Background
Glioblastoma (GBM) is the most aggressive primary tumor of the central nervous system and accounts for ~ 50% of all adult gliomas [
1]. Current management is based on cytoreduction through a combination of surgery, radiation therapy, and chemotherapy. Despite this multidisciplinary approach to treatment, the prognosis of patients with GBM remains poor with a median overall survival of 9–15 months and a 2-year survival rate of 9–26% [
2].
Radiotherapy, which kills cancer cells by causing DNA damage, is a highly cost-effective single-modality treatment [
3]. However, studies have also reported that radiation can induce even more aggressive behavior in cancer cells [
4‐
6], which further contributes to its failure in the treatment of GBM patients. Studies have found that epithelial–mesenchymal transition (EMT) is involved in the aggressive invasion of cancer cells after radiation [
7‐
9]. EMT, a vital process in embryonic development, is believed to be utilized by cancer cells to increase mobility and invasiveness during metastasis [
10,
11]. A hallmark of EMT is the downregulation of E-cadherin and the upregulation of the mesenchymal markers, such as N-cadherin and β-catenin. It has been well documented that cells that have undergone EMT withstand external insults better, leading to increase resistance to chemotherapy and radiotherapy [
12,
13].
STAT3 is a cell proliferation-related transcription factor that regulates numerous apoptosis-related proteins including Bcl-2, Bcl-xL, Mcl-1, and cyclin D1 [
14]. The activity of STAT3 has also been reported to correlate with the development of GBM [
15]. STAT3 also displays an important role in EMT, and thus, downregulating the protein leads to reversal of EMT progress in cancer cells [
16,
17]. Recently, STAT3 inhibitors have been shown to enhance the radiosensitivity of cancer cells [
18,
19] and to reduce the malignant invasive ability induced by radiation [
20,
21]. Thus, STAT3 inhibitors are promising candidates in combination with radiotherapy in the treatment of malignant cancers.
As a first-in-class small molecule inhibitor of survivin, YM155 selectively inhibited survivin expression at both mRNA and protein levels in the nanomolar range and exhibited anticancer activity in several types of neoplasms [
22,
23]. Studies have also demonstrated that YM155 has a clear radiosensitizing effect in diverse cancers, including non-small cell lung cancer and esophageal squamous cell carcinoma [
24,
25]. However, the mechanisms engaged by YM155 to radiosensitize GBM have not been fully investigated. In the present study, we examined the radiosensitizing effects of YM155 on GBM cells. We found that YM155 reversed EMT in glioma cells and prevented radiation-induced in vitro and in vivo invasion, and that YM155 might elicit these activities through inhibition of STAT3.
Methods
Cell culture
Human glioma cell lines U87 and U251 were purchased from the cell bank of the Chinese Academy of Sciences and were cultured in Dulbecco’s modified Eagle’s medium (ThermoFisher Scientific; Waltham, MA, USA) containing 10% fetal bovine serum (FBS; ThermoFisher Scientific), glutamine (4 mM), penicillin (10 U/mL), and streptomycin (100 mg/mL).
Cell viability assay
Cell viability was assessed using Cell Counting Kit-8 (CCK-8, Dojindo; Kumamoto, Japan). U251 and U87 cells (1.0 × 104 cells/well) were seeded into 96-well, flat-bottomed plates with DMEM containing 10% FBS and incubated at 37 °C overnight. YM155 (Selleck Chemicals; Houston, TX, USA) was dissolved in DMSO and diluted to working concentrations in culture medium. After 48 h, the cells were incubated for an additional 1 h at 37 °C with 10 μL of CCK-8 in 100 μL of serum-free DMEM. The absorbance at 450 nm was measured using a microplate reader (Bio-Rad; Hercules, CA, USA).
EdU proliferation assay
U251 and U87 cells (2.5 × 104 cells/well) were seeded into 24-well, flat-bottomed plates. After incubation at 37 °C overnight, cells were treated with increasing concentrations of YM155 for an additional 48 h in DMEM with 10% serum, and subsequently stained with EdU using the EdU incorporation assay kit (Ribobio; Guangzhou, China) according to the manufacturer’s instructions. EdU positive cells were counted from at least five random fields under fluorescence microscopy (Leica DMi8; Wetzlar, Germany).
