The reagents and kits used in the present study were purchased as follows: PN (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany); DDP (Shandong Dezhou Taikang Pharmaceutical Co., Ltd., Dezhou, China); RPMI-1640 (Sigma-Aldrich; Merck KGaA); fetal bovine serum (Hangzhou Evergreen Biological Engineering Materials Co., Ltd., Hangzhou, China); Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) detection kit (Nanjing KGI Biological Technology Development Co., Ltd., Nanjing, China); MTT (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China); enhanced chemiluminescence (ECL; GE Healthcare, Chicago, IL, USA); and DAPI (Beyotime Institute of Biotechnology, Haimen, China). The SGC-7901/DDP drug-resistant gastric cancer cell line was purchased from Shanghai Bogoo Biotechnology Co., Ltd. (Shanghai, China). The antibodies were obtained from the following: Goat anti-rabbit and anti-mouse immunoglobulin G secondary antibodies (GE Healthcare Life Sciences, Chalfont, UK); primary antibodies directed against phosphorylated-signal transducer and activator of transcription 3 (p-STAT3), apoptosis regulator Bcl-xL and Bcl-2 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA); β-actin (Sigma-Aldrich; Merck KGaA); and apoptosis regulator BAX (Bax), caspase-3, cleaved caspase-3, cellular tumor antigen p53, caspase-9, cleaved caspase-9, cyclin-dependent kinase inhibitor 1 (p21), cyclin D1 and STAT3 (Wanleibio Biotechnology Co., Ltd., Shanghai, China). Antibodies are detailed in Table I.

General electrophoresis, horizontal and vertical electrophoresis tank, and semi-dry transfer blotter were purchased from Bio-Rad Laboratories, Inc., (Hercules, CA, USA). The Ti-U manual fluorescence/inverted phase contrast microscope was obtained from Nikon Corporation (Tokyo, Japan); BioSpectrum^® Imaging system from UVP Inc., (Upland, CA, USA); microplate reader from Tecan Group Ltd. (Männedorf, Switzerland); and Alpha Automatic Image Analysis system from Bio-Techne (ProteinSimple; Minneapolis, MN, USA).

The human gastric cancer drug-resistant SGC-7901/DDP cell line was cultured in RPMI-1640 supplemented with 10% fetal bovine serum at 37°C with 5% CO₂.

MTT was used to measure the effect of PN treatment on SGC-7901/DDP cell proliferation and sensitivity to DDP. SGC-7901/DDP cells in the exponential growth phase were harvested and made into single cell suspensions at a concentration of 5×10⁴ cells/ml. The cells were seeded in 96-well plates (100 µl/well) and incubated for 24 h at 37°C. The SGC-7901/DDP cells were treated with various concentrations of PN (0, 2.5, 5, 10, 12.5 and 15 µmol/l) and DDP (0, 1.25, 2.5, 5, 10 and 15 µg/ml) with five-replicates/concentration. A total of 20 µl of 5 g/l MTT solution was added to each well at 24, 48 and 72 h, and then incubated for 4 h at 37°C. The supernatant was discarded and 150 µl dimethyl sulfoxide was added to each well. The plates were oscillated at a low speed for 2 min at room temperature (23–25°C) until all crystals were fully dissolved. The absorbance of each well was measured at a wavelength of 490 nm using a spectrophotometer. The growth inhibitory rate (%) was calculated using the following formula: (1-(absorbance of experimental well/absorbance of blank)) ×100%.

In order to prevent excessive cell death, based on the results from the above experiments, the combinations of less toxic concentrations of PN (5 and 10 µmol/l) and DDP (1.25 and 2.5 µg/ml) were selected. Therefore, the SGC-7901/DDP cells were treated with the following combinations: 5 µmol/l PN+1.25 µg/ml DDP; 5 µmol/l PN+2.5 µg/ml DDP; 10 µmol/l PN+1.25 µg/ml DDP; or 10 µmol/l PN+2.5 µg/ml DDP. The MTT assay was performed at 24 h following the treatments. The Zhenjun Jin method (11) was used to determine the synergy q=E(AB)/(EA+EB-EA × EB) EAB refers to growth inhibitory rate of combined treatment; and EA and EB refer to the growth inhibitory rate with either A or B treatment. q<0.85 indicates mutual antagonism of two drugs; q=0.85–1.15 indicates a simple additive effect of the two drugs; q>1.15 indicates that the two drugs have a synergistic effect.

