Human ovarian cancer SKOV3 cells were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China), and cultured in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin, and 100 U/ml streptomycin (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA), in a humidified atmosphere containing 5% CO₂ at 37°C. Casticin (purity ≥98%) was manufactured by Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China) as yellow crystals (molecular weight, 374.3 Da). Cyclopamine was manufactured by Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Rabbit anti-Gli-1 antibody (1:1,000; cat. no. 2534) and Twist-related protein 1 (Twist1) antibodies (1:1,000; cat. no. 46702) (both from Cell Signaling Technology, Inc., Danvers, MA, USA), and rat anti-E-cadherin (1:1,000; cat. no. SAB4503751) and mouse monoclonal anti-N-cadherin (1:1,000; cat. no. C2542) (both from Sigma-Aldrich; Merck KGaA) antibodies were used in the present study. Goat anti-rabbit lgG, horseradish peroxidase (HRP)-linked antibody (cat. no. 7074; 1:1,000 dilution) and goat anti-mouse lgG, HRP-linked antibody (cat. no. 7076; 1:1,000 dilution) were both purchased from Cell Signaling Technology, Inc.

Cell proliferation and growth were assessed by MTT and Soft agar assays. For MTT assay, SKOV3 cells (10,000/well) were seeded in 96-well plates and incubated for 24 h prior to treatment with increasing casticin (0.0, 1.0, 2.0, 4.0 and 8.0 µM) or cyclopamine amounts (0.0, 12.5, 25.0, 50.0 and 100.0 µM) for 48 h. Subsequently, 20 µl MTT (5 mg/ml) was added for further 4 h. Dimethyl sulfoxide was used to dissolve the resulting formazan crystals; absorbance was read at 490 nm on Bio-Rad 550 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The half-maximal inhibitory concentration (IC₅₀; causing 50% cell growth inhibition) values were determined. Cell growth inhibition rate was derived as: [(A_{490 control cells} - A_{490 treated cells})/A_{490 control cells}] × 100.

Soft agar assay was performed using SKOV3 cells (10,000/well) treated with or without casticin (2.0 µM) or cyclopamine (25.0 µM) for 48 h and then cells were plated in 0.35% agarose overlying a 0.6% agar layer. Cells were incubated at 37°C for 27 days; at the end of the experiment, colonies were counted under an inverted light microscope at a ×4 magnification (Olympus Corportation, Tokyo, Japan).

When SKOV3 cells reached an optimal confluence (80–90%) in a 6-well culture plate, the monolayer was gently and slowly scratched with a clean 100 µl pipette tip across the center of the plate. Following two washes with PBS, casticin (2.0 µM) or cyclopamine (25.0 µM) were added at IC₅₀ values. The artificial wounds were imaged at 0 and 24 h, respectively, under an inverted light microscope at a ×10 magnification (CKX41; Olympus Corporation, Tokyo, Japan).

For the invasion assay, 1×10⁵ SKOV3 cells treated with casticin or cyclopamine were seeded in the top chamber onto the Matrigel-coated membrane (24-well insert; pore size, 8 µm; Corning Life Sciences, Corning, NY, USA). Cells were plated in DMEM medium without serum or growth factors in the top chamber, and DMEM medium supplemented with 10% FBS was used in the lower chamber. The cells were incubated for 48 h and then cells that invaded via the pores were removed with a cotton swab. Cells on the lower surface of the membrane were fixed with 4% paraformaldehyde for 10 min at 25°C and stained with 10% Giemsa for 30 min at room temperature. The number of cells invading via the membrane was counted under a light microscope (magnification, ×20; three random fields per well).

SKOV3 Cells were treated with casticin (2.0 µM) or cyclopamine (25.0 µM) for 0, 3, 6, 12 and 24 h, then RNA was extracted and cDNA prepared as previously reported (29). RT-qPCR was conducted with 1 µl cDNA on MyiQ real-time PCR Instrument (Bio-Rad Laboratories, Inc.) using iQ SYBR Green PCR Supermix (Bio-Rad Laboratories, Inc.), following the manufacturer's instructions. The thermocycling conditions were as follows: 95°C for 3 min, 95°C for 15 sec, and 60°C for 20 sec, for 40 cycles. Relative gene expression levels were determined by the 2^−ΔΔCq method (30) with GAPDH as an internal reference. The following primers were used: Gli-1 forward, 5′-TTCCTACCAGAGTCCCAAGT-3′ and reverse, 5′-CCCTATGTGAAGCCCTATTT-3′; Twist1 forward, 5-CGGACAAGCTGAGCAAGATT-3′ and reverse, 5′-CCTTCTCTGGAAACAATGAC-3′; N-cadherin forward, 5′-CGTGAAGGTTTGCCAGTGTG-3′ and reverse, 5′-GCGTTCTTTATCCCGCCGTT-3′; E-cadherin forward, 5′-CCTCAGGTCATAAACATCATTG-3′ and reverse, 5′-CGCCTCCTTCTTCATCATAGTAA-3′; GAPDH forward, 5′-GGTGGTCTCCTCTGACTTCAACA-3′ and reverse, 5′-GTTGCTGTAGCCAAATTCGTTGT-3′.

