In total, 109 microarray and clinicopathological data sets of sarcoma from Gene Expression Omnibus (GEO) dataset GSE14827 (OS), GSE13433 (alveolar soft part sarcoma, ASPS), GSE8167 (gastrointestinal stromal tumor, GIST), GSE20196 (synovial sarcoma, SYN), and GSE20559 (liposarcoma, LPS) and from E-MEXP-1922 (leiomyosarcoma, LMS) were obtained from the NCBI and ArrayExpress websites, respectively. Nine microarray data sets of normal osteoblasts (GSE9451 and GSE10311) were retrieved from the NCBI website. All these datasets were in Affymetrix U133 Plus 2.0 platform. To decrease intrasubtype heterogeneity, not all retrieved samples were included in the analysis. For OS, only tumors without subsequent metastasis were selected; for GIST, only those with exon 11 mutation were included; for SYN, only tumor with SYT-SXX type 1 fusion gene and non-poorly differentiated histology were put into analysis; and for LPS, only well-differentiated (WD) and dedifferentiated (DD) tumors were chosen due to their similar genetic background. Gene expression data were normalized using dChip (28,29). The expression levels of individual genes were obtained through Z-score transformation, and the differences between different subtypes were subsequently compared using the Student's t-test.

Three OS cell lines, U2OS, MG63 and SJSA were selected for in vitro study and maintained in either Dulbecco's modified Eagle's medium (DMEM), Iscove's modified Dulbecco's medium, or DMEM/F12 base media with 10% fetal bovine serum. GSK461364 was purchased from Calbiochem (San Diego, CA, USA). The following antibodies were used for immunoblotting: PLK1 (#4513; 1:1,000), phospho-cdc25C (Ser216) (pCDC25C) (#4901; 1:1,000), phospho-Histone H3 (Ser10) (pHisH3) (#9701; 1:1,000), and phospho-Histone H2A.X (Ser139) (pH2AX) (#2577; 1:1,000), all from Cell Signaling Technology, and anti-actin (ABS 24–100; 1:10,000).

Two methods were used in the cell viability assay, the first being the TACS™ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay (Trevigen, Gaithersburg, MD, USA). Approximately 2,000–20,000 cells/100 μl/well were seeded in 96-well plates overnight. Subsequently, reagents at different concentrations were added in triplicates. The plates were incubated for the desired time at 37°C, pulsed with 10 μl MTT reagent, and incubated for an additional 4 h at 37°C. Furthermore, detergent reagent of 100 μl/well was added and mixed thoroughly to dissolve the blue crystals. Absorbance of the converted dye was measured using a Vmax microplate reader (Molecular Devices, Sunnyvale, CA, USA) at 570 nm (test) and 650 nm wavelengths. Cell survival was calculated using the following equation: % survival = (mean experimental absorbance/mean control absorbance) × 100 (30).

The possible synergistic effect of GSK461364 and chemotherapy was measured using the combination index (CI) calculated using CalcuSyn software (Biosoft, Ferguson, MO, USA) (31). CI >1 was defined as antagonism, CI = 1 as additivity, and CI <1 as synergy; the experiment was performed in triplicate.

Cell viability was also measured through a trypan blue exclusion assay (32). OS cells were plated at 5×10⁴ cells/well in 24-well plates; a variable concentration of GSK461364 was added to each well the following day, and the plates were incubated at 37°C for 72 h. Subsequently, the cells were trypsinized and mixed with trypan blue. Viable cells with intact cell membranes and not absorbing trypan blue were counted using a light microscope. All experiments were performed in triplicate.

Annexin V staining was used to detect apoptosis in cell lines treated with GSK361364. The cells were washed once with 1X phosphate-buffered saline (PBS) following drug treatment and resuspended in 100 μl staining solution [containing Annexin V fluorescein and propidium iodide in an Annexin V-binding buffer, Annexin V-fluorescein isothiocyanate (FITC); BD Biosciences, San Diego, CA, USA]. Subsequently, the cells were incubated at room temperature for 15 min and diluted in 1X Annexin V-binding buffer. The percentages of apoptotic cells were measured through flow cytometry.

Cell cycle analysis was performed through flow cytometry, as previously described (33). Briefly, cells were trypsinized, washed twice with PBS, and fixed with 70% ethanol at −20°C overnight, following which the fixed cells were washed twice with cold PBS, suspended in 420 μl PBS, added with 50 μl 10 mg/ml RNase A (Sigma), and shaken at 37°C for 15 min. Subsequently, 20 μl of 0.2 mg/ml propidium iodide (PI) was added and cells were retained at room temperature for 1 h. Flow cytometry was performed using FACSCalibur (Becton-Dickinson and Co., Oxford, CA, USA) to measure relative DNA content based on red fluorescence levels. The percentages of the cells in the different phases of the mitotic cell cycle were calculated using CellQuest software (Becton-Dickinson).

Monolayers of cultured cells were rinsed in PBS and scraped into lysis buffer [25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS (Thermo Fisher Scientific)] containing protease and phosphatase inhibitor cocktail (1:100 dilution; Thermo Fisher Scientific). Lysates were incubated for 30 min at 4°C and subsequently clarified through centrifugation for 30 min at 13,200 rpm at 4°C. Protein concentrations were determined with the Pierce BCA protein assay kit (Thermo Fisher Scientific). Protein extracts (20–50 μg/lane) were electrophoretically separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, transferred to polyvinyl difluoride membranes (Millipore), and blotted with specific antibodies. Immunoreactive bands were detected using enhanced chemiluminescence (Millipore) and exposed to X-ray film.

