EJ human bladder carcinoma cells were purchased from ATCC (Manassas, VA, USA) and maintained in Invitrogen RPMI-1640 medium (Thermo Fisher Scientific, Inc., Carlsbad, CA, USA) with 10% fetal bovine serum (Sigma-Aldrich; Merck Millipore, Billerica, MA, USA), 1% non-essential amino acids and 1% penicillin/streptomycin in a humidified incubator at 37°C supplied with 5% CO₂.

Rottlerin (Sigma-Aldrich; Merck Millipore) was dissolved in dimethyl sulfoxide (DMSO) as a stock solution of 20 mM and stored at −20°C. Human microtubule-associated protein 1 light chain 3 (LC3; cat. no. ABC432; working dilution, 1:1,000) antibody was purchased from Sigma-Aldrich (Merck Millipore). Human caspase-3 (cat. no. ab13585; dilution, 1:1,000) and human poly (ADP-ribose) polymerase (PARP; cat. no. ab75607; dilution, 1:400) antibodies were purchased from Abcam (Cambridge, MA, USA). The human anti-β-actin antibody (cat. no. sc-130065; dilution, 1:1,000) was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Horseradish peroxidase-conjugated donkey anti-rabbit (cat. no. NA934; dilution, 1:1,000) and anti-mouse (cat. no. N1034; dilution, 1:10,000) IgG secondary antibodies were obtained from GE Healthcare Life Sciences (Uppsala, Sweden).

Cell viability was measured by MTT assay. EJ cells (5,000/well) were seeded into 96-well plates and cultured in a CO₂ incubator overnight. Rottlerin at various concentrations was then added. Following incubation periods of 24, 48 and 72 h, 10 µl of MTT solution (Sigma-Aldrich; Merck Millipore), dissolved in autoclaved phosphate-buffered saline (PBS) at 5 mg/ml, was added. After 4 h, 150 µl of DMSO was added to dissolve the formazan crystals, and the absorbance (optical density; OD) of each well was measured using an automatic microplate reader (Thermo Fisher Scientific, Inc.) at 490 nm. Each experiment was repeated three times. Cell viability results were calculated as a percentage, as follows: Cell viability % = (OD_treatment-OD_blank)/(OD_control-OD_blank)×100.

TEM was used to determine the appearance of autophagosomes in treated and control EJ cells. Cells treated with rottlerin (2 µM) or without for 48 h were harvested by trypsinization, washed with ice-cold PBS and fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, then post-fixed in 1% osmium tetroxide buffer. Following dehydration in a graded series of ethanol, the cells were embedded in Spurr resin. Ultra-thin sections (60 nm) were cut on an ultramicrotome and were subsequently observed under a JEM-1230 transmission electron microscope.

Cells were treated with rottlerin (2 µM) and washed with ice-cold PBS at 48 h post-treatment, and cell lysate was isolated using Protein Extraction Reagent (Beyotime Institute of Biotechnology, Nantong, China). Equal amounts of cell lysate protein (30 µg) were separated by gel electrophoresis on a 6–12% gradient SDS-PAGE gel and transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat dried milk in PBS for 1 h at room temperature prior to the application of the specific primary antibodies for incubation overnight at 4°C. Subsequently the membranes were washed three times with PBS containing Tween-20, and incubated with secondary antibodies at room temperature for 1 h. The blots were detected with an ECL kit (Beyotime Institute of Biotechnology) using a Storm 840 PhosphorImager system (Molecular Dynamics, Inc., Sunnyvale, CA, USA). The images were further analyzed using Image Quant TL 8.1 software (GE Healthcare Life Sciences).

Apoptosis was analyzed by flow cytometry using propidium iodide (PI) and an annexin V-fluorescein isothiocyanate FITC staining kit (BD Biosciences, Franklin Lakes, NJ, USA). Cells were detached by trypsinization at 37°C for 5 min. Detached cells were rinsed twice with RPMI-1640 medium and cold PBS, then separated by centrifugation at 168 × g for 5 min at room temperature. Cells were resuspended in 1X binding buffer at a concentration of 1×10⁶ cells/ml. Cell/buffer solution (100 µl; 1×10⁵ cells) was transferred to a round-bottom tube and incubated with 5 µl of PI and annexin V-FITC for 15 min at room temperature in darkness. Finally, 400 µl of 1X binding buffer was added to each sample tube and evaluated by flow cytometry (FACSCalibur; BD Biosciences) according to the manufacturer's instructions.

