyphenolic flavonoid isolated from the milk thistle, Silybum marianum L. Gaertn., has been used since ancient times in traditional European medicine, and is well-known for its hepatoprotective and anti-oxidant activity (von Schonfeld et al., 1997; Maheshwari et al., 2003). Silibinin could inhibit the transformation in rat tracheal epithelial cells from an exposure to the carcinogen benzo(a)pyrene (Steele et al., 1990), and reduce tumor promoter-caused induction of epidermal ornithine decarboxylase activity (Agarwal et al., 1994). In our previous study, silibinin inhibited the invasion of human lung cancer cells via decreased production of u-PA and MMP-2 (Chu et al., 2004). Moreover, a recent study showed that silibinin treatment caused significant growth inhibition through G0/G1 or G2 arrest, together with marked induction of cellular apoptosis by activation of a caspase cascade in different cancer cells (Agarwal et al., 2003; Mallikarjuna et al., 2004). Although it was quite clear that silibinin may inhibit the growth of various cancers by inducing cells toward apoptosis and anti-proliferation, the precise impact and related molecular mechanism of silibinin on cancer metastasis were still uncertain. In this study, the effects of silibinin on cell invasion were examined in vitro on SCC-4 cells, and we also investigated whether silibinin inhibited cell metastasis in vivo.

	MATERIALS & METHODS

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Cell Culture and Silibinin Treatment
SCC-4, a human tongue SCC obtained from ATCC (Manassas, VA, USA), was cultured in Dulbecco’s modified Eagle’s medium supplemented with a nutrient mixture, F-12 Ham’s medium (Life Technologies, Grand Island, NY, USA), 10% fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 400 ng/mL hydrocortisone.

Cell Invasion and Motility Assays
After a treatment with silibinin (0, 25, 50, 75, and 100 µM) for 24 hrs, cells were harvested and seeded in a Boyden chamber (Neuro Probe, Cabin John, MD, USA) with 10⁴ cells/well in serum-free medium, and then incubated for 48 hrs at 37°C. For the invasion assay, 10 µL Matrigel (25 mg/50 mL; BD Biosciences, Bedford, MA, USA) was applied to 8-µm-pore-size polycarbonate membrane filters, and the bottom chamber of the apparatus contained standard medium. Following incubation, filters were then air-dried for 5 hrs in a laminar flow hood. The invaded cells were fixed with methanol and stained with Giemsa. Cell numbers were counted under a light microscope. The motility assay was carried out as described for the invasion assay, with no coating of Matrigel (Chen et al., 2006).

Cell-Matrix Adhesion Assay
After a pre-treatment with silibinin (0, 25, 50, 75, and 100 µM) for 24 hrs, cells were plated into 24-well dishes coated with 150 µL type I collagen (10 µg/mL) overnight and cultured for 1 hr. Afterward, non-adherent cells were removed by PBS washes, and adherent cells were fixed in ethanol. After a staining with 0.1% crystal violet, fixed cells underwent lysis in 0.2% Triton X-100, and the absorbance at 550 nm was measured (Chen et al., 2006).

Determination of MMP-2 and u-PA Activities by Zymography
The activities of MMP-2 in medium were measured by gelatin-zymography protease assays as previously described (Chu et al., 2004). Briefly, collected media were prepared with SDS sample buffer without boiling or reduction, and subjected to 0.1% gelatin-8% SDS-PAGE electrophoresis. After electrophoresis, gels were washed with 2% Triton X-100 and then incubated in reaction buffer (40 mM Tris-HCl, pH 8.0, 10 mM CaCl₂, 0.02% NaN₃) for 12 hrs at 37°C. The gel was then stained with Coomassie brilliant blue R-250.

Visualization of u-PA activity was performed as previously described (Chu et al., 2004). Briefly, 2% w/v casein and 20 µg/mL plasminogen were added to 8% SDS-PAGE gels, and then electrophoresis and zymography were performed as described in the gelatin zymography.

