姜黄素联合顺铂对非小细胞肺癌细胞A549放疗增敏作用的初步研究

A preliminary study on radiosensitization effect of curcumin plus cisplatin on non-small cell lung cancer A549 cells

目的探讨姜黄素联合顺铂对非小细胞肺癌细胞A549放射增敏的影响。方法采用MTT法观察不同浓度姜黄素（10、20、50、100、200μmol/L）和顺铂（1、2、5、10、20 mg/L）作用24、48及72 h后的细胞存活率,根据实验设计分为单纯照射（R）组、姜黄素＋照射（C＋R）组、顺铂＋照射（P＋R）组及姜黄素＋顺铂＋照射（C＋P＋R）组,采用克隆形成实验检测4组经不同剂量X线（0、2、4、6、8、10 Gy）照射后的存活分数（SF）并通过单击多靶模型拟合细胞存活曲线计算增敏比（SER）,分别采用人工划痕、Transwell试验及Western blotting检测以上4组处理24 h后的细胞迁移、侵袭及表皮生长因子受体（EGFR）的蛋白水平。结果随姜黄素（10-200μmol/L范围内）和顺铂（1-20 mg/L范围内）浓度增加,A549细胞的细胞存活率降低,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）;C＋P＋R组在照射剂量为2-10 Gy时的细胞SF均低于其余3组,而C＋R组在4-10 Gy,P＋R组在2-10 Gy时的细胞SF均低于R组,差异有统计学意义（P〈0.05）,C＋R组、P＋R组及C＋P＋R组相对于R组的SER依次为1.24、1.31和1.96;C＋P＋R组的迁移距离、穿膜细胞数量及EGFR蛋白水平均低于其余3组,而C＋R组和P＋R组的以上指标亦低于R组,以上差异均有统计学意义（P〈0.05）。结论姜黄素联合顺铂可抑制A549细胞增殖并具有放射增敏作用,同时抑制其迁移和侵袭,可能与EGFR相关信号通路受抑制有关。

Objective To explore the radiosensitization effect of curcumin plus cisplatin on non-small cell lung cancer A549 cells. Methods Cell viability at 24,48 and 72 h after treatment with different concentrations（ 10,20,50,100,200 μmol / L） of curcumin or（ 1,2,5,10,20 mg / L） of cisplatin were determined by MTT. According to the experimental protocol,the below experiments were carried out in irradiation（ R） group,curcumin ＋ irradiation（ C ＋ R） group,cisplatin ＋ irradiation（ P ＋ R） group and curcumin ＋ cisplatin ＋ irradiation（ C ＋ P ＋ R） group. The colony formation assay was employed to observe the surviving fraction（ SF） of above four groups after X-ray irradiation of 0,2,4,6,8,10 Gy. The artificial scratch,Transwell test and Western blotting were employed to detect the cell migration,invasion and protein level of epidermal growth factor receptor（ EGFR） at 24 h after treatment in four groups. Results The cell viability of A549 cells gradually decreased with the increasing concentration of curcumin ranging from 10 to200 μmol / L and cisplatin ranging from 1 to 20 mg / L in a dose- and time-dependent manner（ P〈0. 05）. The SF of C ＋ P ＋ R group were lower than the remaining 3 groups under the dose of 2-10 Gy（ P〈0. 05）. Compared with the R irradiation group,there was lower SF in C ＋ R group under the dose of 4-10 Gy and P ＋ R group under the dose of 2-10 Gy with significant difference（ P〈0. 05）. Compared with R group,the sensitizing enhancement ratio were 1. 24,1. 31 and 1. 96 in C ＋ R group,P ＋ R group and C ＋ P ＋ R group,respectively.There were lower migration distance,transmembrane cell number and EGFR protein level in C ＋ P ＋ R group versus the remaining 3groups（ P〈0. 05）. Compared with the R group,the above indicators were also lower in C ＋ R group and P ＋ R group under the dose of2-10 Gy with significant difference（ P〈0. 05）. Conclusion Curcumin plus cisplatin can inhibit the proliferation of A549 cells with radiosensitizing effect and suppress its migration and invasion possibly via inhibition of EGFR signaling pathways.