The p-AKT, AKT, XIAP, COX-2, p65 and cyclin B1 antibodies were obtained from Cell Signaling. The Bcl-2 and Bax antibodies were purchased from Santa Cruz Biotechnology, and the anti-β-actin antibody was purchased from Sigma. Thymoquinone (Sigma) was dissolved in dehydrated alcohol to create a 20 mmol/l stock solution.

TFK-1 cells, which are moderately differentiated, and HuCCT1 cells, which are poorly differentiated, were kindly provided by the Cancer Cell Repository, Tohoku University, Japan. The cells were grown in monolayers in RPMI-1640 medium, supplemented with antibiotics, 10% fetal bovine serum (FBS) (all from Gibco) and 2 mmol/l glutamine at 37°C with 5% CO₂ in a humidified atmosphere.

To assess cell viability, both the TFK-1 and HuCCT1 cells were seeded in 96-well plates at a density of 3×10³ cells/well and allowed to adhere and grow overnight in RPMI-1640 medium containing 10% heat-inactivated FBS. Cell Counting Kit-8 (CCK-8; Dojindo Lab-Orgemcitabineries, Kumamoto, Japan) was used to determine the viability of the cells. The cells were then cultured with TQ (0, 10, 20, 40, 60, or 80 μM) for 24, 48 or 72 h. Cell viability was measured using the CCK-8 kit according to the manufacturer’s instructions, as previously described (22). Three independent experiments were performed.

The CycleTest™ Plus DNA Reagent kit (BD Biosciences, San Jose, CA, USA) was used to identify the percentage of cells in the G0–G1, S and G2-M phases of the cell cycle. Subconfluent CCA cells were plated at a density of 1×10⁶ cells/well in 6-well plates. After treatment with TQ (0–60 μM) for 48 h, the supernatant was discarded, and the cells were washed with phosphate-buffered saline (PBS), then centrifuged. The cells were washed twice with ice-cold PBS, and 3 ml of ice-cold 70% ethanol was added. The cells were then incubated for 1 h at 4°C. The cells were washed twice with PBS, and 10 mg/ml RNase A was added. Propidium iodide (PI) was added to the tubes at a final concentration of 0.05 mg/ml, and the cells were incubated at 4°C for 30 min in the dark. Flow cytometric analysis was performed using FACScan (Becton-Dickinson) to detect the percentage of cells in the various phases of the cell cycle, as previously described (22). The experiments were repeated three times.

The PI/Annexin V-FITC apoptosis detection kit (BD Biosciences) was used to assess the number of apoptotic CCA cells after treatment with TQ; the kit was used according to the manufacturer’s instructions. Briefly, following treatment, the cells were harvested with trypsin, washed in PBS and counted. The cells were then resuspended in binding buffer, and 5 ml of Annexin V and 5 ml of PI were added. The cells were then incubated at room temperature for at least 15 min in the dark. The percentage of apoptotic cells was analyzed using flow cytometry (Epics Altra II; Beckman Coulter, USA) as previously described (23). The experiments were repeated three times.

Nuclear extract (5 μg) was incubated with 1 μg of poly(deoxyinosinic-deoxycytidylic acid) in binding buffer for 30 min at 4°C. DNA-binding activity was confirmed using a biotin-labeled oligonucleotide bio-NF-κB probe (5′-AGTTGAGGGGACTTTCCCAGGC-3′) using an EMSA kit according to the manufacturer’s instructions (Viagene, Beijing, China). The probe was resolved on a 4% polyacrylamide gel containing 0.25X TBE (Tris/borate/EDTA) buffer and visualized using a Cool Imager imaging system (IMGR002; Viagene).

The method was described previously (24). Briefly, the cells were washed twice in PBS, sonicated in lysis buffer and homogenized; alternatively, tumor tissues were excised, minced and homogenized in protein lysate buffer. Debris was removed by centrifugation. Samples (20 μg) of the total protein lysate were resolved on 12% polyacrylamide SDS gels and were electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% skim milk in TBST buffer (TBS plus 0.1% Tween-20), incubated with the appropriate primary Ab and subsequently incubated with an alkaline phosphatase-conjugated secondary Ab. The blots were developed using 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (Tiangen Biotech Co., Ltd., Beijing, China). The membranes were then washed, and the protein bands were visualized following exposure of the membrane to X-ray film. β-actin was used as a loading control.