Senescence-associated β-galactosidase (SA-β-gal) activity was detected using the cellular senescence assay kit (Millipore) according to the manufacturer's instructions. U2OS cells were treated with GSK461364 for 72 h; the adherent cells were fixed and stained with X-gal in a staining solution at pH 6.0 and washed twice with 1X PBS. The percentage of SA-β-gal-positive cells (the number of positive cells relative to the total number of cells) was quantified by counting 100 cells in 3 randomly chosen fields per dish by using an Olympus IX51.

DcR2 expression was detected through flow cytometry. OS cells treated with GSK461364 for 72 h were washed twice with 1X PBS and incubated with Alexa Fluor 488-labeled anti-DcR2 (R&D Systems, Minneapolis, MN, USA) for 30 min at room temperature in the dark. After washing twice with 1X PBS and resuspending in 1X PBS, the mean of fluorescence intensity on the cell surface was measured through flow cytometry using FACSCalibur (Becton-Dickinson).

RNAs were extracted from the cell lines using TRIzol^® reagent (Invitrogen) according to the manufacturer's instructions and reverse transcribed with 1 μg RNA by using SuperScript^® III First-Strand Synthesis system (Invitrogen) for reverse transcription polymerase chain reaction (RT-PCR). The copy number for both interleukin (IL)-1α and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was measured through qRT-PCR by using Maxima SYBR-Green/ROX qPCR Master Mix (Thermo Fisher Scientific) and a LightCycler^® 480 system (Roche). The primer sequences used in the reaction were as follows: IL-1α forward, 5′-CCGTGAGTTTCCCAGAAGAA-3′ and IL-1α reverse, 5′-ACTGCCCAAGATGAAGACCA-3′; GAPDH forward, 5′-GCCAAGGTCATCCATGACAACT-3′ and GAPDH reverse, 5-GAGGGGCCATCCACAGTCTT-3′ (34). The PCR cycling conditions were as follows: 95°C for 5 min followed by 45 cycles of 95°C for 30 sec, 55°C for 30 sec followed by 72°C for 40 sec. The gene expression levels were calculated as previously described (35).

We explored the expression level of PLK1 among normal osteoblasts and OS as well as among other types of sarcoma. The expression level of PLK1 was compared between 27 OS and 9 normal osteoblasts. As depicted in Fig. 1A, PLK1 was significantly overexpressed in OS compared with normal osteoblasts. We further compared the expression of PLK1 in OS with other types of sarcoma. As depicted in Fig. 1B, the transcript level of PLK1 was significantly higher in OS compared with other types of sarcoma. These results indicated that PLK1 was overexpressed in OS.

An in vitro model was used to explore the potential of PLK1 as a therapeutic target in OS. GSK461364, a potent PLK1 inhibitor, has demonstrated favorable activity in various types of cancers (27). OS cells were treated with GSK461364 and the levels of PLK1 and pCDC25C (a downstream effector of PLK1) were measured through western blotting. Except for U2OS in time-dependent studies, decreased level of PLK1 and pCDC25C were noted in all three GSK461364-treated OS cell lines (Fig. 2). Moreover, we demonstrated that all OS cell lines treated with GSK461364 exhibited a dose- and time-dependent increase in pHisH3, an indicator of mitotic arrest (Fig. 2).

Furthermore, we explored the effects of GSK461364 on cell cycle progression in OS cell lines. As depicted in Fig. 3, flow cytometric analysis of DNA content in cells treated with GSK461364 for 24 h demonstrated marked accumulation of cells at G2/M DNA content in all 3 OS cell lines.

Cell viability assays were used to detect the possible cytotoxic activity of GSK461364 against OS cell lines. As depicted in Fig. 4, GSK461364 displayed cytotoxicity against all the three OS cell lines when assessed using either an MTT assay (Fig. 4A), or a trypan blue exclusion assay (Fig. 4B). Moreover, a significant induction of apoptosis in OS cell lines was revealed on co-staining with PI and FITC-labeled Annexin V (Annexin V-FITC; Fig. 4C).

Cellular senescence can be induced by cell cycle inhibition. To explore whether GSK461364 induces senescence in OS cells, a SA-β-gal assay was used in the U2OS cells following treatment with GSK461364 for 72 h. A significant increase in SA-β-gal activity in the U2OS cells was revealed following GSK461364 treatment (Fig. 5A). In addition, the expression of DcR2, a well-known senescence biomarker (34,36), was dose-dependently enhanced following GSK461364 administration (Fig. 5B). Furthermore, the expression of IL-1α, a cytokine associated with the senescence-associated secretory phenotype (SASP) (34,36), was upregulated in the U2OS cells treated with GSK461364, as measured through qRT-PCR (Fig. 5C). These results indicate that GSK461364 treatment induces cellular senescence in OS cells.

We evaluated the possible synergistic effect of GSK461364 with other chemotherapies. As depicted in Figs. 6 and 7, except for the U2OS cells treated with doxorubicin, GSK461364 showed no significant synergistic effect with DNA damaging agents, such as doxorubicin or cisplatin, or with the topoisomerase inhibitor topotecan. However, it showed a significant synergistic effect with paclitaxel in both U2OS and MG63 cells.

Furthermore, we explored the underlying mechanism of the synergistic effect of GSK461364 and paclitaxel. As depicted in Fig. 8, a combination of GSK461364 and paclitaxel produced significantly increased mitotic arrest, as indicated by the increased pHisH3 level. By contrast, a combination of GSK461364 with other chemotherapeutic agents reduced mitotic arrest. No significant changes were observed in the pH2AX level, an indicator of DNA damage.