Resveratrol and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) bromide were purchased from Sigma (St. Louis, MO, USA) and dissolved at a concentration of 100 mmol/l in 100% dimethyl sulfoxide (DMSO) as a stock solution, stored at −20°C. The final DMSO concentration did not exceed 0.1% throughout the study. Annexin V-FITC and primary antibody for human hypoxia-inducible factor-1α (HIF-1α) was purchased from BD Transduction Laboratories (San Diego, CA, USA). Antibodies for p70S6K, p-4E-BP-1, caspase-3, Bcl-2, and β-actin were purchased from Santa Cruz Biotechnology, Inc., (Santa Cruz, CA, USA). Antibodies for cyclins A, B, D, E and cyclin-dependent kinases (CDK)/p34 were from New England Biolabs (Beverly, MA, USA). Horseradish peroxidase (HRP) conjugated secondary antibodies were obtained from Pierce (Rockford, IL, USA).

CNE-2Z, a poorly differentiated human NPC cell line, and CNE-1, a well differentiated human NPC cell line, were cultured in RPMI-1640 complete culture medium (Gibco, USA) supplemented with 15% heat-inactivated fetal bovine serum (FBS; Gibco), penicillin (100 U/ml) and streptomycin (100 μg/ml) at 37°C in a humidified incubator with 5% CO₂ (10).

The effect of resveratrol on the cell growth of CNE-1 and CNE-2Z cells was examined using the MTT assay. Cancer cells were plated in 96-well plates at 10⁴ cells/well. Following pretreatment with different concentrations of resveratrol for 24 or 48 h, viable cells were determined using the MTT Assay kit (Chemicon, Temecula, CA, USA) according to the manufacturer’s protocol. At indicated time-points, 20 μl of MTT solution (5 mg/ml in PBS) were added into each well and incubated for 4 h. Then, 100 μl of DMSO was added to dissolve the crystals. The plate was then left to stand for 10 min at room temperature and the corresponding absorbance was measured at 490 nm. Each experiment was performed in triplicate.

For the apoptosis analysis, NPC cells were plated into 6-well plates (2×10⁵ cells/well) and cultured for 24 h. Then cells were incubated with 50 μM resveratrol for 48 h. Subsequently, the trypsinized cells together with the medium were collected and centrifuged. The pelleted cells were washed and resuspended in PBS for flow cytometric analysis using the Annexin V-FITC/propidium iodide (PI) Apoptosis Detection kit according to the manufacturer’s protocols (BD Biosciences).

For the cell cycle analysis, NPC cells were plated into 6-well plates (2×10⁵ cells/well) and cultured for 24 h. Then cells were incubated with 50 μM resveratrol for 48 h. The cells were collected and washed with PBS 2 times, followed by resuspending in 1 ml PBS. The cells were fixed with ethanol (70%) for 2 h, centrifuged, washed with PBS, and resuspended in 1 ml of PI solution (0.1% Triton X-100, 200 μg/ml RNase, 20 μg/ml PI in 10 ml of PBS) and incubated at room temperature for 30 min. Cell cycle distribution was analyzed by flow cytometry.

Treated and untreated NPC cells were lysed with RIPA lysis buffer containing 20 mmol/l β-mercaptoethanol, 250 μmol/l sodium orthovanadate, 1 mmol/l PMSF and complete protease inhibitor cocktail (Sigma), and incubated at 4°C for at least 1 h. The lysates were ultra-sonicated and centrifuged at 14,000 × g for 15 min. The supernatants were collected and stored at −70°C. Protein concentrations were determined by BCA methods. Protein (100 μg) was separated on 8–10% polyacrylamide-SDS gel and electroblotted onto nitrocellulose membranes (Bio-Rad). After blocking with TBS/5% skim milk, the membrane was incubated overnight at 4°C with specific primary antibodies (1:1,000–1:2,000) followed by incubation with HRP-conjugated secondary antibodies (1:5,000) for 1 h at room temperature, and signals were detected by ECL (10).

Six to eight-week-old female nude mice (BALB/c nu/nu) were purchased from the Animal Center of Southern Medical University (Guangzhou, China) and were housed in a sterile environment with a light/dark cycle of 12/12 h and were allowed free access to food and water for 1 week. All animal studies were performed following international guidelines on animal welfare and were approved by the Institutional Animal Care and Use Committee (IACUC) of Guangdong Medical College. CNE-2Z (1×10⁶) cells in 0.1 ml PBS were subcutaneously (s.c.) injected into the right hind flank of the mice. One week after inoculation of tumor cells (∼100 mm³), the tumor-bearing mice were randomly divided into the control and resveratrol (20 mg/kg body weight) treatment groups (n=5/group), whereby resveratrol dissolved in 200 μl of 10% DMSO in PBS was intraperitoneally (i.p.) injected into mice once a day for 3 weeks. Control animals were injected with the vehicle (10% DMSO in PBS). Tumor sizes were measured every other day with a caliper and were calculated as 1/2× length × width² (V=LW²/2 mm³). At the end of the experiments, the mice were sacrificed and tumor tissues were excised for further analysis.

Data are expressed as the means ± SD and were analyzed with the commercially available statistical software package, SPSS 13.0 (SPSS). One-way ANOVA or Student’s t-test was performed. P<0.05 was considered to indicate a statistically significant difference.

