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The present study describes the microbial transformation of anabolic drugs, metenolone acetate (1), and epiandrosterone (6). Three new metabolites, 6β,17β-dihydroxy-1-methyl-3-oxo-5α-androst-1-en (2), 5α,15α-dihydroxy-1-methyl-3-oxo-1-en-17-yl acetate (3), 15β-hydroxy-1-methyl-3-oxo-5α-androst-1,4-dien-17-yl acetate (4), and a known metabolite, 17β-hydroxy-1-methyl-4-androstadiene-3-one (5) were obtained by biotransformation of metenolone acetate (1) via Trametes hirsuta mushroom. Metabolites 7, and 8 were obtained from the incubation of epiandrosterone (6) with Cunninghamella blakesleeana. While bioconversion of compound 6 with Aspergillus alliaceus yielded seven known metabolites 9-15. Modern spectroscopic techniques were employed for the structure elucidation of biotransformed products. All compounds were evaluated for their aromatase inhibitory activity. Among them, new metabolite 3 exhibited a significant human placental aromatase activity with an IC 50  = 19.602 ± 0.47 µM, as compared to standard anti-cancer drug exemestane (IC 50  = 0.232 ± 0.031 µM), whereas, metabolite 5 (IC 50 = 0.0049 ± 0.0032 µM) exhibited a very potent activity. While substrate 6, and metabolites 2, 7, and 9 were found inactive. Aromatase plays a key role in the biosynthesis of estrogen hormone, responsible for cancer cell proliferation. Its inhibition is therefore targeted for the treatment of ER + breast cancer. Further structural modifications (lead optimization) of compound 3 can lead to more potent aromatase inhibition for possible treatment of ER + breast cancer., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

• Formestane is a substance prohibited in sports by WADA. • There are differences on formestane metabolism depending on the administration route. • Metabolic ratios allow to discriminate the foremestane administration route. • Applying metabolic ratio may avoid unnecessary IRMS confirmation analyses. Formestane (4-hydroxyandrost-4-ene-3,17-dione, 4OH-AED) is an aromatase inhibitor prohibited in sports. In recent years, it has been demonstrated that it can also originate endogenously by the hydroxylation in C4 position of androstenedione. Thus, the use of isotope ratio mass spectrometry (IRMS) is mandatory according to the World Antidoping Agency (WADA) to discriminate endogenous from synthetic origin. In a previous work and after oral administrations of formestane (4OH-AED), the ratio between the main formestane metabolite (4α-hydroxyepiandrosterone; 4OH-EA) and formestane parent compound could help to identify the endogenous origin, avoiding unnecessary and costly IRMS confirmations. In the present work, we investigated whether the same criteria could also be applied after transdermal applications. Six volunteers were transdermally treated once with formestane. Urine samples were collected for 120 h postadministration and analyzed by gas chromatography coupled to mass spectrometry (GC–MS and GC–MS/MS). Formestane and its major metabolites were monitored. The kinetic profile of formestane and its main metabolites was found different between oral and transdermal application. A shift on the excretion of the metabolites compared to formestane itself that can be observed after the oral administration, is absent after the transdermal one. This makes that a simple criteria cannot be applied to differentiate the endogenous from the synthetic origin based on metabolic ratios. The ratio between 4-hydroxyepiandrosterone and 4-hydroxyandrosterone (4OH-A) can be used to differentiate the route of administration. Ratios higher than one (4OH-EA/4OH-A > 1) are diagnostic of an oral administration. This allows to correctly interpret the 4OH-EA/4OH-AED ratio as proposed in our previous investigation. The results of this work demonstrate that the use of appropriate biomarkers (metabolic ratios) helps to reach correct conclusions without using complex and costly instrumentation approaches. [ABSTRACT FROM AUTHOR]

Placenta produces progesterone and estradiol for maintaining pregnancy. Two critical enzymes are responsible for their production: 3β-hydroxysteroid dehydrogenase 1 (HSD3B1) that catalyzes the formation of progesterone from pregnenolone and aromatase that catalyzes the production of estradiol from testosterone. Fungicide ziram may disrupt the placental steroid production. In the present study, we investigated the effects of ziram on steroid formation in human placental cell line JEG-3 cells and on HSD3B1 and aromatase in the human placenta. Ziram did not inhibit progesterone production in JEG-3 cells and HSD3B1 activity at 100 μM. Ziram was a potent aromatase inhibitor with the half maximal inhibitory concentration (IC 50 ) value of 333.8 nM. When testosterone was used to determine the mode of action, ziram was found to be a competitive inhibitor. Docking study showed that ziram binds to the testosterone binding pocket of the aromatase. In conclusion, ziram is a potent inhibitor of human aromatase. [ABSTRACT FROM AUTHOR]

