Human ovarian cancer SKOV3 cells were purchased from the Chinese Academy of Medical Sciences (Beijing, China) and were maintained in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc., Carlsbad, CA, USA), supplemented with 10% (v/v) fetal calf serum (Gibco; Thermo Fisher Scientific, Inc.), 100 mg/ml streptomycin and 100 U/ml penicillin (each from Genview, Galveston, TX, USA). The cells were incubated at 37°C in an atmosphere containing 5% CO₂. Myricetin was purchased from Sigma-Aldrich (St. Louis, MO, USA) and was dissolved in dimethylsulfoxide (DMSO) for storage at −20°C.

Cell viability was determined using a 3-(4,5-dimetrylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Beyotime Institute of Biotechnology, Haimen, China). SKOV3 cells, during the exponential growth phase, were seeded into 96-well culture plates in 100 µl RPMI-1640 at a density of 8×10³ cells/well. Following 24 h incubation, the indicated dose of myricetin were added for a further 24 h incubation in four parallel wells. The MTT assays were performed as follows: 20 µl MTT solution [(5 mg/ml in phosphate-buffered saline (PBS)] was added to each well and the cells were incubated at 37°C for 4 h, following which 150 µl DMSO (Beijing Chemical Industry Co., Ltd., Beijing, China) was added to each well. The cells were agitated for 10 min prior to measuring the absorbance at 570 nm using a microplate reader (680; Bio-Rad Laboratories, Inc., Hercules, CA, USA). The growth inhibition rate was calculated as follows: Inhibition (%) = [1 − (absorbance of experimental group / absorbance of control group)] × 100. The mean value of four replicate wells was calculated for each treatment group.

Whole-cell protein extracts from SKOV3 cells were prepared using cell lysis buffer [50 mM Tris-hydrochloride (HCl; pH 7.5); 150 mM NaCl; 1 mM Na₂ EDTA; 1 mM EDTA; 1% Triton; 2.5 mM sodium pyrophosphate; 1 mM β-glycerophosphate; 1 mM Na₃VO₄; 1 mM NaF; 1 µg/ml leupeptin; 1 mM PMSF]. The protein extracts were quantified using a Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Inc.). For western blot analysis, protein lysates (30–50 µg) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto Immobilon-P Membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk powder in buffer [10 mM Tris-HCl (pH 7.6), 100 mM NaCl and 0.1% Tween-20] for 2 h at room temperature and were subsequently incubated with the appropriate primary antibodies overnight at 4°C. Anti-GRP-78 rabbit anti-human polyclonal antibody (cat no. sc-13968; dilution, 1:200) was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), mouse anti-human anti-C/EBP homologous protein (CHOP) monoclonal antibody (cat no. ab11419; dilution, 1:1,000) from Abcam (Hong Kong, China), rabbit anti-human anti-γ-H₂AX monoclonal antibody (cat no. 9718; dilution, 1:1,000) from Cell Signaling Technology (Beverly, MA, USA), and mouse anti-human anti-β-actin monoclonal antibody (cat no. 60008-1-Ig; dilution, 1:1,000) from Proteintech Group, Inc. (Chicago, IL, USA). Following incubation, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (Thermo Fisher Scientific, Inc.) at a dilution of 1:2,000 for 1 h at room temperature. Immunodetection was performed using enhanced chemiluminescence reagents and images were captured using a Syngene Bio Imaging system (Synoptics, Cambridge, UK). The protein levels were normalized against those of β-actin, and the ratios of the normalized protein are presented as the mean ± standard deviation from three independent experiments. Protein levels were quantified by densitometry using Quantity One software version 4.4.02 (Bio-Rad Laboratories, Inc.).

