Background
Survivin is the smallest member of the inhibitor of apoptosis protein (IAP) family with a molecular mass of 16.5 kD. Survivin functions to inhibit mitochondrial pathway of apoptosis and regulate cell division as a component of the spindle checkpoint machinery [
1]. Overexpression of survivin was detected in many types of cancers including esophageal squamous cell carcinoma (ESCC) but rarely in normal differentiated adult tissues [
2]–[
4]. Furthermore, elevated levels of survivin have been associated with poor prognosis in several human cancers [
5]–[
9]. In ESCC patients high levels of survivin expression indicated poor prognosis as well as high resistance to radiotherapy and chemotherapy [
10],[
11]. Accordingly, the suppression of survivin expression with the use of antisense oligonucleotides or ribozymes effectively overcame apoptosis resistance in different types of cancer cells and sensitized cancer cells to radiation and chemotherapeutic agents in vitro and in vivo [
12]–[
16].
It is thought that survivin enhances tumor cell survival primarily through the suppression of apoptosis-related cell death via direct inhibition of caspase-related proteins. However, it has become increasingly clear that the role of survivin in response to ionizing radiation far exceeds a simple inhibition of apoptotic pathway. The underlying molecular mechanisms seem to be multifaceted and involve broader cellular adaptation processes within separate subcellular compartments. Chakravarti
et al. are the first to report on novel caspase-independent mechanisms through which survivin enhances tumor cell survival upon radiation exposure, including the regulation of double-strand DNA break repair and cell metabolism [
12]. Recently, Reichert
et al. found a direct relationship between survivin and components of the DNA-double strand break (DSB) repair machinery following irradiation in radiation resistant glioblastoma cells [
17].
In the nucleus, survivin is selectively expressed at G
2/M phase of the cell cycle and localized to microtubules of the mitotic spindle, thus performing the role of the regulator of cell division [
18]. Connor
et al. showed that survivin underwent cell cycle-dependent phosphorylation on Thr34 by a Cdc2/cyclin B1 complex, which was required to prevent from caspase-9-dependent apoptosis of cells traversing mitosis and preserve cell viability at cell division [
19]. Thus we speculated that the attenuation of survivin expression is expected to impact DNA damage induced G
2/M checkpoint.
YM155 was identified as a first-in-class small molecule inhibitor of survivin. YM155 selectively inhibited survivin expression at both mRNA and protein levels at subnanomolar range and exhibited anticancer activity in preclinical models of several types of cancers [
20]–[
22]. However, the effectiveness of YM155 with ESCC has not been confirmed. In the present study, we employed two ESCC cell lines Eca109 and TE-13 to evaluate the radiosensitizing effects of YM155 on ESCC, with a special emphasis on its interference with cell cycle checkpoint.
Discussion
In the present study, we have shown that survivin inhibition by YM155 enhanced radiation-induced inhibition of growth of esophageal cancer cells and xenografts. Radiosensitization by YM155 in ESCC is associated with the abrogation of radiation-induced G2 checkpoint as well as the attenuation of homologous-recombination-mediated DNA damage repair.
Iwasa
et al. indicated that radiosensitization of NSCLC cells by YM155 was associated with increased activity of caspase-3, suggesting that YM155 sensitized tumor cells to radiation partly by enhancing radiation-induced apoptosis [
23]. Our findings now show that abrogation of G
2 checkpoint in response to radiation is an additional mechanism of sensitization by survivin inhibitor in esophageal cancer cells. We found that YM155 at nanomolar concentration was sufficient to abrogate G
2 arrest. It is known that shortening of the G
2 checkpoint leads to decreased repair of radiation-induced damage that could take place before cell division [
24],[
25]. In support of this possibility, we found that YM155 significantly increased persistent γ-H2AX expression and suppressed RAD51 recruitment in the nuclei of irradiated ESCC cells. The impairment in DSB repair could enhance the apoptosis induced by irradiation. Consistently, we demonstrated that YM155 enhanced the apoptosis and promoted the cleavage of caspase-3 and PARP in irradiated ESCC cells.