[Display omitted] • A steroidal spiro morpholinone was prepared in six steps starting from estrone. • The spiro morpholinone and its hydroxychloroacetamide derivative precursor are potent antiproliferative agents. • Docking simulation shows key interactions between the synthesized compounds and the human aromatase enzyme. Estrogens play a pivotal role in the development of estrogen-dependent breast cancer and other hormone-dependent disorders. A common strategy to overcome the pathological effects of estrogens is the use of aromatase inhibitors (AIs), which bind to the enzyme and prevent the union with the natural substrate, decreasing the amount of estrogens produced. Several AIs have been developed, including inhibitors with a steroidal backbone and a nitrogen heterocycle in their structure. Encouraged by the notable results presented by current and clinical steroidal drugs, herein we present the synthesis of a steroidal spiro morpholinone derivative as a plausible aromatase inhibitor. The morpholinone derivative was synthesized over a six-step methodology starting from estrone. The title compound and its hydroxychloroacetamide derivative precursor were evaluated for their antiproliferative profile against estrogen-dependent and independent solid tumor cell lines: A549, HBL-100, HeLa, SW1573, T-47D and WiDr. Both compounds exhibited a potent antiproliferative activity in the micromolar range against the six cancer cell lines, with the hydroxychloroacetamide derivative precursor being a more potent inhibitor (GI 50 = 0.25–2.4 µM) than the morpholinone derivative (GI 50 = 2.0–11 µM). Furthermore, both compounds showed, in almost all cases, better GI 50 values than the steroidal anticancer drugs abiraterone and galeterone. Docking simulations of the derivatives were performed in order to explain the experimental biological activity. The results showed interactions with the iron heme (derivative 3) and important residues of the steroidal binding-site (Met374) for the inhibition of human aromatase. A correlation was found between in vitro assays and the score obtained from the molecular docking study. [ABSTRACT FROM AUTHOR]

Bioconversion of the aromatase inhibitor formestane (4-hydroxyandrost-4-ene-3,17-dione) ( 1 ) by the fungus Rhizopus oryzae ATCC 11145 resulted in a new minor metabolite 3,5α-dihydroxyandrost-2-ene-4,17-dione ( 2 ) and the known 4β,5α-dihydroxyandrostane-4,17-dione ( 3 ) as the major product. The structural elucidation and bioactivities of these metabolites are reported herein. Molecular modeling studies of the interactions between these metabolites and the aromatase protein indicated that acidic (D309), basic (R115), polar (T310), aromatic (F134, F221, and W224), and non-polar (I133, I305, A306, V369, V370, L372, V373, M374, and L477) amino acid residues contribute important interactions with the steroidal substrates. These combined experimental and theoretical studies provide fresh insights for the further development of more potent aromatase inhibitors. [ABSTRACT FROM AUTHOR]

Breast cancer’s hormonal dependence is well known and has been so for a long time. However in the last two decades great advances have been made in understanding the local metabolism of steroids within tissue. In the form of aromatase inhibition this is already one of the mainstays of breast cancer therapy. This review aims to summarise briefly what is known in terms of the metabolism of C18 steroids but perhaps more importantly to touch on the new developments regarding the importance of the metabolism of androgens and glucocorticoids in breast tissue. It is our hope that this review should provide the reader with a “birds eye view” of the current state of knowledge regarding localised steroid metabolism in the breast. [ABSTRACT FROM AUTHOR]

Plasma estrogen measurement with use of radioimmunoassays has been instrumental in the development of aromatase inhibitors for endocrine therapy of postmenopausal breast cancer. However, due to low plasma estrogen concentrations in postmenopausal women, direct radioimmunoassays lack the sensitivity required. While certain laboratories have developed highly sensitive assays for research purposes revealing plasma estrogen suppression consistent with results from tracer studies, such assays are time and labor-consuming due to need for pre-analytical chromatographic purification, sample concentration and sometimes conversion of precursors to products. While novel chromatographic methods involving mass spectrometry analysis are likely to replace such radioimmunoassays in the future, so far a limited number of laboratories have developed suitable assays with a detection limit (around 1 pM) that is required for analyzing plasma estrogen levels in patients during treatment with potent aromatase inhibitors. [ABSTRACT FROM AUTHOR]

There are many questions which cannot be answered without a very sensitive estradiol assay. A recombinant cell bioassay (RCBA) for estradiol was developed in 1994. The sensitivity of the bioassay is 0.02–0.2 pg/ml (0.07–0.7 pmol/L), more than 20 times more sensitive than commercial RIAs and 10 times more sensitive than newer mass spectrometry assays. The RCBA for estradiol opened the door to study low levels of estradiol equivalents (EE) across the physiological spectrum of life from prepubertal children through menopause and across the spectrum from normal physiology, in boys as well as girls, to pathology, including: premature thelarche; estradiol suppression in children treated with GnRH analogues for precocious puberty; aromatase inhibition in boys with growth hormone deficiency; the differences between oral and transdermal routes of estrogen administration in girls with Turner’s syndrome; women with breast cancer treated with aromatase inhibitors; and women with urogenital atrophy treated with low dose vaginal estrogen. A bioassay also allows study of endocrine disruptors, like phytoestrogens and other environmental compounds, which are relevant to public health and alternative medicine options. This paper reviews the assay and the last 20 years of applications. A bioassay for estrogen has a role because measuring biological effect is theoretically useful, increasing the understanding of physiology in addition to biochemical levels, giving different information than other assays, and opening the door to measure very low levels of estrogen activity in both humans and the environment. [ABSTRACT FROM AUTHOR]

