Experimental Study On The NF-κB Signaling Pathway In Radio-induced Apoptosis Of Adenoid Cystic Carcinoma Cells

Background:Adenoid cystic carcinoma(ACC) is among the most common subtypes of malignant epithelial tumors occuring in salivary gland. Its charaeristic including low rate of locoregional lymph node metastasis, high rate of recurrence, great tendency of perineural invasion and early development of haematogenous metastasis. The perineural invasion and haematogenous metastasis after10years is50%and30-40%respectively, and single surgery treatment can not eliminate tumor completely. Radiotherapy is advised by its significant improvement in reducement of local recurrence compared with surgery alone. Because of the relative low radio-sensitivity in adenoid cystic carcinoma, however, further investigation is strongly in need. How to improve the sensitivity of malignant tumor to radiotherapy by means of gene therapy has inevitably led our concerns. The abarrently activation Nuclear factor κB (NF-κB) signaling pathway plays a crucial role in the anti-apoptosis mechanism,and closely is related to the radio-resistance function of tumor cells. Thereafter, inhabitation of this pathway via genetic engineering technology may significantly improve the sensitivity and curative effect of radiotherapy.Objectives:The adenoid cystic carcinoma cell line(ACC-2cell) was transiently transfected with IκBα-M gene, and irradiated by high-energy X ray. The irradiated ACC-2cells were divided into six different dose groups including0,2,4,6,8,10Gy, and cells were harvested at different time point (1,3,6,10,24,48h) to proceed immunocytochemistry to determine the nuclear translocation of NF-κB p65protein, and Western blot to determine the quantitive expression of NF-κB p65protein, and flow cytometric analysis and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay(TUNEL) to determine the apoptosis changes of different dose-and time-group.Method:The adenoid cystic carcinoma cell line(ACC-2cell) was cultivated at37℃in a humidified5%CO2incubator. Cells under the exponential phase of growth were transiently transfected with pBabe-SR-IκBa pasmid by the application of Lipofectamine2000. Western blot was applied to determined the expression change of IκBa protein after transfection. The transfected ACC-2cells were irradiated by different doses of high-energy irradiation (0,2,4,6,8,10Gy). Immunocytochemistry was applied to investigate the nuclear translocation of NF-κB p65at protein different time points after irradiation(1,3,6,10,24,48h), and Image Pro-Plus image analytical software was applied to measure the average nuclear gray scale. Western blot was applied to investigate the quantitive expression changes of NF-κB p65protein at protein at indicated time points. Flow cytometric analysis and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay(TUNEL) were applied to investigate the apoptosis rate of indicated dose-and time-groups.Results:1. Compared to the transfected and non-transfected pBabe plasmid group,the ACC-2cells for transfection of the pBabe-SR-IκBa plasmid expressed endogenous IKBa protein as well as SR-IκBa protein. The average gray scale3h after receiving the same doses of irradiation increased in pBabe-SR-IκBa plasmid transfection group.2. Immunocytochemistry shows that the average nuclear gray scale of different dose group in pBabe-SR-IκBa plasmid transfection group was lower than that of OGy group. Western blot analysis shows that the quantitive expression of NF-κB p65protein of different dose group in pBabe-SR-IκBa plasmid transfection group was higher than that of0Gy group. Flow cytometric analysis and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay show corresponding changes in cell apoptosis, which means that the apoptosis rate of different dose groups were higher than that of0Gy group.3. Immunocytochemistry and Western blot analysis show that, at6-10h time point after receiving different doses irradiation, the average nuclear gray scale reached the deepest valley, and the quantitive expression of NF-κB p65protein reached the peak. At the same time point, cells in3h group shows decreased average nuclear gray scale and increased expression of NF-κB p65protein with the increase of irradiation dose(P<0.05).4. Flow cytometric analysis and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay show that, with the increased expression of NF-κB p65protein, the apoptosis rate of irradiated cells exhibited reverse tendency(P<0.05), which indicated the anti-apoptosis function of NF-κB p65protein.Conclusions:The NF-κB signaling pathway was inhibited after transfection of mutated IicBa gene.The expression of NF-κB p65protein possesses dose-time dependent effects in radio-induced apoptosis. The expression of NF-κB p65protein inhibited cell apoptosis.