Abstract 5668: Vorinostat exposure results in differential sensitivity and nuclear structure normalization in an in vitro model of esophageal adenocarcinoma progression
Authors: Nandakumar Vivek , Hernandez Kathryn F. , Senechal Patti , Slaydon Miranda , Davies Paul C.W , Johnson Roger H. , Bussey Kimberly J. , Meldrum Deirdre R. 
Published: August 2012

Esophageal adenocarcinoma (EA) has a poor prognosis with a five-year survival rate of less than 20%. Barrett's esophagus (BE), often consequent to chronic acid reflux, contributes to a 15-fold increased risk for EA. BE is a multi-stage condition that may progress through metaplasia and increasingly severe dysplasia to EA. The progression from normal squamous epithelium through BE to EA is characterized by genetic and nuclear structure aberrations. Histone deacetylase (HDAC) inhibitors are becoming increasingly popular as anticancer therapeutics. Vorinostat (Zolinza) is a pan-HDAC inhibitor currently approved to treat cutaneous T-cell lymphoma. It is under investigation in at least 81 actively recruiting clinical trials for various other cancer types including gastrointestinal cancers. We investigated the effects of vorinostat treatment on cell lines representative of normal squamous (EPC2), metaplasia (CPA) and adenocarcinoma (Flo1) of the esophageal epithelium. We assessed variations in viability and three-dimensional (3D) nuclear morphology upon drug treatment, comparing the results against vehicle (DMSO) control and untreated cells of all three types. A 12-point, three-fold, serial dilution drug-dose-response (DDR) experiment performed at 24, 48 and 72 hour time points indicated a differential response among the cell types. Flo1 cancer cells were most sensitive to the drug, with an IC50 value at 48 hours of 0.7928 micromolar, versus 7.536 micromolar for CPA and 21.49 micromolar for EPC2. After 24 hours of vorinostat exposure, assessment of apoptotic activity with the Caspase 3/7 Glo assay revealed increased apoptosis in Flo1 but not the other cells. To understand the morphological correlates of vorinostat treatment, we used micro-optical computed tomography to acquire 3D images of 200 individual, hematoxylin-stained, untreated and treated (48 hours at the Flo1 IC50 dose) cells of each cell type with sub-micron, isotropic spatial resolution. We applied computer vision techniques to quantify 3D nuclear morphology and coarse chromatin organization. Treatment with vorinostat normalized the nuclear structure of cancer and metaplastic cells, reverting them to a more nearly-normal phenotype: Flo1 cells showed reduced cell and nuclear volumes and decreased chromatin clumpiness after vorinostat treatment. The same morphological trends were present but less pronounced in CPA cells. EPC2 cells showed minimal changes in response to treatment. These results support further exploration of vorinostat for the treatment of patients with BE and EA. Our data indicate that vorinostat sensitivity increases, with a corresponding increase in apoptosis, as cells progress from normal to esophageal adenocarcinoma. This increased sensitivity to the drug is accompanied by a normalization of nuclear architecture in surviving abnormal cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5668. doi:1538-7445.AM2012-5668