|Year : 2018 | Volume
| Issue : 1 | Page : 46-51
The role of NRAGE subcellular location and epithelial–mesenchymal transition on radiation resistance of esophageal carcinoma cell
Xiaojing Chang, Xiaoying Xue, Yafang Zhang, Ge Zhang, Huandi Zhou, Yanling Yang, Yuge Ran, Zhiqing Xiao, Xiaohui Ge, Huizhi Liu
Department of Radiotherapy, The Second Hospital of Hebei Medical University, Shijiazhuang, China
|Date of Web Publication||8-Mar-2018|
Prof. Xiaoying Xue
Department of Radiotherapy, The Second Hospital of Hebei Medical University, Shijiazhuang 050000
Source of Support: None, Conflict of Interest: None
Background: Neurotrophin receptor-interacting MAGE homolog (NRAGE) has been considered as a tumor suppressor. In the previous study, we established human esophageal carcinoma resistance cell line TE13R120 and found the difference of NRAGE expression between TE13 and TE13R120 cells by gene microarray. Herein, we further discuss the possible molecular mechanism of NRAGE on participating the radiation sensitivity of esophageal carcinoma cells.
Materials and Methods: We used colony formation assay to measure the surviving fraction and relevant radiobiological parameters. NRAGE expression was estimated by immunofluorescence and Western blot. Tumor growth factor-β (TGF-β) was used for inducing epithelial–mesenchymal transition (EMT) in TE13 cells to detect the relationship between NRAGE and EMT; the capacity of cell migration was also assessed by wound healing assay.
Results: TE13R120 cells were showed significantly radioresistance compared with TE13. The D0, Dq, and N value of TE13R120 were all higher than those of TE13 (2.499, 1.991, and 2.219 vs. 2.242, 0.854, and 1.645), as well as SF2 (0.734 vs. 0.538). Results of immunofluorescences showed that NRAGE was mainly expressed in the nucleus of TE13R120 cells, but in TE13 cells, it was mainly in cytoplasm. In addition, EMT phenotype was observed in TE13R120 cells and TGF-β-induced EMT in TE13 cells, E-cadherin expression was decreased, but vimentin was upregulated. Furthermore, TE13 cells have a rising tendency in NRAGE nucleus expression after treatment with TGF-β. Results of wound healing assay showed that the cell migration of TE13R120 and TGF-β-induced EMT in TE13 cells were remarkably enhanced.
Conclusions: Our results indicate that NRAGE subcellular localization is related to radiation resistance of esophageal carcinoma cell and EMT may be involved in NRAGE subcellular location.
Keywords: Epithelial–mesenchymal transition, esophageal carcinoma, neurotrophin receptor-interacting MAGE homolog, radiation resistance, subcellular location
|How to cite this article:|
Chang X, Xue X, Zhang Y, Zhang G, Zhou H, Yang Y, Ran Y, Xiao Z, Ge X, Liu H. The role of NRAGE subcellular location and epithelial–mesenchymal transition on radiation resistance of esophageal carcinoma cell. J Can Res Ther 2018;14:46-51
|How to cite this URL:|
Chang X, Xue X, Zhang Y, Zhang G, Zhou H, Yang Y, Ran Y, Xiao Z, Ge X, Liu H. The role of NRAGE subcellular location and epithelial–mesenchymal transition on radiation resistance of esophageal carcinoma cell. J Can Res Ther [serial online] 2018 [cited 2019 Nov 12];14:46-51. Available from: http://www.cancerjournal.net/text.asp?2018/14/1/46/226752
| > Introduction|| |
Esophageal carcinoma is one of the most common fatal cancers in the world, especially in China; it is the fourth leading cause of cancer-related deaths; to date, the prognosis remains poor., Radiotherapy is an important form of local and regional therapy for malignant tumors, particularly for the cervical and upper thoracic esophageal carcinoma and radiation therapy is the preferred. The curative effect depends on the radiation sensitivity of tumor tissues. However, radiotherapy has been reported to be related with a high rate of local recurrence and metastasis, the reason mainly due to the radiation resistance of cancer cells, which leads to radiotherapy failure., Therefore, how to improve the radiation sensitivity of esophageal cancer, increasing the local control and radiation effect will be the hot spot in the field of tumor radiobiology research in the future.
