Abstract
Necrostatin-1 (Nec-1) inhibits necroptosis by allosterically inhibiting the kinase activity of receptor-interacting protein 1 (RIP1), which plays a critical role in necroptosis. RIP1 is a crucial adaptor kinase involved in the activation of NF-κB, production of reactive oxygen species (ROS) and the phosphorylation of mitogen activated protein kinases (MAPKs). NF-κB, ROS and MAPKs all play important roles in apoptotic signaling. Nec-1 was regarded as having no effect on apoptosis. Here, we report that Nec-1 increased the rate of nuclear condensation and caspases activation induced by a low concentration of shikonin (SHK) in HL60, K562 and primary leukemia cells. siRNA-mediated knockdown of RIP1 significantly enhanced shikonin-induced apoptosis in K562 and HL60 cells. Shikonin treatment alone could slightly inhibit the phosphorylation of ERK1/2 in leukemia cells, and the inhibitory effect on ERK1/2 was significantly augmented by Nec-1. We also found that Nec-1 could inhibit NF-κB p65 translocation to the nucleus at a later stage of SHK treatment. In conclusion, we found that Nec-1 can promote shikonin-induced apoptosis in leukemia cells. The mechanism by which Nec-1 sensitizes shikonin-induced apoptosis appears to be the inhibition of RIP1 kinase-dependent phosphorylation of ERK1/2. To our knowledge, this is the first study to document Nec-1 sensitizes cancer cells to apoptosis.
Highlights
Necroptosis is a recently discovered, regulated form of programmed necrosis initiated by the activation of tumor necrosis factor alpha (TNFα) and/or Fas that is distinct from caspase-dependent apoptotic cell death [1]
Subsequent studies showed that Nec-1 allosterically inhibits the kinase activity of receptor-interacting protein 1 (RIP1) by interacting with the T-loop without affecting other functional domains, which is essential in death receptor (DR) triggered necroptosis [3], but has no effects on another pro-necrotic kinase receptor-interacting protein 3 (RIP3) [4]
Our results indicated that inhibition of RIP1 kinase activity by Nec-1 or knockdown of RIP1 expression using siRNA could promote shikonin-induced apoptosis in leukemia cells
Summary
Necroptosis is a recently discovered, regulated form of programmed necrosis initiated by the activation of tumor necrosis factor alpha (TNFα) and/or Fas that is distinct from caspase-dependent apoptotic cell death [1]. Necrostatin-1 (Nec-1) is a small molecule inhibitor originally identified in a chemical library screen as a potent and specific inhibitor of necroptosis It does not protect against caspase-dependent apoptosis, or against other programmed cell death, such as autophagy [2]. Because of its relatively specific effect against the pro-necrotic activity of RIP1, Nec-1 has become a popular tool to investigate the role of necrosis in different experimental models of cell injury. Nec-1 inhibits the kinase activity of RIP1 and has no effect on the apoptotic signaling pathway. Further investigation indicated that Nec-1 enhanced shikonin-induced apoptosis through inhibition of RIP1 and ERK1/2 activation
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