Surfactin (from B. subtilis), 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and other reagents not referred were purchased from Sigma-Aldrich (St. Louis, MO, USA). BD BioCoat™ Matrigel™ Invasion Chambers were obtained from BD Biosciences (San Jose, CA, USA). SB203580, SP600125 and LY294002 were purchased from A.G. Scientific (San Diego, CA, USA). The FuGene 6 transfection reagent was purchased from Roche Diagnostics (Indianapolis, IN, USA). Antibodies against phosphorylated p38 (p-p38), phosphorylated c-Jun N-terminal kinase (p-JNK), phosphorylated extracellular signal-regulated kinase (p-ERK), phosphorylated Akt (p-Akt), phosphorylated inhibitory protein κB (p-IκB)-α, MMP-2 and MMP-9 were purchased from Cell Signaling Technology (Beverly, MA, USA), and antibodies against ERK, JNK, p38, Akt, c-Jun, c-Fos, nuclear factor-κB (NF-κB), inhibitory protein κB-α (IκB-α) and histone H1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

The human breast cell lines, MCF-7 and MDA-MB-231, were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were grown in RPMI (Gibco BRL, Carlsbad, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C in a humidified incubator with 5% CO₂.

The effect of surfactin on cell viability was determined using an established MTT assay. Briefly, cells (5×10⁴ cells/24-well) were seeded in wells and incubated at 37°C for 24 h to allow attachment. The attached cells were treated with TPA in the presence or absence of surfactin for 24 h at 37°C. The cells were washed with phosphate-buffered saline (PBS), after which MTT (62.5 μg/ml) was added and the cells were incubated at 37°C for 30 min. Following incubation, formazan crystals were dissolved with dimethylsulfoxide (150 μl/well) and detected at 570 nm using a microplate reader (Wallac 1420, PerkinElmer Life Sciences, Boston, MA, USA).

For the cell migration assay, cells were seeded into a 24-well culture dish until 90% confluent. The cells were then maintained in serum-free medium for 12 h. The monolayers were carefully scratched using a 200-μl pipette tip. The cellular debris was subsequently removed by washing with PBS, and the cells were incubated in medium without serum. The migrated cells were fixed in cold 75% methanol for 30 min and washed three times with PBS. The cultures were photographed at 0 and 24 h to monitor the migration of cells into the wounded area, and the closure of the wounded area was calculated.

A cell invasion assay was conducted using BioCoat Matrigel Invasion Chambers according to the manufacturer’s instructions. Briefly, the Matrigel coating was re-hydrated in 0.5 ml DMEM for 30 min immediately before the experiments. Cells (5×10⁴) suspended in 0.5 ml of serum-free medium were then added to the upper chamber of Matrigel-coated filter inserts. After treatment with surfactin for 1 h, 0.5 ml of serum-free medium containing TPA was added to the bottom well as a chemoattractant. The chambers were then incubated for 24 h. After incubation, cells on the upper side of the chamber were removed using cotton swabs. Cells that had migrated were then fixed and stained with 2% ethanol containing 0.2% crystal violet powder. Invading cells were enumerated under a light microscope using a x10 objecive lens.

The activity of MMP-2 and MMP-9 in the conditioned medium was determined by gelatin zymography protease assays. Briefly, cells (2×10⁵) were seeded in 6-well plates and allowed to grow to 80% confluency. The cells were then maintained in serum-free medium for 12 h prior to being treated with surfactin and TPA for 24 h. The conditioned medium was subsequently collected, cleared by centrifugation and mixed with 2X sodium dodecyl sulphate (SDS) sample buffer, followed by electrophoresis on polyacrylamide gels containing 0.1% (w/v) gelatin. Following electrophoresis, the gels were incubated in renaturing buffer (2.5% Triton X-100) with gentle agitation to remove the SDS and were then incubated in developing buffer (50 mM Tris-HCl buffer, pH 7.4, and 10 mM CaCl₂) overnight at 37°C to allow digestion of the gelatin. Finally, the gels were stained with SimplyBlue SafeStain (Invitrogen Corp., Carlsbad, CA, USA) until clear bands indicative of gelatin digestion appeared.

Cells were harvested in ice-cold lysis buffer consisting of 1% Triton X-100, 1% deoxycholate and 0.1% sodium SDS. The protein content of the cell lysates was then determined using Bradford reagent (Bio-Rad, Hercules, CA, USA). Proteins in each sample were resolved by 12% SDS-polyacrylamide gel electrophoresis, transferred onto a polyvinylidene difluoride membrane, and exposed to the appropriate antibodies. The proteins were visualized with an enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ, USA) using horseradish peroxidase-conjugated secondary antibodies. Images were acquired using an ImageQuant 350 analyzer (Amersham Biosciences).

