Dihydrotanshinone I-Induced CYP1 Enzyme Inhibition and Alteration of Estradiol Metabolism.

Dihydrotanshinone I (DHTI) is a pharmacologically active component occurring in the roots of the herbal medicine Salvia miltiorrhiza Bunge. This study investigated DHTI-induced inhibition of CYP1A1, CYP1A2, and CYP1B1 with the aim to determine the potential effects of DHTI on the bioactivation of estradiol (E2), possibly related to preventive/therapeutic strategy for E2-associated breast cancer. Ethoxyresorufin as a specific substrate for CYP1s was incubated with human recombinant CYP1A1, CYP1A2, or CYP1B1 in the presence of DHTI at various concentrations. Enzymatic inhibition and kinetic behaviors were examined by monitoring the formation of the corresponding product. Molecular docking was further conducted to define the interactions between DHTI and the three CYP1s. The same method and procedure were employed to examine the DHTI-induced alteration of E2 metabolism. DHTI showed significant inhibition of ethoxyresorufin O-deethylation activity catalyzed by CYP1A1, CYP1A2 and CYP1B1 in a concentration-dependent manner (IC50 = 0.56, 0.44, and 0.11 µM, respectively). Kinetic analysis showed that DHTI acted as a competitive type of inhibitor of CYP1A1 and CYP1B1, whereas it noncompetitively inhibited CYP1A2. The observed enzyme inhibition was independent of NADPH and time. Molecular docking analysis revealed hydrogen bonding interactions between DHTI and Asp-326 of CYP1B1. Moreover, DHTI displayed preferential activity to inhibit 4-hydroxylation of E2 (a genotoxic pathway) mediated by CYP1B1. Exposure to DHTI could reduce the risk of genotoxicity induced by E2. SIGNIFICANCE STATEMENT: CYP1A1, CYP1A2, and CYP1B1 enzymes are involved in the conversion of estradiol (E2) into 2-hydroxyestradiol (2-OHE2) and 4-hydroxyestradiol (4-OHE2) through oxidation. 2-OHE2 is negatively correlated with breast cancer risk, and 4-OHE2 may be a significant initiator and promoter of breast cancer. The present study revealed that dihydrotanshinone I (DHTI) competitively inhibits CYP1A1/CYP1B1 and noncompetitively inhibits CYP1A2. DHTI exhibits a preference for inhibiting the genotoxicity associated with E2 4-hydroxylation pathway mediated by CYP1B1, potentially reducing the risk of 4-OHE2-induced genotoxicity.

Comparative Inhibitory Effects of Natural Biflavones from Ginkgo against Human CYP1B1 in Recombinant Enzymes and MCF-7 Cells.

Human cytochrome P450 1B1 (CYP1B1) is an extrahepatic enzyme overexpressed in many tumors and associated with angiogenesis. Ginkgetin, isoginkgetin, sciadopitysin, and amentoflavone, the primary biflavones found in Ginkgo biloba, have excellent anti-inflammatory and anti-tumor effects. However, the effect of biflavones on CYP1B1 activities remains unknown. In this study, 7-ethoxyresorufin O-deethylation (EROD) was used to characterize the activities of CYP1 families. The impacts of four ginkgo biflavones on CYP1B1 activity and the cellular protein expression of CYP1B1 were systematically investigated. The results showed that amentoflavone with six hydroxyl substituents exhibited the most potent selective inhibitory effect on CYP1B1 activity with IC50 of 0.054 µM in four biflavones. Sciadopitysin, with three hydroxyl and three methoxy substituents, had the weakest inhibitory activity against CYP1B1. Ginkgetin and isoginkgetin, both with four hydroxyl and two methoxy substituents, showed similar inhibitory intensity towards CYP1B1 with IC50 values of 0.289 and 0.211 µM, respectively. Kinetic analysis showed that ginkgetin and amentoflavone inhibited CYP1B1 in a non-competitive mode, whereas sciadopitysin and isoginkgetin induced competitive or mixed types of inhibition. Notably, four ginkgo biflavones were also confirmed to suppress the protein expressions of CYP1B1 and AhR in MCF-7. Furthermore, molecular docking studies indicated more hydrogen bonds formed between amentoflavone and CYP1B1, which might explain the strongest inhibitory action towards CYP1B1. In summary, these findings suggested that biflavones remarkably inhibited both the activity and protein expression of CYP1B1 and the inhibitory activities enhanced with the increasing hydroxyl substitution, providing new insights into the anti-tumor potentials of biflavones.

