Human BCa cell lines T24 and J82 were obtained from the ATCC (American Type Culture Collection, Manassas, VA, USA) and cultured in DMEM supplemented by 10% FBS (Invitrogen, Carlsbad, CA, USA). Cells were cultured in incubators (Thermo Scientific, Germany) in an atmosphere with 5% CO₂ at 37°C.

To obtain stable, cisplatin-resistant cell lines, we monitored the IC₅₀ of cisplatin and obtained values of 58 µM for T24 and 49 µM for J82. Second, the cisplatin-resistant index (RI) was evaluated by MTT; the RIs of the cell lines were 21.35 and 28.75 for T24 and J82, respectively. The cultured parental T24 and J82 cells were supplemented with 20 µM cisplatin. The medium was refreshed every two days to remove the dead cells, and the cells were washed three times with sterile PBS (pH 7.2). This treatment was administered for more than three months to obtain stable cisplatin-resistant T24/J82 cells (tagged with T24^R/J82^R).

To inhibit NF-κB signaling, pyrrolidine dithiocarbamate (PDTC) (Sigma-Aldrich, USA) (19), an inhibitor of the NF-κB pathway, was used; the final concentration was 10 µM in the medium for the last 24 h before analysis.

Wound healing assays were carried out by scratching a 6-well dish with a 10-µl pipette tip when the dish was at 80% confluence (including parental cell and cisplatin-resistant cell lines). The width of the scratches was compared at 0, 6, 12 and 24 h after scratching.

Pretreated cells were harvested at 80% confluency and washed three times with cold PBS. Total cellular protein lysates were prepared with RIPA buffer [50 mM Tris (pH 8.0), 150 mM NaCl, 0.1% SDS, 1% NP40 and 0.5% sodium deoxycholate] containing proteinase inhibitors [1% inhibitor cocktail and 1 mM PMSF, both from Sigma, (St. Louis, MO, USA)]. Then, 30 µg of protein was separated on 10% SDS-PAGE gels and transferred to nitrocellulose membranes. The membranes were blocked at room temperature for 1 h with 5% skim milk in Tris-buffered saline (pH 7.6, TBS). Polyclonal primary antibodies were applied at different dilutions (Table I) in 5% skim milk in TBS at 4°C overnight, followed by TBST (with Tween-20) washes. Membranes were incubated with fluorescent secondary antibodies (LI-COR, Rockford, IL, USA) coupled to the first antibody at room temperature in the dark for 1 h, then washed with TBST, dried with neutral absorbent paper and scanned with the Odyssey Detection system (lI-COR). MG-132 (Sigma-Aldrich) was used to inhibit the proteasome-dependent degradation when necessary (10 µM, 4 h before the protein harvest). GAPDH was used as a loading control (for total cell fraction).

Cellular total RNA was isolated using TRIzol reagent (Invitrogen) and quantified by absorbance at 260 nm. RNA (2 µg) was reverse transcribed using Revert Aid™ First Strand cDNA Synthesis kit (MBI Fermentas, St. Leon-Rot, Germany) strictly according to the manufacturer's protocol. For real-time PCR, we used the SYBR Premix Ex Taq™ II system (Takara Biotechnology Co., ltd., Dalian, China) and the Bio-Rad CFX96TM Real-time system (Bio-Rad, CA, USA). Primers are listed in Table II. Briefly, 12.5 µl of SYBR Premix Ex Taq II, 1 µl of primer (F and R, respectively), 200 ng of cDNA and 9.5 µl of distilled and deionized water were mixed together, followed by two-stage, pre-denaturation at 95°C, 30 sec, one repeat; and PCR reaction, at 95°C, 5 sec followed by 60°C, 30 sec, 30 repeats; and the third stage as dissociation, 95°C, 15 sec followed by 60°C, 30 sec, and another 95°C, 15 sec. GAPDH was used as the loading control.

Cell viability was assessed using a tetrazolium-based assay. Pretreated cells were incubated in the absence or presence of cisplatin/doxorubicin for the indicated times, and then washed once with PBS and incubated with 0.5 mg/ml of MTT at 37°C for 1 h. The reagent was reduced by living cells to form an insoluble blue formazan product. After incubation, cells were lysed with DMSO. Colorimetric analysis using a 96-well microplate reader was performed at a wavelength of 490 nm. The experiments were performed in triplicate.

