Antibodies were obtained from the following commercial sources: antibodies against survivin, XIAP, Smac and β-actin were obtained from Epitomics (Burlingame, CA, USA), and the caspase-3 antibody was from Abcam (Cambridge, MA, USA). Anti-retinoblastoma antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Thymoquinone was purchased from Sigma (St. Louis, MO, USA) and was dissolved in DMSO to make 20 mmol/l stock solution. The 0.1% DMSO alone was set as the control group.

Human osteosarcoma cell line SaOS-2 was purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). Mouse osteoblastic cell line MC3T3-El was preserved in our laboratory. SaOS-2 and MC3T3-El cells were maintained in modified Eagle’s medium containing 10% fetal bovine serum (FBS), 0.5% penicillin-streptomycin, and 1% glutamine at 37°C with 5% CO₂. Human umbilical vein endothelial cells (HUVECs; obtained from ATCC) were cultured in gelatin-coated plates with M199 medium containing 20% FBS, endothelial cell growth supplement (50 μg/ml, Sigma) and antibiotics, and incubated at 37°C in 5% CO₂ in air.

Morphological changes of SaOS-2 cells were observed under a fluorescence microscope (Olympus, Tokyo, Japan) by the Hoechst staining method. SaOS-2 cells were seeded at a density of 2×10⁵ cells per well onto a 12-well plate for 24 h, followed by incubation with vehicle alone (0.1% DMSO) or 80 μmol/l thymoquinone for 24 h. Following treatment, cells were fixed with 3.7% formaldehyde for 15 min, permeabilized with 0.1% Triton X-100 and stained with 5 mg/ml of Hoechst 33258 for another 5 min at 37°C. The cells were then washed with PBS and observed under a fluorescence microscope.

SaOS-2 cells were seeded at a density of 3×10³ cells per well in 96-well culture plates. After overnight incubation, the medium was removed and replaced with fresh medium containing different concentrations of thymoquinone (20, 40 and 80 μmol/l). Following a 24-h incubation, cell viability was determined by CCK-8 assay (Dojin Laboratory, Kumamoto, Japan) according to the manufacturer’s instructions. Briefly, CCK-8 solution was added to cells in 96-well plates, the cells were then incubated at 37°C for 60 min, and absorbance was measured at 570 nm using an MRX Revelation 96-well multiscanner (Dynex Technologies, Chantilly, VA, USA). This experiment was repeated three times.

The measurement of phosphatidylserine redistribution in a plasma membrane was conducted according to the protocol outlined by the manufacturer of the Annexin V-FITC/PI apoptosis detection kit (Abcam). After exposing the cells to increasing concentrations of thymoquinone (20, 40 and 80 μmol/l) for 24 h at 6-well plates, harvested 1×10⁵ cells were incubated with 5 μl of Annexin V-FITC and 5 μl of PI for 15 min at room temperature in the dark and then analyzed by flow cytometry using the Cell Quest program (Becton-Dickinson, San Jose, CA, USA).

The angiogenesis of HUVEC induced by Matrigel was assessed by the modified methods previously described (12). Briefly, Matrigel (Becton-Dickinson) was dissolved at 4°C overnight, and each well of pre-chilled 48-well plates was coated with 100 μl Matrigel and incubated at 37°C for 45 min. HUVECs (2×10⁴ cells) were seeded onto the Matrigel in 250 μl M199 supplemented with 20% FBS and incubated with various concentrations of thymoquinone (40, 80 and 160 nmol/l) at 37°C for 24 h in a humidified 5% CO₂ atmosphere. Endothelial cell tube formation was photographed and the light micrograph images were stored in a computer. Tubular structures were quantified by manual counting and percent inhibition was expressed using untreated wells as 100%.

At the end of this incubation, cells were washed with ice-cold PBS and lysed in NP40 lysis buffer (20 mmol/l Tris-HCl (pH 7.4), 100 mmol/l NaCl, 1% NP40, 0.5% sodium deoxycholate, 5 mmol/l MgCl₂, 0.1 mmol/l phenylmethylsulfonyl fluoride, and 10 mg/ml of protease inhibitor mixture). Protein was extracted using Mammalian Protein Extraction Reagent (Pierce Inc., Rockford, IL, USA) and its concentration was determined by BCA (Pierce) assay. Proteins (30 μg) were separated in 10–15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyphorylated difluoride (PVDF) membrane. Membranes were incubated with primary antibody overnight at 4°C and then with the respective secondary antibodies. Immunoreactive bands were detected by the enhanced chemiluminescence (ECL) kit for western blotting detection with hyper-ECL film. The same membrane was reprobed with the anti-β-actin antibody, which was used as an internal control for protein loading.

