The BxPC-3 human pancreatic cancer cell line cells were purchased from the Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China). Gardiquimod was obtained from InvivoGen (San Diego, CA, USA). 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxyme-thoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) was obtained from Promega Corporation (Madison, WI, USA). Antibodies against β-actin (cat. no. sc-8432; mouse monoclonal IgG1), cyclin E (cat. no. sc-247; mouse monoclonal IgG2b), cyclin B1 (cat. no. sc-594; rabbit polyclonal IgG), B-cell lymphoma (Bcl)-2 (cat. no. sc-7382; mouse monoclonal IgG1) and B-cell-associated X protein (Bax; cat. no. sc-493, rabbit polyclonal IgG) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Fluorescein isothiocyanate (FITC)-Annexin V and propidium iodide (PI) were obtained from BioSharp (Hefei, China). Primers (Table I) were purchased from Sangon Biotech Co. Ltd. (Shanghai, China). Trypsin, Triton X-100, RNase A, radioimmunoprecipitation (RIPA) assay buffer, phenylmethylsulfonyl fluoride (PMSF), bicinchoninic acid (BCA) assay kit and SDS-polyacrylamide gel electrophoresis (PAGE) gels were obtained from Sigma-Aldrich (St. Louis, MO, USA) and Tris-buffered saline with Tween-20 (TBST) was purchased from Sunshine Biotechnology Co., Ltd. (Nanjing, China). Fetal bovine serum (FBS), streptomycin and penicillin were obtained from Invitrogen Life Technologies (Shanghai, China).

EDTA anticoagulated venous blood samples (2 ml) were collected from healthy volunteers and PBMCs were isolated using lymphocyte separation medium (Tianjin Haoyang Biological Manufacture Co., Ltd., Tianjin, China). The expression of TLR7 in PBMCs was analyzed and served as a positive control. Written informed consent was obtained from the volunteers.

The BxPC-3 cells were cultured for 2 days in RPMI-1640 medium supplemented with 10% FBS, 100 U/ml streptomycin and 100 U/ml penicillin at 37°C in a 5% CO₂ atmosphere. Upon reaching 70% confluence, the cells were passaged for use in the following experiments.

BxPC-3 cells were seeded (3×10³ cells/well) in 24-well plates and cultured in FBS-free media for 3 days. Total cellular RNA was extracted from the cells using TRIzol (Invitrogen Life Technologies, Carlsbad, CA, USA). RT-qPCR analysis of TLR7 and GAPDH were performed, as previously described (29). Briefly, samples were amplified in an Applied Biosystem 7500 Real-Time PCR System (Applied Biosystems Life Technologies, Foster City, CA, USA) for 40 cycles with the following conditions: Denaturation at 95°C for 15 sec, and annealing and extension at 60°C for 40 sec. The primer sequences used are presented in Table I and the relative expression of TLR7 was calculated using 2^−ΔΔCt.

The BxPC-3 cells were seeded (3×10³ cells/well) in 96-well plates and cultured in FBS-free RPMI-1640 for 1 day. The cells were continually cultured at 37°C in a 5% CO₂ atmosphere and treated with different concentrations (0, 1.5, 3 and 5 µg/ml) of gardiquimod for various time-periods (0, 12 and 30 min, 1, 3, 6, 12, 24 and 48 h), following which 20 µl MTS reagent was added. The cells were incubated for 2 h and absorbance was measured at 490 nm using a Spectrophotometer Multiskan GO (Thermo Fisher, Vantaa, Finland). The experiment was repeated a minimum of three times.

The BxPC-3 cells were seeded in 12-well plates and treated with 3 µg/ml gardiquimod for different time-periods, between 1 and 5 days, in FBS-free RPMI-1640 at 37°C in a 5% CO₂ atmosphere. The cells were detached with trypsin and washed with phosphate-buffered saline (PBS), and were then BxPC-3 cells were suspended at a density of 3×10⁵/ml and fixed in 70% alcohol for 30 min. Subsequent to washing with PBS, 5 µl Triton X-100, 3 µl RNase A (20 ug/ml) and 1 µl PI dye (50 ug/ml) were added to the cells. Following mixing at room temperature and maintenance in the dark for 30 min, flow cytometric analysis was performed. For analysis of apoptosis in the cells, 5 µl FITC-Annexin V and 5 µl PI were added to the resuspended cells at room temperature for 15 min. Then the cells were detected by flow cytometry using a BD FACSVerse™ flow cytometer (BD Biosciences, San Jose, CA, USA). The cells in the lower left, lower right, upper right and upper left quadrant signified viable, early apoptotic, late apoptotic or necrotic cells, respectively. The experiment was repeated three times.

