Mi Qiuwen, Su Bao-Ning, Chai Heebyung, Cordell Geoffrey A, Farnsworth Norman R, Kinghorn A Douglas, Swanson Steven M
Program for Collaborative Research in the Pharmaceutical Sciences, Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60612, USA.
Anticancer Res. 2006 Mar-Apr;26(2A):947-52.
Rocaglaol is a cytotoxic cyclopenta[b]benzofuran isolated from the bark of Aglaia crassinervia. It exhibited in vitro cytotoxic activity against Lu1, LNCaP and MCF-7 cells with ED50 values of 13.8, 23.0 and 9.2 nM, respectively. DAPI staining indicated that LNCaP cells treated with rocaglaol underwent apoptosis. In order to determine whether rocaglaol-induced apoptosis is mediated by the mitochondrial pathway, apoptosis-related mitochondrial-associated proteins were studied. Rocaglaol treatment induced Bax expression through 12 to 72 h of exposure, while Bcl-xl expression was slightly decreased through 48 h, and decreased more significantly by 72 h. Cleaved caspase-9 expression was detected at 72 h, and cleaved caspase-7 was increased through 48 to 72 h. Consequently, the large fragment (89 kDa) of PARP resulting from caspase cleavage was detected at 12, 24 and 48 h, and especially at 72 h. Cleaved PARP expression was also detected at 72 h. Since rocaglaol caused dose-dependent G2/M phase arrest of LNCaP cells as indicated by flow cytometric analysis, the protein levels of cell cycle-related genes were measured. Rocaglaol treatment (230 nM) did not change cyclin B after 24- to 60-h treatment. The expression of cdc2 was not changed and phospho-cdc2 (Tyr 15) increased after 36-, 48- and 60-h treatment. In addition, protein phosphatase Cdc25C, which functions as a mitotic activator by dephosphorylation of Cdc2, decreased in a time-dependent manner after rocaglaol treatment. Taken together, these results suggest that rocaglaol is a potent anticancer drug that induces apoptosis of LNCaP cells through the mitochondrial pathway and its G2/M-phase cell cycle arrest is associated with the down-regulation of Cdc25C and the dephosphorylation of Cdc2.
罗卡劳尔是一种从粗脉米仔兰树皮中分离得到的具有细胞毒性的环戊[b]苯并呋喃。它在体外对Lu1、LNCaP和MCF-7细胞表现出细胞毒性活性，其半数有效剂量（ED50）值分别为13.8、23.0和9.2 nM。4',6-二脒基-2-苯基吲哚（DAPI）染色表明，用罗卡劳尔处理的LNCaP细胞发生了凋亡。为了确定罗卡劳尔诱导的凋亡是否由线粒体途径介导，对凋亡相关的线粒体相关蛋白进行了研究。罗卡劳尔处理在12至72小时的暴露时间内诱导了Bax表达，而Bcl-xl表达在48小时内略有下降，在72小时时下降更为显著。在72小时时检测到裂解的半胱天冬酶-9表达，并且在48至72小时内裂解的半胱天冬酶-7增加。因此，在12、24和48小时，尤其是在72小时时检测到由半胱天冬酶裂解产生的聚（ADP-核糖）聚合酶（PARP）的大片段（89 kDa）。在72小时时也检测到裂解的PARP表达。由于流式细胞术分析表明罗卡劳尔导致LNCaP细胞出现剂量依赖性的G2/M期阻滞，因此测量了细胞周期相关基因的蛋白水平。在24至60小时的处理后，罗卡劳尔处理（230 nM）未改变细胞周期蛋白B。在36、48和60小时的处理后，细胞分裂周期蛋白2（cdc2）的表达未改变，而磷酸化的cdc2（酪氨酸15）增加。此外，通过使Cdc2去磷酸化而作为有丝分裂激活剂发挥作用的蛋白磷酸酶Cdc25C在罗卡劳尔处理后呈时间依赖性下降。综上所述，这些结果表明罗卡劳尔是一种有效的抗癌药物，它通过线粒体途径诱导LNCaP细胞凋亡，并且其G2/M期细胞周期阻滞与Cdc25C的下调和Cdc2的去磷酸化有关。
