Differential Interactions of Flavonoids with the Aryl Hydrocarbon Receptor In Silico and Their Impact on Receptor Activity In Vitro

Abstract

The molecular mechanisms underlying the observed anticancer effects of flavonoids remain unclear. Increasing evidence shows that the aryl hydrocarbon receptor (AHR) plays a crucial role in neoplastic disease progression, establishing it as a potential drug target. This study evaluated the potential of hydroxy flavonoids, known for their anticancer properties, to interact with AHR, both in silico and in vitro, aiming to understand the mechanisms of action and identify selective AHR modulators. A PAS-B domain homology model was constructed to evaluate in silico interactions of chrysin, naringenin, quercetin apigenin and agathisflavone. The EROD activity assay measured the effects of flavonoids on AHR's activity in human breast cancer cells (MCF7). Simulations showed that chrysin, apigenin, naringenin, and quercetin have the highest AHR binding affinity scores (-13.14 to -15.31), while agathisflavone showed low scores (-0.57 and -5.14). All tested flavonoids had the potential to inhibit AHR activity in a dose-dependent manner in the presence of an agonist (TCDD) in vitro. This study elucidates the distinct modulatory effects of flavonoids on AHR, emphasizing naringenin's newly described antagonistic potential. It underscores the importance of understanding flavonoid's molecular mechanisms, which is crucial for developing novel cancer therapies based on these molecules.

Keywords: AHR; TCDD; antagonist; cancer therapy; naringenin; polyphenols.

Figure 1

AHR PAS-B ligand-binding domain homology model. (A) Schematic structure of human AHR domains. Numbers at the domain boundaries refer to the amino acids of human proteins. (B) Three-dimensional representation of the PAS-B domain of human AHR. The model reveals the presence of four alpha helices along with a region characterized by antiparallel beta sheets. (C) Three-dimensional representation of 3 potential PAS-B ligand domain predicted binding sites. Amino acids of the binding pockets are highlighted in red. The binding regions were named based on which docking system they originated from. AHR PAS-B site 1: described in the work of Leclair et al., (2020) [23]; AHR PAS-B site 2: described in the work of SZÖLLÖSI et al. (2016) [28]; 3: calculated by CASTp (TIAN et al., 2018) [29] using the search radius of 1.4 Å. Three-dimensional models were built on the SWISS-MODEL platform.

Figure 2

Flavonoids interact differently with the AHR binding domain (PAS-B). (a) Heat map showing the flavonoids chrysin (CHRY), apigenin (API), naringenin (NAR), quercetin (QUER), and agathisflavone (FAB) and their putative interactions with amino acids of the PAS-B domain (site 2). (b) Heat map showing the flavonoids CHRY, API, NAR, QUER, and FAB and their putative interactions with amino acids of the PAS- B domain (site 3). (c) Two-dimensional diagram of flavonoid structures and proposed hydroxyl group positions of CHRY, API, NAR, QUER, and FAB hydrogen bond interaction with AHR residues in the AHR binding site model (PAS-B). Orange-colored areas indicate hydrogen bonds with AHR residues at AHR binding sites (PAS-B). Residue names colored in orange indicate interactions with residues at model AHR binding site 2 (PAS-B), and names colored in blue indicate interactions with residues at AHR binding site 3 (PAS-B).

Figure 3

Flavonoids modulate the canonical AHR activity induced by TCDD. MCF7 cells were pretreated with the flavonoids (A) chrysin (1, 5, 10, 50 µM), (B) apigenin (1, 5, 10, 50 µM), (C) naringenin (5, 10, 20, 30 µM), (D) quercetin (1, 5, 10, 20 µM), and (E) agathisflavone (10, 20, 30 µM) for 2 h and exposed to agonist (TCDD 5 nM) for 6 h to measure the induction of CYP1A1 activity using an EROD activity assay. The CYP1A1 activity was analyzed considering the positive control condition (TCDD 5 nM). The results were compared to the control (100%) n = 3. The significance was evaluated by a one-way ANOVA test followed by the Tukey test; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.