Butein was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Butein was dissolved in dimethylsulfoxide (DMSO) and stored at −20°C until use. The concentration of DMSO used was <1% in the control and the butein-containing medium.

Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). Propidium iodide (PI) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma (St. Louis, MO, USA). Stock solutions of PI and MTT were prepared by dissolving 1 mg of each compound in 1 ml of phosphate-buffered saline (PBS). The solution was stored at 4°C in the dark and used within 1 month. Caspase-3, -8 and -9 Colorimetric Activity Assay kits were purchased from R&D Systems (Minneapolis, MN, USA). Apoptotic Cells Detection kit (Annexin V-FITC/ethidium homodimer III staining) was purchased from PromoKine Systems (Heidelberg, Germany).

For western blot analysis, the antibodies used were: a mouse monoclonal anti-human Bcl2, mouse monoclonal anti-human Bax, mouse monoclonal anti-human MMP-2, and mouse monoclonal anti-human MMP-9, all purchased from Cell Signaling Technology (Beverly, MA, USA). Mouse monoclonal anti-human AKT, mouse monoclonal anti-human phosphorylated (p)-AKT, mouse monoclonal anti-human PI3K, mouse monoclonal anti-human (p)-PI3K, mouse monoclonal anti-human mTOR, mouse monoclonal anti-human (p)-mTOR, were obtained from Santa Cruz Biotechnology, Inc. Mouse monoclonal anti-human β-actin and anti-mouse immuno globulin G-horseradish peroxidase-conjugated secondary antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA).

The HeLa human cervical cancer cell line was purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences, Shanghai Institute of Cell Biology (Shanghai, China). Cultured cells were grown in DMEM supplemented with 10% fetal calf serum and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin) at 37°C in a humidified atmosphere containing 5% CO₂.

Cell viability was determined by MTT assay as previously described (31). Briefly, HeLa cells were cultured in 96-well plates and treated with different concentrations of butein (0–100 μM) for 72 h. Cell viability was determined via an MTT assay. Absorbance at 570 nm was measured in an ELISA plate reader (Molecular Devices Corp., Sunnyvale, CA, USA).

HeLa cells were seeded in 6-well plates at a density of 1×10³ cells/well. After being cultured for 24 h, the cells were treated with the indicated concentrations of butein (0, 15, 25 and 50 μM). The cells were then washed with drugs-free medium and allowed to grow for 14 days in drugs-free conditions. After 14 days, the cells were fixed in paraformaldehyde and stained with Giemsa for 15 min, and images of the colonies were captured by a digital camera (Olympus, Tokyo, Japan). The percentage of colony formation was calculated by adjusting control (untreated cells) to 100%.

The cells were seeded at a density of 1×10⁶ cells/well in 6-well plates in complete medium for 16 h. At the end of treatment with the indicated concentration of butein, the cells were collected and fixed with ice-cold 70% ethanol overnight at −20°C. After centrifugation at 3,000 × g for 5 min, the cell pellets were added into 4 μg/ml PI solution containing 1% Triton X-100 and 100 μg/ml RNase at 37°C for 30 min. The samples were analyzed by a flow cytometer (FACSCalibur), and the percentage of cell-cycle phases was quantified using ModFit LT software 2.0 (BD Biosciences, San Jose, CA, USA).

The cells were plated at a density of 5×10⁵ cells/60-mm Petri dish in complete medium for 16 h, and then treated with the indicated concentrations of butein for 24 h. Apoptotic cells were assessed using an Apoptotic Cells Detection kit according to the manufacturer’s instructions. The cells were collected and resuspended in 500 μl of binding buffer, and 5 μl of Annexin V-FITC and 5 μl of PI were added. The cells were cultured for 10 min at room temperature. The samples were analyzed by flow cytometer using CellQuest software (BD Biosciences).

In addition, the expression of Bax and Bcl-2 were determined by western blotting 24 h after treatment with the different concentrations of butein.

Activities of caspase-3, -8 and -9 were detected as previously described (21). Briefly, HeLa cells were seeded in 6-well plates at a density of 4×10⁵ cells/well and cultured for 24 h, then treated with indicated concentration of butein for 2 h. The cells were washed with cold PBS and collected by centrifugation at 1,500 rpm for 5 min at 4°C. The cells were lysed using a cell extraction buffer (BioSource International, Camarillo, CA, USA) with protease inhibitor cocktail (Sigma) and PMSF (BioSource), incubated on ice for 30 min and vortexed for 30 sec. The samples were centrifuged at 10,000 rpm for 10 min at 4°C to remove the supernatant. The caspase-3, -8 and -9 levels in cell lysates were measured using the caspase-3, -8 and -9 colorimetric kit according to the manufacturer’s instructions. The optical density was read using a microplate reader (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 405 nm. The relative caspase activity of the untreated group (0 μM butein treatment) was taken as 100.