Migration/invasion ability was demonstrated by Boyden chamber assay. Chambers with 8-µm-diameter pores were obtained from Millipore (Millipore, Switzerland). For the migration assay, 0.2 ml of FBS-free DMEM medium suspension with 10,000 cells was added to the upper chamber in a 24-well plate, and 0.8 ml of FBS-free DMEM was added to the lower chamber. After 12 h of incubation, the chambers were washed with PBS (pH 7.4) three times to remove the cells in the upper chamber, fixed with 4% formalin for 15 min, and then stained with crystal violet (0.01% in ethanol) for 25 min followed by washing three times with PBS. The cells were counted using an inverted microscope, five images were randomly taken at 200× magnification, and the average number of cells was analyzed. For the invasion assay, the cell suspension (10,000 cells/well) in the upper chamber contained 0.2 ml mixture of FBS-free DMEM/Matrigel at an 8/1 ratio (Matrigel, Sigma, USA). Cells were incubated for 36 h, and the remainder of the protocol was conducted in a similar manner to the migration assay.

A 5-bromo-2-deoxyuridine (BrdU) incorporation assay was used to analyze tumor proliferative ability. Briefly, pretreated cells were plated on 8-well glass plates (Millipore) until 50–70% confluency. BrdU was added to the medium (3 µg/ml), followed by 4 h of incubation and rinsing 3X with PBS over 10 min to remove residual free BrdU. Cells were then fixed with 4% paraformaldehyde for 45 min, followed by rinsing 5X with PBS over 20 min. Then, 0.1% Triton X-100 was used to permeabilize the cell membrane for 15 min, and 2 N HCl was added for 25 min to separate DNA into single strands and thus allowing primary antibody access to the incorporated BrdU. Cells were then rinsed 3X with PBS over 10 min, and non-specific epitopes were blocked by 10% BSA for 20 min. Anti-BrdU antibody (1:200) in 10% BSA was added and incubated overnight at 4°C. Cells were rinsed 5X with PBS, followed by incubation with TRITC-labeled secondary antibody for 1 h at room temperature, and finally rinsed 3X with PBS to remove the free antibody. The fluorescence intensity of TRITC was monitored with a SuperMicro Orifice Plate spectrophotometer (BioTek, USA) at 547 nm.

After the designated treatment, the pretreated cells were washed three times with cold PBS (pH 7.4), followed by fixing with 4% paraformaldehyde for 15 min, permeabilization in 0.5% Triton X-100 for 10 min, and incubation in 1% BSA blocking solution for 1 h. Fixed cells were incubated overnight at 4°C with rabbit anti-human-P65 in 1% BSA. Cells were washed and incubated with mouse anti-rabbit TRITC (Red) IgG antibody (Santa Cruz Biotechnology, USA) diluted 1:100 in blocking buffer for 1 h. Nuclei were stained with DAPI for 5 min. Cells were examined with a fluorescence microscope equipped with narrow bandpass excitation filters to individually select for red and blue fluorescence. Cells were observed through the Image-Pro Plus system™ mounted on a fluorescent microscope (Olympus, Japan). Each experiment was repeated three times.

ANOVA test was used to analyze the statistical discrepancy in >3 groups. Student's t-test was used to detect any statistically significant difference between 2 groups. P-values <0.05 were considered significant.

Chemoresistance is considered a vital obstacle in the battle against BCa and leads to the failure of BCa chemotherapy (20). Cisplatin, which is one of the major reagents in the chemotherapeutic regime for BCa, is recommended as first-line treatment in the clinic (21). Therefore, cisplatin resistance is ubiquitous in BCa patients, and dissecting the underlying mechanism potentially brings benefits to BCa patients. Cisplatin was used to treat BCa cell lines T24/J82 to obtain stable cisplatin-resistant cell lines (tagged by T24^R and J82^R, respectively), as indicated in the Materials and methods. Compared with parental T24/J82, T24^R/J82^R manifested cisplatin-resistance demonstrated by MTT assay (Fig. 1A). In addition, the in vitro analysis suggested that the wound-healing time was shorter for these cisplatin-resistant BCa cells (Fig. 1B), accompanied by enhanced proliferation (Fig. 1C) and migration/invasion (Fig. 1E) ability.

The enhanced capacity of migration/invasion and proliferation of T24^R/J82^R prompted us to monitor the expression of related genes, including EMT markers and matrix metalloproteinase (MMP). Both western blot analysis (Fig. 2A) and real-time PCR (Fig. 2B) indicated the elevated expression of EMT-related markers, e.g., N-cadherin, vimentin, MMP2 and MMP9, with decreased expression of E-cadherin. ABCB1, also called MDR1, plays a vital role in BCa chemoresistance (20). Therefore, the expression of ABCB1 was monitored in T24^R/J82^R by western blot analysis and real-time PCR. ABCB1 was elevated in T24^R/J82^R vs. T24/J82, as shown by blot analysis (Fig. 2C) and real-time PCR (Fig. 2B).

