Inhibition of Tumor Necrosis Factor-induced Cell Death in MCF7 by a Novel Inhibitor of Neutral Sphingomyelinase

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 M. It did not inhibit acid SMase at up to at least 150 M. 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 M) partially inhibited TNF-induced sphingomyelin (SM) hydrolysis, and 20 M of the compound was protected completely from the loss of SM. The addition of 10 -20 M 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 M 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 M, had no effect on this pathway of ceramide generation. In addition, GW4869 did not significantly impair TNF-induced NF-B 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 NSMase activation is a necessary step for the full development of the cytotoxic program induced by TNF.

Mitochondrial Superoxide Production and Nuclear Factor Erythroid 2-Related Factor 2 Activation in p75 Neurotrophin Receptor-Induced Motor Neuron Apoptosis

“…1 A, dashed lines). NGF-induced motor neuron death was blocked by manumycin A (10 nM) and GW4869 (100 nM), specific inhibitors of nSMase (Arenz et al, 2001;Luberto et al, 2002) (Fig. 1 A).…”

Isc1p Plays a Key Role in Hydrogen Peroxide Resistance and Chronological Lifespan through Modulation of Iron Levels and Apoptosis

“…In both conditions, the increased cell death was associated with apoptotic and oxidative stress markers ( Figure 11). Interestingly, the neutral sphingomyelinase 2, the mammalian orthologue of Isc1p, has been implicated in H 2 O 2 -and TNF-induced apoptosis (Luberto et al, 2002;Levy et al, 2006). However, it has also been suggested that ceramides containing different fatty acids may have distinct impacts in cell physiology, by differentially affecting the biophysical properties of the membrane lipid bilayer or by interacting with specific downstream components in signaling pathways (Cutler and Mattson, 2001).…”

Neutral Sphingomyelinase-3 Is a DNA Damage and Nongenotoxic Stress-Regulated Gene That Is Deregulated in Human Malignancies

“…Furthermore, ceramide has been shown to function as a key mediator of apoptosis brought on by stress-inducing therapeutic agents, such as anthracyclines (11)(12)(13)(14), ionizing radiation (15,16), tumor necrosis factor (TNF)-related apoptosis-inducing ligand (17), and paclitaxel (18). Reciprocally, the inhibition of ceramide production by compounds such as the ceramide synthase inhibitor fumonisin B1 or by small interfering RNA approaches has been shown to abrogate stress-induced apoptosis (11,(19)(20)(21). Collectively, these findings have outlined (a) a critical inhibitory role for ceramide during tumorigenesis and (b) its ability to sensitize malignant cells to the effects of radiation and chemotherapeutic agents.…”