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. Quantitative 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.

Graphical Abstract

INTRODUCTION

The use of botanical dietary supplements (BDS), such as hops (Humulus lupulus L., Cannabaceae), have steadily increased in part due to the findings of the Women’s Health Initiative study.¹ This study found that traditional hormone therapy, involving conjugated estrogens and medroxyprogesterone, led to a 26% increase in the incidence of breast cancer.² Women taking hop supplements tend to use them as an alternative to hormone therapy, as a sleep inducing remedy, and for their cytoprotective properties.^{3, 4} Hop extract contains prenylated chalcones and flavanones as bioactive compounds, which according to the long-term research in our Botanical Center can elicit multiple women’s health resilience promoting properties: cytoprotection attributed to the chalcone xanthohumol (XH), estrogenicity and aromatase (P450 19A1) inhibition effected by 8-prenylnaringenin (8-PN), and aryl hydrocarbon receptor (AhR) activation by 6-prenylnaringenin (6-PN) (Figure 1).^{1, 5-11}

Figure 1: Hop bioactive compounds of interest.³⁴.

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.

Increased local estrogen levels in the mammary gland of postmenopausal women, are associated with a greater risk of breast cancer.^12-14 Estrogen carcinogenesis, or the role of estrogen in carcinogenesis, can be divided mainly into two pathways, hormonal and chemical.^{5, 15} Chemical estrogen carcinogenesis involves the metabolism of estrogens to reactive quinones that can lead to DNA damage and, subsequently, genotoxicity and mutagenesis comprising processes involved in tumor initiation.⁵ This can occur in the breast, as estrogens (estradiol/estrone, E₂/E₁) are produced locally in the mammary gland.^{5, 9} Steroidal estrogens can be metabolized by P450 1A1 into their non-genotoxic 2-hydroxylated form termed the 2-OHE₂/E₁ estrogen detoxification pathway (Figure 2). However, the hormonal pathway involves steroidal estrogens that can also be metabolized into the genotoxic 4-hydroxylated form (4-OHE₂/E₁) by P450 1B1, also located in the breast tissue.^{9, 14, 16-19}

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. ^{19, 20}

Both P450 1A1 (CYP1A1-mediated) and P450 1B1 (CYP1B1-mediated) are transcribed by the transcription factor aryl hydrocarbon receptor (AhR) and are important enzymes responsible for estrogen metabolism in mammary tissue.⁵ However, P450 1B1 estrogen metabolism is considered particularly genotoxic due to the formation of the reactive 4-OHE-O-quinone that can form DNA adducts.^19-22 In accordance, epimediological studies indicated a reduced risk for breast cancer with enhanced levels of 2-hydroxylated estrogens in postmenopausal women.^{19, 21-23} Targeting a reduction in estrogen chemical carcinogenesis has clinical relevance for breast cancer prevention.^{19, 21-23} Identifying plant-derived extracts and compounds which preferentially activate the benign, P450 1A1 pathway, has plausible health benefits, as they may reduce factors related to estrogen chemical carcinogenesis. To this note, we have previously shown that the unique hop flavanone, 6-PN (Figure 1), acts as an agonist of AhR to preferentially induce CYP1A1 over CYP1B1.⁹

AhR has also been shown to be important in the attenuation of estrogen receptor alpha (ERα) responses by inducing degradation of ERα.^{24, 25} In regard to estrogen carcinogenesis, the crosstalk interactions of ERα and AhR may be of particular relevance.^{16, 26, 27}. One example of ERα-AhR crosstalk involves E₂-activated ERα epigenetic downregulation of the benign CYP1A1 (P450 1A1) estrogen detoxification pathway.^{28, 29} Past studies revealed that agonist-activated AhR induces the proteasomal degradation of ERα.^{28, 29} This degradation of ERα has been shown to preferentially upregulate CYP1A1 transcription, potentially reversing ERα-mediated downregulation of CYP1A1.^{28, 29} Reversing this crosstalk could result in a preferential increase in 2-hydroxylated estrogens and possibly contribute to a reduction in estrogen chemical carcinogenesis.

Epigenetic regulation of proteins have been an evolving target for understanding crosstalk mechanisms, including those involved in this ERα-AhR crosstalk.^30-33 These epigenetic ERα-mediated regulations likely contribute to selective transcription of AhR-mediated genes, such as CYP1A1, but not CYP1B1.^{30, 31} Among others, DNA methyltransferases (DNMTs) have been implicated in selective epigenetic silencing of CYP1A1 by ERα.³⁰ Therefore, reducing ERα-mediated recruitment of DNMTs to CYP1A1 may be important for preferential AhR-activation of the P450 1A1 estrogen detoxification pathway.

