SV-40 immortalized human urothelial cell line (SV-HUC-1) was obtained from the Chinese Academy of Type Culture Collection Cell Bank (Shanghai, China). Curcumin was purchased from Sigma-Aldrich (St. Louis, MO, USA; purity, 99.0%). Benzidine (4,4′-diaminobiphenyl; ≥98.0%, RT), dimethyl sulfoxide (DMSO) and methanol were obtained from Merck (Reading Township, NJ, USA). Growth media (F-12K medium, Kaighn's Medium; Wisent Inc., Montreal, QC, Canada). Fetal bovine serum (FBS), phosphate-buffered saline (PBS), antibiotics and trypsin were obtained from HyClone Laboratories (Logan, UT, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich. Phosphorylated ERK5 (p-ERK5), phosphorylated c-jun (p-c- jun), phosphorylated c-fos (p-c-fos), E-cadherin, N-cadherin and vimentin were obtained from Cell Signaling Technology (Beverly, MA, USA). The antibody for ZO-1 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The GAPDH antibody was obtained from Biogot Technology (Nanjing, China). XMD8-92 was purchased from Selleck Chemicals (Huston, TX, USA). Sources of other materials are noted accordingly in the text.

SV-HUC-1 cells were cultivated with F-12K growth medium with addition of 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin under the atmosphere of 5% CO₂ at 37°C in an incubator. Cells were seeded in 10-cm² culture plates. The medium was changed every day until cells reached 80–90% confluence, and then was treated with various concentrations of benzidine, XMD8-92 or curcumin.

SV-HUC-1 cells were seeded in 96-well plate at the density of 2×10³ cells/well. Then, the medium mixed with benzidine or curcumin at different concentrations was used to cultivate cells and changed every day for 4 days (12,22,23). Ten microliters of methylthiazoletetrazolium assay solution (5 mg/ml) was added to each well and the plates were further incubated for 4 h at 37°C. Medium containing MTT was removed and the precipitants were solubilized in DMSO. Absorbance was measured at 490 nm using a microplate reader (Titertek Instruments, Incorp., Huntsville, AL, USA). All measurements were performed in triplicate.

Wound healing assay was applied to detect change of migratory ability. Medium without benzidine or within benzidine at concentration of 0.005 or 0.05 μM was used for pretreating SV-HUC-1 cells for 4 days. Then, cells were seeded in 6-well plate at 4×10⁵/well and cultivated for 24 h with serum-free medium before wounding. Afterwards, manual scrape was made to the cell monolayer to create a wound with a pipette tip. After washing the plate with PBS, each well was covered with medium to acquire images at the time-points of 0, 12, 24 and 36 h.

The invasion assays were performed in a 24-well Boyden chamber with an 8-μm pore size polycarbonate membrane (Millipore, Billerica, MA, USA) coated with Matrigel to form a matrix barrier. Pretreated cells without or with benzidine (0.005 and 0.05 μM) for 4 days, were suspended and 1×10⁴ cells in 150 μl serum-free medium were added to the upper compartment of the chamber. Moreover, 800 μl F-12K medium supplemented with 10% FBS was added into the lower compartment. After culturing in the incubator for 48 h, cells remaining on the membranes inside the chamber were wiped off with a cotton-tipped swab, while cells adhered to lower membranes were fixed with methanol stained with crystal violet and finally photographed under a microscope. Then the number of invaded cells per microscope field was counted and analyzed.

SV-HUC-1 cells were harvest after a 4-day culture. Cells were washed with cryopreserved PBS and lysed with RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA). Concentrations of extracted proteins were measured with BCA protein assay (Pierce, Rockford, IL, USA). Then, the proteins were diluted to equal concentrations, boiled for 5 min and separated by 7.5–10% SDS-PAGE, transferred onto polyvinylidene difluoride membranes (Millipore). Afterwards, membranes were blocked in 5% fat-free dry milk in Tween-20 Tris-buffered saline (TBST) and then incubated in primary antibodies and secondary antibodies. Protein bands were subsequently developed using High-sig ECL western blotting substrate kit (Tanon Science and Technology, Co., Ltd., Shanghai, China). GAPDH served as loading control.

