Modulation of RIP1 ubiquitylation and distribution by MeBS to sensitize cancer cells to tumor necrosis factor α-induced apoptosis.

Overexpression of anti-apoptosis protein cIAP1 due to its genetic amplification is found in certain cancers such as esophageal squamous cell carcinoma, hepatocellular carcinoma, cervical cancer and lung cancer, and plays a significant role in resistance to cancer therapy. We previously reported that a class of small molecules represented by (-)-N-[(2S, 3R)-3-amino-2-hydroxy-4-phenyl-butyryl]-L-leucine methyl ester (MeBS) activates auto-ubiquitylation of cIAP1 for proteasomal degradation, and enhances apoptosis of various cancer cells. However, the molecular mechanism of how MeBS sensitizes cancer cells to apoptosis via downregulation of cIAP1 is not well understood. Here, we show that ubiquitylation and distribution of RIP1, a protein ubiquitylated by cIAP1, is modulated by MeBS. Upon tumor necrosis factor (TNF)α stimulation, ubiquitylated RIP1 associates with the TNF-receptor (TNFR) complex, whereas non-ubiquitylated RIP1 associates with caspase8. MeBS reduces the ubiquitylated RIP1 in the TNFR complex and increases non-ubiquitylated RIP1 bound to caspase8. Downregulation of RIP1 by siRNA reduces apoptosis induced by TNFα plus MeBS treatment. These results indicate an important role of RIP1 in apoptosis induced by combined treatment with TNFα and MeBS, suggesting that MeBS sensitizes cancer cells to apoptosis by modulating RIP1 ubiquitylation and distribution.