Analysis Of Nasopharyngeal Carcinoma Radioresistance By Proteomics

Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in southern China with the highest incidence and mortality rate in the world. It poses one of the most serious public health problems in this area. Radiotherapy is the primary treatment for NPC. Although the radiotherapeutic equipments and technique are improved continuously, the radiotherapeutic efficacy of NPC remains unfavorable. The main reason for the failure of NPC radiotherapy is that there are a certain percentage of radioresistant cells in the NPC tissues, which not only have higher radioresistance, but also are more aggressive after radiotherapy, prone to lymph node and distant metastasis. Therefore, the radioresistance remains a serious obstacle to successful treatment in some NPC cases.However, the molecular mechanism underlying radioresistance of NPC has not been elucidated yet. The finding of radioresistant proteins will be useful for revealing the mechanism of NPC radioresistance, and providing the scientific basis for predicting NPC sensitivity as well as targets and personalized therapy, which plays an important role in improving efficacy and prognosis of NPC.To identify proteins involved in NPC resistance, we established a radioresistant subclone cell line (CNE2-IR) derived from NPC cell line CNE2 by treating the cells with five rounds of sublethal ionizing radiation (11Gy). Proteomics was then performed to compare protein profiles of CNE2-IR and CNE2 cells. Two-dimensional gel electrophoresis (2-DE) was performed to separate the total proteins of CNE2-IR and CNE2 cells, PDQuest software was applied to analyze the differential protein spots between the CNE2-IR and CNE2 cell lines, these differential spots were identified by both MALDI-TOF-MS and ESI-Q-TOF-MS,and the differential proteins identified by proteomic approach were confirmed by Western blot.Highly reproductive and resolvable 2-DE maps of CNE2-IR and CNE2 cells were established. A total of 34 differential proteins was identified by MALDI-TOF MS and ESI-Q-TOF MS.Among them,14-3-3σand Maspin was significantly downregulated, and GRP78 and Mn-SOD was significantly upregulated in the radioresistant CNE2-IR as compared with control CNE2. The differential expression levels of the four proteins (14-3-3σ, Maspin, GRP78 and Mn-SOD) between the CNE2-IR and CNE2 cell lines were confirmed by Western blot.To investigate whether the differentially expressed proteins serve as predictive molecular markers for radiosensitivity of NPC, immunohistochemistry was performed to detect the expression levels of 14-3-3σ, Maspin, GRP78 and Mn-SOD proteins in the 39 radioresistant and 51 radiosensitive NPC tissues. The ability of 14-3-3σ, Maspin, GRP78 and Mn-SOD in distinguishing the radiosensitive and radioresistant NPC tissues was evaluated by the ROC (receiver operating characteristic) analysis. The results showed that 14-3-3σand Maspin was significantly downregulated, and GRP78 and Mn-SOD was significantly upregulated in the radioresistant NPC tissues as compared with radiosensitive NPC tissues.The downregulation of 14-3-3σand Maspin and upregulation of Mn-SOD and GRP78 were significantly correlated with NPC radioresistance.As a panel, the four proteins achieved a sensitivity of 90% and a specificity of 88% in discriminating radiosensitive from radioresistant NPC.To address the question whether the expression levels of the four proteins may associate with the development of NPC radioresistance, we selected 14-3-3σ, one of the four differential proteins to be further functionally studied. pcDNA3-14-3-3σplasmid was transfected into the CNE-2-IR cells to upregulate the expression of 14-3-3σ, and the effect of 14-3-3σ-upregulation on CNE-2-IR radiosensitivity was measured by using a clonogenic survival assay. The results indicated that the radioresistance can be partially reversed by overexpression of 14-3-3σin the CNE2-IR cells.Our data showed that 14-3-3σ, Maspin, GRP78 and Mn-SOD proteins are potential biomarkers for predicting NPC radiosensitivity.Their dysregulation may be involved in the development of NPC radioresistance.