Total cellular RNA was isolated using RNA Spin Mini RNA isolation kits (GE Healthcare, Buckinghamshire, UK), according to the manufacturer’s instructions. Total RNA (1 μg) was reverse-transcribed using Maxime RT PreMix (Intron Biotechnology, Seongnam, Korea) and anchored oligo-(dT)₁₅-primers. The amplification sequence protocol was as follows: 25 cycles of 95°C for 30 sec, 55°C for 30 sec and 72°C for 1 min. The PCR products were subjected to 1.5% agarose gel electrophoresis and images were captured by an ImageQuant 350 analyzer (Amersham Biosciences). Real-time PCR was performed using the SYBR-Green Master Mix (Applied Biosystems, Foster City, CA, USA) in a Chromo4 instrument (Bio-Rad). The relative amount of target mRNA was determined using the Ct method by normalizing target mRNA Ct values to those for GAPDH (ΔCt) (18). Real-time PCR was conducted by subjecting the samples to the following conditions: 95°C for 5 min followed by 40 cycles of 95°C for 30 sec, 55°C for 20 sec and 72°C for 30 sec. The following primers were used for PCR: MMP-9 sense, 5′-TTCCCTGGAGACCTGAGAACC-3′ and antisense, 5′-CGGCAAGTCTTCCGAGTAGTTT-3′; MMP-2 sense, 5′-GATGGCACCCATTTACACCT-3′ and antisense, 5′-CACAGTCCGCCAAATGAA-3′; and GAPDH sense, 5′-AGGTGGTCTCCTCTGACTTC-3′ and antisense, 5′-TACCAGGAAATGAGCTTGAC-3′.

MCF-7 cells were cultured directly on glass coverslips in 35 mm-diameter dishes. Cells were fixed with −20°C methanol for 10 min. To investigate the cellular localization of NF-κB, we treated cells with a 1:100 dilution of polyclonal antibody against NF-κB for 24 h. The cells were then extensively washed with PBS, after which they were further incubated with a 1:1,000 dilution of secondary fluorescein isothiocyanate-conjugated donkey anti-rabbit IgG antibody for 4 h at room temperature. Cell nuclei were stained with 1 μg/ml of 4′,6-diamidino-2-phenylindole (DAPI), and then analyzed by confocal microscopy using an LSM 510 Meta microscope (Zeiss, Jena, Germany).

To detect the in vivo association of nuclear proteins with human MMP-9 promoter, ChIP analysis was conducted as described previously (19) with some modifications. Briefly, 2×10⁷ cells were incubated in culture medium containing 1% formaldehyde for 10 min at room temperature, after which the cross-linking reaction was quenched by adding glycine to a final concentration of 0.125 M. The isolated nuclei were then digested with 10 U of MNase at 37°C for 15 min followed by sonication to produce chromatin of primarily mononucleosome size. Fragmented chromatin was then reacted with antibodies for 3 h at 4°C. Protein-DNA complexes were recovered using protein A agarose beads, washed and eluted with elution buffer. Crosslinks were reversed at 65°C in 0.25 M NaCl overnight and the DNA was then digested with proteinase K for 2 h at 50°C. The immunoprecipitated DNAs were subsequently isolated and used for PCR. PCR primers specific for the MMP-9 promoter [including NF-κB/activator protein-1 (AP-1) cluster, GenBank accession no. AF538844] were as follows: sense, 5′-CACTTCAAAGTGGTAAGA-3′ antisense, 5′-GAAAGTGATGGAAGACTCC-3′.

We used a dual-luciferase reporter assay system (Promega, Madison, WI, USA) to determine promoter activity. Briefly, cells were transfected with NF-κB luciferase reporter plasmid (20) or AP-1 luciferase reporter plasmid (Stratagene, Grand Island, NY, USA) using FuGENE-6 reagent (Roche Applied Science, Indianapolis, IN, USA) according to the manufacturer’s instructions. Renilla luciferase control plasmid pRL-CMV (Promega) was co-transfected as an internal control to evaluate transfection efficiency. Twenty four hours after transfection, the cells were incubated with the indicated reagents for 1 h and then treated with TPA for 24 h. Luciferase activity was assayed using the dual-luciferase assay kit (Promega) according to the manufacturer’s instructions. Luminescence was measured with a GloMax™ 96 microplate luminometer (Promega).

