Nastiuk Kent L, Yoo Kiwon, Lo Karen, Su Kevin, Yeung Patricia, Kutaka Julia, Danielpour David, Krolewski John J
Department of Pathology and Laboratory Medicine, School of Medicine, University of California Irvine, Medical Sciences I D450, Irvine, CA 92697-4800, USA.
Mol Cancer Res. 2008 Feb;6(2):231-42. doi: 10.1158/1541-7786.MCR-07-0386.
Androgen withdrawal induces the regression of human prostate cancers, but such cancers eventually become androgen-independent and metastasize. Thus, deciphering the mechanism of androgen withdrawal-induced apoptosis is critical to designing new therapies for prostate cancer. Previously, we showed that in the rat, castration-induced apoptosis is accompanied by a reduction in the expression of the apical caspase inhibitor FLICE-like inhibitory protein (FLIP). To test the functional role of FLIP in inhibiting prostate epithelial cell apoptosis, we employed the rat prostate epithelial cell line NRP-152, which differentiates to a secretory phenotype in a low-mitogen medium and then undergoes apoptosis following the addition of transforming growth factor beta1 (TGFbeta1), mimicking androgen withdrawal-induced apoptosis. FLIP levels decline with TGFbeta1 treatment, suggesting that apoptosis is mediated by caspase-8 and indeed the caspase inhibitor crmA blocks TGFbeta1-induced apoptosis. Small interfering RNA-mediated knockdown of FLIP recapitulates and enhances TGFbeta1-induced cell death. NRP-152 cells stably transfected with constitutively expressed FLIP were refractory to TGFbeta1-induced apoptosis. TGFbeta1-induced caspase-3 activity is proportional to the level of cell death and inversely proportional to the level of FLIP expression in various clones. Moreover, neither caspase-3 nor PARP is cleaved in clones expressing high levels of FLIP. Furthermore, insulin, which inhibits differentiation, increases FLIP and inhibits TGFbeta-induced death in a FLIP-dependent manner. Although neither Fas-Fc, sTNFRII-Fc, nor DR5-Fc blocked TGFbeta1-induced cell death, there is a significant increase in tumor necrosis factor mRNA following TGFbeta stimulation, suggesting both an unexpected role for tumor necrosis factor in this model system and the possibility that FLIP blocks another unknown caspase-dependent mediator of apoptosis.
雄激素撤除可诱导人前列腺癌消退，但此类癌症最终会变得雄激素非依赖并发生转移。因此，阐明雄激素撤除诱导细胞凋亡的机制对于设计前列腺癌新疗法至关重要。此前，我们发现，在大鼠中，去势诱导的细胞凋亡伴随着顶端半胱天冬酶抑制剂FLICE样抑制蛋白（FLIP）表达的降低。为了测试FLIP在抑制前列腺上皮细胞凋亡中的功能作用，我们使用了大鼠前列腺上皮细胞系NRP-152，该细胞系在低丝裂原培养基中分化为分泌表型，然后在添加转化生长因子β1（TGFβ1）后发生凋亡，模拟雄激素撤除诱导的细胞凋亡。FLIP水平随TGFβ1处理而下降，表明细胞凋亡由半胱天冬酶-8介导，事实上半胱天冬酶抑制剂crmA可阻断TGFβ1诱导的细胞凋亡。小干扰RNA介导的FLIP敲低可重现并增强TGFβ1诱导的细胞死亡。稳定转染组成型表达FLIP的NRP-152细胞对TGFβ1诱导的细胞凋亡具有抗性。TGFβ1诱导的半胱天冬酶-3活性与细胞死亡水平成正比，与各个克隆中FLIP表达水平成反比。此外，在表达高水平FLIP的克隆中，半胱天冬酶-3和PARP均未被切割。此外，抑制分化的胰岛素以FLIP依赖的方式增加FLIP并抑制TGF诱导的死亡。尽管Fas-Fc、sTNFRII-Fc和DR5-Fc均未阻断TGFβ1诱导的细胞死亡，但TGFβ刺激后肿瘤坏死因子mRNA显著增加，提示肿瘤坏死因子在该模型系统中具有意外作用，且FLIP可能阻断另一种未知的半胱天冬酶依赖性凋亡介质。
