br Sulforhodamine B SRB assay br Cell proliferation was
2.4. Sulforhodamine B (SRB) assay
Cell proliferation was determined by sulforhodamin B (SRB, Sigma, St. Louis, USA) assay. The cancer cells were seeded at a concentration of 3 × 104 cells/well in 48-well tissue culture plates and incubated with various concentrations of AC and TRAIL for 24 h. After treatment, medium was aspirated and 10% trichloro-acetic CHIR99021 was added. After 1 h incubation at 4 °C, the plate was washed five times with D.W and air-dried. The cells were stained with 0.4% (w/v) SRB at room tem-perature for 1 h and then washed five times using 1% acetic acid. Bound SRB was solubilized with 10 mM Tris, and the absorbance was mea-sured at 540 nm using a microplate reader (Molecular Devices, Inc. US).
The influence of z-vad-fmk (caspases inhibitor), N-PM (AIF in-hibitor), and LY294002 (PI3K and AKT inhibitor) on cell viability was also determined by SRB assay. The cells were seeded at a densities of 1 × 105 cells per well in a 24-well plate, and then cultured for 24 h in DMEM. The cells were pre-incubated with 10 μM of z-VAD-fmk or 2 μM of N-PM or 10 μM of LY294002 for 2 h and then treated with the in-dicated concentrations of AC and TRAIL for 24 h. SRB assay was con-ducted as described above.
2.5. Annexin V staining by flow cytometry
The apoptotic cell death was determined using MuseⓇ Annexin V and dead cell reagent, according to the manufacturer's protocol. 1 × 105 RC-58T/h/SA#4 cells were seeded into 24 well plates and in-cubated for 24 h. Treatments were given with AC and TRAIL for 24 h. The cells were trypsinized and washed with PBS. Added 100 μL of the reagent to microcentrifuge tubes and then added 10 μL of cell suspen-sion to each tube and incubated for 20 min at room temperature. The cells were analyzed using a Muse cell analyzer (Merck KGaA, Darmstadt, Germany). The flow cytometry data was obtained from
Fig. 1. Cell growth inhibitory eﬀects on prostate cancer and prostate epithelial cells. Cells were treated with various concentrations of AC and/or TRAIL for 24 h. (A) RWPE-1 prostate epithelial cell and (B) RC-58T/h/SA#4 primary prostate cancer viability were measured by SRB assay. (C) Morphological changes were observed under an inverted microscope after 24 h of treatment. Significant diﬀerences were compared with the control at *p < 0.05 and **p < 0.01 using one-way
5000 events (gated cells) per sample. The percentages of cells shown in the figures were calculated from the mean fluorescence intensity in each of the four quadrants. In addition, the coeﬃcient of variation from the mean fluorescence was less than 10%.
2.6. Detection of morphological apoptosis
Characteristic apoptotic morphological changes were assessed by fluorescent microscopy using bis-benzimide (Hoechst 33258) staining. Briefly, the cells were seeded in 6-well plates at a density of 1 × 106 cells per well, followed by treatment with AC and TRAIL for 24 h. After harvesting, the cells were washed twice with PBS and then stained with 200 μL of bis-benzimide (5 μg/mL) for 10 min at room temperature. Then, 10 μL of this suspension was placed on a glass slide and covered with a cover slip. The cells were examined using a
fluorescence microscope (Olympus Optical Co. Ltd. Japan) to determine nuclei fragmentation and chromatin condensation.
2.7. Analysis of DNA fragmentation
Fig. 2. Combined treatment with AC and TRAIL induces apoptotic cell death in RC-58T/h/SA#4 primary prostate cancer cells. Cells were treated with 5 μM AC and/or 100 ng/mL of TRAIL for 24 h. (A) Apoptotic cell population was quantified by Annexin V staining assay using a Muse cell analyzer. (B) DNA fragmentation was observed by 2% agarose gel electrophoresis. (C) Nuclear condensation was detected by Hoechst staining assay. Significant diﬀerences were compared with the control at *p < 0.05 and **p < 0.01 using one-way ANOVA.
electrophoresis was performed on 2% agarose containing ethidium bromide. The resulting DNA bands were examined using a UV Trans illuminator Imaging System.
2.8. Mitochondria isolation
Mitochondria fraction was isolated from cell lysate using a Mitochondria isolation kit (Pierce, Rockford, USA). Briefly, 800 μL of Reagent A and 10 μL of Reagent B were added to a 2 × 106 cell pellet and incubated on ice for 2 min. The resulting pellets were centrifuged at 700×g for 10 min at 4 °C. The supernatant was then transferred to a new tube and centrifuged at 12,000×g for 15 min at 4 °C. The super-natant (cytosol fraction) was added to a new tube, and the pellet con-taining mitochondria received 500 μL of Reagent C followed by cen-trifugation (12,000×g, 5 min, 4 °C). The mitochondria pellets were lysed by lysis buﬀer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 50 mM NaF, 30 mM Na4P2O7, 1 mM PMSF, and 2 μg/mL of aprotinin) for 30 min on ice. Protein expression in the mitochondria fraction was analyzed by Western blot assay.