Abstract 1621: Tolcapone, a catechol-O-methyltransferase inhibitor, alone and in combination with oxaliplatin induces cell death in neuroblastoma
Authors: Hayes David E. , Zhao Ping , Voydanoff Austin , Nagulapally Abhinav , Bond Jeff , Sholler Giselle 
Published: August 2015

Abstract
Background: Neuroblastoma (NB) is an aggressive childhood cancer that arises from neural crest cells of the sympathetic nervous system. These cells contain cathecholamines which may be a therapeutic target. Catechol-O-methyltransferase (COMT) metabolizes catechol-containing compounds, including dopamine. COMT inhibitors, including tolcapone, are used as an adjunctive treatment for Parkinson's disease, as they increase dopamine within cells. Increase in dopamine and other catecholamines within cells may result in cell toxicity.
Methods: Cell viability was measured using Calcein AM fluorescent assay at tolcapone doses 1.5625 μM - 200 μM, both alone and in combination with oxaliplatin at doses 1.5 μM - 6 μM. Western blot analysis was used to measure cleaved poly ADP ribose polymerase (PARP) and cleaved and full caspase-3 levels. ATP level per cell was measured using CyQuant fluorescent DNA assay combined with the Cell Titer GLO luminescent cell viability assay. Expression of COMT was confirmed using microarray analysis and immunofluorescence. Reactive oxygen species (ROS) levels were measured using DCFDA - Cellular Reactive Oxygen Species Detection Assay Kit. IncuCyte ZOOM machine was used in conjugation with SYTOX Green dye to measure cell death and CellPlayer™ Kinetic Caspase-3/7 Apoptosis Assay Reagent to measure apoptosis over 48 hours. Microarray analysis using U133+ RNA expression profiles were used to evaluate gene expression changes after cells were treated with tolcapone for 12 hours.
Results: Tolcapone IC50 values ranged from 13.33 μM to 156 μM in seven different NB cell lines. Concentrations of 25 μM and 50 μM increased levels of cleaved PARP and cleaved caspase-3 over 48, 72, and 96 hours treated. Intracellular ATP decreased significantly with concentrations ranging from 12.5-200 μM treated over 48 hours and ROS levels increased significantly with concentrations ranging from 12.5-200 μM treated over 24 hours. IncuCyte Zoom analysis displayed dose dependent levels of cell death and presence of cleaved caspase-3 and 7 with increasing concentrations ranging from 1.5625 μM -200 μM. The combination of tolcapone at 25 μM and oxaliplatin at 3.5 μM show synergy in cell viability assays.
Conclusion: There is no curative therapy for relapsed/refractory NB patients. The preclinical evidence suggests that patients with overexpression of COMT may respond to tolcapone via catecholamine induced ROS and cytotoxicity, especially when combined with oxaliplatin. Therefore, tolcapone and oxaliplatin may be a potential new therapy for children with NB with plans to be evaluated in a Phase I/II trial.
Citation Format: David E. Hayes, Ping Zhao, Austin Voydanoff, Abhinav Nagulapally, Jeff Bond, Giselle Sholler. Tolcapone, a catechol-O-methyltransferase inhibitor, alone and in combination with oxaliplatin induces cell death in neuroblastoma. [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 1621. doi:10.1158/1538-7445.AM2015-1621