The human SW1990 and PANC-1 PC cell lines were obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China) and were routinely cultured in Roswell Park Memorial Institute (RPMI)-1640 medium and Dulbecco's modified Eagle's medium (DMEM; both from Gibco; Thermo Fisher Scientific, Inc.). All cell lines were supplemented with 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin solution at 37°C in a humidified atmosphere of 5% CO₂. The nutrient medium was replaced every 2-3 days and the cells were subcultured when they reached 70-80% adherence to the bottom of the culture plate, followed by digestion with tryptase. EVO (purity >99%; Fig. 1A) was purchased from Sigma; Merck KGaA and dissolved in dimethyl sulfoxide (DMSO; Nacalai Tesque, Kyoto, Japan) at 0.2 mol/l to produce the stock solution. The final DMSO concentration in the media did not exceed 0.1%. LY294002 (Akt inhibitor), U0126 [extracellular signal-regulated kinase (ERK)1/2 inhibitor] and SB203580 (p38 inhibitor) were obtained from Merck KGaA. Fluorine-18-labeled fluorodeoxyglucose (18F-FDG) was provided by Zhejiang University (Hangzhou, China).

Rabbit monoclonal antibodies against phosphory-lated (p)-Akt (Ser473) (D9E) (cat. no. CST 4060), Akt (C67E7; cat. no. CST 4691), p-ERK (Thr202/Tyr204) (D13.14.4E) (cat. no. 4370), ERK (137 F5) (cat. no. 4695), p-p38 (Thr180/Tyr182) (D3F9) (cat. no. 4551), p38 (D13E1) (cat. no. 8690), phosphorylated signal transducer and activator of transcription activator 3 (p-STAT3; Tyr705) (D3A7) (cat. no. 9145), STAT3 (79D7) (cat. no. 4904), P62 (D5E2) (cat. no. 8025) and LC3 (D3U4C) (cat. no. 12741) were purchased from Cell Signaling Technology, Inc. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; cat. no. sc-47724) and HRP AffiniPure Goat Anti-Rabbit IgG (H+L, cat. no. A32731) were obtained from Santa Cruz Biotechnology, Inc.

The cells were seeded into 96-well plates at a density of 5×10³ cells per well in 100 µl of the respective medium overnight and were then divided into five groups, each comprising five wells. The cells were treated with different concentrations (0, 0.5, 1.0, 2.5, 5.0 and 10.0 µM) of the EVO at 4°C, and then cultured the cells at 37°C for 48 h. Wells treated with 0.1% mass fraction dimethyl sulfoxide (DMSO) without the drug were used as the control. The blank retainer group was set at the same time. At 1 h prior to the end of incubation, 10 µl CCK-8 (Beyotime Institute of Biotechnology) was added to each well. The plates were mixed for 10 min on a gyratory shaker, and absorbance at the 450 nm wavelength was measured with an ELISA reader (BioTek ELx800, BioTek Instruments, Inc.).

Following treatment, the cells exposed to phosphatase inhibitors were collected, total protein was extracted, the concentration was measured in all samples using the Bradford Protein Assay kit, and the protein samples (30-100 µg) were separated using 10-15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated proteins were then transferred onto a polyvinylidene fluoride membrane, which was blocked for 1.5 h in blocking buffer consisting of 5% milk in Tris-buffered saline (TBS) containing 0.05% Tween-20, and then incubated with the different primary antibodies (1:400) overnight at 4°C. The membrane was washed with TBS containing 0.1% Tween-20, incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:1,000) for 1 h at 25°C. Primary and secondary antibodies were diluted in Tris-buffered saline (TBS) containing 0.05% Tween-20. The immunoreactive bands were then visualized using enhanced chemiluminescence kits. The density of the immunoreactive bands was analyzed using the Image J software (v1.8.0; National Institute of Health).

