Human ovarian adenocarcinoma cells (SKOV3) and the corresponding cisplatin-resistant variant (SKOV3/DDP) were acquired from the Chinese Academy of Sciences. Cells were cultured in 1640 medium containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) at 37˚C and 5% CO₂. SKOV3 and SKOV3/DDP cells received 50 µM apigenin (Selleck Chemicals LLC; cat. no. S2262) for 24 h at 37˚C.

An MTT assay was used to determine the relative sensitivity of SKOV3 and SKOV3/DDP cells to cisplatin, and to establish a model of chemoresistance to cisplatin in OC cells. The IC₅₀ value of cisplatin (Selleck Chemicals LLC; cat. no. S1166) was 2 µM in SKOV3 cells and 10 µM in SKOV3/DDP cells in this experiment (data not shown). Cells were seeded in 96-well plates (10⁴ cells/well) and cultured in a 5% CO₂ humidified incubator at 37˚C until 70% of the culture surface was occupied. Cisplatin at a concentration of 2 µM was added to the SKOV3 cells and at a concentration of 10 µM was added to SKOV3/DDP cells in triplicate and the cells incubated for a further 24 h at 37˚C. The complete 1640 media was replaced with serum-free media containing 0.5 mg/ml MTT and the cells were incubated for another 4 h at 37˚C. Once the plates had dried, 100 µl DMSO was added to each well and the OD readings were measured at 570 nm using the Microplate reader (Multiskan FC; Thermo Fisher Scientific, Inc.). Using a concentration vs. percentage cellular growth inhibition graph, a regression equation was derived and the IC₅₀ values of cisplatin were determined for SKOV3 and SKOV3/DDP cells.

For the colony formation assay, a sample comprising 1,500 cells was plated into 6-well plates and incubated in 1640 media with 10% FBS at 37˚C for 1 week. After 1 week, cells were fixed with 4% paraformaldehyde at 4˚C overnight and stained with 0.1% crystal violet at room temperature for 10 min, and visible colonies were manually counted. Wells were measured in triplicate for each group.

Cells were resuspended in complete 1640 medium (Qiagen GmbH), and a Click-iT^® EdU cell proliferation assay (Qiagen GmbH) was performed. After 48 h of culture at 37˚C, cells were incubated for 2 h with 10 µmol EdU at 37˚C. Digestion was carried out using 0.05% trypsin and cells were washed with PBS. Next, cells were fixed for 15 min using 100 µl Click-iT fixative at 25˚C and centrifuged at 37˚C for 5 min at 1,000 x g, after which the cells were washed with PBS. Permeabilization was performed for 15 min using 100 µl permeabilization and washing agent (Qiagen GmbH; 0.2%) at room temperature. Cells were then incubated at room temperature for 30 min in the dark with 500 µl reaction solution, composed of 496 µl PBS, 4 µl buffer additive (component F; Qiagen GmbH), 1 mM CuSO₄ and 10 µM Alexa Fluor 488. Then, 3 ml permeabilization and washing agent was added, and cells were centrifuged at 37˚C for 5 min at 1,000 x g before being washed with PBS. Permeabilization and washing agent (500 µl) was added to the resuspension, and cell proliferation was assessed using a Beckman Coulter FC 500 MCL/MPL flow cytometer with FlowJo software (version 7.6.1; FlowJo LLC).

Cell death triggered by apigenin in OC cells was investigated using an Annexin V and PI double staining apoptosis detection kit (cat. no. TA5354; BioLegend, Inc.) with FITC tags. After 24 h of 50 μmol apigenin treatment at 37˚C, the cells were trypsinized and incubated for 15 min with 300 µl Annexin V/PI staining solution at room temperature. Cells were then evaluated using a flow cytometer to detect cell apoptosis.

Transmembrane ΔΨm was determined using the JC-1 assay, as previously described (17). Cells (1x10⁴ cells/well) were seeded in a 96-well plate and incubated overnight at 37˚C. Medium was removed and 5 µg/ml JC-1 dye (cat. no. C2006; Beyotime Institute of Biotechnology) was added for 20 min at room temperature. Cells were then washed and incubated in PBS for 10 min at room temperature. ΔΨm was measured using a fluorescence plate reader. In healthy mitochondria, JC-1 generated J-aggregates, which are manifested as red signals (20). In the case of mitochondria depolarization, JC-1 is present in the cytoplasm as monomers and is manifested as green signals (21). The transformation from red to green signals suggested ΔΨm depolarization.

TRIzol^® (Thermo Fisher Scientific, Inc.) was used to isolate total RNA as per the manufacturer's instructions, and the isolated RNA was purified using a RNeasy Mini kit (cat. no. 74104; Qiagen GmbH). RT was performed to obtain cDNA using a Superscript III kit (Thermo Fisher Scientific, Inc.) for 42˚C 30 min and 85˚C for 5 min. The temperature protocol was 42˚C for 2 min followed by 37˚C for 15 min and 85˚C for 5 sec before cooling to 4˚C. qPCR was performed on the product using the SYBR-Green PCR Supermix kit (Bio-Rad Laboratories, Inc.). Thermocycling conditions using the LightCycler^® 96 (Roche Molecular Systems, Inc.) were as follows: 95˚C for 30 sec followed by 40 cycles of 95˚C for 5 sec and 60˚C for 60 sec. Primers used were as follows: Myeloid cell leukemia-1 (Mcl-1) forward, 5'-TGTCTTGTGACCGCAATGGT-3' and reverse, 5'-GTTGGACAGGTCAAGGCTTT-3'; and GAPDH forward, 5'-CCACCCATGGCAAATTCCATGGCA-3' and reverse, 5'-TCTAGACGGCAGGTCAGGTCCACC-3'. All procedures were carried out in triplicate, with ≥3 independent runs. Expression was detected using RT StatMiner (Integromics, Inc.), and GAPDH served as an internal reference. Fold change was determined by relative quantification (2^-ΔΔCq) (22).

