Cancer Res Treat. 2003 Aug;35(4):341-8. doi: 10.4143/crt.2003.35.4.341.
Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL)/APO-2L is a member of the TNF family that can kill a wide variety of tumor cells, but not normal cells. This study was designed to investigate the down stream target proteins in TRAIL-mediated apoptosis of human liver, Chang cells.
The expressions of DR4/DR5 in hepatoma cells, including Chang, HepG2 and Hep3B cells, were determined by RT-PCR. Cell viability was measured by MTT assay and apoptosis was assessed by DNA fragmentation assay. The catalytic activity of caspase- family proteases, including caspase-3 and -9, was tested by using fluorogenic biosubstrates. Expression of apoptotic mediators, including procaspase-3 and PARP proteins, was measured by Western blotting. The expression profile of proteins in Chang cells by using two-dimensional (2-D) gel electrophoresis and MALDI-TOF.
The results demonstrated that TRAIL (100 ng/ml) induced the apoptotic death of Chang cells, as characterized by the ladder-pattern fragmentation of genomic DNA. TRAIL increased the enzymatic activity of caspase- 3, corresponding to the time of appearance of cleaved PARP and caspase-9. In 2-D gel electrophoresis and MALDI- TOF analysis, the comparison of control versus apoptotic cells in the protein expressions revealed that signal intensity of 7 spots were decreased, whereas 6 spots were increased among 300 spots. These spots were resolved and identified as a protein information by MALDI-TOF.
We suggested that TRAIL induces the apoptotic death of Chang cells via proteome alterations inducing caspase cascade.
肿瘤坏死因子（TNF）相关凋亡诱导配体（TRAIL）/APO-2L 是 TNF 家族的一员，能够杀死多种肿瘤细胞，但不会杀死正常细胞。本研究旨在探讨 TRAIL 介导的人肝癌 Chang 细胞凋亡的下游靶蛋白。
采用 RT-PCR 检测肝癌细胞（包括 Chang、HepG2 和 Hep3B 细胞）中 DR4/DR5 的表达。采用 MTT 法检测细胞活力，采用 DNA 片段化检测法评估细胞凋亡。采用荧光生物底物检测 caspase 家族蛋白酶（包括 caspase-3 和 caspase-9）的催化活性。采用 Western blot 法检测凋亡介质（包括 procaspase-3 和 PARP 蛋白）的表达。采用二维（2-D）凝胶电泳和 MALDI-TOF 分析 Chang 细胞的蛋白表达谱。
结果表明，TRAIL（100ng/ml）诱导 Chang 细胞凋亡，表现为基因组 DNA 的梯状片段化。TRAIL 增加了 caspase-3 的酶活性，与 cleaved PARP 和 caspase-9 的出现时间相对应。在 2-D 凝胶电泳和 MALDI-TOF 分析中，与凋亡细胞相比，对照细胞的蛋白表达比较显示 7 个点的信号强度降低，而 300 个点中有 6 个点增加。这些斑点通过 MALDI-TOF 被解析并鉴定为蛋白质信息。
我们认为 TRAIL 通过诱导 caspase 级联反应引起蛋白质组改变诱导 Chang 细胞凋亡。
