A human gastric cancer cell line, SGC7901, and a normal gastric cell line, GES-1, were obtained from the cell bank of Xiangya Medical School, Central South University (Changsha, China). The cells were cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS; HyClone; GE Healthcare, Logan, UT, USA), penicillin (100 U/ml) and streptomycin (100 µg/ml). The cells were incubated at 37°C in a humidified atmosphere of 5% CO₂.

Fisetin and dimethyl sulfoxide (DMSO) were supplied by Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). The stock solution of fisetin in DMSO was stored at −15°C. The inhibitor for activation of ERK 1/2, PD98059, was obtained from Selleck Chemicals LLC (Shanghai, China).

SGC7901 and GES-1 cell lines were distributed at a density of 2×10⁵ cells per well into 96-well culture plates and incubated at 37°C overnight. Then, the medium was replaced with fresh DMEM containing 0 (control), 1, 5, 10, 15 and 20 µM concentrations of fisetin. After 48 h of incubation under a humidified atmosphere of 5% CO₂ at 37°C, 200 µl CCK-8 solution (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was added to each well. The plates were incubated at 37°C for a further 4 h. Then, a microplate reader was used to measure the absorbance at 450 nm. The experiments were performed independently in triplicate.

Apoptosis induction in gastric carcinoma cells following treatment with fisetin was analyzed using an Annexin V/Propidium Iodide (PI; BD Biosciences, Franklin Lakes, NJ, USA) apoptosis kit, according to the manufacturer's protocol. SGC-7901 and GES-1 cells were treated with 0 (control), 5, 10 and 15 µM concentrations of fisetin for 48 h under a humidified atmosphere of 5% CO₂ at 37°C. The cells were harvested by trypsinization, washed three times with phosphate-buffered saline (PBS), and resuspended in binding buffer at a concentration of 2×10⁷ cells/ml. The cells were then treated with Annexin V-fluorescein isothiocyanate (5 µl) and PI (10 µl) at room temperature in the dark for 10 min. The cells were analyzed using a flow cytometer (FACSAria III; BD Biosciences, Franklin Lakes, NJ, USA). CellQuest software version 3.3 (BD Biosciences) was used for analysis of flow cytometry. The experiments were performed in triplicate for each concentration.

SGC7901 cells at a density of 2.5×10⁵ cells/well were distributed into 6-well plates and subjected to incubation for 48 h. RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% FBS was used for cell culture and incubation was performed at 37°C in an atmosphere of 5% CO₂. The medium was then replaced with fresh medium containing fisetin (15 µM) in DMSO. Following 48 h incubation at 37°C, the cells were subjected to trypsinization and subsequent washing with cold PBS. The cells were then fixed with 70% ethyl alcohol at 4°C for at least 4 h, followed by addition of 20 µl RNase (Thermo Fisher Scientific,) and 20 µl PI (Sigma-Aldrich; Merck KGaA). The cells were then incubated for 30 min at 37°C before analysis using a FACSCalibur flow cytometer (BD Biosciences) and CellQuest software version 3.3 (BD Biosciences).

The phosphorylation of ERK 1/2 and expression of caspase-7, pro-caspase-7, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bcl-x), BH3 interacting domain death agonist (Bid) and Bcl-2-like protein 11 (Bim) was analyzed using a western blot assay. Effect of PD98059 (ERK 1/2 inhibitor) at 100 µM on activation of ERK ½ was also analyzed using this assay. The SGC7901 cells were treated with 15 µM fisetin for 48 h at 37°C under a humidified atmosphere of 5% CO₂. Following incubation, the cells were treated with radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) under ice-cold conditions for 45 min. The cell lysates were subjected to centrifugation at 12,000 × g for 15 min at 4°C. The concentration of proteins in the cell lysates was determined using a bicinchoninic acid assay. The proteins were separated using 10% SDS-PAGE by loading 3 µl protein per lane and subsequently transferred to polyvinylidene difluoride membranes. In the membranes, non-specific sites were blocked with non-fat milk containing Tris-buffered saline with Tween-20. The membranes were incubated with rabbit primary monoclonal antibodies against ERK (cat. no. 137F5; dilution 1:1,000) and p-ERK (cat. no. D13.14.4E; dilution 1:1,000; both from Cell Signaling Technology, Inc., Danvers, MA, USA) at 4°C for overnight. The other antibodies used were against Bcl-2 (cat. no. ab7973), Bcl-x (cat. no. ab77566), Bid (cat. no. ab32060), Bim (cat. no. ab32158), β-actin (cat. no. ab8226) and α-tubulin (cat. no. ab7291; all dilution 1:1,000, Abcam, Cambridge, UK). The membranes were washed and incubated with goat anti-rabbit HRP-conjugated polyclonal secondary antibodies (cat. no. 12–348; dilution 1:2,000, Merck KGaA) for 1 h at room temperature. The bands were visualized using an enhanced chemiluminescence blotting detection system (FluorChem E; ProteinSimple, San Jose, CA, USA).

