Metabolism Of Estradiol-17β Research Articles

Abstract LB296: Human CYP1B1 enhances cancer progression through induction of Sp1 via DNMT-mediated epigenetic regulation of miR-375

Abstract Human cytochrome P450 1B1 (CYP1B1) is a major catalytic enzyme for metabolism of 17β-estradiol (E2) to catechol estrogens, mainly carcinogenic metabolite 4-hydroxyestradiol (4-OHE2). To identify the unknown molecular mechanism for cancer progression induced by CYP1B1, we studied the role of CYP1B1 in human tumor cells, MCF-7, MDA-MB-231, and HeLa cells. Suppression of CYP1B1 by shRNA significantly reduced cancer cell viability and invasion. Clinical data from cancer patients showed that high level of CYP1B1 expression resulted in relatively lower survival rate. Interestingly, overexpression of CYP1B1 markedly activated Wnt/β-catenin signaling and EMT process, and suppression of specificity protein 1 (Sp1) by siRNA, an well-known transcription factor, demolished the promoting effects of CYP1B1 on cancer progression. To investigate the correlation between CYP1B1 and Sp1, we analyzed the clinical data from breast and cervical cancer patients and the results showed the positive correlation between two genes. Furthermore, TMS (tetramethoxystilbene), a specific CYP1B1 inhibitor, diminished the induction of Sp1 by CYP1B1, and 4-OHE2 exceedingly promoted the expression level of Sp1. Surprisingly, miR-375, known as an oncogenic microRNA, substantially decreased by 4-OHE2 and CYP1B1 overexpression in CYP1B1-transgenic (TG) mice. Methylation specific assay revealed that CYP1B1 suppressed miR-375 expression through induction of hyper-methylation on pre-miR-375. In addition, epigenetic regulation of miR-375 by CYP1B1 was executed via induction of DNA methyltransferases (DNMTs), DNMT1, 3a, and 3b. Enhanced expression level of Sp1 by 4-OHE2 was significantly deteriorated by 5-aza-dC, a DNMT inhibitor. Taken together, our data suggest that CYP1B1 enhances cancer progression through induction of cancer cell proliferation and invasion via DNMT-mediated epigenetic regulation of miR-375 and further promotion of Sp1 expression. Citation Format: Young-Jin Chun, Yeo-Jung Kwon, Tae-Uk Kwon. Human CYP1B1 enhances cancer progression through induction of Sp1 via DNMT-mediated epigenetic regulation of miR-375 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB296.

Impact of Human SULT1E1 Polymorphisms on the Sulfation of 17β-Estradiol, 4-Hydroxytamoxifen, and Diethylstilbestrol by SULT1E1 Allozymes.

Previous studies have revealed that sulfation, as mediated by the estrogen-sulfating cytosolic sulfotransferase (SULT) SULT1E1, is involved in the metabolism of 17β-estradiol (E2), 4-hydroxytamoxifen (4OH-tamoxifen), and diethylstilbestrol in humans. It is an interesting question whether the genetic polymorphisms of SULT1E1, the gene that encodes the SULT1E1 enzyme, may impact on the metabolism of E2 and these two drug compounds through sulfation. In this study, five missense coding single nucleotide polymorphisms of the SULT1E1 gene were selected to investigate the sulfating activity of the coded SULT1E1 allozymes toward E2, 4OH-tamoxifen, and diethylstilbestrol. Corresponding cDNAs were generated by site-directed mutagenesis, and recombinant SULT1E1 allozymes were bacterially expressed, affinity-purified, and characterized using enzymatic assays. Purified SULT1E1 allozymes were shown to display differential sulfating activities toward E2, 4OH-tamoxifen, and diethylstilbestrol. Kinetic analysis revealed further distinct Km (reflecting substrate affinity) and Vmax (reflecting catalytic activity) values of the five SULT1E1 allozymes with E2, 4OH-tamoxifen, and diethylstilbestrol as substrates. Taken together, these findings highlighted the significant differences in E2-, as well as the drug-sulfating activities of SULT1E1 allozymes, which may have implications in the differential metabolism of E2, 4OH-tamoxifen, and diethylstilbestrol in individuals with different SULT1E1 genotypes.

