Abstract 1914: A novel celecoxib derivative that lacks COX-2 inhibition but displays potent colon tumor cell growth and PDE5 inhibitory activity
Authors: Sigler Sara C. , Ramirez-Alcantara Veronica , Keeton Adam B. , Abdel-Halim Mohammad , Abadi Ashraf H. , Piazza Gary A. 
Published: August 2015

Abstract
Background: The use of celecoxib for cancer chemoprevention is not recommended because of modest efficacy and potentially fatal cardiovascular toxicity due to COX-2 inhibition. There is evidence that celecoxib inhibits tumor growth by a COX-2 independent mechanism, suggesting that efficacy and safety could be improved by chemical optimization towards the underlying target. We synthesized a novel series of celecoxib derivatives that lack COX-2 inhibitory activity, yet display higher potency to inhibit colon tumor cell growth. Here we study a potential mechanism involving phosphodiesterase 5 (PDE5) inhibition, elevation of intracellular cGMP levels, PKG activation and suppression of β-catenin signaling.
Methods: The IMAP fluorescence polarization PDE assay was used to measure PDE activity. Colon tumor cell lines (HT-29 and HCT-116) were used to measure growth inhibitory activity using the Cell Titer Glo Assay. COX inhibitory activity was measured using a fluorescent assay. Intracellular cGMP levels in live cells were measured using a cGMP biosensor assay, while cGMP levels in cell and tissues lysates were measured by EIA assay. PKG activity was measured by western blotting using a phosphospecific antibody to VASP. Levels of β-catenin, survivin, and cyclin D1 were determined by western blotting. TCF/LEF transcriptional activity was measured using a luciferase reporter assay. Cell cycle arrest was measured using an IXM imager and MetaXpress software. Oral bioavailability was determined based on drug concentrations in plasma and colon mucosal tissue after treatment by oral gavage in C57BL/6 mice.
Results: Celecoxib inhibits HT-29 and HCT-115 colon tumor cell growth and PDE5 activity with IC50 values of 34.3, 35.3, and 37.0 μM, while a derivative, RF26, had values of 5.8, 6.6, and 0.001 μM, respectively. RF26 was highly selective for PDE5 compared with other PDE isozymes. Celecoxib displays an IC50 of 0.058 μM for COX-2 inhibition, while RF26 lacks COX-2 inhibitory activity. RF26 increases cGMP levels, activates PKG, and decreases β-catenin levels and activity demonstrated by decreased survivin and cyclin D1 protein levels and TCF/LEF transcriptional activity. The mechanism of growth inhibition involves G1 cell cycle arrest. In mice, RF26 displays promising pharmacokinetics and drug levels in the target tissue that increase cGMP levels.
Conclusions: A novel series of celecoxib derivatives was identified that lack COX-2 inhibitory activity; yet display high potency to inhibit colon tumor cell growth. PDE5 inhibition appears to be an important mechanism for this class of compounds, which is consistent with previous observations from our group implicating cGMP/PKG activation as an anticancer target. These findings may lead to the development of novel celecoxib derivatives with improved anti-tumor efficacy and reduced risk of toxicity. Supported by NIH grants 1R01CA155638 and 1R01CA131378 (Piazza).
Citation Format: Sara C. Sigler, Veronica Ramirez-Alcantara, Adam B. Keeton, Mohammad Abdel-Halim, Ashraf H. Abadi, Gary A. Piazza. A novel celecoxib derivative that lacks COX-2 inhibition but displays potent colon tumor cell growth and PDE5 inhibitory activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1914. doi:10.1158/1538-7445.AM2015-1914