A lot of studies have shown that some tumour-related genes contribute to the radioresistance of many types of cancers.,, Neurotrophin receptor-interacting MAGE homolog (NRAGE), also named Dlxin-1 and MAGE-D1, belongs to MAGE family and is a p75 neurotrophin receptor-interacting protein. NRAGE is expressed in most developing and adult tissues and plays a pivotal role in the growth and differentiation of normal cells, especially in nervous system.,, Studies found that NRAGE regulated the switch between differentiation and apoptosis of neural progenitors and P19 cell through the BMP signaling pathway., A newly published study also reported that NRAGE promoted the proliferation and weakened the odontoblastic differentiation of mouse dental pulp cells through the NF-κB signaling.
Recently, some studies found that NRAGE might be involved in the metastatic progression of tumor cells. To date, numerous studies support the tumor suppressor role of NRAGE as it induces cell apoptosis and suppresses cell metastasis.,, Studies reported that NRAGE regulated the activation of NF-κB through interacting with XIAP-TAB1-TAK1 signaling and noncanonical BMP pathway, then inducing cell apoptosis., However, some studies found that NRAGE was overexpressed in esophageal cancer, hepatocellular carcinoma, and promoted cell growth., In previous work, we established human esophageal cancer resistance cell line TE13R120 through repeated X-ray irradiation on TE13 and found that NRAGE expression was much higher in TE13R120 compared with TE13 cell line by gene microarray technology, and further clinical study by IHC was indicated that NRAGE was overexpressed in the invalid samples of radiotherapy., Furthermore, the nuclear localization of NRAGE was observed to be more obvious in TE13R120 compared with TE13 cell line. Taken together, these results indicate that NRAGE expression, especially its nuclear localization may be relevant to the resistance of esophageal carcinoma.
An increasing number of studies have observed that radiation could induce epithelial–mesenchymal transition (EMT) in many types of tumors.,, A newly published study reported that enrichment of EMT pathway was observed with radioresistant phenotype through gene set enrichment analysis. In addition, Kumar et al. reported that ankyrin-G sequestered NRAGE in cytoplasm, but EMT could downregulate ankyrin-G, then enhanced the nuclear localization of NRAGE. Herein, we speculate radiation-induced EMT of esophageal carcinoma resistance cell line, then regulated the nuclear localization of NRAGE.
| > Materials and Methods|| |
Human esophageal carcinoma cell line TE13 was obtained from the Department of the First Surgery of Okayama University (Okayama, Japan), human esophageal carcinoma resistance cell line TE13R120 was developed by radiation treatment, both of the two cell lines were maintained as previous recommended. Cells were cultured in RPMI 1640 (Invitrogen; USA) which contains 10% fetal calf serum (FCS) and incubated in surroundings of 5% CO2 at 37°C.
Irradiation was performed using 6 MV X-rays which generated by a linear accelerator (Elekta Synergy, England Elekta Limited). The field of radiation was 20 cm × 20 cm and source-skin distance was 100 cm.
Clonogenic survival assay
TE13 and TE13R120 cells were plated in 100 mm dishes and irradiated with 0, 2, 4, 6, 8, and 10 Gy. After irradiation, cells were cultured for 2 weeks, then fixed with absolute ethanol containing 1% methyl violet for 20 min, and counted the number of surviving colonies (defined as a colony with >50 cells). The plating efficiency (PE) and the cell survival fraction (SF) were calculated as follows: PE = (number of colonies in the control group/number of inoculated cells) × 100% and SF = (number of colonies in the experimental group/number of inoculated cells)/PE. The SF was calculated by the GraphPad Prism 5.0 software (GraphPad Software, Inc. USA) based on the multitarget/single-hit model (SF = 1 − [1 − e −D/D0]N). Radiobiological parameters including the average lethal dose (D0), quasi-threshold dose (Dq), and the extrapolation number (N) were also calculated.