Bioactive Compounds From Coptidis Rhizoma Alleviate Pulmonary Arterial Hypertension by Inhibiting Pulmonary Artery Smooth Muscle Cells' Proliferation and Migration.

ABSTRACT: Pulmonary arterial hypertension (PAH) is a devastating disorder characterized by excessive proliferation and vasoconstriction of small pulmonary artery vascular smooth muscle cells (PASMCs). Coptidis rhizoma (CR) because of the complexity of the components, the underlying pharmacological role and mechanism of it on PAH remains unknown. In this article, the network pharmacological analysis was used to screen the main active constituents of CR and the molecular targets that these constituents act on. Then, we evaluated the importance of berberine and quercetin (biologically active components of CR) on the proliferation and migration of PASMCs and vascular remodeling in experimental models of PAH. Our results showed that berberine and quercetin effectively inhibited the proliferation and migration of hypoxia-induced PASMCs in a manner likely to be mediated by the suppression of MAPK1, NADPH oxidase 4 (NOX4), and cytochrome P450 1B1 (CYP1B1) expression. Furthermore, berberine and quercetin treatment attenuates pulmonary hypertension, reduces right ventricular hypertrophy, and improves pulmonary artery remodeling in monocrotaline-induced pulmonary hypertension in rat models. In conclusion, this research demonstrates CR might be a promising treatment option for PAH, and the network pharmacology approach can be an effective tool to reveal the potential mechanisms of Chinese herbal medicine.

Role of the Aryl Hydrocarbon Receptor (AhR) in Mediating the Effects of Coffee in the Colon.

SCOPE: This study investigates the mechanism of action and functional effects of coffee extracts in colonic cells, on intestinal stem cell growth, and inhibition of dextran sodium sulfate (DSS)-induced intestinal barrier damage in mice. METHODS AND RESULTS: Aqueous coffee extracts induced Ah receptor (AhR) -responsive CYP1A1, CYP1B1, and UGT1A1 gene expression in colon-derived Caco2 and YAMC cells. Tissue-specific AhR knockout (AhRf/f x Lgr5-GFP-CreERT2 x Villin-Cre), wild-type (Lgr5-CreERT2 x Villin-Cre) mice are sources of stem cell enriched organoids and both coffee extracts and norharman, an AhR-active component of these extracts inhibited stem cell growth. Coffee extracts also inhibit DSS-induced damage to intestinal barrier function and DSS-induced mucosal inflammatory genes such as IL-6 and TGF-ß1 in wild-type (AhR+/+ ) but not AhR-/- mice. In contrast, coffee does not exhibit protective effects in intestinal-specific AhR knockout mice. Coffee extracts also enhanced overall formation of AhR-active microbial metabolites. CONCLUSIONS: In colon-derived cells and in the mouse intestine, coffee induced several AhR-dependent responses including gene expression, inhibition of intestinal stem cell-enriched organoid growth, and inhibition of DSS-induced intestinal barrier damage. We conclude that the anti-inflammatory effects of coffee in the intestine are due, in part, to activation of AhR signaling.

Cumin Prevents 17ß-Estradiol-Associated Breast Cancer in ACI Rats.

Breast cancer (BC) is a leading cause of cancer deaths in women in less developed countries and the second leading cause of cancer death in women in the U.S. In this study, we report the inhibition of E2-mediated mammary tumorigenesis by Cuminum cyminum (cumin) administered via the diet as cumin powder, as well as dried ethanolic extract. Groups of female ACI rats were given either an AIN-93M diet or a diet supplemented with cumin powder (5% and 7.5%, w/w) or dried ethanolic cumin extract (1%, w/w), and then challenged with subcutaneous E2 silastic implants (1.2 cm; 9 mg). The first appearance of a palpable mammary tumor was significantly delayed by both the cumin powder and extract. At the end of the study, the tumor incidence was 96% in the control group, whereas only 55% and 45% animals had palpable tumors in the cumin powder and extract groups, respectively. Significant reductions in tumor volume (660 ± 122 vs. 138 ± 49 and 75 ± 46 mm3) and tumor multiplicity (4.21 ± 0.43 vs. 1.16 ± 0.26 and 0.9 ± 0.29 tumors/animal) were also observed by the cumin powder and cumin extract groups, respectively. The cumin powder diet intervention dose- and time-dependently offset E2-related pituitary growth, and reduced the levels of circulating prolactin and the levels of PCNA in the mammary tissues. Mechanistically, the cumin powder diet resulted in a significant reversal of E2-associated modulation in ERα, CYP1A1 and CYP1B1. Further, the cumin powder diet reversed the expression levels of miRNAs (miR-182, miR-375, miR-127 and miR-206) that were highly modulated by E2 treatment. We analyzed the composition of the extract by GC/MS and established cymene and cuminaldehyde as major components, and further detected no signs of gross or systemic toxicity. Thus, cumin bioactives can significantly delay and prevent E2-mediated mammary tumorigenesis in a safe and effective manner, and warrant continued efforts to develop these clinically translatable spice bioactives as chemopreventives and therapeutics against BC.

