Regorafenib Induces Apoptosis and Inhibits Metastatic Potential of Human Bladder Carcinoma Cells

Materials and Methods

Chemicals and agents. Regorafenib was kindly provided by Bayer Corporation (Whippany, NJ, USA). RPMI-1640 medium, fetal bovine serum (FBS), L-glutamine, and penicillin streptomycin (PS) were purchased from Gibco/Life Technologies (Carlsbad, CA, USA). 3,3’-Dihexyloxacarbocyanine Iodide (DiOC₆) was bought from Enzo Life Sciences (Farmingdale, NY, USA). RNase was bought from Fermentas (St. Leon-Rot, Baden-Wurttemberg, Germany). Propidium iodide (PI), CaspGLOW™ Fluorescein Active Caspase-3 Staining Kit, and CaspGLOW™ Red Active Caspase-8 Staining Kit were obtained from Biovision (Mountain View, CA, USA). MTT was purchased from Sigma-Aldrich (St. Louis, MO, USA). Matrigel and Transwell (8-μm pore size) were purchased from Selleck Chemicals (Houston, TX, USA) and Corning Life Sciences (Tewksbury, MA, USA), respectively. Primary antibody to X-linked inhibitor of apoptosis protein (XIAP) was bought from Thermo Fisher Scientific (Fremont, CA, USA). Primary antibody to β-actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary antibody to induced myeloid leukemia cell differentiation protein (MCL1) was bought from BioVision (Milpitas, CA, USA). Primary antibody to matrix metallopeptidase 9 (MMP9) was obtained from EMD Millipore (Billerica, MA, USA). Cellular FLICE (FADD-like IL1β-converting enzyme)-inhibitory protein (C-FLIP) were bought from Cell Signaling Technology, Inc. (Danvers, MA, USA). Secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA, USA).

Cell culture. TSGH 8301 human bladder carcinoma cells were used for this study and obtained from Professor Jing-Gung Chung at the Department of Biological Science and Technology, China Medical University, Taichung, Taiwan, ROC. TSGH 8301 cells were incubated in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37°C in a humidified incubator containing 5% CO₂ (9).

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. TSGH 8301 cells were seeded into 96-well plates at 1×10⁴ cells/well, incubated overnight, and then treated with different concentration of regorafenib (0-50 μM in 0.1% dimethylsulfoxide) for 24 and 48 h. The effect of regorafenib on cell viability of TSGH 8301 cells was evaluated with MTT assay as previously described (5).

Detection of the sub-G₁ cell population. TSGH 8301 cells were seeded into 6-well plates at 5×10⁵ cells/well, incubated overnight, and then treated with 30 μM regorafenib for 24 and 48 h. Propidium iodide (PI) buffer (40 μg/ml PI, 100 μg/ml RNase and 1% Triton X-100 in PBS) was used to identify cell-cycle distribution. The effect of regorafenib on sub-G₁ cell population was analyzed by flow cytometry (FACSCalibur; Becton-Dickinson, Franklin Lakes, NJ, USA) as previously described (8).

Detection of active caspase-3. TSGH 8301 cells were seeded into 6-well plates at 5×10⁵ cells/well incubated overnight, and then treated with 30 μM regorafenib for 24 and 48 h. Asp(OCH₃)-Glu(OCH₃)-Val-Asp(OCH₃)-fluoromethyl ketone (DEVD-FMK) was conjugated by fluorescein isothiocyanate (FITC) to make a working solution (1 μl FITC-DEVD-FMK in 300 μl PBS), which was used to monitor expression of active caspase-3. The effect of regorafenib on active caspase-3 was analyzed by using flow cytometry (FACSCalibur; Becton-Dickinson) as described by Chiang et al. (10).

