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

Authors

  • Scott N. Pinchot MD,

    1. Endocrine Surgery Research Laboratory, Department of Surgery and the University of Wisconsin Carbone Cancer Center, Madison, Wisconsin
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  • Renata Jaskula-Sztul PhD,

    1. Endocrine Surgery Research Laboratory, Department of Surgery and the University of Wisconsin Carbone Cancer Center, Madison, Wisconsin
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  • Li Ning MD,

    1. Endocrine Surgery Research Laboratory, Department of Surgery and the University of Wisconsin Carbone Cancer Center, Madison, Wisconsin
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  • Noel R. Peters MS,

    1. University of Wisconsin Carbone Cancer Center Small Molecule Screening Facility, University of Wisconsin, Madison, Wisconsin
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  • Mackenzie R. Cook BS,

    1. Endocrine Surgery Research Laboratory, Department of Surgery and the University of Wisconsin Carbone Cancer Center, Madison, Wisconsin
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  • Muthusamy Kunnimalaiyaan PhD,

    1. Endocrine Surgery Research Laboratory, Department of Surgery and the University of Wisconsin Carbone Cancer Center, Madison, Wisconsin
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  • Herbert Chen MD

    Corresponding author
    1. Endocrine Surgery Research Laboratory, Department of Surgery and the University of Wisconsin Carbone Cancer Center, Madison, Wisconsin
    • H4/722 Clinical Science Center, 600 Highland Avenue, Madison, WI 53792
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    • Fax: (608) 263-7652


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.

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