BI2536, a potent and selective inhibitor of polo-like kinase 1, in combination with cisplatin exerts synergistic effects on gastric cancer cells
- Authors:
- Published online on: January 25, 2018 https://doi.org/10.3892/ijo.2018.4255
- Pages: 804-814
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Copyright: © Lian et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Gastric cancer is a malignant tumor that is common worldwide and has a poor prognosis (1,2). The 5-year survival rate of patients with gastric cancer is <10% (3). The majority of patients are diagnosed at an advanced stage (4), and few efficacious treatment options are available for patients with this late stage of the disease (5). Surgical therapy combined with adjuvant chemotherapy is the primary treatment option for gastric cancer. It has been demonstrated that the single administration of traditional chemotherapeutic drugs, such as cisplatin and fluorouracil is only 10–20% efficacious in the treatment of gastric cancer (6). Even when combined with new drugs, such as docetaxel, irinotecan and oxaliplatin, the optimum reaction rate is <50% (7). Currently, an early diagnosis coupled with a good treatment strategy is considered an effective approach for the treatment of gastric cancer. The use of biomarkers has been confirmed to be a less invasive method for gastric cancer diagnosis (8). Moreover, targeted therapies for the treatment of gastric cancer have attracted increasing attention (9). However, there is still a lack of effective targeted therapies for the treatment of this disease.
Polo-like kinases (PLKs) are associated with oncogenesis in several types of cancer (10). PLKs exist in 4 isoforms, PLK1-4; however, only one of these isoforms, PLK1, is involved in centrosome maturation, chromosome segregation, bipolar spindle formation and cytokinesis execution (11). It has been reported that PLK1 exhibits oncogenic potential in gastric cancer (12). The inhibition of PLK1 following transfection with PLK1 siRNA and folate deficiency have been shown to synergistically inhibit the growth of gastric cancer cell lines (13). Moreover, a high PLK1 expression and DNA aneuploidy have been shown to correlate with a poor prognosis in patients with gastric cancer (14). PLK1 plays a key role in carcinogenesis and represents a promising target in the treatment of cancer (15,16). PLK1 inhibitors have recently emerged as a feasible strategy for the treatment of cancer (11). BI2536 is a highly selective and potent inhibitor of PLK1, which always participates in mitotic progression (17). Preclinical studies have indicated that BI2536 can disrupt spindle assembly, leading to mitotic arrest and the apoptosis of human cancer cell lines (18,19). However, the effects of BI2536 on the regulation of gastric cancer development have not yet been documented, at least to the best of our knowledge.
In the present study, the pivotal roles of BI2536 and cisplatin in regulating gastric cancer cell viability, migration, invasion and apoptosis were investigated. Differentially expressed proteins in gastric cancer cells treated with BI2536 (IC50) for 48 h, as well as the signaling pathways of these differentially expressed proteins were analyzed by protein pathway array (PPA). The aim of this study was to determine whether BI2536 exerts an antitumor effect on gastric cancer and whether it can synergistically inhibit the malignant behavior of gastric cancer cells when used in combination with cisplatin. Our findings may provide new insight into the targeted therapy for this disease.
Materials and methods
Drugs and treatments
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.
Cell culture
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.
3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay
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.
Colony formation assay
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 assay
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).
Cell cycle analysis
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 analysis
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).
Western blot analysis
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).
Protein pathway array (PPA) analysis
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).
Statistical analysis
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.
Results
PLK1 is upregulated in SGC-7901/DDP gastric cancer cells
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.
BI2536 enhances the inhibitory effects of cisplatin on the viability and colony-forming ability of the SGC-7901/DDP 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).
BI2536 enhances the inhibitory effects of cisplatin on the invasive ability of the SGC-7901/DDP cells
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).
BI2536 significantly induces G2/M arrest in the SGC-7901/DDP cells
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).
BI2536 promotes cisplatin-induced SGC-7901/DDP cell apoptosis
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).
BI2536 induces the differential expression of signaling proteins between the SGC-7901 and SGC-7901/DDP cells
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).
Discussion
Cisplatin is a common and effective anticancer drug; however, its use is limited due to its related side-effects, such as renal, gastrointestinal and neurological toxicities (21). Therefore, to improve the antitumor efficacy of cisplatin and reduce cisplatin-induced side-effects, further studies are warranted in order to aid the develelopment of small-molecule drugs. In the present study, we combined the PLK1 inhibitor, BI2536, with cisplatin to treat gastric cancer cells and to determine whether BI2536 and cisplatin can synergistically inhibit the malignant behavior of gastric cancer cells. The results revealed that BI2536 enhanced the cisplatin-induced inhibitory effects on SGC-7901/DDP cell viability and invasion. BI2536 induced G2/M arrest in the SGC-7901/DDP cells by decreasing the expression of p-Cdc25C and increasing the expression of p-Cdc2 and cyclin B1. BI2536 promoted cisplatin-induced SGC-7901/DDP cell apoptosis. Moreover, BI2536 induced the differential expression of 68 proteins between the SGC-7901 and SGC-7901/DDP cells, and these differentially expressed proteins were involved in sevral cell functions and signaling pathways, such as the Wnt/β-catenin and MEK/ERK/RSK1 signaling pathways.
