Downmodulation of Bcl-2 Sensitizes Metastatic LNCaP-LN3 Cells to Undergo Apoptosis via the Intrinsic Pathway
Downmodulation of Bcl-2 Sensitizes Metastatic LNCaP-LN3 Cells to Undergo Apoptosis via the Intrinsic Pathway
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DOI：
10.1002/pros.21091
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发表时间：
2010-05-01
期刊：
影响因子：
2.8
通讯作者：
Pettaway, Curtis A.
中科院分区：
文献类型：
--
作者：
Song, Renduo;Harris, Loleta D.;Pettaway, Curtis A.
背景。我们探讨了转移性LNCaP-LN3细胞（LN3）下调Bcl-2蛋白后凋亡的机制。LNCaP、LNCaP-Pro5 （Pro5）和LN3细胞分别在5%炭脱血清（CSS）或R1881（合成雄激素）和比卡鲁胺（合成抗雄激素）中培养，观察其生长抑制情况。测定雄激素受体（AR）和Bcl-2的表达水平。LN3细胞转染小干扰RNA Bcl-2 （siRNA Bcl-2）或对照siRNA寡核苷酸。计算细胞凋亡率和增殖率。用+/-环孢素A （CsA）测定处理细胞和对照细胞的细胞色素c定位。下调Bcl-2后，检测Caspases 9、3和poly （adp -核糖）聚合酶裂解（PARP）；Bcl-2调节联合多西紫杉醇化疗对体外细胞生长的抑制作用。LN3细胞在体外去势条件下保持生长。AR蛋白扩增不能解释LN3细胞的抗去势生长。LN3细胞中的Bcl-2蛋白水平明显高于Pro5细胞，并被siRNA Bcl-2有效下调。随后观察到细胞色素c介导的细胞凋亡增加和增殖减少，CsA逆转了这一现象。转染siRNA bcl -2的LN3细胞显示出高水平的caspase 9、3和PARP切割。LN3细胞暴露于多西紫杉醇导致凋亡增加，同时下调Bcl-2显著增强了这一作用。在转移性去势抵抗LNCaP-LN3细胞中，Bcl-2下调通过细胞色素c依赖途径导致凋亡，这一途径在多西他赛治疗下得到增强。中华医学会医学分会，2010。(C) 2009 Wiley-Liss, Inc。
BACKGROUND. We explored the mechanisms of apoptosis after Bcl-2 protein down-modulation in metastatic LNCaP-LN3 cells (LN3).METHODS. 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.RESULTS. 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.CONCLUSIONS. 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. Prostate 70: 571-583, 2010. (C) 2009 Wiley-Liss, Inc.