The A2780 and OVCAR3 human ovarian cancer cells were purchased from ATCC (Manassas, VA, USA) and cultured in RPMI-1640 medium (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) supplemented with 10% fetal bovine serum (Sigma-Aldrich; Merck KGaA), penicillin (100 U/ml; Sigma-Aldrich; Merck KGaA) and streptomycin (100 µg/ml; Sigma-Aldrich; Merck KGaA), at 37°C in a humidified chamber.

Cell viability was determined spectrophotometrically using an MTT assay. A2780 and OVCAR3 cells were seeded into 96-well plates at a density of 1×10⁴ cells/ml. The cells were cultured to ~70% confluency and exposed to various concentrations of myricetin (0, 5, 10, 25, 50 and 100 µM; Sigma-Aldrich; Merck KGaA) for 48 h, at 37°C, under the described cell culture conditions. Following treatment, MTT (Sigma-Alrich; Merck KGaA) solution was added to each well at a concentration of 2 mg/ml and incubated at 37°C for 2 h. The formazan that formed was dissolved using DMSO and the absorbance was measured at 570 nm using a microplate reader and Multiskan EX software, v3.4 (Multiskan EX, Lab systems, Helsinki, Finland).

Role of apoptosis in myricetin-induced cytotoxicity in A2780 and OVCAR3 cells was determined using a single stranded-DNA Apoptosis ELISA kit (EMD Millipore, Billerica, MA, USA) (21). Cultured A2780 and OVCAR3 cells (1×10⁴ cells/ml) were treated with 25 µM myricetin for 48 h at 37°C, and induction of apoptosis in untreated (control) and myricetin-treated cells were determined according to the manufacturer's protocol. The extent of apoptosis was determined from the absorbance measured at 405 nm using a microplate reader and Multiskan EX software, v3.4 (Multiskan EX, Lab systems).

The migratory properties of A2780 and OVCAR3 cells in the presence and absence of myricetin were determined using a Boyden chamber assay with track-etched polyethylene terephthalate membranes and an 8.0-µm diameter pore size (Corning Incorporated, Corning, NY, USA). A2780 and OVCAR3 cells (1×10⁴ cells/ml) were treated with various concentrations of myricetin (0, 10 and 25 µM) for 48 h under the described cell culture conditions and the migration assay was performed according to the manufacturer's protocol.

Cultured (at 37°C) A2780 and OVCAR3 cells were treated with 25 µM of myricetin. Total protein from the cell extracts was isolated in ice-cold lysis buffer consisting of 150 mM NaCl, 20 mM Tris-HCl, 1% NP-40, 20 µg/ml leupeptin, 20 µg/ml aprotinin, 1 mM ortho-vanadate and 2 mM PMSF, pH 7.4. After treatment, cells were incubated in the lysis buffer at 4°C for 30 min and the cell suspension was subsequently centrifuged at 2,000 × g for 15 min at 4°C. Protein concentration was estimated using a Bradford reagent (Sigma-Alrich; Merck KGaA). Protein from each cell sample (~50 µg) was loaded into each well and separated by 10% SDS-PAGE, and then transferred to a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membrane was blocked in Super Block blocking buffer (Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 1 h at room temperature and subsequently incubated with various primary antibodies, including rabbit polyclonal anti-Bax (dilution, 1:500; N20, #sc-493; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), rabbit polyclonal anti-Bcl-2 (dilution, 1:500; N-19, sc-7382; Santa Cruz Biotechnology, Inc.), rabbit monoclonal anti-cleaved caspase-3 (Asp175) antibody (dilution, 1:1,000; #9661; Cell Signaling Technology, Inc., Danvers, MA, USA), mouse monoclonal anti-Mdr-1 antibody (dilution, 1:500; G-1; sc-13131, Santa Cruz Biotechnology, Inc.). After blocking membranes were washed three times in 0.25% TBST buffer for 10 min and incubated with secondary antibodies, including goat anti-mouse immunoglobulin G (IgG)-horseradish peroxidase (HRP; #sc-2005; Santa Cruz Biotechnology, Inc.) and mouse anti-rabbit IgG-HRP (#sc-2357; Santa Cruz Biotechnology, Inc.) for 30 min at room temperature. The secondary antibodies were used at a dilution of 1:10,000. Images of the membranes were captured using the Super Signal ULTRA Chemiluminescent Substrate (Pierce; Thermo Fisher Scientific, Inc.) using a KODAK Image Station 4000 (Kodak, Rochester, NY, USA).