Surgical procedures and administration of drugs to the animals were in accordance with the Institutional Animal Ethics Guidelines. Tumors were fixed by subcutaneous injection of 4×10⁶ HuCCT-1 tumor cells into the flanks of the mice. The tumor volumes were approximated according to the formula: π/6xa²xb, where a is the short axis, and b is the long axis. When the tumors reached ~100 mm³ after approximately two weeks, the mice were randomly assigned to four groups, each containing seven mice. The mice received either daily doses of PBS or 2, 4 or 8 mg/mouse TQ given daily by intragastric intubation. The doses and methods were based on our preliminary experiments and previous reports (15). The treatments lasted for 20 days, during which time the sizes of the tumors were documented. The mice were euthanized three days after the last treatment, and the tumors were excised, weighed and fixed in 10% buffered formalin for immunohistochemistry and apoptotic assays.

Immunohistochemistry analysis was performed using an anti-Ki67 Ab. Briefly, after deparaffinization, rehydration and antigen retrieval, the permeabilized sections (4 μm) were blocked with 3% bovine serum albumin (BSA) for 2 h and incubated overnight with primary Abs. The sections were subsequently cultivated for 30 min with IgG, mounted and examined under a light microscope or a fluorescence microscope.

Histological analysis of DNA fragmentation was performed to identify the apoptotic cells. Tumor sections were stained with the TUNEL reagent (Roche, Shanghai, China), and the TUNEL-positive cells were counted in 10 randomly selected ×400 high-power fields. The apoptosis index was calculated according to the following formula: The number of apoptotic cells/total number of nucleated cells × 100%.

The data obtained are expressed as the mean values ± standard deviation of at least three separate experiments. Statistical significance was determined using Student’s t-test, and P<0.05 was considered to indicate a statistically significant difference.

To determine the effects of TQ on cell growth, the CCA cell lines (TFK-1 and HuCCT1) were treated with varying concentrations (0–80 μM) of TQ for 24, 48 and 72 h, and cell viability was assessed using the CCK-8 assay. As shown in Fig. 1B and C, TQ treatment resulted in a dose- and time-dependent inhibition of cell growth in both the CCA cell lines tested. The inhibitory concentration IC₅₀ values were ~62.91, 33.47 and 23.17 μM for the TFK-1 cells and 48.27, 27.51 and 20.28 μM for the HuCCT1 cells for the 24, 48 and 72 h treatments, respectively. Of note, TQ inhibited the poorly differentiated HuCCT1 cell line more than the moderately differentiated TFK-1 cell line. These results indicate that TQ has potent antiproliferative effects in CCA cell lines.

Based on the preliminary assays in which we evaluated the effect of TQ on the growth of CCA cells, the 0, 20, 40 and 60 μM doses of TQ were selected for further in vitro mechanistic studies. To explore the underlying mechanism of the TQ-induced growth inhibition of the cells, the effect of TQ on the cell cycle distribution was studied using flow cytometric analysis of the cellular DNA content. As shown in Fig. 2, treatment with TQ for 48 h resulted in a significant dose-dependent arrest of the CCA cells in the G2/M phase of the cell cycle. The G2/M phase cell cycle distribution was 4.05, 9.40, 11.76 and 18.55% in the TFK-1 cells (Fig. 2A) and 5.51, 12.25, 16.14 and 24.02% in the HuCCT1 cells when treated with 0, 20, 40 and 60 μM TQ (Fig. 2B), respectively. This increase in the percentage of cells in G2/M phase was accompanied by a concomitant reduction in the percentage of cells in G0/G1 phase.

In addition to the cell cycle arrest, morphological observation of the TQ-treated CCA cells indicated that the TQ-induced growth inhibition may also be associated with the induction of apoptosis. Therefore, the apoptosis-inducing effect of TQ in CCA cells was assessed. The cells were treated with various concentrations of TQ (0–60 μM). Then, the cells were stained with Annexin V/PI and subjected to flow cytometry to determine the amount of apoptotic cells. As shown in Fig. 3, treatment with TQ for 48 h resulted in a significant dose-dependent enhancement in both the number of early and late apoptotic CCA cells. The percentages of apoptotic cells were 8.5, 18.0, 26.1 and 67.3% in the TFK-1 cells (Fig. 3A) and 5.9, 21.7, 31.9 and 75.7% in the HuCCT1 cells treated with 0, 20, 40 and 60 μM TQ (Fig. 3B), respectively. Taken together, our results suggest that the TQ-induced growth inhibition was partially due to the induction of apoptosis.

