NF-κB-independent actions of sulfasalazine dissociate the CD95L- and Apo2L/TRAIL-dependent death signaling pathways in human malignant glioma cells

Death receptor-mediated apoptosis of human malignant glioma cells triggered by CD95 ligand (CD95L) or Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) share several features, including processing of multiple caspases and mitochondrial cytochrome c release. We here report that CD95L-induced cell death is inhibited by sulfasalazine (SS) in all of four human glioma cell lines, both in the absence and presence of cycloheximide (CHX). Coexposure to CD95L and SS prevents the CD95L-evoked processing of caspases 2, 3, 8 and 9, the release of cytochrome c from mitochondria, and the loss of BCL-xL protein. This places the protective effect of SS proximal to most known events triggered by the CD95-dependent signaling pathway in glioma cells. CD95L promotes the accumulation of nuclear factor kappa B (NF-κB) in the nucleus and induces the DNA-binding activity of NF-κB assessed by electrophoretic mobility shift assay. The total levels of p50, p65 and IκBα remain unchanged, but the levels of phosphorylated IκBα and of nuclear p65 increase, in response to CD95L. IκBα phosphorylation as well as nuclear NF-κB translocation and DNA binding are blocked by SS. However, unlike SS, dominant-negative IκBα (IκBdn) does not block apoptosis, suggesting that SS inhibits CD95L-mediated apoptosis in an NF-κB-independent manner. In contrast to CD95L, the cytotoxic effects of Apo2L/TRAIL are enhanced by SS, and SS facilitates Apo2L/TRAIL-evoked caspase processing, cytochrome c release, and nuclear translocation of p65. These effects of SS are nullified in the presence of CHX, suggesting that the effects of SS and CHX are redundant or that enhanced apoptosis mediated by SS requires protein synthesis. IκBdn fails to modulate Apo2L/TRAIL-induced apoptosis. Similar effects of SS on CD95L- and Apo2L/TRAIL-induced apoptosis are observed in MCF-7 breast and HCT116 colon carcinoma cells. Interestingly, HCT cells lacking p21 (80S14p21−/−) are only slightly protected by SS from CD95L-induced apoptosis, but sensitized to Apo2L/TRAIL-induced apoptosis, indicating a link between the actions of SS and p21. Thus, SS modulates the death cascades triggered by CD95L and Apo2L/TRAIL in opposite directions in an NF-κB-independent manner, and SS may be a promising agent for the augmentation of Apo2L/TRAIL-based cancer therapies.