Casticin was purchased from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China). Casticin has a molecular weight of 374.3, appears as yellow crystals and has a purity of 98.0%. Casticin was prepared in dimethylsulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) as a 10-mmol/l stock solution and diluted in medium to the indicated concentration prior to use. Mouse monoclonal antibodies against FOXM1, survivin and β-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Mouse anti-human monoclonal antibodies FOXO3a and phospho-FOXO3a-Thr32 were purchased from Millipore (Bedford, MA, USA). The horseradish peroxidase-conjugated goat anti-mouse secondary antibody was purchased from Santa Cruz Biotechnology, Inc. Lipofectamine™ 2000 was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). The protease inhibitor cocktail, MTT, and all other chemicals were obtained from Sigma-Aldrich.

The MDA-MB-231 and MCF-7 cell lines were purchased from the China Centre for Type Culture Collection (Wuhan, China) and were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen Life Technologies) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 4 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. The cells were incubated at 37°C in a humidified atmosphere of 5% CO₂.

The cell apoptosis ELISA detection kit (Roche, Palo Alto, CA, USA) was used to detect apoptosis in cells treated with casticin according to the manufacturer’s instructions. Briefly, cells were seeded in 96-well plates at a density of 1×10⁴ cells/well. When cells reached 70–80% confluence, testing agents were added to the culture medium containing 10% FBS. After 48 h of culture, the cytoplasm of the cells that was extracted from the control or treatment groups was transferred to 96-well plates, which were pre-coated with streptavidin and previously incubated with a biotinylated mouse anti-histone monoclonal antibody and peroxidase-tagged mouse anti-human DNA monoclonal antibody for 2 h at room temperature. The absorbance was measured at 405 nm under the EXL-800-type enzyme-linked immunosorbent apparatus (Bio-Tek, Winchester, VA, USA).

The cells were seeded at a density of 4×10⁶ cells/well in 100 ml culture flasks for 24 h and then treated with various concentrations (0.1, 0.5 and 1.0 μM) of casticin for 48 h. PI staining for DNA content was performed as described previously (22). Briefly, the cells were collected and prepared as a single cell suspension by mechanical blowing with PBS (Hyclone), washed twice with cold PBS, fixed with 700 ml/l alcohol at 4°C for 24 h, stained with PI (Sigma-Aldrich) and cell apoptosis was detected using flow cytometry (FACS420, BD Biosciences, Franklin Lakes, NJ, USA).

The cells were seeded at a density of 4×10⁶ cells/well in 250 ml culture flasks for 48 h and then treated with DMEM containing various concentrations (0.1, 0.5 and 1.0 μM) of casticin or DMSO and 10% FBS for 24 h. The assay was performed as previously described (22). Briefly, cells were washed twice with PBS and DNA was extracted with Apoptotic DNA Ladder Detection kit (Bodataike Company, Beijing, China) according to the manufacturer’s instructions. Extracted DNA was maintained at 4°C overnight. Subsequently, 8.5 μl of the DNA sample was combined with 1.5 μl of 6× buffer solution (New England Biolabs Inc., Ipswich, MA, USA), electrophoresed on 20 g/l agarose gel containing ethidium bromide (BBI Solutions, Madison, WI, USA) at 40 V, and observed using the DBT-08 gel image analysis system (VWR International Ltd., East Grinstead, UK).

Control non-specific small interfering RNA (siRNA; 5′-UUCUCCGAACGUGUCACGUdTdT-3′) was purchased from Qiagen, Inc. (Valencia, CA, USA). FOXO3A-targeted siRNA (5′-ACUCCGGGUCCAGCUC CAC-3′) was purchased from Santa Cruz Biotechnology, Inc. The cells were seeded in six-well plates and transfected at 50% confluence with either 200 nmol/l of control non-specific siRNA or FOXO3a-specific siRNA using Oligofectamine™ reagent (Invitrogen Life Technologies) according to the manufacturer’s instructions. After 24 h of transfection, the cells were treated with DMSO (control) or 0.5 μM casticin for 48 h. The cells were then collected and processed for western blotting and histone/DNA ELISA.