The PC cells (5×10⁵ cells/well) were seeded in 6-well plates and allowed to adhere overnight without serum-starvation. At 80-90% confluence, a 'reference line' scratch was made at the bottom of the plate using a sterile 100-µl pipette tip. Following washing three times with phosphate-buffered saline (PBS), the cells were incubated with EVO (1, 5 and 10 µM) or the vehicle (DMSO) medium to detect cells in the absence of cell growth migration. Following processing for 0 and 24 h, a digital camera following analysis with an MX4R Inspection microscope (Canon, Inc., magnification, ×100) was used to capture micrographs of the cells that had migrated across the reference line in different fields. Compared with the control, the rate of movement was quantified according to the migrated distance of the cells from the reference line to the center. All experiments were repeated three times.

To determine the cell colony-forming units, 500 cells per well were seeded into 6-well plates with 2 ml of culture medium overnight. The cells were pretreated with EVO, mixed with DMSO for 8-12 h, washed twice with PBS, and then transferred to fresh medium to allow the cells to grow for 7 days. The colonies were washed with PBS and then fixed with 4% methanol for 15 min at room temperature. The cells were washed then twice with PBS and stained with 1% crystal violet (25% methanol) for 10 min at room temperature. An image of the stained cell colonies was captured using a digital camera following analysis with an MX4R Inspection microscope. The cell size in the field of view is magnified 10 times. Three independent experiments were conducted for each assay.

Apoptosis was assessed using an Annexin V/propidium iodide (PI) kit (BD Biosciences Pharmingen). Exponentially growing cells were seeded in 6-well plates and then treated with EVO (1,5 and 10 µM) or DMSO (vehicle control, final concentration of 0.1%) for 48 h. Treated cells were harvested at room temperature and stained with Annexin V and PI for 15 min and analyzed using flow cytometry with a FACSCalibur instrument (BD Biosciences). The exported data was processed and analyzed using FlowJo Software version 7.6.1 (FlowJo LLC).

The PC cells were cultured in a nutrient-deficient medium for 24 h (17). The well-attached cells (5×10⁵ cells/well) were placed directly onto coverslips to allow them to grow. The coverslips containing logarithmic growth-phase cells were placed in a 6-well plate, fixed in 4% paraformaldehyde (1 ml per well) for 30 min and washed with PBS three times for 5 min each. The cells were then permeabilized with PBS containing 1% Triton X-100 (PBST) for 15 min, washed three times with PBS for 5 min each, and blocked with blocking solution (normal donkey serum; Gibco; Thermo Fisher Scientific, Inc.) for 30 min in 4°C. Non-specific staining was assessed by replacing the LC3 antibody (D3U4C, cat. no. 12741) with PBS. In the control group, only DMSO was added. The coverslips were subsequently incubated with anti-LC3 (1:400) overnight at 4°C and rinsed with PBST three times (flushing for 5 min each time). For indirect immunofluorescence, the coverslips were further incubated with HRP AffiniPure Goat Anti-Rabbit IgG (H+L, cat. no. A32731; 1:50) in the dark at room temperature for 1 h and then rinsed with PBST three times. Primary and secondary antibodies were diluted in Tris-buffered saline (TBS) containing 0.05% Tween-20. Following each 5-min rinse, the coverslips were rinsed again with distilled water, one drop of the encapsulated tablet was dropped, the cover-slips were mounted and then examined using fluorescence microscopy.

Following the indicated treatments, the cells were fixed with 4% parafor-maldehyde for 20 min and were washed thoroughly with PBS between each reaction to remove any residual solvent. The cells were stained with DAPI for 15 min in 4°C and viewed under a fluorescence microscope equipped with an ultraviolet (UV) filter.

Female BALB/c nude mice, (age, 4-6 weeks; weight, weight 20-25 g) were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. for tumor implantation. All mice were maintained in a sterile environment under constant temperature (25°C) and humidity, with a 12-h light/dark cycle, and free access to food and water. Their care adhered to the laboratory animal regulations of the Ministry of Science and Technology of the People's Republic of China (http://www.most.gov.cn/kytj/kytjzcwj/200411/t20041108_32465.hgrowthtm). The feed and drinking water of nude mice were purchased from Beijing Keao Xieli Feed Co., Ltd.

