The Mitochondrial Target Protein Study Of Emodin Acylate Derivative B Radiosensitization Effect On Nasopharyngeal Carcinoma Cells

Objective:To study the functional changes of mitochondrial on nasopharyngeal carcinoma cells. iTRAQ-2D-LC-MALDI-TOF/TOF/MS was used to analyze the different proteins, and comfirm the mitochondrial target protein of nasopharyngeal carcinoma cells during Emodin Acylate Derivative B radiosensitization treatment.Method:(1) Mitochondria ultrastructure changes was observed by ransmission electron microscope;(2) Mitochondria membrane potential changes was detected by rhodaminel23combine laser scanning confocal microscope;(3) Intracellular Ca2+changes was detected by Fluo-3AM combine laser scanning confocal microscope;(4)Mitochondria protein was extracted by a mitochondria proteins extract kit;(5) mitochondria proteome was quantitatively analyzed by iTRAQ-2D-LC/MALDI-TOF/TOF/MS and proteins was reported with confidence>95%by ProteinPilot2.0in NCBInr database;(6) intracellular location, molecule function and bioprocess of the differentially expressed protein were analysed in gene ontology, David and NCBI database, and proteins interaction network was analysed in String database of the different proteins to definite the target protein on nasopharyngeal carcinoma cells mitochondrial during Emodin Acylate Derivative B radiosensitization treatment. Results:(1) Tansmission electron microscope results showed that cells were eumorphism and fulled of integral organelles, and mitochondrial inner membrane was also integral and clearly in the control group; little change was happened in2Gy group and10μg/ml Acylate Derivative B group; cell expansion, mitochondrial swelling and ridge breakage was observed in the2Gy combine10μg/ml Acylate Derivative B group.(2) The fluorescence intensity was40.63±2.36in the control group after rhodamine123staining; they were36.73±2.62and26.56±1.29in2Gy group and10μg/ml Acylate Derivative B group respectively, which have a statistical significance compare to the control group(p<0.05). The fluorescence intensity was19.14±3.84in the2Gy combine10μg/ml Acylate Derivative B group, which have a statistical significance compare to2Gy group and10μg/ml Acylate Derivative B group (P<0.05) and have a evident statistical significance compare to the control group (P<.01)(3) The fluorescence intensity was1164.17±68.69in the control group after Fluo-3AM staining; they were1391.83±33.35and1406.0±48.02in2Gy group and10μg/ml Acylate Derivative B group respectively, which have a statistical significance compare to the control group (P<0.05). The fluorescence intensity was1940.08±55.74in the2Gy combine10μg/ml Acylate Derivative B group, which had a statistical significance compare to2Gy group and10μg/ml Acylate Derivative B group (P<0.05) and have a evident statistical significance compare to the control group (P<0.01) (4) Extracted mitochondrial showed glaucous graininess and linar after Janus green B staining; protein concentration were measured by BCA method with a coefficient of correlation was0.99, protein concentration of control group,2Gy group,10μ/ml Acylate Derivative B group and2Gy combine10μg/ml Acylate Derivative B groups were:2.07,2.10,1.93and2.07μg/μl respectively.(5) iTRAQ-2D-LC/MALDI-TOF/TOF/MS was performanced successfully and998kinds of protein was identified (>95%confidence), their molecular weight range was between555.62KD and6.92KD.(6) There were101kinds of expressed protein were proved to have a significance difference in the three group compare to the control group.The proteins about response to wounding and defense was most which had a percentage of19.8%. Through the protein interaction regulation network, HspA8, Cox7C and ATP5Alwere key point protein on nasopharyngeal carcinoma mitochondrial during Emodin Acylate Derivative B radiosensitization treatment.Conclusion:(1) Radiosensitization effect of10μg/ml Emodin Acylate Derivative B on CNE-1may be relate to mitochondria damaging, membrane potential decreasing, and Ca2+overloading in cells.(2)101kinds of different protein are identified by the iTRAQ-2D-LC/MALDI-TOF/TOF/MS method on nasopharyngeal carcinoma mitochondrial after Emodin Acylate Derivative B radiosensitization treatment, their molecule function and bioprocess are mainly involved in defense response and oxidation-reduction reaction. (3) Through classification and bioinformatics analysis to the different proteins, key point proteins of HspA8, Cox7C and ATP5A1may become the target protein of Emodin Acylate Derivative B radiosensitization effect.