The C6 rat glioma cell line was obtained from the American Type Culture Collection (ATCC; USA). The culture medium used in the experiments was Dulbecco's modified Eagle's medium (DMEM; Thermo Scientific, Logan, UT, USA) containing 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic. The cells were incubated at 37°C in a humidified atmosphere of 5% Co₂. BITC, PEITC, SFN and N-methyl-β-phenethylamine (NMPEA) were obtained from Sigma (St. Louis, MO, USA).

C6 cells were seeded in a 96-well plate and allowed to attach for 24 h. Media were then discarded and replaced with 100 µl of new media containing various concentrations of ITCs and cultured for 24 h. 3-[4,5-Dimethylthiazol2-yl]-2.5-diphenyltetrazolium bromide (MTT; Roche Applied Science, Indianapolis, IN, USA) was added to each well. The amount of formazan deposits was quantified according to the supplier's protocol after 4 h of incubation with MTT reagent at 37°C in a 5% Co₂ incubator. The half maximal inhibitory concentrations (IC₅₀) were determined as the concentration of the test mixture that gave a 50% reduction in absorbance compared to that of the control.

Zymography was performed using the procedure described by Lee et al with minor modification (24). C6 cells were seeded in 6-well culture plates and incubated until they reached 80% confluency. Fresh serum-free medium was then added with ITCs to each dish, and further cultured for 24 h. Conditioned medium, so obtained, was electrophoresed on polyacrylamide gels containing 0.1% (w/v) gelatin. Gels were washed at room temperature for 30 min with 2.5% Triton X-100 and then incubated at 37°C for 24 h in a buffer containing 10 mM CaCl₂, 0.01% NaN₃ and 50 mM Tris-HCl (pH 7.5). Gels were then stained with 0.2% Coomassie Brilliant Blue (Bio-Rad, Hercules, CA, USA) and photographed on a light box. Proteolysis was detected as a white zone in a dark blue field.

The intracellular concentration of ROS in C6 glioma cells was measured using an oxidation-sensitive fluorescent probe dye, 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA; Invitrogen Molecular Probes, Eugene, OR, USA). DCF-DA diffuses into cells, where it is hydrolyzed by intracellular esterase to polar 2′,7′-dichlorodihydrofluorescein. This non-fluorescein analog gets trapped inside the cells and is oxidized by intracellular oxidants to a highly fluorescent 2′,7′-dichlorofluorescein level. C6 glioma cells were treated with ITCs and PMA for 12 h, after which the cells were incubated with 10 µM DCF-D at 37°C for 30 min. The fluorescence of 2′,7′-dichlorofluores-cein was detected in equivalent quantities of proteins using a multi-plate reader, VICTOR3 (excitation, 530 nm, emission, 485 nm; Perkin-Elmer, Waltham, MA, USA).

C6 cells were suspended in lysis buffer (50 mM Tris, 150 mM NaCl, 5 mM EDTA, 1 mM DTT, 1% Nonidet P-40, 100 µM phenylmethylsulfonyl fluoride, 20 µM aprotinin and 20 µM leupeptin, adjusted to ph 8.0) at 4°C for 30 min, followed by centrifugation at 12,000 rpm for 10 min. In addition, to separate the proteins in cells buffer A [10 mM HEPES (pH 7.9), 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM DTT, 300 mM saccharose, 0.1% NP-10, 0.5 mM phenylmethylsulfonyl fluoride] was used. After incubation for 5 min on ice, the samples were centrifuged at 1,000 rpm at 4°C for 1 min and the pellet was separated. A separate pellet was dissolved in buffer B [20 mM HEPES (ph 7.9), 20% glycerol, 100 mM KCl, 100 mM NaCl₂, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride]. After incubation for 15 min on ice, the samples were centrifuged at 1,000 rpm at 4°C for 5 min. Total protein concentration was determined using the Bradford assay (Bio-Rad). Total protein (30 µg) was separated on 6 to 12% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH, USA) using standard SDS-polyacrylamide gel electrophoresis procedure. The membranes were blocked in 5% skim milk in TBS-T for 1 h at room temperature. The membranes were then incubated with the primary antibody overnight at 4°C, washed three times with TBS-T, incubated with goat anti-mouse IgG or goat anti-rabbit IgG secondary antibodies for 1 h at room temperature and then washed with TBS-T three times. Signals were detected using enhanced chemiluminescence (ECL; Amersham Life Science Corporation, Arlington heights, IL, USA) film and ChemiDOC XRS (Bio-Rad). Primary antibodies used in this study were MMP-9 and MMP-2 purchased from Millipore (Billerica, MA, USA). Phospho-ERK, phospho-JNK, phospho-p38, phospho-AKT, phospho-FAK, NF-κB, c-Jun, and c-Fos were purchased from Santa Cruz biotechnology, Inc. (Santa Cruz, CA, USA).

Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. For RT-PCR, cDNA was synthesized from 1 µg of total RNA using Bioneer AccuPower PCR PreMix kit (Daejeon, Korea) according to the manufacturer's protocol. The cDNA was amplified by PCR with the following primers: MMP-9, 5′-AAACCTCCAACCTCACGGAC-3′ (sense) and 5′-GAAAGGCGTGTGCCAGTAGA-3′ (antisense); TIMP-1, 5′-CTGCAACTCGGACCTGGTTA-3′ (sense) and 5′-GTGCACAAATCTGGATTCCG-3′ (antisense); and β-actin, 5′-ATGTGGATAAAGCCGTCAGTGG-3′ (sense) and 5′-CTGGAGTGTCCATGGGACAG-3′ (antisense). PCR products were analyzed by agarose gel electrophoresis and visualized by treatment with ethidium bromide.

Matrigel-coated filter inserts (8-µm pore size) that fit into 24-well migration chambers were obtained from Becton-Dickinson (Franklin Lakes, NJ, USA). Cells were then plated on the upper chamber. The lower chamber was filled with culture media containing various drugs. Cells in the chamber were incubated for 24 h at 37°C and cells that invaded the lower membrane surface were fixed with methanol and stained with hematoxylin and eosin. The cells that passed through the Matrigel and were located on the underside of the filter were counted. Random fields were counted by light microscopy (×400 magnification).

This assay was performed using the procedure described by Lin et al with minor modification (25). Cells were seeded in 6-well plates and incubated until they reached 80% confluency. Monolayers were scratched with a 200-µl pipette tip to create a wound, and cells were then washed twice with serum-free culture media to remove floating cells. Media were then replaced with fresh serum-free media. Cells were subjected to the indicated treatment for 24 h, and cells were photographed at 24 h.

All in vitro results are representative of at least three independent experiments performed in triplicate. The significance of differences between the experimental and control values was analyzed by the Newman-Keuls test using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA). P-values of <0.05 were deemed to indicate a significant difference.

Before investigating the anticancer pharmacological potential of ITCs on C6 glioma cells, the effect of ITCs on cell viability was first examined. As shown in Fig. 1, a cytotoxic effect on C6 glioma cells was exhibited by BITC (IC₅₀, 32.8 µM), whereas PEITC (IC₅₀, 34.3 µM) showed comparable cytotoxic activity and SFN (IC₅₀, 50.8 µM) exhibited relatively less cytotoxicity. NMPEA, which is a structural analog of PEITC without ITC functionality (26), was used as the negative control. NMPEA did not significantly affect the cell viability below a concentration of 20 µM. There was no obvious reduction in the cell viability of the C6 glioma cells after treatment with all ITCs drugs at doses <10 µM. Based on these results, a concentration of 10 µM of the ITCs was used in the following experiments. The MMP-9 secretion in the C6 glioma cells was induced by PMA in a dose-dependent manner and a time-dependent manner (Fig. 2A), whereas MMP-2 did not change (data not shown). In the following experiments, the C6 glioma cells were treated with 50 nM PMA for 24 h. Then the inhibitory effects of ITCs on MMP-9 and MMP-2 activity were determined via gelatin zymography assay. ITCs at a concentration of 10 µM decreased the MMP-9 activity in the C6 glioma cells but did not change MMP-2 activity (Fig. 2B).

