Structure-dependent activation of gene expression by bis-indole and quinoline-derived activators of nuclear receptor 4A2

Abstract

Bis-indole derivatives including 1,1-bis(3'-indolyl)-1-(4-chlorophenyl)methane (DIM-C-pPhCl) and substituted quinolines such as chloroquine (CQ) and amodiaquine (AQ) are nuclear receptor 4A2 (NR4A2, Nurr1) ligands, and they exhibit anti-inflammatory activities in mouse and rat models of Parkinson's disease, respectively. However, computational modeling demonstrates that the quinoline derivatives interact with the ligand-binding domain, whereas the bis-indoles preferentially interact with a C-terminal cofactor binding site of NR4A2. In this study, the effects of DIM-C-pPhCl and related analogs were compared with CQ/AQ as inducers of NR4A2-responsive genes including vasoactive intestinal peptide, osteopontin, proopiomelanocortin, and neuropilin 1 in Panc1 and Panc28 pancreatic cancer cells. The results demonstrate that, among the bis-indole analogs, their relative potencies as inducers were structure-gene- and cell context dependent. In contrast, CQ and AQ were significantly less potent than the bis-indole derivatives and, for some of the NR4A2-regulated genes, CQ and AQ were inactive as inducers. These results demonstrate that although bis-indole and quinoline derivatives have been characterized as activators of NR4A2-dependent gene expression, these two classes of compounds exhibit different activities, indicating that they are selective NR4A2 modulators.

Keywords: NR4A2 ligand; bis-indole-derived; quinoline-derived.

Figure 1

Transactivation of NR4A2 by bis-indole analogs. Six structurally related bis-indole analogs (A) were used in the transactivation assays. Panc1 (B) and Panc28 (C) cells were cotransfected with UAS-luciferase reporter constructs and GAL4-NR4A2 expression constructs and then the cells were treated with 5, 10 and 15 μM bis-indole analogs. Luciferase activity fold induction was determined as described in the Materials and Methods. Results are expressed as mean ± SD for at least three independent determinations for each treatment. *, P < 0.01, treatment vs. solvent control (DMSO).

Figure 2

Transactivation of NR4A2 by substituted quinolines. Chloroquine and amodiaquine (A) were used in the transactivation assays. Panc1 and Panc28 cells were cotransfected with UAS-luciferase reporter constructs and GAL4-NR4A2 expression constructs followed by treatment with 200 μM CQ or 100 μM AQ (B). Panc1 (C) and Panc28 (D) cells were transfected with only UAS-luciferase plasmid and cells were treated with 7.5 and 15 μM bis-indole analogs, 200 μM CQ or 100 μM AQ. Luciferase activity fold induction was determined as described in the Materials and Methods. Results are expressed as mean ± SD for at least three independent determinations for each treatment. *, P < 0.01, treatment vs. solvent control (DMSO).

Figure 3

Effects of bis-indole analogs and quinoline derivatives on VIP gene expression. Panc1 (A and B) and Panc28 (C and D) cells were treated with bis-indole analogs (5, 10 and 15 μM), CQ (100, 150 and 200 μM) or AQ (50, 75 and 100 μM). Relative expression levels of VIP were determined by quantitative PCR analysis as described in Materials and Methods. Results are expressed as means ± SD for at least six independent determinations for each treatment. The asterisk (*) indicates significant gene induction (P < 0.01) of the highest concentration treatment vs. solvent control (DMSO).

Figure 4

Effects of bis-indole analogs and quinoline derivatives on OPN gene expression. Panc1 (A and B) and Panc28 (C and D) cells were treated with bis-indole analogs (5, 10 and 15 μM), CQ (100, 150 and 200 μM) or AQ (50, 75 and 100 μM). Relative expression levels of OPN were determined by quantitative PCR analysis as described in Materials and Methods. Results are expressed as means ± SD for at least six independent determinations for each treatment. The asterisk (*) indicates significant gene induction (P < 0.01) of the highest concentration treatment vs. solvent control (DMSO).

Figure 5

Effects of bis-indole analogs and quinoline derivatives on POMC and NRP1 gene expression. Panc1 (A, B, C and D) and Panc28 (E and F) cells were treated with bis-indole analogs (5, 10 and 15 μM), CQ (100, 150 and 200 μM) or AQ (50, 75 and 100 μM). Relative expression levels of POMC and NRP1 were determined by quantitative PCR analysis as described in Materials and Methods. Results are expressed as means ± SD for at least six independent determinations for each treatment. The asterisk (*) indicates significant gene induction/repression (P < 0.01) of the highest concentration treatment vs. solvent control (DMSO).