Thirty days oral Aframomum melegueta extract elicited analgesic effect but influenced cytochrome p4501BI, cardiac troponin T, testicular alfa-fetoprotein and other biomarkers in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Pain is the commonest symptom of a disease and the percentage of persons manifesting one form of pain is growing globally. Aframomum melegueta (AM) is commonly used by traditional doctors as medication for many ailments such as body pains and rheumatism because it possesses anti-inflammatory, anti-allergenic, antiviral, anti-ageing and anti-tumour phytochemical agents. AIM OF THE STUDY: Traditionally a botanical remedy in the management of pain was assessed. A common tropical plant Aframomum melegueta (AM) was evaluated for the amelioration of pain. For further pharmacologic understanding sensitive marker were used to assess the effect of the extract on the organ as a multifaceted approach to the evaluation of safety and analgesic efficacy. MATERIALS AND METHOD: Sensitive biomarkers such as troponin-T (CTnT), cardiac troponin-I (CTnI), interleukin-beta (IL-ß), interleukin-6 (IL-6), tumor necrosis factor-alfa (TNF-α) were evaluated using the enzyme-linked immunosorbent assay (ELISA) method and electrocardiographic parameters were also evaluated. The dynamics of concentrations of the various subfamilies of cytochrome were also assessed using ELISA in the evaluation of thirty-day oral AM, while histopathological changes of organs were also observed. RESULTS: Thirty-day oral AM doses 40 mg/kg and 80 mg/kg showed analgesic potential but influenced IL-6 level, IL-1ß, TNF-α and P-LCR. Electrocardiographic parameters showed the extract had arrhythmogenic effects the other cardiac parameters influenced was CTnT. The testicular alfa-fetoprotein and prostate specific antigen were also influenced. There were also some histopathological changes. CONCLUSIONS: The extract showed analgesic, anti-oxidant and anti-inflammatory potential with possible adverse effects consistent with testicular and prostate cancers, cardiovascular complication, hepatic congestion and cholestasis.

Overexpression of MECP2 attenuates cigarette smoke extracts induced lung epithelial cell injury by promoting CYP1B1 methylation.

MECP2 (Methyl-CpG-binding protein 2) has been shown to have a critical role in regulating DNA methylation against smoke exposed lung injury. However, the biological function of MECP2 and the underlying molecular mechanism remains elusive. Human bronchial epithelial (16HBE) and alveolar type II epithelial cells (AECII) were exposed to increasing concentrations of cigarette smoke extracts (CSE) solution to establish CSE-induced lung epithelial cell injury models. Our findings revealed that MECP2 was down-regulated, while CYP1B1 was up-regulated in CSE-induced lung epithelial cell injury models by quantitative real time PCR, western blotting and immunofluorescence staining. Down-regulated CYP1B1 was ascribed to the demethylation of its promoter by methylation-specific PCR (MSP). The in vitro experiments further showed that MECP2 overexpression significantly attenuated CSE-triggered cell growth attenuation, cell cycle arrest, apoptosis and ROS generation in lung epithelial cells by CCK-8 and flow cytometry assays. In molecular level, we further demonstrated that MECP2 overexpression obviously suppressed the expression of CYP1B1 through enhancing DNA methylation. Therefore, our data suggest that MECP2 protects against CSE-induced lung epithelial cell injury possibly through down-regulating CYP1B1 expression via elevating its methylation status.

Carvedilol serves as a novel CYP1B1 inhibitor, a systematic drug repurposing approach through structure-based virtual screening and experimental verification.

