Increase in antioxidant response genes by thioredoxin system inhibition

2301

The redox protein thioredoxin (Trx-1) is a target for anticancer therapy because of the important role Trx-1 redox signaling plays in cell growth and proliferation. Tumor cells frequently show high levels of Trx-1 and are resistant to chemotherapy and programmed cell death. Patients with elevated tumor Trx-1 show aggressive tumor growth, inhibited apoptosis and decreased survival. PX-12 (1-methylpropyl 2-imidazolyl disulfide) and pleurotin are inhibitors of Trx-1 and thioredoxin reductase, respectively. cDNA microarray has been used to evaluate the changes in gene expression by HT-29 human colon cancer cells in response to 50μM PX-12 and 20μM pleurotin. A custom cDNA microarray containing approximately 549 genes known to be important in redox regulation, cancer growth and apoptosis was used for this analysis. Although the mechanism of inhibition of Trx-1 signaling differs for PX-12 and pleurotin, there were 26 genes which showed similar changes. Of these 26 genes, 7 have previously been reported to be dependent on the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) for their expression. Nrf2 binds to the antioxidant responsive element (ARE) found in the promoter region of many antioxidant and phase 2 metabolism genes, which are thought to be important in the cellular defense against reactive oxygen species (ROS). An additional 3 genes were found to have an ARE consensus sequence within their promoters. PX-12 and pleurotin at therapeutic concentrations increased the levels of Nrf2 in the nucleus measured by Western blotting, while the cytoplasmic levels were unchanged. PX-12 and pleurotin treatment of HT-19 cells also increased the binding measured by an EMSA of proteins in nuclear extracts to an ARE consensus sequence. Transfection of Trx-1 inhibited the binding. A potential mechanism of Nrf2 activation is through phospho-PRKR-like endoplasmic reticulum kinase (p-PERK). Levels of p-PERK were dramatically increased following PX-12 treatment, but were not increased by pleurotin. Phosphoinositide-3-kinase (PI3K) and protein kinase C (PKC) have also been shown to activate Nrf2. The PI3K inhibitor wortmannin, and the PKC inhibitor calphostin C did not block the increase in nuclear Nrf2 by PX-12 or pleurotin. The results suggest that inhibition of Trx-1 signaling either by inhibiting thioredoxin reductase or Trx-1 itself cause increased nuclear Nrf2 and increased binding to the ARE leading to increased expression of ARE regulated antioxidant genes. For PX-12 an increase in PERK activity may contribute to the activation of Nrf2. The increases in ARE/Nrf2 dependent genes by Trx-1 signaling inhibitors may have implications for new approaches to chemotherapy and chemoprevention of human tumors.