Using β -sitosterol and stigmasterol as precursor materials, a concise and efficient hemisynthesis of aromatase inhibitors: testololactone and testolactone was accomplished in a well-established reaction scheme. It involves highly effective Oppaneur oxidation of both β -sitosterol as well as stigmasterol to generate the required enone moiety in ring ‘A’ of the desired steroid system. The Oppaneur oxidation products of both β -sitosterol and stigmasterol were then subjected to oxidative cleavage of the side chain to produce 4-androstene-3,17-dione. Baeyer–Villiger oxidation of 4-androstene-3,17-dione using m -CPBA yielded testololactone. Dehydrogenation of 4-androstene-3,17-dione using phenylselenyl chloride in ethyl acetate followed by selenoxide elimination with H 2 O 2 in dichloromethane furnished androstenedienone. Baeyer–Villiger oxidation of the resulting androstenedienone yielded the desired testolactone (overall yield 33%). This expeditious reaction scheme may be exploited for the bulk production of aromatase inhibitors (especially testolactone marketed under the brand name Teslac) from the most abundant and naturally occurring phytosterols like β -sitosterol. [ABSTRACT FROM AUTHOR]

Highlights: [•] Introduction of a lower concentration limit of 25ng/mL. [•] A reference population was used to determine endogenous 13C values for formestane. [•] Excretion studies with testosterone and androstenedione. [ABSTRACT FROM AUTHOR]

Highlights: [•] Design and synthesis of new 7α-allylandrostanes as strong aromatase inhibitors. [•] Structure–activity relationships were carried-out. [•] The 1-ene analogues are more potent than the 3-deoxo analogues. [•] The C-17 carbonyl group is required to achieve maximum aromatase inhibition. [Copyright &y& Elsevier]

Abstract: Long term exposure to estrogens is associated with an increased risk of breast cancer. The precise mechanisms responsible for estrogen mediated carcinogenesis are not well understood. The most widely accepted theory holds that estradiol (E2), acting through estrogen receptor alpha (ERα), stimulates cell proliferation and initiates mutations arising from replicative errors occurring during pre-mitotic DNA synthesis. The promotional effects of E2 then support the growth of cells harboring mutations. Over a period of time, sufficient numbers of mutations accumulate to induce neoplastic transformation. Laboratory and epidemiological data also suggest that non-receptor mediated mechanisms resulting from the genotoxic effects of estrogen metabolites are involved in breast cancer development. This manuscript critically reviews existing data implicating both ER-dependent and -independent mechanisms. The weight of evidence supports the possibility that both mechanisms are involved in the carcinogenic process. In addition, estrogen metabolites likely modulate stem cell functionality and cancer progression. The roles of ER dependent and independent actions in the carcinogenic process are pertinent to the consideration of breast cancer preventative agents as anti-estrogens block only receptor mediated pathways whereas the aromatase inhibitors block both. [Copyright &y& Elsevier]

Abstract: Estrogens, responsible for the growth of hormone-dependant breast cancer are biosynthesized from androgens involving aromatase enzyme in the last rate limiting step. Inhibition of aromatase is an efficient approach for the prevention and treatment of breast cancer. Novel 4-phenylthia derivatives (2, 3 and 7) have been synthesized as aromatase inhibitors. The synthesized compounds (2, 3 and 7) exhibited noticeable enzyme inhibiting activity. Kinetics study of these compounds (2, 3, and 7) showed negligible inhibition of the enzyme under conditions conducive for irreversible inhibition of the enzyme. Introduction of unsaturation at C-4, C-1 & 4 or C-4 & 6 (compounds 5, 9 and 11) was observed to not be an effective strategy for entrancing aromatase inhibiting activity in 17-oxo-16β-carbonitrile derivatives. The d-seco derivatives (13–15 and 17) having unsaturation at C-4, C-1 & 4 or C-4 & 6 along with carbonitrile function in ring-D showed complete loss of aromatase inhibiting activity. [Copyright &y& Elsevier]

Abstract: As a part of our investigations into the structural–activity relationship studies of a novel class of medicinally active 16-substituted steroids, several new 16-imidazolyl substituted steroidal derivatives have been synthesized and pharmacologically evaluated in the current study. The new steroidal analogues 5, 6, 8, 9, 11 and 12 exhibited moderate cytotoxic effects in sixty cancer cell lines derived from nine cancers types. The imidazolyl substituted steroidal derivatives 6 (DPJ-RG-1241) and 7 (RB-401) were obtained as the powerful inhibitors of aromatase with IC50 =0.18μM and IC50 =0.168μM, respectively, approximately 1.2 and 1.4times more potent in comparison to standard drug exemestane. The bis-quaternary steroids 13 and 14 displayed potent skeletal muscle relaxant properties. An affinity constant of 0.007μM was observed for compound 14 on frog rectus abdominis muscle preparation and 13 displayed a very high anticholinesterase activity Ki =25nM, approximately 115-fold higher in comparison to standard drug galanthamine (Ki =2.9μM). [Copyright &y& Elsevier]

