Selaginella tamariscina was purchased from herb stores and dried whole plants (100 g) were extracted twice with 500 ml of 50% ethanol in distilled water. The pooled extracts were filtered and concentrated at 70°C using a rotary evaporator under low pressure. The concentrated crude extract was frozen at −80°C for 2-3 days and then it was freeze-dried in a lyophilizer and stored at −20°C. The extraction yield was 2.8% (w/w) and the chemical profile of Selaginella tamariscina extract (STE) was analyzed by using high-pressure liquid chromatograms (HPLC)-mass spectrometer [19]. Briefly, Selaginella tamariscina were analysed by HPLC-mass spectrometer using a HPLC (Hitachi L-6200 with an L-4500 Diode Array detector) with a PE Sciex Qstar Pulsar ESI-TOF mass spectrometer. Samples (10 µl) were injected onto a Merck LiChrospher 100 RP-18 column (4 x 250 mm). The column was equilibrated in 0.05% acetic acid/water (solution A) and elution of the components was achieved by increasing the concentration of solution B (100% acetonitrile) from 0 to 100% in 30 min at a flow rate of 1 ml/min. Absorbance was monitored at 254 nm. The molecular masses of the peaks were determined from electrospray ionisation mass spectra using multiply-charged ion profile [19]. The extract was dissolved in dimethyl sulfoxide (DMSO) (Sigma Co., USA) and was prepared at different concentrations for the subsequent experiments.

HSC-3, a human tongue squamous cell carcinoma cell line obtained from ATCC (Manassas, VA, USA), was cultured in Dulbecco’s modified Eagle’s medium (Life Technologies, Grand Island, NY, USA), 10% fetal bovine serum (Hyclone Laboratories, Logan, UT, USA), 2 mM glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. All cell cultures were maintained at 37 ^oC in a humidified atmosphere of 5% CO₂. For STE treatment, appropriate amounts of stock solution of STE were added into culture medium to achieve the indicated concentrations and then incubated with cells for indicated time periods, whereas dimethyl sulfoxide solution without STE was used as blank reagent.

For cell viability experiment, a microculture tetrazolium (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay was performed to determine the cytotoxicity of STE. HSC-3 cells were seeded in 24-well plates at a density of 5 x 10⁴ cells/well and treated with STE at a concentration between 0–100 µg/mL at 37 ^oC for 24 h. After the exposure period, the media was removed, and cells were washed with phosphate buffered saline (PBS) and then incubated with 20 µL MTT (5 mg/mL) (Sigma chemical Co., St. Louis, MO, USA) for 4 h. The viable cell number per dish is directly proportional to the production of formazan, which can be measured spectrophotometrically at 563 nm following solubilization with isopropanol.

HSC-3 cells (1×10⁵ cells/well) were plated in 6-well plates for 24 h, wounded by scratching with a pipette tip, then incubated with DMEM medium containing 0.5% FBS and treated with or without STE (0, 25, 50, 75 and 100 µg/mL) for 0, 12 and 24 h. Cells were photographed using a phase-contrast microscope (×100).

Cell migration and invasion were assayed according to the methods described by Yang et al. [19]. After a treatment with STE (0, 25, 50, 70 and 100 µg/mL) for 24 h, surviving cells were harvested and seeded to Boyden chamber (Neuro Probe, Cabin John, MD, USA) at 10⁴ cells/well in serum free medium and then incubated for 24 hours at 37 ^oC. For invasion assay, 10 µL Matrigel (25 mg/50 mL; BD Biosciences, MA, USA) was applied to 8 µm pore size polycarbonate membrane filters and the bottom chamber contained standard medium. Filters were then air-dried for 5 h in a laminar flow hood. The invaded cells were fixed with 100% methanol and stained with 5% Giemsa. Cell numbers were counted under a light microscope. The migration assay was carried out as described in the invasion assay with no coating of Matrigel.

The activities of MMP-2 in conditional medium were measured by gelatin zymography protease assays. Briefly, collected media of an appropriate volume (adjusted by vital cell number) 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 2.5% Triton X-100 and then incubated in reaction buffer (40 mM Tris–HCl, pH 8.0; 10 mM CaCl₂ and 0.01% NaN3) for 12 h at 37 ^oC. Then gel was stained with Coomassie brilliant blue R-250.

For total cell lysates preparation, cells were rinsed with PBS twice and scraped with 0.2 mL of cold RIPA buffer containing protease inhibitors cocktail, and then vortexed at 4 ^oC for 10 min. Cell lysates were subjected to a centrifugation of 10,000 rpm for 10 min at 4 ^oC, and the insoluble pellet was discarded. The protein concentration of total cell lysates was determined by Bradford assay.

