Eeyarestatin I (ESI)-induced ERAD inhibition exhibits anti-cancer activity through multiple mechanisms in human colorectal cancer cells

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a cellular process for maintenance of protein homeostasis in the ER and aberration of ERAD regulation leads to abnormal function of ER. As an inhibitory compound to ERAD, Eeyarestatin I (ESI) exhibits anti-cancer activity. In this study, we elucidated the anti-cancer mechanisms of ESI-induced ERAD inhibition in human colorectal carcinoma cells. Cellular viability of three different types of human colorectal cancer cells decreased in a dose-dependent manner by treatment with ESI. Treatment of ESI to human colorectal cancer cells led to significant increase of ubiquitin accumulation, G2/M phase cell cycle arrest, apoptosis, ER stress and autophagy. In addition, ESI treatment reduced transcriptional activity of nuclear factor kappa B (NF-κB), and increased phosphorylation of c-Jun NH₂-terminal kinase (JNK) and intracellular production of reactive oxygen species (ROS). Decrease of cell viability and ROS release were JNK-dependent and apoptosis was ROS-dependent. On the other hand, treatment of the cells with ESI downregulated the expression of translocon-associated protein (TRAP) subunits including TRAPα, β, γ and δ, which was JNK- and ROS-dependent. In summary, ESI-induced ERAD inhibition triggers ER stress, G2/M cell cycle arrest, ROS-dependent apoptosis, and autophagy in human colorectal cancer cells. We are the first to identify TRAPs as novel target ER membrane proteins that are downregulated by ERAD inhibition in human colorectal cancer cells.