Recent studies have shown that 6-prenylnaringenin (6-PN), the A-ring prenyl regioisomer of the phytoestrogen, 8-PN, contained in hop extract acts as an AhR agonist, upregulating CYP1A1 transcription, and thereby is a suitable bioactive marker of standardized hop extracts.⁹ However, little else is known about how hops and 6-PN in particular may alter ERα-AhR crosstalk under estrogenic conditions.^{9, 34} Considering our prior research on hops, we hypothesize that 6-PN in a standardized hop extract contributes to a large degree in the ability of a hop extract to promote the estrogen detoxification pathway in vitro through activation of AhR under estrogenic conditions in a model with ER+ MCF-7 cells.^{3, 9, 34} The present paper presents evidence substantiating this hypothesis by elucidation of a new mechanism. This mechanism involves the ability of both hop extract and 6-PN to activate AhR, degrade ERα, and reverse the E₂-activated and ERα-mediated downregulation of CYP1A1 in ER+ breast cancer cells, collectively resulting in upregulation of the estrogen detoxification pathway. An understanding of how 6-PN contributes to the overall bioactivities of the hop extract, and how hop extracts may be optimized to reduce carcinogenic metabolism of estrogens, represents significant progress in the understanding of BDS that are widely used to promote resilience in women’s health.

EXPERIMENTAL PROCEDURES

Chemicals and Reagents

The chemically standardized clinical spent hop (Humulus lupulus L., Cannabaceae) extract, containing 33.20 % XH, 1.22 % 6-PN, 1.11 % isoxanthohumol, and 0.28 % 8-PN, was originally obtained from Hopsteiner (New York, NY, USA, and Mainburg, Germany).³⁵ This extract was characterized by LC-MS/MS, LC-UV, and ¹H NMR (qHNMR), and has been used for previous publications, including a Phase 1 clinical trial in postmenopausal women.^{4, 9, 35} All presented biological assays also utilized compounds purchased from Sigma-Aldrich (St. Louis, MO, USA) as follows: DMSO, E₂, ICI 182 780 (ICI; Fulvestrant), (+/−)-6-PN, (+/−)-8-PN, XH. Solid TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) was purchased from AccuStandard (New Haven, CT, USA). 6-PN and 8-PN were previously analyzed and showed a purity of > 95%, while XH had a purity of > 90%.^34-36

Cell Culture and Conditions

MCF-7 WS8 cells were originally provided by Dr. V.C. Jordan and are generally referred to as MCF-7 cells in this paper. These cells were cloned from MCF-7 cells as previously described and were chosen due to increased ER expression and estrogen sensitivity in protein expression and cell growth assays.^{37, 38} Otherwise, no significant phenotypic differences have been observed between the parental MCF-7 and the MCF-7 WS8 cell line.³⁸ Cells used in experiments were kept within 15 passages and grown in RPMI 1640 media without phenol red containing 10% fetal bovine serum, 1% nonessential amino acids, 1% glutaMAX, 1% AB/AM, and 6 ng/mL insulin (Thermo Fisher, Waltham, MA, USA). The MCF-7 WS8 cell line used for these studies was authenticated as previously published.²⁸ The MCF-7 WS8 cells were 93% similar to the MCF-7 cells from ATCC, but contain an allele deletion (D5S818:12), indicating a slight difference between the MCF-7 WS8 subclone and the ATCC MCF-7 cell line.^{28, 37} Cell treatments consisted of either 10 nM TCDD, 10 nM E₂, 5 μg/mL hops extract, or 1 μM compounds unless otherwise stated. A cell viability assay (MTT assay) in MCF-7 cells showed IC₅₀ values greater than 20 μM for 6-PN, 8-PN, and XH, greater than 1 μM for TCDD, and an IC₅₀ value of 32.8 μg/mL for the hop extract (Figure S3).

Quantification of in-cell protein expression

In-Cell Western™ (ICW) was used as a high-throughput assay for quantification of ERα and β-actin expression, comparable to a traditional western. MCF-7 cells were plated at 50,000 cells/well and incubated at 37 °C in 5% CO₂ for 2 days before treating overnight. 2 hour pretreatments were performed with the AhR antagonist, CH-223191 (Sigma-Aldrich, St. Louis, MO, USA), or the proteasome inhibitor, MG-132 (Calbiochem, San Diego, CA, USA). LI-COR (Lincoln, NE) protocol for In-Cell Western™ was performed for fixing, staining, and detection of ERα (F10; Santa Cruz Biotechnology, Dallas, TX, USA) and β-actin (13E5; Cell Signaling Technology, Danvers, MA, USA). Wells of equal pixel area were quantified with ImageStudioLite before subtracting background and normalizing to β-actin. The final data was represented relative to the negative control DMSO, which was set at 100%.

