Genistein (4′,5,7-trihydroxyisoflavone), 2-chloro-5-nitrobenzanilide (GW9662), dimethyl sulfoxide (DMSO), Triton X-100, RNase A and propidium iodide (PI) were all purchased from Sigma-Aldrich Corp. (Sigma-Aldrich Corp., St. Louis, MO, USA). Dissolving genistein and GW9662 in DMSO were recommended. According to the product information, genistein was dissolved to prepare a 10 mg/ml stock solution and GW9662 was reconstituted as a 26 mg/ml stock solution. The solutions were then diluted to appropriate concentrations with a culture medium. Dulbecco’s modified Eagle’s medium (DMEM), penicillin-streptomycin, trypsin-EDTA, trypsin and fetal bovine serum (FBS) were all obtained from Gibco/Life Technologies (Carlsbad, CA, USA). Rabbit polyclonal antibodies specific for PPARγ, phosphatase and tensin homolog (PTEN), P21^WAF1/CIP1, Cyclin B1, B-cell lymphoma-2 (BCL-2), survivin and mouse monoclonal antibody specific for β-actin were purchased from Assay Biotechnology (Sunnyvale, CA, USA). Horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit secondary antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

The MG-63 OS cell line was purchased from the China Center Type Culture Collection (CCTCC, Shanghai, China). The cells have been maintained under standard culture conditions in DMEM, supplemented with 10% (vol/vol) FBS and 1% pencillin-streptomycin at 37°C, under a humidified atmosphere of 5% CO₂ in our laboratory. The medium was replaced every 3 days before full confluency.

Cells were seeded with a density of 2–4×10³/well in 96-well plates, which was allowed to attach overnight. The medium was replaced with a fresh medium containing different concentrations of genistein (0–120 μmol/l or GW9662 0–100 μmol/l), diluted from the stock solution. Since the final DMSO concentration of genistein in the medium was >0.1%, a DMSO (solvent) concentration of 0.32% (vol/vol) was used for vehicle control. After 24, 48 and 72 h of incubation, 10 μl of Cell Counting Kit-8 (CCK-8) (Dojindo, Tokyo, Japan) solution was added. Absorbance at 450 nm was measured by the Microplate Reader (Thermo Labsystems, Helsinki, Finland). The relative folds of drug resistance were analyzed by comparing with IC₅₀.

As stated above, cells were plated and allowed to attach overnight; the medium was then removed and replaced with a fresh medium containing various doses of genistein (40, 60 and 80 μmol/l), with 1 μmol/l of GW9662, for 48 h. The effect of GW9662 on cell viability was examined by CCK-8 method, as described above.

The proliferation assay of MG-63 cells was performed using a Cell-Light™ 5-ethynyl-2′-deoxyuridine (EdU) DNA Cell Proliferation kit (Ruibo Biotech, Guangzhou, China), based on the manufacturer’s protocols (44–46). MG-63 cells were cultured in 24-well plates at 2–3×10⁴/well with different concentrations of genistein (40, 60 and 80 μmol/l), with or without GW9662 (1 μmol/l), for 48 h. Then, the cells were incubated with 100 μmol/l of EdU for another 2 h. Finally, the cells were fixed with 4% formaldehyde in phosphate buffered solution (PBS) buffer solution, for 30 min. After labeling, five random × 100 fields were photographed in each well. The EdU-positive cells were counted, averaged and compared. Assays were performed three times using triplicate wells.

To measure the apoptosis rate of the cells, we used the Annexin V-FITC/PI doublefluorescence apoptosis detection kit (Biouniquer Technology, Shanghai, China). Briefly, the cells were seeded in 6-well plates (2×10⁵ cells/ml) and exposed to genistein (40, 60 and 80 μmol/l), with or without GW9662 (1 μmol/l), for 48 h. The cells were washed 2 times by PBS buffer solution at 4°C, diluted with 500 μl of Annexin V binding liquid and 5 μl of Annexin V-FITC was added at 4°C for 15 min in the dark. Finally, 10 μl of PI was added at 4°C for 5 min in the dark. Samples were analyzed using a FACScan flow cytometer (Becton-Dickinson, San Jose, CA, USA) - within 1 h after staining.

MG-63 cells were plated in T₂₅ tissue culture flasks at 2×10⁶/flask in a culture medium. After 24 h, cells were washed in PBS buffer and treated with a fresh medium, with or without 1 μmol/l of GW9662 or various concentrations of genistein (40, 60 and 80 μmol/l). Following 48 h of genistein treatment, the cells were detached and trypsinized and washed two times in PBS buffer solution at 4°C. Then, the cells were fixed and permeabilized with 70% ethanol on ice for 2 h. Cells were washed again in 0.5 ml of PBS buffer solution and stained for 30 min with 1 ml of DNA fluorochrome solution at normal temperature, containing 200 μg of PI, 0.1% of Triton X-100 and 2 mg of RNase A. Then 2×10⁴ cells were counted and analyzed using a FACScan flow cytometer (Becton-Dickinson).

