ABSTRACT
The Notch receptor serves a fundamental role in the regulation of cell fate determination through intracellular signal transmission. Mutation of the Notch receptor results in abnormal active signaling, leading to the development of diseases involving abnormal cell growth, including malignant tumors. Therefore, the Notch signaling pathway is a useful pharmacological target for the treatment of cancer. In the present study, a compound screening system was designed to identify inhibitors of the Notch signaling targeting Notch intracellular domain (NICD). A total of 9,600 compounds were analyzed using the Michigan Cancer Foundation7 (MCF7) human breast adenocarcinoma cell line and the SHSY5Y human neuroblastoma cell line with the reporter assay system using an artificial protein encoding a partial Notch carboxylterminal fragment fused to the Gal4 DNAbinding domain. The molecular mechanism underlying the inhibition of Notch signaling by a hit compound was further validated using biochemical and cell biological approaches. Using the screening system, a potential candidate, Notch signaling inhibitor1 (NSI1), was isolated which showed 50% inhibition at 6.1 µM in an exogenous Notch signaling system. In addition, NSI1 suppressed the nuclear translocation of NICD and endogenous gene expression of hairy and enhancer of split1, indicating that NSI1 specifically targets Notch. Notably, NSI1 suppressed the cell viability of MCF7 cells and another human breast adenocarcinoma cell line, MDAMB231 exhibiting constitutive and high Notch signaling activity, whereas no significant effect was observed in the SHSY5Y cells bearing a lower Notch signaling activity. NSI1 significantly suppressed the viability of SHSY5Y cells expressing exogenous human Notch1. These results indicate that NSI1 is a novel Notch signaling inhibitor and suggest its potential as a useful drug for the treatment of diseases induced by constitutively active Notch signaling.
