Tang Peter S, Tsang Melanie E, Lodyga Monika, Bai Xiao-Hui, Miller Abigale, Han Bing, Liu Mingyao
Division of Cellular and Molecular Biology, University Health Network Toronto General Research Institute, Toronto, Ontario, Canada.
J Cell Physiol. 2006 Nov;209(2):457-67. doi: 10.1002/jcp.20751.
Caspase-independent cell death has drawn increasing attention. In the present study, we found that lipopolysaccharide (LPS) accelerated spontaneous death of human lung epithelial A549 cells in a serum- and cell density-dependent manner: while serum starvation has been demonstrated to induce apoptosis in the same cell line, LPS-induced cell death was only observed in the presence of serum; in addition, the cell death was not observed when the cells were seeded at 10- or 100-fold lower density. The apoptotic features were demonstrated by TUNEL assay, DNA laddering and Annexin V staining. However, treatment of cells with two commonly used pan-caspase inhibitors, zVAD.fmk or BOC-D.fmk, failed to block cell death. In contrast, two cathepsin B inhibitors, Ca074-Me or N-1845, reduced cell death significantly. A time-dependent activation of cathepsin B, but not caspase 3, was observed in both control and LPS-treated cells. Although LPS did not further activate cathepsin B or its release, it increased expression and translocation of apoptosis inducing factor from mitochondria to the nucleus, and increased release of cytochrome c from mitochondria. LPS-induced cell death was significantly attenuated by either N-acetyl-L-cysteine or pyrrolidine-dithiocarbamate, both free radical scavengers. Disruption of lipid raft formation with filipin or methyl-beta-cyclodextrin also reduced apoptosis significantly, suggesting that lipid raft-dependent signaling is essential. These data imply that confluent cells undergo spontaneous cell death mediated by cathepsin B; LPS may accelerate this caspase-independent cell death through release of mitochondrial contents and reactive oxygen species.
不依赖半胱天冬酶的细胞死亡已引起越来越多的关注。在本研究中，我们发现脂多糖（LPS）以血清和细胞密度依赖性方式加速人肺上皮A549细胞的自发死亡：虽然血清饥饿已被证明可诱导同一细胞系发生凋亡，但LPS诱导的细胞死亡仅在有血清存在时才观察到；此外，当细胞以低10倍或100倍的密度接种时未观察到细胞死亡。通过TUNEL检测、DNA梯状条带分析和膜联蛋白V染色证实了凋亡特征。然而，用两种常用的泛半胱天冬酶抑制剂zVAD.fmk或BOC-D.fmk处理细胞未能阻断细胞死亡。相反，两种组织蛋白酶B抑制剂Ca074-Me或N-1845显著减少了细胞死亡。在对照细胞和LPS处理的细胞中均观察到组织蛋白酶B的时间依赖性激活，但未观察到半胱天冬酶3的激活。虽然LPS没有进一步激活组织蛋白酶B或促进其释放，但它增加了凋亡诱导因子从线粒体到细胞核的表达和转位，并增加了细胞色素c从线粒体的释放。N-乙酰-L-半胱氨酸或吡咯烷二硫代氨基甲酸盐这两种自由基清除剂均可显著减轻LPS诱导的细胞死亡。用制霉菌素或甲基-β-环糊精破坏脂筏形成也显著减少了凋亡，这表明脂筏依赖性信号传导至关重要。这些数据表明，汇合细胞会经历由组织蛋白酶B介导的自发细胞死亡；LPS可能通过释放线粒体内容物和活性氧来加速这种不依赖半胱天冬酶的细胞死亡。