Quantification of AhR activity

A luciferase driven assay was used to obtain AhR activity. MCF-7 cells were plated in 24 well plates and transfected at 70% confluency. Transfection occurred over 5 hours using Lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA), 1 μg/well of xenobiotic response element (XRE) luciferase plasmid (pGL4.43) (Promega, Madison, WI, USA), and 0.1 μg/well of beta-galactosidase plasmid (pSV-β-Gal) (Promega, Madison, WI, USA). Cells were treated with E₂/compounds 18 hours and lysed with 110 μL of lysis buffer (25 mM glycyl glycine, 15 mM MgSO4, 4 mM EGTA, 1 mM DTT, and 2% Triton X-100). Gen5 Microplate Reader and Imager Software was used on a BioTek SYNERGY™ multi-mode reader system (BioTek, Winooski, VT, USA) with 50 μL luciferin substrate (Sigma-Aldrich, St. Louis, MO, USA) per sample. Equal amounts of lysate were mixed with 50 μL of ortho-nitrophenyl-β-galactoside (ONPG) assay buffer (200 mM NaH₂PO₄, 2 mM MgCl₂, 7.3% β-mercaptoethanol, and 21 mM ONPG) and incubated for 4 hours. Absorbance at 420 nm was obtained using the BioTek SYNERGY™. Data calculated using mean RLU over the corresponding β-Gal absorbance and normalized to the averaged DMSO for the XRE-Luciferase induction.

Quantification of P450 1A1 and P450 1B1 mRNA

Real-time reverse transcription PCR (qRT-PCR) was used to quantify CYP1A1 and CYP1B1 in MCF-7 cells. These cells were plated at a density of 40,000 cells/well and incubated at 37 °C in 5% CO₂ for 2 days before 24 hours treatment. Lysis and qRT-PCR was carried out according to manufacturer’s protocol for Ambion Cells to CT™ 1-Step TaqMan™ kit from Thermo Fisher Scientific. HPRT1 (Hs02800695_m1) was used as a control for the genes of interest, CYP1A1 (Hs00153120_m1) or CYP1B1 (Hs00164383_m1) (Thermo Fisher, Waltham, MA, USA). CT (cycle threshold) was obtained with a StepOnePlus™ fluorescence detection system (Thermo Fisher, Waltham, MA, USA) and the comparative CT method (ΔΔCT) was used to express fold induction relative to DMSO.

Quantification of protein expression from lysate

Traditional Western blots employed a previously published protocol with minor changes.¹⁵ Briefly, MCF-7 cells were treated with compounds for 24 hours before collection and sonication. Protein concentration was determined using BCA assay (Pierce™ BCA Protein Assay Kit, Thermo Fisher, Waltham, MA, USA), and 30 μg of protein was added to each well of a 10 % bis-tris gel with LDS sample buffer (Thermo Fisher, Waltham, MA, USA). Gel was transferred using the iBlot™ gel transfer device with PVDF membrane iBlot™ transfer stacks (Thermo Fisher, Waltham, MA, USA). Primary antibodies (1:1000) (ERα, PA1-308; CYP1A1, PA1-340; Thermo Fisher, Waltham, MA, USA) (β-actin, 4967S, Cell Signaling, Danvers, MA, USA) were added to blocked membranes and incubated at 4 °C overnight. Secondary antibody was added (1:1000) (Horseradish Peroxidase-linked anti-rabbit IgG, 7074S, Cell Signaling, Danvers, MA, USA) to membranes for 1 hour at room temperature. Then, SuperSignal™ West Femto (Thermo Fisher, Waltham, MA, USA) was added and imaged on a FluorChem™ system (Protein Simple, Santa Clara, CA, USA). Quantification used ImageJ software to determine ratios of protein of interest over β-actin were obtained, which were represented as relative to 100 % DMSO.

Quantification of CYP1A1 associated with a DNMT1 pulldown

A Chromatin immunoprecipitation (ChIP) assay was performed in MCF-7 cells grown in estrogen free media on 10 cm plates until 90% confluency before treating with vs. without E₂ and compounds for 12 hours. Cross-linking, precipitation, pulldowns, washing, and decross-linking were all done according to previous published protocols, with the exception of antibodies used.³⁹ DynaBeads™ Protein A (Thermo Fisher, Waltham, MA, USA) were rotated in cold for 2 hours with either anti-DNMT1 antibody (ab13537, ABCAM, Cambridge, United Kingdom), anti-DNMT3B antibody (ab2851, ABCAM, Cambridge, United Kingdom), or normal mouse IgG antibody (sc-2025, Santa Cruz Biotechnology, Dallas, TX, USA). QIAquick (Qiagen, Hilden, Germany) PCR Purification Kit was used according to manufacturer’s protocol to obtain DNA samples. Primers used are listed in the supporting information (Figure S2). SYBR Green I (Thermo Fisher, Waltham, MA) was used to perform qPCR. Data was analyzed using the fold induction method, before normalizing to IgG control.

