The effects of a phenylbutyrate-derived histone deacetylase inhibitor (HDAC-42) on acid-induced gene expression patterns of SEG-1 human esophageal adenocarcinoma cells

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Over the last thirty years, incident cases of esophageal adenocarcinoma (EAC) have increased 8-fold throughout the Western world. The precise reason for the rapid increase remains to be fully elucidated, but increasing rates have been linked to chronic acid reflux and obesity. Furthermore, acid-pulsing SEG-1 human EAC cells reportedly results in down-regulation of apoptosis-related genes followed by an up-regulation of cell proliferation-associated genes. Thus, this experiment was undertaken to evaluate the potential of a histone deacetylase inhibitor (HDAC-42) to modulate acid-induced changes in gene expression profiles of SEG-1 EAC cells. Earlier work in our laboratory found this agent to inhibit cell proliferation, induce apoptosis and arrest growth of both esophageal squamous and adenocarcinoma cell lines. In the current experiment SEG-1 EAC cells were pretreated with either vehicle or HDAC-42 [1.6 μM] for 24 hrs, exposed to either acidified DMEM at pH 3.5 or media alone for 20 minutes, acid was neutralized, and RNA was harvested 6 hrs later. Total RNA was extracted using TRIzol methods, cDNA was generated, transcribed into cRNA, and hybridized to Affymetrix human U133 2.0 plus oligonucleotide microarrays. Initially, WEDGE++ analysis was conducted to determine significant differentially expressed genes by treatment and acid exposure. Next, EASE analysis software was utilized to identify biological themes or Gene Ontology categories significantly over-represented within the significantly up and down-regulated genes relative to the entire microarray. Treatment of SEG-1 cells with HDAC-42 resulted in over-representation of 10 gene categories with cellular process, cell communication, endosome, protein binding, and signal transduction representing the five most significantly over-represented categories derived from the significantly up-regulated probe sets. 113 categories of genes were significantly over-represented among the significantly down-regulated genes following HDAC-42 treatment. Mitotic cell cycle, cell cycle, M-phase, nucleus, nuclear division and cell proliferation were the top significantly over-represented categories. Acid-pulsing SEG-1 cells did not result in significant over-representation of gene categories suggesting that acid treatment of EAC cells does not target specific categories of genes. In addition, HDAC-42 pretreatment followed by acid pulsing did not result in significant over-representation of gene categories; although, large numbers of probe sets were significantly up or down-regulated. Real-time PCR based methods are being utilized to validate gene expression changes and further explore HDAC-42 targeted pathways which are also altered in esophageal cancer progression. The markers Ki-67, ODC1, BIRC5, TOPAII, E2F5, TFIP2, and EP300 have been validated.