Background
Nowadays, esophageal cancer has been identified as the sixth most common cause of cancer-related death all over the world, and more than one third of esophageal cases are diagnosed at advanced stage [
1]. Esophageal squamous cell carcinoma (ESCC) accounts for more than 90% of this cancer in China [
2]. Although there are three major treatment modalities for esophageal cancer including surgery, radiotherapy and chemotherapy, radiotherapy is widely applied to the patients who are not candidates for operations [
3‐
5]. However, intrinsic radioresistance is developed in large portion of cancer cells of esophageal carcinoma patients, resulting in the failure of treatment, therefore, it is urgent to develop methods to increase the radiation sensitivity to reduce the mortality of esophageal cancer.
Raltitrexed, also named ZD1694 or Tomudex, has been considered as a specific and long-lasting inhibitor against thymidylate synthase (TS). As we know, TS plays a critical role in DNA synthesis and repair. Several clinical studies have indicated that TS is increased in tumor tissues, suggesting TS is involved in tumorigenesis or progression. Raltitrexed, as an TS inhibitor, is well known to be effective in the treatment of colorectal cancer. Meanwhile, preclinical studies reported that raltitrexed also showed antitumor effect in other kinds of cancers due to its direct anti-proliferation and anti-apoptosis effects, including colorectal cancer, malignant mesothelioma, head and neck cancer, hepatic cancer and gastric cancer [
6‐
11]. Raltitrexed has also been considered as an antimetabolic drug, which could induce DNA damage [
12] and cell cycle arrest [
13]. However, there is little known about the effect of raltitrexed in improving the radiosensitivity of ESCC and according molecular mechanism. In the present study, we investigated raltitrexed-mediated radiosensitization and the underlying molecular mechanism.
Materials and methods
Reagents, cell culture, and irradiation
Raltitrexed, generously provided by Jiangsu Zhengda Tianqing Pharmaceutical Co., Ltd. (Nanjing, China), was dissolved in dimethyl sulfoxide (DMSO, Sigma, St. Louis, USA) as a stock concentration of 1 mol/l. Two human esophageal cancer cell lines, Kyse150 and TE-13, were obtained from Shanghai institute of Biological Science, China. Cells were cultured in RPMI 1640 (Hyclone, Thermo Scientific, MA) supplemented with 10% fetal bovine serum (Hyclone), 100 U/ml penicillin and 100 mg/l streptomycin at 37 °C in a humidified atmosphere of 5% CO2. Irradiation was delivered at a dose rate of 566 cGy/min, using an X-ray medical linear accelerator (Elekta Precise, Sweden). Cells were irradiated at a single dose at room temperature.
Cell counting kit 8 assay
The cytotoxicity effect of raltitrexed was measured by Cell counting kit 8 (CCK-8) assay (Beyotime Technology, Shanghai, China). Briefly, cells were plated in 96-well plates at a density of 4 × 103 cells/well. Then, fresh medium was added with or without raltitrexed after 24, 48, and 72 h incubation. Following the treatment, CCK-8 reagent (10 μl/well) was added into each well and cells were incubated with CCK-8 reagent for 1 h. The absorbance ratio of each sample was measured at 450 nm using a microplateReader (BioTek Elx800, Winooski, VT, USA). All experiments were repeated at least three times.
Cell proliferation assay after radiation
For combination treatment, TE-13 and Kyse150 cells were plated at a concentration of 6 × 103 cells/well in 96-well plates for18 h. Then cells were treated with raltitrexed (4 nmol/l [nM]) for 24 h and different radiation doses (0, 2, 4, 6, 8 Gy). 48 h later, CCK-8 assay Kit was used to assess cell proliferation. The OD ratio was used to calculate the cell proliferation capacity. All experiments were repeated three times.
Clonogenic survival assay
Cells were seeded at varying cell densities in six-well plates after trypsinization, which were treated with raltitrexed (4 or 8 nM) or DMSO as control for 24 h, and then subjected to X-rays of 0, 2, 4, 6, 8 Gy. The cells were cultured at 37 °C for another 10 days, following fixing with 75% ethanol for 15 min and then staining with crystal violet for 10 min. The colonies with more than 50 cells were counted. The mean inactivation dose in raltitrexed treated cells divided the mean inactivation dose in control cells as the survival enhancement ratio (SER) that combined with single-hit multi-target model to make cell survival curves. All experiments were repeated three times.
