The human pancreatic cancer PANC-1 cell line was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). PANC-1 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum (FBS), 100 µg/ml streptomycin and 100 U/ml penicillin (all from Invitrogen, Carlsbad, CA, USA). The PANC-1 cells were seeded on 6-well culture plates to 50–70% confluency in complete medium containing 5% FBS for 24 h, and then changed to serum-free medium for 24 h before treatment with SSBE (Lot no. 20101017; Xuancheng Baicao Plant Industry and Trade Co., Ltd., Anhui, China). The extraction protocol of SSB was carried out according to a previous study (16).

Viability of the PANC-1 cells treated with SSBE were measured using the CCK-8 cell proliferation and cytotoxicity assay kit (Dojindo, Shanghai, China) according to the manufacturer's instructions. At the indicated time points, 10 µl of this reagent at different concentrations was added to each well containing 100 µl of the cell suspension (5×10³ cells) and incubated for an additional 1 h. The absorbance at 450 nm was monitored, and the percent viability of the cells was calculated by comparison to that of the untreated control cells. All the experiments were repeated at least three times.

The PANC-1 cells treated with SSBE were seeded for 24 h, and then were collected by centrifugation. For apoptosis analysis, resuspended cells were incubated with Annexin V-FITC and propidium iodide (PI) at room temperature for 5 min in the dark. Analysis of Annexin V-FITC binding by flow cytometery (Ex, 488 nm; Em, 530 nm; BD FACSVerse™) (BD Biosciences, Frankin Lakes, NJ, USA) was accomplished using FITC signal detector and PI staining by the phycoerythrin emission signal detector. For cell cycle analysis, resuspended cells were fixed in 70% ethanol at 4°C overnight and permeated in 0.1% Triton X-100 at 4°C for 30 min. After incubating with PI at 4°C for 30 min, PCCs were analyzed using flow cytometry and the proportion of nuclei in each phase of the cell cycle was determined using ModFit LT 3.2 software (Verity Software House, Topsham, ME, USA).

The PANC-1 cells were cultured with SSBE on 6-well plates containing glass slides, and were washed in phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) at 4°C for 30 min. After permeabilization in 0.1% Triton X-100 for 10 min, specimens were washed in PBS, and then the substrate was blocked with 10% FBS to eliminate the nonspecific fluorescence. Immunofluorescence staining was performed according to a previous study (17) using antibodies against proliferating cell nuclear antigen (PCNA; 1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), c-Myc (1:100; Biogot Technology, Shanghai, China), α-SMA (1:200), p21 (1:200), Ptch1 (1:200) and Smo (1:200) (all from Santa Cruz Biotechnology) as the primary antibodies at 4°C overnight. After washing in PBS three times, the cell preparations were incubated with DyLight 488 (green)/594 (red)-labeled secondary antibodies (Sigma-Aldrich) for 1 h at room temperature. After being washed in PBS, the cell preparations were dropped in appropriate acacia and covered with a slide. Immunocytochemical studies were semi-quantitatively or quantitatively assessed by two independent investigators in a blinded manner.

Western blot analyses were examined according to a previous study (18). Whole proteins from PANC-1 cells were collected and the protein concentrations were determined using a bicinchoninic acid protein assay kit (Beyotime Biotechnology). Whole proteins (20 µg) from each sample were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Solarbio, Beijing, China). After treatment with 5% skim milk at 4°C overnight, the membranes were incubated with various antibodies for 1 h, and then incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (Beyotime). Bound antibodies were visualized using chemiluminescence detection on autoradiographic film. Primary antibodies were as follows: polyclonal anti-Bcl-2 (1:400), Bax (1:400), Bcl-2 (1:400), caspase-3 (1:400), caspase-8 (1:400) (all from Beyotime), p53 (1:500; Biogot), p21 (1:200; Biotechnology) and c-Myc (1:500; Biogot). Quantification was performed by measuring the intensity of signals using Image-Pro Plus (version 6.0), and normalized to that for GAPDH (1:10,000; Cell Signaling Technology, Danvers, MA, USA).

Male nude mice (BALB/c) weighing 18–22 g and 6–8 weeks old were purchased from the Experimental Animal Centre of Wenzhou Medical University (Wenzhou, China). Mice were housed in a temperature-, humidity- and light-controlled environment, and fed a standard mouse chow and water. Mice were fasted on the day prior to the experiments being conducted. Before the experiments, all mice were anesthetized by an intraperitoneal injection with 0.2% pentobarbital natrium. The left neck of the experimental mice (n=12) were subcutaneously injected with 5×10⁶ PANC-1 cells in 10 µl of PBS and then received daily intragastric administration of SSBE (10 and 100 mg/kg·day) for 30 days. Model mice (n=12) received an injection of 5×10⁶ PANC-1 cells and daily gastric tube of solvent (PBS), and the healthy group (n=6) was treated with PBS only. Tumors were monitored daily until they became cumbersome or necrotic. Tumor volume (V) was measured every other day based on the formula: V = length² × width, where length was always the longest dimension. After the experiments, all mice were euthanized by immersion in an ice-water bath and then burned. The animal study protocols including the method involving animal euthanasia were approved by the Institutional Animal Care and use Committee of Wenzhou Medical university, China. The methods were also performed according to the guidelines approved by the Institutional Review Board of Wenzhou Key Laboratory of Surgery, China.

