Differential Effects of Glycyrrhiza Species on Genotoxic Estrogen Metabolism: Licochalcone A Downregulates P450 1B1, whereas Isoliquiritigenin Stimulates It

Abstract

Estrogen chemical carcinogenesis involves 4-hydroxylation of estrone/estradiol (E1/E2) by P450 1B1, generating catechol and quinone genotoxic metabolites that cause DNA mutations and initiate/promote breast cancer. Inflammation enhances this effect by upregulating P450 1B1. The present study tested the three authenticated medicinal species of licorice [Glycyrrhiza glabra (GG), G. uralensis (GU), and G. inflata (GI)] used by women as dietary supplements for their anti-inflammatory activities and their ability to modulate estrogen metabolism. The pure compounds, liquiritigenin (LigF), its chalcone isomer isoliquiritigenin (LigC), and the GI-specific licochalcone A (LicA) were also tested. The licorice extracts and compounds were evaluated for anti-inflammatory activity by measuring inhibition of iNOS activity in macrophage cells: GI ≫ GG > GU and LigC ≅ LicA ≫ LigF. The Michael acceptor chalcone, LicA, is likely responsible for the anti-inflammatory activity of GI. A sensitive LC-MS/MS assay was employed to quantify estrogen metabolism by measuring 2-MeOE1 as nontoxic and 4-MeOE1 as genotoxic biomarkers in the nontumorigenic human mammary epithelial cell line, MCF-10A. GG, GU, and LigC increased 4-MeOE1, whereas GI and LicA inhibited 2- and 4-MeOE1 levels. GG, GU (5 μg/mL), and LigC (1 μM) also enhanced P450 1B1 expression and activities, which was further increased by inflammatory cytokines (TNF-α and IFN-γ). LicA (1, 10 μM) decreased cytokine- and TCDD-induced P450 1B1 gene expression and TCDD-induced xenobiotic response element luciferase reporter (IC50 = 12.3 μM), suggesting an antagonistic effect on the aryl hydrocarbon receptor, which regulates P450 1B1. Similarly, GI (5 μg/mL) reduced cytokine- and TCDD-induced P450 1B1 gene expression. Collectively, these data suggest that, of the three licorice species that are used in botanical supplements, GI represents the most promising chemopreventive licorice extract for women's health. Additionally, the differential effects of the Glycyrrhiza species on estrogen metabolism emphasize the importance of standardization of botanical supplements to species-specific bioactive compounds.

Figure 1. Hypothesis: effect of botanicals on inflammation-stimulated estrogen chemical carcinogenesis

Inflammation potentiates estrogen chemical carcinogenesis through activation of transcription factor NF-κB which up-regulates estrogen metabolizing enzyme P450 1B1 that is classically regulated by AhR (figure has been simplified for clarity). P450 1B1 increases the estrogen metabolite 4-OHE₁ which redox cycles with the genotoxic estrogen quinone 4-OHE₁-Q to form ROS. E₂ (not shown for clarity) is similarly metabolized to genotoxic metabolites. The hypothesis is that, to prevent estrogen carcinogenesis, several steps in the inflammation-stimulated estrogen carcinogenesis pathway could be modulated by chemopreventive botanicals, as indicated with green arrows.

Figure 2

Key bioactive marker compounds in licorice.

Figure 3. Licorice extracts/compounds differentially modulated iNOS activity in macrophage cells

Nitrite levels from macrophage RAW 264.7 cells were detected with Griess reagent after 24 h treatment with LPS (1 μg/mL) and A) GG, GU, and GI and B) licorice compounds LigF, LigC, and LicA. Results are the means ± SEM of three independent experiments analyzed by one-way ANOVA with Dunnett’s multiple comparison post-test, *p < 0.001.

Figure 4. Licorice extracts/compounds differentially modulated estrogen metabolism

MCF-10A cells treated for 3 days with E₂ (1 μM) and A) GG, GU, and GI (5 μg/mL) or B) various doses of GI. C) After 3 days treatment of MCF-10A cells with E₂ (1 μM) and LigF, LigC, and LicA, media were collected and analyzed for 2-MeOE₁ and 4-MeOE₁ metabolites by LC-MS/MS. Results are the means ± SEM of three independent experiments analyzed by one-way ANOVA with Dunnett’s multiple comparison post-test, *p < 0.001. D) Representative SRM chromatograms with transition 534.4 – 171.2 showing 2-MeOE₁ and 4-MeOE₁ peaks after 3 days treatment of MCF-10A cells with E₂ (1 μM) and LigF, LigC, and LicA (10 μM).

Figure 4. Licorice extracts/compounds differentially modulated estrogen metabolism

MCF-10A cells treated for 3 days with E₂ (1 μM) and A) GG, GU, and GI (5 μg/mL) or B) various doses of GI. C) After 3 days treatment of MCF-10A cells with E₂ (1 μM) and LigF, LigC, and LicA, media were collected and analyzed for 2-MeOE₁ and 4-MeOE₁ metabolites by LC-MS/MS. Results are the means ± SEM of three independent experiments analyzed by one-way ANOVA with Dunnett’s multiple comparison post-test, *p < 0.001. D) Representative SRM chromatograms with transition 534.4 – 171.2 showing 2-MeOE₁ and 4-MeOE₁ peaks after 3 days treatment of MCF-10A cells with E₂ (1 μM) and LigF, LigC, and LicA (10 μM).

