The DU145 human prostate cancer cell line was supplied by American Type Culture Collection (Manasas, VA, USA) and was grown in monolayer culture in Dulbecco’s modified Eagle’s medium-F12 (DMEM-F12; Biological Industries, Kibbutz Beit-Haemek, Israel) supplemented with 10% heat-inactivated fetal calf serum (Gibco, Invitrogen Life Technologies, Paisley, UK), 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich, St Louis, MO, USA). Cells in semi-confluent flasks were harvested using 0.05% trypsin (Sigma-Aldrich), centrifuged (Nuve NF200; Laboratory and Sterilization Technology, Ankara, Turkey) following the addition of DMEM-F12 for trypsin inactivation, and re-suspended in culture medium. The antibodies used were anti-caspase-3 (1:100 diluted; 3510-100, BioVision, Inc., Milpitas, CA, USA), anti-caspase-8 (1:100 diluted; 250576, Abbiotec, USA), anti-p53 (1:100 diluted; 3036R-100, BioVision, Inc.) and goat anti-rabbit immunoglobulin G-fluorescein isothiocyanate (FITC) (1:100 diluted; sc-2012, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

For FACS (FACSAria; BD Biosciences, San Jose, CA, USA), the cells were detached using non-enzymatic cell dissociation solution (Sigma-Aldrich) and ~5×10⁴ cells were incubated with an antibody (diluted 1:100 in FACS wash with 0.5% bovine serum albumin; 2 mM NaN₃ and 5 mM EDTA) for 15 min at 4°C. An isotype and concentration-matched phycoerythrin (PE)-labeled control antibody (Miltenyi Biotec Ltd., Woking, Surrey, UK) was used and the samples were labeled with PE-labeled CD133/1 (clone AC133/1; Miltenyi Biotec Ltd.) and FITC-labeled CD44 (clone G44-26; BD Biosciences). After 3–5 min, the cells were washed with the FACS wash, and subsequently the cells were re-suspended. The cells were organized into a CD133^high/CD44^high subpopulation to become CSCs.

The investigated drug, flavopiridol, was applied to monolayer cultures of CD133^high/CD44^high human prostate CSCs at the following final concentrations: 100, 300, 500 and 1000 nM, except for the control cells, to which nutrient medium was applied. The cultures were incubated for 24, 48 and 72 h. The half maximal inhibitory concentration (IC₅₀) value of the drug was determined as 500 nM for monolayer cells. Dead cells were analyzed prior and subsequent to exposure to increasing flavopiridol doses using the Muse™ Count and Viability kit and the Muse™ Annexin V and Dead Cell Assay kit (Muse™Cell Analyzer; Millipore, Billerica, MA, USA) according to the manufacturer’s instructions.

For the spheroid cultures, the CD133^high/CD44^high human prostate CSCs were grown as a monolayer and re-suspended with trypsin. The clonogenic potential of the different phenotypic populations was analyzed in a 3D non-adherent culture condition. The liquid overlay technique was used for the constitution of spheroids (16). Briefly, the cells were counted, re-suspended and plated with 1×10⁴ cells per well in a 6-well plate (plates coated with 3% Noble agar) (Difco Laboratories, Inc.; BD Diagnostic Systems, Detroit MI, USA) and incubated at 37°C under 95% air/5% CO₂. Fresh medium was added every 2–3 days to remove cellular debris and the spheroids that were not well-formed. Two weeks after initiation, the plates were inspected for colony (sphere) growth.

Flavopiridol was applied to the spheroids to measure the IC₅₀ doses at the beginning of the multicellular tumor spheroid formation, incubated at 37°C and protected from light. Flavopiridol plus DMEM was replaced every three days. In the second set of experiments, flavopiridol was added at the IC₅₀ dose to day-15 mature spheroids and incubated for 24, 48 and 72 h. The number and diameter of colonies within each well was counted each day under the microscope (Olympus BX-51; Olympus, Hamburg, Germany) and the images were captured for the representative fields.

CD133^+high/CD44^+high prostate CSCs were treated as indicated above and were harvested and fixed in 4% paraformaldehyde for 30 min. Subsequently, the cells were rendered permeable with 0.1% Triton X-100 for 10 min at room temperature, and blocked with phosphate-buffered saline containing 5% bovine serum albumin for 2 h. Following incubation with antibodies against caspase-3, caspase-8, p53 and bcl-2 overnight at 4°C, the cancer cells were treated with FITC-conjugated secondary antibody for 3 h at room temperature. The cells were counterstained with 4′,6-diamidino-2-phenylindole and assessed by a fluorescence microscope equipped with a camera (Olympus BX-51 and the Olympus C-5050 digital test). Statistical analysis was tested by one-way analysis of variance, followed by Tukey’s or Dunett’s post hoc test. P<0.05 was considered to indicate a statistically significant difference.