U251 and U87 cells (3 × 103 cells/well) were plated in six-well plates. The adherent cells were treated with 5 nM YM155 or DMSO as control for 24 h before receiving one dose of 4 Gy at a dose rate of 1.8 Gy/min and subsequently incubated for 14 days. Colonies were rinsed with PBS, fixed in 4% paraformaldehyde, stained with 0.1% crystal violet and counted. Colony numbers were counted using the gel documentation system EAGLE EYETM II (UVP, LLC., Upland, CA, USA).
Annexin V apoptosis assay
Apoptosis was evaluated using the FITC-annexin V/propidium iodide assay kit (BD Biosciences; San Jose, CA, USA). After treatment with YM155 or radiation, cells were collected by trypsin–EDTA, pelleted (1000 rpm for five flow cytometry in a minute), washed in ice-cold PBS, resuspended in the reagent containing annexin V-FITC and 1 µg/mL propidium iodide and Secondary antibodies were horseradish peroxidase-conjugated anti-mouse or anti-rabbit antisera incubated in the dark for 15 min. Cells were analyzed by flow cytometry using a C6 flow cytometer (BD Biosciences). A minimum of 20,000 cells per sample were analyzed.
Western blot analysis
GBM cells were seeded in 6-well plates. After incubation overnight, cells were treated as indicated (YM155, radiation or combination treatment, or transfection) and then harvested at 48 h. Protein lysates (20 µg) were prepared in RIPA buffer, run on polyacrylamide gel electrophoresis, and transferred to PVDF membranes. Membranes were blocked with 5% skimmed milk in Tris-buffered saline containing 0.1% Tween-20, and subsequently incubated with primary and indicated secondary antibodies. Proteins on western blots where visualized using the Chemiluminescent Reagents Kit (Millipore, Billerica, MA, USA). Chemiluminescent signals were detected with the ChemiDoc XRS+ (Bio-Rad, Hercules, CA, USA) and quantified using Image Lab 3.0 software (Bio-Rad). Immunoblot analysis was performed according to the manufacturer’s instructions.
The following antibodies were used for Western blotting: phospho-histone H2A.X (Ser139), E-cadherin, N-cadherin, slug, β-catenin, Zeb1, STAT3, phospho-stat3 (Tyr705), Cyclin D1, and c-Myc (Cell Signaling Technology; Danvers, MA, USA); BRCA1 and phospho-stat3 (Ser727) (Abcam; Cambridge, MA, USA); GAPDH and Rad51 (Santa Cruz; Dallas, TX, USA). Secondary anti-mouse or anti-rabbit conjugated with horseradish peroxidase (Sigma-Aldrich; St. Louis, MO, USA).
Homologous recombination (HR) assay
HR assays were performed with a kit (Norgen Biotek; Thorold, ON, Canada) according to the manufacturer’s instructions. Briefly, U251 or U87 cells were transfected with positive control plasmid or two HR dl plasmids (dl-1 and dl-2) on day 3 of DMSO or YM155 (5 nM) treatment. After 24 h, DNA was isolated using the Wizard genomic DNA purification kit (Promega; Madison, WI, USA). The recombined region was detected by qPCR performed with the supplied primers on a Roche LightCycler 480 II (Roche Applied Science; Indianapolis, IN, USA).
Acti-stain 568 phalloidin staining
U251 and U87 cells cultured on coverslips were treated with DMSO or YM155 (5 nM) for 48 h. Cells were rinsed with PBS, fixed in 4% paraformaldehyde, and permeabilized with 0.3% Triton X-100. Cells were stained with acti-stain 568 phalloidin (Cytoskeleton; Denver, CO, USA) to label F-actin, and DAPI (Invitrogen; Carlsbad, CA, USA) was used to stain nuclei. Cells were examined under fluorescence microscopy (Leica DMi8).
