B16F10 murine melanoma cells, which are highly metastatic to the lungs of C57BL/6J mice, and HT1080 human fibrosarcoma cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM; Lonza, Walkersville, MD, USA) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (FBS) and 100 U/ml penicillin/100 μg/ml streptomycin (both from Gibco, Invitrogen, Carlsbad, CA, USA) at 37°C in a humidified 5% CO₂ incubator. For animal experiments, specific pathogen-free female C57BL/6J mice were purchased from Taconic Farms lnc. (Samtako Bio Korea, O-San, Korea) and maintained in our animal facility for 1 week before use. Mice were housed under specific pathogen-free conditions at a temperature of 24±1°C and humidity of 55±5% in a barrier facility with a 12-h light-dark cycle. Animal experimental procedures were approved by the Korea Institute of Oriental Medicine Care and Use Committee (reference nos. 12-094 and 12-111) and performed in accordance with the Korea Institute of Oriental Medicine Care Committee Guidelines.

Anti-IκBα, anti-phospho-IκBα (Ser32/36), anti-NF-κB p65, anti-MMP-9 and anti-tubulin antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-TATA sequence-binding protein (TBP) was obtained from Lifespan Biosciences, Inc. (Seattle, WA, USA). A cytotoxicity detection lactate dehydrogenase (LDH) kit and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Roche Diagnostics GmbH (Mannheim, Germany) and Sigma Chemical Co. (St. Louis, MO, USA), respectively.

The herbs for preparing KIOM-C, as mentioned above, were purchased from Korea Medicine Herbs Association (Yeongcheon, Korea). The identification of all herbs was confirmed by Professor Ki Hwan Bae of the College of Pharmacy, Chungnam National University (Daejeon, Korea), and all voucher specimens were deposited in the herb bank of the Korea Institute of Oriental Medicine (KIOM, Daejeon, Korea). A total of 2456.5 g KIOM-C formula was placed in 15 liters distilled water and then heat-extracted for 3 h at 115°C in an extractor (Cosmos-600 Extractor; Gyeonseo Co., Inchon, Korea), filtered using standard testing sieves (150-μm; Retsch, Haan, Germany) and then concentrated to dryness in a lyophilizer. Freeze-dried KIOM-C powder (50 mg) was dissolved in 1 ml distilled water, filtered through a 0.22-μm disk filter, and stored at −20°C until use.

Cytotoxicity was evaluated using MTT and LDH release assays. Briefly, cells seeded in 96-well culture plates at a density of 5×10³ cells/well were incubated with specific KIOM-C concentrations between 10 and 250 μg/ml. After a 48-h treatment, cells were incubated with 10 μl MTT solution (5 mg/ml in PBS) for an additional 4 h. Formazan precipitates were dissolved with dimethyl sulfoxide (DMSO), and then the absorbance was measured at 570 nm with the Infinite^® M200 microplate reader (Tecan Group Ltd., Switzerland). In addition, LDH release into the culture supernatant from KIOM-C-treated cells was determined by a commercial cytotoxicity detection kit according to the manufacturer’s instructions.

To determine anchorage-independent cell growth, cells (1×10⁴) suspended in 3 ml of medium containing 0.3% agar and 10% FBS were applied to the solidified bottom agar containing 0.6% agar and 10% FBS. During 3 weeks of incubation, colonies on soft agar were observed under a phase-contrast microscope and photographed.

Two hundred cells were seeded in a 12-well culture plate in 1 ml 10% FBS/DMEM and incubated to allow attachment. After adding KIOM-C at the specified concentrations, cells were incubated for 10 days, and colonies were stained with 0.2% crystal violet/20% methanol (wt/vol) solution.

Cells were pre-incubated with 25 μg/ml mitomycin C (Sigma Chemical Co.) for 30 min, and injury lines were drawn on a confluent monolayer of cells. After washing with DMEM, cells were allowed to migrate in the presence of KIOM-C, and migration was observed under a phase-contrast microscope at specific time points.

Cells were pre-incubated for 12 h in serum-free DMEM with KIOM-C at the specified concentrations and then stimulated with 5 nM PMA for an additional 24 h. The equivalent volumes of the conditioned medium were electrophoresed on an 8% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) containing 0.1% gelatin. Gels were washed thoroughly with washing buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2.5% Triton X-100) and then incubated in activation buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM CaCl₂, 0.02% NaN₃, 1 μM ZnCl₂) at 37°C. The gels were stained with Coomassie Brilliant Blue R-250 staining solution (Bio-Rad Laboratories, Hercules, CA, USA) and destained (10% isopropanol, 10% acetic acid). MMPs were detected as clear bands against a dark blue background.