For the radiosensitization of cancer cells, pharmacological agents with the ability to inhibit specific checkpoint components, particularly at the G
2/M transition, have gained interest recently. UCN-01, AZD7762 and SB-218078 have demonstrated synergy with ionizing radiation to inhibit cancer growth by abrogating G
2/M checkpoint through selective inhibition of Chk1 [
26]–[
28]. Caffeine and pentoxifylline were radiosensitizers that disrupted radiation-induced G
2/M checkpoint by inhibiting the activation of ATM and ATR [
9],[
11]. DNA damage induced G
1/S arrest and subsequent Non-homologous end joining (NHEJ) repair are p53-dependent and, thus, are deficient in many tumor cells [
29]. Wang
et al. showed that UCN-01 was much less active in abrogating γ-ray-induced G
2 checkpoint in MCF-7 cells that had normal p53 function, but it preferentially increased the cytotoxicity of cisplatin in MCF-7 cells defective for p53 function [
28]. In addition, Chk1 inhibition preferentially sensitizes HCT116 p53−/− cells (versus HCT p53+/+) to gemcitabine and radiation [
30],[
31]. Therefore, the cells harboring mutations in p53 are deficient in G1 checkpoint and depend on p53-independent G2 checkpoint for DNA damage repair, rendering them more sensitive to G2 checkpoint abrogation [
32]. Similarly, in this study we found that radiation caused typical G
2/M arrest in both ESCC cell lines, indicating aberrant function of p53 in these cell lines. However, further studies are needed on more types of cells with identified p53 status to evaluate the influence of p53 function on YM155 mediated abrogation of G
2 checkpoint and enhancement of radiation cytotoxicity.
Although this is the first study demonstrating G2 checkpoint abrogation by a survivin suppressant during radiosensitization, some efforts have previously been made to investigate the contribution of survivin inhibition to cell cycle transition. Transfection of siRNA directed against survivin caused specific G0/G1 phase arrest in hepatocellular and lung cancer cells [
33],[
34]. However, in gastric cancer, survivin-specific siRNA caused cells accumulation in the G2/M phase and diminished the number of cells in the G0/G1 phase [
35]. Yamanaka et al. reported that the concomitant treatment of YM155 relieved docetaxel-induced cell cycle arrest at the G2/M phase and synergistically enhanced the cytotoxic activity of docetaxel [
36]. Accordingly, our present findings demonstrate that YM155 abrogates the G2 arrest induced by ionizing radiation, thus resulting in preferential cancer cell death. YM155 probably abrogates G2 checkpoint by inhibiting the Wee1/Mik1 kinases that suppress Cdc2 activation or by activating the Cdc25C phosphatase that, in turn, activates Cdc2 [
37].
Methods
Cell culture and reagents
The human esophageal squamous cell carcinoma cell lines Eca109 and TE13 were obtained from Shanghai Cell Bank (Chinese Academy of Sciences, Shanghai, China). The cells were cultured in RPMI 1640 (Hyclone, Thermo Scientific, MA) supplemented with 10% fetal bovine serum (Hyclone) at 37°C under a humidified atmosphere of 5% CO2. YM155 monobromide was purchased from SelleckChem (Houston, TX). For the in vitro experiments, YM155 was dissolved in DMSO and diluted in medium to a final DMSO concentration of ≤0.1%. For the in vivo experiments, YM155 was dissolved and diluted in saline immediately before administration. Mitotic inhibitor nocodazole was purchased from Beyotime institute of biotechnology (Shanghai, China).
Irradiation conditions
X-ray radiation was delivered by a 6 MV linear accelerator (Elekta, Stockholm, Sweden) at a dose rate of 250 cGy/min with a source-to-target distance of 100 cm. For tumor irradiation, animals were anesthetized with isoflurane and positioned to place the tumor in the center of 1.0 × 1.0 cm radiation field, with the rest of mice shielded from radiation.
Cell viability assay
Cells were seeded in 96-well plates overnight, then treated with YM155 at various concentrations (0, 1, 2, 5, 8, 10, 25, 50, 100 nmol/L). Twenty-four and forty-eight hours later, 10 microliters of CCK-8 solution (Cell counting kit-8, Dojindo Molecular Technologies, Gaithersburg, MD, USA) was added to each well. Absorbance was determined at 450 nm after 3 hours of incubation. The viability of cells was calculated as following: Viability = (ODtest groupODblank group)/(ODcontrol groupODblank group) × 100 %, and IC50 (half maximal inhibitory concentration) was calculated from the dose–response curves. All experiments were repeated in triplicate.
Western blot analysis
Cells were harvested and homogenized in RIPA lysis buffer and centrifuged at 12,000 rpm for 20 min at 4°C. Protein concentrations of the supernatants were determined using BCA Protein Quantification Kit (Vazyme biotech co., Itd.). Western blot analysis was performed as previously described [
38], using rabbit polyclonal antibodies to human survivin and c-IAP1 (R&D systems, MN), XIAP and Cyclin B1 (Cell signaling, MA), phosphor-Cdc25C, Cdc2, phospho-Cdc2, cleaved PARP and cleaved caspase-3 (Santa Cruz, CA).
