Study On Promoting Action Of The Apoptosis And The Radiotherapy Sensitivity Of Aspirin And Rapamycin On Nasopharyngeal Carcinoma SUNE Cell Line

Background:Nasopharyngeal carcinoma(NPC) is one of the most common malignant tumors in our country,mainly happened in South of China.There is apparent race and family assembling in NPC disease incidence.It is estimated that about 80%of NPC in the world took place in our country.The incidence of NPC holds the first place in cephalo-trachelian tumor,and the age of onset is between three to eighty-four,mainly in 30-50 age range.The incidence of male is two or three times as high as that of female.Now,NPC has become one of the top ten life-threatening malignant tumors in our country.The great majority of NPC is poorly differentiated squamous-cell carcinoma, and much of them is temperate sensitive to radiation,while their proximal structure has a higher tolerance to radioactive ray.So the major treatment to NPC nowadays is radiotherapy combined with chemotherapy.But there are different radiosensitivity existing in different patients,so that the therapeutic efficacy is different.There are many factors could effect cellular radiosensitivity.Apoptosis is one of the important factors.Sine apoptosis has been found by Kerry in 1972,more apoptosis controlling signal thansduction pathway like NF-κB,mTOR have been found.So that the apoptosis mechanism of NPC cells is promoted.Many investigations demonstrated that it could provoke the loss of control of the cell growth and even provke the tumor generation or tumor apoptosis if certain link which controls the cell proliferation and differentiation could generate dysfunc -tion.Among so many signal transduction pathways mediated the tumor cell apoptosis,the NF-κ3,mTOR signal thansduction pathways shows importance to the regulation of the apoptosis.The activation of this signal transduction pathway could inhibit the cell apoptosis induced by much stimulus,promote the progression of the cell cycle and promote the cell survival and proliferation and also participate in the vasiformation.It takes an important part in the tumor formation and participates in tumor invasion and transfusion.It shows the important pole in the generation and development of the mapigant tumor through inducing the tumor cells survival,differentiation and vasiformation.Research has found that aspirin are important inhibitors of NF-κB and rapamycin are specific inhibitors of mTOR.Rapamycin and aspirin are made use of inhibiting mTOR,NF-κB activity,interfering with nasopharyngeal carcinoma cell signal transduction pathway,thereby inducing apoptosis-mediated radiotherapy of nasopharyngeal carcinoma cells,thus increasing the effect of radiotherapy for nasopharyngeal carcinoma.It will provide a theoretical foundation and experimental evidence for soluting radiation resistance.However,because of time,we still limited to experimental junior level,we will make a further study in the future. PART ONE:Study on promoting Action of the Apoptosis and the Radiotherapy Sensitivity of Aspirin on Nasopharyngeal Carcinoma SUNE Cell LineObjective:To explore the promoting action of the apoptosis and the radiotherapy sensitivity of aspirin on nasopharyngeal carcinoma SUNE cell line and its mechanism.Methods:1.Inhibitory effect of SUNE cell proliferation were detected and concentration had no effect on proliferation of SUNE cell lines was selected with MTT.2.On the role of the concentration no effect on proliferation in SUNE cells,the growth inhibition rate of SUNE on different radiation doses(2Gy,4Gy,8Gy) was discovered.3.The apoptosis rate of each group was compared by flow cytometry assay.4.NF-κB expression of each group was finded by immunofluorescence technique and flow cytometry.Results:1.Aspirin in vitro inhibited SUNE cell growth and there is a clear dose-effect relationship.There was not significant difference on the cell inhibition rate with the control group,when the concentration of ASA was not more than 2mmol / L. (P＞0.05)2.There was significant statistical difference between simple ASA treatment group and the control group.The cell growth inhibition rate increased with radiation dose increase,but the irradiation+ASA group was significantly higher than the alone irradiation group. 