Figure 4. Licorice extracts/compounds differentially modulated estrogen metabolism

MCF-10A cells treated for 3 days with E₂ (1 μM) and A) GG, GU, and GI (5 μg/mL) or B) various doses of GI. C) After 3 days treatment of MCF-10A cells with E₂ (1 μM) and LigF, LigC, and LicA, media were collected and analyzed for 2-MeOE₁ and 4-MeOE₁ metabolites by LC-MS/MS. Results are the means ± SEM of three independent experiments analyzed by one-way ANOVA with Dunnett’s multiple comparison post-test, *p < 0.001. D) Representative SRM chromatograms with transition 534.4 – 171.2 showing 2-MeOE₁ and 4-MeOE₁ peaks after 3 days treatment of MCF-10A cells with E₂ (1 μM) and LigF, LigC, and LicA (10 μM).

Figure 5. Licorice extracts/compounds differentially modulated inflammation-stimulated P450 1B1 gene expression in MCF-10A cells

P450 1B1 mRNA expression analyzed after 24 h via qPCR in MCF-10A cells with cytokines (TNF-α and IFN-γ, 10 ng/mL each) (black bars) and without cytokines (open bars). A) AhR antagonist CH-223191 (100 nM) and NF-κB pathway inhibitor IKK inhibitor VII (2 μM). MCF-10A cells treated with B) GG, GU, and GI (5 μg/mL) C) LigF, LigC, and LicA (1 μM). Results with extracts/compounds were analyzed by Student’s t-test, *p < 0.05 (GG, GU, LigC) and *p < 0.001 (GI and LicA) to compare treatment groups to DMSO and cytokine controls. D) Dose-response for LicA (open squares) and LicA + TNF-α and IFN-γ (black squares). Results are the means ± SEM of three independent experiments analyzed by one-way ANOVA with Dunnett’s multiple comparison post-test, *p < 0.001.

Figure 5. Licorice extracts/compounds differentially modulated inflammation-stimulated P450 1B1 gene expression in MCF-10A cells

P450 1B1 mRNA expression analyzed after 24 h via qPCR in MCF-10A cells with cytokines (TNF-α and IFN-γ, 10 ng/mL each) (black bars) and without cytokines (open bars). A) AhR antagonist CH-223191 (100 nM) and NF-κB pathway inhibitor IKK inhibitor VII (2 μM). MCF-10A cells treated with B) GG, GU, and GI (5 μg/mL) C) LigF, LigC, and LicA (1 μM). Results with extracts/compounds were analyzed by Student’s t-test, *p < 0.05 (GG, GU, LigC) and *p < 0.001 (GI and LicA) to compare treatment groups to DMSO and cytokine controls. D) Dose-response for LicA (open squares) and LicA + TNF-α and IFN-γ (black squares). Results are the means ± SEM of three independent experiments analyzed by one-way ANOVA with Dunnett’s multiple comparison post-test, *p < 0.001.

Figure 6. LicA down-regulated TCDD-induced P450 1B1 mRNA expression, P450 1A/1B activity, and XRE-luciferase activity

A) P450 1B1 mRNA expression was analyzed via qPCR after 24 h treatment of MCF-10A cells with TCDD (10 nM) and licorice compounds (10 μM). B) P450 1A/1B activity measured via EROD assay in MCF-10A cells after treatment with compounds (10 μM) 1 h prior to treatment with TCDD (10 nM) for an additional 48 h. C) HepG2 cells were incubated with TCDD (10 nM) and LicA for 24 h before analysis of XRE-luciferase reporter activity. Results are the means ± SEM of three independent experiments analyzed by one-way ANOVA with Dunnett’s multiple comparison post-test, *p < 0.001.

Figure 6. LicA down-regulated TCDD-induced P450 1B1 mRNA expression, P450 1A/1B activity, and XRE-luciferase activity

A) P450 1B1 mRNA expression was analyzed via qPCR after 24 h treatment of MCF-10A cells with TCDD (10 nM) and licorice compounds (10 μM). B) P450 1A/1B activity measured via EROD assay in MCF-10A cells after treatment with compounds (10 μM) 1 h prior to treatment with TCDD (10 nM) for an additional 48 h. C) HepG2 cells were incubated with TCDD (10 nM) and LicA for 24 h before analysis of XRE-luciferase reporter activity. Results are the means ± SEM of three independent experiments analyzed by one-way ANOVA with Dunnett’s multiple comparison post-test, *p < 0.001.

Figure 7. LicA only moderately inhibited P450 1A/1B activity in MCF-10A cells and did not show P450 1A1 or 1B1 inhibition selectivity

A) MCF-10A cells were pretreated with TCDD (10 nM) for 48 h to induce P450 enzymes and thereafter pre-incubated with LigF/LigC/LicA (1 μM, open bars) and (10 μM, striped bars) for 5 min at 37°C before ethoxyresorufin and NADPH was added for 20 min. B) Human recombinant P450 1A1 and C) P450 1B1 protein with reductase were incubated with ethoxyresorufin, NADPH and LigF/LigC/LicA (1 μM, open bars) and (10 μM, striped bars) or 2,3′,4,5′-tetramethoxystilbene (TMS) (2 μM) at 37°C for 20 min. EROD activity was measured and results were analyzed by one-way ANOVA with Dunnett’s multiple comparison post-test, * p < 0.001.