Abstract 4668: Human CYP1B1 prevents TRAIL-DR4-mediated cancer cell apoptosis through downregulation of miR-375

Abstract Human cytochrome P450 1B1 (CYP1B1) is an important enzyme that catalyzes metabolism of 17β-estradiol (E2) to catechol estrogens, including carcinogenic metabolite 4-hydroxyestradiol (4-OHE2). CYP1B1 may play a crucial role in estrogen-mediated carcinogenesis since CYP1B1 shows tumor-specific expression pattern in various hormone-responsive tissues. However, the detailed mechanism of CYP1B1-dependent carcinogenesis remains still unclear. Here, we investigated the association between TRAIL, DR4, and CYP1B1 expression in human tumor cells, including MCF-7, MDA-MB-231, and HeLa cells. CYP1B1 can inhibit the expression and release of TRAIL simultaneously with the expression of the death receptor DR4/5, known as TRAIL receptors, to prevent apoptosis. Furthermore, we found that CYP1B1 represses the transcription of DR4 by inducing DNA methylation in the promoter region of DR4. In addition, we revealed that CYP1B1 regulates TRAIL and DR4 through activation of Sp1 transcription factor. CYP1B1 and its enzymatic product, 4-OHE2, promote Sp1 expression levels in both mRNA and protein levels. Interestingly, CYP1B1 elevated Sp1 through downregulation of miR-375, which is a candidate tumor suppressive microRNA and transcriptional inhibitor of Sp1. Taken together, our data suggest that CYP1B1 inhibits TRAIL-DR4-mediated cancer cell apoptosis through induction of Sp1 through repression of miR-375, which promotes cancer cell survival. Citation Format: Yeo-Jung Kwon, Dong-Jin Ye, Huoung-Seok Baek, Sangyun Shin, Tae-Uk Kwon, Eunah Cho, Hong-Gyu Ahn, Youngjin Chun. Human CYP1B1 prevents TRAIL-DR4-mediated cancer cell apoptosis through downregulation of miR-375 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4668.

Isolation of Trametes hirsuta La-7 with high laccase-productivity and its application in metabolism of 17β-estradiol

Estrogens, which are extensive in the eco-environments, are a category of high-toxic emerging contaminants that induce metabolic disorders and even carcinogenic risks in wildlife and humans. Here we investigate whether fungus-secreted laccase can be used as a green catalyst to eliminate a representative estrogen, 17β-estradiol (E2). A white-rot fungus Trametes hirsuta La-7 with high laccase-productivity, was isolated from pig manure-contaminated soil. Extracellular laccase activity expressed by strain La-7 was 65.4 U·mL−1 for a 3 d inoculation under the optimal fermentation parameters. The concentrated-crude laccase from Trametes hirsuta La-7 (CC-ThLac) was capable of effectively metabolizing E2 at pH 4–6, and the apparent pseudo first-order reaction rate constant and half-life values were respectively 0.027–0.055 min−1 and 25.86–12.67 min (R2 > 0.98). The mass measurement of high-resolution mass spectrometry in combination with 13C-isotope labeling identified that the main by-products of E2 metabolism were dimers, trimers, and tetramers, which are consistent with radical-driven C–C and/or C–O–C covalent coupling pathway, involving the initial enzymatic production of phenoxy radical intermediates and then the successive oxidative-oligomerization of radical intermediates. The formation of oligomers dramatically reduced the estrogenic activity of E2. Additionally, CC-ThLac also exhibited high-efficiency metabolism capability toward E2 in the natural water and pig manure, with more than 94.4% and 91.0% of E2 having been metabolized, respectively. These findings provide a broad prospect for the clean biotechnological applications of Trametes hirsuta La-7 in estrogen-contaminated ecosystems.

Metabolism of 17β-estradiol by Novosphingobium sp. ES2-1 as probed via HRMS combined with 13C3-labeling

This study investigated the biodegradation and metabolic mechanisms of 17β-estradiol (E2) by Novosphingobium sp. ES2-1 isolated from the activated sludge in a domestic sewage treatment plant (STP). It could degrade 97.1% E2 (73.5 μmol/L) in 7 d with a biodegradation half-life of 1.29 d. E2 was initially converted to estrone (E1), then to 4-hydroxyestrone (4-OH-E1), before subsequent monooxygenation reactions cleaved 4-OH-E1 into a metabolite with long-chain ketones structure (metabolite P8). However, when 4-OH-E1 was cleaved through the 4,5-seco pathway, the resulting phenol ring cleavage product could randomly condense with NH3 to yield a pyridine derivative, accompanied by the uncertain loss of a carboxy group at C4 before the condensation. The derivative was further oxidized into the metabolites with both pyridine and long-chain ketones structure (metabolite N5) through a similar formation mechanism as for P8 performed. This research presents several novel metabolites and shows that E2 can be biodegraded into the metabolite with long-chain structure through three optional pathways, thereby reducing E2 contamination.