Reverse-transcription/Polymerase Chain-reaction (RT-PCR)
For reverse transcription, a 2-µg quantity of total RNA was used as a template in a 20-µL reaction containing 4 µL dNTPs (2.5 mM), 2.5 µL Oligo dT (10 pmole/µL), and 200 U RTase. The appropriate primers [5'-GGCCCTGTCACTCCTGAGAT-3' and 5'-GGCATCCAGGTTATCGGGGA-3' for MMP-2 (473 bp), 5'-CTCCTGCTCCCCCTGCTCACG-3' and 5'-CTCACCCCCAT AAAGTTGCTG-3' for MT1-MMP (827 bp), 5'-TTGCGGCC ATCTACAGGAG-3' and 5'-ACTGGGGATCGTTATACATC-3' for u-PA (351 bp), 5'-GGATCCAGCCACTGGAAA GGCAACATG-3' and 5'-GGATCCGTGCCGGACCACAAA GAGGAA-3' for PAI-1 (254 bp), 5'-GGCGTTTTGCAATGC AGATGTAG-3' and 5'-CACAGGAGCCGTCACTTCTCTTG-3' for TIMP-2 (496 bp), and 5'-CGGAGTCAACGGATTTGG TCGTAT-3' and 5'-AGCCTTCTCCATGGTTGGTGAAGAC-3' for GAPDH (305 bp)] were used for PCR amplifications. PCR was performed with Platinum Taq polymerase (Invitrogen) under the following conditions: 25 cycles of 94°C for 1 min, 55°C (u-PA and PAI-1) or 63°C (MMP-2, MT1-MMP, TIMP-2, and GAPDH) for 1 min, 72°C for 2 min followed by 10 min at 72°C.

Immunoblotting
Samples of cell lysates or nuclear fractions were separated in a 12.5% polyacrylamide gel and transferred onto a nitrocellulose membrane as previously described (Chu et al., 2004). The blot was subsequently treated by standard procedures and probed with ERK1/2, p38, JNK1/2, and Akt, the total and phosphorylated proteins (Biosource, Camarillo, CA, USA), and NF-B, c-Jun, c-Fos, C23 (BD Transduction Laboratories, San Diego, CA, USA), TIMP-2 (Serotec, Oxford, UK), and PAI-1 (American Diagnostics Inc., Greenwich, CT, USA) antibodies. The protein expression was detected by chemiluminescence with an ECL Plus detection kit (Amersham Life Sciences, Inc., Piscataway, NJ, USA).

Measurement of Lung Metastasis and Tumor Growth in LLC-bearing Mice
C57BL/6 male mice (National Taiwan University Animal Center, Taiwan), 6 wks old, were implanted with 2 x 10⁶ LLC cells (0.1 mL/mouse) via a subcutaneous injection. On the following day (Day 1), mice were randomly divided into two groups (n = 5 for each group) to be fed by oral gavage with corn oil (control) and silibinin (200 mg/day/kg of body weight). The growth of tumors was measured daily during the study. After 30 days, animals were killed, and the primary tumors were isolated and weighed. Using a blunt-end 18-gauge needle, we perfused the lungs with 1 mL of 5% India ink through the trachea. The lungs were isolated, and the metastatic nodules on their surfaces were counted with a black-stained background by microscopy (Sigounas et al., 2004).

Statistical Analysis
Statistical significances of difference throughout this study were calculated by Student’s t test (SigmaStat 2.0, Jandel Scientific, San Rafael, CA, USA).

	RESULTS

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Inhibition of Invasion, Motility, and Adhesion of SCC-4 Cells by Silibinin
Results of the [3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] assay (MTT assay) showed that a 24-hour treatment of silibinin at various concentrations (0-100 µM) exhibited no cytotoxicity in SCC-4 cells (data not shown). This concentration range was then applied to all subsequent experiments. In addition, the same procedures were performed on normal human gingival fibroblasts, and similar results were obtained (data not shown).

To screen for the preventive effectors against cancer metastasis, we examined the inhibitory effect of silibinin on invasion and motility. Silibinin significantly reduced the invasion and motility of SCC-4 cells in a concentration-dependent manner, with 89% and 66.4% of inhibition, respectively, after treatment by 100 µM silibinin (Figs. 1A, 1B). Moreover, a slightly inhibitory effect of silibinin on cell-collagen interactions was observed (Fig. 1C).

View larger version (12K): [in this window] [in a new window]   Figure 1. The effects of silibinin on cell invasion, motility, and cell-matrix adhesion of SCC-4 cells. Cells were treated with silibinin at a concentration of 0, 25, 50, 75, or 100 µM for 24 hrs, and were then subjected to analyses for invasion (A), motility (B), and cell-matrix adhesion (C) as described in MATERIALS & METHODS. Data represent the mean ± SD of at least 3 independent experiments. Statistical significance was determined by Student’s t test (*P < 0.05; **P < 0.01; ***P < 0.001).

