Shimada Keiji, Nakamura Mitsutoshi, Ishida Eiwa, Kishi Munehiro, Yonehara Shin, Konishi Noboru
Second Department of Pathology, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521, Japan.
Jpn J Cancer Res. 2002 Oct;93(10):1164-74. doi: 10.1111/j.1349-7006.2002.tb01219.x.
Fas-associated death domain (FADD) plays an important role as an adapter molecule in Fas (CD95/APO-1)-mediated apoptosis and contributes to anticancer drug-induced cytotoxicity. We treated three human prostate cancer cell lines with etoposide, a toposiomerase II inhibitor with activity against various tumors including prostate cancer. We found that the overexpression of FADD sensitizes etoposide-induced apoptosis through a rapid activation of c-Jun NH(2)-terminal kinase (JNK) and, subsequently, of caspase 3. In addition, phosphorylation of FADD at serine 194 coincided with this sensitization. Treatment with the caspase 3 inhibitor, N-acetyl-Asp-Glu-Val-Asp-aldehyde (DEVD-CHO), or overexpression of either mitogen-activated protein kinase kinase (MKK) 7 or Bcl-xL canceled FADD-mediated sensitization to etoposide-induced apoptosis. Moreover, treatment with the caspase 8 inhibitor, benzyloxy-carbonyl-Val-Ala-Asp-fluoromethylketone (z-IETD-fmk), or overexpression of viral FLICE/caspase-8-inhibitory protein (FLIP) from equine herpesvirus type 2 E8 also had an inhibitory effect, supporting a major involvement of a caspase 8-dependent mitochondrial pathway. Interestingly, FADD was phosphorylated, and etoposide-induced JNK/caspase activation and apoptosis were enhanced in the cells arrested at G2/M transition, but not in those overexpressing mutant FADD, in which 194 serine was replaced by alanine. Our results demonstrate that phosphorylated FADD-dependent activation of the JNK/caspase pathway plays a pivotal role in sensitization to etoposide-induced apoptosis in prostate cancer cells.
Fas相关死亡结构域（FADD）作为衔接分子在Fas（CD95/APO-1）介导的细胞凋亡中发挥重要作用，并参与抗癌药物诱导的细胞毒性作用。我们用拓扑异构酶II抑制剂依托泊苷处理了三种人前列腺癌细胞系，该抑制剂对包括前列腺癌在内的多种肿瘤具有活性。我们发现，FADD的过表达通过快速激活c-Jun氨基末端激酶（JNK），随后激活caspase 3，使依托泊苷诱导的细胞凋亡敏感化。此外，FADD在丝氨酸194处的磷酸化与这种敏感化同时发生。用caspase 3抑制剂N-乙酰天冬氨酸-谷氨酸-缬氨酸-天冬氨酸醛（DEVD-CHO）处理，或有丝分裂原活化蛋白激酶激酶（MKK）7或Bcl-xL的过表达，均可消除FADD介导的对依托泊苷诱导的细胞凋亡的敏感化作用。此外，用caspase 8抑制剂苄氧羰基-缬氨酸-丙氨酸-天冬氨酸-氟甲基酮（z-IETD-fmk）处理，或过表达来自马疱疹病毒2型E8的病毒FLICE/caspase-8抑制蛋白（FLIP）也具有抑制作用，支持caspase 8依赖性线粒体途径的主要参与。有趣的是，在停滞于G2/M期转换的细胞中，FADD被磷酸化，依托泊苷诱导的JNK/caspase激活和细胞凋亡增强，但在过表达将194位丝氨酸替换为丙氨酸的突变型FADD的细胞中则不然。我们的结果表明，JNK/caspase途径的磷酸化FADD依赖性激活在前列腺癌细胞对依托泊苷诱导的细胞凋亡的敏感化中起关键作用。