Cytochrome P450 1B1 (CYP1B1) is a promising target for prevention and therapy of cancer, particularly those with drug resistance, stimulating cancer cell survival, and promoting cancer resistance. In view of the extreme complexity and high risk in drug discovery and development, a drug repurposing strategy was applied in the present study to find potential CYP1B1 inhibitors through structure-based virtual screening in the FDA database. Intriguingly, after a thorough assessment of docking scores, binding affinities, as well as binding modes, six compounds were highlighted for further verification. In fact, both carvedilol and indacaterol showed inhibitory activity towards human CYP1B1 with the IC50 of 1.11 µM and 59.52 µM, respectively, according to EROD assay; however, neither docking score nor the detailed binding mode of carvedilol in the hit pose dictated to be a superior CYP1B1 inhibitor to indacaterol, which called for the necessity to re-access the binding mode of carvedilol. Thus, the top two representative docking poses of carvedilol were re-assessed. Indeed, compared to the one hit in the virtual screening (due to a false positive Glide gscore), the other docking pose exhibited ideal performance in both molecular dynamics (MD) simulation, binding free energy, and density functional theory (DFT) calculation evaluations. This identification of the exact binding pose of carvedilol is not only essential for a better understanding of the mechanism underlying its activity, but also contributes to uncovering the structure-activity relationship of CYP1B1 inhibitors. Of note, carvedilol exhibited direct cytotoxicity against both human lung adenocarcinoma epithelial cell line A459 and its Taxol-resistant subline (A549/Taxol). In particular, it showed superior toxicity towards A549/Taxol cells that overexpressed CYP1B1, which further supported its potential to be an effective CYP1B1 inhibitor.

Ultraviolet A light induces DNA damage and estrogen-DNA adducts in Fuchs endothelial corneal dystrophy causing females to be more affected.

Fuchs endothelial corneal dystrophy (FECD) is a leading cause of corneal endothelial (CE) degeneration resulting in impaired visual acuity. It is a genetically complex and age-related disorder, with higher incidence in females. In this study, we established a nongenetic FECD animal model based on the physiologic outcome of CE susceptibility to oxidative stress by demonstrating that corneal exposure to ultraviolet A (UVA) recapitulates the morphological and molecular changes of FECD. Targeted irradiation of mouse corneas with UVA induced reactive oxygen species (ROS) production in the aqueous humor, and caused greater CE cell loss, including loss of ZO-1 junctional contacts and corneal edema, in female than male mice, characteristic of late-onset FECD. UVA irradiation caused greater mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) damage in female mice, indicative of the sex-driven differential response of the CE to UVA, thus accounting for more severe phenotype in females. The sex-dependent effect of UVA was driven by the activation of estrogen-metabolizing enzyme CYP1B1 and formation of reactive estrogen metabolites and estrogen-DNA adducts in female but not male mice. Supplementation of N-acetylcysteine (NAC), a scavenger of reactive oxygen species (ROS), diminished the morphological and molecular changes induced by UVA in vivo. This study investigates the molecular mechanisms of environmental factors in FECD pathogenesis and demonstrates a strong link between UVA-induced estrogen metabolism and increased susceptibility of females for FECD development.

Antiproliferative effect of 2-Hydroxy-6-tridecylbenzoic acid from ginkgo biloba sarcotestas through the aryl hydrocarbon receptor pathway in triple-negative breast cancer cells.

This study aims to isolate the potential antiproliferative and cytotoxic compounds from ginkgo biloba sarcotestas (GBS) and investigates the underlying mechanism in human MDA-MB-231 and mouse 4T-1 triple-negative breast cancer cells. Our results showed that 2-Hydroxy-6-tridecylbenzoic acid was isolated by cytotoxicity-guided fractionation where different fractions were assessed using MTT assay against MDA-MB-231 and 4T-1 cells. Colony formation assay showed that 2-Hydroxy-6-tridecylbenzoic acid significantly inhibited cell proliferation. The inhibition was associated with the enhancement of cytochrome P450 (CYP) 1B1 expression in a dose- and time-dependent manner and no significant change of CYP1A1 expression by qPCR and Western blot assays in MDA-MB-231 and 4T-1 cells. The mechanism was further demonstrated by the activation of aryl hydrocarbon receptor (AhR) pathway with the upregulation of AhR, AhR nuclear translocator (ARNT) and AhR-dependent xenobiotic response elements (XRE) activity. These findings may have implications for development of anticancer agents containing 2-Hydroxy-6-tridecylbenzoic acid as functional additives.

Nitidine Chloride-Induced CYP1 Enzyme Inhibition and Alteration of Estradiol Metabolism.

