Dulbecco's modified Eagle's medium (DMEM) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Penicillin-streptomycin solution and fetal bovine serum (FBS) were purchased from Hyclone (South Logan, UT, USA). Trypsin-EDTA (0.05%) was obtained from Gibco-BRL (Grand Island, NY, USA). Antibodies specific for phosphorylated STAT3 (Tyr705), STAT3, MMP9, and β-actin, together with secondary antibody reagents (goat anti-mouse and rabbit IgG-horseradish peroxidase), were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against MMP2 were purchased from Enogene Biotech Co. (NY, USA), with an anti-Jak2 antibody obtained from Millipore (Billerica, MA, USA). An anti phosphor Jak2 antibody (Tyrosine residue 1007/1008) was purchased from Cell Signaling Technology (Beverly, MA, USA). Silibinin was purchased from Sigma Co. (diluted in DMSO).

MDA-MB-231 human breast cancer cells were cultured in DMEM containing 10% FBS, 2 mM glutamine, and 100 U/ml penicillin and streptomycin. Cell culture was at 37°C, in a 5% CO₂ gassed incubator. For each experiment, cells were resuspended in medium at a density of 2.5×10⁵ cells/ml. Unless otherwise specified, cells were treated with 200 µM silibinin for 24 h.

Cell viability was assayed by MTT assay. Briefly, cells were resuspended in DMEM one day before drug treatment, at a density of 3×10³ cells per well in 96-well culture plates. Culture medium was replaced with fresh medium containing dimethyl sulfoxide (DMSO) as a vehicle control. Cells were incubated with increasing concentrations of silibinin (from 50 to 350 µM). Following drug treatment, MTT (5 mg/ml) was added, with culture dishes incubated for 4 h at 37°C. The resulting formazan product was dissolved in DMSO and its absorbance read at 550 nm on an Ultra Multifunctional Microplate Reader (Tecan, Durham, NC, USA). All measurements were performed in triplicate, with experiments repeated at least three times.

Whole cell lysates from MDA-MB-231 cells treated (or not) with silibinin were prepared on ice using radioimmunoprecipitation (RIPA) lysis buffer containing phosphatase and protease inhibitors. Cells were disrupted by aspiration through a 23-gauge needle and centrifuged at 15,000 rpm for 10 min at 4°C to remove cellular debris. Protein concentrations were measured using the Bradford method (Thermo Fisher Scientific, MA, USA). Equal amounts of protein were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose membranes. The blots were blocked for 1 h with 5% skimmed milk in TBS-T buffer. Membranes were then probed overnight at 4°C with the relevant primary antibody, followed by washing with TBS-T and incubated for 1 h with the horseradish peroxidase-conjugated secondary antibodies. Detection was performed using the ECL Plus detection kit [Amersham Pharmacia Biotech (Piscataway, NJ, USA)] and a LAS-4000 imaging device (Fujifilm, Japan). Blot stripping was with Restore western blot stripping buffer.

Total RNA was extracted using the RNeasy Mini kit and QIAprep Spin Miniprep kit (Qiagen) according to the manufacturer's protocol, with RNA quantified spectrophotometrically at 260 nm. Then, RT-PCR analyses for MMP2, MMP9, and 18S RNA were performed. cDNA was synthesized from total RNA using a first strand cDNA synthesis kit (Bioneer, Korea) and oligo d(T) primers. The RT-PCR Premix kit with primers for MMP2, MMP9, and 18S amplification, were synthesized by Bioneer (Daejeon, Korea). PCR amplification to generate a 472-bp MMP2 fragment was with the following primers: MMP2 sense, 5′-GGCCCTGTCACTCCTGAGAT-3′; antisense, 5′-GGCATCCAGGTTATCGGGGA-3′. The PCR conditions were 94°C for 5 min, and then 32 cycles of 94°C for 30 sec, 58°C for 30 sec, 72°C for 45 sec, followed by 72°C for 7 min. PCR amplification to generate a 455-bp MMP9 fragment involved the following primers and PCR conditions: MMP9 sense, 5′-CCTGCCAGTTTCCATTCATC-3′; antisense, 5′-GCCATTCACGTCGTCCTTAT-3′, 94°C for 5 min, and then 30 cycles of 94°C for 40 sec, 60°C for 40 sec, 68°C for 50 sec, followed by 72°C for 7 min. Finally, a 489-bp amplified 18S mRNA fragment was generated using the following primers and PCR conditions: 18S sense, 5′-CGGCTACCACATCCAAGGAA-3′ and antisense, 5′-CCGGCGTCCCTCTTAATC-3′, 94°C for 5 min, and then 30 cycles of 94°C for 40 sec, 60°C for 40 sec, and 68°C for 50 sec, followed by 72°C for 7 min. PCR products were resolved by electrophoresis on a 1% agarose gel with PCR products visualized by ethidium bromide staining.

