抗人DR5功能性抗体mDRA-6诱导人白血病Jurkat细胞凋亡的机制

Caspase-dependent molecular mechanisms of anti-human DR5 monoclonal antibody mDRA-6 inducing apoptosis of human leukemia Jurkat cells

背景与目的:肿瘤坏死因子相关凋亡诱导配体(tumor necrosis factor-related apoptosis-inducing ligand,TRAIL)及其死亡受体的功能性单克隆抗体具有杀伤肿瘤细胞的活性。我们研制了抗人死亡受体5(death receptor,DR5)功能性单克隆抗体(mDRA-6),本研究对其诱导Jurkat细胞凋亡的Caspase分子机制进行初步探讨。方法:mDRA-6作用后,琼脂糖凝胶电泳检测Jurkat细胞的DNA ladder;MTT法检测细胞存活情况;Annexin V-FITC/PI双染流式细胞术定量分析细胞凋亡情况;观察Caspase-10、-9、-8、-3等的抑制剂对mDRA-6诱导细胞凋亡的抑制作用;免疫印迹技术检测凋亡信号蛋白Caspase-10、-9、-8、-3,多聚ADP核糖聚合酶(poly ADP-ribose polymerase,PARP)、BH3相关结构域死亡激动剂(BH3interactingdomain death agonist,Bid)、短缩的Bid(truncated Bid,tBid)、细胞色素C(cytochrome c,Cyto C)等活化裂解的情况。结果:琼脂糖凝胶电泳显示DNA呈现明显梯状带型;mDRA-6对Jurkat细胞具有明显的诱导凋亡作用且呈量-效关系。2.0μg/mL mDRA-6作用Jurkat细胞0.25h、0.5h、1h、2h,其凋亡率分别为16.2%、28.3%、69.2%、78.2%;Caspase-8抑制剂能明显抑制mDRA-6诱导的细胞凋亡,抑制率为77.9%,Caspase-3和Caspase-9抑制剂作用的抑制率分别为54.2%、8.7%,而Caspase-10的抑制剂无抑制作用;免疫印迹技术检测显示Caspase-8、-3、-9均呈现随时间延长酶原逐渐减少、活化片段增加的现象,PARP的降解片段亦增加,Bid激活降解为tbid,Cyto C大量释放,而Caspase-10酶原无明显改变、无活化片段出现。结论:mDRA-6诱导Jurkat细胞凋亡主要是通过激活Caspase路径和线粒体路径来完成的。

Background and Objective： 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. Methods. After exposure to different doses of mDRA-6, DNA fragmentation of Jurkat cells was detected by agarose gel electrophoresis, cell proliferation was detected by Ml-r 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）, cytochrome c （Cyto c） were analyzed by Western blot. Results： 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 μg/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 ZlF, 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, abundant 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. Conclusion： mDRA-6 can induce apoptosis of Jurkat cells via the Caspase-dependent and mitochondrial pathways.