Epithelial to mesenchymal transition (EMT) is a process by which cancer cells increase invasiveness and motility by changing their phenotype from epithelial to mesenchymal. The mechanism of hydrogen peroxide (H2O2)-induced EMT remains elusive. We hypothesize that H2O2 production in different subcellular compartments will trigger EMT at different intensities. We used a chemo-genetic approach to produce hydrogen peroxide in the cytoplasm, nucleus and mitochondria of A549 lung adenocarcinoma cells to see how EMT progresses in each case. Cells were transfected with one of three different plasmids containing specific localization sequences for cytoplasm, nucleus and mitochondria. These plasmids have the peroxide generating enzyme D-Amino-Acid Oxidase (DAAO) which generates H2O2 by metabolizing D-Alanine. We also used the inhibitors of peroxide metabolism, 3-aminotriazole (3-AT) and auranofin (Au) to increase H2O2 concentrations in these compartments. Preliminary studies show that cytoplasmic peroxide generation induces EMT significantly after 7 days. The epithelial marker E-Cadherin was reduced and the mesenchymal marker N-Cadherin was increased significantly in our immunoassays. The extent of change in EMT markers was greater upon the addition of 3-AT and Au. However, there was no significant change in EMT markers when H2O2 was generated in the nucleus or mitochondria even in the presence of peroxide metabolism inhibitors. We are investigating the molecular mechanisms responsible for cytoplasmic peroxide-induced EMT.
