Thymoquinone (C10H12O2) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO). Stock solutions were stored at −20°C. Antibodies against transforming growth factor-β (TGF-β), Smad2, Smad3, E-cadherin, vimentin, Slug and β-actin were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma Chemical Co. (St. Louis, MO, USA).

Human prostate cancer cell lines DU145 and PC3 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). These two cell lines were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), 100 µg/ml streptomycin and 100 U/ml penicillin (Invitrogen, Carlsbad, CA, USA) at 37°C in a humidified incubator with 5% CO₂.

A modified MTT assay was used to evaluate cell proliferation. In brief, DU145 and PC3 cells were seeded at a density of 1.2×10⁴/well in 96-well plates with 90% density and subsequently exposed to increasing concentrations of thymoquinone for 24 h. Then, 20 µl MTT dye solution (5.0 mg/ml) was mixed with 180 µl medium and added to each well. After incubation at 37°C for 4 h, the culture medium was wiped out and the cells were lysed with DMSO to dissolve the formazan crystals. The optical density (OD) of each well was evaluated at 490 nm wavelength using a 96-well microplate reader (Bio-Rad, Hercules, CA, USA). The growth inhibitory rate was calculated as: [(OD 490control cells - OD 490treated cells)/OD 490control cells] × 100. The experiments were performed in triplicate.

Wound healing assay was performed to detect the effect of thymoquinone on cell migration. Prostate cancer DU145 or PC3 cells were seeded onto 6-well plates. When the cell density reached above 90%, scratch wounds were made across the monolayer using the tip of a 200-µl pipette. Then, the wounded cultures were incubated in a serum-free medium with thymoquinone treatment at different times (0, 24 h), and five random fields (magnification, ×100) were subsequently chosen from each scratch wound and observed by microscopy to evaluate the migratory capacity. The experiments were performed in triplicate.

Transwell migration assay is another method to evaluate cell migratory ability. It was performed using prostate cancer DU145 and PC3 cells after thymoquinone treatment. Cells (4×10⁴) with 200 µl serum-free medium were seeded onto the upper chamber, while 800 µl of medium supplemented with 10% fetal calf serum was added to the lower chamber. After certain incubation at 37°C, the adherent cells on the top chambers were wiped off with a cotton swab. The migrated cells on the lower surface of the filter were then fixed with 4% paraformaldehyde and stained with 0.1% crystal violet (Beyotime, Shanghai, China). Cells were then counted in five randomly chosen visual fields using a microscope at a magnification of ×100. The experiments were performed in triplicate.

The impact of thymoquinone on the invasion of prostate cancer cells was evaluated by Matrigel invasion assay using a Millicell chamber (Millipore, Billerica, MA, USA). The membrane (polycarbonic membrane, 8 µm pore size) in the top chamber was pretreated with 50 µl Matrigel (Matrigel, serum-free medium 1:5). After incubation at 37°C for 5 h, the cells (10×10⁴) in 200 µl serum-free medium were treated with thymoquinone according to the instructions of the Transwell migration assay.

DU145 and PC3 cells were treated with different concentrations of thymoquinone and the total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Complementary DNA (cDNA) was subsequently synthesized using a PrimerScript RT reagent kit (Takara, Dalian, China). Then, the relative levels of target gene messenger RNA (mRNA) transcript were measured by quantitative real-time PCR assay (qRT-PCR) using the SYBR-Green Master Mix. The sequences of primers for the PCR amplification were forward, 5′-GGCCAGATCCTGTCCAAGC-3′ and reverse, 5′-GTGGGTTTCCACCATTAGCAC-3′ for TGF-β (201 bp); 5′-CGTCCATCTTGCCATTCACG-3′ and reverse, 5′-CTCAAGCTCATCTAATCGTCCTG-3′ for Smad2 (182 bp); 5′-TGGACGCAGGTTCTCCAAAC-3′ and reverse, 5′-CCGGCTCGCAGTAGGTAAC-3′ for Smad3 (90 bp); forward 5′-CGAGAGCTACACGTTCACGG-3′ and reverse, 5′-GGGTGTCGAGGGAAAAATAGG-3′ for E-cadherin (119 bp); forward, 5′-GACGCCATCAACACCGAGTT-3′ and reverse, 5′-CTTTGTCGTTGGTTAGCTGGT-3′ for vimentin (238 bp); forward, 5′-CGAACTGGACACACATACAGTG-3′ and reverse, 5′-CTGAGGATCTCTGGTTGTGGT-3′ for Slug (87 bp); forward, 5′-CATGTACGTTGCTATCCAGGC-3′ and reverse, 5′-CTCCTTAATGTCACGCACGAT-3′ for β-actin (250 bp). All the experiments were performed in triplicate and calculated on the basis of the ΔΔCt method. The n-fold change in mRNA expression was evaluated according to the method of 2^−ΔΔCt.