STAT3 DNA binding activity was detected using EMSA (Promega Corp., Madison, WI, USA). The electrophoretic mobility shift assay (EMSA) kit, oligonucleotide probes (STAT3), and reporter lysis buffer were from Promega. MDA-MB-231 cells were grown to ~90% confluence with nuclear protein extracts prepared using the Nuclear Extract kit (Affymetrix, CA, USA). EMSA was performed using the EMSA gel shift kit (Panomics, Fremont, CA, USA), according to the manufacturer's protocol. Briefly, nuclear protein was subject to hybridization to a double stranded, biotin-labeled oligonucleotide probe, containing the consensus-binding site for STAT3 (sense strand, 5′-GATCCTTCTGGGAATTCCTAGATC-3′). The protein-DNA complexes were resolved on a 6% non-denaturing PAGE gel and then transferred to a Pall Biodyne B nylon membrane (Pall Life Sciences, Pensacola, FL, USA). Signal detection was with streptavidin-HRP and a chemiluminescent substrate.

MDA-MB-231 cells (1×10⁵) were cultured in 6-well plates, and grown to 70% confluence. Cells were then transfected with the ON-TARGETplus SMARTpool siRNA, targeting STAT3, or ON-TARGETplus non-target siRNA (Dharmacon, Chicago, IL, USA), as a control. Transfection was with the DharmaFECT transfection reagent (Dharmacon, Lafayette, CO, USA), according to the manufacturer's instructions. Following transfection for 48 h, cells were cultured in serum-free medium for a further 24 h, and then exposed to 200 µM silibinin for 24 h. Levels of STAT3, p-STAT3, MMP2, and β-actin were quantified by western blotting.

MDA-MB-231 cells were cultured in 6-well plates at a concentration of 1×10⁵ cells/well in DMEM media (i.e., serum containing), and then incubated for 24 h in a humidified chamber. After forming a confluent monolayer, the cell layer was scratched with a pipette tip and washed with PBS to remove cell debris. Cells were untreated (controls) or exposed to 100 or 200 µM silibinin. Images of the scratch sites (wounds) were captured at 0 and 24 h, and the average area of the wound calculated using ImageJ software.

The Transwell invasion assay was performed using Matrigel pre-coated, ready to use invasion chambers (BD Biocoat, MA, USA). Cells suspended at 5×10⁴ were added to the inserts. Drug-containing media was then added to the receiver plate, and the inserts placed onto it. After a 24-h incubation in a humidified chamber at 37°C, the cells that had invaded the apical surface of the inserts were identified using crystal violet. The plates were then incubated in ambient conditions for 24 h followed by a wash and then fixation using formaldehyde. The cells on the upper surface were removed using a cotton swab and the invaded cells quantified using a microscope. Cells were counted in four fields of view.

All experiments were performed at least three times with results expressed as means ± SEM. Statistical analyses were with ANOVA and Student's t-test (SAS 9.3 program). One-way analysis of variance (ANOVA) was performed with Duncan's multiple range test. A p-value of <0.05 was considered to be statistically significant.

The effect of silibinin on cell proliferation was examined by the MTT assay. The human triple-negative breast cancer cell line MDA-MB-231 was exposed to increasing concentrations of silibinin (50, 100, 150, 200, 250, 300 and 350 µM) for a period of 24 h. The number of silibinin-treated cells during the logarithmic phase of growth was compared to that of control cells. Exposure to silibinin substantially decreased viability in a dose-dependent manner, with IC₅₀ values ranging from 200 to 250 µM following a 24-h exposure (Fig. 1). Therefore, a 200 µM concentration of silibinin was taken as the IC₅₀, with this concentration used in subsequent experiments.

We previously reported that STAT3 is a potent signaling molecule associated with tumor development and progression (29). In this study, our aim was to analyze the role of silibinin in the expression of JAK2/STAT3-related proteins. Initially, MDA-MB-231 cells were exposed to silibinin for 24 h at different doses, and then for varying time periods. Whole cell lysates were prepared using 1X RIPA lysis buffer and immunoblotted using antibodies with Jak2, STAT3, and their respective phospho forms. As seen in Fig. 2A, silibinin suppressed the expression of Jak2, STAT3, and phospho proteins in a dose-dependent manner. Compared with the control group, 200 µM silibinin provoked an almost 50% reduction in the expression of these proteins (Fig. 2B). A time course experiment (15 min to 6 h) showed that the expression of STAT3 and phospho proteins continuously decreased in a time-dependent manner. Concurrent with the dose-dependent inhibition experiments, the time-dependent analysis also showed a 50% reduction in the expression and phosphorylation of Jak2/STAT3 signaling proteins (Fig. 2D). These data suggest that silibinin has the capacity to regulate the activity of the Jak2/STAT3 signaling pathway.

