Human ovarian adenocarcinoma cancer cells from the SKOV3 cell line were obtained from the cell bank of the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). Cells were cultured in the RPMI 1640 medium (Invitrogen; Thermo Fisher Scientific, Inc. Waltham, MA, USA) including 10% fetal bovine serum (FBS; HyClone; GE Healthcare Life Sciences, Logan, UT, USA) in an environment of 5% CO₂ at 37°C. For cell cycle analysis, the cells were synchronized to G₀ by serum starvation for 72 h prior to treatment with GA.

Propidium iodide (PI) and RNAase used in the cell cycle analysis were obtained from Sigma-Aldrich (St. Louis, MO, USA). The cell counting kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies Inc. (Kumamoto, Japan). The p65 DNA activity kit was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Caspase-3, caspase-9, p65, phospho-p65 and β-actin antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA).

The cell count kit analysis was detected by the protocol of methods as described previously (13). i) After cultivation in an incubator, the cell suspension was inoculated in 96-well plates (100 µl/well), ii) 10 µl of CCK-8 reagent was added to each well. iii) The culture plate was incubated in the incubator for 2 h. iv) The absorbance value was measured at 450 nm using the Multiskan™ GO Microplate Spectrophotometer (Thermo Fisher Scientific, Inc.). v) Calculation of cell survival rate was carried out using the formula: Cell survival (%) = (V_drug - V_control) /(V_drug=0 - V_control) × 100%.

SKOV3 cells were treated with varying concentrations of GA for 24 h. After the cells were harvested, 70% alcohol was used to fix the cells overnight. The fixed cells were washed with phosphate-buffered saline (PBS), then 150 µl RNAase (0.1 mg/ml) was added for 30 min at 4°C. Following this, 120 µl PI (0.1 mg/ml) was added and annexin V staining was carried out for 10 min, at 4°C. Finally, the cells were analyzed for DNA content distribution by flow cytometry (FCM).

SKOV3 cells were harvested following treatment with varying concentrations of GA. Protein separation was carried out by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) as described previously (14,15). The specific antibodies caspase-3 (1:800) and caspase-9 (1:800) came from the Apoptosis Antibody Sampler kit (cat. no. 9915T; Cell Signaling Technology, Inc.), the specific antibodies p65 (1:800) and phospho-p65 (1:800) came from the NF-κB p65 Antibody Sampler kit (cat. no. 4767T; Cell Signaling Technology, Inc.), and were used to detect the protein levels of indicated cells. β-actin served as an internal reference.

p65 DNA activity analysis was performed following the previously defined protocol (16). i) 10 µg of nuclear extract was added to the cell culture plate, which had already been incubated with 100 µl of p65 DNA sequence overnight at 4°C). ii) Each well was washed five times with 200 µl wash buffer. iii) 100 µl diluted goat anti-rabbit secondary antibody (1:1,500; cat. no. 7074; Cell Signaling Technology, Inc.) was added for 1 h. iv) Each well was washed five times with 200 µl wash buffer. v) 100 µl of developing solution per well was added for 45 min. vi) 100 µl of stop solution per well was added for 5 min. vii) Absorbance was measured at 450 nm.

Equal numbers of SKOV3 cells (2×10⁶) were harvested at the log growth phase. Tumor xenografts were established by subcutaneous fat pad injections into athymic mice. The tumor volumes were monitored as described previously (17). After sixty days, the mice were sacrificed.

GA therapy was initiated ten days after subcutaneous fat pad injections were performed. 3.2 µM of GA was administered to the mice intraperitoneally every 2 days. All animal procedures were performed according to the 1998 Guide for the Care and Use of Medical Laboratory Animals (Ministry of Health, Beijing, China), and with the ethical approval of the Dongda Hospital Animal Care and Use Committee (Xi'an, China; permit no. SYXK 2013–0024) and the Ethical Committee of Southeast University (Nanjing, China; permit no. 2013008).

All statistical analyses were performed using Graph Pad software (version 5; GraphPad Software Inc., La Jolla, CA, USA). A one-way ANOVA statistical test was also carried out. P<0.05, was considered to indicate a statistically significant difference.

The SKOV3 cells were treated with varying concentrations of GA (0, 0.8, 1.6, 3.2, 6.4, 12.8, 25.6, 51.2 µM). After 24 h, the cell survival rate was analyzed using the CCK-8 assay. The results demonstrated that GA inhibited SKOV3 cell growth in a dose-dependent manner (Fig. 1A). The SKOV3 cells were then treated with 3.2 µM GA, and cell survival rate was analyzed at set times (0, 30, 60, 120 and 240 min) by CCK-8. GA was also found to inhibit SKOV3 cell growth in a time-dependent manner (Fig. 1B).

To further study the mechanism of GA on the SKOV3 cells, cell cycle analysis was performed using FCM. The cells were treated with varying concentrations of GA (0, 3.2, and 6.4 µM). After 24 h, the cells were harvested. The results of the cell cycle analysis indicated that a higher proportion of the cells treated with GA were arrested in the S-phase compared to control cells (Fig. 2). This demonstrates that treatment of GA inhibited cell growth by arresting ovarian cancer cells in the S-phase.

To study whether caspase activity was involved in the GA-induced growth arrest in SKOV3 cells, protein levels of active caspase-3 and active caspase-9 were measured before and after GA treatment. The preceding results suggested that the vehicle could not exert an influence on the survival of SKOV3 cells, therefore western blot analysis was performed to compare the levels of active caspase-3 and caspase-9 before and after GA treatment (Fig. 3). The results obtained show that GA treatment increased the expression of active caspase-3 and active caspase-9, whilst overall capsase-3 and capsase-9 levels remained unchanged, demonstrating that GA inhibited cell growth by upregulating caspase-3 and caspase-9 activity.

Evidence from previous studies suggests that the NF-κB signaling pathway is closely related to tumor formation via different mechanisms including cell apoptosis, cell cycle arrest, cell differentiation and migration (11). In the current study, phosphorylated p65 (p-p65) expression decreased after treatment with GA. Furthermore, p65 DNA binding activity markedly decreased when treated with increasing concentrations of GA (Fig. 4). Taken together, these results suggest that GA inhibited the cell survival of ovarian cancer cells by suppressing p65 DNA binding activity.

Finally, tumor xenograft experiments were conducted in nude mice in order to detect whether GA could suppress tumor growth in vivo. The results showed that the tumors in GA-treated mice were significantly smaller than in control mice or mice treated with a vehicle (Fig. 5; P<0.05). These results indicated that GA inhibited tumor growth in a mouse model.