The PANC-1 human pancreatic cancer cell line and MDA-MB-231 and MCF-7 breast cancer cell lines, obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) containing penicillin (50 U/ml), streptomycin (50 U/ml; all Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and 10% fetal bovine serum (FBS) from ATCC. DMSO was purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Anti-CDC20 (cat. no. sc-13162) and anti-β-actin (cat. no. sc-47778) antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-FLAG M2 antibodies were from Sigma-Aldrich (Merck KGaA; cat. no. F3165).

Triterpenes were prepared from the pulverized sclerotium of P. cocos (Fujian, China). The method of preparation and identification follows as reported in our previous study (27). The quantification of HPLC analysis demonstrated that PTE contained 55.7% pachymic acid (PA), 31.7% DPA and 4.1% PPAC. The PTE, PA, DPA and PPAC were dissolved in DMSO at a concentration of 50 mg/ml and 50 mM, respectively and then stored at −20°C.

The PANC-1 or MDA-MB-231 cells were transfected with human CDC20 siRNA (cat. no. sc-156154) or control siRNA-A (cat. no. sc-37007) using siRNA Transfection Reagent (cat. no. sc-29528) from Santa Cruz Biotechnology, Inc. as previously described (27). CDC20 siRNA was used at 0.2 µM and control siRNA-A was used at 0.9 µM. After 48 h at 37°C of transfection, the cells were harvested and CDC20 knockdown was verified by western blot analysis.

The FLAG-tagged plasmid DNA with CDC20 and FLAG-tagged plasmid DNA were provided by Professor Michele Pagano (NYU School of Medicine, New York, NY, USA). The PANC-1 or MCF-7 cells were transiently transfected with FLAG-tagged plasmid DNA with CDC20 or control FLAG-tagged plasmid DNA using Lipofectamine™ LTX Reagent according to the manufacturer’s instructions (Invitrogen, Thermo Fisher Scientific, Inc.). After 24 h of trans fection, the cells were harvested and the overexpression of CDC20 was verified by western blot analysis.

The sub-confluent (80-90%) PANC-1 cells were treated with PTE (30 and 60 µg/ml), PA (30 and 60 µM), DPA (30 and 60 µM), or PPAC (30 and 60 µM) for 24 h at 37°C. Whole protein extracts isolated from cells were prepared using radioimmunoprecipitation assay buffer (Sigma-Aldrich; Merck KGaA). A total of 25 µg protein per lane was separated on gels and transferred to a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was blocked with 5% bovine serum albumin (cat. no. A7906; Sigma-Aldrich; Merck KGaA) in Tris-buffered saline buffer with Tween-20 (TBTS) buffer for 1 h at room temperature, followed by the incubation of the anti-CDC20, anti-FLAG and anti-β-actin antibodies (diluted 1:100) overnight at 4°C as previously described (28). The PVDF membrane was then incubated with horseradish peroxidase-conjugated anti-mouse secondary antibodies (cat. no. NA-931; diluted 1:5,000; Amersham Biosciences, Buckinghamshire, UK) for 1 h at room temperature and protein expression was visualized by the ECL Plus Western Blotting Detection system (Amersham Biosciences). The western blots were scanned with HP Scanjet 5470c scanner (Hewlett Packard, Palo Alto, CA, USA) and the optical densities of proteins were quantified with UN-SCAN-IT software (version 7.0; Silk Scientific, Inc., Orem, UT, USA).

Cell migration of the PANC-1 cells treated with PTE (30 and 60 µg/ml) or PPAC (30 and 60 µM) was assessed in Transwell chambers according to an established method (29). The PANC-1 cells (0.2×10⁶) suspended in serum-free medium were added to the upper chamber of an insert, and the insert was placed in a 24-well plate containing medium with 10% FBS. The migration assays were performed for 24 h at 37°C. Data points represent the mean ± standard deviation (SD) of three individual filters within one representative experiment repeated at least twice. The changes in cell migration in the different cell lines were examined. For the metastatic MDA-MB-231 breast cancer cell line, migration can be evaluated following 3 h of incubation. For the migration of the non-metastatic MCF-7 human breast cancer cell line, incubation for 24 h is necessary (29).