The tumor specimens were fixed in formalin and embedded in paraffin and then cut into 4-µm sections and stained with hematoxylin and eosin (H&E; Yuanye Biotechnology, Shanghai, China). Slides were examined and images were captured using a DM4000 B LED microscope system and a DFC420C 5M digital microscope camera (both from Leica Microsystems, Wetzlar, Germany).

Total RNA was extracted from the PANC-1 cells or tumor tissues using TRIzol reagent (Invitrogen), and reverse-transcribed to cDNA templates using a ReverTra Ace qPCR RT kit (Toyobo, Osaka, Japan). qRT-PCR was performed using SYBR-Green Real-Time PCR Master Mix Plus (Toyobo). Quality was analyzed on agarose gels, and quantities were measured using a Varioskan Flash (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Sequence-specific primers for TP53, c-Myc, E-cadherin, α-SMA, Bcl-2, Bax, Bad, survivin, CCND1, Smo, Ptch1 and Gli1 (Table I) were synthesized by Invitrogen and GAPDH was used as an endogenous reference gene. Samples were analyzed in triplicate, and the melting curve was examined to verify that a single product was amplified. For quantitative analysis, all samples were analyzed using the ΔΔCT value method.

Data are presented as the mean ± standard error of the mean. All statistical analyses were performed using a Statistical Package for Social Sciences (version 16.0; SPSS, Inc., Chicago, IL, USA). A two-sided Student's t-test was used to analyze differences between the two groups. A one-way analysis of variance was used when >2 groups were present. A P-value of <0.05 was considered to indicate a statistically significant result.

To investigate whether SSBE exerts a protective effect on pancreatic cancer, we first evaluated cell proliferation in the SSBE-treated PCCs. As shown in Fig. 1A, evidence from phase contrast microscopy showed that the injury of PANC-1 cells induced by SSBE was significantly aggravated, and cell number was decreased. CCK-8 assay revealed that this inhibitory effect of SSBE on cell proliferation was concentration-dependent, but not time-dependent (Fig. 1B). In addition, immunofluorescence staining indicated that SSBE treatment significantly downregulated the expression of PCNA (Fig. 1C). Thus, these findings suggested that SSBE has a marked inhibitory effect on the proliferation of PCCs.

Secondly, we examined the effect of SSBE on cellular apoptosis. In the SSBE-treated PANC-1 cells, marked cellular apoptosis was observed. As shown in Fig. 1D, the number of apoptotic cells (including early and late apoptotic and necrotic cells) in the controls was 8.77%, but reached 14.35% in the SSBE (100 µg/ml)-treated PANC-1 cells, suggesting that SSBE significantly increased the number of apoptotic cells in a concentration-dependent manner. In addition, western blot analyses showed that SSBE treatment increased the protein expression of Bax, caspase-3 and caspase-8, and decreased the protein expression of Bcl-2 (Fig. 1E). Moreover, upregulated gene expression of Bax and Bad, and downregulated gene expression of Bcl-2 were observed in the PANC-1 cells treated with SSBE (Fig. 1F). Thus, these data indicated that SSBE-induced apoptosis of PCCs may be through a mitochondrial-mediated pathway. This induction of apoptosis of PCCs after SSBE treatment was also identified by the downregulation of mRNA expression of survivin (also called baculoviral inhibitor of apoptosis repeat-containing 5), a member of the inhibitor of apoptosis (IAP) family (Fig. 1F) (19).

In SSBE-treated PANC-1 cells, the protein expression of c-Myc, determined by western blotting was significantly decreased and the expression of p53 was increased (Fig. 2A). Reduction in c-Myc expression was also confirmed by immunofluorescence staining (Fig. 2B). In addition, downregulated mRNA expression of Myc and upregulated expression of TP53 were also observed in the PANC-1 cells following SSBE treatment (Fig. 2C). These findings indicated that SSBE inhibits c-Myc expression and promotes p53 expression in PCCs. Previous studies have shown that c-Myc (encoded by the gene Myc) has an important role in apoptosis, cell cycle progression and cellular transformation (20). Enhanced expression of c-Myc induces the unregulated expression of many genes, some of which are involved in cell proliferation. Similarly, as a tumor-suppressor protein, p53 (encoded by the gene Tp53) plays a crucial role in cellular apoptosis, genomic stability and inhibition of angiogenesis (21). Low expression of p53 induces G2/M transition in various cancer cells. In the present study, SSBE inhibited gene and protein expression of c-Myc and promoted the expression of p53, thereby inducing the cellular apoptosis of PCCs and inhibiting proliferation.