We first observed the effects of resveratrol on the proliferation of human NPC cells. CNE-1 and CNE-2Z cells were treated with increasing concentrations of resveratrol for 24 or 48 h followed by MTT assay. Our results indicated that resveratrol treatment led to a time- and dose-dependent reduction in the proliferation rate of both types of NPC cells, whereby at the concentrations of 50 and 100 μM, resveratrol exhibited more pronounced inhibitory effects on the proliferation of CNE-2Z cells than on that of CNE-1 cells (Fig. 1A and B). Additionally, we showed that resveratrol treatment inhibited colony formation of CNE-2Z cells in a dose-dependent manner (Fig. 1C). These results suggest that resveratrol has the potential to inhibit the proliferation of NPC cells with different differentiation grades.

Next, we investigated the effect of resveratrol on apoptosis in NPC cells. CNE-2Z cells were exposed to different concentrations of resveratrol for 48 h, followed by determination of apoptosis using the Annexin V-FITC/PI staining kit and flow cytometric analysis. Compared with controls, resveratrol treatment resulted in a dose-dependent induction of apoptosis of CNE-2Z cells (Fig. 2), suggesting that resveratrol potently induces apoptosis of NPC cells.

We then explored the potential mechanisms underlying resveratrol-induced apoptosis and inhibition of proliferation in NPC cells. To this end, CNE-2Z cells were treated with different concentrations of resveratrol for 16 h (overnight), and the expression of several important genes involved in the regulation of apoptosis was determined by western blot analysis. Resveratrol treatment significantly inhibited the protein expression of 2 anti-apoptotic genes, Bcl-2 and HIF-1α (10,11), in CNE-2Z cells (Fig. 3A). On the other hand, resveratrol treatment led to a dose-dependent increase in the expression of caspase-3 which plays a critical role in apoptosis induction (10,11). Since the pAkt1/p70S6K/p-4E-BP-1 signaling pathway plays an essential role in the maintenance of proliferation and survival of cancer cells (12), we then explored whether resveratrol can block the activation of this signaling pathway. Our results demonstrated that treatment of CNE-2Z cells with resveratrol led to dose-dependent decrease in the phosphorylated levels of pAkt1, p70S6K as well as p-4E-BP-1 (Fig. 3B). These findings suggest that resveratrol inhibits proliferation and induces apoptosis of NPC cells possibly by blocking PI3K/Akt signaling pathways.

We then explored whether resveratrol had any effect on the cell cycle distribution of NPC cells. CNE-1 and CNE-2Z cells were exposed to 10 and 50 μM of resveratrol for 24 h and cell cycle distribution was analyzed with flow cytometry. Compared with controls, treatment of CNE-1 cells with resveratrol at 10 μM showed no obvious effects on the cell cycle distribution (P>0.05), while at 50 μM, resveratrol treatment significantly reduced the percentage of G1-phase cells (from 72.5±9.4 to 42.1±8.6%; P<0.01) and increased the percentage of S-phase cells (from 13.1±6.1 to 45.8±7.2%; P<0.05) (Fig. 4A, upper panel). Additionally, we showed that resveratrol exhibited stronger regulatory effects on cell cycle distribution in CNE-2Z cells. Compared with controls, treatment of CNE-2Z cells with resveratrol even at 10 μM led to a significant reduction in the percentage of G1-phase cells (from 70.3±8.3 to 52.6±6.5%; P<0.05) and a marked increase in the percentage of S-phase cells (from 12.8±4.1 to 32.3±7.8%; P<0.05), but had no obvious effects on the percentage of G2/M-phase cells (P>0.05) (Fig. 4A, lower panel). At 50 μM, resveratrol treatment further decreased the percentage of G1-phase cells (36.2±7.9%; P<0.01) but simultaneously increased the percentage of cells at both the S-phase (40.5±8.2%; P<0.01) and G2/M-phases (16.6±5.2 to 25.7±6.9%; P<0.05) (Fig. 4A, lower panel). These findings suggest that resveratrol treatment was able to arrest the cell cycle progression of NPC cells at the S- and G2/M-phases.

To further explore the molecular mechanisms involved in resveratrol-induced alterations in cell cycle distribution of NPC cells, CNE-2Z cells were treated with increasing concentrations of resveratrol for 24 h. Then, the protein expression of several cyclins involved in cell cycle regulation was determined by western blot analysis. We found that resveratrol treatment markedly inhibited the expression of cyclin A, B, D and E, and CDK/p34 proteins in CNE-2Z cells, thus further confirming the effects of resveratrol on cell cycle progression in NPC cells.

To further validate the anticancer effect of resveratrol on NPC, in vivo studies on xenografts of CNE-2Z cells in nude mice were performed. Our results showed that i.p. administration of resveratrol (20 mg/kg body weight) significantly suppressed the growth of xenografted tumors from Day 12 after resveratrol administration (P<0.05) (Fig. 5A and B). Histological analysis by H&E staining indicated that resveratrol-treated tumors appeared to be more organized with fewer cellular components and more collagenous structures (Fig. 5C). Collectively, these results provide substantial evidence that resveratrol possesses potent anticancer effects on human NPC.