CYP1B1 prevents proteasome-mediated XIAP degradation by inducing PKCε activation and phosphorylation of XIAP

Cytochrome P450 1B1 (CYP1B1) is a key enzyme that catalyzes the metabolism of 17β-estradiol (E2) into catechol estrogens, such as 2-hydroxyestradiol (2-OHE2) and 4-hydroxyestradiol (4-OHE2). CYP1B1 is related to tumor formation and is over-expressed in a variety of cancer cells. In particular, CYP1B1 is highly expressed in hormone-related cancers such as breast cancer, ovarian cancer, or prostate cancer compared to other cancers. However, the detailed mechanisms involving this protein remain unclear. In this study, we demonstrate that CYP1B1 affects X-linked inhibitor of apoptosis protein (XIAP) expression. When CYP1B1 was over-expressed in cells, there was significant increase in the XIAP protein level, whereas the XIAP mRNA level was not affected by CYP1B1 expression. Treatment with 4-OHE2, mainly formed by CYP1B1 activity, also increased XIAP protein levels, whereas treatment with 2-OHE2 did not have a significant effect. Treatment with 4-OHE2 significantly prevented proteasome-mediated XIAP degradation. In addition, phosphorylation of XIAP on serine 87, which is known to stabilize XIAP, was up-regulated by 4-OHE2, indicating that 4-OHE2 affects XIAP stability through XIAP phosphorylation. We also found that phosphorylation of protein kinase C (PKC)ε, which is required for XIAP phosphorylation, increased when cells were treated with 4-OHE2. In summary, our data show that CYP1B1 may play an important role in preventing ubiquitin-proteasome-mediated XIAP degradation through the activation of PKCε signaling in cancer cells.

Cholestasis-induced bile acid elevates estrogen level via farnesoid X receptor–mediated suppression of the estrogen sulfotransferase SULT1E1

The liver is the main site of estrogen metabolism, and liver disease is usually associated with an abnormal estrogen status. However, little is known about the mechanism underlying this connection. Here, we investigated the effects of bile acid (BA)-activated farnesoid X receptor (FXR) on the metabolism of 17β-estradiol (E2) during blockage of bile flow (cholestasis). Correlations between BA levels and E2 concentrations were established in patients with cholestasis, and hepatic expression profiles of key genes involved in estrogen metabolism were investigated in both WT and FXR-/- mice. We found that the elevated E2 level positively correlated with BA concentrations in the patients with cholestasis. We further observed that bile duct ligation (BDL) increases E2 levels in mouse serum, and this elevation effect was alleviated by deleting the FXR gene. Of note, FXR down-regulated the expression of hepatic sulfotransferase SULT1E1, the primary enzyme responsible for metabolic estrogen inactivation. At the molecular level, we found that FXR competes with the protein acetylase CREB-binding protein (CBP) for binding to the transcription factor hepatocyte nuclear factor 4α (HNF4α). This competition decreased HNF4α acetylation and nuclear retention, which, in turn, repressed HNF4α-dependent SULT1E1 gene transcription. These findings suggest that cholestasis induces BA-activated FXR activity, leading to downstream inhibition of SULT1E1 and hence impeding hepatic degradation of estrogen.

Novel p-carborane-containing multitarget anticancer agents inspired by the metabolism of 17β-estradiol

The female hormone 17 β-estradiol (E2) is synthesized from estrone by steroid sulfatase (STS), and metabolized into 2-methoxyestradiol (2-ME), whereby the biological activity of the latter is substantially different from that of E2. Based on the metabolic pathways of E2, a carborane-containing 2-ME mimic (1c) and its derivatives (1 and 2) were designed and synthesized as novel multitarget anticancer agents. Bissulfamate 1f exhibited potent STS-inhibitory activity and tubulin-polymerization-inhibitory activity. Moreover, the cell-growth-inhibitory (CGI) activity of 1f was similar to that of 2-ME in a panel screening against 39 human cancer cell lines. Accordingly, 1f should be a promising perspective therapeutic agent for hormone-dependent breast tissue.