The cells were seeded onto coverslips in 24-well plates at a density of 5×10⁴ cells/well 24 h prior to treatment. Following exposure to 40 µg/ml myricetin for 0, 6, 12 and 24 h, the cells were fixed with 4% paraformaldehyde for 30 min at room temperature, stained with the nuclear stain Hoechst 33342 (2 µg/ml; Sigma-Aldrich) for 2 min at room temperature, washed with PBS and examined using a confocal laser microscope (FV1000; Olympus, Tokyo, Japan) to reveal chromatin condensation. The expression levels of GRP-78, active Caspase 3 and γ-H₂AX were examined using an indirect immunofluorescence method. Briefly, after the cells were cultured, treated and fixed, as previously described, they were subsequently permeabilized with 0.1% Triton X-100 for 5 min, blocked with bovine serum albumin for 30 min and incubated with primary antibodies against GRP-78, active caspase 3 and γ-H₂AX (dilution, 1:100) overnight at 4°C. Following incubation, the cells were incubated with fluorescein isothiocyanate/Texas Red-conjugated secondary antibodies (dilution, 1:400; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature, stained with Hoechst 33342 (2 µg/ml) for 2 min at room temperature, washed with PBS three times, and examined using the Olympus FV1000 confocal laser microscope.

The data are representative of the results from three independent experiments. Statistical analysis was performed using one-way analysis of variance. The Tukey post-hoc test was used to determine the significance of all pairwise comparisons of interest. P<0.05 was considered to indicate a statistically significant difference.

Our previous results provided us with the appropriate dose range for myricetin treatment (unpublished data). SKOV3 cells were treated with the indicated doses of myricetin for 24 h. Myricetin inhibited the viability of SKOV3 cells in a dose-dependent manner (Fig. 1A). In addition, changes in cell morphology were also apparent following myricetin treatment. Myricetin-treated cells appeared more rounded and shrunken compared with the control group (Fig. 1B). The present study, therefore, hypothesized that myricetin may induce apoptosis in SKOV3 cells, and this was examined in detail using confocal microscopy.

Based on the above MTT results, SKOV3 cells were treated with 40 µg/ml myricetin for 0, 6, 12 or 24 h, stained with Hoechst 33342, and examined using confocal microscopy. The nuclei of myricetin-treated cells appeared more condensed, when compared with the untreated cells (Fig. 2A).

Caspase 3 is considered to be the primary executor of apoptosis, and cleaved Caspase 3 (active Caspase 3) is used as a biomarker for apoptosis. As shown in Fig. 2B, increasing concentrations of myricetin generated higher levels of red fluorescence. This indicated that myricetin induced the activation of Caspase 3 in a time-dependent manner (Fig. 2b). Together, these results indicated that myricetin triggered apoptosis in SKOV3 cells.

GRP-78 is an ER chaperone molecule, which increases following ER stress (19). In order to determine whether myricetin induced ER stress, confocal microscopy was used to detect the expression of GRP-78 in myricetin-treated cells. Myricetin was found to increase the mean fluorescence intensity of GRP-78 in SKOV3 cells, which became notable following 24 h of treatment (Fig. 3A).

Increased and sustained ER stress can cause an apoptotic response. To determine whether the response to myricetin treatment caused ER stress-associated apoptosis, the levels of CHOP were investigated (20). Western blot analysis indicated that the protein levels of CHOP markedly increased at 6, 12 and 24 h following myricetin treatment (Fig. 3B and C). Consistent with the data from the confocal experiments, GRP-78 levels also increased in a time-dependent manner (Fig. 2B and C). These results indicated that myricetin triggers ER stress-associated apoptosis.

Cisplatin is one of the classic chemotherapeutic drugs known to induce a marked apoptotic response by inhibiting DNA replication and damage (21,22). It was therefore hypothesized that myricetin can also induce DNA DSBs. It has been reported that following a DSB, histone H₂AX is rapidly phosphorylated (becoming γ-H₂AX) near the site of the DSB, and is involved in the recruitment of other factors that contribute to lesion repair. The levels of γ-H₂AX have been shown to be downregulated when the DNA is repaired. If the lesion remains unrepaired (or the repair process is delayed), then the levels of γ-H₂AX remain high; therefore, the levels of γ-H₂AX comprise an ideal indicator of the degree of unrepaired DSBs, which can then contribute to cell death (23,24).

Firstly, using confocal microscopy, the expression of γ-H₂AX was detected in cells treated with either cisplatin (6 µg/ml) or myricetin (40 µg/ml). Cisplatin-treated cells were used as the positive control. The results revealed that both cisplatin and myricetin increased the formation of the γ-H₂AX foci (Fig. 4A). Secondly, western blotting was used to detect the protein levels of γ-H₂AX in myricetin-treated cells. It was revealed that myricetin increased the levels of γ-H₂AX at 6, 12 and 24 h treatment (Fig. 4B and C).