Wogonin, whose molecular formula is C₁₆H₁₂O₅ (Fig. 1A), was isolated from Scutellariae radix. An air-dried material (60 g) was extracted with hot methanol. Following concentration, the crude extract was chromatographed on silica gel to yield a crude crystalline fraction, which was recrystallized from methanol to yield 5,7-dihydroxy-8-methoxyflavone (wogonin; 350 mg) (26). Wogonin was kindly provided by Dr Ahn (Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea). Unless otherwise indicated, samples containing wogonin of ≥98% purity were used in all experiments. Prior to experiments, wogonin was dissolved in dimethyl sulfoxide (DMSO) and diluted with medium. The final DMSO concentration did not exceed 0.1% throughout the study. Fetal bovine serum (FBS) and RPMI-1640 were obtained from Thermo Fisher Scientific, Inc. (Waltham, MA, USA), and MK886, U75302 and LY255283 were acquired from Cayman Chemical Co. (Ann Arbor, MI, USA). LPS (Escherichia coli serotype O55:B5), bovine serum albumin, and DMSO were acquired from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Antibodies to 5-LO were obtained from BD Transduction Laboratories, Inc. (BD Biosciences, Franklin Lakes, NJ, USA), and antibodies to phosphorylated (p-) ERK, ERK, MMP-9 and β-actin were acquired from Cell Signaling Technology, Inc. (Danvers, MA, USA). All other chemicals were obtained from standard sources and were of molecular biological grade or higher.

The human breast cancer cell line MDA-MB-231 was obtained from the Korean Cell Line Bank (Seoul, Korea). The MDA-MB-231 cells were maintained in RPMI-1640 containing 10% heat-inactivated FBS and antibiotic-antimycotic solution (Thermo Fisher Scientific, Inc.) at 37°C in a humidified 5% CO₂ atmosphere.

Cell viability was assessed by cell counting assays. Briefly, 5×10⁴ cells per well were plated in a 12-well plate and incubated in complete culture medium prior to drug exposure. After 24 or 48 h of treatment, aliquots were removed, and viable cells were counted in triplicate by trypan blue (Sigma-Aldrich; Merck KGaA) exclusion. Based on the results of the cytotoxicity assay, a nontoxic concentration of wogonin was selected to use on the MDA-MB-231 cells in subsequent experiments.

Total RNA was extracted from the cells with Easy-Blue (Intron, Sungnam, Korea), according to the manufacturer's instructions. A portion (2 μg) of the RNA was subjected to reverse transcription with M-MLV reverse transcriptase (Beams Bio, Gyunggi, Korea), and then, semi-quantitative PCR analysis was performed with a PCR PreMix kit (Intron, Sungnam, Korea) and specific forward and reverse primers (Genotech, Daejeon, Korea), according to the manufacturer's instructions. The sequences of the primers used for PCR amplification are listed in Table I. The specificity of all primers was confirmed by sequencing the PCR products. All data were normalized to corresponding data for GAPDH. The thermocycling conditions were as follows: Initial denaturation at 95°C for 5 min, followed by amplification (number of cycles, temperature and duration per step for each gene are listed in Table I), and a final extension phase at 72°C for 10 min. The reaction products were separated by electrophoresis on a 1.5% agarose gel, stained with EtBr, and then visualized by a ChemiDoc™ XRS+ System (Bio-Rad Laboratories, Hercules, CA, USA).

The cells were washed with ice-cold PBS, scraped into a lysis buffer [20 mM Tris-HCl, pH 7.5; 150 mM NaCl; 0.5% Nonidet P-40; 5 mM EDTA; 1% Triton X-100; and protease inhibitors (100 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 2 μg/ml leupeptin, and 2 μg/ml aprotinin)], maintained at 4°C and then heated at 95°C for 5 min. Protein concentrations were determined using the Bradford method. A total of 50 μg protein was loaded into each lane, and then, the proteins were separated by 8% SDS-PAGE prior to being transferred electrophoretically to a polyvinyli-dene fluoride membrane at 100 V for 60 min. The membrane was exposed to TBS containing 0.05% Tween-20 and 5% dried nonfat milk for 1 h and then incubated with primary antibodies against 5-LO (cat. no. 610695; 1:1,000; BD Transduction Laboratories Inc.; BD Biosciences), MMP-9 (cat. no. CST 3852), p-ERK (cat. no. 9101S), total ERK (cat. no. 9102) and β-actin (cat. no. 4970S) (1:1,000; all from Cell Signaling Technology, Inc.). Secondary antibodies were anti-rabbit (cat. no. 7074S; 1:2,000) or anti-mouse antibodies (cat. no. 7076S, 1:2,000) both from Cell Signaling Technology, Inc., and then, the proteins were visualized using enhanced chemiluminescence reagent (Amersham; GE Healthcare, Chicago, IL, USA), according to the manufacturer's recommendations.