Clonogenic survival assay
Exponentially growing cells were trypsinized as single-cell suspension and diluted serially to appropriate densities and seeded in triplicate in six-well plates. After cell adhesion, they were treated with 0.1% DMSO (control) or YM155 (5 nM or 10 nM) for 24 h, and then subjected to 0, 2, 4, 6 or 8 Gy X-rays irradiation. The cells were then washed with PBS, cultured in drug-free medium for 14 days, fixed with methanol, and stained with Giemsa. Only colonies containing more than 50 cells were scored. SF (surviving fraction) of each irradiation group was corrected by the PE (plating efficiency) of the nonirradiated control. The cell survival curves were fitted according to a multi-target single-hit model and the survival enhancement ratio (SER) was calculated as the ratio of the mean inactivation dose in control cells divided by the mean inactivation dose in YM155-treated cells. The experiment was repeated for three times.
Flow cytometry for cell cycle
Eca109 or TE13 cells were harvested after 24 h or 48 h treatment with YM155 (10 nM) and/or 8 Gy X rays, respectively. After washing with ice-cold PBS, the cells were fixed with ice-cold 70% ethanol and stored at −20°C for 1 h. Before analysis by flow cytometry, the cells were washed with PBS, resuspended in a staining solution containing 20 μl RNase A solution and 400 μl propidium iodide staining solution (Vazyme biotech co., Itd). Next, cell cycle distribution assessment was performed using a fluorescence-activated cell sorter (BD FACS Calibur).
Cdc2-Associated H1 Kinase assay
A total of 4 × 107 cells were lysed in 400 μl lysis buffer (50 mM Hepes/NaOH, pH7.4, 150 mM NaCl, 1% Triton X-100, 1 mM dithiothreitol, protease/phosphatase inhibitor cooktail) on ice for 20 min. The cell lysate was centrifuged at 15,000 rpm for 10 min at 4°C and concentration of protein in the supernatant was determined using a BCA protein quantification kit (Beyotime, Shanghai, China). Cellular extracts were cleared by incubation with protein A or protein G-agarose (Cell Signaling, MA) for 30 min at 4°C. After centrifuging at 2,500 rpm for 5 min at 4°C, the supernatant was incubated with 4 μg Cdc2 antibody (Cell Signaling, MA) and 20 μl protein A/G agarose beads for 2 h at 4°C. The protein-agarose beads and bound immune complexes were then pelleted by centrifugation and immunoprecipitates were washed twice with lysis buffer and twice with wash buffer (50 mM Hepes/NaOH, pH 7.4, 10 mM MgCl2, 1 mM dithio-threitol) and then subjected to a Cdc2 kinase assay using histone H1 peptide (Merck Millipore, Darmstadt) as a substrate. The immunocomplex was incubated with 40 μg wash buffer containing 10 μg of histone H1, 10 mM adenosine triphosphate (ATP) for 10 min at 30°C, and then subjected to immunoblotting using phosphoserine/threonine/tyrosine antibody (Abcam, Cambridge, MA).
Immunofluorescence
Immunfluorescence detection of phospho- H2AX and RAD51 foci was performed to monitor DNA double-strand breaks (DSBs) formation and homologous recombination repair (HRR). Cells cultured on coverslips were treated with 10 nM YM155 and irradiated with a dose of 4 Gy to assure a discrimination of individual nuclear foci in immunofluorescence staining. At indicated time points, the cells were fixed by 4% paraformaldehyde for 20 min at room temperature and permeabilized with 0.1% Triton X-100 for 10 min at 4°C. After blocking with Immunol Staining Blocking Buffer (Beyotime, Shanghai, China) for 1 h at room temperature, cells were incubated with antibody for phospho-H2AX (Ser139) (Millipore/Upstate, Temecula, CA) and RAD51 (EMD Millipore, Billerica, MA) at 4°C overnight, followed by staining with Fluorescein (FITC)-conjugated goat anti-mouse IgG (Jackson Immunoresearch, PA) and Rhodamine (TRITC)-conjugated goat anti-rabbit IgG (Jackson Immunoresearch, PA) for 1.5 h at room temperature. Finally, the samples were counterstained with 2 μg/ml DAPI and mounted in 3 μl of mounting medium (Beyotime). Three random fields each containing 50 cells were examined at a magnification of ×100 under a Zeiss LSM5 confocal laser-scanning microscope (Carl Zeiss, Jena, Germany). Nuclei containing ≥10 immunoreactive foci were scored as positive for γ-H2AX, and ≥5 foci as positive for RAD51.
Apoptosis analysis
Annexin V-FITC/PI dual staining was performed by using apoptosis detection kit from Keygen Biotech (Nanjing, China) according to the manufacturer’s instruction. Flow-cytometric analysis for induction of apoptosis was performed as previously described [
38].
