The Mechanism Of The Thermotherapy,chemotherapy Combined With Radiotherapy On Nasopharyngeal Carcinoma Xenograft Models

Obiective: Based on the preliminary study on the role of TPT with chronoradiotherapy on nasopharyngeal carcinoma xenograft models, to explore the functions and relevant mechanisms of the TPT with radiotherapy and thermotherapy on nasopharyngeal carcinoma xenograft models can lay foundation for further study of Chronomodulated radiotherapy and thermotherapy. Methods: 170 BALB/c nu/nu mice were feed synchronally under uniform lighting conditions comprising 12 hours for light and 12 hours for dark at least 3 weeks to establish a unified biological rhythms. Human nasopharyngeal poorly differentiated cells(CNE-2) were inoculated subcutaneously into the lateral thigh of each nude mice, and the human nasopharyngeal carcinoma xenografted model were established. Three weeks after successful transplantation, all nude mice bearing NPC xenografts were randomly divided into 7 groups: control group, TPT chemotherapy group(TPT), radiotherapy(RT) group, thermotherapy group, and TPT radiosensitizing group( TPT+RT), radiotherapy-thermotherapy group, TPT+RT+thermotherapy group, and each group has 22 mice. Select radiotherapy dose and tome according to previous research. The radiotherapy is 18 Gy single radiotherapy(the object is 60 CO away from the radiotherapy machine; 22:00 is chosen to bethe radiotherapy time). TPT(10mg/kg) were injected introperitoneally 30 minutes before radiotherapy. Thermotherapy refers to water bath which is partially heated to make the tumor temperature reach 43℃ and keep this temperature for 60 min after radiotherapy. RT group, TPT+RT group and RT+ thermotherapy group were given single radiotherapy of 18 Gy.Control group didn’t receive any treatment. Half of mice in each group were executed 1 hour after treated. The growth curve of the remaining mice were observed and the regrowth delay time(TGD) was recorded.(The part of the experience will be observed by another graduate student.)Tumor specimens obtained according to the protocol were used for three parts: The first part were tested the expressions of HP-1, Bax, Bcl-2, HSP70 and Ki67 using immunohistochemistry,, and the second part was used to examine the DNA quantities of cell cycle via flow cytometry. The third part was assayed the apoptosis rate by TUNEL. All the data from quantitative assays was expressed as the mean±standard deviation. Statistical analyses were performed using one-way ANOVA and SNK. The difference was considered statistically significant when p <0.05. All statistical analyses were carried out with SPSS 19.0 software. Results: 1. The study found that thermotherapy can improve the sensitivity of radiotherapy and concurrent chemoradiation. The effective rates had significant difference among chemoradiation-thermotherapy group and chemoradiation group, the P value is less than 0.05. Theeffective rates had significant difference among radiotherapythermotherapy group and RT group, the P value is less than 0.05. The SER of thermotherapy combined with radiotherapy is 1.32, greater than 1, suggesting that thermotherapy has radiosensitizing effect, and the SER of chemotherapy,thermotherapy plus radiotherapy is 1.86, greater than 1, suggesting that thermotherapy has chemoradiosensitizing effect.(The result can be obtained according to the data by the student who is studying on the phenomenon of the experience).2.Thermotherapy can reduce tumor cell proliferation ability after radiation and chemotherapy. A.HE found that differentiation degree of chemoradiation- thermotherapy group was higher, and nuclear division was less with lower proliferation ability. The nuclear division of rates had significant difference among chemor- adiation-thermotherapy group and other six groups, when the P value is less than 0.05. In radiotherapy- thermotherapy group, nuclear membrane was not clear with degeneration of cytoplasm vacuoles. The nuclear division of rates had significant difference among thermotherapy plus radiotherapy group and RT group, when the P value is less than 0.05. B. IHC staining found radiotherapy- thermotherapy group had good effects. The expressions of Ki67 had significant difference between radiotherapy- thermotherapy group and other six groups, when the P value is less than 0.05. The expressions of Ki67 had significant difference between thermotherapy plus radiotherapy group and RT group, when theP value is less than 0.05.. Each experimental group compared with the negative group, in which P values were less than 0.05, was statistically significant.. The expressions of Ki67 had no significant difference between thermotherapy group and TPT group, when the P value is more than 0.05.The expressions of HSP70 had significant difference between thermotherapy group group and other six groups, when the P value is less than 0.05. The expressions of HSP70 had significant difference between radiotherapy- thermotherapy group and RT group, when the P value is less than 0.05. The expressions of HSP70 had no significant difference between RT and TPT group, when the P value is more than 0.05. The expressions of Bax had significant difference between chemoradiation-thermotherapy group and other six groups, when the P value is less than 0.05. The expressions of Bax had significant difference between radiotherapy- thermotherapy group and RT group, when the P value is less than 0.05. The expressions of Bax had no significant difference among RT group 、TPT group、TPT+RT group and control group, when the P value is more than 0.05. The expressions of Bcl-2 had significant difference between chemoradiation- thermotherapy group and other six groups, when the P value is less than 0.05. The expressions of Bcl-2 had no significant difference among RT group、TPT group、TPT+RT group and control group,when the P value is more than 0.05. The expressions of Bcl-2 had significant difference betweenradiotherapy- thermotherapy group and RT group when the P value is less than 0.05. The expressions of HP-1 had significant difference between chemoradiation-thermotherapy group and other six groups, when the P value is less than 0.05. The expressions of HP-1 had significant difference between radiotherapy-thermotherapy group and RT group,when the P value is less than 0.05.The expressions of HP-1 had no significant difference between RT group and TPT group,.when the P value is more than 0.05.Each experimental group compared with the negative group, when the P values were less than 0.05, was statistically significant. 3. The mechanism study found that: A. thermotherapy can sensitize radiation and chemotherapy by increasing tumor cell apoptosis and regulating expression of related apoptosis gene.The apoptosis index had significant difference between chemoradiation-thermotherapy group and other six groups, when the P value is less than 0.05. The apoptosis index had significant difference between radiotherapy- thermotherapy group and RT group, when the P value is more han 0.05.B. The thermotherapy can sensitize radiation and chemotherapy by reducing tumor hypoxia. The level of tumor hypoxia had significant difference between chemoradiation-thermotherapy group and other six groups, when the P value is less than 0.05. The apoptosis index had significant difference between radiotherapy- thermotherapy group and RT group, when the P value is less than 0.05.C.Flow cytometry which was used to detect theDNA contents of the cell cycle, showed that thermotherapy can reduce the cell proportion of S phase, thus reducing tumor cell transcription and inhibiting tumor growth. The proportion of S-phase cells had significant difference between chemoradiation-thermotherapy group and other six groups, when the P value is less than 0.05. The proportion of S-phase cells had significant difference between radiotherapy- thermotherapy group and RT group, when the P value is more than 0.05. Conclusions: Thermotherapy can improve the TPT and radiotherapy sensitivity of transplantation tumors in nasopharyngeal carcinoma.Its chronomodulated radiosensitization mechanisms might be related to the following factors:, hypoxia,cell proliferationcell,cycle redistribution and apoptosis,...