Human thyroid squamous cell carcinoma SW579 cells (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were cultured in DMEM (Gibco Life Technologies, Carlsbad, CA, USA) in an incubator at 37°C with 5% CO₂. Once the cells reached 80% confluence, the cells were removed with trypsin and dissolved in DMEM (containing 10% calf serum) to a final concentration of 2×10⁸/l. The cells were counted and seeded in corresponding culture plates for subsequent experiments.

Cells were treated with hyperoside (Sigma, St. Louis, MO, USA) and the cell viability was detected using the MTT colorimetric assay (Sigma), and the cells were seeded in 96-well plates at a concentration of 1×10⁵/ml with 100 µl DMEM/well. After 24 h, hyperoside was added (to final concentrations of 0, 2.5, 5, 10 and 20 µg/ml, respectively). Five wells were replica-plated for each concentration and the experiment was repeated six times independently. The control group cells were not treated with hyperoside. After incubating the cells at 37°C with 5% CO₂ for 24, 48 and 72 h, respectively, 10 µl MTT was added into each well to a final concentration of 5 mg/ml, after 4 h, the optical density (OD) at 570 nm was measured using a microplate reader. The cell inhibition rate was calculated according to the formula: inhibition rate (%) = (OD value of the control group - OD value of the experimental group/OD value of the control group) × 100%.

After hyperoside treatment for 24 h (with 0, 5, 10 and 20 mg/ml hyperoside), any morphological changes were observed and images were captured with an inverted microscope (Nikon, Tokyo, Japan).

SW579 cells in the logarithmic growth phase were seeded in 6-well plates. After 12 h, the cells were treated with 0, 5, 10 and 20 µg/ml hyperoside, respectively. After 24 h, the cells were washed three times with PBS, digested with trypsin and then centrifuged. The specific protocol for the Annexin V-FITC apoptosis detection kit (Biyuntian Biotechnology Research Institute, Jiangsu, China) was carried out as per the manufacturers instructions. Briefly, after centrifugation the cells were resuspended in 0.3 ml of binding buffer. Then, 5 µl of Annexin V and 5 µl of PI were added, and the cells were incubated at room temperature in the dark for 15 min. Binding buffer (0.2 ml) was added to each sample and cell apoptosis was detected by flow cytometry (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).

Cells were seeded in 6-well plates with 1×10⁴ cells/well. After 24 h, the supernatant was aspirated and cells were treated with 0, 5, 10 and 20 µg/ml hyperoside, respectively. After 24 h, the cells were collected and total RNA was extracted according to the protocol in the RNA extraction kit (Invitrogen Life Technologies, Carlsbad, CA, USA). The concentration and purity of total RNA were detected by UV-Vis spectrophoto-meter (Hitachi, Tokyo, Japan) (A260/A280 >1.8 indicating pure RNA). cDNA was obtained via reverse transcription from RNA and the expression of Fas and FasL mRNAs was detected by RT-PCR based on the instruction of the RT-PCR kit (Invitrogen Life Technologies). Primers were designed by Takara Bio (Dalian, China) (Table I). Reaction conditions were as follows: 94°C for 5 min; then 94°C for 30 sec, 57°C for 30 sec, 72°C for 30 sec, a total of 35 cycles of amplification; and a final elongation at 72°C for 5 min. The Ct value was automatically calculated by software, the Ct values were all normalized against the abundance of the GAPDH control RNA, and the relative quantification of gene expression was calculated by the 2^−ΔCt method according to the formula: ΔCt (target gene) = Ct (target gene) - Ct (control gene).

Cells were seeded in 6-well plates with 10⁴ cells/well. After 24 h, the supernatants were aspirated and the cells were treated with 0, 5, 10 and 20 µg/ml hyperoside, respectively. After 24 h, the cells were collected and lysed with cell lysis buffer (Biyuntian Biotechnology Research Institute), and then centrifuged for 15 min at high speed and low temperature. After centrifugation at 10,050 × g for 15 min the supernatant containing the soluble fraction was collected. The extracted protein concentrations were determined by BCA kit (Biyuntian Biotechnology Research Institute), 50 µg of protein was separated on SDS-PAGE, and the separated protein was transferred to a PVDF membrane. The membrane was incubated in blocking buffer for 1 h at room temperature and then primary rabbit monoclonal survivin antibody (dilution, 1:500; cat. no. ab76424; Abcam, Cambridge, MA, USA) was added and the membrane was incubated over-night at 4°C. After washing the membrane with TTBS, the secondary goat anti-rabbit (HRP) IgG antibody (dilution, 1:2,000; cat. no. ab6721; Abcam) was added; and the membrane was incubated at room temperature for 1 h. ECL was then added on the membrane and blots were developed in the dark. Images were recorded with a gel imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA), GAPDH was used as the internal reference and grayscale values were analyzed and compared. All the antibodies were purchased from Wuhan Sanying Biotechnology (Wuhan, China).

SPSS 17.0 software (IBM Corp., Armonk, NY, USA) was used for statistical analyses. Data are presented as means ± standard deviation, and analyzed using one-way ANOVA. Differences with a P<0.05 were considered statistically significant.

After cells were treated with 0, 2.5, 5, 10 and 20 µg/ml hyperoside, the proliferation of SW579 cells was significantly inhibited in all groups. The proliferation inhibition rates were significantly increased with the increase in concentration and time (P<0.05) showing an obvious dose-dependent mechanism (Table II). However, this study aimed to examine the mechanism of apoptosis, and since 2.5 µg/ml hyperoside did not elicit a clear inhibitory effect on cell proliferation, subsequent experiments were carried out with 5 µg/ml hyperoside as the smallest concentration used. The duration of treatment was 24 h.

As shown in Fig. 1, after 24 h in the presence of 5, 10 and 20 µM hyperoside, respectively, the cells underwent significant morphological changes in a dose-dependent manner involving cell shrinkage, loose adherence, a decrease in cell number and an increase in the number of cell death.

As shown in Fig. 2, flow cytometry results showed the apoptotic rates as 3.23±0.52, 27.34±3.18, 33.93±3.78 and 46.63±4.75% in the control 5, 10 and 20 µg/ml hyperoside groups, respectively. Compared with the control group, the apoptotic rates of all the other groups were significantly increased (P<0.01).

As shown in Fig. 3, the normalized expression of Fas and FasL mRNAs in each hyperoside group was significantly higher than that in the control group, and increased along with the increasing concentrations of hyperoside treatment.

As shown in Fig. 4, the expression of survivin protein in each hyperoside group was significantly lower than that in the control group and decreased in a step-wise manner with each increase in the hyperoside concentration applied (P<0.01).