In many anticancer treatments, the G2/M checkpoint is an effective target site for molecular targeted therapy and chemotherapy sensitization (22,23). There are data to suggest that mammalian PLK1 plays a regulatory role at the cell cycle G2 checkpoint (24,25). PLK1 has been implicated in mitotic entry via the activation of Cdc25C (26). PLK1 has also been identified as a target that can sensitize cells to traditional chemotherapeutic drugs in the treatment of cancer (27,28). In addition, a high degree of G2/M arrest induced by PLK1 inhibition has been found to be associated with radiosensitization in various cancer cell lines (29). The combination of MS275 and BI2536 has been shown to synergistically inhibit cell growth and to induce G2/M phase arrest in A549 non-small cell lung cancer cells (30). Gleixner et al demonstrated that the inhibitory effect of BI2536 on CML cell growth was associated with mitotic arrest, particularly G2/M arrest, and consecutively resulted in apoptosis (31). In this study, BI2536 enhanced the cisplatin-induced inhibitory effects on SGC-7901 cell viability and invasive ability. BI2536 induced G2/M arrest in the SGC-7901/DDP cells by decreasing the expression of p-Cdc25C and increasing the expression of p-Cdc2 and cyclin B1. BI2536 promoted cisplatin-induced SGC-7901/DDP cell apoptosis. Taken together, we speculate that the combination of cisplatin and BI2536 can synergistically inhibit cell growth, induce G2/M phase arrest, and consecutively induce the apoptosis of SGC-7901/DDP cells.
Furthermore, we applied PPA analysis to examine the differentially expressed proteins between the SGC-7901 and SGC-7901/DDP cells following treatment with BI2536 (IC50) for 48 h. A total of 68 proteins were found to be differentially expressed, which were involved in signaling pathways, such as the Wnt/β-catenin and MEK/ERK/RSK1 signaling pathways. It has been reported that Wnt/β-catenin signaling plays a key role in regulating the self-renewal of gastric cancer stem cells, and salinomycin treatment may be used for the treatment of gastric cancer by targeting Wnt/β-catenin signaling (32). The inhibition of the Wnt/β-catenin pathway by niclosamide has been shown to result in decreased cellular proliferation and increased cell death in gastric cancer (33). In addition, ERK/RSK1 activation by growth factors can delay the cell cycle at the G2 phase, thus reducing mitotic aberrations and maintaining genomic integrity (34). Notably, PLK1 is involved in mitotic arrest via the inhibition of the MEK/ERK/RSK1 cascade (35). Although the association between BI2536 and the Wnt/β-catenin or MEK/ERK/RSK1 signaling pathways has not yet been verified experimentally, our results provide an important indication pertaining to BI2536 likely promoting the chemotherapeutic sensitivity of SGC-7901/DDP cells to cisplatin via the involvement of the Wnt/β-catenin or MEK/ERK/RSK1 signaling pathways.
The strengths of our study were that BI2536 and cisplatin synergistically inhibited the malignant behavior of the SGC-7901/DDP (cisplatin-resistant) gastric cancer cells, which may provide a broader perspective for improving the chemotherapeutic sensitivity of cancer cells to cisplatin. Despite the clear strength of our study, however, some limitations merit further consideration. Firstly, there were no significant effects of BI2536 treatment alone on cell viability, migration and apoptosis, which limited the clinical application of BI2536. Secondly, the synergistic effects of BI2536 and cisplatin were not verified using gastric cancer primary cells or an in vivo xenograft model of SGC7901 and SGC7901/DDP cells. Further research is still required in order to verify the synergistic interaction between BI2536 and cisplatin in gastric cancer primary cells. Thirdly, we did not analyze PLK1 expression according to the information of the The Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia (CCLE) databases. Further studies are required to investigate the role of PLK1 in SGC7901 and SGC7901/DDP gastric cancer cells using siRNA-mediated gene knockdown. Fourthly, signaling pathways were only analyzed by PPA. The expression of Wnt/β-catenin and MEK/ERK/RSK1 signaling pathway-related proteins were not determined by qPCR or western blot analysis in treated samples. Fifthly, we only used MTT assay to determine changes in cell viability, which only monitored the ATP-dependent metabolic activity. To better detect the synergisstic effects of BI2536 and cisplatin on cell proliferation, BrdU DNA proliferation assay should also be performed to monitor the number of cellular divisions and DNA synthesis. Finally, we only analyzed the differentially expressed proteins between the SGC-7901 and SGC-7901/DDP cells following treatment with BI2536 (IC50) for 48 h. The key mechanisms involved in the combined effects of BI2536 and cisplatin treatment in regulating the malignant behavior of gastric cancer cells remain largely unknown. Therefore, further studies are still required in order to verify our observations.
In conclusion, the findings of the present study suggest that BI2536 and cisplatin synergistically inhibit the malignant behavior of SGC-7901/DDP (cisplatin-resistant) gastric cancer cells. BI2536 may enhance the chemotherapeutic sensitivity of SGC-7901/DDP cells to cisplatin via the involvement of the Wnt/β-catenin or MEK/ERK/RSK1 signaling pathways. The development of a PLK1 inhibitor may thus be an effective strategy for the treatment of gastric cancer.
Acknowledgments
This study was supported by the National Natural Science Foundation of China (grant nos. 81572355 and 81702363).
Notes
[1] Competing interests
The authors declare that they have no competing interests.
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