Since treatment with TRAIL normally acti vates caspase dependent apoptosis, we actively inhibited caspases/apoptosis by addition of the broad spectrum cas pase inhibitor zVAD fmk. This treatment is hepatocellular carcinoma not only ex perimentally required to suppress apoptosis, but in addition potentiates programmed necrosis by inhibiting caspase 8, which acts as a negative regulator of programmed necrosis and which otherwise would prevent the induction of programmed necrosis by TRAIL. Furthermore, all cells were additionally treated with non toxic concentrations of the protein biosynthesis Inhibitors,Modulators,Libraries inhibitor cycloheximide that we had previously found to sensitize for programmed necrosis. As depicted in Figure 1a, treatment with TRAIL/ zVAD/CHX induced programmed necrosis in eight out of 14 tested tumor cell Inhibitors,Modulators,Libraries lines.

The tumor cell lines U 937, Mz ChA 1, BxPC 3 and HT 29 exhibited the highest sensitivity, followed by Colo357, Panc89, PancTu I and A818 4 cells. The remaining cell lines, i. e. CCRF CEM, MKN 28, SK OV 3, KNS 62, Pt45P1, and SK MEL 28 displayed only a marginal or no response to treatment with TRAIL/ zVAD/CHX. We obtained Inhibitors,Modulators,Libraries essentially the same results in control assays when we induced programmed necro sis with TNF/zVAD/CHX. As the only ex ception, CCRF CEM cells were resistant to TRAIL/ zVAD/CHX but clearly sensitive to TNF/zVAD/CHX induced programmed necrosis. In the course of the above experiments, the issue arose whether cell death under the above conditions occurred exclusively by programmed necrosis or whether the combination of TRAIL/zVAD or TNF/zVAD with other cytotoxic agents such as CHX might still result in a net increase in caspase activity and thus in residual apop tosis.

Arguing against this assumption, we have previ ously shown in several studies that no features of apoptosis are detectable in the presence of 20 or 50 uM zVAD fmk for both TRAIL and TNF in duced cell death in multiple cell systems. In particular, neither caspase 8 nor caspase 3 activity was detectable in our previous studies, dying cells displayed a necrotic nu clear and cellular morphology, Inhibitors,Modulators,Libraries cell death was dependent on RIPK1 and could be blocked by the RIPK1 inhibitor necrostatin 1, no early release of phosphatidylserine or early loss Inhibitors,Modulators,Libraries of ��m occurred, and the DNA repair enzyme PARP 1 was not cleaved by activated caspase 3 to its apop totic signature 89 kDa fragment. Moreover, in control experiments that we additionally performed for this study, the 17-AAG clinical tumor cell lines U 937 and HT 29 did not display apoptotic membrane blebbing when treated with TRAIL/ zVAD/CHX or TNF/zVAD/CHX.