The 20 µg samples of total cell lysates or nuclear fractions were separated by SDS-PAGE on 10% polyacrylamide gels and transferred onto a nitrocellulose membrane using the Mini-Protean Tetra Electrophoresis System as described previously [22]. The blot was subsequently incubated with 5% non-fat milk in Tris-buffered saline (20 mM Tris, 137 mM NaCl, pH 7.6) for 1 h to block non-specific binding and then overnight with polyclonal antibodies against MMP-2, MMP-9, TIMP-1, TIMP-2, three MAPKs (ERK 1/2, JNK 1/2 and p38), or Akt with the specific antibodies for unphosphorylated or phosphorylated forms of the corresponding ERK 1/2, JNK 1/2, p38 and Akt. Blots were then incubated with a horseradish peroxidase goat anti-rabbit or anti-mouse IgG for 1 h. Afterwards, signal was detected by using enhanced chemiluminescence (ECL) commercial kit (Amersham Biosciences) and relative photographic density was quantitated by scanning the photographic negatives on a gel documentation and analysis system (AlphaImager 2000, Alpha Innotech Corporation, San Leandro, CA, USA).

Total RNA was isolated from oral cancer cells using Trizol (Life Technologies, Grand Island, NY) according to the manufacturer’s instructions. Quantitative real-time PCR analysis was carried out using Taqman one-step PCR Master Mix (Applied Biosystems). 100 ng of total cDNA was added per 25 µl reaction with MMP-2, MMP-9 or GAPDH primers and Taqman probes. The MMP-2, MMP-9 and GAPDH primers and probes were designed using commercial software (ABI PRISM Sequence Detection System; Applied Biosystems). Quantitative real-time PCR assays were carried out in triplicate on a StepOnePlus sequence detection system. The threshold was set above the non-template control background and within the linear phase of target gene amplification to calculate the cycle number at which the transcript was detected.

HSC-3 cells were seeded at a concentration of 5 x10⁴ cells per well in 6-well cell culture plates. After 24 h of incubation, pGL3-basic (vector), MMP-2 or MMP-9 promoter plasmid were co-transfected with a β-galactosidase expression vector (pCH110) into cells using Turbofect (Fermentas, Carlsbad, CA). After 12 h of transfection, cells were treated with vehicle or STE (0 or 100 µg/mL) for 24 h. The cell lysates were harvested, and luciferase activity was determined using a luciferase assay kit. The value of the luciferase activity was normalized to transfection efficiency and monitored by β-galactosidase expression.

Chromatin immunoprecipitation analysis was performed as described previously [23,24]. DNA immunoprecipitated with anti-CREB, anti-SP1 or anti c-fos was purified and extracted using phenol-chloroform. Immunoprecipitated DNA was analyzed by PCR or quantitative PCR by using specific primers as described previously [23].

For all of the measurements, analysis of variance followed by Scheffe posteriori comparison was used to assess the differences between control and cells treated with various concentration of STE. A difference at p < 0.05 was considered to be statistically significant and the experiments were repeated three times.

HSC-3 cell viability in the presence of various concentrations (0-100 µg/mL) of Selaginella tamariscina for 24 hours is shown in Figure 1A. Even the highest concentration, 100 µg/mL, did not have a cytotoxic effect on the HSC-3 cells. We used 0-100 µg/mL of Selaginella tamariscina to conduct the following experiments. Figure 1B shows the results of using a scratch-wound assay to calculate the migration ability of HSC-3 cells treated with various concentrations of Selaginella tamariscina. The results demonstrate that Selaginella tamariscina significantly reduced cell motility both time- and dose-dependently (p<0.001) (Figure 1B and 1C).

To examine the effects of Selaginella tamariscina on cell migration and invasion, we used a Boyden chamber assay to detect cell motility. Figure 2A shows that Selaginella tamariscina significantly inhibited migration in a concentration-dependent manner for 24 hours. Similarly, Figure 2B indicates that the invasiveness of HSC-3 cells was also reduced after incubation with different concentrations (0-100 µg/mL) of Selaginella tamariscina for 24 hours.