Briefly, prostate cancer DU145 and PC3 cells were collected after thymoquinone treatment, and lysed on ice for 10 min with a lysis buffer [10 mmol/l Tris-HCl (pH 7.4), 150 mmol/l NaCl, 0.1% sodium dodecyl sulfate (SDS), 1 mmol/l ethylenediaminetetraacetic acid, 1 mmol/l ethylene glycol tetraacetic acid, 0.3 mmol/l phenylmethylsulfonyl fluoride, 0.2 mmol/l sodium orthovanadate, 1% NP-40, 10 mg/ml leupeptin and 10 mg/ml aprotinin]. After centrifugation and denaturation, the clarified protein lysates (~30–60 µg) were subjected to SDS-polyacrylamide gel electrophoresis (10 or 15%) and transferred to polyvinylidene membranes (Millipore). Membranes were subsequently probed with antibodies against TGF-β, Smad2, Smad3, E-cadherin, vimentin, Slug and β-actin overnight at 4°C. The immunoreactive bands were then washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature (25°C). Ultimately, the protein bands were visualized with ECL substrate and exposed to X-ray film.

TGF-β cDNA was cloned into the pcDNA3.1 vector. When prostate cancer DU145 or PC3 cells reached 70–80% confluency for plasmid transfection, the cells were then transfected with X-tremeGENE HP DNA transfection reagent (Roche, Mannheim, Germany) for 48 h following the manufacturer's instructions, and prepared for the subsequent turnover experiments.

All statistical analyses were evaluated by GraphPad Prism (version 5.0) software (GraphPad Software, Inc., La Jolla, CA, USA), and Student's t-test (two-sided) was used for comparisons involving only two groups. A value of P<0.05 was identified as a statistical significant difference.

Firstly, the chemical structure of thymoquinone is presented in Fig. 1A. In view of the metastatic potential, DU145 and PC3 cells were chosen for the next research. Subsequently, to exclude the effect of thymoquinone on growth inhibition in prostate cancer cells, a modified MTT assay was used. The results demonstrated that the growth of prostate cancer DU145 and PC3 cells at a density >90% was notably repressed upon thymoquinone treatment at concentrations ≥10.0 µM (Fig. 1B-E). Thymoquinone at 10 µM was used as the adequate concentration in the subsequent research, to exclude interference from growth inhibition by thymoquinone.

To explore the effect of thymoquinone on migratory and invasive abilities in prostate cancer cells, a wound healing assay was used to verify the antimetastatic effect of thymoquinone. The results indicated that the scratch width was much wider upon thymoquinone treatment than that in the control group in the DU145 and PC3 cells, suggesting the effective antimetastatic activity of thymoquinone on prostate cancer cells (Fig. 2A and B). To further confirm the effects of thymoquinone on the metastatic phenotype of prostate cancer cells, Transwell migration and Matrigel invasion assays were carried out. We found that the migratory ability of DU145 cells was significantly decreased by thymoquinone treatment (P<0.05) (Fig. 2C). Similar results were noted in the prostate cancer PC3 cells treated with thymoquinone (Fig. 2D). Next, we explored the effect of thymoquinone on the invasiveness of prostate cancer cells and our findings indicated that thymoquinone significantly suppressed the invasive capability of the DU145 and PC3 cells (P<0.05) (Fig. 2C and D).

Taken together, the results from the wound healing and Transwell assays revealed that thymoquinone had a strong antimetastatic effect on human prostate cancer cells.

EMT has been widely reported to be associated with the ability of migration and invasion in various cancers. In view of that, we first examined the expression levels of EMT markers including E-cadherin, vimentin and Slug by quantitative real-time PCR after treatment of DU145 and PC3 cells with 2.5, 5.0 and 10 µM thymoquinone for 24 h. We found that the mRNA level of E-cadherin was notably upregulated upon thymoquinone treatment in a concentration-dependent manner, whereas the expression levels of vimentin and Slug at mRNA levels were significantly downregulated (P<0.05)(Fig. 3A and B). Consistent with the above results, western blot analysis showed that thymoquinone decreased the expression levels of the mesenchymal markers including vimentin and Slug, while treatment with thymoquinone increased the expression of E-cadherin (Fig. 3C and D) in the DU145 and PC3 cells. The results revealed that thymoquinone reversed EMT in prostate cancer cells.

Studies have shown that the TGF-β signaling pathway is implicated in cancer invasion, metastasis and angiogenesis (16). To validate the underlying mechanism involved in the antimetastatic effect of thymoquinone in prostate cancer cells, we detected the expression levels of TGF-β, Smad2 and Smad3 in the prostate cancer cells upon thymoquinone treatment. Notably, thymoquinone downregulated TGF-β, Smad2 and Smad3 expression at the mRNA level in a concentration-dependent manner (Fig. 4A and B). In accordance with the above results, the protein levels of TGF-β, Smad2 and Smad3 in the DU145 and PC3 cells were significantly reduced following thymoquinone treatment (P<0.05) (Fig. 4C and D).

To further explore whether the TGF-β signaling pathway is involved in the antimetastatic effect of thymoquinone on prostate cancer cells, the TGF-β plasmid was transiently transfected into DU145 and PC3 cells to overexpress TGF-β. Intriguingly, overexpression of TGF-β partially restored the migratory and invasive capacities of the prostate cancer cells, which was inhibited by thymoquinone (Fig. 5A and B). These findings suggested that TGF-β may play a crucial role in the antimetastatic activity of thymoquinone in prostate cancer. Subsequently, we found a marked reversal of the elevated E-cadherin level, and reduced vimentin, Smad2 and Smad3 levels following the combined treatment of thymoquinone and TGF-β overexpression, compared with thymoquinone treatment alone (Fig. 5C and D). These results revealed that the antimetastatic capacity of thymoquinone in prostate cancer may be mediated by the TGF-β signaling pathway.