Following treatment, the cells were suspended in 400 μl of ice-cold lysis buffer [1 mol/l HEPES (pH 7.9), 1 mol/l KCl, 0.5 mol/l EDTA, 0.1 mol/l EGTA, 0.1 mol/l DTT, 0.1 mol/l PMSF, 2 μg/ml aprotinin, 2 μg/ml leupeptin, and 0.5 mg/ml benzamidine] for 15 min. The cells were allowed to swell on ice for 20 min and then 4.8 μl of 10% Nonidet P-40 was added to every 400 μl cell suspension, vortexed, and centrifuged for 1 min at 4°C. The nuclear pellet was resuspended in 30 μl nuclear extraction buffer [2 mol/l HEPES (pH 7.9), 0.4 mol/l NaCl, 1 mol/l EDTA, 0.1 mol/l EGTA, 0.1 mol/l DTT, 0.1 mol/l PMSF, 2 μg/ml aprotinin, 2 μg/ml leupeptin, and 0.5 mg/ml benzamidine] and incubated on ice with intermittent mixing. The tubes were then centrifuged at 10,000 g for 20 min at 4°C, and the supernatant (nuclear extract) was quantified using the BCA protein assay. Electrophoretic mobility shift assay (EMSA) was performed by incubating 10 μg of nuclear proteins with IRDye™-700 labeled NF-κB oligonucleotide. The incubation mixture included 2 μg of poly(deoxyinosinic-deoxycytidylic acid) in a binding buffer. The DNA-protein complex formed was separated from free oligonucleotide on 8.0% native polyacrylamide gel using buffer containing 50 mmol/l Tris, 200 mmol/l glycine (pH 8.5), and 1 mmol/l EDTA and then visualized by Imager apparatus. Equal protein loading was ensured by immunoblotting 10 μg of nuclear protein with anti-Rb antibody.

Male athymic BALB/c nu/nu mice (4–6 weeks old) were obtained from Wenzhou Medical College. All animals were maintained in the standard mouse plexiglass cages in a room maintained at constant temperature and humidity under 12-h light and darkness cycle. The food, water, and bedding for these immunocompromised mice were sterilized and changed at least once weekly.

The spontaneously metastatic mouse model was developed as previously described (16). Briefly, the left tibia was wiped with 70% ethanol and a 27-gauge needle coupled to a Hamilton syringe was inserted through the tibial plateau with the knee flexed, and 1×10⁵ SaOS-2 cells, resuspended in 10 μl PBS, were injected into the marrow space of the proximal tibia. As a control, all animals were injected with controled 0.1% DMSO in the contralateral tibia. After 1 week of implantation, mice were randomized into 2 groups (n=6): a) vehicle alone (control); b) thymoquinone (6 mg/kg given daily by intragastric intubation for 15 days) (6). Serial primary tumor volumes were excised and the final tumor volume was measured using the formula: π × (d/2)³, where d is the diameter of the tumor. Mice were sacrificed 5 days after the last treatment. Half of the tumor tissue was formalin fixed and paraffin embedded for immunohistochemistry and routine H&E staining. The other half was snap-frozen in liquid nitrogen and stored at −80°C. H&E staining confirmed the presence of tumor(s) in each tissue.

Xenograft tissues of all mice from both the control and the treated groups were harvested at the end of the treatment and fixed with formaldehyde. The fixed tissue was sectioned and immunostaining was performed using primary antibodies specific for Ki-67, CD34 and NF-κB with appropriate dilutions and using normal host serum for negative controls, followed by staining with appropriate HRP-conjugated secondary antibodies. Results were expressed as percentage of Ki-67⁺ cells ± SE per ×40 magnification. A total of ten ×40 fields were examined and counted from three tumors of each of the treatment groups. Areas of greater vessel density were then examined under higher magnification (x100) and counted. Any distinct area of positive staining for CD34 was counted as a single vessel. Results were expressed as the mean number of vessels ± SE per high-power field (x100). A total of 20 high-power fields were examined and counted from three tumors of each of the treatment groups. The slides were developed in diaminobenzidine and counterstained with a weak solution of haematoxylin solution stain. H&E was carried out on paraffin-embedded tissue sections. The stained slides were dehydrated and mounted in permount and visualized on an Olympus microscope (Olympus, Japan). Images were captured with an attached camera linked to a computer.

Three independent experiments were performed, and data are represented as the mean ± SD for the absolute values or percent of controls. SPSS15.0 software was used for statistical analysis. Statistically significant differences between values obtained under different experimental conditions were determined using the 2-tailed unpaired Student’s t-test or χ² test. P<0.05 was considered to indicate statistically significant differences.

In order to investigate whether thymoquinone exerts anti-viability effects, SaOS-2 cells were treated with increasing concentrations of thymoquinone (20, 40 and 80 μmol/l) for 24 h, and then the viability of the cells was examined by CCK-8 assays. As shown in Fig. 1B, the viability of SaOS-2 cells was decreased dose-dependently in the presence of thymoquinone.

