Hydroxylated Chalcones as Aryl Hydrocarbon Receptor Agonists: Structure-Activity Effects

Abstract

Hydroxylated chalcones are phytochemicals which are biosynthetic precursors of flavonoids and their 1,3-diaryl-prop-2-en-1-one structure is used as a scaffold for drug development. In this study, the structure-dependent activation of aryl hydrocarbon receptor (AhR)-responsive CYP1A1, CYP1B1, and UGT1A1 genes was investigated in Caco2 colon cancer cells and in non-transformed young adult mouse colonocytes (YAMC) cells. The effects of a series of di- and trihydroxychalcones as AhR agonists was structure dependent with maximal induction of CYP1A1, CYP1B1, and UGT1A1 in Caco2 cells observed for compounds containing 2,2'-dihydroxy substituents and this included 2,2'-dihydroxy-, 2,2',4'-trihydroxy-, and 2,2',5'-trihydroxychalcones. In contrast, 2',4,5'-, 2'3',4'-, 2',4,4'-trihydroxy, and 2',3-, 2',4-, 2',4'-, and 2',5-dihydroxychalcones exhibited low to non-detectable AhR activity in Caco2 cells. In addition, all of the hydroxychalcones exhibited minimal to non-detectable activity in YAMC cells, whereas 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced CYP1A1, CYP1B1, and UGT1A1 in Caco2 and YAMC cells. The activity of AhR-active chalcones was confirmed by determining their effects in AhR-deficient Caco2 cells. In addition, 2,2'-dihydroxychalcone induced CYP1A1 protein and formation of an AhR-DNA complex in an in vitro assay. Simulation and modeling studies of hydroxylated chalcones confirmed their interactions with the AhR ligand-binding domain and were consistent with their structure-dependent activity as AhR ligands. Thus, this study identifies hydroxylated chalcones as AhR agonists with potential for these phytochemicals to impact AhR-mediated colonic pathways.

Keywords: Ah receptor; CYP1A1; CYP1B1; UGT1A1; cell; chalcones; colon; computational modeling; gene-expression; structure-activity.

Figure 1.

AhR responsiveness of trihydroxychalcones in Caco2 cells. Cells were treated with DMSO and 10 nM TCDD and different concentrations of chalcones for 18 h and effects on CYP1A1 (A), CYP1B1 (B), and UGT1A1 (C) gene expression were determined by real-time PCR as outlined in the Materials and Methods section. Results of gene expression studies (Figs. 1–5) are expressed as means ± SD for at least 3 separate experiments and significant (p < .05) induction is indicated.

Figure 2.

AhR responsiveness of trihydroxychalcones in YAMC cells. Cells were treated with DMSO or 10 nM TCDD and different concentrations of chalcones for 18 h and effects on CYP1A1 (A), CYP1B1 (B), and UGT1A1 (C) gene expression were determined by real-time PCR as outlined in the Materials and Methods section.

Figure 3.

AhR responsiveness of dihydroxychalcones in Caco2 cells. Cells were treated with DMSO or 10 nM TCDD and different concentrations of chalcones for 18 h and effects on CYP1A1 (A), CYP1B1 (B) and UGT1A1 (C) gene expression were determined by real time PCR as outlined in the Materials and Methods section.

Figure 4.

AhR-responsiveness of dihydroxychalcones in YAMC cells. Cells were treated with DMSO or 10 nM TCDD and different concentrations of chalcones for 18 h and effects on CYP1A1 (A), CYP1B1 (B), and UGT1A1 (C) gene expression were determined by real-time PCR as outlined in the Materials and Methods section.

Figure 5.

AhR responsiveness of 2,2′-dihydroxychalcone analogs in Caco2 cells expressing AhR and in Caco2-AhRKO cells where the AhR is silenced. Cells were treated with DMSO or 10 nM TCDD and different concentrations of chalcones for 18 h and effects on CYP1A1 (A), CYP1B1 (B), and UGT1A1 (C) gene expression were determined by real-time PCR as outlined in the Materials and Methods section.

Figure 6.

2,2′-Dihydroxychalcone activation of AhR transformation and induction of CYP1A1 in Caco2 cells. A, Wild-type and mutant oligonucleotides derived from the human AhR (hAhR) promoter were incubated with whole cell lysates from Caco2 cells treated with TCDD (10 nM) and 2,2′-dihydroxychalcone (0.1 and 1 μM) and binding was determined in a colorimetric Episeeker DNA-protein assay as outlined in the Materials and Methods section. B, Caco2 cells were treated with DMSO (NC), 10 nM TCDD, or 2,2′-dihydroxychalcone for 24 h and whole cell lysates were analyzed by Western blots as outlined in the Materials and Methods section.

Figure 7.

Molecular graphics images of representative snapshots extracted from the lowest free energy binding modes of the human AhR with the (A) inactive 2′,4,4′-trihydroxychalcone and (B) the active 2,2′,4′-trihydroxychalcone. In both panels, the ligand is shown in thick licorice representation. AhR is shown in transparent cyan new cartoon representation, and a portion of the interacting residues is shown in thin licorice representation. Hydrogen bonds (and polar attractions) are shown in dotted lines. Both compounds form hydrogen bonds or polar interactions with Ser365 and however 2,2′,4′- and 2′,4,4′- chalcone form two simultaneous hydrogen bonds and a single hydrogen bond, respectively, with Gln383. Comparable modeling analysis of the interactions of 2,2′,4′- and 2′,4,4′-chalcone with the mouse AhR gave similar interactions to those observed with human AhR (data not shown).