Cell viability was determined following incubation with PTE (30 and 60 µg/ml) or PPAC (30 and 60 µM) for 24 h by staining with Trypan blue (0.4%) at 22°C for 5 min. The cells were then viewed using an inverted light microscope at a magnification of ×40, as previously described (30). This method is used to assess cytotoxicity in a variety of cell lines. Data are presented as the mean ± SD from three independent experiments.

All statistical analyses were performed using GraphPad Prism 5 (GraphPad Software, Inc., San Diego, CA, USA). Data are presented as the mean ± SD. Statistical comparisons were performed using one-way analysis of variance with the significance level adjusted to P<0.05 using repeated t-tests with the Bonferroni correction.

To evaluate whether CDC20 was involved in the process of cancer metastasis, CDC20 was silenced with siRNA as described above. As shown in Fig. 1A and B, the knockdown of CDC20 effectively suppressed the migration of chemoresistant PANC-1 pancreatic cancer cells by >40%. In the metastatic MDA-MB-231 breast cancer cell line, ~40% of cell migration was significantly inhibited by CDC20 knockdown (Fig. 1C and D). These results indicate that CDC20 is an important target of cancer metastasis. However, the use of additional siRNAs is planned in future investigations to assess whether the inhibitory effects of siRNA CDC20 are not caused by the off-target effect.

In order to gain further insight into the functional role of CDC20 associated with cancer metastasis, FLAG-tagged plasmid DNA with CDC20 was transfected into the PANC-1 cell line and MCF-7 breast cancer cell line, respectively, to induce the overexpression of CDC20. Induction of the expression of CDC20 markedly promoted the migration of PANC-1 and MCF-7 cells compared with the control (Fig. 2A-D). By contrast, transfection with FLAG-tagged plasmid DNA had no effect on cell migration, showing that promotion of the metastasizing capacities of pancreatic and breast cancer was closely associated with CDC20. The use of a mutant CDC20 expression vector is planned in future investigations to further confirm the crucial role of CDC20 in cell migration.

As it is suggested that CDC20 serves an important role in cancer metastasis, the present study aimed to identify novel CDC20 inhibitors from natural compounds. The PANC-1 cells were treated with PTE (30 and 60 µg/ml), a triterpene mixture extracted from P. cocos, or three triterpenes PA, DPA and PPAC (30 and 60 µM), which were purified from PTE, for 24 h. Western blot analysis was then performed. As shown in Fig. 3, PTE suppressed the expression of CDC20 in PANC-1 cells dose-dependently. Among the purified triterpenes, PPAC was the most effective compound in downregulating the expression of CDC20, and DPA exerted moderate inhibition at a high dose. However, PA had no effect on the expression of CDC20. Different experimental methods resulted in different expression levels of CDC20 in the control PANC-1 cells. In Fig. 1, the endogenous expression of CDC20 was determined by CDC20 antibody. In Fig. 2, the overexpression of CDC20 was determined by FLAG antibody in cells which were transiently transfected with FLAG-CDC20 plasmid DNA. Therefore, the same cell line can exhibit different expression levels of CDC20.

To determine whether the suppression of CDC20 by triterpenes from P. cocos is associated with the metastasizing capacities in chemoresistant pancreatic cancer, the PANC-1 cells were treated with PTE (30 and 60 µg/ml) or PPAC (30 and 60 µM) for 24 h and cell migration was evaluated, as described above. In accordance with expectations, PTE (Fig. 4A and B) and PPAC (Fig. 5A and B) significantly inhibited the migration of the chemoresistant PANC-1 pancreatic cancer cells in a dose-dependent manner. The inhibition of cell migration was not caused by the toxic effects of the tested triterpenes, since PTE or PPAC did not affect the viability of PANC-1 cells (Figs. 4C and 5C). These results further demonstrated that triterpenes from P. cocos inhibited the migration of pancreatic cancer associated with CDC20. Although cell motility analysis in Transwell chambers is an established method to asses cell migration, a scratch assay and animal experiments will be performed in future experiments to verify these results in vitro and in vivo and to reduce the limitation of the present study.