Shi was purchased from the Shanghai Technology Institute of Yuanye. The chemical structure of Shi is shown in Fig. 1.

The colon cancer cell lines, HCT116 and SW620, were purchased from the American Type Culture Collection. HCT116 and SW620 cells were cultured in McCoy's 5A and L-15 medium, respectively, containing 10% FBS (HyClone; GE Healthcare Life Sciences), 100 U/ml penicillin and 100 mg/ml streptomycin (both Amresco; VWR International, LLC). All cells were cultured at 37°C in a humidified atmosphere with 5% CO₂.

Both HCT116 and SW620 cells (8×10³ cells/well) were seeded in 96-well plates. Shi (0, 1, 2.5, 5, 7.5 and 10 µM) was added to the cells at the different concentrations stated and incubated for 24 and 48 h. Subsequently, MTT solution (10 mg/ml) was added. Cells were cultured for a further 2 h at 37°C, and 100 µl DMSO was added. The optical density of each well was measured at 570 nm and the inhibition rate was calculated using the following equation: Inhibition rate (%)=(average A570 of the control group-average A570 of the experimental group)/(average A570 of the control group-average A570 of the blank group) ×100. All MTT assays were repeated at least three times. The control group was the cells treated with DMSO alone, and the blank group was the wells without cells added.

HCT116 and SW620 cells were cultured in a 6-well plate at a density of 1×10³ cells/well, and treated with Shi (0, 5 or 10 µM). After three weeks, cells were stained with crystal violet staining solution for 30 min at room temperature (Beyotime Institute of Biotechnology) and the visible colonies were counted under an optical light microscope at ×4 magnification (IX70; Olympus Corporation).

HCT116 cells were cultured in McCoy's 5A medium and SW620 cells were cultured in L-15 without FBS. The cells were cultured in 6-well plates and grown to 100% confluence. A 200 µl pipette was used to create the wound, then PBS was used to wash the cell debris, and serum-free medium was added. Cells were treated with 10 µM Shi, and wound closure was observed using an inverted light microscope at a ×4 magnification imaged using a digital camera. The extent of wound healing is defined as the ratio of the difference of wound area. All the experiments were repeated three times.

The HCT116 and SW620 cells (3×10⁵ cells/well) were grown in 6-well plates, treated with various concentrations of Shi and transfected with YAP cDNA or YAP small interfering (si)RNA or empty vector using lipofectamine^® 3000 according to the manufacturer's protocol. YAP siRNA oligonucleotides were purchased from GenePharma (Shanghai, China) and the sequences were; forward, 5′-GGUGAUACUAUCAACCAAATT-3′; and reverse, 5′-UUUGGUUGAUAGUAUCACCTT-3′. After 48 h of transfection with the lentiviral vectors (Biofeng, Guangzhou, China), successfully transfected cell lines were selected for using puromycin (Santa Cruz Biotechnology, Inc.) for 21 days.

Cells were harvested using RIPA lysis buffer (Beyotime Institute of Biotechnology) and protein concentration was determined using a bicinchoninic acid assay kit (Beyotime Institute of Biotechnology). Soluble proteins were extracted from the cell lysate using 150 mM NaCl, 50 mM Tris, pH 8.0, 30% Acrylamide-Bis-acrylamide, 10% SDS, 1 mM PMSF, 1 mM sodium vanadate (Beyotime, Shanghai, China). A total of 25 µg protein were loaded into 10 or 15% SDS gels and resolved using SDS-PAGE. Subsequently, resolved proteins were transferred to PVDF membranes (EMD Millipore) and blocked in 5% skimmed milk for 2 h at 37°C. The membranes were incubated with primary antibodies at 4°C overnight and then washed with TBS-Tween-20. The primary antibodies used were: Anti-GAPDH (1:1,000; cat. no. AF0006; Beyotime Institute of Biotechnology); anti-LC3 (1:1,000; cat. no. 3868T; CST Biological Reagents Co., Ltd.); anti-P62 (1:1,000; cat. no. no. 5114T; CST Biological Reagents Co., Ltd.); anti-YAP (1:1,000; cat. no. 14074; CST Biological Reagents Co., Ltd.), anti-phospho-(p-)YAP (S127) (1:1,000; cat. no. 4911; CST Biological Reagents Co., Ltd.). Subsequently, membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (1:5,000; cat. nos. E030120-01 and E030110-01; EarthOx Life Sciences) at room temperature for 1 h. Signals were visualized using a SuperSignal^® West Pico Trial kit (Pierce; Thermo Fisher Scientific, Inc.). ImageJ 1.43U (National Institutes of Health) was used for densitometry analysis.