The 2×10⁶ cells that were treated for 48 h with genistein (40, 60 and 80 μmol/l) and/or GW9662 (1 μmol/l) were washed twice in an ice-cold PBS buffer solution; and the cells were resuspended in 200 μl of ice-cold lysis buffer solution (50 mmol/l Tris-HCl, 150 mmol/l NaCl, 0.02% NaN₃ and 1% NP₄₀) for 30 min. The lysates were clarified by centrifugation for 15 min at 14,000 rpm at 4°C. The protein concentration of the supernatants was determined by using a bicinchoninic acid protein assay (Pierce, Rockford, IL, USA). Equal amounts of lysate protein were separated by 8–12% SDS-polyacrylamide gel electrophoresis, which were then transferred onto polyvinylidene fluoride membranes. After being blocked with 10% non-fat dry milk for 1 h at room temperature, the membranes were washed three times with a PBS Tween-20 (PBST) buffer solution and incubated with monoclonal antibodies or phosphorylated antibodies overnight, at 4°C. Subsequently, the membranes were washed three times with PBST buffer solution and incubated with peroxidase-conjugated secondary anti-mouse or anti-rabbit antibodies for one hour at room temperature. The bands were visualized using enhanced chemiluminescence detection and exposure to film.

RNA extraction and cDNA conversion were performed. MG-63 cells in the logarithmic growth phase were placed into 6-well plates with a density of 2×10⁵/ml; and the cells were treated with different genistein concentrations (40, 60 and 80 μmol/l), with or without 1 μmol/l of GW9662, for two days. Total RNA was isolated with a TRIzol reagent (Takara Biotech, Dalian, China). cDNA was generated from total RNA by using a PrimeScript RT Master Mix kit (Takara Biotech). Quantitative real-time PCR was performed using LightCycler™ 480 Real-Time system (Roche Applied Science, IN, USA) and SYBR Premix Ex Taq™ kit (Tli RNaseH Plus) (Takara Biotech). The threshold cycle (C_t) value of each gene was measured from each RT sample. The C_t value of β-actin was used as an endogenous reference for normalization purposes. Thus, the obtained C_t value was normalized to negative control and was expressed as fold changes. The primers and reaction conditions used for the amplification are listed in Table I.

Statistical analysis was performed using SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). The data were expressed as means ± SD and the statistical differences between multiple groups were calculated by one-way ANOVA, followed by Dunnett’s test. P<0.05 values were considered statistically significant. The results from the in vitro studies are representative of at least three independent experiments.

To determine the cytotoxicity of genistein on OS cells, we tested the effects of various doses of genistein on the viability of MG-63 cell line using CCK-8 assay. The cells were treated for 24, 48 and 72 h; and cell inhibition was found to be significant at 48 and 72 h (data at 24 h was not significant). As shown in Fig. 1A, cell growth was reproducibly inhibited by genistein treatment in a dose-dependent manner. After 48-h treatment with 20, 40, 60, 80, 100 and 120 μmol/l of genistein, the cell survival rates were 103.4±2.7, 76.7±3.7, 54.6±6.6, 40.0±3.5, 33±4.8 and 11.1±0.5, respectively. After 72 h of MG-63 cell treatments with different genistein concentrations, the cell survival rates were 122.3±7.1, 87.2±5.6, 48.8±1.9, 26.7±2.9, 17.7±2.3 and 9.2±0.5, respectively, inhibiting cell growth (Fig. 1A). Based on the data, the conditions used in 48-h treatments of genistein were selected for further studies.

Subsequently, we evaluated the effects of GW9662 - a potent specific antagonist of PPARγ. It was found that low concentrations of GW9662 (0.01–1 μmol/l) played a weak role in impairing the proliferation of MG-63 cells (Fig. 1B). Similarly, Seargent et al (47) reported that GW9662 could directly prevent tumor cells by the PPARγ-independent pathway. As a single agent, these results indicated that GW9662 was a growth inhibitor of OS cells. Therefore, 1 μmol/l of GW9662 was chosen for our studies, due to its minimal effects on MG-63 cells.

To assess whether the inhibition of genistein-induced cells are related to PPARγ activation (Fig. 1C), we investigated the effects of combining genistein and GW9662 on cell viability by CCK-8 assay. The cells were treated with different concentrations of genistein (20, 40, 60, 80, 100 and 120 μmol/l), with or without GW9662 (1 μmol/l), for 48 h. MG-63 cells showed comparable cell growth augmentation, when treated with genistein and GW9662. However, genistein treatment alone caused a significant increase in cell growth inhibition.

Therefore, to investigate the effects of PPARγ activation by genistein, our subsequent studies primarily focused on the MG-63 cell line, simulating different treatments for 48 h.