SGC-7901/DDP cells in the exponential growth phase were seeded in 6-cm petri dishes at a density of 5×10⁵ cells/dish. The medium was changed 12 h following seeding and the cells were treated with 0, 7.5 or 15 µmol/l of PN for 48 h at 37°C. The supernatant was discarded and the cells were digested with EDTA-free trypsin, washed twice with PBS, and fixed with 70% ethanol at −20°C overnight. Cell were washed twice with PBS, treated with RNaseA (a final concentration of 250 µg/ml) and incubated in a 37°C water bath for 30 min. The cells were stained with 5 µl PI in a dark room for 50 min at room temperature, and then the cell cycle distribution was measured using a FACSCalibur flow cytometer and FlowJo 7.6.1 (BD Biosciences, Franklin Lakes, NJ, USA).

SGC-7901/DDP cells in the exponential growth phase were seeded in 6-cm petri dishes at a density of 5×10⁵ cells/dish. After culturing for 12 h, cells were treated with 0, 7.5, or 15 µmol/l PN for 48 h at 37°C. Cells were digested with EDTA-free trypsin, washed twice with PBS, stained for 15 min at room temperature in the dark using the Annexin V-FITC Apoptosis Detection kit and then the apoptosis rate was detected.

SGC-7901/DDP cells in the exponential growth phase were seeded in 24-well culture plates at a density of 5×10⁴ cells/well, and cultured for 24 h at 37°C. Cells were treated with 0, 7.5, or 15 µmol/l of PN for 24 h at 37°C. Cells were fixed with 0.1% paraformaldehyde for 30 min, washed with PBS for 5 min and stained with DAPI for 10 min at room temperature (23–25°C). The cells were observed using a fluorescence microscope (magnification, ×100) at an excitation wavelength of 359 nm.

SGC-7901/DDP cells in the exponential growth phase were seeded in 10 cm dishes at a density of 5×10⁵ cells/ml and cultured for 24 h at 37°C. Cells were treated with different concentrations of PN (final concentration of 0, 7.5 or 15 µmol/l) and collected 48 h after treatment. The total protein was extracted using radioimmunoprecipitation assay buffer (P0013J; Beyotime Institute of Biotechnology) and phenylmethanesulfonyl fluoride (ST506; Beyotime Institute of Biotechnology). The protein quantity was determined using a bicinchoninic acid assay kit (P0011; Beyotime Institute of Biotechnology). Protein samples were boiled at 100°C for 5 min. A total of 30 µg protein/lane was loaded and subjected to SDS-PAGE (10% gel). The proteins were transferred to polyvinylidene difluoride membranes, blocked with 0.5% skim milk for 1 h (23–25°C) and incubated with the appropriate primary antibody at 4°C overnight. The membranes were washed with TBS-Tween 20 three times (5 min/wash) and then incubated with the corresponding secondary antibody at room temperature for 2 h. The membranes were washed with TBST and detected using an ECL luminescent liquid with cytometry. The quantitative analysis was performed with ImageJ software (version 1.45; National Institutes of Health, Bethesda, MD, USA. The relative expression of the target protein was calculated as the ratio of grey values of the target protein to the β-actin internal control.

SGC-7901/DDP cells in the exponential growth phase were seeded in 6-well plates at a density of 5×10⁵ cells/well and cultured until 70% confluence was achieved. A scratch was made in each well using a 100-µl pipette tip. The cells were treated with various concentrations of serum-free PN (0, 2.5 or 5 µmol/l). Images were acquired at 24, 48 and 72 h after treatment. The scratch width was measured and the mobility was calculated as follows: (Distance/scratch width) ×100%.

In order to assess the invasive ability of cells, 120 µg Matrigel was added to a Transwell chamber. The gel was diluted with two volumes of serum-free RPMI-1640 and incubated at 37°C for 30 min until the gel solidified. RPMI-1640 containing 10% FBS was added to the lower chamber as a chemoattractant. SGC-7901/DDP cells in the exponential growth phase were digested with EDTA-free trypsin and viable cell counts were performed by eye. A 200-µl suspension containing 5×10⁴ cells was added to each Transwell chamber and cultured for 48 h at 37°C. The experiment was performed in triplicate. The liquid in the upper chamber was discarded. The non-invasive cells and Matrigel on the membrane surface were removed using a wet cotton swab. The membrane was rinsed with saline, slightly dried, fixed in formalin (4%) for 15 min and stained with crystal violet for 12 min (23–25°C). The penetrating cell count was performed in three different fields at a magnification of ×100 using a light microscope.

Data are presented as the mean ± standard deviation. Statistical analysis was performed using SPSS (version 10.0; SPSS, Inc., Chicago, IL, USA) using one-way analysis of variance. Comparisons between groups were measured using the post-hoc test. Multiple comparisons amongst groups were performed using the Student-Newman-Keuls test. P<0.05 was considered to indicate a statistically significant difference.

