The human LN229 and U251 cell lines were purchased from the Institute of Biochemistry and Cell Biology, the Chinese Academy of Sciences. All cells were maintained at 37°C in a 5% CO₂ incubator in Dulbecco's modified Eagle's medium (DMEM; Gibco-BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Invitrogen). NCTD was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Cells were plated in 96-well plates at a density of 5,000 cells/well. Cells were allowed to attach overnight in growth medium. After 24 h, the cells were treated with NCTD. After incubation for 72 h, cellular proliferation was measured using the MTS assay and absorbance was measured at 490 nm. Proliferation data are presented as mean ± SD.

For cell cycle analysis by flow cytometry, NCTD-treated and NC cells in the log phase of growth were harvested, washed with phosphate-buffered saline (PBS), fixed with 90% ethanol overnight at 4°C, and then incubated with RNase at 37°C for 30 min. Nuclei of the cells were stained with propidium iodide for an additional 30 min. A total of 20,000 nuclei were examined in a FACSCalibur flow cytometer (Becton-Dickinson), and DNA histograms were analyzed using Modifit software. Three independent experiments were performed, and the data are presented as the mean ± SD.

Forty-eight hours after treatment with NCTD and PBS, apoptosis in the cultured cells was evaluated using Annexin V labeling. For the Annexin V assay, an Annexin V-FITC-labeled Apoptosis Detection kit (Abcam) was used according to the manufacturer's instructions. Three independent experiments were performed, and the data are presented as the mean ± SD.

Transwell invasion assay filters (Costar) were coated with Matrigel (3.9 mg/ml, 60–80 µl) on the upper surface of a polycarbonate membrane (diameter 6.5 mm and pore size 8 µm). After incubating at 37°C for 30 min, the Matrigel had solidified and served as the extracellular matrix for the tumor cell invasion analysis. The Transwell migration assay was carried out without the presentation of Matrigel on the upper surface of the polycarbonate membrane. A total of 600 µl of conditioned medium derived from the tumor cells was used as a source of chemoattractant and was placed in the bottom compartment of the chamber. Harvested cells (5×10⁴) in 100 µl of serum-free DMEM were added into the upper compartment of the chamber. After 24 h of incubation at 37°C with 5% CO₂, the medium was removed from the upper chamber. The non-invaded cells on the upper side of the chamber were scraped off with a cotton swab. The cells that had invaded from Matrigel into the pores of the inserted filter were fixed with 100% methanol, and stained with crystal violet. The number of cells invading through the Matrigel was counted using three randomly selected visual fields from the central and peripheral portions of the filter by an inverted microscope at x200 magnification. Each assay was repeated three times.

Total RNA was isolated using TRIzol reagent (Invitrogen) and cDNA was synthesized using the High Capacity cDNA Archive kit (Applied Biosystems) according to the manufacturer's instructions.

WIF-1 and cyclin B1 mRNA were quantified by qRT-PCR using SYBR Premix Ex Taq (Takara Bio, Inc.). The primers were: WIF-1 forward, 5′-CCGAAATGGAGGCTTTTGTA-3′ and reverse, 5′-TGGTTGAGCAGTTTGCTTTG-3′; cyclin B1 forward, 5′-GCAACCTCCAAGCCCGGACTG-3′ and reverse, 5′-GCAACCTCCAAGCCCGGACTG-3′.

The PCR conditions included an initial denaturation step of 95°C for 2 min, followed by 35 cycles of 95°C for 10 sec, 56°C for 20 sec and 72°C for 20 sec, and a final elongation step of 72°C for 10 min. All qRT-PCRs were performed in duplicate. Quantitation relative to the endogenous control was carried out using Applied Biosystems 7500 Fast System SDS software.

Genomic DNA from the cell lines and normal lung tissue was extracted using the DNeasy kit (Qiagen), according to the manufacturer's instructions. Bisulfite modification of genomic DNA was performed using a methylation kit (EZ DNA Methylation kit; Zymo Research, Orange, CA, USA). Bisulfite-treated genomic DNA was amplified using either a methylation-specific or an unmethylation-specific primer set. HotStarTaq DNA polymerase (Qiagen) was used in the experiments.

Sequences of the methylation-specific primers were 5′-GGGCGTTTTATTGGGCGTAT-3′ (forward) and 5′-AAACCAACAATCAACGAAC-3′ (reverse). Sequences of the unmethylation-specific primers were 5′-GGGTGTTTTATTGGGTGTAT-3′ (forward) and 5′-AAACCAACAATCAACAAAAC-3′ (reverse).

Extraction of proteins from the cultured cells was followed by immunoblotting with the relevant antibodies. Each experiment was repeated at least three times.

Immunofluorescence staining was conducted with LN229 and U251 cells cultured on coverslips. The cells were fixed in 4% paraformaldehyde and permeabilized for 10 min in buffer containing 0.1% Triton X-100. The relevant antibodies were then added at the dilutions recommended by the manufacturers. DAPI reagent was used to stain the glioma cell nuclei, and the cells were visualized using an FV-1000 laser-scanning confocal microscope and analyzed using IPP5.1 (Olympus, Tokyo, Japan).

Quantitative variables are expressed as mean ± SD and analyzed by one-way analysis of variance (ANOVA) and the Student's t-test. All differences were considered to be statistically significant at the level of P<0.05. Statistics were performed using the SPSS Graduate Pack version 11.0 statistical software (SPSS, Inc., Chicago, IL, USA).

