MCF-7 cells were obtained from the American Type Culture Collection (Manassas, USA). The cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics. TPA and anti-β-actin antibodies were obtained from Sigma-Aldrich (St. Louis, USA). FBS, high-glucose DMEM, and phosphate-buffered saline were obtained from Gibco-BRL (Gaithersburg, USA).

The mature fruits of C. tiglium L. were purchased from Kwangmyungdang Medicinal Herbs Co., Ltd. (Ulsan, Korea) and authenticated by professor G.S.L. The fruits were powdered and extracted with 70% aqueous ethanol under reflux for 2 hours. The solvent was removed by evaporation and the extract was freeze-dried. The yield of the final extract was 11.48% (w/w).

The effect of CFE on MCF-7 cell viability was determined by using EZ-Cytox Enhanced Cell Viability Assay Kit (DOGEN, Seoul, Korea) in accordance with the manufacturer's instructions. Briefly, 3×10⁴ cells/well were treated with 1, 5, 10, 30, 50, or 100 µg/mL CFE for 24 hours. Thereafter, EZ-cytox was added (10 µL/well), followed by an incubation period of 30 minutes at 37℃. The absorbance at 450 nm was measured by using an ELISA reader (Sunrise™; Tecan, Männedorf, Switzerland).

The cells were incubated for 24 hours with TPA, which was followed by treatment with 10 or 30 µg/mL CFE for 1 hour. The cells were lysed with ice-cold radioimmunoprecipitation assay (RIPA) buffer (Thermo, Rockford, USA), and the protein lysates (20 µg) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated proteins were transferred to polyvinylidene difluoride (PVDF) membranes using Western blotting apparatus. The membranes were blocked for 2 hours with 2% bovine serum albumin or 5% skim milk and then incubated with 1 µg/mL primary antibody overnight at 4℃. The primary antibodies used for detection were: protein kinase C (PKC)α, PKCβ, PKCδ, p38, phosphorylated p38 (p-p38), c-Jun N-terminal kinase (JNK), p-JNK, extracellular signal-regulated kinase (ERK), p-ERK, p-c-Jun (Cell Signaling Technology, Beverly, USA), sodium potassium ATPase (Na-K ATPase) (Abcam, Cambridge, UK), MMP-9, p50, p65, and proliferating cell nuclear antigen (PCNA) (Santa Cruz Biotechnology, Dallas, USA). Horseradish peroxidase conjugated IgG was used as a secondary antibody and the protein expression levels were evaluated by signal analysis using a MINI HD6 image analyzer (Uvitec, Cambridge, UK).

First, the medium was collected and mixed with sample buffer. The gel for electrophoresis contained 0.1% (w/v) gelatin. The gel was washed with 2.5% Triton X-100 solution at 18℃ to 23℃ (room temperature) for 30 minutes and then incubated at 37℃ in 5 mM CaCl₂, 0.02% Brij, and 50 mM Tris-HCl (pH 7.5) for 16 hours. The gel was stained using Coomassie brilliant blue solution and then photographed using an image analyzer (Fujifilm, Tokyo, Japan). Proteolysis was identified by the presence of white zones in dark blue fields. Densitometric analysis was performed by using Multi Gauge Software for Image Analysis (Fujifilm).

Total RNA was isolated using TRIzol® Reagent (Life Technologies, Grand Island, USA). The cDNA was synthesized from 1 µg total RNA by using the PrimeScript™ RT reagent kit (TaKaRa, Shiga, Japan). The RNA expression levels of MMP-9 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were determined using the ABI PRISM 7900 sequence detection system and SYBR® Green (Applied Biosystems, Foster City, USA). The following primers (2 µM) were used: MMP-9 (NM 004994), sense 5´-CCTGGAGACCTGAGAACCAATCT-3´, antisense 5´-CCACCCGAGTGTAACCATAGC-3´; and GAPDH (NM 002046), sense 5´-ATGGAAATCCCATCACCATCTT-3´, antisense 5´-CGCCCCACTGATTTTGG-3´.

The cells were treated with CFE in the presence or absence of TPA for 3 hours and then pelleted by centrifugation at 1,800×g for 4 minutes. The isolation of the cytoplasmic and nuclear extracts was performed using the NE-PER® Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Rockford, USA).

Affinity purification of the MCF-7 nuclear protein was performed as described above. The MCF-7 nuclear protein was incubated at room temperature in 10 mM Tris-HCl, 2.5% (v/v) glycerol, 5 mM MgCl₂, 50 ng/µL poly (dI-dC), and 0.05% (v/v) Nonidet P-40 binding buffer, pH 7.5. Then, 1 ng of activator protein-1 (AP-1) electrophoretic mobility gel shift assay (EMSA) probe was added (Panomics Inc., Fremont, USA). After incubation for 20 minutes at room temperature, the samples were resolved on a 4% polyacrylamide gel in 0.5× Tris-borate buffer. The proteins were electrotransferred onto a 0.45-µm nylon membrane (Hybond-N+; Amersham, UK) at 100 V for 45 minutes at 4℃ and then crosslinked to the membrane with CL-1000 ultraviolet crosslinker (UVP Inc., Upland, USA). The blots were developed by using a Chemiluminescent Nucleic Acid Detection Module Kit (Thermo Scientific).

