Du Yao-Wu, Liu Guang-Chao, Wang Jing, Zhao Yue-Ping, Li Shu-Lian, Chen Ju-Gao, Jiang Qi, Cai Jing, Ma Yuan-Fang
Institute of Immunology, Medical College, Henan University, Kaifeng, Henan, PR China.
Ai Zheng. 2009 Feb;28(2):112-6. Epub 2009 Feb 27.
Both tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and some monoclonal agonistic antibodies against TRAIL receptors have antitumor activity. We have previously prepared a novel monoclonal agonistic antibody against human death receptor 5 (DR5) and designated it as mDRA-6. This study was to explore the Caspase-dependent molecular mechanisms of mDRA-6 inducing apoptosis of human leukemia Jurkat cells.
After exposure to different doses of mDRA-6, DNA fragmentation of Jurkat cells was detected by agarose gel electrophoresis, cell proliferation was detected by MTT assay, and cell apoptosis was detected by flow cytometry after Annexin V-FITC/PI double staining. Jurkat cells were further treated with the inhibitors for Caspase-10, -9, -8 and -3. The active cleavage products of Caspase-10, -9, -8, -3 and poly ADP-ribose polymerase (PARP), BH3 interacting domain death agonist (Bid), truncated Bid (tBid) and cytochrome c (Cyto c), were analyzed by western blot.
After mDRA-6 treatment, DNA fragmentation was detected in Jurkat cells. mDRA-6 inhibited cell proliferation in a dose-dependent manner. When treated with 2.0 microg/mL mDRA-6, the apoptosis rates of Jurkat cells were 16.2% at 0.25 h, 28.3% at 0.5 h, 69.2% at 1 h and 78.2% at 2 h. Interestingly, the mDRA-6-induced apoptosis was repressed by 77.9% by Caspase-8 inhibitor ZIF, 54.2% by Caspase-3 inhibitor ZDF, and 8.7% by Caspase-9 inhibitor ZLF, but was not repressed by Caspase-10 inhibitor ZAF. After mDRA-6 exposure, the proenzymes of Caspase-8, -9 and -3 were reduced and their active cleavage products were increased along with the increase of exposure time, the cleavage products of PARP were also increased, Bid was degraded to tBid, and an abundance of Cyto c was released from mitochondria, but the proenzyme of Caspase-10 showed no change and no cleavage products of Caspase-10 were detectable.
mDRA-6 can induce apoptosis of Jurkat cells via the Caspase-dependent and mitochondrial pathways.
肿瘤坏死因子相关凋亡诱导配体（TRAIL）以及一些针对TRAIL受体的单克隆激动抗体均具有抗肿瘤活性。我们之前制备了一种新型的抗人死亡受体5（DR5）单克隆激动抗体，并将其命名为mDRA-6。本研究旨在探讨mDRA-6诱导人白血病Jurkat细胞凋亡的半胱天冬酶依赖性分子机制。
用不同剂量的mDRA-6处理后，通过琼脂糖凝胶电泳检测Jurkat细胞的DNA片段化，用MTT法检测细胞增殖，用Annexin V-FITC/PI双染后通过流式细胞术检测细胞凋亡。用半胱天冬酶-10、-9、-8和-3的抑制剂进一步处理Jurkat细胞。通过蛋白质印迹分析半胱天冬酶-10、-9、-8、-3和聚ADP核糖聚合酶（PARP）、BH3相互作用结构域死亡激动剂（Bid）、截短的Bid（tBid）和细胞色素c（Cyto c）的活性裂解产物。
mDRA-6处理后，在Jurkat细胞中检测到DNA片段化。mDRA-6以剂量依赖性方式抑制细胞增殖。用2.0μg/mL mDRA-6处理时，Jurkat细胞的凋亡率在0.25小时为16.2%，在0.5小时为28.3%，在1小时为69.2%，在2小时为78.2%。有趣的是，半胱天冬酶-8抑制剂ZIF可使mDRA-6诱导的凋亡受到77.9%的抑制，半胱天冬酶-3抑制剂ZDF可使其受到54.2%的抑制，半胱天冬酶-9抑制剂ZLF可使其受到8.7%的抑制，但半胱天冬酶-10抑制剂ZAF不能抑制。mDRA-6作用后，随着作用时间的增加，半胱天冬酶-8、-9和-3的酶原减少，其活性裂解产物增加，PARP的裂解产物也增加，Bid降解为tBid，并且有大量的Cyto c从线粒体释放，但半胱天冬酶-10的酶原没有变化，也未检测到半胱天冬酶-10的裂解产物。
mDRA-6可通过半胱天冬酶依赖性和线粒体途径诱导Jurkat细胞凋亡。