Quantification of in vitro 2- or 4-methoxyestrone metabolites

E₂ metabolism was studied in MCF-7 cells that were treated with E₂ (10 nM) and hop extract (5 μg/mL), 6-PN (1 μM), or TCDD (10 nM) for 72 hours and analyzed by UHPLC-MS/MS. For the last 24 hours, cells were treated or cotreated with E₂ (1 μM). The samples were extracted, derivatized with dansyl chloride, purified, and analyzed according to a previously published protocol.² Standard curves with dansylated 2- or 4- MeOE₁ (methoxyestrone) and samples were analyzed on an LC-Agilent 1200 nano flow system (Agilent, Santa Clara, CA) coupled to a QTrap 5500 (AB Sciex, Framingham, MA). Deuterated (d4) 4-methoxyestrone (CDN, Pointe-Claire, Quebec) was used as an internal standard. Using Analyst software (AB Sciex, Framingham, MA), the area under the curve of qualifier ions for multiple reaction monitoring conditions (534.4–171.2 for dansylated MeOE₁, and 538.3−171.2 for dansylated MeOE1-d4) was used to calculate the amount of 2-MeOE₁, and 4-MeOE₁.

Statistics

Data from at least three independent biological triplicates (except Western blot) were expressed as mean plus/minus the standard error of the mean (+/− SEM) for all assays. Using GraphPad Prism version 8 (San Diego, CA, USA), significance was determined using one or two-way ANOVA with Dunnett’s post-test for control and treatment group comparison. Significance was assigned using a single asterisk (*) for any p ≤ 0.05.

RESULTS

Hop extract and 6-PN induce ERα degradation through an AhR-dependent mechanism.

Quantification of ERα protein was analyzed using In-Cell Western™ in MCF-7 cells with the hop extract and the prenylated hop compounds all demonstrating significant increased degradation of ERα (Figure 3A). E₂ significantly decreased ERα protein, confirming previous results.²⁴ This is an example of E₂ acting as a protective modulator through compound-induced ER protein-downregulation in breast cancer cells.⁴⁰ This degradation in the presence of E₂ was potentiated for treatment with hop extract, 6-PN, and XH, as well as with the positive control, the AhR agonist, TCDD (Figure 3A). 8-PN did not potentiate ERα degradation in the presence of E₂, nor did the selective ER degrader/downregulator (SERD), ICI (Figure 3A). This suggests that the phytoestrogen, 8-PN, and ICI use a mechanism similar to that of E₂ and likely compete with E₂ at the ERα binding site. However, the data also indicate that 6-PN, XH, and hop extract at least in part partake in a different mechanism of action than E₂ or the phytoestrogen, 8-PN, suggesting multiple pathways of ERα degradation by hop constituents. To analyze whether the compounds/extracts reduce ER through a proteasome degrading mechanism, the cells were pretreated with the proteasome inhibitor, MG-132 (Figure 3B). In the presence of the proteasome inhibitor known to inhibit ERα degradation, the ERα protein-reducing effects seen by the test compounds, extract, and controls were significantly attenuated (Figure 3B).²⁴ This indicates that hop extract and its bioactive compounds facilitate ERα degradation through a proteasomal pathway. AhR activation is known to lead to proteasomal degradation of ERα.²⁴ To analyze the involvement of AhR in hops-mediated ERα downregulation, the cells were pretreated with the AhR antagonist, CH-223191. After pretreatment with the AhR antagonist, sustained and significantly higher levels of ERα expression were observed for treatments with TCDD, 6-PN, and hop extract (Figure 3C), thereby indicating involvement of AhR. TCDD-induced ERα expression was fully restored by the AhR antagonist; however, cotreatment with the AhR antagonist did not fully restore 6-PN and hop extract-induced ERα degradation. The specificity of the AhR antagonist for dioxins over flavonoids may be a cause for the lack of complete restoration of ERα expression for 6-PN when compared to TCDD treatment (Figure 3C).^{41, 42}

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.

