Identification and validation of Notch pathway activating compounds through a novel high‐throughput screening method

Abstract

BACKGROUND: Carcinoids are neuroendocrine (NE) tumors with limited treatment options. Notch activation has been shown to suppress growth and hormone production in carcinoid cells. METHODS: The purpose of this study was to provide a process for identifying Notch activating compounds via high‐throughput screening (HTS) and to validate the effects of the strongest hit from the 7264 compounds analyzed: resveratrol (RESV). RESULTS: Treatment of carcinoid cells with RESV resulted in up‐regulation of the Notch signaling pathway as measured by suppression of its downstream target achaete‐scute complex‐like 1. Luciferase reporter assays incorporating the centromere‐binding factor 1 binding site also confirmed the functional activity of RESV‐induced Notch. Because activation of the Notch pathway has been shown to suppress carcinoid proliferation, RESV treatment of carcinoid cells led to a dose‐dependent inhibition of cellular growth. Immunoblotting revealed phosphorylation of cdc2 (Tyr15) and up‐regulation of p21Cip1/Waf, markers of cell cycle arrest, with RESV treatment. Flow cytometry confirmed the mechanism of RESV‐induced growth inhibition is S phase cell cycle arrest. Furthermore, because Notch has been shown to inhibit bioactive hormone production from NE tumors, RESV also suppressed expression of the NE peptides/hormones chromogranin A and serotonin. RNA interference assays demonstrated that the hormone suppressing capacity of RESV was due to up‐regulation of the Notch2 isoform. CONCLUSIONS: HTS can be used to identify novel Notch activating compounds, which may have the potential to suppress carcinoid tumor growth and the associated endocrinopathies. Cancer 2011. © 2010 American Cancer Society.

Functional characterization of human pluripotent stem cell-derived arterial endothelial cells

“…S5A). Consistent with their previously described roles (14,18,19), we found that VEGFA, WNT3A, and resveratrol [RESV, a NOTCH agonist (20)] promoted arterial specification (SI Appendix, Fig. S5 B-F).…”

Tumor-Suppressor Role of Notch3 in Medullary Thyroid Carcinoma Revealed by Genetic and Pharmacological Induction

“…Thus, we sought to identify a small molecule which induces Notch3 in MTC cells. We had previously developed a high throughput screen for Notch activating compounds (34). Utilizing this system, we identified a synthetic compound, AB3, which appeared to have this property.…”

Ehmt2/G9a controls placental vascular maturation by activating the Notch pathway

“…Our finding that Notch effectors are downregulated in G9a-mutant labyrinth endothelium and in the chorionic villi of human placentae affected by IUGR opens the possibility that pharmacological activation of the Notch pathway in the placenta might be a potential strategy to prevent or treat abnormal maturation of the placental vasculature. Valproic acid, which is used to control epileptic seizures and psychiatric disorders, was found to activate the Notch pathway resulting in increased activity of Rbpj (Pinchot et al, 2011). The use of valproic acid during pregnancy has limitations as it can promote a higher risk of neural tube closure defects in babies when administered during the first trimester of pregnancy (Jentink et al, 2010).…”