Abstract
A high throughput screen for neutral, magnesium-dependent sphingomyelinase (SMase) was performed. One inhibitor discovered in the screen, GW4869, functioned as a noncompetitive inhibitor of the enzyme in vitro with an IC(50) of 1 microm. It did not inhibit acid SMase at up to at least 150 microm. The compound was then evaluated for its ability to inhibit tumor necrosis factor (TNF)-induced activation of neutral SMase (N-SMase) in MCF7 cells. GW4869 (10 microm) partially inhibited TNF-induced sphingomyelin (SM) hydrolysis, and 20 microm of the compound was protected completely from the loss of SM. The addition of 10-20 microm GW4869 completely inhibited the initial accumulation of ceramide, whereas this effect was partially lost at later time points (24 h). These data therefore support the inhibitory action of GW4869 on N-SMase not only in vitro but also in a cellular model. The addition of GW4869 at both 10 and 20 microm did not modify cellular glutathione levels in response to TNF, suggesting that the action of GW4869 occurred downstream of the drop in glutathione, which was shown previously to occur upstream of the activation of N-SMase. Further, whereas TNF treatment also caused a 75% increase of de novo synthesized ceramide after 20 h of incubation, GW4869, at either 10 or 20 microm, had no effect on this pathway of ceramide generation. In addition, GW4869 did not significantly impair TNF-induced NF-kappaB translocation to nuclei. Therefore, GW4869 does not interfere with other key TNF-mediated signaling effects. GW4869 was able, in a dose-dependent manner, to significantly protect from cell death as measured by nuclear condensation, caspase activation, PARP degradation, and trypan blue uptake. These protective effects were accompanied by significant inhibition of cytochrome c release from mitochondria and caspase 9 activation, therefore localizing N-SMase activation upstream of mitochondrial dysfunction. In conclusion, our results indicate that N-SMase activation is a necessary step for the full development of the cytotoxic program induced by TNF.
Highlights
A high throughput screen for neutral, magnesium-dependent sphingomyelinase (SMase) was performed
The results show that GW4869 is a selective inhibitor of neutral SMase (N-SMase), especially among enzymes known to act on SM
Effects of GW4869 on Cellular Activation of N-SMase—To verify the effect of GW4869 on N-SMase activity in cells, MCF7 breast cancer cells were treated with 3 nM tumor necrosis factor (TNF), since in this cell line it has been previously shown that treatment with TNF would cause activation of N-SMase as evaluated by changes in SM and ceramide [13]
Summary
After ϳ21 h of treatment with TNF, the medium was collected, and the plates were washed once with PBS that was combined with the medium and centrifuged at 2000 ϫ g for 5 min at 4 °C to collect floating cells. The pellet was resuspended in 400 l of lysis buffer (10 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride) and incubated for 10 min on ice. Just prior to centrifugation, 20 l of 10% Nonidet P-40 was added, and the suspension was mixed by pipetting up and down three times. Preparation of Cytosolic Extracts—At the indicated time points, plates were washed once with ice-cold PBS, and floaters in the medium and from the washes were collected by centrifugation. Adherent cells were scraped in lysis buffer
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