View larger version (45K): [in this window] [in a new window]   Figure 2. Effects of silibinin on the protein and mRNA levels of proteases and their endogenous inhibitors. Cells were treated with 0, 25, 50, 75, or 100 µM silibinin for 24 hrs. For protein levels, cells were subjected to gelatin zymography, casein zymography, and Western blotting to analyze the activities and levels of MMP-2 (A), u-PA (B), TIMP-2, and PAI-1 (C), respectively, as described in MATERIALS & METHODS. Determined activities of these proteins were subsequently quantified by densitometric analysis, with control being 100%, as shown below the gel data. For mRNA levels (D), total RNAs were extracted and subjected to semi-quantitative RT-PCR for MMP-2, TIMP-2, MT1-MMP, u-PA, and PAI-1, with GADPH being an internal control. Data represent the mean ± SD of at least 3 independent experiments. Statistical significance was determined by Student’s t test (*P < 0.05; **P < 0.01; ***P < 0.001).

The Inhibition of ERK1/2 Phosphorylation by Silibinin
Since we have shown that treatment of SCC4-cells with silibinin inhibited the cell invasion and activities of MMP-2 and u-PA, the underlying mechanisms were further investigated. Silibinin significantly inhibited the activation of ERK1/2, whereas it had no significant effect on p38, JNK1/2, and Akt activity (Fig. 3A). Moreover, no significant change in the total amount of ERK1/2, p38, and JNK1/2 proteins was observed (data not shown).

View larger version (41K): [in this window] [in a new window]   Figure 3. An inhibitory effect of silibinin on the phosphorylation of ERK1/2. (A) Cells were treated with the indicated dose of silibinin (0, 25, 50, 75, or 100 µM) for 24 hrs, and then cell lysates were subjected to SDS-PAGE followed by Western blotting and immuno-probing with anti-phospho-ERK1/2, anti-phospho-p38, anti-phospho-JNK1/2, or anti-phospho-Akt antibodies. Protein reactivity was visualized with an ECL detection system. For determination of the effects of MEK inhibitor (U0126) and silibinin on the activity of MMP-2 and u-PA and cell invasion, cells were plated in culture dishes and pre-treated with U0126 (10 or 20µM) for 30 min, and then incubated in the presence or absence of silibinin (25 µM) for 24 hrs.Afterward, the culture media were subjected to gelatin and casein zymography to analyze the activities of MMP-2 (B) and u-PA (C), respectively, and cells were subjected to analyses for invasion (D) as described in MATERIALS & METHODS. Determined activities of these proteins were subsequently quantified by densitometric analysis, with control being 100% as shown below the gel data. Nuclear extracts were subjected to SDS-PAGE followed by Western blotting and immunoprobing with anti-NF-B, c-Jun, c-Fos, or C23 antibodies (E) as described in MATERIALS & METHODS. Data represent the mean ± SD of at least 3 independent experiments. (*P < 0.05; **P < 0.01).

To examine the effect of silibinin on the downstream effectors of the ERK1/2 signaling pathway, we analyzed the binding activities of NF-B and AP-1 by EMSA and the expression of NF-B, c-Jun, and c-Fos, with C23 being the internal control, in nuclear extracts by immuno-blotting. The results indicated that those nuclear levels (Fig. 3E) and binding activities (data not shown) of NF-B, c-Jun, and c-Fos were strongly decreased by silibinin.

The Anti-metastatic Effects of Silibinin in vivo
To verify the in vivo anti-metastatic effects of silibinin, we treated LLC-bearing C57BL/6 mice with corn oil or silibinin. Lung metastases of animals treated with silibinin decreased by 61.0% compared with those in the corn-oil-treated group (Fig. 4A). Small solid tumors were observed 8 days after the cell inoculation, and a 1.7-fold reduction was seen on Day 29 in silibinin-treated animals, compared with control animals (Fig. 4B). Moreover, by Day 30, silibinin feeding induced a 2.0-fold reduction in tumor mass (Fig. 4C) without any apparent signs of toxicity, as evidenced by body weight monitoring (Fig. 4D) throughout the experiment.

