The Mechanism Of The Chronomodulated Radiosensitization Of Topotecan On Nasopharyngeal Carcinoma Xenograft Models

Obiective:To investigate the mechanism of chronomodulated radiosensitization of topotecan on human nasopharyngeal carcinoma in nude mice through the cellular level and molecular level. Methods:240BALB/c nu/nu mice were feed synchronally under uniform lighting conditions comprising12hours for light and12hours for dark at least3weeks 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 into4groups:control group, TPT chemotherapy group(TPT), radiotherapy(RT) group and TPT radiosensitizing group (TPT+RT), and each group has48mice. The therapeutic results of the treated mice at four different time points, including the early rest period (3HALO), the late rest period (9HALO), the early active period (15HALO), and the late active period (21HALO) of the mice was observed. TPT (10mg/kg) were injected introperitoneally30minutes before four time points. RT group and TPT+RT group were given single radiotherapy of18Gy at the four time points respectively. Control group didn’t receive any treatment. Half of mice in each group were executed1hour after radiotherapy. The growth curve of the remaining mice were observed and the regrowth delay time(TGD) was recorded. Tumor specimens obtained according to the protocol were used for three parts: the first part were tested the expressions of HP-1and y-H2AX using immunohistochemistry, then semi-quantitative analysis was established by image analysis software (IPWIN60), the second part was used to test the expression of topoisomerase I using DNA agarose gel electrophoresis, and the third part was used to examine the DNA quantities of cell cycle and apoptotic rate via flow cytometry. 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 SPSS13.0software. Results:1. Different treatment factors for nasopharyngeal carcinoma xenografts produce different degrees of inhibition. According to Overall assay, TPT+RT group obtained the best inhibitory effect. Treatment with TPT+RT group at15HALO showed the greatest efficacy (15HALO>21HALO>9HALO>3HALO).2. Immunohistochemistry results show that the HP-1expression was15HALO<21HALO<9HALO<3HALO in control group and3HALO group has statistical significance compared to15HALO group which means the level of tumor hypoxia varied in a time dependent manner. At the same time point, the expressions of HP-1and y-H2AX had significant difference between RT group/TPT+RT group and control group (P<0.01). At the different time point, the expressions of HP-1in RT group and TPT+RT group:3HALO>9HAL0>21HALO>15HALO; The expressions of y-H2AX in RT group and TPT+RT group:15HALO21HALO>9HALO>3HALO. There were significant difference between15HAL0and3HAL0(P<0.01).3. The level of topoisomerase I varied according to circadian time using DNA agarose gel electrophoresis,15HALO>21HALO>9HALO>3HALO. There were significant difference between15HAL0and3HALO.4. To detect cell cycle, apoptosis index was increased and proportion of S-phase cells was decreased by flow cytometry. There were statistic significance between the control group and each other group. Conclusions:This study suggested that TPT combined with chronomodulated radiotherapy can enhance the radiosentivity of nasopharyngeal carcinoma xenografts, and TPT+RT group at15HAL0has the best therapeutic effect. Topotecan is a promising chronomodulation radiosensitizer. Its chronomodulated radiosensitization mechanisms might be related to the following factors:tumor hypoxic state at different time points, cell cycle redistribution and apoptosis, the damage of DNA double-strand, the circadian rhythm of DNA topoisomerase I expression.