The cytochrome P450 (P450) 1 family is an important phase I enzyme involved in carcinogen activation. Nitidine chloride (NC) is a pharmacologically active alkaloid with polyaromatic hydrocarbon found in the roots of Zanthoxylum nitidum (Roxb.) DC, a traditional medicinal herb widely used in China. We examined the inhibitory effects of NC on CYP1A1, 1B1, and 1A2. NC significantly inhibited CYP1A1- and 1B1-catalyzed ethoxyresorufin O-deethylation activity (IC50 = 0.28 ± 0.06 and 0.32 ± 0.02 µM, respectively) in a concentration-dependent manner, but only showed slight inhibition of CYP1A2 activity (IC50 > 50 µM). Kinetic analysis revealed that NC competitively inhibited CYP1B1 with a K i value of 0.47 ± 0.05 µM, whereas NC caused a mixed type of inhibition on CYP1A1 with K i and K I values of 0.14 ± 0.04 and 0.19 ± 0.09 µM, respectively. The observed enzyme inhibition neither required NADPH nor revealed time dependency. Molecular docking manifested the generation of strong hydrogen-bonding interactions of Ser116 in CYP1A1 and Ser127 in CYP1B1 with methoxy moiety of NC. Additionally, NC-induced alteration of estradiol (E2) metabolism was also investigated in the present study. Hydroxyestradiols, including 2-hydroxyestradiol [(2-OHE2) nontoxic] and 4-hydroxyestradiol [(4-OHE2) genotoxic] generated in recombinant enzyme incubation systems and cultured MCF-7 cells were analyzed, and NC was found to preferentially inhibit the nontoxic 2-hydroxylation activity of E2 mediated by CYP1A1. In conclusion, NC was a mixed type inhibitor of CYP1A1 and a competitive inhibitor of CYP1B1. The remarkable inhibition on E2 2-hydroxylation might increase the risk of 4-OHE2-induced genotoxicity. SIGNIFICANCE STATEMENT: CYP1 enzymes catalyze oxidative metabolism of a variety of compounds and are known to play a crucial role in the development of cancer. CYP1A1 and CYP1A2 are responsible for hydroxylation of estradiol (E2) at the C-2 position, resulting in the formation of 2-OHE2, which is proposed to be a detoxification pathway. However, CYP1B1-mediated hydroxylation of E2 at the C-4 position has been suggested to be a tumor initiator. The present study found that nitidine chloride is a mixed type inhibitor of CYP1A1 and a competitive inhibitor of CYP1B1. NC preferentially inhibited the nontoxic E2 2-hydroxylation pathway mediated by CYP1A1, which might increase the risk of 4-OHE2-induced genotoxicity and cause severe drug-drug interactions.

Potential of Teucrium chamaedrys L. to modulate apoptosis and biotransformation in colorectal carcinoma cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Teucrum chamaedrys L. is one of the known medicinal plants, useful for treatment of various health problems, especially digestive. In this study, we investigated methanol, ethyl-acetate and acetone extracts of T. chamaedrys in respect to their anticancer properties in SW480 colorectal cancer cells. MATERIALS AND METHODS: Cytotoxicity and proapoptotic potential were assessed by MTT cell viability assay and AO/EB double staining. Molecular mechanisms of induced apoptosis were determined by monitoring Fas receptor protein expression through immunofluorescence, Caspase 8 and 9 activity, as well as concentrations of O2.- spectrophotometrically. Additionally, mRNA expression of biotransformation enzymes (CYP1A1, CYP1B1, GSTP1) and membrane transporters (MRP1 and MRP2) involved in drug resistance were investigated by qPCR method. Qualitative analysis of individual phenolic compounds was performed by reversed phase HPLC-MS analysis. RESULTS: Methanol extract shows the best cytotoxicity and selectivity compared to ethyl-acetate and acetone extracts, mainly causing apoptosis of SW480 cells, without affecting normal HaCaT keratinocytes. The increased expression of Fas receptor protein and caspase 8 activity indicate that the death receptor-mediated pathway plays a crucial role in the observed apoptosis. The increased caspase 9 activity and O2.- concentration suggest that mitochondria are also involved in the apoptosis. T. chamaedrys methanol extract inhibits mRNA expression of CYP1A1, CYP1B1, GSTP1, MRP1 and MRP2 in SW480 cells. CONCLUSIONS: Induction of apoptosis and inhibition of CYP1A1, CYP1B1, GSTP1, MRP1 and MRP2 mRNA expression implies that T. chamaedrys can serve as a valuable source of bioactive compounds as dietary supplements or selective anticancer agents, with the ability to induce apoptosis and modulate drug resistance in colorectal cancer cells.

Rooibos (Aspalathus linearis) and honeybush (Cyclopia species) modulate the oxidative stress associated injury of diesel exhaust particles in human umbilical vein endothelial cells.

