CDA-Ⅱ抑制组织因子表达对肺癌细胞放疗增敏的作用及机制探讨

Effect of CDA-Ⅱinhibition of tissue factor expression on radiosensitivity of lung cancer cells.

目的探讨尿多酸肽(CDA-Ⅱ)抑制组织因子表达对肺癌细胞放疗增敏的作用及其机制。方法体外实验分组:对照组(仅含有A549、H1395细胞)、不同剂量CDA-Ⅱ组(0.03、0.06、0.12、0.25、0.50、1.00、2.00、3.00 mg/L)总共9个分组,每个分组同时处理24、48 h后检测细胞活力;行克隆增敏实验探究CDA-Ⅱ对肺癌细胞增敏影响;将细胞分为空白组(不予药物及射线处理)、射线组(仅予8 Gy照射处理)、射线+0.06 mg/L CDA-Ⅱ组(予8 Gy射线+0.06 mg/L CDA-Ⅱ药物处理)、射线+0.06 mg/L CDA-Ⅱ组(予8 Gy射线+0.12 mg/L CDA-Ⅱ药物处理),流式检测细胞凋亡;蛋白质印迹(Western blotting)分别检测不同处理组细胞内组织因子和丝裂原活化蛋白激酶(MAPK)通路蛋白磷酸化水平变化,进而阐明放疗增敏机制。结果CDA-Ⅱ时间剂量依赖性的抑制肺癌细胞增殖活性,24 h半数抑制浓度(IC50)为A5490.195 mg/L,H13950.32 mg/L;单击多靶模型分析克隆形成表明:CDA-Ⅱ抑制组织因子表达能够提高肺癌细胞放射敏感性,并呈剂量依赖性。0.06、0.12 mg/L CDA-Ⅱ组增敏比(SER)为(A549:1.17、1.28;H1395:1.08、1.25);流式实验证实:药物CDA-Ⅱ联合射线处理后细胞的凋亡率较单纯射线处理组升高,且随着CDA-Ⅱ的剂量增加而增加;Western blotting结果显示,与空白对照组、仅予药物处理组和单纯射线组相比较,药物联合射线组组织因子、MAPK通路相关蛋白ERK、JNK及p38磷酸化水平降低。结论CDA-Ⅱ抑制组织因子表达能提高肺癌细胞放疗敏感性,其机制可能与MAPK途径相关。

Objective To investigate the effect and mechanism of uropolypeptide(CDA-Ⅱ)inhibiting tissue factor expression on radiosensitivity of lung cancer cells.Methods In vitro:Establish experimental groups:control group(only containing A549 and H1395 cells),different doses of CDA-Ⅱgroup(0.03,0.06,0.12,0.25,0.50,1.00,2.00,3.00 mg/L),a total of 9 groups,In each group,the cell viability was tested at the two time nodes of 24 h and 48 h;the clone sensitization experiment was conducted to explore its effect on lung cancer cell sensitization;the cells were divided into control group(no drugs and radiation treatment),radiation group(only 8 Gy radiation treatment),radiation+0.06 mg/CDA-Ⅱgroup(for 8 Gy radiation+0.06 mg/L CDA-Ⅱdrug treatment),radiation+0.06 mg/CDA-Ⅱgroup(for 8 Gy radiation+0.12 mg/L CDA-Ⅱdrug Treatment),flow cytometric detection of cell apoptosis;Western blot detection of different treatment groups(blank control group,0.06 mg/L CDA-Ⅱtreatment group,0.12 mg/L CDA-Ⅱtreatment group,simple radiation group,0.06 mg/L CDA-ⅡThe changes in phosphorylation levels of tissue factor and mitogen-activated protein kinase(MAPK)pathway protein in cells in the treatment+radiation group,0.12 mg/L CDA-Ⅱtreatment+radiation group,and then clarified the mechanism of radiotherapy sensitization.Results CDA-Ⅱinhibited the proliferation of lung cancer cells in a dose-dependent manner.The 24 h half inhibitory concentration(IC50)was 0.195 mg/L for A549 and 0.32 mg/L for H1395.Clicked on the multi-target model analysis showed that the clone formation showed that CDA-Ⅱinhibited the expression of TF.It can improve the radiosensitivity of lung cancer cells in a dose-dependent manner.The sensitization ratio(SER)of 0.06,0.12 mg/L CDA-Ⅱwas(A549:1.17,1.28;H1395:1.08,1.25);flow cytometry experiments confirmed that the cell apoptosis rate after treatment with the drug CDA-Ⅱcombined with radiation was higher than that of simple radiation the treatment group increased,and increased with the increase of the dose of CDA-Ⅱ;Western blotting results showed that compared with the blank control group,the drug-only treatment group and the radiation-only group,the drug combined radiation group tissue factor,the levels of MAPK pathway related protein ERK,JNK and p38 phosphorylation was reduced.Conclusion CDA-Ⅱinhibition of tissue factor expression can increase the radiotherapy sensitivity of lung cancer cells,and its mechanism may be related to the MAPK pathway.