Arctigenin and bovine serum albumin (BSA) were bought from Santa Cruz Biotechnology (Dallas, TX, USA) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Pierce™ BCA Protein Assay Kits were purchased from Thermo Scientific (Waltham, MA, USA). Dimethyl sulfate (DMSO) was obtained from Duksan Pure Chemicals (Ansan, Korea) and protease inhibitor cocktail and phosphatase inhibitor cocktail were from GenDEPOT (Barker, TX, USA). Dulbecco's modified Εagles medium (DMEM), antimycotic/antibiotic solution and fetal bovine serum (FBS) were obtained from Welgene (Daegu, Korea), Tris-base and glycine were from BioShop Canada Inc. (Burlington, ON, Canada). Polyvinylidenefluoride (PVDF) membranes were purchased from Pall Life Sciences (Port Washington, NY, USA). Antibodies for ERK1/2 (cat. no. 4695), p-ERK1/2 (cat. no. 4370), JNK1/2 (cat. no. 9258), p-JNK1/2 (cat. no. 4668), P38 MAPK (cat. no. 8690), p-P38 MAPK (cat. no. 4511), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB; cat. no. 8242), p-NF-κB (cat. no. 3033), c-Jun (cat. no. 9265), c-Fos (cat. no. 2250), COX-2 (cat. no. 12282), GAPDH (cat. no. 5174), and histone H3 (cat. no. 14269) were obtained from Cell Signaling Technology (Beverly, MA, USA). β-actin (cat. no. sc-69879) was from Santa Cruz Biotechnology (Dallas, TX, USA); HRP-conjugated anti-rabbit IgG (cat. no. NCI1460KR) and -mouse IgG (cat. no. NCI1430OKR) from Thermo Scientific Fisher Scientific, Inc (Rockford, IL, USA); sodium dodecyl sulfate (SDS) from Amresco (VWR Life Science, Radnor, PA, USA), and 30% polyacrylamide solution from SERVA (Heidelberg, Germany). Collagen type I and matrigel were bought from Corning Life Sciences (Bedford, MA, USA), and hematoxylin and eosin were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).

4T-1 mouse TNBC cells were purchased from the Korean Cell Line Bank (Seoul, Korea) and routinely cultured in DMEM containing 10% FBS and 1% antibiotic-antimycotic solution at 37°C in a 5% CO₂ incubator. The effect of arctigenin on cell viability was evaluated using an MTT assay. Briefly, 2×10³ cells/well were seeded into 96-well plates incubated for 24 h at 37°C, treated with 0, 25, 50, 100 or 200 µM arctigenin and cultured for an additional 24, 48 or 72 h. Cell viabilities were determined by measuring absorbances at 570 nm using a Spectramax M2e (Molecular Devices, Sunnyvale, CA, USA).

Cells were seeded in 6-well plates coated with collagen type I (Corning Life Sciences, Bedford, MA, USA), grown until confluent, and scratched with a blue tip to create wounds. They were then treated with culture media containing 0, 50, 100, 150 or 200 µM arctigenin. Optical microscopic images were captured from two different areas of each well at 0 and 48 h after wounding.

The effect of arctigenin on the invasiveness of 4T-1 mouse TNBC cells was determined using transwell chambers (Corning Life Sciences) inserts in 24-well plates. Lower faces of polycarbonate filters (transwell inserts) were coated with matrigel for 1 h at 37°C, and then, 3×10⁴ cells were seeded into matrigel-coated transwell chambers and 750 µl of culture media was added to lower chambers. After 24 h, cells were treated with conditioned media containing 2 or 10% FBS and 0, 50, 100, 150 or 200 µM arctigenin and incubated at 37°C in 5% CO₂ atmosphere for 24 h. Cells that migrated across membranes were then fixed and stained using hematoxylin and eosin (H&E) and photographed under an inverted microscope at ×200.