A total of 40 mice were used, and the food, water and bedding of the immunocompromised mice were sterilized and replaced at least once per week. PANC-1 PC cells (5×10⁶) in log phase were collected in 50 µl serum-free DMEM per mouse and orthotopically implanted into anesthetized athymic nude mice. In brief, a small laparotomy was performed on the left upper quadrant of each mouse. Anesthesia of the mice was induced with 3% isoflurane and maintained with 2% isoflurane. The PANC-1 cells were injected subcapsularly in a region of the pancreas just beneath the spleen. Successful subcapsular intra-pancreatic injection of the cancer cells was identified by the appearance of a fluid bleb without intraperito-neal (i.p) leakage (18). All surgical procedures were performed under sterile conditions using a 10X microscope. At 1 week post-implantation, the mice were medially randomized into four groups to receive i.p. injections of the vehicle (0.9% sodium chloride) or different concentrations of EVO (10, 20 and 30 mg/kg) (19) three times per week for 2 weeks.

PET imaging was performed 4 weeks after the nude mice were treated. The mice were fasted 1 day before imaging and injected via the tail vein with 0.1 mCi 18F-FDG/mouse. Anesthesia of the mice was induced using 3% isoflurane and maintained with 2% isoflurane, placed in the prone position, and scanned along the long axis of the Micro PET scanner. Data were collected following the administration of 18F-FDG-PET for 10 min and the ingestion time was 1 h after tracer injection. Static acquisition in 3D was performed using a micro PET imaging system (R4, Concorde Microsystems, Inc.). For quantitation, the regions of interest (ROIs) method and standard uptake values (SUVs) were used and statistically analyzed using micro PET ASIPro6.0.5.0 (Concorde Microsystems, Inc.). The ROI perimeters were drawn around the tumor and the area around the coronal section showing the best tumor depiction. All images were displayed using the same color scale. The highest and average intakes (percentage injected doses) in the tumor and surrounding ROIs, respectively were recorded and calculated (20). The tumor/non-tumor (T/N) ratio, which indicates tumor growth metabolism, of the semi-quantitative analysis was calculated using the following formula: T/N ratio = maximum pixel uptake in the tumor ROI / average pixel uptake in the surrounding ROI. The SUVs for ROIs of different groups were calculated as follows: SUV = radioactivity concentration in ROI (mCi/mL) x body weight (g) / injection dose (mCi), and compared. At the end of the experiment, the mice were sacrificed by cervical dislocation 1 week after the final PET imaging. The tumor samples were weighed and fresh frozen in liquid nitrogen and 10% formalin prior to analysis. The mice were sacrificed 8 weeks after implantation.

The formalin-fixed paraffin-embedded tumor tissue samples were sectioned, deparaffinized, rehydrated, blocked with 10% goat serum (Gibco; Thermo Fisher Scientific, Inc.) in PBS for 1 h (4°C), and then immunostained. The sections (25×75 mm) were incubated with p-Akt (Ser473, D9E, cat. no. CST 4060), p-ERK (Thr202/Tyr204) (D13.14.4E, cat. no. 4370) and p-p38 (Thr180/Tyr182, D3F9, cat. no. 4551) in a moist chamber overnight at 4°C. Following washing with PBS, the sections were incubated with an HRP-conjugated secondary antibody for an additional 30 min at room temperature. Following staining with hematoxylin, the sections were evaluated using a microscope (Olympus BX51, Olympus Corporation). Non-specific primary antibody staining was assessed by substituting PBS for the primary antibody, and at least 10 fields were randomly selected from each section for evaluation. The images were analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc.).

The data are presented as the mean ± standard error of the mean and the experiments were repeated three times (n=3). The statistical package for the social sciences (SPSS) 13.0 software (SPSS, Inc.) was used for statistical analysis. Differences in mean values between the groups were initially analyzed using one-way analysis of variance.

The inhibitory effects of EVO on PANC-1 and SW1990 PC cell lines were evaluated following incubation with different concentrations for 48 h. As shown in Fig. 1B and C, EVO dose-dependently reduced the viability of SW1990 and PANC-1 cells, indicating that EVO markedly inhibited the growth of PC cells.

The colony-forming ability characterizes the independent survival and environmental adaptation of cancer cells. Therefore, the effect of EVO on the clonogenic survival of PC cells was evaluated using a colony formation assay in plates. As shown in Fig. 2A and B, EVO dose-dependently inhibited the clonogenic survival of PANC-1and SW1990 cells after 7 days of treatment. These findings indicated that EVO potently inhibited PC cell growth in vitro and may be useful for the treatment of PC.