The endocrine disrupting alkylphenols and 4,4′-DDT interfere with estrogen conversion and clearance by mouse liver cytosol

Endocrine disrupting chemicals (EDCs) are ubiquitous compounds known for negative impacts on reproductive functions and for increasing cancer risk. EDCs are believed to cause the harmful effects in part through their inappropriate low-affinity binding to steroid receptors and other possible non-receptor mediated paradigms, however there is a need to further elucidate other mechanisms involving the direct and indirect impact of EDCs on reproductive functions.We examined the metabolism of 17β-estradiol (E2) and estrone (E1) by cell-free hepatic cytosol in the presence of alkylphenols (nonylphenol/NP and 4-tert-octylphenol/tOP), Dichlorodiphenyltrichloroethane (4,4′-DDT) and other EDCs. Tandem liquid chromatography mass spectrometry was utilized to quantitatively assess the impact of each EDC on estrogen clearance, inter-conversions and downstream metabolism by mouse liver cytosol.The results revealed that NP and tOP (0.1–3μg/mL) significantly reduced the hepatic cytosol clearance and biotransformation of estrogens with inclination for accumulating E2, the stronger estrogen form, than E1. Alkylphenols also caused up to a 34-fold increase in the E2/E1 ratio possibly by suppressing the hepatic E2→E1 conversion by 17β-hydroxysteroid dehydrogenase (17βHSD) types 2, 4 while displaying a weaker inhibition of E1→E2 conversion by type 1, 17βHSD. On the other hand, the pesticide 4,4′-DDT was a weaker inhibitor of clearance of estrogens by the cytosol preparations when compared to alkylphenols, whereas chemicals such as phthalates and atrazine were ineffective.Our data suggest that exposure to NP, tOP and DDT can indirectly increase the estrogenic load by suppressing the hepatic clearance of estrogens and by elevating the E2/1 ratio and could therefore increase the risk of reproductive lesions.

Metabolism of androstenone, 17β-estradiol and dihydrotestosterone in primary cultured pig hepatocytes and the role of 3β-hydroxysteroid dehydrogenase in this process.

Steroids metabolism plays an important role in mammals and contributes to quality of pig meat. The main objective of this study was to identify metabolites of androstenone, 17β-estradiol and dihydrotestosterone using primary cultured pig hepatocytes as a model system. The role of 3β-hydroxysteroid dehydrogenase (3βHSD) in regulation of steroid metabolism was also validated using trilostane, a specific 3βHSD inhibitor. Steroid glucuronide conjugated metabolites were detected by liquid chromatography time of flight mass spectrometry (LC-TOF-MS). 3βHSD enzyme was essential for metabolism of androstenone to 5α-androst-16-en-3β-ol, which then formed androstenone glucuronide conjugate. Metabolism of 17β-estradiol was accompanied by formation of glucuronide-conjugated estrone and glucuronide-conjugated estradiol. The ratio of the two metabolites was around 5∶1. 3βHSD enzyme was not involved in 17β-estradiol metabolism. 5α-Dihydrotestosterone-17β-glucuronide was identified as a dihydrotestosterone metabolite, and this metabolism was related to 3βHSD enzyme activity as demonstrated by inhibition study.

Chronic exposure of killifish to a highly polluted environment desensitizes estrogen-responsive reproductive and biomarker genes

Reproductive and endocrine disruption is commonly reported in aquatic species exposed to complex contaminant mixtures. We previously reported that Atlantic killifish (Fundulus heteroclitus) from the chronically contaminated Newark Bay, NJ, exhibit multiple endocrine disrupting effects, including inhibition of vitellogenesis (yolk protein synthesis) in females and false negative vitellogenin biomarker responses in males. Here, we characterized the effects on estrogen signaling and the transcriptional regulation of estrogen-responsive genes in this model population. First, a dose–response study tested the hypothesis that reproductive biomarkers (vtg1, vtg2, chg H, chg Hm, chg L) in Newark Bay killifish are relatively less sensitive to 17β-estradiol at the transcriptional level, relative to a reference (Tuckerton, NJ) population. The second study assessed expression for various metabolism (cyp1a, cyp3a30, mdr) and estrogen receptor (ER α, ER βa, ER βb) genes under basal and estrogen treatment conditions in both populations. Hepatic metabolism of 17β-estradiol was also evaluated in vitro as an integrated endpoint for adverse effects on metabolism. In the third study, gene methylation was evaluated for promoters of vtg1 (8 CpGs) and vtg2 (10 CpGs) in both populations, and vtg1 promoter sequences were examined for single nucleotide polymorphism (SNPs). Overall, these studies show that multi-chemical exposures at Newark Bay have desensitized all reproductive biomarkers tested to estrogen. For example, at 10ng/g 17β-estradiol, inhibition of gene induction ranged from 62% to 97% for all genes tested in the Newark Bay population, relative to induction levels in the reference population. The basis for this recalcitrant phenotype could not be explained by a change in 17β-estradiol metabolism, nuclear estrogen receptor expression, promoter methylation (gene silencing) or SNPs, all of which were unaltered and normal in the Newark Bay population. The decreased transcriptional sensitivity of estrogen-responsive genes is suggestive of a broad effect on estrogen receptor pathway signaling, and provides insight into the mechanisms of the endocrine disrupting effects in the Newark Bay population.

