Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Gibco/Life Technologies (Carlsbad, CA, USA). Fetal bovine serum (FBS), penicillin, streptomycin, and all other tissue culture reagents were obtained from Thermo Scientific HyClone (Logan, UT, USA). Paclitaxel and propidium iodide (PI) were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

DU145 cells were cultured in DMEM with 10% FBS (v/v) and penicillin (100 U/ml)/streptomycin (100 μg/ml). Cultures were maintained in a humidified incubator at 37°C in a 5% CO₂ incubator. When the cells reached 30% confluence, they were synchronized at the late G1 phase if necessary by the addition of mimosine (0.5 mM) for 20 h and then released into fresh medium.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma) tests were performed to evaluate the cytotoxic effects in vitro. Briefly, DU145 cells were plated in 96-well plates (8×10³ cells/well). Following treatment with NCTD for 48 h at the indicated concentrations, MTT was added to each well (100 μg/well) followed by incubation for an additional 4 h. The produced insoluble formazan was dissolved with 200 μl DMSO and the optical density (OD) was measured using an ELISA reader (Thermo Labsystems, Helsinki, Finland) at wavelengths of 570 and 630 nm. The experiments were carried out in triplicate.

The DU145 cells were seeded onto 22-mm diameter coverglasses placed in 6-well plates (3×10⁵ cells/coverglass). One hour prior to fixing the cells, 10 μM BrdU (Sigma Chemicals) were added to the cultures. The cells were rinsed and fixed in 4% phosphate-buffered paraformaldehyde for 10 min. Following aspiration, the cells were rinsed 3 times in PBS for 5 min and 0.2% Triton X-100 was added to the specimens for 10 min. The specimens were then incubated in 4 M HCl after being rinsed 3 times in PBS for 5 min. After neutralization using PBS, the specimens were blocked in goat serum for 60 min. The blocking solution was aspirated and the specimens were incubated in diluted primary mouse-monoclonal antibody to BrdU (1:1,000, Cell Signaling Technology Inc., Beverly, MA, USA) overnight at 4°C. After rinsing 3 times in PBS for 5 min, the specimens were incubated in fluorochrome-conjugated secondary antibody diluted in PBS at room temperature in the dark and observed under fluorescent microscope. At least 1,000 cells/treatment using at least 2 cover-glasses/treatment were counted, and the number of positive cells was recorded. Labeling indexes were calculated as the number of positively stained cells divided by the number of total cells.

The cells were harvested and suspended in ice-cold lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM PMSF and 10 U/ml aprotinin) for 20 min, then centrifuged at 12,000 rpm for 10 min at 4°C. Total proteins were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with TBS containing 0.1% Triton X-100 and 5% non-fat milk for 1 h at room temperature, then incubated with rabbit-anti-human monoclonal antibody against Cdc6 (1:1,000; Cell Signaling Technology Inc., Danvers, MA, USA), rabbit-anti-human monoclonal against Mcm2 (1:1,000, Cell Signaling Technology), goat polyclonal antibody against Mcm3 (1:500, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), mouse-anti-human monoclonal antibody against Mcm6 (1:500, Santa Cruz Biotechnology, Inc.) and rabbit-anti-human monoclonal against GAPDH (1:2,000, Cell Signaling Technology) at 4°C overnight. After washing, the membranes were incubated with HRP-conjugated Ig at room temperature for 1 h. Signal detection was carried out with an ECL system (Millipore, Billerica, MA, USA).

The cells were harvested and resuspended in tubes with extraction buffer (100 mM KCl, 50 mM HEPES-KOH pH 7.5, 2.5 mM MgCl₂, 50 mM Na₄P₂O₇, 0.1 mM NaVO₃, 0.5% Triton X-100) containing protease inhibitors, then set on ice for 5 to 10 min for incubation. The tubes were flicked to mix the solution every 2–3 min during incubation. Subsequently, 30% ice-cold sucrose containing protease inhibitors was added to the bottom of the tubes. The tubes were then spinned in a microfuge, 12–15 krpm, 10 min, 4°C and the supernatants were transferred to new tubes. The pellets were washed with EB buffer and flicked to dislodge the pellets from the wall of the tubes and vortexed briefly for resuspension, followed by spinning in a microfuge, 12–15 krpm, 5 min, 4°C. The supernatants were saved and combined (this is the nonchromosomal fraction) from this and the previous step. The pellets were resuspended with EB buffer (the pellets are the chromatin-binding fraction). The supernatants and the pellets were then used for western blot analysis.

