outlined in the Materials and Methods. doi:10.1371/journal.pone.0128308.g003 cells with siNR4A1 decreased expression of survivin, bcl-2 and EGFR and this was accompanied by increased PARP cleavage, a marker of apoptosis. 7 / 17 Inhibition of Renal Cell Adenocarcinoma by NR4A1 Antagonists 8 / 17 Inhibition of Renal Cell Adenocarcinoma by NR4A1 Antagonists Fig 4. NR4A1 plays a role in expression of Sp-regulated growth promoting and survival genes. Cells were transfected with siNR4A1 and whole cell lysates were analyzed by western blots as outlined in the Materials and Methods. Cells were treated with DIM-C-pPhOH or DIM-C-pPhCO2Me and after 24 hr, whole cell lysates were analyzed by western blots. Western blot analysis of tumor lysates from athymic nude mice bearing ACHN xenografts and treated with vehicle or DIM-C-pPhOH was also determined. Band intensities were quantitated relative to -actin and significantly decreased staining intensities are indicated. doi:10.1371/journal.pone.0128308.g004 Similar results were observed in both RCC cell lines after A-83-01 Treatment with DIM-C-pPhOH or DIM-C-pPhCO2Me, confirming that the NR4A1 antagonists inhibited NR4A1-regulated expression of survivin, bcl-2 and EGFR in ACHN and 786-O cells as previously reported in pancreatic, lung and colon cancers. Thus, cells transfected with siNR4A1 or treated with C-DIM/NR4A1 antagonists induced multiple markers of apoptosis including increased Annexin V staining, cleavage of caspases 3, 7, 8 and PARP and this was accompanied by downregulation of the anti-apoptotic genes survivin and bcl-2. In addition, we also observed decreased expression of survivin, bcl-2, EGFR and induced PARP cleavage in tumor lysates from nude mice bearing ACHN cells as a xenograft and treated with DIM-CpPhOH . siNR4A1 and C-DIM/NR4A1 antagonist 
induce stress in RCC cells It has been shown that NR4A1 maintains low levels of stress in cancer cells by PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19709857 regulating expression of TXNDC5 and IDH1 that in turn maintain high levels of cellular reducing agents. Transfection of ACHN and 786-O cells with siNR4A1 decreased expression of TXNDC5 and IDH1 in both cell lines and this was accompanied by increased expression of CHOP, ATF4 and phospho-PERK which are markers of ER stress. Treatment of ACHN and 786-O cells with DIM-C-pPhOH or DIM-C-pPhCO2Me also decreased expression of TXNDC5 and IDH1 and induced markers of ER stress. Moreover, siNR4A or treatment with the C-DIM/NR4A1 antagonists also induced ROS as determined using the cell permeant probe CM-H2DCFCA. In addition, DIM-C-pPhOH also decreased expression of TXNDC5 and IDH-1 and induced CHOP expression in tumors from athymic nude mouse xenografts, demonstrating that C-DIM/NR4A1 antagonists-dependent inhibition of RCC cell and tumor growth is due, in part, to induction of stress. siNR4A1 and C-DIM/NR4A1 antagonists inhibit mTOR NR4A1 binds and inactivates p53, and treatment with siNR4A1 or C-DIM/NR4A1 antagonists results in p53-dependent induction of sestrin 2 which activates AMPK and inhibits mTOR. Knockdown of NR4A1 by RNAi in ACHN cells that express wild-type p53 resulted in the induction of sestrin 2, activation of AMPK, and inhibition of phospho-mTOR; this was also accompanied by decreased activation/phosphorylation of mTOR-regulated p70S6K, S6RP and 4EBP1. Treatment of ACHN cells with the NR4A1 PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19710274 antagonists DIM-C-pPhOH and DIM-C-pPhCO2Me also induced sestrin 2, activated AMPK and inhibited activation of mTOR and downstream kinases and these results a