Abstract: Exemestane is an aromatase enzyme complex inhibitor. Its metabolism in humans is not fully described and there is only one known metabolite: 17β-hydroxyexemestane. In this work, excretion studies were performed with four volunteers aiming at the detection of new exemestane metabolites in human urine by gas chromatography coupled to mass spectrometry (GC–MS) after enzymatic hydrolysis and liquid–liquid extraction. Urine samples collected from four volunteers were analyzed separately. The targets of the study were mainly the 6-exomethylene oxidized metabolites. Two unreported metabolites were identified in both free and glucuconjugated urine fractions from all four volunteers, both of them were the result of the 6-exomethylene moiety oxidation: 6ξ-hydroxy-6ξ-hydroxymethylandrosta-1,4-diene-3,17-dione (metabolite 1) and 6ξ-hydroxyandrosta-1,4-diene-3,17-dione (metabolite 2). Furthermore, only in glucoconjugated fractions from all volunteers, one metabolite arising from the A-ring reduction was identified as well, 3ξ-hydroxy-5ξ-androst-1-ene-6-methylene-17-one (metabolite 3). The molecular formulae of all these metabolites were ascertained by the determination of exact masses using gas chromatography coupled to high resolution mass spectrometry (GC–HRMS). Moreover, all metabolites were confirmed using an alternative derivatization with methoxyamine and MSTFA/TMS-imidazole. [Copyright &y& Elsevier]

Abstract: Lung cancer is the leading cause of cancer mortality in both women and men worldwide but gender differences exist in their clinical and biological manifestations. In particular, among life time non-smoker, female are far more likely to develop lung carcinoma than male. Recent studies demonstrated that estrogens are synthesized in situ in both male and female lung cancers through aromatase, suggesting that sex steroid may contribute to the pathogenesis and development of lung carcinoma. In addition, human lung carcinomas have been recently demonstrated to be frequently associated with expression of estrogen receptors in both male and female patients and a lower expression of aromatase was reported to be associated with better prognosis. Preclinical studies further demonstrated that aromatase inhibitor (AI) suppressed the lung tumor growth both in vitro and in vivo. These findings all suggest a potential role of intratumoral aromatase in biological behavior of non-small cell lung cancer (NSCLC), the most common form of human lung malignancy. Therefore, AIs may become viable therapeutic options for disease management in NSCLC patients but further studies are definitely required to obtain a better understanding of the potential roles of intratumoral aromatase expression as a predictive biomarker for clinical outcome in these NSCLC patients. [Copyright &y& Elsevier]

Abstract: Treatment with anti-estrogens or aromatase inhibitors (AI) is the main therapeutic strategy used against estrogen receptor ERα-positive breast cancer. Resistance to these therapies presents a major challenge in the management of breast cancer. Little is known about ERβ in breast carcinogenesis. Our aim in this study is to examine potential novel strategies utilizing ERβ activity to overcome AI resistance. We provide evidence that ERβ agonist can reduce the growth of AI-resistant breast cancer cells. Our data further confirm that therapeutic activation of ERβ by DPN, an ERβ agonist, blocks letrozole-resistant tumor growth in a xenograft model. Interestingly, DPN exerted tumor growth inhibition only in the presence of the AI letrozole, suggesting that combination therapy including ERβ activators and AI may be used in the clinical setting treating AI resistant breast cancer. An increase in ERβ levels, with diminished ERα/ERβ ratio, was observed in the tumors from mice treated with DPN/letrozole combination compared to single agents and control. Decreased Cyclin D1 and increased CyclinD1/CDK inhibitors p21 and p27 levels in DPN/letrozole treated tumors were observed, suggesting that the combination treatment may inhibit tumor growth by blocking G1/S phase cell cycle progression. Our data show a decrease in MAPK phosphorylation levels without affecting total levels. In addition to providing evidence suggesting the potential use of ERβ agonists in combination with letrozole in treating AI resistant breast cancer and prolonging sensitivity to AI, we also provide mechanistic evidence supporting the role of ERβ in altering the expression profile associated with resistance. [Copyright &y& Elsevier]

Abstract: Phase III aromatase inhibitors (AIs) are proving successful in the treatment of hormone-dependent postmenopausal breast cancer. Side-effects associated with total body aromatase inhibition have prompted new research into the development of breast-specific AIs. The identification of tissue- and disease-specific usage of aromatase promoters has made the inhibition of aromatase at the transcriptional level an interesting approach. We have previously demonstrated that AMPK-activating drugs, including metformin, were potent inhibitors of aromatase expression in primary human breast adipose stromal cells (hASCs). This study examines the promoter-specific effects of metformin on inhibiting aromatase expression in hASCs. Tumour-associated promoters PII/PI.3 were activated using forskolin (FSK)/phorbol ester (PMA), whereas normal adipose associated promoter PI.4 was activated using dexamethasone (DEX)/tumour necrosis factor-α (TNFα). Results demonstrate that metformin significantly decreased the FSK/PMA-, but not the DEX/TNFα-mediated expression of total aromatase at concentrations of 10, 20, and 50μM (P ≤0.05). Using PCR to amplify promoter-specific transcripts of aromatase, it appears that the inhibition of the FSK/PMA-mediated expression of aromatase is due to decreases in PII/PI.3-specific transcripts, whereas no effect of metformin is observed on any promoter-specific transcript, including PI.4, in DEX/TNFα-treated hASCs. This report therefore supports the hypothesis that metformin would act as a breast-specific inhibitor of aromatase expression in the context of postmenopausal breast cancer. [Copyright &y& Elsevier]