Cell cycle analysis
TE-13 and Kyse150 cells were incubated in six-well plates (2 × 105 per well) in RPMI-1640 medium without serum for 18 h and divided into the four groups, respectively: control, raltitrexed (4 nM), irradiation (4 Gy), and irradiation with raltitrexed (4 Gy + 4 nM). Each group was repeated in three wells. After a 24 h exposure to raltitrexed followed by a 24 h exposure to X-ray, all cells were harvested and washed with cold Phosphate Buffered Saline (PBS), and then fixed with 70% ethanol at 4 °C overnight. Before detecting, all samples were washed with 1× PBS, then incubated with 6 ul of 1 g/l RNase A, 1 ml of 1 mg/ml Propidium iodide (PI), and 400 ul of PBS at room temperature and protected from light for 15 min. Deoxyribonucleic acid (DNA) content and cell cycle distribution were analyzed using flow cytometry (FCM, BD FACS Calibur). All experiments were repeated for three times.
Apoptosis analysis
TE-13 and Kyse150 cells were seeded in six-well plates (1 × 105 per well) for 12 h and divided into the four groups: control, raltitrexed (4 nM), irradiation (8 Gy), and irradiation with raltitrexed (4 nM + 8 Gy). All cells were harvested and washed with cold PBS after 24 h exposure to raltitrexed followed by 24 h exposure to X-ray. Apoptotic cells were distinguished by Annexin V-FITC/PI dual staining using apoptosis detection kit from Keygen Biotech (Nanjing, China) and analyzed by flow cytometry (FCM, BD FACS Calibur). All experiments were repeated for three times.
Immunofluorescence staining for phosphor-Histone H2AX (γ-H2AX) detection
DNA double strand breaks (DSBs) and DSB repair capacity were determined using immunofluorescence staining for γ-H2AX foci. TE-13 and Kyse150 cells were seeded on cover slips and then treated with either raltitrexed or DMSO (control). After 24 h of incubation, the cells were irradiated with 6 Gy of γ-rays and followed with 4% paraformaldehyde for 20 min at 2, 4, 24 h after irradiation. And then samples were permeabilized in 0.1% TritonX-100 for 10 min at 4 °C followed by washing with PBS and block with 5% bovine serum albumin at 37 °C for 1 h. Antibody against γ-H2AX (1:250) and 0.25 mg/ml DAPI (Beyotime, Jiangsu, China) were added for 5 min. Images were captured by a charge coupled device camera. For each group, γ-H2AX foci were counted in at least 50 cells per filed [
14,
15]. Experiments were repeated for three times.
Western blot analysis
TE-13 and Kyse150 cells were cultured and divided into eight groups: the control group, raltitrexed (4 nM) group, irradiation (4 Gy) groups (2 h, 4 h, 24 h) and raltitrexed combined with irradiation groups (2 h, 4 h, 24 h). Cells were collected and lysed with RIPA lysis buffer (Beyotime Biotechnology, Jiangsu, China). Protein concentrations were determined using BCA Protein Quantification Kit (Beyotime Technology, Shanghai, China). An equal amount of protein (50 ug) for each sample was resolved using 5% or 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS/PAGE) with MOPs running buffer and electrophoretically transferred onto a polyvinylidene difluoride (PVDF) membranes (IPVH00010, Millipore, Massachusetts, USA). The membrane was blocked with 5% bovine serum albumin at 37 °C for 1 h and sequentially blotted with primary antibodies (rabbit polyclonal or monoclonal antibodies to human Cyclin B1, Cdc25C, phosphor-Cdc25CS216 [pCdC25C], Cdc2, phospho-Cdc2Thr14/Tyr15[pCdc2], P53, P21Waf1/Cip1, γ-H2AX, Bax, c-Caspase-3, and folylpolyglutamate synthase [FPGS] 1:1000) and GAPDH antibody (1:2000), After washing with cold PBS, peroxidase-conjugated goat anti Rabbit IgG (1:5000) secondary antibody was added and followed by ECL detection. Experiments were repeated for three times.
