叉头蛋白转录因子3a／p27激酶抑制蛋白1及氧化应激介导紫铆因抗肺腺癌的机制

Butein induces lung adenocarcinoma A549 cell apoptosis via activating forkhead box O 3a/p27 kinase inhibitor protein 1 and cell oxidative stress

目的观察紫铆因（Butein）的抗肺腺癌作用及叉头蛋白转录因子3a（FOX03a）／p27激酶抑制蛋白1（p27kipl）和氧化应激在该过程中的作用。方法常规培养A549细胞，分别给予25、50、100μmol／L的Butein处理24h后，检测细胞活力、细胞迁移能力、半胱氨酰天冬氨酸特异性蛋白酶（Caspase）-3活性、烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶活性、活性氧（ROS）含量、总谷胱甘肽（GSH）水平，从而明确Butein通过激活氧化应激抑制A549细胞。Westernblot法检测FOX03a、p27kipl、B淋巴细胞瘤-2（bcl-2）、bcl-2相关x蛋白（bax）的表达水平。进而采用小干扰RNA（siRNA）干扰法敲减FOX03a表达并检测FOX03a、p27kipl以及bax、bcl-2的表达水平，从而明确FOX03a／p27kipl通路在该过程中的作用。结果25、50、100μmol／L的Butein处理A549细胞24h后，细胞活力分别下降到（78．3±4．6）％、（63．5±4．8）％和（40．1±4．1）％（t=10．330，P=0．000），细胞划痕间距增大到（120．0±8．4）％、（150．0±10．6）％和（175．0±11.1）％（t=4．777，P=0．001）。Butein处理后Caspase-3、NADPH氧化酶的活性增强，ROS含量增多，总GSH减少。Westernblot结果显示Butein激活了FOX03a／p27kipl通路，上调了bax／bcl-2的比值。siRNA处理后阻断了FOX03a／p27kip1通路，逆转了Butein引起的bax的增多和bcl-2的减少。结论Butein能显著抑制肺腺癌A549细胞增殖和迁移，该作用可能是通过激活FOX03a／p27kipl信号通路和细胞内氧化应激进而诱导细胞凋亡实现的。

Objective To investigate the role of Butein on lung adenocarcinoma A549 ceils and the role of forkhead box O 3a （ FOXO3a）/p27 kinase inhibitor protein 1 （ p27kipl ） and cell oxidative stress in this process. Methods A549 cells were treaded with 25, 50 and 100μmol/L Butein, then the cell viability, cell migration, Caspase- 3 activity, NADPH oxidase activity, Reactive oxygen species （ROS） and total glutathione （GSH） were detected. Further expressions of FOXO3a, p27kipl, B- cell lymphoma- 2 （bcl- 2） and bcl -2 associated X protein （bax） were detected by Western blotting. In addition, small interference RNA was used to knock down FOXO3a expression in A549 cell. Then the cells were divided into four groups, which were the control group, the FOXO3a siRNA group, the Butein 100 μmol/L group and FOXO3a siRNA ± Butein 100 μmol/L group. Expression of FOXO3a, p27kipl, bc1-2 and bax were detected. Results After treatment of Butein （25, 50, 100 μmol/L）, cell viability were reduced from 100% to （78.3±4.6）%, （63.5 ±4.8）% and （40.1 ±4.1）% （t=10.330, P= 0.000） and the distance between cell boundary were increased from 100% to （120.0 ± 8.4）%, （150.0±10.6）% and （175.0±11.1）% （t=4.777, P=0.001）. In addition, Butein treatment in-creased Caspase - 3 activity, NADPI-I oxidase activity and ROS production, and reduced total GSH concen- tration. Furthermore, Butein treatment increased FOXO3a, p27kipl and bax expression and reduced bcl- 2 expression. FOXO3a siRNA treatment blocked FOXO3a expression and p27kipl transcription, and reversed Butein induced A549 cell apoptosis by increasing bcl -2 and reducing bax expression. Conclu- sion Butein can induce A549 cell apootosis via activating FOXO3a/p27kipl and cell oxidative stress.