Department of Urology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
Prostate. 2010 May 1;70(6):571-83. doi: 10.1002/pros.21091.
We explored the mechanisms of apoptosis after Bcl-2 protein downmodulation in metastatic LNCaP-LN3 cells (LN3).
LNCaP, LNCaP-Pro5 (Pro5) and LN3 cells were cultured in 5% charcoal-stripped serum (CSS) or in R1881 (synthetic androgen) and bicalutamide (synthetic anti-androgen) and growth inhibition was assessed. Expression levels of androgen receptor (AR) and Bcl-2 were determined. LN3 cells were transfected with small interfering RNA Bcl-2 (siRNA Bcl-2) or control siRNA oligonucleotides. Rates of apoptosis and proliferation were obtained. Cytochrome c localization in treated and control cells was assessed +/- cyclosporine A (CsA). Caspases 9, 3, and poly (ADP-ribose) polymerase cleavage (PARP) were measured upon downmodulation of Bcl-2; and cell growth inhibition in vitro after Bcl-2 modulation combined with docetaxel chemotherapy was determined.
LN3 cells maintained growth under castrate conditions in vitro. AR protein amplification did not explain castrate-resistant LN3 cell growth. Bcl-2 protein levels in LN3 cells were significantly higher than in Pro5 cells, and were effectively downmodulated by siRNA Bcl-2. Subsequently increased apoptosis and decreased proliferation mediated by cytochrome c was noted and this was reversed by CsA. siRNA Bcl-2-transfected LN3 cells exhibited elevated levels of caspases 9, 3, and PARP cleavage. Exposure of LN3 cells to docetaxel led to increased apoptosis, and simultaneous downmodulation of Bcl-2 substantially enhanced this effect.
Downmodulation of Bcl-2 in metastatic castrate-resistant LNCaP-LN3 cells led to apoptosis via a cytochrome c-dependent pathway that was enhanced with docetaxel treatment.
我们探索了 Bcl-2 蛋白下调后转移性 LNCaP-LN3 细胞（LN3）凋亡的机制。
在 5%去铁蛋白血清（CSS）或 R1881（合成雄激素）和比卡鲁胺（合成抗雄激素）中培养 LNCaP、LNCaP-Pro5（Pro5）和 LN3 细胞，并评估生长抑制情况。检测雄激素受体（AR）和 Bcl-2 的表达水平。LN3 细胞用小干扰 RNA Bcl-2（siRNA Bcl-2）或对照 siRNA 寡核苷酸转染。获得凋亡和增殖率。用环孢素 A（CsA）处理和对照细胞，评估细胞色素 c 的定位。下调 Bcl-2 后测定半胱天冬酶 9、3 和多聚（ADP-核糖）聚合酶裂解（PARP）；并测定 Bcl-2 调节与多西紫杉醇化疗相结合后体外细胞生长抑制作用。
LN3 细胞在体外去势条件下维持生长。AR 蛋白扩增并不能解释 LN3 细胞的去势抵抗性生长。LN3 细胞中的 Bcl-2 蛋白水平明显高于 Pro5 细胞，并且可以被 siRNA Bcl-2 有效下调。随后观察到细胞色素 c 介导的凋亡增加和增殖减少，而 CsA 则逆转了这种作用。siRNA Bcl-2 转染的 LN3 细胞表现出半胱天冬酶 9、3 和 PARP 裂解水平升高。用多西紫杉醇处理 LN3 细胞导致凋亡增加，同时下调 Bcl-2 则大大增强了这种作用。
下调转移性去势抵抗性 LNCaP-LN3 细胞中的 Bcl-2 可通过细胞色素 c 依赖性途径诱导凋亡，与多西紫杉醇治疗联合使用可增强该作用。
