Abstract
Accumulated evidence has indicated that necrotic cell death is not an incidental event but proceeded in a programmed manner. Recently, necrostatin-1 (Nec-1) is developed to protect cells from necrotic death that is mediated through death receptor (DR) signaling. Nec-1 acts on RIP1 kinase activity as the first-in-class inhibitors of RIP1 that is associated with necrosis but irreverent to NF-κB activation. Our previous study has demonstrated that cadmium (Cd) induces necrosis in CHO K1 cells and the signaling pathway has been established. Notably, the Cd-induced necrotic cell death can be attenuated by Nec-1. In this study, we used this established necrotic pathway as a template for us to examine the target sites for Nec-1 action. Two treatments causing necrosis were also studied in parallel for comparisons. Treating U937 cells with TNF-α/z-VAD-fmk or ethacrynic acid (EA) caused necrotic death which could be rescue by Nec-1. However, Nec-1 didn’t protect EA-treated DLD-1 cells from necrosis. Notably, RIP1 activity abolished by GA did not inhibited Cd- or EA-induced necrosis. Systematic analyses revealed that Nec-1 was ineffective to modulate intracellular calcium contents. There was also lack of consistent and conclusive result for Nec-1 in regulating the activity of calpain, a downstream protease activated by calcium. The most significant effect of Nec-1 was on cells that mitochondrial membrane potential (MMP) reduced after giving necrosis inducing factors. The MMP can be restored by Nec-1 and cells rescued. Addition of RIP1 activity inhibitor, geldanamycin (GA), did not rescue cells from Cd-induced necrosis, indicating that Cd did not cause necrosis through RIP1 pathway. Either Cd, EA or death ligand elevated reactive oxygen species (ROS). However, Nec-1 had no effect on altering the ROS level. Nec-1 by itself stimulated NF-κB activity in a dose and time-dependent manner. Additionally, Nec-1 was able to restore the activity that was inhibited by necrosis-inducing agents. Analysis of IκB level showed an acceleration of IκB degradation by Nec-1 treatment. Using IKK specific inhibitor, PS-1145, to block its activity abolished the Nec-1-induced IκB degradation and NF-κB activation. These results suggest that Nec-1 can act as an NF-κB activator by enhancing the IKK activity. Activation of NF-κB may participate partly in protecting cells from necrosis.