The in vitro migration and invasion assays were performed using a Transwell chamber with a 10-mm diameter and an 8-μm pore size polycarbonate membrane (Corning Costar, Cambridge, MA, USA). In brief, after filling the lower chamber with 600 μl 10% FBS/DMEM, cells (1×10⁵/100 μl) in serum-free DMEM were added to each upper chamber and incubated for 12–36 h at 37°C. Cells attached to the upper surface of the filters were removed by wiping with a cotton swab, and the filters were stained with 0.2% crystal violet/20% methanol (wt/vol) solution. The invasion assay was conducted using the Transwell chamber after coating with 20 μl of a 1:2 mixture of Matrigel:DMEM (Matrigel; BD Biosciences, Bedford, MA, USA) as the intervening invasive barrier.

Whole cell lysates and nuclear/cytosolic extracts were prepared using M-PER Mammalian Protein Extraction Reagent and NE-PER Nuclear and Cytosolic Extraction Reagent (Pierce Biotechnology, Inc., Rockford, IL, USA), respectively, according to the manufacturer’s instructions. Protein concentration was determined using the bicinchoninic acid (BCA) assay. After immunoblotting, proteins were visualized using a Power Opti-ECL Western blotting detection reagent (Animal Genetics, Inc., Korea) and an ImageQuant LAS 4000 mini (GE Healthcare, Piscataway, NJ, USA). Band intensities were calculated using ImageJ software (National Institutes of Health, USA).

Total RNA was extracted using PureHelix™ RNA extraction solution and reverse transcribed to cDNA using HelixCript™ 1st Strand cDNA Synthesis kit (NanoHelix Co., Daejeon, Korea). cDNA aliquots corresponding to 1 μg RNA were analyzed by semi-quantitative PCR using the following primers; hMMP-9, 5′-TCTTCCCTGGAGACTGAGAA-3′ and 5′-GGCAAGTCTTCCGAGTAGTTT-3′; GAPDH, 5′-TCATGACCACAGTCCATGCC-3′ and 5′-TCCACCACCCTGTTGCTGTA-3′.

The female C57BL/6J mice were divided into 3 groups (n=5 for each group), and B16F10 cells (3×10⁵ cells/0.2 ml) were injected via the tail veins. The amount of KIOM-C for human adults with an average body weight of 60 kg is ~50–150 g/day, and the yield of powdered extraction is ~20.7% (wt/wt). Therefore, KIOM-C at doses of 170 or 510 mg/day/kg of body weight and saline (controls) were orally administered to mice for 17 days. The mice were sacrificed, their lungs were fixed in Bouin’s solution (Sigma) and the number of B16F10 colonies on the surface of the lung was determined by visual inspection.

Statistical significance of the difference between groups was analyzed using the Student’s t-test with the SigmaPlot 8.0 software, and a p-value <0.05 was considered to indicate a significant result. Data are presented as the means ± standard deviation (SD), and all experiments were repeated at least 3 times.

To determine the non-cytotoxic concentration of KIOM-C, B16F10 cells were treated with various KIOM-C concentrations for 48 h followed by MTT and LDH assays. Compared to the control cells, the viability and morphology did not significantly change in response to KIOM-C, suggesting that KIOM-C at a concentration ranging from 25 to 250 μg/ml was non-cytotoxic to B16F10 cells (Fig. 1A and B); thus we used this concentration range for KIOM-C in all subsequent experiments. We further investigated whether KIOM-C at non-cytotoxic doses influences the malignant phenotype in terms of anchorage-dependent colony formation at low density. As shown in Fig. 1C (upper panel), untreated control B16F10 cells displayed rapid proliferation and formed sizable colonies from one cell, whereas KIOM-C treatment suppressed colony-forming activity involving a lower number of sizable colonies and a reduced colony size in a dose-dependent manner. Anchorage-independent growth, the ability of cells to form colonies in a semi-solid medium, which is closely correlated with metastatic potential (14), was also significantly decreased in the KIOM-C-treated cells when compared to the untreated control cells by ~60–95% at concentrations of 25–250 μg/ml (Fig. 1C, lower panel). The inhibition of colony formation was not due to KIOM-C cytotoxicity, as shown in Fig. 1A and B.

To investigate the effect of KIOM-C on metastatic activity, we first assessed migration activity using a wound healing assay. Control B16F10 cells migrated across the wound area, leading to ~25 and 70% healing at 24 and 48 h, respectively, whereas KIOM-C treatment at 100 and 250 μg/ml significantly suppressed wound migration to 60 and 55% at 48 h, respectively (Fig. 2A). Next, we performed Transwell migration and invasion assays. As shown in Fig. 2B, the migration activity of serum-induced B16F10 cells was significantly decreased following KIOM-C treatment in a dose-dependent manner. In addition, the invasiveness, which is determined by the ability of cells to invade a Matrigel barrier, was also considerably suppressed in KIOM-C-treated cells. Treatment with KIOM-C at 250 μg/ml prevented serum-induced migration and invasion by ~55% compared to the untreated control cells (Fig. 2B).

