Apigenin (4′,5,7-trihydroxyflavone), HIF-1α (hypoxia-inducible factor 1-α) inhibitor (EF-24), 7-aminoactinomycin D (7-AAD), rhodamine 123 and nicardipine were purchased from Sigma Chemical Co. (St. Louis, MO, USA). These compounds were dissolved in dimethyl sulfoxide (DMSO) or ethanol, and the final concentration of DMSO or ethanol in the controls and in each sample did not exceed 0.1%. The STAT3 inhibitor (S3I-201) was purchased from Calbiochem (San Diego, CA, USA). JAK (Janus kinase) inhibitor I was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Annexin V, Alexa Fluor^® 488 Conjugate was purchased from Thermo Fisher Scientific Korea (Seoul, Korea). An EZ-western chemiluminescent detection kit was obtained from Daeillab Service Co. (Seoul, Korea).

MCF-7 (ATCC, American Type Culture Collection, Manassas, VA, USA) and MCF-7/ADR (a gift from Professor Hwa Jeong Lee, Ewha Womans University, Seoul, Korea) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) and RPMI-1640 medium, respectively, containing 50 U/ml penicillin, 50 mg/ml streptomycin and 10% fetal bovine serum (FBS; Welgene, Daegu, Korea) at 37°C in an atmosphere of 5% CO₂.

Primary antibodies directed against cleaved caspase-8, poly(ADP-ribose) polymerase (PARP) and P-gp (MDR1) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Primary antibodies directed against STAT3 and phospho-STAT3 (Tyr705) were purchased from Upstate-Millipore (Billerica, MA, USA). The anti-tubulin antibody came from Sigma Chemical Co. Horseradish peroxidase (HRP)-conjugated secondary antibodies (mouse and rabbit) were obtained from Calbiochem, and anti-goat secondary antibody was from Jackson ImmunoResearch (West Grove, PA, USA).

MCF-7 and MCF-7/ADR cells were plated in 96-well culture plates at a density of 3×10³ cells/well and incubated for 24 h at 37°C. Then, they were treated with adriamycin (0–20 µg/ml), apigenin (0–100 µM), STAT3 inhibitor (0–500 µM), JAK inhibitor I (0–10 µM), or HIF-1α inhibitor (0–100 µM) for 48 or 72 h. After incubation, MTT reagents (0.5 mg/ml) were inserted to each well, and the plates were incubated in a humidified incubator at 37°C for 2 h. At the end of the incubation period, the medium was removed, the resulting formazan was dissolved in DMSO, and the optical density was determined at 570 nm using an ELISA plate reader.

For the colony formation assay, MCF-7 and MCF-7/ADR cells were plated in 6-well culture plates at a density of 5×10² cells/well. After 24 h, the cells were treated with different concentrations of apigenin (0–80 µM) or vehicle and maintained for 10 days at 37°C. Medium was changed every 3 days. Finally, the plates were stained with hematoxylin, and colony numbers were counted.

MCF-7 and MCF-7/ADR cells treated with apigenin (0–80 µM) were harvested with 0.25% trypsin and washed once with phosphate-buffered saline (PBS). After the cells were centrifuged, they were fixed in cold 95% ethanol with 0.5% Tween-20 and stored at −20°C for at least 30 min. The cells were incubated in 50 µg/ml of propidium iodide (PI) (including 1% sodium citrate and 50 µg/ml RNase A) at room temperature in the dark for 30 min. The analysis of apoptotic cells was performed with a FACScan flow cytometer (Becton-Dickinson, Mountain View, CA, USA), and the data were analyzed using CellQuest software.

Apoptotic cell death was measured by an Annexin V-FITC and 7-AAD assay. Cells were stained with Annexin V-FITC and 7-AAD for 30 min at room temperature in the dark. The apoptotic cell population was analyzed with a FACSCalibur™ flow cytometer by measuring the signal in the FL-1 and FL-3 channels.

