Rao P V Lakshmana, Jayaraj R, Bhaskar A S B, Kumar Om, Bhattacharya R, Saxena Parag, Dash P K, Vijayaraghavan R
Division of Pharmacology and Toxicology, Defence Research and Development Establishment, Jhansi Road, Gwalior 474002, India.
Biochem Pharmacol. 2005 Mar 1;69(5):855-65. doi: 10.1016/j.bcp.2004.11.010. Epub 2005 Jan 22.
The mechanism of ricin-induced apoptosis in human cervical cancer cell line HeLa was studied. The present study demonstrated that ricin induces apoptosis of human cervical cancer cells (HeLa) in a time dependent manner with an IC(50) for cell viability of 1 microg/ml. Ricin treatment resulted in a time dependent increase in LDH leakage, DNA fragmentation, percent apoptotic cells, generation of reactive oxygen species and depletion of intracellular glutathione levels. DNA agarose gel electrophoresis showed typical oligonucleosomal length DNA fragmentation. Additionally, DNA diffusion assay was performed to confirm DNA damage and apoptosis. Ricin activated caspase-3 as evidenced by both proteolytic cleavage of procaspase-3 into 20 and 18 kDa subunits, and increased protease activity. Caspase activity was maximum at 4h and led to the cleavage of 116 kDa poly(ADP-ribose) polymerase (PARP), resulting in the 85 kDa cleavage product. Ricin-induced caspase-3 activation also resulted in cleavage of DNA fragmentation factor-45 (DFF45/ICAD) and DFF40 or caspase-activated DNase in HeLa cells. Activation of caspase-3, cleavage of PARP and DNA fragmentation was blocked by pre-treatment with caspase-3 specific inhibitor Ac-DEVD-CHO (100 microM) and broad-spectrum caspase inhibitor Z-VAD-FMK (40 microM). Ricin-induced DNA fragmentation was inhibited by pre-treatment with PARP inhibitors 3-aminobenzamide (100 microM) and DPQ (10 microM). Our results indicate that ricin-induced cell death was mediated by generation of reactive oxygen species and subsequent activation of caspase-3 cascade followed by down stream events leading to apoptotic mode of cell death.
研究了蓖麻毒素诱导人宫颈癌细胞系HeLa凋亡的机制。本研究表明，蓖麻毒素以时间依赖性方式诱导人宫颈癌细胞（HeLa）凋亡，细胞活力的半数抑制浓度（IC50）为1微克/毫升。蓖麻毒素处理导致乳酸脱氢酶（LDH）泄漏、DNA片段化、凋亡细胞百分比、活性氧生成以及细胞内谷胱甘肽水平的时间依赖性增加。DNA琼脂糖凝胶电泳显示出典型的寡核小体长度的DNA片段化。此外，进行了DNA扩散试验以确认DNA损伤和凋亡。蓖麻毒素激活了半胱天冬酶-3，这可通过前体半胱天冬酶-3蛋白水解裂解为20 kDa和18 kDa亚基以及蛋白酶活性增加来证明。半胱天冬酶活性在4小时时最高，并导致116 kDa聚（ADP-核糖）聚合酶（PARP）的裂解，产生85 kDa的裂解产物。蓖麻毒素诱导的半胱天冬酶-3激活还导致HeLa细胞中DNA片段化因子-45（DFF45/ICAD）和DFF40或半胱天冬酶激活的脱氧核糖核酸酶的裂解。半胱天冬酶-3特异性抑制剂Ac-DEVD-CHO（100微摩尔）和广谱半胱天冬酶抑制剂Z-VAD-FMK（40微摩尔）预处理可阻断半胱天冬酶-3的激活、PARP的裂解和DNA片段化。PARP抑制剂3-氨基苯甲酰胺（100微摩尔）和DPQ（10微摩尔）预处理可抑制蓖麻毒素诱导的DNA片段化。我们的结果表明，蓖麻毒素诱导的细胞死亡是由活性氧的产生介导的，随后激活半胱天冬酶-3级联反应，接着是导致细胞凋亡死亡模式的下游事件。