Cellular Metabolism of Estradiol in Models for Select Molecular Subtypes of Clinical Breast Cancer

The mitogenic ovarian steroid hormone 17β-estradiol (E2) is associated with progression of Estrogen Receptor Positive (ER+) breast cancer, and ER represents a major therapeutic target for endocrine therapy. In addition to the ER dependent signal transduction, cellular oxidative metabolism of the ER ligand E2 generates several metabolites with pleotropic growth modulatory effects on breast cancer cells, providing valuable leads for novel therapeutic approach. The molecular classification of clinical breast cancer has defined cancer subtypes based on differential expression of the genes for hormones and growth factor receptors, thereby facilitating subtype targeted therapeutic interventions. However, de novo or acquired resistance to conventional endocrine and targeted small molecule based treatment limits the therapeutic efficacy and promotes therapy resistant disease progression. These aspects emphasize the need for identification of new efficacious non-toxic lead compounds. The present review summarizes critical experiments conducted to i) develop and optimize human tissue derived cell culture models for select molecular subtypes of clinical breast cancer, ii) determine the status of homeostatic growth control, cellular metabolism of 17β-estradiol (E2) and cancer risk in the developed models, and iii) evaluate the therapeutic efficacy and identify possible mechanisms of action of select herbal extracts/phyto-chemical. Additionally, this review discusses the evidence for the role of E2 metabolism in breast carcinogenesis and therapy. The data generated from the cell culture experiments demonstrate that the models for select molecular sub-types exhibit aberrant hyperproliferation, altered cellular metabolism of E2 and enhanced cancer risk. Select mechanistically distinct herbal extracts and natural phyto-chemical at their respective maximum cytostatic concentrations modulate cellular metabolism of E2 favoring generation of anti-proliferative metabolites and inhibit anchorage independent growth, thus reducing cancer risk. Collectively, these data validate the present mechanism based cell culture approach to identify and prioritize novel efficacious lead compounds for subtype targeted therapy of clinical breast cancer.

Abstract 263: Differential Effects of 17ß-Estradiol and 16a-Hydroxyestrone in Oxidative Stress and Proliferative Responses in Human Pulmonary Artery Smooth Muscle Cells - Implications in Pulmonary Arterial Hypertension

Women develop pulmonary arterial hypertension (PAH) more frequently than men. This may relate, in part, to metabolism of 17β-estradiol (E2), leading to formation of the deleterious metabolite, 16α-hydroxyestrone (16α OHE1), which plays a role in the remodelling of pulmonary arteries. Molecular mechanisms whereby 16αOHE1 influences PASMC remodelling are unclear but ROS may be important, since oxidative stress has been implicated in the pathogenesis of PAH. We hypothesised that E2 and 16αOHE1 leads to Nox-induced ROS production, which promotes PASMC damage. Cultured PASMCs were stimulated with either E2 (1nM) or 16αOHE1 (1nM) in the presence/absence of EHT1864 (100μM, Rac1 inhibitor) or tempol (antioxidant; 10μM). ROS production was assessed by chemiluminescence (O2-) and Amplex Red (H2O2). Antioxidants (thioredoxin, peroxiredoxin 1 and NQ01), regulators of Nrf2 (BACH1, Nrf2) and, marker of cell growth (PCNA) were determined by immunoblotting. E2 increased O2- production at 4h (219 ± 30% vs vehicle; p<0.05), an effect blocked by EHT1864 and tempol. E2 also increased H2O2 generation (152 ± 4%; p<0.05). Thioredoxin, NQ01 and peroxiredoxin1 (71 ± 6%; 78 ± 9%; 69 ± 8%; p<0.05 respectively) levels were decreased by E2 as was PCNA expression (72 ± 2%; p<0.05). 16αOHE1 exhibited a rapid (5 min) and exaggerated increase in ROS production (355 ± 41%; p<0.05), blocked by tempol and EHT1864. This was associated with an increase in Nox4 expression (139 ± 11% vs vehicle, p<0.05). 16αOHE1 increased BACH1, (129 ± 3%; p<0.05), a competitor of Nrf2, which was decreased (92 ± 2%). In contrast, thioredoxin expression was increased by 16aOHE1 (154 ± 22%; p<0.05). PCNA (150 ± 5%) expression was also increased after exposure to 16αOHE1. In conclusion, E2 and 16αOHE1 have differential effects on redox processes associated with PASMC growth. Whereas E2 stimulates ROS production in a slow and sustained manner without effect on cell growth, 16αOHE1 upregulates Nox4 with associated rapid increase in ROS generation and downregulation of antioxidant systems, affecting proliferation. Our findings suggest that E2 -derived metabolites may promote a pro-proliferative PASMC phenotype through Nox4-derived ROS generation. These deleterious effects may impact on vascular remodeling in PAH.