MCF-7 cells (5×10⁷) were pretreated with 30 µg/mL CFE for 1 hour and then incubated with TPA for 1 hour at 37℃. The membrane proteins were prepared from cells using the MEM-PER™ plus kit (Thermo Scientific).

The invasion assay was conducted in 24-well chamber dishes (8-µm pore size) coated with 20 µL of Matrigel that was rehydrated in 0.5 mL DMEM for 30 minutes prior to the experiments. Cells (2×10⁵) and chemoattractant (conditioned medium; 0.5 mL) were added to the upper and lower chamber of the dishes, respectively. After incubation, the invading cells were fixed and stained with a Toluidine blue solution. The analyzed data were presented as the mean±standard error from three individual experiments performed in triplicate.

The statistical analyses of data were performed using analysis of variance and Duncan test using the Microsoft 2010 Excel program (Microsoft Corp., Redmond, USA). Values of p<0.05 were considered statistically significant.

To evaluate the cytotoxicity of CFE on MCF-7 cells, the viability of untreated and CFE-treated cells was compared. At all tested concentrations, CFE treatment for 24 hours did not result in any significant changes in cell viability. CFE also did not affect MCF-7 cell proliferation at 100 µg/mL for up to 72 hours (Figure 1A and Supplementary Figure 1A, available online). Thus, the optimal nontoxic concentrations (10 and 30 µg/mL) of CFE were used for all subsequent experiments.

To examine the effect of CFE on TPA-induced MMP-9 expression, MCF-7 cells were analyzed by Western blot analysis, real-time quantitative polymerase chain reaction (RT-qPCR), and gelatin zymography. Western blot analysis revealed that CFE treatment blocked the upregulation of TPA-induced MMP-9 protein expression in MCF-7 cells. Zymography was performed to determine the effects of CFE on TPA-induced MMP-9 secretion and showed that TPA increased MMP-9 secretion in MCF-7 cells. However, CFE significantly diminished TPA-induced MMP-9 secretion (Figure 1B). RT-qPCR revealed that TPA induced an increase in MMP-9 expression levels in MCF-7 cells, whereas CFE blocked TPA-induced MMP-9 upregulation in a dose-dependent manner (Figure 1C). These results indicated that CFE exerted potent inhibitory effects against the TPA-induced upregulation of MMP-9 expression in MCF-7 cells.

To determine the effects of CFE on TPA-induced PKC activation, we analyzed the PKC isotype levels in the membrane fraction. CFE inhibited the membrane localization of PKCδ induced by TPA (Figure 2), which suggested that CFE inhibited TPA-induced MMP-9 expression via the suppression of PKCδ activation in MCF-7 cells.

To further understand the signaling pathway involved in the inhibitory action of CFE on TPA-induced MMP-9 activation, we used Western blotting to evaluate the effect of CFE on the TPA-induced activation of mitogen-activated protein kinase (MAPK). The treatment with CFE showed inhibitory effects on p38 and JNK, but not on ERK (Figure 3A). These results suggested that the p38 and JNK pathways were involved in the suppression of TPA-induced MMP-9 expression by CFE.

It has been reported that the MMP-9 promoter has nuclear factor-κB (NF-κB) and AP-1 binding sites; these are known to be involved in MMP-9 gene induction by TPA [1011]. To clarify the transcription mechanism by which CFE inhibited MMP-9 expression, the effect of CFE on the TPA-induced activation of NF-κB and AP-1 was evaluated using Western blotting. As shown in Figure 3B, TPA upregulated the expression of p65 and p50 (NF-κB subunits) and p-c-Jun (AP-1 subunit) in the nuclear fraction. The pretreatment of cells with CFE resulted in the inhibition of TPA-induced p-c-Jun expression, but not that of NF-κB activation. Additionally, the DNA binding activity of AP-1 was confirmed. The treatment with CFE suppressed TPA-induced AP-1 DNA binding activity (Figure 3C). These results indicated that CFE specifically blocked AP-1 activation in MCF-7 cells.

The upregulation of MMP expression is known to contribute significantly to cancer invasion [1213]. In this study, we investigated the inhibitory effects of CFE on the invasive ability of MCF-7 cells induced by TPA by performing a Matrigel invasion assay. The cells pretreated with 20 nM TPA were subsequently incubated with CFE for 24 hours. TPA-induced MCF-7 cell invasion was suppressed by CFE treatment (Figure 4). Additionally, CFE slightly inhibited the migration and invasion of MDA-MB-231 cells, a highly metastatic cell line, at nontoxic concentration (Supplementary Figure 2, available online).