The cell invasion and migration assays were carried out using Transwell inserts (Corning, Corning, NY, USA) according to the manufacturer’s instructions. For the invasion assay, filters were precoated with Matrigel (BD Biosciences, Bedford, MA, USA) for 30 min. Approximately 5×10⁴ cells were placed in the upper chamber in serum-free DMEM, and 500 μl of DMEM containing 20% FBS and the indicated concentrations of butein (0, 15, 25 and 50 μM) were placed in the lower chamber in the invasion and migration assays. After 24 h of treatment, the cells on the upper side of the filters were mechanically removed by wiping with a cotton swab. Cells migrating to the lower surface of filter were fixed in 70% ethanol for 15 min and stained with 0.2% crystal violet for 15 min. The invaded or migrated cells were photographed and quantified by counting the number of stained cells in five randomly selected fields under a light microscope (Olympus).

Generation of intracellular ROS was examined by flow cytometry as previously described (32). Briefly, 5×10⁴ HeLa cells were seeded in 60-mm dishes overnight, and exposed to different concentrations of butein (0, 15, 25 and 50 μM) for 1 h. The cells then were stained with 2 μM hematoxylin and eosin (H&E) or 5 μM H₂DCFDA at 37°C for 30 min. The cells were collected and fluorescence was analyzed using a flow cytometer. The cells were also pretreated with 10 mM NAC prior to butein exposure and analysis of ROS generation.

At the end of treatment, the cells were lysed in the ice-cold whole cell extract buffer containing protease inhibitors (Sigma) for 30 min, and centrifuged at 10,000 rpm for 10 min at 4°C. The protein concentration was measured by BCA protein assay kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. Equal amounts of total proteins (20 μg) were subjected to electrophoresis using 8–12% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE). After electrophoresis, the proteins were transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% dry milk in TBS-T overnight at 4°C and then incubated with specific primary antibody at room temperature for 2 h. After washing with TBS-T twice, the membranes were incubated with anti-mouse immunoglobulin G-horseradish peroxidase-conjugated secondary antibodies, followed by washing three times with TBS-T. The protein bands were then visualized on the X-ray film using the enhanced chemiluminescence detection system (PerkinElmer Life and Analytical Sciences, Boston, MA, USA). Blots were stripped and reprobed with anti-β-actin for the loading variations. Quantity One software (Bio-Rad) was used to analyze the intensity of bands on X-ray film by semi-quantification.

Twenty female BALB/c nude mice (6 weeks old) were obtained from the Experiments Animal Center of Changchun Biological Institute (Changchun, China), and maintained under specific pathogen-free conditions based on the guidelines established by the Institutional Animal Care and Use Committee of Jilin University. HeLa cells (2×10⁶) mixed with 10% Matrigel (R&D Biosystems) were subcutaneously injected into the right flank of each mouse. Tumor-bearing mice were randomly divided into two groups with 10 animals each. Animals received either intraperitoneal (i.p.) injection of 1% polysorbate resuspended in deionized water (control group) or butein (5 mg/kg in 100 μl) on alternate days for 4 weeks. The mice were weighed weekly to determine any toxicity associated with butein treatments, and the tumors were measured using digital vernier calipers. The volume was calculated as: (π/6 × length × width × height). There was no mortality during the treatment regimen. At the end of the study, the animals were euthanized by CO₂ asphyxiation, the tumor tissues were harvested and weighed. Sections of each tumor tissue were wax-embedded and H&E stained to study cell apoptosis in vivo by TUNEL using In Situ Cell Death Detection kit (Roche Diagnostics, Branchburg, NJ, USA) according to the manufacturer’s instructions.

For the in vitro and in vivo studies, the statistical significance was evaluated by the two-tailed Student’s t-test and ANOVA, and data are presented as the mean ± SD (standard deviation). In vitro assessments were performed in three independent experiments to confirm the results. Statistical analyses were performed using the GraphPad Prism version 5.01 (GraphPad Software, San Diego, CA, USA). P<0.05 was considered to indicate a statistically significant result.

To examine the proliferation effects of butein, HeLa cells were treated with different dose of butein (0–100 μM) for 72 h, and the percentage of viable cells was determined. Results shown in Fig. 1B indicated that butein decreased cell viability of HeLa cells in a dose-dependent manner. The IC₃₀ values (the effective dose that inhibits 30% growth) and IC₅₀ values (the effective dose that inhibits 50% growth) for treatment of HeLa cells by butein were 14.89 and 24.76 μM, respectively. Based on the results we selected the concentrations of 15 μM (IC₃₀), 25 μM (IC₅₀) and 50 μM (2×IC₅₀) salidroside for subsequent experiments.