Cell migration and invasion assays
Cell migration was assessed in a wound healing assay. A silicone culture insert (Ibidi GmbH; Martinsried, Germany) was inserted into each well of a 4-well μ-slide (Ibidi GmbH), and U251 and U87 cells (70 μL at 4 × 105 cells/mL) were added into each half of the culture inserts. After 24 h, the culture insert was removed, which resulted in a wound. Cells were rinsed with culture medium for three times, and DMSO or YM155 (5 nM) were added to the wells. Cell migration into the wound was examined at 0, 6, 12 and 24 h under bright field microscopy (Leica DMi8).
Invasion was measured using the Transwell Matrigel assay. Cells were seeded into 6-well plates. After treatment, cells were trypsinized, counted, and plated into a BD Biocoat Matrigel Invasion Chamber (pore size: 8 μm, 24-well; BD Biosciences) in serum free medium. The chemo-attractant was 10% FBS-containing medium (10%) added to the bottom wells. Invaded cells were fixed after 24 h, stained with crystal violet, and counted.
Phospho-kinase antibody array
Phospho-kinases were analyzed using the Human Phospho-Kinase Array Kit (R&D Systems; Minneapolis, MN, USA) according the manufacturer’s instructions. Protein extracts were prepared from U251 cells treated with DMSO or YM155 (5 nM) for 48 h. Cell lysates were diluted and incubated overnight with the array membrane. The array was rinsed to remove unbound protein, incubated with an antibody cocktail, and developed using streptavidin–horseradish peroxidase and chemiluminescent detection reagents.
Cell transfection
Cells were transfected twice with the same siRNA at a 24-h interval with lipofectamine 2000 (Invitrogen). The final concentration of siRNAs was 20 nM. Sequences for the siRNAs used were the following: survivin, 5′-GCATTCGTCCGGTTGCGCT-3′; STAT3, 5′-GUUCAUCUGUGUGACACCATT-3′; nontargeting siRNA controls, 5′-UUCUCCGAACGUGUCACGUTT-3′ (Genepharma, Shanghai, China). The STAT3 cDNA (KIAA1524) was purchased from the Addgene plasmid repository (
http://www.addgene.org/). After transfection, U251 and U87 cells were cultured in the presence of G418 (0.8 mg/mL) for 8 weeks to select for stable transfectants. GBM cells that stably expressed STAT3 were used for treatment with YM155 and radiation.
In vivo experiments
All animal protocols were approved by the ethics committee at the Shandong University (Jinan, China) and conducted according to the national regulations in China. Nude mice were anesthetized with 4% chloral hydrate (300 mg/kg) and placed in a stereotactic frame. Using aseptic surgical procedures, an incision was made in the parietal scalp, and a small burr hole was drilled 2.5 mm lateral to the bregma. U251 (1 × 106 cells/mouse) were implanted 2.0 mm into the right striatum using a Hamilton syringe (Hamilton Co.; Reno, NV, USA). Two weeks later, mice were randomly divided into four groups (6 mice/group). Groups 1 and 2 were given two intratumoral injections of DMSO or YM155 (5 mg/kg) per week in a total of five injections. Group 3 was given three doses of localized irradiation (5 Gy) at days 15, 20, and 25 after implantation. Group 4 was irradiated three times following intratumoral injections of YM155 (5 mg/kg) per week in a total of 5 injections. Mice were sacrificed when central nervous system symptoms (such as poor ambulation, lethargy, and hunched posture) or weight loss of > 20% body mass developed. The mice were anesthetized with chloral hydrate and perfused transcardially with 4% paraformaldehyde in PBS. Whole brains were removed, post-fixed overnight in 4% paraformaldehyde in PBS, coronally sectioned into five slices, and paraffin embedded.
For subcutaneous GBM model, U87 cells were harvested by trypsinization, resuspended at 107 cells/mL in a 1:1 solution of PBS/Matrigel (BD Biosciences, USA), and injected subcutaneously into the right shoulder of the nude mouse. Then mice (n = 24) were divided into four groups as described above. 4 days later, groups 1 and 2 were given intratumoral injections of DMSO or YM155 (5 mg/kg) every other day in a total of 5 injections. Group 3 was given three doses of localized irradiation (5 Gy) at days 5, 10, and 15 after implantation. Group 4 was irradiated three times following intratumoral injections of YM155 (5 mg/kg) per week in a total of 5 injections. Then tumor tissues were isolated 24 days after injection. Tumors were measured in three dimensions (a, b, c), and volume was calculated as abc × 0.52.
