目的 探讨伊立替康(CPT-11)对食管癌EC109细胞的放射增敏作用及核因子(NF)κB活性的影响.方法 采用MTT法观察不同浓度CPT-11(20、50、100、200μmol/L)作用24、48及72 h后的细胞存活率以筛选低细胞毒性药物浓度进行后续实验;根据实验设计分为对照组、单纯照射组和CPT-11+照射组,采用克隆形成实验检测经不同剂量X线(0、2、4、6、8和10 Gy)的存活分数(SF)并通过单击多靶模型拟合细胞存活曲线计算增敏比(SER),采用流式细胞技术检测各组处理48 h后的凋亡率和caspase-3活化率,Foci焦点形成实验检测各组的DNA损伤情况,Western blotting检测各组NF-κB p65和IκB-α的蛋白水平,实时定量PCR(qPCR)检测各组的Rad51水平.结果 在20~200μmol/L范围内,随CPT-11浓度增加,EC109细胞的增殖抑制率升高,抑制效应呈浓度和时间依赖方式,差异有统计学意义(P<0.05).选取与CPT-1120%抑制浓度(IC20)接近的浓度(25μmol/L)进行增敏实验.CPT-11+照射组的SF低于单纯照射组,且CPT-11+照射组的D0为1.39±0.14,单纯照射组的D0为2.46±0.17,SER为1.77.与对照组和单纯照射组比较,CPT-11+照射组的凋亡率、caspase-3活化率及Foci焦点数目均升高,而Rad51水平降低,差异有统计学意义(P<0.05).CPT-11+照射组的NF-κB p65水平低于其余两组,且IκB-α水平高于其余两组(P<0.05).结论 CPT-11可抑制食管癌EC109细胞增殖并具有放射增敏作用,同时诱导凋亡,可能与抑制NF-κB途径有关.%Objective To investigate the effect of irinotecan ( CPT-11) on radiosensitization and nuclear factor ( NF)-κB of esophageal cancer cells EC109. Methods MTT assay was used to observe the cell survival rates after treatment with different concen-trations of CPT-11 (20, 50, 100, 200μmol/L) for 24, 48 and 72 h as to screen the concentration with low cytotoxicity drug for subse-quent experiments. According to the experimental design, the EC109 cells were divided into the control group, the irradiation group and the CPT-11+irradiation group. The survival fraction ( SF) of different doses of X-rays ( 0, 2, 4, 6, 8 and 10 Gy) was detected by clone formation assay and the cell survival curve was calculated by using the multiple target model to calculate the enhancement ratio ( SER) . The activation rate of caspase-3 and apoptotic rate were detected by flow cytometry after 48 h. Foci focus formation was applied to detect the DNA damage of each group. Western blotting was used to detect the protein level of NF-κB p65 and IκB-αin each group. The real-time quantitative polymerase chain reaction( qPCR) was applied to detect the Rad51 level in each group. Results CPT-11 in the range of 20~200 mol/L can decrease the proliferation rate of EC109 cells. The inhibitory effect was exerted in a dose and time de-pendent manner and the difference was statistically significant ( P<0. 05) . Then, the concentration of CPT-11 close to 20% inhibition concentration(IC20)(25 μmol/L) was selected in the following sensitivity test. The SFs of CPT-11+irradiation group were lower than those of the irradiation group, and the D0 of CPT-11+group was 1. 39±0. 14, lower than 2. 46±0. 17 of irradiation group with SER of 1. 77. Compared with the control group and the irradiation group, the apoptotic rate, caspase-3 activation rate and the number of Foci focus were all increased, while the Rad51 level was decreased, and the difference was statistically significant (P<0. 05). Compared with other two groups, there were lower level of NF-κB p65 and higher level of IκB-α in CPT-11+irradiation group ( P<0. 05) . Con-clusion CPT-11 can inhibit the proliferation, has the function of radiation sensitization and induce apoptosis of EC109 cells, which may be related to the inhibition of NF-κB pathway.
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