Human prostate adenocarcinoma cell lines, PC-3 and DU-145, were purchased from American Type Culture Collection (Rockville, MD, USA). RPMI 1640 medium, fetal bovine serum (FBS), penicillin, streptomycin and 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) were purchased from GIBCO/BRL Life Technologies (Grand Island, NY). Control siRNA, antibodies of PARP-1, Bax (6A7), Bcl-2 (C-2), Bcl-xL, Bak (G-23), Mcl-1 (22), Rad51, α -tubulin (B-7), DNA-PKcs (H-163), anti-mouse and anti-rabbit IgGs were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Antibodies of γ-H2A.X (Ser139), Bid, caspase-8, cleaved caspase-9 and Ku80 were from Cell Signaling Technologies (Boston, MA). Caspase-3 was from Imgenex, Corp.
(San Diego, CA). Antibodies of p-Chk2 (Thr68) and p-DNA-PKcs (Thr2609) (10B1) were from Abcam PLC, Inc. (Massachusetts, US). Antibody of RPA32 (12F3.3) was from GeneTex Inc. Antibody of PDE5 was from OriGene Technologies, Inc. (Rockville, MD, USA). PDE5 siRNA was from GE Healthcare Dharmacon Inc. (Chicago, USA).
Doxorubicin, camptothecin, etoposide, sildenafil, vardenafil, tadalafil, propidium iodide (PI) and all other chemical compounds were purchased from Sigma–Aldrich (St. Louis, MO, USA).
2.2. Methods
2.2.1. Cell culture
HRPC cell lines, PC-3 and DU-145, were cultured in RPMI 1640 medium with 5%
FBS (v/v), penicillin (100 units/ml) and streptomycin (100 g/ml). Cultures were maintained in a 37℃ incubator with 5% CO 2. When cells were 90%-100% confluent, cells were detached by using 0.05 % trypsin-EDTA for passaging.
2.2.2. PI staining and flow cytometric analysis
Cells with the indicated treatments were harvested by trypsinization, fixed with 70% (v/v) alcohol at -20°C for at least 30 minutes and washed with PBS. After centrifugation, cells were resuspended with 0.5 ml PI solution containing Triton X-100 (0.1% v/v), RNase (100 μg/ml) and PI (80 μg/ml). DNA content was analyzed with FACScan and CellQuest software (Becton Dickinson, Mountain View, CA).
2.2.3. Western blotting Sample preparation
After the indicated treatment, the cells were trypsinized and lysed with 60 l ice-cold lysis buffer (20 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 10 g/ml leupeptin, 1mM Na3VO4, 1mM NaF and
1mM dithiothreitol)
.
The lysate was incubated on ice for 10 minutes and then clarified by centrifugation at 14,000 rpm (13,000 g) at 4°C for 20 minutes. After the centrifugation, supernatant (cell extract) was collected to determine protein concentration using Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). To prepare sample for loading into gels, 5 sample buffer (0.3 M Tris pH 6.8, 10 % SDS, 50 % glycerol, 10 % β -mercaptoethanol, 0.02 % bromophenol blue) was added to the cell extract in a ratio of 1:4, and samples were mixed thoroughly then being heated at 95℃ for 5 minutes. Samples can be stored at -20°C for future use.Protein separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
The polyacrylamide gel used in a single electrophoresis run can be divided into stacking gel and separating gel. The acrylamide percentage (6~14%, pH 8.8) of the
separating gel depends on the sizes of the target proteins in the sample. After the gelation of the separating gel in the casting frame, stacking gel (acrylamide 5%, pH 6.8) was added on top of the separating gel and a gel comb was inserted into the stacking gel.
The fully gelated polyacrylamide gels were then set up into the vertical gel electrophoresis tank, and the running buffer (25 mM Tris-base, 192 mM glycine, 3.5 mM SDS, pH 8.3) was poured into the inner and outer chamber of tank. The protein marker and the equal amount of proteins (30 g) from each prepared sample were loaded into wells, and the gel was ready to be run at 60 V (for protein stacking) ~ 90 V (for protein separation) for about 2.5 hours.
