The Syudy Of Radiosensitisation In Differentiation Type Of Nasopharyngeal Carcinoma Cells By Regulation Of RAC1/NADPH Signaling Pathway

Objective:RAC1 is one of the most potential targets in the radiosensitisation process for nasopharyngeal carcinoma cells. After activation or inhibition the RAC1/NADPH signaling pathway, we explore the relationship between RACl/NADPH signaling pathway and radiosensitisation in nasopharyngeal carcinoma (NPC) CNE-1 and CNE-2 cells and to reveal the possibility of GXHSWAQ-4 compound as a potential radiosensitizer for nasopharyngeal carcinoma radiotherapy.Method:A well differentiated squamous cell line of nasopharyngeal carcinoma CNE-1 and a poorly differentiated squamous cell line of CNE-2 were chosen as the research objects in this study. PMA was selected for the RAC1 activator, NSC23766 was used as RAC1 inhibitor, the GXHSWAQ-4 compound was the test substance and the irradiation dose was 2Gy. After a series of different treatments, the following indicators were determined.1. The activation of RAC1-GTP of each group of cells was detected with RAC1-GTP pulldown assay.2. The expression level of total RAC1, p47, p67, p38, AP-1, JNK1/2, phosphor-p38, phosphor-AP-1 and phosphor-JNK were determined by western blot.3. The distribution and location of the activated RAC1 in CNE-1 and CNE-2 cells were observed by immunofluorescence.4. The activity of NADPH oxidase of each groups of cells was detected by NBT assay.5. The concentration of ROS and the apoptosis rates of each groups of cells was detected by flow cytometer.6. The viability of the cells without irradiation treatment was determined by MTT assay.Results:1. When PMA concentration was 100 nmol.L-1 and reaction time was 30 min, the activation effect of NADPH oxidase activity was the best; when NSC23766 concentration was 25μmol.L-1 and reaction time 1 h, the inhibitory effect of NADPH oxidase activity was the best.2. Compared with the control or 2Gy cells, the expression of RAC1-GTP increased in the cells treated with PMA or PMA combine with 2Gy; Rac1 activators appeared to recruit Rac1 to the protruding edge of cells; The expression of total protein RAC1, p47, p67 which are the key proteins of RAC1/NADPH signaling pathway and phospho-p38, phospho-AP-1 and phospho-JNK, which are the proteins of downstream signaling pathway JNK/AP-1 were up-regulated. The activity of NADPH oxidase, reactive oxygen concentration and the apoptosis rate were increased. Among most of the above indicators, the degree of change in CNE-1 cells was greater than CNE-2 cells. The results of cells that treated with NSC23766 or NSC23766 combine with 2Gy was opposite. The above indexes were statistically significant difference.3. After the cells treated with GXHSWAQ-4 compound combine with 2Gy irradiation, compared with 2Gy cells, the expression of RAC1-GTP, total protein RAC1, p67, phospho-p38, phospho-AP-1 and phospho-JNK were up-regulated. The activity of NADPH oxidase, reactive oxygen concentration and the apoptosis rate were all increased.Conclusion:1. PMA and NSC23766 can effectively activate and inhibit the activity of RAC1-GTP, thus regulating RAC1/NADPH signaling pathway.2. Regulation the RAC1/NADPH signaling pathway can alter the activiation of downstream JNK/AP-1 signaling pathway, thereby changing the radiosensitisation in CNE-1 and CNE-2 cells.3. GXHSWAQ-4 combine with 2Gy can increase the radiosensitisation in nasopharyngeal carcinoma CNE-1 and CNE-2 cells. GXHSWAQ-4 compound may be a good radiosensitizer for nasopharyngeal carcinoma radiotherapy.