Cell culture and reagents
Human prostate cancer cell line PC-3 was obtained from the American Type Culture Collection (ATCC). The cells were cultured in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco/Invitrogen, Australia), and 1% penicillin/streptomycin (Invitrogen) at 37 °C in a humidified incubator in the presence of 5% CO2. Docetaxel (DOX) Injection and GSI (PF-03084014) were purchased from Sigma-Aldrich (cat. No 01885) and Selleckchem (cat. No S8018), respectively.
PCSCs
PCSCs has been enriched from PC-3 cell line as previously described [6]. PCSCs were cultured in EF20 medium composed of Neurobasal medium (Invitrogen) supplemented with 3 mM L-Glutamine (Mediatech), 20 ng/mL human EGF (R&D Systems), 20 ng/mL human FGF-2 (PeproTech), 1 × B27 supplement (Invitrogen), 0.5 × N2 supplement (Invitrogen), 2 mg/mL heparin (Sigma), and 0.5 × penicillin G-streptomycin sulfate-amphotericin B complex (Mediatech). For passaging, spheres were centrifuged, treated with Accutase (Innovative Cell Technologies) at 37℃ for 5 min, dissociated by pipetting, washed with PBS, and resuspended in EF20 medium [13].
Quantitative real-time PCR
Total RNA was extracted from PC-3 parental cells and PCSCs using TRIzol Plus RNA Purification Kit (Invitrogen) according to the manufacture’s protocol. Using RevertAid First Strand cDNA Synthesis Kit (Fermentas), mRNA was reverse-transcribed into cDNA. Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) was performed using the SYBR Green Master Mix (Fermentas). The primers were as follows: Notch-1 (forward 5’-CCTGTCTGAGGTCAATGAGT-3’; reverse 5’-GTAGCCACTGGTCATGTCTT-3’); Oct-4 (forward 5’- CGAAAGAGAAAGCGAACCAG-3’; reverse 5’- GCCGGTTACAGAACCACACT-3’); Nanog (forward 5'- AAGGTCCCGGTCAAGAAACAG-3’; reverse 5’-CTTCTGCGTCACACCATTGC-3’); GAPDH (forward 5’- AGAAGGCTGGGGCTCATTTG-3’; reverse 5’- AGGGGCCATCCACAGTCTTC-3’). In order to compare the relative expression of the mRNA in different samples, the comparative delta Ct (threshold cycle number) was calculated.
Cell susceptibility assay
PC-3 parental cells and PCSCs were seeded into 96-well plates at 3,000 cells/well for overnight. Cells were treated for 72 hr with DOX (0.001, 0.01, 0.1, 1, 10, and 100 nM), or GSI (0.01, 0.03, 0.1, 0.3, 1, 3, 10, and 30 µM), or DOX (0.001, 0.01, 0.1, 1, 10, and 100 nM) and GSI (5 µM). 5-(3-Carboxymethoxyphenyl)-2-(4,5-dimethylthiazoly)-3-(4-sulfophenyl)tetrazolium, inner salt (MTS) assays (Promega) were performed according to the manufacturer’s instructions, and dose-response curves were plotted (non-linear fit; GraphPad Prism).
Flow cytometric analysis
Flow cytometric analysis was used to detect the apoptosis (using FITC Annexin V Apoptosis Detection Kit, BD Pharmingen TM) and cell cycle distribution (using Propidium Iodide Staining, Sigma) of PC-3 PCSCs treated with (1) vehicle (control, DMSO); (2) DOX (10 nM); (3) GSI (5 µM); (4) DOX (10 nM) + GSI (5 µM) for 72 hr. Flow cytometric analysis was performed using a Beckman Coulter FC 500 MCL/MPL counter fitted with a 488 nm laser.
Sphere formation assay
PC-3 PCSCs were dissociated and passed through a 100- µm filter to produce single-cell suspensions. One thousand cells were cultured in 100 µL EF20 media/well in 96-well plates, and treated with (1) vehicle (control, DMSO); (2) DOX (10 nM); (3) GSI (5 µM); (4) DOX (10 nM) + GSI (5 µM). Then 14 days later, sphere (diameters ≥ 50 µm) size and numbers in each well were measured using microscopy. Sphere formation ratio (%) was calculated using the following formula: sphere formation ratio (%) = (numbers of wells with spheres) ÷ (total wells seeded with PCSCs) × 100.
In vivo studies
Six- to eight-week-old male BALB/c nude mice were purchased from Hunan SLK Laboratory Animal Center (Hunan, China) and housed under standard conditions. After one week of adaptation, the animals were used for in vivo studies. All experimental protocols were approved by the Animal Care Committee of Wuhan University. PC-3 PCSCs (5 × 104 in 100 µL) were injected subcutaneously into the flank of the mice. Tumor volume (V) was calculated following the formula: V (mm3) = a2 × b × 0.52, where a and b are the shortest and longest diameters, respectively. Tumor growth was measured using Vernier calipers. Three to 4 weeks after inoculation, when the tumor volumes were approximately 100–200 mm3, the mice were randomly assigned to different treatment groups (7 mice per group): (1) control (DMSO); (2) DOX (10 mg/kg, i.p., weekly for 4 weeks); (3) GSI (150 mg/kg, daily, p.o., 7-days-on/7-days-off schedule for 4 weeks based on a previous report [14]); (4) DOX + GSI (same dosage as above for 4 weeks). There was no significant difference in mean tumor volumes and body weight across all groups at the beginning of treatment. Tumor size and body weight were measured twice a week. At the end of treatment, tumor specimens were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5-µm thick slides. The expression of Notch-1 (1:150, Santa Cruz Biotechnology) was evaluated by immunofluorescent staining according to the manufacturer’s instructions. Sections were examined for positive staining that was quantified as previously described [13].
Statistical analysis
All experiments were repeated at least in triplicate. Collected data were analyzed using GraphPad Prism 5.0 software (GraphPad Software, Inc., San Diego, CA), and results were displayed as mean ± SD. Comparison between two groups was analyzed with unpaired Student’s t test (two tailed) and differences among more than two groups were determined by a one-way ANOVA followed by Newman-Keuls test. Difference with p < 0.05 was considered statistically significant.