In vitro metabolism of hydroxylated polybrominated diphenyl ethers and their inhibitory effects on 17β-estradiol metabolism in rat liver microsomes

Hydroxylated polybrominated diphenyl ethers (OH-PBDEs) have emerged as contaminants of environmental concerns because they pose potential risks to human and animal health. The purpose of this study was to investigate the in vitro metabolism of OH-PBDEs and their potential inhibition against 17β-estradiol (E2) metabolism. Rat liver microsomes were used as a source of P450 enzymes in an in vitro metabolism study of OH-PBDEs. Inhibition of E2 metabolism and kinetic study were performed by incubating with rat liver microsomes in the presence of OH-PBDEs. The obtained data clearly demonstrated that OH-PBDEs, especially those congeners with lower bromination, could be metabolized to bromophenol and diOH-PBDEs. The less metabolic rate of OH-PBDEs was observed with the increasing number of bromine substituents. OH-PBDEs with hydroxyl group and bromine adjacent to the ether bridge showed faster metabolic rates. In addition, the results showed non-competitive inhibition of E2 metabolism by OH-PBDEs with IC(50) values in the range from 13.7 to 55.2 μM. The most potent OH-PBDE inhibitor was found to be 3'-OH-BDE-100. The inhibitory potencies for OH-PBDEs were significantly higher than those of parent PBDE and methoxylated metabolites, providing the evidence that PBDEs exerted estrogenic activity in part by their hydroxylated metabolites. OH-PBDEs exhibited large differences in their capacity to be metabolized and to inhibit E2 metabolism in rat liver microsomes. The finding might increase our understanding of healthy risk associated with PBDEs in human and wildlife.

P3-09-04: Comparative Preventive Efficacy of Aqueous Extracts from Lycium Barbarum Bark and Fruit on Estrogen Receptor Positive Human Mammary Carcinoma MCF-7 Cells.

Abstract Background: Chemotherapy, selective estrogen receptor modulators and aromatase inhibitors represent major treatment options for estrogen receptor positive (ER+) clinical breast cancer. These modalities frequently exhibit acquired tumor resistance and adverse systemic toxicity. Natural herbs are increasingly being used in the integrative support of breast cancer patients undergoing conventional treatment modalities and for prevention purposes. We previously reported favorable findings for the fruit of Lycium barbarum (LB), known commonly as goji berry, in the prevention for ER+ breast cancer (Nutrition & Cancer 61:408–414, 2009). The present study compares the preventive efficacy of non-fractionated aqueous extracts from the bark of LB (LBB) and the fruit of LB (LBF) on a cell culture model for ER + breast cancer. Material and Methods: The ER+ human mammary carcinoma derived MCF-7 cells represented the model. Anchorage dependent growth, anchorage independent colony formation and cellular metabolism of 17β-estradiol (E2) represented the mechanistic bases for efficacy. Results: MCF-7 cells grown in serum depleted medium retained their responsiveness to E2, exhibiting E2 stimulated promotion of anchorage dependent growth and anchorage independent colonies. LBB was much more potent than LBF as evidenced by a 95% reduction of IC50 from 0.38% for LBF to 0.02% for LBB, and a 95% reduction of maximum cytostatic concentration (MCC) from 1% for LBF to 0.05% for LBB. At their respective MCC, LBB treatment and LBF treatment produced a 93% and an 89% decrease, respectively, in the number of anchorage independent colonies. Also, LBB produced a 6.8 fold increase in 2-hydroxyestrone (2-OHE1), a 40% decrease in 16a-hydroxyestrone (16a-OHE1) and a 3.7 fold increase in estriol (E3) formation. The corresponding values for LBF were 3.9, 33 and 10.5. LBB treatment resulted in a 16.3 fold increase in 2-OHE1:16a-OHE1 ratio and a 2 fold increase in E3:16a-OHE1 ratio, while LBF treatment produced a 6.0 fold increase and a 2.9 fold increase, respectively, in these endocrine biomarkers. These data suggest that the preventive efficacy of LBB may predominantly be due to up-regulation of the anti-proliferative 2-OHE1 formation, while accelerated conversion of the pro-mitogenic 16α-OHE1 to the mitogenically inert E3 is the case with LBF. Discussion: Growth inhibitory profiles of LBB and LBF may in part be due to their distinct chemical composition and their complementary actions on E2 metabolism. The superior sensitivity of LBB relative to LBF, together with its non-toxic nature, suggests the feasibility of its use in the prevention of human breast cancer. A prevention trial to assess its efficacy and to establish relevant dose schedule is warranted. This study validates a mechanism based approach to identify and prioritize efficacious herbal extracts for the prevention of ER+ breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-09-04.