Hop extract and 6-PN act on AhR to activate XRE-motifs

Upon AhR binding to its cognate ligand, the complex is translocated into the nucleus and ultimately associates with the xenobiotic response element (XRE), initiating, among others, gene transcription of the P450 1 family.^{9, 43} To test whether hop extract or its bioactive compounds activate AhR in ER+ breast cancer cells, transiently transfected MCF-7 cells with XRE-luciferase were analyzed for luciferase activity. E₂ slightly lowered XRE activation, albeit insignificantly (Figure 4A). In the presense of E₂, both 6-PN and hop extract, exhibited significant luciferin output, reversing the reducing effect of E₂ on the ability for AhR to bind and to activate its cognate XRE-motifs (Figure 4A). Similarly, hop extract and 6-PN significantly activated AhR-dependent XRE-motifs without E₂, although to a slightly greater extent (Figure 4B).

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.

Hop extract and 6-PN induce preferential transcription of CYP1A1 over CYP1B1

Reverse transcription PCR (qRT-PCR) was used to analyze changes in relative transcription for P450 1A1 and P450 1B1 genes in the presence of E₂ and hop extract or hop constituents of interest. The current data confirms that E₂ alone significantly downregulates CYP1A1 without affecting CYP1B1 transcription (Figure 5A).²⁸ Similarly, treatment with the potent phytoestrogen, 8-PN alone, significantly downregulated CYP1A1 (Figure 5A). In contrast, the present studies also confirmed that the hop extract and 6-PN significantly induce CYP1A1 (Figure 5C).⁹ Additionally, E₂-mediated downregulation of CYP1A1 mRNA levels was reversed by hop extract, 6-PN, and the positive control, TCDD, leading to a significant induction of CYP1A1 mRNA (Figure 5B). 8-PN or XH cotreated with E₂ showed no significant change in CYP1A1 or CYP1B1 gene expression (Figure 5B).

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).

Hop extract and 6-PN decrease ERα expression and increase P450 1A1 expression

Quantification of relative protein expression from MCF-7 cells utilized traditional Western blot (Figure 5). Similar to the In-Cell Western™ blot data (Figure 3), ERα expression was reduced for all treatments when compared to DMSO, while this decrease in ERα expression was significant for treatments with TCDD, 6-PN, 8-PN, and hop extract (Figure 6A). Confirming the gene expression analysis of CYP1A1 (Figure 5C), P450 1A1 (CYP1A1) protein expression was significantly increased for treatments with TCDD, 6-PN, and hop extract when compared to DMSO (Figure 6B). Interestingly, hop extract exhibited the greatest decrease in ERα and increase in P450 1A1 protein, respectively.

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.

Hop extract and 6-PN reverse E₂ associated DNMT1 recruitment to CYP1A1

Because only the hop extract and 6-PN degraded ERα through AhR activation, showed enhanced XRE-reporter activity, and increased CYP1A1 and P450 1A1 levels (Figures 3-6), a potential epigenetic mechanistic link controlling CYP1A1 was analyzed for 6-PN and hop extract treatment. A Chromatin Immunoprecipitation (ChIP) pulldown with an anti-DNMT1 or anti-DNMT3B antibody was performed for treatment groups with E₂ alone and in combination with hop extract, 6-PN, or TCDD. An anti-DNMT1 ChIP was also done without E₂. Using qPCR, purified DNA was amplified with CYP1A1 promoter specific primers (Figure S2). ³⁰ An anti-DNMT1 pulldown for E₂ showed a significant increase in gene amplification compared to DMSO suggesting involvement of activated ERα in recruitment of DNMT1 to CYP1A1, (Figure 7A). Anti-DNMT1, cotreatment of E₂ with hop extract or 6-PN, when compared to E₂ alone, showed a significant decrease in the amount of DNMT-associated CYP1A1 that was precipitated similar to the positive control, TCDD (Figure 7A). This trend of DNMT1 downregulation at CYP1A1 continued for hop extract and 6-PN without E₂ (Figure 7B). This shows that methylation and downregulation of CYP1A1 by DNMT1 and ERα is reversed through AhR activating 6-PN and hop extract. This effect was not observed using an anti-DNMT3B antibody for precipitation (Figure 7C).

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.

Hop extract and 6-PN preferentially induce non-genotoxic estrogen metabolism

The UHPLC-MS/MS approach previously used for the analysis of oxidative estrogen metabolism modulation by botanicals was applied to quantify the amount of 2- and 4-methoxyestrones.⁹ In the presence of 1 μM E₂, only TCDD increased the amount of 4-methoxyestrone significantly when compared to E₂ alone (Figure 8C). However, the non-genotoxic 2-methoxyestrone was significantly increased for E₂ cotreatments with TCDD, 6-PN, and hop extract (Figure 8B). Moreover, the ratio of 2- to 4-methoxyestrone was significantly increased for TCDD, 6-PN, and hop extract (Figure 8A). These results were in accordance with previous results for 6-PN in MCF-7 and MCF-10A cells.⁹

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.

