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The novel antiangiogenic VJ115 inhibits the NADH oxidase ENOX1 and cytoskeleton-remodeling proteins

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Summary

Targeting tumor vasculature represents a rational strategy for controlling cancer. (Z)-(+/−)-2-(1-benzylindol-3-ylmethylene)-1-azabicyclo[2.2.2]octan-3-ol (denoted VJ115) is a novel chemical entity that inhibits the enzyme ENOX1, a NADH oxidase. Genetic and small molecule inhibition of ENOX1 inhibits endothelial cell tubule formation and tumor-mediated neo-angiogenesis. Inhibition of ENOX1 radiosensitizes tumor vasculature, a consequence of enhanced apoptosis. However, the molecular mechanisms underlying these observations are not well understood. Herein, we mechanistically link ENOX1-mediated regulation of cellular NADH concentrations with proteomics profiling of endothelial cell protein expression following exposure to VJ115. Pathway Studios network analysis of potential effector molecules identified by the proteomics profiling indicated that a VJ115 exposure capable of altering intracellular NADH concentrations impacted proteins involved in cytoskeletal reorganization. The analysis was validated using RT-PCR and immunoblotting of selected proteins. RNAi knockdown of ENOX1 was shown to suppress expression of stathmin and lamin A/C, proteins identified by the proteomics analysis to be suppressed upon VJ115 exposure. These data support the hypothesis that VJ115 inhibition of ENOX1 can impact expression of proteins involved in cytoskeletal reorganization and support a hypothesis in which ENOX1 activity links elevated cellular NADH concentrations with cytoskeletal reorganization and angiogenesis.

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Acknowledgements

Supported in part by grants from U.S. National Institutes of Health/National Cancer Institute grants RO1CA140409, T32CA093240, Vanderbilt-Ingram Cancer Center grant P30 CA68485, and the Vanderbilt Academic Venture Capital fund for proteomics. We are thankful to Drs. V.J. Sonar and Y.T.R. Reddy for the synthesis of VJ115.

Conflict of interest statement

The authors declare that they have no conflict of interest

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Authors and Affiliations

  1. Department of Radiation Oncology, Vanderbilt University, Nashville, TN, 37232, USA

    Amudhan Venkateswaran, Soumya Sasi, Girish Rachakonda, Michael L. Freeman & Konjeti R. Sekhar

  2. Department of Biochemistry, Vanderbilt University, Nashville, TN, 37232, USA

    David B. Friedman

  3. Proteomics Laboratory, Vanderbilt University, Nashville, TN, 37232, USA

    David B. Friedman

  4. Department of Biomedical Engineering, Vanderbilt University, Nashville, TN, 37232, USA

    Alexandra J. Walsh & Melissa C. Skala

  5. Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR, 72205, USA

    Peter A. Crooks

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  1. Amudhan Venkateswaran
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  2. David B. Friedman
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  4. Melissa C. Skala
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Corresponding authors

Correspondence to Michael L. Freeman or Konjeti R. Sekhar.

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Supplemental Fig. 1

The relative change in protein expression over time for 20 polypeptides from 2D-DIGE study whose abundance changed by 20 % or more (p < 0.05) after VJ115 treatment. (DOC 86 kb)

Supplemental Fig. 2

A) Western blot analysis was employed to confirm the down-regulation of phosphor-c-Jun expression. B) Immunoblot of phosphorylation status of p70S6K and its down-stream target, ribosomal protein S6 in HUVECs exposed to VJ115. (DOC 70 kb)

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Venkateswaran, A., Friedman, D.B., Walsh, A.J. et al. The novel antiangiogenic VJ115 inhibits the NADH oxidase ENOX1 and cytoskeleton-remodeling proteins. Invest New Drugs 31, 535–544 (2013). https://doi.org/10.1007/s10637-012-9884-9

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  • DOI: https://doi.org/10.1007/s10637-012-9884-9

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