BI2536 (cat. no. 50-873-3) and cisplatin (cat. no. 50-901-13218) were purchased from Thermo Fisher Scientific (Waltham, MA, USA) and diluted in dimethyl sulfoxide (DMSO) in accordance with the manufacturer's instructions.

The human gastric cancer cell lines, AGS, BGC-823, Hs746T, N87, KATOIII, SGC-7901 and SGC-7901/DDP (a cisplatin-resistant cell line), were obtained from the Molecular Pathology Laboratory at Mount Sinai Medical Center (New York, NY, USA). The BGC-823, SGC-7901 and SGC-7901/DDP cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA). The AGS cells were grown in Ham's F12 medium. The Hs746T cells were cultured in DMEM containing 10% FBS. The KATOIII cells were maintained in IMDM mixed with 20% FBS. All media contained penicillin (100 U/ml) and streptomycin (100 U/ml), and all cells were cultured at 37°C in a humidified incubator at 5% CO₂.

MTT assay was used to evaluate cell viability. In brief, the cells (5,000 cells/well) at the logarithmic growth phase were seeded in 96-well plates. Following 24 h of incubation, the cells were treated with various concentrations of cisplatin (1, 2, 4, 8, 16, 32 and 64 µM) and BI2536 (1, 2, 4, 8, 16, 32 and 64 nM) for 72 h at 37°C. Subsequently, 20 µl of MTT solution (5 mg/ml, pH 7.4) were added to each well, followed by incubation of the cells at 37°C for a further 4 h. After terminating the reaction, some of the supernatant was discarded, and 150 µl of DMSO were added to dissolve the crystals. The absorbance (570 nm) was then measured using a microplate reader (serial no. 155489; Bio-Tek Instruments, Inc., Winooski, VT, USA). Each experiment was performed in triplicate. Furthermore, the half maximal inhibitory concentration (IC₅₀) of cisplatin and BI2536 was further calculated by the modified Kou-type method (20): lgIC₅₀ = Xm-I [P-(3-Pm-Pn)/4], in which Xm indicates lg maximum dose, I indicates lg (maximum dose/adjacent dose), P indicates the sum of positive response rate, Pm indicates the largest positive response rate and Pn indicates the smallest positive response rate.

The cells were digested with 0.25% trypsin and split into individual cells. Subsequently, 50, 100 and 200 cells were seeded into 10-ml culture dishes and maintained under standard culture conditions for 2–3 weeks. When the colonies were visible to the naked eye, the culture dish was washed twice with phosphate-buffered saline (PBS). The colonies were then fixed with 4% paraformaldehyde for 15 min, followed by staining with crystal violet (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for 20 min. Under a microscope (Nikon Eclipse TS100; Nikon Instruments, Badhoevedorp, The Netherlands) the colonies that comprised at least 10 cells were counted.

Cell invasion was evaluated using Transwell chambers (8-µm pore size; Corning Inc., Corning, NY, USA) coated with serum-free RPMI-1640 medium containing Matrigel (Sigma-Aldrich, Shanghai, China). In brief, the SGC-7901 and SGC-7901/DDP cells (5×10⁴ cells) were grown in the upper chamber containing medium with 10% FBS, and BI2536 (IC₁₀) and cisplatin (IC₅₀) were then added to treat the cells. The lower chamber was filled with RPMI-1640 medium containing 20% FBS as a chemoattractant. Following incubation for 24 h at 37°C, the non-invading cells were removed using cotton swabs, and the invading cells were stained with 1% crystal violet for 30 min. The invading cells in different fields were then counted using a light microscope (Nikon Model Eclipse TS100LED MV; Nikon Corp., Tokyo, Japan).

The cells (1×10⁵ cells/ml) were collected, washed twice with ice-cold PBS, and fixed with 75% ice-cold ethanol. After washing with ice-cold PBS again, the cells were suspended in 300 µl of PBS and 20 µl of RNase A was then added, followed by incubation of the cells for 30 min at 37°C. Subsequently, the cells were stained with 400 µl of propidium iodide (PI) for 45 min in the dark. Cell cycle analysis at 488 nm was performed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA).

