Chen Yen-Chou, Shen Shing-Chuan, Lee Woan-Ruoh, Hsu Foun-Lin, Lin Hui-Yi, Ko Ching-Huai, Tseng Shi-Wen
Graduate Institute of Pharmacognosy Science, Taipei Medical University, 250 Wu-Hsing Street, Taipei, Taiwan.
Biochem Pharmacol. 2002 Dec 15;64(12):1713-24. doi: 10.1016/s0006-2952(02)01386-2.
Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is an active constituent of Rheum palmatum, and showed inhibitory activity on lipopolysaccharide-induced NO production in our previous study. However, the apoptosis-inducing activity of emodin has remained undefined. Among three structurally related anthraquinones, including emodin, physcion, and chrysophanol, emodin showed the most potent cytotoxic effects on HL-60 cells, accompanied by the dose- and time-dependent appearance of characteristics of apoptosis including an increase in DNA ladder intensity, morphological changes, appearance of apoptotic bodies, and an increase in hypodiploid cells. Emodin at apoptosis-inducing concentrations causes rapid and transient induction of caspase 3/CPP32 activity, but not caspase 1 activity, according to cleavage of caspase 3 substrates poly(ADP-ribose) polymerase and D4-GDI proteins, the appearance of cleaved caspase 3 fragments being detected in emodin- but not physcion- or chrysophanol-treated HL-60 cells. A decrease in the anti-apoptotic protein, Mcl-1, was detected in emodin-treated HL-60 cells, whereas other Bcl-2 family proteins including Bax, Bcl-2, Bcl-XL, and Bad remained unchanged. The caspase 3 inhibitor, Ac-DEVD-CHO, but not the caspase 1 inhibitor, Ac-YVAD-CHO, attenuated emodin-induced DNA ladders, associated with the blockage of PARP and D4-GDI cleavage. Free radical scavenging agents including NAC, catalase, SOD, ALL, DPI, L-NAME and PDTC showed no preventive effect on emodin-induced apoptotic responses, whereas NAC, CAT and PDTC prevented HL-60 cells from ROS (H(2)O(2))-induced apoptosis through inhibition of caspase 3 cascades. Induction of catalase, but not SOD, activity was detected in emodin-treated HL-60 cells by in gel activity assays, and H(2)O(2)-induced intracellular peroxide level was significantly reduced by prior treatment of emodin in HL-60 cells. Our experiments provide evidence that emodin is an effective apoptosis inducer in HL-60 cells through activation of the caspase 3 cascade, but that it is independent of ROS production.
大黄素（1,3,8 - 三羟基 - 6 - 甲基蒽醌）是掌叶大黄的一种活性成分，在我们之前的研究中显示出对脂多糖诱导的一氧化氮产生具有抑制活性。然而，大黄素的凋亡诱导活性尚未明确。在三种结构相关的蒽醌（包括大黄素、大黄素甲醚和大黄酚）中，大黄素对HL - 60细胞显示出最有效的细胞毒性作用，同时伴随着凋亡特征的剂量和时间依赖性出现，包括DNA梯状条带强度增加、形态变化、凋亡小体出现以及亚二倍体细胞增加。根据半胱天冬酶3底物聚（ADP - 核糖）聚合酶和D4 - GDI蛋白的裂解情况，凋亡诱导浓度的大黄素会快速且短暂地诱导半胱天冬酶3/CPP32活性，但不会诱导半胱天冬酶1活性，在大黄素处理而非大黄素甲醚或大黄酚处理的HL - 60细胞中检测到裂解的半胱天冬酶3片段的出现。在大黄素处理的HL - 60细胞中检测到抗凋亡蛋白Mcl - 1减少，而其他Bcl - 2家族蛋白包括Bax、Bcl - 2、Bcl - XL和Bad保持不变。半胱天冬酶3抑制剂Ac - DEVD - CHO，但不是半胱天冬酶1抑制剂Ac - YVAD - CHO，减弱了大黄素诱导的DNA梯状条带，这与PARP和D4 - GDI裂解的阻断相关。包括NAC、过氧化氢酶、超氧化物歧化酶、ALL、DPI、L - NAME和PDTC在内的自由基清除剂对大黄素诱导的凋亡反应没有预防作用，而NAC、CAT和PDTC通过抑制半胱天冬酶3级联反应防止HL - 60细胞发生ROS（H₂O₂）诱导的凋亡。通过凝胶内活性测定在大黄素处理的HL - 60细胞中检测到过氧化氢酶活性的诱导，但未检测到超氧化物歧化酶活性，并且在HL - 60细胞中预先用大黄素处理可显著降低H₂O₂诱导的细胞内过氧化物水平。我们的数据表明大黄素通过激活半胱天冬酶3级联反应在HL - 60细胞中是一种有效的凋亡诱导剂，但它与ROS产生无关。
