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Involvement of AMP-activated protein kinase in mediating pyrrolo-1,5-benzoxazepine–induced apoptosis in neuroblastoma cells

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Summary

Neuroblastoma, a paediatric malignancy of the sympathetic nervous system, accounts for 15 % of childhood cancer deaths. Despite advances in understanding the biology, it remains one of the most difficult paediatric cancers to treat partly due to the development of multidrug resistance. There is thus a compelling demand for new treatment strategies that can bypass resistance mechanisms. The pyrrolo-1,5-benzoxazepine (PBOX) compounds are a series of novel microtubule-targeting agents that potently induce apoptosis in various tumour models. We have previously reported that PBOX compounds induce apoptosis in drug sensitive and multidrug resistant neuroblastoma cells and synergistically enhance apoptosis induced by chemotherapeutics such as carboplatin. In this study we present further data concerning the molecular basis of PBOX-induced apoptosis in neuroblastoma. We demonstrate that PBOX-6 induced AMP-activated protein kinase (AMPK) activation and downstream acetyl-CoA carboxylase phosphorylation. Increased reactive oxygen species (ROS) appeared to serve as the upstream signal for AMPK activation as pretreatment of cells with the antioxidant N-acetylcysteine inhibited both AMPK activation and PBOX-induced apoptosis. Furthermore, activation of AMPK by PBOX-6 was found to inhibit mTOR complex 1 (mTORC1) signalling. Finally, we demonstrate the efficacy of PBOX-6 in an in vivo xenograft model of neuroblastoma. This study provides new insights into understanding the molecular and cellular mechanisms involved in PBOX-induced cell death in neuroblastoma and further supports their future use as novel anti-cancer agents for the treatment of neuroblastoma.

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Abbreviations

ACC:

Acetyl Co-A carboxylase

AMPK:

5’ adenosine monophosphate-activated protein kinase

CML:

Chronic myeloid leukaemia

DTT:

Dithiothreitol

eEF2:

Eukaryotic elongation factor 2

FAC:

Fluorescence associated cell sorter

FBS:

Foetal bovine serum

H202 :

Hydrogen peroxide

Mcl-1:

Myeloid cell leukemia 1

MDR:

Multidrug resistance

MTA:

Microtubule targeting agent

mTOR:

Mammalian target of rapamycin

NAC:

N-acetylcysteine

ROS:

Reactive oxygen species

PARP:

Poly (ADP-ribose) polymerase

PBS:

Phosphate buffered saline

PBOX:

pyrrolo-1,5-benzoxazepine

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Acknowledgments

Stefania Butini would like to thank Istituto Toscano Tumori. This study was funded by the National Children’s Research Centre, Our Lady’s Children’s Hospital, Crumlin, Dublin, Ireland.

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Authors and Affiliations

  1. School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, 152-160 Pearse Street, Dublin 2, Ireland

    Jennifer C. Lennon, D. Clive Williams & Daniela M. Zisterer

  2. The National Children’s Research Centre, Crumlin, Dublin, Ireland

    Jennifer C. Lennon

  3. European Research Centre for Drug Discovery & Development (NatSynDrugs), University of Siena, Siena, Italy

    Stefania Butini & Giuseppe Campiani

  4. Our Lady’s Children’s Hospital, Crumlin, Dublin 12, Ireland

    Anne O’Meara

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  1. Jennifer C. Lennon
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  2. Stefania Butini
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  3. Giuseppe Campiani
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  4. Anne O’Meara
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  5. D. Clive Williams
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Correspondence to Jennifer C. Lennon.

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Lennon, J.C., Butini, S., Campiani, G. et al. Involvement of AMP-activated protein kinase in mediating pyrrolo-1,5-benzoxazepine–induced apoptosis in neuroblastoma cells. Invest New Drugs 34, 663–676 (2016). https://doi.org/10.1007/s10637-016-0366-3

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