The cells (1×10⁶) were seeded in 100-mm culture dishes, allowed to attach by overnight incubation and treated with DMSO (control) or 0.5 μM casticin for the specified time periods. Cell lysates were prepared as previously described (22). Lysates were cleared by centrifugation at 16,873 × g for 30 min. Lysate proteins were resolved by 10 or 12.5% SDS-PAGE (Millipore) and transferred to polyvinylidene fluoride membranes. The membranes were incubated with Tris-buffered saline containing 0.05% Tween 20 and 5% (w/v) non-fat dry milk. The membranes were then treated with the desired primary antibody for 1 h at room temperature or overnight at 4°C. Following treatment with the horseradish peroxidase-conjugated goat anti-mouse secondary antibody the immunoreactive bands were visualized using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Piscataway, USA). The blots were stripped and re-probed with anti-actin antibody to normalize for differences in protein loading. Changes in the level of the desired protein were determined by densitometric scanning of the immunoreactive band and corrected for the β-actin loading control. Immunoblotting for each protein was performed at least twice using independently prepared lysates to ensure reproducibility of the results.

The data were analyzed using SPSS software, version 15.0 (SPSS Inc., Chicago, IL, USA). Data are expressed as the means ± standard deviation. The means of multiple groups were compared with one-way analysis of variance, after the equal check of variance, and the comparisons among the means were performed using the least significant difference method. Statistical comparison was also performed with Dunnett’s two-tailed t-test when appropriate. P<0.05 was considered to indicate a statistically significant difference.

It has previously been reported that casticin inhibits the growth of MCF-7 human breast cancer cells and induces G2/M cell cycle arrest (9). Thus, whether casticin exerts any effect on apoptosis of the estrogen-responsive MCF-7 or the estrogen-independent MDA-MB-231 breast cancer cell lines was investigated. ER-positive MCF-7 cells were originally isolated from pleural effusion of a stage IV invasive ductal carcinoma. These cells are aneuploid with high chromosomal instability and are defective for the G1 and mitotic spindle checkpoints. However, the cells express wild-type p53 (23). The MDA-MB-231 cell line, which was derived from a stage IV invasive ductal carcinoma, is ER-negative, partially proficient for all cell cycle checkpoints and expresses mutant p53 (23).

After 48 h of exposure, casticin significantly induced histone/DNA fragmentation in a concentration-dependent manner in MCF-7 (Fig. 1A) and MDA-MB-231 cells (Fig. 1B). Agarose gel electrophoresis revealed a typical ladder pattern of internucleosomal DNA fragmentation in MDA-MB-231 cells treated with 0.5 and 1.0 μM casticin (Fig. 1C). Flow cytometry analysis showed that casticin treatment resulted in increased sub-G1 population in MCF-7 (Fig. 1D and F) and MDA-MB-231 (Fig. 1E and G) cells (P<0.05) in a concentration-dependent manner. Overall, these findings suggest that casticin induces breast cancer cell apoptosis.

Previous research, including a study by Wang et al (24), has demonstrated that FOXM1 is a novel target of natural active compounds (24,25). Thus, whether FOXM1 is a downstream signaling target of casticin in breast cancer cells was investigated. Dose titration of casticin in the MCF-7 and MDA-MB-231 cell lines was performed, and the effects on FOXM1 and its downstream target survivin were assayed (Fig. 2A and B). A dose of 0.5 μM was selected for subsequent experiments. The MCF-7 and MDA-MB-231 cells were then treated with 0.5 μM casticin for 0, 6, 12 and 24 h. Western blot analysis revealed that casticin treatment decreased FOXM1 expression and this coincided with a decrease in the FOXM1 target, survivin (Fig. 2C and D). Collectively, these findings suggest that FOXM1 is a cellular target of casticin in breast cancer cells.

FOXO3a is an upstream regulator of FOXM1. Additionally, the antiproliferative and apoptotic effects of genistein, an isoflavone derived from soybeans, were partly mediated through the regulation of Akt/FOXO3a signaling (26). Thus, phosphorylated FOXO3a protein was examined in order to determine whether differences in the expression or activity of signaling regulators may enhance the effect of casticin on FOXM1. Western blot analysis revealed that treatment with casticin led to a decrease in FOXO3a phosphorylation and a corresponding reduction in FOXM1 and its target, survivin (Fig. 3A and B). These findings suggest that the casticin-induced repression of FOXM1 may be associated with FOXO3a activation.

In order to determine the importance of FOXO3a in the cellular response to casticin, the MCF-7 and MDA-MB-231 cells, which express high protein levels of FOXO3a, were transfected with specific siRNAs. As shown in Fig. 4A and B, FOXM1 and survivin proteins were increased in FOXO3a-knockdown cells. The decrease of FOXO3a significantly attenuated the apoptotic effects of casticin in breast cancer cells (Fig. 4C and D). These findings support the hypothesis that casticin induces breast cancer cell apoptosis by inducing FOXO3a activity, which represses FOXM1.