DISCUSSION

The scope of this current research involves elucidating the mechanism responsible for the preferential transcription of CYP1A1 over CYP1B1 by the standardized hop extract and 6-PN (Figure 5). Although it has been shown previously that 6-PN acts as an agonist of AhR, it is unknown in how far the involment of ERα-AhR crosstalk plays a role in this preferential CYP1A1 transcription (Figure 2).⁹ Thus, it was hypothesized that the standardized hop extract and its constituent, 6-PN, degrade ERα through AhR activation to facilitate preferential transcription of CYP1A1. The fact that AhR-mediated degradation of ERα was confirmed for some constituents in these studies, but not all compounds is indicative that hop compounds, including 6-PN, promote ERα degradation through multiple pathways (Figure 3). This degradation would likely have a favorable effect on estrogen metabolism, as ERα degradation leads to an increase in P450 1A1 transcription (Figure 2), seen for ICI in previous studies.²⁸ Interestingly, hop extract and 6-PN both significantly decrease ERα and increase P450 1A1 expression (Figure 3, 5, and 6).

Induction of AhR pathways is dependent upon XRE recognition on genes such as CYP1A1 and CYP1B1, yet the extent of individual CYP1A1 or CYP1B1 activation varies and can depend upon specific ligands, cofactors, epigenetic influences, or cell types.^{9, 28, 31, 44} AhR activation can also vary based on its ligands, due to the different confirmations of the AhR receptor.^{41, 42} This variability was confirmed by the observed full restoration of ERα expression was fully restored for treatments with an AhR antagonist and TCDD (Figure 3C). However, the AhR antagonist only exhibited partial ERα restoration of 6-PN- and hop extract-induced ERα degradation (Figure 3C). This is likely due to different AhR binding pockets for dioxins and flavonoids and the specificity of the antagonist for the dioxin binding site.^{41, 42} The genotoxic CYP1B1 estrogen metabolism pathway, for example, is dominant for E₂ or estrogenic ligands, such as genistein, from red clover or soy.^{28, 44} However, preferential transcription of CYP1A1, and consequently metabolism by the estrogen detoxification pathway, occurs with endogenous AhR ligands, such as 6-formylindolo[3,2-b]carbazole (FICZ), exogenous agonists, such as TCDD or the SERD, ICI.^45-47 The results seen in the XRE-luciferase assay in MCF-7 cells (Figure 4) indicate significant AhR activity for 6-PN and hops, which is in accordance with the CYP1A1 induction data (Figure 5). However, the stark increase seen in the qRT-PCR data is less apparent in the XRE-luciferase assay due to decreased specificity, as XRE motifs are associated with multiple genes, and each gene has specific cofactors that influence its expression.⁴³ The same can be concluded for the effect of E₂ on the change in XRE activity, as the downregulation of CYP1A1 by E₂ is more likely dependent on epigenetic factors such as DNMT1 in the direct promoter region of CYP1A1, than XRE in general.²⁸ Additionally, results from these investigations are supported by previous studies showing 6-PN exhibits significant XRE-activation in MCF-7 cells.⁹

Prior studies have been shown that DNMT-mediated methylation of CYP1A1 occurs through recruitment by and association with ERα.³⁰ For this study, the decrease in DNMT1 recruitment by 6-PN, hop extract, and TCDD with or without E₂ (Figure 7) correlates with the ability to activate CYP1A1 (Figure 5, 6) and reverse E₂-mediated activity (Figure 5, 7). ^{30, 31} DNMTs are associated with the inhibition of P450 1A1 transcription, and E₂ increases the recruitment of both ERα and DNMTs to the CYP1A1 promoter.³⁰ Previous studies have also shown increased XRE methylation in the CYP1A1 promotor region with combinatorial E₂/TCDD treatment compared to TCDD treatment alone.³⁰ Additionally, combinations of 10 nM TCDD/E₂ have shown sustained degradation of ERα starting after 3 hours of treatment and maintained at least for 24 hours.²⁴ All these results suggest the involvement of DNMTs in E₂-mediated repression of AhR-induced CYP1A1 expression, although responses can vary for cell types or ligands and doses used.^30-32