Harvested cells were lysed in modified RIPA buffer containing 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris (pH 8.0), 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM NaF, 1 mM Na₃VO₄ and a protease inhibitor mixture]. The lysates were cleared by centrifugation at 13,000 rpm for 15 min at 4°C. The supernatants were collected and stored at −70°C until use. The protein concentration was quantified using a Bio-Rad Bradford protein assay (Bio-Rad, Hercules, CA, USA). Total proteins (30 µg) were electrophoresed using 6–15% reducing SDS-polyacrylamide gels and transferred to nitrocellulose membranes. After blocking with 0.1% Tween-20 in PBS (PBST) containing 1% skim milk and 1% BSA for 1 h, the membranes were incubated overnight at 4°C with the indicated primary antibodies. After washing in 1X PBST for 15 min (3 times × 5 min), the membranes were incubated with diluted enzyme-linked secondary antibodies. After washing in 1X PBST for 1 h (4 times × 15 min), the protein bands were detected using the EZ-western chemiluminescent detection kit and visualized by exposing the membranes to X-ray film.

MCF-7 and MCF-7/ADR cells (2×10⁴ cells/well) were plated in 8-well chamber slides, incubated overnight at 37°C and treated with apigenin (80 µM) for 24 h. The cells were fixed with 4% paraformaldehyde for 30 min and treated with 3% hydrogen peroxide (H₂O₂) in methanol for 20 min. The cells were washed with PBS, blocked with 5% BSA in PBS for 1 h and incubated with an anti-STAT3 primary antibody (1:100 dilution) for 24 h at 4°C. After the cells were rinsed with PBS, they were incubated with anti-rabbit biotin-conjugated secondary antibody for 1 h at room temperature. Finally, the cells were treated with Vectastain ABC reagent (Vector Laboratories, Inc. Burlingame, CA, USA) for 30 min at 4°C and stained with diaminobenzidine tetra chloride (DAB) and hematoxylin. The cells were mounted with mounting medium and subsequently analyzed by microscopy.

RNA was isolated from cells using easy-blue RNA extraction kit (iNtRON Biotech, Sungnam, Korea) according to the manufacturer's instructions. Isolated RNA content was measured using the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA). Total cellular RNA (2 µg) from each sample was reverse transcribed using cDNA synthesis kit (Takara, Otsu, Shiga, Japan). PCR was conducted in a 20-µl reaction mixture consisting of DNA template, 10 pM of each gene-specific primer, 10X Taq buffer, 2.5 mM dNTP mixture, and 1 U of Taq DNA polymerase (Takara). PCR was performed using the specific primer and primer sequences are shown in Table I.

The levels of VEGF (vascular endothelial growth factor) and MMP-9 (matrix metalloproteinases-9) were determined by sandwich ELISA using the BD Pharmingen human ELISA set (Pharmingen, San Diego, CA, USA). Briefly, plates were coated with capture antibody in ELISA coating buffer (Sigma) and incubated overnight at 4°C. Plates were washed with PBS-Tween-20 (0.05%) and subsequently blocked (10% FBS in PBS) for 1 h at 20°C. Serial dilutions of standard antigen or sample in dilution buffer (10% FBS in PBS) were added to the plates and plates were incubated for 2 h at 20°C. After washing, biotin-conjugated anti-mouse IgE and streptavidin-conjugated horseradish peroxidase (SAv-HRP) were added to the plates and plates were incubated for 1 h at 20°C. Finally, tetramethylbenzidine (TMB) substrate solution was added to the plates and after 15 min incubation in the dark, 2 N H₂SO₄ solution was added to stop the reaction. Optical densities were measured at 450 nm on an automated ELISA reader (Versa Max, Molecular Devices, Sunnyvale, CA, USA).

MCF-7 and MCF-7/ADR cells were treated with apigenin (0–80 µM) or nicardipine (positive control, 12.5 µM) for 24 h and incubated for another 1 h with 1 µg/ml of rhodamine 123. Accumulation of rhodamine 123 in the cells was analyzed by flow cytometry.

All experiment results were expressed as the means ± standard deviations (SD) of at least three separate tests. Student's t-test was used for single variable comparisons, and a P-value <0.05 was considered statistically significant. Statistical analyses were performed using PRISM software (GraphPad Software Inc., La Jolla, CA, USA).

Before investigating the effect of apigenin on the growth of adria- mycin-resistant cells, we determined whether MCF-7/ADR cells display resistance to adriamycin. Fig. 1A demonstrates that adriamycin decreased the growth rate of MCF-7 cells, while it did not affect the growth rate of MCF-7/ADR cells, confirming that the MCF-7/ADR cell line is adriamycin-resistant. Fig. 1B indicates that apigenin significantly suppressed the proliferation of both MCF-7 and MCF-7/ADR cells in a dose-dependent manner after 72 h of treatment. This growth inhibition induced by apigenin was confirmed by microscopic observation (Fig. 1C). Apigenin also induced morphological changes in these cells; the shape of the cells became round and shrunken. The changes were common both to MCF-7 and MCF-7/ADR (Fig. 1C). These results indicate that apigenin suppresses the growth of adriamycin-resistant cells.