The effects of butein on the cell colony formation of HeLa cells were also investigated. It was found that butein significantly inhibited the colony formation of HeLa cells in a dose-dependent manner (P<0.05, Fig. 1C).

To determine whether the growth inhibitory effect of butein in HeLa cells was associated with the induction of the cell cycle, we monitored cell-cycle progression using flow cytometry following treatment with the indicated concentrations of butein (0, 15, 25 and 50 μM). Exposure to butein resulted in an increase of G2/M phase cells, accompanied by a decrease in G1 phase cells (Fig. 2A). The effect observed at 50 μM butein was the greatest, with ~48% of cells being in the G2/M phase, compared to ~9% in the control condition.

The effects of butein on the cell apoptosis of HeLa cells were also examined. HeLa cells were treated with butein at different concentrations of 0, 15, 25 and 50 μM for 48 h, and cell apoptosis was analyzed by flow cytometry (Fig. 2B). It was found that butein significantly increased total apoptotic rate of HeLa cells in a dose-dependent manner (P<0.05). Consequently, our results demonstrated that treatment with butein inhibits HeLa cell growth by inducing G2/M transition and cell apoptosis.

To determine the potential mechanism of butein, the effects of cell apoptosis, apoptosis-related protein, Bax and Bcl-2 expression were examined by western blot analysis. The results revealed a significant upregulation of Bax expression and downregulation of Bcl-2 expression in HeLa cells treated with butein in a dose-dependent manner (Fig. 2C). Butein upregu-lated the Bax/Bcl-2 ratio in HeLa cells in a dose-dependent manner (Fig. 2D), which triggered the release of cytochrome c from the mitochondria and stimulated pro-caspase-3, thereby promoting cell apoptosis.

To examine the contribution of caspases in the butein-induced apoptosis, the role of caspase-3, -8 and -9 was investigated after treatment with different concentration butein (0, 15, 25 and 50 μM). It was found that treatment with butein resulted in a significant increase in the activities of caspase-3, -8 and -9 of HeLa cells in a dose-dependent manner (P<0.05, Fig. 3A–C).

To investigate the effect of butein on migration and invasion of HeLa cells, the cells were seeded on Transwell with uncoated (for migration) or Matrigel-coated (for invasion) filters. After 48 h of treatment, we examined the migration activity and invasive potential of HeLa cells, and found that butein at 15, 25 or 50 μM significantly inhibited the migration and invasion of HeLa cells (Fig. 4A and B). These results suggested that butein suppressed the migration and invasion of HeLa cells in a dose-dependent manner.

To determine the potential mechanism of butein effect on cell migration and invasion, MMP-2 and MMP-9 expression was determined by western blotting following treatment with butein. It was found that butein significantly reduced MMP-2 and MMP-9 expression of HeLa cells in a dose-dependent manner (Fig. 4C and D).

Previous findings have shown that generation of ROS plays an important role in regulating cell apoptosis and cell cycle (27,32). To determine whether butein affected the ROS level, ROS generation was determined using the DCFDA staining method following treatment with the indicated concentrations of butein. As shown in Fig. 5A, treatment with butein significantly induced ROS generation in HeLa cells in a dose-dependent manner. ROS generation was reduced by pretreatment with the antioxidant agent NAC (10 mM) (Fig. 5A).

It is well known that the PI3K/AKT/mTOR signaling pathways play keys role in cell proliferation, survival, motility and angiogenesis. We evaluated the effect of butein on this signaling pathway. Measurements of the phosphorylation/activation pattern of PI3K, AKT and mTOR were carried out by western blotting 24 h after treatment with the indicated concentration of butein. Treatment of HeLa cells with butein caused a dose-dependent inhibition in the expression of phosphorylated PI3K, AKT and mTOR, while the total protein levels of PI3K, AKT and mTOR in different concentrations of butein group were not altered (Fig. 5B).

We performed a HeLa xenograft mouse study to test the effectiveness of butein in preventing tumor growth. Results showed that treatment with butein (5 mg/kg) on alternate days for 4 weeks caused a significant inhibition of HeLa tumor xenograft growth in the butein treatment group compared to the control group (untreated group) (Fig. 6A–C). We did not observe any gross signs of toxicity and/or possible side-effects/mortality during the butein treatment relative to the control group, suggesting butein is a safe anticancer agent.

In addition, we determined cell apoptosis of tumor tissue by TUNEL. The results of TUNEL showed that butein obviously induced the cell apoptosis of tumor tissue compared to the control group (Fig. 6D). These data suggested that butein treatment suppressed the tumor growth of cervical cancer in vivo.