Lipopolysaccharide accelerates caspase-independent but cathepsin B-dependent death of human lung epithelial cells.
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.
Tang
Peter S
PS
Division of Cellular and Molecular Biology, University Health Network Toronto General Research Institute, Toronto, Ontario, Canada.
Tsang
Melanie E
ME
Lodyga
Monika
M
Bai
Xiao-Hui
XH
Miller
Abigale
A
Han
Bing
B
Liu
Mingyao
M
D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002401:Cathepsin B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002452:Cell Count; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects; D004847:Epithelial Cells / Q000166:cytology* / Q000187:drug effects*; D016166:Free Radical Scavengers / Q000378:metabolism; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008168:Lung / Q000166:cytology* / Q000187:drug effects; D021962:Membrane Microdomains / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects; D044967:Serum / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors
D061945:Caspase Inhibitors; D016166:Free Radical Scavengers; D008070:Lipopolysaccharides; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D002401:Cathepsin B