Detection of active caspase-8. TSGH 8301 cells were seeded into 6-well plates at 5×10⁵ cells/well, incubated overnight, and then treated with 30 μM regorafenib for 24 and 48 h. Z-Ile-Glu(OMe)-Thr-Asp(OMe)-FMK (IETD-FMK) was conjugated by sulforhodamine (Red-IETD-FMK) to make a working solution (1 μl Red-IETD-FMK in 300 μl PBS), which was used to monitor expression of active caspase-8. Effect of regorafenib on active caspase-8 was analyzed by using flow cytometry (FACSCalibur; Becton-Dickinson) as described by Chen et al. (11).

Detection of mitochondrial membrane potential (Ψ_m). TSGH 8301 cells were seeded into 6-well plates at 5×10⁵ cells/well, incubated overnight, and then treated with 30 μM regorafenib for 24 and 48 h. DiOC₆ solution (4 μM DiOC₆ in 500 μl PBS) was used to detect ΔΨ_m. The effect of regorafenib on ΔΨ_m was evaluated by using flow cytometry (FACSCalibur; Becton-Dickinson) as described by Wang et al. (12).

Migration assay. Transwell insert with 8 μm pore size was purchased from Corning (Tewksbury, MA, USA). TSGH 8301 cells were seeded into 10 cm diameter dishes at 3×10⁶ cells, incubated overnight, and then treated with different concentration of regorafenib for 48 h. After treatment, 1×10⁶ cells were collected by centrifugation and resuspended in 1 ml serum free RPMI-1640 then 100 μl cell suspension was put into the apical chamber of the transwell insert, and then incubated for 48 h. One hundred microliters of RPMI-1640 with 10% serum was added to the basolateral chamber. After 24-h incubation, the migrated cells on the transwell membrane were fixed with a mixture of methanol and acetic acid (3:1) for 15 min and then stained by 0.5% crystal violet solution. Migrated cells were photographed under a light Nikon ECLIPSE Ti-U microscope at ×100 and then quantified using ImageJ software version 1.50 (National Institutes of Health, Bethesda, MD, USA) (13).

Invasion assay. Transwell insert with 8 μm pore size was coated with 50 μl matrigel solution (25 μl matrigel in 25 μl serum-free RPMI-1640) and incubated overnight at 37°C. TSGH 8301 cells were seeded into 10 cm diameter dishes with 3×10⁶ cells, incubated overnight, and then treated with different concentration of regorafenib for 48 h. After treatment, 1×10⁶ cells were collected by centrifugation and resuspended in 1 ml serum-free RPMI-1640 then 100 μl of cell suspension was put into the apical chamber of the transwell insert and incubated for 48 h. One hundred microliters of RPMI-1640 with 10% serum was added to the basolateral chamber. After the incubation period, matrigel on the transwell membrane was removed by sterile cotton swab. The invaded cells on the transwell membrane were fixed with mixture of methanol and acetic acid (3:1) for 15 min and then stained by 0.5% crystal violet solution. The invaded cells were photographed under a light Nikon ECLIPSE Ti-U microscope at ×100 and then quantified by using ImageJ software version 1.50 (National Institutes of Health) (14).

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Figure 1.

Effect of regorafenib on cell viability of bladder cancer TSGH 8301 cells. Cells were treated with different concentration of regorafenib for 24 and 48 h. Cell viability was evaluated with MTT assay. Significantly different at *p<0.05 and **p<0.01 as compared to the control group. Results are presented as the mean±standard error.

Western blotting assay. TSGH 8301 cells (3×10⁶) were seeded into 10 cm diameter dishes, incubated overnight, and then treated with 30 μM regorafenib for 24 and 48 h. Total cell proteins from each group were extracted by lysis buffer (50 mM Tris-HCl pH 8.0, 120 mM NaCl, 0.5% NP-40, and 1 mM phenylmethanesulfonyl fluoride). The effect of regorafenib on protein expression levels of MMP-9, XIAP, MCL1, and c-FLIP was determined with western blotting assay as described by Chen et al. (15). Protein bands were quantified by using ImageJ software version 1.50 (National Institutes of Health).

Statistical analysis. Student's t-test was used to test significance of difference of means between treatment group and control. Results are presented as mean±standard error. Statistical significance was achieved if p<0.05. Three independent repeats of each experiment were performed.