Abstract
BackgroundThe X-linked Inhibitor of Apoptosis (XIAP) has attracted much attention as a cancer drug target. It is the only member of the IAP family that can directly inhibit caspase activity in vitro, and it can regulate apoptosis and other biological processes through its C-terminal E3 ubiquitin ligase RING domain. However, there is controversy regarding XIAP's role in regulating tumor cell proliferation and survival under normal growth conditions in vitro.MethodsWe utilized siRNA to systematically knock down XIAP in ten human tumor cell lines and then monitored both XIAP protein levels and cell viability over time. To examine the role of XIAP in the intrinsic versus extrinsic cell death pathways, we compared the viability of XIAP depleted cells treated either with a variety of mechanistically distinct, intrinsic pathway inducing agents, or the canonical inducer of the extrinsic pathway, TNF-related apoptosis-inducing ligand (TRAIL).ResultsXIAP knockdown had no effect on the viability of six cell lines, whereas the effect in the other four was modest and transient. XIAP knockdown only sensitized tumor cells to TRAIL and not the mitochondrial pathway inducing agents.ConclusionsThese data indicate that XIAP has a more central role in regulating death receptor mediated apoptosis than it does the intrinsic pathway mediated cell death.
Highlights
The X-linked Inhibitor of Apoptosis (XIAP) has attracted much attention as a cancer drug target
Efficient siRNA-mediated knockdown of XIAP protein levels XIAP protein levels were monitored in 10 human tumor cell lines derived from different tumor types (Table 1 and Figure 1)
The relatively high XIAP expression in SW-620 cells was consistent with published work, where it was shown that these cells are resistant to TNF-related apoptosis-inducing ligand (TRAIL) mediated apoptosis [23]
Summary
All cell lines were maintained in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen) at 37° with humidified air containing 5% CO2. Graphing of data to determine XIAP levels was performed using Graphpad PRISM® software. % XIAP = (fluorescence XIAP band of sample-bkgrd/ fluorescence Hsp band of sample-bkgrd) ÷ (fluorescence XIAP band of untreated cells-bkgrd/fluorescence Hsp of untreated cells-bkgrd) Viability Assays After transfection, cells were immediately diluted in RPMI 1640 supplemented with 10% fetal bovine serum and added to clear bottom white wall 96 well plates at a concentration of 2500 cells/well in a 100 μL volume. For experiments to determine TRAIL sensitivity, soluble human recombinant KillerTRAILTM (Alexis Biochemicals, San Diego) was added at 32 hr after transfection; cells were incubated for an additional 24 hr prior to addition of ATPliteTM
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