Abstract B07: Treatment of a chemoresistant human neuroblastoma cell-line (BE-2c) with experimental ozonide antimalarials
Authors: Coulter Don W. , McGuire Timothy R. , Sharp John G. , Dong Yuxiang , Wang Xiaofang , McIntyre Erin , Chen Xiaoyu , Vennerstrom Jon 
Published: May 2016

Abstract
Objective: To evaluate the anti-tumor activity of a class of experimental antimalarial drugs in an MYCN amplified chemoresistant neuroblastoma cell line (BE-2c) intended to model poor prognostic neuroblastoma.
Methods: Etoposide cytotoxicity was studied at concentrations (250 ng/ml,500 ng/ml, 1 mcg/ml, 2 mcg/ml, and 3 mcg/ml) to confirm chemoresistance to a commonly used cytotoxic agent in the treatment of neuroblastoma. Concentrations chosen for the ozonide antimalarials were those that have been achieved in-vivo when studied in a malarial model in rodents. Ozonide antimalarial drugs were tested in a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay which measures cell viability colorimetrically. A series of 13 ozonide compounds were evaluated for activity using a concentration response scheme (0, 250 ng/ml, 500 ng/ml, 1 mcg/ml, 5 mcg/ml, and 10 mcg/ml) that allowed the generation of IC50 for each agent based on mean MTT absorption values from 10 replicates at each concentration. The most active ozonide (OZ513) identified was then studied in a cell cycle flow cytometry analysis using propidium idodide with measurement of the A0 peak which estimates apoptosis. A neurosphere assay was also used which is considered enriched for “cancer stem cells” and is able to model movement of drug into a small avascular tumor was used to further evaluate the activity of OZ513. Spheres were counted and characterized after treatment and compared to non-treatment controls. Because it has been postulated that the anti-cancer mechanism of action of the ozonide antimalarials is the modulation of cancer cell metabolism (autophagy, disruption of oxidative metabolism) the effect of OZ513 on mitochondrial oxidative metabolism and glycolysis was evaluated in BE-2c cells using Seahorse metabolic analysis.
Results: Etoposide had no cytotoxic activity at any of the concentrations studied. OZ513 antimalarials were consistently active in BE-2c cell culture with the MTT assay generating IC50s on two separate experiments of 0.03 and 0.05 mcg/ml. The MTT results were confirmed using trypan blue staining to directly measure cell death. The Ao peak on PI flow cytometry increased after treatment with OZ513 indicating an increase in apoptosis. In addition, 5 mcg/ml of OZ513 increased G1, S, and G2 aneuploid fraction on propidium iodide cell cycle analysis. OZ513 disrupted BE-2c neurospheres confirming their activity in these multicellular tumorspheres. Metabolic studies demonstrated little effect on mitochondria based oxidative metabolism or glycolysis. Structure activity relationship suggests that the alcohol of the ozonide structure is required for optimal activity.
Conclusion: The antimalarial agent OZ513 has promising activity against the chemoresistant neuroblastoma cell-line BE-2c by increasing apoptosis by mechanisms that remain unclear, but increased cell cycle aneuploidy with treatment of cells with 5 mcg/ml of OZ513 indicates defects in mitosis and cytokinesis.
Citation Format: Don W. Coulter, Timothy R. McGuire, John G. Sharp, Yuxiang Dong, Xiaofang Wang, Erin McIntyre, Xiaoyu Chen, Jon Vennerstrom. Treatment of a chemoresistant human neuroblastoma cell-line (BE-2c) with experimental ozonide antimalarials. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B07.