In the present study, all experiments were repeated at least three times and the results were analyzed using GraphPad Prism v5.0 (GraphPad Software, Inc.) and SPSS v17.0 (SPSS, Inc.). Data are presented as the mean ± standard deviation. Results were analyzed using an unpaired Student's t-test or one-way ANOVA followed by a Bonferroni's post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

Shi is a natural plant extract which exhibits antiproliferative effects on cancer cell growth (8). The chemical structure of Shi is shown in Fig. 1. The effect of Shi on HCT116 and SW620 cells was investigated using an MTT assay, and the results showed that as Shi concentration or treatment time was increased, cell viability of HCT116 and SW620 cells appeared to be reduced (Fig. 2A and B). Colony formation assays showed a significant decrease in colony formation following Shi treatment and this appeared to be dose-dependent (Fig. 2C and D). Subsequently wound-healing assays were performed to examine the effects of Shi on colon cancer cells migration. When the cell density reached 100%, wounds were created with a pipette tip, and 10 µM Shi was added. After 24 h, wound closure in the HCT116 and SW620 cells was ~35 and 55%, respectively. Shi treatment significantly slowed wound closure in HCT116 (50%) and SW620 (80%) cells compared with the respective control (Fig. 2E and F). All these results showed that Shi exhibited cytotoxic effects on HCT116 and SW620 cells.

Increasing evidence suggests that autophagy is an important mechanism of death in cancer cells and may be of importance in the development of tumors (11). To evaluate whether autophagy was involved in Shi-induced cell death, the expression levels of LC3-II and p62 were evaluated in Shi-treated cells. HCT116 and SW620 cells were treated with 0, 2.5, 5 or 10 µM Shi for 24 h, or with 10 µM Shi for 0, 6, 12 or 24 h. After autophagy begins, the C-terminal LC3 is cleaved to produce the LC3II protein, which then translocates to autophagosomes (12). The expression of LC3-II was detected using western blot analysis and the results suggested that increasing Shi concentrations resulted in significantly upregulated LC3-II expression (Fig. 3A-D). The degradation of p62 (also known as sequestosome 1) is considered an accurate indicator of autophagy. Therefore, the expression of p62 in colon cancer cells treated with Shi was investigated. As the concentration of Shi used or the treatment time was increased, degradation of P62 became more notable in HCT116 and SW620 cells and the difference was significant compared with the control group (P<0.05) (Fig. 3A-D). In conclusion, these findings suggest that Shi induced autophagy in colon cancer cells. In addition, the changes in expression of YAP was also studied. The expression of YAP in HCT116 and SW620 cells treated with Shi was downregulated as the concentration or treatment with Shi was increased, and the difference was significant compared with the control group (P<0.05; Fig. 3E-H). Meanwhile, the serine¹²⁷ phosphorylation of YAP in HCT116 and SW620 cells was also detected (Fig. 3E-H), suggesting that Shi could upregulate YAP phosphorylation. All these data concluded that Shi decreased the protein expression of YAP.

To detect whether YAP serves a critical role in the inhibitory effect of Shi on colon cancer, HCT116 and SW620 cells were transfected with the YAP-specific cDNA and subsequently treated with 10 µM Shi for 24 h (Fig. 4A and B). The results showed that overexpression of YAP promoted colon cancer cell growth and reversed the inhibition of Shi on cell growth to a certain extent. In addition, as shown in the Fig. 4C and D, YAP overexpression decreased the percentage of autophagy in HCT116 cells and SW620 cells. Additionally, LC3II protein expression levels were increased and p62 levels were decreased (Fig. 4C and D) in both cell lines. Densitometry analysis of LC3-II, P62 and YAP expression levels are shown in Fig. 4E-H. These results showed that the anticancer activity of Shi is partly caused by the inactivation of YAP.

To investigate whether the cytotoxicity of Shi is related to the regulation of YAP, viability assays were performed in HCT116 and SW620 cells. The HCT116 and SW620 cells were transfected with YAP-specific siRNA. The results show that downregulation of YAP decreased cell viability compared with the control group and caused colon cancer cells to become more sensitive to Shi. Shi and YAP siRNA only compared with control group and YAP siRNA + Shi group compared with YAP siRNA group (Fig. 5A and B). Moreover, it was found that the expression of LC3-II increased and expression of p62 decreased in the Shi and YAP siRNA only group compared with the control group, and in the YAP siRNA + Shi group compared with YAP siRNA group (Fig. 5C and D). Therefore Shi and YAP siRNA showed stronger induction of autophagy compared with siRNA transfection or Shi alone. Densitometry analysis of LC3-II, p62 and YAP expression levels are presented in Fig. 5E-H. The results showed that the synergistic effects of YAP-specific siRNA and Shi increased Shi-induced autophagy in colon cancer cells.