Proteomic Analysis Of P53-mediated Radiobiological Effects In Nasopharyngeal Carcinoma

Backgroud and Objective: Nasopharyngeal Carcinoma (NPC) is one of the common malignant tumors in southern China. Radiation is the primary therapy method for NPC, but the overall efficacy is unfavorable, and 5-year survival rate after radiotherapy of NPC is still 40%-50%. p53 gene is a very important tumor-suppressor gene. p53 can induce cell cycle arrest and apoptosis, involves in DNA damage and repair, and also mediates radiobiological effects, so it is closely associates with the radiosensitivity of cancer. But mutant p53 gene does not possess these functions, and even decreases the radiosensitivity or involves in the radiation resistance of cancer. Numerous studies showed that there was the overexpression or accummulation of p53 protein in over 60% NPC and in amost all NPC cell lines, and overexpressed p53 protein in NPC might be dysfunction or inactivation. But till now it is unclear if the overexpressed p53 protein in NPC affects the radiosensitivity, and the molecular mechanisms of p53-mediated radiotherapy of NPC need to be further elucidated. Therefore, investigating the effects of overexpressed p53 protein on the radiosensitivity and searching for the proteins associated with the p53-mediated radiosensitivity of NPC will help to reveal the mechanisms of p53-mediated radiotherapy in NPC.Methods: (1) The effect of p53 knockdown on the radiobiological characteristics of nasopharyngeal carcinoma cell line CNE2: NPC cell line CNE2sip53 stably transfected by p53 siRNA vector and control cell line CNE2/pSUPER transfected with empty vector were used in this study. A clonogenic survival assay was performed to obtain an irradiation dose-survival curve and a survival fraction (SF), and then calculate radiobiological parameters, including SF2, D₀,α,βand sensitization enhancement ratios (SER). MTT assay was performed to determine the effects of irradiation on the cell growth, and Hochest33342 and flow cytometry were performed to determine the effects of irradiation on cell cycle distribution and apotosis respctevely. (2) Searching for the proteins associated with the p53-mediated radiosensitivity of NPC by proteomics: Two-dimensional gel electrophoresis (2-DE) was performed to separate the total proteins from CNE2sip53 and CNE2p/SUPER cells before and after radiation respctevely, PDQuest software was used to analyze the images of 2-DE, and MALDI-TOF-MS was used to identify the differentially expressed proteins in CNE2sip53 and CNE2p/SUPER cells before and after radiation. Western blotting was used to validate the differential proteins HSP70, Tcp-1β, Annexin A2 and 14-3-3σin CNE2 sip53 and CNE2p/SUPER cells before and after radiation.Results: (1) SF of CNE2sip53 cells is significantly higher than that of CNE2/pSUPER. When compared with CNE2/pSUPER, SF2 and D0 were significantly increased, whereasα,βand SER were significantly decreased in CNE2sip53 cells. (2) The number of apoptotic cells and the inhibition of cell growth induced by radiation in CNE2sip53 cells were significantly decreased as compared with CNE2/pSUPER cells. (3) Irradiation could arrested CNE2/pSUPER cells at G1 phases while arrested CNE2sip53 cells at G2 phases. (4) 2-DE patterns of CNE2sip53 and CNE2p/SUPER cell line before and after radiation were established respectively. Nineteen differential proteins identified by MALDI-TOF-MS between radiated and un-radiated CNE2/pSUPER cells, and fifteen differential proteins identified by MALDI-TOF-MS between radiated and un-radiated CNE2sip53 cells. The differential expression levels of HSP70, Tcp-1β, Annexin A2 and 14-3-3σbetween radiated and un-radiated CNE2/pSUPER and CNE2sip53 cells were validated by Western blot analysis. (5) Fourteen different proteins in the differential expression proteins between the radiated and un-radiated CNE2/pSUPER and CNE2sip53 cell lines were found, including Pyrophosphatase 1,Annexin A1,14-3-3 protein sigma,Proteasome (prosome, macropain) subunit,Prohibitin,Peroxiredoxin-2,Glutathione transferase,GRP78 precursor,Cytokeratin 18,Ras-related protein,Glutathione transferase omega 1-1,NM23-H1,Calmodulin-related protein and Electron transfer flavoprotein subunit alpha. Visant software analysis showed that seven of the fourteen proteins were directly or indirectly related to p53.Conclusion: (1) The overexpressed p53 protein in NPC possess biological function and is involved in the process of cell damage and apoptosis induced by radiation. (2) 2-DE patterns of radiated and un-radiated CNE2sip53 and CNE2p/SUPER cell line were established. Nineteen differential proteins between radiated and un-radiated CNE2/pSUPER cells, and fifteen differential proteins between radiated and un-radiated CNE2sip53 were identified by MALDI-TOF-MS, which provide the important reference information for investigating the radiobiologiy of NPC. (3) The fourteen different proteins in the differential proteins between the radiated and un-radiated CNE2/pSUPER and CNE2sip53 cell lines might be associated with the p53-mediated radiotherapy of NPC, which provides experimental evidences for elucidating the mechanisms of p53-mediated NPC radiotherapy.