BACKGROUND: Previous evidence show foods and beverages rich in polyphenolic compounds to have favourable effects on the cardiovascular system. HYPOTHESIS: The current study assessed the modulation of oxidative stress and associated inflammation induced by diesel exhaust particles (DEP - SRM 2975) by pre-treatment of human umbilical vein endothelial cells (HUVECs) with aqueous extracts of rooibos [fermented (FR) as well as green form (GR)] and honeybush [fermented form (FH)]. STUDY DESIGN: HUVEC are either exposed to DEP (10 µg/ml) for 4 h or pre-treated with 40 and 60 µg/ml of FR or GH or FR, or 50 µg/ml orientin (OR) for 6 h prior to DEP exposure. METHODS: In vitro antioxidant capacity of the extracts was assessed and the polyphenol contents were also assessed by HPLC. ROS, cell viability, lactate dehydrogenase leakage, lipid peroxidation, GSH:GSSG ratios, conjugated diene and protein carbonyl levels were determined as indices of oxidative stress and cytotoxicity. RT-qPCR and western blot were used to assess inflammatory cytokines and antioxidant genes expression. RESULTS: DEP caused a dose and time-dependent increase in ROS production, significant (p < 0.001) increase in protein carbonyl (PC) formation, thiobarbituric acid reactive substances and conjugated dienes levels (p < 0.01) and a significant reduction in glutathione (GSH) redox status. Pre-incubation with either the herbal extracts or orientin attenuated these effects. The significant increase in IL-1α, IL-6, IL-8, VCAM-1 and ATF4 gene expression caused by DEP (10 µg/ml) were also attenuated by the presence of the FR, GR and FH extracts, and OR . Pre-treatment with the rooibos extracts or flavone orientin enhanced cell viability, reduced LDH leakage, enhanced mRNA expression of NQO1 and Nrf2, but repressed CYP1B1 mRNA induced by DEP. Western blot showed both the herbal tea extracts and orientin to enhance NQO1 and γGSC protein induction by DEP. CONCLUSION: Taken together, the herbal extracts offer protection against DEP-induced oxidative stress and inflammatory response.

Oroxylin A, a methylated metabolite of baicalein, exhibits a stronger inhibitory effect than baicalein on the CYP1B1-mediated carcinogenic estradiol metabolite formation.

Human cytochrome P450 1B1 (CYP1B1)-mediated formation of 4-hydroxyestradiol (4-OHE2) from 17ß-estradiol plays an important role in the progression of human breast cancer, while the biotransformation of 17ß-estradiol to 2-hydroxyestradiol mediated by cytochrome P450 1A1 (CYP1A1) is considered as a less harmful pathway. In this study, inhibitory effects of flavonoids baicalein and oroxylin A, a metabolite of baicalein in human body, on CYP1A1 and 1B1 activities were investigated in vitro. The inhibition intensities of baicalein and oroxylin A towards CYP1B1 were greater than towards CYP1A1 with a mixed mechanism. In addition, oroxylin A showed a stronger inhibitory effect than baicalein towards the CYP1B1-mediated 17ß-estradiol 4-hydroxylation, with the IC50 values of 0.0146 and 2.27 µM, respectively. Docking studies elucidated that oroxylin A had a stronger binding affinity than baicalein for CYP1B1. In MCF-7 cells, compared with baicalein-treated groups, oroxylin A with lower doses decreased and increased the formation of 4-OHE2 and 2-hydroxyestradiol, respectively, with a preferential induction of mRNA of CYP1A1 over CYP1B1. In conclusion, this study demonstrated that oroxylin A showed a stronger inhibitory effect than baicalein on CYP1B1-mediated 4-OHE2 formation in MCF-7 cells, providing crucial implications for their possibly preventive/therapeutic potential against breast cancer via inhibition of CYP1B1, particularly of oroxylin A.

Phytoestrogens and their synthetic analogues as substrate mimic inhibitors of CYP1B1.

Phytoestrogens are class of natural compounds that shares structural similarity with estrogen and has the capacity to alter the fertilization in mammals. Till early 1990s, the natural phytoestrogens as well as their synthetic analogues were explored for their fertility modulating activity. During late 1990s, two findings renewed the interest on phytoestrogens as means to control hormone induced cancer: (i) revelation of overexpression of CYP1B1 in breast & ovarian cancer and (ii) protection offered by alphanapthoflavone (ANF) against hormone induced cancer. The objective of the review is to summarize the CYP1B1 inhibitory activity of phytoestrogens and their synthetic analogues reported till date. This review is an attempt to classify phytoestrogens and their synthetic analogues on their chemical architecture rather than simply by their chemical class (flavones, stilbenes etc.). This provides a broader sense to cluster many chemical classes under a particular chemical architecture/framework. Accordingly, we divided the phytoestrogen into three different classes based on two aryl groups (Ar) separated by linker (X), which may be either cyclic (c) or linear (l). The number in subscript to X denotes number of atoms: (i) Ar-cX4-Ar, (ii) Ar-lX3-Ar and (iii) Ar-lX2-Ar. This provides an opportunity to cluster flavones, quinolines and quinazolinones under Ar-cX4-Ar class, while biphenyl ureas and chalcones under Ar-lX3-Ar class. We believe in coming years many chemical scaffolds may be clustered under this framework.