Abstract: Despite effective treatments for oestrogen receptor-positive breast cancers, drug resistance is common and remains a significant clinical challenge. Targeting tumour vasculature by blockade of the vascular endothelial growth factor (VEGF) has proved successful in a variety of cancers. Phase III clinical trials of bevacizumab in combination with chemotherapy showed some efficacy in breast cancer. Concomitant targeting of the VEGF and oestrogen signalling pathways has the potential to provide enhanced therapeutic benefit in oestrogen receptor-positive breast cancer, and this strategy is under evaluation in clinical trials. This article summarises the rationale for this approach and clinical studies so far. [Copyright &y& Elsevier]

Abstract: Evidence that aromatase expression in tumor-associated breast stroma is elevated, provides a rationale for use of aromatase inhibitors (AIs) in breast cancer treatment. However, regulation of local aromatase expression in cancer-free breast stroma is poorly understood. Recent clinical work indicates that stromal cells in dense breast tissue tend to express higher levels of aromatase than their counterpart from non-dense tissue. Consistent with the clinical observation, our cell culture-based study indicated that cell density, cell shape, and extracellular matrix (ECM) significantly induced stromal aromatase expression via a distinct signal transduction pathway. In addition, we identified a number of cell surface markers that are commonly associated with aromatase-expressing stromal cells. As mammographic density is one of the strongest and most prevalent risk factors for breast cancer, these findings provide a potential mechanistic link between alterations in tissue composition of dense breast tissue and increased stromal aromatase expression. Further exploration of the in vitro model system may advance understanding of an important problem in breast cancer biology. [Copyright &y& Elsevier]

Abstract: Aromatase is the rate-limiting enzyme in estrogen biosynthesis. As a cytochrome P450, it utilizes electrons from NADPH–cytochrome P450 reductase (CPR) to produce estrogen from androgen. Estrogen is a key factor in the promotion of hormone-dependent breast cancer growth. Aromatase inhibitors (AIs) are drugs that block estrogen synthesis, and are widely used to treat estrogen-dependent breast cancer. Structure–function experiments have been performed to study how CPR and AIs interact with aromatase to further the understanding of how these drugs elicit their effects. Our studies have revealed a strong interaction between aromatase and CPR, and that the residue K108 is situated in a region important to the interaction of aromatase with CPR. The published X-ray structure of aromatase indicates that the F221, W224 and M374 residues are located in the active site. Our site-directed mutagenesis experiments confirm their importance in the binding of the androgen substrate as well as AIs, but these residues interact differently with steroidal inhibitors (exemestane) and non-steroidal inhibitors (letrozole and anastrozole). Furthermore, our results predict that the residue W224 also participates in the mechanism-based inhibition of exemestane, as time-dependent inhibition is eliminated with mutation on this residue. Together with previous research from our laboratory, this study confirms that W224, E302, D309 and S478 are important active site residues involved in the suicide mechanism of exemestane against aromatase. [Copyright &y& Elsevier]

Abstract: Oestrogens in breast cancers are derived from both uptake from the circulation and in situ synthesis. Third generation aromatase inhibitors (AIs) effectively block aromatase activity within the breast. The effects of AIs on the molecular biology of breast cancers may be monitored in patients given neoadjuvant therapy. Changes in tumour gene expression associated with AIs is influenced by time of drug exposure and gene expression profiles may provide important information on tumour response/ resistance to AIs. [Copyright &y& Elsevier]

Abstract: Aromatase inhibitors (AIs) have become the front-line choice for treatment of ER+ breast cancer. Nevertheless, although patients are responsive initially, they may acquire resistance and become unresponsive to further treatment. In addition, approximately 25% of breast cancers do not express the estrogen receptor (ERα) and consequently, are innately resistant to endocrine therapy. We have investigated the mechanisms associated with this lack of treatment response using xenograft models. We found that in cells and tumors that acquired resistance to the AI letrozole therapy, expression of the ER was reduced whereas growth factor signally was enhanced, including a marked increase in HER2 expression. Treatment with trastuzumab (HER2 antibody) resulted in a significant down-regulation of HER2 and p-MAPK as well as restoration of ERα expression. Thus, when trastuzumab was added to letrozole treatment at the time of tumor progression, there was significantly prolonged tumor suppression compared to trastuzumab or letrozole alone. This suggests that inhibition of both HER2 and ERα signaling pathways are required for overcoming resistance and restoring treatment sensitivity. ER negative tumors are innately resistant to endocrine therapy. Repression of the ERα has been found to be due to epigenetic modifications such as increased methylation and histone deacetylation. We found that entinostat (ENT), a histone deacetylase inhibitor (HDACi), activated not only expression of ERα but also aromatase in MDA-MB-231 ER-negative breast cancer cells, resulting in their ability to respond to estrogen and letrozole. Treatment with ENT in combination with letrozole significantly reduced tumor growth rate in xenografts compared to control tumors (p <0.001). ENT plus letrozole treatment also prevented the colonization and growth of MDA-MB-231 cells in the lung with a significant reduction (p <0.03) in both visible and microscopic foci. These results provide a strong indication for possible use of AIs in combination with HDAC inhibitors for the treatment of ER-negative breast cancer. [Copyright &y& Elsevier]