The ability of Selaginella tamariscina to suppress the migratory and invasive abilities of HSC-3 cells by decreasing MMP-2 and MMP-9 expression was evaluated using gelatin zymography. Figure 3A shows that the enzyme activity of MMP-2 and MMP-9 was suppressed by Selaginella tamariscina in a concentration-dependent manner. The highest concentration of Selaginella tamariscina, 100 µg/mL, inhibited MMP-2 and MMP-9 activity by 57% and 51%, respectively (Figure 3B). Selaginella tamariscina also substantially reduced MMP-2 and MMP-9 protein expression when detected using western blotting (Figure 3C). Thus, we suggest that the anti-metastatic ability of Selaginella tamariscina at least partially inhibited MMP-2 and MMP-9 expression. Investigation of the effects of STE on the protein expression of the MMPs endogenous inhibitor, TIMP-1 and TIMP-2, showed that STE induced TIMP-1 and TIMP-2 upregulation in a concentration-dependent manner (Figure 3C and 3D)

The effects of Selaginella tamariscina on MMP-2 and MMP-9 mRNA expression were also examined. A low level of MMP-2 and MMP-9 mRNA expression was observed at the highest dose of Selaginella tamariscina (100 µg/mL) for 6 hours (Figure 4A and 4B). To further investigate how Selaginella tamariscina regulates the transcriptional activity of MMP-2 and MMP-9, we conducted a luciferase reporter assay in which both Selaginella tamariscina and the control cells were transfected with an MMP-2 and MMP-9 promoter construct. Figure 4C shows that the MMP-2 promoter activity was reduced by Selaginella tamariscina in a dose-dependent manner. Similarly, approximately 50% inhibition of MMP-9 promoter activity was evident at 100 µg/mL of Selaginella tamariscina (Figure 4D). These observations suggest that Selaginella tamariscina regulates MMP-2 and MMP-9 activity at the transcriptional level.

HSC-3 cells were treated with STE (0, 25, 50, 75 and 100 µg/mL) for 24 h and then subjected to quantitative real-time PCR to analyze the mRNA expression of MMP-2 (A), or MMP-9 (B). (C) MMP-2 or (D) MMP-9 promoter reporter assay to analyze the promoter activity of MMPs. Luciferase activity, determined in triplicates, was normalized to β-galactosidase activity. The values represented the means ± SD of at least three independent experiments. *p < 0.05 as compared with the vehicle group.

Previous studies have shown that MMP promoters are regulated by several transcription factors, such as AP-1, NFκB, CREB, and SP-1 [23,25,26]. We performed a chromatin immunoprecipitation (ChIP) assay to evaluate the involvement of transcription factors in the inhibitory effects of Selaginella tamariscina on MMP-2 and MMP-9 activity (Figure 5A and 5B). ChIP assay and quantitative real-time PCR showed that Selaginella tamariscina substantially suppressed binding of CREB and SP-1 to the MMP-2 promoter (Figure 5A). Figure 5B indicates that Selaginella tamariscina considerably inhibited AP-1, but not the NF-κB DNA-binding to the MMP-9 promoter. These results indicate that Selaginella tamariscina inhibited MMP-2 and MMP-9 expression by regulating the binding activity of transcription factors on the cis-element of MMP promoters.

HSC-3 cells were treated with STE 100 µg/mL for 24 h and then the nuclear fraction was prepared as described in "Materials and Methods". ChIP analysis of the association of various transcription factors with the MMP-2 (A) or MMP-9 (B) promoter region in HSC-3 cells. The values represented the means ± SD of at least three independent experiments. *p < 0.05 as compared with the vehicle group.

To further investigate the underlying mechanisms of the upstream signaling pathways of MMP-2 and MMP-9, we used western blotting to evaluate the effects of Selaginella tamariscina on the MAPK and Akt pathways. Figure 6A–6C reveal that the MAPK pathway, which includes ERK, JNK, and p38 protein kinases, was not notably inhibited. However, Selaginella tamariscina reduced phosphorylation of Akt in a dose-dependent manner (Figure 6D). Thus, we suggest that the activation of the Akt signaling pathway is required for Selaginella tamariscina to suppress MMP-2 and MMP-9.

HSC-3 cells were cultured in various concentrations of STE (0, 25, 50, 75 and 100 µg/mL) for 24 hours, and then the cell lysates were subjected to SDS–PAGE followed by western blots with (A) anti-ERK1/2, (B) anti-JNK, (C) anti-p38 and (D) anti-Akt (total and phosphorylated) antibodies as described in Materials and Methods. Determined activities of these proteins were subsequently quantified by densitometric analyses with that of control being 100% as shown just below the gel data. The values represented the means ± SD of at least 3 independent experiments. *p< 0.05 as compared with the vehicle group.