Fulvene-5 potently inhibits NADPH oxidase 4 and blocks the growth of endothelial tumors in mice
Sulochana S. Bhandarkar, Marisa Jaconi, Levi E. Fried, Michael Y. Bonner, Benjamin Lefkove, Baskaran Govindarajan, Betsy N. Perry, Ravi Parhar, Jamie Mackelfresh, Allie Sohn, Michael Stouffs, Ulla Knaus, George Yancopoulos, Yvonne Reiss, Andrew V. Benest, Hellmut G. Augustin, Jack L. Arbiser
Sulochana S. Bhandarkar, Marisa Jaconi, Levi E. Fried, Michael Y. Bonner, Benjamin Lefkove, Baskaran Govindarajan, Betsy N. Perry, Ravi Parhar, Jamie Mackelfresh, Allie Sohn, Michael Stouffs, Ulla Knaus, George Yancopoulos, Yvonne Reiss, Andrew V. Benest, Hellmut G. Augustin, Jack L. Arbiser
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Research Article Dermatology

Fulvene-5 potently inhibits NADPH oxidase 4 and blocks the growth of endothelial tumors in mice

Abstract

Hemangiomas are the most common type of tumor in infants. As they are endothelial cell–derived neoplasias, their growth can be regulated by the autocrine-acting Tie2 ligand angiopoietin 2 (Ang2). Using an experimental model of human hemangiomas, in which polyoma middle T–transformed brain endothelial (bEnd) cells are grafted subcutaneously into nude mice, we compared hemangioma growth originating from bEnd cells derived from wild-type, Ang2+/–, and Ang2–/– mice. Surprisingly, Ang2-deficient bEnd cells formed endothelial tumors that grew rapidly and were devoid of the typical cavernous architecture of slow-growing Ang2-expressing hemangiomas, while Ang2+/– cells were greatly impaired in their in vivo growth. Gene array analysis identified a strong downregulation of NADPH oxidase 4 (Nox4) in Ang2+/– cells. Correspondingly, lentiviral silencing of Nox4 in an Ang2-sufficient bEnd cell line decreased Ang2 mRNA levels and greatly impaired hemangioma growth in vivo. Using a structure-based approach, we identified fulvenes as what we believe to be a novel class of Nox inhibitors. We therefore produced and began the initial characterization of fulvenes as potential Nox inhibitors, finding that fulvene-5 efficiently inhibited Nox activity in vitro and potently inhibited hemangioma growth in vivo. In conclusion, the present study establishes Nox4 as a critical regulator of hemangioma growth and identifies fulvenes as a potential class of candidate inhibitor to therapeutically interfere with Nox function.

Authors

Sulochana S. Bhandarkar, Marisa Jaconi, Levi E. Fried, Michael Y. Bonner, Benjamin Lefkove, Baskaran Govindarajan, Betsy N. Perry, Ravi Parhar, Jamie Mackelfresh, Allie Sohn, Michael Stouffs, Ulla Knaus, George Yancopoulos, Yvonne Reiss, Andrew V. Benest, Hellmut G. Augustin, Jack L. Arbiser

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Figure 7

Treatment with vehicle control and fulvene-5 in vivo.

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Treatment with vehicle control and fulvene-5 in vivo. For each treatment...
For each treatment condition, 3 mice were subcutaneously injected on day 1 with 106 bEnd.3 cells and monitored for tumor development. From day 2 to day 12, mice were treated with fulvene-5 by making a stock solution of 4 mg fulvene-5 compound dissolved in 100 μl of 100% ethanol plus 1.1 ml of intralipid and then sonicated for 15 seconds. The treatment stock solution (330 μl) was injected intraperitoneally into each of 3 mice of the fulvene-5 treatment group. For control animals, a stock solution was made of 100 μl of 100% ethanol plus 1.1 ml of intralipid solution, and 330 μl of stock solution was injected intraperitoneally into each of 3 mice of the control group. Injections were continued for 2 weeks. On day 9 and day 12, tumors were measured in all 6 animals, and there was a significant difference in tumor volume between the control and fulvene-5–treated mice. No toxicity was noted following injection. Animals were euthanized on day 14, secondary to tumor burden in the control animals. Photos represent average tumor burden in each of the 2 groups (A), and tumor volume (mm3) is graphically depicted (B). Error bars represent SEM. P < 0.05 versus control.