Crosstalk between ERα and AhR is complex, with this study revealing a role for DNMT1 in E₂-mediated inhibition of CYP1A1. DNMT proteins can catalyze the 5-methylation of cytosine in a promoter, inhibiting gene transcription.^{30, 34} Isoforms of DNMTs may be highly important in discerning epigenetic influence on crosstalk. DNMT1 and DNMT3 have shown relevance in epigenetic crosstalk between AhR and ERα, but DNMT2 to our knowledge has not been reported to be involved.^{30, 48} DNMT3B has been implicated to drive an ERα-dependent methylation pattern at CYP1A1.³⁰ In past studies, the ERα cofactor SRC-1 has been associated with increased expression of DNMT1 and DNMT3A.⁴⁸ Histone variant H2A.Z has shown importance in TCDD-induced transcription of CYP1A1 by providing a stable physical environment and recruiting RNA polymerase II.³⁰ Thymine-DNA glycosylase (TDG) removes oxidated 5-methylcytosine, a key role in reversing epigenetic CYP1A1 inhibition, and critical for TCDD-activated AhR transcription of CYP1A1.³¹ Histone deacetylase 1 (HDAC1) is associated with the promoter region of CYP1A1 during inhibition, and its removal is needed for AhR-mediated transcription of CYP1A1.⁴⁹ Recently HDAC inhibitors have been implicated in activation of AhR responsive gene induction.⁵⁰ Additionally, trimethylation of lysine 4 on histone 3 (H3K4me3) in the promoter of CYP1A1 is enriched by TCDD, common for active gene transcription.³¹ ERα recruits DNMT to methylate the promoter of CYP1A1, and AhR agonists can cause the demethylation of affected DNA through complex epigenetic influences not yet fully elucidated. As epigenetics is an evolving field; it is likely that additional epigenetic effects for AhR agonists remain to be discovered, and that the influence of hop extract on these and other epigenetic factors has yet to be studied.

The present studies suggest ERα-AhR crosstalk is in part regulated by DNMT1, whose key role in E₂-mediated CYP1A1 inhibition is reversed by AhR-active 6-PN and the standardized hop extract. Although the dose of 6-PN (1 μM) used for analysis was approximately 5 times the amount of 6-PN found in the hop extract (179 nM in 5 μg/mL), the hop extract exhibited an activity similar to that of 6-PN in assays tested. This apparent discrepancy is a ubiquitous phenomenon for botanical extracts and herbal medicines, and may be interpreted as a prototypical example of chemical potentiation relating to over-additive effects of phytoconstituents and, specifically, chemical potentiation between 6-PN and congeneric prenyl-phenolic hop constituents.^{51, 52} Although multiple compounds may act on the same enzyme, other hop compounds may indirectly cause chemical potentiation, influencing the pharmacodynamic profile of 6-PN through activity on additional targets, an example of polypharmacology.³⁵ For example, the prenylated chalcone and Michael acceptor, XH (4.68 μM in 5 μg/mL hop extract), is the most abundant and therefore most prominent constituent of the hop extract. Interestingly, the polypharmacological properties of hops depend greatly on XH, which exhibits resilience promoting properties in vitro through activation of the NRF2 antioxidant cascade, inhibition of inflammatory NF-κB signaling, and reduction of cell viability.^{34, 53} The nature of XH as a Michael acceptor permits for KEAP1 alkylation, enhancing nuclear NRF2 concentration, and allowing for potential increase in AHR gene transcription.⁵⁴ This is one example of a mechanism by which XH may potentiate the ability of hops and its preparations to promote estrogen detoxification.

In contrast, the polypharmacology of the investigated clinical hop extract on estrogen metabolism likely also includes antagonistic effects of 6-PN-mediated transcription of CYP1A1, for example, by the downregulating effect of the phytoestrogen, 8-PN (41.1 nM in 5 μg/mL hop extract). Yet, 8-PN concentrations may increase in vivo due to metabolic reactions mediated by intestinal gut microbiota or via phase I metabolism of isoxanthohumol to 8-PN.⁵ At the same time, the isoxanthohumol concentration can increase through cyclization of XH, providing more isoxanthohumol for subsequent 8-PN formation.^{55, 56} Additionally, 8-PN has been shown to be roughly five times more bioavailable than 6-PN when purely administered in vivo.⁵⁷ However, 8-PN may play a valuable role by inhibiting aromatase, the enzyme responsible for conversion of certain androgens to estrogens.^{8, 58} Additional prenylated phenols found in hops, such as isoxanthohumol and desmethylxanthohumol, may also contribute to the overall polypharmacology of hop extracts.³⁴ In summary, optimizing the bioactivity of a hop product for estrogen chemical detoxification seems to depend on 6-PN, but still involves many other compounds, their chemical transformations and reactions, as well as their pharmacodynamic interactions - as could be expected for a botanical agent.

