Department of Cellular Biochemistry, Institute of Hematology and Blood Transfusion, U Nemocnice 2, 128 20 Prague 2, Czech Republic.
J Cell Biochem. 2011 Nov;112(11):3334-42. doi: 10.1002/jcb.23263.
The effects of the pan-caspase inhibitor Q-VD-OPh on caspase activity, DNA fragmentation, PARP cleavage, 7A6 exposition, and cellular adhesivity to fibronectin were analyzed in detail in three different apoptotic systems involving two cell lines (JURL-MK1 and HL60) and two apoptosis inducers (imatinib mesylate and suberoylanilide hydroxamic acid). Q-VD-OPh fully inhibited caspase-3 and -7 activity at 0.05 µM concentration as indicated both by the measurement of the rate of Ac-DEVD-AFC cleavage and anti-caspase immunoblots. Caspase-8 was also inhibited at low Q-VD-OPh concentrations. On the other hand, significantly higher Q-VD-OPh dose (10 µM) was required to fully prevent the cleavage of PARP-1. DNA fragmentation and disruption of the cell membrane functionality (Trypan blue exclusion test) were both prevented at 2 µM Q-VD-OPh while 10 µM inhibitor was needed to inhibit the drug-induced loss of cellular adhesivity to fibronectin which was observed in JURL-MK1 cells. The exposition of the mitochondrial antigen 7A6 occurred independently of Q-VD-OPh addition and may serve to the detection of cumulative incidence of the cells which have initiated the apoptosis. Our results show that Q-VD-OPh efficiency in the inhibition of caspase-3 activity and DNA fragmentation in the whole-cell environment is about two orders of magnitude higher than that of z-VAD-fmk. This difference is not due to a slow permeability of the latter through the cytoplasmic membrane.
我们详细分析了泛半胱天冬酶抑制剂 Q-VD-OPh 对三种不同凋亡系统中半胱天冬酶活性、DNA 片段化、PARP 切割、7A6 暴露以及细胞对纤维连接蛋白黏附性的影响，这三种系统涉及两种细胞系（JURL-MK1 和 HL60）和两种凋亡诱导剂（伊马替尼甲磺酸盐和琥珀酰亚胺基羟胺）。0.05μM 的 Q-VD-OPh 浓度可完全抑制 caspase-3 和 -7 活性，这一点既可以通过 Ac-DEVD-AFC 切割速率的测量，也可以通过抗半胱天冬酶免疫印迹来证实。低浓度的 Q-VD-OPh 也可以抑制半胱天冬酶-8。另一方面，要完全阻止 PARP-1 的切割，则需要更高浓度的 Q-VD-OPh（10μM）。在 2μM Q-VD-OPh 时，可防止 DNA 片段化和细胞膜功能障碍（台盼蓝排斥试验），而在 JURL-MK1 细胞中，需要 10μM 的抑制剂来抑制药物诱导的细胞对纤维连接蛋白黏附性的丧失。线粒体抗原 7A6 的暴露与 Q-VD-OPh 的添加无关，可能用于检测已经启动凋亡的细胞的累积发生率。我们的结果表明，Q-VD-OPh 在全细胞环境中抑制 caspase-3 活性和 DNA 片段化的效率比 z-VAD-fmk 高约两个数量级。这种差异不是由于后者穿过细胞质膜的渗透性较慢所致。