Lysates were homogenized with a RIPA lysis buffer (cat. no. P0013K; Beyotime Institute of Biotechnology), and proteins were quantified using a Bradford assay (Bio-Rad Laboratories, Inc.). Samples containing 25 µg of protein were subjected to SDS-PAGE on 8-15% Tris-HCl polyacrylamide gels (Bio-Rad Laboratories, Inc.) and were then transferred to PVDF membranes (EMD Millipore). The blots were incubated overnight with primary antibodies against Mcl-1 (1:1,000; cat. no. ab32087, Abcam), cyclin B1 (1:1,000; cat. no. ab32053; Abcam), Bcl-2 (1:1,000; cat. no. ab32124; Abcam), cleaved-caspase 3 (1:1,000; cat. no. ab13847; Abcam), cyclin D (1:1,000; cat. no. ab16663; Abcam), cyclin E (1:1,000; cat. no. ab71535; Abcam), Bax (1:1,000; cat. no. ab32503, Abcam) and β-actin (1:1,000; cat. no. ab17946; Abcam) in Tris-buffered saline/0.1% Tween 20 at 4˚C. The membranes were then incubated with a secondary antibody (1:500; cat. no. ab6802; Abcam) conjugated with horseradish peroxidase at room temperature for 1.5 h. Enhanced chemiluminescence plus detection reagent (Pierce; Thermo Fisher Scientific, Inc.; cat. no. 32109) was used to examine the immunoreactive bands. ImageJ software (v1.51; National Institutes of Health) was used for densitometry.

Data are presented as the mean ± SEM. Differences among various groups were assessed using ANOVA, followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistical significance difference.

The impact of apigenin on cell growth was investigated in SKOV₃ and cisplatin-resistant SKOV3/DDP cells. The cytotoxic effects of apigenin were identified via colony formation testing, and it was found that the addition of 50 µmol apigenin to these cells decreased both the number and size of the colonies compared with the control group (Fig. 1A-C). Moreover, apigenin inhibited the proliferation of SKOV3 and SKOV3/DDP cells compared with the control group (Fig. 1D-F), and the combination of apigenin + cisplatin exerted a significantly greater inhibitory effect on cell proliferation.

Cyclin-dependent proteins, such as cyclin D, B1 and E, are crucial regulators of cell proliferation (23). Therefore, the present study investigated the impact of apigenin on the expression levels of cyclin-dependent proteins in SKOV3 and SKOV3/DDP cells. It was demonstrated that apigenin signficantly downregulated cyclin D, B1 and E compared with the control group (Fig. 2A-H). Thus, the present results suggested apigenin inhibited SKOV3 and SKOV3/DDP proliferation by suppressing cyclin-dependent translations.

SKOV3 and SKOV3/DDP cells were treated with apigenin for 24 h, and the apoptotic rate was examined by Annexin V-PI flow cytometry. The present results indicated that apigenin significantly promoted early apoptosis or necrosis and late apoptotic cell death in both cell types (Fig. 3A-D).

To investigate the involvement of apigenin on cell death, its effect on apoptotic-associated proteins was assessed in SKOV3 and SKOV3/DDP cells. It was found that apigenin downregulated the expression of the antiapoptotic protein Bcl-2 and upregulated the expression levels of the proapoptotic proteins Bax and cleaved caspase-3 (Fig. 4A-H). Therefore, the present results suggested that apigenin triggered SKOV3 and SKOV3/DDP apoptosis by enhancing the expression of proapoptotic proteins, while suppressing that of antiapoptotic proteins.

Our previous study showed the influence of apigenin on mitochondria-modulated cell death (24). Therefore, the present study examined whether apigenin-induced mitochondrial malfunction was a dominant contributor to cell death using a JC-1 assay. It was identified that mitochondria in control cells exhibited red signals, thus suggesting complete ΔΨm. However, apigenin triggered ΔΨm depolarization, demonstrated by the presence of green signals and reduction in the ratio of J-aggregates/J-monomers (Fig. 5A-C). Collectively, the present results indicated that apigenin triggered mitochondrial malfunction to induce apoptosis.

Mcl-1 is an essential factor in malignant cell growth, cell proliferation and apoptosis (25). The present study examined the changes in Mcl-1 expression in SKOV3 and SKOV3/DDP cells in order to understand the mechanisms underlying apigenin-induced apoptosis. It was found that Mcl-1 expression was significantly inhibited by apigenin compared with control cells (Fig. 6A and B). Moreover, Mcl-1 protein expression was downregulated by apigenin in SKOV3 and SKOV3/DDP cells (Fig. 6C-E). The combination of apigenin + cisplatin further promoted the inhibitory effect on both mRNA and protein expression levels of Mcl-1, thus indicating that the downregulation of Mcl-1 by apigenin may be involved in apoptosis and cell cycle arrest of SKOV3 and SKOV3/DDP cells.