## 2 Results  
In this study, we used three DIM-3,5 compounds, which include the 3,5-dichlorophenyl (DIM-3,5), 3,5-dibromophenyl (DIM-3,5-Br\({}_{2}\)) and 3-chloro-5-trifluoromethylphenyl (DIM-3-CI-5-CF\({}_{3}\)) analogs, exhibiting similar KD values for binding NR4A2 (5.5-12.0 uM) and lower KD values for binding NR4A1 (3.1-7.7 uM) [30]. Results illustrated in Figure 1A show that the three DIM-3,5 analogs decrease the growth of RKO cells, and significant growth inhibition is observed at concentrations ranging from 5 to 10 umol/L. Similar results were observed in SW480 (Figure 1B) and HCT116 (Figure 1C) colon cancer cells, and DIM-3,5-Br\({}_{2}\) was the least active compound in RKO and HCT116 cells. The effects of these compounds on NR4A1 and NR4A2 expression and on previously identified NR4A1-responsive genes were also determined in RKO and SW480 cells. The DIM-3,5 analogs did not affect NR4A2 expression in either cell line (Figure 1D,E), whereas 7.5 or 15 umol/L of DIM-3,5 compounds decreased levels of NR4A1 protein. Both Sp1 and Sp4 form complexes with NR4A1 (i.e., NR4A1/Sp) to regulate expression of some genes [23], and the DIM-3,5 dual NR4A1/2 ligands decreased levels of Sp1 and Sp4 proteins. DIM-3,5 compounds also decreased expression of NR4A1-responsive gene products G9a, \(\beta\)1-integrin and bcl-2 [23; 24; 25] in RKO and SW480 cells (Figure 1F,G), and Parp cleavage was also observed. The quantitation of Western blot results is summarized in Figure 1H,I. The effects of the knockdown of NR4A1, NR4A2 or their combination was also investigated, and Figure 2A shows that oligonucleotides targeting NR4A1 (siNR4A1) and NR4A2 (siNR4A2) decreased RKO cell viability, with comparable results observed in SW480 (Figure 2B) and HCT116 (Figure 2C) cells. Figure 2D illustrates the differential effectiveness of the oligonucleotides in downregulating NR4A1 and NR4A2, and SW480 and RKO cells were selected for cell proliferation (Figure 2A-C) and Western blot (Figure 2E) experiments. RNA interference was also used to examine the effects of siNR4A1 and siNR4A2 on Sp1, Sp4 and several prototypical NR4A1-responsive genes (Figure 2E). The results show that both siNR4A1, siNR4A2 and their combination decrease expression of these gene products, suggesting that G9a, bcl2 and \(\beta\)1-integrin are co-regulated by both NR4A1 and NR4A2. In addition, we also observed that the knockdown of NR4A1 and NR4A2 decreased expression of Sp1 and Sp4 (Figure 2A). Previous studies have shown that NR4A regulates pro-reducant genes and the knockdown of NR4A1 or treatment with CDIMs induces ROS [23], and ROS activate pathways that downregulate Sp transcription factors. This has also been observed for other ROS-inducing compounds which decrease expression of Sp and Sp-regulated genes [31; 32]. The effects of DIM-3,5-Br\({}_{2}\), DIM-3-CI-5-CF\({}_{3}\) and DIM-3,5-CI\({}_{2}\) on NR4A1- and NR4A2-dependent transactivation were determined in RKO (Figure 3A), SW480 (Figure 3B) and HCT116 (Figure 3C) colon cancer cell lines transfected with GAL4-NR4A1 or GAL4-NR4A2 chimeras and UAS-Luc, which contains five yeast GAL4 response elements. The three CDIM compounds decreased transactivation in RKO cells expressing GAL4-NR4A1, and this is consistent with the inverse NR4A1 agonist activities observed for these compounds in inhibiting expression of various gene products. In contrast, the three CDIM compounds induced luciferase activity in RKO cells expressing GAL4-NR4A2. Although this enhanced transactivation did not parallel the observed growth inhibition by DIM-3,5 compounds or NR4A2-dependent downregulation of gene products, the results are comparable with previous studies using GAL4-NR4A2 and other NR4A2 ligands. The GAL4-NR4A2-dependent transactivation is cell context-dependent and is induced in colon and pancreatic cancer cells [30; 33] and decreased in U87 MG glioblastoma cells [30], whereas CDIM compounds that bind NR4A2 decrease cell viability in colon, pancreatic cancer and glioblastoma cells [23; 24; 25; 30].