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Notch2 activation is protective against anticancer effects of zerumbone in human breast cancer cells

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Abstract

We showed previously that zerumbone (ZER), a sesquiterpene isolated from subtropical ginger, inhibited in vitro (MCF-7 and MDA-MB-231cells) and in vivo (MDA-MB-231 cells) growth of human breast cancer cells in association with apoptosis induction. Here, we investigated the role of Notch receptors in anticancer effects of ZER (cell migration inhibition and apoptosis induction) using breast cancer cells. Western blotting was performed to determine protein expression changes. Effect of ZER on transcriptional activity of Notch was assessed by luciferase reporter assays. Transfection with small hairpin RNA or small interfering RNA was performed for knockdown of Notch2 or Presenilin-1 protein. Cell migration and apoptosis were quantitated by Boyden chamber assay and flow cytometry, respectively. Exposure of MDA-MB-231, MCF-7, and SUM159 cells to ZER resulted in increased cleavage of Notch2 in each cell line. On the other hand, levels of cleaved Notch1 and Notch4 proteins were decreased following ZER treatment. Increased cleavage of Notch2 in ZER-treated cells was accompanied by induction of Presenilin-1 protein and transcriptional activation of Notch. Inhibition of cell migration as well as apoptosis induction resulting from ZER exposure was significantly augmented by knockdown of Notch2 protein. ZER-mediated cleavage of Notch2 protein in MDA-MB-231 cells was markedly attenuated upon RNA interference of Presenilin-1. Knockdown of Presenilin-1 protein also resulted in escalation of ZER-induced apoptosis. The present study indicates that Notch2 activation by ZER inhibits its proapoptotic and anti-migratory response at least in breast cancer cells.

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Abbreviations

DMSO:

Dimethyl sulfoxide

GAPDH:

Glyceraldehyde 3-phosphate dehydrogenase

HES-1:

Hairy and enhancer of split-1

HEY-1:

Hairy/enhancer-of-split related with YRPW motif protein 1

PBS:

Phosphate-buffered saline

PARP:

Poly(ADP-ribose)-polymerase

RT-PCR:

Reverse transcription-polymerase chain reaction

shRNA:

Small hairpin RNA

siRNA:

Small interfering RNA

ZER:

Zerumbone

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Acknowledgments

This investigation was supported by USPHS grant RO1 CA142604-05 and CA129347-07, awarded by the National Cancer Institute of the National Institutes of Health. This research project used the Flow Cytometry Facility supported in part by the Cancer Center Support Grant P30 CA047904 from the National Cancer Institute of the National Institutes of Health. The funder had no role in 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 and Chemical Biology, University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, 5117 Centre Avenue, Pittsburgh, PA, USA

    Anuradha Sehrawat & Shivendra V. Singh

  2. Department of Biochemical Science & Technology, Kagoshima University, Korimoto 1-21-24, Kagoshima, 890-0065, Japan

    Kozue Sakao

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  1. Anuradha Sehrawat
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  2. Kozue Sakao
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  3. Shivendra V. Singh
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Correspondence to Shivendra V. Singh.

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Sehrawat, A., Sakao, K. & Singh, S.V. Notch2 activation is protective against anticancer effects of zerumbone in human breast cancer cells. Breast Cancer Res Treat 146, 543–555 (2014). https://doi.org/10.1007/s10549-014-3059-7

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  • DOI: https://doi.org/10.1007/s10549-014-3059-7

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