Analysis of Antioxidant Responsive Gene Expression Changes by Thioredoxin System Inhibitors and Hypoxia

Gene expression profiling measured by cDNA microarray offers a powerful tool for identifying cancer drug mechanism of action, defining drug specificity and for identifying new cancer drug targets. An anti-tumor inhibitor of the thioredoxin-1 (Trx-1) redox system, PX-12 (1-methylhydroxypropyl 2-imidazoloyl disulfide), is currently in Phase I clinical trials and was used as a model for targeted therapy to evaluate the differences between cells growing in culture and the same cells grown as xenografts in immunocompromised mice. Without drug treatment, MCF-7 breast cancer and HT-29 colon cancer cells growing as xenografts in severe combined immune deficient (scid) mice showed marked changes in gene expression compared to the cells in culture, with 42% of genes showing more than a two fold difference in expression. Following treatment with PX-12, the gene changes observed in common accounted for approximately 1% of the total genes changed vivo. After elimination of the effects of Trx-1 inhibitors on known targets in vivo and in vitro, the common increase in genes involved in antioxidant response pathway was chosen for further investigation. Gene expression changes observed during microarray experimentation were validated by real-time RT-PCR. Binding and activation of Nrf2 (nuclear factor (erythroid-derived 2)-like 2) to the antioxidant responsive element (ARE) was evaluated and found to be increased by treatment with Trx-1 inhibitors and hypoxia. Potential mechanisms accounting for the increase of ARE-dependent genes, including activation of protein kinase C, PI3K (phosphoinositide-3-kinase) and p-PERK (phospho-PRKR-like endoplasmic reticulum kinase) and an increase in cellular reactive oxygen species (ROS), determined that the increase in ROS due to hypoxia and Trx-1 system inhibition was sufficient to activate the pathway. Decreasing ROS by addition of an antioxidant was able to reverse the effect of activation of this pathway. The increased expression of ARE/Nrf2 dependent genes by these mechanisms may have important implications for chemotherapy and chemoprevention of human tumors