Evidence for Chemopreventive and Resilience Activity of Licorice: Glycyrrhiza Glabra and G. Inflata Extracts Modulate Estrogen Metabolism in ACI Rats.

Women are increasingly using botanical dietary supplements (BDS) to reduce menopausal hot flashes. Although licorice (Glycyrrhiza sp.) is one of the frequently used ingredients in BDS, the exact plant species is often not identified. We previously showed that in breast epithelial cells (MCF-10A), Glycyrrhiza glabra (GG) and G. inflata (GI), and their compounds differentially modulated P450 1A1 and P450 1B1 gene expression, which are responsible for estrogen detoxification and genotoxicity, respectively. GG and isoliquiritigenin (LigC) increased CYP1A1, whereas GI and its marker compound, licochalcone A (LicA), decreased CYP1A1 and CYP1B1 The objective of this study was to determine the distribution of the bioactive licorice compounds, the metabolism of LicA, and whether GG, GI, and/or pure LicA modulate NAD(P)H quinone oxidoreductase (NQO1) in an ACI rat model. In addition, the effect of licorice extracts and compounds on biomarkers of estrogen chemoprevention (CYP1A1) as well as carcinogenesis (CYP1B1) was studied. LicA was extensively glucuronidated and formed GSH adducts; however, free LicA as well as LigC were bioavailable in target tissues after oral intake of licorice extracts. GG, GI, and LicA caused induction of NQO1 activity in the liver. In mammary tissue, GI increased CYP1A1 and decreased CYP1B1, whereas GG only increased CYP1A1 LigC may have contributed to the upregulation of CYP1A1 after GG and GI administration. In contrast, LicA was responsible for GI-mediated downregulation of CYP1B1 These studies highlight the polypharmacologic nature of botanicals and the importance of standardization of licorice BDS to specific Glycyrrhiza species and to multiple constituents.

The citrus flavonone hesperetin attenuates the nuclear translocation of aryl hydrocarbon receptor.

The environmental polycyclic aromatic hydrocarbons (PAH) and dioxins are carcinogens and their adverse effects have been largely attributed to the activation of AhR. Hesperetin is a flavonone found abundantly in citrus fruits and has been shown to be a biologically active agent. In the present study, the effect of hesperetin on the nuclear translocation of AhR and the downstream gene expression was investigated in MCF-7 cells. Confocal microscopy indicated that 7, 12-dimethylbenz[α]anthracene (DMBA) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) -induced nuclear translocation of AhR was deterred by hesperetin treatment. The reduced nuclear translocation could also be observed in Western analysis. Reporter-gene assay further illustrated that the induced XRE transactivation was weakened by the treatment of hesperetin. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay demonstrated that the gene expressions of CYP1A1, 1A2, and 1B1 followed the same pattern of AhR translocation. These results suggested that hesperetin counteracted AhR transactivation and suppressed the downstream gene expression.

Quercetin attenuates the hyperoxic lung injury in neonatal mice: Implications for Bronchopulmonary dysplasia (BPD).

Quercetin (QU) is one of the most common flavonoids that are present in a wide variety of fruits, vegetables, and beverages. This compound possesses potent anti-inflammatory and anti-oxidant properties. Supplemental oxygen is routinely administered to premature infants with pulmonary insufficiency. However, hyperoxia is one of the major risk factors for the development of bronchopulmonary dysplasia (BPD), which is also termed chronic lung disease in premature infants. Currently, no preventive approaches have been reported against BPD. The treatment of BPD is notably limited to oxygen administration, ventilatory support, and steroids. Since QU has been shown to be effective in reducing inflammation and oxidative stress in various disease models, we hypothesized that the postnatal QU treatment of newborn mice will protect against hyperoxic lung injury by the upregulation of the phase I (CYP1A/B) and/or phase II, NADPH quinone reductase enzymes. Newborn C57BL/6J mice within 24 h of birth with the nursing dams were exposed to either 21% O2 (air) and/or 85% O2 (hyperoxia) for 7 days. The mice were treated, intraperitoneally (i.p.) once every other day with quercetin, at a concentration of 20 mg/kg, or saline alone from postnatal day (PND) 2-6. The mice were sacrificed on day 7, and lung and liver tissues were collected. The expression levels of CYP1A1, CYP1B1, NQO1 proteins and mRNA as well as the levels of MDA-protein adducts were analyzed in lung and liver tissues. The findings indicated that QU attenuated hyperoxia-mediated lung injury by reducing inflammation and improving alveolarization with decreased number of neutrophil and macrophage infiltration. The attenuation of this lung injury correlated with the upregulation of CYP1A1/CYP1B1/NQO1 mRNA, proteins and the down regulation of NF-kB levels and MDA-protein adducts in lung and liver tissues. The present study demonstrated the potential therapeutic value of quercetin in the prevention and/or treatment of BPD.