Abstract: In postmenopausal breast cancers, the increase in aromatase expression observed in tumour associated adipose stromal cells is mediated via the upregulation of promoter II (PII) transcription. Factors such as PGE2 which are secreted from breast carcinomas induce PII expression. The orphan nuclear receptor LRH-1/NR5A2 is one of the critical downstream transcriptional mediators of this effect. The aim of the current study was to determine whether LRH-1 could bind directly to PII and whether the suppression of LRH-1 expression could inhibit aromatase expression in human adipose stromal fibroblasts. Chromatin immunoprecipitation demonstrated endogenous LRH-1 occupancy on PII under basal conditions and with treatment with forskolin and phorbol 12-myristate 13-acetate (PMA). To assess the impact of LRH-1 knockdown on FSK/PMA mediated PII expression, cells were transfected with shRNA targeted against LRH-1 (shLRH-1) and treated with forskolin and PMA. A decrease in LRH-1, PII and total aromatase mRNA transcripts was observed in shLRH-1 transfected cells compared to controls under basal and treatment conditions. The results of this study support the hypothesis that suppression of LRH-1 may potentially be beneficial in the tissue specific regulation of aromatase expression in post menopausal breast cancer. [Copyright &y& Elsevier]

Abstract: Considerable heterogeneity exists amongst oestrogen receptor positive (ER+ve) breast cancer in both its molecular profile and response to therapy. Attempts to better define variation amongst breast tumours have led to the definition of four main “intrinsic” subtypes of breast cancer with two of these classes, Luminal A and B, composed almost entirely of ER+ve cancers. In this study we set out to investigate the significance of intrinsic subtypes within a group of ER+ve breast cancers treated with neoadjuvant anastrozole. RNA from tumour biopsies taken from 104 postmenopausal women before and after 2 weeks treatment with anastrozole was analyzed on Illumina 48K microarrays. Gene-expression based subtypes and risk of relapse (ROR) scores for tumours pre- and post-treatment were determined using the PAM50 method. Amongst pre-treatment samples, all intrinsic subtypes were found to be present, although luminal groups were represented most highly. Luminal A and B tumours obtained similar benefit from treatment, as measured by the proportional fall in the proliferation marker Ki67 upon treatment (mean suppression=75.5% vs 75.7%). Tumours classified as basal and Her2-like showed poor reductions in Ki67 upon treatment. Residual Ki67 staining after two weeks remained higher in the Luminal B group. ROR score was significantly associated with anti-proliferative response to AI and with clinical response. These results suggest that in the short-term, Luminal A and B tumours may gain similar benefit from an AI but that the higher residual Ki67 level seen in Luminal B is indicative of poorer long term outcome. [Copyright &y& Elsevier]

Abstract: Aromatase (CYP19A1) is an integral membrane enzyme that catalyzes the removal of the 19-methyl group and aromatization of the A-ring of androgens. All human estrogens are synthesized from their androgenic precursors by this unique cytochrome P450. The crystal structure of active aromatase purified from human placenta has recently been determined in complex with its natural substrate androstenedione in the high-spin ferric state of heme. Hydrogen bond forming interactions and tight packing hydrophobic side chains closely complement puckering of the steroid backbone, thereby providing the molecular basis for the androgenic specificity of aromatase. In the crystal, aromatase molecules are linked by a head-to-tail intermolecular interaction via a surface loop between helix D and helix E of one aromatase molecule that penetrates the heme-proximal cavity of the neighboring, crystallographically related molecule, thus forming in tandem a polymeric aromatase chain. This intermolecular interaction is similar to the aromatase-cytochrome P450 reductase coupling and is driven by electrostatics between the negative potential surface of the D–E loop region and the positively charged heme-proximal cavity. This loop-to-proximal site link in aromatase is rather unique—there are only a few of examples of somewhat similar intermolecular interactions in the entire P450 structure database. Furthermore, the amino acids involved in the intermolecular contact appear to be specific for aromatase. Higher order organization of aromatase monomers may have implications in lipid integration and catalysis. [Copyright &y& Elsevier]

Abstract: Oestrogens regulate normal behaviour and have been implicated in modulating pathological behaviour such as obsessive compulsive disorder and neurological disorder such as Parkinsonism. Therefore, by regulating the expression of the oestrogen-synthesising enzyme, aromatase, we may identify what behaviour is regulated by oestrogen. Inhibition of aromatase either genetically or pharmacologically has been reported to induce sexual behaviour impairment, compulsive behaviour and susceptibility to neurodegeneration. [Copyright &y& Elsevier]