The role of apoptosis in EVO-induced cell death was then evaluated using Annexin V-fluorescein isothiocyanate and PI staining. The PANC-1 and SW1990 cells were treated with EVO (1.0, 5.0 and 10 µM) for 48 h and then stained with Annexin V and PI for the analysis of apoptosis using flow cytometry. As shown in Fig. 3A and B, the exposure of cells to EVO at various concentrations for 48 h led to a dose-dependent increase in apoptosis. To determine whether EVO-induced cell death was related to apoptosis, DAPI staining was performed to analyze the changes in nuclear morphology. The results revealed condensed and fragmented nuclei in both cell types following 24 h of EVO (10 µM) treatment (Fig. 3C).

Previous data showed that the AKT/PI3K signaling pathway serves an important role in the occurrence and development of various tumor cells (19). Therefore, the present study performed western blot analysis of PC cells to examine whether EVO inhibits the expression of AKT, ERK1/2 and P38. As shown in Fig. 4A and B, EVO inhibited the phosphorylation of AKT, ERK1/2 and P38 in a dose-dependent manner. The role of p38, AkT and erk1/2 in EVO-induced cell viability was also investigated. PANC-1 and SW1990 cells pretreated with LY294002, SB203580 or U0126 exhibited decreased cell viability compared with those treated only with EVO (Fig. 4C).

Treatment with EVO significantly reduced the levels of apoptotic marker STAT3 in PC cells (Fig. 5A and B). In addition, EVO reduced the expression of LC3-II and enhanced that of P62, autophagy markers in both cell lines, indicating that autophagy was inhibited (Fig. 5A and B). The distribution of LC3 was observed using cellular immunostaining (Fig. 5C). Treatment with EVO decreased the number of LC3 puncta, also indicating that autophagy was inhibited (Fig. 5C and D).

To evaluate the inhibitory effects of EVO on cell migration, a scratch wound model was used. The wound areas were marked and images were captured at 0 and 24 h. Compared with the control PANC-1 and SW1990 cells, those treated with EVO at different concentrations (1.0, 5.0 and 10 µM) migrated into the wound with a significantly decreased distance between the edges after 24 h, as shown in Fig. 6A and B.

The survival rate was of the mice was 100% and all tumors were observed in the coronal plane of the micro PET. The arrows in Fig. 7A show the tumor localization. It was found that the drug decreased the T/NT ratio in a concentration-dependent manner. The T/NT ratios of the EVO 10, 20 and 30 mg/kg groups (2.95±0.090, 1.83±0.260 and 1.31±0.142, respectively) were lower than those of the control group (0.9% sodium chloride, 3.52±0.095, P<0.05, Fig. 7B). The SUVs of the control and the 10, 20 and 30 mg/kg EVO groups were 5.21±0.33, 4.21±0.16, 3.11±0.26 and 2.36±0.35, respectively (Fig. 7C). The regional average intake of the tracer was downregulated in an EVO concentration-dependent manner compared with that of the in vivo control group (P<0.05).

The effects of EVO on orthotopic xenografts in nude mice were investigated (Fig. 8A). The tumor weights (Fig. 8B and C) of the EVO 10, 20 and 30 mg/kg groups, were 0.82±0.13, 0.67±0.18 and 0.23±0.17 g, respectively, compared with that of the control group (1.58±0.27 g). As the concentration of EVO increased, the body weight of nude mice also increased. In addition, the volume of tumors in the nude mice decreased with increasing drug concentration (Fig. 8D). These results showed that EVO inhibited tumor growth in the nude mice in a concentration-dependent manner.

The detection of p-AKT, p-ERK and p-P38 indicated the inhibition of PC cell proliferation in the treatment group (Fig. 9A and B). The expression levels of p-AKT, p-ERK, and p-P38 were microscopically examined at ×400 magnification. Compared with those in the control group, the expression levels of p-AKT, p-ERK and p-P38 in the tumor tissues decreased significantly in a concentration-dependent manner (Fig. 9B).