Immunohistochemical analysis
Sections were cut (10 µm) from paraffin embedded tissues and stained with hematoxylin–eosin reagent or incubated with primary antibodies as indicated. The following primary antibodies were used for immunohistochemistry (IHC): phospho-stat3 (Tyr705) (Cell Signaling Technology), and phospho-stat3 (Ser727) (Abcam). Five representative images from each section were taken at 400× magnification using bright field microscopy (Leica DMi8). For each image, positive staining positive nuclei were counted using ImageJ software (National Institutes of Health; Bethesda, MD, USA). Positively staining cells are presented as a percentage of total number of cells counted.
Statistical analysis
Unpaired t-tests were performed using SPSS software 13.0 (SPSS Inc., 2005; Chicago, IL, USA). Results are presented as the mean ± SE. P values < 0.05 were considered statistically significant.
Discussion
Radiotherapy is a mainstay treatment modality after surgical resection in GBM patients. It mainly kills cancer cells by causing DNA damage. However, many factors, such as DNA damage repair, cell cycle arrest and autophagy, lead to radioresistance in tumors [
28]. Even worse is the discovery that radiation might actually enhance malignant progression in cancer cells, further contributing to the failure of radiotherapy in the clinic [
4‐
6]. Accumulating evidence supports YM155 as a radiosensitizer of high efficiency [
24,
25]. Besides, previous study showed that YM155 inhibited invasion in glioma cells [
26]. Here, we not only found that YM155 increased the radiosensitivity of GBM cells by impairing HR, but we also observed YM155 inhibited invasion induced by radiation. The changes in the morphology and actin cytoskeletal organization of U251 and U87 were reminiscent of the process of EMT. These changes were also evident in the levels of several EMT markers indicating that YM155 reversed EMT in GBM cells. Such results render YM155 a molecule of interest in the treatment of human glioma, especially in the context of radiotherapy.
However, YM155 activity was mediated not only through survivin, the putative target of the drug; inhibition of survivin through siRNA knockdown, for example, did not induce EMT changes in the morphology of GBM cells. Using a phospho-kinase antibody array kit, we also identified STAT3 (decreased phosphorylation at Ser727) as a potential mediator of YM155 activity. STAT3 is a transcription factor, contributing to diverse biological processes, including tumor cell proliferation, migration, invasion and survival. Thus, STAT3 is an ideal target for cancer therapy [
29,
30]. Our results are consistent with previous observations that STAT3 also plays an important role in promoting EMT [
8,
16] and thus invasion and migration of tumor cells. We found that knockdown with STAT3 siRNA induced an epithelial-like morphology in U251 and U87 cells, which was consistent with changes in EMT markers at the molecular level. STAT3 knockdown also attenuated increased invasion of glioma cells induced by radiation. Finally, stable overexpression of STAT3 enabled invasion of GBM cells, despite treatment with YM155.
We identified YM155 as a promising radiosensitizer in GBM based on its ability to impair HR and reverse EMT. While YM155 does not easily penetrate the blood brain barrier [
31], which is not like the antipsychotic we have reported before [
32], we chose to administer YM155 intratumorally and found that the drug also inhibited invasion in vivo. More studies are needed to demonstrate efficacy in vivo combined with radiation. Nanotechnology may serve as the platform to enable further investigation of YM155 in animal models and ultimately in the clinic [
33].
Authors’ contributions
XZ and CQ designed the experiments. CQ, JW and XGL supervised the study. XZ, XHW, RX, JXJ, YYX, MZH and YZW performed the experiments. BH, AJC, QZ and WJL assisted with the performance of experiments. XZ, XHW and RX analyzed the data. XZ, JW, XGL, and CQ wrote the paper. All authors read and approved the final manuscript.