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J Cell Physiol. 2006 Nov;209(2):457-67.

Lipopolysaccharide accelerates caspase-independent but cathepsin B-dependent death of human lung epithelial cells.

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Division of Cellular and Molecular Biology, University Health Network Toronto General Research Institute, Toronto, Ontario, Canada.


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.

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