The human bladder cancer cell lines T24, 253J and HTB9 were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). The cisplatin-resistant cell line T24R2 was generated by serial desensitization (20). The cells were maintained in RPMI-1640 medium (Gibco; Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin (Gibco; Invitrogen) in a humidified atmosphere of 95% air and 5% CO₂ at 37°C. Motesanib was purchased from Selleck Chemicals (Houston, TX, USA; Fig. 1). It was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and diluted to obtain the working concentration. The final concentration of DMSO in the culture media was 0.1% (v/v). Media containing 0.1% DMSO were used as a control. Cisplatin was obtained from JW Pharmaceutical (Seoul, Korea).

Bladder cancer cells were seeded at 2×10³ in 96-well plates. After 24 h, the cells were incubated with motesanib, cisplatin, and a combination of the two for 48 and 72 h. At the end of the drug exposure, 10 µl of the Cell Counting Kit-8 (CCK-8) solution (Dojindo Molecular Technologies, Inc., Gaithersburg, MD, USA) was added to each well. After 4 h of incubation, the optical density of each well was measured at 450 nm using a microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA). Cell viability was calculated as the percentage of viable cells in the total population.

The synergistic effect between motesanib and cisplatin was determined based on a combination index (CI) using CalcuSyn software (version 2.1; Biosoft, Cambridge, UK). The CI indicates synergism at <1.0, antagonism at >1.0, and additive effects at 1.0.

T24 and T24R2 cells were plated at 4×10² in 6-well plates, incubated with either motesanib or cisplatin, and combined for 48 h. The cells were cultured for another 10–14 days in motesanib- and cisplatin-free medium. The colonies were fixed with methanol and stained with 0.1% crystal violet solution. The plates were photographed, and colonies >0.2 mm in diameter were counted.

T24R2 cells were cultured at 3×10⁵/60 mm dish, grown for 24 h, and then incubated with motesanib or cisplatin alone and combined for 48 h. The cells were trypsinized, fixed in 70% ethanol, and stained with propidium iodide (PI; Sigma-Aldrich; Merck KGaA) solution for 30 min at 37°C. The cell cycle distribution was determined on a FACSCalibur instrument (BD Biosciences, San Jose, CA, USA). The resultant data were analyzed with the BD CellQuest Pro software (BD Biosciences).

Total RNA from T24R2 cells was extracted using the RNeasy Mini Kit (Qiagen, Inc., Valencia, CA, USA). cDNA was produced from 1 µg RNA using the Omniscript RT kit (Qiagen, Inc.) according to the manufacturer's instructions. Real-time PCR was performed for target genes using the Power SYBR-Green PCR Master Mix (Applied Biosystems, Warrington, UK) with a 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The specific primer sequences are presented in Table I.

Cells were lysed with radio immunoprecipitation assay buffer, consisting of 50 mM Tris-HCl (pH 8.0), 150 mM sodium chloride, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and 1 mM phenylmethylsulfonyl fluoride. Protein concentrations from cell extracts were assessed using a bicinchoninic acid protein assay kit (Pierce; Thermo Fisher Scientific, Inc.). Equal amounts of total protein (30 µg) were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (8–12% SDS-PAGE) and transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked in 5% (w/v) non-fat dry milk for 1 h at room temperature, and then incubated with primary antibodies (dilution 1:1,000) against poly(ADP-ribose) polymerase (PARP, cat. no. 9542); cleaved caspases-3 (cat. no. 9664), −8 (cat. no. 9496), and −9 (cat. no. 9505); cytochrome c (cat. no. 4272); Bcl-2 (cat. no. 15071); Bad (cat. no. 9268); cyclin E1 (cat. no. 4129); phosphoinositide 3-kinase (PI3K; cat. no. 4257); phospho-PI3 kinase p85 (Tyr458)/p55 (Tyr199; cat. no. 4228); protein kinase B (Akt, cat. no. 4685); phospho-Akt (Ser473; cat. no. 4060); extracellular signal-regulated kinases (Erk; cat. no. 4695); phosphorylated-p44/42 MAPK (Erk1/2) (Thr202/Tyr204; cat. no. 4376); VEGF (cat. no. 2445); VEGFR-1 (cat. no. 2893); VEGFR-2 (cat. no. 2479) (Cell Signaling Technology, Inc., Beverly, MA, USA); and VEGFR-3 (cat. no. sc-28297) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at 4°C overnight. After incubation with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (dilution 1:10,000; cat. no. sc-516102) or anti-rabbit IgG (dilution 1:5,000; cat no. sc-2004) for 1 h, protein expression was detected with the Enhanced Chemiluminescence Western Blot substrate kit (Pierce; Thermo Fisher Scientific, Inc.). The blots were analyzed using ImageJ 1.48v software (NIH; National Institutes of Health, Bethesda, MD, USA).