Recent studies have shown that STAT3 activation regulates MMP2 expression (18). Upon activation, STAT translocates to the nucleus and binds specific response elements in target gene promoters in order to exert transcriptional control. Therefore we assayed levels of STAT3 and pSTAT3 in nuclear extracts prepared from silibinin-treated and control, non-treated TNBC cells. As shown in Fig. 3A, there was a reduction in the level of STAT3 and pSTAT3 in nuclear extracts derived from the 200 µM silibinin-treated groups. We then analyzed the MMP2 gene promoter for the presence of a GAS element, the DNA-binding sequence for the STAT family of transcription factors. Having located this element [in a domain upstream of the transcription start site (TSS) (Fig. 3B)] DNA-binding was then studied using EMSA. These data revealed that exposure to silibinin provokes an inhibition of STAT3-DNA binding (Fig. 3C), indicating that silibinin, via STAT3, transcriptionally regulates MMP2 gene expression.

In the above section we showed that silibinin suppressed Jak2/STAT3 signaling in a dose- and time-dependent manner, and inhibited STAT3-DNA binding. We hypothesized that the inhibition of STAT3-DNA binding should provoke a downregulation of MMP2 gene expression. In order to confirm this, the expression of STAT3 downstream targets were assayed at both the mRNA (Fig. 4A), and protein levels (Fig. 4C). Transcriptional level analyses revealed that treatment with silibinin led to a dose-dependent decrease in the transcription of both MMP2 and MMP9 (Fig. 4A). The silibinin-treated groups manifested an ~40–60% inhibition of MMP2 mRNA expression when compared to non-treated groups (Fig. 4B). Similar to the transcriptional data, at the protein level, dose-dependent reductions in the expression of both MMP2 and MMP9 were evident (Fig. 4C) Membranes were then probed overnight at 4°C with the relevant primary antibody, followed by washing with TBS-T and incubated for 1 h with the horseradish peroxidase-conjugated secondary antibodies with silibinin-treated groups incurring a 40–60% inhibition of MMP2 protein expression (Fig. 4D).

In order to confirm crosstalk between STAT3 and MMP2 expression, STAT3 was knocked-down using an siSTAT3 construct, and our analyses repeated. STAT3 knock-down cells were exposed (or not, for controls) to silibinin for a predetermined time, then whole cell lysates prepared and analyzed for their expression of MMP2. STAT3 knock-down effectively eliminated STAT3 expression, with levels of MMP2 also markedly reduced (Fig. 5A). The relative expression of protein, with respect to vehicle controls, was performed to confirm the effect of STAT3 on the inhibition of MMP2 expression following exposure to silibinin (Fig. 5B). These data showed that silibinin could inhibit cell migration and invasion via inhibition of MMP2 in a STAT3-dependent manner.

In the previous section we showed that knock-down of STAT3 lead to a decline in the expression of MMP2. MMP2 is a major determinant of cancer invasion, including cell migration and invasion (30). The ability of silibinin to inhibit cancer cell migration was subsequently studied with the help of a wound-healing assay (Fig. 6A). Here the relative inhibition of wound closure was used as a proxy measure for migration. The non-treated control group showed a relatively high degree of cell migration and wound closure, which was diminished in the silibinin-treated group (Fig. 6B). The crossing of the extracellular membrane is an essential step for the dissemination of cancer cells from the primary tumor to distant locations. The ability of silibinin to inhibit this invasion process was studied using a Matrigel invasion assay. Silibinin-treated cells showed a promising inhibition of invasion compared to untreated controls. The role of STAT3 in silibinin inhibition of invasion was then studied using STAT3 knock-down cells. The results confirmed that silibinin and STAT3 play an important role in inhibiting the invasive capacity of TNBC cells (Fig. 6C). Compared with the control group, both 200 µM silibinin-treated, and the STAT3 knock-down group, showed a 50% reduction in migration rate (Fig. 6D). These results confirmed that silibinin inhibits cell migration and invasion by inhibiting STAT3 phosphorylation and MMP2 expression.