Anti-proliferative effects of Chinese herb Cornus officinalis in a cell culture model for estrogen receptor‑positive clinical breast cancer

Selective estrogen receptor modulators and a combination of mechanistically distinct chemotherapeutic agents represent conventional therapeutic interventions for estrogen receptor-positive (ER+) clinical breast cancer. Long-term treatment with these agents is associated with acquired tumor resistance and other adverse side effects that impact on patient compliance. Herbal medicines are being widely used in complementary and alternative medicine. However, long-term safety and efficacy of the use of herbal medicines, as well as their interaction with conventional endocrine and chemotherapeutic drug regimens remain largely unknown. The present study utilized a human cell culture model for ER+ clinical breast cancer to examine the potential therapeutic efficacy of an aqueous extract prepared from the fruit of popular Chinese herb Cornus officinalis (CO), also known as Fructus cornii. The human mammary carcinoma-derived MCF-7 cell line represented the model. Status of anchorage-independent growth and cellular metabolism of 17β-estradiol (E₂) represented the quantitative end-point biomarkers for efficacy. MCF-7 cells adapted for growth in serum-depleted medium (0.7% serum, <1 nM E₂) retained their endocrine responsiveness as evidenced by growth promotion by physiological levels of E₂, and growth inhibition by the selective ER modulator tamoxifen at the clinically achievable concentrations. Treatment of MCF-7 cells with CO resulted in inhibition of E₂-stimulated growth in a dose-dependent manner. Similarly, CO treatment also produced a dose-dependent progressive reduction in the number of anchorage-independent colonies, indicating effective reduction of the carcinogenic risk. Treatment of MCF-7 cells with CO at a maximally effective cytostatic concentration resulted in a 5.1-fold increase in the formation of the anti-prolifertive E₂ metabolite 2-hydoxyestrone (2-OHE₁), a 63.6% decrease in the formation of the pro-mitogenic metabolite 16α-hydroxestrone (16-αOHE₁) and a 9.1% decrease in the formation of mitogenically inert metabolite estrone (E₃). These alterations led to a 14.5-fold increase in the 2-OHE₁:16α-OHE₁, and a 3.3-fold increase in the E₃:16α-OHE1 ratios. These data validate a rapid cell culture-based mechanistic approach to prioritize efficacious herbal medicinal products for long-term animal studies and future clinical trials on ER+ clinical breast cancer.

2-Methoxyestradiol: New perspectives in colon carcinoma treatment

Colon carcinoma represents a major problem in oncology, since this type of cancer responds poorly to conventional chemotherapy. Many groups are actively involved in the search of new experimental strategies to bypass this problem. We investigated the effects of 2-methoxyestradiol (2-ME), which derives from the NADPH-dependent cytochrome P450 metabolism of 17β-estradiol. This compound has raised much interest in the past few decades for its inhibitory effects on the growth of cancer cells of different origin; however, little is known about its use on colon carcinoma-derived cell lines. In the present study, we investigated the effects of 2-ME on cell proliferation and cell cycle of two human colon carcinoma cell lines, namely HCT116 and SW613-B3. Our results showed a net anti-proliferative effect of 2-ME on both cell lines, which is accompanied by cell cycle arrest; moreover, we demonstrated that 2-ME is able to induce apoptosis as well as autophagy. This body of evidence points out that 2-ME could be considered as a promising tool against colon carcinoma.