Glycyrrhiza glabra extract and quercetin reverses cisplatin resistance in triple-negative MDA-MB-468 breast cancer cells via inhibition of cytochrome P450 1B1 enzyme.

The development of multi-drug resistance to existing anticancer drugs is one of the major challenges in cancer treatment. The over-expression of cytochrome P450 1B1 enzyme has been reported to cause resistance to cisplatin. With an objective to discover cisplatin-resistance reversal agents, herein, we report the evaluation of Glycyrrhiza glabra (licorice) extracts and its twelve chemical constituents for inhibition of CYP1B1 (and CYP1A1) enzyme in Sacchrosomes and live human cells. The hydroalcoholic extract showed potent inhibition of CYP1B1 in both Sacchrosomes as well as in live cells with IC50 values of 21 and 16 µg/mL, respectively. Amongst the total of 12 constituents tested, quercetin and glabrol showed inhibition of CYP1B1 in live cell assay with IC50 values of 2.2 and 15 µM, respectively. Both these natural products were found to be selective inhibitors of CYP1B1, and does not inhibit CYP2 and CYP3 family of enzymes (IC50 > 20 µM). The hydroalcoholic extract of G. glabra and quercetin (4) showed complete reversal of cisplatin resistance in CYP1B1 overexpressing triple negative MDA-MB-468 breast cancer cells. The selective inhibition of CYP1B1 by quercetin and glabrol over CYP2 and CYP3 family of enzymes was studied by molecular modeling studies.

Red Clover Aryl Hydrocarbon Receptor (AhR) and Estrogen Receptor (ER) Agonists Enhance Genotoxic Estrogen Metabolism.

Many women consider botanical dietary supplements (BDSs) as safe alternatives to hormone therapy for menopausal symptoms. However, the effect of BDSs on breast cancer risk is largely unknown. In the estrogen chemical carcinogenesis pathway, P450 1B1 metabolizes estrogens to 4-hydroxylated catechols, which are oxidized to genotoxic quinones that initiate and promote breast cancer. In contrast, P450 1A1 catalyzed 2-hydroxylation represents a detoxification pathway. The current study evaluated the effects of red clover, a popular BDS used for women's health, and its isoflavones, biochanin A (BA), formononetin (FN), genistein (GN), and daidzein (DZ), on estrogen metabolism. The methoxy estrogen metabolites (2-MeOE1, 4-MeOE1) were measured by LC-MS/MS, and CYP1A1 and CYP1B1 gene expression was analyzed by qPCR. Nonmalignant ER-negative breast epithelial cells (MCF-10A) and ER-positive breast cancer cells (MCF-7) were derived from normal breast epithelial tissue and ER+ breast cancer tissue. Red clover extract (RCE, 10 µg/mL) and isoflavones had no effect on estrogen metabolism in MCF-10A cells. However, in MCF-7 cells, RCE treatments downregulated CYP1A1 expression and enhanced genotoxic metabolism (4-MeOE1/CYP1B1 > 2-MeOE1/CYP1A1). Experiments with the isoflavones showed that the AhR agonists (BA, FN) preferentially induced CYP1B1 expression as well as 4-MeOE1. In contrast, the ER agonists (GN, DZ) downregulated CYP1A1 expression likely through an epigenetic mechanism. Finally, the ER antagonist ICI 182,780 potentiated isoflavone-induced XRE-luciferase reporter activity and reversed GN and DZ induced downregulation of CYP1A1 expression. Overall, these studies show that red clover and its isoflavones have differential effects on estrogen metabolism in "normal" vs breast cancer cells. In breast cancer cells, the AhR agonists stimulate genotoxic metabolism, and the ER agonists downregulate the detoxification pathway. These data may suggest that especially breast cancer patients should avoid red clover and isoflavone based BDSs when making choices for menopausal symptom relief.