BLT2-specific (5′-CCACGCAGUCAACCUUCUG-3′) (27) and pre-designed control (scrambled) small interfering (si) RNAs (cat. no. SN-1003) were obtained from Bioneer (Daejeon, Korea). The siRNAs were introduced into the cells by transfection in Opti-MEM (cat. no. 31985070; Thermo Fisher Scientific, Inc.) for the indicated times using Oligofectamine (cat. no. 12252-011; Thermo Fisher Scientific, Inc.).

The invasive potential of MDA-MB-231 cells was assessed using BioCoat Matrigel Invasion Chambers (BD Biosciences), as described (5,13). Cells (3.5×10⁴) were harvested with RPMI-1640 supplemented with 0.5% FBS and seeded in rehydrated Matrigel inserts containing the same media. RPMI-1640 supplemented with 5% FBS, which served as a chemoattractant, was added to the lower chamber. MDA-MB-231 cells were incubated at 37°C for approximately 24 h, after which the cells on the upper surface of each filter were removed, and the remaining cells were fixed in methanol and stained with hematoxylin-eosin (H&E). The cells in 10 randomly selected high-power (×40) fields were then counted with a CKX41 microscope (Olympus Corporation, Tokyo, Japan) equipped with a DP71 digital camera (Olympus Corporation). Each sample was assayed in triplicate.

Conditioned media were immediately frozen and lyophilized. ELISA kits for human IL-8 and LTB4 were obtained from BD Biosciences (cat. no. 550799) and Enzo Life Sciences (cat. no. ADI-900-068; Farmingdale, NY, USA), respectively. Each assay procedure was conducted according to the manufacturer's instructions for each kit. The concentrations of the mediators were determined at 450 nm for IL-8 and 405 nm for LTB4, using an Epoch microplate spectrophotometer (BioTek Instruments, Winooski, VT, USA).

This study was approved by the Ethics Committee of Korea University, and all experimental animals used herein were handled according to the guidelines approved by the Institutional Animal Care and Use Committee of Korea University. All animals were maintained under a 12-h light/dark cycle and housed at a density of 5 mice per static polycarbonate microisolator cage on disposable bedding. Wire-lid food hoppers within the cages were filled to capacity with rodent chow, and water was supplied by a bottle. For the spontaneous metastasis assays, cultured MDA-MB-231 cells were treated with LY255283 (10 μM), wogonin (20 μM) or DMSO, prior to being treated with LPS (1 μg/ml) for 24 h, as described previously (13). Then, six-week-old female nude (BALB/C) mice (Daehan Biolink, Chungbuk, Korea) were injected unilaterally in the fourth right mammary fat pad with cultured MDA-MB-231 (3.5×10⁶) cells in 100 μl of PBS; the cells were injected subcutaneously at the base of the nipple. Wogonin (20 mg/kg), LY255283 (2.5 mg/kg) or DMSO was injected intraperitoneally three times at 5-day intervals beginning immediately following cell implantation. The animals were sacrificed 14 weeks post-cell implantation, and the number of metastatic nodules on the surface of the small bowel was determined.

The data are representative of three independent experiments, and the results are presented as the mean ± standard deviation. Comparisons between groups were performed with one-way analysis of variance, followed by Tukey's post-hoc test. SPSS software (IBM SPSS Statistics for Windows, version 21.0; IBM Corp., Armonk, NY, USA) was used for statistical analysis. P<0.05 was considered to indicate a statistically significant difference.

Before studying the anti-invasive activity of wogonin, the effect of wogonin on cell viability was assessed. MDA-MB-231 cells were treated with wogonin at the indicated concentrations (10 or 20 μM). The viability of MDA-MB-231 cells was not significantly affected in 24 or 48 h of treatment with wogonin at concentrations up to 20 μM (Fig. 1B), suggesting that wogonin exhibits no cytotoxicity at these doses in MDA-MB-231 cells. To determine whether wogonin has any effect on the LPS-enhanced invasive potential of MDA-MB-231 cells, Matrigel-coated Transwell chambers were used. Similar to previous studies (13), MDA-MB-231 cell invasiveness was increased in the LPS-treated group compared with the control group (Fig. 2A). However, wogonin significantly suppressed LPS-induced invasiveness in a dose-dependent manner. Specifically, treatment with wogonin at 10 and 20 μM inhibited cell invasion by ~98% compared with the control (Fig. 2A). Previous studies have demonstrated that IL-8 and MMP-9 expression is required for breast cancer cell invasiveness and metastasis (5,13,28). To understand the signaling mechanism by which wogonin suppressed the LPS-induced invasiveness of the MDA-MB-231 cells, the mRNA expression levels of IL-8 and MMP-9 were examined by RT-PCR (Fig. 2B). IL-8 and MMP-9 expression was indeed markedly increased by LPS treatment, and 20 μM wogonin clearly suppressed LPS-induced IL-8 and MMP-9 overexpression, in a dose-dependent manner (Fig. 2B). This effect was also confirmed at the protein level, by ELISA for IL-8 (Fig. 2C) and by western blotting for MMP-9 (Fig. 2D). Taken together, these results suggest that wogonin attenuated cell invasiveness by downregulating the expression of IL-8 and MMP-9 in LPS-stimulated MDA-MB-231 cells.