Emerging evidence noted the importance of NF-κB signaling in tumorigenesis and cancer metastasis (22,23), as well as chemoresistance (24), leading us to link the cisplatin-induced phenomenon to this signaling pathway. As indicated in Fig. 2D, immunofluorescence staining suggested that T24^R/J82^R cells significantly manifested P65 nuclear translocation, suggesting the activation of this signaling pathway. In a parallel experiment, nuclear lysates from T24^R/J82^R indicated the accumulation of P65 in nuclei, as demonstrated by blot analysis (data not shown). Thus, we concluded that NF-κB signaling was overactivated in T24^R/J82^R cells vs. parental T24/J82 cells.

Silibinin has been used clinically to treat various liver diseases and has been marketed as a dietary supplement (25). Previous studies had noted that silibinin inhibited tumor growth by suppressing MMPs (26,27), VEGF, HIF-1α (25) and the process of EMT (28,29). In the present study, silibinin was used to treat BCa cell T24^R/J82^R, followed by Boyden chamber assay, BrdU incorporation and immunofluorescence staining to observe the malignant behavior. As expected, the enhanced proliferation capacity of T24^R/J82^R cells was attenuated in the presence of 100 µM silibinin (Fig. 3B), accompanied by decreased migration/invasion ability (Fig. 3C and E). In addition, the nuclear translocation of NF-κB was significantly inhibited in the presence of silibinin in T24^R/J82^R cells (Fig. 3D).

Previous results suggested that silibinin could inhibit cisplatin-induced migration/invasion and proliferation. In addition, immunofluorescence staining indicated that silibinin could inhibit the activity of the NF-κB signaling pathway. According to the previous studies, this phenomenon was accompanied by an alteration of related genes and might be involved in a related signaling pathway. Therefore, we monitored the expression of EMT-related markers and ABCB1 in the presence of silibinin. As indicated in Fig. 4A and B, silibinin treatment led to significant inhibition of EMT, e.g., decreased expression of vimentin, N-cadherin, MMP2, and MMP9, with increased E-cadherin expression. In addition, silibinin suppressed the expression of ABCB1 in T24^R/J82^R (Fig. 4C and D). As indicated in Fig. 4, the effects of silibinin on T24^R/J82^R manifested in a dose-dependent manner, at doses from 100 µM, 200 µM to 400 µM, but revealed the most powerful role of PDTC.

Previous reports indicated that forced inhibition of NF-κB signaling in BCa cells resulted in the reversal of EMT (30). Herein, in parallel, we used a specific inhibitor of NF-κB signal to repeat this process. In accordance with previous reports, our data indicated that PDTC effectively attenuated the activation of NF-κB signaling (data not shown), accompanied by decreased expression of EMT-related markers (Fig. 4A and B). In addition, as mentioned above, ABCB1 was one of the target genes of NF-κB signaling, and inhibiting this pathway led to the decreased expression of ABCB1 (Fig. 4C and D).

The previous results suggested that silibinin manifested more powerful inhibitory roles on T24^R/J82^R cells than on parental T24/J82 cells. Moreover, as stated above, NF-κB signaling was inactivated in parental T24/J82 cells, suggesting that PDTC had no visible inhibitory roles on the malignancy and proliferative capacity of parental T24/J82 cells. This led us to ask whether the inhibitory roles of silibinin on T24^R/J82^R involved NF-κB signaling or whether they were unrelated phenomena. Our results suggested that in T24^R/J82^R cells, silibinin had a stronger inhibitory role than did PDTC in the tumor cell expression of EMT markers (Fig. 4A and B), ABCB1 (Fig. 4C), cisplatin sensitivity (Fig. 5A and B), proliferation capacity (Fig. 5C) and migration/invasion capacity (Fig. 5D), suggesting that the inhibitory roles of silibinin were partially independent of NF-κB signaling. Combined with the NF-κB signal inhibition induced by silibinin, we concluded that silibinin suppressed BCa cell line T24^R/J82^R in an NF-κB-dependent and -independent manner.

Our data indicated that inhibition of NF-κB signaling by PDTC in T24^R/J82^R cells led to the attenuation of malignancy and reversal of EMT. In contrast, NF-κB signaling was suppressed in parental T24/J82 cells. In parallel, as indicated in Fig. 5C and D, PDTC had no visible effect on the migration/invasion and proliferation of parental T24/J82.