Each experiment was repeated at least three times and all results are expressed as the means ± SE. Statistical analysis was performed using SPSS software (version 18.0) to determine significant differences. We used either one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test for comparison between three or more groups. A value of p<0.05 was considered to indicate a statistically significant difference.

To verify the effects of surfactin on cell viability, non-aggressive MCF-7 and aggressive MDA-MB-231 human breast cancer cells were treated with surfactin in the absence or presence of TPA for 24 h. When compared with the untreated control cells, MCF-7 and MDA-MB-231 cells treated with surfactin at concentrations between 0 and 10 μM exhibited no cytotoxicity, regardless of whether they were treated with TPA or not (Fig. 1). Thus, this concentration range of surfactin was applied in all the subsequent experiments.

In order to investigate the effects of surfactin on the invasive potency of breast cancer cells, we carried out a wound-healing assay in non-aggressive MCF-7 and aggressive MDA-MB-231 human breast cancer cells. When confluent monolayers of cells were treated with TPA for 24, the MCF-7 and MDA-MB-231 cells readily closed the gap over 24 h, whereas the untreated cells did not. In the wound-healing assay, 10 μM of surfactin caused a decrease in the number of cells migrating into the wound area (Fig. 2).

Considering that invasion across the basement membrane by cancer cells is a critical process in tumor metastasis, we used Transwell invasion assay to investigate the effects of surfactin on cancer cell invasion. When MCF-7 and MDA-MB-231 cells were treated with TPA, the cells were able to invade freely through the Matrigel. The numbers of MCF-7 and MDA-MB-231 cells that passed through Matrigel were remarkably decreased by treatment with surfactin (Fig. 3), and the inhibition rates were approximately 68 and 84%, respectively.

Tumor metastasis is a multi-step and complex process that includes the proteolytic digestion of the ECM, cell migration to the circulation system, as well as colonization at metastatic sites. Therefore, we examined the effects of surfactin on clonogenicity with soft agar colony formation assays. As shown in Fig. 4, surfactin inhibited the anchorage-independent growth of MCF-7 and MDA-MB-231 cells and the inhibition rates were approximately 70 and 61%, respectively.

The upregulation of MMP-9 has been reported to play an essential role in invasion and metastasis in breast cancer cells (6). Thus, we examined whether the inhibitory effect of surfactin against breast cancer cell invasion is associated with the regulation of MMP-9 expression and enzyme activity by western blot analysis and gelatin zymo graphy assay. To accomplish this, MCF-7 cells were treated with surfactin 1 h prior to the addition of TPA and then incubated for a further 24 h. The medium from the control cells contained weak proteolytic activity at 92 kDa corresponding to MMP-9 and high proteolytic activity at 72 kDa corresponding to MMP-2. Treatment with TPA for 24 h dramatically upregulated MMP-9 activity, while the activity of MMP-2 was not affected by TPA or surfactin. TPA-induced MMP-9 secretion (in conditioned medium) was also dramatically inhibited in the presence of surfactin in a dose-dependent manner (Fig. 5A and B). Furthermore, the treatment of breast cancer cells with surfactin decreased the TPA-stimulated intracellular expression of MMP-9 in a dose-dependent manner, while the level of MMP-2 was not affected by TPA or surfactin (Fig. 5A and B).

To determine whether the inhibition of MMP-9 expression by surfactin was due to a reduced level of transcription, we performed RT-PCR and real-time PCR. The treatment of breast cancer cells with surfactin significantly inhibited the levels of TPA-stimulated MMP-9 mRNA in a dose-dependent manner, whereas surfactin together with TPA had little effect on the MMP-2 mRNA levels (Fig. 5C and D). These results indicate that surfactin suppresses TPA-induced MMP-9 expression through the inhibition of its transcriptional activity. Surfactin had similar inhibitory effects on MMP-9 expression in MDA-MB-231 cells (data not shown).

To confirm the involvement of MMP-9 in breast cancer cell invasion, we examined the effects of MMP-9 siRNA via a Matrigel invasion assay. MMP-9 siRNA reduced the expression of MMP-9 and the knockdown of MMP-9 markedly decreased the invasion of MCF-7 cells (Fig. 5E and F).

Since the physiological activity of MMP-9 is closely related to that of TIMPs, specific endogenous inhibitors of MMP-9 (21), we investigated the potential effects of surfactin on TIMP-1 and TIMP-2 expression. As shown in Fig. 5G, the expression levels of TIMP-1 and TIMP-2 were not altered by surfactin. When the MCF-7 cells were co-treated with surfactin and TPA, TIMP-1 and TIMP-2 were slightly upregulated by TPA, although their expression level was not altered by surfactin (Fig. 5H). Similar effects of surfactin on TIMP-1 and TIMP-2 expression were observed in the MDA-MB-231 cells (data not shown).