Cell apoptosis was assessed by flow cytometry after Annexin V and PI staining (BD Pharmingen, San Diego, CA, USA). In brief, the cells (1×10⁶ cells/ml) were harvested and resuspended in 1X Annexin V-binding buffer. Subsequently, 5 µl of Annexin V-FITC was added, and the cells were incubated for 15 min away from light, followed by the addition of 10 µl of PI and incubation of the cells for 5 min at 4°C. Cell apoptosis was then analyzed using a FACSCalibur flow cytometer (BD Biosciences).

The cells were lysed with 1X cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA). Using a Pierce BCA protein assay kit (Pierce, Rochford, IL, USA), the protein concentration was adjusted to 1 µg/µl. An equal amount of protein extract was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The blots were then transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The membranes were then blocked in 5% non-fat milk in 1X TBST containing 100 mM NaCl, 20 mM Tris-HCl (pH 7.5), and 0.1% Tween-20 for 1 h. Primary antibodies to PLK1 (1:1,000; cat. no. sc-5585; Santa Cruz Biotechnology, Inc.), p-Cdc2 (1:1,000; cat. no. 9111; Cell Signaling Technology), cyclin B1 (1:1,000; cat. no. sc-594), p-cdc25c (1:1,000; cat. no. sc-327) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:1,000; sc-32233) (all from Santa Cruz Biotechnology, Inc.) were added, followed by incubation of the membranes overnight at 4°C. GAPDH served as an internal control. Subsequently, the membranes were probed with horseradish peroxidase (HRP)-labeled secondary antibodies (1:10,000; cat. no. sc-2370 or sc-2371, Santa Cruz Biotechnology, Inc.) at room temperature for 1 h. After washing with 1X TBST buffer, the bands were detected with a chromogenic substrate using the enhanced chemiluminescence (ECL) method and analyzed using the Quantity One software package (Bio-Rad).

The cells were lysed with 1X cell lysis buffer, and equal amounts of protein extracts were separated by 10% SDS-PAGE, as described above. The blots were then transferred onto nitrocellulose membranes (Bio-Rad). After blocking in 3% bovine serum albumin (BSA) for 1 h, the membranes were fixed on a western blotting manifold (Mini-PROTEAN II Multiscreen apparatus, cat. no. 170-4017; Bio-Rad) containing 20 channels. A total of 286 protein-specific or phosphorylation-specific antibodies (Table I) were used in the multiplex immunoblot. To each channel (1–19), a mixture of two antibodies dissolved in the blocking buffer was added, followed by incubation of the membranes overnight at 4°C; BSA without any antibody was added to channel 20. Following incubation with HRP-conjugated secondary anti-rabbit (1:10,000; cat. no. sc-2371) or anti-goat (1:10,000; cat. no. sc-2370) or anti-mouse antibodies (1:10,000; cat. no. sc-2345) (all from Santa Cruz Biotechnology, Inc.) for 1 h, Immun-Star™ HRP Peroxide Buffer and Immun-Star™ HRP Luminol Enhancer (cat. no. 94547; Bio-Rad) were added followed by incubation of the membranes for 4 min. Chemiluminescence signals were then analyzed with the ChemiDoc XRS system (Bio-Rad). The same membranes was then washed twice with 1X TBST buffer and used to detect other primary antibodies, as described above. The signal intensity of each protein was analyzed using Quantity One software 4.5.0 (Bio-Rad). To reduce the variations caused by total protein loading amount, transferring and blotting efficiency, 'global median subtraction' was used to normalize the background subtracted intensity. The normalized expression of each protein = the average intensity of each protein in all samples × (the signal intensity of each protein/the total intensity of all proteins in the same blot membrane).

All in vitro experiments were repeated 3 times and PPA was performed twice. All measurement data are expressed as the means ± SD. The differences between groups were calculated using the Student's t-test or one-way ANOVA. Further comparison between groups was performed using a Tukey post-hoc test. Statistical analyses were performed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA). Unsupervised hierarchical clustering analysis was performed using the BRB ArrayTools Software V3.3.0. The significant pathway for the differentially expressed proteins was analyzed using Ingenuity Pathway Analysis (IPA) software. A value of P<0.05 was considered to indicate a statistically significant difference.

As shown in Fig. 1, PLK1 was upregulated in the SGC-7901. DDP (cisplatin-resistant) gastric cancer cells compared with the SGC-7901 cells. Thus, we further explored the function of the PLK1 inhibitor, BI2536, in gastric cancer cells.