3.NF-κB positive expression signal(green) are mainly distributed in cytoplasm in SUNE cells before irradiation(8Gy).The positive signal in the nucleus enhanced significantly after irradiation(8Gy)..But the positive signal in the ASA+irradiation group declined than in the simple irradiation group significantly.The amount of cells of the positive expression of NF-κB in the simple irradiation group significantly reduced in the cytoplasm by flow cytometry,while that of the irradiation + ASA group,within the cytoplasm NF-κB expression than simple irradiation group significantly heightened.4.There was not significant difference between the control group and ASA group by LSD.The apoptosis rate of the irradiation + ASA group increased significantly than the simple irradiation group,and there was a statistical significance.Conclusions:1.Aspirin in vitro can inhibit SUNE cell growth and there is a clear dose-effect relationship.There was not significant difference on the cell inhibition rate with the control group,when the concentration of ASA was not more than 2mmol / L.2.Aspirin significantly increases the apoptosis of nasopharyngeal carcinoma cells. Aspirin has a role of increasing radiosensitivity in nasopharyngeal carcinoma cells.3.NF-κB-p65 protein is mainly distributed in the cytoplasm and is inactivation state.The majority of cells are death after irradiation,but NF-κB-p65 protein in the live cells translocates from cytoplasm to the nucleus,and NF-κB is activated.4.Aspirin can inhibit NF-κB transfering from cytoplasm to nucleus.5.After aspirin inhibited NF-κB activation,cell apoptosis was significantly increased.Inhibition of NF-κB activation may be involved in increasing the radiation sensitivity of nasopharyngeal carcinoma cells. PART TWO:Study on promoting Action of the Apoptosis and the Radiotherapy Sensitivity of Rapamycin on Nasopharyngeal Carcinoma SUNE Cell LineObjective:To observe the promoting action of the apoptosis and the radiotherapy sensitivity of rapamycin on nasopharyngeal carcinoma SUNE cell Line and it's mechanism.Methods:1.The expression level of Akt,mTOR were discovered in 60 cases of nasopharyngeal carcinoma by immunohistochemical.2.Inhibitory effect of proliferation of SUNE cell were detected and concentration had no effect on proliferation of SUNE cell lines was selected with MTT.3.On the role of the RPM concentration no effect of proliferation in SUNE cells,the growth inhibition rate of SUNE on different radiation doses(2Gy,4Gy,8Gy) was discovered.4.The apoptosis rate in each group was compared by flow cytometry assay.5.The expression of mTOR was discovered by immunofluorescence.6.Akt,p-Akt protein was contrived by West blotting.Results:1.In 60 cases of nasopharyngeal carcinoma,Akt had 49 cases of positive expression, the positive rate was 81.7%,and positive signals was mainly in the cytoplasm of nasopharyngeal carcinoma,mTOR had 45 positive expression cases,positive expression rates was 75%,and positive signal mainly located in cytoplasm of nasopharyngeal carcinoma,too.Both positive were 40 cases.After statistical analysis, Akt,mTOR were correlated the intensity of positive expression significantly.2.Rapamycin in vitro inhibited SUNE cell growth and there is a clear dose-effect relationship.There was not significant difference on the cell inhibition rate with the control group,when the concentration of RPM was not more than 1 nmol / L.3.There was significant statistical difference between simple RPM treatment group and the control group.The cell growth inhibition rate increased with radiation dose increase,but the irradiation+RPM group was significantly higher than the alone irradiation group.4.There was no significant difference between the control group and RPM group.(P=0.000) The apoptosis rate of the irradiation + RPM group increased significantly than the simple irradiation group,and there was a statistical significance.5.mTOR positive expression signal(green) are mainly distributed in cytoplasm in SUNE cells.After dealing with radiation(8Gy) and rapamycin(1nmol / L),the majority of cell were death,but positive signals in the cytoplasm of the live cells did not change significantly.Western blot technique detected that the total amount of Akt were unchanged before and after irradiation(8Gy) and rapamycin(1nmol / L), and phosphorylation of Akt decreased significantly after rapamycin treatment.It explained that radiation had no effect on expression of Akt,mTOR.Inhibiting the phosphorylation of Akt was involved in promoting nasopharyngeal carcinoma cells apoptosis by rapamycin.Conclusions:1.The expression of mTOR,Akt in nasopharyngeal carcinoma is high,and both of them have significant positive correlation.2.Rapamycin in vitro inhibited SUNE cell growth and there is a clear dose-effect relationship.There was no significant difference on the cell inhibition rate with the control group,when the concentration of RPM was not more than 1nmol / L.3.Rapamycin significantly increases the apoptosis of nasopharyngeal carcinoma cells.Rapamycin has a role of increasing radiosensitivity in nasopharyngeal carcinoma cells.4.The mTOR protein is mainly distributed in the cytoplasm of nasopharyngeal carcinoma cells.Although the majority of cell deceased after irradiation and rapamycin treatment,but mTOR protein expression in the live cells have no significant diversification.5.The total amount of Akt were unchanged before and after irradiation and rapamycin,and phosphorylation of Akt decreased significantly after rapamycin treatment.Inhibiting the phosphorylation of Akt was involved in promoting apoptosis of nasopharyngeal carcinoma cells and enhancing radiosensitivity.6.The up-regulation phosphorylation of Akt is involved in the radiation resistance of nasopharyngeal carcinoma cells.