Li X, Marani M, Mannucci R, Kinsey B, Andriani F, Nicoletti I, Denner L, Marcelli M
Department of Medicne, Baylor College of Medicine, Veterans Affairs Medical Center, Houston 77030, USA.
Cancer Res. 2001 Feb 15;61(4):1699-706.
We have reported previously that among human prostate cancer cell lines LNCaP but not PC-3 cells undergo apoptosis after treatment with the protein kinase inhibitor staurosporine (STS). We have now further investigated this model to uncover the molecular mechanism causing resistance to STS-induced apoptosis in PC-3 cells. S-100 lysates of both cell lines showed biochemical changes typical of apoptosis after the addition of cytochrome c and dATP, suggesting that the postmitochondrial phase of apoptosis was intact. Upon addition of STS, the proapoptotic molecules Bax and Bad became predominantly mitochondrial in both cell lines. This, in turn, was followed by loss of mitochondrial transmembrane potential, translocation of cytochrome c to the cytosol, activation of caspase-9, -3, and -7, and cleavage of the apoptotic targets, DNA fragmentation factor and poly(ADP-ribose) polymerase, in LNCaP but not in PC-3 cells. Components of the mitochondrial permeability transition pore, adenine nucleotide transporter and voltage-dependent anion channel, were normally expressed in the correct subcellular fraction of both cell lines. Overexpression of the proapoptotic proteins Bax and Bad, fused to a green fluorescent protein but not of green fluorescent protein alone, induced apoptosis in >80% of PC-3 cells. These experiments suggested that a factor protecting the mitochondria of PC-3 cells mediates resistance to STS-induced apoptosis. A wide search among the antiapoptotic Bcl-2 family members was performed, and Bcl-X(L) was found to be overexpressed in PC-3 cells. Experiments down-regulating Bcl-X(L) expression by using the tyrosine kinase inhibitor genistein, sodium butyrate, or an antisense Bcl-X(L) oligonucleotide restored sensitivity to apoptosis in PC-3 cells. Thus, Bcl-X(L) overexpression is one of the mediators of resistance to STS-induced apoptosis in the prostate cancer cell line PC-3.
我们之前报道过，在人前列腺癌细胞系中，蛋白激酶抑制剂星形孢菌素（STS）处理后，LNCaP细胞会发生凋亡，而PC-3细胞不会。我们现在进一步研究该模型，以揭示PC-3细胞对STS诱导凋亡产生抗性的分子机制。添加细胞色素c和dATP后，两种细胞系的S-100裂解物均显示出凋亡典型的生化变化，这表明凋亡的线粒体后阶段是完整的。添加STS后，促凋亡分子Bax和Bad在两种细胞系中主要定位于线粒体。继而，LNCaP细胞出现线粒体跨膜电位丧失、细胞色素c转位至细胞质、caspase-9、-3和-7激活以及凋亡靶点DNA片段化因子和聚（ADP-核糖）聚合酶的裂解，而PC-3细胞未出现这些情况。线粒体通透性转换孔的成分、腺嘌呤核苷酸转运体和电压依赖性阴离子通道在两种细胞系的正确亚细胞组分中均正常表达。促凋亡蛋白Bax和Bad与绿色荧光蛋白融合而非单独的绿色荧光蛋白过表达，可诱导>80%的PC-3细胞凋亡。这些实验表明，保护PC-3细胞线粒体的一个因子介导了对STS诱导凋亡的抗性。我们对抗凋亡Bcl-2家族成员进行了广泛搜索，发现Bcl-X(L)在PC-3细胞中过表达。使用酪氨酸激酶抑制剂染料木黄酮、丁酸钠或反义Bcl-X(L)寡核苷酸下调Bcl-X(L)表达的实验恢复了PC-3细胞对凋亡的敏感性。因此，Bcl-X(L)过表达是前列腺癌细胞系PC-3对STS诱导凋亡产生抗性的介导因素之一。