Aiming for a reduction in estrogen chemical carcinogenesis through an induction of the beneficial 2-hydroxylation pathway has clinical importance for breast cancer prevention and resilience in women.²¹ Therefore, the induction of the 2-hydroxylation estrogen detoxification pathway by hop extract and 6-PN and the increased ratio of 2- over 4-methoxyestrone (Figure 8) indicates a protective and resilient effect against breast cancer.⁹ However, in vivo studies are warranted to substantiate these in vitro results. Although the favorable 2-hydroxylation of estrogens is mainly catalyzed by P450 enzymes in the breast tissue, P450 1B1 can also catalyze the 2-hydroxylation in the breast tissue, and P450 1A2 is the main liver enzyme conducting the 2-hydroxylation of estrogens.^{5, 22, 59} Additionally, while P450 1B1 is the predominant enzyme in the breast tissue catalyzing the genotoxic metabolism of estrogen to its 4-hydroxy catechol, this metabolism pathway can also occur through P450 1A1 in breast tissue and through P450 3A4 and P450 1A2 in the liver.^{22, 59} However, P450 1A1 and P450 1B1 are primarily responsible for the initiation of estrogen detoxification and the carcinogenic metabolism of catechol estrogens in the local mammary tissue, respectively.^{19, 21-23}

The Phase I enzyme, NAD(P)H-quinone oxidoreductase 1 (NQO1) plays an important role in estrogen detoxification.^{5, 22} NQO1 can chemically reduce the genotoxic 4-OHE-o-quinone to the catechol.^{5, 34} Also, Phase II enzymes play an important role in detoxification of estrogens.^{5, 22} Sulfotransferases and glucuronosyltransferases aid in estrogen detoxification through increasing water solubility for eventual excretion.^{5, 22} The reactivity of catechol estrogens is reduced through methylation by catechol-O-methyltransferase (COMT), and is associated with a reduction in breast cancer risk.^{5, 9, 22, 34} The 2- and 4-methoxyestrone metabolites formed by COMT are stable and can be assessed as biomarkers of their respective catechol estrones, indicative of estrogen detoxification or genotoxic estrogen metabolism, respectively.⁹ Ultimately, the estrogen metabolite profiles are important for the clinical influence of hops and its botanical preparations.

Chemical and biological analysis and multi-constituent standardization of extract polypharmacology is especially important for optimal safety and bioactivity of hop supplements, as they are used by women worldwide. Botanicals are complex agents and may interact differently in various populations depending on the constituent ratio in the extract. As shown by the present study, a hop formulation optimizing the activation of estrogen detoxification pathways with 6-PN, in particular, may enhance resiliency in women. To begin this process, an understanding of ERα-AhR crosstalk and the pharmacology of individual hop compounds was necessary. The present study revealed that 6-PN and a standardized hop extract decreased ERα-mediated epigenetic inhibition of CYP1A1, and preferentially upregulated the AhR-dependent estrogen detoxification pathway through attenuation of DNMT1-mediated repression of CYP1A1 transcription. This suggests that 6-PN drives the ability of the hop extract to influence ERα-AhR crosstalk and increases activity of the estrogen detoxification pathway. Additional hop constituents provide chemical potentiation of estrogen detoxification for 6-PN, and support the concept of polypharmacology in hops. Supplementing the present in vitro outcomes, in vivo studies are needed for further mechanistic insight and analysis of reasonable expectaction for translational outcomes. Based on our investigations, it can be hypothesized that a 6-PN-rich hop extract may enhance resilience against breast cancer in preclinical outcomes, through the attenuation of ERα-mediated epigenetic inhibition of AhR-dependent CYP1A1 transcription, accompanied with preferential 2-hydroxylation of estrogens by P450 1A1 in breast tissue. This hypotheses should be suitable for subsequent translational studies of hops BDS.

ABBREVIATIONS

6-PN

6-prenylnaringenin

8-PN

8-prenylnaringenin

AhR

aryl hydrocarbon receptor

BDS

botanical dietary supplements

ChIP

chromatin immunoprecipitation

CT

cycle threshold

DNMT

DNA (cytosine-5)-methyltransferase

E₁

estrone

E₂

estradiol

ERα

estrogen receptor alpha

ICI

Fulvestrant

ICW

In-Cell Western™

IgG

immunoglobulin G

MCF-7

Michigan Cancer Foundation-7 (breast cancer cells)

P450 1A1/1B1

cytochrome P450 1A1/1B1

qRT-PCR

quantitative reverse transcription polymerase chain reaction

SERD

selective estrogen receptor degrader/downregulator

ST

sulfurtransferase

TCDD

2,3,7,8-tetrachlorodibenzo-p-dioxin

UGT

uridine 5’-diphospho-glucuronyltransferase

XH

xanthohumol

XRE

xenobiotic response element