RNA extraction and real-time quantitative reverse transcription- polymerase chain reaction
Total RNA was extracted from cultured cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized using the Expand Reverse Transcriptase Kit (Takara, Japan). Reverse transcription reaction conditions: 42°C, 45 min, 95°C, 5 min. Real-time polymerase chain reaction (PCR) was performed in a 25 μl final volume containing 12.5 μl SYBR Green (Takara, Japan), 2 μl of each primer, and 8.5 μl of diethylpyrocarbonate water. PCR amplification condition was as follows: One cycle at 95°C for 30 s, 40 cycles at 95°C for 5 s, and 60°C for 30 s. Primers used as follows: NRAGE 5'-GCTCGGTCTCCTCTTGGTGATTC-3'(sense), 5'-GGCACTCGTCTGTAGTCCAGGTATT-3'(antisense), E-cadherin: 5'-CAGCGTGTGTGACTGTGAA G-3' (sense), 5'-AAACAGCAAGAGCAGCAG AA-3'(antisense), vimentin: 5'-CCTGCAGGATGAGATTCAGA-3' (sense), 5'-GGCAGAGAA ATCCTGCTCTC-3'(antisense), and GAPDH as 5'-CATGAGAAGTATGACAACAGCCT-3'(sense), 5'-AGTCCTTCCACGATACCAAAGT-3' (antisense). Each real-time PCR reaction was done at least thrice.
Whole-cell lysates were prepared from TE13 and TE13R120 cells. Cytoplasm, nucleus, and total proteins were, respectively, extracted according to the instruction of Vazyme biotech (Nanjing, China) and the concentration of protein was determined according to the instructions of bicinchoninic acid concentration assay kit. Samples were transferred to polyvinylidene difluoride membranes (Millipore Corp, Bedford, MA, USA) and incubated with primary anti-NRAGE (1:1000, Santa Cruz, USA), E-cadherin (1:500, Santa Cruz, USA), vimentin (1:500, Santa Cruz, USA), and GAPDH antibody. After overnight at 4°C, the membranes were washed with Tris-buffered saline, then incubated with a horseradish peroxidase-conjugated antibody against rabbit IgG (Sigma-Aldrich, St. Louis, MO, USA) for 2 h at room temperature. Immunoreactive protein bands were visualized with an ECL detection kit (Thermol Biotech Inc., Rockford, IL, USA). Each experiment was repeated three times.
Immunofluorescence was done on human esophageal carcinoma cells which were plated in 24-well plates (Costar Corp, Corning, NY, USA). After overnight incubation, TE13 and TE13R120 cells were fixed in 4% paraformaldehyde for 15 min and washed with PBS for three times, then incubated with antihuman NRAGE monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or an isotype control (MAB002 R and D Systems) overnight, followed by incubation with a 1:100 dilution of mouse antimurine TRICT-conjugated IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2 h in the dark. Nuclei were visualized with Hochest 33258 counterstain and examined using a fluorescence microscope (Olympus BX-40).
The morphological alteration for TE13 cells after tumor growth factor-β (TGF-β) treatment was observed by phase contrast (Zeiss S 100 TV) and microscopy (Zeiss EM 100). To analyze the effect of TGF-β on cell morphology, TE13 cells were grown to 70% confluence in RPMI 1640 containing 10% FCS. Cells were starved for 24 h in serum-free medium, then treated with TGF-β (10 ng/ml) or medium alone for 48 h. After 48 h incubation, the treated and untreated cells were fixed in 2% glutaraldehyde and 2% paraformaldehyde in 0.1M cacodylate buffer (pH 7.4) for at least 90 min.
Wound healing assay
The wounding healing assay was performed in 6-well plate, approximately 1 × 105 cells were plated in each well. When cell confluence reached about 90%–100%, the wound was generated by scratching the surface with a 200 μl pipette tip. Wound healing was observed after 24 h, and representative scrape lines for each cell type were photographed using phase-contrast microscope (Olympus, Japan).
For statistical analysis, SPSS version 17.0 (SPSS, Chicago, IL, USA) and the GraphPad Prism 5.0 software were used throughout. Data were presented as mean ± standard deviation, P < 0.05 was considered statistically significant.
| > Results|| |
TE13R120 showed radioresistance compared with TE13 following different radiation doses
A colony-forming assay was performed to detect the radiosensitivity of cells. As shown in [Figure 1]a and [Figure 1]b, irradiation killed the cells logarithmically and the colony counting was obviously less in TE13 cell line than those of TE13R120. The radiosensitivity parameters were summarized in [Table 1]. The D0 which means the inverse of the slope of the radiation survival curve, Dq which means quasi-threshold dose, N value which is the back extrapolation of the slope to the ordinate, and SF2 which is the SF at 2 Gy of TE13R120 were all higher than those of TE13. Furthermore, results of wound healing assay showed that TE13R120 cells showed retarded wound closing compared to TE13 cells [Figure 2].