The effect of arctigenin on MMP-9 activity was evaluated by gelatin zymography. Briefly 2×10⁵ cells/well were seeded into 6-well plates and allowed to attach for 24 h. Cells were then serum-starved for 4 h and treated with serum-free media supplemented with various concentrations of arctigenin (0, 50, 100, 150 or 200 µM) for 24 h. Conditioned media were then transferred to new conical tubes and centrifuged to remove cell debris. The supernatants were objected by 8% SDS-polyacrylamide gel electrophoresis (PAGE) containing 0.1% (v/v) gelatin under non-reducing conditions. The gel was then washed with 2.5% Triton X-100 for 1 h at room temperature to remove SDS and gelatinase reactions were performed in reaction buffer (50 mM Tris-HCl, pH 7.5, 10 mM CaCl₂, 0.04% NaN₃) at 37°C for 24 h. The gel was then stained with Coomassie staining solution (0.05% Coomassie brilliant blue R, 45% methanol, and 10% acetic acid) and destained at room temperature. Densitometric analysis was performed using ImageJ.

4T-1 mouse TNBC cells (2×10⁵ cells/well) were seeded into 6-well plates, allowed to attach for 24 h, and cultured in serum-free DMEM containing 0, 25, 50, 100, 150 or 200 µM arctigenin for an additional 24 h. The cells were then collected by trypsinization for RNA extraction, which was performed using the easy-BLUE™ Total RNA extraction kit (iNtRON Biotechnology, Inc., Sungnam, Korean). Extracted total RNA was quantified using a NanoDrop spectrophotometer (Schimazu Scientific Instruments, Kyoto, Japan), and cDNA was synthesized from 1 µg of total RNA in 1X Goscript reaction buffer containing 2 mM MgCl₂, 0.5 mM and Goscript™ Reverse Transcriptase (all from Promega, Madison, WI, USA). RT-q PCR was conducted using Q Green SYBR Green Master Mix Kits (Cellsafe, Suwon, Korea) using an Eco™ Real-Time PCR machine (Illumina, San Diego, CA, USA). cDNA amplification reactions were performed as follows: Pre-heating for 5 min at 95°C, 45 cycles at 95°C for 10 sec, 60°C for 15 sec and 72°C for 20 sec. Relative mRNA expressions were calculated automatically using the 2^−ΔΔCq method and Eco™ Software v3.1.7 (Illumina, Inc.) (43). The primer sequences used for RT-q PCR were as follows: MMP-9, forward, 5′-TGTCTGGAGATTCGACTTCA-3′ and reverse, 5′-TGAGTTCCAGGGCACACCA-3′; MMP-3, forward, 5′-CTTTGAAGCATTTGGGTTTCTCTAC-3′ and reverse, 5′-AGCTATTGCTCTTCAATATGTGGGT-3′; COX-2, forward, 5′-CCTGCTGCCCGACACCTTCA-3′ and reverse, 5′-AGCAACCCGGCCAGCAATCT-3′; β-actin, forward, 5′-CATCCGTAAAGACCTCTATGCCAAC and reverse, 5′-ATGGAGCCACCGATCCACA-3′.

4T-1 mouse TNBC cells were seeded into 6-well plates at 2×10⁵ cells/well and allowed to attach for 24 h. The cells were then serum-starved for 4 h, treated with conditioned-media containing various concentration of arctigenin (0, 25, 50, 100, 150 or 200 µM) for 24 h, and washed twice with ice-cold phosphate buffered saline. Hypertonic buffer (20 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl₂] containing protease inhibitor cocktail and phosphatase inhibitor cocktail (GenDEPOT, Barker, TX, USA) was then added to each well. The cells were detached with a rubber policeman (SPL Life Sciences, Pocheon, Korea), transferred to 1.5 ml microtubes, kept on ice for 15 min, treated with 10% NP-40 (final NP-40 concentration 0.125%) with vortex-mixing for 10 sec at the highest setting, and left on ice for 10 min. Cell mixtures were then centrifuged at 3,000 rpm for 10 min at 4°C, supernatants (cytosolic fractions) were removed and pellets were lysed with Cell Extraction Buffer (Invitrogen, Carlsbad, CA, USA) containing phosphatase and protease inhibitor cocktail for 30 min on ice, lysates were centrifuged at 14,000 × g for 30 min at 4°C, and supernatants (nuclear fractions) were collected. Cytosolic and nuclear fractions were stored at −80°C until required.