SKOV3 cells (1×10⁶) treated with casticin or cyclopamine (at IC₅₀) for 48 h were lysed and analyzed by western blot, as previously reported (31), using the aforementioned antibodies; β-actin was employed as an internal control. Protein bands were visualized by enhanced chemiluminescence kit (GE Healthcare, Chicago, IL, USA), and visualized using Tanon-5500 chemiluminescence instrument (Tanon Inc., Shanghai, China). Data were quantified by ImageJ 1.49v software (National Institutes of Health, Bethesda, MD, USA).

Experiments were repeated ≥3 times, with data expressed as the mean ± standard deviation. All data were analyzed by SPSS 16.0 for Windows (SPSS Inc., Chicago, IL, USA). One-way analysis of variance was used to perform group comparisons, with a least significant difference test for post hoc multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

The effects of casticin and cyclopamine on SKOV3 cell viability were determined by the MTT and soft agar assay. The MTT assay revealed that casticin (Fig. 1A) or cyclopamine (Fig. 1B) inhibited SKOV3 cell growth in a dose-dependent manner. IC₅₀ values of casticin and cyclopamine were 2.18±0.37 and 27.61±2.16 µM, respectively. Thus, 2.0 µM casticin and 25 µM cyclopamine were used to perform the soft agar assay. As presented in Fig. 1C, the SKOV3 cells treated with casticin or cyclopamine formed ~100% fewer colonies than the control.

Wound-healing assays were performed to assess the effect of casticin and cyclopamine on the migration of SKOV3 cells. As presented in Fig. 2, treatment with casticin (2.0 µM) or cyclopamine (25.0 µM) resulted in wider wounds compared with the control group after 24 h of incubation. Subsequently, quantification was conducted, which revealed that the migration rate of the casticin (72%±1.73) and cyclopamine groups (53.7%±4.37) were significantly lower compared with the control (100%; P<0.05). To analyze whether casticin (2.0 µM) or cyclopamine (25.0 µM) affected cell invasion further, a transwell invasion assay was performed. The results demonstrated that there was a marked difference in the invasion rate of cells that underwent treatment with casticin (2.0 µM) or cyclopamine (25.0 µM; 28.8±7.1 or 35.8±4.4, respectively) compared with the control group (100%; data not shown). These findings indicated that casticin and cyclopamine inhibited the migration and invasion of the human ovarian cancer SKOV3 cell line.

To assess whether casticin and cyclopamine affect the mRNA expression of effectors of Hh signaling and EMT markers, SKOV3 cells were treated for 24 h with complete medium containing 2.0 µM casticin and 25.0 µM cyclopamine, respectively, and were analyzed by RT-qPCR. Compared with the control group, the mRNA expression levels of Twist1 and N-cadherin were markedly decreased (~32 and ~17%, respectively) in the casticin (2.0 µM) groups, and decreased by ~30 and ~38% in the cyclopamine (25.0 µM) groups (Fig. 3A-C). However, the mRNA expression levels of E-cadherin were increased ~4-fold for casticin-treated cells and ~2-fold for cyclopamine-treated cells (Fig. 3D). In addition, the mRNA expression levels of the Hh signaling effector, Gli-1, were also decreased by 35 and 26% in the casticin (2.0 µM) and cyclopamine (25.0 µM) groups, respectively. These results indicated the inhibitory effects of casticin and cyclopamine on Hh signaling and the EMT process.

Whether casticin and cyclopamine affect Hh signaling pathway effectors and EMT markers was investigated at the protein level. After 24 h of treatment, E-cadherin protein expression levels were markedly increased in the casticin (2.0 µM) and cyclopamine (25.0 µM) groups compared with in the control; however, the protein expression levels of Gli-1, Twist1 and N-cadherin were markedly reduced by casticin (2.0 µM) and cyclopamine (25.0 µM) treatments, respectively, in comparison with the control group (Fig. 4A). These findings indicated that casticin and cyclopamine suppressed Hh signaling and the EMT process.

To determine the time course of casticin activity on mRNA expression, SKOV3 cells were exposed to 2.0 µM casticin at 0, 3, 6, 12, and 24 h, and Gli-1, Twist1, E-cadherin, and N-cadherin mRNA expression levels were quantitated by RT-qPCR. Gli-1, Twist1 and N-cadherin mRNA expression levels were decreased as time progressed (Fig. 5A-C), with significant alterations in expression levels detected at 24 h, which were 19, 14 and 16% of the expression at 0 h, respectively. E-cadherin levels increased (Fig. 5D) as incubation time increased.

To evaluate the time course of casticin action on protein expression, Gli-1, Twist1, E-cadherin and N-cadherin protein expression levels were determined. The results of the present study were consistent with those of the RT-qPCR analysis: Time-dependent increases in E-cadherin protein levels, concomitant with reduced Gli-1, Twist1 and N-cadherin levels were observed (Fig. 6). Notably, the alterations were most pronounced at 24 h, indicating that casticin may inhibit EMT and Hh signaling to a greater degree over time.