DU145 human prostate cancer cells were separated with FACS as the CD133^high/CD44^high population (sorted cells) (Fig. 1). The purity of the CSCs samples were tested with CD133 and CD44 antibodies. The sorting rate analysis and purity of the cells was evaluated sequentially and the rate was 96.7±5.4% for the sorted cells. In order to confirm the flow cytometry analyses, the cells were re-evaluated following sorting and the analyses were repeated after one passage. The results showed that the cell purity following sorting was 85%. Immunofluorescence staining yielded a cell purity of >85% in all the samples (Fig. 1).

Treated cells were subjected to flavopiridol and are shown in Fig. 2. Flavopiridol reduced the cell viability of CSCs in a dose-dependent manner. According to the data, there were no significant decreases in cell viability at the low doses (100 and 300 nM) of flavopiridol treatment for 24 h (P>0.05) when compared to the control. There was no statistically significant change between 100 nM when compared to 300 nM for 72 h (P=0.093). However, flavopiridol treatment caused significant growth inhibition at 500 (P=0.018) and 1000 nM (P<0.001) when compared to the control at 24 h. Treatment for 48 and 72 h significantly decreased the cell viability of the population at 300, 500 and 1000 nM (P<0.001) (Fig. 3). Therefore, 500 nM was chosen as the optimal dose to be used in the subsequent experiments.

G₀/G₁ analysis showed that there were statistically significant changes between 100 and 500 nM (P<0.005), 100 and 1000 nM (P<0.001), 300 and 1000 nM (P<0.001), and 500 and 1000 nM (P<0.001) (Fig. 4). In this phase, CSCs were effectively influenced by 1000 nM treatment and G₀/G₁ arrest was observed. Flavopiridol effected the S phase of prostate CSCs at the 500 nM dose only when compared to the other doses (P=0.046). Of note, flavopiridol significantly influenced the cells in the G₂/M phase, particularly in high-dose treatments. Statistically significant differences were apparent between the control and 500 (P<0.001) and 1000 nM (P<0.001). Dose-dependent G₂/M-phase arrest was also observed between 100 and 1000 nM (P<0.002), 300 and 500 nM (P=0.022), and 300 and 1000 nM (P<0.001) (Fig. 5).

Flavopiridol induced apoptosis in a dose-dependent manner as measured by the Muse™ Annexin V and Dead Cell assay (Fig. 6). Apoptosis was evaluated as early apoptotic cells, late apoptotic cells and dead cells. In this analysis the number of dead cells were significantly decreased, whereas the apoptotic cells increased. Regarding this analysis, it should be noted that flavopiridol induced apoptotic cell death in CSCs mainly as early and late apoptosis, and even the percentage of live cell numbers significantly decreased. Early and late apoptotic cell numbers were increased and were significant different (P<0.001) (Fig. 7A).

The total apoptotic cells were quantified and this demonstrated that flavopiridol induced apoptosis in a dose-dependent manner. Increased flavopiridol treatment induced apoptosis and the maximum effective concentration was 1000 nM when compared to 100, 300 and 500 nM (Fig. 7B).

Immunofluorescence staining for caspase-3, caspase-8 and p53 supports these results and revealed the apoptotic pathway. Decreased immunoreactivity was observed in pretreated prostate CSCs (Fig. 8). Flavopiridol treatment with an IC₅₀ dose (500 nM) resulted in a significant increase in immunofluorescence staining of caspase-3, caspase-8 and p53 (Fig. 9).

The ability of sphere formation was markedly suppressed in a dose-dependent manner from the beginning of the spheroid constitution and also mature spheroids (Fig. 10). The early and mature spheroids (15 day) were incubated for 24, 48 and 72 h and cells were treated with flavopiridol. The number and diameter of colonies within each well was counted each day under the microscope. Decreased spheroid diameter was observed following flavopiridol treatment. According to statistical analysis, an inhibition of early spheroid formation was observed in all groups (24, 48 and 72 h) at the IC₅₀ treatment dose (IC₅₀=500 nM) (P<0.001) (Fig. 11). In mature spheroids, there was no significant statistical difference between days 15 and 18 in the flavopiridol treatment group (P=0.931). Besides this group, spheroid formation inhibition was also observed in the other groups and this was significantly different (P<0.001) (Fig. 11). Notably, the downregulation of cell proliferation significantly enhanced the flavopiridol-mediated induction of cell apoptosis and inhibition of sphere formation.