Xenograft tumor radiosensitivity studies
Animal experiments protocol was approved by Ethics Committee of Nanjing Medical University. Five to six week-old male BALB/C nude mice (Nanjing OGpharma technology lt. co) were injected with the suspension of 5 × 106 TE-13 cells in 0.1 ml PBS into the right sub axillary. After 1–2 weeks post-injection, twenty-four nude mice with established tumors (all about 90 mm3) were divided into four groups: control group treated with (a) vehicle (PBS) alone; (b) Raltitrexed alone (7.5 mg/kg/day, continuous infusion at day 0–4 and day 7–11); (c) a single dose of 8 Gy IR; or (d) Raltitrexed plus IR (a single fraction of 8 Gy IR delivered on day 0 after Raltitrexed administration). Body weight and tumor diameter were measured every other day, and tumor volume was calculated according to the formula: (length [L] × width [W]2)/2. The tumor volume was recorded before and after treatment to evaluate the relative tumor volume (RTV) by Vt/V0 (Vt represents tumor volume, V0 represents pretreatment tumor volume). And the relative tumor control rate (T/C (%)) was determined by following formula: (RTV of Treatment group/ RTV of control group) × 100. After 19 days observation, tumors were harvested, fixed in 10% formalin, embedded in paraffin (FFPE), and mounted onto glass slides with 5 μm-thick sections for immunohistochemistry (IHC) assay.
Immunohistochemistry
Slides were deparaffinized in xylene followed by rehydration in graded ethanol, rinsing twice with PBS, endogenous peroxidase blocking in 3% hydrogen peroxide for 15 min. Then the specimens were incubated with monoclonal antibody to PCNA (1:200, Abcam, Ltd., Cambridge, United Kingdom) or Ki-67 (1:200, Cell Signaling, Beverly, MA, USA) overnight at 4 °C and detected with horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody for 1 h at room temperature or overnight at 4 °C. Next, the slides were visualized by incubation with 3,3-diaminobenzidine (DAB) (Dako, Hamburg, Germany), counterstained with hematoxylin (37%) and photographed using a Zeiss Axiovert A1 light microscope.
Statistical analysis
All statistical analysis was performed using the GraphPad Prism software package version 6.0. All data were collected from three independent experiments and expressed as the mean ± standard deviation (SD). The Student’s t test or one-factor ANOVA was used to explore significant statistical differences between groups. P value less than 0.05 (p < 0.05) was considered statistically significant.
Discussion
Our study demonstrated that raltitrexed could enhance the radiosensitivity of ESCC cells in vitro and in vivo. The viability and proliferation of ESCC cells were decreased in a time and dose-dependent manner by raltitrexed incubation. And the apoptosis was increased by raltitrexed combined with irradiation. Furthermore, we found G2/M phase arrest contributed to radiosensitization of raltitrexed through the upregulation of Cdc2/Cyclin B1 complex and the increased expression of γ-H2AX foci, which indicated the inhibition of DNA-damage repair. Besides, we observed that raltitrexed sensitized TE-13 xenograft tumor to irradiation. Therefore, raltitrexed could be used as a radiosensitizer to treat ESCC.
To prevent cells from entering into a new phase, a number of cell cycle checkpoints have been made to ensure proper cell cycle progression. It is widely accepted that the cell cycle phase determined different degrees of radiosensitivity of cells to radiation. G2 and M phases are the critical time points that cells are most sensitive to irradiation and most radioresistant time point was S phase cell cycle. Cdc2/Cyclin B1 complex activity has been considered as an import role in G2/M phase transition. Several studies have indicated that G2/M cell cycle arrest showed decreased the expression of Cdc2/Cyclin B1 [
18,
19]. Increased Cdc2/Cyclin B1 activity can promote radiation-induced cell apoptosis by inducing G2/M transition [
20,
21]. However, cyclin B1 is also related to radioresistance [
22]. In this work, we detected the changes in the cell cycle progression by using flow cytometry and western blot. The results showed that radiation alone induced G2/M-phase arrest in TE-13 and Kyse150 cells, while combined with raltitrexed, the G2/M arrest was significantly increased. Meanwhile, the protein expression of Cyclin B1, pCdc2, pCdc25c were increased after exposing to raltitrexed combined with irradiation. These results may be related to the transition from G2 phase to M phase. G1/S and G2/M transition are the two major checkpoints of cell cycle, on the occasion of DNA damage or incomplete DNA replication, the checkpoints can prevent inappropriate cell cycle progression [