Next, we investigated the effect of apigenin on the clonogenic survival of MCF-7/ADR cells using a colony formation assay. As shown in Fig. 2, apigenin strongly inhibited colony formation in both MCF-7 and MCF-7/ADR cells in a dose-dependent manner. The effect of apigenin was stronger in MCF-7 cells than in MCF-7/ADR cells, as evidenced by the relative absence of colony formation in MCF-7 cells and the presence of colonies in MCF-7/ADR cells with 20 µM treatment. These results indicate that MCF-7/ADR cells are drug-resistant and that apigenin overcomes this resistance.

To study whether apigenin blocks cell proliferation by promoting changes in cell cycle progression, the effect of apigenin on the cell cycle profile was assessed in MCF-7 and MCF-7/ADR cells. For this purpose, cells were treated with apigenin (0–80 µM) for 72 h and then analyzed for cell cycle stage by flow cytometry. The results demonstrated that apigenin induced an increase in the sub-G₀/G₁ apoptotic population in both cells (Fig. 3A). In the Annexin V assays, we found that apigenin increased the number of apoptotic cells in both cell lines (Fig. 3B). Moreover, we observed that apigenin induces not only apoptosis but necrosis. Consistent with these results, we also found that apigenin altered the apoptotic protein machineries; apigenin upregulated the levels of cleaved caspase-8 and PARP in both cell lines (Fig. 3C). Therefore, our data indicate that apigenin induces apoptosis in chemotherapy-resistant breast cancer cells.

Because P-gp (MDR1), multidrug resistance-associated proteins (MRP), and breast cancer resistance protein (BCRP), are important contributors to drug resistance, we checked whether apigenin regulates their expression. RT-PCR demonstrated that apigenin reduced the mRNA expression of MDR1, MRP1, MRP3, MRP5 and BCRP (Fig. 4). Western blot analysis indicated that P-gp (MDR1) was not induced in MCF-7 cells in the presence or absence of apigenin, while apigenin decreased the protein expression of P-gp (MDR1) at 80 µM in MCF-7/ADR cells (Fig. 4). These results indicate that apigenin inhibits drug resistance by reducing the expression of drug resistance proteins in MCF-7/ADR cells.

Because drug resistance is associated with drug efflux by the cells, we performed a rhodamine 123 assay to investigate whether apigenin recovers drug accumulation in MCF-7/ADR cells. As expected, rhodamine 123 accumulation was high in the MCF-7 cell line, which is a drug-sensitive cell line (Fig. 5). In contrast, rhodamine 123 was ejected from the drug-resistant MCF-7/ADR cells, but apigenin recovered rhodamine 123 accumulation (Fig. 5). These results indicate that apigenin inhibits drug resistance by suppressing drug efflux in MCF-7/ADR cells.

Since STAT3 is related to oncogenic signaling, and it is known that STAT3 deactivation reverses chemotherapeutic resistance (27), we investigated whether apigenin inhibits the STAT3 signaling pathway in MCF-7/ADR cells. We found that apigenin reduced the expression of p-STAT3 in both MCF-7 and MCF-7/ADR cells (Fig. 6A). Immunocytochemical study demonstrated that apigenin decreased the nuclear localization of STAT3 in both MCF-7 and MCF-7/ADR cells (Fig. 6B). ELISA demonstrated that apigenin decreased the production of intracellular VEGF and MMP-9, which are STAT3 target genes in both MCF-7 and MCF-7/ADR cells (Fig. 6C and D). Moreover, the STAT3 inhibitor S3I-201, the JAK inhibitor I, and the HIF-1α inhibitor EF-24 induced cell growth inhibition in both MCF-7 and MCF-7/ADR cells, as shown in Fig. 7A-C. Fig. 7D demonstrates that both apigenin (80 µM) and the STAT3 inhibitor S3I-201 reduced the expression of p-STAT3, P-gp (MDR1) and survivin (a STAT3 target gene) in MCF-7/ADR cells. These results indicate that apigenin suppresses drug resistance by inhibiting the STAT3 signaling pathway in MCF-7/ADR cells. Therefore, STAT3 may be a promising therapeutic target for overcoming drug resistance in breast cancer.