Tu1672 Involvement of p21-Activated Kinase 4 (PAK4) in Gastrin-Induced Migration of Gastric Cancer Cells

Abstract

Gastro-esophageal reflux disease complicated by Barrett's esophagus (BE) is a major risk factor for esophageal adenocarcinoma (EA). The mechanisms whereby acid reflux may accelerate the progression from BE to EA are not fully understood. Acid and reactive oxygen species (ROS) have been reported to cause DNA damage in Barrett's cells. We have previously shown that NADPH oxidase NOX5-S is responsible for acid-induced H2O2 production in Barrett's cells and in EA cells. In this study we examined the role of intracellular calcium in acid-induced DNA damage in a Barrett's EA cell line FLO. FLO cells were exposed acid (pH 5.0) for one hour, washed and cultured in regular culture medium for additional 24 hours. DNA damage was detected by a Comet Assay. We found that pulsed acid treatment significantly increased tail length from 1.7±0.2 to 10.1±0.6 pixels (t test, P<0.01), tail area from 51.3±7.2 to 312.7±23.5 pixels (t test, P<0.01), and tail moment from 0.7±0.1 to 3.1±0.3 (t test, P<0.01), suggesting that pulsed acid treatment increases DNA damage in FLO EA cells. In addition, acid treatment significantly increased intracellular Ca2+ concentration by 216.2±14.4% control in Fura-2/AM-loaded FLO cells, an increase which was significantly decreased by calcium-free medium plus EGTA or by thapsigargin, and almost blocked by Ca2+ free medium with EGTA and thapsigargin. Acid-induced increase in tail length, tail area, tail moment and histone H2AX phosphorylation was significantly decreased by blockade of intracellular Ca2+ increase, by NADPH oxidase inhibitor diphenylene iodonium and by knockdown of NOX5-S with NOX5 siRNA. Conversely, overexpression of NOX5-S significantly increased tail length, tail area, tail moment and histone H2AX phosphorylation. Moreover, intracellular calcium increase induced by calcium ionophore A23187 significantly increased tail length from 3.0±0.7 to 11.8±1.1 pixels (t test P<0.0001), tail area from 63.3±21.1 to 234.1±25.5 pixels (t test P<0.0001), and tail moment from 0.5±0.3 to 1.4±0.3 (t test, P<0.03), suggesting that intracellular calcium increase may cause DNA damage in FLO EA cells. We conclude that pulsed acid treatment causes DNA damage via increase of intracellular calcium and activation of NOX5-S. It is possible that in Barrett's esophagus acid reflux increases intracellular calcium, activates NOX5-S and increases ROS production, which causes DNA damage, thereby contributing to the progression from BE to EA. Supported by NIH NIDDK R01 DK080703.