View larger version (18K): [in this window] [in a new window]   Figure 4. The in vivo anti-metastatic effects of silibinin. After subcutaneous implantation of LLC cells, C57BL/6 mice were treated with corn oil or silibinin as described in MATERIALS & METHODS, and then analyzed for the number of lung metastases (A), the growth of tumors (B), the weight of the primary tumor (C), and body weight (D). The values represent the means ± SD. Comparisons were performed by Student’s t test (*P < 0.05).

	DISCUSSION

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Many reports have addressed the importance of interactions between cells and ECM, which could enhance cell invasion, proliferation, and ECM degradation (Liotta et al., 1980; Azzam and Thompson, 1992). As shown in this study, attachment of SCC-4 cells to the collagen was slightly decreased after pre-treatment with silibinin. After the tumor cell detaches from neighboring cells, the ECM must be proteolytically degraded by matrix-degrading proteinases for tumor cell metastasis. Many studies have revealed that enhanced production of MMPs or u-PA correlates with the metastasis and invasion in various cancers, including OSCC (Kurahara et al., 1999; Geletu et al., 2004), and their expression and activity against matrix macromolecules have been shown to overbalance their endogenous inhibitors. TIMP-2 and PAI-1 have been shown to suppress tumor growth and metastatic potential in cell and animal model systems (Valente et al., 1998; Whitley et al., 2004; Lee et al., 2005). In this study, our findings showed that silibinin could lead to decreased enzyme activities of MMP-2 and u-PA, while levels of TIMP-2 and PAI-1 were enhanced. This suppression of MMP-2 or u-PA activities and enhancement of TIMP-2 or PAI-1 were consistent with the results of RT-PCR analysis. Moreover, silibinin was also able to inhibit the transcription of MT1-MMP. Since MT1-MMP is responsible for activation of MMP-2 (Sato et al., 2005), it was possible that such inhibition may contribute to a decreased activity of MMP-2. Analysis of these data suggested that silibinin affected MMP-2, MT1-MMP, u-PA, TIMP-2, and PAI-1 expression at the transcriptional levels, following which the inhibitory mechanism of MMPs and u-PA expression by silibinin was further examined.

The role of MAPKs in the regulation of MMP-2 or u-PA expressions in carcinoma cells has been well-studied (Rao, 2003). It has been shown that inhibition of p-ERK1/2 may lead to a reduction in the expression of MMP-2 or u-PA, as well as in the invasion of tumor cells (Westermarck and Kahari, 1999; Lev et al., 2004). Indeed, as shown in Fig. 3, silibinin could inhibit the phosphorylation of ERK1/2, and the involvement of the MAPK pathway was further supported by the use of the MEK inhibitor in our experimental model, to show that a treatment with U0126 could lead to an inhibition of MMP-2 or u-PA secretion, as well as a reduction in cell invasion.

Finally, with LLC-bearing mice, a well-established animal model for metastasis (Sigounas et al., 2004), it was suggested that silibinin may have a potential inhibitory effect on the cell invasion and metastasis of SCC-4 in vivo, without showing any apparent sign of toxicity, as demonstrated by body weight profile. In the future, the effects of silibinin on the metastasis of SCC-4 cells in vivo will be examined further. In conclusion, we have shown that silibinin exerted an inhibitory effect on several essential steps of metastasis, including cell invasion, cell-matrix interaction, and cell motility. In addition, silibinin could regulate the activities of invasion-associated proteases and their natural inhibitors, which may be mediated by inactivation of the p-ERK1/2 signaling pathway and regulation of transcription factors such as AP-1 and NF-B. As evidenced from the above results, silibinin may be a powerful candidate in the development of preventive agents for cancer metastases.

	ACKNOWLEDGMENTS

 
This study was supported by grants from the National Science Council, Republic of China (NSC 93-2320-B-040-061 and NSC 93-2313-B-166-001).

	FOOTNOTES

 
^a authors contributing equally to this work

Received July 27, 2005; Last revision October 20, 2005; Accepted November 2, 2005

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This Article Abstract Figures Only Full Text (PDF) Services Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (9) Citing Articles via Google Scholar Google Scholar Articles by Chen, P.-N. Articles by Chu, S.-C. Search for Related Content PubMed PubMed Citation Articles by Chen, P.-N. Articles by Chu, S.-C.