Cytochrome P450-mediated 17β-estradiol metabolism in zebrafish (Danio rerio)

Cytochrome P4501 (CYP1) and CYP3A proteins are primarily responsible for the metabolism of 17beta-estradiol (E(2)) in mammals. We have cloned and heterologously expressed CYP1A, CYP1B1, CYP1C1, CYP1C2, CYP1D1, and CYP3A65 from zebrafish (Danio rerio) to determine the CYP-mediated metabolism of E(2) in a non-mammalian species. Constructs of each CYP cDNA were created using a leader sequence from the bacterial ompA gene to allow appropriate expression in Escherichia coli without 5' modification of the gene. Membrane vesicles were purified, and functional CYP protein was verified using carbon monoxide difference spectra and fluorescent catalytic assays with the substrates 7-ethoxyresorufin and 7-benzyloxy-4-(trifluoromethyl)-coumarin. Rates of in vitro E(2) metabolism into 4-hydroxyE(2) (4-OHE(2)), 2-hydroxyE(2) (2-OHE(2)), and 16alpha-hydroxyE(1) (16alpha-OHE(1)) metabolites were determined by gas chromatography/mass spectrometry. The 2-OHE(2) metabolite was produced by all CYPs tested, while 4-OHE(2) was only detected following incubation with CYP1A, CYP1B1, CYP1C1, and CYP1C2. The 16alpha-OHE(1) metabolite was only produced by CYP1A. The highest rates of E(2) metabolism were from CYP1A and CYP1C1, followed by CYP1C2. CYP1B1, CYP1D1, and CYP3A65 had low rates of E(2) metabolism. E(2) metabolism by zebrafish CYP1A, CYP1C1, and CYP1C2 produced similar ratios of 4-OHE(2) to 2-OHE(2) as previous studies with mammalian CYP1As. CYP1B1 formed the highest ratio of 4-OHE(2) to 2-OHE(2) metabolites. Contrary to mammals, these results suggest that fish CYP1A and CYP1C proteins are primarily responsible for E(2) metabolism, with only minor contributions from CYP3A65 and CYP1B1. Similar to mammals, 2-OHE(2) is the predominant metabolite from CYP-mediated E(2) metabolism in fish, suggesting that all vertebrate species produce the same major E(2) metabolite.

In vitro and in vivo analysis of fatty acid effects on metabolism of 17β-estradiol and progesterone in dairy cows

Some studies have reported improved reproductive performance with dietary fat supplementation. This study examined effects of fatty acids with different lengths, or desaturation, or both, on metabolism of estradiol (E2) and progesterone (P4) in bovine liver slice incubations (experiments 1 and 2) and in vivo (experiment 3). In experiment 1, effects of fatty acids C16:0 (palmitic acid), C16:1 (palmitoleic acid), C18:1 (oleic acid), and C18:3 (linolenic acid) were evaluated at 30, 100, and 300 μM on P4 and E2 metabolism in vitro. In experiment 2, stearic acid (C18:0) and C18:3 were evaluated in the same incubation conditions. In experiment 1, all of the fatty acids had some significant inhibitory effect on metabolism of P4, E2, or both (300 μM C16:0 on E2; 100 μM C16:1 on E2; 300 μM C16:1 on both P4 and E2; 300 μM C18:1 on P4; and 100 and 300 μM C18:3 on both P4 and E2). In experiment 2, C18:3 (100 and 300 μM) but not C18:0 decreased P4 and E2 metabolism. Overall, the most profound increase (∼60%) in half-life of P4 and E2 was observed with incubations of 300 μM C18:3 in both in vitro experiments. Based on these in vitro results, in experiment 3 linseed oil (rich in C18:3) was supplemented into the abomasum and acute effects on metabolism of E2 and P4 were evaluated. Cows (n=4) had endogenous E2 and P4 minimized (corpus luteum regressed, follicles aspirated) before receiving continuous intravenous infusion of E2 and P4 to analyze metabolic clearance rate for these hormones during abomasal infusion of saline (control) or 70mL of linseed oil every 4h for 28h. Linseed oil infusion increased C18:3 in plasma by 46%; however, metabolic clearance rate for E2 and P4 were similar for control cows compared with linseed-treated cows. Thus, in vitro experiments indicated that E2 and P4 metabolism can be inhibited by high concentrations of C18:3. Nevertheless, in vivo, linseed oil did not acutely inhibit E2 and P4 metabolism, perhaps because insufficient C18:3 concentrations (increased to ∼8 μM) were achieved. Further research is needed to determine the mechanism(s) of fatty acid inhibition of P4 and E2 metabolism and to discover practical methods to mimic this effect in vivo.

Intracellular metabolism of 17β-estradiol in cultured transgenic V79 cells: Metabolite profile and impact on cellular stress response

Intracellular metabolism of 17β-estradiol in cultured transgenic V79 cells: Metabolite profile and impact on cellular stress response