To determine whether the antitumor effect was associated with PI3K/Akt signaling, the TFK-1 and HuCCT-1 cells were treated as described above for 48 h and were then subjected to western blot analysis. As shown in Fig. 4, treatment with TQ resulted in the downregulation of p-Akt in both of the CCA cell lines, whereas the levels of total Akt protein were not altered (Fig. 4A). Furthermore, the downregulation of p-Akt was associated with the downregulation of XIAP and Bcl-2, as well as the upregulation of BAX, which is further evidence of the induction of apoptosis in cells exposed to TQ for 48 h (Fig. 4B). These results suggest that PI3K/Akt signaling was, at least partially, involved in the effect.

To investigate whether TQ abrogates the constitutively active NF-κB in the CCA cells, we used a DNA binding assay to determine whether NF-κB, a downstream target of the PI3K/Akt signaling pathway, was also involved in the effect. After the above treatments, nuclear extracts were obtained and the NF-κB DNA-binding activity was determined using an EMSA assay; additionally, total protein extracts were used to determine the expression of the downstream NF-κB genes by western blot analysis. As shown in Fig. 4C, treatment with TQ resulted in constitutive NF-κB activity. In both cell lines, TQ treatment significantly reduced the DNA-binding activity of NF-κB, and this decrease was correlated with the increased inhibitory effect on cell viability and increased apoptosis. To confirm our hypothesis, we tested whether the inhibitory effect of TQ on NF-κB may result in the downregulation of NF-κB regulated genes. As shown in Fig. 4B, in both cell lines, TQ treatment resulted in significantly decreased expression of COX-2, VEGF and cyclin B1, which are known to be regulated by NF-κB. These results are consistent with the increased growth inhibition and apoptosis-inducing effects, suggesting that in vitro TQ inhibits NF-κB DNA-binding activity and the expression of its downstream gene products, which are believed to be partially responsible for the enhanced cell killing.

To evaluate the role of TQ in tumor proliferation in vivo, we examined the ability of TQ to suppress the growth of HuCCT-1 xenografts in nude mice. HuCCT-1-derived xenograft tumors were allowed to develop and grow to a size of ~100 mm³; once the tumors had reached this size, TQ was administered at daily doses of 2, 4 or 8 mg/mouse by intragastric intubation for 20 days. As shown in Fig. 5A, the tumors in the mice treated with various concentrations of TQ decreased in volume after 20 days of treatment, and this reduction in volume was significantly different from that of the control tumors. These results suggested that TQ may largely inhibit the growth of the tumor xenografts. In general, the tumors in the control group grew continuously during the experimental period, whereas tumor growth in the TQ-treated mice was suppressed significantly (Fig. 5C). However, there was no apparent change in liver (Fig. 5J), spleen (Fig. 5K) or body weight (Fig. 5B) in the animals, indicating that TQ is a potential therapeutic agent for the treatment of CCA and is relatively non-toxic to mice. Ki-67 staining for cell proliferation was performed in the tumors that were removed from the animals. The relative number of Ki-67-positive tumor cells was substantially less in the tumors from mice treated with TQ than in the control tumors (Fig. 5G and H). Additionally, as shown in the representative images, the tumor xenografts from the TQ-treated mice showed a marked increase in the number of TUNEL-positive apoptotic cells compared to the control group (Fig. 5G and I). Quantification of TUNEL-stained samples showed a 2- to 3-fold increase (P<0.05) in the number of TUNEL-positive cells in the TQ-treated groups compared to the control group.

We next evaluated the expression of p-Akt in the HuCCT-1 xenografts from the control and TQ-treated mice. The expression of p-Akt and its downstream gene products was assessed in the tumor tissues using western blot analysis, and the results showed that TQ treatment resulted in a decrease in the expression of p-Akt, XIAP and Bcl-2, as well as an increase in the expression of BAX (Fig. 5E). These results suggest that the PI3K/Akt signaling pathway is partially involved in the effects of TQ on CCA cells in vivo.

We investigated whether the antitumor effect of TQ in mice was associated with the inhibition of NF-κB activation. The EMSA results showed that TQ had an effect on NF-κB activation in the tumor samples (Fig. 5F). Accordingly, we observed the expression of downstream NF-κB target genes (as mentioned above) in these tumors samples. As shown in Fig. 5D, TQ treatment resulted in a significant decrease in the expression of all these proteins in the HuCCT-1 xenografts compared to the control treatment.