For nuclear staining, the cells were treated with or without 50 or 100 μM NCTD for 48 h, washed twice with cold PBS, and fixed with 4% paraformaldehyde for 15 min. The fixed cells were permeabilized with 0.2% Triton X-100 and then incubated with 4, 6-diamidino-2-phenylindole (DAPI) at the concentration of 5 μg/ml for 5 min. A fluorescent microscope was used for imaging. At least 1,000 cells/treatment were counted, and the number of cells with signs of mitotic catastrophe was recorded. Labeling indexes were calculated as the number of cells with signs of mitotic catastrophe divided by the number of total cells.

Following treatment with or without hydroxyurea (HU) or NCTD/HU for 24 h, the cells in each group were collected by Trypsin digestion. The cells were fixed with 70% alcohol and then centrifuged again for 10 min at 8,000 rpm. The supernatant was discarded and 500 μl PI staining solution (5 μg/ml RNase, 0.1% Triton X-100, 0.1 mM EDTA, 50 μg/ml PI) was added. The cells were then incubated away from light for 30 min at 4°C. The cell cycle and apoptosis were then measured by flow cytometry (Becton-Dickinson, San Jose, CA, USA).

DU145 cells were treated with NCTD in combination with paclitaxel at various concentrations for 48 h. MTT assays were performed to detect the cell viability in vitro. Briefly, the cells were seeded on 96-well plates in 10% FBS-containing medium at a density of 4,000 cells/well and incubated at 37°C for 24 h prior to exposure to the drugs. The cells were then treated with NCTD, paclitaxel, or a combination of both for 48 h at the indicated concentrations. The combination ratio of NCTD (μM) and paclitaxel (nM) was 1:1 and the concentration range is presented in Table I. Cell viability was determined following treatment with the drugs for 48 h by MTT assay. Each experiment was performed in quintuplicate wells for each drug concentration and independently carried out 3 times. By combining both agents at graded concentrations, the combined effects of growth inhibition were obtained and analyzed using Calcusyn1 software. The combination index (CI) was calculated as follows: CI = (D)1/(Dx)1 + (D)2/(Dx)2 + (D)1(D)2/(Dx)1(Dx)2; where (Dx)1 is the dose of drug 1 required to produce an X% effect alone, (D)1 is the dose of drug 1 required to produce the same X% effect in combination with drug 2, (Dx)2 is the dose of drug 2 required to produce an X% effect alone, and (D)2 is the dose of drug 2 required to produce the same X% effect in combination with drug 1.

The effects of the combination treatment were then transformed and displayed as FA-CI plots. The combined effects were determined as follows: CI <1, synergy; CI = 1, zero interaction (strictly additive effects); and CI >1, antagonism.

Average values are expressed as the means ± standard deviation (SD). Statistical significance between different groups was determined using the Student’s t-test. A P-value <0.05 was considered to indicate a statistically significant difference.

To evaluate the effects of NCTD inhibition on the proliferation of DU145 PCa cells, MTT assay was carried out. In the NCTD-treated group, a dose-dependent suppression of cell proliferation was observed. NCTD effectively inhibited the proliferation of DU145 cells, with an IC₅₀ of ~200 μM (Fig. 1).

We investigated whether NCTD inhibits the proliferation of DU145 cells by suppressing DNA synthesis. The BrdU incorporation assays revealed that ~47% of the PCa cells in the control group were able to incorporate BrdU, while in the NCTD-treated group, the number of BrdU-positive cells decreased to 31% (Fig. 2A and B). NCTD significantly inhibited DNA replication (P<0.05).

To further characterize the inhibitory effect of NCTD on DNA replication, we examined the effects of NCTD on pre-RC proteins. Total proteins from NCTD-treated DU145 cells were analyzed by western blot analysis. Of the pre-RC components, Cdc6, Mcm3 and Mcm6 are the most frequently reported proteins that are overexpressed in tumors. Our results revealed that Cdc6 and Mcm6 were significantly degraded in a dose-dependent manner following treatment with NCTD, as compared with the control group (Fig. 3A and B).

