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NMR based metabonomics study of NPY Y5 receptor activation in BT-549, a human breast carcinoma cell line

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Abstract

Overexpression of neuropeptide Y (NPY) and its receptors has been found in various cancers. In our previous study, we demonstrated expression of NPY Y5 receptor (Y5R) in various breast cancer cell lines along with Y1 receptor. In Y5R expressing BT-549 cells, NPY induced cell proliferation that was blocked by Y5R-selective antagonist CGP1683A (CGP). Here, NMR-based metabonomics was used to monitor the metabolic profile of BT-549 cells in the presence of NPY and CGP to assess the effect of Y5R activation and inhibition during NPY-induced cell proliferation. To study changes in intra and extra cellular metabolites in response to various treatments, 1D 1H-NMR spectra of both hydrophilic cell extracts and growth medium were recorded from BT-549 with three treatments: (1) NPY, (2) CGP, and (3) CGP followed by NPY (CGP/NPY). Principal component analysis and statistical significance analysis indicated changes in intracellular concentrations of seven metabolites in hydrophilic cell extracts with NPY treatment: decreases in lactate, succinate, myo-inositol, and creatine, and increases in acetate, glutamate, and aspartate. A significant increase in intracellular lactate level and attenuation of other metabolites to baseline was detected in CGP/NPY group. Also, significant decreases in lactate and increases in pyruvate were observed in growth medium from NPY treated cells. Based on the metabonomics analysis, Y5R activation induces cell proliferation by increasing the rate of glycolysis, glutaminolysis, and TCA cycle. Inhibition of Y5R by CGP counteracts NPY-induced changes in cellular metabolites. These changes may play a role in cell proliferation and migration by NPY through Y5R activation.

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Abbreviations

α-KGDH:

Alpha-ketoglutarate dehydrogenase

AST:

Aspartate transaminase

cAMP:

Cyclic adenosine monophosphate

CGP:

Y5R-selective antagonist (CGP1683A)

CGP/NPY:

Co-treatment of CGP followed by NPY

DMEM/F12:

Dulbecco’s modified eagle medium: nutrient mixture F-12

DMSO:

Dimethyl sulfoxide

EDTA:

Ethylenediaminetetraacetic acid

FBS:

Fetal bovine serum

FH:

Fumarate hydratase

LDH:

Lactate dehyrogenase

NMR:

Nuclear magnetic resonance

NOESY:

Nuclear Overhauser effect spectroscopy

NPY:

Neuropeptide Y

PC:

Principal components

PCA:

Principal components analysis

PYY:

Peptide YY

SDH:

Succinate dehydrogenase

TCA:

Cycle, tricarboxylic acid cycle

TSP:

Trimethylsilylproprionate

Y5R:

NPY Y5 receptor

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Acknowledgements

MAK would like to acknowledge support of Miami University and the Ohio Board of Regents for funding to establish the Ohio Eminent Scholar Laboratory where the work was performed. MAK would also like to acknowledge support from Bruker Biospin, Inc that enabled development of the statistical significance analysis software used in the analysis of the data reported in this paper. MW would like to acknowledge support of a Miami University Cell, Molecular, and Structural Biology Fellowship. This study is supported in part by Shriners Hospital for Children Grant #864006 (AB) and Department of Veteran Affairs grant #1Io1BX000263 (AB).

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, Miami University, 106 Hughes Laboratories, Oxford, OH, 45056, USA

    Miki Watanabe, Junho Cho, Kenneth B. Lewis & Michael A. Kennedy

  2. Department of Surgery, University of Cincinnati Medical Center, Cincinnati, OH, USA

    Sulaiman Sheriff & Ambikaipakan Balasubramaniam

  3. Shriners Hospital for Children, Cincinnati, OH, 45229, USA

    Nijiati Kadeer & Ambikaipakan Balasubramaniam

  4. Cincinnati Veterans Affairs Medical Center, Cincinnati, OH, 45220, USA

    Ambikaipakan Balasubramaniam

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  1. Miki Watanabe
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  2. Sulaiman Sheriff
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  3. Nijiati Kadeer
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  4. Junho Cho
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  5. Kenneth B. Lewis
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  6. Ambikaipakan Balasubramaniam
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  7. Michael A. Kennedy
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Corresponding author

Correspondence to Michael A. Kennedy.

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Miki Watanabe and Sulaiman Sheriff contributed equally to this study.

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Watanabe, M., Sheriff, S., Kadeer, N. et al. NMR based metabonomics study of NPY Y5 receptor activation in BT-549, a human breast carcinoma cell line. Metabolomics 8, 854–868 (2012). https://doi.org/10.1007/s11306-011-0380-6

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  • DOI: https://doi.org/10.1007/s11306-011-0380-6

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