The induction of cellular apoptosis of PCCs and inhibition of proliferation may be a result of cell cycle arrest. Thus, we examined the effect of SSBE on the cell cycle. Flow cytometric analysis showed that the percentage of G0/G1 phase PCCs in the SSBE-treated PCCs was significantly decreased compared with this percentage in the controls, and the percentage of G2/M-phase cells was increased (Fig. 2D), indicating that SSBE treatment induced excessive accumulation and cell cycle arrest of PCCs at the G2/M phase. Further study showed that the SSBE-induced cell cycle arrest of PCCs was through the downregulation of the expression of cyclin D1 (encoded by the gene CCND1) (Fig. 2E) and upregulation of the expression of cell cycle inhibitor p21 (Fig. 2F). Cyclin D1 is a protein required for progression through the G1 phase of the cell cycle (22). As a regulatory subunit of cyclin-dependent kinases CDK4, the cyclin D1-CDK4 complex promotes passage through the G1 phase by inhibiting the retinoblastoma protein (pRb), and enables the activation of cyclin E-CDK2 complex by sequestering CDK inhibitory protein p21 (23). SSBE-induced changes in gene and protein expression of PCCs drive cell cycle arrest at the G2/M phase.

EMT is a process whereby epithelial cells lose their epithelial phenotypes such as E-cadherin, and regain new characteristic features of mesenchymal molecules including α-smooth muscle actin (α-SMA) (24). EMT is critical for tumor invasion and metastasis (25). In the present study, the gene and protein expression levels of α-SMA in PANC-1 cells were downregulated by SSBE treatment (Fig. 3A and B). Additionally, the expression of E-cadherin was upregulated in the SSBE-treated cells (Fig. 3B). Thus, SSBE inhibited the EMT process of PCCs, resulting in the reduction in the ability for invasion and metastasis.

As mentioned above, SSBE has an inhibitory effect on the proliferation of PCCs. This inhibition of proliferation may be associated with the activation of proliferation-related signaling. Thus, we examined the activity of the Hedgehog signaling pathway in the SSBE-treated PANC-1 cells. Our results showed that SSBE treatment increased the protein expression of Ptch1 as determined by immunofluorescence staining (Fig. 4A) and the gene expression as determined by qRT-PCR (Fig. 4B). As a crucial receptor of canonical Hedgehog signaling, enhanced Ptch1 expression inhibits the activation of signaling. In addition to these, SSBE also decreased the expression of Smo and Gli1 (Fig. 4A and B), which are targets of activated Hedgehog signaling, suggesting that activity of the Hedgehog pathway in PCCs was reduced by SSBE treatment. As a result, SSBE inhibits the proliferation of PCCs.

Since SSBE treatment exerts its inhibitory effect on the activation of the Hedgehog signaling pathway in PCCs, we ascertained whether regulation of Hedgehog signaling is involved in SSBE-mediated inhibition. In the present study, exogenous recombinant protein sonic Hedgehog (Shh) was used to activate Hedgehog signaling in the SSBE-treated PANC-1 cells. We found that Shh abolished SSBE-mediated upregulated expression of Ptch1 and downregulated expression of Gli1 (Fig. 5A), indicating that Hedgehog signaling was reactivated, resulting in the reduction of p27 and α-SMA (Fig. 5B and C). p27, also called Kip1, regulates the cell cycle by inhibiting the checkpoint kinase cdk2/cyclin E and blocking cell cycle progression through G1/S transition. Downregulated expression of p27 abolishes cell cycle arrest, and thereby promotes proliferation and inhibits apoptosis. Thus, our in vitro experiment indicated that the Hedgehog signaling pathway plays an important role in SSBE-mediated inhibition of PCC proliferation and EMT.

To assess whether similar anticancer effects also occur in vivo, SSBE (10 and 100 mg/kg·day) was administered continuously for 30 days to mice subjected to injection of PCCs. Fig. 6A shows the morphological changes in the experimental groups as a result of SSBE treatment. H&E staining identified the pathological results of pancreatic cancer in tissues of the model group (Fig. 6B) and the tissue mass of the model group was significantly increased when compared with the vehicle group (Fig. 6C). Following SSBE treatment, the mass of the tumor tissues was significantly decreased in a concentration-dependent manner (Fig. 6C). Thus, these findings suggest that SSBE exerts its inhibitory effect on tumor growth. Further study showed that the mRNA expression of Ptch1 was decreased in the model group, and the expression of Smo was increased, indicating that Hedgehog signaling was activated in the occurrence and development of pancreatic cancer (Fig. 6D). This activation of Hedgehog signaling in the model group was inhibited by SSBE administration. Therefore, these findings reconfirmed that SSBE-mediated antitumor activity may be through suppression of Hedgehog signaling.