In G1 phase cells, Cdc6 resides in the nucleus and is essential for pre-RC formation and replication initiation (23). The end result of pre-RC formation is the loading of Mcms onto origin DNA (24). To examine the inhibitory effect of NCTD on the assembly of pre-RC, the cells were incubated with mimosine for 20 h and then released into fresh medium with or without NCTD (NCTD was added 8 h prior to the release of mimosine). The cells were collected at 0, 3 and 6 h after the release of mimosine. The chromatin binding of Cdc6, Mcm2 and Mcm6 was examined by chromatin-binding assay. Our results indicated that NCTD significantly reduced the chromatin binding of Cdc6 and Mcm2 in the cells at the 6 h time point (Fig. 4A–C). The chromatin binding of Mcm6 was significantly decreased in cells at all time points (Fig. 4A and D). The results revealed that NCTD disturbed the assembly of Cdc6, Mcm2 and Mcm6 onto chromatin.

Previous studies have shown that Cdc6 is not only required for G1 origin licensing, but is also crucial for proper S phase DNA replication and participates in the S-M checkpoints (25). As shown in a previous study, the loss of Cdc6 did not activate the ATR-dependent DNA damage response and induced aberrant mitosis (25). It has been reported that human Cdc6 physically interacts with ATR, in a manner that is stimulated by phosphorylation by Cdk, and the presence of Cdc6 during the S phase is essential for ATR to bind to chromatin in response to replication inhibition (15). In our study, NCTD effectively degraded Cdc6 in DU145 cells (Figs. 3 and 4). Thus, there is a possibility that NCTD can inhibit ATR binding to chromatin by the degradation of Cdc6. Our results revealed that few ATR chromatin-binding fractions were detected in the control group, suggesting that the ATR-dependent checkpoint pathway was not activated without DNA replication stress (Fig. 5A and B). In the low-dose NCTD-treated groups (50 μM), ATR was detected in chromatin, suggesting that DNA replication was inhibited and ATR was activated. However, under higher doses of NCTD (100 and 200 μM), ATR disappeared from chromatin. In addition, the chromatin binding of Cdc6 signficantly decreased in a dose-dependent manner following treatment with NCTD (Fig. 5A and C). These results suggested that NCTD (lower dose) activates the ATR-dependent checkpoint pathway by inducing DNA replication stress. However, at the same time, NCTD (higher dose) also attenuates the ATR-dependent checkpoint activity by inhibiting ATR binding to chromatin, which may be due to the Cdc6 degradation.

ATR plays a crucial role in the S-M checkpoint pathway by signaling to Chk1 and conferring G2/M arrest when the cells are under replication stress (26). This surveillance warrants cells will not transit into mitosis before the completion of the S phase (15,27). To further characterize the inhibitory effect of NCTD on the S-M checkpoint, we examined the influence of NCTD on HU-induced S phase arrest. The results revealed that HU effectively induced S phase arrest (31%) and the cells did not progress into the G2/M phase (0%). In the NCTD group, 26% of the cells still progressed into the G2/M phase under HU stress. Of note, there was a significant accumulation of cells in the sub-G1 phase in the NCTD/HU-treated group, suggesting that the aberrant mitosis led to apoptosis (Fig. 6).

In our experiments, DNA replication was inhibited by NCTD. Simultaneously, the chromatin binding of ATR and hence, the S-M checkpoint was also prevented. Thus, it is rational to suggest that treatment with NCTD may induce aberrant mitosis by simultaneously disturbing DNA replication and disabling the S-M checkpoint. The results from DAPI nuclear staining revealed that following treatment with NCTD, the cells underwent mitotic catastrophe, indicated by the presence of cells with micronuclei and giant multinucleated cells (28). The percentages of DU145 cells exposed to 50 or 100 μM NCTD which underwent mitotic catastrophe were ~11 and 17%, respectively, significantly higher than those of the control group (Fig. 7A and B) (P<0.05).

As a microtubule-poisoning drug, paclitaxel can induce mitotic catastrophe and is effective for PCa therapy. As opposed to the NCTD-induced mitotic catastrophe, paclitaxel interferes with the normal breakdown of microtubules during cell division (29,30). Therefore, a combination of these 2 drugs, may produce synergistic effects. In this study, we investigated whether NCTD and paclitaxel exert synergistic effects. As shown by our results, a strong synergistic anticancer effect on DU145 cells was observed with the combination treatment of NCTD and paclitaxel at a low concentration range (CI <1) (Fig. 8). Our findings provide justification for the further development of the combined treatment with NCTD and paclitaxel for the treatment of PCa.