Xenograft tumor radiosensitivity studies
Animal experiments were approved by Ethics Committee of Nanjing Medical University. Five to six week-old male BALB/C nude mice were provided by Nanjing Medical University Animal Center. A suspension of 1 × 106 Eca109 cells in 0.1 mL PBS was injected s.c. into one site of the right leg of nude mice. Tumors were allowed to grow for 7 days before treatment. Twenty-four nude mice with established tumors (all ~250 mm3) were divided into four groups and treated with (a) vehicle (PBS) alone; (b) a single dose of 8 Gy IR; (c) YM155 alone (5 mg/kg as a 7-day continuous infusion); or (d) YM155 plus IR (a single fraction of 8 Gy IR delivered on day 3 of drug treatment). Body weight and tumor diameter were measured three times per week, and tumor volume was determined according to the formula: (length[L] × width[W]2)/2. The efficacy of each treatment was evaluated by the volume change during the treatment period. Growth delay time (GD) was calculated as the time for treated tumors to reach double in volume minus the time for control tumors to reach double in volume. The enhancement factor (EF) was then determined as follows: EF = (GDIR + YM155–GDYM155)/GDIR. The first day of treatment was designated as day 0, and observation continued until the day 20. At the end of observation, mice were sacrificed and tumors were fixed in 10% formalin, embedded in paraffin, and cut into 5 μm-thick sections for immnohistochemistry (IHC) and terminal deoxynucleotidyltransferase (dUTP) nick-end labeling (TUNEL) assay.
Immunohistochemistry
Paraffin-embedded tumor tissue sections were deparaffinized in xylene, rehydrated in graded ethanol, and rinsed twice with PBS. Endogenous peroxidase activity was blocked by incubating sections with 3% hydrogen peroxide in the dark for 15 min. The sections were then incubated overnight at 4°C with polyclonal antibody to survivin (1:50; Abcam, Ltd., Cambridge, United Kingdom) or cleaved caspase-3 (1:100; Cell Signaling, Beverly, MA, USA). After washes, slides were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody for 1 h at room temperature. Finally, the slides were visualized by incubation with 3, 3′-diaminobenzidine (DAB) (Dako, Hamburg, Germany) and counterstained with hematoxylin (37%). The sections were analyzed under a Zeiss Axiovert A1 light microscope. The intensity for survivin immunoreactitity in tumor cells was evaluated using Image-Pro Plus 6.0 software. Mean intensity was calculated by dividing the integral optical density (IOD) by area. The cleaved caspase-3 labeling index was calculated by dividing the number of activated caspase-3-positive cells by the total number of nuclei (per 1000 tumor cells from four microscopic visual fields).
TUNEL assay
Paraffin-embedded tumor tissue were deparaffinized, rehydrated and blocked in 3% hydrogen peroxide as above. The slides were permeabilized with TritonX-100 in 0.1% sodium citrate for 10 min. TUNEL staining was performed using the in situ cell death detection Kit-POD (Roche Molecular Biology, Mannheim, Germany) according to the manufacture’s protocol. Peroxidase activity was visualized using the liquid DAB substrate chromogen system (Dako), followed by a haematoxylin counterstaining. The percentage of apoptosis was calculated by dividing the number of TUNEL-positive cells by the total number of nuclei (per 1000 tumor cells from four microscopic visual fields).
Statistical analysis
All data were expressed as the mean ± standard deviation (SD). Statistical differences between groups were determined by the unpaired Student’s t test or one-factor ANOVA. All statistical analysis was performed using SPSS statistics 17.0 software (SPSS, Inc., Chicago, IL, USA). A P value < 0.05 was considered statistically significant.
Acknowledgements
This work was supported by funds from the College Graduates Research and Innovation Project of Jiangsu Province (no. CXZZ12_0588), the Natural Science Foundation of China (no. 81272504), the Innovation Team (no. LJ201123), Jiangsu Provincial Natural Science Fund (no. BK2011854), “333” Project of Jiangsu Province (no. BRA2012210), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) (no. JX10231801), the Key Academic Discipline of Jiangsu Province “Medical Aspects of Specific Environments”, and the Six Major Talent Peak Project of Jiangsu Province (no. 2013-WSN-040).
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
QQ, CHY, LJ, ZLL, and SXC conceived and designed the experiments; QQ and CHY performed the experiments; QQ, CHY, ZJC, and SXC contributed reagents and wrote the manuscript; CJ, YX, ZC, LJ, XLP, and ZHC provided critical comments on the design of the study, analysis, and of the interpretation of data; all authors supplied data, critically revised, and gave final approval of the article.