汉防己甲素对人鼻咽癌CNE1和CNE2细胞株的放疗增敏作用

Effect of tetrandrine on radiosensitivity of nasopharyngeal carcinoma cells

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1.5μmol/L;对CNE2细胞为1.8μmol/L)、4 Gy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G_2/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G_2/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cleaved caspase-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pERK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/CDC25C/CDK1/cyclin B1通路、去除放疗导致的G2/M期阻滞有关。

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-CDC25 C,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 G_2/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 pCDC25 C 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-CDC25 C,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-CDC25 C 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 G_2/M arrest via activation of ERK/CDC25C/CDK1/cyclin B1 pathways.