The data were analyzed using GraphPad Prism version 4.0 (GraphPad Software, Inc., La Jolla, CA, USA). All data are presented as the mean ± standard deviation. Statistically significant differences between the groups were determined using a paired Student's two-tailed t-test. P<0.05 was considered to indicate a statistically significant result.

Treatment of the A2780 and OVCAR3 human ovarian cancer cells with various concentration of myricetin resulted in the induction of cytotoxicity. Following a 48-h incubation, a gradual loss of cell viability was observed in myricetin-treated A2780 cells, with the IC50 concentration determined to be ~25 µM (Fig. 1B). A similar inhibition of cellular growth was observed in myricetin-treated OVCAR3 cells following a 48-h incubation period, with the IC50 concentration calculated as ~25 µM (Fig. 1C).

To elucidate the mechanisms underlying myricetin-induced cytotoxicity in ovarian cancer cells, the involvement of apoptosis in myricetin-treated cells was investigated. The induction of apoptosis was evaluated using the single-stranded DNA Apoptosis ELISA kit. Myricetin treatment was observed to induce apoptosis in A2780 and OVCAR3 cells. In A2780 cells treated with 25 µM myricetin, an ~2.5-fold increase in the apoptotic signal was observed, compared with untreated cells, whereas under similar treatment conditions, the apoptotic signal was increased by ~4-fold in OVCAR3 cells compared with untreated cells (*P<0.05, control vs. myricetin-treated cells; Fig. 2A). The results suggest that apoptosis is involved in myricetin-induced cytotoxicity in certain ovarian cancer cells.

Furthermore, it was also revealed that the expression of the pro-apoptotic protein B-cell lymphoma-2 (Bcl-2)-associated X-protein (BAX) was significantly upregulated, and the expression of the anti-apoptotic protein Bcl-2 was downregulated, in myricetin-treated ovarian cancer cells compared with untreated cells (*P<0.05, control vs. myricetin-treated cells; Fig. 2B). Cleaved caspase-3 is central to the intrinsic apoptotic signaling pathway, and was also revealed to be upregulated in myricetin-treated cells compared with untreated cells (*P<0.05, control vs. myricetin-treated cells). Therefore, the results suggested that apoptosis has an important role in myricetin-induced cell death.

The migratory properties of A2780 and OVCAR3 cells were determined in the absence and presence of myricetin using a Boyden Chamber assay. Cell migration was observed to be markedly inhibited by myricetin, in a dose-dependent manner. In A2780 cells that were treated with 10 µM myricetin the extent of migration was inhibited by ~18%, whereas in the presence of 25 µM myricetin migratory activity was inhibited by ~60% compared with untreated cells (*P<0.05, control vs. myricetin-treated cells; Fig. 3A). A similar inhibitory pattern was observed in myricetin-treated OVCAR3 cells. In the presence of 10 µM myricetin, OVCAR3 cell migration was inhibited by ~28%, and in the presence of 25 µM myricetin it was inhibited by ~65% compared with untreated cells (*P<0.05, control vs. myricetin-treated cells).

In order to investigate whether myricetin is able to enhance the chemotherapeutic potential of paclitaxel (PTX), the ovarian cancer cells were treated with a sub-lethal concentration of myricetin (5 µM) for 48 h, and then incubated with a sub-lethal concentration of paclitaxel (100 nM). As previously demonstrated (Fig. 1), 5 µM myricetin induces <25% inhibition of cell viability in A2780 and OVCAR3 cells. By contrast, 100 nM paclitaxel was not observed to induce significant cytotoxicity in the two cell types. However, when the myricetin-treated cells were further incubated with 100 nM paclitaxel, a marked reduction in cell viability was observed (Fig. 4A and B). The combination therapy of myricetin and paclitaxel was, therefore, effective in these cell lines, causing an ~50% loss of cell viability (Fig. 4A and B).

As MDR-1 expression in ovarian cancer cells has been indicated to be associated with paclitaxel-resistance, the status of MDR-1 in the myricetin-treated cells was further investigated using western blot analysis. It was observed that, in the myricetin-treated A2780 and OVCAR3 cells, MDR-1 expression was significantly downregulated compared with untreated cells (*P<0.05, control vs. myricetin-treated cells; Fig. 4C), which may be associated with the enhancement of paclitaxel efficacy in certain ovarian cancer cells.