Cell proliferation was evaluated by detecting immunofluorescence for EdU. As shown in Fig. 1D and E, 48-h genistein treatments (40, 60 and 80 μmol/l) potently decreased the number of proliferating MG-63 cells. In contrast, combining different concentrations of genistein (40, 60 and 80 μmol/l) and GW9662 (1 μmol/l) did not significantly affect the proliferation of MG-63 cells. Therefore, the inhibition of PPARγ with GW9662 significantly reduced the genistein-induced anti-proliferation activites of MG-63 cells. These data suggest that the result of activating the PPARγ mechanism partially caused genistein to lose its cell proliferation ability.

Using fluorescence-activated cell sorting to quantify apoptosis, we observed that genistein as a single agent induced apoptosis in MG-63 cells dose-dependently (Fig. 2). DNA electrophoresis showed a more significant DNA ladder in MG-63 cells that underwent a combined treatment of genistein and GW9662. Flow cytometry experiments also showed that GW9662 can enhance genistein-induced OS cell apoptosis. The induction of genistein-induced apoptosis was dose-dependent and was unrelated to PPARγ.

To detect if the suppressive effect of genistein can be caused by the specific perturbation of cell cycle-related events, the DNA content of genistein-treated and combination-treated MG-63 cells were measured using flow cytometry after PI staining of the nuclei. The G₁ and S cell population significantly decreased in a dose-dependent manner and after 48-h exposure to genistein (Fig. 3), while cells were arrested in the G₂/M phase of the cell cycle. Incubating with GW9662 reversed the effects of the G₂/M cell cycle arrest caused by genistein.

PPARγ activation inhibits cell growth and causes both differentiation and apoptosis in a variety of cancer cell types (25). Therefore, in our present study, we used GW9662, a PPARγ inhibitor that can block PPARγ functions, to determine whether genistein prevents OS cell growth via the PPARγ pathway. Although GW9662 (1 μmol/l) induced a decrease in cell growth, GW9662 treatment partially reversed the inhibitory effects of genistein based on the cell number; the related MG-63 cell protein expression was also changed.

To elucidate the effect of genistein in OS cell growth, proteins related to the PPARγ pathway were examined by western blot analysis. As shown in Fig. 4A and B, a stable level of PPARγ protein was observed in MG-63 cells after exposure to different genistein concentrations; however, the PPARγ protein expression was not altered by the GW9662 inhibitor. PTEN is a downstream target protein that plays a notable role in malignancy growth and causes the activation of the phosphoinositide-3 kinase/AKT (PI3K/Akt) signaling pathway (48). Fig. 4A and C shows that genistein (40, 60 and 80 μmol/l) promoted the expression of the PPARγ target protein, PTEN, in a concentration-dependent manner. GW9662 treatment significantly decreased the PTEN expression caused by genistein; suggesting possible PPARγ pathway implications on the mechanisms responsible for the effects of genistein on OS cell growth.

Fig. 4D and E shows the real-time quantitative RT-PCR results of PPARγ-related genes (PPARγ and PTEN). There was a prominent increase in PPARγ gene expression levels, as well as in the PTEN genes of MG-63 cells treated with genistein. GW9662 significantly inhibited the expression of these two genes induced by genistein. With β-actin constitutively expressed, its levels were not affected by genistein or the combined treatment. Thus, the real-time quantitative RT-PCR results support the previous observations of our western blot assay.

To further understand the downstream mechanism of the PPARγ pathway by genistein treatment, G₂/M regulatory molecules were analyzed. Since genistein treatment perturbed the G₂/M phase of the cell cycle, as assessed by flow cytometry, we examined the intracellular protein expression of the cell cycle regulating components at the G₂/M boundary, such as P21 ^WAF1/CIP1 and Cyclin B1. As shown in Fig. 5A and B, a dramatic dose-dependent increase in the P21^WAF1/CIP1 protein levels were observed with genistein, compared with the co-treatments. In contrast, Cyclin B1 levels (Fig. 5A and C) were reduced by either the reagent or the combined treatment, the former causing a more dramatic effect in a dose-dependent manner.

Several lines of evidence indicate that protein BCL-2 and survivin, related to the protein of PTEN (49,50), were generally considered to be apoptosis inhibitors. These were frequently overexpressed in several types of human cancers, including OS. Parallel to the above observations, BCL-2 and survivin levels by genistein were decreased, as indicated by the significantly decreased levels of BCL-2 and survivin, even with the presence of GW9662 (Fig. 5D–F). While the inhibition of PPARγ repressed apoptosis-related protein levels, GW9662 did not reverse the genistein-induced effects on the promoted apoptosis, which was consistent with earlier findings.

Taken together, the data suggest that the PPARγ pathway was associated with the effects of genistein. Furthermore, genistein could significantly induce OS cell cycle arrest by activating PPARγ, while it is probable for genistein-induced apoptosis to be independent from the PPARγ pathway.