The BxPC-3 cells were cultured in FBS-free RPMI-1640 at 37°C in a 5% CO₂ atmosphere, following which gardiquimod (3 ug/ml) was added and the cells were cultured for 0, 12 and 30 min, 1, 3, 6, 12, 24 and 48 h. Subsequent to washing with cold PBS, the cells were incubated in RIPA assay buffer containing 1 mM PMSF fluoride for 30 min in an ice-water bath. Subsequent to protein quantification using a BCA method, 100 µg proteins were loaded onto 12% SDS-PAGE gels and the separated proteins were transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membrane was blocked in TBST containing 5% non-fat milk for 2 h at room temperature with agitation. The membranes were then incubated with primary antibodies against β-actin, cyclin B1, cyclin E, Bcl-2 and Bax (1:500) overnight at 4°C. Subsequent to washing with TBST, the membrane was incubated in horseradish peroxidase-linked secondary antibody/TBST solution for 2 h at room temperature. The bound secondary antibodies were detected using chemiluminescence substrate (Pierce Biotechnology, Inc., Woburn, MA, USA) and were visualized by exposure to X-ray film. The band intensity was determined using a Gel Image Analysis system and were normalized to β-actin.

The BxPC-3 cells were cultured in 24-well dishes (5×10⁴ cells/well) for 24 h at 37°C in a 5% CO₂ atmosphere. A 10-µl pipette tip was used to scratch three perpendicular lines on the bottom of dishes. Following washing with PBS, gardiquimod (3 µg/ml) was added to the cells for different time periods (1–5 days) at 37°C in a 5% CO₂ atmosphere. The distance that the cells migrated into the scratched region was quantified using the ocular micrometer of a Nikon Ecolipse TS100 microscope (Nikon Corporation, Tokyo, Japan; 10X objective lens, 1 unit = 10 mm). The assessment was repeated three times.

All experiments were repeated a minimum of three times and the representative results are presented. The data are presented as means ± standard deviation. Student's t-test, analysis of variance and the χ² analysis were conducted using SPSS software, version 13.0 (SPSS, Inc., Chicago, IL, USA) and P<0.05 was considered to indicate a statistically significant difference.

In the present study, total RNA and protein were extracted from the BxPC-3 cells, following which RT-qPCR and western blot analysis were used to analyze the mRNA and protein expression levels of TLR, respectively. Peripheral blood mononuclear cells (PBMCs) were used as a positive control sample. Compared with the PBMCs, the expression of TLR7 in the BxPC-3 cells was reduced at the mRNA (Fig. 1A) and protein (Fig. 1B) levels.

Treatment of gardiquimod inhibited the proliferation of the BxPC3 cells. The inhibition ratio was 10.41% at a low concentration (1.5 µg/ml) and 20.12% at a high concentration (5 µg/ml; P<0.05; Fig. 1C). The time course analysis indicated that the inhibition ratio gradually increased between 7.01 and 28.55% between days 1 and 5 following the addition of 3 µg/ml gardiquimod (P<0.05; Fig. 1D).

Analysis of the cell cycle analysis using flow cytometry demonstrated that the percentage of BxPC-3 cells treated with gardiquimod in the G₁ phase gradually increased between 76.41 and 82.97% (Fig. 2). In addition, the percentage of cells in the S phase reduced between 19.13 and 10.09% as the duration of treatment increased (Fig. 2).

The effect of gardiquimod on the apoptosis of BxPC-3 cells was also examined (Fig. 3). The percentage of cells in the early apoptotic phase increased between 11.5% at day 1 and 19.32% at day 5. There was a statistically significant difference between the control group (Fig. 3A) and the groups treated with gardiquimod for >3 days (P<0.05; Fig. 3D–F).

Based on the results of the MTS and flow cytometry, gardiquimod was found to inhibit the proliferation and induce the apoptosis of the BxPC-3 cells. In order to investigate the underlying mechanism, western blot analysis was performed to analyze changes in the expression levels of cyclin B1, cyclin E, Bcl-2 and Bax in the BxPC-3 cells treated with gardiquimod. As shown in Fig. 4A, treatment with gardiquimod reduced the expression levels of cyclin B1, cyclin E and Bcl-2 in the BxPC-3 cells and, following treatment for 12 h, the expression levels of these three proteins markedly reduced. By contrast, gardiquimod induced the expression of Bax.

Cancer cells can migrate from their original sites and invade into adjacent normal tissues or into the blood or lymph vessels, and form additional neoplasms. In the present study, a scratch/migration assay was used to detect the effects of gardiquimod on the migration of BxPC-3 cells. In the control group, without gardiquimod treatment, the BxPC-3 cells occupied almost the entire scratched space at day 5, however, in the groups treated with gardiquimod, ~60% of the space present at day 0 remained unoccupied at day 5 (Fig. 4B).