Transferring the protein from the gel to the membrane
When performing a wet transfer, the separating gel was put into the a “transfer sandwich” (from cathode to anode pad: sponge-filter paper-gel-PVDF membrane-filter paper-sponge), which was placed in a tray with transfer buffer (25 mM Tris-base, 192 mM glycine, 20 % methanol, pH 8.3). Transferring the protein from the gel to the membrane at 65~75 V (about 200~250 mA) for 2~2.5 hours.
Immunoblotting
After an overnight incubation in PBST/5% nonfat milk at 4 ℃, the membrane was washed with PBS/0.1% Tween 20 (PBST) for three times and immuno-reacted with the indicated first antibody (1:1000~1:3000 dilutions) for 2 h at room temperature. After four washings with PBST, the anti-mouse or anti-rabbit IgG-HRP secondary antibody (1:5000 dilution) was applied to the membranes for 1 h at room temperature. The membranes were washed with PBST for 1 h and the detection of signal was performed with an enhanced chemiluminescence detection kit (Amersham Biosciences). The light signal was captured by X-ray films. The relative protein levels were quantified using the Bio-Rad Quantity One software (Hercules, CA, USA).
2.2.4. Measurement of reactive oxygen species (ROS)
Cells pretreated with or without the antioxidant NAC (1 mM) or trolox (0.3 mM) were then incubated with 0.1 % DMSO (control), doxorubicin or/and sildenafil for the indicated times. Thirty minutes before the termination of the incubation period, DCFH-DA (final concentration of 10 mM) was added to the cells and incubated for the last 30 min at 37 ℃ . Cells with different drug treatments were then harvested respectively for the detection of ROS production (%) by measuring the percentage of DCF fluorescence-positive cells in the collected total cells (10000 events) using FACScan flow cytometric analysis.
2.2.5. Comet assay
After treatment, the cells were pelleted and resuspended in ice-cold PBS. The resuspended cells were mixed with 1.5% low melting point agarose. This mixture was loaded onto a fully frosted slide that had been pre-coated with 0.7% agarose and a coverslip was then applied to the slide. After the gelation of the cell mixture, the coverslip was removed. The slides were then submerged in pre-chilled lysis solution (1% Triton X-100, 2.5 M NaCl, and 10 mM EDTA, pH 10.5) for 30 minutes at 4°C.
After soaking with pre-chilled unwinding and electrophoresis buffer (0.3 N NaOH and 1 mM EDTA) for 30 minutes, the slides were subjected to electrophoresis for 15 minutes at 0.5 V/cm (25 mA). After electrophoresis, slides were stained with 1X SYBR Gold (Molecular Probes) and nuclei images were visualized and captured at 200X magnifications with an Zeiss AxioImager A1 fluorescent microscope (Zeiss, Germany) equipped with a CCD camera (Optronics, Goleta, CA). Over one hundred of cells in each sample were scored to calculate the average of comet tail moment (Tail moment =
%DNAtail × Lengthtail) using TriTek CometScoreTM software.
2.2.6. Nuclear extraction
Nuclear extracts were prepared by sequential cell lysis and nuclear lysis. Cell pellet was suspended in 200 l buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 0.5 mM dithiothreitol and 0.2 mM PMSF. The cells were subjected to vigorous vortex for 20 seconds, ice-cold incubation for 10 min to disrupt the cell membrane and centrifuged at 2000 rpm for 2 min. The pelleted nuclei were washed twice with 100 l buffer containing 10 mM HEPES, 50 mM NaCl, 25% glycerol and 0.1 mM EDTA without resuspension the pellet. After removal of the washing buffer by centrifugation at 2000 rpm for 5 min, the pelleted nuclei were lysed with 30 l buffer containing 20 mM HEPES (pH 7.9), 25% glycerol, 1.5 mM MgCl2 , 420 mM NaCl, 0.2 M EDTA, 0.5 mM dithiothreitol and 0.2 mM PMSF. After 20 minutes on ice, the lysates were centrifuged at 14,000 rpm for 5 min. The supernatants containing the solubilized nuclear proteins were used for Western blotting.
