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Withaferin A causes activation of Notch2 and Notch4 in human breast cancer cells

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Abstract

Ayurvedic medicine plants continue to draw attention for the discovery of novel anticancer agents. Withaferin A (WA) is one such small-molecule constituent of the ayurvedic medicine plant Withania somnifera with efficacy against cultured and xenografted human breast cancer cells. However, the mechanism underlying anticancer effect of WA is not fully understood. This study was undertaken to determine the role of Notch signaling in anticancer effects of WA using human breast cancer cells as a model. Notably, Notch signaling is often hyperactive in human breast cancers. Exposure of MDA-MB-231 and MCF-7 human breast cancer cells to pharmacological concentrations of WA resulted in cleavage (activation) of Notch2 as well as Notch4, which was accompanied by transcriptional activation of Notch as evidenced by RBP-Jk, HES-1A/B, and HEY-1 luciferase reporter assays. On the other hand, WA treatment caused a decrease in levels of both transmembrane and cleaved Notch1. The WA-mediated activation of Notch was associated with induction of γ-secretase complex components presenilin1 and/or nicastrin. Inhibition of MDA-MB-231 and MDA-MB-468 cell migration resulting from WA exposure was significantly augmented by knockdown of Notch2 as well as Notch4 protein. Activation of Notch2 was not observed in cells treated with withanone or withanolide A, which are structural analogs of WA. The results of this study indicate that WA treatment activates Notch2 and Notch4, which impede inhibitory effect of WA on breast cancer cell migration.

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Abbreviations

WA:

Withaferin A

WE:

Withanone

WLA:

Withanolide A

DMSO:

Dimethyl sulfoxide

DAPI:

4′,6-Diamidino-2-phenylindole

PBS:

Phosphate-buffered saline

siRNA:

Small interfering RNA

shRNA:

Small-hairpin RNA

uPA:

Urokinase-type plasminogen activator

uPAR:

uPA receptor

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Acknowledgments

Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under Award Number R01CA142604. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funder had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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None of the authors has any conflict of interest.

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Authors and Affiliations

  1. Department of Pharmacology & Chemical Biology, University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, 2.32A Hillman Cancer Center Research Pavilion, 5117 Centre Avenue, Pittsburgh, PA, 15213, USA

    Joomin Lee, Anuradha Sehrawat & Shivendra V. Singh

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  1. Joomin Lee
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  2. Anuradha Sehrawat
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  3. Shivendra V. Singh
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Correspondence to Shivendra V. Singh.

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Lee, J., Sehrawat, A. & Singh, S.V. Withaferin A causes activation of Notch2 and Notch4 in human breast cancer cells. Breast Cancer Res Treat 136, 45–56 (2012). https://doi.org/10.1007/s10549-012-2239-6

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  • DOI: https://doi.org/10.1007/s10549-012-2239-6

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