Kim Sang Soo, Cho Hee-Ju, Cho Jeong-Man, Kang Jung Yoon, Yang Hyun-Won, Yoo Tag Keun
Eulji Medi-Bio Research Institute, Seoul 139-871, Republic of Korea.
ScientificWorldJournal. 2013 Jun 19;2013:174392. doi: 10.1155/2013/174392. Print 2013.
We evaluated effect of dual gene silencing of Hsp27 and c-FLIP in doxazosin-induced apoptosis of PC-3 cell. After transfection using Hsp27 and c-FLIP siRNA mixture (dual silencing), doxazosin treatment was done at the concentrations of 1, 10, and 25 μ M. We checked apoptosis of PC-3 cells with and TUNEL staining. We also checked interaction between Hsp27 and C-FLIP in the process of apoptosis inhibition. Spontaneous apoptotic index was 5% under single gene silencing of Hsp27 and c-FLIP and 7% under dual silencing of Hsp27 and c-FLIP. When doxazosin treatment was added, apoptotic indices increased in a dose-dependent manner (1, 10, and 25 μ M): nonsilencing 10, 27, and 52%; Hsp27-silencing: 14, 35, and 68%; c-FLIP silencing: 21, 46, and 78%; dual silencing: 38, 76, and 92%. While c-FLIP gene expression decreased in Hsp27- silenced cells, Hsp27 gene expression showed markedly decreased pattern in the cells of c-FLIP silencing. The knockout of c-FLIP and Hsp27 genes together enhances apoptosis even under 1 μ M, rather than low concentration, of doxazosin in PC-3 cells. This finding suggests a new strategy of multiple knockout of antiapoptotic and survival factors in the treatment of late-stage prostate cancer refractory to conventional therapy.
我们评估了热休克蛋白27（Hsp27）和细胞凋亡抑制蛋白（c-FLIP）双基因沉默对多沙唑嗪诱导的PC-3细胞凋亡的影响。使用Hsp27和c-FLIP siRNA混合物转染（双沉默）后，分别用1、10和25μM浓度的多沙唑嗪进行处理。我们通过TUNEL染色检测PC-3细胞的凋亡情况。我们还检测了凋亡抑制过程中Hsp27和C-FLIP之间的相互作用。在Hsp27和c-FLIP单基因沉默下，自发凋亡指数为5%，在Hsp27和c-FLIP双基因沉默下为7%。加入多沙唑嗪处理后，凋亡指数呈剂量依赖性增加（1、10和25μM）：非沉默组分别为10%、27%和52%；Hsp27沉默组为14%、35%和68%；c-FLIP沉默组为21%、46%和78%；双沉默组为38%、76%和92%。在Hsp27沉默的细胞中c-FLIP基因表达下降，而在c-FLIP沉默的细胞中Hsp27基因表达显著下降。即使在1μM而非低浓度的多沙唑嗪作用下，同时敲除c-FLIP和Hsp27基因也能增强PC-3细胞的凋亡。这一发现提示了一种新的策略，即多重敲除抗凋亡和生存因子，用于治疗对传统疗法难治的晚期前列腺癌。