The LN229 and U251 cells were exposed to 0, 3.75, 7.5, 15, 30, 60 and 120 µM NCTD and PBS for 48 h. The results indicated that the cellular viability decreased with increasing NCTD concentrations (Fig. 1). The 50% growth inhibition (IC₅₀) of NCTD was 44 µM for LN229 and 10 µM for U251 cells after 48 h, respectively.

To further characterize the growth arrest, LN229 and U251 cell lines were treated with 44 and 10 µM NCTD, and after 48 h they were subjected to propidium iodide-staining and FACS analysis. Treatment with NCTD resulted in accumulation of cells in the G2 phase, with ~42.33±0.514% of NCTD-treated LN229 and 28.28±2.74% of NCTD-treated U251 cells in the G2 phase compared with 9.09±0.09% PBS-treated LN229 and 16.80±0.57% PBS-treated U251 cells (Fig. 2A) (P<0.05). Cyclin B1 level was decreased in the cell lines, as shown using western blotting (Fig. 3). These analyses indicate that NCTD induced cell cycle arrest at the G2 phase.

NCTD also inhibited glioma cell survival. As shown in Fig. 2B, compared with the NC groups (4.47±1.13 and 4.55±2.11%) in the LN229 and U251 cells, the treatment of NCTD caused a significant increase in apoptotic death (26.40± and 32.00±5.00%) (P<0.05). The Bcl-2 protein level decreased and the cleaved caspase-3 protein level increased in the NCTD-treated cell lines, as shown using western blotting (Fig. 3).

The intrinsic pathway of apoptosis is controlled by Bcl-2 family proteins, and cell death depends on the balance between pro-apoptotic (Bax) and anti-apoptotic (Bcl-2, Bcl-xl and Mcl-1) proteins. We also investigated the treatment effect on such proteins. As shown in Fig. 3, treatment with NCTD had some effect on Bax expression. However, treatment with NCTD resulted in a significant downregulation of Bcl-2. Cleaved caspase-3 release was detected by western blot analysis after treatment of NCTD. The activity of pro-caspase-3 cleaved to caspase-3 increased after treatment of NCTD. Together, this analysis demonstrated that NCTD induced downregulation of Bcl-2 as previously reported but had little effect on Bax. Activation of cleaved caspase-3 indicated that the compound induced the intrinsic and extrinsic apoptotic pathways.

To test whether NCTD regulates tumor cell invasion and migration, Transwell assays were performed. The assays showed that NCTD markedly inhibited Transwell invasion and migration of glioma cells (Fig. 2C and D). In regards to the LN229 cells, the number of cells invading through the Matrigel in the NCTD group was 38.30±2.87 which was significantly decreased compared with the number of invading cells in the NC group (110.00±8.20) (P<0.05). Regarding the U251 cells, the invasive activity was also inhibited in the NCTD group (41.3±3.30), when compared with that of the NC group (90.67±8.16) (P<0.05). Regarding the LN229 cells, the number of cells migrating through the polycarbonate membrane in the NCTD group was 55.67±4.19, significantly decreased when compared with the number of invading cells in the NC group (157.00±2.94) (P<0.05). Regarding the U251 cells, the invasive activity was also inhibited in the NCTD group (47.33±1.70), compared with that of the NC group (95.00±4.08) (P<0.05). The above data showed that NCTD suppressed the invasion and migration of the human glioma cells.

qRT-PCR assay was also performed to analyze the expression of WIF-1 at the transcription level. We found that the WIF-1 transcript was downregulated in the LN229 and U251 cell lines in the NC group. In contrast, it was upregulated in the NCTD-treated cells (Fig. 4A). The results showed that WIF-1 expression in the LN229 and U251 cell lines in the NC group (10.00±0.00) was significantly lower compared with the expression level in NCTD-treated cells (50.23±5.00 and 61.00±6.56) (P<0.05). To examine whether the methylation status of the promoter correlates with the expression of WIF-1, methylation-specific PCR was carried out (Fig. 4B). Promoter demethylation was observed in the NCTD-treated cells. In contrast, aberrant methylation was observed in the NC group. To detect the expression level of WIF-1, western blotting was performed in the LN229 and U251 cell lines (Fig. 4C). The level of WIF-1 expression was significantly lower in the LN229 and U251 cell lines in the NC group than those levels in the NCTD-treated cells, but expression of DNA methyl-transferase-1 (DNMT1) was not influenced (Fig. 4C).

qRT-PCR results determined that the relative expression level of cyclin B1 in the NCTD-treated LN229 cells was 20.40±5.43% (P<0.05), and 35.54±5.99% in the NCTD-treated U251 cells (P<0.05), compared with the NC groups, respectively (Fig. 4D).

β-catenin, Wnt-target proteins and their expression in many cell lines have been described. Wnt signaling has been proven to be associated with various disease pathologies. NCTD was reported to act as a negative regulator of Wnt signaling. Western blotting of total protein extracts from the LN229 and U251 cells revealed that the total β-catenin content was reduced after treatment with NCTD (Fig. 3). The level of protein cyclin B1, a known Wnt downstream target, was also significantly reduced (Fig. 3). Immunofluorescence assays in the LN229 and U251 cell lines revealed altered nuclear location of β-catenin. After treatment of NCTD, β-catenin was mainly located in the cytoplasm, while β-catenin was mainly located in the nucleus in the NC cells (Fig. 5). This showed that the location of β-catenin in cells shifted from the nucleus to the cytoplasm when the cells were treated with NCTD.