Human bladder cancer cell lines T24 and UMUC-3 [American Type Culture Collection (ATCC) Manassas, VA, USA] were maintained in RPMI-1640 supplemented with 10% fetal bovine serum at 37°C in a 5% CO₂ atmosphere. WP1066 (Calbiochem, La Jolla, CA, USA) used in the present study was dissolved in 50 mM of dimethyl sulfoxide (DMSO) and stored at −20°C. The antibodies used in the present study were antibodies against phospho-STAT3 (p-STAT3, 1:1,000), STAT3 (1:1,000), phospho-ERK (p-ERK, 1:1,000), ERK (1:1,000), cleaved caspase-3 (1:1,000), cleaved PARP (1:1,000), Bcl-2 (1:1,000), Bcl-xL (1:1,000) (all from Cell Signaling Technology, Inc., Danvers, MA, USA), and β-actin (1:3,000; Chemicon International, Inc., Temecula, CA, USA). Horseradish-peroxidase-conjugated secondary antibodies and an enhanced chemiluminescence system were obtained from Amersham Pharmacia Biotech (Piscataway, NJ, USA).

Cells that had been treated with the indicated concentrations of WP1066 or the corresponding amount of DMSO for the indicated time-points were lysed in RIPA buffer (10 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 1% sodium deoxycholate, 0.1% SDS, 1.2% aprotinin, 5 µM leupeptin, 4 µM antipain, 1 mM phenylmethylsulfonyl fluoride and 0.1 mM Na₃VO₄). Equal amounts of the resulting lysates were separated using 10% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with a solution containing 5.0% skim milk, and then incubated with a primary antibody overnight at 4°C. They were then incubated with a secondary antibody coupled to horseradish peroxidase (Amersham, Arlington Heights, IL, USA). The reactive proteins were visualized using enhanced chemiluminescence according to the manufacturer's recommendations. The relative intensities of protein expression were calculated using the ImageJ program.

Following incubation overnight in 6-well plates (5×10⁴ cells/well, in triplicate), the cells were treated with WP1066 at the indicated concentrations. The total number of cells in 3 independent wells in each group were counted at the indicated time-points after incubation using a hemocytometer, and the mean value of 4 fields was recorded. The results were expressed as the mean ± SE of the total number of cells in each well in each group. Cell viability was assessed by MTS assay using the CellTiter 96 AQ_ueous Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI, USA) according to the manufacturer's instructions. Briefly, the cells were incubated overnight in 96-well plates (1.5×10⁴ cells/well), and were then treated for 24 h with the indicated concentrations of WP1066 or with the corresponding amount of WP1066-free DMSO. Two hours after adding MTS the plates were read at 490 nm on a microplate autoreader. The results were expressed as the mean optical density (OD) of the 6-well set for each group, and the plates were assessed twice with similar results.

Cells were incubated for 24 h with the indicated concentrations of WP1066 or the corresponding amount of DMSO. Propidium-iodide stained nuclear fractions were obtained and the cell cycle data were acquired using a flow cytometer with CellQuest software (Becton-Dickinson, Heidelberg, Germany). Apoptotic cells were assessed by double-staining with FITC-conjugated Annexin V and propidium-iodide. This staining was carried out using the Annexin V apoptosis detection kit (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

Cells were grown to confluence on 6-well tissue culture plates and a wound was made by scraping the middle of the cell monolayer with a P200 pipette tip. After floating cells were removed by extensive washing with ice-cold phosphate-buffered saline (PBS), fresh complete medium containing WP1066 or the corresponding amount of DMSO was added. Migration and cell movement throughout the wound area were examined after 12 h. The ImageJ program was used to estimate and compare the covered areas (17).

An invasion assay was performed using Matrigel-coated Transwell inserts (BD Biosciences, San Diego, CA, USA) with 8-µm pores in 24-well plates. Briefly, a suspension of 1×10⁵ cells in 500 µl serum-free medium was added to the insert, and 750 µl serum-free medium supplemented with WP1066 or the corresponding amount of DMSO was added to the bottom of the well. After the plates were incubated for 12 h at 37°C, the inserts were fixed in methanol, the filters were stained with 1% toluidine blue in 1% borax, and in 3 independent experiments the number of cells that invaded through the Matrigel-coated Transwell inserts was counted in at least 10 fields/well.