Cells were trypsinized, washed twice with cold PBS, fixed in ice-cold 70% ethanol and stored at 4°C. Prior to analysis, cells were washed again with PBS, suspended in 50 g/ml PI and 0.25 g/ml RNase A, and further incubated for 30 min in the dark. Cytometric analyses were performed using flow cytometer and CellQuest v1.0.2 software (BD Biosciences). For each determination, ~10,000 cells were counted.

The clonogenic assay was performed to evaluate in vitro cell survival following treatment with rottlerin. Cells were seeded in 6-well plates (2×10⁵/well) and treated with rottlerin (2 µM) at 24 h after seeding. Untreated cells were used as a negative control for this analysis. After a further 24 h incubation, cells were trypsinized and washed once with PBS, then counted and seeded in 6-well plates (200 cells/well). Each experiment was repeated three times. All experimental samples were incubated in an environment of 5% CO₂ at 37°C for 14 days to form appropriately large clones consisting of ≥50 cells. Cells were subsequently stained with crystal violet staining solution (Beyotime Institute of Biotechnology). The visible colonies (≥50 cells) were counted and typical images were captured using a Nikon camera.

All statistical analyses were performed with SPSS software version 17.0 (SPSS, Inc., Chicago, IL, USA). Results are presented as the mean ± standard error of the mean. Different test conditions were compared using a one-way analysis of variance or Student's t-test, with P<0.05 considered to indicate a statistically significant difference.

Using an MTT assay, the effects of rottlerin on EJ bladder cancer cells were determined. The cells were treated with 0–16 µΜ rottlerin for 24, 48 or 72 h prior to the assessment of cell viability. As shown in Fig. 1, rottlerin had a growth inhibitory effect on EJ cells in a dose-dependent and time-dependent manner, indicating that rottlerin has antitumor effects in this cell type; however, concentrations of 0, 0.5 and 1 µΜ rottlerin had non-significant effects on the cells, and 2 µΜ was therefore selected for further experiments.

To evaluate the activation of autophagy by rottlerin, the expression levels of the autophagy-related protein LC3 were assessed by western blotting analysis and autophagosome formation was observed by TEM. During autophagosome formation, the microtubule-associated LC3-I is converted to the membrane-bound form LC3-II (17). In EJ cells, the expression level of LC3 increased following treatment with rottlerin (Fig. 2A) as compared with the control cells, and quantification of the protein expression levels demonstrated that this difference was significant (P=0.014) (Fig. 2B). These results indicate that autophagy is activated by rottlerin. To further confirm this finding, TEM studies were performed, revealing a greater amount of autophagosomes in rottlerin-treated EJ cells, whereas no autophagosomes were observed in the untreated control group (Fig. 2C). Collectively these data suggest that autophagy is induced by rottlerin in EJ cells.

To assess apoptosis, a PI and annexin V-FITC assay was performed. The results revealed that a marked increase in apoptosis occurred following a 48-h treatment (Fig. 3A). Quantification of the apoptosis rates demonstrated a significant increase in apoptosis in cells treated with rottlerin (P<0.01) (Fig. 3B). Cell cycle distribution was also analyzed by flow cytometry (Fig. 4A and B). EJ cells were predominantly blocked in G1 phase, indicating that the cell cycle distribution of EJ cells was affected by rottlerin. These results indicate that, after 48 h treatment, the percentage of cells arrested in G1 phase was increased from 54.4±2.5 to 69.7±1.1% (P=0.005).

Rottlerin induces apoptosis in certain cancer cell lines (18), and the activation of a caspase cascade is important in apoptotic induction and execution (19). To confirm the involvement of caspases in rottlerin-induced apoptosis, the expression levels of pro-caspase-3 and PARP were measured by western blot. The results indicated that pro-caspase-3 and PARP levels were not increased in rottlerin-treated cells (Fig. 5A-C), and the cleaved forms of caspase-3 and PARP were also not detected (Fig. 5A). These findings indicate that caspases are not involved in rottlerin-induced apoptosis in EJ cells.

As shown in Fig. 6, EJ cells exposed to rottlerin 48 h treatment exhibited a prominent reduction in the clonogenic capacity compared with untreated cells (P=0.003).