Background. Increased level of reactive oxygen species (ROS), which is known to stimulate cell proliferation through signal transduction, has been observed in a wide spectrum of human cancers. Previous studies suggested that there is an association between ROS generation and oncogenic signal involving Ras, which seems to affect the activity of the plasma membrane associated redox enzyme NADPH oxidase (NOX). However, the mechanisms responsible for such oxidative stress in Ras-transformed cancer cells are still unclear, and the potential therapeutic implications remain to be investigated. Methods and Results. By using a variety of biochemical and molecular biology methods including flow cytometry analysis of intracellular ROS, immuoflorescence microscopy and electrophoresis analysis of protein expression, enzyme assay of NOX activity, and matrigel assays for cell motility and invasion, we investigated the biochemical and molecular events involved in Ras transformation and production of ROS. We found that the K-Ras transformed pancreatic cells produced significantly higher level of ROS, which stimulated cell growth and motility, and was associated with an upregulation of superoxide dismutase (SOD) expression, likely as an protective mechanism in response to the ROS stress. Further studies revealed that K-Ras transformation led to increased level of PtdIns(3,4,5)P3 and activation of AKT. The increase of PtdInsP3 seemed to promote the assembling of active NOX enzyme complex, leading to increased production of superoxide radical. Interestingly, capsaicin, a compound previously known to interact with NOX, was able to induce a dramatic accumulation of ROS and cause a preferential cytotoxicity in the transformed cells compared to the non-malignant cells, as evidenced by morphological change, inhibition of motility and invasiveness, and suppression of cell proliferation. Conclusion. Our results suggested that Ras oncogenic signal induced consititutive activation of NOX by activation of PI3-kinase pathway in cancer cells, leading to an increase in free radical generation and preferential vulnerability to further ROS stress by targeting NOX with capsaicin.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]