Gelatinolytic activity of MMP-2 and MMP-9 was analyzed by gelatin gel zymography using a commercially available kit (code no. AK45; Cosmo Bio Co., Ltd., Tokyo, Japan). In brief, following incubation overnight in 6-well plates (5×10⁴ cells/well), the cells were treated with WP1066 at the indicated concentrations in serum-free medium for 12 h, and then the conditioned medium was collected. Equal amounts of protein from the medium were separated on pre-casted gels containing gelatin. The gels were washed in denaturing buffer for 60 min at room temperature and then incubated at 37°C overnight in developing buffer. They were then stained with 0.25% Coomassie brilliant blue R-250 for 4 h at room temperature and destained in distilled water containing 30% methanol and 10% glacial acetic acid. The areas where the gelatin substrate had been degraded by gelatinases developed into white lines on a dark background. The relative intensities of gelatinolytic activity were calculated using the ImageJ program.

Results are expressed as the mean ± SE for 3 independent experiments. Variables for different groups were compared using Student's t-test or analysis of variance (ANOVA), and P<0.05 was considered to indicate a statistically significant result.

We first tested whether WP1066 inactivated STAT3 in bladder cancer cells. It has been reported that 5–10 µM WP1066 inhibited STAT3 phosphorylation effectively in other cancer cell lines (18,19), thus, we incubated T24 and UMUC-3 cells with 2.5, 5 and 10 µM WP1066 for 24 h and examined the phosphorylation of STAT3 at Tyr705. WP1066 inhibited STAT3 phosphorylation in both cell lines in a dose-dependent manner, and 10 µM WP1066 completely abolished it (Fig. 1). Conversely, phosphorylation of ERK, which often acts contrary to STAT3 (18,20,21), was instead increased by WP1066 treatment in a dose-dependent manner (Fig. 1).

We next examined the ways in which the viability of bladder cancer cells was affected when STAT3 was inhibited by WP1066. We treated T24 and UMUC-3 cells with 1–10 µM WP1066 for 24 h, and examined their viability by MTS assay. WP1066 significantly suppressed the viability of both T24 and UMUC-3 cells, the former at concentrations >2.5 µM and the latter at concentrations >1 µM (P<0.001; Fig. 2A). In addition, the number of cells present after treatment with 5 or 10 µM WP1066 for 72 h was significantly smaller than that present after incubation without WP1066 (P<0.001; Fig. 2B). We further examined the effect of WP1066 on cell cycle progression by flow cytometry. T24 and UMUC-3 cells were treated with 5 or 10 µM WP1066 for 24 h, and their cell cycles were analyzed. In both cell lines the sub-G1 population, which indicates the percentage of apoptotic cells, was significantly increased by WP1066 (P<0.001 in both cell lines; Fig. 3A). To confirm the induction of apoptosis by WP1066 treatment, T24 and UMUC-3 cells incubated for 24 h with 5 or 10 µM WP1066 were double-stained with Annexin V and propidium iodide and analyzed by flow cytometry. The percentage of the population that was Annexin-positive and propidium iodide-negative, which are apoptotic cells, was significantly greater for cells treated with WP1066 than for control cells (P<0.001 in both cell lines; Fig. 3B). STAT3 has been shown to regulate the expression of apoptosis-related proteins such as Bcl-2 and Bcl-xL (22,23). We further examined the effects of WP1066 on these apoptosis-related molecules and found that the Bcl-2 and Bcl-xL in T24 cells were decreased with treatment of WP1066, however they were not altered in UMUC-3 cells (Fig. 4). In contrast, cleaved caspase-3 and cleaved PARP were evident in both T24 and UMUC-3 cells treated with WP1066, which is also evidence that WP1066 induced apoptosis (Fig. 4).

Previous studies have shown that STAT3 promotes cell motility and invasiveness by increasing the activation of matrix metalloproteinases (MMPs), including MMP-2 and MMP-9 (24,25). To assess the effect of WP1066 on the motility and invasiveness of bladder cancer cells, we examined wound healing and used Matrigel invasion assays. Monolayers of T24 and UMUC-3 cells were scraped with the tip of a sterile pipette to create a uniform wound, and then treated with DMSO or 5 or 10 µM WP1066 for 12 h before the covered area was assessed. We chose this treatment time since no significant decrease in the total number of cells was found for either T24 or UMUC-3 cells until 12 h after WP1066 treatment. In both cell lines, however, the covered area was significantly smaller for WP1066-treated cells than control cells (Fig. 5). In addition, the Matrigel invasion assay demonstrated that the number of T24 and UMUC-3 cells invading through the chamber was significantly decreased by WP1066 (Fig. 6). We further examined the effect of WP1088 treatment on the activities of MMP-2 and MMP-9 by gelatin zymography. Treatment with 10 µM WP1066 inhibited the activity of MMP-2 and MMP-9 completely in UMUC-3 cells (Fig. 7). These results revealed that WP1066 decreased the motility and invasiveness of bladder cancer cells by inhibiting MMP activity.