Cells were allowed to cover the gold electrodes in each well of the array prior to exposure to CE

re consistent with previous studies in other cancer cells expressing wild-type p53. The 786-O cell line expresses a mutant p53; however, transfection of these cells with siNR4A1 also resulted in the induction of sestrin 2 and inactivation of mTOR and mTOR signaling. Moreover, similar results were observed after treatment of 786-O cells with the NR4A1 antagonists DIM-C-pPhOH and DIM-C-pPhCO2Me. It has previously been reported that sestrin 2 is induced by other factors including oxidative stress, and Fig 7D shows that induction of sestrin 2 by siNR4A1, DIM-C-pPhOH and DIM-C-pPhCO2Me was attenuated 9 / 17 Inhibition of Renal Cell Adenocarcinoma by NR4A1 Antagonists Fig 5. siNR4A1 and C-DIM/NR4A1 antagonists induce stress in RCC cells. Cells were transfected with siNR4A1 or treated with DIM-C-pPhOH or DIM-C-pPhCO2Me and whole cell lysates were analyzed by western blots. After treatment of cells as described in –, ROS was measured using the cell permeant CM-H2DCFDA probe as outlined in the Materials and Methods. Significant induction of ROS is indicated. Tumor lysates from athymic nude mice bearing ACHN cells as xenografts and treated with vehicle control or DIM-C-pPhCO2Me were analyzed by western blots and individual bands were quantitated. Significant induction of ROS is indicated. doi:10.1371/journal.pone.0128308.g005 10 / 17 Inhibition of Renal Cell Adenocarcinoma by NR4A1 Antagonists 11 / 17 Inhibition of Renal Cell Adenocarcinoma by NR4A1 Antagonists Fig 6. siNR4A1 and NR4A1 antagonist inhibit mTOR in ACHN cells. Cells were transfected with siNR4A1 and treated with DIMI-C-pPhOH and PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19713490 DIM-C-pPhCO2Me, and whole cell lysates were analyzed by western blots as outlined in the Materials and Methods. doi:10.1371/journal.pone.0128308.g006 after cotreatment with the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19709857 antioxidant glutathione. Thus, induction of ROS by NR4A1 antagonist-mediated downregulation of TXNDC5 and IDH1 also results in the induction of sestrin 2 in cells expressing mutant p53, suggesting that NR4A1 antagonists inhibit mTOR by both p53-dependent and-independent induction of sestrin2. These studies demonstrate that NR4A1 is a target for NR4A1 antagonists in RCC cells; this leads to inhibition of several NR4A1-regulated pro-oncogenic pathways including mTOR and these results are comparable to those observed in pancreatic, lung and colon cancer cells and tumors. Discussion NR4A LY-411575 receptors are immediate-early genes that are induced by diverse stressors in multiple tissues/cell lines and these receptors are important for maintaining cellular homeostasis. The function of NR4A1 in cancer cells has also been extensively investigated and in solid tumors, there is evidence that this receptor exhibits unique functions that are dependent on its subcellular location. NR4A1 is a nuclear receptor; however, treatment of some cancer cell lines with apoptosis-inducing agents results in the induction and nuclear export of NR4A1 which associates with mitochondrial bcl-2 to form a pro-apoptotic complex. Thus, mitochondrial NR4A1 exhibits tumor suppressor-like activity, and both paclitaxel and peptide mimetics of NR4A1 that bind bcl-2 also activate apoptosis in cancer cells. In contrast, results of RNAi studies suggest that nuclear NR4A1 is pro-oncogenic and plays a role in cancer cell proliferation and survival, and studies in this laboratory have identified at least three pathways and associated genes that contribute to the functions of NR4A1 in cancer cells. Thus, NR4A1 is unique among orphan n