As shown in Fig. 2A and B, cisplatin and BI2536 significantly inhibited the viability of the 7 gastric cancer cell lines in a dose-dependent manner. The highest chemosensitivity to cisplatin was observed in the BGC-823 and SGC-7901 cells, the IC₅₀ values of which were 2 and 6 µM, respectively. The least chemosensitivity to cisplatin was exhibited by the Hs746T and SGC-7901/DDP cells, the IC₅₀ values of which were 30 and 60 µM, respectively. Notably, BI2536 (IC₁₀) significantly enhanced the inhibitory effects of cisplatin on the viability of the gastric cancer cells, particularly by improving the chemosensitivity of SGC-7901/DDP to cisplatin (Fig. 2C). Therefore, a colony formation assay was then performed using the SGC-7901 and SGC-7901/DDP cells in order to verify the effects of BI2536 and cisplatin on cell viability. As shown in Fig. 2D and E, BI2536 (IC₅) alone did not inhibit colony formation compared with the controls (P>0.05); however, cisplatin (IC₁₀) significantly inhibited colony formation (P<0.05), particularly in the SGC-7901 cells (P<0.01). Following co-treatment with BI2536 (IC₅) and cisplatin (IC₁₀), the results revealed that BI2536 (IC₅) significantly enhanced the inhibitory effects of cisplatin on the colony-forming ability of the SGC-7901/DDP cells (P<0.01), but not that of the SGC-7901 cells (P>0.05).

We further determined the effects of BI2536 and cisplatin on gastric cancer cell invasion (Fig. 3). The results revealed that BI2536 (IC₁₀) did not inhibit the invasive ability of the SGC-7901 and SGC-7901/DDP cells (P>0.05), although cisplatin (IC₅₀) significantly inhibited the invasive ability of the cells (P<0.05). Moreover, following treatment with a combination of BI2536 (IC₁₀) and cisplatin (IC₅₀), only the inhibitory effects of cisplatin on the invasiveness of the SGC-7901/DDP cells, but not that of the SGC-7901 cells (P>0.05), were enhanced (P<0.01).

In the cell cycle analysis, the SGC-7901 cells were treated with 1, 5 and 10 nM BI2536 for 72 h, and the SGC-7901/DDP cells were treated with 5, 10 and 20 nM BI2536 for 24 h. The results of flow cytometry revealed that BI2536 significantly induced G₂/M arrest in both the SGC-7901 and SGC-7901/DDP cells (P<0.05) (Fig. 4A and B). We further determined the expression of key proteins involved in the G₂/M cell cycle, including p-Cdc2, cyclin B1 and p-Cdc25C by western blot analysis (Fig. 4C and D). We found that PLK1 expression was not significantly altered following treatment with various concentrations of BI2536 in both the SGC-7901 and SGC-7901/DDP cells (P>0.05). Notably, compared with the control group, BI2536 treatment resulted in the decreased expression of p-Cdc25C and in the increased expression of p-Cdc2 and cyclin B1 in the SGC-7901/DDP cells in a dose-dependent manner (P<0.01) (Fig. 4D), while the expression levels of these proteins exhibited no significant changes in the SGC-7901 cells (P>0.05).

Flow cytometry was also performed to determine the effects of BI2536 on gastric cancer cell apoptosis. Following treatment with various concentrations of BI2536 for 24 h, the proportions of SGC-7901 and SGC-7901/DDP cells undergoing early apoptosis were all significantly increased (P<0.05) (Fig. 5A and B). Furthermore, we found that cisplatin significantly induced SGC-7901 and SGC-7901/DDP cell apoptosis when used in combination with BI2536 (IC₂₀) (P<0.05) (Fig. 5C and D). Notably, BI2536 (IC₂₀, 20 nM) significantly promoted cisplatin-induced SGC-7901/DDP cell apoptosis (P<0.05) (Fig. 5D).

We applied PPA analysis to analyze the differentially expressed proteins between the SGC-7901 and SGC-7901/DDP cells following treatment with BI2536 (IC₅₀) for 48 h. We found that 68 proteins were differentially expressed when compared with the controls (Fig. 6A). IPA analysis also revealed that the differentially expressed proteins induced by BI2536 treatment were involved in many cell functions and signaling pathways, such as cell death, cell development, tumorigenesis, the cell cycle, DNA duplication/recombination/repair, cellular movement, and in the Wnt/β-catenin and mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK)/ribosomal S6 kinase 1 (RSK1) signaling pathways (Fig. 6B).