To further confirm the influence of ITCs on MMP-9 expression, western blotting was performed. ITCs inhibited MMP-9 expression, whereas MMP-2 expression was not reduced in the C6 glioma cells (Fig. 2C). NMPEA had no effect on both MMP-9 activity and expression (Fig. 2B and C). ITCs also reduced the PMA-induced MMP-9 mRNA level in the C6 glioma cells, whereas NMPEA had no affect (Fig. 2D). Moreover, since the activity of MMP-9 is regulated by the endogenous tissue inhibitor of metalloproteinase-1 (TIMP-1) (27), the expression level of TIMP-1 was measured via RT-PCR. ITCs did not alter TIMP-1 mRNA expression. These results suggest that the sulfur-containing functional group of the ITCs directly inhibited the MMP-9 transcription level, as NMPEA had no effect on PMA-induced C6 glioma cells. In addition, since ROS generation is known to trigger PMA-mediated induction of cell migration (28), the effects of ITCs on intracellular ROS concentrations were measured. The C6 glioma cells exposed to PMA had increased ROS levels compared with the untreated cells (Fig. 2E). However, ITCs did not affect the PMA-induced ROS generation in the C6 glioma cells, suggesting that the inhibitory effects of ITCs on MMP-9 were not related to ROS generation.

MAPK is one of the pathways involved in the modulation of PMA-induced MMP-9 expression (29). To investigate the effects of ITCs on the MAPK expression associated with migration and invasion in C6 glioma cells, western blotting was performed. As shown in Fig. 3A, the ITCs suppressed the PMA-induced phosphorylation of JNK, but ERK and p38 were not changed. As the FAK/JNK and FAK/ERK signaling pathways control MMP secretion in carcinoma cells (30,31), it was determined whether the ITCs suppress PMA-induced phosphorylation of FAK in C6 glioma cells. ITCs with a concentration of 10 µM inhibited the PMA-induced phosphorylation of FAK (Fig. 3b), and NMPEA had no effect. To confirm that FAK modulates the PMA-induced MAPK pathways, C6 glioma cells were treated with 10 µM PF573228 (FAK inhibitor). The PMA-induced JNK phosphorylation was decreased by PF573228. However, PMA-induced phosphorylation of ERK and p38 were not changed (Fig. 3C). In addition, co-treatment with ITCs and PF573228 only blocked PMA-induced JNK phosphorylation in C6 glioma cells. We further analyzed the effect of PF573228 on MMP-9 expression and activity in PMA-induced C6 glioma cells. The MMP-9 expression and activity were blocked by PF573228 and co-treatment with ITCs and PF573228 (Fig. 3D). We suggest that ITCs suppress MMP-9 activity and expression via blocking FAK-dependent JNK phosphorylation.

NF-κB and AP-1 are both important transcription factors associated with the modulation of cell migration and invasion (12). As shown in Fig. 4A, PMA induced the nuclear translocation of the NF-κB subunit p65 and the AP-1 subunit c-fos. ITCs blocked the nuclear translocation of p65 and c-fos. It was further confirmed that FAK regulates the nuclear translocation of p65 and c-fos using PF573228. The nuclear translocation of p65 and c-fos was blocked by PF573228, and co-treatment with ITCs and PF573228 (Fig. 4B). These data suggest that ITCs regulate the transcriptional activation of MMP-9 by reducing the PMA-induced nuclear translocation of the NF-κB subunit p65 and the AP-1 subunit c-fos.

A wound-healing experiment was performed to evaluate the effect of ITCs on C6 glioma cell migration. C6 glioma cells were grown and then wounded by scraping. As illustrated in Fig. 5A and B, PMA induced the migration of the C6 glioma cells. BITC, PEITC and SFN at the concentration of 10 µM decreased C6 glioma cell migration by 88, 76 and 68% compared with PMA, respectively. To further determine the inhibitory effect of ITCs on invasion, C6 glioma cells were treated with 10 µM of ITCs, a Matrigel-based Transwell invasion assay was performed. BITC, PEITC and SFN reduced the C6 glioma cell invasion by 83, 70 and 61%, respectively (Fig. 5C and D). However, NMPEA did not affect PMA-induced cell migration and invasion, suggesting that ITCs suppressed the migration and invasion of C6 glioma cells by inhibiting MMP-9 expression and the sulfur-containing functional group plays an important role in the anti-metastatic effects of ITCs.