Total RNA was isolated by RNAiso Plus according to the manufacturer's instructions. (Takara Bio, Shiga, Japan). Then, total RNA was transcribed into cDNA using the AMV reverse transcriptase (Takara) following the manufacturer's protocol. qRT-PCR was performed using the Power SYBR-Green Master Mix (Takara) and an ABI 7300 Real-Time PCR Detection system (Applied Biosystems, Foster City, CA, USA). The primers used were as follows: E-cadherin, forward, 5′-TCGACACCCGATTCAAAGTGG-3′ and reverse, 5′-TTCCAGAAACGGAGGCCTGAT-3′; ZO-1, forward, 5′-GCAGCCACAACCAATTCATAG-3′ and reverse, 5′-GCAGACGATGTTCATAGTTTC-3′; vimentin, forward, 5′-CCTTGACATTGAGATTGCCA-3′ and reverse, 5′-GTATCAACCAGAGGGAGTGA-3′; N-cadherin forward, 5′-ATCAAGTGCCATTAGCCAAG-3′ and reverse, 5′-CTGAGCAGTGAATGTTGTCA-3′; and GAPDH, forward, 5′-GCTGCCCAACGCACCGAATA-3′ and reverse, 5′-GAGTCAACGGATTTGGTCGT-3′. All of the primers were synthesized by Invitrogen (Carlsbad, CA, USA). The levels of mRNA expression for each gene were normalized by its respective GAPDH. Fold changes in gene expression were calculated by a comparative threshold cycle (Ct) method using the formula 2^−(ΔΔCt).

Pretreated SV-HUC-1 cells were transferred into 35-mm Glass bottom dishes (Cellvis, Mountain View, CA, USA) at the density of 5×10⁴/dish. After fixing and washing, dishes were stained with rabbit E-cadherin (1:150 dilution) and vimentin (1:100 dilution) antibodies at 4°C overnight and then incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit secondary antibody for 1 h. To stain the nuclei, 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) was added for 10 min. Images were captured using fluorescence microscopy (Zeiss, LSM700B; Carl Zeiss AG, Germany).

Statistical analyses were performed with the SPSS 16.0. All data were expressed as mean ± standard deviation (SD). One-way ANOVA was used for comparison of statistical differences among multiple groups, followed by the LSD significant difference test. Unpaired Student's t-test was also used for the comparison between the two groups. P-values of P<0.05 or P<0.01 were considered significantly different.

Occupational exposure to aromatic amine is a major cause of bladder cancer, and benzidine-induced EMT is important in benzidine-associated malignant generation. To investigate the cell viability change in this process, cells were treated with benzidine (0.001, 0.005, 0.01, 0.05, 0.1, 1, 5, 25 and 100 μM) for 4 days and were detected by MTT assay. The results showed that the cell viability decreased <80% when the cells were exposed to 25 μM benzidine or to higher concentrations (Fig. 1A). Therefore, concentrations range from 0.001 to 0.1 μM were selected for the following experiments.

EMT process was assessed by alterations in the cell morphology, migratory and invasive capacity, and expression levels of epithelial and mesenchymal markers. Benzidine induced morphological change from epithelial to spindle-like mesenchymal shape, as shown by morphological examination of SV-HUC-1 cells following benzidine treatment for 4 days (Fig. 1B). Migratory ability was enhanced as wound healed in the treated groups compared with the control group (Fig. 1C). In invasion assay, cells transferred through the Matrigel at about the number of 10 per field in the control group. While in treated groups, cells increased to about 12 and 30 times, respectively, indicating bezidine induced elevated invasive capacity (Fig. 1D).