Abstract: Background: The third generation aromatase inhibitors (AIs) have become an established component of postmenopausal estrogen receptor positive breast cancer therapy. Unfortunately, up to half of AI-users experience the AI-induced musculoskeletal syndrome (AIMSS) (arthralgia, carpal tunnel syndrome, start pains, stiffness, etc.), which can severely impact quality of life and treatment compliance. We have previously demonstrated that loss of hand grip strength is part of AIMSS and involves tenosynovial changes and fluid retention in joints. Review of literature and hypothesis generating findings: Our presentation during this AI-symposium focuses on available literature regarding AIMSS with new data from a prospective study generating a hypothesis for its pathogenesis. Profound estrogen deprivation as a consequence of AI-use is thought to be the underlying reason but the exact pathway remains unknown. A potential hypothesis is that the growth hormone/insulin like growth factor-I (GH/IGF-I) pathway may be involved. This possibility is based on the non-linear association between body mass index (BMI) and loss of hand grip strength that we observed. It appears that in lean and overweight women, hand grip strength decreases most following intake of an AI. This observation suggests an underlying biological process which probably evolves through the GH/IGF-I pathway, controlled by sex steroids. Conclusion: Estrogen deprivation leads to incapacitating AIMSS and hampers treatment compliance. In our search for the missing link between ‘lowering postmenopausal estrogens’ and ‘arthralgia’ we here report on AI-induced changes in grip strength by BMI which we believe are hypothesis generating for an effect of AIs on the GH/IGF-I axis. This needs to be explored prospectively. [Copyright &y& Elsevier]

Abstract: Aromatase inhibitors (AI) have improved the treatment of oestrogen receptor positive (ER+) breast cancer. Despite the efficacy of these agents over 40% of patients relapse with endocrine resistant disease. Here we describe an in vitro model of acquired resistance to long-term oestrogen deprivation (LTED). The LTED cells retain expression of the ER and appear hypersensitive to oestrogen as a result of altered kinase activity. Furthermore analysis of temporal changes in gene expression during the acquisition of resistance highlight growth factor receptor pathways as key mediators of this adaptive process. [Copyright &y& Elsevier]

Abstract: Novel pyrazole and isoxazole derivatives (6–9) were synthesized as a aromatase inhibitors. Pyrazole was synthesized from hydrazine hydrate and isoxazoles from hydroxylamine hydrochloride under different conditions. Molecular docking studies were carried out for the synthesized compounds. The best score was obtained for the compound (9) followed by compound (6) while compound (8) afforded poorest of the score. Aromatase inhibitory activity for compound (6) having pyrazole ring at 2,3 position showed highest activity followed by nitrile derivative (9). Isomeric forms of isoxazole (7 and 8) showed very poor activity compared to fadrozole and aminoglutethimide. Preliminary kinetic studies have shown that both of the active compounds (6 and 9) are reversible inhibitors of the enzyme. [Copyright &y& Elsevier]

Abstract: This article describes the origins and evolution of “antiestrogenic” medicines for the treatment and prevention of breast cancer. Developing drugs that target the estrogen receptor (ER) either directly (tamoxifen) or indirectly (aromatase inhibitors) has improved the prognosis of breast cancer and significantly advanced healthcare. The development of the principles for treatment and the success of the concept, in practice, has become a model for molecular medicine and presaged the current testing of numerous targeted therapies for all forms of cancer. The translational research with tamoxifen to target the ER with the appropriate duration (5 years) of adjuvant therapy has contributed to the falling national death rates from breast cancer. Additionally, exploration of the endocrine pharmacology of tamoxifen and related nonsteroidal antiestrogen (e.g. keoxifene now known as raloxifene) resulted in the laboratory recognition of selective ER modulation and the translation of the concept to use raloxifene for the prevention of osteoporosis and breast cancer. However, the extensive evaluation of tamoxifen treatment revealed small but significant side effects such as endometrial cancer, blood clots and the development of acquired resistance. The solution was to develop drugs that targeted the aromatase enzyme specifically to prevent the conversion of androstenedione to estrone and subsequently estradiol. The successful translational research with the suicide inhibitor 4-hydroxyandrostenedione (known as formestane) pioneered the development of a range of oral aromatase inhibitors that are either suicide inhibitors (exemestane) or competitive inhibitors (letrozole and anastrozole) of the aromatase enzyme. Treatment with aromatase inhibitors is proving effective and is associated with reduction in the incidence of endometrial cancer and blood clots when compared with tamoxifen and there is also limited cross resistance so treatment can be sequential. Current clinical trials are addressing the value of aromatase inhibitors as chemopreventive agents for postmenopausal women. [Copyright &y& Elsevier]

Abstract: Starting from D-seco derivatives of 5-androstene 1–3, the D-homo lactones, 4 and 5, were synthesized. By the Oppenauer oxidation and/or by dehydration of 4 and 5 with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) or 2,3,5,6-tetrachloro-1,4-benzoquinone (chloranil), the corresponding D-lactones 6–12 were obtained. The structures of 6 and 10 were unambiguously proved by the appropriate X-ray structural analysis. Anti-aromatase assay showed that tested compounds possess inhibition potency, however, two to four times smaller (IC50 from 0.2 to 0.7μM, respectively) in comparison to aminoglutethimide (AG). [Copyright &y& Elsevier]

