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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

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).

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).

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).

Journal of Dental Research, Vol. 85, No. 3, 220-225 (2006)
DOI: 10.1177/154405910608500303


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