After treating 4T-1 mouse TNBC cells for 24 h with various concentration of arctigenin (0, 25, 50, 100, 150 or 200 µM), they were lysed with RIPA lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS and 2 mM ethylenediaminetetraacetic acid] (Biosesang, Seongnam, Korea) containing protease inhibitor cocktail and phosphatase inhibitor cocktail (GenDEPOT, Barker, TX, USA), and centrifuged at 13,000 rpm for 10 min at 4°C. Supernatants (whole cell lysates) were transferred to microtubes and stored at −80°C until required. Total protein in whole cell lysates was quantified using the BCA method, and same amounts of total protein in whole cell lysates were subjected to SDS-PAGE. After transferring proteins to PVDF membranes, membranes were blocked with 1% BSA in Tris-buffered saline (TBS)-Tween (50 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20) for 1 h at room temperature, probed with primary antibodies diluted 1:3,000 with 1% bovine serum albumin in TBS-Tween solution overnight at 4°C, washed three times in TBS-Tween, and reacted with secondary antibodies (dilution 1:5,000) for 1 h at room temperature in TBS-Tween. Target proteins were visualized using a homemade chemiluminescent substrate and photographed using a Luminescent Image Analyzer LAS-4000 (Fujifilm, Tokyo).

One-way analysis of variance followed by the Tukey's post hoc test was used to determine the significances of differences. The analysis was performed using SPSS Ver. 20.0 software (SPSS, Inc., Chicago, IL, USA), and results are presented as means ± SDs. Statistical significance was accepted for P-values of <0.05.

We firstly evaluated the effect of arctigenin on the viability of 4T-1 mouse TNBC cells using an MTT assay. As shown in Fig. 1A, arctigenin (100 and 200 µM at 48 and 72 h) slightly reduced cell viability. Furthermore, arctigenin inhibited cell migration and invasiveness as determined by the matrigel invasion and wound healing assays in a concentration-dependent manner, respectively. Lignans should enters into cells with simple diffusion or a low affinity transporter (44). Therefore, these results suggest arctigenin should inhibit invasion and migration by 4T-1 mouse TNBC cells and we postulated the effect was mediated via the regulation of signaling molecules by arctigenin entered into the cells with simple diffusion or a low affinity transporter.

Due to its important role on metastasis in breast cancer and the associations between MMP-9 activity and cell migration and invasiveness, we evaluated the effects of arctigenin on MMP-9 activity and its mRNA level. Arctigenin was found to reduce both the protein and mRNA levels of MMP-9 dose-dependently (Fig. 2A and B), which suggested that the suppression of MMP-9 expression by arctigenin was responsible for its inhibition of cell migration and invasion.

Active MMP-9 is produced by cleavage of the prodomain in pro-MMP-9 mediated by MMP-3 and MMP-9 and as mentioned above, its activity is also positively associated with MMP-3 activity. We found arctigenin at 150 or 200 µM suppressed MMP-3 transcription (Fig. 2C). Mehner et al showed metastatic potential and MMP-3 expression are associated in breast carcinoma (45), and Chu et al found breast cancer tumorigenesis and metastasis were prevented by MMP-3 knockdown by mir-519d (46). These reports indicate MMP-3 activity is closely linked with its gene expression. Therefore, our observation suggest arctigenin might inhibit MMP-9 activity by downregulating MMP-3 expression in 4T-1 mouse TNBC cells.

COX-2 is another important factor of metastasis in breast cancer and MMP-9 activity is positively associated with COX-2 expression. We found arctigenin dose-dependently downregulated COX-2 protein and mRNA levels (Fig. 3), which suggests that arctigenin might also reduce cancer cell growth and metastatic potential by inhibiting COX-2 expression in 4T-1 mouse TNBC cells.

Because arctigenin appeared to inhibit 4T-1 TNBC cell migration and invasion by suppressing MMP-9, MMP-3, and COX-2. Therefore, we investigated the effect of arctigenin on ERK1/2 and JNK1/2 signaling pathways, which are key regulators of the expressions of MMP-9, MMP-3, and COX-2. The results obtained showed that arctigenin inhibited the phosphorylations of ERK1/2 and JNK1/2 but not p38 MAPK (Fig. 4). Arctigenin did not change the whole cell expressions of c-Jun and c-Fos (AP-1 subunits), but attenuated their nuclear expressions and reduced AP-1 transcriptional activity (Fig. 5). Consequently, our results suggest that anti-metastatic activity of arctigenin is governed by reduction in nuclear c-Jun and c-Fos levels and associated inhibitions of the phosphorylations of ERK1/2 and JNK1/2.