To confirm the inhibitory effect of KIOM-C on tumor metastasis in vivo, we evaluated the ability of B16F10 cells to colonize the lungs of C57BL/6J mice after intravenous injection. As shown in Fig. 3, the incidence of metastatic black colonies was significantly decreased by KIOM-C administration in a dose-dependent manner. In particular, administration with 510 mg/kg KIOM-C resulted in ~71.47% inhibition of lung metastases as compared to the control group. To evaluate whether repeated administration of KIOM-C elicits systemic toxicity, mice were medicated with saline only (control) and KIOM-C at doses of 170 and 510 mg/kg. During the 2-week experimental period, the administration of KIOM-C did not cause death or abnormal behavior and did not affect weight gain in mice (Table I). Organ weight changes were not significantly different between the KIOM-C-treated groups and the controls (Table II). In addition, no critical differences in the serological parameters between KIOM-C-treated and control groups were observed (Table III). The ratios of GOT/GPT and BUN/CRE were not significantly altered in the KIOM-C-treated group when compared to levels in the control, suggesting that KIOM-C administration did not cause hepatic and renal damage. The hematological parameters of KIOM-C-treated mice were also similar to the control mice (Table IV). The numbers of RBCs and the Hb level, an indicator of RBC count balance and anemia, were not altered by KIOM-C. The numbers of WBCs and other parameters were within the normal ranges. These data indicate that KIOM-C administration efficiently suppressed pulmonary metastasis of melanoma cells when compared to the metastasis noted in the controls, but without any adverse effect during treatment based on serological and hematological findings.

We first examined the cytotoxicity of KIOM-C on HT1080 cells by MTT and determined that the non-cytotoxic concentrations ranged from 25 to 250 μg/ml which were used for subsequent experiments (Fig. 4A). Incubation with specified concentrations of KIOM-C substantially inhibited colony formation (Fig. 4B) and wound migration (Fig. 4C) in a dose-dependent manner. In particular, KIOM-C at 250 μg/ml almost completely suppressed colony formation and wound migration. As shown in Fig. 4D, the ability of HT1080 cells to migrate and invade across the Transwell was markedly increased following PMA stimulation to ~3.5- and 4.5-fold, respectively. In contrast, treatment with KIOM-C attenuated migration and invasion by ~50% when compared to the control cells under the PMA-stimulated conditions.

Since MMP-2 and -9 play essential roles in facilitating cancer metastasis by degrading the surrounding ECM (2,6), we examined whether KIOM-C modulates the expression and activity of these MMPs in order to elucidate the inhibitory mechanism of KIOM-C on migration and invasion. As shown in Fig. 5A, mRNA expression of MMP-9 was significantly decreased by KIOM-C treatment in HT1080 cells both under a resting and PMA-stimulated condition. In addition, KIOM-C treatment significantly decreased secreted MMP-9 expression and gelatinolytic activity in the resting state. PMA, a potent inducer for MMP-9 activation (15), increased MMP-9 expression and activity 15.3- and 26.1-fold, respectively, in the control cells. In the KIOM-C-treated cells, the increase in MMP-9 expression and activity in response to PMA was significantly lower than these values in the control cells (Fig. 5B and C). PMA stimulation also increased the secretion of active MMP-2 in the control cells, which was confirmed by western blotting and gelatin zymography (15); however, KIOM-C did not prevent the PMA-induced conversion of latent pro-MMP-2 into its active form (data not shown). In addition, in B16F10 cells, KIOM-C treatment also significantly decreased MMP-9 activity and MMP-9 expression (Fig. 5D). Previous reports have demonstrated that transcription factors such as NF-κB and activator protein-1 (AP-1) are significantly involved in the regulation of the MMP-9 gene expression and enhanced tumor invasion in various types of cells (15–17). To elucidate whether the inhibitory effect of KIOM-C on MMP-9 expression and invasion is linked to NF-κB activity, we examined the levels of IκBα and phospho-IκBα, along with p65 nuclear translocation. As shown in Fig. 6A, PMA stimulation in control HT1080 cells immediately increased the level of IκBα phosphorylation, accompanied by IκBα degradation. In addition, the p65 subunit was rapidly translocated from the cytosol to the nucleus following PMA stimulation (Fig. 6B). However, in the KIOM-C-treated HT1080 cells, the increase in IκBα phosphorylation and p65 nuclear translocation in response to PMA stimulation was insignificant when compared to the control cells, collectively suggesting that KIOM-C inhibits migration and invasion by reducing MMP-9 activity via suppression of NF-κB activation.