2.2.7. DNA fragmentation assay
The DNA fragmentation was determined using the Cell Death Detection ELISAPLUS kit (Roche, Mannheim, Germany). The assay was based on the quantitative in vitro determination of cytoplasmic histone-associated DNA fragments (mono- and oligo-nucleosomes) in cells after the induction of cell death. After treated with the indicated agents, cells were lysed and centrifuged, and the supernatant was used for the detection of nucleosomal DNA fragments according to the manufacturer’s protocol.
2.2.8. Small interfering RNA (siRNA) transfection
PC-3 cells were seeded into a 6-well plate with 30% confluence for each well and grown for 24 hours to 50% confluence. Each well was washed twice with PBS and 1
mL of serum-free Opti-MEM (Life Technologies, Ground Island, NY) was added.
Aliquots containing control or PDE5 siRNA in serum-free Opti-MEM were transfected into cells using Lipofectamine 2000 according to the instructions. The sequences of PDE5 siRNA are GAAGACAGCUCCAAUGACA, GAAAUCAGGUGCUGCUUGA,
GAUGACAGCUUGUGAUCUU and GGAAACGGUGGGACAUUUA. After
transfection for 5 hours, cells were washed twice with PBS and incubated in 10%
FBS-containing RPMI-1640 medium for 48 hours. The cells were then treated with or without doxorubicin for 48 hours, and the level of protein of interest was detected using Western bot analysis.
2.2.9. Immunofluorescence staining of nuclear Rad51 foci
PC-3 cells were grown on coverslips placed in a 6-well plate (1.8 cells/well).
All procedures for immunofluorescence staining were conducted at room temperature.
Following treatment with 1 μ M doxorubicin or/and 10 μ M sildenafil for 24 hours and 8 hours of cell recovery in drug-free medium, PC-3 cells were washed twice with PBS and fixed with 4 % paraformaldehyde in PBS for 20~30 min. After fixation, cells were washed three times with PBS, permeabilized with 0.1% Triton X-100 for 10 minutes followed by three times of wash with PBS and then blocked with 5% BSA/PBS for 1 hour. To examine the nuclear Rad51 foci, cells were subsequently stained with the anti-Rad51 antibody (1:200 dilution in 2.5% BSA/PBS) for 1 hour with gentle agitation and washed three times with PBS. Cells were next incubated with the FITC-conjugated secondary antibody for 1 hour (1:100 dilution in 2.5% BSA/PBS) with gentle agitation.
After washing cells three times with PBS, nuclear staining was performed using 0.15 μg/ml DAPI for 5~10 minutes. Cells on coverslip were finally washed three times with PBS. The air-dried coverslips were next mounted onto glass slides using prolong®
diamond antifade mountant (Thermo Fisher Scientific inc., Waltham, MA, USA). The slides were then kept in the dark at 4℃ for at least one day to dry the antifade mountant.
The immunofluorescent images of nuclear Rad51 foci and nuclei were captured at 630X magnifications (63x/1.4 NA oil immersion objective lens) using Zeiss AxioImager A1 fluorescent microscope (Zeiss, Germany) equipped with a CCD camera (Optronics, Goleta, CA). At least 100 cells were examined in each sample, and the percentage of cells containing over five Rad51 foci in each sample was estimated.
2.2.10. DNA end-binding activity of Ku80 protein
Assessment of DNA end-binding activity of Ku80 was carried out by using a Ku70/Ku80 DNA Repair kit (Active Motif). Briefly, equivalent amounts of nuclear proteins (4 g) were loaded into an oligonucleotide coated 96-well plate. Then, Ku80 proteins contained in nuclear extract specifically bound to the oligonucleotide.
Anti-Ku80 antibody provided by this kit detected DNA bound-Ku80. Addition of the secondary HRP-conjugated antibodies and developing solution provided a colorimetric readout (λ = 450 nm) quantified by spectrophotometry.
2.2.11. Data Analysis
Data are presented as mean standard deviation (SD) for the indicated number of separate experiments. Statistical analysis of data was performed with one-way analysis of variance followed by Bonferroni t-test and p-values < 0.05 were considered significant.