Ovarian cancer (OC) is one of the prominent causes of mortality in female patients diagnosed with gynecologic malignancies. While it has previously been demonstrated that apigenin inhibits cell growth in colon and breast cancer cells, the effect of apigenin in OC cells is not fully understood. Therefore, the aim of the present study was to investigate the impact of apigenin on cell death and resistance to cisplatin in OC cells. It was found that apigenin inhibited proliferation, hindered cell cycle progression and promoted SKOV3 cell apoptosis. Moreover, these effects were also observed in cisplatin-resistant SKOV3/DDP cells. Furthermore, apigenin reduced the mitochondrial transmembrane potential, and elevated the ratios of cleaved caspase-3/caspase-3 and Bax/Bcl-2 in the two cell types. Reverse transcription-quantitative PCR and western blotting results demonstrated that apigenin significantly downregulated Mcl-1 at the transcriptional and translational levels in SKOV3 and SKOV3/DDP cells, which was responsible for its cytotoxic functions and chemosensitizing effects. Collectively, the present results identified the impact of apigenin on OC cell death and resistance to cisplatin, and the potential molecular mechanisms. However, additional studies are required to further elucidate the underlying mechanisms.
Ovarian cancer (OC) is one of the most lethal gynecological malignancies, and is the 5th largest contributor to malignancy-related mortality in female patients worldwide (
Apigenin is present in many kinds of food, such as fruit, seasonings and vegetables. Apigenin is a part of the average daily diet (
Therefore, the aim of the present study was to investigate the impact of apigenin on OC and identify the mechanisms during chemoresistance modulation.
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 (
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) (
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 (
Cyclin-dependent proteins, such as cyclin D, B1 and E, are crucial regulators of cell proliferation (
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 (
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 (
Our previous study showed the influence of apigenin on mitochondria-modulated cell death (
Mcl-1 is an essential factor in malignant cell growth, cell proliferation and apoptosis (
OC is a leading contributor to gynecological malignancy-related mortality (
Mitochondria-modulated cell death contributes to OC and resistance to cisplatin (
Antiapoptotic proteins defend against permeabilization of the outer mitochondrial membrane, and resistance to cell death in OC is related to the significant upregulation of Bcl-2 (
Collectively, the present results suggested that apigenin triggered apoptosis and counteracted cisplatin-triggered resistance in OC cells via Mcl-1. Moreover, the present results indicated that apigenin/Mcl-1 may serve as a potential treatment strategy against OC by enhancing sensitivity.
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No funding was received.
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YYQ and APC conceived the study and designed the experiments. ZXD, YSY and FFR performed experiments and contributed toward data collection. MY and SBY analyzed the data and interpreted the results. YYQ wrote the manuscript. APC contributed to the critical revision of the article. All authors read and approved the final manuscript.
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The authors declare that they have no competing interests.
Apigenin inhibits the proliferation of SKOV3 and SKOV3/DDP cells. SKOV3 and SKOV3/DDP cells were treated for 24 h with 50 µM Api, 2 µM cisplatin or 50 µM Api + 2 µM cisplatin. (A) Colonies were observed by crystal violet staining. Quantitative assessment of (B) SKOV3 and (C) SKOV3/DDP colonies. (D) Evaluation of cell proliferation by 5-Ethynyl-2'-deoxyuridine staining. Difference in proliferation between (E) SKOV3 and (F) SKOV3/DDP cells. Data are presented as the mean ± SEM. *P<0.05, **P<0.01 vs. Con. Api, apigenin; Con, control; EdU, 5-Ethynyl-2'-deoxyuridine.
Apigenin downregulates cyclin-dependent proteins in SKOV3 and SKOV3/DDP cells. SKOV3 and SKOV3/DDP cells were treated for 24 h with 50 µM Api, 2 µM cisplatin or 50 µM Api + 2 µM cisplatin. (A) Representative immunoblots and quantification of (B) cyclin D, (C) cyclin B1 and (D) cyclin in SKOV3 cells. (E) Representative immunoblots and quantification of (F) cyclin D, (G) cyclin B1 and (H) cyclin E in SKOV3/DDP cells. Data are presented as the mean ± SEM. **P<0.01 vs. Con. Api, apigenin; Con, control.
Apigenin triggers SKOV3 and SKOV3/DDP cell apoptosis. SKOV3 and SKOV3/DDP cells were treated for 24 h with 50 µM Api, 2 µM cisplatin or 50 µM Api + 2 µM cisplatin. (A) Annexin V-PI flow cytometry was performed to evaluate SKOV3 cell apoptosis. (B) Quantification of apoptotic SKOV3 cells. (C) Annexin V-PI flow cytometry was performed to evaluate SKOV3/DDP cell apoptosis. (D) Quantification of apoptotic SKOV3/DDP cells. Data are presented as the mean ± SEM. *P<0.05, **P<0.01 vs. Con. Api, apigenin; Con, control.
Apigenin regulates the expression of apoptotic-associated proteins in SKOV3 and SKOV3/DDP cells. SKOV3 and SKOV3/DDP cells were treated for 24 h with 50 µM Api, 2 µM cisplatin or 50 µM Api + 2 µM cisplatin. (A) Representative immunoblots and quantification of (B) Bcl-2, (C) Bax and (D) cleaved caspase-3 in SKOV3 cells. (E) Representative immunoblots and quantification of (F) Bcl-2, (G) Bax and (H) caspase-3 in SKOV3/DDP cells. Data are presented as the mean ± SEM. **P<0.01 vs. Con. Api, apigenin; Con, control.
Apigenin triggers the depolarization of ΔΨm in SKOV3 and SKOV3/DDP cells. (A) ΔΨm was determined by JC-1 fluorescent probe. Images were acquired and observed using fluorescence microscopy. Ratio of red/green signal (ΔΨm) in (B) SKOV3 and (C) SKOV3/DDP cells. Data are presented as the mean ± SEM. *P<0.05, **P<0.01 vs. Con. Api, apigenin; Con, control.
Apigenin downregulates Mcl-1 expression in SKOV3 and SKOV3/DDP cells. Mcl-1 transcription was detected by reverse transcription-quantitative PCR in (A) SKOV3 and (B) SKOV3/DDP cells. (C) Representative immunoblots and quantification of Mcl-1 in (D) SKOV3 and (E) SKOV3/DDP cells. Data are presented as the mean ± SEM. *P<0.05, **P<0.01 vs. Con. Api, apigenin; Con, control.