Western blot analysis exhibited the decrease of protein levels of epithelial markers E-cadherin and ZO-1. On the contrary, benzidine exposure enhanced the protein expression of mesenchymal markers vimentin, N-cadherin, MMP-2 and MMP-9 (Fig. 2A). Subsequent RT-PCT of mRNA levels of epithelial and mesenchymal markers showed similar changes. Expression levels of E-cadherin mRNA at 0.005 and 0.05 were 55.3% (P<0.05), 24.1% (P<0.01). In addition, vimentin mRNA levels at 0.005 and 0.05 were 186.3% (P<0.05) and 194.2% (P<0.05) (Fig. 2B). Results of immunofluorescent staining manifested changes not only in morphology but also in protein levels of epithelial and mesenchymal markers as described (Fig. 2C). Taken together, our results suggested that benzidine could induce EMT in SV-HUC-1 cells.

ERK5 is a member of MAPKs family, which was thought to be onco- signaling. AP-1 is a downstream transcriptional factor of ERK5. To detect whether ERK5/AP-1 pathway was activated after SV-HUC-1 cells were exposed to benzidine for 4 days, protein levels of ERK5/AP-1 markers, p-ERK5, p-c-jun and p-c-fos were measured. The results showed notable increasing of p-ERK5 with dose-dependent manner and simultaneously restrained the level of total ERK5, indicating the upregulation effect of benzidine on ERK5 activity (Fig. 3A). Moreover, benzidine increased protein levels of p-c-jun and p-c-fos (Fig. 3B).

To further explore the correlation between benzidine-induced EMT and activation of ERK5/AP-1, XMD8-92 (5 μM), a highly specific ERK5 inhibitor was used. Consistent with previous research, we found the inhibitor significantly suppressed the ERK5/AP-1 activity (Fig. 4A). Then we observed effects of XMD8-92 on benzidine-induced EMT process. Results showed that inhibitor alone had no effect on cell phenotype, while benzidine-induced SV-HUC-1 morphological changes were reversed after cells were exposed to benzidine combined with the inhibitor (Fig. 4B). Inhibitor resulted in attenuation of benzidine-enhanced cell migration and invasion ability (Fig. 4C and D).

Moreover, molecular changes were detected. Results indicated that suppression of ERK5 activity leads to downregulation of epithelial markers E-cadherin, ZO-1 as well as upregulation of mesenchymal markers vimentin and N-cadherin in protein and mRNA levels (Fig. 4E and F). Collectively, our results indicated that benzidine promoted SV-HUC-1 EMT via upregulating the ERK5/AP-1 signal pathway.

To determine whether curcumin could interfere with benzidine-induced EMT, we first conducted MTT assay to screen appropriate concentration for subsequent experiments. The data showed that curcumin at doses <1 μM had no influence on cell viability, and cell viability was decreased <85% at concentrations >2.5 μM and had significance (Fig. 5A). Therefore, curcumin at 1 μM was chosen for the following experiments. Results showed that curcumin alone had no effect on cell morphology. The benzidine-mediated cell morphological changes were reversed by curcumin (Fig. 5B). Benzidine-enhanced capacity of migration and invasion were also detected to be weakened by curcumin (Fig. 5C and D).

Subsequently, we measured epithelium and mesenchymal markers at molecular levels. Results indicated that treatment with curcumin elevated protein and mRNA levels of E-cadherin and ZO-1, reduced vimentin and N-cadherin, which demonstrated the suppression of curcumin in benzidine-induced EMT (Fig. 5E and F). Furthermore, we detected ERK5/AP-1 activity as cells exposed to curcumin and found that benzidine- activated ERK5/AP-1 was inhibited by curcumin (Fig. 5G). In summary, these findings confirmed that curcumin was able to reverse benzidine-triggered SV-HUC-1 cell EMT via suppression of the ERK5/AP-1 signal pathway.