All concentrations of PN, except 2.5 µmol/l, significantly inhibited SGC-7901/DDP cell proliferation in a time- and concentration-dependent manner compared with untreated cells (Fig. 1). DDP inhibited SGC-7901/DDP cell proliferation in a similar time- and concentration-dependent manner (Fig. 2). To investigate the changes in the sensitivity of SGC-7901/DDP cells to the DDP, SGC-7901/DDP cells were treated with combinations of PN and DDP as described above. The rate of proliferation inhibition in each group is presented in Fig. 3. The q value for each group was calculated using the Zhenjun Jin method. With the exception of the minimal concentration group (5 µmol/l PN+1.25 µg/ml DDP), the q values for all other groups were >1.15, indicating that the two drugs have a synergistic effect.

SGC-7901/DDP cells were treated with different concentrations of PN (0, 7.5 or 15 µmol/l) and the cell cycle distribution was analyzed 48 h following treatment (Fig. 4). The ratio of G₀/G₁ in the untreated and 7.5, and 15 µmol/l PN groups was 27.2±1.05, 32.7±1.50 and 35.8±1.39%, respectively; the percentage of S-phase cells was 63.98±2.74, 60.49±2.11, and 58.94±3.10%, respectively. Furthermore, the percentage of G₂/M phase cells was 8.82±0.57, 6.78±0.73 and 4.72±0.46%, respectively. Compared with the untreated control group, the population of S and G₂/M phase cells in the treatment groups significantly decreased but not significantly, and G₁ phase cells significantly increased in a concentration-dependent manner (P<0.05; Fig. 4).

SGC-7901/DDP cells were treated with 7.5 and 15 µmol/l of PN and the cell apoptosis rates were detected 48 h following treatment (Fig. 5). The early apoptosis rates in the 7.5 and 15 µmol/l treated groups were 6.87±0.63 and 17.7±1.15%, respectively. Compared with the control group (5.30±1.31%), the early apoptosis rate was markedly increased in the 7.5 µmol/l group and significantly increased in the 15 µmol/l group (P<0.05) compared with that in the untreated control group. The late apoptosis rates were 2.88±0.54, 3.4±0.61 and 4.18±0.78% following no treatment, and treatment with 7.5 and 15 µmol/l PN, respectively. No statistically significant differences were identified between these groups (data not shown).

SGC-7901/DDP cells were treated with 7.5 or 15 µmol/l PN and observed using DAPI staining after 24 h (Fig. 6). The cells in the untreated control group possessed normally shaped nuclei (round or oval) with uniform staining and evenly distributed nuclear chromatin. The shrinkage, cohesion and fragmentation (spherical or particulate matter) of nuclei were observed 48 h after PN treatment. The number of apoptotic cells increased in a PN concentration-dependent manner, as indicated by the number of dense fluorescent particles (Fig. 6). The SGC-7901/DDP cell apoptosis rates were 0.88±0.05, 10.56±0.82 and 18.85±1.13% in the untreated, 7.5, and 15 µmol/l PN groups, respectively. The increases in apoptosis rates in the 7.5 and 15 µmol/l groups were significant compared with the untreated control group (P<0.05; Fig. 6).

Among the apoptosis-associated proteins, following PN treatment (7.5 or 15 µmol/l) Bax and p53 expression was significantly upregulated, Bcl-xL and Bcl-2 was significantly downregulated, and cleaved caspase-3 and −9 was significantly increased in a dose-dependent manner as compared with the expression in the untreated control groups (all P<0.05; Fig. 7). Among the cell cycle-associated proteins, cyclin D1 expression was significantly decreased, p21 was significantly increased, and P-STAT3 activation was significantly inhibited (all P<0.05; Fig. 7).

The effect of PN on SGC-7901/DDP cell migration is presented in Fig. 8. The mobility rate of the untreated control group at 24, 48 and 72 h were 11.85±2.55, 25.57±2.75, and 43.32±2.52%, respectively. The mobility rate of the 2.5 µmol/l PN group at 24, 48 and 72 h were 8.09±0.83, 22.70±2.92, and 31.11±3.85%, respectively. The mobility rate of the 5 µmol/l PN group at 24, 48 and 72h were 3.13±0.76, 7.62±1.17, and 16.54±3.02%, respectively. The differences between the 2.5 and 5 µmol/l PN groups, and the untreated control group at each time point were statistically significant (P<0.05).

The effect of PN on invasion ability is presented in Fig. 9. The number of transmembrane cells in the untreated control, and 2.5 and 5 µmol/l PN groups were 737±33.57, 344±12.81, and 127±8.02, respectively. The differences between the treated groups and the untreated control group were statistically significant (P<0.05).