Aromatase (P450arom, CYP19) catalyzes the aromatization reaction that converts androgens to estrogens. Although human P450arom has been readily purified from placenta, its hydrophobic properties and instability has hampered detailed characterization. Utilizing a N-terminal replacement (MARQSFGRGKL, derived from CYP2C11), we successfully modified this unstable enzyme into stable forms. Based on a known polymorphism, we created two constructs, NmA264C and NmA264R having cysteine or arginine at position 264. The recombinant P450arom NmA264R was expressed in Escherichia coli (350–400 nmol/L culture) primarily by coexpression with molecular chaperones GroES/GroEL while NmA264C was expressed (240 nmol/L culture) only in the presence of chloramphenicol. Although NmA264C was recovered only in the membrane fraction, approximately 14% of NmA264R was recovered in the cytosolic fraction, suggesting that NmA264R is more hydrophilic than NmA264C. NmA264R was highly purified to the specific content 13.6 nmol P450/mg protein. Purified P450arom NmA264R converted androstenedione to estrone with Vmax 12.4 nmol/(min nmol) and Km 0.26 μM, and testosterone to estradiol with Vmax 52.2 nmol/(min nmol) and Km 10.9 μM. Because of the increased stability of NmA264R, we could unambiguously determine properties of human P450arom by optical and electron paramagnetic resonance spectroscopy. The purified protein was a typical low-spin form, which was converted to a high-spin form when androstenedione was added. The rhombicity of substrate-bound forms was higher than that reported for other P450s, an interesting characteristic of human P450arom. The highly stable and active P450arom NmA264R sets the stope for detailed structure/function analyses of this important member of the P450 superfamily. [Copyright &y& Elsevier]

Abstract: The third-generation aromatase inhibitors are an important class of drugs for use in adjuvant therapy for postmenopausal women with resected estrogen receptor positive breast cancer. Multiple large prospective randomized trials have established their value in this setting and provided guidance for their use in clinical management. This review will outline the trials that have provided evidence on the value of the aromatase inhibitors in the adjuvant setting as well as the ongoing trials that will expand our knowledge of how to use them most effectively. [Copyright &y& Elsevier]

Abstract: Endocrine therapy has been the most effective treatment modality for hormone receptor positive breast cancer. However, its efficacy has been limited by either de novo or acquired resistance. Recent data indicates that activation of the phosphatidylinositol 3-kinase (PI3K) signaling is associated with the poor outcome luminal B subtype of breast cancer and accompanied by the development of endocrine therapy resistance. Importantly, inhibition of PI3K pathway signaling in endocrine resistant breast cancer cell lines reduces cell survival and improves treatment response to endocrine agents. Interestingly, mutations in PIK3CA, the alpha catalytic subunit of the class IA PI3K, which renders cells dependent on PI3K pathway signaling, is the most common genetic abnormality identified in hormone receptor positive breast cancer. The synthetic lethality observed between estrogen deprivation and PI3K pathway inhibition in estrogen receptor positive (ER+) breast cancer cell lines provides further scientific rational to target both estrogen receptor and the PI3K pathway in order to improve the outcome of ER+ breast cancer. [Copyright &y& Elsevier]

• Steroid synthases were detected in the C 2 C 12 myoblasts cultured in vitro. • Mechanical stretch was the external factor that affected the expression of the estrogen synthesis-related enzymes. • Cyclic mechanical stretching promotes cell proliferation and estrogen production. Skeletal muscle contains estrogens and estrogen synthesis-related enzymes. However, it has not been reported whether myoblasts cultured in vitro also express these enzymes. The purpose of the current study was to address these issues and to explore the effects of mechanical stretch on the enzyme system. The in vitro cultured C 2 C 12 mouse myoblasts were divided into the control, stretch, testosterone and stretch plus testosterone groups. Cells in the stretch and stretch plus testosterone groups were mechanically stretched with the Flexercell cell stress loading device at an amplitude of 10% and in a frequency of 0.5 Hz for 8 h. Cells in the testosterone and stretch plus testosterone groups were incubated with 100 nM testosterone for 24 h before distraction. Following the treatments, cell proliferation and estradiol levels, as well as the expressions of 17β-hydroxysteroid (17β-HSD), 3β-hydroxysteroid (3β-HSD) and aromatase were analyzed. Compared to the control, the cell proliferation in all experimental groups increased significantly, the estradiol levels in the mechanically stretched groups were significantly higher, and, moreover, the estradiol levels were positively correlated with the cell proliferation (r = 0.615, p < 0.01). Additionally, analyses of aromatase protein and mRNA showed that, compared to the control, their levels were significantly increased upon stretching and testosterone exposure. Similarly, the protein and mRNA levels of both 3β-HSD and 17β-HSD in the stretched cells differed significantly from the control. In the presence of aromatase and 5α-reductase inhibitors, the protein and mRNA levels of these enzymes altered significantly compared to the control. Conclusions: Steroid synthases were detected in the C 2 C 12 myoblasts cultured in vitro , the synthesized estrogen was closely related to the cell proliferation, and mechanical stretch was the external factor that affected the expression of the estrogen synthesis-related enzymes. [ABSTRACT FROM AUTHOR]