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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。
Objective: To investigate the mechanism of radiosensitization of human nasopharyngeal carcinoma cell line CNE1 and CNE2 by tetrandrine (Tet), the largest non-toxic dose. METHODS: CNE1 and CNE2 cells were treated with combination of maximum nontoxic doses of Tet (1.5 μmol / L for CNE1 cells and 1.8 μmol / L for CNE2 cells), 4 Gy radiation and the maximum non-toxic dose of Tet. Flow Cytometry The cell cycle distribution of each group was detected. 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 maximum non-toxic dose of Tet combined with radiotherapy could up-regulate the expression of γ-H2AX in CNE1 and CNE2 cells. The proportion of G 2 / M phase in CNE 1 and CNE 2 cells in radiotherapy group was (18.09 ± 0.42)% and (18.48 ± 1.32)% respectively, and the proportion of G 2 / M phase in CNE 1 and CNE 2 cells was 15.88 ± 1.04% (13.80 ± 0.82)% respectively, which was significantly different from radiotherapy group (P <0.05). Combined treatment increased the protein level of cleaved caspase-3 induced by radiotherapy (P <0.05). The protein levels of p-CDC25C and p-CDK1 increased with the increase of Tet concentration (P <0.05) and the expression of CDK1 did not change significantly after treatment with different concentrations of Tet on CNE1 and CNE2 cells. The maximum non-toxic dose Tet did not affect the expression of p-CDC25C , P-CDK1 and CDK1 protein levels. In CNE1 and CNE2 cells, the combined treatment can significantly reduce the protein level of p-CDC25C and p-CDK1 induced by radiotherapy (P <0.05), and up-regulate the expression of cyclin B1 after radiotherapy, but has no obvious effect on the expression of total CDK1. The combined treatment could significantly inhibit the pERK protein level induced by radiotherapy (P <0.05). Conclusion: The maximum nontoxic dose of Tet can increase the DNA fragmentation and apoptosis of CNE1 and CNE2 cells induced by radiotherapy. The mechanism of radiosensitization may be related to the regulation of the pathway of ERK / CDC25C / CDK1 / cyclin B1 by Tet and the removal of G2 / M phase block related.