|Figure 1: (a) Cell survival curves of TE13 and TE13R120 cells plotted by the multitarget single-hit model. (b) Representative images of the clonogenic survival assay of TE13 and TE13R120 cell lines|
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|Table 1: Comparison of the radiation biological parameters between TE13 and TE13R120 cell line|
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|Figure 2: Cell migration capacity was examined by wound healing assay. Results showed that after 24 h treatment with TGF-β, the cell migration was remarkably enhanced in TE13 after TGF-β treatment and TE13R120 cells group, compared with TE13 cells group.|
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TE13R120 demonstrated epithelial-to-mesenchymal transition
Phenotypic alterations were studied by inverted microscope. Results showed TE13R120 lost the epithelial cell polarity, presented long spindle, the number of cell–cell contacts was reduced [Figure 3]a. Results of real-time PCR showed that epithelial marker protein E-cadherin level was decreased (0.15-fold), but the mesenchymal marker protein vimentin was upregulated (2.60-fold) in TE13R120 cells compared with TE13 cells (1 fold as the control) [Figure 3]b. Results of Western blot confirmed our data on E-cadherin and vimentin mRNA expression [Figure 3]c.
|Figure 3: (a) The phenotypic alterations of TE13, TE13 after TGF-β treatment, and TE13R120 cells were studied by inverted microscope. (b) The mRNA expression of E-cadherin and vimentin in TE13 cells group, TE13 after TGF-β treatment, and TE13R120 cells group. (c) The protein expression of E-cadherin and vimentin in TE13 cells group, TE13 after TGF-β treatment, and TE13R120 cells group|
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Nuclear level of NRAGE was upregulated in TE13R120 cells
Immunofluorescence was used to detect the expression position of NRAGE. Results showed that specific NRAGE nuclear immunofluorescences were observed in TE13R120 cells, but in TE13 cells, NRAGE was mainly expressed in perinuclear cytoplasm [Figure 4]a. Results of Western blot confirmed that the nuclear protein level of NRAGE was increased in TE13R120 cells compared with TE13 cell line [Figure 4]b.
|Figure 4: (a) The expression position of NRAGE in TE13, TE13 after TGF-β treatment, and TE13R120 cell lines. NRAGE nuclear immunofluorescences were observed in TE13R120 cells and TE13 cell after TGF-β treatment, but in TE13 cells, NRAGE was mainly expressed in perinuclear cytoplasmic. (b) The expression of NRAGE in total protein, cytoplasm, and nuclear protein of TE13 cells, TE13 after treatment with TGF-β, and TE13R120 cells|
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The subcellular location of NRAGE was related to EMT
Cell morphology was analyzed before and after TGF-β treatment in TE13 cell lines. As shown in [Figure 3]a, treatment with TGF-β-induced EMT in TE13 cell lines. Results of real-time PCR and western blot both showed the decreased of E-cadherin expression (0.18-fold) which is a marker protein for epithelial differentiation in TGF-β-treated TE13 cells after 48 h. In contrast, the mesenchymal marker protein vimentin, a cytoskeletal intermediate filament protein, was weakly expressed before TGF-β treatment but highly expressed after treatment with TGF-β-induced EMT in TE13 cells (2.72-fold). Furthermore, after treatment with TGF-β, TE13 was presented characteristics of the mesenchymal cells, and NRAGE expression was increased in nuclear [Figure 4]b. Results of wound healing assay showed that after treatment with TGF-β in TE13 cells led to retarded wound closing compared to NC groups so was in TE13R120 cells [Figure 2].
| > Discussion|| |
Radiotherapy is a vital local treatment of malignant tumor and could be used to treat almost all cancers, but radioresistance of cancer cells lead to local control failure and recurrence, which is the main reason for radiotherapy failure. It is reported that radiological parameters, such as D0Dq, and SF2 values, which are inversely correlated with radiosensitivity, are obviously increased, and cell growth and aggressivity are obviously enhanced, it suggests that tumor cells showed radioresistance.,
In our previous study, human esophageal carcinoma resistance cell line TE13R120 was established through X-ray irradiation; results showed that D0 values of TE13 and TE13R120 were 1.29 Gy and 2.31 Gy, SF2 which is the SF at 2 Gy were 0.50 and 0.63, respectively. The gene chip analysis showed that NRAGE expression was upregulated after irradiation compared with TE13 cells. Moreover, the nuclear localization of NRAGE was more obvious in TE13R120 compared with TE13 cell line. Recent studies also found that its localization in correlation with participating the multiple, important, biological functions of cells. We thought that NRAGE may be related to radioresistant of esophageal carcinoma cell.