We further investigated the mechanism of MMP-9 transcriptional regulation by surfactin. MMP-9 expression is known to be regulated by the interaction of transcription factors, such as NF-κB and AP-1 with binding elements in the MMP-9 gene promoter (22,23). Therefore, we examined the effect of surfactin on NF-κB and AP-1 activity in TPA-stimulated breast cancer cells. MCF-7 cells were transiently transfected with NF-κB-Luc reporter or AP-1-Luc reporter plasmid, and the reporter activities were found to be regulated by surfactin. The TPA-induced increases in NF-κB and AP-1 reporter activities were suppressed by surfactin in a dose-dependent manner (Fig. 6A and B).

The MMP-9 promoter contains cis-acting regulatory elements for transcription factors, including two Ap-1 sites (-79 and -533) and one NF-κB site (-600); therefore, we used a ChIP assay to determine the effects of surfactin on the binding activities of NF-κB and AP-1 with the MMP-9 promoter. Chromatin was extracted and immunoprecipitated using anti-NF-κB or AP-1 antibodies, and the MMP-9 promoter region (NF-κB/AP-1 cluster -739 to -358) was amplified by PCR (Fig. 6C) and real-time PCR (Fig. 6D). The in vivo binding of NF-κB and AP-1 to the MMP-9 promoter increased in response to TPA; however, the TPA-induced NF-κB and AP-1 binding activities were significantly inhibited by surfactin.

To elucidate whether surfactin can affect the nuclear trans-location of NF-κB and AP-1 transcription factors, MCF-7 cells were treated with various concentrations of surfactin in the presence of TPA for 1 h, and nuclear extracts were prepared and examined by western blot analysis. While TPA induced the nuclear translocation of AP-1 (c-Jun and c-Fos) and NF-κB p65, surfactin inhibited the nuclear translocation of AP-1 and NF-κB in a dose-dependent manner (Fig. 6E). The effect of surfactin on the nuclear translocation of NF-κB p65 was confirmed by immunofluorescence confocal microscopy (Fig. 6F).

In unstimulated cells, NF-κB is present in the cytosol bound to IκB. In response to stimulation, IκBs are rapidly phosphorylated by IκB kinases (IKKs) and then ubiquitinated and degraded by the 26S proteasome complex. The free NF-κB dimers translocate to the nucleus, bind to the κB motif of the target genes and stimulate their transcription. Since IκB phosphorylation and degradation is the predominant pathway for NF-κB activation, we determined the levels of IκB-α and the phosphorylation of IκB-α proteins in cytosolic extract. Whereas the phosphorylation and degradation of IκB-α were stimulated by TPA, surfactin suppressed these effects (Fig. 6E). Surfactin also had similar inhibitory effects on NF-κB and AP-1 activities in the MDA-MB-231 cells (data not shown). These results indicate that surfactin inhibits TPA-stimulated AP-1 and NF-κB activities in breast cancer cells.

MMP-9 gene expression may be activated by a number of signal transduction pathways, including those involving PI-3K/Akt and mitogen-activated protein kinases (MAPKs) such as ERK, c-Jun N-terminal kinase (JNK) and p38, which are also upstream modulators of AP-1 or NF-κB (23–26). To evaluate the effect of surfactin on these signaling pathways, we monitored the activities of these kinases by examining their phosphorylated form. While TPA increased the level of PI-3K/Akt, ERK, JNK and p38 MAPK phosphorylation, surfactin specifically inhi bited the TPA-induced phosphorylation of PI-3K/Akt and ERK in a dose-dependent manner, but not that of JNK and p38 (Fig. 7A). The phosphorylation of theses kinases was not affected by surfactin alone (Fig. 7B). To confirm the signal transduction pathways involved in TPA-stimulated MMP-9 expression, the effects of specific kinase inhibitors on the expression of MMP-9 in MCF-7 cells were analyzed by gelatin zymography and western blot analysis. The TPA-induced MMP-9 expression and secretion were inhibited by inhibitors of PI-3K/Akt (LY294002), ERK (PD98059), JNK (SP600125) and p38 (SB203580) (Fig. 7C). Surfactin had similar inhibitory effects on PI-3K/Akt and ERK activities in MDA-MB-231 cells (data not shown).