汉防己甲素对人鼻咽癌CNE1和CNE2细胞株的放疗增敏作用

摘要

AIM:To investigate the mechanism of the radiosensitizing effect of maximum non-cytotoxic doses of tetrandrine (Tet) on nasopharyngeal carcinoma cell lines CNE1 and CNE2.METHODS:The cells were treated with maximum non-cytotoxic doses of Tet (for CNE1 cells at 1.5 μmol/L and for CNE2 cells at 1.8 μmol/L),irradiation at 4 Gy,or combination of irradiation and maximum non-cytotoxic doses of Tet.The cell cycle distribution was analyzed by flow cytometry.The protein levels of γ-H2AX,cleaved caspase-3,p-CDC25C,CDK1,p-CDK1,cyclin B1,ERK and p-ERK were determined by Western blot.RESULTS:The expression of γ-H2AX was increased in CNE1 cells and CNE2 cells after combined treatment with irradiation and maximum non-cytotoxic doses of Tet.The percentages of CNE1 cells and CNE2 cells at G2/M phase in irradiation group were (18.09 ±0.42)％ and (18.48 ± 1.32)％,respectively,which were decreased to (15.88 ± 1.04) ％ and (13.80 ± 0.82) ％ in combined treatment group,respectively (P ＜ 0.05).Combined treatment enhanced the increase in the protein level of cleaved caspase-3 caused by irradiation.The protein levels of pCDC25C and p-CDK1 were increased in a dose-dependent manner by Tet treatment (P ＜ 0.05),while the expression of CDK1 showed no difference among different doses of Tet treatments.The protein levels of p-CDC25C,p-CDK1 and CDK1 showed no difference after the treatment with maximum non-cytotoxic doses of Tet.The combined treatment with irradiation and the maximum non-cytotoxic doses of Tet decreased the protein levels of p-CDC25C and p-CDK1 (P ＜0.05),increased the expression of cyclin B1,and had no influence on the expression of CDK1 （ P ＜0.05).The combined treatment resulted in an increase in the protein level of p-ERK1 (P ＜ 0.05).CONCLUSION:The maximum non-cytotoxic doses of Tet enhance the DNA damage and apoptosis in CNE1 cells and CNE2 cells caused by irradiation,and the mechanism might be associated with ending of G2/M arrest via activation of ERK/CDC25C/CDK1/cyclin B1 pathways.%目的:探讨最大非毒性剂量汉防己甲素(tetrandrine,Tet)对人鼻咽癌细胞株CNE1和CNE2的放疗增敏机制.方法:分别采用最大非毒性剂量Tet(对CNE1细胞为1.5 μmol/L;对CNE2细胞为1.8 μmol/L)、4Gy放疗和最大非毒性剂量Tet联合放疗处理CNE1和CNE2细胞;流式细胞术检测各组细胞周期分布,Western blot检测各组细胞γ-H2AX、cleaved caspase-3、p-CDC25C、CDK1、p-CDK1、cyclin B1、ERK和p-ERK的蛋白水平.结果:最大非毒性剂量Tet联合放疗后可上调CNE1和CNE2细胞中γ-H2AX的表达.放疗组CNE1和CNE2细胞G2/M期的比例分别为(18.09±0.42)％和(18.48±1.32)％,联合处理组CNE1和CNE2细胞G2/M期的比例降为(15.88±1.04)％和(13.80±0.82)％,与放疗组比较差异有统计学意义(P＜0.05).联合处理可增加放疗所致的cleavedcaspase-3的蛋白水平(P＜0.05).不同浓度Tet处理CNE1和CNE2细胞后,p-CDC25C和p-CDK1的蛋白水平随Tet浓度增加而升高(P＜0.05),CDK1的表达无明显改变;最大非毒性剂量Tet不影响p-CDC25C、p-CDK1和CDK1的蛋白水平.在CNE1和CNE2细胞中,联合处理可明显降低放疗引起的p-CDC25C和p-CDK1的蛋白水平(P＜0.05),上调放疗后cyclin B1的表达,而对总CDK1的表达无明显调节作用;联合处理可显著抑制放疗所致的p-ERK蛋白水平(P＜0.05).结论:最大非毒性剂量Tet可以增加放疗引起的CNE1和CNE2细胞的DNA断裂及细胞凋亡,其放疗增敏的机制可能与Tet调控ERK/CDC25 C/CDK1/cyclin B1通路、去除放疗导致的GJM期阻滞有关.