Next, the results demonstrated that LPS stimulation resulted in upregulated BLT2 mRNA levels in MDA-MB-231 breast cancer cells (Fig. 3A), consistent with previous reports (13). Semiquantitative RT-PCR revealed that LPS markedly increased BLT2 mRNA levels in a time-dependent manner, but had little impact on BLT1 expression (Fig. 3A). Thus, the hypothesis that wogonin may suppress BLT2 expression was examined. Wogonin clearly suppressed LPS-induced BLT2 mRNA expression in a dose-dependent manner (Fig. 3B), but did not have an effect on BLT1 expression (Fig. 3C). Previous reports suggested that the BLT2 cascade contributes to the production of IL-8 and MMP-9 (3,13,29). Furthermore, BLT2 depletion using siRNA has been reported to attenuate the LPS-induced invasive activity of MDA-MB-231 cells (13). In agreement with these results, BLT2 depletion using siRNA, or the selective BLT2 antagonist LY255283, clearly attenuated LPS-induced IL-8 and MMP-9 mRNA and protein expression in MDA-MB-231 cells (Fig. 3D–F); however, the BLT1 antagonist U75302 did not exert such effects (data not shown). These results suggest that BLT2 may be the effector for the wogonin anti-invasion activity.

Recent studies have suggested that 5-LO expression was increased in response to LPS stimulation in MDA-MB-231 cells (13,30). Thus, whether wogonin affects the expression of 5-LO and its metabolite, LTB4, was examined in LPS-treated MDA-MB-231 cells. 5-LO expression was markedly inhibited by wogonin treatment in LPS-stimulated MDA-MB-231 cells (Fig. 4A). In addition, wogonin significantly inhibited LPS-induced production of the 5-LO metabolite LTB4 (Fig. 4B). Under the same experimental conditions, LPS-induced IL-8 and MMP-9 expression was suppressed by pretreatment with the 5-LO-activating protein (FLAP) inhibitor, MK886 (Fig. 4C–E). Taken together, these results suggest that wogonin inhibits 5-LO and thus attenuates the production of its metabolite, LTB4, and the subsequent synthesis of IL-8 and MMP-9.

Previous reports have demonstrated that ERK lies downstream of BLT2 and upstream of MMP-9 in MDA-MB-231 cells (24,30). Exposure to LPS increased ERK phosphorylation in a time-dependent manner (Fig. 5A). By contrast, LPS-induced ERK phosphorylation was markedly inhibited by wogonin in a dose-dependent manner (Fig. 5B). Treatment with PD98059, an ERK inhibitor, suppressed LPS-stimulated IL-8 and MMP-9 expression at both the transcript (Fig. 5C) and protein levels (Fig. 5D and E) and inhibited LPS-stimulated invasiveness (Fig. 5F). These data suggest that wogonin inhibits IL-8/MMP-9 production in LPS-stimulated MDA-MB-231 cells, most likely through ERK activation.

Previously, BLT2 inhibition has been demonstrated to significantly reduce LPS-induced metastasis in an orthotopic breast cancer model (13). Thus, the effect of wogonin on breast cancer metastasis induced by LPS exposure was investigated in vivo. To study the effect of wogonin on the LPS-driven metastasis of MDA-MB-231 cells in vivo, the mouse orthotopic injection tumor metastasis model was used. MDA-MB-231 cells were pretreated with wogonin (20 μM) or DMSO, and then, they were treated with LPS (1 μg/ml) for 24 h prior to being implanted into the mammary fat pads of mice. At 14 weeks post-implantation, metastatic nodules were counted on the small bowel of the mice. The number of nodules in the small bowel was significantly reduced by wogonin, compared with the mice treated with LPS alone (Fig. 6A and B). Taken together, these results indicate that wogonin exerted an inhibitory effect on LPS-induced metastasis in vivo.