To further examine the cytotoxic effects of thymoquinone on SaOS-2 cells, the cells were treated with 40 μmol/l of thymoquinone for 24 h and then observed under fluorescence microscope by Hoechst 33258 staining. Results showed that there was an increase in the number of irregular nuclear, fragmented nucleus, convoluted nucleus and giant nucleus after treatment with thymoquinone (Fig. 1A), suggesting that the DNA fragmentation was occurring in these cells.

To determine whether the induction of cell death by thymoquinone could be linked to apoptosis in SaOS-2 cells, we used a method that allows one to detect concurrently viable, necrotic, early apoptotic and late apoptotic cells based on distinct double-staining patterns with a combination of FITC-conjugated Annexin V and PI.

Results clearly demonstrated that treatment with thymoquinone (Fig. 1C) for 24 h increased the percentage of early apoptotic cells in a dose-dependent manner (Fig. 1D). These data indicate that thymoquinone exerts cytotoxic effects which may be mediated by apoptosis on SaOS-2 cells.

Since the final event during angiogenesis is the organization of endothelial cells in a three-dimensional network of tube, we performed a tube formation assay to investigate the effect of thymoquinone on the capillary-like structure formation of HUVECs. Endothelial cells plated on Matrigel align themselves forming cords, and the tube-like structure formation was maximal within 15 h (Fig. 1E). Treatment of cells with thymoquinone resulted in significant inhibition of tubule formation of HUVECs on Matrigel (Fig. 1F). Our results clearly demonstrate that thymoquinone is effective in controlling the tube formation of endothelial cells in vitro.

Next, we analyzed whether thymoquinone could abrogate constitutively expressed NF-κB in osteosarcoma cells. SaOS-2 cells were treated with varying doses of thymoquinone (20, 40 and 80 μmol/l) for 24 h and subjected to gel shift assay (EMSA). As shown in Fig. 2A, EMSA revealed that thymoquinone induced a concentration-dependent decrease in NF-κB DNA binding activity in SaOS-2 cells, which was further comfirmed by a supershift experiment. These observations provide strong evidence that thymoquinone is effective in downregulating NF-κB DNA-binding activity.

We assessed the expression of the NF-κB-regulated genes XIAP and survivin, the overexpression of which has been linked to tumor survival, chemoresistance, and radioresistance, and VEGF, which plays an important role in angiogenesis. Western blotting revealed that thymoquinone induced a concentration-dependent decrease in the levels of these molecules compared with the control treatment in SaOS-2 cells. Western blotting also showed that thymoquinone increased the expression of caspase-3 and Smac (Fig. 2B).

For the in vivo experiment, 12 mice were divided into two groups as described in Materials and methods. The results showed that after 15 days of treatment with thymoquinone, thymoquinone did not produce significant non-tumor toxicity in tumor-bearing mice. The average mouse body weight of the control group decreased from 22.28±1.22 to 21.24±1.32 g, whereas that of the thymoquinone-treated group increased from 22±1.5 to 24.4±1.2 g (Fig. 3C). The slight decrease of the control group is due to the growth of tumors in the xenograft mice. Next, we determined the mean tumor volume immediately following euthanization in all mice. The mean tumor volume was 74±25 mm³ in the treated group, vs 126±41 mm³ in the negative control tumors, resulting in a significant difference of tumor volume in the xenograft model (P<0.05).

H&E evaluation of the tumors from all groups showed high-grade carcinoma associated with tumor apoptosis and necrosis (Fig. 3A). However, there were significant differences in the pattern of necrosis, inflammatory response and fibrosis among the two groups. In the control group, the tumor was largely viable with high mitosis and minimal intratumoral stroma, whereas the peripheral tumor was largely viable and consisted of large nests of neoplastic cells with minimal intratumoral stroma. By contrast, in the group receiving thymoquinone treatment there was marked tumor destruction throughout the entire tumor.

We next examined the expression of the cell proliferation marker Ki-67 and the microvessel density marker CD34 in tumor tissues from the two groups. The results in Fig. 3A showed that thymoquinone significantly downregulated the expression of Ki-67 and CD34 in tumor tissues compared with the control group. For Ki-67, the proliferation index of control was 77.2±5.1%, but the group receiving thymoquinone treatment was 49.3±4.5%. For CD34, the microvessel density was reduced from 192.3±10.2 to 98.6±14.1 after the treatment of thymoquinone. Furthermore, immunohistochemical analysis revealed that the expression of NF-κB was significantly decreased in tumors derived from mice treated with thymoquinone compared with untreated mice. Tumors also revealed downregulation of a few important NF-κB-regulated molecules such as survivin, XIAP and VEGF proteins, which is consistent with our in vitro results.