Data are presented as the mean ± standard deviation of ≥3 experiments performed independently. Statistical analysis was performed with the SPSS 13.0 statistical software (SPSS, Inc. Chicago, IL, USA). A one-way analysis of variance was used, followed by Dunnett's test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

SGC7901 and GES-1 cells were incubated with various concentrations (1, 5, 10, 15 and 20 µM) of fisetin for 48 h and proliferation was examined. Fisetin treatment at 1, 5, 10, 15 and 20 µM concentration significantly reduced the proliferation rate of SGC7901 cells to 98, 72, 51, 12 and 11%, respectively compared to 100% in control after 48 h (P<0.05; Fig. 1). The proliferation rate of GES-1 cells was found to be 100, 99, 99, 98 and 98% respectively at 1, 5, 10, 15 and 20 µM concentrations of fisetin compared with 100% in untreated cultures (Fig. 1).

Treatment of SGC7901 cells with various concentrations (5, 10 and 15 µM) of fisetin for 48 h induced cell death in a dose-dependent manner (Fig. 2). Flow cytometry revealed a notable increase in the proportion of apoptotic cells at 15 µM concentration of fisetin after 48 h compared with the untreated control cells (Fig. 2). The percentage of apoptotic cells increased to 87% following treatment with 15 µM fisetin compared with 2% in the control for 48 h.

The effect of fisetin on SGC7901 cell cycle progression was determined using flow cytometry. The results indicated that the percentage of cells in the G2/M and S phases of control cell cultures was 18.23 and 9.14%, respectively (Fig. 3). Following treatment of SGC7901 cells with 15 µM fisetin for 48 h, the proportion of SGC7901 cells at the G2/M and S phases was 23.72 and 8.65%, respectively (Fig. 3). Thus, fisetin treatment increased the proportion of cells at G2/M phase with simultaneous reduction of cells at S phase.

Western blot analysis indicated a marked increase in the expression level of caspase-7 following treatment of SGC7901 cells with 1, 5, 10 and 15 µM fisetin for 48 h (Fig. 4). However, the procaspase-7 expression level was slightly decreased by fisetin treatment (Fig. 4). In SGC7901 cells, fisetin treatment led to a marked decrease in the expression level of anti-apoptotic proteins Bcl-2 and Bcl-x. The expression of Bim was increased and that of Bid decreased following treatment of SGC7901 cells with fisetin for 48 h (Fig. 4).

Treatment of SGC7901 cells with fisetin for 48 h resulted in marked reduction of the activation of ERK 1/2 in a concentration-dependent manner (Fig. 5).

The inhibitory effect of fisetin on activation of ERK 1/2 was further demonstrated using an ERK 1/2 inhibitor, PD98059. The results indicated a marked decrease in the proliferation of SGC7901 cells compared with control cells, following treatment with 100 µM PD98059 (P<0.002). The reduction by PD98059 administration was similar to that observed following fisetin (15 µM) treatment for 48 h (Fig. 6). Furthermore, PD98059 was observed to markedly reduce the activation of ERK 1/2 in SGC7901 cells (Fig. 7). Inhibition of ERK 1/2 activation by PD98059 produced similar results to fisetin treatment (15 µM) for 48 h (Fig. 7).