6-Prenylnaringenin from Hops Disrupts ERα-Mediated Downregulation of CYP1A1 to Facilitate Estrogen Detoxification

Abstract

Botanical dietary supplements (BDS) containing hops are sold as women's health supplements due to the potent hop phytoestrogen, 8-prenylnaringenin (8-PN), and the cytoprotective chalcone, xanthohumol. Previous studies have shown a standardized hop extract to beneficially influence chemical estrogen carcinogenesis in vitro by fostering detoxified 2-hydroxylation over genotoxic 4-hydroxylation estrogen metabolism. In this study, hop extract and its bioactive compounds were investigated for its mechanism of action within the chemical estrogen carcinogenesis pathway, which is mainly mediated through the 4-hydroxylation pathway catalyzed by CYP1B1 that can form gentoxic quinones. Aryl hydrocarbon receptor (AhR) agonists induce CYP1A1 and CYP1B1, while estrogen receptor alpha (ERα) inhibits transcription of CYP1A1, the enzyme responsible for 2-hydroxylated estrogens and the estrogen detoxification pathway. An In-Cell Western MCF-7 cell assay revealed hop extract and 6-prenylnaringenin (6-PN) degraded ERα via an AhR-dependent mechanism. Reverse transcription PCR and xenobiotic response element luciferase assays showed hop extract and 6-PN-mediated activation of AhR and induction of CYP1A1. A reduction in estrogen-mediated DNA (cytosine-5)-methyltransferase 1 (DNMT1) downregulation of CYP1A1 accompanied this activity in a chromatin immunoprecipitation assay. Ultimately, hop extract and 6-PN induced preferential metabolism of estrogens to their detoxified form in vitro. These results suggest that the standardized hop extract and 6-PN activate AhR to attenuate epigenetic inhibition of CYP1A1 through degradation of ERα, ultimately increasing 2-hydroxylated estrogens. A new mechanism of action rationalizes the positive influence of hop BDS and 6-PN on oxidative estrogen metabolism in vitro and, thus, potentially on chemical estrogen carcinogenesis. The findings underscore the importance of elucidating various biological mechanisms of action and standardizing BDS to multiple phytoconstituents for optimal resilience promoting properties.

Figure 1:. Hop bioactive compounds of interest.

From left to right: 8-PN, an ER agonist and aromatase inhibitor; 6-PN, an AhR agonist; and XH, a cytoprotective compound activitating NRF2/ARE pathways.

Figure 2:

The influence of the standardized hop extract on oxidative estrogen metabolism: estrogen detoxification- and genotoxic metabolism pathways. A. Epigenetic regulation decreases the estrogen detoxification pathway in the presence of E_2. B. Hop extract standardized to 6-PN inhibit estrogen-mediated epigenetic downregulation of CYP1A1 in MCF-7 cells. C The constitutive activity of AhR on CYP1B1 exhibits little change by hop extract. ^,

Figure 3.

Percent degradation of ERα by hop extract and 6-PN is partially mediated by AhR. MCF-7 cells were treated with either 1 μM of 6-PN, 8-PN, XH, or ICI, 10 nM TCDD, or 5 μg/mL hops; A. with and without 10 nM E₂. B. with and without 2 hour pretreatment with the specific proteasome inhibitor, MG-132 (10 μM). C. with and without 2 hour pretreatment with AhR antagonist, CH-223191 (10 μM). After 24 hours ICW was performed, normalized to DMSO, and analyzed by two-way ANOVA with Dunnett’s multiple comparison post-test. n ≥ 3, mean +/− SEM. Test groups were compared to DMSO (◆) or E₂ (◻) controls (A), or within treatment groups for E₂ and DMSO (A), +/− MG-132 (B) and +/− CH-223191 (C) */^◆/◻p < 0.05.

Figure 4.

Hop extract and 6-PN induce XRE luciferase activity. MCF-7 cells were treated with 1 μM 6-PN, 8-PN, or XH, 5 μg/mL hops, or 10 nM TCDD together with 10 nM E₂ (A) and without E₂ (B) for 18 hours. Luciferase output normalized for cell activity then to DMSO. One-way ANOVA for comparisons to E₂ (A) or to DMSO alone (B). n ≥ 3, mean +/− SEM. *p < 0.05.

Figure 5.

In the presence of E₂, hop extract, and 6-PN preferentially upregulate CYP1A1. MCF-7 cells were treated with E₂ (10 nM), 8-PN (1 μM), or a combination of the two (A), or 1 μM 6-PN, 8-PN, or XH, 5 μg/mL hop extract, or 10 nM TCDD together with 10 nM E₂ (B) and without E₂ (C) for 24 hours. ΔΔCT method for qRT-PCR of CYP1A1 and CYP1B1 mRNA was done using HPRT1 as a control gene. Data was normalized to DMSO before two-way ANOVA with Dunnett’s multiple comparison test. n ≥ 3, mean +/− SEM. *p < 0.05. Comparisons made to DMSO (A,C) or to E₂ (B).

Figure 6.

Traditional Western blots confirm ERα downregulation and P450 1A1 upregulation by hop extract and 6-PN. Western blot quantification of relative ERα (A) and P450 1A1 (CYP1A1) (B) in MCF-7 cells treated 24 hours with 0.1% DMSO, 10 nM TCDD, 1 μM 6-PN, 8-PN, or XH, or 5 μg/mL hop extract. One-way ANOVA was used to discern significance, n ≥ 3, mean +/− SEM. *p < 0.05. (C) Representative traditional Western blot image of an experiment for P450 1A1 (CYP1A1), ERα, and β-actin.

Figure 7:. Treatments with hop extract or 6-PN attentuate E₂-induced DNMT1 at CYP1A1.

MCF-7 cells were treated with 1 μM 6-PN, 5 μg/mL hops, 10 nM TCDD and/or 10 nM E₂ for 12 hours. Cells were analyzed using a ChIP assay for DNMT1 with E₂ (A), without E₂ (B), or for DNMT3B with E₂ (C) pulldowns followed by qPCR of the CYP1A1 promoter. Data were first normalized to IgG (1) and fold induction method was used with a one-way ANOVA compared to DMSO. n ≥ 3, mean +/− SEM. *p < 0.05.

Figure 8:

LC-MS/MS analysis of 2- or 4-methoxyestrone (2/4-MeOE₁) metabolites from MCF-7 cells treated with 1 μM E₂ alone or in combination with 10 nM TCDD, 1 μM 6-PN, or 5 μg/mL hop extract for 24 hours. Data were normalized to E₂ for fold induction of 2-MeOE₁ (B), 4-MeOE₁ (C), and the ratio of these values for 2- over 4-MeOE₁ shown in (A). One-way ANOVA was used to discern significance, n = 3, mean +/− SEM. *p < 0.05.