In the current study, we reused colony formation assay to measure the surviving fraction and the relevant radiobiological parameters. Results showed that the cloning formation capacity was increased in TE13R120 cell line compared with TE13. Meanwhile, the D0, Dq, N, and SF2 of TE13R120 were all significantly higher than those of TE13. The results were similar with that of our previous study. In addition, results of wound healing assay showed that the cell migration of TE13R120 was remarkably enhanced compared with TE13cell line. It confirms that TE13R120 cells showed higher radioresistance compared with TE13 cells.
EMT is characterized by loss of epithelial morphology and the acquisition of mesenchymal characteristics in tumor progression and then obtains the metastatic potential. To date, it is well-known radiation that could induce EMT in numerous tumors and demonstrate radioresistance of cancer cells.,,, In this study, we found that TE13R120 lost the epithelial cell polarity, presented long spindle which was the characteristics of the mesenchymal cells. The mRNA and protein level of epithelial marker protein E-cadherin were both decreased, but the mesenchymal marker protein vimentin was upregulated in TE13R120 cells compared with TE13 cells. This suggests that EMT may induce radioresistance of esophageal carcinoma cells.
We further used immunofluorescence to detect subcellular location of NRAGE. Results showed that NRAGE was mainly expressed in cytoplasm in TE13 cells but mainly located in nuclear in TE13R120 cells. Results of Western blot further confirmed the nuclear level of NRAGE was increased in TE13R120 cells, compared with TE13 cells. It suggests that the subcellular location of NRAGE may contribute to the radiation sensitivity of esophageal carcinoma cell.
TE13R120 cells demonstrated the characteristics of EMT; meanwhile, the nuclear level of NRAGE was increased in TE13R120 cells. We speculated that subcellular location of NRAGE may be related to EMT. To confirm it, TGF-β was further used to induce EMT in TE13 cells. Results demonstrated that after treatment with TGF-β, TE13 cells showed characteristics of the mesenchymal cells, E-cadherin level was decreased, but the mesenchymal marker protein vimentin was upregulated, whereas NRAGE nuclear expression was also increased. Meanwhile, results of wound healing assay showed that the cell migration was remarkably enhanced after treatment with TGF-β. This result was same with Kumar et al., who reported that ankyrin-G sequestered NRAGE in the cytoplasm, but oncogenic EMT could enhance the nuclear localization of NRAGE, then regulated TBX2, repressing the tumor suppressor gene p14ARF, promoted the metastasis of tumor. It suggests that EMT may induce radioresistance through regulating the subcellular localization of NRAGE.
A newly published study reported that NRAGE was overexpressed in esophageal carcinoma tissue and was positively correlated to PCNA, which was only expressed in normal proliferation cells and tumor cells. In our study, NRAGE was overexpressed in the nuclear of TE13R120 cells; we speculate if NRAGE transferred into nuclear after radiation treatment, then interacted with PCNA, inducing a series of biological effect, resulting in radioresistance. It is our further study.
| > Conclusions|| |
We reconfirmed the radioresistance of TE13R120 cells and found that TE13R120 cells showed EMT phenotypes, epithelial marker protein E-cadherin level was decreased, but the mesenchymal marker protein vimentin was upregulated, whereas NRAGE nucleus expression was increased in TE13R120 cells. Our findings indicate that radiation induces the EMT of esophageal carcinoma cell line, maybe participate in the nuclear localization of NRAGE, then further contributed to the radiation resistance of esophageal carcinoma cells. Therefore, further in vitro and in vivo studies are warranted to elucidate the mechanism of NRAGE on participating the radiation sensitivity of esophageal carcinoma cells.
The authors would like to thank Department of Pathology Basic Medical Science of Hebei Medical University for supports.
Financial support and sponsorship
This study was supported in part by a grant from the NationalNatural Science Foundation of Hebei province of China(#C2009001151) and the Foundation of The Second Hospital of Hebei Medical Universi(#2h2201505).
Conflicts of interest
There are no conflicts of interest.
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