23]. The G2/M checkpoint is known to involve a number of proteins, including P53, P21, Cyclin B1, Cdc2, Cdc25C, Chk1 and so on [
24,
25]. P53 is a tumor suppressor, which can induce cell apoptosis, cell cycle arrest and DNA repair in response to a variety of intrinsic or extrinsic factors [
26]. In certain situations, P53 may actually lead damaged cells to self-destruct in order to prevent damaged genetic goods handed down [
26,
27]. Reportedly, P53 is required for P21 induction following exposure to radiation [
28] and P21 can be regulated independently of P53 in certain situations. In our study, we found that the protein expression of P53 was positive in Kyse150 cells and was obviously increased after treatment, especially in the case of raltitrexed combined with Radiation, while in TE-13 cells, P53 was negative and we also observed the increased expression of P21 at 24 h after treatment, obviously later than in Kyse150 cells. The increased P53 and P21 can inhibit DNA damaged cells from entering into mitosis. At the beginning of cell mitosis, Cdc25C is activated and modulates Cdc2/Cyclin B1 complex. The complex of Cdc2/Cyclin B1 at G2/M transition keeps an inactive state by phosphorylation of Cdc2 at Thr-14 and Tyr-15. Our results found that in both TE-13 and Kyse150 cell lines, Cdc2 was remarkably phosphorylated at 4 h after irradiation treatment, especially higher in the combined group, which indicated G2 checkpoint arrest. Moreover, the expression of pCdc2 decreased quickly at 24 h exposure in Kyse150 cell line, which suggested that cells were unable to sustain stable G2 arrest. Meanwhile, the activation of Cdc25C may result in the start of mitosis for cells, which initiates cell death program due to the uncompleted DNA repair. In the TE-13 cell line, this process may be delayed, which can partly explained why the apoptosis rate in TE-13 cells was less than that in Kyse150 cells at 24 h after treatment and was more than in Kyse150 cells at 48 h after treatment.
Raltitrexed has been considered as a specific inhibitor of TS [
29] and could predominantly enter the cell through the reduced folate carrier (RFC) and then undergo polyglutamation by FPGS enzyme. The polyglutamated compound stayed in cells and acted as an anticancer agent. Previous studies have found that raltitrexed could inhibit HepG2 cell proliferation via G0/G1 arrest [
7], inducing the apoptosis of cancer cells [
6]. And we observed that raltitrexed could increase radiation-induced G2/M arrest and apoptosis in ESCC cells, and the latter was confirmed by the increase of c-caspase-3 expression. C-caspase-3 has been identified as an executer of apoptosis that was associated with two signaling pathways, including the mitochondrial and the cell death receptor pathway. And the activation of Caspase-3 is essential for DNA fragmentation. Therefore, the expression of c-Caspase-3 was measured to show the cytotoxic responsiveness as an index. Based on our results, we conclude that apoptosis induction was a possible mechanism for increased radiosensitivity of ESCC cells that was induced by raltitrexed. In this study, we also observed an increasing expression of phosphor-H2AX after raltitrexed treatment in TE-13 and Kyse150 cells, which suggested increased DNA damage. As we known, DNA DSBs are associated with cell cycle arrest and/or death due to unrepaired molecular lesions [
30]. Cell death may involve in a single unrepaired or misrepaired DNA DSB of a functional gene section [
31]. A previous study suggested that raltitrexed treatment in HCT-8 cells resulted in DNA fragmentation and was accompanied with elevated P53 protein expression [
32]. In our study, we found elevated P53 accompanied with overexpression of γ-H2AX in Kyse150 cell line.
Above all, our study demonstrated for the first time that raltitrexed could significantly enhance the radiosensitivity of ESCC cells via inducing G2/M arrest by activating the Cdc2/Cyclin B1 pathway and increasing cell apoptosis. Moreover, raltitrexed could sensitize TE-13 xenograft tumor to irradiation. These results suggested that raltitrexed could be a potent radiosensitization agent for ESCC treatment in the future. In this study, we also observed different protein expression of P53 in TE-13 and Kyse150 cells, showing that P53 was negative in TE-13 while Kyse150 was positive. There are probably other mechanisms involved in the radiosensitization effects of raltitrexed in ESCC cell lines, which needs further investigation.
Authors’ contributions
WXD and XCS designed the study. WXD, JXM, JP, SL, HJW and SZ did the experiments. All the authors wrote and revised the manuscript. All authors read and approved the final manuscript.