The data are presented as the mean ± standard deviation of three independent experiments. Statistical analysis was performed using SPSS statistical software package (IBM SPSS statistics 20; IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference as determined by ANOVA followed by Tukey's multiple-range test.

Bladder cancer cell lines were exposed to motesanib (0, 0.1, 1, 5, 10, 25, 50, 100 and 250 µM), with cell viability assessed by CCK-8 assay. As revealed in Fig. 2, treatment with motesanib for 48 and 72 h inhibited the proliferation of bladder cancer cells in a dose-dependent manner when compared with that of non-treated cells (control). Highly sensitive cell line T24R2 was selected at 48 h for further investigation. Compared with the individual drug, the drug combination of 50 µM motesanib with 2.5 µg/ml cisplatin induced significant inhibition of cell proliferation (Fig. 3).

To evaluate the antiproliferative effect of motesanib combined with cisplatin on T24R2 cells, a clonogenic assay was performed. The colony-forming ability of T24R2 cells was significantly inhibited by 56.9 and 28.3% when treated with 50 µM motesanib or 2.5 µg/ml cisplatin only, respectively, in comparison with that of the non-treated control (Fig. 4). Particularly, the combination of 50 µM motesanib and 2.5 µg/ml cisplatin demonstrated improved suppression of clonogenic formation, compared to motesanib or cisplatin alone. These data indicated that motesanib and cisplatin treatment could synergistically suppress the proliferation of bladder cancer cells.

Flow cytomety was performed to assess changes in the cell cycle and apoptosis in bladder cancer cells. As revealed in Fig. 5, the combined treatment of 50 µM motesanib and 2.5 µg/ml cisplatin significantly increased the sub-G1 cell percentage. These results revealed that the combination treatment increased the sub-G1 population, corresponding to apoptotic cells, in T24R2 bladder cancer cells. In addition, cisplatin alone and combination treatment primarily increased the S-phase cell percentage on compared with the non-treated control. Thus it was demonstrated that the combined treatment of motesanib and cisplatin strongly altered the cell cycle progression of the bladder cancer cell line.

To examine whether VEGFR and PDGFR are affected by the combined treatment of motesanib and cisplatin, VEGFR and PDGFR mRNA expression was determined by real-time PCR. The combined treatment of motesanib and cisplatin significantly reduced the mRNA expression levels of VEGFR-1, −3 and PDGFR-α compared to those of the non-treated control (Fig. 6). VEGFR-2 mRNA expression was also decreased by motesanib alone and combination treatment of motesanib and cisplatin. However, the change was not significant. Moreover, VEGFR-1, −2, and −3 protein levels were also significantly reduced by combination treatment of motesanib and cisplatin (Fig. 7).

To confirm the antitumor effect of the combined treatment of motesanib and cisplatin, western blot analysis was performed. The expression levels of caspases-3, −8, and −9; fragmented PARP; and cytochrome c were markedly enhanced by the combined treatment of motesanib and cisplatin in T24R2 cells (Fig. 8). Furthermore, the expression of the anti-apoptotic protein Bcl-2 was markedly reduced by combined treatment in T24R2 cells, whereas the expression of the pro-apoptotic protein Bad was increased. In addition, the expression of cyclins as an index of S-phase arrest was assessed (21). The combined treatment of motesanib and cisplatin markedly decreased cyclin E1 (Fig. 9). Phosphorylation of PI3K, Akt, and Erk were significantly suppressed by the combined treatment in the bladder cancer cells. Total PI3K, Akt, and Erk did not exhibit any significant changes. In addition, both motesanib and cisplatin individually resulted in lower levels of VEGF compared to those of the non-treated control. These results indicated that the combined treatment of motesanib and cisplatin inhibited the growth of bladder cancer cells via an apoptosis-related mechanism, and disrupted cell survival by inhibiting the PI3K/Akt signaling pathway.