Human PC cell lines Panc-1 and Bxpc-3 were purchased from American Type Culture Collection (ATCC; Rockville, MD, USA). The Panc-1 cells were cultured in Dulbecco's modified Eagle's medium (Gibco, Rockville, MD, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin G and 100 U/ml streptomycin in a humidified incubator containing 5% CO2 in air at 37°C. The Bxpc-3 cells were cultured in RPMI-1640 medium (Gibco) containing supplements as above. Deguelin was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO) to make 0.1 mmol/l stock solution and stored at −20°C. The final concentration of DMSO was <0.1%.

Cell growth-inhibitory curves of 7 days were observed by Cell Counting Kit-8 (CCK-8; Mai Bio, Ltd. Shanghai, China) assay. Briefly, cells were plated at a density of 2×103 cells/well in 96-well microtiter plates. Following treatment, 20 µl CCK-8 solution was added to each well and further incubated for 1–3 h. Then absorbance was measured with an absorbance reader (Bio-Tek ELx800, Winooski, VT, USA) at a wavelength of 450 nm.

The Annexin V-FITC apoptosis detection kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) was used for flow cytometric analysis of apoptosis. Cells were treated with deguelin for 48 h on a six-well plate, then 5×105 cells were harvested and washed twice with phosphate-buffered saline, then resuspended in 500 µl binding buffer. Annexin V-FITC (5 µl) and propidium iodide were added to each sample and incubated for 15 min in the dark. The stained cells were analyzed directly by flow cytometry using the CellQuest program (Becton Dickinson, San Jose, CA, USA).

Cell migration in vitro was assessed by wound healing assay. The culture insert (Ibidi, Martinsried, Germany) was placed in a clean and dry six-well plate in advance. Suspended cells (70 µl) were loaded at a density of 6×105/ml. When a confluent layer was formed, medium with 0.5% FBS was added the well after the culture insert was removed. Acquired images at 0, 12 and 24 h of follow-up incubation were analyzed online at wimasis.com.

Transwell migration assays were performed using polycarbonate membrane Transwell inserts (Corning Incorporated, Corning, NY, USA) with an 8.0-µm pore size and 6.5-mm membrane diameter on 24-well plates. The lower chambers were filled with 600 µl medium (10% FBS), and the upper chambers were loaded with cells at a density of 1×105 cells/well) in 200 µl medium (2% FBS). Following incubation for 24 h, the membranes were fixed in 4% paraformaldehyde for 20 min and stained with hematoxylin and eosin for 10 min, respectively. Cells on the upper surface of the membranes were removed with cotton swabs. Cells adhered to the lower surface were observed under the microscope.

Cells were lysed with RIPA buffer (Sigma-Aldrich) supplemented with a complete protease inhibitor cocktail (Roche, Basel, Switzerland). Following centrifugation at 13,000 × g for 30 min, the supernatant was collected and protein concentration was determined using a bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL, USA). Protein (40 µg) was separated in 8–10% sodium dodecyl sulphate-polyacrylamide gel and transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA). The transblotted membranes were blocked in Tris-buffered saline with Tween-20 (10 mM Tris-HCL pH 7.4, 150 mM NaCl and 0.1% Tween-20) with 5% skimmed milk for 1.5 h at room temperature, then incubated with primary antibodies overnight at 4°C, followed by horseradish peroxidase-conjugated secondary antibodies. The immunoreactive bands were visualized using enhanced chemiluminescence (Biological Industries, Beit Haemek, Israel) and exposed to X-ray films. All the antibodies were procured from Cell Signaling Technology, Inc. (Danvers, MA, USA).

The results were expressed as the means ± standard error. All experiments were performed in triplicate. The one-way ANOVA or two-tailed Student's t-test were used to determine the statistical significance between two unpaired groups. P<0.05 was considered to indicate a statistically significant difference. Analyses were performed using SPSS 13.0 statistical software package (SPSS Inc., Chicago, IL, USA).

To assess the effects of deguelin on PC cell growth, Bxpc-3 and Panc-1 cells were treated with deguelin at increasing concentrations (0–20 µM) for a week. Deguelin inhibited the growth of the two cell lines in a dose- and time-dependent manner under no interference of the solvent (DMSO; Fig. 1A and B). These results indicate that deguelin was an effective inhibitor of PC cell proliferation, and Bxpc-3 cells were more sensitive to deguelin compared with Panc-1 cells.

We subsequently investigated whether deguelin induced apoptosis in PC cells. Flow cytometry revealed that treatment with deguelin for 48 h induced apoptosis in the two cell lines. Exposure to deguelin (5 µM) induced apoptosis by up to 18.1% in Bxpc-3 and 15.1% in Panc-1 cells. The higher concentration (10 µM) induced more apoptosis (36.1% in Bxpc-3, 23.1% in Panc-1 cells) (Fig. 2A). Western blot analysis demonstrated that the expression of two apoptosis-related proteins, cleaved caspase-3 and PARP, significantly increased with the concentration of deguelin (Fig. 2B). These data suggested that deguelin induces apoptosis through the activation of caspase-3 and the cleavage of PARP.

To verify whether deguelin inhibits the migration and invasion ability of PC cells, wound healing assay and Transwell cell invasion assay were performed. The data from wimasis.com demonstrated that the cell-covered area of deguelin-treated cells was smaller than that of untreated cells after 12 and 24 h (Fig. 3A and B).

The results of the Transwell assay revealed that cell invasion in the deguelin-treated group was notably decreased compared with that in the control group (Fig. 3C and D). The results suggested that deguelin inhibited the invasion and migration capability of the two PC cell lines.

In order to verify our theory on the mechanism of deguelin influencing PC, the expression levels of MMP-2 and MMP-9 were measured. Bxpc-3 and Panc-1 cells were treated with various concentrations of deguelin for 48 h, and then total protein was extracted for western blot analysis. We noted that the levels of MMP-2 and MMP-9 protein were gradually decreased with the increase in drug concentration (Fig. 4A). The results revealed that deguelin inhibits metastasis in PC by suppressing the activation of MMP-2 and MMP-9.

To further investigate whether deguelin regulates PC through Hh signaling, we sought to examine the protein expression levels of several key members of this pathway. The expression of Gli1 was downregulated by the treatment of deguelin in Bxpc-3 and Panc-1 cells. Conversely, the expression of SUFU was upregulated. PTCH1 and PTCH2 were able to suppress Hh earlier than SUFU, and their levels were increased in line with our expectations. Furthermore, as the ligand located at the starting point of the Hh signaling pathway, Shh is important for the regulation of Hh. The spliceosome of Shh was increased following treatment with deguelin (Fig. 4B). In sumary, the results regarding the change of ligands in Hh indicated that deguelin inhibited the Hh signaling pathway.